Inhibition of excitatory neurotransmitter activity by hydrogen sulfide in bovine retina, in vitro.

摘要

Hydrogen sulfide (H2S) has been known as a toxic gas and environmental pollutant for decades. However, it was recently reported to be an endogenously produced gasotransmitter and signaling molecule that can reduce intraocular pressure (IOP) in normotensive and glaucomatous rabbits in the anterior segment of the eye. Initial studies support a modulatory role for the fast H2S donor compounds, NaHS and Na2S on excitatory neurotransmitter release in the posterior segment of the mammalian eye, prompting further research on its pharmacological effects in these ocular tissues. SO far, no studies have evaluated the pharmacological actions of H2S donor drugs that produce the gas by different mechanisms in mammalian retina. In the present study, I investigated the effect of different categories of H2S donor drugs, L-cysteine, a substrate for H2S biosynthesis; N-acetyl-cysteine (NAC), a precursor to L-cysteine and GYY 4137, a slow-releasing H2S donor on potassium (K+; 50 mM)-induced glutamate release (measured as [3H]D-aspartate) in bovine isolated retina using the superfusion method. Furthermore, I investigated the ability of various H2S donor drugs to protect bovine retinal neurons from glutamate-induced toxicity using the MTT assay.;The H2S donor drugs, L-cysteine (100 nM to 10 muM), NAC (10 muM to 1 mM) and GYY 4137 (10 nM to 10 muM) caused a concentration-dependent inhibition of K+-induced [3H]D-aspartate release without affecting basal tritium overflow. Interestingly, L-cysteine exhibited the highest potency on the excitatory neurotransmitter release, achieving an inhibition of 54.28 +/- 3.61% (n=4, p<0.005) at the 10 muM concentration of the drug. Whereas, NAC exhibited a maximum inhibition of 29.63 +/- 14.70% (n=5, p<0.01) at 1 mM concentration while GYY 4137 achieved a maximum inhibition of 21.48 +/- 3.52% at the 10 muM concentration of the drug. At an equimolar concentration of 10 muM, the rank order of activity of the three H2S donor drugs was as follows: L-cysteine > GYY 4137 > NAC. Taken together, these results affirm superior modulatory role for L-cysteine, the substrate for endogenous biosynthesis of H2S over the other H2S donor drugs used in this study on the excitatory neurotransmitter release.;There is evidence that endogenous H2S biosynthesis contributes to the effect of H2S donor drugs in ocular tissues. In the present study, the Cystathionine beta-synthase (CBS) inhibitor, aminooxyacetic acid (AOA; 3 mM) did not have an effect on basal K+-induced [ 3H]D-aspartate release. However, it completely reversed the inhibitory effects elicited by L-cysteine (1 muM and 10 muM) and GYY 4137 (1 muM and 10 muM) on the excitatory neurotransmitter release, suggesting that endogenous biosynthesis of H2S is involved in the inhibitory action elicited by the two H2S donor drugs.;The gasotransmitter, NO has been shown to interact with the pharmacological actions of H2S in ocular tissues. In this study, both the non-specific inhibitor of the enzyme nitric oxide synthase (NOS), L-Arginine-N-Nitro Methyl Ester (L-NAME) and inhibitor of inducible (iNOS), aminoguanidine had no effect on K+-induced [3H]D-aspartate release in bovine isolate retina. However, L-NAME (300 muM) abolished the inhibitory effects of L-cysteine (1 muM and 10 muM) and GYY 4137 (1 muM and 10 muM) on the neurotransmitter release. Similarly, aminoguanidine (10 muM) reversed the inhibitory effect of L-cysteine on [3H]D-aspartate release, suggesting that NO is involved in the regulation of excitatory neurotransmitter release by H2S donor drugs bovine isolated retina.;Excessive levels of glutamate are associated with excitotoxicity and neuronal cell death. Since the H2S donor drugs attenuate excitatory neurotransmitter release in the eye, it is conceivable that H2S could confer neuroprotection to retinal neurons.;In conclusion, H2S donor drugs attenuated K+-induced [3H]D-aspartate release in isolated bovine retinae with the following rank order of activity: L-cysteine > NAC > GYY 4137. Unlike NAC, the pharmacological activity of L-cysteine and GYY 4137 was dependent on the on the in situ release of H2S and activation of KATP channels. NO was involved in the pharmacological actions of L-cysteine. Furthermore, the H2S donor drugs protected retinal neurons from glutamate-induced toxicity, suggesting a potential therapeutic role for these compounds in the management of neurodegenerative conditions. (Abstract shortened by UMI.).