Vitamin D, calcium-binding protein and calcium absorption in the intestines

The author presents his own and literature data concerning the action of vitamin D and its analogs on the biosynthesis of calcium-binding protein (CaBP) in the chick intestinal mucosa. The mechanism of involvement of this protein in calcium transport across the intestinal epithelium is discussed. CaCP is considered as a specific test-substance for vitamin D. A close correlation between the CaCP content in the intestinal mucosa and the vitamin antirachitic effect is demonstrated. The quantitative estimates of the biological activity of various forms of vitamin D, their metabolites and synthetic analogs with respect to the CaCP content are given.     

Vitamin D-dependent calcium binding protein in the chick brain

A radioimmunoassay for chick CaBP has been used to measure the distribution of the protein in the chick central nervous system. The cerebellum contained 7.4 μg/mg protein, six times more than the medulla, and eleven times more than the cerebral cortex. Most of the CaBP was found in the supernate, after ultracentrifugation, with only trace amounts in the secretosome, microsome and other fractions. Slices of cerebellum from 19 and 21 days $\text{in}$$\text{ovo}$ chicks were maintained in organ culture. Treatment with kainic acid resulted in a loss of CaBP from the slices while metabolites of vitamin D were without effect.     

Vitamin D metabolism and its possible role in the developing chick embryo.

The relationship between bone formation and vitamin D metabolism was investigated in the developing chick embryo. Fertilized White Leghorn eggs were incubated at 38 degrees C in an incubator for 21 days. The fresh weight and calcium content of embryonic tibiae began to increase at day 12 and attained maximal values at day 19. Bone alkaline phosphatase and citrate decarboxylation activities, both of which represent osteoblastic activity, also began to increase at days 10-12, reached maximal values at day 19 and sharply declined thereafter. Both bone enzyme activities were highly correlated with CA2+-binding activity in the chorioallantoic membrane measured by the Chelex 100 assay. When mesonephric and metanephric homogenates were incubated with 25-hydroxy[3H]cholecalciferol, a marked and concomitant increase occurred in the metanephric 1 alpha- and 24-hydroxylase activity after day 14. The production of 1 alpha, 25-dihydroxycholecalciferol attained a maximal value at day 19 and decreased thereafter, whereas that of 24,25-dihydroxycholecalciferol continued to increase until hatching. The production rate of 1 alpha, 25-dihydroxycholecalciferol by the metanephros coincided with the changes in Ca2+-binding activity in the chorioallantoic membrane and osteoblastic activity. Since both intestinal calcium absorption and bone mineral mobilization do not occur in embryonic life, these results support the idea that 1 alpha, 25-dihydroxycholecalciferol may be involved directly in bone formation or induction of a calcium-binding protein in the chorioallantoic membrane.     

Vitamin D and calcium dysregulation in the polycystic ovarian syndrome.

Over the past 30 years, numerous studies in invertebrates and vertebrates have established a role of calcium in oocyte maturation as well as in the resumption and progression of follicular development. Polycystic ovarian syndrome (PCO) is characterized by hyperandrogenic chronic anovulation, theca cell hyperplasia, and arrested follicular development. The aim of this observational study was to determine whether vitamin D and calcium dysregulation contribute to the development of follicular arrest in women with PCO, resulting in reproductive and menstrual dysfunction. Thirteen premenopausal women (mean age 31 +/- 7.9 years) with documented chronic anovulation and hyperandrogenism were evaluated. Four women were amenorrheic and nine had a history oligomenorrhea, two of whom had dysfunctional bleeding. Nine had abnormal pelvic sonograms with multiple ovarian follicular cysts. All were hirsute, two had alopecia, and five had acanthosis nigricans. The mean 25 hydrovitamin D was 11.2 +/- 6.9 ng/ml [normal (nl): 9-52], and the mean 1,25 dihydroxyvitamin D was 45.8 +/- 18 pg/ml. with one woman with a 1,25 dihydroxyvitamin D <5 pg/ml (nl: 15-60). The mean intact parathyroid hormone level was 47 +/- 19 pg/ml (nl: 10-65), with five women with abnormally elevated parathyroid hormone levels. All were normocalcemic (9.3 +/- 0.4 mg/dl). Vitamin D repletion with calcium therapy resulted in normalized menstrual cycles within 2 months for seven women, with two experiencing resolution of their dysfunctional bleeding. Two became pregnant, and the other four patients maintained normal menstrual cycles. These data suggest that abnormalities in calcium homeostasis may be responsible, in part, for the arrested follicular development in women with PCO and may contribute to the pathogenesis of PCO.     

Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells

A slowly inactivating inward calcium current was identified in the rat osteosarcoma cell line ROS 17/2.8 using a combination of ion flux and electrophysiological techniques. Voltage dependence, dihydropyridine sensitivity, divalent cation selectivity, and single channel properties identified this current as a high threshold, "L-type" calcium current. Ion flux experiments using 45Ca2+ confirmed that calcium uptake through these channel represents a major pathway for calcium entry into osteosarcoma cells. In resting cells, i.e. at negative membrane potentials, stimulation of both calcium current and rapid 45Ca2+ influx could be elicited by concentrations of 1,25-(OH)2-vitamin D3 between 0.1 and 3 nM. At these concentrations, 1,25-(OH)2-vitamin D3 shifted the threshold for activation of inward calcium current to more negative potentials. At higher concentrations (5-10 nM), inhibitory effects became predominant. These opposing effects are functionally similar to those of the dihydropyridine BAY K 8644. Other vitamin D3 metabolites (25-(OH)-D3 and 24,25-(OH)2-D3) exhibited less potent stimulatory effects and greater inhibition of calcium current than 1,25-(OH)2-D3. These results suggest that (i) vitamin D3 acts as a potent modulator of calcium channel function in osteosarcoma cells, and (ii) intracellular Ca2+-dependent signaling processes may be affected acutely by physiological concentrations of vitamin D3 metabolites.     

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

Summary Thein vivo andin 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>25-(OH)D₃>1,24,25-(OH)₃D₃>24,25-(OH)₂D₃>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 theV _max of calcium uptake; the affinity for calcium (K _m=0.54mm) 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 of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)₂D₃. These findings suggest that thein 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 effect of bile, bile acids and detergents on calcium absorption in the chick

1. Bile from rachitic or normal chicks causes an immediate increase in the intestinal absorption of soluble calcium in rachitic and vitamin D(3)-treated chicks as tested in vivo by intestinal-loop and oral-dosing methods. 2. This effect is apparently solely due to the taurine-conjugated bile acids present in the bile and is independent of the action of vitamin D. 3. Chick bile and bile acids can increase the solubility and the absorption of calcium presented as sparingly soluble calcium hydrogen phosphate. 4. In addition, bile is necessary to some extent at least for the intestinal absorption of vitamin D(3) in the chick and this would indirectly enhance the absorption of calcium. 5. Thus bile is capable of a threefold action in the absorption of calcium in the chick. It is suggested that the direct action on sparingly soluble forms of calcium is of considerable physiological importance since most of the calcium in the normal bird's diet would be in this form. 6. Bile acids enhance the absorption of calcium in all regions of the small intestine of the chick. 7. Of a range of bile acids and detergents tested for enhancement of calcium absorption, various taurine-conjugated bile acids and sodium lauryl sulphate, an anionic detergent, are effective. A non-ionic detergent (Tween 80) and a cationic detergent (Zephiran) are without effect. 8. The ability of a substance to increase directly the intestinal absorption of soluble calcium appears to depend to some extent on an anionic detergent action, i.e. the ability to form a salt or complex soluble to some extent in both aqueous and lipid phases. 9. In chicks the immediate deposition of calcium ((45)Ca) in the bones closely reflects any increase in plasma calcium radioactivity regardless of the cause of the increase and regardless of the vitamin D(3) status. Although sodium lauryl sulphate can increase markedly the calcium absorption from the gut and the immediate deposition in the bones it has no significant effect on rickets. 10. Some of the implications of these findings are discussed.     

Determination of Vitamin D-dependent calcium absorption by 45Ca gavage in the rat

Precise determination of Vitamin D-dependent intestinal calcium absorption in longitudinal studies is problematic. We have assessed Vitamin D-dependent intestinal calcium absorption by ⁴⁵Ca gavage. Rats were gavaged with a 1 mL solution containing ⁴⁵Ca (CaCl₂, 9.3 MBq/mL) maintained at 37 °C. Total Ca concentration of the gavage fluid was optimised by comparing the absorption curves for fluids made up to 0.025, 2.025, 4.025 and 40.025 mmol/L with ⁴⁰CaCl₂. The effect of varying dietary Ca on fractional Ca absorption was determined in rats fed semi-synthetic diets containing either 0.05%, 0.2%, 0.4% or 1.0% Ca for 50 days. Serum 1,25 dihydroxyvitamin D (1,25D) was determined by radioimmunoassay. Total gavage Ca of 0.025 mmol/L achieved the highest peak fractional absorption and was adopted for all future experiments. Fifty days after allocation to the diets both fractional Ca absorption and 1,25D were highest in rats fed 0.05% Ca and lowest in those fed 1.0% Ca (absorption, P < 0.05 and 1,25D, P < 0.05). There was a strong logarithmic relationship between 1,25D and fractional Ca absorption (R² 0.69, P < 0.001). Weekly repetition of the procedure did not cause a fall in haematocrit over 7 weeks. Radiocalcium (⁴⁵Ca) absorption by gavage provides a simple measure of Vitamin D-dependent Ca absorption for repetitive use in longitudinal studies.     

Comparison of effects of verapamil, low calcium diet and betamethasone on duodenal calcium absorption efficiency in the chick

The results indicate that oral administration of verapamil for 2 weeks to the chick is followed by an increase in the efficiency of the duodenal absorption of calcium. In these chicks both a decrease in serum calcium level and an increase in the activity of renal 1 alpha-hydroxylase were observed. The increased calcium absorption following prolonged treatment with verapamil resembles that induced by a low calcium diet. The mechanism of both responses presumably involves an increased production of 1,25-dihydroxyvitamin D3. Both verapamil- and low calcium diet-induced adaptations are capable of overcoming the inhibitory action of betamethasone on intestinal calcium absorption. No effects on calcium absorption were noted if verapamil was administered intraperitoneally which suggests that verapamil exerts its action directly on the intestinal mucosa.     

Sodium deoxycholate inhibits chick duodenal calcium absorption through oxidative stress and apoptosis

High concentrations of sodium deoxycholate (NaDOC) produce toxic effects. This study explores the effect of a single high concentration of NaDOC on the intestinal Ca(2+) absorption and the underlying mechanisms. Chicks were divided into two groups: 1) controls and 2) treated with different concentrations of NaDOC in the duodenal loop for variable times. Intestinal Ca(2+) absorption was measured as well as the gene and protein expressions of molecules involved in the Ca(2+) transcellular pathway. NaDOC inhibited the intestinal Ca(2+) absorption, which was concentration dependent. Ca(2+)-ATPase mRNA decreased by the bile salt and the same occurred with the protein expression of Ca(2+)-ATPase, calbindin D(28k) and Na(+)/Ca(2+) exchanger. NaDOC produced oxidative stress as judged by ROS generation, mitochondrial swelling and glutathione depletion. Furthermore, the antioxidant quercetin blocked the inhibitory effect of NaDOC on the intestinal Ca(2+) absorption. Apoptosis was also triggered by the bile salt, as indicated by the TUNEL staining and the cytochrome c release from the mitochondria. As a compensatory mechanism, enzyme activities of the antioxidant system were all increased. In conclusion, a single high concentration of NaDOC inhibits intestinal Ca(2+) absorption through downregulation of proteins involved in the transcellular pathway, as a consequence of oxidative stress and mitochondria mediated apoptosis.     

维生素D_3与雄性生殖

维生素D3的经典作用是调节体内钙、磷的代谢平衡和维持骨的健康,有证据显示维生素D3也可调节动物生殖。维生素D代谢酶可合成和降解活性维生素D3及其代谢中间产物,活性维生素D3通过结合至目标组织的维生素D受体(vitamin D receptor,VDR)以发挥其生物学作用。VDR和维生素D代谢酶在雄性生殖系统中表达,表明维生素D3在雄性生殖生物学中起关键作用。本文对维生素D3与雄性生殖的研究进展作一综述,以期为推进维生素D3影响雄性生殖的分子机制和临床治疗男性不育的研究提供理论依据。     

Effect of 1,25-dihydroxyvitamin D3 on intestinal calcium absorption in strontium-fed rats.

These studies investigated the initial stimulation of intestinal calcium absorption in the rat by 1,25-dihydroxyvitamin D₃. To produce a functional vitamin D₃-deficiency, rats were fed a diet containing 2.4% strontium. After 10 days on the diet, intestinal calcium uptake, as measured by everted gut sacs, was significantly depressed. Strontium-fed rats were dosed orally with 1,25-dihydroxyvitamin D₃, and changes in intestinal calcium uptake, intestinal alkaline phosphatase activity, and intestinal calcium-binding protein were measured as a function of time after dose. Calcium uptake was significantly increased in the proximal 2.5 cm of the duodenum at 4 h and along the whole duodenum by 7 h. Intestinal alkaline phosphatase activity, measured in a Triton extract of the mucosal homogenate and in isolated brush border complexes, was also increased by 7 h. Using both gel electrophoresis and immunodiffusion against a specific antiserum, an increase in intestinal calcium-binding protein was detected in intestinal supernate at 4 h after dosing. Almost no calcium-binding protein was detectable in strontium-fed rats dosed with propylene glycol only. These time studies are consistent with a role for both alkaline phosphatase and calcium-binding protein in the 1,25-dihydroxyvitamin D₃-stimulated uptake of calcium by the intestine. In addition, the usefulness of strontium feeding for producing a functional vitamin D₃ deficiency in rats is demonstrated.