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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. [ABSTRACT FROM AUTHOR]

We have evidence that F-isoprostanes (F-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F-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-F-IsoP on K-evoked [H] 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-F-IsoP inhibited K-evoked [H] d-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9 % at 0.1 µM (IC = 1 nM). The prostanoid receptor antagonists, AH 6809 (EP/DP; 10 µM), SC 51322 (EP; 10 µM) and SC 19220 (EP; 1 µM) partially reversed 5- epi-5-F-IsoP-mediated inhibition of K-induced [H] d-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3 μM) unmasked a biphasic action of 5- epi-5-F-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-F-IsoP (0.1 μM) on neurotransmitter release. In conclusion, 5- epi-5-F-IsoP attenuates K-induced [H] d-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5- epi-5-F-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 F-IsoP on isolated bovine retina. [ABSTRACT FROM AUTHOR]

We have evidence that 15-F-isoprostanes (15-F-IsoPs) regulate excitatory neurotransmitter release in ocular tissues. Although 5-F-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-F-IsoP epimer pair, 5-F-IsoP (C5-OH in β-position) and 5- epi-5-F-IsoP (C5-OH in α-position), on K-evoked [H]D-aspartate release in isolated bovine retina. We further examined the role of prostanoid receptors on the inhibitory action of 5- epi-5-F-IsoP on [H]D-aspartate overflow. Isolated bovine retina were prepared for studies of K-evoked release of [H]D-aspartate using the superfusion method. 5- epi-5-F-IsoP (0.01 nM to 1 μM), attenuated K-evoked [H]D-aspartate release in a concentration-dependent manner, with the inhibitory effect of 26.9% ( P < 0.001; IC = 0.2 μM) being achieved at 1 μM concentration. Its 5-( S)-OH-epimer, 5-F-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 (IC value of 3 nM). Although the prostanoid-receptor antagonists, AH 6809 (10 μM; EP/DP) and BAY-u3405 (10 μM; DP/Tx) exhibited no effect on 5- epi-5-F-IsoP (10 nM-1 μM)-mediated inhibition, SC-19220 (1 μM; EP) completely reversed 5- epi-5-F-IsoP (0.1 μM and 1 μM)-induced attenuation of K-evoked [H]D-aspartate release. Similarly, both SC-51322 (10 μM; EP) and AH 23848 (1 μM; EP) reversed the inhibitory action elicited by 5- epi-5-F-IsoP (0.1 μM) on the neurotransmitter release. We conclude that the 5-F-IsoP epimer pair, 5-F-IsoP and 5- epi-5-F-IsoP, attenuate K-induced [H]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-F-IsoP on excitatory neurotransmitter release. [ABSTRACT FROM AUTHOR]

The role of enzymes and receptors of the prostanoid pathway in the inhibitory effect of 8-isoprostaglandin E2 (8-isoPGE2) on endogenous amino acid neurotransmitter levels was examined, ex vivo. Freshly isolated bovine eyeballs were injected intravitreally with IsoPs, incubated in Krebs buffer for 30 min and retina prepared for HPLC-ECD detection of amino acids. 8-isoPGE2 attenuated retinal glutamate and its metabolite, glutamine and glycine in a concentration-dependent manner. The non-selective cyclooxygenase (COX)-inhibitor, flurbiprofen, COX-2 selective inhibitor, NS-398 and thromboxane (Tx) synthase inhibitor, furegrelate had no effect on both basal amino acid levels and the inhibitory effects of 8-isoPGE2 (1–100 μM) on the retinal amino acids. Whereas the TP-receptor antagonist SQ-29548(10 μM) exhibited no effect, SC-19220(EP1; 30 μM), AH-6809(EP1–3; 30 μM) and AH-23848(EP4; 30 μM) reversed the inhibitory effects of 8-isoPGE2 (0.01–100 μM) on glutamate, glutamine and glycine levels. We conclude that prostanoid EP-receptors regulate the inhibitory effect of 8-isoPGE2 on basal levels of endogenous amino acids in bovine retina, ex vivo. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H2S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on amino acid neurotransmission (using [3H] d-aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [3H] d-aspartate at 37°C. Release of [3H] d-aspartate was elicited by high potassium (K+ 50 mM) pulse. Both NaHS and Na2S donors caused an inhibition of K+-evoked [3H] d-aspartate release from isolated bovine retinae without affecting basal [3H] d-aspartate efflux yielding IC50 values of 0.006 and 6 μm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [3H] d-aspartate from isolated porcine retinae with an IC50 value of 8 μM. The inhibitory action of NaHS on [3H] d-aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine γ-lyase (CSE). Our results indicate that H2S 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 H2S. [ABSTRACT FROM AUTHOR]

Peroxides can enhance field-stimulated [3H]norepinephrine ([3H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional α2-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [3H]NE and prepared for studies of neurotransmitter release using the superfusion method. α2-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [3H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H2O2 (300 μM) had no effect on inhibition of evoked [3H]NE release caused by the α2-adrenergic agonists. However, H2O2 (300 μM) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 μM). In contrast, yohimbine (1 μM) did not prevent the enhancement of evoked [3H]NE overflow induced by H2O2 (300 μM). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional α2-adrenoceptors. [ABSTRACT FROM AUTHOR]