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Previously we reported that uptake of Ca2+ in cells isolated from rat duodenum declined in senescence. In this paper we examined the possible mechanisms for this age-related defect. Duodenal vitamin D-dependent calcium-binding protein decreased steadily from 3-12 months (mo), followed by a minimal decline at 24 mo. On the contrary, Ca2+ uptake was not different in 3-, 6-, and 12-mo-old rats. A significant decline of Ca2+ uptake was observed at 24 mo. ATP contents in duodenal cells from 6- and 24-mo-old rats were not different. This suggests that the metabolic status of the duodenal cells was not the cause of the change in Ca2+ uptake. Ca2+ uptake activity was significantly lower in brush border membrane vesicles isolated from 24-mo-old rats than in those from 6-mo-old rats. The decrease in Ca2+ uptake activity in old rats was not due to a change in the Ca2(+)-binding capacity of the membranes. Kinetic analysis shows that the Vmax, the apparent maximum uptake capacity of membrane vesicles, decreased in senescent rats, whereas the Km, the apparent affinity to Ca2+, was unchanged. Since duodenal Ca2+ influx at the brush border was regulated by 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], we tested the effect of 1,25-(OH)2D3 administration on the uptake activity in isolated membrane vesicles. After 1,25-(OH)2D3 treatment, Ca2+ uptake activity in brush border membranes prepared from senescent rats was only slightly lower than that in membranes from adult rats. We conclude that the decline in the influx of Ca2+ at the brush border membrane was the main cause of the decrease in duodenal Ca2+ uptake activity in aging. This defect was probably due to the low serum 1,25-(OH)2D3 concentration and not the result of impaired response to 1,25-(OH)2D3.

Renal cells isolated from vitamin D-deficient chicks had an increased Na+-dependent phosphate uptake when preincubated with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Phosphate uptake in the absence of Na+ and methyl alpha-glucoside uptake dependent on Na+ were not affected. Phosphate uptake was stimulated 15% by 0.010 pM 1,25-(OH)2D3. Maximal enhancement of 30% was obtained with 100 pM. The uptake when fully stimulated by preincubation in vitro approximated the uptake of cells isolated from chicks that were previously repleted with 1,25-(OH)2D3 in vivo. Cells from repleted chicks were not stimulated additionally when preincubated with 1,25-(OH)2D3 in vitro. The increase in phosphate uptake could be measured after a 1-hr preincubation period; full response required at least 2 hr. Phosphate uptake induced by 1,25-(OH)2D3 was blocked by cycloheximide and actinomycin D. Enhancement of phosphate uptake was relatively specific for the 1,25-(OH)2D3 analog of vitamin D3. The potency order was 1,25-(OH)2D3 greater than 25-(OH)D3 = 1-(OH)D3 greater than 24,25-(OH)2D3 greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of the phosphate uptake system; the affinity for phosphate was unaffected. 3H-Labeled 1,25-(OH)2D3 was taken up by the isolated renal cells. It was estimated that the stimulation of phosphate uptake might be initiated by very few molecules of 1,25-(OH)2D3 per cell. It is proposed that 1,25-(OH)2D3 contributes importantly to the mechanisms by which phosphate transport is regulated in the kidney.

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.

The in vitro incubation of chick renal cells with parathyroid hormone (PTH) resulted in the inhibition of Na+-dependent phosphate uptake when the cells were isolated from 1,25-dihydroxycholecalciferol 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]-repleted chicks but not when the cells came from vitamin D-deficient animals. Na+-independent phosphate and Na+-dependent alpha-methylglucoside uptakes were not affected by PTH and the vitamin D status of the bird. The activation of chick renal cell adenylate cyclase by PTH was significantly blunted when the enzyme was from vitamin D-deficient animals relative to the activation of the enzyme from repleted cockerels. This alteration was due to a change in maximum velocity of the system rather than an effect on the affinity for hormone. The response of adenylate cyclase to other hormones, e.g., prostaglandin E2, and activators, e.g., 5' -guanylyl-imidodiphosphate and forskolin, was not affected by the vitamin D status of the animal. PTH had little effect in activating protein kinase in cells from vitamin D-deficient chicks. In cells from vitamin D-sufficient birds, PTH caused a fourfold increase in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Dibutyryl cAMP inhibited Na+-dependent phosphate uptake by cells from 1,25-(OH)2D3-repleted animals, but the cyclic nucleotide had no effect on phosphate uptake in cells from vitamin D-depleted chicks. This finding suggests that the loss of PTH receptor sites known to be concomitant with the secondary hyperparathyroidism associated with vitamin D deficiency is only a partial explanation for the failure of PTH to inhibit phosphate uptake in cells from vitamin D-deficient animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal cells from Vitamin D-deficient and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-repleted chicks were isolated by a collagenase-hyaluronidase procedure. Exclusion of trypan blue and respiratory measurements indicate that the cells were functionally intact and metabolically active. The uptakes of phosphate and alpha-methylglucoside were stimulated markedly by Na+ in the extracellular medium. Phosphate uptake in the presence of Na+ was saturable with respect to phosphate concentration; half-maximal activity was obtained with approximately 0.2 mM. Three hours after 1,25-(OH)2D3 was injected into vitamin D-deficient chicks the Na+-dependent phosphate uptake by the isolated cells had increased about 40%, i.e., 2.00 compared with 1.44 nmol.min-1.mg protein-1. Phosphate uptake in the presence of K+ in the extracellular medium and alpha-methylglucoside uptake in the presence or absence of Na+ were unchanged. In a secondary response found 17 h after 1,25-(OH)2D3 injection, Na+-dependent phosphate uptake decreased. Serum concentrations of phosphorus and calcium were not measurably changed in the 3-h repleted bird, but both levels were increased 17 h after treatment. Administration of phosphate into vitamin D-deficient chicks, so that the serum concentration of phosphorus was raised to that of the 17-h 1,25-(OH)2D3 repleted animal, effected a comparable decrease in phosphate uptake. Serum calcium levels were not altered by this treatment. The actions of parathyroid hormone in stimulating adenylate cyclase and in inhibiting phosphate uptake were notably blunted in the vitamin D-deficient chick. Sensitivity to parathyroid hormone was not restored until several days after 1,25-(OH)2D3 repletion. These findings suggest that the initial response to 1,25-(OH)2D3, to increase renal phosphate uptake, and the secondary response, to decrease phosphate uptake, were by parathyroid hormone-independent processes. The results also indicate that the isolated renal cell represents an excellent model for studying the mechanism by which 1,25-(OH)2D3 regulates phosphate transport in the kidney.

Uptake of Ca2+ in cells isolated from rat duodenum declined in the senescent rats. This age-related change was not due to an alteration in the rate of Ca2+ efflux or in the size of the cell. The decrease appeared specific, as alpha-methyl glucoside uptake was not altered. Cell population, as monitored by sucrase activity for villus cells, was not different between duodenal cells isolated from 6- and 24-month-old rats. Kinetic analysis shows the Vmax, the apparent maximum uptake capacity, decreased in the cells from senescent rats whereas the Km, the apparent affinity to Ca2+, was unchanged. Serum levels of 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] were determined as a function of age; the levels of 25OHD were not significantly different in 3-, 6-, 12-, and 24-month-old rats. On the other hand, serum 1,25-(OH)2D decreased throughout the age range studied. Since duodenal Ca2+ uptake is closely regulated by 1,25-(OH)2D3, we tested the hypothesis that low serum 1,25-(OH)2D in the senescent rats may have contributed to the decline in duodenal Ca2+ uptake. In vivo administration of 1,25-(OH)2D3 to senescent rats significantly enhanced Ca2+ uptake activity in the isolated duodenal cells. After 1,25-(OH)2D3 treatment, Ca2+ uptake activity in cells isolated from senescent rats was only slightly less than that in cells from adult rats. We conclude that duodenal Ca2+ uptake declined in the senescent rats, and this age-related change was most likely due to the low serum level of 1,25-(OH)2D and not the result of a decrease in any duodenal response to 1,25-(OH)2D3.