Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system.
Transcellular Ca2+ transport in the distal nephron involves passive Ca2+ influx at the apical membrane, diffusion through the cytosol and active extrusion across the opposing basolateral membrane. The molecular identity of the apical Ca2+ entry step is still elusive, but its regulatory aspects have been analyzed in the present study. To this end, rabbit connecting and cortical collecting tubular cells were cultured on permeable and transparent supports and the apical Ca2+ influx was deduced from Mn2+ quenching of Ca2+ independent Fura-2 fluorescence, while the intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously. In parallel experiments, transcellular Ca2+ transport was determined isotopically as 45Ca2+ flux from the apical to basolateral compartment. Decreasing the apical pH from 7.4 to 5.9 inhibited transcellular 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% inhibition 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+ transport and apical Ca2+ influx were reduced by the same magnitude as in the presence of Ca2+, that is by 53 +/- 6% and 45 +/- 4%, respectively. Stimulation of adenylyl cyclase with forskolin (10(-5) M) increased transcellular Ca2+ transport by 108 +/- 40%, stimulated apical Ca2+ influx by 120 +/- 17% and increased [Ca2+]i by 110 +/- 2%. In conclusion, the apical Ca2+ influx is regulated by apical pH, intracellular cAMP and basolateral Na+/Ca2+ exchanger activity, and is coupled in an 1:1 fashion to the rate of transepithelial Ca2+ transport.
Raber
G
G
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Willems
P H
PH
Lang
F
F
Nitschke
R
R
van Os
C H
CH
Bindels
R J
RJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D018929:Cell Culture Techniques; D016257:Fura-2; D017136:Ion Transport; D008345:Manganese / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009399:Nephrons / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits
D015220:Calcium Channels; D008345:Manganese; D002118:Calcium; D016257:Fura-2