Inhibitory action of novel hydrogen sulfide donors on bovine isolated posterior ciliary arteries.

In the present study, we investigate the inhibitory effect of novel H2S donors, AP67 and AP72 on isolated bovine posterior ciliary arteries (PCAs) under conditions of tone induced by an adrenoceptor agonist. Furthermore, we examined the possible mechanisms underlying the AP67- and AP72-induced relaxations. Isolated bovine PCA were set up for measurement of isometric tension in organ baths containing oxygenated Krebs solution. The relaxant action of H2S donors was studied on phenylephrine-induced tone in the absence or presence of enzyme inhibitors for the following pathways: cyclooxygenase (COX); H2S; nitric oxide and the ATP-sensitive K(+) (KATP) channel. The H2S donors, NaSH (1 nM - 10 µM), AP67 (1 nM - 10 µM) and AP72 (10 nM - 1 µM) elicited a concentration-dependent relaxation of phenylephrine-induced tone in isolated bovine PCA. While the COX inhibitor, flurbiprofen (3 µM) blocked significantly (p < 0.05) the inhibitory response elicited by AP67, it had no effect on relaxations induced by NaSH and AP72. Both aminooxyacetic acid (30 µM) and propargylglycine (1 mM), enzyme inhibitors of H2S biosynthesis caused significant (p < 0.05) rightward shifts in the concentration-response curve to AP67 and AP72. Furthermore, the KATP channel antagonist, glibenclamide (300 µM) and the NO synthase inhibitor, l-NAME (100 µM) significantly attenuated (p < 0.05) the relaxation effect induced by AP67 and AP72 on PCA. We conclude that H2S donors can relax pre-contracted isolated bovine PCA, an effect dependent on endogenous production of H2S. The inhibitory action of only AP67 on pre-contracted PCA may involve the production of inhibitory endogenous prostanoids. Furthermore, the observed inhibitory action of H2S donors on PCA may depend on the endogenous biosynthesis of NO and by an action of KATP channels.

Interaction between hydrogen sulfide, nitric oxide, and carbon monoxide pathways in the bovine isolated retina.

PURPOSE: Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) are physiologically relevant gaseous neurotransmitters that are endogenously produced in mammalian tissues. In the present study, we investigated the possibility that NO and CO can regulate the endogenous levels of H2S in bovine isolated neural retina. METHODS: Isolated bovine neural retina were homogenized and tissue homogenates were treated with a NO synthase inhibitor, NO donor, heme oxygenase-1 inhibitor, and/donor. H2S concentrations in bovine retinal homogenates were measured using a well-established colorimetric assay. RESULTS: L-NAME (300 nM-500 µM) caused a concentration-dependent decrease in basal endogenous levels of H2S by 86.2%. On the other hand, SNP (10-300 µM) elicited a concentration-related increase in H2S levels from 18.3 nM/mg of protein to 65.7 nM/mg of protein. ZnPP-IX (300 nM-10 µM) caused a concentration-dependent increase in the endogenous production of H2S whereas hemin (300 nM-20 µM) attenuated the basal levels of H2S. CONCLUSION: We conclude that changes in the biosynthesis and availability of both NO and CO can interfere with the pathway/s involved in the production of H2S in the retina. The demonstrated ability of NO, CO and H2S to interact in the mammalian retina affirms a physiological/pharmacological role for these gaseous mediators in the eye.

Serotonin-2B/2C Receptors Mediate Bovine Ciliary Muscle Contraction: Role in Intraocular Pressure Regulation.

PURPOSE: To study the pharmacological profile of the serotonin (5-hydroxytryptamine [5-HT]) receptor subtype mediating contractions in bovine isolated ciliary muscles. METHODS: Ciliary muscle strips were isolated from bovine eyeballs and mounted in organ baths containing aerated (95% O2, 5% CO2) Krebs buffer solution maintained at 37°C. Each muscle strip was attached at 1 end to a Grass Force-displacement Transducer connected to a Polyview Computer System for recording changes in isometric tension. After an equilibration period, ciliary muscle strips were exposed to selective agonists and antagonists of 5-HT receptors. RESULTS: Both selective and nonselective agonists for 5-HT produced concentration-dependent contractions of isolated ciliary muscles with the following rank order of potency: BW723C86>α-methyl-5-HT>MK-212>>8-hydroxy-DPAT>quipazine>R-DOI>>5-HT>>tryptamine. The selective 5-HT2 receptor antagonists, M-100907 (5-HT2A), RS-127445 (5-HT2B), and RS-102221 (5-HT2C), produced noncompetitive inhibition of the contractile effects of selective agonists yielding antagonist potency (pKB) values of 251 ± 27.2 nM (n = 4), 52.5 ± 6.3 nM (n = 4), and 79.4 ± 9.5 nM (n = 4), respectively. CONCLUSION: On the basis of the profile of activity of selective agonists and antagonists, we conclude that the 5-HT2B and 5-HT2C receptor subtypes appear to be the predominant serotonin receptors that mediate the contractile action of this amine in bovine isolated ciliary muscles.

Pharmacology of Serotonin Receptors Causing Contraction of Isolated Bovine Posterior Ciliary Arteries: Role in Ocular Blood Flow.

PURPOSE: To determine the serotonergic (5HT) receptor subtype mediating the contraction of bovine posterior ciliary arteries (BPCAs) in vitro. METHODS: Longitudinal isometric tension was measured in BPCA strips (4-5 mm) mounted in 25 mL organ baths containing oxygenated Krebs solution at 37°C. Cumulative contractile concentration-response (C-R) curves were generated for various 5-HT agonists to assess their potencies and maximal degrees of contraction. Multiple agonist C-R curves were also constructed in the presence and absence of receptor-selective antagonists to determine antagonist potencies using Schild plots. RESULTS: Selective and nonselective agonists for 5-HT receptors elicited concentration-dependent contractile responses in BPCAs with the following rank order of potency: MK-212 > BW723C86 > α-methyl-5-HT >5-methoxy-α-5-methyl-5-HT >> R-DO1 > >5-HT >> cabergoline >> 5-methoxy-dimethyl-tryptamine >> 2-methyl-5-HT >> tryptamine. Interestingly, both 8-OH-DPAT (5HT1A agonist) and quipazine (5HT3 agonist) did not elicit contractions in BPCAs. The contractions produced by BW723C86 (5-HT2B agonist) were antagonized by 5-HT receptor blockers, RS-127445 (5-HT2B antagonist), and M-100907 (5-HT2A antagonist), yielding antagonist pA2 values of 7.5 ± 0.12 (n = 4) and 6.2 ± 0.17 (n = 4), respectively. Furthermore, contractions elicited by MK-212 (5-HT2C agonist) was blocked by RS-102221 (5-HT2C antagonist), although noncompetitively. CONCLUSIONS: On the basis of the pharmacological profile of selective agonists and antagonists, we conclude that serotonin-induced contractions of the BPCA are mediated primarily by a combination of 5HT2C and/or 5HT2B receptors. It appears that 5-HT1A and 5-HT3 receptors are not involved in the contractile action of BPCAs to serotonin.

Regulation of Aqueous Humor Dynamics by Hydrogen Sulfide: Potential Role in Glaucoma Pharmacotherapy.

Hydrogen sulfide (H2S) is a gaseous transmitter with well-known biological actions in a wide variety of tissues and organs. The potential involvement of this gas in physiological and pathological processes in the eye has led to several in vitro, ex vivo, and in vivo studies to understand its pharmacological role in some mammalian species. Evidence from literature demonstrates that 4 enzymes responsible for the biosynthesis of this gas (cystathionine ß-synthase, CBS; cystathionine γ-lyase, CSE; 3-mercaptopyruvate sulfurtransferase, 3MST; and d-amino acid oxidase) are present in the cornea, iris, ciliary body, lens, and retina. Studies of the pharmacological actions of H2S (using several compounds as fast- and slow-releasing gas donors) on anterior uveal tissues reveal an effect on sympathetic neurotransmission and the ability of the gas to relax precontracted iris and ocular vascular smooth muscles, responses that were blocked by inhibitors of CSE, CBS, and KATP channels. In the retina, there is evidence that H2S can inhibit excitatory amino acid neurotransmission and can also protect this tissue from a wide variety of insults. Furthermore, exogenous application of H2S-releasing compounds was reported to increase aqueous humor outflow facility in an ex vivo model of the porcine ocular anterior segment and lowered intraocular pressure (IOP) in both normotensive and glaucomatous rabbits. Taken together, the finding that H2S-releasing compounds can lower IOP and can serve a neuroprotective role in the retina suggests that H2S prodrugs could be used as tools or therapeutic agents in diseases such as glaucoma.

Effects of Hydrogen Sulfide-Releasing Compounds on Aqueous Humor Outflow Facility in Porcine Ocular Anterior Segments, Ex Vivo.

PURPOSE: To investigate the pharmacological actions of hydrogen sulfide (H2S)-releasing compounds l-cysteine and sodium hydrosulfide (NaHS) on aqueous humor (AH) outflow facility in porcine ocular anterior segment. METHODS: Porcine ocular anterior segments were perfused with Dulbecco's modified Eagle's medium at a constant pressure of 7.35 mmHg. After stable outflow baseline, explants were exposed to NaHS or l-cysteine. The increase in outflow generated by the H2S-releasing compounds was measured in the absence and presence of inhibitor of H2S biosynthesis (aminooxyacetic acid; AOAA), blocker of KATP channels (glibenclamide), and inhibitor of adenylyl cyclase (SQ 22536). Hematoxylin and eosin (H&E) staining was used to assess trabecular meshwork (TM) morphology. RESULTS: l-cysteine elicited a concentration-dependent increase in AH outflow facility, reaching maximal effect at 100 nM (150.6% ± 17.2% of basal level). This increase in outflow induced by l-cysteine was significantly (P < 0.001) antagonized by AOAA (30 µM) and glibenclamide (100 µM). AOAA and glibenclamide had no significant action on baseline outflow, whereas SQ 22536 (100 µM) increased outflow for only an hour. In addition, NaHS produced a concentration-dependent increase in AH outflow, with a maximal effect at 10 µM (151.4% ± 22.9% of basal level). Likewise, the increase in outflow caused by NaHS was significantly (P < 0.04) blocked by glibenclamide and SQ 22536. H&E staining revealed that l-cysteine or NaHS did not alter TM conformation. CONCLUSION: H2S-releasing compounds can increase outflow facility in porcine ocular anterior segment. The stimulatory action of these compounds on outflow is mediated, in part by endogenously produced H2S, KATP channels, and adenylyl cyclase.