Protein-coupled Ca2 Research Articles

Abstract P122: Downregulation and Inhibition of PKCα Causes Increased Ca 2+ -Sensing Receptor-Mediated Vascular Relaxation in Aged Dahl Salt-Sensitive Rats

The G protein-coupled Ca 2+ -Sensing Receptor (CaSR) mediates extracellular calcium (Ca 2+ e ) - induced relaxation of mesenteric arteries under tension and may therefore play a role in blood pressure regulation. This presents CaSR as promising target for therapeutic drug design for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is an important regulator of CaSR signaling and can phosphorylate the receptor leading to desensitization. In this study, we investigated the effects of CaSR mutation and downregulation of PKCα in CaSR-mediated relaxation of phenylephrine (PE)-contracted mesenteric arteries from aged Dahl salt-sensitive rats, fed a low salt (0.1% NaCl) diet, using automated wire myography. We have observed in our laboratory, that there is significant decrease in relaxation response of PE-contracted mesenteric arteries to Ca 2+ e . as animals get older. The results show minimal relaxation responses of arteries to Ca 2+ e and that downregulation of CaSR expression had no significant effect. PKCα downregulation (siRNA) and inhibition of PKC (Gö 6976) showed greater than 80% increase in maximum Ca 2+ -induced relaxation, with significant decreases in EC 50 values compared to untreated controls. The results indicate that PKCα plays an important role in the regulation of CaSR -mediated relaxation of mesenteric arteries. Therefore, inhibition of PKC may lead to increased CaSR sensitivity to Ca 2+ indicating a possible therapeutic mechanism for intervention in age-related vascular dysfunction.

PERK regulates Gq protein-coupled intracellular Ca2+ dynamics in primary cortical neurons.

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca2+ dynamics in insulin-secreting β-cells underlying glucose-stimulated insulin secretion, and modulates Ca2+ signals-dependent working memory, we explored the role of PERK in regulating Gq protein-coupled Ca2+ dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca2+ release, but decreased receptor-operated extracellular Ca2+ influx. Impaired Gq protein-coupled intracellular Ca2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca2+ dynamics in cortical pyramidal neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0268-5) contains supplementary material, which is available to authorized users.

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 μm. The myography technique, first described by Mulvay and Halpern(1), was based on the method proposed by Bevan and Osher(2). The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses(3).The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors. We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca(2+)(e;)). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca(2+)-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca(2+)(e;). Tissues from Wistar rats showed higher Ca(2+)-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca(2+)(e;)-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS. The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side(6,7,8).

Common genetic variation in the 3′-untranslated region of gonadotropin-releasing hormone receptor regulates gene expression in cella and is associated with thyroid function, insulin secretion as well as insulin sensitivity in polycystic ovary syndrome patients

Gonadotropin-releasing hormone receptor (GNRHR) is a member of the G protein-coupled Ca(2+)-dependent family of receptors. It interacts with GnRH, whose signaling plays an important role in thyroid-stimulating hormone (TSH) secretion and insulin activity. There has been no study on the genetic effect of GNRHR on TSH secretion and insulin action in polycystic ovary syndrome (PCOS). We decided to investigate whether naturally occurring genetic variation at the human GNRHR locus is associated with thyroid function, insulin secretion and insulin sensitivity in PCOS. We undertook a systematic search for polymorphisms in GNRHR by resequencing the gene and then genotyped common single-nucleotide polymorphisms across the locus in 261 PCOS patients well-phenotyped for several metabolic traits to determine associations. A test for association of common genetic variants with susceptibility to PCOS was carried out in a large cohort of 948 subjects. Finally, we experimentally validated the marker-on-trait associations using GNRHR 3'-UTR region/reporter analysis in 293T cells. The 3'-UTR variant rs1038426 was associated with serum thyroid concentration (P=0.007), change of insulin levels during oral glucose tolerance test (P=0.004) and insulin sensitivity index (P=0.014). In a functional study, 3'-UTR variant T allele increased reporter expression by a transfected luciferase reporter/GNRHR 3'-UTR expression plasmid. In conclusion, our results strongly suggest that common genetic variant in GNRHR contributes to the phenotypic expression of PCOS. The findings suggest novel pathophysiological links between the GNRHR locus and thyroid function and insulin secretion in PCOS.

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.

Transient Resensitization Interrupting the Development of Carbachol-induced Desensitization in Smooth Muscle of Guinea-pig Taenia Caeci: Ca<sup>2+</sup>-dependent Termination of Resensitization

It is important to clarify developmental mechanisms of desensitization because of their great significance in regulation of cellular responsiveness. We have found that carbachol-induced desensitization to carbachol develops in three successive phases in the presence of extracellular Ca(2+) in the smooth muscle of guinea pig taenia caeci: fast desensitization within 15 s, transient resensitization reaching a peak at 1 min and the subsequent re-development of desensitization to terminate resensitization for up to 30 min. In contrast, in the absence of extracellular Ca(2+), desensitization develops without resensitization. To further clarify the roles of Ca(2+) in the formation of the transient resensitization phase, we examined the developmental process of carbachol-induced desensitization in the absence of extracellular Ca(2+), following the induction of desensitization by a 15-s treatment with carbachol in the presence of extracellular Ca(2+). Desensitization to carbachol occurred due to pretreatment with 10(-4) M carbachol for 15 s in normal physiological solution, and continued pretreatment with carbachol in Ca(2+)-free solution containing 0.2 mM EGTA induced resensitization followed by the obscure progress of re-desensitization for up to 30 min resulting in a long-lasting phase of resensitization. These results suggest that resensitization is promptly terminated by the Ca(2+)-dependent development of subsequent desensitization for further regulation of cellular responsiveness via G(q) protein-coupled Ca(2+)-mobilizing receptors against sustained stimuli.

Free Cholesterol Alters Lipid Raft Structure and Function Regulating Neutrophil Ca2+ Entry and Respiratory Burst: Correlations with Calcium Channel Raft Trafficking

Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl beta-cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca(2+) mobilization. Methyl beta-cyclodextrin had no effect on Ca(2+) store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca(2+) entry. Free cholesterol at very low concentrations regulated Ca(2+) entry into PMN via nonspecific Ca(2+) channels in a biphasic fashion. The specificity of cholesterol regulation for Ca(2+) entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca(2+) signaling and respiratory burst suggest that Ca(2+) influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca(2+) entry. Ca(2+) entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.

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.

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1beta on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1beta (10 ng/ml), contractions elicited by high K+ and carbachol were inhibited in a time-dependent manner. IL-1beta more strongly inhibited the carbachol-induced contractions than high K+ with decreasing myosin light chain phosphorylation. In the alpha-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5'-3-O-(thio)triphosphate increased the Ca2+ sensitivity of contractile elements, and this G protein-coupled Ca2+ sensitization was significantly reduced in the IL-1beta-treated ileum. Among the functional proteins involved in the smooth muscle Ca2+ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1beta, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1beta treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1beta-treated muscles. These results suggest that long term treatment with IL-1beta decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1beta to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1beta.

P44/42 MAPK activation is necessary for receptor activator of nuclear factor-κB ligand induction by high extracellular calcium

Although extracellular calcium (Ca 2+ o) has been suggested to modulate bone remodeling, the exact mechanism is unclear. This study was performed to explore the signaling pathways of high Ca 2+ o that are responsible for controlling the expression of receptor activator of NF-κB ligand (RANKL) in mouse osteoblastic cells. As previously reported, high Ca 2+ o increased RANKL expression. However, the G protein-coupled Ca 2+ o-sensing receptor (CaSR) was not detected in the primary cultured mouse osteoblastic cell. The inhibition of the pertussis-sensitive G protein, phospholipase C, protein kinase C, intracellular calcium mobilization, p38 MAPK, or phosphoinositide 3-kinase did not block RANKL induction caused by high Ca 2+ o. In contrast, the inhibition of p44/42 MAPK pathway reduced the RANKL expression induced by high Ca 2+ o. Moreover, high Ca 2+ o activated p44/42 MAPK and MEK1/2. These results suggest that RANKL induction by high Ca 2+ o might not be mediated by CaSR and its putative downstream signaling pathways, but the pathway employing p44/42 MAPK is involved in the high Ca 2+ o-induced RANKL expression in mouse osteoblastic cells.

Cell-Type Specific Messenger Functions of Extracellular Calcium in the Anterior Pituitary

Calcium can serve not only as an intracellular messenger, but also as an extracellular messenger controlling the gating properties of plasma membrane channels and acting as an agonist for G protein-coupled Ca2+-sensing receptors. Here we studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of the extracellular Ca2+ concentration ([Ca2+]e) induced transient elevations in the intracellular Ca2+ concentration ([Ca2+]i), and elevations in [Ca2+]e above physiological levels decreased [Ca2+]i in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of [Ca2+]i responses depended on the [Ca2+]e and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated Ca2+ influx. Changes in [Ca2+]e also affected GH and PRL secretion. The PRL secretory profiles paralleled the [Ca2+]i profiles in lactotrophs, whereas GH secretion was also stimulated by [Ca2+]e independently of the status of voltage-gated Ca2+ influx. [Ca2+]e modulated GH secretion in a dose-dependent manner, with EC50 values of 0.75 and 2.25 mm and minimum secretion at about 1.5 mm. In a parallel experiment, cAMP accumulation progressively increased with elevation of [Ca2+]e, whereas inositol phosphate levels were not affected. These results indicate the cell type-specific role of [Ca2+]e in the control of Ca2+ signaling and secretion.

Cell-type specific messenger functions of extracellular calcium in the anterior pituitary.

Calcium can serve not only as an intracellular messenger, but also as an extracellular messenger controlling the gating properties of plasma membrane channels and acting as an agonist for G protein-coupled Ca(2+)-sensing receptors. Here we studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of the extracellular Ca(2+) concentration ([Ca(2+)]e) induced transient elevations in the intracellular Ca(2+) concentration ([Ca(2+)]i), and elevations in [Ca(2+)]e above physiological levels decreased [Ca(2+)]i in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of [Ca(2+)]i responses depended on the [Ca(2+)]e and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated Ca(2+) influx. Changes in [Ca(2+)]e also affected GH and PRL secretion. The PRL secretory profiles paralleled the [Ca(2+)]i profiles in lactotrophs, whereas GH secretion was also stimulated by [Ca(2+)]e independently of the status of voltage-gated Ca(2+) influx. [Ca(2+)]e modulated GH secretion in a dose-dependent manner, with EC(50) values of 0.75 and 2.25 mM and minimum secretion at about 1.5 mM. In a parallel experiment, cAMP accumulation progressively increased with elevation of [Ca(2+)]e, whereas inositol phosphate levels were not affected. These results indicate the cell type-specific role of [Ca(2+)]e in the control of Ca(2+) signaling and secretion.

Human Ca2+ Receptor Extracellular Domain: ANALYSIS OF FUNCTION OF LOBE I LOOP DELETION MUTANTS

The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.

Humoral coding and decoding.

Humoral communication systems are dynamically regulated. Most hormones are released in a pulsatile or burst-like manner into the bloodstream. It is well known that information coded in the frequency and amplitude of secretory pulses allows for the differential regulation of specific target cell function and structure. However, despite intensive study of transmembrane signalling relatively little is known about how the temporal dynamics of extracellular humoral stimuli specifically regulates the temporal pattern of intracellular signalling pathways, such as Ca2+-dependent signalling. Repetitive spikes of Ca2+ encode this information in their amplitude, duration and frequency, and are in turn decoded into the pattern of gene expression and phosphorylation of target proteins. Using a mathematical model for G protein-coupled Ca2+ signalling and information-theoretic approaches to stimulus reconstruction we have systematically quantified the amount of information coded in the Ca2+-signal about the dynamics of the stimulus, which allows us to explore the temporal bandwidth of transmembrane signalling. These in silico approaches permit us to differentiate the amount of information coded in the frequency, temporal precision, amplitude and the complete Ca2+-signal. This may open an avenue to the quantification of information flow and processing in the intra- and intercellular coding and decoding machinery.

Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD).

A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl- channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl- channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl- conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNF receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma x rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be abolished by prior treatment with a blocker of volume-regulatory K+ or Cl- channels, suggesting that AVD is caused by normotonic activation of ion channels that are normally involved in RVD under hypotonic conditions. Therefore, it is likely that G protein-coupled receptors involved in RVD regulation and death receptors triggering AVD may share common downstream signals which should give us key clues to the detailed mechanisms of volume regulation and survival of animal cells. In this Topical Review, we look at the physiological ionic mechanisms of cell volume regulation and cell death-associated volume changes from the facet of receptor-mediated cellular processes.

Ca2+-sensing receptor-mediated regulation of volume-sensitive Cl- channels in human epithelial cells.

Since extracellular Ca2+ or Mg2+ has been reported to modulate swelling-activated Cl- currents, we examined the expression of the G protein-coupled Ca2+-sensing receptor (CaR) and its involvement in the regulation of volume-sensitive Cl- channels in a human epithelial cell line (Intestine 407). Reverse transcriptase-polymerase chain reaction and immunoblotting analysis showed that Intestine 407 cells express CaR mRNA and protein. The swelling-activated whole-cell Cl- current was voltage-independently augmented by extracellular Ca2+ or Mg2+. In addition, Ca2+ or Mg2+ voltage-dependently accelerated the inactivation kinetics of the Cl- current. Neomycin, spermine and La3+ augmented volume-sensitive Cl- currents. However, these CaR agonists failed to affect depolarization-induced inactivation. Intracellular application of GTPgammaS, but not GDPbeta]S, increased the amplitude of the swelling-induced Cl- current without affecting the basal current. The upregulating effect of Ca2+ on the Cl- current amplitude was abolished by either GTPgammaS or GDPbetaS. In contrast, GTPgammaS and GDPbetaS failed to affect the inactivation kinetics of the Cl- current and the accelerating effect of Ca2+ thereon. The Cl- current amplitude was enlarged by stimulation with forskolin, dibutyryl cAMP and IBMX. During the cAMP stimulation, extracellular Ca2+ failed to increase the Cl- current but did accelerate depolarization-induced inactivation. It is concluded that stimulation of the CaR induces upregulation of volume-sensitive Cl- channels via a G protein-mediated increase in intracellular cAMP in the human epithelial cell. However, the accelerating effect of extracellular divalent cations on the inactivation kinetics of the Cl- current is induced by a mechanism independent of the CaR and cAMP.

Physiology and pathophysiology of the extracellular calcium-sensing receptor

The system governing extracellular calcium (Ca 2+ o) homeostasis maintains near constancy of Ca 2+ o so as to ensure continual availability of calcium ions for their numerous intracellular and extracellular roles. In contrast to the intracellular ionized calcium concentration (Ca 2+ i), which varies substantially during intracellular signaling via this key second messenger, Ca 2+ o remains nearly invariant. Yet there must be a mechanism that senses small changes in Ca 2+ o so as to set into motion the homeostatic responses that return Ca 2+ o to its normal level. The recent identification and molecular cloning of the mechanism through which parathyroid cells and a number of other cell types sense Ca 2+ o, a G protein-coupled Ca 2+ o-sensing receptor (CaR), has proven unequivocally that extracellular calcium ions serve in an informational capacity. The CaR permits Ca 2+ o to function in a hormone-like role as an extracellular first messen-ger through which parathyroid, kidney, and other cells communicate with one another via the CaR. The identification of inherited human hypercalcemic and hypocalcemic disorders arising from inactivating and activating mutations of the CaR, respectively, has provided additional proof of the essential, nonredundant role of the CaR in mineral ion homeostasis. Moreover, CaR-active drugs are currently in clinical trials for the treatment of primary and uremic hyperparathyroidism, disorders in which there are acquired, tissue-specific reductions in CaR expression and, in turn, defective Ca 2+ o-sensing by pathological parathyroid cells. No doubt further studies of Ca 2+ o-sensing by the CaR will reveal additional functions of Ca 2+ o, not only as a systemic “hormone” but also in local, paracrine, and autocrine signaling through this novel Ca 2+ o-sensing receptor.

Localization of the extracellular Ca(2+)-sensing receptor and PTH/PTHrP receptor in rat kidney.

Using a strategy based on homology to the bovine parathyroid Ca(2+)-sensing receptor previously identified by us (5), we have recently isolated an extracellular, G protein-coupled Ca2+/ polyvalent cation-sensing receptor, RaKCaR (22), from rat kidney. The localization and physiological role(s) of this receptor in the kidney are not well understood. In the present study, we assessed the distribution of mRNAs for RaKCaR and the parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor along the rat nephron by in situ hybridization and reverse transcriptase-polymerase chain reaction of microdissected nephron segments. Our results show that transcripts for both receptors coexpress at glomeruli, proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb, distal convoluted tubule, and cortical collecting duct. In addition, RaKCaR (but not PTH/PTHrP receptor) transcripts were found in the medullary thick ascending limb and outer medullary and inner medullary collecting ducts. These findings raise the possibility of roles for RaKCaR not only in the regulation of divalent mineral reabsorption but also in water reabsorption and urinary concentration. Taken together, our results provide new insights in understanding the effects of hypercalcemia on hormone-stimulated salt and water transport.

Expression of G protein alpha-subunits in bovine parathyroid.

A G protein-coupled Ca(2+)-sensing receptor was recently cloned from bovine parathyroid and shown to mediate divalent cation regulation of PTH secretion. To define which G proteins might be coupled to the Ca(2+)-sensing receptor in parathyroid cells, we determined which G protein alpha-subunit messenger RNAs (mRNAs) are expressed in the parathyroid. We also considered the possibility that a novel parathyroid-specific G alpha might be present. We, therefore, used the reverse transcription-polymerase chain reaction to study the expression of G alpha subunits in a bovine parathyroid mRNA preparation. Degenerate primers, corresponding to two regions conserved in every G alpha subunit, the G3 and G4 sequences, were used to amplify G alpha complementary DNA fragments that were subcloned and sequenced. We found that mRNAs corresponding to G alpha s, G alpha i2, G alpha 11, G alpha 12, and G alpha z are the predominant G alpha mRNAs expressed in the bovine parathyroid. No novel G alpha mRNA was identified. Northern blots confirmed the expression of the cloned G alpha subunits and showed lower expression of G alpha o and G alpha i1 mRNAs. Immunoblots confirmed abundant expression of G alpha s, G alpha i2, and G alpha 11 and provided evidence for expression of G alpha i1 and G alpha i3, but not G alpha o. G alpha q mRNA was not identified by the degenerate primer reverse transcription-polymerase chain reaction strategy, but the immunoblot detected G alpha q protein, albeit at considerably lower levels than G alpha 11. The abundance of G alpha 11 relative to G alpha q in bovine parathyroid is consistent with but does not prove a role for G alpha 11 in coupling the Ca(2+)-sensing receptor to phospholipase C.

Ca2+ Receptor-Dependent Regulation of Cellular Functions

It has been supposed that a cell-surface Ca2+ receptor enables parathyroid cells to detect and respond to extracellular Ca2+. The recent cloning of a G protein-coupled Ca2+ receptor solidifies this view. Ca2+ receptors regulate the activity of various cells and provide novel molecular targets for new pharmaceuticals.