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The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener
Abstract
Hydrogen sulfide (H(2)S) has been traditionally viewed as a toxic gas. It is also, however, endogenously generated from cysteine metabolism. We attempted to assess the physiological role of H(2)S in the regulation of vascular contractility, the modulation of H(2)S production in vascular tissues, and the underlying mechanisms. Intravenous bolus injection of H(2)S transiently decreased blood pressure of rats by 12- 30 mmHg, which was antagonized by prior blockade of K(ATP) channels. H(2)S relaxed rat aortic tissues in vitro in a K(ATP) channel-dependent manner. In isolated vascular smooth muscle cells (SMCs), H(2)S directly increased K(ATP) channel currents and hyperpolarized membrane. The expression of H(2)S-generating enzyme was identified in vascular SMCs, but not in endothelium. The endogenous production of H(2)S from different vascular tissues was also directly measured with the abundant level in the order of tail artery, aorta and mesenteric artery. Most importantly, H(2)S production from vascular tissues was enhanced by nitric oxide. Our results demonstrate that H(2)S is an important endogenous vasoactive factor and the first identified gaseous opener of K(ATP) channels in vascular SMCs.
... To the best of our knowledge, H2S was first identified as an activator of ATP-sensitive potassium (KATP) channels and, subsequently, voltage-gated potassium (Kv7.4) channels [11,12]. This action is correlated with the hyperpolarization of the cell membrane and then with vasorelaxation. ...
... KATP-mediated vasorelaxant effect in aortic rings of Wistar and SHR rats [94]. KATP-mediated vasorelaxant effect in rat aortic rings incubated with H2S (180 µM) [12]. KATP-mediated vasorelaxant effect on rat mesenteric arteries with H2S (100 µmol/L) [95]. ...
... In the aorta, the mesenteric artery, and the tail artery, the expression of the SUR2B and Kir6.1 proteins was lower in hypertensive rats compared with normotensive ones, but treatment with NaHS significantly upregulated the levels of these proteins [94]. Zhao et al. also showed the glibenclamide-dependent vasorelaxant response of H2S [12]. The Snyder group, in 2011, first explored the mechanism through which NaHS stimulates the KATP channels and found S-persulfidation at cysteine-43 on the Kir6.1 subunit [95]. ...
Irisin is a myokine secreted under the influence of physical activity and exposure to low temperatures and through different exogenous stimuli by the cleavage of its precursor, fibronectin type III domain-containing protein 5 (FNDC5). It is mainly known for maintaining of metabolic homeostasis, promoting the browning of white adipose tissue, the thermogenesis process, and glucose homeostasis. Growing experimental evidence suggests the possible central role of irisin in the regulation of cardiometabolic pathophysiological processes. On the other side, hydrogen sulfide (H2S) is well recognized as a pleiotropic gasotransmitter that regulates several homeostatic balances and physiological functions and takes part in the pathogenesis of cardiometabolic diseases. Through the S-persulfidation of cysteine protein residues, H2S is capable of interacting with crucial signaling pathways, exerting beneficial effects in regulating glucose and lipid homeostasis as well. H2S and irisin seem to be intertwined; indeed, recently, H2S was found to regulate irisin secretion by activating the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)/FNDC5/irisin signaling pathway, and they share several mechanisms of action. Their involvement in metabolic diseases is confirmed by the detection of their lower circulating levels in obese and diabetic subjects. Along with the importance of metabolic disorders, these modulators exert favorable effects against cardiovascular diseases, preventing incidents of hypertension, atherosclerosis, heart failure, myocardial infarction, and ischemia–reperfusion injury. This review, for the first time, aims to explore the role of H2S and irisin and their possible crosstalk in cardiovascular diseases, pointing out the main effects exerted through the common molecular pathways involved.
... 3mercaptopyruvate is derived from two sources as follows: L-Cys via cysteine aminotransferase (CAT) (CAT/3-MPST pathway) and D-cysteine via amino acid oxidase (DAO) (DAO/3-MPST pathway) (20). A study reported that H 2 S exhibited specificity to vascular smooth muscle (21), which may be related to differences in the mechanisms of H 2 S production. Given that endogenous H 2 S is synthesized through various pathways, the distribution of H 2 S synthases in various parts of male reproductive organs ( Figure 1) may indicate the source and production mode of H 2 S. Srilatha et al. (22) detected the presence of endogenous H 2 S in smooth muscle tissue homogenates from the CC of rabbits. ...
... The four primary types of potassium (K + ) channels expressed in arterial smooth muscle cells include Ca 2+ -activated (KCa), adenosine triphosphate (ATP)-sensitive (K ATP ), inwardly rectifying (Kir), and voltage-gated (Kv) channels (62). The relaxation effects of H 2 S on vascular SMCs isolated from rats have been shown to rely on the K ATP channel (21). The four K + channels have also been detected in HCC (63). ...
As an important gas signaling molecule, hydrogen sulfide (H2S) affects multiple organ systems, including the nervous, cardiovascular, digestive, and genitourinary, reproductive systems. In particular, H2S not only regulates female reproductive function but also holds great promise in the treatment of male reproductive diseases and disorders, such as erectile dysfunction, prostate cancer, varicocele, and infertility. In this review, we summarize the relationship between H2S and male reproductive organs, including the penis, testis, prostate, vas deferens, and epididymis. As lower urinary tract symptoms have a significant impact on penile erection disorders, we also address the potential ameliorative effects of H2S in erectile dysfunction resulting from bladder disease. Additionally, we discuss the regulatory role of H2S in cavernous smooth muscle relaxation, which involves the NO/cGMP pathway, the RhoA/Rho-kinase pathway, and K⁺ channel activation. Recently, various compounds that can alleviate erectile dysfunction have been reported to be at least partly dependent on H2S. Therefore, understanding the role of H2S in the male reproductive system may help develop novel strategies for the clinical treatment of male reproductive system diseases.
... The mechanism by which H 2 S induces uterine artery dilation is not fully understood. Previous research in mesenteric vascular beds has implicated the activation of ATP-sensitive potassium (K ATP ) channels and large conductance Ca 2+ -activated voltage-dependent potassium (BK Ca ) channels in mediating H 2 S-induced vasodilation [15][16][17]. However, the contribution of K ATP channels to uterine vasodilation associated with pregnancy seems limited [15]. ...
... This H 2 S-induced mesenteric vasodilation was lost in endothelium-denuded arteries [23], suggesting the presence of endothelium is essential for H 2 S-induced BK Camediated mesenteric vasodilation. In contrast, relaxation of the rat aorta and human uterine artery induced by H 2 S appears less dependent on the endothelium [17,19,20], indicating H 2 S directly acts on vascular smooth muscle cells to induce vasodilation. Although BK Ca channels in vascular smooth muscle cells are implicated in pregnancy-induced uterine artery dilation [19,[21][22][23], their involvement in H 2 S-induced vasodilation in rat arteries remains uncertain, and if their sensitivity differs between pregnancy and the nonpregnant state is unknown. ...
Hydrogen sulfide (H2S) is a cardiovascular signaling molecule that causes vasodilation in vascular smooth muscle cells, but its mechanism is unclear. We examined how H2S affects mesenteric and uterine arteries without endothelium in nonpregnant and pregnant rats and the underlying mechanisms. H2S donors GYY4137 and NaHS relaxed uterine arteries more than mesenteric arteries in both pregnant and nonpregnant rats. GYY4137 and NaHS caused greater relaxation in the uterine artery of pregnant versus nonpregnant rats. High extracellular K⁺ abolished NaHS relaxation in pregnant uterine arteries, indicating potassium channel involvement. NaHS relaxation was unaffected by voltage-gated potassium channel blockers, reduced by ATP-sensitive potassium channel blockers, and abolished by calcium-activated potassium (BKCa) channel blockers. Thiol-reductant dithiothreitol also prevented NaHS relaxation. Thus, H2S has region-specific and pregnancy-enhanced vasodilator effects in the uterine arteries, mainly mediated by BKCa channels and sulfhydration.
... In the late 1980's and 1990's, pioneering work by several research groups led to the discovery of H 2 S as an endogenous gasotransmitter with pharmacological and physiological roles in the mammalian cells [1][2][3]. Subsequent studies have also reported its roles in cytoprotection [4][5][6][7][8], neuroprotection [9,10], smooth muscle relaxation [11,12], vasorelaxation and regulation of blood pressure [12][13][14][15][16][17][18], anti-inflammation [5,[19][20][21][22][23][24], cellular respiration [25][26][27][28][29], etc. Owing to its critical role in mammalian pathophysiology, H 2 S has found applications in several conditions as a therapeutic molecule [30][31][32]. ...
Hydrogen sulfide (H 2 S) is a multifaceted gasotransmitter molecule which has potential applications in many pathological conditions including in lowering intraocular pressure and providing retinal neuroprotection. However, its unique physicochemical properties pose several challenges for developing its efficient and safe delivery method system. This study aims to overcome challenges related to H 2 S toxicity, gaseous nature, and narrow therapeutic concentrations range by developing polymeric microparticles to sustain the release of H 2 S for an extended period. Various formulation parameters and their interactions are quantitatively identified using Quality-by-Design (QbD) approach to optimize the microparticle-based H 2 S donor (HSD) delivery system. Microparticles were prepared using a solvent-evaporation coacervation process by using polycaprolactone (PCL), soy lecithin, dichloromethane, Na 2 S.9H 2 O, and silicone oil as polymer, surfactant, solvent, HSD, and dispersion medium, respectively. The microparticles were characterized for size, size distribution, entrapment efficiency, and H 2 S release profile. A Main Effects Screening (MES) and a Response Surface Design (RSD) model-based Box-Behnken Design (BBD) was developed to establish the relationship between critical process parameters (CPPs) and critical quality attributes (CQAs) qualitatively and quantitatively. The MES model identified polymer to drug ratio and dispersion medium quantity as significant CPPs among others, while the RSD model established their quantitative relationship. Finally, the target product performance was validated by comparing predicted and experimental outcomes. The QbD approach helped in achieving overall desired microparticle characteristics with fewer trials and provided a mathematical relationship between the CPPs and the CQAs useful for further manipulation and optimization of release profile up to at least 30 days.
Graphical Abstract
... 89,90 H 2 S treatment increases K ATP channel currents. 91,92 Additionally, H 2 S treatment induces polysulfidation of K ATP channel subunit, Sulphonylurea 2B at Cys24 and Cys1455. 93 Polysulfidation of mitoK ATP would be involved in the cardioprotective effect against IR injury. ...
... Hydrogen sulfide (H 2 S) is now recognized as an endogenous signaling gasotransmitter in various species 22,23 . Cystathionine β-synthase (CBS), which catalyzes the condensation of homocysteine and cysteine to produce H 2 S 24 . ...
Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at “CTGGGG”. In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.
... [3][4][5][6][7][8] For example, the observed vasorelaxation from H 2 S stems from the reaction of H 2 S with oxidized cysteine (Cys) residues of ATP-sensitive potassium channels to produce persulfides, which opens these channels and results in downstream effects of decreasing blood pressure. [9][10][11] As the fundamental understanding of H 2 S and other RSS in biology continues to expand, tools are needed to monitor and perturb RSS in living systems to advance investigations into the biological functions of RSS. ...
Hydrogen sulfide (H2S) is an important reactive sulfur species that is involved in many biological functions, and H2S imbalances have been indicated as a potential biomarker for various diseases. Different H2S donors have been developed to deliver H2S directly to biological systems, but few reports include donors with optical responses that allow for tracking of H2S release. Moreover, donor systems that use the same chemistry to deliver H2S across a palette of fluorescent responses remain lacking. Here we report five thiol‐activated fluorescence turn‐on COS/H2S donors that utilize blue, yellow, orange, red, and near infrared‐emitting dyes functionalized with an H2S‐releasing sulfenyl thiocarbonate scaffold. Upon treatment with thiols, each donor provides a fluorescence turn‐on response (3–310‐fold) and high H2S release efficiencies (>60%). Using combined electrode and fluorescence experiments, we directly correlate the measured H2S release with the fluorescence response. All donors are biocompatible and release H2S in live cell environments. In addition, we demonstrate that the NIR donor allows for imaging H2S release in live rats via subcutaneous injection of the donor loaded into an alginate gel, which to the best of our knowledge is the first in vivo tracking of H2S release from a fluorogenic donor in non‐transparent organisms.
... [3][4][5][6][7][8] For example, the observed vasorelaxation from H 2 S stems from the reaction of H 2 S with oxidized cysteine (Cys) residues of ATP-sensitive potassium channels to produce persulfides, which opens these channels and results in downstream effects of decreasing blood pressure. [9][10][11] As the fundamental understanding of H 2 S and other RSS in biology continues to expand, tools are needed to monitor and perturb RSS in living systems to advance investigations into the biological functions of RSS. ...
Hydrogen sulfide (H2S) is an important reactive sulfur species that is involved in many biological functions, and H2S imbalances have been indicated as a potential biomarker for various diseases. Different H2S donors have been developed to deliver H2S directly to biological systems, but few reports include donors with optical responses that allow for tracking of H2S release. Moreover, donor systems that use the same chemistry to deliver H2S across a palette of fluorescent responses remain lacking. Here we report five thiol‐activated fluorescence turn‐on COS/H2S donors that utilize blue, yellow, orange, red, and near infrared‐emitting dyes functionalized with an H2S‐releasing sulfenyl thiocarbonate scaffold. Upon treatment with thiols, each donor provides a fluorescence turn‐on response (3–310‐fold) and high H2S release efficiencies (>60 %). Using combined electrode and fluorescence experiments, we directly correlate the measured H2S release with the fluorescence response. All donors are biocompatible and release H2S in live cell environments. In addition, we demonstrate that the NIR donor allows for imaging H2S release in live rats via subcutaneous injection of the donor loaded into an alginate gel, which to the best of our knowledge is the first in vivo tracking of H2S release from a fluorogenic donor in non‐transparent organisms.
... Hydrogen sul de (H 2 S) is the sulfur derivative that garners the most attention in the context of colonic health. In the gastrointestinal system, the H 2 S pathway supports epithelial, immune, and enteric nervous system health through various mechanisms, including posttranslational modi cation of protein cysteine residues, activation of K ATP channels, and serving as an inorganic fuel for colonocytes [8][9][10] . However, excessive exposure to H 2 S can be detrimental to the host, damaging the intestinal epithelium and leading to chronic in ammation, as well as disrupting the balance between cellular proliferation and apoptosis 11 . ...
Background
H2S imbalances in the intestinal tract trigger Crohn's disease (CD), a chronic inflammatory gastrointestinal disorder characterized by microbiota dysbiosis and barrier dysfunction. However, a comprehensive understanding of H2S generation in the gut, and the contributions of both microbiota and host to systemic H2S levels in CD, remain to be elucidated. This investigation aimed to enhance comprehension regarding the sulfidogenic potential of both the human host and the gut microbiota.
Results
Our analysis of a treatment-naive CD cohorts' fecal metagenomic and biopsy metatranscriptomic data revealed reduced expression of host endogenous H2S generation genes alongside increased abundance of microbial exogenous H2S production genes in correlation with CD. While prior studies focused on microbial H2S production via dissimilatory sulfite reductases, our metagenomic analysis suggests the assimilatory sulfate reduction (ASR) pathway is a more significant contributor in the human gut, given its high prevalence and abundance. Subsequently, we validated our hypothesis experimentally by generating ASR-deficient E. coli mutants ∆cysJ and ∆cysM through the deletion of sulfite reductase and L-cysteine synthase genes. This alteration significantly affected bacterial sulfidogenic capacity, colon epithelial cell viability, and colonic mucin sulfation, ultimately leading to colitis in murine model. Further study revealed that gut microbiota degrade sulfopolysaccharides and assimilate sulfate to produce H2S via the ASR pathway, highlighting the role of sulfopolysaccharides in colitis and cautioning against their use as food additives.
Conclusions
Our study significantly advances understanding of microbial sulfur metabolism in the human gut, elucidating the complex interplay between diet, gut microbiota, and host sulfur metabolism. We highlight the microbial ASR pathway as an overlooked endogenous H2S producer and a potential therapeutic target for managing CD.
... Garlic, as well as the four Allium ampeloprasum var. Holmense samples here examined, are known to contain several compounds capable of releasing the gasotransmitter H 2 S that, as demonstrated by Zhao et al. (Zhao et al., 2001), in vascular smooth muscle cells stimulates ATP-dependent K + channels causing membrane hyperpolarization and vasodilation. The gasotransmitter can also modify the activity of other K + channels, such as K V 7, intracellular pH, the activity of phosphodiesterases and, consequently, that of cGMP. ...
... Several other enzymes can also produce polysulfides, including su doreductase (SQR), myoglobin, neuroglobin, catalase, superoxide d myeloperoxidase, and cysteinyl tRNA synthetase (CARS) [17][18][19][20][21][22][23]. The tion of H2S with NO also produces polysulfides that have been predicted of synergistic effect of H2S and NO on vascular relaxation [6,[24][25][26][27][28][29][30][31][32]. ...
Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) produced by enzymes play a role as signalling molecules regulating neurotransmission, vascular tone, cytoprotection, inflammation, oxygen sensing, and energy formation. H2Sn, which have additional sulfur atoms to H2S, and other S-sulfurated molecules such as cysteine persulfide and S-sulfurated cysteine residues of proteins, are produced by enzymes including 3-mercaptopyruvate sulfurtransferase (3MST). H2Sn are also generated by the chemical interaction of H2S with NO, or to a lesser extent with H2O2. S-sulfuration (S-sulfhydration) has been proposed as a mode of action of H2S and H2Sn to regulate the activity of target molecules. Recently, we found that H2S/H2S2 regulate the release of neurotransmitters, such as GABA, glutamate, and D-serine, a co-agonist of N-methyl-D-aspartate (NMDA) receptors. H2S facilitates the induction of hippocampal long-term potentiation, a synaptic model of memory formation, by enhancing the activity of NMDA receptors, while H2S2 achieves this by activating transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes, potentially leading to the activation of nearby neurons. The recent findings show the other aspects of TRPA1 channels—that is, the regulation of the levels of sulfur-containing molecules and their metabolizing enzymes. Disturbance of the signalling by H2S/H2Sn has been demonstrated to be involved in various diseases, including cognitive and psychiatric diseases. The physiological and pathophysiological roles of these molecules will be discussed.
... NO has been proven to increase endogenous H2S production by elevating CSE and CBS expression in VSMCs [44,45]. In the present study, it was found that the H2S concentration increased significantly in the PCL/KAT-Cu mat when only GSNO was supplemented (Fig.3 B), indicating that the PCL/KAT-Cu graft could promote H2S production in HUASMCs under a NO atmosphere. ...
... Stimulation operated by 25 mM KCl evoked an active tone sufficiently high to allow the study of myorelaxant agents; moreover, depolarization induced by 25 mM KCl is not particularly marked (see [42]) and does not represent a hurdle to the analysis of potassium channel stimulators [43]. In fact, both L-cysteine, a metabolic precursor of H 2 S that regulates smooth muscle tone by activating K ir 6.1 channels [44,45], and the K ir 6.1 channel activator pinacidil relaxed the high KCl-induced contraction, the latter reverting also the spontaneous tone of the preparations. Furthermore, the specific K ir 6.1 channel blocker glibenclamide shifted the concentration-response curve to both L-cysteine and pinacidil to the right, displaying a competitive antagonism. ...
Flavonoids, ubiquitously distributed in the plant world, are regularly ingested with diets rich in fruit, vegetables, wine, and tea. During digestion, they are partially absorbed in the stomach. The present work aimed to assess the in vitro effects of quercetin and ten structurally related flavonoids on the rat gastric fundus smooth muscle, focussing on ATP-dependent K+ (Kir6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through Kir6.1 channels (IKir6.1) were recorded with the patch-clamp technique and the mechanical activity of gastric fundus smooth muscle strips was studied under isometric conditions. Galangin ≈ tamarixetin > quercetin > kaempferol > isorhamnetin ≈ luteolin ≈ fisetin > (±)-taxifolin inhibited pinacidil-evoked, glibenclamide-sensitive IKir6.1 in a concentration-dependent manner. Morin, rutin, and myricetin were ineffective. The steric hindrance of the molecule and the number and position of hydroxyl groups on the B ring played an important role in the activity of the molecule. Molecular docking simulations revealed a possible binding site for flavonoids in the C-terminal domain of the Kir6.1 channel subunit SUR2B, in a flexible loop formed by residues 251 to 254 of chains C and D. Galangin and tamarixetin, but not rutin relaxed both high K+- and carbachol-induced contraction of fundus strips in a concentration-dependent manner. Furthermore, both flavonoids shifted to the right the concentration-relaxation curves to either pinacidil or L-cysteine constructed in strips pre-contracted by high K+, rutin being ineffective. In conclusion, IKir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion.
... Hydrogen sulfide (H 2 S) is reported to elicit several physiological and pharmacological functions in mammalian cells [1][2][3][4][5]. In addition to modulating neuronal messages in the brain [6,7], H 2 S also mediates critical processes such as neurotransmission [6,8], cytoprotection [9][10][11][12][13], neuroprotection [14,15], smooth muscle relaxation [16,17], vasorelaxation and regulation of blood pressure [7,[17][18][19][20][21][22], antiinflammation [10,[23][24][25][26][27][28] and cellular respiration [29][30][31][32][33]. ...
Background: Hydrogen sulfide (H 2 S), an endogenous gasotransmitter, has potential applications in several conditions. However, its quantification in simulated physiological solutions is a major challenge due to its gaseous nature and other physicochemical properties. Aim: This study was designed to compare four commonly used H 2 S detection and quantification methods in aqueous solutions. Methods: The four techniques compared were one colorimetric, one chromatographic and two electrochemical methods. Results: Colorimetric and chromatographic methods quantified H 2 S in millimolar and micromole ranges, respectively. The electrochemical methods quantified H 2 S in the nanomole and picomole ranges and were less time-consuming. Conclusion: The H 2 S quantification method should be selected based on the specific requirements of a research project in terms of sensitivity, response time and cost-effectiveness.
... In the present study, we cannot exclude the possibility that endogenous H 2 S production directly excites neurons. However, previous studies focusing on the direct effect of H 2 S on neurons have found that H 2 S can activate the K ATP channel and cause hyperpolarization 4,[39][40][41] . In this study, we found that the effects of increasing the excitability of neurons by blocking the inhibitory synaptic transmission on the respiratory pattern completely differed from those of H 2 S synthesis inhibition. ...
Hydrogen sulfide (H 2 S), which is synthesized in the brain, modulates the neural network. Recently, the importance of H 2 S in respiratory central pattern generation has been recognized, yet the function of H 2 S in the medullary respiratory network remains poorly understood. Here, to evaluate the functional roles of H 2 S in the medullary respiratory network, the Bötzinger complex (BötC), the pre-Bötzinger complex (preBötC), and the rostral ventral respiratory group (rVRG), we observed the effects of inhibition of H 2 S synthesis at each region on the respiratory pattern by using an in situ arterially perfused preparation of decerebrated male rats. After microinjection of an H 2 S synthase inhibitor, cystathionine β-synthase, into the BötC or preBötC, the amplitude of the inspiratory burst decreased and the respiratory frequency increased according to shorter expiration and inspiration, respectively. These alterations were abolished or attenuated in the presence of a blocker of excitatory synaptic transmission. On the other hand, after microinjection of the H 2 S synthase inhibitor into the rVRG, the amplitude of the inspiratory burst was attenuated, and the respiratory frequency decreased, which was the opposite effect to those obtained by blockade of inhibitory synaptic transmission at the rVRG. These results suggest that H 2 S synthesized in the BötC and preBötC functions to limit respiratory frequency by sustaining the respiratory phase and to maintain the power of inspiration. In contrast, H 2 S synthesized in the rVRG functions to promote respiratory frequency by modulating the interval of inspiration and to maintain the power of inspiration. The underlying mechanism might facilitate excitatory synaptic transmission and/or attenuate inhibitory synaptic transmission.
Hydrogen sulfide (H2S) is a gaseous signaling molecule produced in the body by three enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). H2S is crucial in various physiological processes associated with female mammalian reproduction. These include estrus cycle, oocyte maturation, oocyte aging, ovulation, embryo transport and early embryo development, the development of the placenta and fetal membranes, pregnancy, and the initiation of labor. Despite the confirmed presence of H2S-producing enzymes in all female reproductive tissues, as described in this review, the exact mechanisms of H2S action in these tissues remain in most cases unclear. Therefore, this review aims to summarize the knowledge about the presence and effects of H2S in these tissues and outline possible signaling pathways that mediate these effects. Understanding these pathways may lead to the development of new therapeutic strategies in the field of women’s health and perinatal medicine.
The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several biochemical processes. Human cystathionine β-synthase is a heme-containing, pyridoxal 5'-phosphate (PLP)-dependent enzyme found in this pathway. The heme group does not participate directly in catalysis, but has a regulatory function, whereby CO or NO binding inhibits the PLP-dependent reactions. In this study, we explore the detailed structural changes responsible for inhibition using quantum chemical calculations to validate the experimentally observed bonding patterns associated with heme CO and NO binding and molecular dynamics simulations to explore the medium-range structural changes triggered by gas binding and propagating to the PLP active site, which is more than 20 Å distant from the heme group. Our results support a previously proposed mechanical signaling model, whereby the cysteine decoordination associated with gas ligand binding leads to breaking of a hydrogen bond with an arginine residue on a neighbouring helix. In turn, this leads to a shift in position of the helix, and hence also of the PLP cofactor, ultimately disrupting a key hydrogen bond that stabilizes the PLP in its catalytically active form.
Recent progress in the molecular design, structural classification, mechanisms of generation, triggered release, structure–activity relationships, and fluorescence response mechanism of fluorescent small molecule donors is discussed.
In this work, we analyzed the role of voltage-gated (KV), calcium-activated (KCa), and inward-rectifier potassium channels (Kir) in the effects of hydrogen sulphide (H2S) donor sodium hydrosulphide (NaHS) on the spontaneous contractile activity of the rat jejunum. Experiments were performed on jejunum segments under isometric contraction conditions. It was shown that NaHS reduced the basal tension of the segments, the amplitude, and the frequency of spontaneous contractions in a dose-dependent manner (10–500 μM); the half-effective concentration (EC50) of the inhibitory effect of NaHS on amplitude was 165 μM. The KV channel blocker 4-AP (200 µM) increased the amplitude of spontaneous contractions and subsequent application of NaHS (200 μM) suppressed the amplitude and frequency of spontaneous activity as well as in the control; the effect on tonic tension was less pronounced. TEA (3 mM), a non-specific blocker, and paxillin (1 µM), a specific blocker of large conductance KСа (ВK) channels, increased the amplitude of spontaneous contractions, while the inhibitory effect of NaHS was completely preserved. The selective blocker of small conductance KCa (SK) channels NS8593 (4 μM) did not affect the tension and the parameters of spontaneous contractions and did not prevent the effects of NaHS. Diazoxide (100 μM), the opener of КATP channels, caused a decrease in the basal tone, the amplitude and frequency of spontaneous contractions. Diazoxide and KATP channel blocker glibenclamide (50 μM) prevented the effects of NaHS on the basal tone. The Kir-channel blocker BaCl2 (30 µM) increased the amplitude of spontaneous contractions and eliminated the inhibitory effects of NaHS on the frequency and amplitude of spontaneous contractions, and the basal tension decrease was less pronounced compared to control. Thus, a decrease in the tonic tension of a rat jejunum preparation under the action of an H2S donor is associated with the activation of Kir, including КATP channels, while the effects of H2S on the amplitude and frequency of spontaneous contractions are mediated by an increase in Ba2+-sensitive conductance.
The intriguing chemistry of chalcogen (S, Se)-containing ligands and their capability to bridge multiple metal centres have resulted in a plethora of reports on transition metal complexes featuring hydrosulfide (HS-) and polychalcogenides (En2-, E = S, Se). While a large number of such molecules are strictly organometallic complexes, examples of non-organometallic complexes featuring HS- and En2- with N-/O-donor ligands are relatively rare. The general synthetic procedure for the transition metal-hydrosulfido complexes involves the reaction of the corresponding metal salts with HS-/H2S and this is prone to generate sulfido bridged oligomers in the absence of sterically demanding ligands. On the other hand, the synthetic methods for the preparation of transition metal-polychalcogenido complexes include the reaction of the corresponding metal salts with En2- or the two electron oxidation of low-valent metals with elemental chalcogen, often at an elevated temperature and/or for a long time. Recently, we have developed new synthetic methods for the preparation of two new classes of binuclear transition metal complexes featuring either HS-, or Sn2- and Sen2- ligands. The new method for the synthesis of transition metal-hydrosulfido complexes involved transition metal-mediated hydrolysis of thiolates at room temperature (RT), while the method for the synthesis of transition metal-polychalcogenido complexes involved redox reaction of coordinated thiolates and exogenous elemental chalcogens at RT. An overview of the synthetic aspects, structural properties and intriguing reactivity of these two new classes of transition metal complexes is presented.
Kidney transplantation has become the preferred treatment option for patients with end-stage renal disease. Although it is a superior alternative to long-term hemodialysis, optimal kidney preservation prior to transplantation remains a major challenge, as the gold standard of renal graft preservation by static cold storage tion in University of Wisconsin (UW) solution on ice at 4 °C contributes to graft injury and long-term post-transplantation complications. An emerging strategy which holds great therapeutic promise against cold renal graft injury and limiting long-term post-transplant complications is supplementation of the cold UW preservation solution with hydrogen sulfide (H2S), the third established member of the gasotransmitter family. This novel pharmacological strategy has been demonstrated experimentally, with compelling success at different preservation temperatures (hypothermic, normothermic and subnormothermic) and preservation techniques such as static cold storage and machine perfusion, suggesting the need for pharmacological modification of existing preservation solutions beyond cold storage to improve the quality of renal graft preservation. This chapter presents an overview of organ graft preservation strategies prior to transplantation, with particular emphasis on the kidney as the most commonly transplantable organ and highly vulnerable to transplantation-induced injury. The chapter also discusses H2S as the future of renal graft preservation for a better transplantation outcome. A section of the chapter also highlights important lessons that can be learned from how nature protects the organs of mammalian hibernators from the damaging effects of repetitive cycle of cold ischemia and reperfusion.
Hydrogen sulfide (H2S), a gas with a characteristic rotten-egg smell, gained historic notoriety for its toxicity and death at high concentrations especially among industrial workers. This is due to its ability to reversibly inhibit the activity of cytochrome c oxidase, a terminal enzyme of the mitochondrial electron transport chain. Recently, however, H2S has risen above its notorious public image and is now seen by researchers as an endogenously produced gaseous signaling molecule that plays an important role in cellular homeostasis and influences several physiological and pathological processes at low physiological and non-toxic concentrations. Its production is catalyzed by two cytosolic enzymes, cystathionine β-synthase and cystathionine γ-lyase, a mitochondrial enzyme, 3-mecaptopyruvate sulfurtransferase, and a peroxisomal enzyme, d-amino acid oxidase. Several recent experimental studies have demonstrated that at low micromolar concentrations, H2S plays a complex and essential role in normal renal function, and dysregulation of its production has been implicated in various renal pathologies. In addition, exogenous H2S administration has been reported to exhibit important therapeutic characteristics that target multiple molecular pathways in common renal pathologies in which reduced levels of renal and plasma H2S were observed. This chapter presents current understanding of H2S in the physiology of the renal system, and lays the foundation for discussion on H2S as a novel pharmacological agent to modify organ transplantation protocol, which are discussed in the subsequent chapters of this book.
Cold ischemia-reperfusion injury (IRI) is an inevitable and unresolved problem that is considered the transplant surgeon’s enemy. It poses a great challenge in solid organ transplantation (SOT), and represents a major factor that increases acute tubular necrosis, decreases graft survival, and delays graft function. This complicates graft quality, post-transplant patient care and organ transplantation outcomes, and therefore undermines the success of SOT. This chapter presents recent advances in research regarding novel pharmacological strategies involving the use of different donor molecules of hydrogen sulfide (H2S), the third established member of the gasotransmitter family, against cold IRI in different experimental models of SOT (kidney, heart, lung, liver, pancreas and intestine). In addition, the author also discusses the molecular mechanisms underlying the effects of these H2S donor molecules in SOT, and suggestions for clinical translation. The findings in this chapter showed that storage of donor organs in H2S-supplemented preservation solution or administration of H2S to organ donor prior to organ procurement and to recipient at the start and during reperfusion is a novel, simple and cost-effective pharmacological approach to minimize cold IRI, limit post-transplant complications and improve transplantation outcomes. In conclusion, experimental evidence demonstrate that H2S donors can significantly mitigate cold IRI during SOT through inhibition of a complex cascade of interconnected cellular and molecular events involving microcirculatory disturbance and microvascular dysfunction, mitochondrial injury, inflammatory responses, cell damage and cell death, and other damaging molecular pathways while promoting protective pathways. Translating these promising findings from bench to bedside will lay the foundation for the use of H2S donor molecules in clinical SOT in the future.
Gasotransmitters are a class of small endogenously produced gaseous signaling molecules. They are dissolved gases in blood and other body fluids, and play important roles in cellular homeostasis and impact physiological and pathophysiological conditions. Nitric oxide was the first gasotransmitter to be identified followed by carbon monoxide, while hydrogen sulfide was recently established as the third member of the gasotransmitter family. Although these gases gained historic notoriety for their toxicity and death, studies over the past few decades have shown that they have risen above their notorious tags and have now emerged as important intracellular mediators of cytoprotection in various pathological conditions such as ischemia-reperfusion injury in organ transplantation. This chapter introduces gasotransmitters as volatile intracellular messenger molecules, with complex interactions with one another in compensatory and reciprocal fashion when the level of one is depleted under pathological conditions. In addition, the chapter also summarizes various therapeutic properties of these gases such as antioxidant, anti-inflammatory, anti-apoptotic and vasodilatory properties, enabling them to mediate donor organ protection during pre-transplant, peri-operative and post-transplant periods, and ultimately contributing to improving organ graft quality and prolonging transplant recipient survival.
In adult fish, neurogenesis occurs in many areas of the brain, including the cerebellum, with the ratio of newly formed cells relative to the total number of brain cells being several orders of magnitude greater than in mammals. Our study aimed to compare the expressions of aromatase B (AroB), glutamine synthetase (GS), and cystathionine-beta-synthase (CBS) in the cerebellum of intact juvenile chum salmon, Oncorhynchus keta. To identify the dynamics that determine the involvement of AroB, GS, and CBS in the cellular mechanisms of regeneration, we performed a comprehensive assessment of the expressions of these molecular markers during a long-term primary traumatic brain injury (TBI) and after a repeated acute TBI to the cerebellum of O. keta juveniles. As a result, in intact juveniles, weak or moderate expressions of AroB, GS, and CBS were detected in four cell types, including cells of the neuroepithelial type, migrating, and differentiated cells (graphic abstract, A). At 90 days post injury, local hypercellular areas were found in the molecular layer containing moderately labeled AroB+, GS+, and CBS+ cells of the neuroepithelial type and larger AroB+, GS+, and CBS+ cells (possibly analogous to the reactive glia of mammals); patterns of cells migration and neovascularization were also observed. A repeated TBI caused the number of AroB+, GS+, and CBS+ cells to further increase; an increased intensity of immunolabeling was recorded from all cell types (graphic abstract, C). Thus, the results of this study provide a better understanding of adult neurogenesis in teleost fishes, which is expected to clarify the issue of the reactivation of adult neurogenesis in mammalian species.
Atherosclerosis is a chronic inflammatory disease in which fats, lipids, cholesterol, calcium, proliferating smooth muscle cells, and immune cells accumulate in the intima of the large arteries, forming atherosclerotic plaques. A complex interplay of various vascular and immune cells takes place during the initiation and progression of atherosclerosis. Multiple reports indicate that tight control of reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) production is critical for maintaining vascular health. Unrestricted ROS and RNS generation may lead to activation of various inflammatory signaling pathways, facilitating atherosclerosis. Given these deleterious consequences, it is important to understand how ROS and RNS affect the signaling processes involved in atherogenesis. Conversely, RSS appears to exhibit an atheroprotective potential and can alleviate the deleterious effects of ROS and RNS. Herein, we review the literature describing the effects of ROS, RNS, and RSS on vascular smooth muscle cells, endothelial cells, and macrophages and focus on how changes in their production affect the initiation and progression of atherosclerosis. This review also discusses the contribution of ROS, RNS, and RSS in mediating various post-translational modifications, such as oxidation, nitrosylation, and sulfation, of the molecules involved in inflammatory signaling.
Hydrogen sulfide (H2S) belongs to the family of gasotransmitters and can modulate a myriad of biological signaling pathways. Among others, its cardioprotective effects, through antioxidant, anti-inflammatory, anti-fibrotic, and proangiogenic activities, are well-documented in experimental studies. Cardiorespiratory failure, predominantly cardiomyopathy, is a life-threatening complication that is the number one cause of death in patients with Duchenne muscular dystrophy (DMD). Although recent data suggest the role of H2S in ameliorating muscle wasting in murine and Caenorhabditis elegans models of DMD, possible cardioprotective effects have not yet been addressed. In this review, we summarize the current understanding of the role of H2S in animal models of cardiac dysfunctions and cardiac cells. We highlight that DMD may be amenable to H2S supplementation, and we suggest H2S as a possible factor regulating DMD-associated cardiomyopathy.
Oxygen (O 2 ), nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H 2 S), and hydrogen (H 2 ) with direct effects, and carbon dioxide (CO 2 ) with complementary effects on the condition of various diseases are known as therapeutic gases. The targeted delivery and in situ generation of these therapeutic gases with controllable release at the site of disease has attracted attention to avoid the risk of gas poisoning and improve their performance in treating various diseases such as cancer therapy, cardiovascular therapy, bone tissue engineering, and wound healing. Stimuli‐responsive gas‐generating sources and delivery systems based on biomaterials that enable on‐demand and controllable release are promising approaches for precise gas therapy. This work highlights current advances in the design and development of new approaches and systems to generate and deliver therapeutic gases at the site of disease with on‐demand release behavior. The performance of the delivered gases in various biomedical applications is then discussed.
Significance:
Aging is a complex process associated with an increased risk of many diseases, including thrombosis. The current review summarizes age-related prothrombotic mechanisms in clinical settings of thromboembolism, focusing on the role of fibrin structure and function modified by oxidative stress.
Recent advances:
Aging affects blood coagulation and fibrinolysis via multiple mechanisms, including enhanced oxidative stress, with an imbalance in the oxidant/antioxidant mechanisms, leading to loss of function and accumulation of oxidized proteins, including fibrinogen. Age-related prothrombotic alterations are multifactorial involving enhanced platelet activation, endothelial dysfunction, and changes in coagulation factors and inhibitors. Formation of more compact fibrin clot networks displaying impaired susceptibility to fibrinolysis represents a novel mechanism, which might contribute to atherothrombosis and venous thrombosis. Alterations to fibrin clot structure/function are at least in part modulated by posttranslational modifications of fibrinogen and other proteins involved in thrombus formation, with a major impact of carbonylation. Fibrin clot properties are also involved in the efficacy and safety of therapy with oral anticoagulants, statins, and/or aspirin.
Critical issues:
Since a prothrombotic state is observed in very elderly individuals free of diseases associated with thromboembolism, the actual role of activated blood coagulation in health remains elusive. It is unclear to what extent oxidative modifications of coagulation and fibrinolytic proteins, in particular fibrinogen, contribute to a prothrombotic state in healthy aging.
Future directions:
Ongoing studies will show whether novel therapies that may alter oxidative stress and fibrin characteristics are beneficial to prevent atherosclerosis and thromboembolic events associated with aging.
Hydrogen sulfide (H2S) was recognized as a gaseous signaling molecule, similar to nitric oxide (-NO) and carbon monoxide (CO). The aim of this review is to provide an overview of the formation of hydrogen sulfide (H2S) in the human body. H2S is synthesized by enzymatic processes involving cysteine and several enzymes, including cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (3MST) and D-amino acid oxidase (DAO). The physiological and pathological effects of hydrogen sulfide (H2S) on various systems in the human body have led to extensive research efforts to develop appropriate methods to deliver H2S under conditions that mimic physiological settings and respond to various stimuli. These functions span a wide spectrum, ranging from effects on the endocrine system and cellular lifespan to protection of liver and kidney function. The exact physiological and hazardous thresholds of hydrogen sulfide (H2S) in the human body are currently not well understood and need to be researched in depth. This article provides an overview of the physiological significance of H2S in the human body. It highlights the various sources of H2S production in different situations and examines existing techniques for detecting this gas.
In prolonged complete unilateral ureteral obstruction, reduced renal blood flow places the kidney in a state of ischemia, which can cause tubular injury and inflammation. Infiltrating inflammatory cells release transforming growth factor-beta 1 (TGF-β1), a cytokine that initiates fibrosis through epithelial-mesenchymal transition (EMT) pathway. Persistent fibrosis can lead to irreversible renal injury and loss of renal function. While surgical intervention can remove the obstruction, relief of obstruction may not fully reverse renal injury. Additionally, patients often encounter long wait times between initial consultation and medical intervention, resulting in the accumulation of renal injury that may cause permanent dysfunction. Currently, accepted pharmacological therapies to mitigate the symptoms of obstructive nephropathy include acetaminophen, cyclooxygenase inhibitors, nonsteroidal anti-inflammatory medications, opioids, and alpha-receptor blockers. However, there is no evidence that they mitigate renal injury. Therefore, identifying potential therapies that could be administered during obstruction may help to improve renal function following decompression. Scientific evidence suggests that endogenously produced gasotransmitters can exhibit anti-inflammatory and antioxidant effects. Nitric oxide, carbon monoxide, and hydrogen sulfide have been identified as gasotransmitters and have been shown to have cytoprotective effects in various models of tissue injury. Studies have shown that treatment with sodium hydrogen sulfide (a hydrogen sulfide donor salt) mitigated TGF-β1 expression, oxidative stress, fibrosis, and inflammation associated with urinary obstruction. More recently, the use of more directed hydrogen sulfide donor molecules, such as GYY4137, has led to significant decreases in inflammation, fibrosis, and expression of EMT markers following urinary obstruction. Taken together, these findings suggest that hydrogen sulfide may be a novel potential therapy against obstructive nephropathy. This chapter focuses on the pathogenesis and treatment of obstructive nephropathy and proposes novel upcoming strategies that could improve patient outcomes.
Hypertension is a major public health problem globally. It is the most common cause of cardiovascular morbidities and mortalities, with a negative impact on renal function. Uncontrolled hypertension causes chronic kidney disease, which progresses to end-stage renal disease and eventually loss of renal function. Unfortunately, the mechanism underlying the pathogenesis of hypertension and its associated nephropathy is still poorly understood. Also worrying is the fact that despite conventional antihypertensive therapies, achievement of blood pressure control and preservation of renal function still remain a worldwide public health challenge in a significant subpopulation of hypertensive patients. This suggests the need for novel pharmacotherapeutic approaches to curb the problem. Hydrogen sulfide (H2S), the third established member of the gasotransmitter family after nitric oxide and carbon monoxide, has been recognized and established to possess antihypertensive and renoprotective properties, which may represent an important therapeutic alternative for hypertensive nephropathy. In this chapter, recent findings from preclinical studies about the therapeutic effect of H2S against hypertensive nephropathy and its future clinical use are discussed. A section of the chapter also discusses recent developments about clinical and translational research on calcium-based nephrolithiasis as a risk factor for hypertensive nephropathy, with a further discussion on H2S as an emerging novel therapy to improve clinical outcome.
Hydrogen sulfide (H2S), a gas with a characteristic rotten-egg smell, gained historic notoriety for its toxicity and death at high concentrations especially among industrial workers. This is due to its ability to reversibly inhibit the activity of cytochrome c oxidase, a terminal enzyme of the mitochondrial electron transport chain. Recently, however, H2S has risen above its notorious public image and is now seen by researchers as an endogenously produced gaseous signaling molecule that plays an important role in cellular homeostasis and influences several physiological and pathological processes at low physiological and nontoxic concentrations. Its production is catalyzed by two cytosolic enzymes, cystathionine β-synthase and cystathionine γ-lyase, a mitochondrial enzyme, 3-mercaptopyruvate sulfurtransferase, and a peroxisomal enzyme, d-amino acid oxidase. Several recent experimental studies have demonstrated that at low micromolar concentrations, H2S plays a complex and essential role in normal renal function, and dysregulation of its production has been implicated in various renal pathologies. In addition, exogenous H2S administration has been reported to exhibit important therapeutic characteristics that target multiple molecular pathways in common renal pathologies in which reduced levels of renal and plasma H2S were observed. Interestingly, whereas the distribution of all four H2S-producing enzymes is subcellular and tissue specific, they are abundantly expressed by endothelial cells, mesangial cells, and podocytes within the glomeruli, as well as in the brush border and cytoplasm of epithelial cells of the renal proximal tubules, distal tubules, and peritubular capillaries. This makes the kidney a rich source of endogenous H2S production. This chapter presents current understanding of H2S in renal physiology and lays the foundation for discussion on H2S as a new therapeutic target for common renal pathologies in the subsequent chapters.
Diabetic kidney disease (DKD) is a chronic renal pathology, which is currently the leading cause of end-stage renal disease. It accounts for 40% of morbidity and mortality among the diabetic population despite optimal management. Its clinical hallmark includes persistent hyperglycemia, hypercreatininemia, uremia, sustained albuminuria, renal hemodynamic changes, and elevated blood pressure. Histologically, DKD presents with excessive accumulation and deposition of extracellular matrix, leading to expansion of mesangial matrix, thickening of glomerular basement membrane, and tubulointerstitial fibrosis. At the molecular level, accumulating evidence suggests that hyperglycemia or high glucose mediates renal injury in DKD via multiple molecular mechanisms such as induction of oxidative stress, upregulation of renal transforming growth factor beta-1 expression, production of pro-inflammatory cytokines, activation of fibroblasts and renin-angiotensin-aldosterone system, and depletion of adenosine triphosphate. Moreover, existing therapies only retard the disease progression but do not prevent or reverse it. Therefore, novel modes of pharmacotherapeutic intervention are in demand to target additional disease mechanisms. A substantial body of experimental evidence demonstrates that hydrogen sulfide (H2S), a gas with a historic notorious label, has recently been established to possess important therapeutic properties that prevent and/or reverse DKD development and progression of DKD by targeting several important molecular pathways, and therefore could be considered a novel pharmacological agent for DKD treatment. The aim of this chapter is to discuss recent experimental findings on the molecular mechanisms underlying the pharmacotherapeutic effects of H2S against DKD development and progression, and its translation from bench to bedside, which could lay the foundation for its future clinical use. A section of the chapter also discusses focal segmental glomerulosclerosis as a mediator of DKD progression to end-stage renal disease, and H2S as a potential novel therapy.
Ischemia-reperfusion injury (IRI) is an unavoidable and unresolved problem that poses a great challenge in kidney transplantation. It represents a major factor that increases acute tubular necrosis, decreases graft survival, and delays renal graft function. This complicates graft quality, posttransplant patient care, and kidney transplantation outcomes and therefore undermines the success of kidney transplantation. In this chapter, we present recent advances in research regarding novel pharmacological strategies involving the use of hydrogen sulfide (H2S), the third established member of the gasotransmitter family, against IRI in different experimental models of involving transplantation of kidney and other transplantable solid organs. Additionally, we also discuss the molecular mechanisms underlying the effects of H2S donor molecules in transplantation and suggestions for clinical translation. Our findings in this chapter showed that storage of renal graft and other solid organ grafts in H2S-supplemented preservation solution or administration of H2S to organ donor prior to organ procurement and to recipient at the start and during reperfusion is a novel, simple, and cost-effective pharmacological approach to minimize cold IRI, limit posttransplant complications, and improve transplantation outcomes. In conclusion, experimental evidence demonstrates that H2S can significantly mitigate IRI during transplantation through inhibition of a complex cascade of interconnected cellular and molecular events involving microcirculatory disturbance and microvascular dysfunction, mitochondrial injury, inflammatory responses, cell damage and cell death, and other damaging molecular pathways while promoting protective pathways. Translating these promising findings from bench to bedside will lay the foundation for the use of H2S in clinical organ transplantation in the future.
Chronic kidney disease (CKD) is a common global health challenge characterized by irreversible pathological processes that reduce kidney function and culminate in the development of end-stage renal disease. It is associated with increased morbidity and mortality in addition to increased caregiver burden and higher financial cost. A central player in CKD pathogenesis and progression is renal hypoxia. Renal hypoxia stimulates induction of oxidative and endoplasmic reticulum stress, inflammation, and tubulointerstitial fibrosis, which in turn promote cellular susceptibility and further aggravate hypoxia, thus forming a pathological vicious cycle in CKD progression. Although the importance of CKD is widely appreciated, including improvements in the quality of existing therapies such as dialysis and transplantation, new therapeutic options are limited, as there is still increased morbidity, mortality, and poor quality of life among CKD patients. Growing evidence indicates that hydrogen sulfide (H2S), a small gaseous signaling molecule with an obnoxious smell, accumulates in the renal medulla under hypoxic conditions and functions as an oxygen sensor that restores oxygen balance and increases medullary flow. Moreover, plasma H2S level has been recently reported to be markedly reduced in CKD patients and animal models. Also, H2S has been established to possess potent antioxidant, anti-inflammatory, and anti-fibrotic properties in several experimental models of kidney diseases, suggesting that its supplementation could protect against CKD and retard its progression. The purpose of this chapter is to discuss current clinical and experimental developments regarding CKD, its pathophysiology, and potential cellular and molecular mechanisms of protection by H2S in experimental models of CKD. A section of the chapter also discusses hyperhomocysteinemia and autosomal dominant polycystic kidney disease, which are forms of CKD, and H2S as an additional/alternative agent for pharmacological treatment or management of these conditions.
Thiosulfate in the form of sodium thiosulfate (STS) is a major oxidation product of hydrogen sulfide (H2S), an endogenous signaling molecule and the third member of the gasotransmitter family. STS is currently used in the clinical treatment of acute cyanide poisoning, cisplatin toxicities in cancer therapy, and calciphylaxis in dialysis patients. Burgeoning evidence shows that STS has antioxidant and anti-inflammatory properties, making it a potential therapeutic candidate molecule that can target multiple molecular pathways in various diseases and drug-induced toxicities. This chapter discusses the biochemical and molecular pathways in the generation of STS from H2S, its clinical usefulness, and potential clinical applications in renovascular hypertension, renal ischemia-reperfusion injury, chronic kidney disease, and uremic pruritus, as well as the molecular mechanisms underlying these clinical applications and a future perspective in kidney transplantation.
We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.
The inference that ATP-sensitive K+ (KATP) channels are involved in arterial responses to the synthetic K+ channel openers, hypoxia, adenosine, and calcitonin gene-related peptide, has relied on the sensitivity of these responses to the sulfonylureas glibenclamide and tolbutamide and to tetraethylammonium (TEA+). The inhibition of KATP currents by glibenclamide, tolbutamide, and TEA+ was investigated in single smooth muscle cells from rabbit mesenteric artery by use of the whole cell patch-clamp technique. The synthetic K+ channel openers pinacidil (half-activation 0.6 microM), cromakalim (half-activation 1.9 microM), and diazoxide (half-activation 37.1 microM) activated K(+)-selective currents that were blocked by glibenclamide. Elevation of pipette (intracellular) ATP concentration decreased K+ currents induced by pinacidil. Half-inhibition of KATP currents by glibenclamide and tolbutamide occurred at 101 nM and 351 microM, respectively. KATP currents were also inhibited by external TEA+, with half-inhibition at 6.2 mM. The results indicate that glibenclamide is an effective inhibitor of KATP channels in arterial smooth muscle and that tolbutamide and TEA+ are much less effective. Furthermore, these results support numerous functional studies that have demonstrated that the vasorelaxations to K+ channel openers are inhibited by < 10 microM glibenclamide but not by < 1 mM TEA+.
Whole‐cell and inside‐out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein.
In whole‐cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri‐ or monophosphates, induced a K‐current that developed gradually over 5 to 15 min. Intracellular 1 m m guanosine 5′‐diphosphate (GDP) induced a slowly developing outward K‐current at − 37 mV that reached a maximum on average of 72 ± 4 pA ( n = 40). Half maximal effect was estimated to occur with about 0.2 m m GDP. Except for ADP, other NDPs had comparable effects. At 0.1 m m , ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 m m but had almost no effect at 10 m m .
In 14% of inside‐out patches exposed to 1 m m GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current‐voltage relationship for the channel was linear in a 60 m m :130 m m K‐gradient and the unitary conductance was 24 pS.
Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP‐induced K‐current ( I K(GDP) ) in the whole‐cell. The K d was 25 n m and the inhibition was fully reversible on wash‐out.
( I K(GDP) ) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward ( I K(GDP) ).
Inclusion of 1 m m ATP in the recording pipette solution reduced ( I K(GDP) ) and also attenuated its decline during long (25 min) recordings.
When perforated‐patch whole‐cell recording was used, metabolic poisoning with cyanide and 2‐deoxy‐ d ‐glucose induced a glibenclamide‐sensitive K‐current. This current was not observed when conventional whole‐cell recording was used. Possible reasons for this difference are discussed.
These K channels appear similar to ATP‐sensitive K channels but we refer to them as nucleotide diphosphate‐dependent K channels (K NDP ) to emphasise what seems to be a primary role for nucleotide diphosphates in their regulation.
We reported previously that a monoclonal antibody against probasin (rat prostatic secretory protein) recognizes a 40-kDa protein localized in rat liver and kidney. The protein (probasin-related antigen, PRB-RA) may participate in a specific differentiated function of these tissues. To clarify the molecular nature of PRB-RA, a series of cDNA clones coding for the protein were isolated from a rat liver expression library using an affinity-purified polyclonal antibody. The amino acid sequence deduced from the determined cDNA sequence included sequences identical with those of proteolytic fragments of PRB-RA, which covered about 70% of the deduced sequence. Northern blot hybridization of poly(A)+ RNA isolated from rat tissues showed the presence of predominant and minor mRNA species of about 2.0 and 4.3 kilobases, respectively, in the liver and kidney. A sequence homology search revealed that PRB-RA is almost completely identical to rat cystathionine gamma-lyase (cystathionase) and that it does not show overall homology with probasin. Three candidates for an epitope common to probasin and PRB-RA were found on close examination of the amino acid sequences of the two proteins. A synthetic peptide, TYFRRI, corresponding to one of the candidates, neutralized the reactivity of the anti-probasin monoclonal antibody to both probasin and PRB-RA on Western blot analysis. These results show that PRB-RA/cystathionase is neither structurally nor functionally related to probasin except for a common epitope and that cystathionase, a cystein-producing enzyme, is localized in urinary tubular epithelial cells in a highly restricted region of the kidney in addition to in liver parenchymal cells.
Hydrogen sulfide (H2S), which is well known as a toxic gas, is produced endogenously from L-cysteine in mammalian tissues. H2S is present at relatively high levels in the brain, suggesting that it has a physiological function. Two other gases, nitric oxide and carbon monoxide, are also endogenously produced and have been proposed as neuronal messengers in the brain. In this work we show the following: (1) an H2S-producing enzyme, cystathionine beta-synthase (CBS), is highly expressed in the hippocampus; (2) CBS inhibitors hydroxylamine and amino-oxyacetate suppress the production of brain H2S; and (3) a CBS activator, S-adenosyl-L-methionine, enhances H2S production, indicating that CBS contributes to the production of endogenous H2S. We also show that physiological concentrations of H2S selectively enhance NMDA receptor-mediated responses and facilitate the induction of hippocampal long-term potentiation. These observations suggest that endogenous H2S functions as a neuromodulator in the brain.
This review examines the properties and roles of the four types of K+ channels that have been identified in the cell membrane of arterial smooth muscle cells. 1) Voltage-dependent K+ (KV) channels increase their activity with membrane depolarization and are important regulators of smooth muscle membrane potential in response to depolarizing stimuli. 2) Ca(2+)-activated K+ (KCa) channels respond to changes in intracellular Ca2+ to regulate membrane potential and play an important role in the control of myogenic tone in small arteries. 3) Inward rectifier K+ (KIR) channels regulate membrane potential in smooth muscle cells from several types of resistance arteries and may be responsible for external K(+)-induced dilations. 4) ATP-sensitive K+ (KATP) channels respond to changes in cellular metabolism and are targets of a variety of vasodilating stimuli. The main conclusions of this review are: 1) regulation of arterial smooth muscle membrane potential through activation or inhibition of K+ channel activity provides an important mechanism to dilate or constrict arteries; 2) KV, KCa, KIR, and KATP channels serve unique functions in the regulation of arterial smooth muscle membrane potential; and 3) K+ channels integrate a variety of vasoactive signals to dilate or constrict arteries through regulation of the membrane potential in arterial smooth muscle.
Free sulfide in rumen preserved with a sulfide antioxidation reducing buffer (SAOB) is determined directly and rapidly with a sulfide ion electrode using a standard addition technique. Acid-labile sulfide in blood preserved in alkaline cadmium acetate is determined by electrode measurement after acid liberation in a Johnson-Nishita apparatus and absorption in 50% SAOB. The sulfide antioxidant reducing buffer SAOB is not recommended for preservation of blood samples because of its desulfuration effect on S-proteins and S-amino acids present in blood.
The present study analyses the influence of hypertension and endothelium on the effect induced by hydrogen peroxide (H 2 O 2 ) on basal tone in aortic segments from normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR) of 6‐month‐old, as well as the possible mechanisms involved.
Single (1 m m ) or cumulative (100 n m –10 m m ) concentrations of H 2 O 2 produced a transient contraction or a concentration‐dependent increase of basal tone, respectively, in segments from WKY and SHR. In both cases, the contractions were higher in intact segments from hypertensive than from normotensive rats, and increased by endothelium removal in both strains. Catalase (1000 u ml ⁻¹ , a H 2 O 2 scavenger) abolished the contraction elicited by 1 m m H 2 O 2 in both strains.
Superoxide dismutase (SOD, 150 u ml ⁻¹ ) and dimethylsulphoxide (DMSO, 7 m m ), scavengers of superoxide anions and hydroxyl radicals, respectively, did not alter H 2 O 2 ‐induced contractions in intact segments from both strains. However, l ‐N G ‐nitroarginine methyl ester ( l ‐NAME, 100 μ m , a nitric oxide synthase inhibitor) increased the response to H 2 O 2 in normotensive rats, although the increase was less than that produced by endothelium removal.
Incubation of segments with 1 m m H 2 O 2 for 15 min and subsequent washout reduced the contractile responses induced by 75 m m KCl in intact segments from SHR and in endothelium‐denuded segments from both strains; this effect being prevented by catalase (1000 u ml ⁻¹ ).
Indomethacin (10 μ m , a cyclo‐oxygenase inhibitor) and SQ 29,548 (10 μ m , a prostaglandin H 2 /thromboxane A 2 receptor antagonist) practically abolished the contractions elicited by H 2 O 2 in normotensive and hypertensive rats.
We conclude that: (1) the oxidant stress induced by H 2 O 2 produces contractions mediated by generation of a product of the cyclo‐oxygenase pathway, prostaglandin H 2 or more probably thromboxane A 2 , in normotensive and hypertensive rats; (2) oxygen‐derived free radicals are not involved in the effect of H 2 O 2 ; (3) in normotensive rats, endothelium protects against H 2 O 2 ‐mediated injury to contractile machinery, determined by the impairment of KCl‐induced contractions; and (4) endothelial nitric oxide has a protective role on the contractile effect induced by H 2 O 2 , that is lost in hypertension.
British Journal of Pharmacology (1998) 125 , 1329–1335; doi: 10.1038/sj.bjp.0702200
Poisoning by hydrogen sulfide has been recognized as an occupational hazard for at least two centuries. The development of alternative sources of energy in North America may increase the incidence of this medical emergency in the future. Until recently, no specific antidote to sulfide was recognized. We have compared sulfide poisoning to cyanide poisoning and documented recent findings that indicate many similarities between the two. The therapeutic induction of methemoglobinemia, as by the intravenous administration of sodium nitrite, has both protective and antidotal effects against sulfide as well as against cyanide in laboratory animals. This procedure has been used successfully in at least one severe human case of sulfide poisoning. Industries at risk should be prepared to initiate this form of therapy in addition to the usual measures for cardiopulmonary resuscitation. No evidence exists to suggest that sulfide poisoning results in an impairment of the oxygen transport capability of blood. On the other hand, some victims of hydrogen sulfide poisoning exhibit frank cyanosis, suggesting that the respiratory tract obstruction is more common in this condition than is generally recognized. Suction of the upper tract and the administration of oxygen may be important ancillary procedures to the administration of sodium nitrite.
The sulphide metabolism of rats fed molybdate up to levels of 1000 ppm molybdenum was examined and large decreases in hepatic sulphite oxidase activity observed; overall sulphide oxidation capacity was also reduced. Molybdate but not tungstate caused increases in the total plasma copper of guinea pigs but in particular the appearance of a new TCA-insoluble fraction. The effect was increased by the addition of 500 ppm sulphur as sulphide, to the molybdate diet whereas the addition of 500 ppm S as sulphate was ineffective. 100 ppm Mo was less effective as thiomolybdate (MoS4=) than as molybdate. The significance of these results in relation to the role of sulphide in the Cu-Mo-S interaction is discussed.
Whole cell patch-clamp recordings were carried out on smooth muscle cells from rat tail artery in short-term culture to verify the existence of and to characterize the calcium channels that are present. Two types of voltage-dependent calcium channels were identified in 55 of 63 cells studied. The T-type calcium channel was activated at -50 mV, and the peak inward current occurred at -10 mV, whereas the L-type channel was activated at -20 mV, and the peak inward current occurred at +10 or +20 mV. The T-type channel current inactivated quickly in contrast to the much slower inactivation of the L-channel current. The voltage dependence of steady-state inactivation of the two channels was similar to that reported for other vascular smooth muscle preparations. An internal solution containing Cs2-aspartate maintained the calcium-channel currents for at least 20 min with only a 5-10% decline. BAY K 8644 had no effect on T-channel currents, but the L-channel current was increased by at least a factor of two. In addition, BAY K 8644 shifted the activation threshold, the peak inward current, and the steady-state inactivation-activation curves of L-type channel currents in the direction of hyperpolarization.
Patch clamp studies of neuroblastoma cells have shown that in the presence of sodium hydrogen sulfide (NaHS; the in vitro precursor of H2S), addition of the sulfonated amino acids, taurine or cysteic acid resulted in reversible abolition of the inward sodium currents. This effect could also be demonstrated by preincubating cells for 3-20 min with 5-10 mM NaHS followed by replacement of the solution with taurine or cysteic acid in sulfide-free saline. Neither NaHS, taurine nor cysteic acid alone had any effect. The sulfhydryl reagents, beta-mercaptoethanol and dithiothreitol, were also found to abolish reversibly the sodium currents. As the effects of the above treatments were nearly identical, the synergistic action of NaHS with taurine or cysteic acid may result from reduction of the disulfide bonds between subunits comprising the sodium channel. The responses to NaHS and taurine, a putative neurotransmitter/neuromodulator, suggest that reductions in sodium channel function may be the mechanism(s) responsible for loss of central respiratory drive during H2S poisoning.
The contribution of cystathionine gamma-lyase, cystathionine beta-synthase and cysteine aminotransferase coupled to 3-mercaptopyruvate sulphurtransferase to cysteine desulphhydration in rat liver and kidney was assessed with four different assay systems. Cystathionine gamma-lyase and cystathionine beta-synthase were active when homogenates were incubated with 280 mM-L-cysteine and 3 mM-pyridoxal 5'-phosphate at pH 7.8. Cysteine aminotransferase in combination with 3-mercaptopyruvate sulphurtransferase catalysed essentially all of the H2S production from cysteine at pH 9.7 with 160 mM-L-cysteine, 2 mM-pyridoxal 5'-phosphate, 3 mM-2-oxoglutarate and 3 mM-dithiothreitol. At more-physiological concentrations of cysteine (2 mM) cystathionine gamma-lyase and cystathionine beta-synthase both appeared to be active in cysteine desulphhydration, whereas the aminotransferase pathway did not. The effect of inhibition of cystathionine gamma-lyase by a suicide inactivator, propargylglycine, in the intact rat was also investigated; there was no significant effect of propargylglycine administration on the urinary excretion of total 35S, 35SO4(2-) or [35S]taurine formed from labelled dietary cysteine.
To determine whether diabetes alters vascular effects mediated by activation of protein kinase C, the contractions induced by phorbol esters were examined in aortic rings from rats with 8- to 12-weeks streptozotocin-induced diabetes and compared with those from age-matched control rats. In diabetic rat aorta, phorbol 12,13-dibutyrate (PDB) (> or = 30 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (300 nM) elicited a delayed, sharply developing rise in tension following an initial gradually developing contraction. In control rat aorta, these agents produced only an initial slowly developing contraction. Both the initial and the delayed contractile responses observed in diabetic aorta were completely abolished by pretreatment with 20 nM staurosporine, and the delayed phase of contraction was not seen in Ca(2+)-free medium or in the presence of 1 microM nifedipine. The concentration-response curves for the contractions induced by PDB revealed that PDB at concentrations > or = 30 nM produced significantly greater responses in diabetic aorta than in control aorta. In control aorta, exposure to Ca(2+)-free medium and pretreatment with 1 microM nifedipine shifted the concentration-response curves for PDB to the right without changing the maximal response. Under these conditions, there were no differences in the curves for PDB in control and diabetic aortas. These results suggest that the appearance of the delayed phase of contraction, possibly due to a delayed opening of Ca2+ channels, during activation of protein kinase C may be responsible for the enhanced contractile responses to phorbol esters in diabetic rat aorta.
1 The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)‐stimulated human neutrophils, and to test the hypothesis that adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E 2 .
2 PGE 2 (0.001 −10 μ m ) inhibited FMLP (100 nM)‐induced O 2 ‐generation from human peripheral blood neutrophils in a concentration‐dependent manner, with an EC 50 of 0.15 ± 0.03 μm, and a maximum effect ranging from 36–84% (mean inhibition of 68.7 ±2.5%, n = 32).
3 The EP 2 ‐receptor agonists, misoprostol, 11‐deoxy PGE 1 , AH13205 and butaprost, all at 10 μ m , inhibited O 2 ‐ generation, causing 95.5 ± 2.9%, 56.8 ± 5.2%, 37.1 ±6.6% and 18.9 ±4.4% inhibition respectively, the latter two being much less effective than PGE 2 . Similarly, the EP 1 ‐receptor agonist, 17‐phenyl PGE 2 (10 μm), and the EP 3 /EP 1 ‐receptor agonist, sulprostone (10 μm), also inhibited O 2 ‐generation, causing 32.2 ± 7.0% and 15.3 ± 3.4% inhibition respectively.
4 The non‐selective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX, 0.25 mM) inhibited the FMLP response by 54.5 ± 5.0%. In addition, IBMX shifted concentration‐effect curves for PGE 2 , misoprostol, 11‐deoxy PGE 1 , butaprost, and AH 13205 to the left, to give EC 50 S of 0.04 ± 0.03 ( n = 13), 0.07 ± 0.03 ( n = 4), 0.08 ± 0.03 ( n = 4), 0.33 ± 0.13 ( n = 4) and 0.41 ± 0.2 μm ( n = 3) respectively, allowing equieffective concentration‐ratios (EECs, PGE 2 = 1) of 11.5, 5.3, 50.7 and 12.7 to be calculated. This agrees well with the relative potencies of these agonists at EP 2 receptors.
5 By contrast, even in the presence of IBMX (0.25 mM), sulprostone and 17‐phenyl PGE 2 were only effective at the highest concentration (10 μm), and gave EECs of > 700 and 486 respectively, suggesting that EP 1 or EP 3 receptors are not involved.
6 The selective type IV phosphodiesterase inhibitor, rolipram at 2 and 10 nM did not inhibit the FMLP response, but at the higher concentration of 50 nM, it decreased the FMLP response by 46.6 ±7.3%. However, rolipram shifted concentration‐effect curves for PGE 2 to the left to give EC 50 S of 0.06 ± 0.022, 0.015 ±0.0, 0.012 ± 0.006 μm at 2, 10 and 50 nM respectively, compared to the control EC 50 of 0.27 ± 0.09 μm for PGE 2 .
7 The EP 4 /TP receptor blocking drug, AH 23848B (10 μ m , 10 min) did not inhibit O 2 ‐ generation by PGE 2 , but was found to potentiate significantly the effect of PGE 2 at the lower concentrations of PGF 2 tested (0.001‐0.1 μm).
8 The adenylate cyclase inhibitor, SQ 22,536 (0.1 mM, 2 min) reduced PGE 2 ‐induced inhibition of O 2 production, giving an EC 50 in the absence of SQ 22,536 of 0.24 ±0.1, and 1.9 ±1.1 μm in its presence.
9 These results suggest that inhibition of superoxide generation by PGE 2 is mediated by stimulation of EP2 receptors and activation of adenylate cyclase, leading to the elevation of intracellular levels of cyclic AMP.
This review examines the properties and roles of the four types of K+ channels that have been identified in the cell membrane of arterial smooth muscle cells. 1) Voltage-dependent K+ (KV) channels increase their activity with membrane depolarization and are important regulators of smooth muscle membrane potential in response to depolarizing stimuli. 2) Ca(2+)-activated K+ (KCa) channels respond to changes in intracellular Ca2+ to regulate membrane potential and play an important role in the control of myogenic tone in small arteries. 3) Inward rectifier K+ (KIR) channels regulate membrane potential in smooth muscle cells from several types of resistance arteries and may be responsible for external K(+)-induced dilations. 4) ATP-sensitive K+ (KATP) channels respond to changes in cellular metabolism and are targets of a variety of vasodilating stimuli. The main conclusions of this review are: 1) regulation of arterial smooth muscle membrane potential through activation or inhibition of K+ channel activity provides an important mechanism to dilate or constrict arteries; 2) KV, KCa, KIR, and KATP channels serve unique functions in the regulation of arterial smooth muscle membrane potential; and 3) K+ channels integrate a variety of vasoactive signals to dilate or constrict arteries through regulation of the membrane potential in arterial smooth muscle.
The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of atherosclerosis. Since monocyte chemoattractant protein 1 (MCP-1) plays a key role in the subendothelial recruitment of monocytes, we tested whether nitric oxide (NO) modulates the expression of MCP-1 in cultured human endothelial cells. Inhibition of basal NO production by NG-nitro-L-arginine (L-NAG) upregulates endothelial MCP-1 mRNA expression (250 +/- 20%) and protein secretion. Exogenous addition of NO dose-dependently decreased MCP-1 mRNA expression and secretion. Changes in MCP-1 mRNA expression and protein secretion were paralleled by corresponding changes in chemotactic activity of cell-conditioned media for monocytes. An MCP-1 antibody reduced monocyte chemotactic activity by 85% and completely abolished the increased monocyte chemotactic activity induced by the inhibition of NO production. Elevation of endothelial cGMP levels had no significant effect on MCP-1 mRNA expression. Inhibition of basal endothelial NO production by L-NAG increased binding activity of a nuclear factor kappa B (NF-kappa B)-like transcriptional regulatory factor, whereas exogenous addition of NO decreased NF-kappa B-like binding activity during stimulation with tumor necrosis factor-alpha. Thus, NO modulates MCP-1 expression and monocyte chemotactic activity secreted by human umbilical vein endothelial cells (HUVECs) in culture. The activation of NF-kappa B-like transcriptional regulatory proteins by inhibition of NO suggests a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis in HUVECs.
Nitric oxide (NO) participates in the regulation of vascular tone and smooth muscle cell proliferation, but little is known of its effect on total protein and matrix synthesis in smooth muscle. We studied the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP, 0.4 to 1.2 mmol/L) and sodium nitroprusside (SNP, 0.1 to 0.5 mmol/L) on total protein (using [3H]leucine) and collagen (using [3H]proline) synthesis in cultured rabbit aortic smooth muscle cells. Both agents caused dose-dependent inhibition of the relative rate of protein (maximum reduction of 87% [SNAP] and 80% [SNP]) and collagen synthesis, as measured by trichloroacetic acid-precipitated label. The magnitudes of percent inhibition of total protein and collagen synthesis were approximately equal. Inhibition of protein synthesis by SNAP and SNP was prevented by hemoglobin (10 mumol/L), suggesting that the protein synthesis inhibition was due to NO release. Inhibition of protein synthesis was reversible after removal of SNAP and SNP and was not caused by damage to the cells. These results suggest that NO may function as a modulator of vascular smooth muscle cell protein synthesis and production of extracellular matrix components.
1. Whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole-cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide-sensitive currents. CGRP increased the glibenclamide-sensitive currents by 3- to 6-fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide-sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current-voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp-cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide-sensitive K+ currents 2.1-, 3.3- and 8.2-fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide-sensitive K+ currents. 5. Dialysis of the cell's interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H-8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP-sensitive K+ channels.
The mechanisms by which hypoxia causes vasoconstriction in vivo are not known. Accumulating evidence implicates the endothelium as a key regulator of vascular tone. Hypoxia induces the expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor in cultured human endothelial cells. We report here that nitric oxide (NO), an endothelial-derived relaxing factor, modifies this induction of ET-1. Whereas low oxygen tension (PO2 = 20-30 Torr) increases ET-1 expression four- to eightfold above that seen at normal oxygen tension (PO2 = 150 Torr), sodium nitroprusside, which releases NO, suppresses this effect. This inhibition of hypoxia-induced ET-1 expression occurs within the first hour of exposure of cells to sodium nitroprusside. Moreover, when the endogenous constitutive levels of NO made by endothelial cells are suppressed using N-omega-nitro-L-arginine, a potent competitive inhibitor of NO synthase, the baseline levels of ET-1 produced in normoxic environments are increased three- to fourfold. The effects of hypoxia and the NO synthase inhibitor on ET-1 expression are additive. The regulation of ET-1 production by NO appears to be at the level of transcription. Similar effects of NO were observed on the expression of the PDGF-B chain gene. PDGF-B expression was suppressed by NO in a hypoxic environment and induced by N-omega-nitro-L-arginine in both normoxic and hypoxic environments. These findings suggest that in addition to its role as a vasodilator, NO may also influence vascular tone via the regulated reciprocal production of ET-1 and PDGF-B in the vasculature.
To investigate inositol phosphate formation and its modulation by the cyclic AMP (cAMP) pathway in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR).
Phenylephrine was used to stimulate inositol phosphate formation in cultured aortic smooth muscle cells from SHR and Wistar-Kyoto (WKY) rats. The smooth muscle cells from passages 6-14 were prelabelled with myo-[2-3H]-inositol (1.9 x 10(5) Bq/ml for 24 h) and inositol phosphate formation was measured after exposure to agonist for 45 min. (-)isoproterenol or forskolin-induced cAMP formation was also evaluated using a radioimmunoassay method.
The basal level of inositol phosphate formation in smooth muscle cells from SHR was higher than that observed in smooth muscle cells from WKY rats. Phenylephrine increased the formation of inositol phosphates in a concentration-dependent manner (0.1-100 mumol/l). In the presence of 100 mumol/l phenylephrine, the increase in inositol phosphate formation was significantly greater in smooth muscle cells from SHR (214 +/- 6%) than that observed in smooth muscle cells from WKY rats (156 +/- 8%). When the cells were pretreated with 1 mmol/l 8-bromoadenosine 3':5'-cyclic monophosphate or with 10 mumol/l forskolin for 45 min, the basal production of inositol phosphates in smooth muscle cells both from SHR and from WKY rats was significantly and similarly decreased by about 20%. In the presence of 1 mmol/l 8-bromoadenosine 3':5'-cyclic monophosphate, 100 mumol/l phenylephrine-induced inositol phosphate formation was similarly decreased by 33 +/- 4 and 30 +/- 3% in smooth muscle cells from SHR and from WKY rats, respectively, whereas, in the presence of 10 mumol/l forskolin, inositol phosphate formation was reduced by 25 +/- 3 and 27 +/- 5%, respectively, in those cells. In contrast, isoproterenol induced less inhibition of phenylephrine-induced inositol phosphate formation in smooth muscle cells from SHR (14 +/- 2%) than it did in those from WKY rats (25 +/- 4.5%). Although there was no significant difference in basal or forskolin-induced cAMP accumulation between smooth muscle cells from SHR and those from WKY rats. (-)isoproterenol-induced cAMP accumulation was significantly lower in smooth muscle cells from SHR.
A marked inhibitory effect of cAMP on the alpha 1-adrenoceptor-mediated inositol phosphate signal transduction pathway was demonstrated in smooth muscle cells of SHR and of WKY rats. Decreased cAMP formation with beta-adrenergic stimulation and increased inositol phosphate formation with alpha-adrenergic stimulation in SHR smooth muscle cells may both contribute to the dominant alpha 1-adrenergic activity observed in SHR.
The effects of noradrenaline (NA) uptake inhibitors on contractions induced by NA, high K ⁺ , and 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA) in rat isolated aorta were investigated.
Protriptyline (0.3 μ m ) and amitriptyline (0.3 μ m ) produced an approximately parallel shift to the right in the dose‐response curve to NA. Protriptyline (>0.3 μ m ), amitriptyline (>0.3 μ m ) and xylamine (0.01‐1 μ m ) significantly reduced the maximal contractile response to NA. The IC 50 values for inhibition of the contractile response to 3 μ m NA were 1.58 μ m for xylamine, 1.70 μ m for amitriptyline and 2.57 μ m for protriptyline.
Protriptyline and amitriptyline dose‐dependently inhibited the high K ⁺ (60 mM)‐induced contraction (IC 50 = 0.69 μ m for protriptyline and IC 50 = 3.15 μ m for amitriptyline). In contrast, xylamine did not affect the high K( ⁺ ‐induced contraction.
Protriptyline and amitriptyline dose‐dependently inhibited TPA (1 μ m )‐induced contraction in calcium‐free solution; xylamine (up to 30 μ m ) was without effect. Staurosporine (10 nM) completely inhibited the TPA‐ and NA‐induced contraction.
Protriptyline (3 μ m ) and amitriptyline (3 μ m ) caused about 54% and 60% inhibition, respectively, of aortic contractions caused by endothelin‐1 (10 nM) in the absence of endothelium. Xylamine (10 μ m ) was without effect.
Inhibitory effects of NA uptake inhibitors on contractions were independent of the presence of endothelium and were unaffected by the K ⁺ channel blockers, tetraethylammonium ions (up to 3 mM) and glibenclamide (up to 30 μ m ).
These results indicate that tricyclic antidepressant drugs such as protriptyline and amitriptyline could act as both postsynaptic adrenoceptor antagonists and direct inhibitors of muscle contraction; whereas, xylamine, a structurally distinct NA uptake blocker might principally exert its action only at α‐adrenoceptors on rat aortic smooth muscle.
1. The mechanisms involved in the 4-aminopyridine (4-AP)-induced block of delayed rectifier K+ current (IK(V)) in vascular smooth muscle cells were studied in cells enzymatically isolated from the rabbit coronary artery. 2. 4-AP inhibited slowly inactivating IK(V) in a dose-dependent manner (concentration producing half-maximal inhibition, K1/2, = 1.37 mM), and shifted the steady-state activation and inactivation curves of IK(V) by +9 and +16 mV, respectively. 3. The time constant of activation was significantly increased by 4-AP at +20 mV; deactivation kinetics were unaffected upon repolarization to -40 mV. The fast (tau f approximately 1 s) and slow (tau s approximately 5 s) time constants of inactivation (0 and +20 mV), and the recovery kinetics (tau r approximately 6 s) at -60 mV were not significantly affected by 0.5 mM 4-AP. However, tau f disappeared in the presence of 2 mM 4-AP while tau s remained unaffected. 4. Use-dependent unblock of IK(V) was revealed at potentials > or = -10 mV from analyses of the voltage dependence of 4-AP-sensitive currents and the frequency-dependent changes ('reverse use dependence') of IK(V) during the application of repetitive steps (-60 to +20 mV for 250 ms at a rate of 0.25 Hz) in control conditions, in the presence of 0.5 mM 4-AP, and after washout of the drug. These results suggested that 4-AP preferentially binds to the channel in the closed state, and unbinding is promoted by transitions to the open state. 5. The channel was modelled as a simple three-state mathematical loop model incorporating single closed, open and inactivated states. The block by 4-AP was modelled as a state-dependent interaction with 4-AP primarily binding to the closed state. Computer simulations support the hypothesis that 4-AP-induced block of the delayed rectifier K+ (KV) channel in the closed state is relieved during membrane depolarization. 6. Closed state binding of 4-AP to the KV channel depolarizes vascular smooth muscle cells by shifting the activation curve of these channels to more positive potentials.
The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (KCa) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K+ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance KCa channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single KCa channels in SMCs. Furthermore, the effect of CO on KCa channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, Gi/Go or Gs) in excised membrane patches. Our results suggest that the direct modulation of high-conductance KCa channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO.
Carbon monoxide (CO) induced a concentration‐dependent relaxation of isolated rat tail artery tissues which were precontracted with phenylephrine or U‐46619. This vasorelaxing effect of CO was independent of the presence of the intact endothelium.
The CO‐induced vasorelaxation was partially inhibited by the blockade of either the cyclicGMP pathway or big‐conductance calcium‐activated K (K Ca ) channels. When both the cyclicGMP pathway and K Ca channels were blocked, the CO‐induced vasorelaxation was completely abolished.
Incubation of vascular tissues with hemin, in order to enhance the endogenous production of CO, suppressed the phenylephrine‐induced vasocontraction in a time‐ and concentration‐dependent manner. The hemin‐induced suppression of the vascular contractile response to phenylephrine was abolished after the vascular tissues were co‐incubated with either oxyhaemoglobin or zinc protoporphyrin‐IX, suggesting an induced endogenous generation of CO from vascular tissues.
The effect of hemin incubation on vascular contractility did not involve the endogenous generation of nitric oxide.
Our results suggest that CO may activate both a cyclicGMP signalling pathway and K Ca channels in the same vascular tissues, and that the endogenously generated CO may significantly affect the vascular contractile responses.
The Hill coefficient is commonly used to estimate the number of ligand molecules that are required to bind to a receptor to produce a functional effect. However, for a receptor with more than one ligand binding site, the Hill equation does not reflect a physically possible reaction scheme; only under the very specific condition of marked positive cooperativity does the Hill coefficient accurately estimate the number of binding sites. The Hill coefficient is best thought of as an "interaction" coefficient, reflecting the extent of cooperativity among multiple ligand binding sites. Several relatively simple, physically plausible reaction schemes are shown here to produce a variety of ligand dose-response curve phenotypes more appropriately suited to modeling ligand-receptor interactions, especially if independent information about the stochiometry of the ligand-receptor interaction is available.
Hydrogen sulfide (H2S), which is well known as a toxic gas, is produced endogenously in mammalian tissues from L-cysteine mainly by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine beta-synthetase and cystathionine gamma-lyase. Recently, we showed that cystathionine beta-synthetase in the brain produces H2S, and that H2S facilitates the induction of hippocampal long-term potentiation by enhancing NMDA receptor activity. Here we show that mRNA for another H2S producing enzyme, cystathionine gamma-lyase, is expressed in the ileum, portal vein, and thoracic aorta. The ileum also expresses cystathionine beta-synthetase mRNA. These tissues produce H2S, and this production is blocked by cystathionine beta-synthetase and cystathionine gamma-lyase specific inhibitors. Although exogenously applied H2S alone relaxed these smooth muscles, much lower concentrations of H2S greatly enhanced the smooth muscle relaxation induced by NO in the thoracic aorta. These observations suggest that the endogenous H2S may regulate smooth muscle tone in synergy with NO.
Carbon monoxide (CO) is an endogenously generated gas that may play an important physiological role in the regulation of vascular tone. The CO-induced vasorelaxation, as a result of a direct action on vascular smooth muscles, has been demonstrated in many cases. Three major cellular mechanisms are proposed to explain the vasorelaxing effect of CO. These include the activation of soluble guanylyl cyclase, stimulation of various types of K channels, and inhibition of the cytochrome P450 dependent monooxygenase system in vascular smooth muscle cells. An interaction between CO and nitric oxide may also significantly contribute to the fine tuning of vascular tone. Furthermore, alterations in either the endogenous production of CO or the vascular responsiveness to CO have been encountered in several pathophysiological situations. A better understanding of the vascular effects of CO and the underlying cellular and molecular mechanisms will pave the way for the establishment of the role played by CO in vascular physiology and pathophysiology.
Calcitonin gene-related peptide (CGRP) is a potent vasodilator that is suggested to act via ATP-sensitive K channels (KATP). In the present study, we examined the actions of CGRP on pressure- and angiotensin II-induced vasoconstriction, using the in vitro perfused hydronephrotic rat kidney. Elevated pressure (from 80 to 180 mmHg) and 0.1 nM angiotensin II elicited similar decreases in afferent diameter in this model. CGRP inhibited myogenic reactivity in a concentration-dependent manner, completely preventing pressure-induced constriction at 10 nM (95 +/- 10% inhibition). These effects were partially attenuated by 10 microM glibenclamide (62 +/- 16% inhibition, P = 0.025), indicating both KATP-dependent and -independent actions of CGRP. In contrast, 10 nM CGRP inhibited angiotensin II-induced vasoconstriction by only 54 +/- 11%, and this action was not affected by glibenclamide (41 +/- 11%, P = 0.31). CGRP also inhibited the efferent arteriolar response to angiotensin II in the absence and presence of glibenclamide. Pinacidil (1.0 microM), a KATP opener also preferentially inhibited pressure- vs. angiotensin II-induced vasoconstriction (97 +/- 5 and 59 +/- 13% inhibition, respectively; P = 0.034). We conclude that the renal vasodilatory mechanisms of CGRP are pleiotropic and involve both KATP-dependent and -independent pathways. The effectiveness of CGRP in opposing renal vasoconstriction and the role of KATP in this action appear to depend on the nature the underlying vasoconstriction. We suggest that this phenomenon reflects an inhibition of KATP activation by angiotensin II.
Intake of nicotine has been related in many cases to acute or chronic hypertension. Using the patch-clamp technique the effect of nicotine on voltage-dependent K+ channel currents in rat tail artery smooth muscle cells was studied. Nicotine at concentrations of 1-100 microM or 0.3-3 mM increased or decreased, respectively, the amplitude of the tetraethylammonium-sensitive K+ currents. Pretreatment of cells with 10 microM dihydro-beta-erythroidine hydrobromide, a nicotinic receptor antagonist, abolished the excitatory effect (n=6), but not the inhibitory effect (n=10), of nicotine on K+ channel currents. The activation of nicotinic receptors with 100 microM 1,1-dimethyl-4-phenylpiperazinium iodide increased K+ channel currents by 27.4+/-3.8% (n=13, P < 0.01). Our results indicate that the excitatory and inhibitory effects of nicotine on K+ channels are respectively mediated by a nicotinic receptor-dependent mechanism and by a direct interaction of nicotine with K+ channels.
In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01-10 micrometers), in the presence of indomethacin (2.8 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation (n = 5). In pressurized arteries, ACh (10 microM), applied intraluminally in the presence of indomethacin (2.8 microM) and L-NAME (100 microM), dilated both PE-stimulated (0.3-0.5 microM; n = 5) and myogenic tone (n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated (n = 5) or myogenic tone (n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation.
Rabbit aortic rings pre-contracted with 1 microm phenylephrine were exposed to organ fluid of isolated guinea pig cochleas which had been subjected to electrical field stimulation (FS, 50 Hz, 50 V, 0.2 ms over 2 min). This resulted in an endothelium-dependent relaxation of the vessel rings sensitive to glibenclamide, an ATP-sensitive K+channel blocker. Tetrodotoxin (1 microm) added to the cochlear fluid blocked the vasorelaxant effect of cochlear FS and it attenuated vasorelaxation when added to aortic rings. The relaxation response paralleled an increase in the level of calcitonin gene-related peptide (CGRP) in both cochlear and vascular organ fluids from undetectable pre-stimulation values to 0.12+/-0.029 and 0.44+/-0.051 n m, respectively. We conclude that CGRP possibly contributes to cochlear nerve stimulation-induced endothelium-dependent vasorelaxation.
The transfer of a nitric oxide group to cysteine sulfhydryls on proteins, known as S-nitrosylation, is increasingly becoming recognized as a ubiquitous regulatory reaction comparable to phosphorylation. It represents a form of redox modulation in diverse tissues, including the brain. An increasing number of proteins have been found to undergo S-nitrosylation in vivo. These proteins are called S-nitrosothiols, and may play an important role in many processes ranging from signal transduction, DNA repair, host defense, and blood pressure control to ion channel regulation and neurotransmission. This review focuses on the importance of the S-nitrosylation reaction and describes some recently identified S-nitrosothiols in various fields of research.
Cyclic nucleotide-gated channels contain four subunits, each with a C-terminal binding site for cGMP or cAMP. The dose-response relation for activation is usually fit with the Hill equation, I/I(max) = [cGMP]n/([cGMP]n + K(1/2)n, where I/I(max) is the fraction of maximal current, K(1/2) is the concentration of cGMP that gives a half-maximal current, and n is the Hill coefficient, taken as the minimum number of ligands required for significant activation. The dose-response relations in multichannel patches are often fit with Hill coefficients of </=2.0, even though other lines of evidence indicate that these channels contain four binding sites and that the binding of three or four ligands is required for significant opening. We have measured dose-response relations for a large number of single cyclic nucleotide-gated channels composed of the bovine rod alpha subunit. We find that the single-channel Hill coefficient is consistently higher than 2.5, with an average of 3.0 +/- 0.37 over 16 patches. In both multichannel and single-channel patches, large variations in K(1/2) have been observed, and are thought to arise from modifications such as phosphorylation. Here we show that mixtures of single channels with high Hill coefficients and variable K(1/2) values will give rise to shallow macroscopic dose-response relations with anomalously low Hill coefficients. This is because activation occurs over a broad range of cGMP concentrations. Thus, dose-response relations from multichannel patches should be interpreted with caution, particularly when detailed mechanistic issues such as cooperativity are being investigated.
The incorporation of sulfide into methylene blue in the presence of acidic N,N-dimethyl-p-phenylenediamine and ferric chloride has been utilized to provide a direct determination for sulfide at concentrations of 2 to 80 mμmoles per milliliter. No distillation step is required. At concentrations of 10−3M, only slight interference by organic thiols was observed. Advantages in sensitivity, specificity, color stability, and convenience are offered over earlier methods.
Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat Rat cystathionine b-synthase: gene organization and alternative splicing Mechanisms of vasodilation by cochlear nerve stimulation. Role of calcitonin gene-related peptide
- M H Stipanuk
- P W Beck
- ±
- C W Sun
- M Alonso-Galicia
- M Swaroop
- K Bradley
- T Ohura
- T Tahara
- M D Roper
- L E Rosenberg
- J P Kraus
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The possible role of hydrogen sul®de as an endogenous smooth muscle relaxant in synergy with nitric oxide Inhibitory effect of noradrenaline uptake inhibitors on contractions of rat aortic smooth muscle
- R Hosoki
- N Matsuki
- H Kimura
Hosoki,R., Matsuki,N. and Kimura,H. (1997) The possible role of hydrogen sul®de as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun., 237, 527±531. Huang,Y. (1996) Inhibitory effect of noradrenaline uptake inhibitors on contractions of rat aortic smooth muscle. Br. J. Pharmacol., 117, 533±539.