ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+ exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of approximately 500 microM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 microM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5'-O-(3-thiotriphosphate), without the need for vanadate, but not by beta,gamma-methyleneadenosine 5'-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis of L-alpha-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 microM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2 increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+ exchanger through a fast phosphorylation chain: a Ca(2+)-independent PIP formation followed by a Ca(2+)-dependent synthesis of PIP2.