Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+.
Agonist-stimulated release of prostacyclin (PGI2) from endothelial cells requires elevation of the concentration of intracellular ionized calcium ([Ca2+]i) above a threshold value, and raised [Ca2+]i provides a sufficient transduction signal to account for the extent of PGI2 production. However, chronic activation of protein kinase C has been reported separately to potentiate PGI2 release, but to depress agonist-induced elevations of [Ca2+]i. We show here that pretreatment with phorbol 12-myristate 13-acetate (PMA) dose-dependently induces PGI2 release over many minutes after a significant lag period without any change in [Ca2+]i. In addition, PMA potentiates the transient release of PGI2 in response to agonists in a complex manner depending on the time of pre-incubation and the concentrations of both PMA and agonist. Concomitant measurement of [Ca2+]i and PGI2 release demonstrates that PMA pretreatment dose-dependently inhibits both the peak [Ca2+]i transient and the subsequent steady-state elevation of [Ca2+]i in response to agonists. Determination of the quantitative [Ca2+]i/PGI2 dose/response relationship, when PGI2 release is driven purely by elevating [Ca2+]i with ionomycin, demonstrates that PMA also enhances the Ca2+-sensitivity of PGI2 release. The observed effects of PMA on PGI2 release can be explained quantitatively by its abilities to lower the threshold [Ca2+]i required for PGI2 synthesis and to depress the peak [Ca2+]i evoked by agonist. We propose that these effects are due respectively to actions of PMA on phospholipase A2 and on a G-protein (Gp) that couples activated receptors to phospholipase C.
Carter
T D
TD
Section of Vascular Biology, MRC Clinical Research Centre, Harrow, Middx., U.K.
Hallam
T J
TJ
Pearson
J D
JD
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D011464:Epoprostenol / Q000378:metabolism*; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate
D011464:Epoprostenol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium