Hector F. De Luca

1,25-Dihydroxyvitamin D3 regulates the synthesis of nerve growth factor in primary cultures of glial cells

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) on nerve growth factor (NGF) synthesis was investigated in primary cultures of astrocytes prepared from brain of neonatal rats. 1,25-(OH)2 D3 elicited a dose-dependent increase of NGF mRNA with a maximal effect at 10(-7) M, which persisted for at least 48 h. Northern blot analysis revealed an expression of the vitamin D3 receptor (VDR) gene in primary glial cells. Treatment of cells with 1,25-(OH)2 D3 led to an increase in the VDR mRNA levels. Similar results were obtained in C6 glioma cells. Exposure of primary glial cells to 10(-8) M 1,25-(OH)2 D3 caused only a 2-fold increase of the levels of cell-secreted NGF after 3 days of treatment. However, a 5-fold increase was observed three days after a second addition of vitamin D3. Likewise, a pretreatment with lower doses of hormone such as 10(-10) M or 10(-9) M enhanced the responsiveness of the cells to a 24 h treatment with 10(-8) M hormone. It appears, therefore, that the duration of the treatment influences the level of synthesis of NGF, possibly as a consequence of the increase of the VDR gene expression. The specificity of 1,25-(OH)2 D3 is supported by the fact that a concentration of 10(-7) M of an another vitamin D3 metabolite, 24,25-(OH)2 D3, had no effect on NGF synthesis. Several lines of evidence indicate that astrocytes constitute the major cell type responsive to 1,25-(OH)2 D3 in primary cultures of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of vitamin D deficiency on skeletal development during early growth in the rat

Abstract To define the role of vitamin D in early development, female weanling rats were grown to maturity on a vitamin D-deficient diet and mated with normal males. At Day 20 of pregnancy the weight and total body calcium of fetuses were determined. At various times after parturition, pups were sacrificed. Plasma samples were analyzed for calcium and phorphorus, and femurs were characterized as to volume, dry weight, ash weight, and total calcium. The results indicate that vitamin D deficiency with its accompanying hypocalcemia does not impair placental transfer of calcium nor weight gain of the fetus. Vitamin D deficiency does appear to increase calcium accumulation in the fetus. After parturition vitamin D is functional in maintaining a normocalcemia as early as 3 days postpartum and its importance increases with age of the neonate. Bone mineralization is clearly disrupted by Day 14 as judged by calcium content per unit bone volume and the severity of the defect increases with age. Both vitamin D and normal concentrations of calcium and phosphorus appear to be essential for proper skeletal development during early growth postpartum.

Intestinal calcium transport: Evidence for two distinct mechanisms of action of 1,25-dihydroxyvitamin D3

Abstract The response of the small intestine in the vitamin D-deficient rat to a single intrajugular injection of 1,25-dihydroxyvitamin D 3 has been studied. The time course of 1,25-dihydroxyvitamin D 3 -induced transport suggests that two separate responses occur. The first or initial response reaches a maximum at 6 h after 1,25-dihydroxy vitamin D 3 administration, decays, and is effectively gone by 12 h postinjection. This response does not appear to be associated with alkaline phosphatase activity. The second or late response first appears roughly 12 h after dosing, reaches a maximum at 24 h, and remains elevated for up to 72 h. This response is accompanied by an elevation of alkaline phosphatase activity and appears to be mediated through the action of 1,25-dihydroxyvitamin D 3 on the absorptive cell during its normal differentiation and migration up the villus.

The role of vitamin D and intestinal phytase in the prevention of rickets in rats on cereal diets.

Abstract A study with rats of the role played by vitamin D in overcoming the rachitogenic properties of high-Ca phytic acid containing diets has shown that phytic acid in a low-Ca cereal ration was hydrolyzed almost completely even when vitamin D was absent. On the other hand, when the Ca content of the ration was increased, the hydrolysis of phytate was decreased. Under these conditions the addition of vitamin D increased the hydrolysis. The phytase and alkaline phosphatase activity of fecal and intestinal extracts from rats, whether they were fed cereal or noncereal, rachitogenic or nonrachitogenic rations, was always increased when vitamin D was given. However, no consistent correlation in a quantitative sense was found between the degree of hydrolysis of dietary phytate and enzyme activity, nor with the severity of rickets. The increased hydrolysis of phytic acid which is effected by the addition of vitamin D to high-Ca cereal diets did not liberate sufficient P in the inorganic form to account by itself for the prevention of rickets. Although less phytate was hydrolyzed by rachitic rats, the inorganic P in the feces was increased due to a decrease in its absorption. It therefore appears that the antirachitic action of vitamin D with cereal diets is due primarily to an improved utilization of inorganic P.

Calcitroic acid: Biological activity and tissue distribution studies

Abstract Calcitroic acid was recently identified as a major metabolite of 1,25-dihydroxyvitamin D 3 (Esvelt, Schnoes, and DeLuca, Biochemistry 18 , 3977, 1979). The metabolite was found to have little, although significant, activity in healing rickets, and causing bone mineral mobilization but elicited no significant elevation in intestinal calcium transport. The compound showed little affinity for either the serum 25-hydroxyvitamin D binding protein or the intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3 . Various tissues of the rat were examined for the presence of calcitroic acid following a 120-ng dose of 1,25-dihydroxy-[3α- 3 H]vitamin D 3 . The metabolite was detected in liver, intestinal mucosa, kidneys, and blood with livers and mucosa containing the highest concentrations. In each of these tissues the calcitroic acid content increased during the period between 4 and 12 h after the dose. The presence of calcitroic acid in femurs was indicated but could not be confirmed. Bile duct cannulation reduced but did not abolish the intestinal calcitroic acid content. In addition to calcitroic acid, other polar metabolites of 1,25-dihydroxyvitamin D 3 were detected in these experiments.

24,24-Difluoro-25-hydroxyvitamin D3-enhanced bone mineralization in rats. Comparison with 25-hydroxyvitamin3 and vitamin D3.

Abstract The biological activity of 24,24-difluoro-25-hydroxyvitamin D 3 was assessed using elevation of serum phosphorus and healing of rickets of vitamin D-deficient rats. Various levels of 24,24-difluoro-25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 3 were administered daily for 2 weeks in the dose range of 6.5 to 3250 pmol after feeding rats a low phosphorus, vitamin D-deficient diet for 3 weeks. Vitamin D 3 was concurrently tested at dose levels of 650 and 3250 pmol. 24,24-Difluoro-25-hydroxyvitamin D 3 is approximately equipotent with 25-hydroxyvitamin D 3 in stimulation of growth, mineralization of rachitic bone, and elevation of serum inorganic phosphorus. Radiological manifestations of rickets were also equally improved by 24,24-difluoro-25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 3 . Compared with vitamin D 3 , these compounds were approximately 5 to 10 times more active in mineralization using rats on a low phosphorus, vitamin D-deficient diet. The functional role, if any, for 24-hydroxylated vitamin D compounds, such as 24,25-dihydroxyvitamin D 3 , therefore remains obscure. It appears that vitamin D compounds that cannot be 24-hydroxylated evoke no disorder in bone mineralization.

1α,25-Difluorovitamin D3: An inert vitamin D analog☆

Abstract 1α,25-Difluorovitamin D 3 has been synthesized by reacting 1,25-dihydroxyvitamin D 3 -3-acetate with diethylaminosulfurtrifluoride followed by hydrolysis. Retention of configuration of the fluoro group in this reaction was demonstrated by physical studies using 1α-fluoro and 1β-fluorovitamin D 3 models. The 1,25-difluorovitamin D 3 compound possessed no vitamin D-like activity demonstrating the importance of 1α- and 25-hydroxylations of vitamin D for activity. However, 1,25-difluorovitamin D 3 had no anti-25-hydroxylation activity and no antivitamin D activity. Since 25-fluorovitamin D 3 has anti-25-hydroxylase activity, it appears the introduction of a fluoro group on the 1 position diminishes interaction of the vitamin D molecule with the 25-hydroxylase system.

In vitro inhibitor studies of vitamin D 25-hydroxylase in rat liver microsomes

The ability of four vitamin D analogs to inhibit the liver microsomal vitamin D-25-hydroxylase was determined. 19-Hydroxy-10(S),19-dihydrovitamin D3,25-fluorovitamin D3, 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide and 25-aza-vitamin D3 were competitive inhibitors with apparent KI values of 44, 137, and 870 nM, and 6.4 microM, respectively. The values for the 19-hydroxy-10(S), 19-dihydrovitamin D3, 25-fluorovitamin D3, and 25-aza-vitamin D3 correspond well to other literature reports with respect to their relative in vivo inhibitory properties. 24-Oxovitamin D3 oxime also proved to be a potent inhibitor but a detailed analysis was prohibited by the lack of material. The 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide was also tested in vivo but had no antagonistic activity when provided at a 2000-fold excess over vitamin D3.

Stimulation of rat intestinal protein synthesis by 1,25-dihydroxyvitamin D3☆

To study general stimulatory effects of 1,25-dihydroxyvitamin D3 on intestinal protein synthesis, slices of duodenal villi from 1,25-dihydroxyvitamin D3-treated and vitamin D-deficient rats were incubated in vitro for 90 min at the surface of medium containing [3H]leucine. Incorporation of the [3H]leucine into TCA-precipitated protein, which was shown to be linear for 12 h and 90% inhibited by cycloheximide, was increased by 50-60% at 26 h after a single injection of 125 ng of 1,25-dihydroxyvitamin D3 (three experiments, P less than 0.001). The increase, which was not due to circadian rhythm fluctuations of the intestine, was in synchrony with the second Ca2+ transport response observed by Halloran and DeLuca (Arch. Biochem. Biophys. 208, 477-486, 1981). However, no significant difference in [3H]leucine incorporation was observed before or during the initial Ca2+ transport response observed by Halloran and DeLuca, i.e., at 1.0, 3.0, and 6.5 h following an injection of 1,25-dihydroxyvitamin D3. The late onset of the 1,25-dihydroxyvitamin D3-induced increase in total protein synthesis implies that it is an indirect rather than a direct effect of the hormone.

The influence of parathyroid hormone on the electron transport mediated reduction of acetoacetate by succinate in rat liver mitochondria

Abstract The parathyroid hormone-dependent phosphate accumulation and the respiration supported reduction of acetoacetate by succinate in rat liver mitochondria were found to be mutually inhibitory. Parathyroid hormone also inhibited the ATP-supported reduction of acetoacetate by succinate in nonrespiring mitochondria. These results provide more direct evidence supporting the hypothesis that the parathyroid hormone-dependent magnesium phosphate accumulation derives its energy from the nonphosphorylated high energy intermediates of oxidative phosphorylation. Magnesium and phosphate were found to strongly influence the reduction of acetoacetate by succinate in both the ATP- and respiration-supported systems. Parathyroid hormone inhibited the respiration supported reduction of acetoacetate in the absence of both magnesium and phosphate. The degree of inhibition by parathyroid hormone was increased by added phosphate but decreased by added magnesium. The role of these ions in the action of parathyroid hormone on mitochondria is discussed.