title: Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.
abstract: The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). 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 Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]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 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (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+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL.
authors.forename: J S
authors.initials: JS
authors.lastname: Johansson
authors.forename: L E
authors.initials: LE
authors.lastname: Nied
authors.forename: D H
authors.initials: DH
authors.lastname: Haynes
mesh_terms: D000634:Aminoquinolines; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002413:Cations, Divalent; D002751:Chlortetracycline / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011863:Radioimmunoassay; D013050:Spectrometry, Fluorescence
chemical_list: D000634:Aminoquinolines; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D000242:Cyclic AMP; D000252:Calcium-Transporting ATPases; C035739:Quin2; D002118:Calcium; D002751:Chlortetracycline