Cyclic AMP stimulates Ca 2+-ATPase-mediated Ca 2+ extrusion from human platelets

Abstract

The effect of cAMP on active Ca 2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca 2+ in the cytoplasmic compartment ([Ca 2+] cy1). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca 2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca 2+] cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147–163). Forskolin, at a maximally effective concentration of 10 μM, was shown to stimulate Ca 2+ extrusion by increasing by a factor of 1.6 ± 0.5 the V m of a saturable component, previously identified with a Ca 2+ Mg 2+-ATPase located in the plasma membrane. Neither the K m (80 nM) or Hill coefficient (1.7 ± 0.3) of the Ca 2+-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na +/Ca 2+ exchanger) over the [Ca 2+] cyt range examined (50–1500 nM), Dibutyryl-cAMP (Bt 2-cAMP, 1 mM) stimulated the Ca 2+ Mg 2+-ATPase component of Ca 2+ extrusion by a factor of 2.0 ± 0.6. Separate experiments showed that 10 μM forskolin reduces the resting [Ca 2+] cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in V m of the pump, countered by a 37–74% increase in the rate constant for passive Ca 2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca 2+] cyt and (b) increasing the rate of Ca 2+ extrusion after the initial influx or triggered release event.