Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout

Hydrogen sulfide (H(2)S) is an endogenous vasodilator in mammals, but its presence and function in other vertebrates is unknown. We generated H(2)S from NaHS and examined the effects on isolated efferent branchial arteries from steelhead (stEBA) or rainbow (rtEBA) trout. H(2)S concentration was measured colorimetrically (CM) and with ion-selective electrodes (ISE) in rainbow trout plasma. NaHS produced a triphasic response consisting of a relaxation (phase 1), constriction (phase 2), and relaxation (phase 3) in both unstimulated vessels and in stEBA precontracted with carbachol (Carb). Phase 1 and phase 3 in stEBA were decreased and phase 2 increased in unstimulated vessels by K(+)(ATP) channel inhibition (glibenclamide), or a cocktail of inhibitors of cyclooxygenase, lipoxygenase, and cytochrome P-450 (indomethacin, esculetin, and clotrimazole). Inhibition of soluble guanylate cyclase with ODQ o NS-2028 inhibited phase 3 in stEBA, although NaHS decreased cGMP production by tEBA. stEBA phase 2 contractions were partially inhibited by the myosin light chain kinase inhibitor, ML-9, but unaffected by L-type calcium channel inhibition (methoxyverapamil), whereas contraction in tEBA was partially inhibited by nifedipine or removal of extracellular calcium. Phase 3 relaxations were more pronounced in stEBA precontracted with Carb and no epinephrine (NE) than those cont acted by KCl or K(2)SO(4). stEBA phase 2 and phase 3 responses were dose dependent (EC(50) = 1.1 +/- 1.2 x 10(-3) M and 6.7 +/- 0.9 x 10(-5) M, respectively; n = 7). NaHS was also vasoactive in steelhead bulbus arteriosus, celiac mesenteric arteries, and anterior cardinal veins. Rainbow trout plasma sulfide concentration was 4.0 +/- 0.3 x 10(-5) M, n = 4 (CM) and 3.8 +/- 0.4 x 10(-5) M, n = 9 (ISE); similar to phase 3 EC(50). Because NaHS has substantial vasoactive effects at physiological plasma concentrations, we propose that its soluble derivative, H(2)S, is a tonically active endogenous vasoregulator in trout.     

Hydrogen sulfide as a vasodilator

Gases such as nitric oxide (NO) and carbon monoxide (CO) play important roles both in normal physiology and in disease. The toxic effects of hydrogen sulphide (H2S) on living organisms have been recognized for nearly 300 years. In recent years, however, interest has been directed towards H2S as the third gaseous mediator, which has been shown to exhibit potent vasodilator activity both in vitro and in vivo most probably by opening vascular smooth muscle K(ATP) channels. Of the two enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthetase (CBS), which utilize L-cysteine as substrate to form H2S, CSE is believed to be the key enzyme which forms H2S in the cardiovascular system. Recent studies have shown an important role of the vasodilator action of H2S in health and disease.     

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.     

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.     

Hydrogen sulfide mediates hypoxic vasoconstriction through a production of mitochondrial ROS in trout gills

Hypoxic pulmonary vasoconstriction (HPV) is an adaptive response that diverts pulmonary blood flow from poorly ventilated and hypoxic areas of the lung to more well-ventilated parts. This response is important for the local matching of blood perfusion to ventilation and improves pulmonary gas exchange efficiency. HPV is an ancient and highly conserved response, expressed in the respiratory organs of all vertebrates, including lungs of mammals, birds, and reptiles; amphibian skin; and fish gills. The mechanism underlying HPV and how cells sense low Po(2) remains elusive. In perfused trout gills (Oncorhynchus mykiss), acute hypoxia, as well as H(2)S, caused an initial and transient constriction of the vasculature. Inhibition of the enzymes cystathionine-β-synthase and cystathionine-γ-lyase, which blocks H(2)S production, abolished the hypoxic response. Individually blocking the four complexes in the electron transport chain abolished both the hypoxic and the H(2)S-mediated constriction. Glutathione, an antioxidant and scavenger of superoxide, attenuated the vasoconstriction in response to hypoxia and H(2)S. Furthermore, diethyldithiocarbamate, an inhibitor of superoxide dismutase, attenuated the hypoxic and H(2)S constriction. This strongly suggests that H(2)S mediates the hypoxic vasoconstriction in trout gills. H(2)S may stimulate the mitochondrial production of superoxide, which is then converted to hydrogen peroxide (H(2)O(2)). Thus, H(2)O(2) may act as the "downstream" signaling molecule in hypoxic vasoconstriction.     

Arterio-venous anastomoses in rainbow trout gill filaments

Summary The origin of arterio-venous anastomoses, connecting the efferent filament artery (EFA) with the central venous sinus (CVS) of gill filaments can be well discerned by scanning electron microscopy in the rainbow trout. Corresponding vessels between the afferent filament artery and the CVS could not be detected with the techniques applied. AVA-specific endothelial cells are characterized by their bulky shape and their microvillous surface. The general morphology of AVA's in Salmo gairdneri is very similar to that of AVA's in Tilapia mossambica (Vogel et al., 1974) but they are much longer in the trout. No filament whorls have been encountered in AVA endothelia of Salmo gairdneri.This study is dedicated to Prof. Dr. W. Graumann, Director of the Institute of Anatomy, University of Tübingen, on the occasion of his 60th birthday. It was supported by the Deutsche Forschungsgemeinschaft     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Localization of angiotensin-converting enzyme in the trout gill

Angiotensin-converting enzyme (ACE) was localized in perfused trout gills by measuring gill extraction of two radiolabeled ACE inhibitors, 125I-351A (an iodinated derivative of lisinopril) and 3H-RAC-X-65, and by autoradiography of gills perfused with 125I-351A. A 125I-351A pulse was preferentially extracted by the arterio-arterial (AA) pathway (61.7% +/- 1.8% extraction; mean +/- SE, N = 4); the arteriovenous (AV) pathway extracted an additional 10%. Extraction by either pathway was reduced by simultaneous perfusion with 10(-5) M unlabeled lisinopril. AA extraction of RAC-X-65 during continuous perfusion was maximal (75% +/- 5%, N = 6) during the first few minutes of perfusion and decreased steadily to 38% +/- 9% by 20 min and to less than 10% by 40 min. AV extraction of RAC-X-65 was negligible. Autoradiography of gills continuously perfused with 125I-351A showed that the radiolabel was concentrated in the respiratory lamellae. The highest grain density was associated with the pillar cells nearest the medial (inner) lamellar margin. Afferent filamental arteries and afferent lamellar arterioles were labeled to a lesser extent. Relatively little label was found on the efferent lamellar arterioles or efferent filamental arteries. 125I-351A binding was not evident in AV vessels. These findings support the hypothesis that the gill is an important site for formation of plasma angiotensin II and they suggest that enzymes associated with mammalian endothelial cells are also common to gill pillar cells.     

NOX4 as an oxygen sensor to regulate TASK-1 activity

When oxygen sensing cells are excited by hypoxia, background K+ currents are inhibited. TASK-1, which is commonly expressed in oxygen sensing cells and makes a background K+ current, is inactivated by hypoxia. Thus TASK-1 is a candidate molecule responsible for hypoxic excitation. However, TASK-1 per se cannot sense oxygen and may require a regulatory protein that can. In the present study, we propose that the NADPH oxidase NOX4 functions as an oxygen-sensing partner and that it modulates the oxygen sensitivity of TASK-1. Confocal imaging revealed the co-localization of TASK-1 and NOX4 in the plasma membrane. In HEK293 cells expressing NOX4 endogenously, the activity of expressed TASK-1 was moderately inhibited by hypoxia, and this oxygen response was significantly augmented by NOX4. Moreover, the oxygen sensitivity of TASK-1 was abolished by NOX4 siRNA and NADPH oxidase inhibitors. These results suggest a novel function for NOX4 in the oxygen-dependent regulation of TASK-1 activity.     

The gill sialic acid content as an index of environmental stress in rainbow trout, Salmo gairdneri, Richardson

The gill sialic acid content in Rainbow Trout exposed to several aqueous concentrations of ammonia NH₃ and/or to a high environmental pH was determined by two different methods. The results showed Warren's method was adequate for the biological material, even without purification of the extracts by Dowex 2.8 resin. A significant increase of the sialic acid concentration occurred in fish exposed to ammonia.     

Hydrogen sulfide as an effective and specific novel therapy for acute carbon monoxide poisoning

Hydrogen sulfide (H₂S) has been recognized as a toxic gas and environment pollutant. So, it is seldom regarded as a therapeutic gas. H₂S has been recognized recently as a novel gaseous messenger and serves as an important neuromodulator in the central nervous system. Many researches have been focused on the protective role of H₂S in treatment of several diseases. Like nitric oxide (NO) and carbon monoxide (CO), which are considered as two gaseous transmitters, H₂S has been regarded as the third one. Recent studies provided evidence that H₂S exerted antioxidant and anti-apoptotic effects, which protected neurons, cardiomyocytes, pancreatic β-cells and vascular smooth muscle cells against oxidative stress by scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS). It has been known that multiple factors, including oxidative stress, free radicals and neuronal nitric oxide syntheses as well as abnormal inflammatory responses are involved in the mechanism underlying the brain injury after acute CO poisoning. Studies have shown that free radical scavengers can display neuroprotective properties. Therefore, we hypothesize that H₂S might be an interesting potential strategy for curing acute CO poisoning.     

Hydrogen sulfide mediates vasoactivity in an O2-dependent manner

Hydrogen sulfide (H(2)S) has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H(2)S is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H(2)S include ATP-sensitive K(+) channels, and H(2)S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O(2) dependent, but this has not been addressed in most studies designed to elucidate the role of H(2)S in vascular function. This is important, since H(2)S reactions can be dramatically altered by the high concentrations of O(2) used in cell culture and organ bath experiments. To test the hypothesis that the effects of H(2)S on the vasculature are O(2) dependent, we have measured real-time levels of H(2)S and O(2) in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H(2)S sensor developed in our laboratory was used to measure H(2)S levels. Here we report that, in rat aorta, H(2)S concentrations that mediate rapid contraction at high O(2) levels cause rapid relaxation at lower physiological O(2) levels. At high O(2), the vasoconstrictive effect of H(2)S suggests that it may not be H(2)S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H(2)S to modulate vascular tone in vivo.     

Hydrogen sulfide is an endogenous potentiator of T cell activation

H(2)S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H(2)S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H(2)S production. Consistent with its increased production, H(2)S is implicated as a mediator of ulcerative colitis both in its genesis or maintenance. As T cells are well established mediators of inflammatory bowel disease, we investigated the effect of H(2)S exposure on T cell activation. Using primary mouse T lymphocytes (CD3+), OT-II CD4+ T cells, and the human Jurkat T cell line, we show that physiological levels of H(2)S potentiate TCR-induced activation. Nanomolar levels of H(2)S (50-500 nM) enhance T cell activation assessed by CD69 expression, interleukin-2 expression, and CD25 levels. Exposure of T cells to H(2)S dose-dependently enhances TCR-stimulated proliferation with a maximum at 300 nM (30% increase, p < 0.01). Furthermore, activation increases the capacity of T cells to make H(2)S via increased expression of cystathionine γ-lyase and cystathionine β-synthase. Disrupting this response by silencing these H(2)S producing enzymes impairs T cell activation, and proliferation and can be rescued by the addition of 300 nM H(2)S. Thus, H(2)S represents a novel autocrine immunomodulatory molecule in T cells.