Two Autocrine Pathways to Regulate Cyclic GMP Synthesis in Cultured Human Retinal Pigment Epithelial Cells

Abstract

Aim: 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 β₁- and α₂-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 β₁- and α₂-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. Conclusion: 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.

Stimulation of Aquaporin-Mediated Fluid Transport by Cyclic GMP in Human Retinal Pigment Epithelium In Vitro

“…1,2 ANP significantly increases intracellular cGMP levels in human RPE, whereas the nitric oxide donor Nanitroprusside (an activator of soluble guanylyl cyclase) is less effective. 30,31 In the rabbit RPE in culture, the application of ANP increases chloride uptake, 32 an effect that is reduced by inhibition of PKG by KT5823. Recent investigation of interferon-gamma signaling via cyclic nucleotides cGMP and cAMP in human RPE demonstrated an increase in fluid transport that was reduced in the presence of either PKA inhibitor H-89 or inhibition of CFTR at the basal membrane.…”