Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Coumestrol induces mitochondrial dysfunction by stimulating ROS production and calcium ion influx into mitochondria in human placental choriocarcinoma cells.

STUDY QUESTION: Does coumestrol inhibit proliferation of human placental choriocarcinoma cells? SUMMARY ANSWER: Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis. WHAT IS KNOWN ALREADY: A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas. STUDY DESIGN, SIZE, DURATION: Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner. PARTICIPCANTS/MATERIALS, SETTING, METHODS: Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing.

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

Species differences in the antidiarrheal and antispasmodic activities of Lepidium sativum and insight into underlying mechanisms.

The aim of this study was to see if the crude extract of Lepidium sativum (Ls.Cr) exhibits species specificity in its antidiarrheal and antispasmodic activities along with insight into the underlying mechanisms using the in-vivo and in-vitro experiments. Ls.Cr inhibited castor oil-induced diarrhea in mice at doses (300 and 1000 mg/kg) three times higher dose than for rats. In isolated rat ileum and jejunum, Ls.Cr completely inhibited carbachol (CCh), low K⁺ (25 mM) and high K⁺ (80 mM)-induced contractions, while in guinea-pig tissues, Ls.Cr caused complete inhibition of only CCh-induced contraction. In rabbit tissues, Ls.Cr completely inhibited CCh and low K⁺-induced contractions sensitive to K⁺ channel antagonists. Pretreatment of guinea-pig and rat tissues with Ls.Cr caused a rightward shift in CCh-induced contractions in a pattern similar to dicyclomine, while in rabbit and rat tissues, Ls.Cr shifted isoprenaline curves to the left similar to papaverine. These data indicate that the antidiarrheal and antispasmodic activities of L. sativum are species dependent, mediating its antispasmodic effect through combinations of multiple pathways including activation of K⁺ channels, and inhibition of muscarinic receptors, Ca⁺⁺ channels and PDE enzyme. Rat tissues showed the highest potency. Based on the results, we recommend using multiple species to know the real pharmacological profile of medicinal products.

Immunomodulatory activity of oenothein B isolated from Epilobium angustifolium.

Epilobium angustifolium has been traditionally used to treat of a number of diseases; however, not much is known regarding its effect on innate immune cells. In this study, we report that extracts of E. angustifolium activated functional responses in neutrophils and monocyte/macrophages. Activity-guided fractionation, followed by mass spectroscopy and NMR analysis, resulted in the identification of oenothein B as the primary component responsible for phagocyte activation. Oenothein B, a dimeric hydrolysable tannin, dose-dependently induced a number of phagocyte functions in vitro, including intracellular Ca(2+) flux, production of reactive oxygen species, chemotaxis, NF-kappaB activation, and proinflammatory cytokine production. Furthermore, oenothein B was active in vivo, inducing keratinocyte chemoattractant production and neutrophil recruitment to the peritoneum after intraperitoneal administration. Biological activity required the full oenothein B structure, as substructures of oenothein B (pyrocatechol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid) were all inactive. The ability of oenothein B to modulate phagocyte functions in vitro and in vivo suggests that this compound is responsible for at least part of the therapeutic properties of E. angustifolium extracts.

Calcimimetic inhibits late-stage cyst growth in ADPKD.

In polycystic kidney disease (PKD), genetic mutations in polycystin 1 and 2 lead to defective intracellular trafficking of calcium, thereby decreasing intracellular calcium and altering cAMP signaling to favor proliferation. We hypothesized that calcimimetics, allosteric modulators of the calcium-sensing receptor, would reduce cyst growth by increasing intracellular calcium. We randomly assigned 20-wk-old male rats with a form of autosomal dominant PKD (heterozygote Cy/+) to one of four groups for 14 to 18 wk of treatment: (group 1) no treatment; (group 2) calcimimetic R-568 formulated in the diet; (group 3) R-568 plus calcium-supplemented drinking water (R-568 plus Ca); or (group 4) Ca-supplemented drinking water with a normal diet (Ca). Severity of PKD did not progress in any of the three treatment groups between 34 and 38 wk. Compared with no treatment, cyst growth was unaffected at 34 wk by all treatments, but cyst volume and fibrosis were lower at 38 wk, with both R-568-treated groups demonstrating a greater reduction than calcium alone. Between 34 and 38 wk, the total kidney weight increased by 78% in the control group (P < 0.001) and by 19% in the Ca group (P < 0.01), but did not increase in the R-568 or R-568 plus Ca groups, suggesting inhibition of disease progression despite equivalent suppression of parathyroid hormone. In summary, treatment of hyperparathyroidism halts late-stage progression of rodent cystic kidney disease. The benefit of R-568 alone suggests calcium-sensing receptor modulation may have additional inhibitory effects on late-stage cyst growth resulting from a direct modulation of intracellular calcium.

Interstitial fibrosis and microvascular disease of the heart in uremia: amelioration by a calcimimetic.

In patients with chronic renal failure, the heart undergoes remodeling, characterized by hypertrophy, fibrosis, and capillary/myocyte mismatch. In this study, we observed the effects of the calcimimetic agent R-568 on microvascular disease and interstitial fibrosis of the heart. Three-month-old male Sprague-Dawley rats were randomized to subtotal nephrectomy (SNX) or sham operation and subsequently received vehicle or R-568 under two experimental protocols, one for 1 month and the other for 3 months. Echocardiography, capillary length density, volume density of interstitial tissue, and immunohistochemistry and western blots (calcium-sensing receptor, collagen I and III, transforming growth factor (TGF)-beta, mitogen-activated protein kinases, and nitrotyrosine) were assessed. After SNX, weight and wall thickness of the left and the right ventricle were elevated. The ratio of heart to body weight and interventricular septum thickness were not changed by R-568 treatment. The left ventricle fractional shortening (by echocardiography) was lower in SNX; this was ameliorated by R-568. Reduced capillary length density and increased interstitial fibrosis in SNX were improved by R-568, which also reduced the expression of TGF-beta, and collagen I and III. The calcimimetic increased the activation of ERK-1/2, normalized p38 and JNK signaling, and prevented oxidative stress. We conclude that lowering parathyroid hormone with a calcimimetic significantly improves cardiac histology and function but not the left ventricular mass in SNX.

Calcification inhibitors and Wnt signaling proteins are implicated in bovine artery smooth muscle cell calcification in the presence of phosphate and vitamin D sterols.

Administration of active vitamin D sterols to treat secondary hyperparathyroidism in patients with chronic kidney disease receiving dialysis has been associated with elevated serum calcium and phosphorus levels, which may lead to increased risk of vascular calcification. However, calcimimetics, by binding to the parathyroid gland calcium-sensing receptors, reduce serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Using cultured bovine aorta vascular smooth muscle cells (BASMCs), an in vitro model of vascular calcification, we compared calcification levels and gene expression profiles after exposure to the phosphate source ss-glycerolphosphate (BGP), the active vitamin D sterols calcitriol and paricalcitol, the calcimimetic R-568, or BGP with the active vitamin D sterols or R-568. Cells exposed to BGP (10 mM) alone or with calcitriol or paricalcitol showed dose-dependent BASMC calcification. No change in calcification was observed in cultures exposed to BGP with R-568, consistent with the observed lack of calcium-sensing receptor expression. Microarray analysis using total cellular RNA from cultures exposed to vehicle or BGP in the absence and presence of 10(-8) M calcitriol or paricalcitol for 7 days showed that cells exposed to BGP with calcitriol or BGP with paricalcitol had virtually identical gene expression profiles, which differed from those of cells treated with BGP or vehicle alone. Several osteoblast- and chondrocyte-associated genes were modulated by BGP and vitamin D exposure. In this study, exposure of BASMCs to phosphate and active vitamin D sterols induced calcification and changes in expression of genes associated with mineralized tissue.

Calcimimetics for secondary hyperparathyroidism in chronic kidney disease patients.

BACKGROUND: Calcimimetic agents have recently been evaluated in the treatment of secondary hyperparathyroidism (SHPT) as add-on therapy to calcitriol and vitamin D analogues and dietary phosphate binders. OBJECTIVES: To evaluate the benefits and harms of calcimimetics for the prevention of secondary hyperparathyroid bone disease (including osteitis fibrosa cystica and adynamic bone disease) in dialysis patients with chronic kidney disease (CKD). SEARCH STRATEGY: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials and conference proceedings were searched for randomised controlled trials (RCTs) evaluating any calcimimetic against placebo or another agent in pre-dialysis or dialysis patients with CKD. SELECTION CRITERIA: We included all RCTs of any calcimimetic agent, cinacalcet HCl (AMG-073, Sensipar), NPS R-467 or NPS R-568 administered to patients with CKD for the treatment of SHPT. DATA COLLECTION AND ANALYSIS: Data were extracted on all relevant patient-centred and surrogate outcomes. Analysis was by a random effects model and results expressed as relative risk (RR) or weighted mean difference (MD) with 95% confidence intervals. MAIN RESULTS: Eight studies (1429 patients) were identified, which compared a calcimimetic agent plus standard therapy to placebo plus standard therapy. The end of treatment values of parathyroid hormone (pg/mL) (MD -290.79, 95% CI -360.23 to -221.34), serum calcium (mg/dL) (MD -0.85, 95% CI -1.14 to -0.56), serum phosphorus (mg/dL) (MD -0.29, 95% CI -0.50 to -0.08) and the calcium by phosphorus product (mg(2)/dL(2))(MD -7.90, 95% CI -10.25 to -5.54) were significantly lower with calcimimetics compared to placebo. No significant effects on patient-based endpoints were demonstrated except for the risk of hypotension which was significantly reduced with calcimimetics compared to placebo (RR 0.53, 95%CI 0.36 to 0.79). AUTHORS' CONCLUSIONS: Calcimimetic treatment of SHPT leads to significant improvements in biochemical parameters that observational studies have shown to be associated with increased mortality, cardiovascular risk and osteitis fibrosa, but patient-based benefits have not yet been demonstrated in trials. For patients with SHPT, the benefits of calcimimetics over standard therapy remain uncertain until further RCTs become available.

Pharmacological activity of calcimimetic NPS R-568 administered intravenously in rats: dose dependency.

Calcimimetics administered orally cause "pharmacological parathyroidectomy" confirmed by a decrease in parathyroid hormone secretion (PTH) and in plasma Ca(2+) concentration. Parathyroids are also the source of parathyroid hypertensive factor (PHF). The aim of this study was to determine the dose-dependent effect of an intravenously (iv) applied calcimimetic, NPS R-568, on plasma Ca(2+) concentration, urinary phosphate excretion and mean arterial blood pressure (MAP) in rats. Clearance experiments were performed on male Wistar rats anesthetized with thiopental and infused iv with saline supplemented with (3)H inulin for glomerular filtration rate (GFR) determination. NPS R-568 was administered iv as a bolus at the doses: 0.5, 1.0, 2.5 and 5.0 mg/kg. Control group of rats received vehicle only. MAP was monitored continuously in the carotid artery. Urine was collected from cannulated urinary bladder. NPS R-568 applied iv dose-dependently decreased plasma Ca(2+) and fractional phosphate excretion (FE(Pi)). In the control group, no significant changes in plasma Ca(2+) and FE(Pi) were observed. The most efficient hypotensive effect vs. control group was induced by the NPS R-568 of a dose of 1.0 mg/kg. Our results indicate that the dose of 1 mg/kg of the calcimimetic NPS R-568 administered iv is sufficient to induce the decrease in plasma Ca(2+) and urinary phosphate excretion accompanied with hypotensive effect in Wistar rats.

Coordinated control of renal Ca(2+) transport proteins by parathyroid hormone.

BACKGROUND: The kidney is one of the affected organs involved in the clinical symptoms of parathyroid hormone (PTH)-related disorders, like primary hyperparathyroidism and familial hypocalciuric hypercalcemia. The molecular mechanism(s) underlying alterations in renal Ca(2+) handling in these disorders is poorly understood. METHODS: Parathyroidectomized and PTH-supplemented rats and mice infused with the calcimimetic compound NPS R-467 were used to study the in vivo effect of PTH on the expression of renal transcellular Ca(2+) transport proteins, including the epithelial Ca(2+) channel transient receptor potential, vanilloid, member 5 (TRPV5), calbindins, and the Na(+)/Ca(2+)-exchanger (NCX1). In addition, the effect of PTH on transepithelial Ca(2+) transport in rabbit connecting tubule/cortical collecting duct (CNT/CCD) primary cultures was determined. RESULTS: Decreased PTH levels in parathyroidectomized rats or NPS R-467-infused mice, resulted in reduced expression of these proteins, which is consistent with diminished Ca(2+) reabsorption, causing the development of the observed hypocalcemia. PTH supplementation of parathyroidectomized rats restored the expression of the renal Ca(2+) transport machinery and serum Ca(2+) levels, independent of serum 1,25-dihydroxyvitamin D(3) levels and renal vitamin D or Ca(2+)-sensing receptor mRNA abundance. Inhibition of the PTH-stimulated transepithelial Ca(2+) transport by the TRPV5-specific inhibitor ruthenium red reduced the PTH-stimulated expression of calbindin-D(28K) and NCX1 in rabbit CNT/CCD primary cultures. CONCLUSION: PTH stimulates renal Ca(2+) reabsorption through the coordinated expression of renal transcellular Ca(2+) transport proteins. Moreover, the PTH-induced stimulation is enhanced by the magnitude of the Ca(2+) influx through the gatekeeper TRPV5, which in turn facilitates the expression of the downstream Ca(2+) transport proteins. Therefore, the renal transcellular Ca(2+) transport proteins, including TRPV5, could contribute to the pathogenesis of PTH-related disorders.

Functional proteins involved in regulation of intracellular Ca(2+) for drug development: the extracellular calcium receptor and an innovative medical approach to control secondary hyperparathyroidism by calcimimetics.

Circulating levels of calcium ion (Ca(2+)) are maintained within a narrow physiological range mainly by the action of parathyroid hormone (PTH) secreted from parathyroid cells. Parathyroid cells can sense small fluctuations in plasma Ca(2+) levels by virtue of a cell surface Ca(2+) receptor (CaR) that belongs to the superfamily of G-protein-coupled receptors. Calcimimetics are positive allosteric modulators that activate the CaR on parathyroid cells and thereby immediately suppress PTH secretion. Pre-clinical studies with NPS R-568, a first generation calcimimetic compound, have demonstrated that daily oral administration inhibits the elevation of plasma PTH levels and parathyroid gland hyperplasia and ameliorates impaired bone qualities in rats with chronic renal insufficiency. The results of clinical trials with cinacalcet hydrochloride, a second generation calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum calcium x phosphorus product levels, a hallmark of an increased risk for cardiovascular death in dialysis patients with end-stage renal disease (ESRD). Thus, calcimimetics have considerable potential as an innovative medical approach to manage secondary hyperparathyroidism associated with ESRD. Indeed, cinacalcet hydrochloride has been approved in several countries and is the first positive allosteric modulator of any G protein-coupled receptor to reach the market.

Magnesium may mediate the favorable impact of whole grains on insulin sensitivity by acting as a mild calcium antagonist.

Recent epidemiology has linked high consumption of whole grains with reduced risk for diabetes, coronary disease, stroke, and various types of cancer; there is reason to suspect that improved insulin sensitivity is largely responsible for this protection. This phenomenon may be partially explained by the lower glycemic indices of some whole grain food products in comparison to their fiber-depleted analogs. Nonetheless, the fact that whole wheat flour promotes insulin sensitivity relative to white flour--and yet has a near-identical glycemic index--suggests that certain nutrients or phytochemicals in whole wheat, depleted by the refining process, promote preservation of insulin sensitivity. Magnesium is a likely candidate in this regard; magnesium deficiency promotes insulin resistance in rodents and in humans, whereas supplemental magnesium has been found to prevent type 2 diabetes in rodent models of this syndrome, and to improve the insulin sensitivity of elderly or diabetic humans. Magnesium-rich diets as well as above-average serum magnesium are associated with reduced diabetes risk in prospective epidemiology, and with greater insulin sensitivity in cross-sectional studies; moreover, other types of magnesium-rich foods--dairy products, legumes, and nuts--have been linked to decreased diabetes risk in prospective studies. The biochemical role of magnesium in support of insulin function is still poorly understood. In light of evidence that magnesium can function as a mild natural calcium antagonist, it is interesting to note suggestive evidence that increases in intracellular free calcium may compromise the insulin responsiveness of adipocytes and skeletal muscle, and may indeed play a pathogenic role in the insulin resistance syndrome. Thus, it is proposed that some or all of the favorable impact of good magnesium status on insulin function may reflect antagonism of the induction or effects of increased intracellular free calcium. Further research concerning the potential health benefits of long-term magnesium supplementation is clearly warranted. These considerations, however, should not detract from efforts to better inform the public regarding the strong desirability of choosing whole grain products in preference to refined grains.

[Calcimimmetic and calcilytics: new perspectives of correction of abnormal parathormone (PTH) secretion]. / Kalcymimetyki i kalcylityki: nowe perspektywy korekcji zaburzonej sekrecji parathormonu (PTH).

Treatment of excessive secretion of parathyroid hormone (PTH) in primary hyperparathyroidism (I degree HPT) as well as in secondary hyperparathyroidism (II degree HPT) in chronic renal insufficiency is symptomatic, short-term acting and far from expectations. Recognition of properties of calcium receptor (CaR) expressed on parathyroid principal cell membranes created possibilities to explore new compounds that could alter directly PTH secretion and provide a novel therapy for direct correction of increased secretion of the hormone in these disorders. Ligands that activate this receptor and inhibit PTH secretion are called calcimimetics. Recently clinical trials with NPS R-568, a calcimimetic of the Ist generation, and AMG 073, a representative of calcimimetics of IInd generation, were completed. Calcimimetics, taken orally, effectively lower increased secretion of PTH and hypercalcemia in I degree HPT, by "pharmacologic parathyroidectomy". Such compounds are also safe and effective in dialysed patients with II degree HPT in chronic renal insufficiency: they decrease PTH plasma level and prevent parathyroid cell hyperplasia. The other compounds, called calcilytics and represented by NPS 2143, inhibit CaR resulting therefore in increase of PTH secretion. Administration of calcilytics would provide a valuable alternative to inhibit progression of osteoporosis. Subcutaneous, pulsative low doses of recombinant PTH (ALX1-11) administration induces increase of bone formation. Such an effect to some extent was obtained by transient increase of endogenous PTH secretion induced by oral administration of calcilytic NPS 2143 to osteopenic ovariectomized rats, especially if it was accompanied by supplementation of estrogens.

Evidence for calcium-sensing receptor mediated stanniocalcin secretion in fish.

The secretion of parathyroid hormone (PTH) and calcitonin (CT) in mammals are both tightly regulated by the prevailing levels of extracellular ionic calcium (Ca(2+)). And, it is now widely recognized that both of these Ca(2+) effects are mediated exclusively through a seven transmembrane calcium sensing receptor or CaR. As in the case of PTH and CT, the secretion of stanniocalcin (STC) in fish is tightly regulated by the levels of extracellular Ca(2+). Fish STC functions as an anti-hypercalcemic hormone such that a rise in extracellular Ca(2+) above the physiological set-point of approximately 1.2 mM provokes an immediate secretory response. Whether or not Ca(2+)-regulated STC secretion in fishes is mediated by similar type of receptor has never been addressed. Here, we have found that Ca(2+)-stimulated STC secretion in salmon is mimicked by CaR mimetics, pharmacological agents that increase the sensitivity of the CaR to calcium. NPS 467, a small organic molecule that acts as a positive allosteric modulator of the CaR and alters calciotropic hormone secretion in mammals, was examined for effects on serum levels of STC in trout. The IP administration of NPS R-467 had time and dose-dependent stimulatory effects on STC secretion that were indistinguishable from those of Ca(2+) loading. The effects of NPS 467 were stereospecific and had no effects on serum CT. NPS 467 induced STC release was also manifested by a downstream physiological response; the inhibition of gill calcium transport. A cDNA clone was amplified from a fish corpuscle of Stannius cDNA library with high homology to the human CaR. RT-PCR revealed that this transcript was also present in gill, kidney, pancreas, brain, muscle and spleen. These findings suggest that Ca(2+)-stimulated STC secretion in fishes is mediated by a calcium ion-sensing receptor similar to that in mammals.

Prevention and treatment of renal osteodystrophy in children with chronic renal insufficiency and end-stage renal disease.

Histologic features associated with secondary hyperparathyroidism remain the predominant skeletal lesion in adults and children with chronic renal failure. When instituted early, vitamin D therapy has been shown to ameliorate the development and progression of the biochemical, radiographic, and histologic evidence of secondary hyperparathyroidism in patients with chronic renal insufficiency. Aggressive parathyroid hormone suppression, however, has led to the increased prevalence of adynamic bone. Adynamic bone has been attributed partly to aggressive calcitriol therapy, administration of high amounts of exogenous calcium either as a phosphate binding agent or during dialysis therapy, presence of diabetes, older age, or previous parathyroidectomy. Several vitamin D analogues are currently being evaluated in patients with chronic renal failure to prevent complications associated with calcitriol therapy. In addition, calcium-free phosphate binding agents and the use of calcimimetic drugs may play a significant role in the effective management of secondary hyperparathyroidism in children with chronic renal failure.

A novel receptor-mediated regulation mechanism of type I inositol polyphosphate 5-phosphatase by calcium/calmodulin-dependent protein kinase II phosphorylation.

D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) are both substrates of the 43-kDa type I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensitive inhibition by 70-85% of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) 5-phosphatase activities was observed in homogenates from rat cortical astrocytes, human astrocytoma 1321N1 cells, and rat basophilic leukemia RBL-2H3 cells after incubation with carbachol. The effect was reproduced in response to UTP in rat astrocytic cells and Chinese hamster ovary cells overexpressing human type I 5-phosphatase. Immunodetection as well as mass spectrometric peptide mass fingerprinting and post-source decay (PSD) sequence data analysis after immunoprecipitation permitted unambiguous identification of the major native 5-phosphatase isoform hydrolyzing Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) as type I inositol polyphosphate 5-phosphatase. In ortho-(32)P-preincubated cells, the phosphorylated 43 kDa-enzyme could be identified after receptor activation by immunoprecipitation followed by electrophoretic separation. Phosphorylation of type I 5-phosphatase was blocked after cell preincubation in the presence of Ca(2+)/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62). In vitro phosphorylation of recombinant type I enzyme by Ca(2+)/calmodulin kinase II resulted in an inhibition (i.e. 60-80%) of 5-phosphatase activity. In this study, we demonstrated for the first time a novel regulation mechanism of type I 5-phosphatase by phosphorylation in intact cells.

[Effects of alfacalcidol on osteoporosis in ovariectomized rats].

OBJECTIVE: To investigate the effects of alfacalcidol on osteoporosis in rats induced by ovariectomy (OVX). METHODS: Fifty-three virgin female Wistar rats of six months old were divided into 6 groups: (1) rats in baseline group (base) were sacrificed what 6 months before OVX, and those in the other 5 groups were either bilaterally OVX or sham-operated (sham) as follows: (2) Sham; (3) OVX baseline (OVXb); (4) OVX end (OVXe); (5) OVX + 17beta-estradiol 20 microgram/kg.(-1) SC. (O + E); and (6) OVX + alfacalcidol 0.1 microgram/kg.(-1) oral (O + VD). Rats in OVXb group were sacrificed 6 weeks after OVX and the others were sacrificed 14 weeks after surgery. Alfacalcidol (TEVA, Israel) was fed orally at 6 weeks after OVX and lasted for 8 weeks. Urine and serum samples were collected before sacrifice to determine the bone turnover markers. The right tibia was processed undecalcified for histomorphometric analysis, and the right femur was prepared for pQCT scanning and bone biomechanical measurement with indentation test. RESULTS: Treatment of OVX rats with alfacalcidol (O + VD) increased tibial cancellous bone volume in histomorphometric analysis (12.0% +/- 1.1% vs 5.8% +/- 1.3%, P < 0.01). pQCT scanning showed that trabecular BMC and BMD were sharply elevated in O + VD group compared with OVXe group, increased 109.2% and 133.1% respectively (P < 0.01). In consistency with these changes in mechanical competency of cancellous bone in distal femur, maximal load was enhanced in O + VD group compared with OVXe group (21.3% +/- 4.0% N vs 7.9% +/- 1.9% N, P < 0.05). All these changes in O + VD group were similar to those in 17beta-estradiol treatment group (O + E). Serum calcium concentration increased in O + VD group. Histomorphometric and biochemical indices of bone resorption reduced in O + VD group. CONCLUSION: Alfacalcidol effectively prevent bone loss in OVX rats and restore partly the trabecula, with evidenced increasing density, contents as well as maximal loads of cancellous bone, and reduce bone resorption.