Abstract
Introduction: :
Hydrogen sulfide (H2S), a colorless gas with the pungent odor of rotten eggs has been reported to produce pharmacological actions in ocular and non-ocular tissues. For instance, we have evidence that H2S donors such as sodium hydrosulfide (NaHS) can increase the production of cyclic AMP in neural retina (Njie-Mbye et al., Neurochem Res 2009).
Purpose: :
In the present study, we investigated the mechanism of action of H2S on cyclic AMP production in rat retinal pigmented epithelial cells (RPE-J).
Methods: :
RPE-J cells were incubated for 30 mins in culture medium containing the cyclooxygenase (COX) inhibitor, flurbiprofen (3 µM) and the cyclic nucleotide phosphodiesterase (PDE) inhibitor, IBMX (2mM). Cells were exposed to the H2S donor, (NaHS 1 nM and 100 nM) in the absence and presence of inhibitors of enzymes of the biosynthetic pathways for H2S production and an antagonist of ATP-sensitive K+ (KATP) channel, glibenclamide. Following drug-treatment, samples were collected and prepared for cyclic AMP content using a well established Enzyme ImmunoAssay methodology.
Results: :
NaHS (10 nM and100 nM) produced a concentration-dependent significant (p < 0.001) increase in cyclic AMP concentrations over basal level at an optimal time of 20 minutes. Both the inhibitor of cystathionine β-synthase (CBS), aminooxyacetic acid (AOA, 30 µM) and the inhibitor of cystathionine γ-lyase (CSE), proparglyglycine (PAG 1 mM) significantly attenuated the increased effect of NaHS (100 nM) on cyclic AMP production by 70% and 50% respectively. The KATP channel antagonist, glibenclamide (100 µM) also caused significant (P < 0.001) inhibition of NaHS (10 nM and 100 nM) induced-increase of cyclic AMP formation in RPE-J cells.
Conclusions: :
NaHS significantly increased cyclic AMP levels in RPE-J cells indicating that this nucleotide serves as a mediator of effects caused by H2S in these cells. Effects elicited by the H2S donor on cyclic AMP formation in RPE-J cells are dependent on intramural biosynthesis of this gas catalyzed by the biosynthetic enzymes, CBS and CSE. Furthermore, KATP channels are involved in mediating the observed effects of the H2S donor on cyclic nucleotide production.
Keywords: second messengers: pharmacology/physiology • retina • enzymes/enzyme inhibitors