The Ca(2+)-ATPase isoforms of platelets are located in distinct functional Ca2+ pools and are uncoupled by a mechanism different from that of skeletal muscle Ca(2+)-ATPase.
Vesicles derived from the dense tubular system of platelets possess a Ca(2+)-ATPase that can use either ATP or acetyl phosphate as a substrate. In the presence of phosphate as a precipitating anion, the maximum amount of Ca2+ accumulated by the vesicles with the use of acetyl phosphate was only one-third of that accumulated with the use of ATP. Vesicles derived from the sarcoplasmic reticulum of skeletal muscle accumulated equal amounts of Ca2+ regardless of the substrate used. When acetyl phosphate was used in platelet vesicles, the transport of Ca2+ was inhibited by Na+, Li+, and K+; in sarcoplasmic reticulum vesicles, only Na+ caused inhibition. When ATP was used as substrate, the different monovalent cation had no effect on either sarcoplasmic reticulum or platelet vesicles. The catalytic cycle of the Ca(2+)-ATPase is reversed when a Ca2+ gradient is formed across the vesicle membrane. The stoichiometry between active Ca2+ efflux and ATP synthesis was one in platelet vesicles and two in sarcoplasmic reticulum vesicles. The coupling between ATP synthesis and Ca2+ efflux in sarcoplasmic reticulum vesicles was abolished by arsenate regardless of whether the vesicles were loaded with Ca2+ using acetyl phosphate or ATP. In platelets, uncoupling was observed only when the vesicles were loaded using acetyl phosphate. In both sarcoplasmic reticulum and platelet vesicles, the effect of arsenate was antagonized by thapsigargin (2 microM), micromolar Ca2+ concentrations, P(i) (5-20 mM), and MgATP (10-100 microM). Trifluoperazine also uncoupled the platelet Ca2+ pump but, different from arsenate, this drug was effective in vesicles that were loaded using either ATP or acetyl phosphate. Trifluoperazine enhanced Ca2+ efflux from both sarcoplasmic reticulum and platelet vesicles; thapsigargin, Ca2+, Mg2+, or K+ antagonized this effect in sarcoplasmic reticulum but not in platelet vesicles. The data indicate that the Ca(2+)-transport isoforms found in sarcoplasmic reticulum and in platelets have different kinetic properties.
Engelender
S
S
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Brasil.
Wolosker
H
H
de Meis
L
L
D001149:Arsenates / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D013379:Substrate Specificity; D014268:Trifluoperazine / Q000494:pharmacology
D001149:Arsenates; D007527:Isoenzymes; D014268:Trifluoperazine; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D000252:Calcium-Transporting ATPases; C025657:arsenic acid; D002118:Calcium