Canine cardiac sarcolemmal vesicles demonstrate rapid initial Na(+)-Ca2+ exchange activity

To identify a rapid, uninhibited rate of exchange activity, we investigated in canine sarcolemmal vesicles the rapid kinetics of Na(+)-Ca2+ exchange. Sarcolemmal vesicles were incubated in 160 mM NaCl and 20 mM HEPES at 25 degrees C (pH 7.4) and actively loaded with 45Ca2+ for 2 minutes by Na(+)-Ca2+ exchange. After further uptake was inhibited by dilution into 0.15 mM Na(+)-free EGTA, sarcolemmal vesicles were immobilized on a rapid filtration apparatus that allowed millisecond resolution of 45Ca2+ fluxes. In the presence of external NaCl (Na+o) but not other monovalent cations (i.e., K+, Li+), a biphasic pattern of Ca2+ release was observed--an initial brief and rapid rate of Ca2+ release followed by a second slower, prolonged phase of Ca2+ release. Semilogarithmic plots of sarcolemmal Ca2+ content versus time were not linear but were consistent with a biexponential rate of Na+o-induced Ca2+ release during the first several seconds of the exchange reaction. The fast phase of Na+o-stimulated Ca2+ release was several thousand-fold more rapid than that in the absence of Na+o. Both phases of Ca2+ release showed a similar Na+o dependence (Km, approximately 12 mM) with evidence of a positive cooperative effect of Na+. Vmax of the fast and slow phases were approximately 37.0 and approximately 0.76 nmol/mg/sec, respectively. Using rapid-reaction techniques, we demonstrated in the present study that the initial velocity of sarcolemmal Na(+)-Ca2+ exchange activity is greater than previously reported in sarcolemmal vesicles and that this exchange process exhibits complex rate behavior with a biphasic pre-steady state kinetic pattern.