Absence of regulatory effects of 1alpha25-dihydroxyvitamin D3 on 25-hydroxyvitamin D metabolism in rats constantly infused with parathyroid hormone

The regulatory role of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2-D3] in metabolism of 25-hydroxyvitamin D was studied in sham-operated (sham) or thyroparathyroidectomized (TPTX) vitamin D-deficient rats into which calcium and parathyroid hormone (PTH) were constantly infused. A single dose of 325 or 650 pmol of 1alpha,25-(OH)2-D3 caused significant inhibition of 1alpha,25-(OH)2-D3 synthesis in D-deficient sham rats. This inhibition by 1alpha,25-(OH)2-D3, however, was not observed in D-deficient TPTX rats into which PTH was constantly infused. These results can be explained by supposing that the major regulatory effect of 1alpha,25-(OH) 2-D3 on 1alpha,25-(OH)2-D3 synthesis is realized mostly, if not all, by suppressing endogenous secretion of PTH.     

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.     

Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D-binding protein and resistance to metabolism

Eldecalcitol [1α,25‐dihydroxy‐2β‐(3‐hydroxypropyloxy)vitamin D₃], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25‐dihydroxyvitamin D₃ (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D‐binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100‐fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [³H]calcitriol was completely degraded within 24 h, [³H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24‐hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high‐DBP affinity which increases the half‐life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues. J. Cell. Biochem. 112: 1348–1352, 2011. © 2011 Wiley‐Liss, Inc.     

Somatostatin and intestinal calcium transport in the rat

In intact rats we studied the influence of low doses of intravenously (i.v.) administered somatostatin (SRIF) on the net absorption and the bidirectional fluxes (lumen-to-plasma, LP; plasma-to-lumen, PL) of calcium in the duodenum, jejunum, ileum and caecum. In the duodenum SRIF inhibited the LP-flux and the net absorption of Ca significantly at infusion rates of 0.75 and 1.0 microgram SRIF . kg-1 . h-1. The PL-flux was not altered by any of the SRIF doses administered. In the other gut segments studied (jejunum, ileum, caecum) neither the net absorption nor the bidirectional Ca fluxes were changed by i.v. SRIF. It is concluded that SRIF in the plasma levels achieved in this study has an influence on the duodenal calcium absorption (CaA) of the rat; questions regarding the mechanisms of this action as well as the physiological significance of our findings are as yet unresolved.     

The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells

Human pancreatic growth hormone releasing factor (1-29)-amide [hpGRF (1-29)-NH2] and the following analogs: [D-Tyr-1]-hpGRF(1-29)-NH2, [D-Ala-2]-hpGRF(1-29)-NH2, [D-Asp-3]-hpGRF(1-29)-NH2, and [N-Ac-Tyr-1]-hpGRF (1-29)-NH2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1-29)-NH2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1-29 amide in the rat. [D-Tyr-1]-hpGRF(1-29)-NH2 was 10 times more potent, [D-Asp-3]-hpGRF(1-29)-NH2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF(1-29)-NH2.     

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.     

Bone calcification rate as a factor of craniofacial transformations in salmonid fish: Insights from an experiment with hormonal treatment of calcium metabolism

Adaptation to different environments can be achieved by physiological shifts throughout development. Hormonal regulators shape the physiological and morphological traits of the evolving animals making them fit for the particular ecological surroundings. We hypothesized that the artificially induced hypersynthesis of calcitonin and parathyroid hormone mutually influencing calcium metabolism could affect bone formation during early ontogeny in fish imitating the heterochrony in craniofacial ossification in natural adaptive morphs. Conducting an experiment, we found that the long-standing treatment of salmonid juveniles with high doses of both hormones irreversibly shifts the corresponding hormone status for a period well beyond the time scale for total degradation of the injected hormone. The hormones program the ossification of the jaw suspension bones and neurocranial elements in a specific manner affecting the jaws position and pharingo-branchial area stretching. These morphological shifts resemble the adaptive variants found in sympatric pelagic and demersal morphs of salmonids. We conclude that solitary deviations in the regulators of calcium metabolism could determine functional morphological traits via transformations in skeletal development.     

Expression of vitamin D receptors in the superior cervical ganglia of rats

We investigated the role of vitamin D in the sympathetic nervous system including the distribution of vitamin D receptors (VDR), 1α-hydroxylase and 24-hydroxylase (CYP24) in neuronal subpopulations and satellite glia in the superior cervical ganglia (SCGs) of rats using immunohistochemistry. VDR immunoreactivity was observed in the cytoplasm and nucleus of nearly all neurons in the SCG. Intensity of VDR fluorescence was significantly greater in the cytoplasm of neuropeptide Y (NPY) negative somata compared to NPY positive neurons. Immunoreactivity for 1α-hydroxylase also was observed in the cytoplasm of all neurons of the SCG, but the intensity of fluorescence was less in the nuclei. To the contrary, the immunoreactivity for CYP24 was stronger in the nuclei, although it was present at lower intensity also in the cytoplasm of neurons. VDR and 1α-hydroxylase immunofluorescence was observed in many non-neuron cells, except satellite glial cells, which exhibited weak CYP24 immunofluorescence. Expression of VDRs and key metabolizing enzymes indicated the importance of vitamin D in the autonomic nervous system and the ability of sympathetic neurons to activate and deactivate vitamin D for its autocrine and paracrine roles.     

Lack of a direct role for cyclic AMP in parathyrin action on phosphate reabsorption by the kidney

Isolated chick kidney proximal tubule cells have been used in a study of the mechanism by which PTH inhibits Na+-dependent Pi transport in the kidney. Treatment with PTH inhibits Pi uptake by the cells by 13% and stimulates cyclic AMP production by 77%. Forskolin, a potent activator of adenyl cyclase, brought about an 11-fold stimulation of cyclic AMP production by the cells, but in contrast to PTH, the drug had no effect on Na+-dependent Pi uptake. These results provide evidence that PTH action on phosphate transport is not mediated by cyclic AMP.     

Role of pituitary hormones in regulating renal vitamin D metabolism in man.

Studies in animals and tissue culture have shown the importance of prolactin and growth hormone in regulating renal 1 alpha-hydroxylase activity and plasma concentrations of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Evidence for a similar role for these hormones in man was sought by using a radioreceptor assay to measure plasma 1,25(OH)2D3 concentrations in 20 normal subjects, 12 patients receiving dialysis, 11 patients with primary hyperparathyroidism, 10 pregnant women, seven women with prolactinoma, and 14 patients with acromegaly. Circulating 1,25(OH)2D3 concentrations were appreciably raised in the patients with primary hyperparathyroidism and the pregnant women (P less than 0.001), slightly but significantly increased in the patients with prolactinoma (P less than 0.05), and greatly raised in those with acromegaly (P less than 0.001). These results suggest that prolactin and growth hormone are important regulators of renal vitamin D metabolism in the physiological conditions of pregnancy, lactation, and growth in man.     

Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 10⁶ cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25–30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)₂D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)₃D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)₂D-3 decreased 1,25(OH)₂D-3 synthesis, and increased both 24,25(OH)₂D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)₂D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.     

Association of vitamin D receptor BsmI (rs1544410) gene polymorphism with the intact parathyroid hormone (iPTH) level among patients with end-stage renal disease

Abstract

Association of vitamin D receptor (VDR) BsmI (rs1544410) gene polymorphism with the intact parathyroid hormone (iPTH) level among patients with end-stage renal disease (ESRD) from the published reports is still conflicting. This meta-analysis was performed to evaluate the relationship between VDR BsmI (rs1544410) gene polymorphism and the iPTH level among patients with ESRD. The association studies were identified from PubMed, and Cochrane Library on 1 March 2014, and eligible investigations were included and synthesized using meta-analysis method. Six reports were recruited into this meta-analysis for the association of VDR BsmI gene polymorphism with iPTH level among patients with ESRD. In this meta-analysis, the iPTH level in ESRD patients carrying BsmI Bb genotype was higher than that in ESRD patients carrying bb genotype in overall populations (Bb versus bb: OR?=?61.40, 95% CI: 19.65–103.16, p?=?0.004). However, the iPTH level in ESRD patients carrying BB genotype was not significant different from that in ESRD patients with Bb genotype and bb genotype in overall populations (BB versus Bb: OR?=??18.30, 95% CI: ?126.28–89.69, p?=?0.74; BB versus bb: OR?=?22.85, 95% CI: ?70.81–116.51, p?=?0.63). Furthermore, the results for Caucasians were similar to those in overall populations. In conclusion, the iPTH level in ESRD patients carrying BsmI Bb genotype was higher than that in ESRD patients carrying bb genotype in overall populations and in Caucasians. However, more studies should be conducted to confirm it.     

Seasonal variation and correlation analysis of vitamin D and parathyroid hormone in Hangzhou,Southeast China

This study aimed to describe the 25‐hydroxyvitamin D (25(OH)D) and parathyroid hormone (PTH) status of Southeast Chinese individuals influenced by season. The secondary aim was to determine the cutoff for sufficient 25(OH)D in a four‐season region. From January 2011 to June 2014, a total of 17 646 individuals were evaluated in our study. The serum levels of PTH were detected simultaneously in 5579 cases. A total of 25(OH)D and intact PTH were measured by the electrochemiluminescent immunoassay. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D and PTH were studied. The mean 25(OH)D concentration in our study was 43.00(30.40) nmol/L. The prevalence of insufficiency (25(OH)D < 50 nmol/L) was 62.87% and that of deficiency (<30 nmol/L) was 28.54%. Mean serum 25(OH)D levels revealed a limited sinusoidal profile throughout the year and were significantly higher in Autumn. On the other hand, PTH levels showed an opposite response to seasonal effects relative to 25(OH)D. Age, BMI and daylight were not significantly correlated with 25(OH)D and serum PTH reached a plateau at higher values of serum 25(OH)D of 42.86 nmol/L. This study demonstrated that Vitamin D insufficiency is highly prevalent in Southeast China. The concentration of 25(OH)D in the male group was generally higher than that in the female group. Seasonal variation was an important aspect of 25(OH)D and PTH concentration. This study revealed that the optimal serum threshold of 25(OH)D for bone health should be between 40 and 50 nmol/L for Southeast Chinese individuals.     

Effects of PTH and Ca2+ on renal adenyl cyclase

The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal-lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity. Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH- or Gpp(NH)p-stimulated activity was given by approximately 50 μM Ca⁺⁺. Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half-maximal stimulation of cAMP formation (1.8 × 10^?8 M) was unchanged by the presence of calcium. These data suggest that calcium acts at some point other than the initial hormone-receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex.     

Calmodulin interacts with the cytoplasmic tails of the parathyroid hormone 1 receptor and a sub-set of class b G-protein coupled receptors

Parathyroid hormone (PTH) binds to its receptor (PTH 1 receptor, PTH1R) and activates multiple pathways. The PTH1R, a class b GPCR, contains consensus calmodulin-binding motifs. The PTH1R cytoplasmic tail interacts with calmodulin in a calcium-dependent manner via the basic 1-5-8-14 motif. Calcium-dependent calmodulin interactions with the cytoplasmic tails of receptors for PTH 2, vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, corticotropin releasing hormone, calcitonin, and the glucagon-like peptides 1 and 2 are demonstrated. The cytoplasmic tails of the secretin receptor and the growth hormone releasing hormone receptor either interact poorly or not at all with calmodulin, respectively. Fluphenazine, a calmodulin antagonist, enhances PTH-mediated accumulation of total inositol phosphates, suggesting that calmodulin regulates signaling via phospholipase C.     

A cell strain cultured from porcine kidney increases cyclic AMP content upon exposure to calcitonin or vasopressin.

Cells originally dispersed from whole juvenile male Hampshire pig kidney and maintained in monolayer culture, increased cyclic AMP content in response to incubation with salmon calcitonin or antidiuretic hormone. Parathyroid hormone and epinephrine did not affect cyclic AMP content. The apparent K_m for arginine vasopressin in the porcine cells was 3.0 nM which is similar to the value obtained in single segments of rabbit kidney tubule. The apparent K_m for salmon calcitonin of 2.7 nM is higher than that reported for the rabbit nephron segments, but comparable to the K_m obtained in rat kidney homogenates. Exposure of the porcine cells to exogenous prostaglandin E₂ did not affect cyclic AMP responses to other hormones. In the cultured porcine kidney cells the pattern of hormone response is similar to that observed in nephron segments prepared from the medullary portion of the thick ascending limb of the loop of Henle, and these findings suggest that the porcine cells may be related to cells present in the medullary region of the kidney tubule.     

Regulation of vitamin D metabolism by calcium and phosphate ions in isolated renal tubules.

The acute and long-term effects of Ca2+ and Pi on vitamin D metabolism were studied in vitro with isolated renal tubules from vitamin D-deficient and vitamin D-supplemented chicks. Ca2+ depletion, achieved by isolating renal tubules in Ca2+-free buffers, led to suppression of 1 alpha-hydroxylase activity. Re-introduction of Ca2+ during incubation caused an acute stimulation of this enzyme. This stimulatory effect of Ca2+ was prevented by prior treatment of Ca2+-depleted renal tubules for 6 h with 1,25-dihydroxycholecalciferol. Ca2+ and Pi produced marked acute affects on 1 alpha-hydroxylase activity, which persisted for the whole 8 h experimental period, in Ca2+-depleted renal tubules from vitamin D-deficient chicks. The effects of either ion were influenced by the concentration of the other. However, the effects of these ions could not be reproduced in either Ca2+-depleted renal tubules from vitamin D-supplemented chicks or in renal tubules from vitamin D-deficient chicks, isolated in Ca2+-containing buffers. Isolation of renal tubules from vitamin D-supplemented chicks in Ca2+-containing buffers and subsequent incubation for 8 h in the presence of increased [Ca2+] led to a modest but statistically significant suppression of 1 alpha-hydroxylase and stimulation of 24-hydroxylase activity. It is concluded that the acute effects of Ca2+ and Pi on 1 alpha-hydroxylase activity of Ca2+-depleted renal tubules from vitamin D-deficient chicks are not regulatory but the results of the experimental conditions. In contrast the long-term effects of Ca2+ on both hydroxylases of renal tubules from vitamin D-supplemented chicks may be of physiological significance.     

Fuzzy cluster analysis of molecular dynamics trajectories.

We propose fuzzy clustering as a method to analyze molecular dynamics (MD) trajectories, especially of proteins and polypeptides. A fuzzy cluster analysis locates classes of similar three-dimensional conformations explored during a molecular dynamics simulation. The method can be readily applied to results from both equilibrium and nonequilibrium simulations, with clustering on either global or local structural parameters. The potential of this technique is illustrated by results from fuzzy cluster analyses of trajectories from MD simulations of various fragments of human parathyroid hormone (PTH). For large molecules, it is more efficient to analyze the clustering of root-mean-square distances between conformations comprising the trajectory. We found that the results of the clustering analysis were unambiguous, in terms of the optimal number of clusters of conformations, for the majority of the trajectories examined. The conformation closest to the cluster center can be chosen as being representative of the class of structures making up the cluster, and can be further analyzed, for example, in terms of its secondary structure. The CPU time used by the cluster analysis was negligible compared to the MD simulation time.