Contribution of Na+‐Dependent and ATP‐Dependent Ca2+ Transport to Smooth Muscle Calcium Homeostasis a

Controversy has been longstanding as to the importance of Na+ -Ca2+ exchange in smooth muscle calcium homeostasis.' Many investigators discounted the importance of this transport system because of its low calcium affinity and assumed that steady-state calcium regulation is dominated by ATP-dependent Ca'+transport. Few studies have measured Naf -dependent Ca2+ flux at submicromolar calcium levels, however. Thus the present studies examined the characteristics of Na+-dependent and ATP-dependent calcium uptake in plasma membrane vesicles derived from pig stomach muscle. Membrane vesicles enriched 15-20-fold for plasma membrane markers were prepared from pig gastric smooth muscle as described previously.' Na+-Ca2+ exchange activity was assessed after 1, 5, or 15 seconds using a standard filtration assay in which Na+-loaded vesicles (preequilibrated with 130 mM NaCl) were diluted either into Na+-containing buffer (no gradient) or into K+-containing buffer (with gradient). Studies were carried out at 12°C and Na+-dependent uptake was defined as the difference in uptake in the presence versus the absence of a transmembrane Na+ gradient. Uptake of label occurred rapidly, plateauing within 5-10 seconds and the Na+dependent component was relatively small, 15-20% of the total uptake. However, this component appeared to reflect true vesicular uptake since it was inhibited by 20 p M dichlorobenzamil (65% reduction, p < 0.01) and by 1 pM monensin (100% reduction,p < 0.001), and since the label was released on reversal of the transmembrane Na+ gradient. Na-dependent Ca2+ uptake was observed over a wide range of Ca" concentrations. The rate of Na+-dependent Ca2+ accumulation increased with increasing Ca2+ levels between 1 and 100 pM, and the apparent KmC,-,) was 50 pM. Between 100 and lo00 nM Ca2+, Na+-dependent Ca2+ uptake showed little Ca2+dependency. However, the uptake observed between pCa 6.8 and 5.0 was appreciable, averaging -6 nmoles/ (min-mg protein) (FIG. 1). This transport was similar in magnitude to that observed for ATP-dependent Ca2+uptake; and as evident in FIGURE 2, Na+-dependent uptake constituted a significant portion of total calcium transport over a wide range of calcium levels. These findings, like those reported recently for bovine aortic smooth m ~ s c l e , ~