Adaptation of intestinal calcium absorption: parathyroid hormone and vitamin D metabolism.

It has already been demonstrated that the adaptation of intestinal calcium absorption of rats on a low calcium diet can be eliminated by thyroparathyroidectomy plus parathyroid hormone administration. This treatment elevates intestinal and plasma levels of 1,25-dihydroxyvitamin D₃ in rats on a high calcium diet while producing no change in rats on a low calcium diet. It therefore appears likely that the modulation of intestinal calcium absorption by dietary calcium is mediated by the parathyroid glands and the renal biogenesis of 1,25-dihydroxyvitamin D₃. Changes in the other unknown vitamin D metabolite levels as a result of dietary calcium are also modified by thyroparathyroidectomy and parathyroid hormone administration, but the effect of these metabolites on intestinal calcium transport is unknown.     

Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1alpha-hydroxy-25-fluorovitamin D3 to 1alpha,24R-dihydroxy-25-fluorovitamin D3.

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D₃ were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D₃, 24R-hydroxy-25-fluorovitamin D₃, or 1α-hydroxy-25-fluorovitamin D₃. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D₃ had increased levels of 1α,25-dihydroxyvitamin D₃; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24R-dihydroxyvitamin D₃ on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D₃ receptor proteins. 1α,24R-Dihydroxyvitamin D₃ binds as well as 1α, 25-dihydroxyvitamin D₃ to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D₃; whereas 1α,24S-dihydroxyvitamin D₃ binds only one-tenth as well as 1α,25-dihydroxyvitamin D₃. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24R-dihydroxy-25-fluorovitamin D₃ and that may rival the potency of 1α,25-dihydroxyvitamin D₃.     

Synthesis of 24S and 24R-hydroxy-[24-3H]vitamin D3 and their metabolism in rachitic rats.

An epimeric mixture of 24-hydroxy-[24-₃H]vitamin D₃ was synthesized by the reduction of 24-ketovitamin D₃ by sodium borotritide. The epimeric mixture was converted to the trimethylsilylether derivatives and subjected to high-pressure liquid chromatography using silica gel columns to separate the 24-hydroxy-[24-₃H]vitamin D₃ isomers. The 24R-hydroxy-[24-₃H] vitamin D₃ induced calcification in rachitic rats while the 24S-hydroxy-[24-₃H] vitamin D₃ had little or no such activity. As both isomers of 24-hydroxy-vitamin D₃ are metabolized to 24,25-dihydroxyvitamin D³, it appears that the 24-hydroxyvitamin D₃-25-hydroxylase does not discriminate between the isomers. Only the R-isomer of 24-hydroxyvitamin D₃ is metabolized to 1,24-dihydroxyvitamin D₃, although only trace amounts of this compound were found 2 days after the administration of 24-hydroxyvitamin D₃. The striking difference in the metabolism of the isomers is the high selectivity of the 1-hydroxylase for R-isomer. It is suggested that the high specificity of biological activity for the R-isomer of 24-hydroxyvitamin D₃ is because of the specificity of the 1-hydroxylation of 24,25-dihydroxyvitamin D₃ for the R configuration.     

Synthesis of vitamin D5: its biological activity relative to vitamins D3 and D2.

The chemical synthesis, spectral characterization, and biological activity of vitamin D₅ in vitamin D-deficient rats is reported. Vitamin D₅ is about 180-fold less active than vitamin D₃ in calcification of rachitic cartilage and about 100- to 200-fold less active in induction of bone-calcium mobilization. In stimulation of intestinal-calcium transport, vitamin D₅ is about 80-fold less active than vitamin D₃. Vitamins D₂ and D₃ appear to be equiactive in all three responses when low doses are administered.     

Metabolism of vitamin D3 in the chick embryo

In agreement with previous reports, chick intestinal calcium-binding protein does not appear in the chick embryo until 1 day after hatching while intestinal alkaline phosphatase begins to appear at 19–20 days of embryonic life. The ability of chick embryo to metabolize vitamin D₃ to 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, and 24,25-dihydroxyvitamin D₃ is present at least by day 18 of embryonic life as demonstrated by in vivo and in vitro techniques. It also illustrates that metabolism of vitamin D₃ was not the limiting factor in the appearance of calcium-binding protein and alkaline phosphatase in intestine. Instead, the uptake of 1,25-dihydroxyvitamin D₃ by the duodenum was very low prior to hatching, even though significant amounts were present in the yolk sac. Injection of a physiological dose of 1,25-dihydroxyvitamin D₃ to chick embryo at 9 days failed to stimulate appearance of calcium binding protein by 18 days of embryonic life. Thus, it appears that either the normal mechanism for transport of 1,25-dihydroxyvitamin D₃ to intestine or its receptors in intestine may not be present prior to day 18–19.A large fraction of radioactive vitamin D₃ injected into the yolk sac was found esterified especially in the embryonic liver. The significance of this is not yet understood.Injection of 1,25-dihydroxyvitamin D₃ at 325 pmoles/per egg at 9 days resulted in 70% mortality of embryos while a 32-pmole dose resulted in no significant increase in mortality. The basis for this toxicity is not yet understood.     

1,24,25-Trihydroxyvitamin D3: a circulating metabolite in vitamin D2-treated bovine

Methods have been developed for the examination of yeast RNA polymerases I, II, and III by electron microscopy. The results enabled us to establish the size and shape of a eucaryotic RNA polymerase for the first time. The enzymes are roughly spherical in shape and compact in appearance. Their measured molecular diameters are 12.7 ± 0.4 and 11.0 ± 1.4 (SD) nm for polymerase I, 12.7 ± 1.1 and 12.2 ± 1.0 (SD) nm for polymerase II, and 13.6 ± 0.6 and 11.5 ± 1.3 (SD) nm for polymerase III.     

Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3.

Kidney homogenates from chicks fed a vitamin D-deficient diet for 10 days and supplemented with 6.5 nmol of vitamin D₃ 48 hr prior to sacrifice metabolized $\text{in}\text{vitro}\text{[}{}^3\text{H}\text{]}$-25-hydroxyvitamin D₃ (25-OH-D₃) to 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified [³H]-24,25-(OH)₂-D₃, 3 similar metabolites (peaks A′, C′ and E′) were produced. On high pressure liquid chromatography, peaks A, C and E migrated to exactly the same respective positions as peaks A′, C′ and E′. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [³H]-25-OH-D₃ or [³H]-24,25-(OH)₂-D₃. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)₂-D₃ in the kidney of chicks supplemented with vitamin D₃.     

1 alpha, 25-difluorovitamin D3: an inert vitamin D analog

1α,25-Difluorovitamin D₃ has been synthesized by reacting 1,25-dihydroxyvitamin D₃-3-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 D₃ models. The 1,25-difluorovitamin D₃ compound possessed no vitamin D-like activity demonstrating the importance of 1α- and 25-hydroxylations of vitamin D for activity. However, 1,25-difluorovitamin D₃ had no anti-25-hydroxylation activity and no antivitamin D activity. Since 25-fluorovitamin D₃ 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.     

Kidney mitochondrial metabolism of 25-hydroxyvitamin D3,: Evaluation of in vitro cation modulation

Oxidative phosphorylation and 1 α,25-dihydroxyvitamin D₃ [lα,25-(OH)₂D₃]synthesis in isolated mitochondria were decreased by the addition of strontium. Calcium effected a similar inhibition of 1α,25-(OH)₂D₃ synthesis which correlated with cation-induced mitochondrial swelling. The ultrastructural changes were found to be a consequence of experimental conditions and not a prerequisite for suppressed 1α,25-(OH)₂D₃ synthesis. Dietary administration of strontium or calcium also resulted in a decreased rate of 1α,25-(OH)₂D₃ synthesis; however, the decrease in 1-hydroxylase activity was accompanied by an induction of mitochondrial 25-hydroxyvitamin D₃ 24-hydroxylase activity. Such an in vivo-prompted mitochondrial response occurred in the absenee of morphological changes or extensive loss of oxidative phosphorylation activity. In contrast, no induction of 24-hydroxylase activity could be observed in acute studies using isolated mitochondria. Therefore, the in vitro action of calcium and strontium does not appear to reflect the in vivo mechanism whereby the cations act to change renal 25-hydroxyvitamin D₃ (25-OHD₃) hydroxylation. Results from in vitro studies corcerning the action of calcium to alter renal 25-OHD₃ metabolism should be interpreted in light of the cation's capacity to decrease oxidative phosphorylation and the subsequent intramitochondrial generation of NADPH.     

The parotid gland: A new target organ for vitamin D action

Radioactively labelled cholecalciferol was administered continuously to rats which were fed a vitamin D-deficient diet. It has been possible to show that all the metabolites of cholecalciferol which normally occur in known target tissues of vitamin D are present in the parotid gland, and the pattern resembled that obtained for the kidney, a known target tissue for vitamin D action.The accumulation of cholecalciferol metabolites in the parotid gland was shown to be functional, as a calcium-binding protein was found to be present in the gland, possessing similar properties to the renal vitamin D- dependent calcium-binding protein.     

The stimulation of 25-hydroxyvitamin D3-1α-hydroxylase by estrogen

Homogenates of kidney from laying Japanese quail incubated in vitro with 25-hydroxy-[26,27-³H] vitamin D₃ produce more 1,25-dihydroxy-[26,27-³H]vitamin D₃ than do homogenates of kidney from mature nonlaying females or males maintained on the same diet and under identical conditions. Instead, the homogenates from male quail or nonlaying female quail convert 25-hydroxyvitamin D₃ to 24,25-dihydroxyvitamin D₃. The administration of 5 mg of estradiol to mature male quail 24 h prior to sacrifice suppressed the 25-hydroxyvitamin D₃-24-hydroxylase and markedly stimulated 25-hydroxyvitamin D₃-1-hydroxylase. The administration of estradiol to male quail caused hypercalcemia, which responded more slowly than did the 1-hydroxylase. As little as 0.1 mg of estradiol/quail was found effective in stimulating the 1-hydroxylase and suppressing the 24-hydroxylase. Other hormones such as follicle stimulating hormone (FSH), cortisone, testosterone, and progesterone, even at high dose levels, produced little or no change in the 25-hydroxyvitamin D₃-1-hydroxylase. Testosterone did, however, suppress the 25-hydroxyvitamin D₃-24-hydroxylase. The stimulation of the 25-hydroxyvitamin D₃-1-hydroxylase by parathyroid hormone was of a smaller magnitude than that of the estradiol, and the effects of the two hormones were additive, suggesting that they function by a different mechanism.     

1,25-Dihydroxyvitamin D3 metabolism. The effect of dietary calcium and phosphorus

Rats maintained on tritiated 1,25-dihydroxyvitamin D₃ as their sole source of vitamin D and placed on diets differing in calcium content had similar intestinal levels of tritiated 1,25-dihydroxyvitamin D₃. Since 1,25-dihydroxyvitamin D₃ administration eliminated adaptation of intestinal calcium transport, it appears that increased production of 1,25-dihydroxyritamin D₃ is responsible for the stimulation of calcium transport by low dietary calcium. When maintained on tritiated 1,25-dihydroxyvitamin D₃, rats fed a low-phosphorus diet had somewhat higher levels of tritiated 1,25-dihydroxyvitamin D₃ in the duodenum and plasma than rats on a normal-phosphorus diet. In addition to stimulating 1,25-dihydroxyvitamin D₃ synthesis, low dietary phosphorus may increase the accumulation of 1,25-dihydroxyvitamin D₃ in both intestine and plasma.     

The effect of vitamin D3 metabolites on membrane proteins of chick duodenal brush borders.

Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Following injection of 25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃, a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears. This change occurs before calcium binding protein can be detected by Chelex assay and prior to the increase in total alkaline phosphatase but correlates closely with increased intestinal calcium absorption in response to the metabolites. The two brush border proteins have been solubilized with n-butanol and partially characterized. The vitamin D-deficient protein has a molecular weight of about 200,000 and has alkaline phosphatase activity but no detectable calcium binding activity. The protein which appears in response to metabolites has a molecular weight of 230,000, binds calcium, and also has alkaline phosphatase activity.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

Inhibition of precartilaginous chick somites by oncogenic virus

Infection of embryonic chicken notochord-somite explants with Rous sarcoma virus inhibited the in vitro differentiation of somites into cartilage. Visual inspection of the explants revealed that viral infection reduced the size of cartilage nodule formation. Formation of the complex of sulfated proteoglycans with hyaluronic acid was inhibited by RSV infection, and sedimentation analysis of the sulfated proteoglycans showed that very little fast sedimenting proteoglycans were synthesized by RSV-infected explants. The infected explants primarily synthesize a slowly sedimenting sulfated proteoglycan which was chondroitinase resistant. These slow-sedimenting sulfated proteoglycans lack the ability to associate with hyaluronic acid and appear to be noncartilaginous. These effects of RSV are apparently due to the src gene of this virus since the mutant td108, which lacks part of the src gene, has no detectable influence on the chondrogenic differentiation of somite explants. Similarly, infection with RAV-2 as well as with uv-irradiated virus had no detectable effect. The inhibition of synthesis of fast sedimenting proteoglycans was observed at 41 degrees C with explants infected with tsNY68, suggesting that residual activity of transforming gene of this virus at the non-permissive temperature is sufficient for this inhibition in the explants.     

Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.