2 VITAMIN-D3-DEPENDENT CALCIUM-BINDING PROTEINS INCREASE CALCIUM REABSORPTION BY DIFFERENT MECHANISMS .1. EFFECT OF CABP-28K

Several clearance and micropuncture studies suggest that vitamin D deficiency decreases calcium (Ca2+) reabsorption by the nephron. In confirmation of these observations, we reported recently that in vitamin D depleted rabbits, Ca2+ transport by both the luminal and the basolateral membranes of the distal tubule was significantly diminished. It is also in the distal tubule that vitamin D stimulates the synthesis of two calcium binding proteins (CaBP) with molecular weights of 28K and 9K. The purpose of this study was to investigate the role of the CaBP 28K in the regulation of Ca2+ transport by the two polar membranes of the distal nephron of the rabbit. Results were compared to those obtained with the corresponding membranes of the proximal tubule. The basolateral and luminal membranes were purified from proximal and distal tubule suspensions separately. Ca2+ uptake was measured by the rapid filtration technique. Preloading the luminal membrane vesicles from proximal and distal tubules with 3 muM CaBP 28K strongly enhanced the initial Ca2+ uptake by the two membranes. When the vesicles, preloaded with CaBP 28K and incubated with 0.5 mm Ca2+, were abruptly diluted in a medium containing 10 muM A 23187 and 2 mm EGTA, the release of Ca2+ was slower than with control vesicles, suggesting that the excess of transported Ca2+ was bound inside the vesicles. The effect of CaBP 28K was dose-dependent with a half-maximal effect at 10(-6) m and a maximal effect at 5 x 10(-6) m. Kinetic analysis of the Ca2+ uptake by the control and the CaBP 28K treated vesicles revealed that the CaBP 28K stimulates exclusively the high affinity system of Ca2+ transport in the distal luminal membrane, increasing the V(max) from 0.36 +/- 0.05 to 0.66 +/- 0.13 pmol/mug/10 seconds (P < 0.001) and leaving the Km Ca2+ unchanged. In contrast, the preincubation of basolateral membrane vesicles from both the proximal and distal tubules with CaBP 28K in the cis-position did not influence the ATP-dependent nor the Na+-dependent Ca2+ uptakes, nor did it influence the uptake of 0.5 mm Ca2+ by these membranes when in trans-position. It is concluded that CaBP 28K, directly or indirectly, participates in the regulation of Ca2+ transport by the luminal membranes. We speculate that the effect of vitamin D in vivo is demonstrable in the distal tubule exclusively because the synthesis of the vitamin D-dependent CaBP 28K occurs only in distal tubules.