RECOVERY FROM TPA INHIBITION OF RECEPTOR-MEDIATED CA2-REGULATION OF PROTEIN-KINASE C-ALPHA IN CHO CELLS EXPRESSING THE CCK-A RECEPTOR( MOBILIZATION IS PARALLELED BY DOWN)

Digital-imaging microscopy of Fura-2-loaded Chinese hamster ovary cell s, stably expressing the cholecystokinin-A receptor, revealed that bot h the C-terminal octapeptide of cholecystokinin (CCK3) and its analogu e JMV-180, which acts as an agonist at the high-affinity CCK-A recepto r, recruited CHO-CCK-A cells dose-dependently in terms of receptor-med iated Ca2+ mobilization. Agonist-evoked cell recruitment was inhibited by short-term (10 min) pretreatment with 0.1 mu M 12-O-tetradecanoylp horbol 13-acetate (TPA). In the case of CCK8, inhibition was overcome with increasing of the hormone concentration. In contrast, increasing of the JMV-180 concentration did not reverse the inhibitory action of TPA. CHO-CCK-A cells gradually regained their responsiveness to JMV-18 0 during prolonged TPA pretreatment. Complete recovery was observed wi thin 1 h following addition of TPA. Western blot analysis using antibo dies directed against the various PKC isotypes revealed that recovery was paralleled by the disappearance of PKC-alpha. Surprisingly, short- term (10 min) TPA pretreatment virtually completely inhibited the form ation of inositol 1,4,5-trisphosphate [lns(1,4,5)P-3] in response to C CK8 concentrations at which the effect on cell recruitment was not aff ected by short term phorbol ester pretreatment. Together with the find ing that JMV-180 does not detectably increase the cellular lns(1,4,5)P -3 content, this suggests a large overproduction of this second messen ger by CCK8 concentrations supramaximal in terms of cell recruitment. Again, full responsiveness was observed after long term TPA pretreatme nt. The present observations are in agreement with the idea that in CH O-CCK-A cells activation of PKC-alpha leads to inhibition of agonist-e voked Ca2+ mobilization through inhibition of receptor-stimulated lns( 1,4,5)P-3 formation.