Pharmacological profile of upacicalcet, a novel positive allosteric modulator of calcium-sensing receptor, in vitro and in vivo.

Upacicalcet (formerly SK-1403/AJT240) is a novel non-peptide calcimimetic agent that acts as a calcium-sensing receptor (CaSR) agonist for the treatment of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). We compared upacicalcet with other calcimimetics (etelcalcetide or cinacalcet) and examined its in vitro and in vivo characteristics in terms of its human CaSR agonistic activity, its efficacy in normal and CKD rats after a single administration, and its effect on gastric emptying in rats. Upacicalcet activated human CaSR depending on the extracellular calcium (Ca2+) concentration without exhibiting an agonistic action when the extracellular Ca2+ level was below the physiological level. On the other hand, etelcalcetide had an agonistic activity even in the absence of physiological levels of extracellular Ca2+. The intravenous administration of upacicalcet to normal and double-nephrectomized rats dose-dependently (0.03-3mg/kg and 0.3-30mg/kg, respectively) decreased the serum intact parathyroid hormone (iPTH) and serum Ca2+ levels; however, the effect of upacicalcet on the reduction in serum Ca2+ disappeared at extracellular Ca2+ levels below the physiologically range, even when administered at a dose higher (100-fold) than the effective dose. Furthermore, upacicalcet did not affect gastric emptying in normal rats when administered up to a dose of 10mg/kg (300-fold higher than the dose affecting serum iPTH levels), while the administration of cinacalcet significantly slowed gastric emptying by approximately 50%. These findings suggest that upacicalcet has potential as an alternative calcimimetic agent with good pharmacological properties and a lower risk of hypocalcemia and gastrointestinal complications.

Calcium-Sensing Receptor Negative Allosteric Modulators Oppose Contraction of Mouse Airways.

Asthma is a heterogeneous chronic airway disease with an unmet need for improved therapeutics in uncontrolled severe disease. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor upregulated in asthma. The CaSR agonist, spermine, is also increased in asthmatic airways and contributes to bronchoconstriction. CaSR negative allosteric modulators (NAMs) oppose chronic airway inflammation, remodeling, and hyperresponsiveness in murine and guinea pig asthma models, but whether CaSR NAMs are effective acute bronchodilators compared with standard of care has not yet been established. Furthermore, the ability of different classes of NAMs to inhibit spermine-induced CaSR signaling or methacholine (MCh)-induced airway contraction has not been quantified. Here, we show CaSR NAMs differentially inhibit spermine-induced intracellular calcium mobilization and inositol monophosphate accumulation in HEK293 cells stably expressing the CaSR. NAMs reverse MCh-mediated airway contraction in mouse precision-cut lung slices with similar maximal relaxation compared with the standard treatment, salbutamol. Of note, the bronchodilator effects of CaSR NAMs are maintained under conditions of ß2-adrenergic receptor desensitization when salbutamol efficacy is abolished. Furthermore, overnight treatment with some, but not all, CaSR NAMs prevents MCh-mediated bronchoconstriction. These findings further support the CaSR as a putative drug target and NAMs as alternative or adjunct bronchodilators in asthma.

Enhancement of allyl isothiocyanate-evoked responses of mouse trigeminal ganglion cells by the kokumi substance γ-glutamyl-valyl-glycine (γ-EVG) through activation of the calcium-sensing receptor (CaSR).

Some γ-glutamyl peptides including glutathione (γ-Glu-Cys-Gly) and γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly= γ-EVG) are reported to increase the intensity of basic tastes, such as salty, sweet, and umami, although they have no taste themselves at tested concentrations. The mechanism of action of γ-glutamyl peptides is not clearly understood, but the calcium sensing receptor (CaSR) may be involved. Glutathione and γ-EVG enhance the pungency of some spices, and the present study investigated the effects of γ-EVG on the responses of trigeminal ganglion (TG) cells to thermosensitiveTRP channel agonists. Single-cell RT-PCR revealed that most CaSR-expressing cells co-expressed TRPV1 (sensitive to capsaicin) and TRPA1 (sensitive to allyl isothiocyanate= AITC). Intracellular Ca2+ imaging showed that pretreatment with γ-EVG excited 7% of trigeminal ganglion (TG) cells and increased the amplitude of their responses to AITC, but not to capsaicin or menthol. The enhancing effect of γ-EVG was prevented by a CaSR inhibitor. The results indicate that γ-EVG increases AITC pungency by activating a subset of trigeminal ganglion cells that co-express CaSR and TRPA1.

Increases in the pungency of allyl isothiocyanate and piperine by CaSR agonists, glutathione and γ-glutamyl-valyl-glycine.

γ-Glutamyl peptides, including glutathione (γ-Glu-Cys-Gly, GSH) and γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), have been shown to increase the intensity of basic tastes, such as salty, sweet, and umami, and flavor, including mouthfulness, but had no taste themselves at the concentrations tested. Although the mechanisms of action of γ-glutamyl peptides currently remain unclear, the involvement of the calcium sensing receptor (CaSR) has been suggested. Since GSH and γ-Glu-Val-Gly increase the pungency of some spices, the present study investigated their effects on the pungency of allyl isothiocyanate (AITC) using a sensory evaluation. GSH and γ-Glu-Val-Gly both significantly increased the pungency of AITC, while anserine, a peptide without CaSR activity, did not. GSH-induced increases in pungency were suppressed by NPS-2143, a CaSR inhibitor. Further, γ-Glu-Val-Gly significantly increased the pungency of piperine. The present results suggest that GSH and γ-Glu-Val-Gly increased the pungency by activating CaSR.

Calcium-sensing receptor (CaSR) agonist R568 inhibits small intestinal motility of mice through neural and non-neural mechanisms.

Gastrointestinal motility (GI) disorder causes symptoms such as dyspepsia, abdominal distention, and constipation and severely affects quality of life. The calcium (Ca2+)-sensing receptor (CaSR) expressed in the digestive tract can be activated by amino acids and participates in GI motility regulation. This study is designed to explore the effect and underlying mechanism of CaSR agonist R568 on the small intestine motility of mice in vivo and ex vivo. R568 was given to male C57BL/6 mice by gavage or incubated with isolated jejunum and ileum segments to observe its effects on GI motility and the involved neurons, neurotransmitters and hormones were detected by fluorescence immunohistochemistry and enzyme-linked immunosorbent assays. The in vivo results showed that the intestinal propulsive rate reduced in response to oral intake of R568. R568 treatment increased the numbers of nitric oxide synthase-positive neurons and nitric oxide release but decreased the choline acetyl transferase-positive neurons and acetylcholine release in the myenteric plexuses. R568 increased the secretion of cholecystokinin in the intestinal tissues and serum but had no effect on the secretion of glucagon like peptide-1. Ex vivo results showed that R568 inhibited the contractility of intestinal strips from the jejunum and ileum. Nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester (L-NAME), M-receptor antagonist-atropine, and tetrodotoxin (TTX) failed to block the effect of R568. CaSR co-expressed with interstitial cells of Cajal (ICCs) in the myenteric plexus suggests the possibility that ICCs mediated the effect of R568. Our findings demonstrate that CaSR activation inhibited intestinal motility, and both the enteric nervous system and non-neural mechanism are involved in this process.

Structural insights into the activation of human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of seven-transmembrane domains, and inter- and intrasubunit conformational changes of seven-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca2+ ions and L-amino acid leading to the activation of the receptor.

Bone responsiveness to parathyroid hormone is negatively associated with parathyroid hormone-lowering drug use in patients undergoing hemodialysis: a cross-sectional study.

BACKGROUND: Parathyroid hormone (PTH) acts on bone to indirectly increase the number and activity of osteoclasts. Thus, PTH has a stimulatory effect on bone resorption and upregulates bone turnover. However, the responsiveness of bone to PTH varies widely among patients receiving dialysis. In fact, relative to the serum PTH level, the level of serum tartrate-resistant acid phosphatase-5b (TRACP-5b), a bone resorption marker derived from osteoclasts, varies as well. This study aimed to examine factors related to bone responsiveness to PTH in patients undergoing chronic hemodialysis (HD). METHODS: This study included patients receiving chronic HD in Kawasaki Municipal Tama Hospital (Kanagawa, Japan) and Yonaha Medical Clinic (Okinawa, Japan) and excluded patients who received HD for less than 6 months, those who received a combination of HD and peritoneal dialysis, and those who had cancer bone metastases or myeloma. The TRACP-5b/intact PTH (iPTH) ratio was created as an index of bone responsiveness to PTH, categorized into tertiles (low, medium, and high), and a cross-sectional study was conducted. P < 0.05 indicated statistically significant differences. RESULTS: One hundred and six patients were analyzed. Age (P = 0.010), body mass index (BMI) (P = 0.003), use of calcium-sensing receptor (CaSR) agonists (P = 0.008), use of vitamin D receptor activators (VDRAs) (P = 0.012), plasma iPTH level (P < 0.001), serum 1,25(OH)2D level (P = 0.003), and serum TRACP-5b level (P < 0.001) were significantly different among the three categories. In the single linear regression analysis, age (P = 0.016), corrected serum calcium level (P = 0.007), and ln [1,25(OH)2D] (P = 0.044) showed a significant positive correlation with ln [TRACP-5b/iPTH], whereas BMI (P = 0.026), use of CaSR agonists (P = 0.001), use of VDRAs (P = 0.009), and serum phosphorus level (P = 0.018) showed a significant negative correlation. Upon conducting multiple linear regression analysis incorporating significant variables in the single linear regression analysis, a significant negative correlation was observed between the TRACP-5b/iPTH ratio and intravenous administration of a CaSR agonist (etelcalcetide) and/or a VDRA (calcitriol or maxacalcitol) in all the adjusted models. CONCLUSIONS: Bone responsiveness to PTH is negatively correlated with the intravenous administration of a CaSR agonist and/or a VDRA in patients undergoing chronic HD.

Development of AC265347-Inspired Calcium-Sensing Receptor Ago-Positive Allosteric Modulators.

The calcium-sensing receptor (CaSR) is a clinical target in the treatment of hyperparathyroidism and related diseases. However, clinical use of approved CaSR-targeting drugs such as cinacalcet is limited due to adverse side effects including hypocalcaemia, nausea and vomiting, and in some instances, a lack of efficacy. The CaSR agonist and positive allosteric modulator (ago-PAM), AC265347, is chemically distinct from clinically-approved CaSR PAMs. AC265347 potently suppressed parathyroid hormone (PTH) release in rats with a lower propensity to cause hypocalcaemia compared to cinacalcet and may therefore offer benefits over current CaSR PAMs. Here we report a structure activity relationship (SAR) study seeking to optimise AC265347 as a drug candidate and disclose the discovery of AC265347-like compounds with diverse pharmacology and improved physicochemical and drug-like properties.

Decreased Calcium-Sensing Receptor Expression Controls Calcium Signaling and Cell-To-Cell Adhesion Defects in Aged Skin.

The calcium-sensing receptor (CaSR) drives essential calcium ion (Ca2+) and E-cadherin‒mediated processes in the epidermis, including differentiation, cell-to-cell adhesion, and epidermal barrier homeostasis in cells and in young adult mice. We now report that decreased CaSR expression leads to impaired Ca2+ signal propagation in aged mouse (aged >22 months) epidermis and human (aged >79 years, donor age) keratinocytes. Baseline cytosolic Ca2+ concentrations were higher, and capacitive Ca2+ entry was lower in aged than in young keratinocytes. As in Casr-knockout mice (EpidCaSR-/-), decreased CaSR expression led to decreased E-cadherin and phospholipase C-γ expression and to a compensatory upregulation of STIM1. Pretreatment with the CaSR agonist N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine normalized Ca2+ propagation and E-cadherin organization after experimental wounding. These results suggest that age-related defects in CaSR expression dysregulate normal keratinocyte and epidermal Ca2+ signaling, leading to impaired E-cadherin expression, organization, and function. These findings show an innovative mechanism whereby Ca2+- and E-cadherin‒dependent functions are impaired in aging epidermis and suggest a new therapeutic approach by restoring CaSR function.

Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors.

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.

Olive Leaf Extract (OLE) impaired vasopressin-induced aquaporin-2 trafficking through the activation of the calcium-sensing receptor.

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.

Matrine, as a CaSR agonist promotes intestinal GLP-1 secretion and improves insulin resistance in diabetes mellitus.

BACKGROUND: Matrine (Mat), a bitter tastes compounds of derived from leguminosae such as Sophora flavescens and S. subprostrata, commonly used to improve obesity and diabetes. PURPOSE: Our study to demonstrate bitter substances can stimulate the Bitter taste receptors (TAS2Rs) or Calcium-sensing receptor (CaSR) to stimulate the secretion of GLP-1 to promote blood glucose regulation. METHODS: The diabetic mice and intestinal secretory cell model were established to evaluate the Mat on glucose metabolism, intestinal insulin secretion and GLP-1 secretion related substances. To clarify the mechanism of Mat in regulating GLP-1 secretion by immunofluorescence, calcium labeling, siRNA, and molecular docking. RESULTS: The results showed that Mat could significantly improve glucose metabolism and increased insulin and GLP-1 secretion in diabetic mice and increased trisphosphate inositol (IP3) levels by affecting the expression of phospholipase C ß2 (PLCß2) and promote an increase in intracellular Ca2+ levels in STC-1 cells to subsequently stimulate the secretion of GLP-1. Knockdown of the bitter taste receptors mTas2r108, mTas2r137, and mTas2r138 in STC-1 cells by siRNA did could not affect the role of Mat in regulating GLP-1. However, the secretion of GLP-1 by Mat could be significantly inhibited by administration of a CaSR inhibitor or siRNA CaSR. Molecular docking analysis showed that Mat could embed CaSR protein and bind to the original ligand of the egg white at the same amino acid site to play the role of an agonist. CONCLUSION: Matrine is a typical bitter alkaloid could be used as an agonist of CaSR to stimulate the secretion of GLP-1 in the intestine, and it may be used as a potential drug for diabetes treatment.

Characterization of Negative Allosteric Modulators of the Calcium-Sensing Receptor for Repurposing as a Treatment of Asthma.

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function.

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor that responds to multiple endogenous agonists and allosteric modulators, including divalent and trivalent cations, L-amino acids, γ-glutamyl peptides, polyamines, polycationic peptides, and protons. The CaSR plays a critical role in extracellular calcium (Ca2+ o) homeostasis, as demonstrated by the many naturally occurring mutations in the CaSR or its signaling partners that cause Ca2+ o homeostasis disorders. However, CaSR tissue expression in mammals is broad and includes tissues unrelated to Ca2+ o homeostasis, in which it, for example, regulates the secretion of digestive hormones, airway constriction, cardiovascular effects, cellular differentiation, and proliferation. Thus, although the CaSR is targeted clinically by the positive allosteric modulators (PAMs) cinacalcet, evocalcet, and etelcalcetide in hyperparathyroidism, it is also a putative therapeutic target in diabetes, asthma, cardiovascular disease, and cancer. The CaSR is somewhat unique in possessing multiple ligand binding sites, including at least five putative sites for the "orthosteric" agonist Ca2+ o, an allosteric site for endogenous L-amino acids, two further allosteric sites for small molecules and the peptide PAM, etelcalcetide, and additional sites for other cations and anions. The CaSR is promiscuous in its G protein-coupling preferences, and signals via Gq/11, Gi/o, potentially G12/13, and even Gs in some cell types. Not surprisingly, the CaSR is subject to biased agonism, in which distinct ligands preferentially stimulate a subset of the CaSR's possible signaling responses, to the exclusion of others. The CaSR thus serves as a model receptor to study natural bias and allostery. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaSR) is a complex G protein-coupled receptor that possesses multiple orthosteric and allosteric binding sites, is subject to biased signaling via several different G proteins, and has numerous (patho)physiological roles. Understanding the complexities of CaSR structure, function, and biology will aid future drug discovery efforts seeking to target this receptor for a diversity of diseases. This review summarizes what is known to date regarding key structural, pharmacological, and physiological features of the CaSR.

Calcium-sensing receptor regulates intestinal dipeptide absorption via Ca2+ signaling and IKCa activation.

Although absorption of di- and tripeptides into intestinal epithelial cells occurs via the peptide transporter 1 (PEPT1, also called solute carrier family 15 member 1 (SLC15A1)), the detailed regulatory mechanisms are not fully understood. We examined: (a) whether dipeptide absorption in villous enterocytes is associated with a rise in cytosolic Ca2+ ([Ca2+ ]cyt ), (b) whether the calcium sensing receptor (CaSR) is involved in dipeptide-elicited [Ca2+ ]cyt signaling, and (c) what potential consequences of [Ca2+ ]cyt signaling may enhance enterocyte dipeptide absorption. Dipeptide Gly-Sar and CaSR agonist spermine markedly raised [Ca2+ ]cyt in villous enterocytes, which was abolished by NPS-2143, a selective CaSR antagonist and U73122, an phospholipase C (PLC) inhibitor. Apical application of Gly-Sar induced a jejunal short-circuit current (Isc), which was reduced by NPS-2143. CaSR expression was identified in the lamina propria and on the basal enterocyte membrane of mouse jejunal mucosa in both WT and Slc15a1-/- animals, but Gly-Sar-induced [Ca2+ ]cyt signaling was significantly decreased in Slc15a1-/- villi. Clotrimazole and TRM-34, two selective blockers of the intermediate conductance Ca2+ -activated K+ channel (IKCa ), but not iberiotoxin, a selective blocker of the large-conductance K+ channel (BKCa ) and apamin, a selective blocker of the small-conductance K+ channel (SKCa ), significantly inhibited Gly-Sar-induced Isc in native tissues. We reveal a novel CaSR-PLC-Ca2+ -IKCa pathway in the regulation of small intestinal dipeptide absorption, which may be exploited as a target for future drug development in human nutritional disorders.

Calcium-sensing receptor promotes tumor proliferation and migration in human intrahepatic cholangiocarcinoma by targeting ERK signaling pathway.

The Calcium-sensing receptor (CaSR) is functionally expressed in the biliary epithelial cells and it has been verified to possess various regulatory functions in several different forms of human cancers. But its pathological role in human intrahepatic cholangiocarcinoma (ICC) development remains obscure. Here, we confirmed that CaSR expression was up-regulated in ICC tumor specimens and cell lines, which was positively correlated with number of tumors, lymph node metastasis and poor prognosis of ICC patients. CaSR activation induced by CaCl2 or Calindol (a selective CaSR agonist) markedly facilitated cell proliferation and migration in ICC cells, while knockdown of CaSR or NPS2143 treatment (a CaSR antagonist) dramatically suppressed the above effects. We also demonstrated that alteration of CaSR activity mediated tumorigenesis and growth of ICC in vivo. Mechanistically, CaSR activation could promote cell cycle progression and induce an upregulation of MMP-2 and MMP-9 expression partly via the simulation of ERK1/2 signaling pathway. And further inhibition of ERK pathway significantly suppressed ICC cell viability and migration capacity. Together, our findings shed novel light on the role of CaSR as an oncogene in ICC progression and indicated that modulation of CaSR might serve as a preventive or therapeutic strategy for ICC.

The calcium sensing receptor modulates H+-ATPase activity in intercalated cells.

Previous studies found that calcium sensing receptor (CaSR) it's expressed in intercalated cells of the collecting duct and that its activation by calcium in the luminal membrane promotes acidification of urine. Therefore, the aim of the study was to analyze the effects of CaSR stimulus on the biochemical activity of the vacuolar H+-ATPase in a cellular model of intercalated cells, MDCK-C11 cells. Biochemical activity of H+-ATPase was performed using cell homogenates and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium ([Ca2+]i) were also determined using Fluo-4. A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300 µM), neomycin (200 µM) and by the calcimimetic R-568 (1 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ concentration ([Ca2+]o) between 10-2 and 2 mM. The calciolytic NPS 2143 (150 nM) significantly reduced the vacuolar H+-ATPase activity observed with 2 mM [Ca2+]o. Inhibition of phospholipase C (PLC) activity with U73122 (5 x 10-7 M) reversed the increase in pump activity observed in the presence of Gd3+. Activation of CaSR by the specific CaSR agonist R-568 produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of thapsigargin. In summary, CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of MDCK-C11 cells, an effect that involves an increase in [Ca2+]i and require PLC activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially reducing the formation of calcium phosphate stones.

GdCl3 attenuates the glomerular sclerosis of streptozotocin (STZ) induced diabetic rats via inhibiting TGF-ß/Smads signal pathway.

Diabetic nephropathy (DN) is the most serious end-stage renal disease which characterized by renal glomerular sclerosis including glomerular hypertrophy, glomerular basement membrane (GBM) thickening, mesangial expansion and renal fibrosis. TGF-ß/Smads signal pathway plays a crucial role in the development of renal fibrosis. In this study, we found that GdCl3 which was an agonist of Calcium-sensing receptor (CaSR) could repress the activation of TGF-ß/Smads signal pathway induced by TGF-ß1 or high glucose and then alleviated the accumulation of extracellular matrix (ECM) in mesangial cells and the kidney of type1 diabetic rats. Further study indicated that GdCl3 could induce the binding of CaSR and TßR II and then both of these two receptors translocated from cell membrane to cytoplasm, in this case, TßR II on the cell membrane was decreased and then desensitized to the stimulation of its ligand TGF-ß1, so that the activation of its downstream factors such as Smad2 and Smad3 were blocked, finally, ECM expression in mesangial cells were inhibited. We concluded that GdCl3 could alleviate the accumulation of ECM in mesangial cells via antagonizing TGF-ß/Smads signal pathway in diabetes mellitus.

Evaluation of Operational Models of Agonism and Allosterism at Receptors with Multiple Orthosteric Binding Sites.

Current operational models of agonism and allosterism quantify ligand actions at receptors where agonist concentration-response relationships are nonhyperbolic by introduction of a transducer slope that relates receptor occupancy to response. However, for some receptors nonhyperbolic concentration-response relationships arise from multiple endogenous agonist molecules binding to a receptor in a cooperative manner. Thus, we developed operational models of agonism in systems with cooperative agonist binding and evaluated the models by simulating data describing agonist effects. The models were validated by analyzing experimental data demonstrating the effects of agonists and allosteric modulators at receptors where agonist binding follows hyperbolic (M4 muscarinic acetylcholine receptors) or nonhyperbolic relationships (metabotropic glutamate receptor 5 and calcium-sensing receptor). For hyperbolic agonist concentration-response relationships, no differences in estimates of ligand affinity, efficacy, or cooperativity were observed when the slope was assigned to either a transducer slope or agonist binding slope. In contrast, for receptors with nonhyperbolic agonist concentration-response relationships, estimates of ligand affinity, efficacy, or cooperativity varied depending on the assignment of the slope. The extent of this variation depended on the magnitude of the slope value and agonist efficacy, and for allosteric modulators on the magnitude of cooperativity. The modified operational models described herein are well suited to analyzing agonist and modulator interactions at receptors that bind multiple orthosteric agonists in a cooperative manner. Accounting for cooperative agonist binding is essential to accurately quantify agonist and drug actions. SIGNIFICANCE STATEMENT: Some orthosteric agonists bind to multiple sites on a receptor, but current analytical methods to characterize such interactions are limited. Herein, we develop and validate operational models of agonism and allosterism for receptors with multiple orthosteric binding sites, and demonstrate that such models are essential to accurately quantify agonist and drug actions. These findings have important implications for the discovery and development of drugs targeting receptors such as the calcium-sensing receptor, which binds at least five calcium ions.

Sweet proteins lysozyme and thaumatin are protein-type agonists for the calcium-sensing receptor.

In addition to the maintenance of Ca2+ homeostasis, the calcium-sensing receptor (CaSR) is involved in many diverse physiological functions in the mammalian body. The receptor works as a kokumi taste receptor in taste buds and as a nutrient sensor in the gut, where it regulates the secretion of glycemic response and appetite-related hormones. To identify novel human CaSR (hCaSR) activators from food ingredients, we conducted a screening using a cell-based hCaSR assay. Hen egg-white lysozyme, which is a sweet protein, was found to be a novel orthosteric agonist of hCaSR with an EC50 value of 592 µM. Lysozyme hydrolysate was not able to activate hCaSR, thus suggesting that the protein structure of lysozyme is necessary for hCaSR activation. Thaumatin, which is another sweet protein, also activated hCaSR with an EC50 value of 71 µM. This is the first report that shows hCaSR activation by proteins with molecular weights exceeding 10,000 Da. These results provide a new avenue for the development of hCaSR activators, which could be applicable in food or drugs that modulate taste perception, appetite, or glucose tolerance, in addition to Ca2+ homeostasis.