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/10¹¹ 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]PIP₂ ([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 PIP₂ 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 (PIP₂) resynthesis, and not by inhibition of the PLC enzyme.