ELEVATION OF CYCLIC-AMP DECREASES PHOSPHOINOSITIDE TURNOVER AND INHIBITS THROMBIN-INDUCED SECRETION IN HUMAN PLATELETS

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-sp ecific phospholipase C (PLC) by ill-defined mechanism(s). Signal trans duction steps downstream of PLC are inhibited by elevated cAMP, sugges ting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [P -32](i)-prelabeled platelets, forskolin increased intracellular cAMP ( 104 nmol/10(11) cells at 10(-5) M forskolin) and [P-32]phosphatidylino sitol 4-phosphate (Delta[P-32]PIP) (30% at 10(-7)-10(-6) M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [P-32]PA, ' overshoots' in [P-32]PIP and [P-32]PIP2 ([P-32]phosphatidylinositol 4, 5-bisphosphate), and the increase in [P-32]PI and secretion of ADP+ATP were abolished by forskolin (10(-7) M). Forskolin stimulated total [P -32]P-i uptake in resting platelets (48%), increased P-32 incorporatio n into PIP (110%), and inhibited P-32 incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the for skolin-induced stimulation of [P-32]P-i uptake. The changes in radioac tive PA, PIP and PIP2 are regarded as being proportional with their ma sses in the prelabeled platelets, while the increase in PI (phosphatid ylinositol) is regarded as a change in specific radioactivity, and hen ce in its synthesis. The results suggest that cAMP elevation inhibits the flux in the polyphosphoinositide cycle through both inhibition of PIP 5-kinase and PI synthesis. The inverse relation between forskolin- produced Delta PIP and [P-32]PA production suggests that the PLC react ion is inhibited by elevated cAMP through reduction of substrate (PIP2 ) resynthesis, and not by inhibition of the PLC enzyme. (C) 1998 Elsev ier Science B.V. All rights reserved.