STIMULATION OF CALCIUM-TRANSPORT BY AMILORIDE IN MOUSE DISTAL CONVOLUTED TUBULE CELLS

This study examined the mechanism by which amiloride dissociates Na and Ca transport in distal convoluted tubules. Control rates of Na uptake averaged 288 nmol/(min mg protein) and were inhibited 39% by mu M amiloride. Amiloride had no effect on Cl uptake. Resting membrane voltage, measured with the voltage-sensitive dye DiOC(6) (3), averaged -70 mV. Amiloride hyperpolarized cells in a reversible manner by 18 mV. Control rates of Ca uptake averaged 2.8 nmol/(min mg protein) and increased by 39% in the presence of amiloride. Alterations of intracellular Ca activity were measured in single cells loaded with Fura2-AM. Control intracellular Ca activity averaged 100 nM. Amiloride increased intracellular Ca activity in a concentration-dependent manner to a maximum of 330 nM at mu M amiloride. Amiloride analogues ethylisopropyl amiloride (EIPA) and dimethylbenzamil (DMB), which preferentially block Na/H and Na/Ca exchange, respectively, had no effect on Na or Ca influx or on intracellular Ca activity. The dihydropyridine Ca channel blocker nifedipine inhibited amiloride-stimulated Ca uptake and the rise of intracellular Ca activity but had no effect on membrane voltage. It is concluded that amiloride blocks Na entry mediated by Na channels. Inhibition of Na entry results in membrane hyperpolarization, which activates Ca entry by dihydropyridine-sensitive Ca channels.