Rodent macrophages metabolize 25-hydroxyvitamin D3 in vitro.
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Animals↗
Biomedical subjects
G E Lester
Publications and source records attributed to G E Lester.
Vitamin D deficiency in rats with normal serum calcium concentrations.
Rats were raised after weaning on a vitamin D-deficient diet which used whole wheat and casein as the major protein source. For at least the first year of life, plasma calcium concentrations of these rats were the same as those of vitamin D-replete rats, and the rate of growth was normal for at least 6 months. The following evidence establishes the vitamin D deficiency of the rats (both male and female) on this diet: (i) plasma levels of 1,25-dihydroxycholecalciferol (1,25-dihydroxyvitamin D3) became undetectable after 6 weeks on the diet; (ii) by 4 months of age, the epiphyseal growth plates of the tibia were significantly enlarged and disorganized; (iii) when subjected to fracture in a dynamic torsion machine, the femur showed marked weakening as indicated by stress analysis; (iv) isolated kidney cells from the deficient rats showed a 3-fold increase in 25-hydroxyvitamin D 1-hydroxylase activity. When mother rats were placed on the vitamin D-deficient diet during lactation, plasma calcium values in the pups decreased and remained low throughout life and there was a stunted body growth pattern. It is concluded that hypocalcemia is not a necessary manifestation of vitamin D deficiency, that the onset of vitamin D deficiency during neonatal life influences the calcium homeostatic system, and that the normocalcemic, vitamin D-deficient animal provides an experimental model in which the effects of vitamin D deficiency can be studied independently of hypocalcemia.
Animals↗
Serum concentrations of calcium and vitamin D metabolites in prosimians.
The concentrations of total calcium, 25-hydroxyvitamin D [25-(OH)D] and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were measured in serum obtained from prosimians, the brown lemurs. The mean serum calcium level was 10.6 mg/dl in male and female lemurs. The mean serum mean 25-(OH)D concentration in serum from male and female lemurs was 27.1 and 31.0 ng/ml, respectively. The mean serum level of 1,25-(OH)2D3 in the female and male lemurs was 65.2 and 65.9 pg/ml, respectively. A small segment of the lemurs had hypercalcemia and elevated serum concentrations of 25-(OH)D or 1,25-(OH)2D3, suggesting the idea that the episodic ingestion of a large quantity of the calcium- and vitamin D-enriched diet normally provided ad libitum might cause hypercalcemia.
Animals↗
25-Hydroxyvitamin D3 metabolism in the pregnant rat: maternal-fetal relationship.
Metabolism of 3H-25OHD3 was studied in pregnant, D-deprived rats and their fetuses at 21 days' gestation. Significant changes in the circulating levels of 3H-24,25(OH)2D3 and 3H-1,25(OH)2D3 in maternal and fetal plasma occurred from 4 to 12 h after the administration of 233 ng of 3H-25OHD3. 3H-1,25(OH)2D3 appeared before 3H-24,25(OH)2D3 in both the mother and fetuses. Maternal plasma contained more 3H-1,25(OH)2D3 than fetal plasma and, contrariwise, fetal plasma contained more 3H-24,25(OH)2D3 than maternal plasma. Maternal nephrectomy studies showed that the in vivo synthesis of 3H-24,25(OH)2D3 at 6 h was dependent on circulating levels of 3H-1,25(OH)2D3. These results revealed aspects of maternal-fetal metabolism of 25OHD3 similar to those in the non-pregnant state as well as others unique to pregnancy.
Animals↗
Vitamin D and pregnancy: the maternal-fetal metabolism of vitamin D.
A model of the maternal-fetal metabolism of vitamin D3 is depicted in Fig. 2. 25-OHD3 of maternal origin is metabolized by the maternal kidneys to the potent metabolite, 1,25-(OH)2D3, which acts on the maternal intestine, kidneys, and skeleton. The maternal kidneys and other organs can produce 24,25-(OH)2D3, although this pathway may be suppressed near the end of gestation. The placenta has selective permeability to the vitamin D3 metabolites, with 25-OHD3 crossing from the mother to the fetus more readily than the dihydroxylated metabolites. The onset of the placental synthesis of 1,25-(OH)2D3 during gestation is unknown. Likewise the regulation of the placental 25-OHD3-1 alpha-hydroxylase is unknown. 1,25-(OH)2D3 of placental origin may enter the maternal or the fetal circulation or act locally on the placenta by inducing the synthesis of proteins involved in the cellular transport of Ca. Perhaps one placenta cell type synthesizes 1,25-(OH)2D3 and another cell type possessing a cytoplasmic receptor for 1,25-(OH)2D3 responds to this metabolite. The function of the 24,25-(OH)2D3 produced by the placenta is unknown. The concentration of free 25-OHD3 and free 1,25-(OH)2D3 in the fetal circulation exceeds the maternal levels due to the differences in the DBP concentrations of the two bloodstreams. The 1,25-(OH)2D3 in the fetal bloodstream may originate from either the placenta or the fetal kidneys. The latter site may not be active in utero due to the hypercalcemia and hyperphosphatemia relative to the maternal levels of these ions. 1,25-(OH)2D3 in the fetal bloodstream acts on those fetal tissues containing cytoplasmic receptors for this metabolite. The intestinal mucosa apparently lacks these receptors until sometime during neonatal life. In contrast, fetal bone cells possess receptors for the 1,25-(OH)2D3. The 24,25-(OH)2D3 in the fetal bloodstream may also be involved in the growth and differentiation of the fetal skeleton. However, the precise role of both metabolites in the fetus remains conjectural.
25-Hydroxyvitamin D 2↗
A modified radioimmunoassay for 1,25-dihydroxycholecalciferol.
A radioimmunoassay for 1,25-dihydroxycholecalciferol which did not cross react with 1,25-dihydroxyergocalciferol is described. IgG fractions were prepared from the serum of rabbits that had been immunized with 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine albumin. Radioligand binding by the IgG fractions was time-, temperature-, and pH-dependent. The IgG fractions had a high affinity for 1,25-dihydroxycholecalciferol but cross reacted with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Vitamin D2 metabolites did not cross react in the assay when amounts up to 9 ng per tube were tested. The determination of 1,25-dihydroxycholecalciferol in human serum required an organic extraction and chromatographic isolation of the metabolite. Radioligand binding was influenced by the presence and concentration of the proteins in the phosphate buffer. The mean concentration of 1,25-dihydroxycholecalciferol in serum from normal adults was 56 (SEM 5.7) ng/L. 1,25-Dihydroxycholecalciferol was not detectable in serum from a nephrectomized subject and the concentration in serum was lower than normal in hypoparathyroid patients. Ingestion of 1,25-dihydroxycholecalciferol by nephrectomized or hypoparathyroid patients restored the concentration of 1,25-dihydroxycholecalciferol in serum to the normal range. The stability of the IgG fraction, the relatively short incubation interval, and the ability to measure 1,25-dihydroxycholecalciferol without interference from 1,25-dihydroxyergocalciferol are unique aspects of this radioimmunoassay.
Adult↗
In vitro synthesis of 1 alpha,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by isolated calvarial cells.
The question of whether the skeleton metabolizes 25-hydroxycholecalciferol [25(OH)D3] to more-polar products was studied. Calvarial cells were dispersed from 16-day old chicken embryos by using collagenase and then grown in culture in serum-free medium. Confluent cell cultures were incubated with 7 nM 25(OH)[3H]D3 for 2 hr, and the vitamin D metabolites were then extracted. At least four polar metabolites were produced. Based on separation by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography, two of these metabolites were identified as 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3]. These metabolites were also produced by cultured kidney cells but not by liver, heart muscle, or skin cells isolated from the same embryos. The specific activities of the calvarial 1- and 24-hydroxylases were similar in magnitude to those in isolated kidney cells. The specific activity of the calvarial 25(OH)D3:1-hydroxylase was inhibited by an 8-hr preincubation with 1,25(OH)2D3, whereas the 24-hydroxylase was enhanced. It is concluded that (i) vitamin D metabolism by isolated cells is organ-specific, (ii) calvarial cells produce active metabolites of vitamin D in significant amounts, (iii) vitamin D metabolism by calvarial cells is regulated by 1,25(OH)2D3, and (iv) locally produced, active metabolites could act locally, thereby adding a new dimension to the regulation of mineral metabolism by vitamin D metabolites.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
Evidence for extra-renal 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in pregnancy.
The kidneys are thought to be the only organs capable of 1 alpha-hydroxylation of vitamin D and its metabolites. We have examined the in vivo conversion of 3H-(25,26)-25-hydroxyvitamin D3(25OHD3) to 3H-(25,26)-1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in vitamin D-deficient, pregnant and nonpregnant rats. As expected, nephrectomy of nonpregnant, vitamin D-deficient rats prevented the conversion of 25OHD3 to 1 alpha,25(OH)2D3. In contrast, nephrectomy of pregnant, vitamin D-deficient rats reduced but did not abolish the formation of 1 alpha,25(OH)2D3 from its precursor. The identity of the radioactive metabolite formed from 3H-25OHD3 which circulated in nephrectomized, pregnant rats was established as 1 alpha,25(OH)2D3 by comigration with synthetic 1 alpha,25(OH)2D3 on high-pressure liquid chromatography. The simultaneous absence of 1 alpha,25(OH)2D3 in the fetal kidneys indicated that the site of 1 alpha-hydroxylation after nephrectomy of the pregnant rat was probably extra-renal in origin. Two sites of 1 alpha-hydroxylation of 25OHD3, one renal and the other extra-renal, either fetoplacental or maternal, may exist in the pregnant, vitamin D-deficient rat.
Animals↗
Stimulation of renin secretion from the intact kidney and from isolated glomeruli by the calcium ionophore A23187.
The ionophore A23187 evoked a dose-dependent release of renin from the isolated perfused cat kidney, which was inhibited by calcium deprivation and adrenergic blockade. The latter finding indicates that the effects of A23187 on the intact kidney are mediated mainly by catecholamine release from sympathetic nerve endings. Ionophore also elicited a concentration-dependent enhancement of renin secretion from a pure preparation of glomeruli isolated from cat kidney; this stimulation was still manifest when the glomeruli were superfused with a calcium-free solution. These findings indicate that A23187 evokes renin secretion from juxtaglomerular cells by mobilizing cellular calcium and support the view that an increase in intracellular calcium is intimately involved in the mechanism of renin secretion.
Animals↗
Evidence for maternal and fetal differences in vitamin D metabolism.
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Animals↗
The role of calcium in renin secretion from the isolated perfused cat kidney.
1. Isolated cat kidneys were perfused in situ with Locke solution and renin release in response to isoprenaline was studied. 2. Perfusion with isoprenaline produced a concentration-dependent enhancement of renin secretion. Increasing the concentration of stimulant also prolonged the duration of the secretory response. 3. After a 10 min exposure to isoprenaline (0-3 micrometer), there was a rapid facilitation of renin release which diminished after 10-30 min, followed by a second transient increase which declined over the next 40-60 min. Cycloheximide did not prevent augmented release when added together with the isoprenaline but did produce a reversible inhibition of the late phase when added 10 min after the isoprenaline. 4. Omission of calcium from the perfusion medium failed to depress the renin release induced by isoprenaline, glucagon, or furosemide. However, during prolonged calcium deprivation, the cycloheximide-sensitive phase of isoprenaline-evoked release was depressed. 5. The calcium antagonist D-600 failed to block the early phase of isoprenaline-induced renin secretion but inhibited the late phase of secretion. 6. Calcium alone elicited an explosive discharge of renin when added after a prolonged period of calcium-free perfusion. 7. These results support the view that extracellular calcium does not play an essential role in the mechanism of renin secretion from the renal juxtaglomerular cells, but that an increased influx of this cation is needed for synthesis and/or mobilization of the enzyme. It is tentatively proposed that the release of calcium from intracellular storage sites may be the signal which triggers renin secretion.
Animals↗
Inhibition of epidermal DNA synthesis by cycloheximide and other inhibitors of protein synthesis.
Cycloheximide, pactamycin and tenuazonic acid inhibit protein synthesis in eukaryotic cells. It is the purpose of these studies to determine whether these agents would be effective in vivo inhibitors of epidermal DNA synthesis. Single I.P. administration of 1.5 mg/kg cycloheximide, 0, 1 or 3 hours prior to a 45 minute pulse of thymidine-3H inhibited epidermal DNA synthesis by 68%, 63% and 88%, respectively. This response was dose related at 3.75 hours after cycloheximide with no effect at 0.375 mg/kg and maximal inhibition at 1.5 mg/kg. Epidermal DNA synthesis was inhibited 18, 29 and 62% at doses of 1.5, 3.0 and 6.0 mg/kg pactamycin administered 3 hours before thymidine-3H. Similarly, at doses of 70 and 140 mg/kg tenuazonic acid, epidermal DNA synthesis was inhibited by 50% and 60%, respectively. These data suggest the possible use of protein synthesis inhibitors in epidermal disorders.
Animals↗
The effect of a nonsteroidal antiinflammatory drug on the healing of ligaments.
Because of the increasing number of ligament sprains being treated with nonsteroidal antiinflammatory drugs (NSAIDs), this study was undertaken to document the effects of one such drug on ligament healing in an experimental setting. Male Sprague-Dawley rats weighing between 400 and 500 g were used to evaluate the effect of the NSAID piroxicam on the healing of an experimental injury to the medial collateral ligament (MCL). The following factors were varied in the experiments: dosage, days of treatment, and the day postinjury when treatment was begun. Piroxicam-treated rats were compared to placebo-treated rats in terms of the drug's effect on the mechanical strength of the healing ligament. The ligaments were mechanically tested in tension to failure at a constant deformation rate of 0.25 mm/sec on a materials testing machine. Administration of piroxicam on Days 1 to 6 postinjury resulted in a 42% increase in strength at Day 14 postinjury for the piroxicam-treated ligaments (P less than 0.01) when compared with the placebo-treated controls. Neither doubling nor halving the standard piroxicam dose significantly altered this increased healing strength. Biochemical analysis of collagen synthesis demonstrated a suggestive, although not statistically significant, increase in collagen synthesis and collagen content in the piroxicam-treated healing ligament. In separate experiments, piroxicam had no effect on the healed ligament at 21 days or on the strength of uninjured ligaments. In conclusion, piroxicam increased the early strength of healing ligaments in the rat when the drug was administered for short periods of time after injury.(ABSTRACT TRUNCATED AT 250 WORDS)
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