Extracellular Calcium-sensing Receptor Research Articles

Regulation of Cellular Signal Transduction Pathways by the Extracellular Calcium-Sensing Receptor

The extracellular calcium-sensing receptor (CaR) is a class III G-protein coupled receptor that coordinates cellular responses to changes in extracellular free Ca(2+) or amino acid concentrations as well as ionic strength and pH. It regulates signalling cascades via recruiting and controlling the activities of various heterotrimeric G-proteins, including G(q/11), G(i/0), and G(12/13), even G(s) in some "unusual" circumstances, thereby inducing changes in the metabolism of membrane lipids, the phosphorylation state of protein kinases and their targets, the activation state of monomeric G-proteins and the levels of intracellular second messengers including cAMP, Ca(2+) ions, fatty acids and other small molecules. According to its site(s) of expression and available signalling pathways, the CaR modulates cell proliferation and survival, differentiation, peptide hormone secretion, ion and water transport and various other processes. In this article we consider the complex intracellular mechanisms by which the CaR elicits its cellular functions. We also consider some of the better understood CaR-regulated cell functions and the nature of the signalling mechanisms that support them.

Extracellular calcium-sensing receptor distribution in osmoregulatory and endocrine tissues of the tilapia

The extracellular calcium-sensing receptor (CaSR) serves an important detector function in vertebrate Ca 2+ homeostasis. In this study, we surveyed using immunohistochemistry the tissue and cellular distribution of the CaSR protein in the Mozambique tilapia ( Oreochromis mossambicus) and the Japanese eel ( Anguilla japonica). Specifically, we examined receptor expression in ion-transporting barrier tissues that may be directly responsive to extracellular Ca 2+ levels, and in tissues that are implicated in endocrine signaling to homeostatic effectors such as Ca 2+-transporting epithelia. In tilapia osmoregulatory tissues, CaSR protein is strongly expressed in proximal segments of renal tubule, but not in distal segments (where Na +,K +-ATPase is prominently expressed) or in glomeruli. The receptor was also localized in the ion-transporting mitochondria-rich cells of gill and in ion- and nutrient-transporting epithelia of middle and posterior intestine. Consistent with our earlier RT-PCR assessment of mRNA expression in tilapia, CaSR protein expression was salinity dependent in some osmoregulatory tissues. In tilapia pituitary gland, CaSR expression was observed in the rostral pars distalis (containing prolactin-secreting cells, and in the pars intermedia (containing somatolactin-secreting and melanocyte-stimulating hormone-secreting cells), with notably greater expression in the latter. In the eel, weak immunostaining was seen in the stanniocalcin-secreting cells of the corpuscles of Stannius. Olfactory lobe CaSR expression suggests an environment-sensing role for the receptor. Altogether, these findings support the involvement of CaSR in piscine Ca 2+ homeostasis at the levels of environmental sensing, of integrative endocrine signaling through both hypercalcemic (prolactin, and perhaps somatolactin) and hypocalcemic (stanniocalcin) hormones, and of direct local regulation of Ca 2+-transporting tissues.

A calcimimetic (R-568), but not calcitriol, prevents vascular remodeling in uremia

Renal insufficiency increases cardiovascular risk, accelerates atherogenesis, and causes vascular wall remodeling. Here we evaluated the effect of the calcimimetic R-568 and non-hypercalcemic doses of calcitriol on vascular structure. Subtotal nephrectomy was produced in Sprague-Dawley rats followed by treatment with R-568, calcitriol, or vehicle for 12 weeks. The aortic wall was significantly thicker in vehicle-treated uremic rats than in those with a sham-operation but R-568-treated uremic rats had a lower value. In contrast, calcitriol increased wall thickness in both the sham-operated and uremic groups. The calcification score, measured by von Kossa staining, and the number of proliferating cells in the intima and media were significantly higher in the calcitriol-treated uremic group. The expression of the calcium sensing receptor was higher in the intima of sham-operated and uremic rats treated with R-568 compared to animals treated with vehicle or calcitriol, while the expression of the vitamin D receptor was upregulated by both calcitriol and R-568. Our study shows that in uremic rats, calcitriol increased while R-568 attenuated media calcification and proliferation of vascular smooth muscle and endothelial cells.

Regulation of mouse lung development by the extracellular calcium‐sensing receptor, CaR

Postnatal lung function is critically dependent upon optimal embryonic lung development. As the free ionized plasma calcium concentration ([Ca(2+)](o)) of the fetus is higher than that of the adult, the process of lung development occurs in a hypercalcaemic environment. In the adult, [Ca(2+)](o) is monitored by the G-protein coupled, extracellular calcium-sensing receptor (CaR), but neither its ontogeny nor its potential role in lung development are known. Here, we demonstrate that CaR is expressed in the mouse lung epithelium, and that its expression is developmentally regulated, with a peak of expression at embryonic day 12.5 (E12.5) and a subsequent decrease by E18, after which the receptor is absent. Experiments carried out using the lung explant culture model in vitro show that lung branching morphogenesis is sensitive to [Ca(2+)](o), being maximal at physiological adult [Ca(2+)](o) (i.e. 1.0-1.3 mM) and lowest at the higher, fetal (i.e. 1.7 mM) [Ca(2+)](o). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high [Ca(2+)](o) on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances intracellular calcium signalling, lung distension and fluid secretion. Conditions which are restrictive either to branching or to secretion can be rescued by manipulating [Ca(2+)](o) in the culture medium. In conclusion, fetal Ca(2+)(o), acting through a developmentally regulated CaR, is an important extrinsic factor that modulates the intrinsic lung developmental programme. Our observations support a novel role for the CaR in preventing hyperplastic lung disease in utero.

The calcium-sensing receptor in taste tissue

Calcium is an essential nutrient that induces a distinctive taste quality, but the sensing mechanism of calcium in the tongue is poorly understood. A recent study linked calcium to T1R3 receptor. Here, we propose another system for calcium taste involving the extracellular calcium-sensing receptor (CaSR). This G protein-coupled receptor that responds to calcium and magnesium cations is involved in calcium homeostasis regulating parathyroid and kidney functions. In this study, CaSR was found in isolated taste buds from rats and mice. It was expressed in a subset of cells in circumvallate and foliate papillae, with fewer cells in the fungiform papillae. This is the first evidence in mammals that locates CaSR in gustatory tissue and provides the basis for better understanding not only calcium taste but also the taste of multiple CaSR agonists.

Le récepteur du calcium : un rôle central dans le métabolisme calcique

The extracellular calcium-sensing receptor (CaSR) enables the parathyroid gland cells to sense the extracellular calcium concentration, to adapt the amount of parathyroid hormone secreted by those glands and, in turn, by its action on kidney and bone, to maintain steady the extracellular calcium concentration. The prominent role of CaSR is illustrated by the fact that CaSR mutations are responsible for disorders in extracellular calcium metabolism. Drugs that either activate or inactivate CaSR will open new therapeutic opportunities in several areas of mineral metabolism.

Calcium-sensing receptor in cancer: good cop or bad cop?

The extracellular calcium-sensing receptor (CaR) is a versatile 'sensor' for di- and polycationic molecules in the body. CaR plays a key role in the defense against hypercalcemia by "sensing" extracellular calcium levels in the parathyroid and kidney, the key organs maintaining systemic calcium homeostasis. Although mutation of CaR gene has so far not been associated with any malignancy, aberrant functions of CaR have implications in malignant progression. One situation is loss of CaR expression, resulting in loss of growth suppressing effects of elevated extracellular Ca(2+) by CaR, reported in parathyroid adenoma and in colon carcinoma. Another situation is activation of CaR, resulting in increased production of parathyroid hormone-related peptide (PTHrP), a primary causal factor in hypercalcemia of malignancy and a contributor to metastatic processes involving bone. CaR signaling and effects have been studied in several cancers including ovarian cancers, gastrinomas, and gliomas in addition to comparatively detailed studies in breast, prostate, and colon cancers. Studies on H-500 rat Leydig cells, a xenotransplantable model of humoral hypercalcemia of malignancy has shed much light on the mechanisms of CaR-induced cancer cell growth and survival. Pharmacological agonists and antagonists of CaR hold therapeutic promise depending on whether activation of CaR is required such as in case of colon cancer or inactivating the receptor is required as in the case of breast- and prostate tumors.

The Extracellular Calcium-Sensing Receptor (CaSR) Is a Critical Modulator of Skeletal Development

The extracellular Ca(2+)-sensing receptor (CaSR) plays a nonredundant role in the functions of the parathyroid gland (PTG) and the kidney. Severe hyperparathyroidism, premature death, and incomplete gene excision in Casr(-/-) mice have precluded the assessment of CaSR function in other tissues. We generated mice with tissue-specific deletion of Casr in the PTG, bone, or cartilage. Deletion of Casr in the PTG or bone resulted in profound bone defects, whereas deletion of Casr in chondrocytes (cartilage-producing cells) resulted in death before embryonic day 13 (E13). Mice in which chondrocyte-specific deletion of Casr was induced between E16 and E18 were viable but showed delayed growth plate development. Our data show a critical role for the CaSR in early embryogenesis and skeletal development.

New Insights in Bone Biology: Unmasking Skeletal Effects of the Extracellular Calcium–Sensing Receptor

Experiments performed in mice in which expression of the extracellular calcium-sensing receptor (CaSR) was completely nullified specifically in parathyroid cells, chondrocytes, or cells of the osteoblast lineage have identified phenotypes that indicate a key role for the CaSR in embryonic development of the skeleton, postnatal bone formation, and osteoblast differentiation that are independent of the calcitropic hormone axis. These long-awaited studies further clarify the signaling relationships between the parathyroid gland, kidney, and metabolic bone disease in patients with mutations in the gene encoding the CaSR, and they provide new insights into understanding the signaling pathways involving the CaSR in skeletal cells.

Switching of G-protein Usage by the Calcium-sensing Receptor Reverses Its Effect on Parathyroid Hormone-related Protein Secretion in Normal Versus Malignant Breast Cells

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galphai in MMECs but coupled to Galphas in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.

CaSR stimulates secretion of Wnt5a from colonic myofibroblasts to stimulate CDX2 and sucrase-isomaltase using Ror2 on intestinal epithelia

To understand whether extracellular calcium-sensing receptor (CaSR) expression on colonic myofibroblast cells (18Co) contributed to epithelial homeostasis, we activated the CaSR with 5 mM Ca(2+), screened by RT-PCR Wnt family members, and measured their secretion. Transcripts for Wnt 1, 2, 2b, 3a, 4, and 7a were either absent or unchanged whereas Wnt3 decreased and Wnt5a increased. We assessed Wnt5a secretion by Western blot. High Ca(2+) (5 mM) substantially increased Wnt5a secretion; small interfering RNA (siRNA) against the CaSR reduced this to constitutive amounts. Expression of Wnt5a plasmid but not Wnt1 or Wnt3a increased caudal homeodomain factor CDX2 transcripts and protein in HT-29 adenocarcinoma cells. Wnt5a increased activity of a sucrase-isomaltase (SI) promoter in Caco-2BBE cells. Wnt5a protein stimulation of CDX2 transcripts and protein and SI reporter were increased by overexpression of wild-type Ror2, a Wnt5a receptor, and reduced with siRNA against Ror2. CaSR activation of HT-29 cells increased Ror2 protein expression. Ror2 protein was expressed in mouse jejunum from crypt base to villus tip and in the colon on surface epithelia. Our results show that activation of a G protein-coupled receptor, the CaSR, stimulates secretion of Wnt5a from myofibroblasts. Stimulation of epithelia by the CaSR increased the expression of a receptor for Wnt5a, the tyrosine kinase Ror2, suggesting existence of a unique paracrine relationship for CDX2 homoeostasis in the intestine and revealing new contributions of CaSR-activated myofibroblasts to intestinal stem cell niche microenvironments.

Antilipolytic effect of calcium-sensing receptor in human adipocytes

The extracellular calcium-sensing receptor (CaSR), a seven transmembrane G-protein-coupled receptor, was cloned in 1993. Its activation was first associated to the regulation of calcium homeostasis; however, the presence in tissues unrelated with this role has revealed its participation in numerous other cell functions. We previously described CaSR expression in human adipocytes, and here we investigated the effect of its activation on adipocyte lipolytic activity by measuring glycerol release to the incubation medium. Treatment of adipocytes with CaSR agonists elicited an inhibitory effect on basal lipolysis, which was prevented by a CaSR antagonist. To further corroborate the antilipolytic effect of CaSR activation, lipolysis was evaluated under conditions that interfere with main antilipolytic regulatory pathways. Cells were preincubated with pertussis toxin (PT, a Gialpha protein inhibitor), the phosphatidylinositol 3 kinase (PI3K) inhibitors wortmannin and LY-294002 as well as the cAMP analog 8Br-cAMP, all of which influenced the antilipolytic effect of CaSR stimulation. In light of the current view of adipose tissue as an organ involved in whole-body metabolic control, the role of the CaSR modulating basal lipolysis elicits great interest, given its metabolic sensing capabilities due to the variety of ligands that regulate its activity, and its potential cross-talk with insulin and adipose tissue-secreted factors.

The calcium-sensing receptor and insulin secretion: a role outside systemic control 15 years on

In the 15 years since the identification and characterisation of the extracellular calcium-sensing receptor (CaR), it has become increasingly apparent that this cationic binding receptor is found in many tissues, not associated with the control of plasma calcium. One of these tissues is the pancreatic islet where insulin secretion provides the basis of energy regulation. It seems inherently unlikely that the islet responds to alterations in systemic calcium and a more plausible and intriguing possibility is that the CaR mediates cell-to-cell communication through local increases in the concentration of extracellular Ca(2+), co-released with insulin. This short article explores this possibility and suggests that this novel mechanism of cell communication, along with direct coupling via gap junctions and other local paracrine regulators helps explain why the glucose responsiveness of the intact islet is greater than the sum of the composite parts in isolation.

The expression and function of Ca2+‐sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes

The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K(+) channels (IK(Ca)) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. Pressure myography, western blotting, sharp microelectrode and K(+)-selective electrode recordings were used to investigate the functional expression of the CaR and IK(Ca) in rat mesenteric arteries. Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K(+)] around the myocytes, and inhibition of this 'K(+) cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK(Ca) expression was also diminished, but IK(Ca)-generated hyperpolarizations mediated by 1-EBIO were unaffected. The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK(Ca) channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.

Intracellular signal transduction by the extracellular calcium-sensing receptor of Xenopus melanotrope cells

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist l-phenylalanine ( l-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G s/PKA nor G q/PKC pathways are involved. However, pertussis toxin (G i/o protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaR-evoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of l-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.

M1647 Extracellular Calcium-Sensing Receptor (CaSR) Stimulates Secretion of the Wnt Antagonist, DKK-1, from Colonic Myofibroblasts to Increase Transepithelial Resistance of T84 Monolayers

M1647 Extracellular Calcium-Sensing Receptor (CaSR) Stimulates Secretion of the Wnt Antagonist, DKK-1, from Colonic Myofibroblasts to Increase Transepithelial Resistance of T84 Monolayers

Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana

Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids. The phosphorylation level of CaS responded strongly to light intensity. The light-dependent thylakoid protein kinase STN8 is required for CaS phosphorylation. The phosphorylation site was mapped to the stroma-exposed Thr380, located in a motif for interaction with 14-3-3 proteins and proteins with forkhead-associated domains, which suggests the involvement of CaS in stress responses and signaling pathways. The knockout Arabidopsis lines revealed a significant role for CaS in plant growth and development.

Extracellular Calcium Matters, Too

During the development of the nervous system, neurons extend processes that navigate the microenvironment by responding to various guidance molecules; many of the signaling pathways involved depend on intracellular Ca 2+ . The extracellular calcium-sensing receptor (CaSR) is found in organs known to regulate Ca 2+ homeostasis; however, CaSR is also found in areas of the brain, such as the hippocampus, and on sympathetic neurons in the peripheral nervous system. This led Vizard et al . to investigate whether CaSR might play a role in neuronal development. The authors used real-time polymerase chain reaction assays and immunofluorescence microscopy to show that the abundance of CaSR in the superior cervical ganglion (SCG) of the mouse peaks on embryonic day 18 (E18) before declining within a few days of birth. This coincides with a transient increase in the concentration of free extracellular Ca 2+ ([Ca 2+ ] o ) in the embryo and with the period during which sympathetic axons innervate their targets. E18 SCG neurons cultured with nerve growth factor developed longer and more branched neurites when exposed to 2.3 mM [Ca 2+ ] o than did those cultured in 0.7 mM [Ca 2+ ] o . Expression in neurons of a dominant-negative CaSR inhibited the effect of [Ca 2+ ] o on neurite growth and branching in vitro. The retinas of Casr −/− mice showed decreased innervation by sympathetic axons compared with those of wild-type mice. Expression of dominant-negative CaSR in hippocampal pyramidal neurons in organ cultures resulted in reduced dendrite outgrowth and branching compared with that in control cultures. As Spitzer discusses in commentary, these data suggest that CaSR controls neurite outgrowth and branching in response to [Ca 2+ ] o , which acts here as a first rather than second messenger. T. N. Vizard, G. W. O’Keeffe, H. Gutierrez, C. H. Kos, D. Riccardi, A. M. Davies, Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor. Nat . Neurosci . 11 , 285-291 (2008). [PubMed] N. C. Spitzer, Calcium: First messenger. Nat . Neurosci . 11 , 243-244 (2008). [PubMed]

Calcium: first messenger

Calcium is a ubiquitous second messenger in the nervous system linking extracellular and intracellular events. Now a study identifies it as a primary messenger controlling axon and dendrite outgrowth during development via activation of extracellular calcium-sensing receptors, better known in the liver and thyroid than in the brain.

Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaSR) monitors the systemic, extracellular, free ionized-calcium level ([Ca(2+)](o)) in organs involved in systemic [Ca(2+)](o) homeostasis. However, CaSR is also expressed in the nervous system, where its role is unknown. We found large amounts of CaSR in perinatal mouse sympathetic neurons when their axons were innervating and branching extensively in their targets. Manipulating CaSR function in these neurons by varying [Ca(2+)](o), using CaSR agonists and antagonists, or expressing a dominant-negative CaSR markedly affected neurite growth in vitro. Sympathetic neurons lacking CaSR had smaller neurite arbors in vitro, and sympathetic innervation density was reduced in CaSR-deficient mice in vivo. Hippocampal pyramidal neurons, which also express CaSR, had smaller dendrites when transfected with dominant-negative CaSR in postnatal organotypic cultures. Our findings reveal a crucial role for CaSR in regulating the growth of neural processes in the peripheral and central nervous systems.