Rapid action of 1,25-dihydroxyvitamin D3 on calcium transport in perfused chick duodenum: Effect of inhibitors

Abstract

Transport of ⁴⁵Ca from the lumen to the venous effluent was studied in duodena of normal, vitamin D₃-replete (+D) chicks perfused through the celiac artery with 130 pM 1,25(OH)₂D₃ or vehicle. Administration of actinomycin D 3 h prior to perfusion did not alter the unstimulated transport rate or diminish the response to exogenous 1,25(OH)₂D₃: After 40 min exposure to the seco-steroid, ⁴⁵Ca in the vascular effluent was 140% of control levels. The anti-microfilament agent cytochalasin b and the ionophore monensin, an inhibitor of Golgi function, similarly failed to suppress 1,25(OH)₂D₃-stimulated calcium transport. In pilot studies, Golgi and basal-lateral membrane fractions were prepared from duodenal epithelium of vitamin D-deficient (-D) chicks treated with vehicle or 650 pmol of 1,25(OH)₂D₃ in vivo 2 h, 10 h, or 15 h before sacrifice, as well as from +D birds. Analyses of Golgi fractions for cathepsin B (CB) activity revealed a biphasic response with time, increasing to 200% of -D levels 2 h after 1,25(OH)₂D₃ administration and in equivalent preparations from +D birds. Less pronounced increases in acid phosphatase activity were observed in the same membrane fractions. In basal-lateral membranes, enhanced CB activity was detectable 10 h after 1,25(OH)₂D₃ in vivo, rose to 155% of -D levels at 15 h, and to 245% of controls in fractions from +D birds, whereas acid phosphatase was 75%, 81%, and 125% of controls, respectively, at these times. Levels of CB in whole homogenates from which membranes were isolated rose to 200% of controls at 2 h and 10 h after 1,25(OH)₂D₃, but declined to 112% of controls in +D chicks. Acid phosphatase levels in the same homogenates were lower than or equivalent to control levels. These findings suggested additional perfusion studies in normal birds. Although vascular perfusion with iodoacetate and 1,25(OH)₂D₃, or leupeptin and 1,25(OH)₂D₃, failed to inhibit the effect of the vitamin D metabolite, 1,25(OH)₂D₃-enhanced calcium transport was abolished by addition of 1 mM leupeptin to the lumenal medium, but not by 1 mM pepstatin. The combined data suggest that lysosomes or their hydrolases may be involved in 1,25(OH)₂D₃-mediated transport of calcium across intestinal epithelial cells.