Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2.

The recently discovered epithelial calcium channels ECaC1 and ECaC2 are thought to play an important role in active calcium absorption in the intestine and kidney. Vitamin D-responsive elements (VDRE) were detected in the promoter sequence of human ECaC1 and regulation of ECaC by the steroid hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has been postulated. In this study we describe the structure of two murine ECaCs genes, each consisting of 15 exons localized on chromosome 6. Murine ECaC2 expression was found in many target tissues of 1,25-(OH)(2)D(3), including skin and osteoblastic cells, while ECaC1 expression is confined to the kidney. By screening the murine promoter sequences, we detected a putative VDRE in ECaC1 and an estrogen response element in ECaC2. However, experiments in mice with a mutant, nonfunctioning vitamin D receptor showed that expression of ECaC1 in the kidney and of ECaC2 in duodenum is regulated by calcium levels, but not by 1,25-(OH)(2)D(3). Also, estrogen-deficient ovariectomized (OVX) mice and OVX mice supplemented with estradiol showed unchanged duodenal ECaC2 expression compared with control mice. We conclude that ECaC expression in the kidney and the intestine is regulated by extracellular calcium but not by vitamin D or estrogen in vivo in mice.

1alpha-hydroxyvitamin D2 is less toxic but not bone selective relative to 1alpha-hydroxyvitamin D3 in ovariectomized rats.

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1alpha-hydroxyvitamin D2 [1alpha(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1alpha(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1alpha(OH)D2 and 1alpha(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 microg of 1alpha(OH)D2 or 1alpha(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1alpha(OH)D2 was significantly less toxic compared with 1alpha(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1alpha(OH)D2 and 1alpha(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1alpha(OH)D2 and 1alpha(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1alpha(OH)D3 and 1alpha(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1alpha(OH)D2 and 1alpha(OH)D3. These data show that although 1alpha(OH)D2 was slightly but significantly less toxic compared with 1alpha(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1alpha(OH)D2 in bone.

High insulin and low IGF-I plasma levels following pancreas transplantation in rats. Implications for bone and mineral metabolism.

Primary disturbances in mineral metabolism and deficiencies in insulin and insulin-like growth factor-I (IGF-I) have been implicated in the pathogenesis of diabetic osteopenia. This prompted us to investigate whether normal bone minerals and bone morphology are preserved after pancreas transplantation. To this end, 8 inbred rats (transplants) were compared with 9 sham-operated rats (controls) 20 months after orthotopic pancreas transplantation. While basal levels of insulin remained unaffected by transplantation, an oral glucose load elicited hyperinsulinemia (integrated incremental response: mean +/- SEM, 62+/-8 nmol l(-1) 60 min in transplants vs. 32+/-6 nmol l(-1) 60 min in controls; p<0.01) in the presence of normal glucose levels. Fecal and urinary excretion and fractional intestinal absorption of calcium, magnesium and phosphorus, net calcium absorption and the respective serum mineral levels were unchanged after transplantation, as were those of the calciotropic hormones. Serum osteocalcin and bone alkaline phosphatase remained unaffected, and urinary excretion of pyridinium and deoxypyridinium were unchanged. Fasting plasma IGF-I concentration was significantly decreased in transplants (930+/-42 ng ml(-1)) vs. control rats (1074+/-49 ng ml(-1); p < 0.05). Despite similar physical and chemical properties of bone in both groups, histomorphometry revealed slight osteopenia in transplant rats, as reflected by a 38% reduction in the cancellous bone area of the proximal tibial metaphysis. Plasma IGF-I levels were significantly correlated with bone mineral apposition rate (r=0.70, p<0.02), osteoblast perimeter (r=0.60, p<0.05) and osteoid perimeter (r=0.60, p<0.05). In conclusion, pancreas transplantation preserves physical and chemical properties of bone, but bone metabolism is not completely normal after transplantation, as evidenced by decreased cancellous bone. This might have resulted from the insulin resistance associated with the lowering of the plasma IGF-I level, which was correlated with the mineral apposition rate.

Skeletal effects of zinc deficiency in growing rats.

There is ample evidence that zinc plays an important role in bone metabolism and zinc deficiency has been implicated as a risk factor in the development of osteoporosis. It was the aim of the present study to investigate the skeletal effects of alimentary zinc deficiency in growing rats using quantitative bone histomorphometry. Twenty-four male Sprague Dawley rats with a mean initial body weight of 101 +/- 2 g were allocated in two groups of 12 rats each and had free access to a semi-synthetic, casein-based, zinc-deficient diet (0.76 mg zinc/kg) or to the same diet supplemented with 60 mg zinc per kg. All rats were sacrificed 42 days after the start of the experiment and the right distal femur was removed for bone histomorphometry. Relative to controls (+Zn), the zinc-deficient rats (-Zn) had a significantly lower body weight and about an 80% reduction in plasma and femur zinc concentration. The histomorphometric evaluation of the distal femoral metaphysis showed that zinc deficiency led to a 45% reduction (p < 0.01) in cancellous bone mass and to a deterioration of trabecular bone architecture, with fewer and thinner trabeculae. The osteopenia in -Zn rats was accompanied by significant reductions in osteoid perimeter (-31%, p < 0.05), osteoblast perimeter (-30%, p < 0.05), and osteoclast number (-38%, p < 0.01) relative to +Zn controls. We conclude that zinc deficiency induced low turnover osteopenia in femoral cancellous bone of growing rats. These results support the hypothesis that zinc deficiency during growth may impair the accumulation of maximal bone mass in humans; additionally, they suggest that zinc deficiency may play a role as a risk factor in the pathogenesis of osteoporosis.

Gastric fundectomy in the rat: effects on mineral and bone metabolism, with emphasis on the gastrin-calcitonin-parathyroid hormone-vitamin D axis.

In humans, gastric surgery results in in osteopenia via mechanisms that are insufficiently understood; surgery-induced changes in the hormonal axes involving the stomach, thyroid, and the parathyroids may play a role. To study this in more detail, we evaluated calcium (Ca), magnesium (Mg), and phosphorus (P) metabolism as well as physical, chemical, and histomorphometric bone parameters in rats rendered hypergastrinemic by fundectomy (FX). In independent experiments, the response to an oral Ca challenge was investigated in intact rats versus FX, and in thyroidectomized versus thyroid-intact FX rats. Sixteen weeks following FX, body weight was approximately 80% that of sham-operated controls. In urine, P excretion was elevated fivefold, the pH was significantly decreased, and cAMP excretion was elevated as compared with controls; serum parathyroid hormone (PTH), calcitonin, 25OHD, Ca, Mg, and P were normal; gastrin and 1,25(OH)2D were elevated. On the basis of bone ash mineral content, FX rats developed significant osteopenia, and histomorphometry indicated only slightly elevated bone turnover and mineralization. Following oral Ca, thyroid-intact FX rats developed hypercalcemia, serum gastrin decreased, and calcitonin increased significantly; in thyroidectomized FX rats, calcitonin remained at baseline levels although there was a similar degree of hypercalcemia; PTH decreased during the hypercalcemic period in both groups. Serum gastrin did not correlate with calcitonin or PTH, and in multivariate regression analysis the only predictor of serum 1, 25(OH)2D was urinary phosphorus. It was concluded that in the FX rat (1) osteopenia is not caused by intestinal Ca malabsorption, vitamin D, Ca deficiency, or secondary hyperparathyroidism; (2) osteopenia may be related to PTH-independent urinary hyperexcretion of P, followed by a rise of serum 1,25(OH)2D; (3) the existence of endocrine axes among gastrin, calcitonin, and PTH cannot be substantiated. FX osteopenia appears to be related to gastric acid abolition, and the reactive hypergastrinemia probably stabilizes the mass and turnover of bone.

Therapeutic efficacy of 1alpha,25-dihydroxyvitamin D3 and calcium in osteopenic ovariectomized rats: evidence for a direct anabolic effect of 1alpha,25-dihydroxyvitamin D3 on bone.

It is an important question for clinical therapy of osteoporosis with vitamin D metabolites whether these compounds exert their beneficial effects on the skeleton indirectly through an increase in intestinal calcium absorption or whether there is also a major direct component of action on bone. In this study, female 6-month-old Fischer rats were either ovariectomized (OVX) or sham operated. One month before surgery, all rats were placed on a diet containing 0.25% calcium and were kept on this diet throughout the study. Beginning 3 months post-OVX, groups of OVX rats orally received vehicle, a calcium supplement, low dose (0.025 microg/kg x day) or high dose (0.1 microg/kg x day) 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or combinations of low and high dose 1,25-(OH)2D3 with the calcium supplement. By 3 months postsurgery, pretreatment OVX controls had lost 74% and 37% of tibial and vertebral cancellous bone, respectively. Two-way factorial ANOVA showed that a 3-month treatment of osteopenic OVX rats with 1,25-(OH)2D3 dose dependently increased vertebral and tibial cancellous bone mass (P < 0.001 and P = 0.021, respectively) and trabecular width (P < 0.001). Furthermore, 1,25-(OH)2D3 increased serum calcium (P = 0.028) and urinary calcium excretion (P < 0.001) and reduced serum PTH levels (P < 0.001), osteoclast numbers (P < 0.001), and urinary collagen cross-links excretion (P < 0.001). Calcium supplementation alone was without therapeutic effect, and there was no significant two-way interaction between the individual treatment effects of 1,25-(OH)2D3 and calcium on bone mass. These data indicate that the anabolic effects of 1,25-(OH)2D3 in osteopenic OVX rats are mediated through a direct activity on bone.

Short-term treatment of rats with high dose 1,25-dihydroxyvitamin D3 stimulates bone formation and increases the number of osteoblast precursor cells in bone marrow.

Using an experimental rat model, this study was undertaken to assess the effects of a short-term application of high dose 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on calcium homeostasis, cancellous bone formation, and numbers of osteoblast precursors in ex vivo bone marrow cultures. For Exp 1 and 2, 6-month-old female rats were sc injected with either 0.2 microg 1,25-(OH)2D3/kg x day or vehicle on days 1, 2, and 3 of the studies. Serum calcium and urinary excretion of calcium were monitored for 12 days in Exp 1. In Exp 2, the rats were ip labeled with five different fluorochromes on days 0, 5, 10, 15, and 20, respectively. Half of the rats in each group were killed on day 7, the rest of the rats were killed on day 24, and the first lumbar vertebrae were processed for histomorphometry. In Exp 3, 0.2 microg 1,25-(OH)2D3/kg BW or vehicle was sc administered to 6-month-old male rats on days 1, 2, and 3, and the number of colony-forming units with the ability to express alkaline phosphatase, to calcify, and/or to synthesize collagen were enumerated sequentially on days 4, 6, 8, 10, 12, and 14 in bone marrow cultures. Short-term 1,25-(OH)2D3 treatment resulted in increased values for serum and urinary calcium during the treatment phase in Exp 1, depressed osteoclast numbers and strongly elevated osteoblast perimeter by day 7, and stimulated mineral apposition rate and bone formation rate in the interval between days 5-15 of Exp 2. Moreover, 1,25-(OH)2D3 administration to rats significantly enhanced the number of mesenchymal precursor cells in bone marrow with the ability to differentiate into an osteoblastic phenotype in ex vivo bone marrow cultures on day 4 of Exp 3. These studies provide evidence that short-term 1,25-(OH)2D3 treatment creates new bone remodeling units and augments osteoblast recruitment and osteoblast team performance in rat cancellous bone.

Osteopenia following total gastrectomy in the rat--state of mineral metabolism and bone histomorphometry.

Total gastrectomy (GX) in humans is frequently followed by osteopenia, but the details are unclear. The present investigations in the rat were aimed at elucidating its pathogenesis. Seventeen weeks after GX, we evaluated Ca, Mg and P metabolism as well as bone parameters, including fluorochrome-based bone histomorphometry. In GX rats, fecal Mg was increased, but intestinal absorption of P, Ca, and Mg was within normal limits, as was the urinary excretion of Ca, Mg, hydroxyproline and the pyridinium cross-links. In contrast, urinary P as well as cyclic AMP were significantly increased. In serum of GX rats, gastrin and 25-hydroxyvitamin D (25-OHD) were decreased, and Ca, Mg, P, parathyroid hormone (PTH), calcitonin, and the bone marker osteocalcin were normal, whereas 1,25-dihydroxyvitamin D [1,25(OH)2D] was significantly increased. GX rats had significantly reduced bone density and mineral content, severe high-turnover osteopenia, characterized by normal width but significantly decreased maturation time of osteoid, increased bone formation rate, and increased numbers of osteoclasts. We concluded that after GX (1) there is high-turnover osteopenia with normal mineralization and other histomorphometric features resembling those seen in states with hyperphosphaturia and subsequent hypervitaminosis D; (2) normal serum PTH levels and several indirect indicators of parathyroid gland function argue against the presence of (secondary) hyperparathyroidism, whereas increased bone mobilization due to elevated 1,25(OH)2D explains the maintenance of homeostasis of serum minerals, especially Ca, at the expense of bone mineral; (3) a complex interplay of mineral-metabolic effectors exists, among which low 25-OHD-PTH-independent renal phosphate losses, and high 1,25(OH)2D are prominent features. The presented animal model is recommended for future research in this area.

Vitamin D metabolites prevent vertebral osteopenia in ovariectomized rats.

The present study investigated the prophylactic effects of vitamin D metabolites and vitamin D metabolite combinations on static and dynamic, tetracycline-based, histomorphometric parameters in the axial skeleton of ovariectomized rats. Forty-three Fischer-344 rats (10 weeks old, 130 g each body weight, BW) were either bilaterally ovariectomized (OVX) or sham-operated (SHAM). The rats were allocated into the following groups: SHAM; OVX; OVX + 7.5 ng 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]/rat/day; OVX + 15 ng 1 alpha,24R,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3]/rat/day; OVX + 75 ng 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]/rat/day; OVX + 7.5 ng 1,25(OH)2D3/rat/day + 15 ng 1,24,25(OH)3D3/rat/day; OVX + 7.5 ng 1,25(OH)2D3/rat/day + 75 ng 24,25(OH)2D3/rat/day. The vitamin D metabolites were fed orally starting 4 weeks after surgery. Urine and blood samples were collected 12 and 16 weeks postovariectomy, respectively. Sixteen weeks after surgery, all rats were sacrificed, and the first lumbar vertebrae were processed undecalcified for histomorphometric analysis. Ovariectomy induced a highly significant reduction (P less than 0.001) of cancellous bone mass in the secondary spongiosa of the lumbar vertebral body. The bone loss in OVX rats was accompanied by a distinct elevation of all histomorphometric parameters of bone formation and resorption. 1,25(OH)2D3 and both vitamin D metabolite combinations significantly raised serum calcium levels and prevented the bone loss by inhibiting the increased bone resorption in OVX rats. In the applied dosage, 1,24,25(OH)3D3 and 24,25(OH)2D3 alone were ineffective in preserving the cancellous bone of the lumbar vertebra in OVX rats.(ABSTRACT TRUNCATED AT 250 WORDS)