The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that "senses" extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT-PCR using four CaR-specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR-specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti-CaR antiserum. In addition, raising Ca2+o from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a "calcimimetic" CaR activator (R-467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca(+)-activated K+ channel having a unitary conductance of 84+/-4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.     

Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor

Calcium (Ca(2+)) has long been recognized as a physiologically indispensable ion owing to its numerous intra- and extracellular roles. More recently, it has become apparent that extracellular calcium (Ca(2+)(o)) also serves as an extracellular first messenger following the cloning of a Ca(2+)(o)-sensing receptor (CaR) that belongs to the superfamily of G protein-coupled receptors (GPCR). The CaR probably functions as a dimer in performing its central role of "sensing" minute alterations in Ca(2+)(o) and adjusting the secretion of parathyroid hormone (PTH) so as to normalize Ca(2+)(o) through the actions of PTH on the effector elements of the mineral ion homeostatic system (e.g., kidney, bone and intestine). Several inherited human conditions are caused by inactivating or activating mutations of this receptor, and mice have been generated with targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in patients with these conditions and in CaR-deficient mice have proven the physiological importance of the CaR in mineral ion homeostasis. An accumulating body of evidence, however, suggests that the CaR also plays numerous roles outside the realm of systemic mineral ion homeostasis. The receptor regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization and apoptosis in a variety of tissues/cells. Finally, the availability of specific "calcimimetic", allosteric CaR activators - which are currently in clinical trials - will probably have therapeutic implications for diseases caused by malfunction of the CaR in tissues not only within, but also outside, the mineral ion homeostatic system.     

Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C

To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca(2+)-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca(2+)] ([Ca(2+)](o))-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca(2+)](o) to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca(2+)](o) in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca(2+)](o) even up to 30 mm. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca(2+)](o)-induced PLC activation and or CaR expression.     

Aminoglycoside antibiotics induce pH-sensitive activation of the calcium-sensing receptor

The aminoglycoside antibiotic (AGA) neomycin is a known agonist of the extracellular calcium-sensing receptor (CaR). To test whether other AGA drugs stimulate the CaR, we studied the relative effects of four AGAs on intracellular Ca(2+) concentration ([Ca(2+)](i)) using CaR-transfected human embryonic kidney (HEK)-293 cells. Gentamicin, tobramycin, and neomycin evoked dose-dependent increases in [Ca(2+)](i) with EC(50) values of 258, 177, and 43 microM, respectively, in CaR-transfected, but not in non-transfected cells. Kanamycin was ineffective at doses <1mM. Thus, AGAs stimulate the CaR with a rank order of potency that correlates positively with the number of their attached amino groups. The CaR is expressed on the apical surface of renal proximal tubule cells, which is also the site of AGA endocytosis and nephrotoxicity. In the current study, reducing extracellular pH from 7.4 to 6.9, to mimic the luminal pH of the proximal tubule, enhanced the sensitivity of the CaR to tobramycin, suggesting that the AGAs may be more potent CaR agonists in the proximal tubule than elsewhere. This pH effect was not observed when stimulating CaR with the non-ionizable agonist, Gd(3+), suggesting that the enhanced AGA effect is due to increased ionization of the drug. Thus, we show that a number of AGA drugs are capable of CaR activation and that their potency most likely relates to the number of their amino side chains and to their pH-dependent charge characteristics. The contribution of CaR activation to the pharmacological/toxicological effects of these AGAs remains to be determined.     

Calcium receptor-mediated intracellular signalling

As a G protein-coupled receptor (GPCR), the extracellular calcium-sensing receptor (CaR) responds to changes in extracellular free calcium concentration by inducing intracellular signalling. These CaR-induced signals then specifically modulate cellular functions such as parathyroid hormone secretion from the parathyroid glands and calcium reabsorption in the kidney and thus to understand how the CaR functions one must understand how it signals. CaR-induced signalling involves intracellular Ca2+ mobilisation/oscillations as well as the activation of various phospholipases and protein kinases and the suppression of cAMP formation. This review will detail the intracellular pathways by which the CaR is believed to elicit its physiological functions and summarises the evidence for cell- and agonist-specific differential signalling.     

Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-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 Galpha(i) 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 Ca(2+)-mobilizing agonists under physiological conditions.     

The role of the calcium-sensing receptor in cancer

The extracellular calcium-sensing receptor (CaR) is a versatile sensor of small, polycationic molecules ranging from Ca2+ and Mg2+ through polyarginine, spermine, and neomycin. The sensitivity of the CaR to changes in extracellular Ca2+ over the range of 0.05-5 mM positions the CaR as a key mediator of cellular responses to physiologically relevant changes in extracellular Ca2+. For many cell types, including intestinal epithelial cells, breast epithelial cells, keratinocytes, and ovarian surface epithelial cells, changes in extracellular Ca2+ concentration over this range can switch the cellular behaviour from proliferation to terminal differentiation or quiescence. As cancer is predominantly a disease of disordered balance between proliferation, differentiation, and apoptosis, disruptions in the function of the CaR could contribute to the progression of neoplastic disease. Loss of the growth suppressing effects of elevated extracellular Ca2+ have been demonstrated in parathyroid hyperplasias and in colon carcinoma, and have been correlated with changes in the level of CaR expression. Activation of the CaR has also been linked to increased expression and secretion of PTHrP (parathyroid hormone-related peptide), a primary causal factor in hypercalcemia of malignancy and a contributor to metastatic processes involving bone. Although mutation of the CaR does not appear to be an early event in carcinogenesis, loss or upregulation of normal CaR function can contribute to several aspects of neoplastic progression, so that therapeutic strategies directed at the CaR could potentially serve a supportive function in cancer management.     

Activation of p42/44 and p38 mitogen-activated protein kinases by extracellular calcium-sensing receptor agonists induces mitogenic responses in the mouse osteoblastic MC3T3-E1 cell line

Recently, substantial evidence has accumulated that the G-protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is expressed in bone marrow-derived cells, including osteoblasts, stromal cells, monocytes-macrophages, and osteoclast precursor cells. Our previous studies have shown that the mouse osteoblastic MC3T3-E1 cell line also expresses the CaR and exhibits mitogenic responses when exposed to various CaR agonists. In this study, in order to understand the signaling pathway(s) mediating this response, we studied the effects of CaR agonists on the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) (Erk1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in MC3T3-E1 cells. Raising the level of Ca(2+)(o) (4.5 mM) or addition of the polycationic CaR agonists, gadolinium (Gd(3+)) (25 microM), neomycin (300 microM) or spermine (1 mM), each stimulated phosphorylation of both p42/44 and p38 MAPKs, but not JNK, as assessed using phospho-specific antibodies to the respective MAPKs. Furthermore, phosphorylation of p42/44 and p38 MAPK were markedly inhibited by their selective and potent inhibitors, PD98059 (50 microM) and SB203580 (10 microM), respectively. Finally, the two inhibitors suppressed [(3)H]thymidine incorporation into DNA in MC3T3-E1 cells at a normal level of Ca(2+)(o) (1.8 mM) as well as when stimulated by high (4.5 mM) Ca(2+)(o), Gd(3+), or neomycin. Thus, in mouse osteoblastic MC3T3-E1 cells, both the p42/44 and p38 MAPK cascades play pivotal roles in CaR-stimulated mitogenic responses.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

Evidence for distinct cation and calcimimetic compound (NPS 568) recognition domains in the transmembrane regions of the human Ca2+ receptor

The Ca(2+) receptor, a member of the family 3 of G protein-coupled receptors (GPCR), responds not only to its primary physiological ligand Ca(2+) but also to other di- and trivalent metals (Mg(2+), Gd(3+)) and the organic polycations spermine and poly-l-Arginine. As has been found for other family 3 GPCRs, the large amino-terminal extracellular domain (ECD) of the Ca(2+) receptor is the primary Ca(2+) binding domain. To examine how the signal is propagated from the ECD to the seven-transmembrane core domain (7TM) we constructed a Ca(2+) receptor mutant (T903-Rhoc) lacking the entire ECD but containing the 7TM. We have found that this structure initiates signaling in human embryonic kidney (HEK) 293 cells stably expressing the construct. One or more cation recognition sites are also located within the 7TM. Not only Ca(2+), but also several other Ca(2+) receptor-specific agonists, Mg(2+), Gd(3+), spermine, and poly-l-Arginine, can activate T903-Rhoc truncated receptor-initiated phosphoinositide hydrolysis in HEK 293 cells. The phenylalkylamine compound, NPS 568, identified as a positive allosteric modulator of the Ca(2+) receptor can selectively potentiate the actions of Ca(2+) and other polycationic agonists on the T903-Rhoc receptor. Similarly, organic polycations synergistically activate T903-Rhoc with di- and trivalent metals. Alanine substitution of all the acidic residues in the second extracellular loop of the T903-Rhoc receptor significantly impairs activation by metal ions and organic polycations in the presence of NPS 568 but not the synergistic activation of Ca(2+) with poly-l-Arginine. These data indicate that although the ECD has been thought to be the main determinant for Ca(2+) recognition, the 7TM core of the Ca(2+) receptor contains activating site(s) recognizing Ca(2+) and Gd(3+) as well as the allosteric modulators NPS 568 and organic polycations that may play important roles in the regulation of receptor activation.     

Effects of calcium-sensing receptor on the secretion of parathyroid hormone-related peptide and its impact on humoral hypercalcemia of malignancy

The extracellular calcium-sensing receptor (CaR) plays a key role in the defense against hypercalcemia by "sensing" extracellular calcium (Ca2+(o)) levels in the parathyroid and kidney, the key organs maintaining systemic calcium homeostasis. However, CaR function can be aberrant in certain pathophysiological states, e.g., in some types of cancers known to produce humoral hypercalcemia of malignancy (HHM) in humans and animal models in which high Ca2+(o), via the CaR, produces a homeostatically inappropriate stimulation of parathyroid hormone-related peptide (PTHrP) secretion from these tumors. Increased levels of PTHrP set a cycle in motion whereby elevated systemic levels of Ca2+(o) resulting from its increased bone-resorptive and positive renal calcium-reabsorbing effects give rise to hypercalcemia, which in turn begets worsening hypercalcemia by stimulating further release of PTHrP by the cancer cells. I review the relationship between CaR activation and PTHrP release in normal and tumor cells giving rise to HHM and/or malignant osteolysis and the actions of the receptor on key cellular events such as proliferation, angiogenesis, and apoptosis of cancer cells that will favor tumor growth and osseous metastasis. I also illustrate diverse signaling mechanisms underlying CaR-stimulated PTHrP secretion and other cellular events in tumor cells. Finally, I raise several necessary questions to demonstrate the roles of the receptor in promoting tumors and metastases that will enable consideration of the CaR as a potential antagonizing/neutralizing target for the treatment of HHM.     

Structure-function relationship of the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaR) originally cloned from bovine parathyroid gland is a G protein-coupled receptor. The physiological relevance of the cloned CaR for sensing and regulating the extracellular calcium concentration has been established by identifying hyper- and hypocalcemic disorders resulting from inactivating and activating mutations, respectively, in the CaR. The cloned CaR has been stably or transiently expressed in human embryonic kidney cells and significant progress has been made in elucidating its regulation and activation process using physiological, biochemical and molecular biological methods. A large collection of naturally occurring CaR mutations offers a valuable resource for studies aimed at understanding the structure-function relationships of the receptor, including functional importance of CaR dimerization. In turn, characterization of these naturally occurring mutations has clarified the pathogenesis of clinical conditions involving abnormalities in the CaR, such as familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

L-amino acids regulate parathyroid hormone secretion

Parathyroid hormone (PTH) secretion is acutely regulated by the extracellular Ca(2+)-sensing receptor (CaR). Thus, Ca(2+) ions, and to a lesser extent Mg(2+) ions, have been viewed as the principal physiological regulators of PTH secretion. Herein we show that in physiological concentrations, l-amino acids acutely and reversibly activated the extracellular Ca(2+)-sensing receptor in normal human parathyroid cells and inhibited parathyroid hormone secretion. Individual l-amino acids, especially of the aromatic and aliphatic classes, as well as plasma-like amino acid mixtures, stereoselectively mobilized Ca(2+) ions in normal human parathyroid cells in the presence but not the absence of the CaR agonists, extracellular Ca(2+) (Ca(2+)(o)), or spermine. The order of potency was l-Trp = l-Phe > l-His > l-Ala > l-Glu > l-Arg = l-Leu. CaR-active amino acids also acutely and reversibly suppressed PTH secretion at physiological ionized Ca(2+) concentrations. At a Ca(2+)(o) of 1.1 mm and an amino acid concentration of 1 mm, CaR-active amino acids (l-Phe = l-Trp > l-His = l-Ala), but not CaR-inactive amino acids (l-Leu and l-Arg), stereoselectively suppressed PTH secretion by up to 40%, similar to the effect of raising Ca(2+)(o) to 1.2 mm. A physiologically relevant increase in the -fold concentration of the plasma-like amino acid mixture (from 1x to 2x) also reversibly suppressed PTH secretion in the Ca(2+)(o) concentration range 1.05-1.25 mm. In conclusion, l-amino acids acutely and reversibly activate endogenous CaRs and suppress PTH secretion at physiological concentrations. The results indicate that l-amino acids are physiological regulators of PTH secretion and thus whole body calcium metabolism.     

Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries

Myogenic tone of small arteries is dependent on the presence of extracellular calcium (Ca(o)(2+)), and, recently, a receptor that senses changes in Ca(2+), the calcium-sensing receptor (CaR), has been detected in vascular tissue. We investigated whether the CaR is involved in the regulation of myogenic tone in rat subcutaneous small arteries. Immunoblot analysis using a monoclonal antibody against the CaR demonstrated its presence in rat subcutaneous arteries. To determine whether the CaR was functionally active, segments of artery (< 250 microm internal diameter) mounted in a pressure myograph with an intraluminal pressure of 70 mmHg were studied after the development of myogenic tone. Increasing Ca(o)(2+) concentration ([Ca(2+)](o)) cumulatively from 0.5 to 10 mM induced an initial constriction (0.5-2 mM) followed by dilation (42 +/- 5% loss of tone). The dose-dependent dilation was mimicked by other known CaR agonists including magnesium (1-10 mM) and the aminoglycosides neomycin (0.003-10 mM) and kanamycin (0.003-3 mM). PKC activation with the phorbol ester phorbol-12,13-dibutyrate (20nM) inhibited the dilation induced by high [Ca(2+)](o) or neomycin, whereas inhibition of PKC with GF109203X (10 microM) increased the responses to Ca(o)(2+) or neomycin, consistent with the role of PKC as a negative regulator of the CaR. We conclude that rat subcutaneous arteries express a functionally active CaR that may be involved in the modulation of myogenic tone and hence the regulation of peripheral vascular resistance.