title: Mechanism of action of hydrogen sulfide on cyclic AMP formation in rat retinal pigment epithelial cells.
abstract: Hydrogen sulfide (H(2)S), a colorless gas with the pungent odor of rotten eggs has been reported to produce pharmacological actions in ocular and non-ocular tissues. We have evidence that H(2)S, using sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) as donors can increase cyclic AMP (cAMP) production in neural retina. In the present study, we investigated the mechanism of action of H(2)S on cyclic nucleotide production in rat retinal pigment epithelial cells (RPE-J). Cultured RPE-J cells were incubated for 30 min in culture medium containing the cyclic nucleotide phosphodiesterase (PDE) inhibitor, IBMX (2 mM). Cells were exposed to varying concentrations of NaHS, the H(2)S substrate (L-cysteine), cyclooxygenase (COX) inhibitors or the diterpene activator of adenylate cyclase, forskolin in the presence or absence of H(2)S biosynthetic enzymes or the ATP-sensitive potassium (K(ATP)) channel antagonist, glibenclamide. Following drug-treatment at different time intervals, cell homogenates were prepared for cAMP assay using a well established methodology. In RPE-J cells, NaHS (10 nM-1 μM) produced a time-dependent increase in cAMP concentrations over basal levels which reached a maximum at 20 min. At this time point, both NaHS (1 nM-100 μM) and L-cysteine (1 nM-10 μM) produced a concentration-dependent significant (p<0.05) increase in cAMP concentrations over basal level. The effects of NaHS on cAMP levels in RPE-J cells was enhanced significantly (p<0.01) in the presence of the COX inhibitors, indomethacin and flurbiprofen. In RPE-J cells, the effects caused by forskolin (10 μM) on cAMP production were potentiated by addition of low concentrations of NaHS. Both the inhibitor of cystathionine β-synthase (CBS), aminooxyacetic acid (AOA, 1 mM) and the inhibitor of cystathionine γ-lyase (CSE), proparglyglycine (PAG, 1mM) significantly attenuated the increased effect of L-cysteine on cAMP production. The K(ATP) channel antagonist, glibenclamide (100 μM) caused inhibition of NaHS induced-increase of cAMP formation in RPE-J cells. We conclude that, H(2)S (using H(2)S donor and substrate) can increase cAMP production in RPE-J cells, and removal of the apparent inhibitory effect of prostaglandins unmasks an excitatory activity of H(2)S on cAMP. Effects elicited by the H(2)S substrate on cAMP formation are dependent on biosynthesis of H(2)S catalyzed by the biosynthetic enzymes, CBS and CSE. In addition to the adenylyl cylcase pathway, K(ATP) channels are involved in mediating the observed effects of the H(2)S on cAMP production.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Texas Southern University, Houston, TX 77004, USA. njiey@tsu.edu
authors.forename: Ya Fatou
authors.initials: YF
authors.lastname: Njie-Mbye
authors.forename: Madhura
authors.initials: M
authors.lastname: Kulkarni
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Opere
authors.forename: Sunny E
authors.initials: SE
authors.lastname: Ohia
mesh_terms: D000480:Alkynes / Q000494:pharmacology; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003541:Cystathionine beta-Synthase / Q000502:physiology; D003542:Cystathionine gamma-Lyase / Q000502:physiology; D003545:Cysteine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006862:Hydrogen Sulfide / Q000494:pharmacology*; D054086:KATP Channels / Q000378:metabolism; D011453:Prostaglandins / Q000378:metabolism; D051381:Rats; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D013440:Sulfides / Q000494:pharmacology
chemical_list: D000480:Alkynes; D016861:Cyclooxygenase Inhibitors; D054086:KATP Channels; D011453:Prostaglandins; D013440:Sulfides; D000625:Aminooxyacetic Acid; C009055:propargylglycine; D000242:Cyclic AMP; D003541:Cystathionine beta-Synthase; D003542:Cystathionine gamma-Lyase; D003545:Cysteine; D005998:Glycine; C033479:sodium sulfide; D006862:Hydrogen Sulfide