Role of protein kinases in mediating [(45)ca(2+)] uptake by human platelets.
It has recently been demonstrated that both initiators and inhibitors (viz iloprost) of aggregation stimulate the uptake of [(45)Ca(2+)] by human platelets. Since it was postulated that this calcium uptake reflects changes associated with signal transduction, we investigated the role of cAMP-dependent protein kinases and protein kinase C (PKC) in mediating [(45)Ca(2+)] uptake by washed human platelets. Phorbol myristate acetate (PMA; a PKC activator), sodium fluoride (NaF; a putative G protein activator), ADP and collagen stimulated the uptake of [(45)Ca(2+)] by platelets in dose-dependent manners. The inert phorbol ester, phorbol 4-α-didecanoate had no effect on [(45)Ca(2+)] uptake. PMA-stimulated and NaF-stimulated [(45)Ca(2+)] uptake was inhibited in concentration-dependent manners by the PKC inhibitor, staurosporine. Staurosporine also inhibited [(45)Ca(2+)] uptake when stimulated with collagen, ADP and to a lesser extent by adrenaline. Staurosporine, however, had no effect on [(45)Ca(2+)] uptake when stimulated with calcium ionophore A23187, dibutyryl cAMP or iloprost. The more specific inhibitor of PKC, chelerythrine, inhibited [(45)Ca(2+)] uptake when stimulated by PMA, collagen and adrenaline but not A23187 or dibutyryl cAMP. H8 (a PKA inhibitor) inhibited iloprost- and dibutyryl cAMP-stimulated (but not A23187-stimulated) [(45)Ca(2+)] uptake. These data indicate that [(45)Ca(2+)] uptake is: (1) mediated by PKC when stimulated by proaggregatory agonists and, (2) that cAMP-dependent protein kinase mediated signal transduction involves a calcium uptake component. Thus, these data demonstrate that the [(45)Ca(2+)] uptake elicited by both stimulators and inhibitors of aggregation reflect events associated with signal transduction, possibly at the plasma membrane and not necessarily changes in intracellular calcium (i.e. calcium influx into the cytosol).
Dept Chemical Pathology and Human Metabolism, Royal Free Hospital & School of Medicine(University of London), Pond Street, London, NW3 2QG, UK.
J
J
Gill
J Y
JY
Jeremy
D P
DP
Mikhailidis