Sarcolemmal calcium transport and drug action

Sarcolemmal-rich microsomal fractions were isolated from rabbit ventricular muscle differential centrifugation and discontinuous sucrose gradient techniques. The fractions were characterized in terms of calcium binding, enzymatic activity, gel electrophoresis, and ultrastructure. Calcium binding and the initial rates of binding were assayed using 45Ca2+ and Millipore filtration in a reaction mixture containing 100 mM KCl, 5 mM MgCl2, 50 mM Tris, Ca2+, and either nitrilotriacetic acid (NTA) or adenosine triphosphate (ATP). The free calcium concentrations of the media were calculated using an ion complexes-in-solutions computer program with either an NTA or an ATP calcium buffering system. The Ca2+-accumulating activity of these sarcolemmal preparations involved more than one type of binding site. One type of site appeared to be rapidly saturable; calcium accumulation at these sites was energy independent. The second type of site required ATP for binding. Calcium accumulation at both sites was temperature and pH sensitive. These results are discussed in terms of the interaction of inotropically active drugs with the different types of binding sites.