Bone turnover in rats treated with 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3

Female rats were given 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 0.25 g per 100 g body weight (bw), 25-hydroxyvitamin D₃ (25(OH)D₃), 1.7 g/100 g bw or 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) 1.7 g/100 g bw, subcutaneously three times a week for 12 weeks. Traditional variables pertaining to calcium homeostasis and growth, i.e. blood and urine calcium (Ca) and phosphate (P), serum levels of vitamin D₃ metabolites parathyroid hormone, (PTH), calcitonin (CT), prolactin (PRL) and growth hormone (GH) were measured every four weeks. This data pool was correlated with bone matrix turnover parameters, i.e. serum levels of alkaline phosphatase (ALP) and urinary hydroxyproline (u-HYP) excretion. After 12 weeks of treatment, 1,25(OH)₂D₃ significantly enhanced serum total and ionized Ca, urine Ca and urine P, and also diminished urine cAMP due to reduced renal function (creatinine clearance). However, 25(OH)D₃ administration had no such impact. 24,25(OH)₂D₃ opposed the effect of 1,25(OH)₂D₃ after 12 weeks by significantly augmenting serum P and diminishing serum levels of total Ca and ionized Ca. Cross sectional group analyses showed that criculating levels of ALP were directly related with serum 1,25(OH)₂D₃ and inversely related to serum 24,25(OH)₂D₃ and CT. Total u-HYP and per cent non-dialysable HYP (ndHYP) were reciprocally and positively correlated with serum PRL, respectively. However, no such relations were observed with serum GH.It appears that rats with elevated circulating levels of 1,25(OH)₂D₃ exhibit increased bone resorption, while augmented 24,25(OH)₂D₃ is associated with the opposite. Apparently, high bone turnover (i.e. reduced total urinary HYP and enhanced ndHYP) is associated with high serum PRL.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Summer/winter differences in the serum 25-hydroxyvitamin D3 and parathyroid hormone levels of Japanese women

Serum 25-hydroxyvitamin D₃ [25(OH)D₃] is produced in the skin in response to exposure to ultraviolet radiation, and is a good indicator of vitamin D nutritional status. The aim of this study was to determine summer/winter differences in serum 25(OH)D₃ and parathyroid hormone (PTH) in Japanese women and how the summer and winter values are related. The subjects were 122 healthy Japanese women aged 45–81 years (average age: 65.7 years). They were medically examined twice, in September 1997 and February 1999. Serum 25(OH)D₃ and intact PTH were determined by high-performance liquid chromatography and a two-site immunoradiometric assay respectively. Lifestyle information was obtained through an interview. The seasonal differences (winter minus summer) in 25(OH)D₃ [Δ25(OH)D₃] and intact PTH concentrations were –18.8 nmol/l (SD 19.2, P<0.0001) and 0.98pmol/l (SD 1.02, P<0.0001) respectively. The correlation coefficient between summer (x) and winter (y) 25(OH)D₃ levels was 0.462 (P<0.0001), with a linearly fitted line of y=0.42x+26.4. This relationship was interpreted as subjects with higher summer 25(OH)D₃ values having greater reductions in winter 25(OH)D₃ concentrations. There were inter-individual differences in Δ25(OH)D₃, although the summer and winter 25(OH)D₃ concentrations were well-correlated. Since Δ25(OH)D₃ was not associated with any of the lifestyle factors, seasonal differences in the 25(OH)D₃ concentrations of an individual appeared to reflect her ability to produce 25(OH)D₃ photochemically in the skin. Sun bathing would be a less effective means of attaining adequate vitamin D nutritional status in a person with a small seasonal difference in 25(OH)D₃, i.e., one with a low 25(OH)D₃ level. Received: 17 December 1999 / Revised: 24 April 2000 / Accepted: 10 May 2000     

Effect of three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 on 25-OH-D3-1 alpha-hydroxylase in isolated mitochondria and on 25-hydroxyvitamin D3 metabolism in cultured kidney cells

Three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)--2-nor-1,3-seco-1,25(OH)2D3 (2-nor analog), 2-oxa-3-deoxy-25-OH-D3 (2-oxa analog), and A-homo-3-deoxy-3,3-dimethyl-2,4-dioxa-25-OH-D3 (A-homo analog)--were tested for their ability to inhibit 25-OH-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) in isolated mitochondria and to alter 25-OH-D3 metabolism in cultured chick kidney cells. The 2-nor and 2-oxa analogs were relatively potent (Kis of 60 and 30 nM, respectively, compared with 170 nM for 1,25(OH)2D3), whereas the A-homo analog was completely ineffective in inhibiting 1 alpha-hydroxylase activity. In contrast, all three analogs were able to repress 1 alpha-hydroxylase and induce 24-hydroxylase activity in cultured chick kidney cells, suggesting that this process is not one of direct action in the mitochondria, but is more likely to be a receptor-mediated one.     

On the regulatory importance of 1,25-dihydroxyvitamin D3 and dietary calcium on serum levels of 25-hydroxyvitamin D3 in rats

The serum level of 25-hydroxyvitamin D3 in rats was found to vary with the dietary intake of calcium. An increase in the dietary intake of calcium was found to be associated with an increase in the concentration of 25-hydroxyvitamin D3 and a decrease in the concentration of 1,25-dihydroxyvitamin D in serum. Intraperitoneal administration of 1,25-dihydroxyvitamin D3 was found to depress the serum concentration of 25-hydroxyvitamin D3 in rats on both medium and high calcium diets. These changes in the serum levels of 25-hydroxyvitamin D3 were not associated with statistically significant changes in the activity of mitochondrial vitamin D3 25-hydroxylase in the liver. Possible mechanisms for the regulation of the level of circulating 25-hydroxyvitamin D3 in serum are discussed.     

Inhibitory effects of 1 alpha, 25-dihydroxycholecalciferol on parathyroid hormone secretion in rats

In an attempt to study the influence of vitamin D metabolites on PTH secretion, serum calcium and urinary excretion of cAMP were sequentially measured in conscious perfused rats, and the effects of a single iv injection of the metabolites on these parameters were examined. Four hours after the administration of 0.25 microgram/kg (0.6 nmol/kg, probably a physiological dose) of 1 alpha, 25-dihydroxycholecalciferol [1 alpha, 25 (OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized rats (approximately 60% of the initial value; P less than 0.05) and this level was sustained for nearly 24 h. Serum concentrations of calcium (total and ionized) did not change. In parathyroidectomized rats which were continuously infused with bovine PTH (1 U/h), the vitamin D metabolite had no significant effect on the urinary excretion of cAMP. 24 R, 25-dihydroxcholecalciferol (12.5 microgram/kg) had no significant effect either on the urinary excretion of cAMP or on serum calcium. These results suggest that in rats, a physiological dose of 1 alpha, 25(OH)2D3 inhibits PTH secretion without causing a significant rise iu serum calcium, reflecting a feed-back mechanism between active vitamin D metabolite, 1 alpha, 25(OH)2D3 and the parathyroid glands.     

Parathyroid hormone is not involved in 1,25-dihydroxyvitamin D regulation of 25-hydroxyvitamin D metabolism in vivo

1,25-Dihydroxyvitamin D₃ administration to vitamin D-deficient rats suppresses accumulation of 1,25-dihydroxy-[3α-³H]vitamin D₃ and stimulates accumulation of 24,25-dihydroxy-[3α-³3H]vitamin D₃ from 25-hydroxy-[3α-³H]vitamin D₃ equally well in the presence and absence of parathyroid glands. These results demonstrate that this regulatory action is not mediated by the parathyroid glands and support conclusions from $\text{in}\text{vitro}$ studies that this represents a direct action of 1,25-dihydroxyvitamin D₃.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.     

Novel regulatory actions of 1 alpha,25-dihydroxyvitamin D3 on the metabolism of polyphosphoinositides in murine epidermal keratinocytes

The in vitro incubation of murine keratinocytes in the presence of 1 alpha,25-dihydroxyvitamin D3 enhanced the rapid hydrolysis of the prelabeled keratinocyte polyphosphoinositides (polyPtdIns) when compared to untreated cells. The rapid hydrolysis of the polyPtdIns and the release of the inositol phosphates (particularly InsP3 and InsP2) precede the onset of differentiation of these cells. These data therefore suggest that 1 alpha,25-dihydroxyvitamin D3 functions in vitro to initiate the rapid generation of InsP3 from cellular polyPtdIns; this in turn may mobilize intracellular Ca2+, thus providing the signal which program the murine keratinocytes from a proliferating mode into a differentiating mode.     

The role of 1,25-dihydroxyvitamin D3 and parathyroid hormone in the regulation of chick renal 25-hydroxyvitamin D3-24-hydroxylase.

A single 325-pmol dose of 1,25-dihydroxyvitamin D₃ given to chicks fed a vitamin D-deficient diet containing 3% calcium and 0.6% phosphorus suppresses renal mitochondrial 25-hydroxyvitamin D₃-1α-hydroxylase and stimulates the 25-hydroxyvitamin D₃-24-hydroxylase as measured by in vitro assay. This alteration in the enzymatic activity takes place over a period of hours. The administration of parathyroid hormone rapidly suppresses the 25-hydroxyvitamin D₃-24-hydroxylase. The alterations in the hydroxylases by parathyroid hormone or 1,25-dihydroxyvitamin D₃ are not related to changes in serum clacium or phosphate but could be related to changes in intracellular levels of these ions. Actinomycin D or cycloheximide given in vivo reduces the 25-hydroxyvitamin D₃-24-hydroxylase activity rapidly which suggests that the turnover of the enzyme and its messenger RNA is rapid (1- and 5-h half-life, respectively). The half-lives of the hydroxylases are sufficiently short to permit a consideration that the regulation by 1,25-dihydroxyvitamin D₃ and parathyroid hormone may involve enzyme synthesis and degradation.     

Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine.

Using a cDNA probe for rat renal 24-hydroxylase, expression of its mRNA was compared in the rat kidney and intestine. Vitamin D-deficient rats received a single injection of 1 alpha,25-dihydroxyvitamin D3. Expression of 24-hydroxylase mRNA was first detected in the kidney at 3-h post-injection and increased thereafter. Similarly, 24-hydroxylase mRNA was expressed in the intestine after 1 alpha,25-dihydroxyvitamin D3 injection. However, the dose level of 1 alpha,25-dihydroxyvitamin D3 required to induce the intestinal 24-hydroxylase mRNA expression was only 1/100 the amount required to induce renal 24-hydroxylase mRNA. Induction of intestinal 24-hydroxylase mRNA expression by 1 alpha,25-dihydroxyvitamin D3 was far more rapid than that of renal 24-hydroxylase mRNA. Thyroparathyroidectomy shortened the time required to induce expression of renal, but not intestinal, 24-hydroxylase mRNA. Administration of either parathyroid hormone or cAMP to vitamin D-deficient rats greatly reduced the expression of 24-hydroxylase mRNA in the kidney but not in the intestine. When rats were fed a vitamin D-repleted diet containing 0.7% (adequate) or 0.03% (low) calcium for 2 weeks, intestinal expression of 24-hydroxylase mRNA could be induced only in the low calcium group. In contrast, renal mRNA expression was preferentially stimulated in the adequate calcium group. These results clearly demonstrate that the expression of 24-hydroxylase mRNA is down-regulated by parathyroid hormone in the kidney but not in the intestine.     

Conformational and biological characterization of human parathyroid hormone hPTH(1-34) analogues containing beta-amino acid residues in positions 17-19

The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.     

Effect of alcohol on renal vitamin D metabolism in chickens.

Intraperitoneal administration of ethanol to young chickens (both vitamin D-replete and vitamin D-deficient) produced a significant impairment of renal 25 hydroxyvitamin D₃ 1α-hydroxylase (EC 1.14.13.13) activity with no significant change in serum calcium or phosphorus. In ethanol treated D-replete chicks the renal 25 hydroxyvitamin D₃ 24-hydroxylase activity was enhanced, and serum 25 hydroxyvitamin D₃ was significantly increased. The alkaline phosphatase levels in the D-deficient ethanol treated chicks were significantly less than the controls. Our data suggest that the impairment of the metabolic effects of vitamin D due to ethanol occurs chiefly via a renal, rather than a hepatic mechanism. Furthermore, 1α -hydroxylated metabolites of vitamin D would appear to be the logical treatment of choice for the bone disease of alcoholism.     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 by blood derived macrophages from a patient with alveolar rhabdomyosarcoma during short-term culture and 1 alpha,25-dihydroxyvitamin D3 after long-term culture

We have examined the ability of blood-derived monocytes and macrophages isolated from a patient with alveolar rhabdomyosarcoma and hypercalcaemia, to form 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3). Adherent monocyte-macrophage cells incubated with 25(OH)D3 over the initial 2 days in culture synthesized 1.9 pmol 24,25(OH)2D3/h/incubation (representing 0.63 pmol/h/10(6) cells), whereas macrophages synthesized 1.03 and 1.15 pmol 1 alpha,25(OH)2D3/h/incubation after 1 and 4 weeks in culture respectively. In a further experiment synthesis of 1 alpha,25(OH)2D3 by long-term cultured macrophages fell from 2.25 to 0.04 pmol/h/incubation following exposure to 10 nM 1 alpha,25(OH)2D3 for 7 days, whereas 24,25(OH)2D3 synthesis was induced (0.46 pmol/h/incubation). The vitamin D3 metabolites were identified by co-chromatography with authentic 24,25(OH)2D3 or 1 alpha,25(OH)2D3 in three different high-performance liquid chromatography systems. Serum 1 alpha,25(OH)2D3 in the patient was markedly suppressed at 5 pg/ml (normal 20-50 pg/ml) indicating that raised 1 alpha,25(OH)2D3 was not the cause of the hypercalcaemia, but rather, that raised calcium may have suppressed renal 1 alpha,25(OH)2D3 synthesis. Administration of APD (3-amino-1-hydroxypropylidine-1,1-bisphosphonate) corrected the hypercalcaemia in the patient suggesting that increased bone resorption was responsible for the raised calcium. The results of this study show for the first time that immature blood derived monocyte-macrophage cells can synthesize 24,25(OH)2D3 before they mature into macrophages able to synthesize 1 alpha,25(OH)2D3.     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.