Calcium-sensing receptor-mediated macrophage polarization improves myocardial remodeling in spontaneously hypertensive rats.

Chronic inflammation is a key element in the progression of essential hypertension (EH). Calcium plays a key role in inflammation, so its receptor, the calcium-sensing receptor (CaSR), is an essential mediator of the inflammatory process. Compelling evidence suggests that CaSR mediates inflammation in tissues and immune cells, where it mediates their activity and chemotaxis. Macrophages (Mφs) play a major role in the inflammatory response process. This study provided convincing evidence that R568, a positive regulator of CaSR, was effective in lowering blood pressure in spontaneously hypertensive rats (SHRs), improving cardiac function by alleviating cardiac hypertrophy and fibrosis. R568 can increase the content of CaSR and M2 macrophages (M2Mφs, exert an anti-inflammatory effect) in myocardial tissue, reduce M1 macrophages (M1Mφs), which have a pro-inflammatory effect in this process. In contrast, NPS2143, a negative state regulator of CaSR, exerted the opposite effect in all of the above experiments. Following this study, R568 increased CaSR content in SHR myocardial tissue, lowered blood pressure, promoted macrophages to M2Mφs and improved myocardial fibrosis, but interestingly, both M1Mφs and M2Mφs were increased in the peritoneal cavity of SHRs, the number of M2Mφs remained lower than M1Mφs. In vitro, R568 increased CaSR content in RAW264.7 cells (a macrophage cell line), regulating intracellular Ca2+ ([Ca2+]i) inhibited NOD-like receptor family protein 3 (NLRP3) inflammasome activation and ultimately prevented its conversion to M1Mφs. The results showed that a decrease in CaSR in hypertensive rats causes further development of hypertension and cardiac damage. EH myocardial remodeling can be improved by CaSR overexpression by suppressing NLRP3 inflammasome activation and macrophage polarization toward M1Mφs and increasing M2Mφs.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

OBJECTIVE: The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. DESIGN AND METHODS: To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. RESULTS: Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. CONCLUSIONS: This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges.

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Inhibition of calcium-sensing receptor by its antagonist promotes gastrointestinal motility in a Parkinson's disease mouse model.

BACKGROUND: The Calcium-sensing receptor (CaSR) participates in the regulation of gastrointestinal (GI) motility under normal conditions and might be involved in the regulation of GI dysmotility in patients with Parkinson's disease (PD). METHODS: CaSR antagonist-NPS-2143 was applied in in vivo and ex vivo experiments to study the effect and underlying mechanisms of CaSR inhibition on GI dysmotility in the MPTP-induced PD mouse model. FINDINGS: Oral intake of NPS-2143 promoted GI motility in PD mice as shown by the increased gastric emptying rate and shortened whole gut transit time together with improved weight and water content in the feces of PD mice, and the lack of influence on normal mice. Meanwhile, the number of cholinergic neurons, the proportion of serotonergic neurons, as well as the levels of acetylcholine and serotonin increased, but the numbers of nitrergic and tyrosine hydroxylase immunoreactive neurons, and the levels of nitric oxide synthase and dopamine decreased in the myenteric plexus in the gastric antrum and colon of PD mice in response to NPS-2143 treatment. Furthermore, the numbers of c-fos positive neurons in the nucleus tractus solitarius (NTS) and cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) increased in NPS-2143 treated PD mice, suggesting the involvement of both the enteric (ENS) and central (CNS) nervous systems. However, ex vivo results showed that NPS-2143 directly inhibited the contractility of antral and colonic strips in PD mice via a non-ENS mediated mechanism. Further studies revealed that NPS-2143 directly inhibited the voltage gated Ca2+ channels, which might, at least in part, explain its direct inhibitory effects on the GI muscle strips. INTERPRETATION: CaSR inhibition by its antagonist ameliorated GI dysmotility in PD mice via coordinated neuronal regulation by both ENS and CNS in vivo, although the direct effects of CaSR inhibition on GI muscle strips were suppressive.

Identification and characterization of a novel CASR mutation causing familial hypocalciuric hypercalcemia.

Context: Although a monoallelic mutation in the calcium-sensing receptor (CASR) gene causes familial hypocalciuric hypercalcemia (FHH), the functional characterization of the identified CASR mutation linked to the clinical response to calcimimetics therapy is still limited. Objective: A 45-year-old male presenting with moderate hypercalcemia, hypocalciuria, and inappropriately high parathyroid hormone (PTH) had a good response to cinacalcet (total serum calcium (Ca2+) from 12.5 to 10.1 mg/dl). We identified the genetic mutation and characterized the functional and pathophysiological mechanisms, and then linked the mutation to calcimimetics treatment in vitro. Design: Sanger sequencing of the CASR, GNA11, and AP2S1 genes was performed in his family. The simulation model was used to predict the function of the identified mutant. In vitro studies, including immunoblotting, immunofluorescence, a cycloheximide chase study, Calbryte™ 520 Ca2+ detection, and half-maximal effective concentration (EC50), were examined. Results: This proband was found to carry a de novo heterozygous missense I554N in the cysteine-rich domain of CASR, which was pathogenic based on the different software prediction models and ACGME criteria. The simulation model showed that CASR I554N mutation decreased its binding energy with Ca2+. Human CASR I554N mutation attenuated the stability of CASR protein, reduced the expression of p-ERK 1/2, and blunted the intracellular Ca2+ response to gradient extracellular Ca2+ (eCa2+) concentration. The EC50 study also demonstrated the correctable effect of calcimimetics on the function of the CASR I554N mutation. Conclusion: This novel CASR I554N mutation causing FHH attenuates CASR stability, its binding affinity with Ca2+, and the response to eCa2+ corrected by therapeutic calcimimetics.

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.

A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects.

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.

Calcium/calcimimetic via calcium-sensing receptor ameliorates cholera toxin-induced secretory diarrhea in mice.

BACKGROUND: Enterotoxins produce diarrhea through direct epithelial action and indirectly by activating the enteric nervous system. Calcium-sensing receptor (CaSR) inhibits both actions. The latter has been well documented in vitro but not in vivo. The hypothesis to be tested was that activating CaSR inhibits diarrhea in vivo. AIM: To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin (CTX) in mice. METHODS: CTX was given orally to C57BL/6 mice to induce diarrhea. Calcium and calcimimetic R568 were used to activate CaSR. To maximize their local intestinal actions, calcium was administered luminally via oral rehydration solution (ORS), whereas R568 was applied serosally using an intraperitoneal route. To verify that their actions resulted from the intestine, effects were also examined on Cre-lox intestine-specific CaSR knockouts. Diarrhea outcome was measured biochemically by monitoring changes in fecal Cl- or clinically by assessing stool consistency and weight loss. RESULTS: CTX induced secretory diarrhea, as evidenced by increases in fecal Cl-, stool consistency, and weight loss following CTX exposure, but did not alter CaSR, neither in content nor in function. Accordingly, calcium and R568 were each able to ameliorate diarrhea when applied to diseased intestines. Intestinal CaSR involvement is suggested by gene knockout experiments where the anti-diarrheal actions of R568 were lost in intestinal epithelial CaSR knockouts (villinCre/Casrflox/flox) and neuronal CaSR knockouts (nestinCre/Casrflox/flox). CONCLUSION: Treatment of acute secretory diarrheas remains a global challenge. Despite advances in diarrhea research, few have been made in the realm of diarrhea therapeutics. ORS therapy has remained the standard of care, although it does not halt the losses of intestinal fluid and ions caused by pathogens. There is no cost-effective therapeutic for diarrhea. This and other studies suggest that adding calcium to ORS or using calcimimetics to activate intestinal CaSR might represent a novel approach for treating secretory diarrheal diseases.

Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

Novel kokumi peptides from yeast extract and their taste mechanism via an in silico study.

Yeast extract, a widely utilized natural substance in the food industry and biopharmaceutical field, holds significant potential for flavor enhancement. Kokumi peptides within yeast extracts were isolated through ultrafiltration and gel chromatography, followed by identification using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two peptides, IQGFK and EDFFVR, were identified and synthesized using solid-phase methods based on molecular docking outcomes. Sensory evaluations and electronic tongue analyses conducted with chicken broth solutions revealed taste thresholds of 0.12 mmol L-1 for IQGFK and 0.16 mmol L-1 for EDFFVR, respectively, and both peptides exhibited kokumi properties. Additionally, through molecular dynamics simulations, the binding mechanisms between these peptides and the calcium-sensing receptor (CaSR) were explored. The findings indicated stable binding of both peptides to the receptor. IQGFK primarily interacted through electrostatic interactions, with key binding sites including Asp275, Asn102, Pro274, Trp70, Tyr218, and Ser147. EDFFVR mainly engaged via van der Waals energy and polar solvation free energy, with key binding sites being Asp275, Ile416, Pro274, Arg66, Ala298, and Tyr218. This suggests that both peptides can activate the CaSR, thereby inducing kokumi activity. This study provides a theoretical foundation and reference for the screening and identification of kokumi peptides, successfully uncovering two novel kokumi peptides derived from yeast extract.

Alterations in gene expression and microbiome composition upon calcium-sensing receptor deletion in the mouse esophagus.

The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) (EsoCaSR-/-) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control and EsoCaSR-/-mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle in EsoCaSR-/-. This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup in EsoCaSR-/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g_Rodentibacter, s_Rodentibacter_unclassified, and s_Lactobacillus_hilgardi were significantly increased in EsoCaSR-/-. Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated in EsoCaSR-/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome.NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.

Exploring Adaptive Phenotypes for the Human Calcium-Sensing Receptor Polymorphism R990G.

Rainforest hunter-gatherers from Southeast Asia are characterized by specific morphological features including a particularly dark skin color (D), short stature (S), woolly hair (W), and the presence of steatopygia (S)-fat accumulation localized in the hips (DSWS phenotype). Based on previous evidence in the Andamanese population, we first characterized signatures of adaptive natural selection around the calcium-sensing receptor gene in Southeast Asian rainforest groups presenting the DSWS phenotype and identified the R990G substitution (rs1042636) as a putative adaptive variant for experimental follow-up. Although the calcium-sensing receptor has a critical role in calcium homeostasis by directly regulating the parathyroid hormone secretion, it is expressed in different tissues and has been described to be involved in many biological functions. Previous works have also characterized the R990G substitution as an activating polymorphism of the calcium-sensing receptor associated with hypocalcemia. Therefore, we generated a knock-in mouse for this substitution and investigated organismal phenotypes that could have become adaptive in rainforest hunter-gatherers from Southeast Asia. Interestingly, we found that mouse homozygous for the derived allele show not only lower serum calcium concentration but also greater body weight and fat accumulation, probably because of enhanced preadipocyte differentiation and lipolysis impairment resulting from the calcium-sensing receptor activation mediated by R990G. We speculate that such differential features in humans could have facilitated the survival of hunter-gatherer groups during periods of nutritional stress in the challenging conditions of the Southeast Asian tropical rainforests.

Calcium sensing receptor: A promising therapeutic target in pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by elevation of pulmonary arterial pressure and pulmonary vascular resistance. The increased pulmonary arterial pressure and pulmonary vascular resistance due to sustained pulmonary vasoconstriction and pulmonary vascular remodeling can lead to right heart failure and eventual death. A rise in intracellular Ca2+ concentration ([Ca2+]i) and enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation contribute to pulmonary vasoconstriction and pulmonary vascular remodeling. Recent studies demonstrated that extracellular calcium sensing receptor (CaSR) as a G-protein coupled receptor participates in [Ca2+]i increase induced by hypoxia in the experimental animals of PH and in PH patients. Pharmacological blockade or gene knockout of CaSR significantly attenuates the development of PH. This review will aim to discuss and update the pathogenicity of CaSR attributed to onset and progression in PH.

Stimulation of the calcium-sensing receptor induces relaxations of rat mesenteric arteries by endothelium-dependent and -independent pathways via BKCa and KATP channels.

Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca2+ concentration ([Ca2+ ]o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca2+ ]o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca2+ ]o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca2+ -activated K+ channels (BKCa , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca2+ ]o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K+ channel (KATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K+ channels (Kir ), Kv7 channels, and small conductance Ca2+ -activated K+ channels (SKCa ) on [Ca2+ ]o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BKCa channels and an endothelium-independent pathway involving stimulation of KATP channels.

Small intestinal CaSR-dependent and CaSR-independent protein sensing regulates feeding and glucose tolerance in rats.

Proteins activate small intestinal calcium sensing receptor (CaSR) and/or peptide transporter 1 (PepT1) to increase hormone secretion1-8, but the effect of small intestinal protein sensing and the mechanistic potential of CaSR and/or PepT1 in feeding and glucose regulation remain inconclusive. Here we show that, in male rats, CaSR in the upper small intestine is required for casein infusion to increase glucose tolerance and GLP1 and GIP secretion, which was also dependent on PepT1 (ref. 9). PepT1, but not CaSR, is required for casein infusion to lower feeding. Upper small intestine casein sensing fails to regulate feeding, but not glucose tolerance, in high-fat-fed rats with decreased PepT1 but increased CaSR expression. In the ileum, a CaSR-dependent but PepT1-independent pathway is required for casein infusion to lower feeding and increase glucose tolerance in chow-fed rats, in parallel with increased PYY and GLP1 release, respectively. High fat decreases ileal CaSR expression and disrupts casein sensing on feeding but not on glucose control, suggesting an ileal CaSR-independent, glucose-regulatory pathway. In summary, we discover small intestinal CaSR- and PepT1-dependent and -independent protein sensing mechanisms that regulate gut hormone release, feeding and glucose tolerance. Our findings highlight the potential of targeting small intestinal CaSR and/or PepT1 to regulate feeding and glucose tolerance.

Calcium-sensing receptor and NF-κB pathways in TN breast cancer contribute to cancer-induced cardiomyocyte damage via activating neutrophil extracellular traps formation.

Cardiovascular disorders are commonly prevalent in cancer patients, yet the mechanistic link between them remains poorly understood. Because neutrophil extracellular traps (NETs) have implications not just in cardiovascular diseases (CVD), but also in breast cancer (BC), it was hypothesized to contribute to CVD in the context of oncogenesis. We established a mouse model using nude mice to simulate liver metastasis of triple-negative BC (TNBC) through the injection of MDA-MB-231 cells. Multiple imaging and analysis techniques were employed to assess the cardiac function and structure, including echocardiography, HE staining, Masson staining, and transmission electron microscopy (TEM). MDA-MB-231 cells underwent treatment with a CaSR inhibitor, CaSR agonist, and NF-κB channel blocker. The phosphorylation of NF-κB channel protein p65 and the expression and secretion of IL-8 were assessed using qRT-PCR, Western Blot, and ELISA, respectively. In addition, MDA-MB-231 cells were co-cultured with polymorphonuclear neutrophils (PMN) under varying conditions. The co-localization of PMN extracellular myeloperoxidase (MPO) and DNA were observed by cellular immunofluorescence staining to identify the formation of NETs. Then, the cardiomyocytes were co-cultured with the above medium that contains NETs or not, respectively; the effects of NETs on cardiomyocytes apoptosis were perceived by flow cytometry. The ultrastructural changes of myocardial cells were perceived by TEM, and ELISA detected the levels of myocardial enzyme (LDH, MDA and SOD). Overall, according to our research, CaSR has been found to have a regulatory role in IL-8 secretion in MDA-MB-231 cells, as well as in the formation of NETs by PMN cells. These findings suggest CaSR-mediated stimulation in PMN can lead to increased NETs formation and subsequently to cytotoxicity in cardiomyocytes, which potentially via activation of the NF-κB signaling cascade of BC cell.

Repurposing calcium-sensing receptor activator drug cinacalcet for ADPKD treatment.

ADPKD is characterized by progressive cyst formation and enlargement leading to kidney failure. Tolvaptan is currently the only FDA-approved treatment for ADPKD; however, it can cause serious adverse effects including hepatotoxicity. There remains an unmet clinical need for effective and safe treatments for ADPKD. The extracellular Ca2+-sensing receptor (CaSR) is a regulator of epithelial ion transport. FDA-approved CaSR activator cinacalcet can reduce cAMP-induced Cl- and fluid secretion in various epithelial cells by activating phosphodiesterases (PDE) that hydrolyze cAMP. Since elevated cAMP is a key mechanism of ADPKD progression by promoting cell proliferation, cyst formation and enlargement (via Cl- and fluid secretion), here we tested efficacy of cinacalcet in cell and animal models of ADPKD. Cinacalcet treatment reduced cAMP-induced Cl- secretion and CFTR activity in MDCK cells as suggested by ∼70 % lower short-circuit current (Isc) changes in response to forskolin and CFTRinh-172, respectively. Cinacalcet treatment inhibited forskolin-induced cAMP elevation by 60 % in MDCK cells, and its effect was completely reversed by IBMX (PDE inhibitor). In MDCK cells treated with forskolin, cinacalcet treatment concentration-dependently reduced cell proliferation, cyst formation and cyst enlargement by up to 50 % without affecting cell viability. Cinacalcet treatment (20 mg/kg/day for 7 days, subcutaneous) reduced renal cyst index in a mouse model of ADPKD (Pkd1flox/flox;Ksp-Cre) by 20 %. Lastly, cinacalcet treatment reduced cyst enlargement and cell proliferation in human ADPKD cells by 60 %. Considering its efficacy as shown here, and favorable safety profile including extensive post-approval data, cinacalcet can be repurposed as a novel ADPKD treatment.

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Cinacalcet Reverses Short QT Interval in Familial Hypocalciuric Hypercalcemia Type 1.

CONTEXT: Familial hypocalciuric hypercalcemia type 1 (FHH-1) defines an autosomal dominant disease, related to mutations in the CASR gene, with mild hypercalcemia in most cases. Cases of FHH-1 with a short QT interval have not been reported to date. OBJECTIVE: Three family members presented with FHH-1 and short QT interval (<360 ms), a condition that could lead to cardiac arrhythmias, and the effects of cinacalcet, an allosteric modulator of the CaSR, in rectifying the abnormal sensitivity of the mutant CaSR and in correcting the short QT interval were determined. METHODS: CASR mutational analysis was performed by next-generation sequencing and functional consequences of the identified CaSR variant (p.Ile555Thr), and effects of cinacalcet were assessed in HEK293 cells expressing wild-type and variant CaSRs. A cinacalcet test consisting of administration of 30 mg cinacalcet (8 Am) followed by hourly measurement of serum calcium, phosphate, and parathyroid hormone during 8 hours and an electrocardiogram was performed. RESULTS: The CaSR variant (p.Ile555Thr) was confirmed in all 3 FHH-1 patients and was shown to be associated with a loss of function that was ameliorated by cinacalcet. Cinacalcet decreased parathyroid hormone by >50% within two hours, and decreases in serum calcium and increases in serum phosphate occurred within 8 hours, with rectification of the QT interval, which remained normal after 3 months of cinacalcet treatment. CONCLUSION: Our results indicate that FHH-1 patients should be assessed for a short QT interval and a cinacalcet test used to select patients who are likely to benefit from this treatment.