Role of hydrogen sulfide production in inhibitory action of L-cysteine on isolated porcine irides.

PURPOSE: To investigate the direct pharmacological actions of L-cysteine, a substrate for the production of H(2)S, on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we examined the underlying mechanism of action of L-cysteine in this smooth muscle. METHODS: Isolated porcine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer solution at 37 degrees C. Longitudinal isometric tension was recorded via a grass FT03 Force-Displacement Transducer and analyzed using the PolyView computer software. The relaxant action of L-cysteine on carbachol-induced tone was studied in the absence and presence of inhibitors of enzymes of the biosynthetic pathways for H(2)S, and prostanoids. In addition, we also examined the effect of ATP-sensitive K(+) (K(ATP)) channel antagonist, glibenclamide on relaxations induced by L-cysteine. RESULTS: L-cysteine (30 nM-1 mM) evoked concentration-dependent relaxations of carbachol-induced tone in isolated porcine irides, reaching a maximum inhibition of 43% at 1 mM. This response was enhanced significantly (P < 0.001) in the presence of the COX inhibitor, flurbiprofen (3 microM). Additionally,in the presence of flurbiprofen, the H(2)S donors, NaHS and Na(2)S, mimicked the relaxations produced by L-cysteine, yielding IC(50) values of 5.8 microM and 180 microM, respectively. Both the inhibitor of cystathionine beta-synthase, AOA (30 microM) and the K(ATP) channel antagonist, glibenclamide (100 microM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to L-cysteine and attenuated the maximum inhibitory response. Conversely, the inhibitor of cystathionine gamma-lyase, PAG (1 mM) blocked only relaxations caused by high concentrations of L-cysteine (> 100 microM). CONCLUSIONS: The inhibitory action of L-cysteine in isolated porcine irides is dependent on the endogenous production of H(2)S by cystathionine gamma-lyase and cystathionine beta-synthase. Furthermore, prostanoids and K(ATP) channels are involved in the inhibitory action of L-cysteine in this tissue.

Regulation of [3H] D-aspartate release from mammalian isolated retinae by hydrogen sulfide.

Hydrogen sulfide (H(2)S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H(2)S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na(2)S as donors) on amino acid neurotransmission (using [(3)H] D: -aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [(3)H] D: -aspartate at 37 degrees C. Release of [(3)H] D: -aspartate was elicited by high potassium (K(+) 50 mM) pulse. Both NaHS and Na(2)S donors caused an inhibition of K(+)-evoked [(3)H] D: -aspartate release from isolated bovine retinae without affecting basal [(3)H] D: -aspartate efflux yielding IC(50) values of 0.006 and 6 microm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [(3)H] D: -aspartate from isolated porcine retinae with an IC(50) value of 8 microM. The inhibitory action of NaHS on [(3)H] D: -aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine gamma-lyase (CSE). Our results indicate that H(2)S donors can inhibit amino acid neurotransmission from both isolated bovine and porcine retinae, an effect that is dependent, at least in part, on intramural biosynthesis of H(2)S.

Effect of hydrogen sulfide on sympathetic neurotransmission and catecholamine levels in isolated porcine iris-ciliary body.

In the present study, we investigated the pharmacological action of hydrogen sulfide (H2S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H2S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [3H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na2S caused a concentration-dependent inhibition of electrically evoked [3H]NE release from porcine iris-ciliary body without affecting basal [3H]NE efflux. The inhibitory action of H2S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H2S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H2S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.

Inhibitory action of hydrogen sulfide on muscarinic receptor-induced contraction of isolated porcine irides.

We investigated the pharmacological actions of hydrogen sulfide (H(2)S) using sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) as donors on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we also investigated the mechanism of action of H(2)S in this smooth muscle. Isolated porcine iris muscle strips were set up in organ baths and prepared for measurement of longitudinal isometric tension. The relaxant action of NaHS or Na(2)S on carbachol-induced tone was studied in the absence and presence of a K(+)-channel inhibitor and inhibitors/activators of enzymes of the biosynthetic pathways for H(2)S, prostanoid and nitric oxide production. In the concentration range, 10 nM to 100 microM, NaHS produced a concentration-dependent relaxation of carbachol-induced tone reaching a maximum of inhibition of 28% at 30 microM. The cyclooxygenase inhibitor, flurbiprofen (1 microM), enhanced relaxations induced by both NaHS and Na(2)S yielding IC(50) values of 7 microM and 70 microM, respectively. With exception of l-NAME (300 muM) inhibitors of cystathionine gamma-lyase, propargylglycine, (PAG) (1 mM) and beta-cyanoalanine, (BCA) (1 mM) and inhibitors of cystathionine beta-synthase, aminooxyacetic acid (AOA) (30 microM) and hydroxylamine (HOA) (30 microM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to NaHS. An activator of cystathionine beta-synthase, SAM (100 microM), enhanced relaxations elicited by low concentrations of NaHS but attenuated responses caused by the higher concentrations of this H(2)S donor. The inhibitor of K(ATP) channel, glibenclamide (100 and 300 microM), blocked relaxations induced by NaHS. We conclude that the observed inhibitory action of NaHS and Na(2)S in isolated porcine irides is dependent on endogenous production of prostanoids and the biosynthesis of H(2)S by cystathionine gamma-lyase and cystathionine beta-synthase. Furthermore, relaxation induced by H(2)S is mediated, at least in part, by K(ATP) channels. Nitric oxide is not involved in the relaxation induced by this gas in the isolated porcine irides.