title: Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells.
abstract: Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840-G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 muM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 muM) and cholecystokinin-(26-33)-peptide amide (CCK8, 0.1 muM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK8-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-alpha and, to a lesser extent, PKC-epsilon. In addition, TPA-induced inhibition of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-alpha. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca2+]i. Taken together, the present findings are in agreement with the idea that PKC-alpha-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca2+]i oscillations and eventually to inhibition of high-affinity Ca2+ signalling through the native CCK receptor in pancreatic acinar cells.
authors.affiliation: Department of Biochemistry, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands.
authors.forename: R L
authors.initials: RL
authors.lastname: Smeets
authors.forename: R V
authors.initials: RV
authors.lastname: Rao
authors.forename: S E
authors.initials: SE
authors.lastname: van Emst-de Vries
authors.forename: J J
authors.initials: JJ
authors.lastname: De Pont
authors.forename: L J
authors.initials: LJ
authors.lastname: Miller
authors.forename: P H
authors.initials: PH
authors.lastname: Willems
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D010179:Pancreas / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D015398:Signal Transduction; D012844:Sincalide / Q000031:analogs & derivatives / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011949:Receptors, Cholecystokinin; C058451:JMV 180; D011493:Protein Kinase C; D019311:Staurosporine; D012844:Sincalide; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2