ACTIVE CALCIUM-ABSORPTION IN PRIMARY CULTURES OF CORTICAL COLLECTING DUCT CELLS

Primary cultures of rabbit cortical collecting duct (CCD) cells demons trated accumulation of Ca at the basolateral (BL) side when cultured o n either impermeable or permeable supports. Cell monolayers cultured o n impermeable plastic surfaces absorbed Ca with such avidity that hydr oxyapatite crystals formed. When cultured on a permeable matrix, the c ells demonstrated significant net apical (A) to BL Ca transport agains t a lumen negative transepithelial potential difference. A steady-stat e BL/A [Ca] ratio of 120 developed across monolayers in 24 h on days 6 through 8 postseeding. Initial rates of unidirectional Ca-45 fluxes o n days 6 through 8 indicated a negligible BL to A flux (5.4 +/- 2.6 nm ol . h-1 . cm-1) compared with A to BL Ca-45 flux (99.4 +/- 19.4 nmol . h-1 . cm-2). Parathyroid hormone applied to the BL side had no signi ficant effect on either unidirectional Ca-45 flux, but the second mess enger analog, 8-bromoadenosine cyclic monophosphate, increased the A t o BL flux by 65%. Inhibiting the Na+ - K+ ATPase with ouabain (10(-4) M) reduced the A to BL flux by 77%; however, a significant net A to BL flux still remained. Apical addition of amiloride (2 x 10(-5) M) did not affect either unidirectional Ca-45 flux. In addition, the inorgani c Ca channel blockers Ni2+ (100 muM and 1 mM), La3+ (100 muM and 1 mM) , and Cd2+ (20 and 50 muM) did not significantly inhibit either unidir ectional Ca-45 flux. These results demonstrate that CCD monolayers act ively absorb Ca and this can be stimulated by cyclic AMP, raising the possibility that apical Ca entry does not involve amiloride-sensitive channels, or typical Ca channels.