We investigated the pharmacological actions of a slow-releasing H2S donor, GYY 4137; a substrate for the biosynthesis of H2S, l-cysteine and its precursor, N-acetylcysteine on potassium (K+; 50 mM)-evoked [3H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K+-evoked [3H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine > GYY 4137 > N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H2S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K+-evoked [3H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of KATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K+-induced [3H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K+-evoked [3H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K+-evoked [3H]D-aspartate release. We conclude that both donors and substrates for H2S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of KATP channels and NO synthase.

PURPOSE In this study, we investigated the effect of a slow-releasing hydrogen sulfide (H2S) donor, GYY 4137, on intraocular pressure (IOP) in normotensive rabbits. Furthermore, we compared the IOP-lowering action of GYY 4137 with those elicited by other H2S-producing compounds, l-cysteine and ACS67 (a hybrid compound of latanoprost with an H2S-releasing moiety). METHODS IOP was measured in New Zealand normotensive male albino rabbits using a pneumatonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY). At 0 h, 50 μL of test compounds were applied topically to 1 eye of each animal, while the contralateral eye received the same quantity of vehicle (saline). IOP was measured hourly until baseline IOP readings were attained and animal eyes monitored for potential side effects (i.e., tearing, hyperemia). RESULTS GYY 4137 (0.1%-2%) produced a dose-dependent decrease in IOP reaching a maximum of 27.8% ± 3.14% (n = 5) after 6 h. Interestingly, a significant contralateral effect was observed in vehicle-treated controls eyes at all doses tested. l-cysteine (5%) and ACS67 (0.005%) also elicited a significant (P < 0.01) decrease in IOP that achieved a maximum of 28.84% ± 1.53% (n = 5) and 23.27% ± 0.51% (n = 5), respectively, after 3 h. All 3 H2S-producing compounds also caused a significant contralateral effect in vehicle-treated control eyes. CONCLUSION We conclude that GYY 4137 and other H2S-producing donors can reduce IOP in normotensive rabbits. However, the profile of IOP-lowering action of GYY 4137 was different from the other H2S donors affirming its ability to act as a slow-releasing gas donor.

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H 2 S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H 2 S-donating moiety; l -cysteine, a substrate for endogenous production of H 2 S and GYY 4137, a slow donor of H 2 S. We also examined the contribution of prostaglandins to the pharmacological actions of the H 2 S donors on release of [ 3 H]-norepinephrine ([ 3 H]NE) triggered by electrical field stimulation. ACS67, l -cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [ 3 H]NE release from isolated bovine iris-ciliary bodies without affecting basal [ 3 H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and l -cysteine on stimulated [ 3 H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-β-synthase and glibenclamide, a K ATP channel blocker reversed the inhibition of evoked NE release induced by the H 2 S donors. We conclude that H 2 S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H 2 S and prostanoids, and is mediated by an action on K ATP channels.

We have evidence that F2-isoprostanes (F2-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F3-IsoPs are generated in mammals, in vivo, their pharmacological actions on neurotransmitter release remain unknown. In this study, we investigated the effect of 5-epi-5-F3t-IsoP on K+-evoked [3H]d-aspartate release in isolated bovine retina using the superfusion method. Furthermore, we examined the role of arachidonic acid metabolites in the regulation of the neurotransmitter release by this novel IsoP. In the concentration range, 0.01 nM–0.1 µM, 5-epi-5-F3t-IsoP inhibited K+-evoked [3H]d-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9 % at 0.1 µM (IC30 = 1 nM). The prostanoid receptor antagonists, AH 6809 (EP1–3/DP; 10 µM), SC 51322 (EP1; 10 µM) and SC 19220 (EP1; 1 µM) partially reversed 5-epi-5-F3t-IsoP-mediated inhibition of K+-induced [3H]d-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3 μM) unmasked a biphasic action of 5-epi-5-F3t-IsoP that was inhibitory at lower (0.1–10 pM) and stimulatory at higher concentrations (≥0.1 nM). The prostanoid pathway antagonists, BAY-u3405 (10 μM; TP/DP-receptors), SQ 29548 (10 μM; TP-receptor) and ozagrel (10 μM; Tx-synthase inhibitor) abolished the stimulatory action of the 5-epi-5-F3t-IsoP (0.1 μM) on neurotransmitter release. In conclusion, 5-epi-5-F3t-IsoP attenuates K+-induced [3H]d-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP1-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5-epi-5-F3t-IsoP that is inhibitory at low concentrations and stimulatory at higher concentrations. Products of the thromboxane synthase pathway may partially account for the stimulatory action of this F3-IsoP on isolated bovine retina.

There is evidence that L‐cysteine, the substrate for biosynthesis of hydrogen sulfide (H2S) can regulate potassium (K+)‐evoked glutamate release from bovine isolated retina. In the present study, we compared the pharmacological effects of the precursor for L‐cysteine, N‐acetyl cysteine (NAC) to that of L‐cysteine on K+‐evoked [3H]D‐aspartate release, and on glutamate‐induced neurotoxicity in bovine isolated retina. Isolated neural retina were incubated in oxygenated Krebs solution containing 200 nM of [3H]D‐aspartate and then prepared for studies of neurotransmitter release. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to assess retinal neuron survival. Both L‐cysteine (0.1 µM to 10 µM) and NAC (10 µM to 1 mM) elicited a concentration‐dependent inhibition of K+‐induced [3H]D‐aspartate release. At an equimolar concentration of 10 µM, both L‐cysteine and NAC reduced [3H]D‐aspartate release by 54.3% (p < 0.001) and 8.3%, respectively. Interestingly, L‐cysteine (1 mM) and NAC (1 µM) attenuated glutamate (12 mM)‐induced neuron degeneration by 31.1% (p<0.05) and 18.4%, respectively. L‐cysteine was more potent in attenuating neurotransmitter release while NAC was more effective in preventing glutamate‐induced toxicity suggesting that these compounds mediate their pharmacological actions via different mechanisms.

Arachidonic acid‐derived F2‐isoprostanes (F2‐IsoPs) can regulate neurotransmitter release in bovine retina (Jamil J. et al, 2012). It is, however, unclear whether eicosapentanoic acid‐derived F3‐IsoPs can produce a similar effect in the retina.PurposeTo investigate the pharmacological actions of 5‐epi‐5‐F3t‐IsoP on K+‐induced [3H]D‐aspartate release from bovine retina. We also examined the role of prostanoid receptors on the F3‐IsoP‐induced response.MethodIsolated neural retina were incubated in oxygenated Krebs solution containing 200 nM of [3H]D‐aspartate and then prepared for studies of neurotransmitter release. Release of [3H]D‐aspartate was evoked by K+ (50 mM) stimuli applied at 90 mins (S1) and at 108 mins (S2) after the onset of superfusion.Results5‐epi‐5‐F3t‐IsoP (0.1 nM – 0.1 μM) elicited an inhibitory action on K+‐evoked [3H]D‐aspartate release in a concentration‐dependent manner, achieving a maximum inhibition of 46.9% at 0.1 μM (IC30 of 1 nM). The effect of 5‐epi‐5‐F3t‐IsoP (0.01 μM) was partially reversed by the prostanoid receptor antagonists, SC 19220 (1 μM; EP1), SC 51322 (10 μM; EP1) and AH 6809 (10 μM; EP1–3/DP1) while AH 23848 (1 μM; EP4/TP1) and SQ 29548 (10 μM; TP) had no effect on the IsoP.Conclusion5‐epi‐5‐F3t‐IsoP can inhibit K+‐evoked [3H]D‐aspartate release in isolated bovine retina, presumably via mechanisms that involve prostanoid receptors.

We have evidence that 15-F2-isoprostanes (15-F2-IsoPs) regulate excitatory neurotransmitter release in ocular tissues. Although 5-F2-IsoPs are abundantly produced in mammals, their pharmacological actions on neurotransmitter release remain unknown. In the present study, we compared the effect of the 5-F2-IsoP epimer pair, 5-F2t-IsoP (C5–OH in β-position) and 5-epi-5-F2t-IsoP (C5–OH in α-position), on K+-evoked [3H]D-aspartate release in isolated bovine retina. We further examined the role of prostanoid receptors on the inhibitory action of 5-epi-5-F2t-IsoP on [3H]D-aspartate overflow. Isolated bovine retina were prepared for studies of K+-evoked release of [3H]D-aspartate using the superfusion method. 5-epi-5-F2t-IsoP (0.01 nM to 1 μM), attenuated K+-evoked [3H]D-aspartate release in a concentration-dependent manner, with the inhibitory effect of 26.9% (P < 0.001; IC25 = 0.2 μM) being achieved at 1 μM concentration. Its 5-(S)-OH-epimer, 5-F2t-IsoP (0.1 nM–1 μM), exhibited an inhibitory biphasic action, yielding a maximal response of 35.7% (P < 0.001) at 10 nM concentration of the drug (IC25 value of 3 nM). Although the prostanoid-receptor antagonists, AH 6809 (10 μM; EP1–3/DP) and BAY-u3405 (10 μM; DP/Tx) exhibited no effect on 5-epi-5-F2t-IsoP (10 nM–1 μM)-mediated inhibition, SC-19220 (1 μM; EP1) completely reversed 5-epi-5-F2t-IsoP (0.1 μM and 1 μM)-induced attenuation of K+-evoked [3H]D-aspartate release. Similarly, both SC-51322 (10 μM; EP1) and AH 23848 (1 μM; EP4) reversed the inhibitory action elicited by 5-epi-5-F2t-IsoP (0.1 μM) on the neurotransmitter release. We conclude that the 5-F2-IsoP epimer pair, 5-F2t-IsoP and 5-epi-5-F2t-IsoP, attenuate K+-induced [3H]D-aspartate release in isolated bovine retina presumably via prostanoid receptor dependent mechanisms. The trans-orientation of the allylic hydroxyl group at position C5 accounts for the apparent biphasic response exhibited by 5-F2t-IsoP on excitatory neurotransmitter release.

Neuroprostanes (nPs) are a series of isoprostane‐like compounds that are spontaneously derived by free‐radical catalyzed peroxidation of the polyunsaturated fatty acid, docosahexanoic acid. Although nPs are elevated in neurodegenerative conditions (Musiek et al., Brain Pathol. 15:149,2005), their biological effects remain largely unknown. We investigated the role of synthetic nPs, CO5‐667, CO5‐668 and CO5‐738 on K+‐induced glutamate release (using [3H]D‐aspartate as a marker) in isolated bovine retina. Isolated neural retina were incubated in oxygenated Krebs solution containing 200nM of [3H] D‐aspartame for 60 mins and then prepared for studies of neurotransmitter release using the superfusion method. Release of [3H]D‐aspartate was evoked by iso‐osmotic concentration of K+ (50mM)‐stimuli applied at 80–88 mins (S1) and 116–124 mins (S2) after the onset of superfusion. In the concentration range, 1 nM to 10 μM, the nPs enhanced K+‐induced [3H]D‐aspartate release from retina without affecting basal tritium overflow. For instance, C05‐668 achieved a maximal excitatory response of about 80% (p<0.01, n=4) at the 10 nM concentration of the nP. At an equimolar concentration of 10 nM, the rank order of activity was as follows: CO5‐668> CO5‐738> CO5‐667. In conclusion, the novel synthetic nPs exert an excitatory effect on K+‐evoked [3H]D‐aspartate release in isolated bovine retina.

We investigated the role of prostanoid receptors in the inhibitory effect of synthetic isoprostane epimers, AG113A and B on K+‐induced glutamate release (using [3H]D‐aspartate as a marker) in isolated bovine retina. Isolated neural retinae were incubated in oxygenated Krebs solution containing 200nM of [3H]D‐aspartate for 60 mins and then prepared for studies of neurotransmitter release using the superfusion method. Release of [3H]D‐aspartate was evoked by iso‐osmotic concentration of K+ (50mM)‐stimuli applied at 80–88 mins(S1) and 116–124 mins(S2) after the onset of superfusion. Both AG113A and B attenuated K+‐induced [3H]D‐aspartate release from retina without affecting basal tritium overflow. AG113A exhibited a biphasic response, with the maximal inhibitory effect of 36%(n=7;p<0.001) being achieved at 0.01ìM while AG113B achieved a maximal inhibitory effect of 26%(n=3;p<0.01) at the 0.1ìM concentration of the isoprostane. The prostanoid antagonists, AH608(EP1‐3/DP1), BAY‐u3405(DP2) and AH23848(EP4) did not reverse the inhibitory effect of AG113A on K+‐induced D‐aspartate release. EP1 antagonists, SC19220 and SC1322 completely reversed the inhibitory effect of AG113A on the neurotransmitter release. Prostanoid EP1‐receptors mediate the inhibitory effect of AG113 on K+‐evoked [3H]D‐aspartate release in isolated bovine retinae.

Neuroprostanes (nPs) are a series of isoprostane (IsoP)‐like compounds that are spontaneously formed in vivo and in vitro by free‐radical catalyzed peroxidation of the polyunsaturated fatty acid (PUFA), docosahexanoic acid. Although nPs are elevated in neurodegenerative conditions and in animal models of neuronal oxidative stress (Musiek et al., Brain Pathol. 15:149,2005), it is unknown whether nPs, like IsoPs, are endogenously produced in mammalian retina. We sought to measure endogenous production of the PUFA metabolites, A4‐nPs and F2‐IsoPs in bovine retinae, in vitro. Freshly isolated bovine retinae were exposed to hydrogen peroxide (H2O2) and cumene hydroperoxide (cuOOH) for up to 6h, in vitro. PUFA metabolites were measured by the stable isotope dilution methods employing GS/negative ion chemical ionization MS. Basal endogenous levels of A4‐nPs and F2‐IsoPs were found to be 35.0± 2.2 and 1.1± 0.001 ng/g tissue, respectively. Exposure of retina to H2O2 (1 mM) for 1h and 6h stimulated production of A4‐nP levels by 15% and 56% while bound F2‐IsoPs were elevated by 44% and 68%, respectively. Similarly, cuOOH (1 mM) enhanced (p<0.001) A4‐nP levels by 408% and 420% and F2‐IsoPs by 831% and 918% after 1h and 6h periods of treatment, respectively. In conclusion, basal levels of nPs exceed that of IsoPs in bovine retina. Furthermore, endogenous production of PUFA metabolites is enhanced by oxidant stress in retina.