Ca2+-sensing Receptor Research Articles

MUPP1 complexes renal K+channels to alter cell surface expression and whole cell currents

We previously found that the Ca(2+)-sensing receptor (CaR) interacts with and inactivates the inwardly rectifying K(+) channel Kir4.2 that is expressed in the kidney cortex and that has a COOH-terminal PDZ domain. To identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two-hybrid cloning with the COOH-terminal 125 amino acids (AA) of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the COOH-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in human embryonic kidney (HEK)-293 cells, Kir4.2 coimmunoprecipitates reciprocally with MUPP1 but not with a Kir4.2 construct lacking the four COOH-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 coimmunoprecipitate reciprocally from rat kidney cortex extracts. Coexpression of MUPP1 with Kir4.2 in HEK-293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Coexpression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared with expression of Kir4.2 alone, whereas expression of Kir4.2DeltaPDZ results in minimal currents and is not affected by coexpression with MUPP1. Immunofluorescence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K(+) as well as other ions.

The full-length calcium-sensing receptor dampens the calcemic response to 1α,25(OH)2 vitamin D3 in vivo independently of parathyroid hormone

1Alpha,25(OH)(2) vitamin D(3) [1,25(OH)(2)D(3)] increases serum Ca(2+) concentration in vivo, an action counteracted by activation of the Ca(2+)-sensing receptor (CaSR), which decreases parathyroid hormone (PTH) secretion and increases renal Ca(2+) excretion. Relatively little is known of the role the CaSR plays in this response through its potentially direct actions in kidney, gut, and bone independently of PTH. We report PTH-independent roles of the CaSR in modulating the response to exogenous 1,25(OH)(2)D(3) in mice with targeted disruption of both the CaSR and PTH genes (C(-)P(-)) compared with that in mice with disruption of the PTH gene alone (C(+)P(-)) or wild-type mice (C(+)P(+)). After intraperitoneal injection of 0.5 ng/g body wt 1,25(OH)(2)D(3), peak calcemic responses were observed at 24 h in all three genotypes in association with 1) a greater increase in serum Ca(2+) in C(-)P(-) mice than in the other genotypes on a Ca(2+)-replete diet that was attenuated by a Ca(2+)-deficient diet and pamidronate, 2) increased urinary Ca(2+)-to-creatinine ratios (UCa/Cr) in the C(+)P(-) and C(+)P(+) mice but a lowered ratio in the C(-)P(-) mice on a Ca(2+)-replete diet, and 3) no increase in calcitonin (CT) secretion in the C(+)P(+) and C(+)P(-) mice and a small increase in the C(-)P(-) mice. PTH deficiency had the anticipated effects on the expression of key genes involved in Ca(2+) transport at baseline in the duodenum and kidney, and injection of 1,25(OH)(2)D(3) increased gene expression 8 h later. However, the changes in the genes evaluated did not fully explain the differences in serum Ca(2+) seen among the genotypes. In conclusion, mice lacking the full-length CaSR have increased sensitivity to the calcemic action of 1,25(OH)(2)D(3) in the setting of PTH deficiency. This is principally from enhanced 1,25(OH)(2)D(3)-mediated gut Ca(2+) absorption and decreased renal Ca(2+) excretion, without any differences in bone-related release of Ca(2+) or CT secretion among the three genotypes that could explain the differences in their calcemic responses.

Identification of a Ca2+-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 microM IBTx, a high-conductance KCa channel blocker indicating a mechanism involving hyperpolarization. NPS R-467 (10 microM) 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.

Recent Updates on the Calcium-Sensing Receptor as a Drug Target

Parathyroid hormone (PTH) secreted from parathyroid cells is a key regulator of circulating levels of calcium ion, which is maintained within a narrow physiological range mainly by the action of a cell surface Ca2+-sensing receptor (CaSR) residing on parathyroid cells. The CaSR belongs to the family C of G-protein-coupled receptors (GPCRs), all members of which have a large extra cellular domain called Venus flytrap, akin to bacterial nutrient sensor. Promiscuity of the CaSR is reflected in its binding to several physiologically relevant di- or polyvalent cations that could elicit cellular signaling, yet the ligand specificity remains poor. This problem has been circumvented by the discovery of calcimimetics, the positive allosteric modulators of the CaSR that activate the CaSR on parathyroid cells, immediately suppressing PTH secretion. Out of several small molecules that act as calcimimetics, cinacalcet is the most extensively studied. The discovery of the CaSR and these specific agonists provides important insights into the discovery of new drugs that will be discussed in this review from the perspective of their structure-activity relationship. The calcimimetic compound cinacalcet has obtained regulatory approval for the treatment of hyperparathyroidism and is the first positive allosteric modulator of any GPCR to reach the market. It has other important uses in dialysis patients with end-stage renal disease, in whom it decreases Ca X P product. Pharmaceutically converse to calcimimetics are calcilytic compounds, which antagonize parathyroid CaSR and stimulate PTH secretion. Antagonism of the CaSR has the potential to yield an anabolic therapy for osteoporosis that awaits clinical validation.

Physiological significance of L-amino acid sensing by extracellular Ca2+-sensing receptors

The calcium-sensing receptor is a multimodal, multimetabolic sensor that mediates the feedback-dependent control of whole body calcium metabolism. Remarkably, in addition to its role in Ca(2+)(o) (extracellular Ca(2+)) sensing, the CaR (Ca(2+)-sensing receptor) also responds to L-amino acids. L-amino acids appear to activate, predominantly, a signalling pathway coupled with intracellular Ca(2+) mobilization, require a threshold concentration of Ca(2+)(o) for efficacy and sensitize the receptor to activation by Ca(2+)(o). Here, we review the evidence that the CaR, like other closely related members of the class 3 GPCR (G-protein-coupled receptor) family including GPRC6A, is a broad-spectrum amino acid-sensing receptor, consider the nature of the signalling response to amino acids and discuss its physiological significance.

Review series on the extracellular Ca2+‐Sensing Receptor

Faithful control of systemic Ca2+ homeostasis is crucial for life for most, if not all, multi-cellular organisms. In humans and many other vertebrates, extracellular Ca2+ is detected by a specific G-protein coupled receptor (GPCR) for divalent cations, the extracellular Ca2+-sensing receptor (CaR) [1]. With this issue, I am pleased to launch a new review series in the Journal of Cellular and Molecular Medicine on this fascinating receptor. The idea that a sensor for extracellular divalent cations existed in mammals was not entirely new. It was apparent from early studies throughout the 1980s that some type of cell-surface receptor coupled to intracellular signal transduction cascades must be present on the parathyroid gland in order to account for the ability of this endocrine organ to sense and respond to minute fluctuations in circulating Ca2+ levels [1-3]. However, the actual cloning of the CaR from the bovine parathyroid by Brown, Hebert, Riccardi and colleagues in 1993 transformed this field [4], permitting a detailed look into the structure-function relationships of the CaR. This also eventually led to the somewhat surprising conclusion that the CaR was expressed at varying levels in numerous tissues apart from the parathyroid gland. With the sequence in hand, it also soon became apparent that naturally occurring mutations in CaR accounted for a number of human disorders of systemic Ca2+ handling. CaR, in fact, is a rare example of a GPCR for which the numerous mutations identified in humans have been unequivocally linked to disease, ranging from severe (even lethal) endocrine dysfunction to largely benign alterations in mineral homeostasis. In the first review article appearing in this issue, Huang and Miller explore the ways in which proteins that interact with CaR can influence the function of the receptor. These authors point out that the simple ‘linear’ model of GPCR signalling in which CaR interacts with heterotrimeric G-proteins in order to modulate second messenger concentrations is difficult to reconcile with the wide variety of biological actions of CaR stimulation observed in different cell types. Huang and Miller suggest that distinct sig-nalling ‘personalities’ can derive from interactions of CaR with specific interacting proteins. Here, they discuss how CaR interacts with inwardly rectifying potassium channels (members of the Kir family), receptor activity modifying proteins (RAMPs), the scaffolding protein filamin and other proteins that form the CaR-based signalling complex. In the second article of this series, Hu and Speigel present a comprehensive look at the structure-function relationships of the CaR. Many of these insights have been obtained through examination of the functional attributes of receptors containing natural mutations identified in patients, as well as through engineered mutations. The similarity of CaR with the metabotropic glutamate receptor type 1, for which a partial crystal structure has been solved, allows further predictions as to the structural and functional domains of CaR. Finally, the actions of specific allosteric modulators of the receptor, many of which are now used clinically (or are being developed) to treat a variety of disorders linked to the CaR, have also revealed important insights into the detailed workings of this interesting receptor. I hope the readers of JCMM will enjoy these two informative articles as much as I have. Aldebaran M. HoferGuest Editor

Structure and function of the human calcium‐sensing receptor: insights from natural and engineered mutations and allosteric modulators

The human extracellular Ca2+-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthos-teric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR.

Constitutive Activity of the Osteoblast Ca2+-Sensing Receptor Promotes Loss of Cancellous Bone

Changes in extracellular [Ca2+] modulate the function of bone cells in vitro via the extracellular Ca2+-sensing receptor (CaR). Within bone microenvironments, resorption increases extracellular [Ca2+] locally. To determine whether enhanced CaR signaling could modulate remodeling and thereby bone mass in vivo, we generated transgenic mice with a constitutively active mutant CaR (Act-CaR) targeted to their mature osteoblasts by the 3.5 kb osteocalcin promoter. Longitudinal microcomputed tomography of cancellous bone revealed reduced bone volume and density, accompanied by a diminished trabecular network, in the Act-CaR mice. The bone loss was secondary to an increased number and activity of osteoclasts, demonstrated by histomorphometry of secondary spongiosa. Histomorphometry, conversely, indicates that bone formation rates were unchanged in the transgenic mice. Constitutive signaling of the CaR in mature osteoblasts resulted in increased expression of RANK-L (receptor activator of nuclear factor-kappaB ligand), the major stimulator of osteoclast differentiation and activation, which is the likely underlying mechanism for the bone loss. The phenotype of Act-CaR mice is not attributable to systemic changes in serum [Ca2+] or PTH levels. We provide the first in vivo evidence that increased signaling by the CaR in mature osteoblasts can enhance bone resorption and further propose that fluctuations in the [Ca2+] within the bone microenvironment may modulate remodeling via the CaR.

Tilapia extracellular Ca 2+ ‐sensing receptor is expressed in osmoregulatory and endocrine tissues

Involvement of the extracellular Ca2+-sensing receptor (CaR) in piscine Ca2+ homeostasis was evaluated in a euryhaline teleost, the Mozambique tilapia (Oreochromis mossambicus; t), by immunohistochemistry using affinity-purified antibodies directed against tCaR. CaR protein expression was visualized by immunoperoxidase staining in serial sections of tissues from fish acclimated to either fresh water (FW, low-Ca2+ environment) or seawater (SW, high-Ca2+ environment). In tilapia osmoregulatory tissues, CaR protein was localized in mitochondria-rich “chloride cells” of gill and in epithelia of middle and posterior intestine in both FW and SW fish. CaR is expressed in proximal segments of renal tubule, with no immunohistochemical staining of distal segments (where Na+/K+-ATPase is expressed), or of glomeruli. Consistent with RT-PCR assessment of mRNA expression, CaR protein expression was higher in kidney of FW tilapia compared with that in kidney of SW fish. In the pituitary, CaR protein expression was observed in cells of the pars intermedia, where both somatolactin- and melanocyte-stimulating hormone-secreting cells exist. These findings support the possible involvement of CaR in piscine Ca2+ homeostasis at the level of ion-transporting tissues, and involvement through endocrine pathways in the pituitary, and perhaps in other tissues. Supported by Japan Society for the Promotion of Science.

Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function

The Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca(2+) metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for approximately 125 aa from the COOH terminus of the inwardly rectifying K(+) channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant CaR(R796W), and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K(+) for the basolateral Na(+)-K(+)-ATPase and thereby contribute to inhibition of Na(+) reabsorption.

The temporal and spatial expression of Claudins in epidermal development and the accelerated program of epidermal differentiation in K14-CaSR transgenic mice

The importance of the epidermal permeability barrier (EPB) in protecting the mammalian species against harmful UV irradiation, microorganism invasion and water loss is well recognized, as is the role of calcium (Ca 2+) in keratinocyte differentiation, cell–cell contact and the EPB. In a previous study, we reported that the overexpression of the Ca 2+-sensing receptor (CaSR) in the undifferentiated basal cells of the epidermis induced a modified epidermal differentiation program including an accelerated EPB formation in transgenic mice, suggesting a role for CaSR signaling in the differentiation of embryonic epidermal cells during development. We now describe the expression profile of claudins (Cldns) and keratin markers in the accelerated EPB formation of K14-CaSR transgenic mice during development as compared to the wild type from E12.5 to newborn stages. Our data show that the transgenic epidermis undergoes an advanced epidermal differentiation program as compared to the wild type as evidenced morphologically as well as by the expression of K14, K1, loricrin, Cldn6, Cldn18 and Cldn11. In addition, we report for the first time the sequential expression of Cldns in epidermal development and describe that the localization of some Cldns change within the epidermis as it matures. Furthermore, we demonstrate that Cldn6 is expressed very early in epidermal morphogenesis, followed by Cldn18, Cldn11 and Cldn1.

Parathyroid-Specific Double Knockout of Gq and G11 α-Subunits Leads to a Phenotype Resembling Germline Knockout of the Extracellular Ca2+-Sensing Receptor

Germline knockout of the extracellular Ca2+ -sensing receptor (CaR) leads to a phenotype that includes severe hypercalcemia, hyperparathyroidism, relative hypocalciuria, skeletal abnormalities, retarded growth, and early postnatal death. To investigate the role of heterotrimeric G proteins in CaR signaling, we used cre/lox technology to delete the respective alpha-subunits of Gq and G11 selectively in parathyroid cells. Mice that were PTH-Cre(+/-); Gnaq(flox/flox); Gna11(-/-) (PTH-Galphaq/Galpha11 -double knockouts) were viable, but showed all the features of germline knockout of the CaR except hypocalcuria. Our results demonstrate the critical role of both Gq and G11 in mediating inhibition of PTH secretion by extracellular Ca2+.

Role of the osteoblast calcium‐sensing receptor in bone formation and mineralization

Functional assessment of the osteoblast Ca2+-sensing receptor (CaR) in vivo using the existing CaR knockout mouse models is complicated by their metabolic disturbances and potential compensation by a spliced CaR variant. To assess the role of the CaR in bone formation and mineralization, we generated transgenic mice with overexpression of constitutively active mutant CaRs (Act-CaR) targeted to mature osteoblasts using a 3.5 kb osteocalcin promoter. The constitutive activity of Act-CaR was confirmed in HEK293 cells by increased sensitivity to extracellular [Ca2+] in promoting inositol phosphate accumulation. Expression of Act-CaR RNA and protein was demonstrated by immunoblotting and quantitative real-time PCR, respectively. In Act-CaR mice, we observed reduced trabecular bone volume, thickness and connectivity assessed by in vivo micro-computed tomography. Cultured bone marrow stromal cells from Act-CaR mice showed increased differentiation indicated by increased alkaline phosphatase activity and matrix mineralization. They also exhibited increased expression of genes that mediate proliferation (cyclin D 1 and 2), cell survival (akt), differentiation (osteopontin), mineralization (alkaline phosphatase, ank, ectonucleotide pyrophosphatase/phosphodiesterase 1) and osteoclastogenesis (RANKL). The increased RANKL expression may play a role in the osteopenic phenotype of the Act-CaRs mice. These findings support a role for osteoblast CaRs in bone cell differentiation and osteoclastogenesis. The work was supported by Department of Veteran Affairs (REAP award and Merit Review) and NIH RO1 DK54793, AG21353.

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids

The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.

Termination of cAMP signals by Ca2+ and Gαi via extracellular Ca2+ sensors

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca2+-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer–dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin–sensitive Gαi and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca2+-mobilizing agonists under physiological conditions.

A Double Mutation in the Extracellular Ca2+-sensing Receptor's Venus Flytrap Domain That Selectively Disables l-Amino Acid Sensing

The extracellular Ca(2+)-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca(2+)-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.

Evidence in Favor of a Calcium-Sensing Receptor in Arterial Endothelial Cells

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Role of ceramide in Ca2+-sensing receptor-induced apoptosis

Increased extracellular Ca(2+) ([Ca(2+)](o)) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, we demonstrate that activation of the CaR leads to apoptosis, which was determined by nuclear condensation, DNA fragmentation, caspase-3 activation, and increased cytosolic cytochrome c. This CaR-induced apoptotic pathway is initiated by CaR-induced accumulation of ceramide which plays an important role in inducing cell death signals by distinct G protein-independent signaling pathways. Pretreatment of wild-type CaR-expressing cells with pertussis toxin inhibited CaR-induced [(3)H]ceramide formation, c-Jun phosphorylation, and caspase-3 activation. The ceramide accumulation, c-Jun phosphorylation, and caspase-3 activation by the CaR can be abolished by sphingomyelinase and ceramide synthase inhibitors in different time frames. Cells that express a nonfunctional mutant CaR that were exposed to the same levels of [Ca(2+)](o) showed no evidence of activation of the apoptotic pathway. In conclusion, we report the involvement of the CaR in stimulating programmed cell death via a pathway involving GTP binding protein alpha subunit (Galpha(i))-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.

Amino Acid-stimulated Ca2+ Oscillations Produced by the Ca2+-sensing Receptor Are Mediated by a Phospholipase C/Inositol 1,4,5-Trisphosphate-independent Pathway That Requires G12, Rho, Filamin-A, and the Actin Cytoskeleton

The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.

Extracellular Ca2+-sensing receptor expression and hormonal regulation in rat uterus during the peri-implantation period

The extracellular Ca2+-sensing receptor (CaR) is a member of the superfamily of G protein-coupled receptors (GPCRs). It is an important mediator of a wide range of Ca2+-dependent physiological responses in various tissues. In reproductive tissues it has been reported to play a significant role in promoting or maintaining placentation. Meanwhile, another Ca2+ regulated gene stanniocalcin-1 (STC-1) has been documented to be involved in decidualization and uterine remodelling. The phenomenon that CaR mediates STC-1's transcription responding to extracellular calcium in fish urges us to suppose that CaR, like STC-1, may also play a role in implantation and decidualization. To resolve this conjecture, we have examined the expression and hormonal regulation of the CaR gene in rat uterus during peri-implantation period. CaR mRNA was expressed at a moderate level in the luminal epithelium of the early stage of pregnancy (from day 1 to day 3). From day 2-3 it began to be expressed more strongly in the stromal cells immediately underneath the luminal epithelium, but decreased to a basal level on day 4. From day 6 to day 9 continuously, both CaR mRNA and protein were highly expressed in the primary decidua. Expression of CaR mRNA and protein in these cells was also observed when a delayed implantation was terminated by estrogen treatment to allow the embryo implantation. In contrast, only basal level expression of the molecules was detected in the cells of animals subjected to a normal-delayed implantation or the pseudopregnant condition. Embryo transplantation experiment confirmed that CaR expression at the implantation site was induced by the implanting blastocyst. Consistent with the normal pregnant process, CaR mRNA and protein in the cells were also induced by an artificial decidualization procedure. Further experiments demonstrated that treatment of the ovariectomized rat with estrogen or/and progesterone stimulated a high level expression of CaR mRNA in the uterine epithelial and glandular epithelium. In conclusion, CaR was specifically induced during the processes of implantation and subsequent decidualization and may play a role in these processes.