Changes In Extracellular Ca2 Research Articles

The Calcium-Sensing Receptor and the Reproductive System.

Active placental transport of maternal serum calcium (Ca2+) to the offspring is pivotal for proper development of the fetal skeleton as well as various organ systems. Moreover, extracellular Ca2+ levels impact on distinct processes in mammalian reproduction. The calcium-sensing receptor (CaSR) translates changes in extracellular Ca2+-concentrations into cellular reactions. This review summarizes current knowledge on the expression of CaSR and its putative functions in reproductive organs. CaSR was detected in placental cells mediating materno-fetal Ca2+-transport such as the murine intraplacental yolk sac (IPYS) and the human syncytiotrophoblast. As shown in casr knock-out mice, ablation of CaSR downregulates transplacental Ca2+-transport. Receptor expression was reported in human and rat ovarian surface epithelial (ROSE) cells, where CaSR activation stimulates cell proliferation. In follicles of various species a role of CaSR activation in oocyte maturation was suggested. Based on studies in avian follicles, the activation of CaSR expressed in granulosa cells may support the survival of follicles after their selection. CaSR in rat and equine sperms was functionally linked to sperm motility and sperm capacitation. Implantation involves complex interactions between the blastocyst and the uterine epithelium. During early pregnancy, CaSR expression at the implantation site as well as in decidual cells indicates that CaSR is important for blastocyst implantation and decidualization in the rat uterus. Localization of CaSR in human extravillous cytotrophoblasts suggests a role of CaSR in placentation. Overall, evidence for functional involvement of CaSR in physiologic mammalian reproductive processes exists. Moreover, several studies reported altered expression of CaSR in cells of reproductive tissues under pathologic conditions. However, in many tissues we still lack knowledge on physiological ligands activating CaSR, CaSR-linked G-proteins, activated intracellular signaling pathway, and functional relevance of CaSR activation. Clearly, more work is required in the future to decode the complex physiologic and pathophysiologic relationship of CaSR and the mammalian reproductive system.

Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria.

The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized. Changes in extracellular Ca(2+) concentration ([Ca(2+) ]e ), as well as modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPγ-S and high [Ca(2+) ]e (5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPγ-S stimulates cell transition from the G0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 µM ATPγ-S and [Ca(2+) ]e (ATP-[Ca(2+) ]e ) for 48 h increases cell number significantly above the control. ATPγ-S treatment in osteogenic medium containing [Ca(2+) ]e stimulates the gene expression of BMP-4, BMP-5, and OPN at 16, 48, and 72 h, respectively, above control. In same conditions, treatment for 6 days with 10 µM UTP or 100 µM UDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statistically significant increase in calcium deposits at 15 and 18 days, for 10 µM ATPγ-S treatment, and at 18 and 22 days, for [Ca(2+) ]e treatment, respect to control but ATP-[Ca(2+) ]e treatment shown a significant greater mineralization at 15 days respect to ATPγ-S, and at 18 days respect to both agonists. In conclusion, we demonstrated that an osteogenic medium containing 10 µM ATPγ-S and 5.35 mM [Ca(2+) ]e enhance osteogenesis and mineralization by rat primary calvarial cells cultures. J. Cell. Biochem. 117: 2658-2668, 2016. © 2016 Wiley Periodicals, Inc.

Human Connexin 26 (Cx26) N14K Mutant Alters Hemichannel Calcium and Voltage Sensitivity

Unpaired hemichannels, those not forming gap junction channels, play an autocrine/paracrine role by releasing transmitters, such as glutamate and ATP, to the extracellular medium. Hemichannels are gated by changes in extracellular Ca+2 concentration and membrane voltage. Several human connexin mutations that produce diseases increase hemichannel opening (e.g., gain of function due to decreased sensitivity to extracellular Ca+2). Using Xenopusoocytes and two-electrode voltage clamp techniques, we studied the voltage and calcium sensitivity of a connexin 26 (Cx26) mutation, N14K, located in the N-terminal domain (NT). The NT is thought to fold into the pore and form part of the pore lining; position N14 is approximately at the “hinge” region at which the NT bends toward the pore. We observe a significant leftward shift in the voltage-dependence N14K (midpoint at - 6.7 mV ± 0.7) mutants with respect to wild type channels (midpoint at + 9.6 mV ± 0.9). Rearrangements of the NT are associated with voltage dependent sub-conductances in wild type Cx26 hemichannels, however, N14K mutant channels did not display voltage dependent sub-conductances. The apparent calcium affinity was also significantly reduced in N14K (KD = 0.74 mM) compared to wild type channels (KD = 0.34 mM). Deactivation kinetics in wild type channels show single mono-exponential relaxations that are accelerated as a function of calcium concentration, but not voltage. The Cx26 mutant N14K displays two slower components. One of these components is both Ca+2 and voltage dependent but the other is not. Overall, our data indicate that under physiological conditions the N14K mutation stabilizes the open state relative to wild type channels by reducing both calcium and voltage sensitivity. R01GM099490/R01GM101950.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

CALCIUM SENSING RECEPTOR DELETION IN THE MOUSE ESOPHAGUS ALTERS THE EXPRESSION OF CELL‐CELL JUNCTIONAL PROTEINS

Calcium sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca+2 concentration. It regulates Ca+2 homeostasis by modulating PTH secretion and Ca+2 reabsorption in the kidney. CaSR also plays a role in glandular and fluid secretion in the GI tract and regulates differentiation and proliferation of keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating KRT5‐CreER;CaSRloxP/loxP compound mutants, where loxP sites flank exon 7 of CaSR gene. For lineage tracing of epithelial cells we bred‐in the reporter allele Rosa26R. Recombination was initiated with multiple tamoxifen injections. We demonstrated exon 7 deletion and Cre expression by PCR analysis of genomic DNA and LacZ staining. qRT‐PCR & Western blot (WB) analyses showed a significant reduction in CaSR mRNA & protein expression as compared to control CaSRloxP/loxP mice. Microscopic examination of esophageal tissues (H&E) did not show gross abnormalities. However, WB data revealed a significant reduction (~25%) in levels of adherens junction proteins E‐cadherin and β catenin and tight junction protein claudin‐1. Levels of Rac/Cdc42, small GTPase proteins involved in actin remodeling, were also reduced. Our data indicate that CaSR plays a role in regulating cell‐cell junctional complexes and is therefore important for the maintenance of the barrier properties in the esophagus.Support: VA Merit & Tulane Enhancement Award

Alginate-coated magnetic nanoparticles for noninvasive MRI of extracellular calcium.

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.

New Calcium‐Selective Smart Contrast Agents for Magnetic Resonance Imaging

Calcium plays a vital role in the human body and especially in the central nervous system. Precise maintenance of Ca(2+) levels is very crucial for normal cell physiology and health. The deregulation of calcium homeostasis can lead to neuronal cell death and brain damage. To study this functional role played by Ca(2+) in the brain noninvasively by using magnetic resonance imaging, we have synthesized a new set of Ca(2+) -sensitive smart contrast agents (CAs). The agents were found to be highly selective to Ca(2+) in the presence of other competitive anions and cations in buffer and in physiological fluids. The structure of CAs comprises Gd(3+)-DO3A (DO3A=1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane) coupled to a Ca(2+) chelator o-amino phenol-N,N,O-triacetate (APTRA). The agents are designed to sense Ca(2+) present in extracellular fluid of the brain where its concentration is relatively high, that is, 1.2-0.8 mM. The determined dissociation constant of the CAs to Ca(2+) falls in the range required to sense and report changes in extracellular Ca(2+) levels followed by an increase in neural activity. In buffer, with the addition of Ca(2+) the increase in relaxivity ranged from 100-157%, the highest ever known for any T1-based Ca(2+)-sensitive smart CA. The CAs were analyzed extensively by the measurement of luminescence lifetime measurement on Tb(3+) analogues, nuclear magnetic relaxation dispersion (NMRD), and (17)O NMR transverse relaxation and shift experiments. The results obtained confirmed that the large relaxivity enhancement observed upon Ca(2+) addition is due to the increase of the hydration state of the complexes together with the slowing down of the molecular rotation and the retention of a significant contribution of the water molecules of the second sphere of hydration.

Role of Calcium‐Sensing Receptor in Esophageal Epithelium

The calcium sensing receptor (CaSR), a G‐protein coupled receptor, senses changes in extracellular Ca+2 and mediates its effects on cellular functions. In cells not directly involved in Ca+2 homeostasis, CaSR plays a role in many cellular processes including differentiation, proliferation and secretion. We investigated the role of CaSR in modulating esophageal barrier function. We used immunohistochemistry, Western blotting and PCR to identify CaSR and cell‐cell adhesion molecules in pig esophageal stratified squamous epithelium (SSE). In primary cultures of SSE, we investigated the effect of CaSR agonist (cinacalcet, CCT) and antagonist, calhex, on cell growth and cell‐cell junction formation. We used MTT assay to monitor proliferation under different conditions that affect CaSR activity. SSE cells exposed to CCT (2.5μM, 24 hours), changed from polygonal to spindle‐shaped cells, a transformation proportional to CCT concentration. Staining with E‐cadherin and β‐catenin antibodies, showed altered distribution of these proteins in CCT treated cells. Our results indicate: 1) CaSR is expressed in supra‐basal cells of esophageal epithelium but not in differentiated apical layers; 2) CaSR activation decreases the rate of growth of SSE in culture; 3) CaSR activity alters expression and distribution of intercellular adhesion proteins and could modulate barrier properties of esophageal epithelium. Support: VA Merit grant and Tulane Enhancement Funds

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca(2+) concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties.

Involvement of liver-intestine cadherin in cancer progression

Cadherins constitute a superfamily of Ca(2+)-dependent cell adhesion molecules that play critical roles in the maintenance of tissue structure and morphogenesis. Their dysregulation is commonly observed in a variety of cancers. Liver-intestine cadherin (LI-cadherin), which was so named in view of its sole expression in the liver and intestine of the rat, is a structurally unique member of the cadherin superfamily, possessing seven cadherin repeats within the extracellular cadherin domain and only 25 amino acids in the cytoplasmic domain. Its adhesive property does not require any interaction with cytoplasmic components such as catenins, and it responds to small changes in extracellular Ca(2+) below the physiological plasma concentration. In humans, the distribution of LI-cadherin is limited to the duodenum, jejunum, ileum, colon, and part of the pancreatic duct. Data accumulated from studies of the biological characteristics of LI-cadherin have shown that it plays an important role in the pathophysiology of human cancers. Here, we review recent information about LI-cadherin and its implications for cancer progression.

Calcium-dependent dynamics of cadherin interactions at cell–cell junctions

Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherin-cadherin interactions are gated by extracellular Ca(2+), which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (τ = 1.17 ± 0.06 s(-1)) upon chelation of extracellular Ca(2+). A cadherin mutant with reduced adhesive activity (W2A) exhibited a faster, more substantial loss of homophilic interactions (τ = 0.86 ± 0.02 s(-1)), suggesting two types of native cadherin interactions--one that is rapidly modulated by changes in extracellular Ca(2+) and another with relatively stable adhesive activity that is Ca(2+) independent. The Ca(2+)-sensitive dynamics of cadherin interactions were transmitted to the cell interior where β-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction.

307 INVOLVEMENT OF THE CALCIUM SENSING RECEPTOR IN GROWTH AND PROLIFERATION OF STEM CELLS FROM EQUINE UMBILICAL CORD MATRIX

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the >8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P < 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the <8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P < 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P < 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P < 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes. Financial support was provided by Fondi di Ateneo 2009; University of Bari Aldo Moro (COD. ORBA09UDWX) (Resp. Sci. Maria Elena Dell’Aquila).

Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression

The Ca(2+)-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca(2+) ([Ca(2+)](o)) levels, maintaining extracellular Ca(2+) homeostasis, and transducing Ca(2+) signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca(2+)-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871-898). Our studies suggest a wrapping around 1-14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca(2+)-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca(2+) responses to changes in extracellular Ca(2+) and regulating cell surface expression of the receptor.

Naturally-Occurring Mutation in the Calcium-Sensing Receptor Reveals the Significance of Extracellular Domain Loop III Region for Class C G-Protein-Coupled Receptor Function

Inactivating mutations of the calcium-sensing receptor (CaSR) cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Most mutations are clustered in the N-terminal and Cys-rich regions of the extracellular domain (ECD) and seven-transmembrane domain. Disease-causing mutations are uncommon in the C terminus of ECD. The aim of the study was to characterize the CaSR mutations causing neonatal severe hyperparathyroidism in a consanguineous family. Parathyroid glands from the index patient were stained for CaSR protein. The CaSR gene was sequenced, mutations were recreated in CaSR cDNA, and HEK293 cells were transfected with the CaSR mutant expression vector. Cellular CaSR targeting was detected by immunoblotting and immunocytochemistry; CaSR activity was assayed by inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry. Immunocytochemistry showed reduced intracellular CaSR in patient parathyroids. An in-frame homozygous deletion/insertion mutation, c.1031 > 1034 (delACAAinsT), replaced His344-Asn345 with a single Leu in CaSR loop III. The mutant reduced cell surface expression of CaSR in transfected HEK293 cells. Inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry revealed blunted signaling responses of the mutant receptor to changes in extracellular Ca(2+) concentration. Deletion of His344-Asn345 in the ECD loop III region affects cell surface targeting of CaSR in transfected cells and in affected parathyroid glands. Absence of conserved Asn345 may interfere with CaSR folding or glycosylation, leading to poor protein targeting to the cell membrane. This loss-of-function mutant indicates that the ECD loop III is required for CaSR activity.

The Epidermal Ca2+ Gradient: Measurement Using the Phasor Representation of Fluorescent Lifetime Imaging

Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca2+ gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca2+ stores than those used in previous studies, and revealed that the bulk of free Ca2+ measured in the epidermis comes from intracellular Ca2+ stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca2+ making a relatively small contribution to the epidermal Ca2+ gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca2+, cease proliferation, and differentiate passively in response to changes in extracellular Ca2+. The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca2+ gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.

Modulation of the cold-activated cation channel TRPM8 by surface charge screening

TRPM8, a cation channel activated by cold and by cooling agents such as menthol and icilin, is critically involved in somatosensory cold sensation. Ion fluxes through TRPM8 are highly sensitive to changes in extracellular Ca(2+) and pH, but the mechanisms underlying this type of modulation are poorly understood. Here we provide evidence that inhibition of TRPM8 currents by extracellular divalent cations and protons is due to surface charge screening. We demonstrate that increasing concentrations of divalent cations or protons cause parallel shifts of the voltage dependence of TRPM8 activation towards positive potentials. These shifts were interpreted using the Gouy-Chapman-Stern theory, yielding an estimate for the density of fixed negative surface charge between 0.0098 and 0.0126 equivalent charges per A(2). These results represent the first description of the effects of surface charge screening on a TRP channel and provide a straightforward explanation for the known effects of extracellular Ca(2+) on cold-sensitive neurons.

Agonists and Allosteric Modulators of the Calcium-Sensing Receptor and Their Therapeutic Applications

The calcium-sensing receptor (CaR) belongs to the G protein-coupled receptor superfamily, with a characteristic structure consisting of seven transmembrane helices, an intracellular C-terminal and an extracellular N terminal domain. The primary physiological function of the CaR is the maintenance of constant blood Ca2+ levels, as a result of its ability to sense very small changes in extracellular Ca2+ (Ca2+(o)). Nevertheless, in addition to being expressed in tissues involved in Ca2+(o) homeostasis, the CaR is also expressed in tissues not involved in mineral homeostasis, suggestive of additional physiological functions. Numerous agonists and modulators of the CaR are now known in addition to Ca2+(o), including various divalent and trivalent cations, aromatic l-amino acids, polyamines, and aminoglycoside antibiotics. The signaling of the CaR is also regulated by extracellular pH and ionic strength. The activated CaR couples mainly to the phospholipase Cbeta and extracellular signal-regulated kinase 1/2 signaling pathways, and it decreases intracellular cAMP levels, leading to various physiological effects. The recent identification of synthetic allosteric modulators of the CaR has opened up a new field of research possibilities. Calcimimetics and calcilytics, which increase and decrease agonist signaling via the CaR, respectively, may facilitate the manipulation of the CaR and thus aid in further investigations of its precise signaling. These allosteric modulators, as well as strontium, have been demonstrated to have therapeutic potential for the treatment of disorders involving the CaR. This review discusses the various agonists and modulators of the CaR, differences in their binding and signaling, and their roles as therapeutics in various diseases.

Identification of a calcium‐controlled negative regulatory system affecting Vibrio cholerae biofilm formation

Vibrio cholerae's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary in V. cholerae's natural aquatic habitats is calcium (Ca(+2)). In this study, we investigated the response of V. cholerae to changes in extracellular Ca(2+) levels. Whole-genome expression profiling revealed that Ca(2+) decreased the expression of genes required for biofilm matrix production. Luria-Bertani (LB) medium supplemented with Ca(2+) (LBCa(2+)) caused V. cholerae to form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca(2+) led to dissolution in biofilms. Transcription of two genes encoding a two-component regulatory system pair, now termed calcium-regulated sensor (carS) and regulator (carR), was decreased in cells grown in LBCa(2+). Analysis of null and overexpression alleles of carS and carR revealed that expression of vps (Vibriopolysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator of vps gene expression.

Pharmacochaperone-Mediated Rescue of Calcium-Sensing Receptor Loss-of-Function Mutants

The calcium sensing receptor (CaSR) is a Family C/3 G protein-coupled receptor that translates changes in extracellular Ca(2+) into diverse intracellular signals. Loss-of-function mutations in human CaSR cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. CaSR must navigate a number of endoplasmic reticulum quality control checkpoints during biosynthesis, including a conformational/functional checkpoint. Here we examine the biosynthesis of 25 CaSR mutations causing familial hypocalciuric hypercalcemia /neonatal severe hyperparathyroidism using immunoprecipitation, biotinylation, and functional assays. We define classes of CaSR mutants based on their biosynthetic profile. Class I CaSR mutants are not rescued to the plasma membrane. To dissect the organellar compartments that class I mutants can access, we engineered a cleavage site for the proprotein convertase furin into the extracellular domain of wild-type CaSR and class I mutants. Based on absence or presence of cleavage fragments, we find most mutants are degraded from the endoplasmic reticulum (no furin-mediated cleavage), whereas others access the Golgi (furin-mediated cleavage) before degradation. Class II CaSR mutants show increased expression and/or enhanced plasma membrane localization upon treatment with MG132 or the pharmacochaperone NPS R-568, permitting assay of functional activity. Of the 10 CaSR mutants that exhibit plasma membrane localization, only two did not show enhanced functional activity after rescue with NPS R-568. The established approaches can be used with current and newly identified CaSR mutations to identify the location of biosynthetic block and to determine the likelihood of rescue by allosteric agonists.

Rapid calcitonin response to experimental hypercalcemia in healthy horses

Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca 2+) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5 mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca 2+ concentrations increased from 6.2 ± 0.3 mg/dL to 9.9 ± 0.5 mg/dL (4 min; P < 0.01). Serum CT increased from 16.7 ± 8.0 pg/mL to 87.1 ± 55.8 pg/mL at 2 min, and 102.5 ± 51.1 pg/mL at 4 min ( P < 0.01). Serum CT returned to baseline by 20 min, whereas serum Ca 2+ returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca 2+ or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca 2+ concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT.