EFFECTS OF PHORBOL ESTER ON CARBACHOL-INDUCED CONTRACTION IN BOVINE CILIARY MUSCLE - POSSIBLE INVOLVEMENT OF PROTEIN-KINASE-C

The aim of the research was to characterize muscarinic receptors of bo vine ciliary muscle and to investigate the desensitization process. Th e role of protein kinase C was analyzed. The results show that muscari nic receptors of bovine ciliary muscle have the pharmacological charac teristics of the M-3 subtype. Acute exposure to phorbol esters (1 mu M phorbol 12,13-dibutyrate, PDB, or 0.1 mu M phorbol 12-myristate 13-ac etate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h ) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensi tization and loss of phorbol ester-induced desensitization. Staurospor ine (3 mu M) and H-7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 mu M), protein kinase C inhibitors, produced a significant potentia tion of the contractile effect of carbachol, reduced the desensitizati on produced by repeated addition of carbachol and suppressed that indu ced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [H-3]quinucli dinyl benzilate. In vitro incubation with PDB and PMA produced, as exp ected, a significant translocation of protein kinase C from the cytoso l to the membrane. The incubation of the ciliary muscle with carbachol , using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ci liary muscle is correlated, at least partially, to the translocation o f protein kinase C from the cytosol to the membrane. The desensitizati on by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the densit y and affinity of muscarinic receptors is not involved. (C) 1997 Elsev ier Science B.V.