Phospholipid metabolism in bradykinin-stimulated human fibroblasts. I. Biphasic formation of diacylglycerol from phosphatidylinositol and phosphatidylcholine, controlled by protein kinase C.

Stimulation of human fibroblasts with bradykinin (BK) results in the generation of diacylglycerol (DG) and phosphatidic acid (PA). Prelabeling of the cells with [3H]arachidonic acid and [14C]palmitic acid allowed us to quantitate these lipid second messengers and to determine their origin, i.e. DGi and PAi from 3H-enriched inositol phospholipids, and DGc and PAc from 14C-enriched phosphatidylcholine, respectively. BK elicited a biphasic DG response: a first peak at 10-15 s, containing DGi, followed by a second peak at 10-30 min, which is mainly DGc. The latter did not result from de novo lipid biosynthesis. BK also generated free [3H]arachidonate and, to a lesser extent, mono[3H]arachidonoylglycerol. BK stimulation rapidly increased PAi, much more so than PAc, suggesting that DGi, rather than DGc, is the preferred substrate for the enzyme DG kinase. Short pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) abolished the BK-induced breakdown of phosphoinositides, but did not affect the second-phase DGc level. PMA alone also elicited DGc formation, but more slowly, suggesting a different mechanism. Down-regulation of protein kinase C (PKC) by long term treatment with phorbol ester, prior to BK stimulation, resulted in (i) enhanced DGi and decreased PAi formation, suggesting that DG kinase activity is positively controlled by PKC; (ii) the unexpected manifestation of rapidly formed DGc; (iii) no change in the DGc levels obtained after 30-min BK stimulation, but complete suppression of PMA-induced DGc formation. In contrast, two inhibitors of PKC, staurosporin and 1-O-hexadecyl-2-O-methylglycerol, inhibited both BK- and PMA-induced DGc formation at 30 min, leaving the rapid response towards BK unaffected. The results suggest that the BK-induced rapid and later-phase DG formation and the PMA-induced DG formation are differentially controlled by PKC via mechanisms that differ in the susceptibility to down-regulation or inhibition of PKC.