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a-d. Perivascular nerves of the basilar artery labelled for NOS (a-c) and a control specimen (d). (a) One NOS-positive (star) and three NOS-negative axon profiles (Ax) are seen close to the smooth muscle (sm). The distance between the NOS-positive profile and the smooth muscle is about 0.5-0.8 ~xm. av, small agranular vesicles; gv, large granular vesicle; m, mitochondria; col, collagen fibres. Scale bar = 0.2 ixm. (b) At least five axon profiles in a nerve bundle are NOS-positive. In one profile/varicosity heavy labelling obscures intracellular structures; labelled microtubules (mr) are seen in intervaricosities. Sch, unlabelled Schwann cell process. Scale bar = 0.2 ~xm. (c) A higher magnification example of NOS-positive varicosity containing small agranular vesicles. The particles of immunoprecipitate (arrows) are attached to the membrane of the vesicles as well as to the axoplasmic side of the axoIemma; immunoprecipitate is also seen 'free' in the axoplasm. Scale bar = 0.1 p,m. (d) Nitric oxide synthase-negative nerve profiles in a control specimen following preabsorption of NOS antiserum with purified NOS. Scale bar = 0.5 ~m. (a) x 35300; (b) @BULLET 48000; (c) x 140600; (d) x 25000.
Source publication
This is the first report on the ultrastructural distribution of nicotinamide adenine dinucleotide phosphate-diaphorase activity and neuronal isoform (Type I) of nitric oxide synthase immunoreactivity in perivascular nerves (axons) and vascular endothelial cells. In the Sprague-Dawley rat cerebral basilar artery, positive labelling for nicotinamide...
Citations
... Chayen et al 10 argued that NOS (or indeed any enzyme) cannot be directly responsible for diformazan production in aldehyde-treated tissues, and that an unidentified 'proteinaceous material' promotes NADPH-dependent reduction of NBT. Key points were that (1) α-NADPH is as effective as β-NADPH in promoting NBT reduction to diformazan 10 , (2) NOS activity is specific to β-NADPH 10 , (3) NOS activity is abolished by paraformaldehyde, and (4) NOS and NADPH diaphorase do not always show a similar distribution in tissues both under optical or electron microscopy scrutiny [11][12][13] . For example, Loesch et al 11,12 found that NOS was associated with membranes of cytoplasmic vesicles in endothelial cells and nerve terminals whereas it was not in the lumen of these vesicles. ...
... Key points were that (1) α-NADPH is as effective as β-NADPH in promoting NBT reduction to diformazan 10 , (2) NOS activity is specific to β-NADPH 10 , (3) NOS activity is abolished by paraformaldehyde, and (4) NOS and NADPH diaphorase do not always show a similar distribution in tissues both under optical or electron microscopy scrutiny [11][12][13] . For example, Loesch et al 11,12 found that NOS was associated with membranes of cytoplasmic vesicles in endothelial cells and nerve terminals whereas it was not in the lumen of these vesicles. In contrast, NADPH diaphorase was contained in the lumen but not membranes of the vesicles suggesting that a soluble factor generates NADPH diaphorase. ...
... Bubbling solutions of NBT with authentic NO in the absence of oxygen and hence NO 2 for 20 min yielded only minor diformazan (solution 11). Bubbling solutions of NBT + β-NADPH or NBT + β-NADPH + paraformaldehyde with NO also yielded minor diformazan (solutions 12,13). This suggests that NO does not promote NADPH-dependent reduction of NBT and that l-Snitrosocysteine-induced facilitation of NADPH-dependent reduction of NBT is not due to decomposition of this S-nitrosothiol to NO. Reduced thiols such as cysteine will directly reduce NBT 49 . ...
NADPH diaphorase is used as a histochemical marker of nitric oxide synthase (NOS) in aldehyde-treated tissues. It is thought that the catalytic activity of NOS promotes NADPH-dependent reduction of nitro-blue tetrazolium (NBT) to diformazan. However, it has been argued that a proteinaceous factor other than NOS is responsible for producing diformazan in aldehyde-treated tissues. We propose this is a NO-containing factor such as an S-nitrosothiol and/or a dinitrosyl-iron (II) cysteine complex or nitrosated proteins including NOS. We now report that (1) S-nitrosothiols covalently modify both NBT and TNBT, but only change the reduction potential of NBT after modification, (2) addition of S-nitrosothiols or β- or α-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus β- or α-NADPH elicited rapid formation of diformazan in the absence or presence of paraformaldehyde, (4) addition of S-nitrosothiols to solutions of NBT plus β- or α-NADP did not produce diformazan, (5) S-nitrosothiols did not promote NADPH-dependent reduction of tetra-nitro-blue tetrazolium (TNBT) in which all four phenolic rings are nitrated, (6) cytoplasmic vesicles in vascular endothelial cells known to stain for NADPH diaphorase were rich in S-nitrosothiols, and (7) procedures that accelerate decomposition of S-nitrosothiols, markedly reduced NADPH diaphorase staining in tissue sections subsequently subjected to paraformaldehyde fixation. Our results suggest that NADPH diaphorase in aldehyde-fixed tissues is not enzymatic but is due to the presence of NO-containing factors (free SNOs or nitrosated proteins such as NOS), which promote NADPH-dependent reduction of NBT to diformazan.
... However, these findings could be either the cause of haemorrhoids or a response to pathological changes. Clearly, NOS1 can be found not only in neural tissue but also in endothelium and vascular smooth muscle [28,29]. ...
... The endothelial cells may adapt to the local environment by up-or down-regulation and/or changes in NOS subtype for generating NO [31,32]. Moreover, the NOS3 expression in neural tissue was reported to be found under normal physiological [28] and under under pathological conditions such as ischaemia-reperfusion state and malignancy [33,34]. ...
Objective:
To study the distribution of nitric oxide synthase (NOS) isoforms and protein levels in human haemorrhoids and rectal tissue Methods: Protein expression of NOS1, NOS2 and NOS3 were compared between haemorrhoids (n=14) and normal rectal submucosa (n=6) using Western blot analysis. Localisation of all NOS isoforms to specific structures was determined by immunohistochemistry.
Results:
Western blot analysis showed median (interquartile range) protein levels of all NOS isoforms were 1.5-2.4 times higher in haemorrhoids than rectal tissue; 121.4 (55.2-165.5) vs 50.0 (25.5-73.7) for NOS1 (p=0.020), 32.2 (23.8-140.6) vs 14.8 (9.6-34.0) for NOS2 (p=0.109), and 80.1 (62.0-139.5) vs 54.3 (48.7 -61.7) for NOS3 (p=0.015). Immunohistochemistry revealed a different distribution and location of all NOS isoforms in vascular and non-vascular structure of haemorrhoids and rectal tissues. The number of haemorrhoid specimens showing positive immunoreactivity of NOS in the vascular endothelium was significantly higher than that in rectal tissue for NOS1 (11/14 (79%) vs 1/6 (17%); p=0.018) and NOS3 (8/14 (57%) vs 0/6 (0%); p=0.042), but not for NOS2 (6/14 (43%) vs 4/6 (67%); p=0.63).
Conclusions:
Haemorrhoids have significantly higher protein levels of NOS1 and NOS3 than rectal tissue. The vascular endothelium of haemorrhoids also has significantly higher positive immunoreactivity of NOS1 and NOS3 than rectal tissue suggesting that blood vessels in haemorrhoids are exposed to higher NO concentrations than those of rectal tissue. Since haemorrhoids exhibit marked vascular dilatation and present with bleeding or swelling, a reduction in NOS - by applying NOS inhibitors - may potentially improve the symptoms of haemorrhoids.
... The exact inductive mechanisms are not fully addressed, however, based on the constituents of cytonemes, it's natural to speculate that NO may act through modulating cytoskeleton behaviors to extend cytomenes. Extensive studies have shown that nitric oxide synthase interacts with tubulin and actin cytoskeleton (Su et al., 2003Kondrikov et al., 2006Kondrikov et al., , 2010 and modulates their activity through tyrosine nitration and S-nitrosylation of target cytoskeletal proteins or cytoskeletonassociated proteins in both animals (Loesch et al., 1994;Aslan et al., 2003;Tedeschi et al., 2005;Thom et al., 2008; and plants (Kasprowicz et al., 2009;Yemets et al., 2011;Yao et al., 2012;Blume et al., 2013;Rodríguez-Serrano et al., 2014). Thus, it's highly likely that these modifications on cytoskeleton, particularly the actin, could eventually impact on cytonemes formation. ...
The reactive oxygen species, generally labeled toxic due to high reactivity without target specificity, are gradually uncovered as signaling molecules involved in a myriad of biological processes. But one important feature of ROS roles in macromolecule movement has not caught attention until recent studies with technique advance and design elegance have shed lights on ROS signaling for intercellular and interorganelle communication. This review begins with the discussions of genetic and chemical studies on the regulation of symplastic dye movement through intercellular tunnels in plants (plasmodesmata), and focuses on the ROS regulatory mechanisms concerning macromolecule movement including small RNA-mediated gene silencing movement and protein shuttling between cells. Given the premise that intercellular tunnels (bridges) in mammalian cells are the key physical structures to sustain intercellular communication, movement of macromolecules and signals is efficiently facilitated by ROS-induced membrane protrusions formation, which is analogously applied to the interorganelle communication in plant cells. Although ROS regulatory differences between plant and mammalian cells exist, the basis for ROS-triggered conduit formation underlies a unifying conservative theme in multicellular organisms. These mechanisms may represent the evolutionary advances that have enabled multicellularity to gain the ability to generate and utilize ROS to govern material exchanges between individual cells in oxygenated environment.
... Different cardioprotective signaling pathways also converge to release NO (29,30). We thus determined how S1P/FTY720 induced NO production that results in cardioprotection. ...
... In contrast, NOS-containing vascular endothelial cells exhibit a characteristic pattern of staining in which the reaction product is restricted to a small number of punctate regions within each endothelial cell (36,37). Other studies (29,40) have shown that this reaction product is present throughout the cytoplasm and in association with the membranes of vesicles, mitochondria, and endoplasmic reticulum. Our study has shown that neonatal cardiomyocytes show an identical pattern of staining following fluorescence using the NO sensitive dye DAF-FM, and thus the punctate staining might represent patches of NOS activity. ...
We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min (n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h (n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1(f/f)), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1(cko)) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.
... nNOS immunoreactivity was identified even in vascular endothelial cells [16]. Immunocytochemical studies indicate that in the nNOS (-/-) mice, eNOS is expressed in CA1 neurons [17]. ...
... Immunocytochemical studies indicate that in the nNOS (-/-) mice, eNOS is expressed in CA1 neurons [17]. The location of eNOS and nNOS are not necessarily divided into vascular endothelial cells and perivascular nerve cells clearly [16,17]. We consider that eNOS (-/-) mice may produce NO from nNOS not only in perivascular nerve cells but also in vascular endothelial cells. ...
The purpose of this study was to clarify the kinetics of nitric oxide (NO) induced by either endothelial NO synthase (eNOS) or neuronal NO synthase (nNOS) after transient global forebrain ischemia. We investigated NO production and ischemic changes to hippocampal CA1 neurons in eNOS knockout (-/-) mice and nNOS (-/-) mice during cerebral ischemia and reperfusion. NO production was continuously monitored by in vivo microdialysis. Global forebrain ischemia was produced by occlusion of both common carotid arteries for 10 minutes. Levels of nitrite (NO(2)(-)) and nitrate (NO(3)(-)), as NO metabolites, in dialysate were determined using the Griess reaction. Two hours after the start of reperfusion, animals were perfused with 4% paraformaldehyde. Hippocampal CA1 neurons were divided into three phases (severely ischemic, moderately ischemic, surviving), and the ratio of surviving neurons to degenerated neurons was calculated as the survival rate. The relative cerebral blood flow (rCBF) was significantly higher in nNOS (-/-) mice than in control mice after reperfusion. Levels of NO(3)(-) were significantly lower in eNOS (-/-) mice and nNOS (-/-) mice than in control mice during ischemia and reperfusion. NO(3)(-) levels were significantly lower in nNOS (-/-) mice than in eNOS (-/-) mice after the start of reperfusion. Survival rate tended to be higher in nNOS (-/-) mice than in control mice, but not significantly. These in vivo data suggest that NO production in the striatum after reperfusion is closely related to activities of both nNOS and eNOS, and is mainly related to nNOS following reperfusion.
... 16,17 This also applies to the control of cerebral circulation where endogenous NO from cerebrovascular endothelium and cerebrovascular autonomic nerves regulates the tone of cerebral arteries. [18][19][20][21][22][23][24][25][26][27] A number of immunohistochemical studies detected both endothelial NOS (eNOS) and neuronal NOS (nNOS) in human and animal large cerebral arteries, for example, basilar, middle cerebral, and anterior cerebral arteries, [28][29][30][31][32][33][34][35] supporting the pharmacological evidence of NO involvement in the regulation of cerebral blood flow. [18][19][20][21] Changes in cerebral blood flow have been reported in patients with DM 36 as has been an increased frequency of cerebrovascular accidents. ...
... Here, the pattern of eNOS-immunolabeling in the nondiabetic control rats was similar to that observed in other studies with healthy and hypertensive rats. 30,[32][33][34]56 Therefore, predominant location of eNOS-immunolabeling throughout the cytoplasm as well as in association with the membranes of intracellular organelles such as endoplasmic reticulum is recognized as characteristic for healthy endothelial cells of the rat basilar artery. Chronic DM altered the subcellular location of eNOS in the endothelial cells compared with nondiabetic controls or rats treated with epoetin delta. ...
Erythropoiesis-stimulating agents (ESAs) are used to treat anemia associated with renal failure. It is now known that these agents also show a broad range of cell- and tissue-protective effects. In the current study, we explored whether an ESA, epoetin delta, affects vascular pathology linked to diabetes mellitus (DM). In a rat model of streptozotocin-induced DM, we investigated, by pre-embedding electron-immunocytochemistry, whether epoetin delta affects DM-induced structural changes in cerebrovascular endothelium of the rat basilar artery and influences the subcellular distribution of endothelial nitric oxide synthase (eNOS). Epoetin delta treatment influenced DM-induced changes to the distribution of eNOS in, and the structure of, the endothelial cell. This may indicate potential beneficial effects of epoetin delta on cerebrovascular endothelium and suggests eNOS as a possible target molecule of epoetin delta in DM.
... These researchers used immunogold electron microscopy, which only showed that there is an eNOS-like protein in the mitochondria in fixed preparations. Supporting evidence for the functional activity of mtNOS was provided via electron microscopy by the detection of NADPH diaphorase activity in mitochondria (20). Later, iNOS-like immunoreactivity was reported in mitochondrial preparations, and these studies also presented data that indicated that NO is indeed generated by isolated mitochondria preparations (21,22). ...
... However, it is also possible that a cellular NOS protein is merely attached to the outer surface of the mitochondrion. Indeed, the earliest studies of mtNOS showed NADPH diaphorase activity in the vicinity of mitochondria and not in the mitochondrial matrix (20,31,32). Henrich and colleagues located eNOS within sensory neurons and found that the enzyme is anchored to juxta-mitochondrial smooth endoplasmic reticulum (33). ...
New discoveries in the last decade significantly altered our view on mitochondria. They are no longer viewed as energy-making slaves but rather individual cells-within-the-cell. In particular, it has been suggested that many important cellular mechanisms involving specific enzymes and ion channels, such as nitric oxide synthase (NOS), ATP-dependent K+ (KATP) channels, and poly-(APD-ribose) polymerase (PARP), have a distinct, mitochondrial variant. Unfortunately, exploring these parallel systems in mitochondria have technical limitations and inappropriate methods often led to inconsistent results. For example, the intriguing possibility that mitochondria are significant sources of nitric oxide (NO) via a unique mitochondrial NOS variant has attracted intense interest among research groups because of the potential for NO to affect functioning of the electron transport chain. Nonetheless, conclusive evidence concerning the existence of mitochondrial NO synthesis is yet to be presented. This review summarizes the experimental evidence gathered over the last decade in this field and highlights new areas of research that reveal surprising dimensions of NO production and metabolism by mitochondria.
... CAPON interacts with synapsin , a component of synaptic vesicles on the presynaptic side of the synapse. Synapsin is enriched in presynaptic densities [156] , a site that is known functionally and by immunocytochemical studies to be enriched in nNOS [157,158]. The coupling of nNOS to synapsin via CAPON may also be important for specifying the targets of S-nitrosylation since the actions of NO on synaptic vesicle release, in part, requires S-nitrosylation, and several synaptic vesicle machinery proteins have been implicated as S-nitrosylation targets [159]. ...
Nitric oxide (NO) is an endogenously-produced small molecule that has critical roles in cellular signaling and a variety of physiological processes in many tissues, including the brain, the vasculature, and the immune system. In several medical disorders, NO has been implicated in disease pathology, in most cases due to persistent activation or overproduction of one of three NO synthase (NOS) isoforms. Although NOS inhibitors that are both potent and cell-permeable have been developed, none is currently used in the treatment of any disorder. One reason that NOS inhibitors fail to have therapeutic efficacy may be linked to their very low isoform-selectivity. An additional possibility is that NOS inhibitors, even if they exhibit isoform selectivity, might indiscriminately affect beneficial and pathological NO signaling pathways. In this review, we discuss emerging approaches in the development of isoform-specific NOS-directed therapeutics including dimerization inhibitors, novel L-arginine (L-Arg) binding site inhibitors, and dimer stabilization. Additionally, we suggest novel strategies for the future including targeting subcellular localization of NOS and protein-protein interactions with NOS effectors.
... The existence of a distinct mitochondrial nitric oxide synthase enzyme (mtNOS) is much debated [5-8]. The initial finding of mitochondrial NADPH diaphorase activity in basilar arteries, a marker of NO synthesis, was followed by several reports which suggested the presence of various NOS isoforms and also NO production within mitochondria [9][10][11][12][13][14][15]. However, some of these early observations were not reproducible by other laboratories and the question of the existence of mtNOS cannot be conclusively answered at this time [6,16-18]. ...
We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.
... Endothelial cells are well known to regulate the haemodynamics of blood vessels by the synthesis and release of the powerful vasorelaxant nitric oxide (Ignarro et al., 1986;Furchgott et al., 1987) and the potent vasoconstrictor endothelin-1 (Yanagisawa et al., 1988). Whether the endothelial cells of the capybara basilar artery can synthesize these vasoactive substances can be confirmed, e.g. by immunocyt- ochemistry to reveal antigenic sites for endthelin-1 or endothelial nitric oxide synthase, the enzyme synthesizing nitric oxide (see Loesch et al., 1993Loesch et al., , 1994Loesch and Burnstock, 1996b). It has been shown, for example, that subpopulations of rabbit endothelial cells of the basilar and posterior communicating arteries have localized presence of endothelin-1, vasopressin and substance P (Loesch et al., 1993). ...
The present study investigated the ultrastructural features of the basilar artery of the largest rodent species, the capybara. The study suggests that the general ultrastructural morphological organization of the basilar artery of the capybara is similar to that of small rodents. However, there are some exceptions. The basilar artery of the capybara contains a subpopulation of 'granular' vascular smooth muscle cells resembling monocytes and/or macrophages. The possibility cannot be excluded that the presence of these cells reflects the remodelling processes of the artery due to animal maturation and the regression of the internal carotid artery. To clarify this issue, more systemic studies are required involving capybaras of various ages.
