Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets

Abstract

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [ 32P] i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol/10 11 cells at 10 −5 M forskolin) and [ 32P]phosphatidylinositol 4-phosphate (Δ[ 32P]PIP) (30% at 10 −7–10 −6 M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [ 32P]PA, ‘overshoots’ in [ 32P]PIP and [ 32P]PIP 2 ([ 32P]phosphatidylinositol 4,5-bisphosphate), and the increase in [ 32P]PI and secretion of ADP+ATP were abolished by forskolin (10 −7 M). Forskolin stimulated total [ 32P]P i uptake in resting platelets (48%), increased 32P incorporation into PIP (110%), and inhibited 32P incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the forskolin-induced stimulation of [ 32P]P i uptake. The changes in radioactive PA, PIP and PIP 2 are regarded as being proportional with their masses in the prelabeled platelets, while the increase in PI (phosphatidylinositol) is regarded as a change in specific radioactivity, and hence 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 ΔPIP and [ 32P]PA production suggests that the PLC reaction is inhibited by elevated cAMP through reduction of substrate (PIP 2) resynthesis, and not by inhibition of the PLC enzyme.