Further Studies on the Inhibition of [₃H]D-Aspartate Release by Hydrogen Sulfide Donors in Isolated Bovine Retina

Abstract

Purpose: We have previously reported that hydrogen sulfide (H₂S) donors can regulate [³H]D-aspartate release in isolated mammalian. In the present study, we compared the effect of H₂S-producing compounds on K⁺-induced glutamate release (using [³H]D-aspartate as a marker) in isolated bovine retina. Furthermore, we examined the mechanism underlying the inhibitory action of H₂S-donors on K⁺-induced [³H]D-aspartate release in bovine retina. in bovine retina.

Methods: Isolated bovine retina were incubated for 60 min. at 37^oC in carbogen-gassed Krebs buffer containing 200 nM of [³H]D-aspartate. Release of [³H]D-aspartate was elicited by iso-osmotic concentration of K⁺ (50mM)-stimuli applied at 80-88 min. (S₁) and 116-124 min. (S₂) after the onset of superfusion. H₂S-donors were added 12-18 min. before S₂ while antagonists were present before and during S₁ and S₂.

Results: The H₂S-producing compounds, GYY 4137 (10 nM-10 µM), NaHS (1 nM-1 µM) and L-cysteine (1-10 µM) elicited a concentration-dependent inhibition of K⁺-induced [³H]D-aspartate release in isolated bovine retina. At an equimolar concentration (1 µM), the rank order of activity was as follows: L-cysteine > NaHS > GYY 4137. The substrate for endogenous H₂S production, L-cysteine was most potent, eliciting a maximum inhibitory action of 54.2% (n=4; p<0.01) at the 10 µM concentration (IC₅₀ of about 5 µM). Whereas, the cystathionine β-synthase inhibitor, aminooxyacetic acid (3 mM) and the ATP-sensitive potassium channel (K_ATP) inhibitor, glibenclamide (300 μM) had no effect (p>0.05) on K⁺-induced [³H]D-aspartate release, they both reversed the inhibitory action of GYY 4137 (10 μΜ) and L-cysteine (1-10 µM) on the neurotransmitter release. Furthermore, the nitric oxide (NO) synthase inhibitor, L-NAME (300 µM) reversed the inhibitory action of GYY 4137 (1-10 µM) on the excitatory neurotransmitter release.

Conclusions: K_ATP-channels, NO pathway and in situ production of H₂S contribute to the inhibitory action of H₂S-producing compounds on excitatory neurotransmitter release in isolated bovine retina.

Keywords: 518 excitatory neurotransmitters • 688 retina • 616 neurotransmitters/neurotransmitter systems