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Nitric oxide and vascular reactivity in developing zebrafish, Danio rerio
Abstract
We used a newly developed digital motion analysis video technique to study the effects of nitric oxide (NO) and epinephrine on the early larval arterial and venous vasculature of zebrafish. Application of the NO donor sodium nitroprusside resulted in a significant increase in both the venous and arterial vessel diameters, whereas N(G)-nitro-L-arginine methyl ester caused a significant decrease in the same diameters. Thus our results show that both the venous and arterial vasculature of the 5- and 6-day-old zebrafish larvae are influenced by endogenously produced NO. By use of immunohistochemistry, NO synthase immunoreactivity was demonstrated in endothelial cells of the dorsal vein. Local application of epinephrine onto the dorsal artery had no effect on vessel diameter. However, if the embryos were preincubated with N(omega)-nitro-L-arginine methyl ester, addition of epinephrine resulted in a significant reduction in both arterial and venous vessel diameters. Thus this study provides increasing evidence that before a functional autonomic innervation of the peripheral vascular system, vascular tone in larval tissue is regulated by a complex interaction of vasoactive substances that are produced locally by vascular endothelial cells
... The pattern of vascular differentiation and growth through the developing tissues, presumably, is mostly determined by genetics, but blood flow is regulated in response to environmental cues and influences virtually all aspects of vasculature development, as discussed in Section 4 (Broemnimann et al., 2016;Goenezen et al., 2012;Johnson et al., 2013). As in the heart, receptors in the vasculature and endogenous signaling molecules become effective modulators of vascular tone, prior to functional innervation of the peripheral nervous system (Fritsche et al., 2000;Pelster et al., 2005). Although autonomic nervous control of the vasculature in fishes is not active until later in development, several chemical cell signals produced by vascular endothelial cells can influence vasculature tone by causing vessel dilation and contraction (Fritsche et al., 2000;Pelster et al., 2005;Sykes et al., 2016). ...
... As in the heart, receptors in the vasculature and endogenous signaling molecules become effective modulators of vascular tone, prior to functional innervation of the peripheral nervous system (Fritsche et al., 2000;Pelster et al., 2005). Although autonomic nervous control of the vasculature in fishes is not active until later in development, several chemical cell signals produced by vascular endothelial cells can influence vasculature tone by causing vessel dilation and contraction (Fritsche et al., 2000;Pelster et al., 2005;Sykes et al., 2016). Most prominently (at least based upon its frequency of study), nitric oxide (NO) is present as vessels develop, and likely plays a prominent role in the control of vascular tone in fish embryos (Bagatto, 2005;Eddy, 2005;Fritsche et al., 2000;Pelster et al., 2005). ...
... Although autonomic nervous control of the vasculature in fishes is not active until later in development, several chemical cell signals produced by vascular endothelial cells can influence vasculature tone by causing vessel dilation and contraction (Fritsche et al., 2000;Pelster et al., 2005;Sykes et al., 2016). Most prominently (at least based upon its frequency of study), nitric oxide (NO) is present as vessels develop, and likely plays a prominent role in the control of vascular tone in fish embryos (Bagatto, 2005;Eddy, 2005;Fritsche et al., 2000;Pelster et al., 2005). Nitric oxide contributes to vascular tone by signaling smooth muscles in the vascular endothelium to relax, causing vasodilation (Gutterman et al., 2016;Heiss et al., 2015;Pelster et al., 2005). ...
Embryonic, larval, and juvenile fish develop in environments that frequently present severe challenges, not just to maintain homeostasis, but to survive. Key to survival is a functional cardiovascular system, which transports respiratory gases, nutrients, and wastes in response to varying tissue needs. This review begins with consideration of how the heart and circulation progressively develop to replace respiration by simple diffusion across the surface area of young fishes, and how the circulation may function initially to aid angiogenesis rather than transport. The morphology and regulation of heart formation, including the heart tube, cardiac chambers and valves, and the cardiac conduction systems is discussed, as is myocardial differentiation. Similarly, the process of angiogenesis and the formation of vascular beds are outlined, with brief mention of the secondary circulation and the lymphatic system in developing fishes. A focus of the chapter is the ontogeny of cardiovascular regulation, including regulation of heart rate, blood pressure, stroke volume, cardiac output, and the peripheral vasculature. The cardiovascular system must respond to environmental variation, so the effects of temperature, oxygenation, and toxicants on the functioning of the cardiovascular system are explored. The chapter concludes with a discussion of ongoing and needed technological advances, and our emerging understanding of potential epigenetic influences on developing fishes.
... Amelio et al. (2008) reported the differential expression of NOS isoforms in the ancestral lungfish (Protopterus dolloi). Fritsche et al. (2000) reported the expression of eNOS in cardiac muscle cells and in dorsal vein of 3 and 5 day post fertilization (dpf) zebrafish larvae. emphasized the importance of NOS/NO in myocardial performance of fish hearts. ...
... Nevertheless, there is evidence from different groups indicating the presence of eNOS in fish on the basis of immunological data. Using monoclonal antibodies, eNOS-like immunoreactivity could be detected in the retina of white bass (Haverkamp et al., 1999), and the presence of eNOS has been reported in developing zebrafish (Fritsche et al., 2000). The eNOS-like immunoreactivity has also been reported for gill cells of Atlantic salmon (Salmo salar) (Ebbesson et al., 2005). ...
... mykiss) (Gallo and Civinini, 2001). Using immunohistochemistry, the expression of eNOS was also detected in cardiac muscle cells and in the dorsal vein of 3 and 5 dpf zebrafish larvae (Fritsche et al., 2000). ...
The nitric oxide (NO), a highly versatile and ubiquitous signaling molecule, is produced in the body by the oxidation' of L-arginine by catalytic action of one of the three isoforms of 'nitric oxide synthase (NOS) in the presence of molecular oxygen and NADPH. In the paradigm of adaptation, one universal regulator controlling the physiological systems and gene expression is NO molecule. NO governs an impressive number of physiological and pathophysiological functions. In mammals, it has been reported to be involved in many different physiological processes including cell proliferation, differentiation, vasodilation, neurotransmission, angiogenesis, apoptosis, and has anti-microbial and anti-tumoral activities and is also involved in secretion of hormones, motility of vascular and non-vascular smooth muscle and immune defence. In recent years~ there has been a growing body of evidences of NOS expression and physiological implication of NO in non-mammalian vertebrates. However, reports on NOS activity and the physiological roles of NO in ectothermic vertebrates such as in fish are relatively less compared to mammalian system. By cloning and sequencing studies, the presence of different isoform of NOS has been demonstrated in a number of fish species, both in adult and early developmental strategies. Various physiological functions of NO have also been demonstrated in certain fish species. Recently, the expression of different isoforms of NOS has also been demonstrated in two Indian air-breathing catfishes (Heteropneustes fossilis and Clarias batrachus) by our group and by few more groups. Further, more expression of NOS along with more production of NO under various environment constraints such as high environmental ammonia, desiccation stress and also under pathological conditions have been demonstrated in these two air-breathing catfish. This chapter reviews about the NO chemistry, its synthesis and also the implication of NO as a signalling molecule in various physiological and pathological conditions with a more emphasis on fish including the Indian air-breathing catfish.
... Even at 20 dpf few VSMCs are found in the caudal vein, probably as venous control of blood pressure (BP) in fish is less critical than in terrestrial animals where gravitational influences are greater (Santoro, Pesce, & Stainier, 2009). Despite this, variation in the degree of vascularization of different vessels in larvae is highly probable (Santoro et al., 2009), and previous studies have revealed responses after application of vasoconstrictors/ dilators (Fritsche, Schwerte, & Pelster, 2000;Watkins et al., 2012). ...
... Significant increases in DA diameter were also detected, and although the VSMC complement of the DA in young larval Zf is thought to be low (Santoro et al., 2009), vasoactive substances have previously been shown to affect their vessel diameter (Fritsche et al., 2000), perhaps via the contribution of endothelial constriction (Watkins et al., 2012). The effect of adrenaline on vertebrate vascular tone is a complex interplay between the predominantly vasoconstrictive effects of α1AR, and the vasodilatory role of β2ARs (Brodde, 1990). ...
... The effect of adrenaline on vertebrate vascular tone is a complex interplay between the predominantly vasoconstrictive effects of α1AR, and the vasodilatory role of β2ARs (Brodde, 1990). With this in mind, Fritsche et al. (2000) failed to detect changes in the dorsal vessels of 5-6 dpf Zf following adrenaline treatment, but blockade of the vasodilatory influence of NO signalling with L-NAME resulted in significant vasoconstriction. The contrasting vasodilation seen here could be explained by differences in the regional distribution of AR subtypes within the developing vasculature, as is the case with the prominence of β2ARs in human splanchnic and skeletal muscle vascular beds (Guimarães & Moura, 2001). ...
... Nos inhibitors did not completely abrogate the NO signal, suggesting that there are additional Nos-independent sources of NO in the developing embryo [85]. NO is involved in the control of vasculature during development [86][87][88] and stimulates angiogenesis and hematopoiesis [87,89]. The canonical NO signaling (NO activating sGC and catalyzing the synthesis of cGMP) targets several proteins, including Bone Morphogenetic Protein-4 (Bmp4), which is involved in determining the positioning of the heart during embryonic development [90]. ...
... NO donors (e.g., SNP) and Nos inhibitors (e.g., L-NAME) affect vascular resistance, confirming its putative vasodilatory function in fish [86,99,100]. The occurrence of perivascular nitrergic neurons, with Nos1 as a likely primary source of NO, innervating the vasculature of some teleost species, is also described [53,101]. ...
Nitric oxide (NO) is a key signaling molecule in almost all organisms and is active in a variety of physiological and pathological processes. Our understanding of the peculiarities and functions of this simple gas has increased considerably by extending studies to non-mammal vertebrates and invertebrates. In this review, we report the nitric oxide synthase (Nos) genes so far characterized in chordates and provide an extensive, detailed, and comparative analysis of the function of NO in the aquatic chordates tunicates, cephalochordates, teleost fishes, and amphibians. This comprehensive set of data adds new elements to our understanding of Nos evolution, from the single gene commonly found in invertebrates to the three genes present in vertebrates.
... mmHg in adult zebrafish) (Michel, 2020). Nevertheless, there seems to be a conserved response of the vasculature to the classical vasodilators and vasoconstrictors, as well as to a number of cardiovascular drugs (Fritsche et al., 2000;Margiotta-Casaluci et al., 2019). Treatment of zebrafish larvae with the NO donor sodium nitroprusside resulted in a significant increase in both arterial and venous vessel diameters, whereas application of NOS inhibitor N G -nitro-L-arginine methyl ester (L-NAME) led to a significant decrease in vessel diameters, which corresponds to responses in mammals. ...
... Treatment of zebrafish larvae with the NO donor sodium nitroprusside resulted in a significant increase in both arterial and venous vessel diameters, whereas application of NOS inhibitor N G -nitro-L-arginine methyl ester (L-NAME) led to a significant decrease in vessel diameters, which corresponds to responses in mammals. Similarly, in larvae pretreated with L-NAME to inhibit endogenously produced NO, addition of adrenaline resulted in vasoconstriction (Fritsche et al., 2000). A comparative analysis of the in vivo cardiovascular responses of zebrafish, rat, dog and human to three cardiovascular drugs, which modulate β-adrenergic and reninangiotensin systems (propranolol, losartan and captopril) showed that zebrafish and human responses were largely comparable in >80% of drug/endpoint combinations, demonstrating the translational power of zebrafish (Margiotta-Casaluci et al., 2019). ...
Mammalian models including non‐human primates, pigs and rodents have been used extensively to study the mechanisms of cardiovascular disease. However, there is an increasing desire for alternative model systems that provide excellent scientific value while replacing or reducing the use of mammals. Here, we review the use of zebrafish, Danio rerio, to study cardiovascular development and disease. The anatomy and physiology of zebrafish and mammalian cardiovascular systems are compared, and we describe the use of zebrafish models in studying the mechanisms of cardiac (e.g. congenital heart defects, cardiomyopathy, conduction disorders and regeneration) and vascular (endothelial dysfunction and atherosclerosis, lipid metabolism, vascular ageing, neurovascular physiology and stroke) pathologies. We also review the use of zebrafish for studying pharmacological responses to cardiovascular drugs and describe several features of zebrafish that make them a compelling model for in vivo screening of compounds for the treatment cardiovascular disease.
LINKED ARTICLES
This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc
... Simultaneously, we observed the increase in perfusion of paw vessels, which also points to vasodilatation. NO plays an important function in the cardiovascular system, not only in mammals but also in fish [38,39]. NOS was detected in endothelial cells of dorsal veins and in the hearts of zebrafish larvae. ...
... The application of NO donors such as sodium nitroprusside and isosorbide dinitrate to zebrafish larvae and rainbow trout alevins resulted in significant vasodilatation and a decrease of HR. L-NAME exerted opposite effects [38,40]. Similarly, we found that L-NAME increased HR, but also completely abolished the effects of PS after 24 and 48 h of exposure in embryonic and larval zebrafish, possibly by competing with L-arginine released from PS. ...
Protamine sulfate (PS) is the only available option to reverse the anticoagulant activity of unfractionated heparin (UFH), however it can cause cardiovascular and respiratory complications. We explored the toxicity of PS and its complexes with UFH in zebrafish, rats, and mice. The involvement of nitric oxide (NO) in the above effects was investigated. Concentration–dependent lethality, morphological defects, and decrease in heart rate (HR) were observed in zebrafish larvae. PS affected HR, blood pressure, respiratory rate, peak exhaled CO2, and blood oxygen saturation in rats. We observed hypotension, increase of HR, perfusion of paw vessels, and enhanced respiratory disturbances with increases doses of PS. We found no effects of PS on human hERG channels or signs of heart damage in mice. The hypotension in rats and bradycardia in zebrafish were partially attenuated by the inhibitor of endothelial NO synthase. The disturbances in cardiovascular and respiratory parameters were reduced or delayed when PS was administered together with UFH. The cardiorespiratory toxicity of PS seems to be charge–dependent and involves enhanced release of NO. PS administered at appropriate doses and ratios with UFH should not cause permanent damage of heart tissue, although careful monitoring of cardiorespiratory parameters is necessary.
... In fish, studies that report the physiological function of NO include, vasodilatation in Danio rerio and Oncorhynchus mykiss (Fritsche et al., 2000;Haraldsen et al., 2002), osmoregulation in certain teleosts (Evans, 2002), ion transport in Salmo salar (Ebbesson et al., 2005), and inhibitory effect on Na + -K + -ATPase activity in gills and kidney of Salmo trutta (Tipsmark and Madsen, 2003). NOS immunoreactivity in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract have been reported in Marthasterias glacialis by Martinez et al. (1994). ...
... However, the gene that encodes eNOS or eNOS nucleotide or protein sequences has not been reported. Nevertheless, studies to reveal protein expression have utilized heterologous mammalian eNOS antibodies to examine eNOS-immunoreactivity in different fish species which includes, vascular endothelial cells of the dorsal vein and in the heart of developing Danio rerio (Fritsche et al., 2000), posterior cardinal vein of Oncorhynchus mykiss (McNeill and Perry, 2006), gills of Salmo salar (Ebbesson et al., 2005), in the endothelial cells of the bulbus arteriosus of hybrid tilapia (Nile tilapia × Mozambique tilapia) (Wang et al., 2007), and the glomerular capillaries of lungfish, Protopterus dolloi (Amelio et al., 2008). Moreover, immunoblot analysis also has shown that the molecular weight of eNOS protein in different fish species is in the range of 135-140 kDa which is similar to that of mammalian eNOS protein (Amelio et al., 2006(Amelio et al., , 2008Cioni et al., 2002;McNeill and Perry, 2006). ...
The present study is concerned with the expression and localization of nitric oxide synthase (NOS) isoforms, nNOS, eNOS and iNOS in the epidermis and the gill epithelium of Chaca chaca by means of immunohistochemical techniques. nNOS immunoreactivity was observed in the outer layer epithelial cells of the epidermis, outer epithelium of gill filaments at their distal regions and in between the secondary lamellae. iNOS positive cells were observed at intervals in the epidermis from basal layer to superficial layer, in outer layers of epithelium of the gill filament and in epithelium of the secondary lamellae. The expression of eNOS is similar to that of iNOS in the gills. In addition, NOS activity was also observed in the taste buds in the epidermis. The expression of different NOS isoforms in C. chaca are associated to increase the adaptability and survivability of the fish in hypoxic condition, help in defence and ion regulation and sensory functions. The study could be useful to understand the expression of NOS isoforms in different fish tissues and their diverse role in relation to the physiology of the fish.
... Thus, the presence of Mb in the endothelial cells suggests its role in regulation of blood flow (Cossins et al., 2009). Autonomic contractile pericytes of the capillaries in mammals can regulate blood flow locally (Peppiatt et al., 2006) by stimulating NO production and breakdown (Fritsche et al., 2000) to cause pericyte relaxation and capillary dilation. It is well known that oxy form of Mb reacts rapidly with NO to yield the met form of Mb and nitrite. ...
... It is well known that oxy form of Mb reacts rapidly with NO to yield the met form of Mb and nitrite. A conversion of nitrite to NO has been reported in zebrafish (Jensen, 2007), and NO generated by the endothelial cells affects vascular function in zebrafish larvae (Fritsche et al., 2000). Therefore, the endothelial Mb might play a role as nitrite reductase (Cossins et al., 2009). ...
Myoglobin (Mb) is one of the most intensively studied intracellular respiratory muscle proteins. Since the discovery of the fascinating fact that Mb is not confined only to oxidative muscle tissues but also is co-localized in different non-muscle tissues of cyprinids, hypoxia tolerant cyprinids have been established as the model teleost. Mb both at mRNA and protein levels have been reported in this study for the first time from a number of muscle and non-muscle tissues of rainbow trout Oncorhynchus mykiss, a hypoxia intolerant species. Mb transcript levels were high in the heart and skeletal muscle, and were comparatively high in the gonad and gill among the non-muscle tissues. Western-blotting by using anti-rainbow trout Mb peptide rabbit antibody detected Mb protein in the muscles and several non-muscle tissues. By both RNA in situ hybridization and immunofluorescence, Mb was localized in the cardiomyocytes and oxidative muscle fibers. On the other hand, Mb both at mRNA and protein levels was restricted to the lamellar epithelial cells of the gill, epithelial layers of hepato-biliary duct, neurons and endothelial cells of brain, ooplasm of gonad, kidney tubules, endothelial cells, and epithelial layer of intestine. Neuroglobin isoform 1 and 2 mRNAs along with Mb mRNA were localized in the granular layer of cerebellum. Considering the previous data reported for cyprinids, the expression sites of Mb in the muscle and non-muscle tissues of teleost could be universal, where Mb concerted with the other globins might play meaningful physiological roles.
... There is still considerable controversy whether or not NO is produced by fish vascular endothelium and acts as a local messenger. NO, or NO donors such as sodium nitroprusside, are vasoconstrictor in Agnathans and Chondrichthyes (Evans, 2001;Evans and Harrie, 2001) and vasodilators in Osteichthyes (Conklin and Olson, 1994;Evans and Harrie, 2001;Fritsche et al., 2000;Jennings et al., 2004;Miller and Vanhoutte, 1986;Olson and Villa, 1991;Olson et al., 2000;Schwerte et al., 1999;Small et al., 1990;Smith et al., 2000). However, while studies in vivo or in perfused organ preparations have supported NO as an endotheliumderived dilator in Osteichthyes (Fritsche et al., 2000;Hylland and Nilsson, 1995;Mustafa and Agnisola, 199a;Schwerte et al., 1999;Soderstrom et al., 1995), studies on isolated blood vessels have failed to support an endothelial source of NO from any fish (Evans and Gunderson, 1998;Farrell and Johansen, 1995;Jennings et al., 2004;Olson et al., 2000) and the possibility remains that NO signaling from the vascular endothelium originated in Amphibia (Miller and Vanhoutte, 1986). ...
... NO, or NO donors such as sodium nitroprusside, are vasoconstrictor in Agnathans and Chondrichthyes (Evans, 2001;Evans and Harrie, 2001) and vasodilators in Osteichthyes (Conklin and Olson, 1994;Evans and Harrie, 2001;Fritsche et al., 2000;Jennings et al., 2004;Miller and Vanhoutte, 1986;Olson and Villa, 1991;Olson et al., 2000;Schwerte et al., 1999;Small et al., 1990;Smith et al., 2000). However, while studies in vivo or in perfused organ preparations have supported NO as an endotheliumderived dilator in Osteichthyes (Fritsche et al., 2000;Hylland and Nilsson, 1995;Mustafa and Agnisola, 199a;Schwerte et al., 1999;Soderstrom et al., 1995), studies on isolated blood vessels have failed to support an endothelial source of NO from any fish (Evans and Gunderson, 1998;Farrell and Johansen, 1995;Jennings et al., 2004;Olson et al., 2000) and the possibility remains that NO signaling from the vascular endothelium originated in Amphibia (Miller and Vanhoutte, 1986). ...
... Furthermore, there is evidence for endothelial NO signalling in teleost fish, which was obtained in perfused vascular beds in which vascular resistance was affected by the NO precursor L-arginine, and inhibition of NO synthase, the enzyme that generates NO (Nilsson and Söderström, 1997;Mustafa et al., 1997;Mustafa and Agnisola, 1998). The only study in teleost fish that has shown anatomically that an endothelial NO synthase is present is that of Fritsche et al. (2000), who showed immunoreactivity to endothelial NOS in developing zebrafish blood vessels. However, in some species of teleost fish, there is now convincing evidence that vasodilatory signalling molecules released by the endothelium are prostaglandins, rather than NO (Olson and Villa, 1991;Kågström and Holmgren, 1997;Park et al., 2000). ...
... This suggests that the dorsal aorta and intestinal vein of A. australis do in fact lack an eNOS. The only study to report the presence of eNOS in teleost blood vessels is that of Fritsche et al. (2000), who demonstrated using immunohistochemistry and a different eNOS antibody to that used in this study, that eNOS immunoreactivity was present in the dorsal vein of the developing zebrafish. Thus, there seems to be conflicting data on the presence of eNOS that could reflect a dichotomy between developing and adult fish. ...
This study investigated the mechanisms by which nitric oxide (NO) regulates the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis. NADPH diaphorase histochemistry and immunohistochemistry using a mammalian endothelial nitric oxide synthase (NOS)antibody could not demonstrate NOS in the endothelium of either blood vessel;however, NOS could be readily demonstrated in the endothelium of the rat aorta that was used as a control. Both blood vessels contained NADPH diaphorase positive nerve fibres and nerve bundles, and immunohistochemistry using a neural NOS antibody showed a similar distribution of neural NOS immunoreactivity in the perivascular nerves. In vitro organ bath physiology showed that a NO/soluble guanylyl cyclase (GC) system is present in the dorsal aorta and the intestinal vein, since the soluble GC inhibitor oxadiazole quinoxalin-1 (ODQ; 10–5 mol l–1)completely abolished the vasodilatory effect of the NO donor, sodium nitroprusside (SNP; 10–4 mol l–1). In addition, nicotine (3×10–4 mol l–1)mediated a vasodilation that was not affected by removal of the endothelium. The nicotine-mediated dilation was blocked by the NOS inhibitor, Nω-nitro-l-arginine (l-NNA;10–4 mol l–1), and ODQ(10–5 mol l–1). More specifically, the neural NOS inhibitor, Nω-propyl-l-arginine(10–5 mol l–1), significantly decreased the dilation induced by nicotine (3×10–4 mol l–1). Furthermore, indomethacin (10–5 mol l–1) did not affect the nicotine-mediated dilation,suggesting that prostaglandins are not involved in the response. Finally, the calcium ionophore A23187 (3×10–6 mol l–1) caused an endothelium-dependent dilation that was abolished in the presence of indomethacin. We propose the absence of an endothelial NO system in eel vasculature and suggest that neurally derived NO contributes to the maintenance of vascular tone in this species. In addition,we suggest that prostaglandins may act as endothelially derived relaxing factors in A. australis.
... After three days post-fertilization (dpf), zebrafish larvae the cardiomyocytes showed immunoreactivity to the antibody used for mammalian eNOS and, in response to the NO donor sodium nitropusside (SNP) and the NOS inhibitor nitro-L -arginine methyl ester ( L -NAME), the main axial vessels react with a significant change in the vessel diameter. Nos2b appears to be the main cause of vasodilation in zebrafish larvae [78]. Nos1 may be most likely the source of NO in perivascular nitrergic neurons that innervate the vasculature of some teleost species [68,71]. ...
Nitric Oxide (NO) is a free radical with numerous critical signaling roles in vertebrate physiology. Similar to mammals, in the teleost system the generation of sufficient amounts of NO is critical for the physiological function of the cardiovascular system. At the same time, NO amounts are strictly controlled and kept within basal levels to protect cells from NO toxicity. Changes in oxygen tension highly influence NO bioavailability and can modulate the mechanisms involved in maintaining the NO balance. While NO production and signaling appears to have general similarities with mammalian systems, the wide range of environmental adaptations made by fish, particularly with regards to differing oxygen availabilities in aquatic habitats, creates a foundation for a variety of in vivo models characterized by different implications of NO production and signaling. In this review, we present the biology of NO in the teleost cardiovascular system and summarize the mechanisms of NO production and signaling with a special emphasis on the role of globin proteins in NO metabolism.
... Studies have shown that a high concentration of AH in the body will be oxidized to produce many superoxide radicals, hydrogen peroxide, and other oxidizing substances (Sathler et al., 2014), which may lead to vascular endothelial oxidative injury and eventually induce thrombosis at last. In addition, it has also been shown that adrenalin-sensitive receptors are present in the vascular system and heart of zebrafish, and a high doses of epinephrine can cause a significant reduction in arterial and venous vessel diameter in 5-6 day old zebrafish (Fritsche et al., 2000), which may lead to embolism due to poor blood flow. ...
Thrombosis is a general pathological phenomenon during severe disturbances to homeostasis, which plays an essential role in cardiovascular and cerebrovascular diseases. Leonurine (LEO), isolated from Leonurus japonicus Houtt, showes a crucial role in anticoagulation and vasodilatation. However, the properties and therapeutic mechanisms of this effect have not yet been systematically elucidated. Therefore, the antithrombotic effect of LEO was investigated in this study. Hematoxylin-Eosin staining was used to detect the thrombosis of zebrafish tail. Fluorescence probe was used to detect the reactive oxygen species. The biochemical indexes related to oxidative stress (lactate dehydrogenase, malondialdehyde, superoxide dismutase and glutathione) and vasodilator factor (endothelin-1 and nitric oxide) were analyzed by specific commercial assay kits. Besides, we detected the expression of related genes (fga, fgb, fgg, pkcα, pkcβ, vwf, f2) and proteins (PI3K, phospho-PI3K, Akt, phospho-Akt, ERK, phospho-ERK FIB) related to the anticoagulation and fibrinolytic system by quantitative reverse transcription and western blot. Beyond that, metabolomic analyses were carried out to identify the expressions of metabolites associated with the anti-thrombosis mechanism of LEO. Our in vivo experimental results showed that LEO could improve the oxidative stress injury, abnormal platelet aggregation and coagulation dysfunction induced by adrenalin hydrochloride. Moreover, LEO restored the modulation of amino acids and inositol metabolites which are reported to alleviate the thrombus formation. Collectively, LEO attenuates adrenalin hydrochloride-induced thrombosis partly via modulating oxidative stress, coagulation cascade and platelet activation and amino acid and inositol metabolites.
... The NO production in the endothelium is mainly catalyzed by eNOS, and NO is reported to modulate the ion transport in the kidneys and gills of trout (Tipsmark &Madsen, 2003) and in the opercular epithelium of killifish (Fundulus heteroclitus) (Evans et al., 2004). eNOS-like immunoreactivity has been detected with heterologous antibodies in zebrafish (Fritsche et al., 2000), tilapia (Cioni et al., 2002), and Atlantic salmon (Ebbesson et al., 2005). However, searching the completed fish genome projects reveals only iNOS and nNOS sequences, and thus eNOS is hypothesized to not exist in fish (Hyndman et al., 2006). ...
Fish have limited ability in endogenous biosynthesis of arginine. Arginine is an indispensable amino acid for fish, and the arginine requirement varies with fish species and fish size. Recent studies on fish have demonstrated that arginine influences nutrient metabolism, stimulates insulin release, is involved in nonspecific immune responses and antioxidant responses, and elevates disease resistance. Specifically, arginine can regulate energy homeostasis via modulating the AMP-activated protein kinase (AMPK) pathway, and also regulate protein synthesis via activating the target of rapamycin (TOR) signaling pathway. The present article reviews pertinent knowledge of arginine in fish, including dietary quantitative requirements, endogenous anabolism and catabolism, regulation of the endocrine and metabolic systems, and immune-regulatory functions under pathogenic challenge. Our findings showed that further data about the distribution of arginine after intake into specific cells, its sub-cellular sensor to initiate downstream signaling pathways, and its effects on fish mucosal immunity, especially the adaptive immune response against pathogenic infection in different species, are urgently needed.
... Further, we demonstrated clear evidence of vessel diameter increment in DA vessels. As previously stated, the DA is directly connected to the heart; thus, the DA diameter is important in the modulation of heartbeats and blood circulation [43,44]. Our results provide proof that treatment with DPHC could dramatically enhance vasodilation in a dose-dependent manner in in vivo models ( Figure 7). ...
Nitric oxide (NO) is released by endothelial cells in the blood vessel wall to enhance vasodilation. Marine polyphenols are known to have protective effects against vascular dysfunction and hypertension. The present study is the first to investigate how diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae affects calcium levels, resulting in enhanced vasodilation. We examined calcium modulation with the well-known receptors, acetylcholine receptor (AchR) and vascular endothelial growth factor 2 (VEGFR2), which are related to NO formation, and further confirmed the vasodilatory effect of DPHC. We confirmed that DPHC stimulated NO production by increasing calcium levels and endothelial nitric oxide synthase (eNOS) expression. DPHC affected AchR and VEGFR2 expression, thereby influencing transient calcium intake. Specific antagonists, atropine and SU5416, were used to verify our findings. Furthermore, based on the results of in vivo experiments, we treated Tg(flk:EGFP) transgenic zebrafish with DPHC to confirm its vasodilatory effect. In conclusion, the present study showed that DPHC modulated calcium transit through AchR and VEGFR2, increasing endothelial-dependent NO production. Thus, DPHC, a natural marine component, can efficiently ameliorate cardiovascular diseases by improving vascular function.
... In mammals NO has been shown to be a potent regulator of vascular tone, and this holds true in zebrafish during vascular development. While a specific vascular source of NO is missing, application of NO donors results in a significant increase in both the venous and arterial vessel diameters and NO scavengers constrict axial vessels suggesting regulation of vascular tone by a complex interaction of vasoactive substances generated locally by vascular endothelial cells (Fritsche et al., 2000;Pelster et al., 2005;North et al., 2009). ...
Since the discovery of new members of the globin superfamily such as Cytoglobin, Neuroglobin and Globin X, in addition to the most well-known members, Hemoglobin and Myoglobin, different hypotheses have been suggested about their function in vertebrates. Globins are ubiquitously found in living organisms and can carry out different functions based on their ability to bind ligands such as O2, and nitric oxide (NO) and to catalyze reactions scavenging NO or generating NO by reducing nitrite. NO is a highly diffusible molecule with a central role in signaling important for egg maturation, fertilization and early embryonic development. The globins ability to scavenge or generate NO makes these proteins ideal candidates in regulating NO homeostasis depending on the micro environment and tissue NO demands. Different amounts of various globins have been found in zebrafish eggs and developing embryos where it's unlikely that they function as respiratory proteins and instead could play a role in maintaining embryonic NO homeostasis. Here we summarize the current knowledge concerning the role of NO in adult fish in comparison to mammals and we discuss NO function during embryonic development with possible implications for globins in maintaining embryonic NO homeostasis.
... Some studies based on antibodies against mammalian NOS3 initially showed immunoreactivity in fish tissues. In particular, apparent Nos3-immunoreactivity has been found in the spinal cord of tilapia [47], in the vascular endothelium of developing zebrafish [75], in gill tissue in the Atlantic salmon (Salmo salar; [76]), in the trout posterior cardinal vein [43], in the heart and endothelial cells of tilapia [77] and in the liver of the Atlantic croaker (Micropogonias undulatus) [41]. Moreover, Nos3-like immunoreactivity has been found in the heart tissues of several teleosts (Anguilla anguilla, Chionodraco hamatus, Protopterus dolloi, Thunnus thynnus thynnus and Trematomus bernacchii [78]). ...
The involvement of nitric oxide (NO) in the modulation of teleost osmoresponsive circuits is suggested by the facts that NO synthase enzymes are expressed in the neurosecretory systems and may be regulated by osmotic stimuli. The present paper is an overview on the research suggesting a role for NO in the central modulation of hormone release in the hypothalamo-neurohypophysial and the caudal neurosecretory systems of teleosts during the osmotic stress response. Active NOS enzymes are constitutively expressed by the magnocellular and parvocellular hypophysiotropic neurons and the caudal neurosecretory neurons of teleosts. Moreover, their expression may be regulated in response to the osmotic challenge. Available data suggests that the regulatory role of NO appeared early during vertebrate phylogeny and the neuroendocrine modulation by NO is conservative. Nonetheless, NO seems to have opposite effects in fish compared to mammals. Indeed, NO exerts excitatory effects on the electrical activity of the caudal neurosecretory neurons, influencing the amount of peptides released from the urophysis, while it inhibits hormone release from the magnocellular neurons in mammals.
... The NOS isoforms that are responsible for enzymatic formation of NO are expressed early in the central nervous system during the embryogenesis period [39]. In zebrafish, nos1 shows distinct spatiotemporal expression starting at 19 hpf in the brain, followed by a major increase in the peripheral body organs, including the eye and the digestive tract at about 72 hpf [40,41]. nos2a is expressed in the gut region at 4 dpf and is inducible [42]. ...
Nitric oxide (NO) is an important signaling molecule that has been implicated in a variety of physiological and pathophysiological processes in living organisms. NO plays an important role in embryonic development in vertebrates and has been reported to influence early organ development and plasticity. Quantifying NO in single embryos and their developing organs is challenging because of the small size of the embryos, the low dynamically changing concentration and the short life-time of NO. Here, we measured the distribution of NO in the intestine of live zebrafish (Danio rerio) embryos in physiological conditions and under the influence of therapeutic agents. NO measurements were performed using a miniaturized electrochemical sensor fabricated on a single carbon fiber (CF) which enables quantitative real time in vivo monitoring, and by fluorescence imaging using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM-DA) dye. NO production was detected in the middle segment the intestine at a level of 3.78 (±0.64) μM, and at lower levels, in the anterior and posterior segments of 1.08 (±0.22) and 1.00 (±0.41) μM respectively. In the presence of resveratrol and rosuvastatin the intestinal NO concentration decreased by 87% and 84%, demonstrating a downregulating effect. These results indicate the presence of variable micromolar concentrations of NO along the intestine of zebrafish embryos and demonstrate the usefulness of CF microelectrodes to measure quantitatively the NO release at the level of a single organ in individual zebrafish embryos. This work provides a unique approach to study in real time the modulatory role of NO in vivo and contributes to further understanding of the molecular basis of embryonic development for developmental biology and drug screening applications.
... However, the endothelium of large systemic vessels lack eNOS, and adults appear to lack NO tone on the vasculature (144,283,424,426). A study on embryonic zebrafish, Danio rerio, documented a NO dilatory tone on the systemic vasculature (200), and a NO tone has also been reported in the embryonic/larval brown trout, Salmo trutta, as indicated by the tachycardiac response to blockade of NO production (150). Therefore, NO may tonically function in the microvascular circulation and/or during early windows of fish ontogeny. ...
Mass transport can be generally defined as movement of material matter. The circulatory system then is a biological example given its role in the movement in transporting gases, nutrients, wastes, and chemical signals. Comparative physiology has a long history of providing new insights and advancing our understanding of circulatory mass transport across a wide array of circulatory systems. Here we focus on circulatory function of nonmodel species. Invertebrates possess diverse convection systems; that at the most complex generate pressures and perform at a level comparable to vertebrates. Many invertebrates actively modulate cardiovascular function using neuronal, neurohormonal, and skeletal muscle activity. In vertebrates, our understanding of cardiac morphology, cardiomyocyte function, and contractile protein regulation by Ca2+ highlights a high degree of conservation, but differences between species exist and are coupled to variable environments and body temperatures. Key regulators of vertebrate cardiac function and systemic blood pressure include the autonomic nervous system, hormones, and ventricular filling. Further chemical factors regulating cardiovascular function include adenosine, natriuretic peptides, arginine vasotocin, endothelin 1, bradykinin, histamine, nitric oxide, and hydrogen sulfide, to name but a few. Diverse vascular morphologies and the regulation of blood flow in the coronary and cerebral circulations are also apparent in nonmammalian species. Dynamic adjustments of cardiovascular function are associated with exercise on land, flying at high altitude, prolonged dives by marine mammals, and unique morphology, such as the giraffe. Future studies should address limits of gas exchange and convective transport, the evolution of high arterial pressure across diverse taxa, and the importance of the cardiovascular system adaptations to extreme environments. © 2017 American Physiological Society. Compr Physiol 7:17-66, 2017.
... In MA, we described an increase in Ach-mediated vasoconstriction indicating the presence of a coupled Ach-NOS-NO dilation. This evidence suggests that there is Ach-NOS-NO-relaxation coupling in MA in both species, such as that shown in Danio rerio in the same vascular area (Fritsche et al., 2000;Holmberg et al., 2006). ...
... In MA, we described an increase in Ach-mediated vasoconstriction indicating the presence of a coupled Ach-NOS-NO dilation. This evidence suggests that there is Ach-NOS-NO-relaxation coupling in MA in both species, such as that shown in Danio rerio in the same vascular area (Fritsche et al., 2000;Holmberg et al., 2006). ...
Previous studies performed in intertidal fish (Girella laevifrons),as well as marine fish (Isacia conceptionis), showed that acetylcholine (ACh) produced contractions mediated by cyclooxygenases that were dependent on the area and potency of contraction in several arterial vessels. Given that the role of nitric oxide is poorly understood in fish, the objective of our study was to evaluate the role of nitric oxide in branchial afferent (ABA), branchial efferent (ABE), dorsal (DA) and mesenteric (MA) arterial vessels from both Girella laevifrons and Isacia conceptionis. We studied afferent and efferent branchial, dorsal and mesenteric arteries that were dissected from 6 juvenile specimens. Isometric tension studies were done using dose response curves (DRC) for Ach (10-13 to 10-3 M) and blockade with L-NAME (10-5 M), and DRC for sodium nitroprusside (SNP, a donor of NO). L-NAME produced an attenuation of the contractile response in the dorsal, afferent and efferent branchial arteries and a potentiation of the contraction in the MA. SNP caused 70% dilation in the mesenteric artery and 40% in the dorsal artery. Our results suggest that Ach promotes precarious dilatation in MA mediated by NO; data that is supported by the use of sodium nitroprusside. In contrast, in the vessels DA, ABA and EBA our results support that the pathway Ach-NO-relaxation is absent in both species.
... Iso is a β1/ β2-adrenoreceptor agonist [15,25,26], whereas sodium nitroprusside (SNP) is a potent nitric oxide donor. Nitric oxide plays a major role in regulating the vascular diameter, endothelial cell migration and angiogenesis [27,28,29,30]. The paralytic agent μ-conotoxin GIIIB was obtained from Alomone Labs, Israel. ...
Introduction
Hemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo.
Materials and Methods
Adult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations.
Results
Under control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 μmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001). Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01) and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03).
Discussion
In this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.
... Further, in the present investigation, it was observed that the intraperitoneal injection of LPS, the bacterial endotoxin, led to induction of iNOS gene leading to enhanced activity of iNOS enzyme as a consequence of more abundance of iNOS protein, and subsequently more synthesis and accumulation of NO in different tissues of singhi catfish. However, the physiological implication of LPS mediated iNOS gene expression in this catfish is yet to be established, similar to the line as suggested in some other fish species such as in brown trout [56], European eel [57], rainbow trout [58] and several other teleosts [59][60][61]. In singhi catfish, the iNOS activity could be detected in liver, kidney, heart, gills, muscle and brain tissues after LPS treatment with maximum activity after 24 h, which was further confirmed by Western blotting using a specific iNOS antiserum. ...
The air-breathing singhi catfish (Heteropneustes fossilis) is frequently being challenged by bacterial contaminants, and different environmental insults like osmotic, hyper-ammonia, dehydration and oxidative stresses in its natural habitats throughout the year. The main objectives of the present investigation were to determine (a) the possible induction of inducible nitric oxide synthase (iNOS) gene with enhanced production of nitric oxide (NO) by intra-peritoneal injection of lipopolysaccharide (LPS) (a bacterial endotoxin), and (b) to determine the effects of hepatic cell volume changes due to anisotonicity or by infusion of certain metabolites, stress hormones and by induction of oxidative stress on production of NO from the iNOS-induced perfused liver of singhi catfish. Intra-peritoneal injection of LPS led to induction of iNOS gene and localized tissue specific expression of iNOS enzyme with more production and accumulation of NO in different tissues of singhi catfish. Further, changes of hydration status/cell volume, caused either by anisotonicity or by infusion of certain metabolites such as glutamine plus glycine and adenosine, affected the NO production from the perfused liver of iNOS-induced singhi catfish. In general, increase of hydration status/cell swelling due to hypotonicity caused decrease, and decrease of hydration status/cell shrinkage due to hypertonicity caused increase of NO efflux from the perfused liver, thus suggesting that changes in hydration status/cell volume of hepatic cells serve as a potent modulator for regulating the NO production. Significant increase of NO efflux from the perfused liver was also observed while infusing the liver with stress hormones like epinephrine and norepinephrine, accompanied with decrease of hydration status/cell volume of hepatic cells. Further, oxidative stress, caused due to infusion of t-butyl hydroperoxide and hydrogen peroxide separately, in the perfused liver of singhi catfish, resulted in significant increase of NO efflux accompanied with decrease of hydration status/cell volume of hepatic cells. However, the reasons for these cell volume-sensitive changes of NO efflux from the liver of singhi catfish are not fully understood with the available data. Nonetheless, enhanced or decreased production of NO from the perfused liver under osmotic stress, in presence of stress hormones and oxidative stress reflected its potential role in cellular homeostasis and also for better adaptations under environmental challenges. This is the first report of osmosensitive and oxidative stress-induced changes of NO production and efflux from the liver of any teleosts. Further, the level of expression of iNOS in this singhi catfish could also serve as an important indicator to determine the pathological status of the external environment.
... Heart rate was slightly increased in the SMA-deficient larvae, most likely in response to vasodilation, and this may have made a small contribution to increased blood flow ( Figure 7C). The effects of the SMA knockdown on vascular function, which are similar to the effects of treatment with the vasodilator sodium nitroprusside (Fritsche et al., 2000), support a role for smooth muscle in regulating vascular resistance in larval zebrafish. ...
Smooth muscle contraction is controlled by the regulated activity of the myosin heavy chain (Myh11) ATPase. Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt) that converts a highly conserved tryptophan to arginine (W512R) in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here we report two novel missense mutations in the Switch-1 (S237Y) and coil-coiled (L1287M) domains of Myh11 that fail to complement mlt. Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter regulation of smooth muscle myosin may be risk factors for diseases affecting the intestine, vasculature and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress.
... Recently, a blood-flow-dependent klf2a-NO signaling cascade has been identified in developing zebrafish embryos (Wang et al., 2011). Endothelial nos isoforms have been well characterized in zebrafish development (Fritsche et al., 2000;Pelster et al., 2005;North et al., 2009;Wang et al., 2011). Inhibition of nos1 (the zebrafish ortholog of mammalian endothelial Nos) expression significantly rescued the bar mutant phenotype as well as endothelial regression ( Figures 7A and 7B). ...
... Shortening fractions were calculated as width [(diastole -systole)/diastole]. Stroke volume was determined as previously described [43]. The perimeter of the ventricle during diastole and systole was outlined on ImageJ and analyzed with a ''fit-to-ellipse'' algorithm, giving the major and minor axes. ...
DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1's role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1^(−/−) mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1^(−/−) mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1's role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1's molecular signaling pathway guided by the cardiac phenotype of tbx1^(−/−) mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1^(−/−) mutants. In addition, both wnt11r^(−/−) mutants and alcama morphants have heart looping and differentiation defects similar to tbx1^(−/−) mutants. Strikingly, heart looping and differentiation in tbx1^(−/−) mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development.
... Results of morphological studies and experimental investigations in teleost fish have shown that NO-produced neurons in CNS involved in different functional mechanisms associated with regeneration [72] differentiation [73, 74], a communication between neurons and glial cells, synaptic transmission and neuromorphogenesis [57, 72, 75]. NO plays special roles in proliferative processes in the matrix areas of fish´s brain which are not limited by embryonic stage of development and continue throughout life [76]. Regulation of neurogenesis in the adult brain is a topic of great interest to researchers and clinicians seeking new therapeutic avenues for treating age and injury-related impairment of neural functioning. ...
The chapter considers the overall organization of the main parts of brain in cyprinoid fish. It is described general cytoarchitectonical acpects of location, elements of neural structure and the system of relations in the most important centers of brainstem, and the forebrain-as the highest integrative center of the fish brain. It is presented a new data about adult neurogenesis and neurochemical (mediator) architectonics of the carp brain. It is described the some zones of neurogenesis in the adult carp brain, comparative data about immunolocalization of hydrogen sulphide producing enzyme the cystathionine β-synthase, NADPH-diaphorase and tyrosine hydroxylase in the different regions of carp brain. It is discussed the involvement of these neurotransmitters and gaseous intermediators in
... Molecular cloning has identified two NOS isoforms in fish which are homologous to the mammalian neuronal NOS (nNOS) and inducible NOS (iNOS) (Hyndman et al., 2006;Wang et al., 2007). Immunoreactivity of endothelial NOS (eNOS) has also been detected in a number fish species (Fritsche et al., 2000;Ebbesson et al., 2005;McNeill and Perry, 2006;Wang et al., 2007;Amelio et al., 2008), a results which is difficult to reconcile given the apparent absence of the eNOS gene in fish. It is well established that NO plays an important role as a neurotransmitter in the ventilatory response to changing environmental O 2 levels in mammals but very little is known about its function on O 2 chemosensing in lower vertebrates such as fish (Summers et al., 1999). ...
Chemoreception in fish is critical for sensing changes in the chemical composition of the external and internal environments and is often the first step in a cascade of events leading to cardiorespiratory and metabolic adjustments. Of paramount importance is the ability to sense changes in the levels of the three respiratory gases, oxygen (O2), carbon dioxide (CO2) and ammonia (NH3). In this review, we discuss the role of piscine neuroepithelial cells (NEC), putative peripheral chemoreceptors, as tri-modal sensors of O2, CO2 and NH3. Where possible, we elaborate on the signalling pathways linking NEC stimulation to afferent responses, the potential role of neurotransmitters in activating downstream neuronal pathways and the impact of altered levels of the respiratory gases on NEC structure and function. Although serotonin, the major neurotransmitter contained within NECs, is presumed to be the principal agent eliciting the reflex responses to altered levels of the respiratory gases, there is accumulating evidence for the involvement of "gasomitters", a class of gaseous neurotransmitters which includes nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S). Recent data suggest that CO inhibits and H2S stimulates NEC activity whereas NO can either be inhibitory or stimulatory depending on developmental age.
Copyright © 2015. Published by Elsevier B.V.
... The teleost cardiovascular system is one of the first organ systems to be fully functional and is capable of responding to changes in environmental oxygen concentration even prior to the development of oxygen sensing cells (Fritsche et al., 2000;Jacob et al., 2002;Pelster et al., 2005). For teleosts, surviving hypoxia, a common event in aquatic ecosystems, requires a balance between oxygen availability and oxygen demand which can be achieved through a variety of physiological responses (Farrell, 2007). ...
... NOS2) isoforms (Lepiller et al., 2009;Olson and Donald, 2009;González-Domenech and Muñoz-Chápuli, 2010;Fago et al., 2012). However: (1) a number of authors show that fish vascular beds are dilated by NO, and thus, provide evidence for an endothelial NO system in this taxa (Mustafa et al., 1997;Nilsson and Söderström, 1997;Mustafa and Agnisola, 1998); (2) several studies (Fritsche et al., 2000;McNeill and Perry, 2006;Wang et al., 2007;Amelio et al., 2008) have identified eNOS immunoreactivity in various fish tissues, including the vascular endothelium, when using heterologous mammalian eNOS antibodies (Olson and Donald, 2009;Imbrogno et al., 2014); and (3) Andreakis et al. (2011) suggest that a neuronal NOS (nNOS) isoform with an 'endotheliallike' consensus may cover some functional features typical of the eNOS isoform. As has previously been reported for mammals (Taniguchi et al., 1999;Oliveira et al., 2009), we showed that NO is involved in A23187-mediated vasodilation of isolated trout coronary arterioles (Fig. 4A), and that this effect was completely dependent on the presence of an intact endothelium. ...
Interleukin (IL)-1ß is associated with hypotension and cardiovascular collapse in mammals during heat stroke, and the mRNA expression of this pro-inflammatory cytokine increases dramatically in the blood of Atlantic cod (Gadus morhua, Linnaeus 1758) at high temperatures. These data suggest that IL-1ß release at high temperatures may negatively impact fish cardiovascular function, and be a primary determinant of upper thermal tolerance in this taxa. Thus, we measured the concentration-dependent response of isolated steelhead trout (Oncorhynchus mykiss, Walbaum 1792) coronary microvessels (< 150 μm in diameter) to recombinant (r) IL-1ß at two temperatures (10 and 20°C). Recombinant IL-1ß induced a concentration-dependent vasodilation with vessel diameter increasing by approximately 8 and 30% at 10(-8) and 10(-7) M, respectively. However, this effect was not temperature dependent. Both vessel denudation and cyclooxygenase blockade (by indomethacin), but not the nitric oxide (NO) antagonist L-NIO, inhibited rIL-1ß's vasodilator effect. In contrast, the concentration-dependent dilation caused by the endothelium-dependent calcium ionophore A23187 was completely abolished by L-NIO and indomethacin, suggesting that both NO and prostaglandin signaling mechanisms exist in the trout coronary microvasculature. These data: 1) are the first to demonstrate a functional link between the immune and cardiovascular systems in fishes; 2) suggest that IL-1ß release at high temperatures may reduce systemic vascular resistance, and thus, the capacity of fish to maintain blood pressure; and 3) provide evidence that both NO and prostaglandins play a role in regulating coronary vascular tone, and thus, blood flow.
© 2015. Published by The Company of Biologists Ltd.
... In different species, nNOS or nNOS-like isoforms may be the major source of NO-mediated action in developmental and plastic processes (Mize et al., 1998;Puenova et al., 2001), including in restricted brain areas with ongoing neurogenesis and neural plasticity in adult mammals (Islam et al., 1998;Moreno-Lopez et al., 2000). In lower vertebrates such as teleosts, NO has been emphasised to play a versatile role in the development of the central nervous system during both embryonic and post-embryonic life stages (Devadas et al., 2001;Fritsche et al., 2000;Ribera et al., 1998). In the brain of adult zebrafish, nNOS mRNA-expressing populations are closely associated with the proliferation zones (Holmqvist et al., 2000a) that generate new cells throughout life (see Ekström et al., 2001;Wulliman and Knipp, 2000). ...
To examine a putative role for neuronal nitric oxide synthase (nNOS) in early vertebrate development we investigated nNOS mRNA expression and cGMP production during development of the zebrafish Danio rerio. The nNOS mRNA expression in the central nervous system (CNS) and periphery showed a distinct spatio–temporal pattern in developing zebrafish embryo and young larvae. nNOS mRNA expression was first detected at 19 h postfertilisation (h.p.f.), in a bilateral subpopulation of the embryonic ventrorostral cell cluster in the forebrain. The number of nNOS mRNA-expressing cells in the brain slowly increased, also appearing in the ventrocaudal cell cluster from about 26 h.p.f., and in the dorsorostral and hindbrain cell cluster and in the medulla at 30 h.p.f. A major increase in nNOS mRNA expression started at about 40 h.p.f., and by 55 h.p.f. the expression constituted cell populations in differentiated central nuclei and in association with the proliferation zones of the brain, and in the medulla and retina. In parts of the skin, nNOS mRNA expression started at 20 h.p.f. and ended at 55 h.p.f. Between 40 and 55 h.p.f., nNOS mRNA expression started in peripheral organs, forming distinct populations after hatching within or in the vicinity of the presumptive swim bladder, enteric ganglia, and along the alimentary tract and nephritic ducts. Expression of nNOS mRNA correlated with the neuronal differentiation pattern and with the timing and degree of cGMP production.
These studies indicate spatio–temporal actions by NO during embryogenesis in the formation of the central and peripheral nervous system, with possible involvement in processes such as neurogenesis, organogenesis and early physiology.
... It is released from circulating erythrocytes in response to low tissue O 2 levels and triggers vasodilation (Allen and Piantadosi, 2006). Similar to adults, NO induces vasodilation in zebrafish larvae (Fritsche et al., 2000); it is thus possible that similar to Hb A , Hb E is also involved in NO transport and production (Pelster et al., 2010). Pelster et al. (Pelster et al., 2010) also suggest that because of its higher O 2 affinity, Hb E may play a role in O 2 storage and buffering in early development. ...
In rainbow trout development, a switch occurs from high-affinity embryonic hemoglobin (Hb) and round, embryonic erythrocytes to lower-affinity adult Hb and oval, adult erythrocytes. Our study investigated the early ontogeny of rainbow trout blood properties and the hypoxia response. We hypothesized that hypoxia exposure would delay the ontogenetic turnover of Hb and erythrocytes because retention of high-affinity embryonic Hb would facilitate oxygen loading. To test this hypothesis we developed a method of efficiently extracting blood from individual embryos and larvae and optimized several techniques for measuring hematological parameters on microliter (0.5 - 2.0 μl) blood samples. In chronic hypoxia (30% of oxygen saturation), stage-matched embryos and larvae possessed half the Hb concentration, erythrocyte counts, and hematocrit observed in normoxia. Hypoxia-reared larvae also had 3 to 6 fold higher mRNA expression of the embryonic Hb α-1, β-1, and β-2 subunits relative to stage-matched normoxia-reared larvae. Furthermore, in hypoxia the round embryonic erythrocytic shape persisted into later developmental stages. Despite these differences, Hb-oxygen affinity (P50), cooperativity, and the Root effect were unaltered in hypoxia-reared O. mykiss. The data support our hypothesis that chronic hypoxia delays the ontogenetic turnover of Hb and erythrocytes but without the predicted functional consequences (i.e. higher than expected P50). These results also suggest that the Hb-oxygen affinity is protected during development in chronic hypoxia to favour oxygen unloading at the tissues. We conclude that in early trout development, the blood-oxygen transport system responds very differently to chronic hypoxia relative to adults, possibly because respiration depends relatively more on oxygen diffusion than convection.
... Contractility has been demonstrated in developing zebrafish vessels by manipulation of nitric oxide (NO) . Administration of sodium nitroprusside (SNP, an NO donor) to 5 dpf zebrafish leads to arterial diameter increase while administration of L-NAME (a NO inhibitor) leads to vasoconstriction (Fritsche et al. 2000). NO also influences angiogenesis. ...
This chapter discusses the developmental physiology of the zebrafish cardiovascular system. The strength of zebrafish as a genetic model system enables the systematic dissection of interactions among genes in an intact vertebrate organism and bolsters its potential for screens of genetic or chemical modifiers of cardiovascular physiology. Phenotype-driven screens for genes that disrupt normal cardiovascular growth or function in the zebrafish offer an opportunity to understand the biology of heart and vascular physiology and pathways that may be disrupted in disease. Cardiac function is essential to the vascular flow, which in turn determines aspects of cardiac morphology. The cardiomyocytes of the zebrafish heart originate in the ventral–marginal zone, involute, and migrate through the lateral plate mesoderm and generate a heart tube precursor at the midline, a cone with the pre-ventricular cells pointing apically. The posterior lateral mesoderm forms vessel and blood precursors. It is adjacent to intermediate mesoderm that forms the kidney.
... They described the ability of stage 49-51 (Nieuwkoop and Faber) larvae to correct a volume-load-induced hypertension, but found no evidence for a baroreflex in these early stages. Vascular reactivity appears to be present very early in developing lower vertebrates: zebrafish (7 dpf, nitric oxide) and Xenopus leavis (nitric oxide and endothelin, stage NF 50-53) (Fritsche, Schwerte, and Pelster 2000;Schwerte, Printz, and Fritsche 2002). Receptors sensing hypoxic conditions and inducing a change in cardiac activity were shown to be present as early as 3 or 4 dpf in zebrafish larvae . ...
High prevalence of the central nervous system disorders necessitates novel methods and approaches for their pharmacological correction. Traditionally used rodent models are limited by high costs of research, complex maintenance and care, and long development. The use of alternative, aquatic model organisms, such as the zebrafish (Danio rerio), in translational neuroscience and medicine allows for fast efficient experimentation with easy maintenance, manipulations, and rapid development. Zebrafish are also sensitive to major classes of physiologically active agents, which makes this model indispensable for preclinical studies of a wide range of small molecules. The similarity of neurochemical systems, the presence of major neurotransmitters, a high degree of genetic and physiological homology with humans, the availability of both larval and adult fish models, and embryonic transparency provide multiple possibilities for using this organism to model CNS pathologies and its genetic and environmental causes.
Short-chain fatty acids (SCFAs) by the gut bacteria have been associated with cardiovascular dysfunction in humans and rodents. However, studies exploring effects of SCFAs on cardiovascular parameters in the zebrafish, an increasingly popular model in cardiovascular research, remain limited. Here, we performed fecal bacterial 16S sequencing and gas chromatography/mass spectrometry (GC-MS) to determine the composition and abundances of gut microbiota and SCFAs in adult zebrafish. Following this, the acute effects of major SCFAs on heart rate and vascular tone were measured in anaesthetized zebrafish larvae using fecal concentrations of butyrate, acetate, and propionate. Lastly, we investigated if co-incubation with butyrate may lessen the effects of angiotensin II (Ang II) and phenylephrine (PE) on vascular tone in anaesthetized zebrafish larvae. We found that the abundance in Proteobacteria, Firmicutes, and Fusobacteria phyla in the adult zebrafish resembled those reported in rodents and humans. SCFA levels with highest concentration of acetate (27.43µM), followed by butyrate (2.19µM) and propionate (1.65µM) were observed in the fecal samples of adult zebrafish. Immersion in butyrate and acetate produced a ~20% decrease in heart rate (HR), respectively, with no observed effects of propionate. Butyrate alone also produced a ~25% decrease in the cross-sectional width of the dorsal aorta (DA) at 60 min ( * P<0.05), suggesting compensatory vasoconstriction, with no effects of either acetate or propionate. In addition, butyrate significantly alleviated the decrease in DA cross-sectional width produced by both Ang II and PE. We demonstrate the potential for zebrafish in investigation of host-microbiota interactions in cardiovascular health.
Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes’ roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.
Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.
Methylparaben (MeP) is widely used as preservative in personal care products, food commodities and pharmaceuticals due to its antimicrobial properties. Its widespread use resulted in the contamination of aquatic environment and raised concerns about the potential adverse effects on human health, especially in the developing organisms. The aim of the present study was to evaluate the embryotoxicity of MeP in zebrafish early-life stages applying the benchmark-dose (BMD) methodology to Fish embryo acute toxicity (FET) tests-OECD guideline 236. Toxic effects were studied by daily evaluation of lethal endpoints, hatching rate and sublethal alterations. Zebrafish fertilized eggs were exposed until 96 h post fertilization (hpf) to five concentrations of MeP: 1 mg/L, 10 mg/L, 30 mg/L, 60 mg/L and 80 mg/L. The lethal concentration 50 (LC 50) was 72.67 mg/L. Indeed, BMD confidence interval (lower bound, BMDL-upper bound, BMDU) was 40.8–57.4 mg/L for lethal endpoints and 16–26.5 mg/L for toxicity index, that includes both lethal and sublethal alterations. Zebrafish embryos exposed to MeP developed sublethal alterations including pericardial edema, yolk edema, blood stasis, reduction in blood circulation, reduced heartbeat and notochord curvature. The number of embryos exposed to the highest concentrations of MeP that reported sublethal alterations increased between 24hpf and 48 hpf-72 hpf-96 hpf. Only zebrafish larvae treated with 30 mg/L of MeP showed behavioural changes. This study highlighted the detrimental effects of MeP on zebrafish early-life stages with attention to its developmental toxicity.
Endothelial dysfunction is an initial pathophysiological mechanism of vascular damage and is further recognized as an independent predictor of a negative prognosis in diabetes-induced micro- and macrovascular complications. Insight into the capability of zebrafish to model metabolic disease like obesity and type II diabetes has increased and new evidence on the induction of vascular pathologies in zebrafish through metabolic disease is available. Here, we raise the question, if zebrafish can be utilized to study the initial impairments of vascular complications in metabolic disorders. In this review, we focus on the advances made to develop models of obesity and type II diabetes in zebrafish, discuss the key points and characteristics of these models, while highlighting the available information linked to the development of endothelial dysfunction in zebrafish and man. We show that larval and adult zebrafish develop metabolic dysregulation in settings of obesity and diabetes, exhibiting pathophysiological mechanisms, which mimic the human condition. The most important genes related to endothelial dysfunction are present in zebrafish and further display similar functions as in mammals. Several suggested contributors to endothelial dysfunction found in these models, namely hyperinsulinaemia, hyperglycaemia, hyperlipidaemia and hyperleptinaemia are highlighted and the available data from zebrafish is summarised. Many underlying processes of endothelial dysfunction in obesity and diabetes are fundamentally present in zebrafish and provide ground for the assumption, that zebrafish can develop endothelial dysfunction. Conservation of basic biological mechanisms is established for zebrafish, but focused investigation on the subject is now needed as validation and particularly more research is necessary to understand the differences between zebrafish and man. The available data demonstrate the relevance of zebrafish as model for metabolic disease and their ability to become a proponent for the investigation of vascular damage in settings of obesity and diabetes.
This review aims to summarize the changes of the NOS/NO system which occur in the lungs, gills, kidney, heart, and myotomal muscle of air breathing fish of the genus Protopterus, i.e. P. dolloi and P. annectens, in relation to the switch from freshwater to aestivation, and vice-versa. The modifications of NOS and its partners Akt and Hsp-90, and HIF-1α, detected by immunohistochemical and molecular biology methods, are discussed together with the apoptosis rate, evaluated by TUNEL. We hypothesize that these molecular components are key elements of the stress-induced signal transduction/integration networks which allow the lungfish to overcome the dramatic environmental challenges experienced at the beginning, during, and at the end of the dry season.
The Deepwater Horizon oil spill of 2010 released a mixture of polycyclic aromatic hydrocarbons (PAHs) into the Gulf of Mexico presenting a complex exposure regime for native species. Concurrently, the Gulf has experienced an increase in hypoxic events due to agricultural runoff from the Mississippi River outflow. This combination presents a unique physiological challenge to native species and a challenge for researchers. The purpose of this study was to determine how the cardiotoxic PAH, phenanthrene interacts with hypoxia to affect the cardiovascular system of larval zebrafish (Danio rerio). We exposed zebrafish larvae to 0, 1, 100, and 1000 μg/L of phenanthrene in combination with normoxia and hypoxia. At late hatching, video of hearts and vessels were used to measure heart rate (ƒH), stroke volume (SV), cardiac output (Q), red blood cell velocity, and caudal vessel diameter. We found that the highest concentration of phenanthrene caused a 58, 80, and 84% decrease in ƒH, Q, and arterial red blood cell velocity in normoxia and an 88, 98, and 99 % decrease in hypoxia, respectively. Co-exposed larvae also experienced higher rates of edema and lordosis in addition to a 33% increase in mortality rate with co-exposure to hypoxia at the 1000 μg/L concentration of phenanthrene. At 12 dpf, baseline swimming behavior was similar between treatments indicating partial recovery from embryonic exposure. This study shows that phenanthrene decreases cardiac parameters, most significantly heart rate and that this effect is exacerbated by simultaneous exposure to hypoxia.
1. Introduction2. Cardiovascular Function and Energetics 2.1. Oxygen Transport by the Cardiovascular System2.2. Responses to Hypoxia2.3. Elasmobranch Cardiac Anatomy2.4. Cardiac Metabolic Biochemistry3. Factors Controlling and Effecting Cardiovascular Function 3.1. Heart Rate and Stroke Volume3.2. Body Fluid Volume and Blood Pressure Regulation4. Signaling Mechanisms Effecting Blood Vessel Diameter 4.1. Gasotransmitters4.2. Endothelins and Prostaglandins (Prostacyclin)4.3. Autonomic Nervous System Signaling Mechanisms (Adrenaline and Noradrenaline)4.4. Other Vascular Signaling Mechanisms (Acetylcholine, Adenosine, CNP, Serotonin, Vasoactive Intestinal Polypeptide, Bombesin, and Neuropeptide Y)4.5. Substances Affecting Gill Blood Flow Patterns5. The Action Potential and Excitation–Contraction (EC) Coupling in Elasmobranch Hearts: The Influences of Environmental, Biochemical, and Molecular Factors 5.1. The Action Potential5.2. EC Coupling5.3. Effects of Catecholamines and Acetylcholine5.4. Effects of Temperature and Acidosis6. Practical Applications: Physiology in the Service of Elasmobranch Conservation 6.1. Global Climate Change and Ocean Acidification6.2. Surviving Interactions with Fishing Gear7. SummaryThe functional characteristics of elasmobranch and teleost cardiovascular systems are similar at routine metabolic rates. Differences do become apparent, however, in cardiovascular function of high-energy-demand species (e.g., mako shark and yellowfin or skipjack tunas) at maximum metabolic rates. Elasmobranchs have an autonomic nervous system separable into parasympathetic and sympathetic components. The vagus nerve has a major role in controlling heart rate, although sympathetic innervation of the heart and blood vessels is absent. Elasmobranchs increase cardiac output primarily by increasing stroke volume which, in turn, is primarily determined by ventricular end diastolic volume. End diastolic volume is determined by filling time and venous filling pressure; with the latter being effected by venous tone and venous capacitance. Blood volume and pressure in elasmobranchs are controlled by endocrine (e.g., renin-angiotensin, kallikrein-kinin, and natriuretic peptides) and paracrine (e.g., endothelins, prostaglandins, the gasotransmitters NO and H2S,) mechanisms. Excitation-contraction (EC) coupling in elasmobranch hearts largely fits the accepted model for vertebrates, although the rise in cytoplasmic calcium is primarily from trans-sarcolemmal sources, which includes Na⁺-Ca²⁺ exchanger (NCX). Some elasmobranchs populations are severely depleted due to the intersection of life history characteristics, unsustainable rates of fisheries-associated mortality, and environmental degradation. To address these issues effectively will require a better understanding of the elasmobranch cardiovascular physiology – including the ability of various species to withstand the physiological consequences of the increasing temperature and expanding hypoxic zones accompanying global climate change, and the severe acidosis and the plasma ionic imbalances resulting from interactions with fishing gear.
Neuronal nitric oxide synthase (nNOS) catalyzes production of nitric oxide in vertebrate brains. Recent findings indicate that endothelial NOS and reactive oxygen species (ROS) are significantly increased during hypoxic stress and are modulated by antioxidants. However, the influence of antioxidants and steroids on nNOS upregulation by hypoxia is largely unknown. In this study, we characterized nNOS cDNA and examined the effects of hypoxia and antioxidant and steroid treatments on nNOS expression in Atlantic croaker hypothalamus. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 and/or 4 weeks) caused significant increases in hypothalamic nNOS mRNA, protein and its neuronal expression. Hypothalamic nNOS expression and superoxide radical (O2−, an index of ROS) production were increased by pharmacological treatment of fish exposed to normoxic conditions with N-ethylmaleimide, an alkene drug which covalently modifies sulfhydryl groups and inhibits aromatase activity. In contrast, treatments with Nω-nitro-l-arginine methyl ester, a competitive NOS-inhibitor, or vitamin E, an antioxidant, prevented the upregulation of O2− production and nNOS expression in hypoxia-exposed (DO: 1.7 mg/L for 4 weeks) fish. Moreover, treatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, increased hypothalamic O2− production and nNOS expression in normoxic control fish; whereas estradiol-17β treatment significantly reduced O2− production and nNOS expression in hypoxia-exposed fish. Double-labeled immunohistochemical results showed that nNOS and aromatase proteins are co-expressed in the hypothalamus. Taken together, the results suggest that upregulation of nNOS and ROS in the croaker hypothalamus in response to hypoxia is influenced by antioxidant and overall estrogen status.
African lungfishes are obligate air breathers, with reduced gills and pulmonary breathing throughout their life. During the dry season they aestivate on land, with the collapse of secondary lamellae of their gills and the establishment of an exclusive aerial ventilation through the vascularization and expansion of their lungs. To date, the mechanisms underlining the respiratory organ remodeling in aestivating lungfishes are unknown. This study aimed to identify key switch components of the stress-induced signal transduction networks implicated in both rapid and medium-long term remodeling of the gills and lungs of the African lungfish Protopterus annectens during aestivation. Through immunofluorescence microscopy and Western blotting, the localization and the expression of Nitric Oxide Synthase (NOS), Akt, Hsp-90 and HIF-1α, were evaluated in both gills and lungs exposed to three experimental conditions: freshwater (FW), 6 months of experimentally induced aestivation (6mAe), and 6 days after arousal from 6 months of aestivation (6mAe6d). After 6mAe, the expression of NOS (p-eNOS antibody), Akt (p-Akt antibody), and Hsp-90 decreased in the gills, while NOS and Hsp-90 expression increased with Akt remained unchanged in the lungs. Upon 6mAe6d, NOS, Akt and Hsp-90 expression in the gills returned to the respective FW values. In the lungs of the aroused fish, NOS and Akt decreased to their respective FW levels, while Hsp-90 expression was enhanced with respect to aestivation. In both respiratory organs, the qualitative and quantitative patterns of HIF-1α expression correlated inversely to those of NOS. Overall, our findings suggest that the molecular components of the NOS/NO system changed in a tissue-specific manner in parallel with organ readjustment in the gills and lungs of P. annectens during aestivation and arousal.
Copyright © 2014 Elsevier Inc. All rights reserved.
Antarctic icefish do not express hemoglobin (Hb). Icefishes possess cardiovascular modifications including increased densities of blood vessels, larger ventricles and increased blood volume compared to red-blooded relatives. In addition to delivering oxygen to tissues, Hb degrades nitric oxide (NO), a small signaling molecule. To investigate the mechanism driving development of icefish cardiovascular characteristics, I present and test the hypothesis that loss of Hb results in increased steady-state levels of NO, triggering downstream signaling pathways such as angiogenesis. I measured NO breakdown products, as a proxy for NO, and found that icefish have higher steady-state levels of NO metabolites in their plasma compared to red-blooded notothenioids. Expression of angiogenesis genes (HIF-1α, PHD2, and VEGF) did not differ significantly between red- and white-blooded notothenioids indicating that, while NO levels are higher in adult icefish, angiogenesis is not active. To investigate whether loss of Hb directly can increase NO and stimulate angiogenesis, hematocrit of red-blooded N. coriiceps was severely reduced using the hemolytic agent, phenylhydrazine HCl. Anemic fish exhibited a significantly higher concentration of NO metabolites in the plasma than did control fish, indicating an increase in NO. Expression of HIF-1α, PHD2, and VEGF mRNA was higher in anemic animals compared to control N. coriiceps, suggesting a causative relationship between loss of Hb and induction of angiogenesis that likely is mediated via NO signaling. In addition to lacking Hb, several species of icefish have lost ability to express myoglobin (Mb), an oxygen-binding protein expressed in the ventricle of most notothenioids. Previous studies have indicated Mb expression was lost on four separate occasions during evolution of the icefish family. Sequencing of the Mb gene from D. hunteri identified a duplicated TATA box previously proposed as the mechanism responsible for loss of Mb in C. aceratus. Sequencing of Mb from all 16 species of icefish identified the duplicated TATA box is present in all but two icefish species. The presence of the duplicated TATA box in Mb-expressing icefish suggests that the loss of Mb in C. aceratus and D. hunteri may occur by a mechanism independent of the duplicated TATA box.
Histopathological changes found in gill structures may be employed to investigate the toxicity of chemical compounds and monitoring of polluted aquatic systems. The main goal of this study was to quantify brachial lesions in Sciades herzbergii and determine the feasibility of using this catfish as a biomonitor to assess the effects of pollutants in São Marcos Bay. Catfish were collected in two sites. The first site (A1) is located near the ALUMAR/ALCOA port, and was used as a potentially contaminated area. The second site (A2) is located near the Crabs Island and was used as a reference area for being in an environmental protection area. Gills were fixed in 10% formalin and standard histological techniques were used in the first right gill arch, with inclusion in paraffin and sections of 5μm thickness. Each slide was analyzed for the presence of branchial lesions. We observed differences between catfish from the two sites. The histopathological changes in animals captured at reference site were lower than branchial lesions in catfish from potentially contaminated area. The most important lesions were: lifting of the lamella epithelium, fusion of some secondary lamellae, blood bosom vasodilatation, lamelar aneurysm, lamelar disorganization, mucous cells and hyperplasia. These results are important as they showed this resident estuarine species may be used for biomonitoring ecosystems in Sao Marcos' Bay, Maranhão, Brazil.
Autonomic innervation, at least of the cardiovascular system, appears late during the development of vertebrate embryos. In the early stages of innervation, control may be achieved by the activity of hormones, including for example the messenger molecule nitric oxide (NO). While NO in adult vertebrates is known to play a key function in many physiological processes, such as control of vascular tone, neurotransmission, macrophage activity and angiogenesis, very little is known about the onset of NO responsiveness during development. In fish, the presence of neuronal NO synthase (nNOS) and inducible NOS (iNOS) have been established by cloning and sequencing. The presence of endothelial NOS (eNOS) is indicated by immunological data, but attempts to identify eNOS at the molecular level have failed so far. nNOS expression, in particular, has been shown to occur at very early developmental stages. Analysis of the effect of NO on the cardiovascular system in zebrafish embryos and larvae revealed almost no effects on cardiac activity during chronic exposure to NO-producing chemicals, whereas vascular reactivity was observed in veins and arteries of the zebrafish in early developmental stages (5–6 days post-fertilization). Chronic exposure to a NO donor also modified the development of the vascular system by inducing an earlier appearance of some blood vessels in the trunk region of the zebrafish larvae. The nervous system and the gut also appear to be organs in which the early expression of NOS is of functional importance.
Independent of species, the cardiovascular system is the first functioning component of developing vertebrate embryos. One of the main hypotheses is the assumption that larval and juvenile stages of fish and amphibians are not just smaller versions of an adult phenotype. In this review, the cardiovascular and respiratory responses to environmental, genetic and epigenetic perturbations are discussed in detail to understand the relationships between cardiac and respiratory performance, haematopoiesis for embryonic or larval stages with special focus on the popular model animal, the zebrafish.
Zebrafish are tiny animals which have many advantages as a model organism in analysis of the cardio-respiratory system. It obtains sufficient amounts of oxygen via bulk diffusion, in contrast to convection-dependent mammals. It is possible to study genetic mutants even with extreme defective phenotypes of the cardio-respiratory system in order to understand its developmental and physiological mechanisms.
It has become apparent that the cardio-respiratory system and its control starts functioning very early during development, long before oxygen uptake becomes diffusion limited in zebrafish. Finally, recent improvements in imaging techniques for the use of fish models relevant for developmental physiology and biomedical research are discussed.
From a Xenopus stage 11 cDNA library, we have cloned a gene, termed X-β1AR, whose sequence is highly homologous to that of the human β1-adrenergic receptor. As shown by RT-PCR assay, X-β1AR RNA is present in the mature oocyte, decreases after fertilization up to stage 6 and then gradually increases during gastrulation. Binding studies performed with radiolabeled ligands reveal that X-β1AR RNA is translated into the receptor protein. Furthermore, noradrenaline and adrenaline are also detected in oocytes and early embryos. The concomitant presence of β1-adrenergic receptors and catecholamines suggest that this ligand-receptor couple could play a role in the very early stages of embryonic development.
Rainbow trout (Oncorhynchus mykiss) eggs and alevins were exposed for up to 4 weeks to the nitricoxide (NO) donor isosorbide dinitrate (ISDN) at concentrations up to 100 mol l–1. Hatching success was over 95%, but increasing concentrations of ISDN significantly delayed hatching, without significant affects on survival. Alevins exposed to ISDN concentrations of 50 or 100 mol l–1 showed significant cardiovascular and developmental changes compared to unexposed alevins. These included lowered heart rate and ventilation rate, doubling of cardiac output, dilation of the vitelline vein and increased in alevin weight. The results indicate a vasodilatory role of NO in trout alevins, and their cardiovascular responses to NO, with reference to the role of the vitelline vein, are discussed.
The analysis of perfusion parameters using the frame-to-frame technique and the observation of small blood vessels in transparent animals using video microscopy can be tedious and very difficult because of the poor contrast of the images. Injection of a fluorescent probe (fluorescein isothiocynate, FITC) bound to a high-molecular-mass dextran improved the visibility of blood vessels, but the gray-scale histogram showed blurring at the edges of the vessels. Furthermore, injection of the fluorescent probe into the ventricle of small zebrafish (Danio rerio) embryos (body mass approximately 1 mg) often resulted in reduced cardiac activity. Digital motion analysis, however, proved to be a very effective tool for analysing the shape and performance of the circulatory system in transparent animals and tissues. By subtracting the two fields of a video frame (the odd and the even frame), any movement that occurred within the 20 ms necessary for the acquisition of one field could be visualised. The length of the shifting vector generated by this subtraction, represented a direct measure of the velocity of a moving particle, i.e. an erythrocyte in the vascular system. By accumulating shifting vectors generated from several consecutive video frames, a complete trace of the routes over which erythrocytes moved could be obtained. Thus, a cast of the vascular system, except for those tiny vessels that are not entered by erythrocytes, could be obtained. Because the gray-scale value of any given pixel or any given group of pixels increased with the number of erythrocytes passing it, digital motion analysis could also be used to visualise the distribution of blood cells in transparent tissues. This method was used to describe the development of the peripheral vascular system in zebrafish larvae up to 8 days post-fertilisation. At this stage, food intake resulted in a clear redistribution of blood between muscle tissue and the gut, and alpha-adrenergic control of peripheral blood flow was established
The role of nitric oxide (NO) in activation of cGMP is well established. It has been proposed that the ratio of cAMP to cGMP may be important in the regulation of preimplantation embryonic growth and differentiation. Therefore, we determined the ability of murine preimplantation embryos to produce NO. In addition, NO as an endogenous smooth muscle relaxant and vasodilator is a candidate for involvement in embryo implantation because this process requires increased vascular permeability and uterine quiescence at the sites of blastocyst apposition. Nitrite assays, an indirect measure of NO production, indicate that preimplantation murine embryos produce NO. This production was reversibly inhibited by culture of embryos in medium containing a nonspecific NO synthase (NOS) inhibitor (NG-nitro-L-arginine). Additionally, inhibition of normal development was observed in embryos cultured with NOS inhibitor. NO levels increased in culture medium when ovariectomized progesterone-treated animals were exposed to estrogen for 1 h in utero. Such hormonal treatment induces implantation. These data indicate that NO levels are regulated by estrogen and may be important in regulation of implantation. In addition, these data demonstrate for the first time that NO production appears to be required for normal embryonic development.
The development of neuroeffector transmission in the embryo and fetal heart requires the appearance and function of presynaptic and postsynaptic components. During ontogenesis, function of the postsynaptic component (transmitter receptor and membrane conductance mechanism) can be demonstrated before the presynaptic component (efferent autonomic nerve) can be detected in the heart. The properties of the postsynaptic receptor have been identified with classical pharmacological methods. Receptor-specific ligands have been used to determine the chemical properties of muscarinic and beta-adrenergic receptors in tissues from adult animals. It is hoped that receptor-specific ligands, particularly those derived from antagonists, can be employed successfully to measure the chemical properties of these receptors in the developing heart. In addition, more information is required to enable the authors to understand the role that ontogenetic changes in membrane ion conductance and intracellular cyclic nucleotides have in determining the actions of neurotransmitter on pacemaker and contracting cardiac cells. The results of morphological studies of the development of the cardiac autonomic nervous system do not always agree with the results obtained in biochemical and histochemical analyses of nerve function. A lack of quantitative information and the observation of divergent results from different laboratories are the difficulties encountered in achieving the synthesis of structural and functional properties of developing nerves. Although neuroeffector transmission has been a useful index of the onset and development of neurosecretory function, chemical determination of the amount of transmitter released by stimuli is required to determine the development of neurosecretory activity and its relationship to the ontogenetic appearance of neuroeffector transmission. Direct measurement of transmitter release would be essential in order to study the development of neuronal interactions, for example, muscarinic inhibition of adrenergic transmitter release. There is no conclusive evidence to support the possibility that changes in transmitter sensitivity are due to the onset of neuroeffector transmission. Experiments in which development of the autonomic nervous system is accelerated or decelerated by various procedures (changing egg incubation temperature, administration of ganglion blocking drugs, nerve growth factor and its antiserum) are essential to determine whether the changes in transmitter sensitivity that occur are causally related to the onset of neurosecretory (or neuronal transport) function. The morphological and functional development of postganglionic adrenergic nerves seems to be regulated by intracellular and extracellular events (see 61, 151). However, it is not known to what extent presynaptic and effector cell development are able to regulate the growth and differentiation of the postganglionic neuron. It has been proposed that trans-synaptic factors regulate the development of the adrenergic nerve terminal and of the effector cells innervated by the adrenergic nerves. Furthermore, it has been speculated that a trophic influence of the effector cell on neuronal input can regulate the biochemical development of postganglionic cholinergic nerves and the retrograde transport of nerve growth factor in adrenergic nerves. The exploration of these problems in the development of cholinergic and adrenergic nerves of the heart is a stimulating challenge.
The notochord of amphibian anuran embryos contains catecholamines during the early developmental stages. In order to determine if these catecholamines are synthesized in situ, the development of their specific histofluorescence was investigated in the notochord alone or the notochord combined with the lateral somitic mesoderm, both explanted at the neurula stage and cultivated in vitro or implanted into the ventral part of early neurulae endoderm. The histofluorescence evolution, on the other hand, was investigated in the notochord alone or combined with myotomes, both explanted after the beginning of catecholamine biosynthesis and cultivated in vitro for one hour, in order to determine the effect of explantation and culture on the accumulation of notochordal catecholamines. The comparative study of catecholamine histofluorescence in these different samples shows that: the notochord is able to perform, on its own, the entire biosynthesis of the catecholamines stored in it during the early developmental stages. The catecholamines generated from isolated notochords tend to diffuse into the culture medium, probably due to a deficiency in the vesicular storage system usually found in the catecholamine-synthesizing cells. This loss of catecholamines in vitro can be obviated by the presence round the notochord of any embryonal tissue (somitic mesoderm, endoderm).
We have identified a previously undescribed intrinsic cardiac adrenergic (ICA) cell type in rodent and human heart. Northern and Western blot analyses demonstrated that ICA cell isolates contain mRNA and protein of enzymes involved in catecholamine biosynthesis. Radioenzymatic catecholamine assays also revealed that the catecholamine profile of adult rat ICA cell isolates differed from that of sympathetic neurons. Unlike sympathetic neuronal cells, isolated ICA cells have abundant clear vesicles on electron microscopy. Endogenous norepinephrine and epinephrine constitutively released by ICA cells in vitro affect the spontaneous beating rate of neonatal rat cardiac myocytes in culture. Finally, ICA cells could be identified in human fetal hearts at a developmental stage before sympathetic innervation of the heart has been documented to occur. These findings support the concept that these cells constitute an ICA signaling system capable of participating in cardiac regulation that appears to be independent of sympathetic innervation.
SYNOPSIS. Cardiovascular variables in developing amphibians are affected by both allometry and organogenesis. Blood pressure, cardiac output, and stroke volume increase during development, whereas peripheral resistance decreases. Invariably, resting heart rate changes as development proceeds from embryo through larva to adult. The pattern of change, however, varies between species and does not even correlate with family. The mechanisms underlying these varying patterns of developmental changes in heart rate of anuran amphibians larvae awaits complete description. Cardiovascular variables in adult vertebrates are influenced by nerves and hormones as well as intrinsic factors. However, information on the development of cardiovascular control systems is scarce, but receptor sensitivity apparently develops before functional innervation becomes apparent. The amphibian heart responds to acetylcholine and adrenaline several stages before functional innervation becomes evident, and in some species the cholinergic and adrenergic sensitivity of the cardiac pacemaker changes with development. As in mammals, acute hypoxia results in different cardiovascular responses depending on the developmental stage of the animal. The different responses reflect the relative maturity of neurohormonal mechanisms operating within the cardiovascular system.'.
SYNOPSIS. Cardiovascular variables in developing amphibians are affected by both allometry and organogenesis. Blood pressure, cardiac output, and stroke volume increase during development, whereas peripheral resistance decreases. Invariably, resting heart rate changes as development proceeds from embryo through larva to adult. The pattern of change, however, varies between species and does not even correlate with family. The mechanisms underlying these varying patterns of developmental changes in heart rate of anuran amphibians larvae awaits complete description. Cardiovascular vari-ables in adult vertebrates are influenced by nerves and hormones as well as intrinsic factors. However, information on the development of cardiovascu-lar control systems is scarce, but receptor sensitivity apparently develops be-fore functional innervation becomes apparent. The amphibian heart responds to acetylcholine and adrenaline several stages before functional innervation becomes evident, and in some species the cholinergic and adrenergic sensi-tivity of the cardiac pacemaker changes with development. As in mammals, acute hypoxia results in different cardiovascular responses depending on the developmental stage of the animal. The different responses reflect the rela-tive maturity of neurohormonal mechanisms operating within the cardiovas-cular system.
Very little is known about the early development of cardiorespiratory regulatory mechanisms in newly hatched amphibian larvae. We tested whether early cardiovascular responses to hypoxia reflect local-flow regulation in tissues and whether regulation of ventilation would improve during larval development. Cardiac output was calculated from heart rate and stroke volume, and buccal pumping rate was measured at 19° -21° C for Xenopus laevis larvae between Nieuwkoop and Faber stages 44 (just after hatching) and 57 (4-1,102 mg) denied access to air at a range of ambient aquatic Po₂ from normoxia (150-155 mmHg) to severe hypoxia (27-45 mmHg). Cardiac output decreased in severe hypoxia in stage 44-49.5 larvae, but not in stage 51-54 larvae, because heart rate decreased significantly in the early larvae, probably a direct effect of O₂ limitation on cardiac metabolism. Stroke volume did not change significantly in hypoxia in either early- or late-stage larvae. Thus there was no evidence of a tissue-mediated increase in cardiac output in hypoxia. Buccal pumping increased by about 50% over normoxic rates in moderate hypoxia in all larvae but sharply decreased in severe hypoxia, decreasing more in younger larvae than older. Younger larvae show significantly more variability in buccal pumping than older larvae, which suggests that regulatory mechanisms are not yet fully developed in early larvae. Cardiac output scales to body mass with a allometric coefficient of 1.15 ± 0.15 (95% confidence limits), significantly higher than literature values for O₂ uptake (0.83), implying that cardiovascular gas transport may be less important (compared to direct diffusion) in very small early-stage larvae than in larger, late-stage larvae.
1. Among the diverse functions of endothelins (ET), their role in the remodelling of blood vessels remains poorly examined. In the present review, we summarize findings obtained in our laboratory and present four independent lines of evidence to support this novel function. We also demonstrate that the motogenic and angiogenic effects of ET are mediated via the ETB receptor and that the functional endothelial nitric oxide synthase (NOS) is requisite for this action.
2. We demonstrated that ET stimulates transmigration of endothelial cells in a modified Boyden chamber and accelerates endothelial wound healing acting via ETB receptors.
3. In genetically engineered Chinese hamster ovary cells expressing either ETB receptor or endothelial NOS or both, application of ET results in accelerated cell migration only when the receptor and the enzyme are coexpressed. Application of antisense oligonucleotides producing a specific knockdown of the endothelial NOS results in the loss of ET ability to stimulate endothelial cell migration in response to ET.
4. Finally, using a novel model of in vivo angiogenesis, we were able to demonstrate that ET enhances formation of new vessels, but this effect requires functional endothelial NOS.
5. The described phenomenon of NO production, serving as a prerequisite for endothelial cell locomotion in response to activation of ETB receptor may explain a host of pathophysiological observations on inadequate angiogenesis despite enhanced generation of ET-1.
6. Based on the contribution of endothelial cell migration to angiogenesis, these data may implicate insufficient NO production in pathological states (e.g. atherosclerosis, heart failure and hypertension) in the inappropriate response to angiogenic stimuli.
Changes in adrenal hormones during the complete developmental cycle from egg to juvenile were investigated in the amphibianXenopus laevis.Whole-body concentrations of the adrenal steroids corticosterone (B), and aldosterone (Aldo) were determined by radioimmunoassay and those of the adrenal catecholamines epinephrine (E), norepinephrine (NE), and dopamine (D) were determined by HPLC. In addition, the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine β-hydroxylase, and phenylethanolamineN-methyltransferase were immunocytochemically localized for the characterization of chromaffin adrenal cells. B and Aldo were not detectable in the whole body before hatching. B levels rose earlier than Aldo levels from stage 36 onward. B had already peaked at stage 46, whereas the largest amounts of Aldo were found at stage 54. After peaking, both steroids decreased gradually to 2.7 ± 0.62 (B) and 0.4 ± 0.1 (Aldo) ng/g body wt (mean ± SEM,n = 10) in juvenile animals. E, NE, and D were detected just after hatching, when E and D showed an early peak at stage 40. E and NE increased moderately during development and demonstrated a sharp increase at the end of metamorphosis from stages 62 onward to 14.4 ± 1.7 (E) and 34.1 ± 4.67 (NE) ng/g body wt (mean ± SEM,n = 6). Interestingly, D levels had a distinct pattern, because concentrations of D remained lower than those of NE and E over nearly the complete development, but showed a dramatic rise during the latest stages, reaching 707 ± 54 ng/g body wt in juveniles. This dramatic shift in catecholamine levels was confirmed by immunocytochemistry in parallel. A large increase in chromaffin cells labeled with tyrosine hydroxylase immunoreactivity occurred in the latest developmental stages. The catabolic rates for all catecholaminesin vivowere similar, which indicates that the different levels are due to various rates of synthesis. Thus, adrenal corticosteroids as well as catecholamines may have regulatory effects during premetamorphosis and metamorphic climax.
The goal of this experiment was to determine whether the type of tight supply-and-demand relationship between cardiac output and metabolic demand for O2 seen in juvenile and adult fish applies during larval development. To this end, we looked at how the heart rate, stroke volume, and cardiac output of rainbow trout (Oncorhynchus mykiss) larvae varied in response to changes in tissue mass and incubation temperature. Previous studies have shown that both factors have a profound influence on metabolic rate. Heart rate and stroke volume were estimated using videomicroscopic methods and used to calculate cardiac output at five or six approximately evenly spaced intervals between hatch (approximately 15 mg wet tissue mass) and 150 accumulated thermal units (degrees C d) posthatch (approximately 50 mg tissue mass) at 5 degrees, 10 degrees, 12 degrees, and 15 degrees C. Cardiac output (range 0.2-20 microL min(-1)) increased significantly in response to increases in both tissue mass and incubation temperature. The increase in cardiac output with tissue mass reflected significant increases in stroke volume as well as heart rate. Temperature only had a significant effect on heart rate (i.e., stroke volume was unaffected). The rate of increase in cardiac output as a result of tissue growth was significantly faster than the rate at which O2 demand increased (the allometric mass exponent [+/-SE] for cardiac output was 1.78 +/- 0.08; literature values for O2 uptake average approximately 1.0), which suggests that the cardiovascular system was less important as a means of delivering O2 to the tissues in small larvae than it was in larger larvae and in juvenile and adult fish. Direct diffusion appeared to be the primary means of O2 delivery in small larvae and embryos.
Histochemical techniques, field stimulation, and application of autonomic drugs were used to study neurotransmission in the heart during ontogenesis of Rana temporaria. Cholinesterase (ChE)-containing fibers, fluorescent chromaffinlike cells, and fluorescent fibers were found first in the heart at tadpole stages 40, 40, and 50, respectively. Inhibitory cholinergic and stimulatory adrenergic responses to field stimulation first appeared at stages 39-40 and 42, respectively. Inhibitory responses to acetylcholine (ACh) and stimulatory responses to epinephrine (Epi) were observed as early as stages 31 and 32. The concentrations producing half-maximal response values for both neurotransmitters increased during development. Indirect evidence was obtained that the subsensitivity of tadpole hearts to ACh was due to increased hydrolysis of ACh by tissue ChE and that the subsensitivity of adult frog heart to Epi could be connected with a maturation of the neuronal uptake mechanism. The pA2 values for atropine and propranolol were 10 times greater in tadpoles than in adults. The main conclusion is that the cholino- and adrenoreactive systems appear in the frog heart cells before they become innervated and the sensitivity of these systems to neurotransmitters does not increase with innervation.
There is increasing evidence that resting pulmonary vascular tone is mediated by the release of endothelium-derived relaxing factors (EDRF). However, the importance of EDRF release during pulmonary hypertension is unknown. Therefore, in eight newborn lambs we studied the effects of both N omega-nitro-L-arginine (an inhibitor of EDRF synthesis) and L-arginine (a precursor of EDRF synthesis) during pulmonary hypertension induced either by the intravenous infusion of U-46619 (a thromboxane A2 mimic) or by hypoxia. After pretreatment with N omega-nitro-L-arginine, the increases in pulmonary arterial pressure produced by U-46619 (102.0 +/- 34.9% vs. 144.8 +/- 28.6%, P less than 0.05) and by hypoxia (35.6 +/- 17.3% vs. 91.4 +/- 24.8%, P less than 0.05) were significantly augmented. However, after pretreatment with L-arginine, the increases in pulmonary arterial pressure produced by U-46619 (107.0 +/- 21.4% vs. 62.6 +/- 22.6%, P less than 0.05) and hypoxia (44.3 +/- 18.3% vs. 9.2 +/- 11.7%, P less than 0.05) were significantly attenuated. These results suggest that during pulmonary hypertension, EDRF is released to limit the increase in pulmonary arterial pressure and that L-arginine availability becomes rate limiting for further EDRF synthesis and release.
To examine mechanisms underlying fetal pulmonary vascular vasodilator responses and maturational changes in endothelial function, we studied effects of endothelium-dependent (acetylcholine, ACh: adenosine diphosphate, ADP; and A23187) and -independent (sodium nitroprusside, SNP) vasodilators on tone of small (third generation) pulmonary artery rings isolated from late-gestation fetal, newborn, and adult sheep. Changes in isometric force of phenylephrine-contracted rings were measured after the cumulative addition of vasodilators in baths aerated with 21% O2 and 5% CO2. Pulmonary artery rings from fetal lambs demonstrated minimal relaxation to ACh, ADP, and A23187, achieving only 17 +/- 3, 14 +/- 5, and 23 +/- 8% relaxation, respectively. In contrast, fetal rings relaxed completely (100%) to SNP. Rings from newborn and adult animals had significantly greater maximal relaxation in response to ACh. ADP, and A23187 than fetal rings (at least P less than 0.05 for each comparison with fetal rings), but responses to SNP were not different. Hemoglobin (10(-5) M), an inhibitor of endothelium-derived relaxing factor, caused less augmentation of phenylephrine contraction in fetal than adult pulmonary artery rings (11 +/- 4% vs. 49 +/- 8%; P less than 0.01). We conclude that in comparison with pulmonary artery rings from postnatal animals, fetal pulmonary artery rings have diminished endothelium-derived relaxation factor activity. We speculate that maturational changes in endothelial cell function contribute to ontogenetic differences in pulmonary vasoreactivity.
Endothelium-derived relaxing factor (EDRF), believed to be nitric oxide or a compound that releases nitric oxide, is a potent vasodilator produced by some arteries in response to acetylcholine (ACh) and bradykinin (BK). ACh and BK are potent dilators of perinatal pulmonary and systemic arteries. The objectives of this study were to determine if EDRF is present in newborn vessels and if EDRF mediates the vasodilator actions of ACh and BK. Arterial rings from newborn guinea pigs, 1 to 3 d old, were obtained from a branch of the main pulmonary artery and the descending aorta for isometric force bioassays. At their optimal resting tension, the rings were preconstricted with phenylephrine 10(-5) M in Krebs-Henseleit solution before adding incremental doses of ACh or BK. If the endothelium was intact, ACh (10(-5) M) relaxed pulmonary arteries and aortas (64 +/- 7%, 72 +/- 9% relaxation, respectively, mean +/- SE). ACh-induced relaxation (ACh 10(-5) M) in the pulmonary artery and aorta, respectively, was significantly (p less than 0.05) attenuated by 1) endothelial removal (11 +/- 9%, 28 +/- 10%) by rubbing the ring lumen; 2) methylene blue, 10(-6) M, (6 +/- 8%, 7 +/- 3%) that inhibits EDRF-associated cGMP production in smooth muscle; and 3) methemoglobin, 10(-5) M, (13 +/- 9%, 17 +/- 7%) that binds EDRF. The results for BK were similar to ACh for the pulmonary artery but BK did not relax the aorta. Indomethacin diminished relaxation of the pulmonary artery and aorta to the submaximal dose (10(-5) M) of ACh but indomethacin did not effect the relaxation to ACh 10(-4) M or BK. We conclude that EDRF is produced in the guinea pig pulmonary artery and descending aorta at birth and that EDRF is a mediator of the vasodilator actions of ACh and BK. Vasodilation by ACh may also involve activation of the cyclooxygenase pathway.
Environmental hyperthermia is a hazard to the poikilothermic chick embryo. We studied effects of hyperthermia on mean vitelline arterial blood pressure and mean dorsal aortic blood flow in stage 18, 21, and 24 chick embryos. The pressure was measured with a servo-null micropressure system, and the blood flow was measured with a 20 MHz pulsed Doppler flowmeter. Temperature was monitored with a needle thermoprobe positioned adjacent to the embryo. Data were obtained at 37 degrees C, after warming to 40 degrees C, and then after cooling to 37 degrees C. At stage 21, the pressure increased from 0.96 +/- 0.05 (+/- SE) to 1.04 +/- 0.06 mm Hg on warming and returned from 1.05 +/- 0.04 to 0.87 +/- 0.04 mm Hg on cooling. Pressure measurements during warming and cooling were performed in two separate groups of embryos because of technical problems. The blood flow, studied using different groups of the embryo from the pressure study, also increased from 0.65 +/- 0.06 to 0.75 +/- 0.06 mm3/s on warming and returned to 0.56 +/- 0.05 mm3/s. The heart rate increased from 173 +/- 2 to 211 +/- 3 at 40 degrees C and returned to 170 +/- 3 at 37 degrees C. Stroke volume (flow/heart rate) did not change during the temperature variation. Vascular resistance, the quotient of pressure to blood flow obtained by a ratio analysis, changed from 1.53 +/- 0.33 (median +/- 95% confidence interval) to 1.42 +/- 0.29 mm Hg/mm3/s on warming and changed to 1.60 +/- 0.32 mm Hg/mm3/s on cooling. Similar results were obtained at stages 18 and 24.(ABSTRACT TRUNCATED AT 250 WORDS)
Stroke volume (SV) and cardiac output (CO) were measured in anesthetized larvae of Xenopus laevis from hatching (3 mg) to the end of metamorphosis (approximately 1 g). CO and SV were calculated from videotaped images of the intact beating heart. SV increased from 2.4 x 10(-3) microliters at 3 mg body mass to 7.6 microliters at 1 g. CO increased from 0.25 microliter/min at 3 mg to 623 microliters/min at 1 g. With use of CO, along with arterial pressures from another study [P.-C. L. Hou and W. W. Burggren. Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R1120-R1125, 1995], peripheral resistance and cardiac work were also calculated. Resistance decreased rapidly from 701 peripheral resistance units (PRU, mmHg.s.mm-3) at 3 mg body mass to 79 PRU at 20 mg and gradually declined toward 0.9 PRU at 1 g. Cardiac work increased from 0.06 dyn.mm at 3 mg body mass to 1.27 dyn.mm at 20 mg and then climbed sharply to 717 dyn.mm at 1 g. The general pattern of change in hemodynamic variables (except heart rate) during larval development is similar in Xenopus laevis and chick embryos, suggesting a common pattern for hemodynamic development in vertebrate embryos/larvae.
The in vivo model of the chick embryo chorioallantoic membrane (CAM) was used to study the involvement of nitric oxide (NO) in angiogenesis. The nitrovasodilator sodium nitroprusside (NaNP) and the amino acid, L-arginine, inhibited angiogenesis, assessed as both collagenous protein biosynthesis and vascular density. NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, increased both collagenous protein biosynthesis and vascular density, indicating that this agent promotes angiogenesis. These results suggest that NO may participate in the regulation of angiogenesis. Manipulation of NO synthesis therefore, may prove to be another approach for controlling angioproliferative diseases.
The influence of acetylcholine (ACh) on cardiac performance of larval (Taylor Kollros [TK] stages II‐XVIII) and postmetamorphic (3–609 g) Rana catesbeiana was analyzed in situ (circulatory system intact) and in vitro (isolated heart or ventricular strip preparations).
Topical application of ACh to the heart in situ resulted in a dose‐dependent decrease in heart rate and in a slight decrease in systolic ventricular pressure in all developmental stages. Injection of acetylcholine in to the ventricle lumen in situ caused a dose‐dependent transient decrease in systolic ventricular pressure, with little heart rate effect. Intraventricular ACh injection also changed the hemodynamic coupling between ventricle and conus arteriosus, generating a biphasic pressure profile in the conus due to sequential contractions of the ventricle and of the conus. In situ the sensitivity of the ventricle to ACh decreased during larval development, with the lowest sensitivity in small postmetamorphic adults.
ACh applied in vitro to cardiac muscle strips or small hearts produced a negative inotropic effect. The ACh dose necessary to induce a 50% reduction in muscle strip contraction force in vitro decreased substantially during larval development, indicating an increase in ACh sensitivity with development.
The effects of ACh both in vitro and in situ were diminished or eliminated by topical application or injection of atropine, suggesting the presence of muscarinic cholinergic receptors. After preincubation with the acetylcholinesterase blocker eserine, injection of ACh in to the conus arteriosus decreased systolic ventricular pressure with a delay of 4–10 seconds, probably representing the minimum blood circulation time.
The observed inotropic and chronotropic responses result from the action of ACh on cardiac muscle, primarily affecting systolic ventricular pressure, and on the cardiac pacemaker, mainly influencing heart rate. These responses occur as early as TK, stage II, indicating a well‐developed set of mechanisms to regulate cardiovascular performance early in development. © 1993 Wiley‐Liss, Inc.
Embryonic cardiovascular function is dynamically regulated at the tissue level. Nitric oxide (NO) regulates vascular tone and influences cardiovascular function in neonatal and mature circulations. However, the role of NO in regulating embryonic cardiovascular function is undefined. We hypothesized that NO released from nitroprusside alters embryonic vascular tone with secondary effects on ventricular function.
We measured ventricular pressure and calculated ventricular volume from area using ellipsoid geometry for 180 s after suffusion of 0.0, 0.1, 1.0, or 2.5 micrograms of nitroprusside in 10 microliters of KHB buffer, or after acute venous hemorrhage in stage 21 chick embryos (n > or = 8 per group). We plotted pressure-volume loops and analyzed standard parameters of cardiovascular function by ANOVA, regression analysis, and ANCOVA.
Following nitroprusside, heart rate was unchanged, end-diastolic volume (EDV), stroke volume (SV), and end-systolic pressure decreased (P < 0.05 at 180 s). For 1.0 microgram of nitroprusside, EDV decreased by 27 +/- 6%, SV decreased by 36 +/- 6%, and end-systolic pressure decreased by 28 +/- 3%. The EDV vs. SV relationship was unchanged with the exception of the 2.5 micrograms nitroprusside dose. Arterial elastance was unchanged with the exception of the 2.5 micrograms nitroprusside dose. The EDV vs. SV relationship and arterial elastance during preload reduction suggest that ventricular afterload did not decrease following NO.
In contrast to the mature circulation, NO reduced preload without decreasing ventricular afterload. Thus, vasoactive agents may have unique roles in the regulation of cardiovascular structure and function during embryogenesis.
Embryonic hemoglobin circulated by the developing heart in the early vertebrate embryo is widely assumed (without substantiation) to perform the same vital role of O2 carriage that it does in fetuses and adults. In order to challenge this assumption, we measured highly O2-dependent physiological variables like O2 consumption, cardiac performance, and initial swim bladder filling in the presence and absence of functional hemoglobin in the embryos and early larvae of the zebra fish, Danio ( = Brachydanio) rerio. Functional ablation of hemoglobin by carbon monoxide or phenylhydrazine did not reduce whole-animal O2 consumption, which was approximately 85 to 90 mumol.g-1.h-1. Similarly, no differences in heart variables like ventricular pressure development or heart rate, which increased from 135 to 175 bpm between stages 36h and 96h (indicating developmental stages 36 and 96 hours after fertilization, respectively), were observed in these experiments. Initial opening of the swim bladder was not influenced in the presence of CO-occupied hemoglobin but was significantly impaired when the embryonic hemoglobin was chemically modified by incubation with phenylhydrazine. That aerobic processes continue without hemoglobin O2 transport indicates the adequacy in the embryo of simple O2 diffusion alone even in developmental stages with extensive convective blood circulation generated by the heart.
Embryonic cardiovascular function has been extensively studied in vivo in the chick embryo. However, the geometry of mammalian and avian hearts differs; the mammalian cardiovascular system is coupled to both yolk sac and placental circulations, and unique murine genetic models associated with structural and functional cardiovascular defects are now available. We therefore adapted techniques validated for the chick embryo to define cardiovascular dimensions and function in the mouse embryo. We bred C3HeB female and C57B1/J6 male mice and ICR pairs for experiments on embryonic days (EDs) 10.5 to 14.5 (n = 130 dams). After maternal anesthesia (pentobarbital, 60 mg/kg IP), laparotomy, and sequential regional hysterotomy, we exposed and then imaged individual embryos at 60 Hz (video) in the ventral and/or left anterior oblique views while maintaining uteroplacental continuity. We measured epicardial chamber dimensions and then calculated right and left ventricular elliptical volumes from ares. In addition, we measured pulsed-Doppler blood velocity across the atrioventricular cushions and ventricular outflow tract. We maintained embryonic temperature with a heated surgical platform, topical oxygenated and warmed buffer, and warming lamps. Embryonic heart rate increased from 124.7 +/- 5.2 to 194.3 +/- 13.2 bpm from EDs 10.5 to 14.5 (P < .01). Right and left ventricular end-diastolic and end-systolic dimensions increased (P < .05 by ANOVA for each). Maximal ventricular mean inflow and outflow velocities increased from 62.33 +/- 4.06 to 106.23 +/- 11.59 and from 55.79 +/- 6.11 to 91.61 +/- 6.93 mm/s, respectively (P < .05 by ANOVA for each). Thus, as has been done for chick and rat embryos, the maturation of murine embryonic cardiovascular function can be quantified in vivo, setting the stage for the investigation of structure-function relations in mouse models of cardiovascular development and disease.
Cardiovascular responses (blood pressure, heart rate, stroke volume, cardiac output, and peripheral vascular resistance) to acute hypoxia (Po2 = 70 mmHg) in developing larvae of Xenopus laevis from Nieuwkoop-Faber (NF) stage 45 and up to newly metamorphosed froglets were investigated. The results revealed two distinct response patterns to acute hypoxia in "early" (NF stages 45-48 and 49-51) and "late" (NF stages 52-53, 54-57, and 58-62) larval Xenopus. The early larvae responded to acute hypoxia with a significantly decreased stroke volume, cardiac output, and blood pressure. Peripheral resistance increased, whereas no change in heart rate occurred. In late larvae, stroke volume and blood pressure increased during acute hypoxia, but an offsetting bradycardia prevented major changes in cardiac output. We conclude that, up to stage 51 of development, hypoxia exerts a direct inhibitory effect on the heart and smooth muscle of the blood vessels, with no Frank-Starling relationship apparent. Older larvae show evidence of both intrinsic and extrinsic regulation of the cardiovascular system in response to acute hypoxia, suggesting that there is a specific point in larval development when cardiovascular regulation during hypoxia is expressed.
From a Xenopus stage 11 cDNA library, we have cloned a gene, termed X-beta1AR, whose sequence is highly homologous to that of the human beta1-adrenergic receptor. As shown by RT-PCR assay, X-beta1AR RNA is present in the mature oocyte, decreases after fertilization up to stage 6 and then gradually increases during gastrulation. Binding studies performed with radiolabeled ligands reveal that X-beta1AR RNA is translated into the receptor protein. Furthermore, noradrenaline and adrenaline are also detected in oocytes and early embryos. The concomitant presence of beta1-adrenergic receptors and catecholamines suggest that this ligand-receptor couple could play a role in the very early stages of embryonic development.
The endothelins (ET) are a family of contractile peptides made up of 21 amino acids. They are synthesised from larger precursors and they are expressed in different tissues. ET-1 is synthesised in endothelial cells by means of a specific endothelin converting enzyme and it is assumed that most of it is secreted into the basolateral compartment. It acts in a paracrine manner on the ETA and ETB2 receptors located on the surface of the vascular smooth muscle to elicit an increase in intracellular calcium and vasoconstriction. The circulating ET-1 can also activate endothelial ETC and ETB1 receptors releasing vascular smooth muscle relaxing factors, such as nitric oxide and prostacyclin. At present, it is generally accepted that ET-1 is a vasodilator in physiological conditions acting on endothelium ETB1 receptors. Nevertheless, in pathological situations such as hypertension, heart failure, acute myocardial infarction, acute renal failure and vasospastic conditions (Raynaud's disease and subarachnoid haemorrhage), ET-1 levels increase and it binds to the receptors present in vascular smooth muscle in such a way that its vasoconstrictor effect is manifested. Currently, experimental and clinical evidence exists to support the importance of the development of drugs that block the production or actions of ET for use in cardiovascular medicine, particularly in conditions in which these peptides are clearly implicated.
Cardiac responses (heart rate, stroke volume, and cardiac output) to cholinergic and adrenergic receptor stimulation were investigated in developing larvae of Xenopus laevis from Nieuwkoop and Faber (NF) stage 33/34 (newly hatched) to NF stage 53 (22 d after hatching). Effects on heart rate (fH), stroke volume (SV), and cardiac output (CO) were analyzed using in situ preparations and video-microscopic techniques to record the continually beating heart. The results show that administration of acetylcholine to the heart decreases heart rate as early as NF stage 40. A significant reduction in SV and CO following acetylcholine administration to the heart was found at NF stages 45-53. Epinephrine had no significant effect on fH, SV, or CO at any of the stages investigated. However, an adrenergic tonus on the heart is present already at NF stage 40 (11%). This tonus increases up to a maximum (44%) at NF stages 45-47, when the maximal heart rate is found during development of X. laevis. We conclude that acetylcholine has a negative chronotropic and possibly also inotropic effect on the heart very early in development of X. laevis. We also hypothesize that the high adrenergic tonus found at NF stages 45-47 is responsible, at least in part, for the peak in heart rate seen at these stages.
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