Two autocrine pathways to regulate cyclic GMP synthesis in cultured human retinal pigment epithelial cells.
OBJECTIVE
To investigate the role of two separate enzymatic pathways [soluble (sGC) vs. particulate (pGC) guanylyl cyclase] in the synthesis of cyclic GMP (cGMP) in cultured human retinal pigment epithelial (RPE) cells.


METHODS
cGMP accumulation was evaluated by quantitative analysis of cGMP immunoreactivity. RPE cells were also stained for inducible nitric oxide synthase (iNOS), ANP and beta(1)- and alpha(2)-subunits of sGC.


RESULTS
We showed nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and iNOS immunoreactivity in RPE cells. Incubation of the cells in the presence of 1 mM IBMX to inhibit phosphodiesterase activity, and the simultaneous inhibition of NOS activity with L-NAME suggested the involvement of sGC in maintaining a low level of cGMP in the RPE cells. The involvement of sGC was further supported by detection of the beta(1)- and alpha(2)-subunits of sGC. Incubation of the cells in the presence of atrial natriuretic peptide (ANP) to stimulate pGC strongly increased cGMP immunoreactivity. We also demonstrated the presence of ANP in all RPE cells.


CONCLUSIONS
Cultured human RPE cells are capable of producing cGMP after stimulation of sGC or pGC. The presence of iNOS and ANP in all cells suggests two different autocrine pathways of stimulating cGMP production in these cells. The possible role of cGMP in the regulation iNOS gene expression and in the regulation of ANP is discussed.
Diederen
Roselie M H
RM
Eye Research Institute Maastricht, Department of Ophthalmology, University of Maastricht, Maastricht, The Netherlands. R.Diederen@NP.unimaas.nl
La Heij
Ellen C
EC
Markerink-van Ittersum
Marianne
M
Hendrikse
Fred
F
de Vente
Jan
J
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D019898:Autocrine Communication / Q000502:physiology*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; C000605660:GUCY1A2 protein, human; D010069:Oxadiazoles; D010726:Phosphodiesterase Inhibitors; D011810:Quinoxalines; D018160:Receptors, Cytoplasmic and Nuclear; D009320:Atrial Natriuretic Factor; D052247:Nitric Oxide Synthase Type II; D009252:NADPH Dehydrogenase; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine; D019331:NG-Nitroarginine Methyl Ester