Enhancement of rat intestinal calcium absorption by vanadate.

Vanadate alters intestinal transport and may have a role in regulating cell function. To determine whether it influences calcium absorption, we tested the effects of acute and chronic vanadate administration on calcium absorption using single-pass perfusion of jejunal and ileal segments of the in vivo rat intestine. Acute vanadate administration increased the lumen-to-mucosa and net fluxes of calcium in both the jejunum and ileum. The increase was largely due to an enhancement of the saturable fluxes of calcium and was observed at 10(-4) M concentration of vanadate, but not at higher or lower concentrations of the oxyanion, except at the highest concentration used, 10(-2) M, where calcium absorption was inhibited. Chronic vanadate administration caused, on the other hand, no changes in calcium absorption. We have demonstrated previously that rat intestinal (Na+ + K+)-ATPase is inhibited by vanadate, an effect that could raise cell sodium and increase the efflux of sodium across the brush border membrane. The results suggest that the vanadate enhancement of calcium absorption may be related to an increased entry of calcium into the mucosa, possibly as a result of an augmented exchange through the Na+/Ca+ antiport system. Alternatively, vanadate may influence access to a calcium channel in the mucosal membrane of the intestinal epithelium, leading to the observed increase in absorption.     

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.     

Strontium and calcium metabolism. Interaction of strontium and vitamin D]

Oral administration of strontium to calcium wellfed rats blocks the intestinal absorption of calcium. When high doses of vitamine D are given over long period, the inhibition of calcium intestinal absorption disapears. Under these conditions the absorption of strontium is increased. It is suggested that there is only one absorption mechanism for these two cations. An overdose of the vitamine D increases the renal elimination of strontium but under these conditions the plasma concentration of the strontium is unchanged. Vitamine D brings about the some action on the bone fixation of the strontium as it does on the bone fixation of calcium. The bone fixation is increased with low dosages. The bone fixation is decreased with high dosages.     

Calcium-dependence of sugar transport in rat small intestine

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.     

Prolactin is an important regulator of intestinal calcium transport

Prolactin has been shown to stimulate intestinal calcium absorption, increase bone turnover, and reduce renal calcium excretion. The small intestine, which is the sole organ supplying new calcium to the body, intensely expresses mRNAs and proteins of prolactin receptors, especially in the duodenum and jejunum, indicating the intestine as a target tissue of prolactin. A number of investigations show that prolactin is able to stimulate the intestinal calcium transport both in vitro and in vivo, whereas bromocriptine, which inhibits pituitary prolactin secretion, antagonizes its actions. In female rats, acute and long-term exposure to high prolactin levels significantly enhances the (i) transcellular active, (ii) solvent drag-induced, and (iii) passive calcium transport occurring in the small intestine. These effects are seen not only in pregnant and lactating animals, but are also observed in non-pregnant and non-lactating animals. Interestingly, young animals are more responsive to prolactin than adults. Prolactin-enhanced calcium absorption gradually diminishes with age, thus suggesting it has an age-dependent mode of action. Although prolactin's effects on calcium absorption are not directly vitamin D-dependent; a certain level of circulating vitamin D may be required for the basal expression of genes related to calcium transport. The aforementioned body of evidence supports the hypothesis that prolactin acts as a regulator of calcium homeostasis by controlling the intestinal calcium absorption. Cellular and molecular signal transductions of prolactin in the enterocytes are largely unknown, however, and still require investigation.     

A calcium dependent post-tetanic hyperpolarization of primary afferent terminals

In decerebrated spinal cats, the effects of iontophoretically applied calcium antagonists, cobalt, manganese and verapamil, and of strontium, which reportedly can act like a calcium agonist, were tested on post-tetanic depression of group I afferent terminal excitability. The actions of these agents on the duration of action potentials in the afferent terminals were determined by a recently described method (8). The calcium antagonists reduced the maximum post-tetanic depression of the antidromic compound action potentials and accelerated the recovery of these potentials from the depression. Strontium, on the other hand, had the opposite effects. The duration of afferent terminal action potentials appeared to increase following a tetanic stimulation. This enhancement in the duration of the action potentials was facilitated by strontium and counteracted by calcium antagonists. These observations indicate that calcium influx into primary afferent terminals is increased following a tetanic stimulation and that post-tetanic hyperpolarization of primary afferent terminals may be, at least partly, dependent on the increased accumulation of calcium in the terminals.     

Effects of vitamin K2 administration on calcium balance and bone mass in young rats fed normal or low calcium diet

OBJECTIVE: The purpose of this study was to examine the effects of vitamin K2 administration on calcium balance and bone mass in young rats fed a normal or low calcium diet. METHODS: Forty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into four groups with 10 rats in each group: 0.5% (normal) calcium diet, 0.1% (low) calcium diet, 0.5% calcium diet + vitamin K2 (menatetrenone, 30 mg/100 g chow diet), and 0.1% calcium diet + vitamin K2. After 10 weeks of feeding, serum calcium and calciotropic hormone levels were measured, and intestinal calcium absorption and renal calcium reabsorption were evaluated. Bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. RESULTS: Feeding a low calcium diet induced hypocalcemia, increased serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels with decreased serum 25-hydrovyvitamin D [25(OH)D] level, stimulated intestinal calcium absorption and renal calcium reabsorption, and reduced cortical bone mass as a result of decreased periosteal bone gain and enlarged marrow cavity, but did not significantly influence cancellous bone mass. Vitamin K2 administration in rats fed a low calcium diet stimulated renal calcium reabsorption, retarded the abnormal elevation of serum PTH level, increased cancellous bone mass, and retarded cortical bone loss, while vitamin K2 administration in rats fed a normal calcium diet stimulated intestinal calcium absorption by increasing serum 1,25(OH)2D level, and increased cortical bone mass. CONCLUSION: This study clearly shows the differential response of calcium balance and bone mass to vitamin K2 administration in rats fed a normal or low calcium diet.     

Intestinal calcium absorption: Molecular vitamin D mediated mechanisms

Rickets and hyperparathyroidism caused by a defective Vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25-(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min after oral gavage in two strains of VDR-knock out (KO) mice (Leuven and Tokyo KO) and observed a threefold lower area under the curve in both KO-strains. Moreover, we evaluated the expression of intestinal candidate genes, belonging to a new class of calcium channels (TRPV), involved in transcellular calcium transport. The calcium transport protein ECaC2 was more abundantly expressed at mRNA level than ECaC1 in duodenum, but both were considerably reduced (ECaC2 > 90%, ECaC1 > 60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was only significantly decreased in the Tokyo KO, whereas PMCA(1b) expression was normal in both VDR-KOs. In Leuven wild type mice, a high calcium diet inhibited (> 90%), and 1,25(OH)(2)D(3) or low calcium diet induced (sixfold) duodenal ECaC2 expression and, to a lesser degree, ECaC1 and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas both ECaC mRNA expressions remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular ECaC2 or TRPV6) is strongly vitamin D dependent and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D dependent active calcium absorption.     

Effect of inhibitors of slow calcium current on resting calcium efflux from cat papillary muscles

Examination of influence of divalent cations (Co2+, Ni2+, Mn2+) and organic blockers (verapamil and D600) on calcium efflux from resting mammalian myocardium shows that they either inhibit or increase transiently 45Ca2+ efflux, depending on the site of action. It seems that those agents whose sites of action are limited to the sarcolemma inhibit Ca2+ efflux. Co2+, Ni2+, verapamil and D600 belong to this group. Mn2+ ions which act also apparently on intracellular structures increase transiently Ca2+ efflux. Such a finding illustrates a diversity in mechanisms of action in a group of agents generally classified as calcium channel blockers.     

Intestinal absorption of calcium from yogurt in lactase-deficient subjects.

Fractional intestinal absorption of calcium (FACa) was measured using radioactive calcium and 200 mg of calcium carrier provided either by yogurt or by CaCl2 in 7 lactase-deficient (L(-] and 7 normal (L(+] subjects. During the control period prior to yogurt consumption, mean calcium intake was 819 mg per day in L(-) and 931 mg per day in L(+) subjects (NS). In both groups of subjects yogurt increased FACa from 20.8 +/- 3.9% to 26.9 +/- 7.2% (P = 0.065) in L(+) subjects and from 20.2 +/- 5.6% to 23.5 +/- 6.4% (P = 0.050) in L(-) subjects. The significant increase in FACa observed in L(-) subjects indicates that yogurt, which is an autodigesting source of lactose, does not impair calcium absorption. FACa increase could reflect the lower dietary calcium intake in L(-) subjects when compared with L(+) subjects, due to avoidance of milk and non-fermented dairy products which could cause intestinal discomfort. It is concluded that yogurt is a well-tolerated and efficient source of calcium in subjects with lactase deficiency.     

Impaired body calcium metabolism with low bone density and compensatory colonic calcium absorption in cecectomized rats

An earlier study reported that cecal calcium absorption contributes less than 10% of total calcium absorbed by the intestine, although the cecum has the highest calcium transport rate compared with other intestinal segments. Thus, the physiological significance of the cecum pertaining to body calcium metabolism remains elusive. Herein, a 4-wk calcium balance study in cecectomized rats revealed an increase in fecal calcium loss with marked decreases in fractional calcium absorption and urinary calcium excretion only in the early days post-operation, suggesting the presence of a compensatory mechanism to minimize intestinal calcium wasting. Further investigation in cecectomized rats showed that active calcium transport was enhanced in the proximal colon but not in the small intestine, whereas passive calcium transport along the whole intestine was unaltered. Since apical exposure to calcium-sensing receptor (CaSR) agonists similarly increased proximal colonic calcium transport, activation of apical CaSR in colonic epithelial cells could have been involved in this hyperabsorption. Calcium transporter genes, i.e., TRPV6 and calbindin-D(9k), were also upregulated in proximal colonic epithelial cells. Surprisingly, elevated serum parathyroid hormone levels and hyperphosphatemia were evident in cecectomized rats despite normal plasma calcium levels, suggesting that colonic compensation alone might be insufficient to maintain normocalcemia. Thus, massive bone loss occurred in both cortical and trabecular sites, including lumbar vertebrae, femora, and tibiae. The presence of compensatory colonic calcium hyperabsorption with pervasive osteopenia in cecectomized rats therefore corroborates that the cecum is extremely crucial for body calcium homeostasis.     

1,25-Dihydroxyvitamin D and not calcium is the major regulator of calbindin-D 9-kDa mRNA levels in vivo.

A possible role of calcium in vivo on intestinal calbindin-D 9-kDa mRNA levels has been studied in rats. In vitamin D-deficient rats, a marked increase in dietary calcium has a small but significant effect on calbindin-D 9-kDa mRNA levels, despite a dramatic increase in serum calcium concentration that clearly resulted from increased intestinal absorption of calcium. On the other hand, vitamin D under all circumstances increased calbindin-D 9-kDa mRNA levels, with the greatest levels found in animals on a low calcium diet where little or no calcium is available for absorption. These results strongly support the idea that 1,25-dihydroxyvitamin D is directly responsible for the induction of calbindin-D 9-kDa.     

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.     

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.     

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line

Background  

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.     

Intestine, bone, and mammary gland contributions to maternal plasma calcium increase after abrupt weaning.

The response of maternal plasma calcium concentration to the abrupt and permanent removal of the suckling pups on Day 13 of lactation was investigated. Maternal plasma calcium did not change at 6 hr or 12 hr following pup removal. At 18 hr and 24 hr after weaning, the maternal plasma calcium concentration increased in mothers consuming either 0.47% calcium or 0.02% calcium diets. At 24 hr after weaning, the plasma calcium increase in mothers consuming low dietary calcium was 55% that of mothers consuming adequate dietary calcium. The contribution of the mammary gland to the plasma calcium increase in rats consuming the low dietary calcium was investigated by removing the mammary glands. Following mammary gland removal, plasma calcium increased 50% compared with mothers that had intact mammary glands. The data suggest that intestinal absorption of calcium and bone calcium mobilization remain stimulated by the lactation process for at least 24 hr after removal of the nursing pups.     

Characterization of dietary phosphorus-dependent duodenal calcium uptake in vitamin D-deficient chicks

The effect of dietary phosphorus on intestinal calcium uptake was examined in duodenal cells isolated from vitamin D-deficient chicks. Cells from chicks on a high phosphorus diet accumulated calcium at a rate 38% higher than cells from animals on a normal phosphorus diet. Diet high in calcium did not affect calcium absorption in duodenal cells. The dietary phosphorus effect on calcium absorption was specific. Uptake of -methyl glucoside was not altered. Increase in calcium absorption by a high phosphorus diet was not due to a change in cellular energy metabolism nor to the content of phosphorus in cells. Kinetically, a high phosphorus diet increased the V _max of calcium uptake; the affinity for calcium was unaffected. The effectiveness of dietary phosphorus to enhance the intestinal calcium uptake could also be demonstrated in brush border membrane vesicles. The increase in calcium uptake was not due to an alteration in membrane binding capacity nor to calcium efflux from vesicles. To test the hypothesis that a high phosphorus diet may affect membrane transport by altering phospholipid metabolism in duodenal cells, we examined the phospholipid content in isolated brush border membranes. The content of phosphatidylcholine, phosphatidylserine, phosphatidyinositol and phosphatidylethanolamine was not altered by the high phosphorus diet. These findings suggest that the vitamin D-independent and dietary phosphorus-dependent effect on intestinal calcium absorption was primarily due to a change in the calcium flux at the luminal side of the cells. However, the precise mechanism is still not clear.     

Influx of Ca2+ via Cav1.3 calcium channels in satellite cells of muscle fibers in rats

Voltage-dependent L-type Ca_v1.3 channels have been detected in satellite cells localized to muscle fibers. It was established that the action of carbachol, which activates nicotinic acetylcholine receptors and causes cell membrane to depolarize, resulted in the activation of these channels. In addition, verapamil and amlodipine, selective L-type calcium channel blockers, suppressed extracellular calcium influx into the cytoplasm. It was noted that in a calcium-free medium, carbachol had no influence on the concentration of calcium in the cytoplasm of satellite cells, whereas adrenaline induced calcium efflux from intracellular stores. In addition, calcium influx into the cytoplasm was not suppressed by verapamil and amlodipine under the action of adrenaline and noradrenalin in a medium with calcium, and an ICI-118551 blocker of β₂-adrenoreceptros significantly decreased the increase in the concentration of calcium in the cytoplasm.     

Parathyroid hormone-related protein: a calcium regulatory factor in sea bream (Sparus aurata L.) larvae

The effects of an N-terminal peptide (amino acids 1-38) of Fugu parathyroid hormone-related protein (PTHrP 1-38) on calcium regulation of larval sea bream were investigated in seawater (36 per thousand) and after transfer to dilute seawater (12 per thousand). Exposure to PTHrP 1-38 evoked a 1.5-fold increase in calcium influx in both full-strength and dilute seawater. Calcium influx in dilute seawater-adapted larvae was roughly one-half that observed in full-strength seawater controls. PTHrP 1-38 also reduced drinking of fish in seawater but, at all concentrations tested, was without effect in dilute seawater. The amount of water imbibed was 55% lower in dilute seawater than in seawater. PTHrP 1-38 exposure affected the calcium influx route: the main contribution of calcium uptake shifted from intestinal absorption to extraintestinal uptake, probably by the induction of a dose-dependent increase in branchial (active) transport. Moreover, seawater-adapted fish exposed to 1 nM and 10 mM PTHrP 1-38 experienced a 2.5-fold reduction in overall calcium efflux. Overall, the calciotropic action of PTHrP 1-38 resulted in a dose-dependent increase in net calcium balance.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.