COORDINATED CONTROL OF APICAL CALCIUM INFLUX AND BASOLATERAL CALCIUM EFFLUX IN RABBIT CORTICAL COLLECTING SYSTEM

Transcellular Ca2+ transport in the distal nephron involves passive Ca 2+ influx at the apical membrane, diffusion through the cytosol and ac tive extrusion across the opposing basolateral membrane. The molecular identity of the apical Ca2+ entry step is still elusive, but its regu latory aspects have been analyzed in the present study. To this end, r abbit connecting and cortical collecting tubular cells were cultured o n permeable and transparent supports and the apical Ca2+ influx was de duced from Mn2+ quenching of Ca2+ independent Fura-2 fluorescence, whi le the intracellular Ca2+ concentration ([Ca2+](i)) was measured simul taneously. In parallel experiments, transcellular Ca2+ transport was d etermined isotopically as Ca-45(2+) flux from the apical to basolatera l compartment. Decreasing the apical pH from 7.4 to 5.9 inhibited tran scellular Ca2+ transport by 53 +/- 1%, whereas apical Ca2+ influx was reduced by 39 +/- 7% and [Ca2+](i) decreased by 18 +/- 3%. Reversal of the Na+/Ca2+ exchanger by iso-osmotic replacement of Na+ by N-methyl- D-glucamine in the basolateral compartment resulted in 50 +/- 5% inhib ition of Ca2+ transport, 46 +/- 3% reduction of apical Ca2+ influx and 60 +/- 3% increase in [Ca2+](i). In the absence of basolateral Ca2+, however, this manoeuvre decreased [Ca2+](i) by 21 +/- 8%, while Ca2+ t ransport and apical Ca2+ influx were reduced by the same magnitude as in the presence of Ca2+, that is by 53 +/- 6% and 45 +/- 4%, respectiv ely. Stimulation of adenylyl cyclase with forskolin (10(-5) M) increas ed transcellular Ca2+ transport by 108 +/- 40%, stimulated apical Ca2 influx by 120 +/- 17% and increased [Ca2+](i) by 110 +/- 2%. In concl usion, the apical Ca2+ influx it; regulated by apical pH, intracellula r cAMP and basolateral Na+/Ca2+ exchanger activity, and is coupled in an 1:1 fashion to the rate of transepithelial Ca2+ transport.