Sodium-Dependent Inactivation of NCX1.3: Aortic Smooth Muscle Cells Versus Transfected CHO Cells

Abstract

Despite the importance of Na/Ca exchange (NCX) activity in Ca homeostasis in blood vessels, there have been few detailed studies of the regulation of NCX activity in smooth muscle cells. Here we investigated the regulation of NCX by allosteric Ca activation and Na-dependent inactivation in rat aortic smooth muscle cells (ASMC) and compared the results with those of the smooth muscle isoform NCX1.3 expressed in CHO cells. Fura-2 loaded ASMC or transfected CHO cells were treated with ATP + thapsigargin to release Ca and prevent subsequent Ca re-accumulation in internal stores. Reverse exchange activity was initiated by applying 0.1mM Ca in Li-PSS or K-PSS. In both ASMC and transfected CHO cells treated with 1 mM ouabain, Ca uptake occurred following a 10-20sec lag period attributable to the positive feedback of allosteric Cai activation. To examine the Na-dependence of NCX activity, cells were treated with gramicidin and preincubated in 10-140 mM Na-PSS before activity was initiated by applying 0.1 mM CaCl2. In ASMC, NCX activity peaked at 20 mM Na but declined at higher Na concentrations; essentially no Ca uptake was seen at 140 mM Na. In contrast, robust activity was seen throughout that entire Na concentration range in transfected CHO cells. At 20 mM Na, Ca uptake in ASMC increased to a peak value and then declined sharply. After removing Ca with EGTA in 20 mM Na, subsequent pulses with 0.1 mM Ca revealed no activity until a 10 min recovery period had occurred. In contrast, NCX activity in the transfected CHO cells recovered within 30 s after EGTA treatment. We conclude that NCX activity in ASMC is much more susceptible to inactivation at high concentrations of Na (Na-dependent inactivation) than in transfected CHO cells.