Abstract
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 µM), NAC (10 µM to 1 mM) and GYY 4137 (10 nM to 10 µM) 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 µM 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 µM concentration of the drug. At an equimolar concentration of 10 µM, 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 β-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 µM and 10 µM) and GYY 4137 (1 µM and 10 µM) 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 effect of AOA on NAC was mixed. Whereas AOA partially reversed the inhibitory effect elicited by NAC (1 mM), it completely abolished NAC (10 µM)-induced inhibition of the excitatory neurotransmitter release in bovine retina. Similar to AOA, the KATP channel blocker, glibenclamide had no effect on K+-induced [3H]D-aspartate release. However, it abolished the inhibitory effects of GYY 4137 (1 µM and 10 µM) and L-cysteine (1 µM and 10 µM) but not NAC (1 mM) on K+-induced [3H]D-aspartate release. These data suggest that KATP channels are involved in the inhibitory action elicited by L-cysteine and GYY 4137 but not NAC on the excitatory neurotransmitter release in bovine retina.|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 µM) abolished the inhibitory effects of L-cysteine (1 µM and 10 µM) and GYY 4137 (1 µM and 10 µM) on the neurotransmitter release. Similarly, aminoguanidine (10 µM) 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 this study, I examined the effect of H2S donor drugs on glutamate-induced toxicity using the MTT assay. Glutamate induced toxicity to retinal neurons in a dose dependent manner, with the 12 mM concentration of the drug eliciting about 50% of retinal neuron death. A comparison of two-hour and six-hour glutamate treatment revealed that, under my experimental conditions, the two-hour treatment period of incubation with glutamate was optimal. I further examined the effect of H2S donor drugs on glutamate (12 mM)-induced toxicity in bovine isolated retina using three classes of H2S donors: substrates for H2S production, L-methionine, L-cysteine and its precursor NAC; the slow releasing H2S donor, GYY 4137; and the fast H2S releasing hybrid of latanoprost-H2S donating moiety, ACS 67. In the concentration range, 10-8 M to 10-3M, L-cysteine achieved a protective effect of 31.2 ± 9.90%; (p<0.05; n=16) at 10-3M concentration. The L-cysteine precursors, L-Methionine (10-8M to 10-5M) and NAC (10-6 to 10-3M) reversed glutamate-induced toxicity by 18.43% ± 2.64 (p<0.05; n=16) at the 10-6M concentration and 22.14 ± 4.25% (p<0.05; n=16) at the 10-8M concentration, respectively. The slow releasing H2S donor, GYY 4137 (10-8M to 10-5M) achieved a protective effect of 22.1 ± 3% (p<0.05; n=16) at the 10-8M concentration. It was also interesting to note that the protective effect elicited by NAC (10-8M) was equivalent to that of GYY 4137 (10-8M). Compared to latanoprost (10-10M to 10-7M), ACS 67 (10-11 M to 10-7M) reversed glutamate-induced toxicity by 28.98 ± 3.82% (p<0.05; n=16) at the 10-11 M concentration while latanoprost exhibited a protective effect of 18.98 ± 0.61% (p<0.05; n=16) at the 10-10M. This suggests that the H2S-moiety contributes to the additional neuroprotective effect of ACS 67. These data affirm the protective role for H2S donors against glutamate-induced toxicity. The exact mechanism underlying the neuroprotective effect of H2S donors in bovine isolated retina remains to be determined. |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.