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Familial hypocalciuric hypercalcemia (FHH) is a lifelong, benign autosomal dominant disease characterized by hypercalcemia, normal to increased parathyroid hormone level, and a relatively low renal calcium excretion. Inactivation of the calcium-sensing receptor in heterozygous patients results in FHH, while in homozygous patients as well as in compound heterozygous or dominant negative heterozygous patients, it may result in neonatal severe hyperparathyroidism (NSHPT). Parathyroid surgery is not indicated in FHH and does not lower plasma calcium unless total parathyroidectomy is performed, in which case hypocalcemia ensues. There is currently no definitive medical treatment available, although pamidronate can be used to stabilize these patients before parathyroidectomy. Some NSHPT patients are asymptomatic subsequently in their lives. In this paper, clinical characteristics of this relatively rare disorder are presented.

The calcium-sensing receptor (CaSR) is a G-protein coupled receptor that is involved in tumor suppression of cancers. However, its role in breast cancer remains largely unknown. The aim of the study was to investigate the expression of CaSR in breast cancers and to evaluate its prognostic significance. We found that the protein levels of CaSR were significantly reduced in cancer lesion compared with its paired non-tumor tissues. By analyzing the expression of CaSR in a 148 cases of breast cancer tissue microarray (TMA) by immunohistochemistry, we found that patients with lower expression of CaSR were significantly associated with poor overall survival, cause-specific survival, and distant metastasis-free survival. The Cox multivariate analysis showed that CaSR was an independent prognostic significance for both overall survival and cause-specific survival of breast cancer patients. Our data confirmed the tumor suppressor role of CaSR and suggested that CaSR is an independent prognostic indicator of breast cancer.

Pathologic dysregulation of extracellular calcium metabolism is difficult to correct. The extracellular Ca(++)-sensing receptor (CaSR), a G protein-coupled receptor that regulates renal Ca(++) handling through changes in paracellular channel permeability in the thick ascending limb, has emerged as an effective pharmacological candidate for managing calcium metabolism. However, manipulation of CaSR at the systemic level causes promiscuous effects in the parathyroid glands, kidneys, and other tissues, and the mechanisms by which CaSR regulates paracellular transport in the kidney remain unknown. Here, we describe a CaSR-NFATc1-microRNA-claudin-14 signaling pathway in the kidney that underlies paracellular Ca(++) reabsorption through the tight junction. With CaSR-specific pharmacological reagents, we show that the in vivo gene expression of claudin-14 is regulated through a transcriptional mechanism mediated by NFATc1-microRNA and associated chromatin remodeling. Transgenic knockout and overexpression approaches showed that claudin-14 is required for CaSR-regulated renal Ca(++) metabolism. Together, our results define an important signaling cascade that, when dysregulated, may mediate Ca(++) imbalance through changes in tight junction permeability.

Pulmonary arterial hypertension (PAH) is defined as an intractable disease characterized by a progressive elevation of pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP), leading to right heart failure and premature death. The five-year survival rate after diagnosis is approximately 57%. Although extensive research has identified some factors associated with the cause of PAH, the etiology and pathogenesis remain unclear. In addition to Ca(2+) channel blockers (nifedipine, diltiazem), three categories of drug have been developed for the treatment of PAH based on the pathological mechanisms: prostacyclin and its analogues (epoprostenol, treprostinil, iloprost), endothelin receptor antagonists (bosentan, ambrisentan), and phosphodiesterase type 5 inhibitors (sildenafil, tadalafil). However, screening of novel types of drug acting on the signal pathway associated with the pathological mechanism underlying PAH is ongoing. We recently found that the extracellular Ca(2+)-sensing receptor (CaSR), which belongs to family C of the G protein-coupled receptor (GPCR) superfamily, is upregulated in pulmonary arterial smooth muscle cells (PASMCs) from patients with idiopathic PAH (IPAH). The upregulated CaSR is necessary for the enhanced Ca(2+) signaling and the augmented cell proliferation in PASMCs from IPAH patients. Most importantly, blockage of CaSR with an antagonist, NPS2143, prevents the development of pulmonary hypertension and right ventricular hypertrophy in animal models of pulmonary hypertension. The use of calcilytics, antagonists of CaSR, may be a novel therapeutic approach for PAH patients.

In forebrain neurons, knockout of synaptotagmin-1 blocks fast Ca(2+)-triggered synchronous neurotransmitter release but enables manifestation of slow Ca(2+)-triggered asynchronous release. Here, we show using single-cell PCR that individual hippocampal neurons abundantly coexpress two Ca(2+)-binding synaptotagmin isoforms, synaptotagmin-1 and synaptotagmin-7. In synaptotagmin-1-deficient synapses of excitatory and inhibitory neurons, loss of function of synaptotagmin-7 suppressed asynchronous release. This phenotype was rescued by wild-type but not mutant synaptotagmin-7 lacking functional Ca(2+)-binding sites. Even in synaptotagmin-1-containing neurons, synaptotagmin-7 ablation partly impaired asynchronous release induced by extended high-frequency stimulus trains. Synaptotagmins bind Ca(2+) via two C2 domains, the C2A and C2B domains. Surprisingly, synaptotagmin-7 function selectively required its C2A domain Ca(2+)-binding sites, whereas synaptotagmin-1 function required its C2B domain Ca(2+)-binding sites. Our data show that nearly all Ca(2+)-triggered release at a synapse is due to synaptotagmins, with synaptotagmin-7 mediating a slower form of Ca(2+)-triggered release that is normally occluded by faster synaptotagmin-1-induced release but becomes manifest upon synaptotagmin-1 deletion., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Extracellular calcium-sensing receptor (CaSR) has been known to play a critical role in the maintainance of systemic Ca(2+) homeostasis. Recent studies have shown that CaSR is also expressed in many tissues that are not directly related to plasma Ca(2+) regulation, such as the central and peripheral nervous system, where the function of this receptor remains to be defined. In this study, we aimed to investigate the expression of CaSR and its potential interaction with transient receptor potential vanilloid receptor type 1 (TRPV1) in rat vagal bronchopulmonary sensory neurons. Our immunohistochemical experiments demonstrated the expression of CaSR in these sensory neurons as well as in trachea and lung parenchyma. Results from our whole-cell patch-clamp recordings in isolated neurons showed that strong activation of CaSR with high concentrations of its agonists, including spermine, NPS R-568 and Ca(2+), inhibited the capsaicin-evoked whole-cell inward current. Blockade of CaSR with its antagonists NPS 2390 and NPS 2143 significantly enhanced the capsaicin-evoked TRPV1 current. These data suggest that CaSR is likely to be involved in the integration of primary bronchopulmonary sensory inputs in physiological and/or pathophysiological conditions.

This paper aims to investigate the effect of store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC) on Ca(2+)-sensing receptor (CaR)-induced extracellular Ca(2+) influx and nitric oxide (NO) generation in human umbilical vein endothelial cells (HUVEC). SOC blocker, non-selective cation channel blocker, ROC agonist and ROC blocker were used separately and combined. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by Fura-2/AM loading. The activity of endothelial nitric oxide synthase (eNOS) and the production of NO were determined by the DAF-FM diacetate (DAF-FM DA). The results showed that increases of [Ca(2+)]i, eNOS activity and NO generation induced by CaR agonist Spermine were all reduced after single blocking the SOC or ROC, respectively (P < 0.05). ROC agonist can partially abolish the ROC blocker's effect (P < 0.05). The above mentioned effects evoked by CaR agonist Spermine were further reduced when blocking both SOC and ROC than single blocking SOC or ROC in HUVEC (P < 0.05). In conclusion, these results suggest that the SOC and ROC participate in the processes of CaR-evoked extracellular Ca(2+) influx and NO generation by a synergistic manner in HUVEC.

A narrow serum calcium level which is essential for many metabolic processes is regulated by the calcium-sensing receptor which regulates parathyroid hormone (PTH) release. Primary hyperparathyroidism is supposed to be the third most common endocrine disorder. Besides nephrolithiasis and an increased incidence of cardiovascular symptoms it is associated with bone loss and an increased risk of fracture. Several different classical bone turnover markers have been shown to be increased. However, there are many uncertainties in pathophysiology of PHPT. Hardly any conclusive data exist on the RANK (receptor activator of nuclear factor-kB)/RANKL (receptor activator of nuclear factor-kB ligand)/OPG (osteoprotegerin) system, cathepsin K, sclerostin, FGF-23 (Fibroblast growth factor-23), Klotho, and DKK 1 (Dickkopf 1) in patients suffering from PHPT.

Purpose of Review: Changes in extracellular calcium concentration affect several functions of the renal tubule. The calcium-sensing receptor (CaSR), initially identified in the parathyroid gland cells, is also expressed in the kidney and was assumed to mediate all effects of extracellular calcium on the renal tubule. The purpose of this review is to critically review the evidence supporting this assumption., Recent Findings: Recent results confirm that, in the kidney, the CaSR is mainly expressed in the thick ascending limb of the loop of Henle. There, it is involved in the control of calcium reabsorption, independently of its action on parathyroid hormone secretion, through an effect on the paracellular pathway permeability. Although extracellular calcium affects transports other than that of calcium, the direct evidence that CaSR is involved in these effects is still lacking in many instances., Summary: As the CaSR in the kidney controls calcium reabsorption and excretion and subsequently affects blood calcium concentration, agonists and antagonists of the CaSR could be used to control blood calcium concentration in patients who have lost their ability to regulate parathyroid hormone secretion. In addition, more work is needed to further decipher the molecular mechanisms through which CaSR determines calcium transport in the loop of Henle.

The presentation of patients with primary hyperparathyroidism is often atypical and ranges from normocalcemic, primary hyperparathyroidism to severe, symptomatic hypercalcemia. G-protein-coupled, calcium-sensing receptor (CaSR), vitamin D receptor (VDR), and fibroblast growth factor receptor (FGFR)/klotho complexes seem to be involved in the development of pHPT. Parathyroid glands from 53 patients with pHPT and normal parathyroid tissue from 7 patients were obtained during parathyroidectomy. Conventional detailed morphological and immunohistochemical analyses of parathyroid glands were performed after dividing each slide in a 3 × 3 array. From morphology, the number of lipocytes was significantly lower in parathyroid tissue glands in the pHPT group (p < 0.001). Protein expressions of klotho, CaSR, and VDR were significantly reduced in the pHPT compared with the control group (p = 0.004, p = 0.007, p < 0.001). No differences were seen between the two groups (p = 0.35) regarding expression of FGFR. Correlations between expression showed significant positively correlations between klotho and CaSR and FGFR and VDR. No correlations between klotho expression and serum calcium levels could be detected (R = -0.13, p = 0.66), but there were positive correlations between expressions of CaSR/serum phosphate and klotho/serum phosphate. Impaired protein expression of CaSR and VDR seem to be involved in the development of pHPT. The role of the FGFR/klotho-axis remains still unclear. Correlations between protein expression of CaSR and serum phosphate and klotho and serum phosphate levels could be detected. Whether these findings give new insights into the pathogenesis of the disease is yet unknown and has to be elucidated.

In fetal mammals, serum levels of both total and ionized calcium significantly exceed those in the adult. This relative fetal hypercalcemia is crucial for skeletal development and is maintained irrespectively of maternal serum calcium levels. Elegant studies by Kovacs and Kronenberg have previously addressed the role of the CaSR in creating and maintaining this relative fetal hypercalcemia, through the regulation of parathyroid hormone-related peptide secretion. More recently we have shown that the CaSR is widely distributed throughout the developing fetus, where the receptor plays major, unexpected roles in ensuring growth and maturation of several organs. In this article, we present evidence for a role of the CaSR in the control of skeletal development, and how fetal hypercalcemia, acting through the CaSR, regulates lung development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Through regulation of excretion, the kidney shares responsibility for the metabolic balance of calcium (Ca(2+)) with several other tissues including the GI tract and bone. The balances of Ca(2+) and phosphate (PO4), magnesium (Mg(2+)), sodium (Na(+)), potassium (K(+)), chloride (Cl(-)), and water (H2O) are linked via regulatory systems with overlapping effects and are also controlled by systems specific to each of them. Cloning of the calcium-sensing receptor (CaSR) along with the recognition that mutations in the CaSR gene are responsible for two familial syndromes characterized by abnormalities in the regulation of PTH secretion and Ca(2+) metabolism (Familial Hypocalciuric Hypercalcemia, FHH, and Autosomal Dominant Hypocalcemia, ADH) made it clear that extracellular Ca(2+) (Ca(2+)o) participates in its own regulation via a specific, receptor-mediated mechanism. Demonstration that the CaSR is expressed in the kidney as well as the parathyroid glands combined with more complete characterizations of FHH and ADH established that the effects of elevated Ca(2+) on the kidney (wasting of Na(+), K(+), Cl(-), Ca(2+), Mg(2+) and H2O) are attributable to activation of the CaSR. The advent of positive and negative allosteric modulators of the CaSR along with mouse models with global or tissue-selective deletion of the CaSR in the kidney have allowed a better understanding of the functions of the CaSR in various nephron segments. The biology of the CaSR is more complicated than originally thought and difficult to define precisely owing to the limitations of reagents such as anti-CaSR antibodies and the difficulties inherent in separating direct effects of Ca(2+) on the kidney mediated by the CaSR from associated CaSR-induced changes in PTH. Nevertheless, renal CaSRs have nephron-specific effects that contribute to regulating Ca(2+) in the circulation and urine in a manner that assures a narrow range of Ca(2+)o in the blood and avoids excessively high concentrations of Ca(2+) in the urine., (Published by Elsevier Ltd.)

The epidermis is a stratified squamous epithelium composed of proliferating basal and differentiated suprabasal keratinocytes. It serves as the body's major physical and chemical barrier against infection and harsh environmental insults, as well as preventing excess water loss from the body into the atmosphere. Calcium is a key regulator of the proliferation and differentiation in keratinocytes. Elevated extracellular Ca(2+) concentration ([Ca(2+)]o) raises the levels of intracellular free calcium ([Ca(2+)]i), promotes cell-cell adhesion, and activates differentiation-related genes. Keratinocytes deficient in the calcium-sensing receptor fail to respond to [Ca(2+)]o stimulation and to differentiate, indicating a role for the calcium-sensing receptor in transducing the [Ca(2+)]o signal during differentiation. The concepts derived from in vitro gene knockdown experiments have been evaluated and confirmed in three mouse models in vivo., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Maintaining a constant level of blood Ca(2+) is essential because of calcium's myriad intracellular and extracellular roles. The CaSR plays key roles in maintaining [Formula: see text] homeostasis by detecting small changes in blood Ca(2+) and modulating the production/secretion of the Ca(2+)-regulating hormones, PTH, CT, FGF23 and 1,25(OH)2D3, so as to appropriately regulate Ca(2+) transport into or out of blood via kidney, intestine, and/or bone. When Ca(2+) is high, the CaSR suppresses PTH synthesis and secretion, promotes its degradation, and inhibits parathyroid cellular proliferation. It has just the opposite effects on the C-cell, stimulating CT when [Formula: see text] is high. In bone, Ca(2+), acting via the CaSR, stimulates recruitment and proliferation of preosteoblasts, their differentiation to mature osteoblasts, and synthesis and mineralization of bone proteins. Conversely, [Formula: see text] inhibits the formation and activity and promotes apoptosis of osteoclasts, likely via the CaSR. These actions tend to mobilize skeletal Ca(2+) during [Formula: see text] deficiency and retain it when Ca(2+) is plentiful., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The calcium sensing receptor (CaSR) is expressed by subpopulations of neuronal and glial cells throughout the brain and is activated by extracellular calcium [Formula: see text] . During development, the CaSR regulates neuronal cell growth and migration as well as oligodendroglial maturation and function. Emerging evidence suggests that in nerve terminals, CaSR is implicated in synaptic plasticity and neurotransmission. In this review, we analyze the roles attributed to CaSR in regulating diverse brain functions, including central regulation of body fluid composition and blood pressure. We also discuss the potential relevance of Ca(2+)-sensing in brain by other family C G protein-coupled receptors. Finally, evidence that the CaSR contributes to the pathogenesis of various brain disorders raises the possibility that pharmacological modulators of the CaSR may have therapeutic benefit., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cardiac injury is a common pathological change frequently accompanied by diabetes mellitus. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiac tissue. However, the functional role of CaSR in diabetic cardiac injury remains unclear. The present study was designed to investigate the relationship between CaSR activation and diabetes-induced cardiac injury. Diabetic model was successfully established by administration of streptozotocin (STZ) in vivo, and cardiomyocyte injury was simulated by 25.5 mM glucose in vitro. Apoptotic rate, intracellular calcium concentration ([Ca²⁺](i)) and the expression of Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal protein kinase (JNK), and p38 were examined. We demonstrated a significant increase in left ventricular end-diastolic pressure (LVEDP) as well as decrease in maximum rate of left ventricular pressure rise and fall (±dp/dtmax), and left ventricular systolic pressure (LVSP), apoptosis of cardiomyocytes was also observed by TUNEL staining. In vitro, 25.5 mM glucose-induced apoptosis was detected by flow cytometry in neonatal rat cardiomyocytes. Further results showed that 25.5 mM glucose significantly increased [Ca²⁺](i), up-regulated the expression of Bax, P-ERK and P-JNK, and suppressed Bcl-2 expression. However, the above deleterious changes were further confirmed when co-treatment with CaSR agonist GdCl₃ (300 µM). But the effects of GdCl₃ were attenuated by 10 µM NPS-2390, a specific CaSR inhibitor. When CaSR was silence by siRNA transfection, the effects of high glucose were inhibited. These results suggest that CaSR activation could lead to the apoptosis of cardiomyocytes in diabetic cardiac injury through the induction of calcium overload, the activation of the mitochondrial, and mitogen-activated protein kinase pathway.

Rationale: An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH)., Objective: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC., Methods and Results: The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt)., Conclusions: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

A hallmark of chronic kidney disease is hyperphosphatemia due to renal phosphate retention. Prolonged parathyroid gland exposure to hyperphosphatemia leads to secondary hyperparathyroidism characterized by hyperplasia of the glands and excessive secretion of parathyroid hormone (PTH), which causes renal osteodystrophy. PTH secretion from the parathyroid glands is controlled by the calcium-sensing receptor (CaSR) that senses extracellular calcium. High extracellular calcium activates the CaSR causing inhibition of PTH secretion through multiple signaling pathways. Cinacalcet is the first drug targeting the CaSR and can be used to effectively control and reduce PTH secretion in PTH-related diseases. Cinacalcet is a positive allosteric modulator of the CaSR and affects PTH secretion from parathyroid glands by shifting the calcium-PTH concentration-response curve to the left. One major disadvantage of cinacalcet is its hypocalcemic side effect, which may be caused by increased CaSR-mediated calcitonin secretion from the thyroid gland. However, multiple studies indicate that PTH and calcitonin secretion are stimulated by different signaling pathways, and therefore it might be possible to develop a CaSR activating drug that selectively activates signaling pathways that inhibit PTH secretion while having no effect on signaling pathways involved in calcitonin secretion. Such a drug would have the same therapeutic value as cinacalcet in lowering PTH secretion while eliminating the side effect of hypocalcemia by virtue of it not affecting calcitonin secretion. The present review will focus on recent advancements in understanding signaling and biased signaling of the CaSR, and how that may be utilized to discover new and smarter drugs targeting the CaSR.

More than 200 naturally occurring mutations have been identified in the human CaSR, which have been linked to diseases involving dysregulation of extracellular Ca(2+) homeostasis. These mutations have classically been termed "loss-" or "gain-of-function" mutations, which is an oversimplification given that amino acid changes can alter numerous molecular properties of a receptor. We thus sought to characterize the effects of 21 clinically relevant mutations, the majority located in the heptahelical domains and extracellular loop regions of the CaSR, using flow cytometry to measure cell surface receptor expression levels, and measurements of intracellular Ca(2+) mobilization and ERK1/2 phosphorylation to monitor receptor signaling. We identified distinct molecular phenotypes caused by these naturally occurring amino acid substitutions, which included combinations of loss- and gain-of-expression and changes in intrinsic signaling capacity. Importantly, we also identified biased signaling in the response of the CaSR to different mutations across the two pathways, indicating that some mutations resulted in receptor conformations that differentially altered receptor-coupling preferences. These findings have important implications for understanding the causes of diseases linked to the CaSR. A full appreciation of the molecular effects of these amino acid changes may enable the development of therapeutics that specifically target the molecular determinant of impairment in the receptor.

Following the discovery of the calcium-sensing receptor (CaSR) in 1993, its pivotal role in disorders of calcium homeostasis such as Familial Hypocalciuric Hypercalcemia (FHH) was quickly demonstrated. Since then, it has become clear that the CaSR has immense functional versatility largely through its ability to activate many different signaling pathways in a ligand- and tissue-specific manner. This allows the receptor to play diverse and crucial roles in human physiology and pathophysiology, both in calcium homeostasis and in tissues and biological processes unrelated to calcium balance. This review covers current knowledge of the role of the CaSR in disorders of calcium homeostasis (FHH, neonatal severe hyperparathyroidism, autosomal dominant hypocalcemia, primary and secondary hyperparathyroidism, hypercalcemia of malignancy) as well as unrelated diseases such as breast and colorectal cancer (where the receptor appears to play a tumor suppressor role), Alzheimer's disease, pancreatitis, diabetes mellitus, hypertension and bone and gastrointestinal disorders. In addition, it examines the use or potential use of CaSR agonists or antagonists (calcimimetics and calcilytics) and other drugs mediated through the CaSR, in the management of disorders as diverse as hyperparathyroidism, osteoporosis and gastrointestinal disease., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Context: Parathyroid oxyphil cells, whose function is unknown, are thought to be derived from chief cells. Oxyphil cells increase in number in parathyroid glands of patients with chronic kidney disease (CKD) and are even more abundant in patients receiving treatment for hyperparathyroidism with calcitriol and/or the calcimimetic cinacalcet., Objective: We examined oxyphil and chief cells of parathyroid glands of CKD patients for differential expression of genes important to parathyroid function., Design/setting/participants: Parathyroid tissue from CKD patients with refractory hyperparathyroidism was immunostained for gene expression studies., Main Outcome Measure: Immunostaining for PTH, PTHrP, calcium-sensing receptor, glial cells missing 2, vitamin D receptor, 25-hydroxyvitamin D-1α-hydroxylase, and cytochrome c was quantified and expression reported for oxyphil and chief cells., Results: Expression of all proteins analyzed, except for the vitamin D receptor, was higher in oxyphil cells than in chief cells., Conclusion: Human parathyroid oxyphil cells express parathyroid-relevant genes found in the chief cells and have the potential to produce additional autocrine/paracrine factors, such as PTHrP and calcitriol. Additional studies are warranted to define the secretory properties of these cells and clarify their role in parathyroid pathophysiology.

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS). Agonists of the extracellular calcium-sensing receptor (CaSR) can reduce toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Here we show that the CaSR is also highly expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, suggesting that CaSR agonists might also function through neuronal pathways. To test this hypothesis, rat colon segments containing intact ENS were isolated and mounted on Ussing chambers. Basal and cyclic nucleotide-stimulated electrolyte secretions were monitored by measuring changes in short-circuit current (I(sc)). CaSR was activated by R-568 and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of I(sc) in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. In the absence of TTX, activation of CaSR with R-568 significantly reduced basal I(sc) and cyclic AMP-stimulated I(sc); it also completely reversed the cAMP-stimulated secretory responses if the drug was applied after the forskolin stimulation. Such inhibitory effects of R-568 were either absent or significantly reduced when serosal TTX was present, suggesting that this agonist exerts its antisecretory effect on the intestine by inhibiting ENS. The present results suggest a new model for regulating intestinal fluid transport in which neuronal and nonneuronal secretagogue actions are modulated by the inhibitory effects of CaSR on the ENS. The ability of a CaSR agonist to reduce secretagogue-stimulated Cl(-) secretion might provide a new therapeutic approach for secretory and other ENS-mediated diarrheal conditions.

To investigate the effect of Ca(2+)-sensing receptor (CaR) on Spermine-induced extracellular Ca(2+) influx and NO generation in human umbilical vein endothelial cells (HUVEC), the small interference RNA (siRNA) specifically targeting CaR gene was designed, synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank. The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot, respectively. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by Fura-2/AM loading. The production of NO and the activity of endothelial nitric oxide synthase (eNOS) were determined by the DAF-FM diacetate (DAF-FM DA). Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection (P < 0.05). At the same time, the Spermine-induced [Ca(2+)](i), eNOS activity and NO generation were also significantly reduced (P < 0.05) in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group. In conclusion, the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca(2+) influx and NO generation in HUVEC.

The extracellular calcium-sensing receptor (CaSR) is the first identified G protein-coupled receptor to be activated by an ion, extracellular calcium (Ca(2+)). Since the identification of the CaSR in 1993, genetic mutations in the CaSR gene, and murine models in which CaSR expression has been manipulated, have clearly demonstrated the importance of this receptor in the maintenance of stable, free, ionized Ca(2+) concentration in the extracellular fluids. These functions have been extensively reviewed elsewhere. However, the distribution pattern and expression of the CaSR in lower vertebrates strongly suggest that the CaSR must play a role that is independent of mineral cation metabolism. This review addresses the involvement of the CaSR in nutrient sensing; its putative and demonstrated functions during conception, embryonic development, and birth; and its contributions to adult physiology and disease, with reference to CaSR-based therapeutics. Recent ongoing developments concerning the role of the CaSR in stem cell differentiation are also reviewed.

The Arabidopsis calcium-sensing receptor CAS is a crucial regulator of extracellular calcium-induced stomatal closure. Free cytosolic Ca(2+) (Ca(2+)(i)) increases in response to a high extracellular calcium (Ca(2+)(o)) level through a CAS signalling pathway and finally leads to stomatal closure. Multidisciplinary approaches including histochemical, pharmacological, fluorescent, electrochemical, and molecular biological methods were used to discuss the relationship of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) signalling in the CAS signalling pathway in guard cells in response to Ca(2+)(o). Here it is shown that Ca(2+)(o) could induce H(2)O(2) and NO production from guard cells but only H(2)O(2) from chloroplasts, leading to stomatal closure. In addition, the CASas mutant, the atrbohD/F double mutant, and the Atnoa1 mutant were all insensitive to Ca(2+)(o)-stimulated stomatal closure, as well as H(2)O(2) and NO elevation in the case of CASas. Furthermore, it was found that the antioxidant system might function as a mediator in Ca(2+)(o) and H(2)O(2) signalling in guard cells. The results suggest a hypothetical model whereby Ca(2+)(o) induces H(2)O(2) and NO accumulation in guard cells through the CAS signalling pathway, which further triggers Ca(2+)(i) transients and finally stomatal closure. The possible cross-talk of Ca(2+)(o) and abscisic acid signalling as well as the antioxidant system are discussed.

The extracellular calcium ([Formula: see text])-sensing receptor (CaSR) was the first GPCR identified whose principal physiological ligand is an ion, namely extracellular Ca(2+). It maintains the near constancy of [Formula: see text] that complex organisms require to ensure normal cellular function. A wealth of information has accumulated over the past two decades about the CaSR's structure and function, its role in diseases and CaSR-based therapeutics. This review briefly describes the CaSR and key features of its structure and function, then discusses the extracellular signals modulating its activity, provides an overview of the intracellular signaling pathways that it controls, and, finally, briefly describes CaSR signaling both in tissues participating in [Formula: see text] homeostasis as well as those that do not. Factors controlling CaSR signaling include various factors affecting the expression of the CaSR gene as well as modulation of its trafficking to and from the cell surface. The dimeric cell surface CaSR, in turn, links to various heterotrimeric and small molecular weight G proteins to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. CaSR signaling is impacted by its interactions with several binding partners in addition to signaling elements per se (i.e., G proteins), including filamin-A and caveolin-1. These latter two proteins act as scaffolds that bind signaling components and other key cellular elements (e.g., the cytoskeleton). Thus CaSR signaling likely does not take place randomly throughout the cell, but is compartmentalized and organized so as to facilitate the interaction of the receptor with its various signaling pathways.

The role of Ca(2+) as a key and pivotal second messenger in cells depends largely on a wide number of heterogeneous so-called calcium binding proteins (CBP), which have the ability to bind this ion in specific domains. CBP contribute to the control of Ca(2+) concentration in the cytosol and participate in numerous cellular functions by acting as Ca(2+) transporters across cell membranes or as Ca(2+)-modulated sensors, i.e., decoding Ca(2+) signals. In this chapter we review the main Ca(2+)-modulated CBP, starting with those intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins and calcineurin. Then, we address intracellular CBP lacking the EF-hand domain: CBP within intracellular Ca(2+) stores (paying special attention to calreticulin and calsequestrin), annexins and proteins that contain a C2 domain, such as protein kinase C (PKC) or sinaptotagmin. Finally, extracellular CBP have been classified in six groups, according to their Ca(2+) binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) γ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca(2+)-dependent (C)-type lectin-like domains; (vi) Ca(2+)-binding pockets of family C G-protein-coupled receptors. For all proteins, we briefly review their structure, location and function and additionally their potential as pharmacological targets in several human diseases.

Receptor activity modifying proteins (RAMPs) are single transmembrane proteins discovered for their role in the regulation of translocation of certain G-protein coupled receptors (GPCRs) to the plasma membrane. Since its discovery in 1998, several pivotal advances have been made in understanding the function of this family of proteins. This chapter provides a basic introduction to RAMPs as well as details on the various chapters in this book.

The kidney handles calcium by filtration and reabsorption. About 60% of the plasma calcium is filterable, and 99% is reabsorbed in the tubule. In the proximal tubule, the reabsorption is passive and paracellular, but in the distal tubule is active and transcellular. Thus, renal tubular cells are exposed to very high concentrations of calcium in both, the extracellular and the intracellular compartments. Extracellular calcium signaling is transmitted by the calcium sensing receptor, located both in the luminal and basolateral sides of tubular cells. This receptor is able to control levels of extracellular calcium and acts in consequence to maintain calcium homeostasis. Furthermore, renal tubular cells possess several calcium channels that regulate some of the cell functions. Among those, voltage gated calcium channels, transient receptor potential channels and N-methyl-D-aspartate receptor channels have been reported to control several functions. Those functions include survival, apoptosis, differentiation, epithelial-mesenchymal transition, and active vitamin D and renin synthesis.

In this study, we sought to determine whether the calcium-sensing receptor (CaSR) is involved in Cyclosporin A (CsA)-induced cardiomyocyte apoptosis and identify its signal transduction pathway. Forty Wistar rats were randomly divided into four groups: the control group, the CsA group (CsA 15 mg/kg/day intraperitoneally, i.p.), the GdCl3 group (GdCI3 10 mg/kg, every other day, i.p.), and the CsA + GdCl3 group (CsA 15 mg/kg/day, i.p. and GdCl3 10 mg/kg, every other day, i.p.). The groups were treated for two weeks. Cardiomyocyte apoptosis and injury were observed by light microscopy, electron microscopy and TUNEL staining. CaSR mRNA expression was determined by RT-PCR, and CaSR protein expression was detected by western blot and immunohistochemistry. The protein expression levels of cytochrome c, cleaved caspase-9, cleaved caspase-3, Bax, and Bcl-2 were detected by western blot and immunohistochemistry. CsA increased the expression of CaSR mRNA and protein and enhanced cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression and cardiomyocyte apoptosis and led to the upregulation of cytochrome c, cleaved caspase-9, cleaved caspase-3, and Bax, as well as the downregulation of Bcl-2. The present in vivo study provides further information on CsA-induced cardiomyocyte apoptosis. We determined for the first time that CaSR is involved in CsA-induced cardiomyocyte apoptosis in the rat through the activation of downstream cytochrome c-caspase-3 pathways. Furthermore, we offer evidence that the Bcl-2 family is involved in this process. These findings could provide novel strategies for the prevention and cure of CsA-induced cardiotoxicity.

Low-calcium intake is associated with increased risk of obesity, but the mechanism underlying this is not clear. We previously reported that the calcium-sensing receptor (CaSR) plays an important role in modulating the expression of rate-limiting lipolysis enzymes in human adipocytes. In the present study, rats were fed diets containing normal [0.50% (NC)], low [0.30% (LC)], or very low [0.15% (VLC)] calcium for 15 wk. Ten rats of each group were killed at wk 5, 10, and 15 of the intervention. The LC-fed rats had greater visceral fat mass, lower serum FFA and glycerol concentrations, and greater CaSR expression in white adipose tissue than did those fed the NC diet at wk 10 and 15. Hormone-sensitive lipase (HSL) and adipose TG lipase (ATGL) protein levels were lower, whereas fatty acid synthase mRNA in white adipose tissue was greater in the LC-fed rats compared with the NC-fed rats. These differences from the NC group were greater in the VLC group than in the LC group at wk 15. In vitro experiments showed that 1,25-dihydroxycholecalciferol stimulated the expression of CaSR through the nuclear vitamin D receptor (nVDR). This resulted in an antilipolytic effect by increasing intracellular calcium, decreasing the intracellular cAMP level, and downregulating HSL and ATGL protein expression in adipocytes. These effects were suppressed by either nVDR or CaSR small-interfering RNA. These results suggest that CaSR affects fat accumulation by mediating antilipolytic pathways in adipose tissue of rats fed low-calcium diets.

Background: The calcimimetic cinacalcet has recently been increasingly used for persistent hyperparathyroidism after renal transplantation. The present study investigated the short-term effects of cinacalcet on urinary electrolyte concentration and arterial blood pressure in kidney transplant patients with persistent hyperparathyroidism., Methods: In a prospective controlled single-center cross-over study, we examined 10 stable kidney transplant patients (mean estimated glomerular filtration rate 51±10 mL/min/1.73 m(2)) who received cinacalcet daily for persistent hyperparathyroidism. Urine specimens were collected at baseline and every 2 hr for a total study period of 6 hr after ingestion of 30 mg cinacalcet and without cinacalcet. Intact parathyroid hormone was determined at baseline and 2 hr later. Using ambulatory blood pressure measurement, arterial blood pressure was determined every 15 min., Results: Intact parathyroid hormone was significantly reduced with cinacalcet as compared with controls (-37±27.7% vs. -9.6±10.3%, P=0.009). With cinacalcet, urinary calcium and magnesium concentration were increased (P=0.042 and P=0.007, respectively) and differed significantly as compared with the control phase without cinacalcet. After 4 hr, an increased urinary sodium concentration was also found compared with the control phase (P=0.039). Systolic blood pressure was reduced with cinacalcet (P<0.001) and differed significantly from control phase (-13.7±9.9 mm Hg vs. -3.2±5.2 mm Hg after 2 hr, P=0.009; -18.1±10.8 mm Hg vs. -1.9±5.2 mm Hg after 4 hr, P=0.001)., Conclusions: In the short term, cinacalcet increases the urinary concentration of calcium, magnesium, and sodium. The observed antihypertensive effect might be beneficial in patients with a high cardiovascular risk after kidney transplantation.

Calcimimetics increase the sensitivity of the parathyroid calcium-sensing receptor to extracellular calcium for efficient control of hyperparathyroidism. Recent studies suggest that there are beneficial effects of calcimimetics beyond the control of bone and mineral homeostasis. Here, we tested whether the calcium-sensing receptor is also expressed and functionally relevant in podocytes. Analysis of microarray data using Gene Set Enrichment Analysis found that the calcimimetic R-568 influenced various pathways related to oxidative stress, cytoskeletal regulation, cell proliferation, and survival in cultured podocytes. R-568 induced a dose- and time-dependent phosphorylation of the ERK1/2-P90RSK-CREB signaling cascade, and induced pro-survival phosphorylation of BAD and Bcl-xl, thus reducing puromycin aminonucleoside (PAN)-induced podocyte apoptosis by half. Moreover, R-568 preserved the actin cytoskeleton in podocytes exposed to PAN and improved recovery from exposure to cytochalasin D, a reversible inhibitor of actin polymerization. In rats, co-administration of R-568 prevented the proteinuria caused by a single dose of PAN and attenuated the glomerulosclerosis and loss of GFR caused by repetitive puromycin treatment. Hence, calcimimetics limit podocyte damage by antiapoptotic and cytoskeleton-stabilizing effects and may constitute a new approach in the prevention and treatment of glomerular disease.

Calcium sensing receptor (CaSR) is a component of the C family of the G protein-coupled receptors. It is ubiquitously expressed in human and mammal cells but is more expressed in parathyroid glands and kidney cells. It is located on the cell plasma membrane and senses the changes of extracellular calcium concentrations. Thus, it may modify cell functions according to serum calcium levels. CaSR has a key role in calcium homeostasis because it allows parathyroid glands and kidney to regulate PTH secretion and calcium reabsorption in order to keep serum calcium concentration within the normal range. CaSR appears as an important player in the regulation of renal calcium handling and body calcium metabolism. Thus, CaSR may protect human tissues against calcium excess. In kidneys, its protective effect includes the stimulation of diuresis and phosphate retention, along with the potential prevention of calcium precipitation and deposition in kidney tubules and interstitium.

Extracellular calcium concentration changes are recognized by Ca++ sensing receptor (CaR), a member of the G-protein-coupled receptor family. Recently, progress has been made in the understanding of CaR functional role in bone cells, notwithstanding a lack of detailed knowledge about the identity of the cation receptors. It is generally agreed that a high extracellular calcium induces osteoblast proliferation and osteoclastogenesis inhibition. Potential implications that may be considered include a role for CaR in osteogenesis, in serum calcium homeostasis regulation, and as a factor coupling bone formation to resorption in bone remodeling. The localization of CaR in bone cells provides further knowledge of the mechanisms operating in the bone remodeling model; in fact, increased calcium gradient in the site of bone resorption favors osteoblast precursors chemotaxis and inhibits osteoclasts through the increase of [Ca++]e. In vitro data indicate that CaR is a physiological regulator of bone cells, regulating the recruitment, differentiation and survival of osteoblasts and osteoclasts. This leads to the concept that the CaR present in bone cells may be targeted by agonists or antagonists to control bone cell metabolism and bone remodeling.

Disorders of mineral metabolism and hypercalcemia are frequent in kidney transplant recipients. Calcium to creatinine (Ca/Cr) clearance ratio was used as a criterion to distinguish between different calcium metabolism disorders. The study comprised 91 (53 men, 38 women) kidney recipients aged 23-70 years, with creatinine clearance (CrCl) >60 ml/min. The following parameters related to mineral metabolism were measured in serum: iPTH, total alkaline phosphatase (tALP), telopeptide (bone degradation marker, CTX), 25(OH)D(3), total and ionized calcium, Ca(++), Pi, creatinine (Cr). Creatinine and Ca were also determined in urine, as well as Ca/Cr clearance ratio. According to the Ca/Cr clearance ratio, patients were divided into three groups as follows: <0.01 (found in disorders caused by reduced calcium-sensing receptor sensitivity, N = 30), 0.01-0.02 (normal value, N = 45), and >0.02 (found in hyperparathyroidism, N = 16). In the group of patients with Ca/Cr clearance ratio <0.01, seven patients had hypercalcemia, and four patients had hypercalcemia and elevated iPTH. It seems that impairment of renal calcium excretion may occur in kidney transplant recipients with good kidney function. Inappropriately low calciuria and impaired sensitivity of calcium-sensing receptor may be pathogenetic factors causing hypercalcemia in kidney transplant recipients.

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

The discovery of the calcium-sensing receptor (CaSR) prompted the identification of substances that affect its function. Cinacalcet, for example, is a drug that allosterically modifies the receptor so as to increase its sensitivity to circulating calcium (thus the name "calcimimetic") and in this way decreases parathyroid hormone secretion. Clinical use of cinacalcet is already approved for the treatment of primary and secondary hyperparathyroidism, but research is ongoing to identify further potential actions of this drug. The effects and functions of the CaSR have been evaluated in different systems and tissues, beyond parathyroid glands, such arterial walls. A complete understanding of the properties of calcimimetics are of obvious clinical interest, since therapeutic indications may be affected accordingly.

Renal failure is a growing problem that involves a large part of the population and has a great social impact, with often incapacitating complications, mainly related to mineral bone disorders (MBD) and cardiovascular diseases. Analysis of the recent scientific literature confirms that a large number of chronic kidney disease (CKD) patients develop an early derangement of the parameters of Ca-P metabolism in which phosphate homeostasis and a reduced endogenous synthesis of calcitriol play a critical role. Recent findings from several large observational studies have also suggested that the benefits of vitamin D receptor activators may extend beyond the traditional parathyroid hormone-lowering effect, and could result in direct cardiovascular and metabolic benefits. Treatment of secondary hyperparathyroidism has become even more complex with the arrival of the calcium-sensing receptor agonist cinacalcet hydrochloride and with the uncovering of novel mechanisms responsible for secondary hyperparathyroidism. The aim of this review is the analysis of some of the recent contributions in the field of CKD-MBD, to update the understanding of the pathogenetic mechanisms and possibly the most appropriate therapeutic approach in this field.

Introduction: In the parathyroid gland, the calcium sensing receptor responds to small changes in circulating levels of Ca(2+), and consequently stimulates or inhibits the secretion of parathyroid hormone (PTH). Thus, ligands potentiating the action of calcium (calcimimetics) lead to decreased PTH secretion and can thus be useful for the treatment of hyperparathyroidism. On the other hand, ligands which antagonize the action of calcium (calcilytics) stimulate PTH secretion, favoring bone tissue regeneration., Areas Covered: This review first discusses the rapid development of calcimimetics (only one of which has been approved for the treatment of hyperparathyroidism) followed by that of calcilytics (none of which has as yet been approved for the treatment of osteoporosis). Peer-reviewed articles generated by these patents are also surveyed., Expert Opinion: The rapid progress in developing a clinically approved calcimimetic has not been matched by an identical success in finding an orally available calcilytic useful for the treatment of osteoporosis. However, the growing importance of osteoporosis as a debilitating disease is a stimulating factor in discovering such compounds.

We discovered structurally novel human calcium-sensing receptor (CaSR) allosteric agonists and compared their pharmacology to phenylalkylamine calcimimetics. 1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol (AC-265347) activated CaSR signaling in cellular proliferation and phosphatidylinositol (PI) hydrolysis assays with potencies of 30 and 10 nM, respectively. (S)-1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(S)-AC-265347], the S-enantiomer of AC-265347, was approximately 10- to 20-fold more potent than (R)-1-benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(R)-AC-265347]. The phenylalkylamines cinacalcet and calindol had activity similar to that of AC-265347 in cellular proliferation assays but less activity in PI assays. All compounds had reduced activity when extracellular Ca(2+) was removed, indicating that they cooperate with Ca(2+) to activate CaSRs, and all activated CaSR isoforms with the N-terminal extracellular domain deleted, indicating that they interact with the transmembrane domains. In both cases, AC-265347 and therefore (S)-AC-265347 were significantly more efficacious than the phenylalkylamines. Mutations E837A(7.39) and I841A(7.43) strongly reduced phenylalkylamine-induced signaling, but not AC-265347- or (S)-AC-265347-induced signaling, suggesting different modes of binding. AC-265347 and (S)-AC-265347 stimulated significantly greater responses than cinacalcet or calindol at each of four loss-of-function human polymorphic CaSR variants. AC-265347 did not inhibit the CYP2D6 cytochrome P450 isozyme, unlike cinacalcet, which is a potent CYP2D6 inhibitor. In rats, AC-265347, (S)-AC-265347, and (R)-AC-265347 each reduced serum parathyroid hormone (PTH) with a rank order potency correlated with their in vitro potencies. AC-265347 and (S)-AC-265347 also reduced plasma ionizable calcium ([Ca(2+)](o)). AC-265347 was orally active, and its plasma concentrations correlated well with its effects on serum PTH. Thus, these highly efficacious CaSR allosteric agonists represent leads for developing therapeutic agents with potential advantages over existing therapies.

Spontaneous release of glutamate is important for maintaining synaptic strength and controlling spike timing in the brain. Mechanisms regulating spontaneous exocytosis remain poorly understood. Extracellular calcium concentration ([Ca(2+)](o)) regulates Ca(2+) entry through voltage-activated calcium channels (VACCs) and consequently is a pivotal determinant of action potential-evoked vesicle fusion. Extracellular Ca(2+) also enhances spontaneous release, but via unknown mechanisms. Here we report that external Ca(2+) triggers spontaneous glutamate release more weakly than evoked release in mouse neocortical neurons. Blockade of VACCs has no effect on the spontaneous release rate or its dependence on [Ca(2+)](o). Intracellular [Ca(2+)] slowly increases in a minority of neurons following increases in [Ca(2+)](o). Furthermore, the enhancement of spontaneous release by extracellular calcium is insensitive to chelation of intracellular calcium by BAPTA. Activation of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor present in nerve terminals, by several specific agonists increased spontaneous glutamate release. The frequency of spontaneous synaptic transmission was decreased in CaSR mutant neurons. The concentration-effect relationship for extracellular calcium regulation of spontaneous release was well described by a combination of CaSR-dependent and CaSR-independent mechanisms. Overall these results indicate that extracellular Ca(2+) does not trigger spontaneous glutamate release by simply increasing calcium influx but stimulates CaSR and thereby promotes resting spontaneous glutamate release.

Although the function of extracellular Ca(2+)-sensing receptor (CaR) is known, the regulatory mechanism of the CaR function remains to be clarified. The purpose of the present study was to investigate the effect of caveolin-1 (Cav-1) on CaR-induced extracellular Ca(2+) influx by using acute caveolae disruption with Filipin or siRNA targeted to the Cav-1 in human umbilical vein endothelial cells (HUVECs). Intracellular Ca(2+) concentration ([Ca(2+)](i)) was detected by Fura-2/AM loading. The results showed that different concentrations of extracellular Ca(2+) failed to increase [Ca(2+)](i), while the CaR agonist Spermine (2 mmol/L) resulted in an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) (P<0.05). No matter in buffer with or without 2 mmol/L Ca(2+), the [Ca(2+)](i) increase induced by Spermine in HUVECs was abolished after inhibition of CaR by a negative allosteric modulator Calhex231 (1 μmol/L) (P<0.05), conversely, the effect of Spermine on the increase in [Ca(2+)](i) in HUVECs was further augmented after acute caveolae disruption with Filipin (1.5 μg/mL) or transfection with siRNA targeted to the Cav-1 (P<0.05). This indicated that Cav-1 produced an inhibition of CaR-induced extracellular Ca(2+) influx. As to the biological mechanism of Cav-1-induced inhibition, immunofluorescence technique showed that both CaR and Cav-1 were present in HUVECs, and confocal microscopy supported the co-localization of CaR and Cav-1 on the plasma membrane. Functionally, the Cav-1 protein expression was decreased in HUVECs transfected with siRNA targeted to the Cav-1 (P<0.05); simultaneously, the CaR membrane protein expression was decreased (P<0.05), whereas CaR total protein level was unaffected (P>0.05). In conclusion, the present study suggests that CaR and Cav-1 co-localize on the plasma membrane in HUVECs and CaR-induced Ca(2+) influx is down-regulated by binding with Cav-1, and the mechanism involves the effect of Cav-1 on CaR localization on the plasma membrane and attenuating the CaR response to the agonist.

Compelling evidence of a cell surface receptor sensitive to extracellular calcium was observed as early as the 1980s and was finally realized in 1993 when the calcium-sensing receptor (CaR) was cloned from bovine parathyroid tissue. Initial studies relating to the CaR focused on its key role in extracellular calcium homeostasis, but as the amount of information about the receptor grew it became evident that it was involved in many biological processes unrelated to calcium homeostasis. The CaR responds to a diverse array of stimuli extending well beyond that merely of calcium, and these stimuli can lead to the initiation of a wide variety of intracellular signaling pathways that in turn are able to regulate a diverse range of biological processes. It has been through the examination of the molecular characteristics of the CaR that we now have an understanding of how this single receptor is able to convert extracellular messages into specific cellular responses. Recent CaR-related reviews have focused on specific aspects of the receptor, generally in the context of the CaR's role in physiology and pathophysiology. This review will provide a comprehensive exploration of the different aspects of the receptor, including its structure, stimuli, signalling, interacting protein partners, and tissue expression patterns, and will relate their impact on the functionality of the CaR from a molecular perspective.

Secondary hyperparathyroidism classically appears during the course of chronic renal failure and sometimes after renal transplantation. Understanding the mechanisms by which parathyroid hormone (PTH) synthesis and secretion are normally regulated is important in devising methods to regulate overactivity and hyperplasia of the parathyroid gland after the onset of renal insufficiency. Rapid regulation of PTH secretion in response to variations in serum calcium is mediated by G-protein coupled, calcium-sensing receptors on parathyroid cells, whereas alterations in the stability of mRNA-encoding PTH by mRNA-binding proteins occur in response to prolonged changes in serum calcium. Independent of changes in intestinal calcium absorption and serum calcium, 1α,25-dihydroxyvitamin D also represses the transcription of PTH by associating with the vitamin D receptor, which heterodimerizes with retinoic acid X receptors to bind vitamin D-response elements within the PTH gene. 1α,25-Dihydroxyvitamin D additionally regulates the expression of calcium-sensing receptors to indirectly alter PTH secretion. In 2°HPT seen in renal failure, reduced concentrations of calcium-sensing and vitamin D receptors, and altered mRNA-binding protein activities within the parathyroid cell, increase PTH secretion in addition to the more widely recognized changes in serum calcium, phosphorus, and 1α,25-dihydroxyvitamin D. The treatment of secondary hyperparathyroidism by correction of serum calcium and phosphorus concentrations and the administration of vitamin D analogs and calcimimetic agents may be augmented in the future by agents that alter the stability of mRNA-encoding PTH.