Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cellsIn vitro

Summary Binding of [³H] 1,25 (OH)₂D₃ and effects of 1,25 (OH)₂D₃ on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [³H] 1,25 (OH)₂D₃ by a 3.5 S macromolecule with DNA binding, K_D 6.2×10⁻¹⁰M and N_max 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10⁻⁸ M 1,25 (OH)₂D₃, but not 25 (OH)D₃, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.     

Effect of vitamin D on growth cartilage cell proliferation in vitro

Disturbed calcification of the growth plate and stunting is a frequent finding in vitamin D-deficiency rickets, vitamin D-dependency rickets and renal osteodystrophy, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of Ic, 25-dihydroxy vitamin D₃ [1,25(OH)₂D₃] receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In tibial growth plates and in primary cultures of tibial growth cartilage of male Sprague-Dawley rats (80 g) specific binding of [³H]-1,25(OH)₂D₃ was noted. Scatchard analysis revealed the presence of a single class of non-interacting binding sites.K _d was 10^–11 M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA-cellulose affinity chromatography. By immunohistology, growth cartilage cells (rabbit tibia) were shown to express nuclear 1,25(OH)₂D₃ receptors, most prominently in the proliferative and early hypertrophic zone. This corresponds to binding data which showed highest binding of 1,25(OH)₂D₃ in the logarithmic growth phase (12,780 molecules/cell versus 4,538 molecules/cell in confluent cells) in primary cultures of growth plate chondrocytes. In the presence of delipidated fetal calf serum 1,25(OH)₂D₃ had a biphasic effect on cell proliferation and density, i.e. stimulation at 10^–12 M and dose-dependent inhibition at 10^–10 M and below. Inhibition was specific and not seen with 24 (R), 25-dihydroxyvitamin D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and specific effects of 1,25(OH)₂D₃ on chrondrocyte proliferation in vitro point to a role for vitamin D in the homeostasis of growth cartilage of the rat.     

1,25(OH)2D3 and dihydrotestosterone interact to regulate proliferation and differentiation of epiphyseal chondrocytes

Growth plate chondrocytes are affected by 1,25(OH)₂D₃ and androgens, which may critically interact to regulate proliferation and differentiation during the male pubertal growth spurt. We investigated possible interactions of 1,25(OH)₂D₃ and the non-aromatizable androgen dihydrotestosterone (DHT) in primary chondrocyte cultures from young male rats. DHT and 1,25(OH)₂D₃ independently stimulated DNA synthesis and cell proliferation in a dose-dependent manner with maximally effective doses of [10^-8 M] and [10^-12 M], respectively. Both DHT and 1,25(OH)₂D₃ stimulated the expression and release of IGF-I, and the proliferative effects of each hormone were prevented by an IGF-I antibody. DHT and 1,25(OH)₂D₃ increased messenger RNAs (mRNAs) of their cognate receptors and of IGF-I receptor mRNA (IGF-I-R). 1,25(OH)₂D₃ also stimulated mRNA of the androgen receptor (AR), whereas DHT did not affect mRNA of the vitamin-D receptor (VDR). Coincubation with both steroid hormones did not stimulate receptor mRNAs more than either hormone alone. The proliferative effects of DHT and 1,25(OH)₂D₃ were completely inhibited by simultaneous incubation with both hormones, despite potentiation of IGF-I synthesis. In contrast, both hormones synergistically stimulated cell differentiation as judged by alkaline phosphatase activity, collagen X mRNA, and matrix calcification in long-term experiments. We conclude that DHT and 1,25(OH)₂D₃ interact with respect to chondrocyte proliferation and cell differentiation. The proliferative effects of both hormones are mediated by local IGF-I synthesis. Simultaneous coincubation with both hormones blunts the proliferative effect exerted by either hormone alone, in favor of a more marked stimulation of cell differentiation.     

Dexamethasone and 1,25(OH)2 vitamin D3 modulate the synthesis of insulin-like growth factor-I in osteoblast-like cells

Summary In the present study, we have shown that insulin-like growth factor-I (IGF-I) was released by primary cultures of rat osteoblast-like (ROB) cells into the conditioned medium (CM). Dexamethasone (DEX) caused a dose-dependent inhibition of the IGF-I. At 10⁻⁸ M, DEX reduced IGF-I level to 70% of the control value (P<0.05); at 10⁻⁷ M DEX, the IGF-I level was further reduced to 60% of the control (P<0.01). The active vitamin D metabolite 1,25-dihydroxycholecalceferol [1,25(OH)₂D₂] slightly increased the IGF-I level, but the increase was not statistically significant. However, in combined treatments of 10⁻⁷ M DEX and 10⁻⁸ M of 1,25(OH)₂D₃, the inhibition of DEX was partially antagonized by the presence of 1,25(OH)₂D₃. Studies with metabolically radiolabeled IGF-I by immunoprecipitation indicated the changes of IGF-I in the CM reflected synthesis of the protein by the cells. The alteration of IGF-I level may mediate some of the actions of these steroid hormones on bone cells.     

Regulation of Murine Osteoblast Macrophage Colony-Stimulating Factor Production by 1,25(OH)2D3

Macrophage colony stimulating factor (MCSF) is important for formation of osteoclasts. We investigated the ability of 1,25(OH)₂D₃ to regulate osteoblast production of MCSF. Mouse calvarial osteoblasts were cultured for 2 days ± 1,25(OH)₂D₃. Since 1,25(OH)₂D₃ decreased osteoblast proliferation by 17.6 ± 1% at 10 nM and 11 ± 4% at 1 nM, the effect of growth rate on MCSF secretion was examined. Limiting cell proliferation by serum did not affect MCSF production. 1,25(OH)₂D₃ (1 nM) increased MCSF production (U/10⁵ cells) maximally by 68 ± 33% (n = 3) with an ED₅₀ for 1,25(OH)₂D₃ of 5 × 10⁻¹¹ M. To investigate effects of 1,25(OH)₂D₃ on MCSF gene regulation, RT-PCR primers were designed to identify the mRNA coding for the membrane-bound isoform of MCSF. Simultaneous RT-PCR of glyceraldehyde-phosphate dehydrogenase (GAP) allowed semiquantitative assessment of MCSF mRNA between treatment groups expressed as the MCSF/GAP RT-PCR product ratio; both MCSF and GAP (+) primers were labeled with ³²P-ATP for phosphorimage quantitation. The membrane-bound MCSF/GAP PCR product ratio was not affected by proliferative rate when growth was limited by [serum]. The MCSF/GAP RT-PCR product ratio was dose dependently increased by 1,25(OH)₂D₃, maximally at 1 nM at 2.2 ± 0.2 = fold (n = 10). 1,25 (OH)₂D₃ also increased the expression of an RT-PCR MCSF/GAP product ratio which represented the secreted isoform of MCSF. The ability of 1,25(OH)₂D₃ to pretranslationally regulate expression of membrane-bound osteoblast MCSF may be important in osteoblast:osteoclast interactions. Received: 12 August 1995 / Accepted: 25 March 1996     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

In Vivo Treatment with Calcitriol (1,25(OH)2D3) Reverses Age-Dependent Alterations of Intestinal Calcium Uptake in Rat Enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D₃; [1,25(OH)₂D₃] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)₂D₃ stimulation of Ca²⁺ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20–24-month-old rats given 1,25(OH)₂D₃ for 3 days (30 ng/100 g bw/day), calcitriol (10⁻¹⁰ M, 3–5 minutes) stimulated Ca²⁺ uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)₂D₃. When the aged rats were injected with 25(OH)D₃, similar Ca²⁺ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1,25(OH)₂D₃-dependent changes in Ca²⁺ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)₂D₃ stimulation of duodenal cell Ca²⁺ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)₂D₃ or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system. Received: 26 December 1997 / Accepted: 12 May 1998     

Lack of In vitro evidence for storage of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 1,25(OH)2D3 binding protein in skeletal matrix

A few studies have reported on the measurement of 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) in bone, using chloroform/methanol extraction and radioreceptor assay. As the significance of bone 1,25(OH)₂D₃ content was not defined in any of these reports, the objective of the current investigation was to determine whether 1,25(OH)₂D₃ may be stored in skeletal matrix. Bone powder samples from the iliac crest were extracted in ethylacetate/cyclohexane and 1,25(OH)₂D₃ isolated from the extract by means of Sephadex LH-20 and high pressure liquid chromatographic separation and subsequently measured by radioimmunoassay (RIA). Within the detection range of the RIA, no 1,25(OH)₂D₃ could be measured, suggesting that 1,25(OH)₂D₃ is not stored in skeletal matrix. Vitamin D bone concentrations previously measured may therefore have reflected plasma contamination. Consistent with this hypothesis, only traces of skeletal 1,25(OH)₂D₃ binding protein were measured when compared with serum values. Although 1,25(OH)₂D₃ may act as a potential local determinant of bone remodeling, there is no evidence supporting a delayed paracrine function by matrix-derived 1,25(OH)₂D₃.     

Effects of phosphate and 1,25(OH)2D3 on in vitro bone collagen synthesis in the hypophosphatemic mouse

Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] (10⁻¹²M–10⁻⁷M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)₂D₃ was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [³H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)₂D₃ inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)₂D₃-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)₂D₃, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)₂D₃ was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)₂D₃ in the Hyp mouse.     

1,25 Dihydroxyvitamin D3 is required for growth-independent expression of alkaline phosphatase in cultured rat osteoblasts

Summary Osteoblastic cells were isolated from periosteum-stripped parietal bones of neonatal rat calvaria, seeded at low density (5,000 cells/35 mm of Falcon dish), and cultured for 6 days in BGJ medium supplemented with 20% of vitamin D-depleted FCS or vitamin D and calcium-depleted FCS, with daily addition of 1,25 dihydroxyvitamin D₃ (10⁻⁹ M) or 24,25-dihydroxyvitamin D₃ (10⁻⁹ M). Plating efficiency, clonal growth (number and size distribution of the colonies formed), and the alkaline phosphatase phenotype were evaluated on days 2 and 6 of culture. (1) Culture for 6 days in media not supplemented with 1,25(OH)₂D₃ led to a significant (P<0.001) loss of the alkaline phosphatase phenotype of the osteoblastic cells; the loss was greater in proliferating cells than in nonproliferating ones and occurred in both 0.12 mM or 1.1 mM ionized calcium concentrations. (2) Daily addition of 1,25(OH)₂D₃ (10⁻⁹ M) but not 24,25(OH)₂D₃ maintained the basal percentage of Alk Pase positive cell units in nonproliferating cells and significantly reduced the loss of this phenotype in proliferating colonies. (3) This effect did not stem from an action of the hormone on cell growth. 1,25(OH)₂D₃ was also found to enhance the adhesiveness of the seeded osteoblasts, irrespective of the medium calcium concentration.     

Effect of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion

Summary The active vitamin D metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in anin vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)₂D₃ and the related metabolite 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)₂D₃. In short-term studies (30–120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24–48 hours, using primary tissue culture in the presence of test agents), neither vitamin D₃ nor 25(OH)D₃ affected PTH secretion or pre-proPTH mRNA over the concentration range 10⁻¹¹–10⁻⁷M. On the other hand, 24,25(OH)₂D₃ produced significant suppression of both pre-proPTH mRNA (77% of control,P<.01) and PTH secretion (75% of control,P<.005) at 10⁻⁷ M. By comparison, 10⁻¹¹ M 1,25(OH)₂D₃ produced levels of suppression (25–30%) of both pre-proPTH mRNA and PTH secretion comparable to 10⁻⁷ M 24,25(OH)₂D₃, while even greater suppression (40–50%) occurred at 10⁻⁹-10⁻⁷ M 1,25(OH)₂D₃. From these studies, we conclude that vitamin D₃ and 25(OH)D₃ do not have significant effects on PTH synthesis and secretion over the range of doses tested. Compared with 1,25(OH)₂D₃, 24,25(OH)₂D₃ exhibits mild suppression at pharmacologic concentrations. The effect of 24,25(OH)₂D₃ prabably occurs through weak interaction of 24,25(OH)₂D₃ with the 1,25(OH)₂D₃ receptor.     

Biochemical characterization of 1,25(OH)2D3 receptors in chick embryonal duodenal cytosol

Summary This study presents measurements of serum vitamin D metabolites, calcium and phosphorus as well as measurements of the equilibrium dissociation constant for duodenal 1,25(OH)₂D₃ receptor in 15-, 18-, 19-, and 20-day chick embryos in comparison to that in 1- and 118-day-old chicks and to vitamin D-deficient chicks. The present results showed that: (a) serum 1,25(OH)₂D and 24,25(OH)₂D levels rise from 15 and 18 to days 19 and 20 of embryonic development while serum phosphate levels are stable; (b) serum calcium levels rise at hatching to adult levels; (c) the duodenal 1,25(OH)₂D₃ receptor is detectable in 15-day-old embryo and has a Kd similar to that of 118-day-old vitamin D-replete chicks; and (d) the activity of 1,25(OH)₂D₃ receptor in chick duodenal cytosol is maximal at hatching.     

Effects of vitamin D-binding protein on bone resorption stimulated by 1,25 dihydroxyvitamin D3

Summary Vitamin D and its metabolites are tightly bound to the serum vitamin D-binding protein (DBP) and only the free hormone is considered to be physiologically active. On the other hand, DBP could interact with cell membranes and even favor its intracellular entry. The present study was undertaken to examine the effects of DBP on bone resorption stimulated by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Forelimb bones from 19-day-old fetal rats were cultured for 5 days in the presence of purified human or rat serum albumin (hSAP or rSAP) and 1,25(OH)₂D₃, with or without human or rat DBP (hDBP or rDBP). Bone resorption was assessed by measuring the release of previously incorporated⁴⁵Ca. We found that the resorptive response to 1,25(OH)₂D₃ was minimally altered by hDBP (5 μM). The minimal effects of hDBP on 1,25(OH)₂D₃ activity on rat bones might be explained by a 6-fold lower affinity of hDBP (1.1×10⁷ M⁻¹) than rDBP (5.9×10⁷ M⁻¹) for 1,25(OH)₂D₃ or by species differences in cellular recognition of DBP. In a homologous rat system, however, rDBP at low (0.5 μM) or physiological (5 μM) concentration significantly decreased 1,25(OH)₂D₃-induced bone resorption. These data therefore support the hypothesis that free rather than DBP-bound 1,25(OH)₂D₃ is physiologically important.     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

Short-term course of 1,25(OH)2D3 stimulates osteoblasts but not osteoclasts in osteoporosis and osteoarthritis

Summary We investigated the effect of short-term, 1,25-dihydroxyvitamin D₃ therapy (4 μg/day for 4 days) on calcium metabolism in 27 postmenopausal women (11 cases with osteoporosis and 16 cases with osteoarthritis). Bone mass at the axial and appendicular skeleton was higher in osteoarthritis than in osteoporosis. Initial values of calcium metabolism were similar. Osteoporotic and osteoarthritic patients responded with a similar significant increase in serum osteocalcin (+61% and +54%, respectively), fasting urinary calcium excretion (+178% and +124%, respectively) and 24 hour calcium excretion (+148% and +142%, respectively). Parathyroid hormone (PTH) levels decreased significantly in both groups (−30% and −18%, respectively). Osteoclastic bone resorption, evaluated by urinary hydroxyproline excretion, was not stimulated in either group. We conclude that in osteoporosis and also in osteoarthritis (1) 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulation of osteoblast function is similar in production of osteocalcin; (2) the vitamin D target tissues react adequately to 1,25(OH)₂D₃ stimulation; (3) short-term high dose of 1,25(OH)₂D₃ does not stimulate bone resorption; and (4) the differences in bone mass between osteoarthritis and osteoporosis are not related to an alteration of the responsiveness to stimulation by 1,25 (OH)₂D₃.     

Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.     

1,25-Dihydroxyvitamin D3 control of free cytosolic Ca2+ in the kidney

Summary Changes in cytosolic calcium ([Ca]i) have been implicated in the regulation of various cellular processes. 1,25-dihydroxyvitamin D₃ (1,25 (OH)₂D₃) has been reported to increase [Ca]i in hepatocytes, Hl-60, parathyroid cells and osteoblasts. It is not clear, however, whether or not 1,25 (OH)₂D₃ increases [Ca]i in their another important target cells, renal cells. In order to adress this issue, the effect of 1,25 (OH)₂D₃ on [Ca]i was examined in a newly established mouse renal cortical slice perifusion system using a fluorescent Ca²⁺ indicator, fura-2. 1,25 (OH)₂D₃ at physiological concentrations increased [Ca]i within 2 min in a dose-dependent manner both in the presence and absence of extracellular Ca²⁺. 24,25 (OH)₂D₃ even at 10⁻⁷M did not affect [Ca]i levels. These results demonstrated that 1,25 (OH)₂D₃ very rapidly increased [Ca]i in another target cells, renal cells and our newly established mouse renal perifusion system may be useful to study the effects of vitamin D metabolites on [Ca]i in renal cells. This study was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare, Japan.     

Somatostatin inhibits duodenal calcium transport mediated by 1,25-dihydroxyvitamin D3 in perfused duodena from normal chicks

We have reported that physiological dose (30pM-650pM) of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] increased the unidirectional movement of⁴⁵Ca²⁺ from the lumen to the venous effluent within a few minutes in perfused duodena from normal chicks, and hypercalcemia inhibited this rapid stimulatory effect on calcium transport mediated by 1,25(OH)₂ D₃. The purpose of the present study was to determine the effect of somatostatin on calcium transport in chicks. The basal Ca²⁺ transport, in the absence of 1,25(OH)₂ D₃, did not change when 10⁻⁸M to 10⁻⁶M of somatostatin was added to the perfusate. The effect of 1,25(OH)₂D₃ on calcium transport, however, was completely abolished on addtion of 10⁻⁶M somatostatin in the perfusate, and partially blocked on addition of 10⁻⁷M somatostatin and 10⁻⁸M somatostatin had no effect on 1,25(OH)₂ D₃ mediated calcium transport. These results suggest that somatostatin may decrease intestinal calcium transport mediated by the rapid direct action of 1,25(OH)₂ D₃.     

In vitro effects of vitamin D3 metabolites on rat calvaria cAMP content

Summary Results from in vitro works suggest that 1,25- and 24,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃ and 24,25-(OH)₂D₃) act on bone via different mechanisms. The present investigation was performed to study the effect of these two metabolites and of their precursor 25-hyxdroxyvitamin D₃ (25-(OH)D₃) on bone cAMP content in vitro. Rats' paired half calvaria were incubated under sterile conditions with one vitamin D₃ derivative (10⁻¹³ to 10⁻⁹ M) or with ethanol (0.005 ml for 15 min to 24 h in 1 ml medium containing 0, 0.2, 1, 2, or 3 mM calcium. In some experiments: (a) cycloheximide (10⁻⁵M) was added simultaneously with the vitamin D₃ metabolites; (b) 1–84 bPTH (5 × 10⁻⁸ M) was added for 5 or 15 min at the end of the 24 h incubation. Calvaria were immersed in 1 ml TCA 5% 4°C and homogenized. The cAMP was extracted with diethylether and measured by a competitive protein binding assay. Results bring further evidence for a particular effect of low doses of 24,25-(OH)₂D₃ (10⁻⁹ to 10⁻¹²M) and of 25-(OH)D₃ (10⁻⁹ to 10⁻¹¹M) on bone, different from that of 1,25-(OH)₂D₃: cAMP content was higher in 24,25-(OH)₂D₃- or 25-(OH)D₃-treated and lower in 1,25-(OH)₂D₃-treated calvaria than in ethanol-treated ones with 1 mM calcium. The 1,25-(OH)₂D₃ effect persisted in calcium-free medium whereas 25-(OH)D₃ and 24,25-(OH)₂D₃ effects could not be observed with 0 mM nor with 3 mM calcium. The required duration of the preincubation (over 1 h) as well as the inhibitory action of cycloheximide may suggest an involvement of protein synthesis in the vitamin D₃ metabolites effects. Neither 1,25-(OH)₂D₃ nor 24,25-(OH)₂D₃ affected the PTH-induced increase in bone cAMP content.