Alterations of duodenal vitamin D-dependent calcium-binding protein content and calcium uptake in brush border membrane vesicles in aged Wistar rats: role of 1,25-dihydroxyvitamin D3

Previously we reported that uptake of Ca2+ in cells isolated from rat duodenum declined in senescence. In this paper we examined the possible mechanisms for this age-related defect. Duodenal vitamin D-dependent calcium-binding protein decreased steadily from 3-12 months (mo), followed by a minimal decline at 24 mo. On the contrary, Ca2+ uptake was not different in 3-, 6-, and 12-mo-old rats. A significant decline of Ca2+ uptake was observed at 24 mo. ATP contents in duodenal cells from 6- and 24-mo-old rats were not different. This suggests that the metabolic status of the duodenal cells was not the cause of the change in Ca2+ uptake. Ca2+ uptake activity was significantly lower in brush border membrane vesicles isolated from 24-mo-old rats than in those from 6-mo-old rats. The decrease in Ca2+ uptake activity in old rats was not due to a change in the Ca2(+)-binding capacity of the membranes. Kinetic analysis shows that the Vmax, the apparent maximum uptake capacity of membrane vesicles, decreased in senescent rats, whereas the Km, the apparent affinity to Ca2+, was unchanged. Since duodenal Ca2+ influx at the brush border was regulated by 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], we tested the effect of 1,25-(OH)2D3 administration on the uptake activity in isolated membrane vesicles. After 1,25-(OH)2D3 treatment, Ca2+ uptake activity in brush border membranes prepared from senescent rats was only slightly lower than that in membranes from adult rats. We conclude that the decline in the influx of Ca2+ at the brush border membrane was the main cause of the decrease in duodenal Ca2+ uptake activity in aging. This defect was probably due to the low serum 1,25-(OH)2D3 concentration and not the result of impaired response to 1,25-(OH)2D3.     

A role for calmodulin in renal production of 1,25-dihydroxyvitamin D3

The conversion of circulating 25-hydroxyvitamin D3 (25OHD3) to its active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in the renal tubule mitochondrion by the enzyme 25OHD3-1 alpha-hydroxylase is closely regulated in vivo according to the physiological need for calcium and phosphorus. The mechanism by which this regulation is achieved at the cellular level has not been clarified, although a number of lines of evidence suggest that calcium ions (Ca2+) are involved. This study was designed to determine whether calmodulin, the ubiquitous cell protein that binds and mediates many of the regulatory functions of Ca2+, plays a role in the regulation of renal vitamin D metabolism. The calmodulin antagonists trifluoperazine (TFP), Janssen R24571, and the naphthalene sulfonamides W5 and W7 inhibited conversion of 25OHD3 to 1,25-(OH)2D3 by isolated renal tubules from vitamin D-deficient chicks in a dose-dependent manner (ED50: TFP, 12 mumol/liter; R24571, 10 mumol/liter; W7, 30 mumol/liter; W5, 75 mumol/liter). TFP did not inhibit production of the alternative metabolite 24,25-(OH)2D3 by chick renal tubules. In a similar manner, TFP, W7, and W5 inhibited conversion of 25OHD3 to 1,25-(OH)2D3 by isolated energized chick renal mitochondria, with no detrimental effect on mitochondrial respiratory indices. Bovine brain calmodulin in a concentration of 1 X 10(-7) mol/liter enhanced 1,25-(OH)2D3 production by isolated chick renal mitochondria in Ca2+ -containing medium, but not in the absence of Ca2+. Preincubating mitochondria with anticalmodulin antiserum resulted in decreased conversion of 25OHD3 to 1,25-(OH)2D3, an effect that was prevented by exogenous calmodulin. These data support the notion of a role for calmodulin in the Ca2+ -mediated control of renal 1 alpha-hydroxylase activity.     

Bone mineral homeostasis in spontaneously diabetic BB rats. I. Abnormal vitamin D metabolism and impaired active intestinal calcium absorption

Calcium homeostasis was investigated in male BB rats with a diabetes duration of 3-4 weeks and compared with that in nondiabetic littermates either fed ad libitum or receiving selective semistarvation or an oral Ca supplement to obtain additional weight-matched and Ca intake-matched control groups. Diabetic rats had markedly increased food and Ca intake, so that their net Ca balance remained positive despite a 13-fold increase in urinary Ca excretion and a disappearance of active duodenal Ca absorption. Decreased duodenal Ca uptake correlated with decreased 1,25-(OH)2D3 levels (89 +/- 15 vs. 160 +/- 13 pg/ml in nondiabetic rats), decreased duodenal 9K Ca-binding protein concentrations (10 +/- 1 vs. 21 +/- 2 micrograms/mg protein), and decreased number of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-binding sites in duodenum, although the binding affinity was above normal. Nondiabetic Ca-supplemented rats exhibited a similar response: decreased 1,25-(OH)2D3 (95 +/- 8 pg/ml) and 9K Ca-binding protein (7 +/- 0.5 micrograms/mg protein) concentrations, decreased active duodenal Ca uptake, increased urinary Ca excretion, and a normal net Ca balance. Plasma vitamin D-binding protein levels were decreased by 62% in diabetic rats, due to a marked decrease in production rate, while the plasma half-time remained normal. The free 1,25-(OH)2D3 index was highest in diabetic rats, suggesting partial vitamin D resistance at the duodenal level. In semistarved rats, 1,25-(OH)2D3 levels and active Ca uptake remained normal, and the free 1,25-(OH)2D3 index was increased, together with suppressed vitamin D-binding protein levels. These studies indicate that nutritional abnormalities may contribute to but cannot totally explain the disturbances in vitamin D metabolism, transport, or action at the intestinal level.     

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 uptake by duodenal mucosal cells of Hyp mice

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] uptake by duodenal mucosal cells of Hyp mice were investigated. In Hyp mice fed a high phosphate diet (1.1% Ca and 2.0% phosphate) for 2 weeks, maximal nuclear 1,25-(OH)2D3 binding by duodenal mucosal cells is significantly increased from 5.01 +/- 0.49 x 10(3) to 8.23 +/- 1.10 x 10(3) sites/cell (P less than 0.05). No significant change was observed in normal mice fed the same diet. The serum phosphate concentration of Hyp mice increased significantly (P less than 0.01), whereas no significant change was found in normal mice. On this regimen, serum calcium, urinary cAMP to creatinine ratio, and cytosolic 1,25-(OH)2D3 receptor number in Hyp mice were not changed significantly. On the basis of these data, we speculate that the recovery of serum phosphate in Hyp mice fed a high phosphate diet affects the recovery of nuclear 1,25-(OH)2D3 uptake by duodenal mucosal cells. The mechanism for this recovery is not related to either the secondary hyperparathyroidism or the change in cytosolic 1,25-(OH)2D3 receptor content but, rather, to increased binding of 1,25-(OH)2D3-receptor complex to nuclei. Hypophosphatemia, therefore, appears to play a role in the vitamin D resistance in Hyp mice.     

Influx of extracellular calcium mediates 1,25-dihydroxyvitamin D3-dependent transcaltachia (the rapid stimulation of duodenal Ca2+ transport)

We investigated the role of extracellular Ca2+ in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] rapid stimulation of intestinal Ca2+ transport (termed transcaltachia) in the perfused duodenal of vitamin D-replete chicks. The carboxylic ionophore ionomycin (2 microM) was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25-(OH)2D3. The increase in duodenal 45Ca2+ transport caused by 1,25-(OH)2D3 was dependent on the presence of medium Ca2+, since it was abolished by prior addition of EGTA and was restored upon the addition of Ca2+. Depolarization of the basal lateral membrane of intestinal epithelial cells with 70 mM K+ caused a rapid increase in 45Ca2+ transport (30% above control values within 2 min and 250% after 20 min of vascular perfusion). The rise was also abolished by prior addition of EGTA. Intracellular calcium concentrations ([Ca2+]i) were measured in isolated duodenal cells from vitamin D-replete chicks using the fluorescent dye fura 2. A 1-min incubation with physiological concentrations of 1,25-(OH)2D3 (130 pM) caused an increase in [Ca2+]i from a basal level of 168 +/- 23 nM to 363 +/- 44 nM. Pretreatment of intestinal epithelial cells with the protein kinase-C activator tetradeconyl-phorbol acetate (100 nM) or the adenylate cyclase activator forskolin (10 microM), both shown to induce acute stimulation of intestinal 45Ca2+ transport in the perfused duodenum, also mimicked the stimulatory effect of 1,25-(OH)2D3 on [Ca2+]i. The increase in [Ca2+]i elicited by the 1,25-(OH)2D3 was due to Ca2+ influx from the extracellular medium, since it was blocked by the Ca2+ chelator EGTA (5 mM) and the Ca2+ channel antagonist nifedipine (1 microM). These results suggest that the acute effects of 1,25-(OH)2D3 on duodenal 45Ca2+ transport are triggered by the influx of Ca2+ through voltage-operated Ca2+ channels and that both protein kinase-C and protein kinase-A play an important role in mediating or modulating 1,25-(OH)2D3 effects on transcaltachia.     

Dual actions of 1,25-dihydroxycholecalciferol on intracellular Ca2+ in GH4C1 cells: evidence for effects on voltage-operated Ca2+ channels and Na+/Ca2+ exchange

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] amplifies the TRH-induced spike phase of increase in cytosolic free calcium ([Ca2+]i). In the present report we describe the results of investigations on the mechanisms of action of 1,25-(OH)2D3 on Ca2+ homeostasis in these cells. Pretreatment with 1 nM 1,25-(OH)2D3 for at least 24 h caused no change in basal uptake of 45Ca2+ compared with that in control cells or in 45Ca2+ uptake induced by the calcium channel agonist Bay K 8644. However, when the cells were depolarized with 50 mM K+, 1,25-(OH)2D3-treated cells showed an up to 90% enhancement of uptake (3-120 min) of 45Ca2+. An enhanced increase in [Ca2+]i was also observed in fura-2-loaded cells. The effect was specific and dose dependent for 1,25-(OH)2D3. The calcium channel antagonists nimodipine and verapamil inhibited completely the enhancing action of 1,25-(OH)2D3 as did the protein synthesis inhibitor cycloheximide. No enhanced uptake of 45Ca2+ into intracellular stores was detected when cells were incubated with 1,25-(OH)2D3. Na+/Ca2+ exchange was determined by measuring exchange of extracellular 45Ca2+ for intracellular Na+. Na+/Ca2+ exchange was dependent on intracellular Na+, was inactive when Li+ replaced Na+, was insensitive to calcium channel antagonists, and showed electrogenic properties. In cells incubated with 1,25-(OH)2D3 for at least 24 h, Na+/Ca2+ exchange was enhanced up to 54% compared with that in control cells. Enhanced exchange was dose dependent and specific for 1,25-(OH)2D3. Ca2+ channel antagonists were without effect while dichlorobenzamil inhibited partially the 1,25-(OH)2D3 enhancement of Na+/Ca2+ exchange. Cycloheximide abolished completely the action of 1,25-(OH)2D3 on Na+/Ca2+ exchange. We conclude that in GH4C1 cells, 1,25-(OH)2D3 enhances membrane calcium transport by modulating voltage-operated Ca2+ channels and activating Na+/Ca2+ exchange by mechanisms requiring new protein synthesis.     

Hydroxylation of 25-hydroxyvitamin D3 by renal mitochondria from rats of different ages

The hydroxylation of 25-hydroxyvitamin D3 (25OHD3) in kidney mitochondria from female rats of different ages was studied. The specific activity of 1 alpha-hydroxylase was highest in mitochondria isolated from the 2-month-old rat (0.47 pmol/10 min X mg protein), falling gradually with age to 0.17, 0.10, 0.07, and 0.06 pmol/10 min X mg protein in 6-, 12-, 18-, and 24-month-old rats, respectively. The alteration in 1 alpha-hydroxylase activity with age was due to a change in the V'm of the system; the K'm for 25OHD3 was unchanged (3.9-4.0 microM). The specific activity of 24-hydroxylase was lowest in mitochondria isolated from the 2-month-old rat (8.2 pmol/10 min X mg protein), increasing to 37.8, 37.4, 38.2, and 55.7 pmol/10 min X mg protein in 6-, 12-, 18-, and 24-month-old rats, respectively. The alteration in 24-hydroxylase activity with age was due to a change in the V'm of the system; the K'm value for 25OHD3 was unchanged (1.1-1.2 microM). The age-dependent decrease in 1 alpha-hydroxylase and concomitant increase in 24-hydroxylase activities observed in mitochondria isolated from kidneys of 2-, 6-, 12-, 18-, and 24-month-old rats could not be attributed to changes in the bioenergetic properties, i.e. the respiratory chain, of the mitochondria. The relative mitochondrial content of the kidney, however, probably decreased with age. These findings support the view that the kidneys of aged rats produce less 1,25-dihydroxyvitamin D3 because of lower mitochondrial 1 alpha-hydroxylase specific activity and reduced number of mitochondria. This would be consistent with the lower levels of vitamin D hormone reported in the serum of senescent rats.     

In vitro stimulation of phosphate uptake in isolated chick renal cells by 1,25-dihydroxycholecalciferol.

Renal cells isolated from vitamin D-deficient chicks had an increased Na+-dependent phosphate uptake when preincubated with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Phosphate uptake in the absence of Na+ and methyl alpha-glucoside uptake dependent on Na+ were not affected. Phosphate uptake was stimulated 15% by 0.010 pM 1,25-(OH)2D3. Maximal enhancement of 30% was obtained with 100 pM. The uptake when fully stimulated by preincubation in vitro approximated the uptake of cells isolated from chicks that were previously repleted with 1,25-(OH)2D3 in vivo. Cells from repleted chicks were not stimulated additionally when preincubated with 1,25-(OH)2D3 in vitro. The increase in phosphate uptake could be measured after a 1-hr preincubation period; full response required at least 2 hr. Phosphate uptake induced by 1,25-(OH)2D3 was blocked by cycloheximide and actinomycin D. Enhancement of phosphate uptake was relatively specific for the 1,25-(OH)2D3 analog of vitamin D3. The potency order was 1,25-(OH)2D3 greater than 25-(OH)D3 = 1-(OH)D3 greater than 24,25-(OH)2D3 greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of the phosphate uptake system; the affinity for phosphate was unaffected. 3H-Labeled 1,25-(OH)2D3 was taken up by the isolated renal cells. It was estimated that the stimulation of phosphate uptake might be initiated by very few molecules of 1,25-(OH)2D3 per cell. It is proposed that 1,25-(OH)2D3 contributes importantly to the mechanisms by which phosphate transport is regulated in the kidney.     

Effects of high doses of vitamin D3 and 1,25-dihydroxyvitamin D3 in lactating rats on milk composition and calcium homeostasis of the suckling pups

Changes in serum Ca and phosphorus and in kidney Ca were determined in lactating rats and their suckling pups after the mothers received high doses of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. High dietary vitamin D3 intake (300 IU/g diet) or daily oral doses of vitamin D3 (1 microgram/g BW) to vitamin I)-replete (+D) lactating rats for 8 or 12 days caused significant increases in serum Ca in the mothers (1-2 mg/dl) and in their suckling pups (1.5 mg/dl). Daily oral doses of 1,25-(OH)2D3 (2 ng/g BW) to +D lactating rats caused a similar increase in serum Ca in the mothers, but did not affect the serum Ca of the pups. The administration of a high dose of 1,25-(OH)2D3 to vitamin D-deficient lactating rats or high doses of vitamin D3 to +D rats, caused no change in milk Ca, Mg, or phosphorus. Milk from +D rats given high doses of [3H]vitamin D3 (1 microgram/g BW) contained mostly [3H]vitamin D3 (85%) and a small amount of [3H]25-hydroxyvitamin D3 (6%). The results indicate that high doses of vitamin D3, but not 1,25-(OH)2D3, given to +D lactating rats can cause hypercalcemia in the suckling pups. The hypercalcemic effect on the pups observed after vitamin D3 treatment of the mother is probably a result of transport of toxic amounts of primarily vitamin D3 into the milk and is not due to altered mineral composition of the milk.     

Vitamin D-induction of secretory responses in rat pituitary tumour (GH4C1) cells

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) selectively enhances prolactin gene expression in GH4C1 clonal rat pituitary tumour cells. Because this effect requires extracellular Ca2+, we studied the effect of 1,25-(OH)2D3 on another Ca2+-dependent process, agonist-induced hormone secretion. Pretreatment with 1,25-(OH)2D3 (1 nmol/l) caused at least 25-fold sensitization of GH4C1 cells to the voltage-sensitive Ca2+ channel agonist BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine -5-carboxylate) as a prolactin secretagogue. This inductive effect of 1,25-(OH)2D3 followed a similar time-course to the enhancement of prolactin production. 1,25-(OH)2D3 had no effect on basal or BAY K 8644-induced 45Ca2+ uptake. The Ca2+-selective divalent cation ionophore 11,19,21-trihydroxy-4,6,8,12,14,18,20- heptamethyl-9-oxo-22-(tetrahydro-5 methyl-5-tetra hydro-5-(1-hydroxyethyl)-5-methyl-2-furanyl)-10,16-docosadienoic acid (ionomycin; 12 nmol/l-1.2 mumol/l) caused no significant increase in prolactin secretion in the absence of 1,25-(OH)2D3, but in cells treated with 1,25-(OH)2D3-(1 nmol/l), it increased prolactin secretion by 73% at 12 nmol/l and by a maximum of 98% at 0.12 mumol/l. These data demonstrate that vitamin D markedly enhances the responsiveness of GH4C1 functional pituitary tumour cells to two secretagogues which acts primarily through Ca2+-dependent mechanisms. They support the proposal that 1,25-(OH)2D3 acts in this cultured cell model either by effecting a redistribution of intracellular Ca2+ or by increasing the response of a Ca2+-sensitive effector system, but not by enhancing agonist-induced Ca2+ uptake.     

Injection of 1,25-(OH)2 vitamin D3 enhances resistance artery contractile properties

The hypothesis that 1,25-dihydroxyvitamin D3 [1,25-(OH)2 vitamin D3] modulates vascular smooth muscle contractile function was tested. 1,25-(OH)2 vitamin D3 (50 ng/day) was administered by intraperitoneal injection over a 3-day period to 13-15-week-old male spontaneously hypertensive and Wistar-Kyoto normotensive rats. On the fourth day, serum was prepared and contractile force generation of isolated mesenteric resistance arteries was examined. Treatment with 1,25-(OH)2 vitamin D3 approximately doubled serum levels of the hormone and increased ionized and total serum Ca2+ and phosphate by 5-10%. No effect on blood pressure was detected. 1,25-(OH)2 vitamin D3 injection in both strains enhanced maximal stress generation to norepinephrine and serotonin by 30-40%, with no effect on apparent sensitivity of the vessels to the agonists. To assess the effect of a maneuver that elevates serum ionized Ca2+ without the addition of exogenous hormone, maximal stress generation was examined in resistance arteries isolated from rats fed diets containing 0.5% or 2% calcium over a 6-7-week period. Maximal stress generation in response to norepinephrine was greater in vessels from rats of both strains maintained on 0.5% calcium. It is concluded that 72-hour in vivo treatment with 1,25-(OH)2 vitamin D3 increases contractile force-generating capacity of resistance arteries without affecting blood pressure. It is proposed that this action of 1,25-(OH)2 vitamin D3 is the result of a direct action of the hormone on the vascular wall.     

Regulatory effect of 1,25-dihydroxycholecalciferol on calcium fluxes in thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) on the regulation of calcium fluxes in rat thyroid FRTL-5 cells. The ATP-induced uptake of 45Ca2+ was decreased in cells pretreated with 1,25(OH)2D3 for 48 h. No effect was seen on basal uptake of 45Ca2+. At least a 24 h incubation period was required for the effect of 1,25(OH)2D3 to be expressed. Pretreatment with 1,25(OH)2D3 for 48 h did not change resting intracellular Ca2+ ([Ca2+]i) in fura-2-loaded FRTL-5 cells. However, the ATP-induced increase in [Ca2+]i was significantly enhanced in cells preincubated with 1,25(OH)2D3. The effect of 1,25(OH)2D3 was abolished in Ca(2+)-free buffer. No difference in the ionomycin-induced increase in [Ca2+]i was observed between control cells and cells pretreated with 1,25(OH)2D3. However, in Ca(2+)-free buffer the ionomycin response was decreased in cells incubated with 1,25(OH)2D3. The ATP-induced change in [Ca2+]i was decreased when ATP was added after ionomycin to cells treated with 1,25(OH)2D3. The results suggest that 1,25(OH)2D3 has a regulatory effect on Ca2+ fluxes in FRTL-5 cells, possibly by acting on Ca2+ sequestration.     

12-O-tetradecanoyl-phorbol-13-acetate decreases influx of extracellular Ca2+ induced by depolarization in GH4C1 cells: effects of pretreatment with 1,25-dihydroxycholecalciferol.

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol (1,25(OH)2D3) causes amplification of both the TRH-induced spike phase in cytosolic free calcium [( Ca2+]i) and the increase in [Ca2+]i induced by depolarization with K+. In the present study we investigated the actions of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Ca2(+)-homeostasis in GH4C1 cells pretreated with 1,25(OH)2D3 for 24 h. In control and 1,25(OH)2D3-pretreated cells, incubation with TPA (0.1-300 nM) for 15 min in the presence of 45Ca2+ did not affect the basal uptake of 45Ca2+. However, if the cells were treated with 50 mM K+, TPA induced a time- and concentration-dependent decrease in depolarization-induced net 45Ca2+ uptake. A maximal decrease of 30-50% was observed with 100-300 nM TPA, 1,25(OH)2D3 pretreated cells being more responsive to the action of TPA than control cells. sn-1-Oleoyl-2-acetyl-glycerol, which mimics the action of TPA on protein kinase C (PKC), did not alter depolarization-induced uptake of 45Ca2+. Two agents which inhibit PKC activity, polymyxin B and K252A, did not prevent the effect of TPA on depolarization-induced uptake of 45Ca2+, whereas staurosporin totally inhibited the action of TPA. In Fura-2 loaded cells pretreated with 1,25(OH)2D3, incubation with 200 nM TPA for 9 min decreased the depolarization-induced spike and plateau phases of change in [Ca2+]i; only the spike phase was decreased in control cells. TPA did not affect basal [Ca2+]i in either group. Treatment with TPA for only 3 min decreased the TRH-induced spike in [Ca2+]i only in 1,25(OH)2D3 pretreated cells; however, after a 5-min treatment with TPA, the TRH-induced spike in [Ca2+]i was decreased in both control and 1,25(OH)2D3 pretreated cells. TPA did not affect the spike in [Ca2+] induced by 50 nM ionomycin. Na+/Ca2+ exchange was not altered by TPA, nor did TPA enhance efflux of 45Ca2+ from cells preloaded with 45Ca2+ for 2.5 h. We conclude that, in GH4C1 cells, TPA modulates plasma membrane calcium flux, probably via an inhibitory action on voltage-operated Ca2+ channels. This inhibitory action may be independent of activation of PKC, and 1,25(OH)2D3 pretreated cells are more responsive to the actions of TPA than are control cells. These results are consistent with our previous findings that 1,25(OH)2D3 enhances voltage-dependent Ca2+ channel activity in GH4C1 cells.     

Production of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by growth zone and resting zone chondrocytes is dependent on cell maturation and is regulated by hormones and growth factors.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 have been shown to promote chondrocyte proliferation and differentiation; resting zone chondrocytes respond primarily to 24,25-(OH)2D3, whereas growth zone chondrocytes respond primarily to 1,25-(OH)2D3. This study determined whether resting zone and growth zone cells produce 24,25-(OH)2D3 or 1,25-(OH)2D3; whether this production is regulated by 1,25-(OH)2D3 (10(-8) M), 24,25-(OH)2D3 (10(-7) M), dexamethasone (10(-7) M), or recombinant human transforming growth factor-beta 1 (11 ng/ml); and whether the metabolites produced are biologically active. Confluent fourth passage rat costochondral growth zone or resting zone chondrocytes were cultured in Dulbecco's Modified Eagle's Medium containing [3H]25-hydroxyvitamin D3 ([3H]25OHD3), 2% fetal bovine serum, and antibiotics. Metabolism of [3H]25OHD3 was measured by analyzing the lipid extracts of the conditioned medium and the cell layer for [3H]1,25OHD3, [3H]1,25-(OH)2D3, and [3H]24,25-(OH)2D3 using flow-through scintillation spectroscopy of HPLC eluates. Chemically synthesized radioinert vitamin D3 metabolites were used as standards, and their migration was determined by absorbance at 254 nm. To ensure that the radioactive peaks were 1,25-(OH)2D3 and 24,25-(OH)2D3, the fractions were rechromatographed into three other HPLC solvent systems. Biological activity was confirmed; the addition of HPLC-purified 1,25-(OH)2D3 produced by growth zone chondrocytes elicited a dose-dependent stimulation of alkaline phosphatase specific activity in growth zone cell cultures, but had no effect on the resting zone cells. There was a time-dependent increase in both [3H]1,25-(OH)2D3 and [3H]24,25-(OH)2D3 in the conditioned medium of both types of cultures. At 24 h, the percent conversion of [3H]25OHD3 to [3H]1,25-(OH)2D3 was 5.3 +/- 1.2, and the percent conversion to [3H]24,25-(OH)2D3 was 1.8 +/- 0.4 in growth zone chondrocyte cultures. No such effect was found in cultures freeze-thawed five times or without cells. When resting zone cells were cultured with [3H]25OHD3, the percent conversion to 1,25-(OH)2D3 and 24,25-(OH)2D3 was 4.5 +/- 1.0 and 1.7 +/- 0.4, respectively. The addition of dexamethasone significantly increased the percent production of 1,25-(OH)2D3 at 6 and 24 h and at 6 h by resting zone and growth zone cells, respectively, compared to the control values. Recombinant human transforming growth factor-beta 1 increased the percent production of 1,25-(OH)2D3 after 1 h in resting zone cells and, after 24 h, the production of 24,25-(OH)2D3 in growth zone cells. Radiolabeled 1,25-(OH)2D3 and 24,25-(OH)2D3 were not detected in the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects

The serum vitamin D2 and vitamin D3 metabolite concentrations and intestinal absorption of vitamin D2 were determined in healthy ambulatory and chronically institutionalized elderly subjects with normal renal function. The 25-hydroxyvitamin D (25OHD) concentrations were normal in all subjects (range, 8-43 ng/ml), although institutionalized subjects had a significantly lower mean value [19.2 +/- 2 (+/- SEM) ng/ml; P less than 0.01] compared with ambulatory subjects (25.3 +/- 2 ng/ml). All but one ambulatory subject had 25OHD3 as the major circulating form, whereas 25OHD2 was the major circulating metabolite in one third of the institutionalized subjects. The mean 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentration in both groups was normal, but nine subjects had levels at or below the lower limit of normal despite normal 25OHD concentrations. Separate assay of 1,25-(OH)2D2 and 1,25(OH)2D3 revealed proportional distributions similar to those for 25OHD2 and 25OHD3. To study the effect of age on the intestinal absorption of vitamin D, we compared serum vitamin D2 concentrations after oral administration of 50,000 IU vitamin D2 in both healthy vitamin D-sufficient elderly subjects and young adults. We found no evidence of malabsorption of vitamin D in the elderly subjects. In summary, elderly subjects in New York, whether institutionalized or not, have normal serum 25OHD concentrations. However, while most elderly subjects have normal serum 1,25-(OH)2D levels, a significant proportion fail to produce normal concentrations of 1,25-(OH)2D, possibly due to age-related disturbances in renal synthesis of the hormone.     

Effect of the X-linked Hyp mutation and vitamin D status on induction of renal 25-hydroxyvitamin D3-24-hydroxylase

The present study was undertaken to evaluate the response of Hyp mice to regulators known to inhibit renal 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and stimulate renal 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). Renal mitochondrial metabolism of 25-hydroxyvitamin D3 (25OHD3) was initially examined in vitamin D- and calcium-deprived normal and mutant mice (with no detectable 24-hydroxylase) treated with either calcium, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or both calcium + 1,25-(OH)2D3. In normal mice, 1,25-(OH)2D3 treatment was more effective than calcium in turning off 1-hydroxylase and turning on 24-hydroxylase activity; serum calcium, however, was similarly increased by both treatments. Although calcium + 1,25-(OH)2D3 did not result in a further change in 25OHD3 metabolism in normal mice, a further elevation in serum calcium was apparent. In Hyp mice, treatment with calcium + 1,25-(OH)2D3 resulted in a greater decrease in 1-hydroxylase and a greater increase in 24-hydroxylase and in serum calcium than treatment with either agent alone. In spite of similar serum calcium levels in both genotypes, 24-hydroxylase was 20-fold, 3-fold, and 8-fold greater in Hyp mice relative to normals treated with calcium, 1,25-(OH)2D3, and calcium + 1,25-(OH)2D3, respectively. Kinetic studies revealed that the maximum velocity (Vmax) for induced 24-hydroxylase was 6-fold greater than normal in Hyp mice whereas the apparent Michaelis-Menten constant (Km) was not different in the two groups of calcium + 1,25-(OH)2D3-treated mice. The effect of 1,25-(OH)2D3 treatment on the above serum and renal parameters was also examined in vitamin D replete normal and Hyp mice. A sharp rise in serum phosphate was observed in 1,25-(OH)2D3-treated Hyp mice whereas normal littermates experienced marked hypercalcemia in response to treatment. Renal 24-hydroxylase was significantly stimulated by 1,25-(OH)2D3 treatment in both normal and Hyp mice and genotype differences were not apparent. The present study demonstrates that vitamin D- and calcium-deprived Hyp mice are more responsive to signals which induce 24-hydroxylase than normal littermates; Vmax for induced 24-hydroxylase is 6-fold greater in Hyp mice than in normal littermates whereas apparent Km is unchanged; the inhibitory control of 1-hydroxylase appears to be intact in the mutant strain; induced 24-hydroxylase is similar in vitamin D replete normal and Hyp mice; and vitamin D status can thus modify the response of both genotypes to treatment with 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)     

1,25-Dihydroxycholecalciferol enhances both the bombesin-induced transient in intracellular free Ca2+ and the bombesin-induced secretion of prolactin in GH4C1 pituitary cells

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances both the synthesis of PRL and the TRH-induced transient increase in cytosolic free calcium ( [Ca2+]i). In the present report we investigated whether 1,25-(OH)2D3 could enhance the effect of the tetradecapeptide bombesin (BBS) in GH4C1 cells. Pretreatment of the cells with 1 nM 1,25-(OH)2D3 for 24 h enhanced the BBS-induced transient increase in [Ca2+]i compared to that in control cells, while having no significant effect on the plateau phase of [Ca2+]i. Addition of the Ca2+ channel blocker nimodipine or chelating extracellular Ca2+ with EGTA did not abolish the enhancement of the BBS response in 1,25-(OH)2D3-pretreated cells. Furthermore, the BBS-induced efflux of 45Ca2+ from cells preequilibrated with 45Ca2+ was larger in cells treated with 1,25-(OH)2D3. Incubating GH4C1 cells with 1,25-(OH)2D3 alone or in combination with BBS for up to 72 h did not stimulate synthesis of PRL. However, the BBS-induced secretion of PRL was enhanced in cells pretreated with 1,25-(OH)2D3 for 24 h compared with that in vehicle-treated control cells. The effect of 1,25-(OH)2D3 on BBS-induced secretion was dose dependent, with 10(-11) M 1,25-(OH)2D3 enhancing the stimulated secretion of PRL. We conclude that in GH4C1 cells, pretreatment with 1,25-(OH)2D3 enhances the BBS-induced transient increase in [Ca2+]i. This effect may be due to a modulation of the availability of sequestered intracellular Ca2+ and/or membrane Ca2+ conductance. Furthermore, pretreatment with 1,25-(OH)2D3 enhanced secretion of PRL stimulated by BBS. The enhanced transient increase in [Ca2+]i may be the factor inducing the enhanced BBS-induced secretion of PRL.     

Extrarenal production of calcitriol in normal and uremic humans

We have previously reported low serum levels of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and increased 1,25-(OH)2D3 production after the administration of 25-hydryoxyvitamin D (25OHD) to anephric humans. Since normal alveolar macrophages are known to synthesize 1,25-(OH)2D3 when stimulated with gamma-interferon or lipopolysaccharide, we determined whether macrophages derived from peripheral blood monocytes could be an extrarenal source of 1,25-(OH)2D3. Our results demonstrated that macrophages from normal individuals synthesize 1,25-(OH)2D3. The apparent Km for 25OHD3 was 6.6 +/- 0.5 nM and the maximum velocity was 47.4 +/- 13.7 fmol 1,25-(OH)2D3/h.microgram DNA. The activity of this enzyme was reduced 37.2 +/- 3.1% by physiological concentrations (96 pmol/L) of 1,25-(OH)2D3 in the incubation medium. Normal macrophages further hydroxylated 1,25-(OH)2D3 to more polar metabolites, and this catabolic activity was significantly enhanced by physiological concentrations of 1,25-(OH)2D3. In chronic renal failure, peripheral macrophages exhibited an enhanced 1 alpha-hydroxylase activity (8.2 +/- 0.8 vs. 4.2 +/- 0.5 fmol 1,25-(OH)2D3/microgram DNA.h in controls) and a decreased capacity to degrade 1,25-(OH)2D3. Exogenous 1,25-(OH)2D3, in physiological concentrations, reduced 1,25-(OH)2D3 synthesis to a degree (23.6 +/- 8.5%) comparable to that observed in normal cells. 1,25-(OH)2D3 production by macrophages did not correlate with the severity of hyperparathyroidism. Moreover, human PTH-(1-34) in supraphysiological concentrations (20,000 and 100,000 ng/L) did not stimulate the 1 alpha-hydroxylase activity of macrophages from either normal or uremic subjects. These results demonstrate that 1) normal peripheral macrophages metabolize 25OHD3 and 1,25-(OH)2D3; 2) macrophages in uremia display higher rates of 1,25-(OH)2D3 synthesis and lower rates of catabolism than normal macrophages; and 3) 1,25-(OH)2D3 deficiency, but not hyperparathyroidism, may play a role in the stimulation of 1,25-(OH)2D3 production by macrophages in chronic renal failure.     

Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro

Renal production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] from 25-hydroxyvitamin D3 (25OHD3) is increased by PTH. The complete mechanism by which PTH modulates renal 25OHD3 metabolism is not known, but there is some evidence that the stimulation of renal cAMP production by PTH may be important. Therefore, we have used forskolin, a direct activator of adenylate cyclase in the intact tissue, to further investigate the role of cAMP in regulating renal 25OHD3 metabolism. The effect of forskolin on renal 25OHD3 metabolism and renal adenylate cyclase activity was measured using isolated renal slices from thyroparathyroidectomized rats previously fed a vitamin D-deficient, low calcium diet. Forskolin added to renal slices in vitro for 4 h increased renal 1,25-(OH)2-D3 production in a concentration-dependent manner. In separate experiments, forskolin was found to increase tissue cAMP in a concentration-dependent manner when added for 5 min. The concentration of forskolin necessary for half-maximal stimulation of adenylate cyclase was 10 microM, and that needed for half-maximal stimulation of 1,25-(OH)2-D3 production was 1 microM. PTH added to renal slices also increased renal 1,25-(OH)2-D3 production, but the effects of PTH and forskolin were not additive. Inclusion of 1,25-(OH)2-D3 in the incubation medium blocked the effect of forskolin on 1,25-(OH)2-D3 production, but it did not block the effect of forskolin on tissue cAMP content. These studies support the concept that forskolin and PTH modulate renal 25OHD3 metabolism though a cAMP-dependent pathway. However, this pathway may be further regulated at sites distal to cAMP production by compounds such as 1,25-(OH)2-D3.     

The role of insulin in the stimulation of renal 1,25-dihydroxyvitamin D synthesis by parathyroid hormone in rats

To evaluate the role of insulin in 1,25-dihydroxyvitamin D [1,25(OH)2D] production in response to PTH, 25-hydroxyvitamin D-1 alpha-hydroxylase activity in kidney homogenates as well as serum 1,25(OH)2D concentration was measured both after dietary calcium (Ca) deprivation and after PTH infusion in control and streptozotocin-diabetic rats. Although serum Ca and phosphate (Pi) levels did not change significantly after dietary Ca deprivation for 1 week, urinary cAMP excretion increased significantly, indicating that dietary Ca deprivation caused secondary hyperparathyroidism without a significant change in serum Ca level. In control rats, renal 1 alpha-hydroxylase activity increased markedly from 0.11 +/- 0.05 to 1.70 +/- 0.46 ng/300 mg tissue/20 min in parallel with the change in serum 1,25(OH)2D level from 121 +/- 8 to 360 +/- 54 pg/ml in response to Ca deprivation. In contrast, serum 1,25(OH)2D level (82 +/- 3 pg/ml) and 1 alpha-hydroxylase activity (0.07 +/- 0.02 ng/300 mg tissue.20 min) were lower in the diabetic rats on a normal Ca diet than those in control rats, and the increase in both 1,25(OH)2D level and 1 alpha-hydroxylase activity in response to Ca deprivation was suppressed in diabetic rats (136 +/- 24 pg/ml and 0.38 +/- 0.12 ng/300 mg tissue.20 min, respectively, after Ca deprivation). Insulin treatment of the diabetic rats restored the baseline levels of serum 1,25(OH)2D (125 +/- 14 pg/ml) and renal 1 alpha-hydroxylase activity (0.21 +/- 0.02 ng/300 mg tissue.20 min) as well as those after Ca deprivation (340 +/- 52 pg/ml and 2.05 +/- 0.30 ng/300 mg tissue.20 min, respectively). Furthermore, when control and diabetic rats were thyroparathyroidectomized and infused with a maximal stimulatory dose of PTH, the increase in serum 1,25(OH)2D and renal 1 alpha-hydroxylase activity in response to PTH was markedly inhibited in diabetic rats. In addition, the baseline levels of serum 1,25(OH)2D and renal 1 alpha-hydroxylase activity in thyroparathyroidectomized diabetic rats were not different from those in control rats. These results are consistent with the conclusion that insulin plays an important role in the regulation of renal 1 alpha-hydroxylase activity and serum 1,25(OH)2D levels in response to PTH.