Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

Abstract

The in vivo and in vitro effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on calcium uptake by isolated chick duodenal cells were studied. In vivo, 1,25-(OH)2D3 given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion. In vitro, pre-incubation of 1,25-(OH)2D3 with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10(-15) M, was maximal at 10(-13) M, and was diminished at higher (10(-11) M) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D3 analogs was 1,25-(OH)2D3 = 1-(OH)D3 greater than 25-(OH)D3 greater than 1,24,25-(OH)3D3 greater than 24,25-(OH)2D3 greater than D3. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)2D3 was five orders of magnitude greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of calcium uptake; the affinity for calcium (Km = 0.54 mM) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)2D3, in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect on calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)2D3. These findings suggest that the in vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)2D3 induction of calcium transport and specific biochemical correlates.

Alternative perspective on intestinal calcium absorption: proposed complementary actions of Cav1.3 and TRPV6

“…Since the polymers were readily hydrolyzed in vivo, it was assumed that enhancement of Ca 2+ absorption was mediated by the principal breakdown product, glucose. Further reports support the view that glucose enhances transcellular Ca 2+ absorption in rats, 104–107 chicks, 108 and human 109 . The glucose effect occurs in streptozotocin‐diabetic rats, 104 which are vitamin D‐deficient, and in rachitic rats, 78,110 leading the authors to conclude that glucose‐induced Ca 2+ absorption is vitamin D‐independent.…”

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

“…The procedure of Ca 2+ uptake by enterocytes described by Liang et al [17] was used with slight variations. An aliquot of 300 μL of cell suspension (about 5 g of protein/L) was incubated with an uptake solution composed of 140 mmol/L KCl, 10 mmol/L HEPES, 2 mmol/L CaCl 2 (pH 7.4) plus 1.85×10 8 Bq/L of 45 CaCl 2 .…”