PHARMACOLOGICAL DIFFERENTIATION BETWEEN INTRACELLULAR CALCIUM-PUMP ISOFORMS

We studied the Ca2+-ATPase isoforms of sarco/endoplasmic reticulum (SE RCA) derived from cerebellum, cardiac muscle, and skeletal muscle, The Mg2+ dependence varied among the three enzyme preparations. The Ca2transport in skeletal muscle vesicles, but not in cerebellar or cardia c vesicles, was activated by free Mg2+ concentrations varying from 0.1 to 0.3 mM. Concentrations of Mg2+ of >1 mM inhibited Ca2+ transport i n all three vesicle preparations but with more pronounced effect in ce rebellar and cardiac vesicles. At 10-80 mu M, trifluoperazine activate d Ca2+ uptake in cerebellar and cardiac vesicles but not in skeletal m uscle vesicles. The activation was due to an increase in the coupling ratio between Ca2+ transport and ATP hydrolysis and was observed only in the presence of ATP concentrations of >100 mu M. The Ca2+ transport in all three vesicle preparations was inhibited by trifluoperazine co ncentrations of >100 mu M. The inhibition promoted by trifluoperazine was prevented by the addition of dimethylsulfoxide (10% v/v) to the me dium. The Ca2+ efflux from loaded vesicles was increased by arsenate a nd even more by trifluoperazine. In skeletal muscle vesicles, the effl ux promoted by arsenate was several-fold faster than that promoted in vesicles derived from cerebellum or cardiac muscle. In skeletal muscle , the enhancement of Ca2+ efflux promoted by both arsenate and trifluo perazine was antagonized by thapsigargin, Ca2+, Mg2+, and K+. These ag ents partly antagonized the enhancement of Ca2+ efflux promoted by tri fluoperazine in cardiac vesicles but had little or no effect in the ce rebellar vesicles. Finally, Mg Pi and Mg ATP, the two substrates that phosphorylate the Ca2+-ATPase, antagonized the effect of trifluoperazi ne in all of the preparations tested. The concentration of ATP needed was in the same range as that of the second K-m value for ATP (50-300 mu M) of the SERCA isoforms. The results indicate that the effect of t he drugs on the cytosolic Ca2+ homeostasis may vary depending on the t arget tissue.