Recovery from TPA inhibition of receptor-mediated Ca2+ mobilization is paralleled by down-regulation of protein kinase C-α in CHO cells expressing the CCK-A receptor

Pfl�gers Archiv European Journal of Physiology

et al. 1998

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.

Section: Discussionsupporting

confidence: 75%

Section: Pkc Isotype Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

See 1 more Smart Citation

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

Smeets

Rao

Pfl�gers Archiv European Journal of Physiology

et al. 1998

“…We have previously shown that activation of PKC by the phorbol ester 12‐ O ‐tetradecanoylphorbol 13‐acetate (TPA) inhibits high‐affinity Ca 2+ signalling through the CCK A receptor in freshly isolated pancreatic acinar cells (Willems et al , 1993b; 1995) and stably transfected CHO‐K1 cells (Smeets et al , 1996). The aim of the present work was to compare modes of signal transduction and regulation of receptor functioning by PCK of pure CCK A and CCK B receptors after functional expression in the same eukaryotic expression system, namely the CHO‐09 cell.…”

Section: Introductionmentioning

confidence: 99%

Protein kinase C‐mediated inhibition of transmembrane signalling through CCK_A and CCK_B receptors

Smeets

Fouraux

et al. 1998

British J Pharmacology

1 The rat CCK A and CCK B receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and e ects of protein kinase C (PKC) thereupon. 3 Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK A cells. By contrast, none of these agonists increased cyclicAMP in CCK B cells. 4 Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca 2+ mobilizing e ect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK A cells. In both cases, the inhibitory e ect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-a-phorbol 12-myristate 13-acetate. 5 During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca 2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKCa, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6 This study demonstrates that following expression in CHO cells (i) both CCK A and CCK B receptors are coupled to Ca 2+ mobilization, (ii) only CCK A receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

“…Western blotting was performed essentially as described previously [27]. Briefly, total cell lysates from CHO-CCK A cells and rat pancreatic acini and the membrane and cytosol fractions prepared from both cell types were subjected to SDS-PAGE.…”

Section: Western Blottingmentioning

confidence: 99%

Rat pancreatic acinar cells express a cytosolic phospholipase D1b isoform that is not regulated by cholecystokinin

Bosch

Harris

Pfl�gers Archiv European Journal of Physiology

et al. 2001

Evidence for the presence of a regulated phospholipase D (PLD) activity in pancreatic acinar cells is conflicting. Such knowledge is important because signal-activated PLD has been implicated in, amongst other things, regulated exocytosis. In this study, freshly isolated rat pancreatic acini were used to identify PLD transcripts by RT-PCR, to assess the presence and subcellular localization of PLD protein by Western blotting and to evaluate the presence of secretagogue-regulated PLD activity by means of the PLD-catalysed transphosphatidylation reaction. Transcripts of PLD1b and PLD2, but not PLD1a, were present in acinar cells. Moreover, a specific anti-human PLD1 antibody demonstrated the expression of substantial amounts of PLD1 protein. Intriguingly, however, the distribution pattern of acinar PLD1 seen following subcellular fractionation was clearly atypical in that immunoreactivity occurred predominantly in the acinar cytosol. Pretreatment of intact acini with a phorbol ester (4beta-phorbol 12-myristate 13-acetate, PMA) to activate PLD1 protein kinase C (PKC) dependently did not change the subcellular distribution of PLD1. Similarly, pretreatment of a broken cell preparation of acini with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) to activate PLD via small GTPases and PMA also did not influence this distribution. In the presence of ethanol, cholecystokinin-(26-33)-peptide amide (CCK8) did not increase the amount of radiolabelled phosphatidylethanol (PtdEth) in intact acini prelabelled with either o-[32P]phosphate or [3H]myristic acid. Similarly, an increased cytosolic Ca2+ concentration evoked by the specific inhibitor of the endoplasmic reticulum Ca2+-ATPase, thapsigargin, did not stimulate acinar PLD activity whereas high-level PKC activation with PMA elicited slight stimulation. In contrast, all three stimuli are known to increase PLD activity readily in Chinese hamster ovary (CHO) cells expressing the rat pancreatic acinar cell CCKA receptor. Finally, the combination of PMA and GTPgammaS did not increase PLD activity following homologous reconstitution of acinar cytosol and membranes, whereas the same manoeuvre resulted in marked stimulation of PLD activity in CHO cells. Heterologous reconstitution experiments revealed that PLD activity in CHO membranes was stimulated readily in the presence of acinar cytosol, indicating that the acinar cytosol contains the necessary factors for PMA/GTPgammaS-induced stimulation of membrane PLD activity. In contrast, CHO cell cytosol did not confer PMA/GTPgammaS-stimulation of PLD activity on acinar membranes, in agreement with the predominantly cytosolic localization of acinar PLD. The present findings show that rat pancreatic acinar cells express a cytosolic PLD1 isoform that is not regulated by the physiologically important secretagogue CCK.