The calcium-sensing receptor has only a parathyroid hormone-dependent role in the acute response of renal phosphate transporters to phosphate intake.

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

Modulation of fibroblast growth factor-23 expression and transepithelial calcium absorption in Caco-2 monolayer by calcium-sensing receptor and calcineurin under calcium hyperabsorptive state.

Fibroblast growth factor (FGF)-23 and calcium-sensing receptor (CaSR) have previously been postulated to be parts of a negative feedback regulation of the intestinal calcium absorption to prevent excessive calcium uptake and its toxicity. However, the underlying mechanism of this feedback regulation remained elusive, especially whether it required transcription of FGF-23. Herein, we induced calcium hyperabsorptive state (CHS) by exposing intestinal epithelium-like Caco-2 monolayer to 30 mM CaCl2 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after which FGF-23 mRNA levels and transepithelial calcium flux were determined. We found that CHS upregulated FGF-23 transcription, which was reverted by CaSR inhibitors (Calhex-231 and NPS2143) but without effect on CaSR transcription. Although 10 nM 1,25(OH)2D3 was capable of enhancing transepithelial calcium flux, the higher-than-normal calcium inundation as in CHS led to a decrease in calcium flux, consistent with an increase in FGF-23 protein expression. Administration of inhibitors (≤10 µM CN585 and cyclosporin A) of calcineurin, a mediator of CaSR action to control transcription and production of its target proteins, was found to partially prevent FGF-23 protein production and the negative effect of CHS on calcium transport, while having no effect on FGF-23 mRNA expression. Direct exposure to FGF-23, but not FGF-23 + PD173074 (FGFR1/3 inhibitor), also completely abolished the 1,25(OH)2D3-enhanced calcium transport in Caco-2 monolayer. Nevertheless, CHS and CaSR inhibitors had no effect on the mRNA levels of calcineurin (PPP3CB) or its targets (i.e., NFATc1-4). In conclusion, exposure to CHS induced by high apical calcium and 1,25(OH)2D3 triggered a negative feedback mechanism to prevent further calcium uptake. CaSR and its downstream mediator, calcineurin, possibly contributed to the regulatory process, in part by enhancing FGF-23 production to inhibit calcium transport. Our study, therefore, corroborated the physiological significance of CaSR-autocrine FGF-23 axis as a local feedback loop for prevention of excessive calcium uptake.

Solid-phase extraction with the functionalization of calcium-sensing receptors onto magnetic microspheres as an affinity probe can capture ligands selectively from herbal extract.

Magnetic solid phase extraction with the functionalization of protein onto micro- or nano-particles as a probe is favorable for the discovery of new drugs from complicated natural products. Herein, we aimed to develop a rapid method by immobilizing halogenated alkane dehalogenase (Halo)-tagged calcium-sensing receptor (CaSR) directly out of crude cell lysates onto the surface of magnetic microspheres (MM) with no need to purify protein. Thereby we achieved CaSR-functionalized MM for revealing adsorption characteristics of agonist neomycin and screening ligands from herbal medicine Radix Astragali (RA). About 43.87 mg CaSR could be immobilized per 1 g MM within 30 min, and the acquired CaSR-functionalized MM showed good stability and activity for 4 weeks. The maximum adsorption capacity of neomycin on CaSR-functionalized MM was determined as 4.70 × 10-4 ~ 3.96 × 10-4 mol/g within 277 ~ 310 K, and its adsorption isotherm characteristics described best by the Temkin model were further validated using isothermal titration calorimetry. It was inferred that CaSR's affinity for neomycin was driven by electrostatic forces in a spontaneous process when the system reached an equilibrium state. Moreover, the ligands from the RA extract were screened, three of which were assigned as astragaloside IV, ononin, and calycosin based on HPLC-MS. Our findings demonstrated that the functionalization of a receptor onto magnetic materials designed as an affinity probe has the capability to recognize its agonist and capture the ligands selectively from complex matrices like herbs.

The CaSR Modulator NPS-2143 Reduced UV-Induced DNA Damage in Skh:hr1 Hairless Mice but Minimally Inhibited Skin Tumours.

The calcium-sensing receptor (CaSR) is an important regulator of epidermal function. We previously reported that knockdown of the CaSR or treatment with its negative allosteric modulator, NPS-2143, significantly reduced UV-induced DNA damage, a key factor in skin cancer development. We subsequently wanted to test whether topical NPS-2143 could also reduce UV-DNA damage, immune suppression, or skin tumour development in mice. In this study, topical application of NPS-2143 (228 or 2280 pmol/cm2) to Skh:hr1 female mice reduced UV-induced cyclobutane pyrimidine dimers (CPD) (p < 0.05) and oxidative DNA damage (8-OHdG) (p < 0.05) to a similar extent as the known photoprotective agent 1,25(OH)2 vitamin D3 (calcitriol, 1,25D). Topical NPS-2143 failed to rescue UV-induced immunosuppression in a contact hypersensitivity study. In a chronic UV photocarcinogenesis protocol, topical NPS-2143 reduced squamous cell carcinomas for only up to 24 weeks (p < 0.02) but had no other effect on skin tumour development. In human keratinocytes, 1,25D, which protected mice from UV-induced skin tumours, significantly reduced UV-upregulated p-CREB expression (p < 0.01), a potential early anti-tumour marker, while NPS-2143 had no effect. This result, together with the failure to reduce UV-induced immunosuppression, may explain why the reduction in UV-DNA damage in mice with NPS-2143 was not sufficient to inhibit skin tumour formation.

Current progress in kokumi-active peptides, evaluation and preparation methods: a review.

Kokumi is a complex sensation characterized by thickness, mouthfulness and continuity. Kokumi-active peptides, which are distributed in many kinds of food, induce a rich and long-lasting mouthfeel of food. Aimed to provide a comprehensive overview of kokumi peptides, this review covers the aspects of preparation and evaluation methods for kokumi peptides, kokumi receptor calcium-sensing receptor (CaSR), as well as structural features of kokumi peptides and derivatives. Apart from extraction and separation from natural and fermented food, preparation of kokumi peptides can be effectively obtained from enzymatic generation. Kokumi peptides are perceived by CaSR in taste cells and the proposed transduction pathway has been described. The evaluation on kokumi-inducing effect of peptides has employed a combination of sensory assessment and CaSR method. The discovered kokumi peptides mainly comprise glutamyl peptides, leucyl peptides and other peptides without specific features. Derivatives of amino acids and peptides including sulphur-containing amino acids, N-acyl-Tyr derivatives, N-acetylated amino acids and Maillard reaction products (MRPs) also work as kokumi enhancers. Based on the summarized developments, great sensory properties and bioactivities enable kokumi peptides as promising protein ingredients in future application.

Homer1 mediates CaSR-dependent activation of mTOR complex 2 and initiates a novel pathway for AKT-dependent ß-catenin stabilization in osteoblasts.

The calcium-sensing receptor (CaSR) is critical for skeletal development, but its mechanism of action in osteoblasts is not well-characterized. In the central nervous system (CNS), Homer scaffolding proteins form signaling complexes with two CaSR-related members of the G protein-coupled receptor (GPCR) family C, metabotropic glutamate receptor 1 (mGluR1) and mGluR5. Here, we show that CaSR and Homer1 are co-expressed in mineralized mouse bone and also co-localize in primary human osteoblasts. Co-immunoprecipitation experiments confirmed that Homer1 associates with CaSR in primary human osteoblasts. The CaSR-Homer1 protein complex, whose formation was increased in response to extracellular Ca2+, was bound to mechanistic target of rapamycin (mTOR) complex 2 (mTORC2), a protein kinase that phosphorylates and activates AKT Ser/Thr kinase (AKT) at Ser473 siRNA-based gene-silencing assays with primary osteoblasts revealed that both CaSR and Homer1 are required for extracellular Ca2+-stimulated AKT phosphorylation and thereby inhibit apoptosis and promote AKT-dependent ß-catenin stabilization and cellular differentiation. To confirm the role of the CaSR-Homer1 complex in AKT initiation, we show that in HEK-293 cells, co-transfection with both Homer1c and CaSR, but neither with Homer1c nor CaSR alone, establishes sensitivity of AKT-Ser473 phosphorylation to increases in extracellular Ca2+ concentrations. These findings indicate that Homer1 mediates CaSR-dependent AKT activation via mTORC2 and thereby stabilizes ß-catenin in osteoblasts.

Parathyroid Cell Proliferation in Secondary Hyperparathyroidism of Chronic Kidney Disease.

Secondary hyperparathyroidism (SHP) is a common complication of chronic kidney disease (CKD) that correlates with morbidity and mortality in uremic patients. It is characterized by high serum parathyroid hormone (PTH) levels and impaired bone and mineral metabolism. The main mechanisms underlying SHP are increased PTH biosynthesis and secretion as well as increased glandular mass. The mechanisms leading to parathyroid cell proliferation in SHP are not fully understood. Reduced expressions of the receptors for calcium and vitamin D contribute to the disinhibition of parathyroid cell proliferation. Activation of transforming growth factor-α-epidermal growth factor receptor (TGF-α-EGFR), nuclear factor kappa B (NF-kB), and cyclooxygenase 2- prostaglandin E2 (Cox2-PGE2) signaling all correlate with parathyroid cell proliferation, underlining their roles in the development of SHP. In addition, the mammalian target of rapamycin (mTOR) pathway is activated in parathyroid glands of experimental SHP rats. Inhibition of mTOR by rapamycin prevents and corrects the increased parathyroid cell proliferation of SHP. Mice with parathyroid-specific deletion of all miRNAs have a muted increase in serum PTH and fail to increase parathyroid cell proliferation when challenged by CKD, suggesting that miRNA is also necessary for the development of SHP. This review summarizes the current knowledge on the mechanisms of parathyroid cell proliferation in SHP.

Calcium-sensing receptor mediates interleukin-1ß-induced collagen expression in mouse collecting duct cells.

The mechanisms that underlie the profibrotic effect of interleukin (IL)-1ß are complicated and not fully understood. Recent evidence has suggested the involvement of the calcium-sensing receptor (CaSR) in tubular injury. Therefore, the current study aimed to investigate whether CaSR mediates IL-1ß-induced collagen expression in cultured mouse inner medullary collecting duct cells (mIMCD3) and to determine the possible downstream signaling effector. The results showed that IL-1ß significantly upregulated the expression of type I and III collagens in a concentration- and time-dependent manner. Moreover, CaSR was expressed in mIMCD3 cells, and its expression was increased by increasing the concentrations and times of IL-1ß treatment. Selective inhibitors (Calhex231 or NPS2143) or the siRNA of CaSR attenuated the enhanced expression of type I and III collagens. Furthermore, IL-1ß increased nuclear ß-catenin protein levels and decreased cytoplasmic ß-catenin expression in cells. In contrast, blockage of CaSR by the pharmacological antagonists or siRNA could partially attenuate such changes in the IL-1ß-induced nuclear translocation of ß-catenin. DKK1, an inhibitor of ß-catenin nuclear translocation, further inhibited the expression of type I and III collagens in cells treated with IL-1ß plus CaSR antagonist. In summary, these data demonstrated that IL-1ß-induced collagen I and III expressions in collecting duct cells might be partially mediated by CaSR and the downstream nuclear translocation of ß-catenin.

Renal Ca2+ and Water Handling in Response to Calcium Sensing Receptor Signaling: Physiopathological Aspects and Role of CaSR-Regulated microRNAs.

Calcium (Ca2+) is a universal and vital intracellular messenger involved in a diverse range of cellular and biological processes. Changes in the concentration of extracellular Ca2+ can disrupt the normal cellular activities and the physiological function of these systems. The calcium sensing receptor (CaSR) is a unique G protein-coupled receptor (GPCR) activated by extracellular Ca2+ and by other physiological cations, aminoacids, and polyamines. CaSR is the main controller of the extracellular Ca2+ homeostatic system by regulating parathyroid hormone (PTH) secretion and, in turn, Ca2+ absorption and resorption. Recent advances highlight novel signaling pathways activated by CaSR signaling involving the regulation of microRNAs (miRNAs). miRNAs are naturally-occurring small non-coding RNAs that regulate post-transcriptional gene expression and are involved in several diseases. We previously described that high luminal Ca2+ in the renal collecting duct attenuates short-term vasopressin-induced aquaporin-2 (AQP2) trafficking through CaSR activation. Moreover, we demonstrated that CaSR signaling reduces AQP2 abundance via AQP2-targeting miRNA-137. This review summarizes the recent data related to CaSR-regulated miRNAs signaling pathways in the kidney.

The calcium-sensing receptor as a mediator of inflammation.

The teleologic link between increased production of pro-inflammatory cytokines resulting from a systemic inflammatory response to a burn injury and consequent stimulation of bone resorption is unclear. While it is known that cytokines can stimulate osteocytic and osteoblastic production of the ligand of the receptor activator of NFκB, or RANKL, it is not certain why this occurs. It was therefore hypothesized that the subsequent osteoclastic bone resorption liberates calcium from the bone matrix and somehow affects the inflammatory response. In this paper we show how the cytokine-mediated inflammatory response following severe burn injury in children results in simultaneous increase in bone resorption and up-regulation of the parathyroid calcium-sensing receptor. The acute bone resorption leads to release of calcium from the bone matrix with consequent calcium accumulation in the circulation. The up-regulation of the parathyroid calcium-sensing receptor suppresses the release of parathyroid hormone resulting in a lowering of blood calcium concentration. The simultaneous occurrences of these processes could regulate blood calcium concentration and if calcium concentration affects the inflammatory response, then the calcium-sensing receptor could, at the very least, modulate the inflammatory response by adjusting the blood calcium concentration. We describe in vitro studies in which we demonstrated that peripheral blood mononuclear cells in culture produce the chemokines MIP-1α and RANTES in proportion to the medium calcium concentration and they produce the chemokine MCP-1 in quantities inversely related to medium calcium concentration. CD14+monocytes in culture will also produce MIP-1α in direct relationship to medium calcium concentration but the correlation coefficient is markedly reduced compared to that with peripheral blood mononuclear cells. These monocytes, which possess the calcium-sensing receptor, do not produce MCP-1 in either direct or inverse relationship to medium calcium concentration. Therefore, it is possible that other peripheral blood mononuclear cells are primarily responsible for the production of chemokines in relation to calcium concentration but these cells have not yet been defined.

Prolonged exposure to 1,25(OH)2D3 and high ionized calcium induces FGF-23 production in intestinal epithelium-like Caco-2 monolayer: A local negative feedback for preventing excessive calcium transport.

Overdose of oral calcium supplement and excessive intestinal calcium absorption can contribute pathophysiological conditions, e.g., nephrolithiasis, vascular calcification, dementia, and cardiovascular accident. Since our previous investigation has indicated that fibroblast growth factor (FGF)-23 could abolish the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-enhanced calcium absorption, we further hypothesized that FGF-23 produced locally in the enterocytes might be part of a local negative feedback loop to regulate calcium absorption. Herein, 1,25(OH)2D3 was found to enhance the transcellular calcium transport across the epithelium-like Caco-2 monolayer, and this stimulatory effect was diminished by preceding prolonged exposure to high-dose 1,25(OH)2D3 or high concentration of apical ionized calcium. Pretreatment with a neutralizing antibody for FGF-23 prevented this negative feedback regulation of calcium hyperabsorption induced by 1,25(OH)2D3. FGF-23 exposure completely abolished the 1,25(OH)2D3-enhanced calcium transport. Western blot analysis revealed that FGF-23 expression was upregulated in a dose-dependent manner by 1,25(OH)2D3 or apical calcium exposure. Finally, calcium-sensing receptor (CaSR) inhibitors were found to prevent the apical calcium-induced suppression of calcium transport. In conclusion, prolonged exposure to high apical calcium and calcium hyperabsorption were sensed by CaSR, which, in turn, increased FGF-23 expression to suppress calcium transport. This local negative feedback loop can help prevent unnecessary calcium uptake and its detrimental consequences.

Activation of calcium-sensing receptor by allosteric agonists cinacalcet and AC-265347 abolishes the 1,25(OH)2D3-induced Ca2+ transport: Evidence that explains how the intestine prevents excessive Ca2+ absorption.

Long-term high-calcium intake and intestinal calcium hyperabsorption are hazardous to the body. It is hypothesized that enterocytes possess mechanisms for preventing superfluous calcium absorption, including secretion of negative regulators of calcium absorption and utilization of calcium-sensing receptor (CaSR) to detect luminal calcium. Herein, Caco-2 monolayers were treated with high doses of 1,25(OH)2D3 to induce calcium hyperabsorption or directly exposed to high apical calcium. The expression of counterregulatory factor of calcium absorption, fibroblast growth factor (FGF)-23, was also investigated in the intestine of lactating rats, which physiologically exhibit calcium hyperabsorption. We found that FGF-23 expression was enhanced in all intestinal segments of lactating rats. In Caco-2 monolayers, high apical calcium and 1,25(OH)2D3 induced FGF-23 secretion into culture media. FGF-23 antagonized 1,25(OH)2D3-induced calcium transport and led to a significant, but small, change in paracellular permeability. Furthermore, high-dose 1,25(OH)2D3 upregulated FGF-23 expression, which was prevented by CaSR inhibitors. Activation of apical CaSR by cinacalcet and AC-265347 abolished 1,25(OH)2D3-induced calcium transport in a dose-dependent manner. In conclusion, the intestinal FGF-23 expression was upregulated in conditions with calcium hyperabsorption, presumably to help protect against excessive calcium absorption, while CaSR probably monitored calcium in the lumen and induced FGF-23 production for preventing superfluous calcium uptake.

Discovery of evocalcet, a next-generation calcium-sensing receptor agonist for the treatment of hyperparathyroidism.

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.

Calcium-sensing receptor (CaSR) modulates vacuolar H+-ATPase activity in a cell model of proximal tubule.

BACKGROUND: The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H+-ATPase in a cellular model of proximal tubule cells, OKP cells. METHODS: 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. RESULTS: A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300 µM) and neomycin (200 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ (Ca2+o) between 10-4 and 2 mM. Gd3+ and neomycin produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of 0.1 µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5 × 10-8 M) reduced the increase in [Ca2+]i induced by neomycin. CONCLUSION: CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of OKP cells, an effect that involves an increase in [Ca2+]i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

Calcium-sensing receptor antagonist NPS2390 attenuates neuronal apoptosis though intrinsic pathway following traumatic brain injury in rats.

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. Previous study indicates that calcium-sensing receptor (CaSR) activation contributes to neuron death in focal cerebral ischemia-reperfusion mice, however, its role in neuronal apoptosis after TBI is not well-established. Using a controlled cortical impact model in rats, the present study was designed to determine the effect of CaSR inhibitor NPS2390 upon neuronal apoptosis after TBI. Rats were randomly distributed into three groups undergoing the sham surgery or TBI procedure, and NPS2390 (1.5 mg/kg) was infused subcutaneously at 30 min and 120 min after TBI. All rats were sacrificed at 24 h after TBI. Our data indicated that NPS2390 significantly reduced the brain edema and improved the neurological function after TBI. In addition, NPS2390 decreased caspase-3 levels and the number of apoptotic neurons. Furthermore, NPS2390 up-regulated anti-apoptotic protein Bcl-2 expression and down-regulated pro-apoptotic protein Bax, and reduced subsequent release of cytochrome c into the cytosol. In summary, this study indicated that inhibition of CaSR by NPS2390 attenuates neuronal apoptosis after TBI, in part, through modulating intrinsic apoptotic pathway.

Role of the calcium sensing receptor in cardiomyocyte apoptosis via mitochondrial dynamics in compensatory hypertrophied myocardium of spontaneously hypertensive rat.

Calcium sensing receptor (CaSR) mediates pathological cardiac hypertrophy. Mitochondria maintain their function through fission and fusion and disruption of mitochondrial dynamic is linked to various cardiac diseases. This study examined how inhibition of CaSR by the inhibitor Calhex231 affected the mitochondrial dynamics in a hypertensive model in rats. Spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were used in this study. Cardiac function and blood pressure was evaluated at the end of the study. SHRs showed increases in the ratio of heart weight to body weight and the levels of CaSR; all of these increases were suppressed by Calhex231. Additionally, Calhex231 treatment of SHRs changed the expression of proteins involved in mitochondrial dynamics. Our results demonstrated that CaSR activation induced cardiomyocyte apoptosis through the mitochondrial dynamics mediated apoptotic pathway in hypertensive hearts.

γ-Glutamyl cysteine and γ-glutamyl valine inhibit TNF-α signaling in intestinal epithelial cells and reduce inflammation in a mouse model of colitis via allosteric activation of the calcium-sensing receptor.

BACKGROUND: The extracellular calcium-sensing receptor (CaSR) is distributed throughout the gastrointestinal tract, and its activation has been shown to promote intestinal homeostasis, suggesting that CaSR may be a promising target for novel therapies to prevent chronic intestinal inflammation such as inflammatory bowel disease (IBD). The γ-glutamyl dipeptides γ-glutamyl cysteine (γ-EC) and γ-glutamyl valine (γ-EV) are dietary flavor enhancing compounds, and have been shown to activate CaSR via allosteric ligand binding. The aim of this study was to examine the anti-inflammatory effects of γ-EC and γ-EV in vitro in intestinal epithelial cells and in a mouse model of intestinal inflammation. RESULTS: In vitro, treatment of Caco-2 cells with γ-EC and γ-EV resulted in the CaSR-mediated reduction of TNF-α-stimulated pro-inflammatory cytokines and chemokines including IL-8, IL-6, and IL-1ß, and inhibited phosphorylation of JNK and IκBα, while increasing expression of IL-10. In vivo, using a mouse model of dextran sodium sulfate (DSS)-induced colitis, γ-EC and γ-EV treatment ameliorated DSS-induced clinical signs, weight loss, colon shortening and histological damage. Moreover, γ-EC and γ-EV reduced the expression of TNF-α, IL-6, INF-γ, IL-1ß, and IL-17, and increased the expression of IL-10 in the colon, in a CaSR-dependent manner. The CaSR-mediated anti-inflammatory effects of γ-EC were abrogated in ß-arrestin2 knock-down Caco-2 cells, and involvement of ß-arrestin2 was found to inhibit TNF-α-dependent signaling via cross-talk with the TNF-α receptor (TNFR). CONCLUSIONS: Thus CaSR activation by γ-EC and γ-EV can aid in maintaining intestinal homeostasis and reducing inflammation in chronic inflammatory conditions such as IBD.

Discovery of novel dihydrobenzofuran cyclopropane carboxylic acid based calcium sensing receptor antagonists for the treatment of osteoporosis.

In a search for novel small molecule calcium-sensing receptor (CaSR) antagonists as oral bone anabolic agents, we discovered dihydrobenzofuran cyclopropane carboxylic acid derivatives, such as 12f (IC50=27.6nM), are highly potent calcium-sensing receptor antagonists. Studies in rats established that compound 12f stimulates parathyroid hormone (PTH) release in a fast-acting, pulsatile manner.

Clinical and outcome comparison of genetically positive vs. negative patients in a large cohort of suspected familial hypocalciuric hypercalcemia.

OBJECTIVE: Biochemical suspicion of familial hypocalciuric hypercalcemia (FHH) might provide with a negative (FHH-negative) or positive (FHH-positive) genetic result. Understanding the differences between both groups may refine the identification of those with a positive genetic evaluation, aid management decisions and prospective surveillance. We aimed to compare FHH-positive and FHH-negative patients, and to identify predictive variables for FHH-positive cases. DESIGN: Retrospective, national multi-centre study of patients with suspected FHH and genetic testing of the CASR, AP2S1 and GNA11 genes. METHODS: Clinical, biochemical, radiological and treatment data were collected. We established a prediction model for the identification of FHH-positive cases by logistic regression analysis and area under the ROC curve (AUROC) was estimated. RESULTS: We included 66 index cases, of which 30 (45.5%) had a pathogenic variant. FHH-positive cases were younger (p = 0.029), reported more frequently a positive family history (p < 0.001), presented higher magnesium (p < 0.001) and lower parathormone levels (p < 0.001) and were less often treated for hypercalcemia (p = 0.017) in comparison to FHH-negative cases. Magnesium levels showed the highest AUROC (0.825, 95%CI: 0.709-0.941). The multivariate analysis revealed that family history and magnesium levels were independent predictors of a positive genetic result. The predictive model showed an AUROC of 0.909 (95%CI: 0.826-0.991). CONCLUSIONS: The combination of magnesium and a positive family history offered a good diagnostic accuracy to predict a positive genetic result. Therefore, the inclusion of magnesium measurement in the routine evaluation of patients with suspected FHH might provide insight into the identification of a positive genetic result of any of the CaSR-related genes.