Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss‐of‐Function Gα11 Mutation

G‐protein subunit α‐11 (Gα₁₁) couples the calcium‐sensing receptor (CaSR) to phospholipase C (PLC)‐mediated intracellular calcium (Ca²⁺_i) and mitogen‐activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss‐of‐function Gα₁₁ mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα₁₁ germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild‐type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα₁₁, which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC‐mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα₁₁ proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα₁₁ protein to impair CaSR‐mediated Ca²⁺_i and extracellular signal‐regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα₁₁ cleft region also impaired signaling by PLC. The loss‐of‐function associated with the Ser220 Gα₁₁ mutant was rectified by treatment of cells with cinacalcet, which is a CaSR‐positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα₁₁ mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα₁₁ germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα₁₁ hydrophobic cleft region for CaSR‐mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss‐of‐function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Identification of a G‐Protein Subunit‐α11 Gain‐of‐Function Mutation,Val340Met,in a Family With Autosomal Dominant Hypocalcemia Type 2 (ADH2)

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain‐of‐function mutations of the G‐protein coupled calcium‐sensing receptor (CaSR), and ADH2 caused by germline gain‐of‐function mutations of G‐protein subunit α‐11 (Gα₁₁). To date Gα₁₁ mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole‐exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα₁₁ mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three‐dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C‐terminal α5 helix, which may affect G‐protein coupled receptor binding and G‐protein activation. In vitro functional expression of wild‐type (Val340) and mutant (Met340) Gα₁₁ proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα₁₁ led to a leftward shift of the concentration‐response curve with a significantly (p < 0.0001) reduced mean half‐maximal concentration (EC₅₀) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild‐type EC₅₀ of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain‐of‐function mutation. A novel His403Gln variant in transforming growth factor, beta‐induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain‐of‐function Gα₁₁ mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα₁₁ C‐terminal region for G‐protein function and CaSR signal transduction. © 2016 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Calcilytic Ameliorates Abnormalities of Mutant Calcium‐Sensing Receptor (CaSR) Knock‐In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)

Activating mutations of calcium‐sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D₃ or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT‐305/MK‐5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT‐305/MK‐5442 suppressed the hypersensitivity to extracellular Ca²⁺ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock‐in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT‐305/MK‐5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1‐34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock‐in mice exhibited low bone turnover due to the deficiency of PTH, and JTT‐305/MK‐5442 as well as PTH(1‐34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.     

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium‐Sensing Receptor‐Mediated Signaling

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β‐catenin), the molecular basis of abnormal calcium sensing by calcium‐sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein‐coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3′,5′‐cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N‐terminally truncated human GPR64 is constitutively active and a 15–amino acid–long peptide C‐terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full‐length GPR64 physically interacts with CaSR and attenuates the CaSR‐mediated intracellular Ca²⁺ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.     

A semimechanistic model of the time‐course of release of PTH into plasma following administration of the calcilytic JTT‐305/MK‐5442 in humans

JTT‐305/MK‐5442 is a calcium‐sensing receptor (CaSR) allosteric antagonist being investigated for the treatment of osteoporosis. JTT‐305/MK‐5442 binds to CaSRs, thus preventing receptor activation by Ca²⁺. In the parathyroid gland, this results in the release of parathyroid hormone (PTH). Sharp spikes in PTH secretion followed by rapid returns to baseline are associated with bone formation, whereas sustained elevation in PTH is associated with bone resorption. We have developed a semimechanistic, nonpopulation model of the time‐course relationship between JTT‐305/MK‐5442 and whole plasma PTH concentrations to describe both the secretion of PTH and the kinetics of its return to baseline levels. We obtained mean concentration data for JTT‐305/MK‐5442 and whole PTH from a multiple dose study in U.S. postmenopausal women at doses of 5, 10, 15, and 20 mg. We hypothesized that PTH is released from two separate sources: a reservoir that is released rapidly (within minutes) in response to reduction in Ca²⁺ binding, and a second source released more slowly following hours of reduced Ca²⁺ binding. We modeled the release rates of these reservoirs as maximum pharmacologic effect (E_max) functions of JTT‐305/MK‐5442 concentration. Our model describes both the dose‐dependence of PTH time of occurrence for maximum drug concentration (T_max) and maximum concentration of drug (C_max), and the extent and duration of the observed nonmonotonic return of PTH to baseline levels following JTT‐305/MK‐5442 administration.     

Parathyroid hormone signaling via Gαs is selectively inhibited by an NH2‐terminally truncated Gαs: Implications for pseudohypoparathyroidism

Pseudohypoparathyroid patients have resistance predominantly to parathyroid hormone (PTH), and here we have examined the ability of an alternative Gαs‐related protein to inhibit Gαs activity in a hormone‐selective manner. We tested whether the GNAS exon A/B‐derived NH₂‐terminally truncated (Tr) αs protein alters stimulation of adenylate cyclase by the PTH receptor (PTHR1), the thyroid‐stimulating hormone (TSH) receptor (TSHR), the β₂‐adrenergic receptor (β₂AR), or the AVP receptor (V2R). HEK293 cells cotransfected with receptor and full‐length (FL) Gαs ± Tr αs protein expression vectors were stimulated with agonists (PTH [10^?7 to 10^?9 M], TSH [1 to 100 mU], isoproterenol [10^?6 to 10^?8 M], or AVP [10^?6 to 10^?8 M]). Following PTH stimulation, HEK293 cells cotransfected with PTHR1 + FL Gαs + Tr αs had a significantly lower cAMP response than those transfected with only PTHR1 + FL Gαs. Tr αs also exerted an inhibitory effect on the cAMP levels stimulated by TSH via the TSHR but had little or no effect on isoproterenol or AVP acting via β₂AR or V2R, respectively. These differences mimic the spectrum of hormone resistance in pseudohypoparathyroidism type 1a (PHP‐1a) and type 1b (PHP‐1b) patients. In opossum kidney (OK) cells, endogenously expressing the PTHR1 and β₂AR, the exogenous expression of Tr αs at a level similar to endogenous FL Gαs resulted in blunting of the cAMP response to PTH, whereas that to isoproterenol was unaltered. A pseudopseudohypoparathyroid patient with Albright hereditary osteodystrophy harbored a de novo paternally inherited M1I Gαs mutation. Similar maternally inherited mutations at the initiation codon have been identified previously in PHP‐1a patients. The M1I αs mutant (lacking the first 59 amino acids of Gαs) blunted the increase in cAMP levels stimulated via the PTHR1 in both HEK293 and OK cells similar to the Tr αs protein. Thus NH₂‐terminally truncated forms of Gαs may contribute to the pathogenesis of pseudohypoparathyroidism by inhibiting the activity of Gαs itself in a GPCR selective manner. © 2011 American Society for Bone and Mineral Research     

α 1‐Adrenoceptor‐mediated Negative Inotropic Effect Caused by Intracellular Ionic Activities in Guinea‐pig Papillary Muscle

In guinea‐pig papillary muscle, phenylephrine (PE), an agonist of α₁‐adrenoceptor (α₁‐AR), led to a transient negative inotropic effect (NIE) and a subsequent sustained positive inotropic effect (PIE). To clarify the ionic mechanisms underlying the NIE, we measured the [Na⁺]i or [pH]i by ion‐selective microelectrodes. PE produced a decrease in the intracellular Na⁺ concentration ([Na⁺]i) and an increase in intracellular pH ([pH]i). During the phase of NIE, PE produced only a (−) change of [Na⁺]i (Δ[Na⁺]i). With a decrease in extracellular Na⁺ or an increase in extracellular Ca²⁺, the PE‐induced NIE was attenuated and PE produced (+)Δ[Na⁺]i. The PE‐induced NIE and (−)Δ[Na⁺]i were definitely strengthened by lowering the bath temperature or increasing the stimulation frequency. 2‐(2,6‐di‐methoxyphenoxyethyl)amino‐methyl‐1,4‐benzidioxane HCl, an antagonist of α_1A‐AR, completely abolished the PE‐induced NIE and (−)Δ[Na⁺]i. Phorbol 12,13‐dibutyrate, an activator of protein kinase C (PKC), decreased the baseline [Na⁺]i and twitch force and increased the baseline [pH]i in mimicry of PE. Pretreatment with 1‐5(isoquinolinesulphonyl)‐2‐methylpiperazine, an inhibitor of PKC, abolished the PE‐induced NIE and (−)Δ[Na⁺]i. During pretreatment with benzamil, an inhibitor of Na⁺/Ca²⁺ exchange, we found that the PE‐induced NIE and (−)Δ[Na⁺]i were reversibly abolished. Our results indicate the PE‐induced NIE may be elicited upon the activation of Na⁺/Ca²⁺ exchange which can be attributed to the (−)Δ[Na⁺]i. (−)Δ[Na⁺]i is mediated through a PKC‐dependent pathway via an activation of α_1A‐AR subtype and its effect could be strengthened remarkably at high [Na⁺]i and [Ca²⁺]i values.     

Sex and age modify biochemical and skeletal manifestations of chronic hyperparathyroidism by altering target organ responses to Ca2+ and parathyroid hormone in mice

We studied mice with or without heterozygous deletion of the Casr in the parathyroid gland (PTG) [^PTGCaSR(+/–)] to delineate effects of age and sex on manifestations of hyperparathyroidism (HPT). In control mice, aging induced a left‐shift in the Ca²⁺/parathyroid hormone (PTH) set point accompanied by increased PTG CaSR expression along with lowered serum Ca²⁺ and mildly increased PTH levels, suggesting adaptive responses of PTGs to aging‐induced changes in mineral homeostasis. The aging effects on Ca²⁺/PTH set point and CaSR expression were significantly blunted in ^PTGCaSR(+/–) mice, who showed instead progressively elevated PTH levels with age, especially in 12‐month‐old females. These 12‐month‐old knockout mice demonstrated resistance to their high PTH levels in that serum 1,25‐dihydroxyvitamin D (1,25‐D) levels and RNA expression of renal Cyp27b1 and expression of genes involved in Ca²⁺ transport in kidney and intestine were unresponsive to the rising PTH levels. Such changes may promote negative Ca²⁺ balance, which further exacerbate the HPT. Skeletal responses to HPT were age‐, sex‐, and site‐dependent. In control mice of either sex, trabecular bone in the distal femur decreased whereas cortical bone in the tibiofibular junction increased with age. In male ^PTGCaSR(+/–) mice, anabolic actions of the elevated PTH levels seemed to protect against trabecular bone loss at ≥3 months of age at the expense of cortical bone loss. In contrast, HPT produced catabolic effects on trabecular bone and anabolic effects on cortical bone in 3‐month‐old females; but these effects reversed by 12 months, preserving trabecular bone in aging mice. We demonstrate that the CaSR plays a central role in the adaptive responses of parathyroid function to age‐induced changes in mineral metabolism and in target organ responses to calciotropic hormones. Restraining the ability of the PTG to upregulate CaSRs by heterozygous gene deletion contributes to biochemical and skeletal manifestations of HPT, especially in aging females. © 2013 American Society for Bone and Mineral Research.     

Robust exponential l2 − l ∞ control for discrete‐time switched system: A cone complement linearization method

The robust exponential l₂ ? l _∞ control problem is considered in this paper for discrete‐time switched systems with both time‐varying delay and norm‐bounded parameter uncertainties. An exponential l₂ ? l _∞ performance index is first introduced for the discrete‐time switched systems. The designed controller is a memory exponential l₂ ? l _∞ controller. By introducing an average dwell time approach and a Lyapunov–Krasovskii functional technique, some sufficient criteria guaranteeing exponential stability are presented, and the desired memory exponential l₂ ? l _∞ controller is established by resorting to a cone complement linearization method. Some results on exponential stability using memoryless exponential l₂ ? l _∞ controller and asymptotical stability with traditional l₂ ? l _∞ performance index are also established. Finally, a numerical example is provided to demonstrate that the proposed approach can lead to less conservatism compared with the developed result using memoryless exponential l₂ ? l _∞ controller and asymptotical stability analysis with traditional l₂ ? l _∞ performance index. Copyright © 2011 John Wiley & Sons, Ltd.     

The methanolic extract of Guibourtia tessmannii (caesalpiniaceae) and selenium modulate cytosolic calcium accumulation,apoptosis and oxidative stress in R2C tumour Leydig cells: Involvement of TRPV1 channels

This study evaluated the effects of the methanolic extract of Guibourtia tessmannii (GT) and selenium (Se) on cell viability, intracellular calcium concentration ([Ca²⁺]_i), apoptosis and oxidative stress through transient receptor potential vanilloid 1 (TRPV1) channel activity in CCL‐97 (R2C) tumour Leydig cells. The cells were divided into nine groups and treated as follows: (a)‐Control, (b)‐Capsazepine (CPZ, 0.1 mM, a TRPV1 channel blocker), (c)‐Capsaicin (CAP, 0.01 mM, a TRPV1 channel activator), (d)‐GT (500 μg/ml), (e)‐GT+CPZ, (f)‐GT+CAP, (g)‐Se (200 nM), (h)‐Se+CPZ and (i)‐Se+CAP. After treatments, cell viability, [Ca²⁺]_i, apoptosis, caspase 3/9, reactive oxygen species (ROS) and mitochondrial membrane depolarisation (MMD) were evaluated. The [Ca²⁺]_i, apoptosis, caspase 3/9, MMD and ROS levels were significantly (p < 0.001) increased in CAP group, but lowered in CPZ group. Interestingly, these parameters were significantly (p < 0.001) improved by GT and Se, compared to the CAP group. Moreover, the co‐administration of GT+CAP or Se+CAP inhibited the cytotoxicity of CAP. Thus, the modulatory properties of GT and Se on Ca²⁺ influx, apoptosis and oxidative stress require the integrity of TRPV1 channel in CCL‐97 Leydig cells. These results suggest that GT and Se might be used in the management of cytotoxicity in the testes, involving TRPV1 channel activity.     

Dose‐Dependent Cannabidiol‐Induced Elevation of Intracellular Calcium and Apoptosis in Human Articular Chondrocytes

Cannabidiol (CBD) is the most abundant non‐psychoactive compound of Cannabis sativa extracts. Cannabinoids have been shown to exhibit anti‐inflammatory, analgesic, antioxidant, neuroprotective, and anti‐tumorigenic effects. In the present study, we investigated the effects of CBD on human articular chondrocytes. Cell viability was determined by Resazurin assays. Apoptosis was analyzed by annexin‐V/7‐actinomycin D (7‐AAD) staining followed by flow cytometry. Caspase 3/7 activity was measured with caspase assays. Intracellular Ca²⁺ ([Ca²⁺]_i) was monitored by time‐lapse fluorescence imaging. The perforated whole‐cell patch‐clamp technique was used for measuring the cell membrane potential. Erk1/2 phosphorylation was assessed by western blot analysis. The chondrocyte cell line C28/I2 and primary chondrocytes showed a reduced viability after treatment with concentrations of CBD greater than 4 µM. This apoptotic effect was accompanied by an increase of caspase 3/7 activity and an increase in the early apoptotic cell population. CBD elevated [Ca²⁺]_i, which was accompanied by depolarization of the cell membrane potential. The increase of [Ca²⁺]_i was abrogated, when Ca²⁺ was omitted from the bath solution, indicating an influx of extracellular Ca²⁺. The cannabinoid receptor 1 (CB1) antagonist AM251 inhibited the Ca²⁺ influx triggered by CBD. Preincubation with AM251 reduced the toxic effects of CBD. By looking for mediators of the apoptotic CBD effect downstream of the CB1 receptor, enhanced Erk1/2 phosphorylation could be detected after CBD treatment. However, this Erk1/2 activation proved to be unaffected by CB1 receptor blockage. The present study demonstrates that CBD promotes apoptosis and [Ca²⁺]_i elevation in human articular chondrocytes via a CB1‐receptor‐mediated mechanism. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 37:2540–2549, 2019     

Calcium-sensing receptor (CaSR) modulates vacuolar H<Superscript>+</Superscript>-ATPase activity in a cell model of proximal tubule

Background

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H⁺-ATPase in a cellular model of proximal tubule cells, OKP cells.

Methods

Biochemical activity of H⁺-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca²⁺]_i were also determined using Fluo-4.

Results

A significant increase of vacuolar H⁺-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd³⁺ (300 µM) and neomycin (200 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca²⁺ (Ca²⁺_o) between 10^?4 and 2 mM. Gd³⁺ and neomycin produced a sustained rise of [Ca²⁺]_i, an effect that disappears when extracellular calcium was removed in the presence of 0.1 µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5?×?10^?8 M) reduced the increase in [Ca²⁺]_i induced by neomycin.

Conclusion

CaSR stimulation induces an increase in the vacuolar H⁺-ATPase activity of OKP cells, an effect that involves an increase in [Ca²⁺]_i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

     

A Calcium Release Activated Calcium Influx in Primary Cultures of Rat Osteoblast-like Cells

Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca²⁺ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca²⁺]_e) by 5 μM thapsigargin (TG) or 2 μM A23187. Readdition of 1.8 mM [Ca²⁺]_e increased the free intracellular Ca²⁺ ([Ca²⁺]_i) after a delay of 30–60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca²⁺]_e and was substantially lowered if readdition of 1.8 mM [Ca²⁺]_e was preceded by, e.g., 0.72 mM [Ca²⁺]_e. CRAC-induced [Ca²⁺]_i peaks were correlated (r= 0.543) with [Ca²⁺]_i peaks during the complete depletion of ICS with A23187. Ca²⁺ influx due to CRAC could be blocked by flufenamic acid (100 μM) but not verapamil (20 μM). Ni²⁺ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca²⁺]_i influx rate. Induction of CRAC enhanced the influx of Mn²⁺ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca²⁺. This Ca²⁺ flux may be activated by transmembraneous gradients of Ca²⁺ and Ni²⁺. Received: 8 September 1997 / Accepted: 27 January 1998     

Identification of a Ca<Superscript>2+</Superscript>-Sensing Receptor in Rat Trigeminal Ganglia,Sensory Axons,and Tooth Dental Pulp

Extracellular Ca²⁺ regulates dentin formation, but little information is available on this regulatory mechanism. We have previously reported that sensory denervation reduces dentin formation, suggesting a role for sensory nerves in tooth mineralization. The G protein-coupled Ca²⁺-sensing receptor (CaR) is expressed in dorsal root ganglia and perivascular sensory nerves in mesenteric arterioles, and activation of these receptors by Ca²⁺ has been shown to induce vascular relaxation. The present study determined CaR expression in tooth dental pulp (DP), sensory axons, and trigeminal ganglion (TG) as well as the effect of increased [Ca²⁺]_e or a calcimimetic on tooth blood flow. The distribution of CaR, studied by immunochemistry, RT-PCR, and Western blot, indicates abundant expression of CaR in sensory axons in the jaws, TG, and DP. Restriction analysis of PCR products with specific endonucleases showed the presence of CaR message in TG and DP, and Western blotting indicates the expression of mature and immature forms of the receptor in these tissues. Pulpal blood flow, measured by laser-Doppler flowmetry, increased by 67% ± 6% (n = 12) following receptor stimulation with 5 mM Ca²⁺, which was completely inhibited by 5 μM IBTx, a high-conductance K_Ca channel blocker indicating a mechanism involving hyperpolarization. NPS R-467 (10 μM) increased blood flow by 85% ± 18% (n = 6), suggesting regulation through the CaR. Our results suggest that the CaR is present in sensory nerves, DP, and TG and that an increase in Ca²⁺ in the DP causes vasodilatation, which may contribute to accumulation of Ca²⁺ during dentin mineralization.     

Thiazide‐Sensitive Na+‐Cl− Cotransporter (NCC) Gene Inactivation Results in Increased Duodenal Ca2+ Absorption,Enhanced Osteoblast Differentiation and Elevated Bone Mineral Density

Inactivation of the thiazide‐sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca²⁺) regulation. We investigated intestinal Ca²⁺ absorption and bone Ca²⁺ metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc^S707X/S707X mice). Compared to wild‐type and heterozygous knockin littermates, Ncc^S707X/S707X mice had increased intestinal absorption of ⁴⁵Ca²⁺ and expression of the active Ca²⁺ transport machinery (transient receptor potential vanilloid 6, calbindin‐D_9K, and plasma membrane Ca²⁺ ATPase isoform 1b). Ncc^S707X/S707X mice had also significantly increased Ca²⁺ content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow‐derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress‐induced focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca²⁺ absorption as well as osteoblast differentiation and bone Ca²⁺ storage, possibly through a FAK/ERK dependent mechanism. © 2014 American Society for Bone and Mineral Research.     

Familial Hypocalciuric Hypercalcemia as an Atypical Form of Primary Hyperparathyroidism

Familial hypocalciuric hypercalcemia (FHH) causes lifelong hypercalcemia with features that overlap with typical primary hyperparathyroidism (PHPT). The incompleteness of this overlap has led to divergent nomenclatures for FHH. I compare two nomenclatures. One sets FHH as an entity distinct from PHPT. The other groups FHH with PHPT but conditions FHH as atypical PHPT. I analyzed selected articles about calcium‐sensing receptors, FHH, PHPT, CASR, GNA11, and AP2S1. FHH usually results from a heterozygous germline inactivating mutation of the CASR, and less frequently from mutation of GNA11 or AP2S1. The CASR encodes the calcium‐sensing receptors. These are highly expressed on parathyroid cells, where they sense serum calcium concentration and regulate suppression of PTH secretion by serum calcium. Their mutated expression in the kidney in FHH causes increased renal tubular reabsorption of calcium (hypocalciuria). Many FHH features are shared with PHPT and thus support FHH as a form of PHPT. These include a driver mutation expressed mainly in the parathyroid cells. The mutation causes a parathyroid cell insensitivity to extracellular calcium in vivo and in vitro, a right‐shift of the set point for suppression of PTH secretion by calcium. Serum PTH is normal or mildly elevated; ie, it is not appropriately suppressed by hypercalcemia. Total parathyroidectomy causes hypoparathyroidism and durable remission of hypercalcemia. Some other features are not shared with PHPT and could support FHH as a distinct entity. These include onset of hypercalcemia in the first week of life, frequent persistence of hypercalcemia after subtotal parathyroidectomy, and hypocalciuria. The features supporting FHH as a form of PHPT are stronger than those favoring FHH as a distinct entity. Classifying FHH as an atypical form of PHPT represents compact nomenclature and supports current concepts of pathophysiology of FHH and PHPT. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.     

Spontaneous Contractions of Intestinal Smooth Muscle Re‐aggregates from the New‐born Rat Triggered by Thromboxane A2

Isolated smooth muscle cells from the small intestine of new‐born rats were prepared by enzymatic digestion. These cells re‐aggregate after 1 day in culture to clusters. The re‐aggregates show spontaneous rhythmical contractions at 37°C with a frequency (13.1 ± 0.8 min⁻¹, n = 49), which is similar to that of the intact smooth muscle layer. The cholinergic agonist carbachol (5 × 10⁻⁵ mol l⁻¹) caused an increase in the frequency of the spontaneous contractions often ending in a permanent contraction. A similar effect was achieved with the thromboxane A₂ (TXA₂) agonist, U‐46619 (10⁻⁵ mol l⁻¹). In contrast, both the TXA₂ receptor blocker, Bay u3405 (5 × 10⁻⁴ mol l⁻¹), as well as the Ca²⁺ channel blocker, verapamil (5 × 10⁻⁵ mol l⁻¹), suppressed the spontaneous contractions. The observed contractility was insensitive against the neuronal blocker tetrodotoxin (10⁻⁶ mol l⁻¹). These analyses of video images were supported by the measurement of relative changes in the intracellular Ca²⁺ concentration with the Ca²⁺‐sensitive dye, fura‐2. Spontaneous contractions were paralleled by spikes in the intracellular Ca²⁺ concentration, which were abolished by Bay u3405, but stimulated by U‐46619 or carbachol. In summary, these results obtained at re‐aggregates of intestinal smooth muscle cells support the hypothesis of a role of TXA₂ in the generation of spontaneous intestinal smooth muscle contractions in vitro.     

PHEX 3′‐UTR c.*231A>G Near The Polyadenylation Signal Is a Relatively Common,Mild, American Mutation That Masquerades as Sporadic or X‐Linked Recessive Hypophosphatemic Rickets

Heritable forms of hypophosphatemic rickets (HR) include X‐linked dominant (XLH), autosomal recessive, and autosomal dominant HR (from deactivating mutations in PHEX, DMP1 or ENPP1, and activating mutations in FGF23, respectively). Over 30 years, we have cared for 284 children with HR. For those 72 deemed sporadic XLH, we preliminarily reported mutation analysis for 30 subjects. Eleven had PHEX mutations. However, the remaining 19 lacked readily identifiable defects in PHEX, DMP1, or FGF23. In 2008, a novel single‐base change near the polyadenylation (pA) signal in the 3′‐UTR of PHEX was identified in XLH by other investigators. This c.*231A > G mutation is 3‐bp upstream of the putative pA signal (AATAAA) in PHEX. Accordingly, we investigated whether this 3′‐UTR defect accounted for HR in any of these 19 sporadic XLH patients. PCR amplification and sequencing of their 3′‐UTR region showed the c.*231A > G mutation in four unrelated boys. Then, among an additional 22 of our 72 “sporadic” XLH patients, one boy and one girl were found to have the 3′‐UTR defect, totaling six patients. Among these 52 sporadic XLH patients with PHEX analysis, 36 were girls and 16 were boys; ie, a ～2:1 gender ratio consistent with XLH. However, finding five boys and only one girl with this 3′‐UTR mutation presented an unexplained gender bias (p = 0.02). Haplotyping for the five boys, all reportedly unrelated, showed a common core haplotype suggesting a founder. Five of their six mothers had been studied clinically and biochemically (three radiologically). Remarkably, the seemingly unaffected mothers of four of these boys carried the 3′‐UTR mutation. These healthy women had normal height, straight limbs, lacked the radiographic presentation of XLH, and showed normal or slight decreases in fasting serum Pi levels and/or TmP/GFR. Hence, PHEX c.*231A > G can masquerade as sporadic or X‐linked recessive HR. © 2014 American Society for Bone and Mineral Research.     

Danshen‐Enhanced Cardioprotective Effect of Cardioplegia on Ischemia Reperfusion Injury in a Human‐Induced Pluripotent Stem Cell‐Derived Cardiomyocytes Model

Myocardial ischemia‐reperfusion (I/R) injury is unavoidable during cardioplegic arrest and open‐heart surgery. Danshen is one of the most popular traditional herbal medicines in China, which has entered the Food and Drug Administration‐approved phase III clinical trial. This study was aimed to develop a human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) model to mimic I/R injury and evaluate the cardioprotective effect of regular cardioplegic solution with Danshen. hiPSC‐CMs were cultured with the crystalloid cardioplegic solution (Thomas group) and Thomas solution with 2 or 10 µg/mL Danshen (Thomas plus Danshen groups). The cells under normoxic culture condition served as baseline group. Then, the cells were placed in a modular incubator chamber. After 45 min hypoxia and 3 h reoxygenation, hiPSC‐CMs subjected to hypoxia/reoxygenation resulted in a sharp increase of reactive oxygen species (ROS) content in Thomas group versus baseline group. Compared with the Thomas group, ROS accumulation was significant suppressed in Thomas plus Danshen groups, which might result from elevating the content of glutathione and enhanced activities of superoxide dismutase and glutathione peroxidase. The enhanced L‐type Ca²⁺ current in hiPSC‐CMs after I/R injury was also significantly decreased by Danshen, and meanwhile intracellular Ca²⁺ level was reduced and calcium overload was suppressed. Thomas plus Danshen groups also presented less irregular transients and lower apoptosis rates. As a result, Danshen could improve antioxidant and calcium handling in cardiomyocytes during I/R and lead to reduced arrhythmia events and apoptosis rates. hiPSC‐CMs model offered a platform for the future translational study of the cardioplegia.     

Regulation of BMP2‐induced intracellular calcium increases in osteoblasts

Although bone morphogenetic protein‐2 (BMP2) is a well‐characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca²⁺ signaling. In this study, intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3‐E1 osteoblast cell line using fluorescence resonance energy transfer‐based biosensors. Increase in [Ca²⁺]_i, FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF‐573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2‐stimulated [Ca²⁺]_i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2‐induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca²⁺]_i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725–1733, 2016.