MECHANISMS OF CALCIUM-TRANSPORT ACROSS THE BASOLATERAL MEMBRANE OF THE RABBIT CORTICAL THICK ASCENDING LIMB OF HENLE LOOP

Although net Ca2+ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca2+ entry step across the luminal membrane is mediated by Ca2+ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca2+ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca2+([Ca2+]i) with fura-2. The resting [Ca2+]i in this segment was 49.8 +/- 4.5 nmol/l. Neither Na+ removal from the bathing solution nor addition of ouabain (0.1 mmol/1) to the bath increased [Ca2+]i, indicating that a Na+/Ca2+ exchanger in the basolateral membrane may not contribute in any major way to [Ca2+]i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na+/Ca2+ exchanger has been reported. In this segment, Na+ removal from the bath increased cell Ca2+ from 148.6 +/- 6.4 nmol/l to 647.6 +/- 132.0 nmol/l, confirming the documented fact. [Ca2+]i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca2+]i. When the bath pH was made alkaline, [Ca2+]i was also increased. This response was abolished when Ca2+ was eliminated from the bath, indicating that the Ca2+ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca2+]i induced by an alkaline bath was inhibited by increased the bath K+ from 5 nmol/l to 50 mmol/l, suggesting that the Ca2+ entry system is voltage-dependent. However, the pH-dependent [Ca2+]i increase was unaffected by 0.1 - 10 mumol/l nicardipine in the bath. We conclude that Ca2+ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca2+rather than a Na+/Ca2+ exchanger secondarily linked to a Na+, K+ pump.