Recovery from TPA inhibition of receptor-mediated Ca2+ mobilization is paralleled by down-regulation of protein kinase C-alpha in CHO cells expressing the CCK-A receptor.
Digital-imaging microscopy of Fura-2-loaded Chinese hamster ovary cells, stably expressing the cholecystokinin-A receptor, revealed that both the C-terminal octapeptide of cholecystokinin (CCKB) and its analogue JMV-180, which acts as an agonist at the high-affinity CCK-A receptor, recruited CHO-CCK-A cells dose-dependently in terms of receptor-mediated Ca2+ mobilization. Agonist-evoked cell recruitment was inhibited by short-term (10 min) pretreatment with 0.1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA). In the case of CCKB, 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-180 during prolonged TPA pretreatment. Complete recovery was observed within 1 h following addition of TPA. Western blot analysis using antibodies 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 formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to CCKB concentrations at which the effect on cell recruitment was not affected by short term phorbol ester pretreatment. Together with the finding that JMV-180 does not detectably increase the cellular Ins(1,4,5)P3 content, this suggests a large overproduction of this second messenger by CCKB concentrations supramaximal in terms of cell recruitment. Again, full responsiveness was observed after long term TPA pretreatment. The present observations are in agreement with the idea that in CHO-CCK-A cells activation of PKC-alpha leads to inhibition of agonist-evoked Ca2+ mobilization through inhibition of receptor-stimulated Ins(1,4,5)P3 formation.
Smeets
R L
RL
Department of Biochemistry, University of Nijmegen, The Netherlands.
Garner
K M
KM
Hendriks
M
M
van Emst-de Vries
S E
SE
Peacock
M D
MD
Hendriks
W
W
de Pont
J J
JJ
Willems
P H
PH
D000818:Animals; D016466:CHO Cells / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D015536:Down-Regulation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000037:antagonists & inhibitors* / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors
D007527:Isoenzymes; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium