Abstract

Cultured retinal pigment epithelium cells prepared from post-mortem adult human eyes are shown to contain muscarinic receptors associated with phosphoinositide turnover. Carbachol at a concentration of 100 microM induced a four-fold increase in 3H-inositol phosphates (more than 76% is in the form of 3H-inositol-1-phosphate) accumulation within 45 min in cells prelabelled with 3H-myoinositol and exposed to 5 mM LiCl. The EC50 of carbachol was approx. 70 microM and the saturation concentration was about 1 mM. The carbachol-induced response was blocked by both atropine and pirenzepine, the former being most effective. Pre-exposure of cells to carbachol resulted in desensitization and a drastic reduction in the subsequent carbachol-induced stimulation of 3H-inositol phosphates. The carbachol response could be attenuated by the biologically active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate, and this was nullified by the protein kinase C inhibitor, staurosporine. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13 dideconoate, did not attenuate the carbachol-induced stimulation of 3H-inositol phosphates. Pertussis toxin failed to influence the carbachol receptor-mediated phosphoinositide turnover. These studies provide clear evidence for the occurrence of muscarinic receptors coupled to phosphoinositide hydrolysis on human retinal pigment epithelium cells.