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.

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.

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.

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.

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.

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.

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.

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 activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas.

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl- channels, and basolateral membrane K+ channels. 30 µM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Identification and target of action of cholecystokinin-releasing peptides from simulated digestion hydrolysate of wheat protein.

BACKGROUND: Wheat protein intake leads to improved appetite control. However, the active components causing appetite in wheat have not been fully clarified. Gut cholecystokinin (CCK) plays a vital role in appetite control. This study aimed to investigate the ability of wheat protein digest (WPD) to stimulate CCK secretion and clarify the active components and target of action. RESULTS: WPD was prepared by a simulated gastrointestinal digestion model. WPD treatment with a concentration of 5 mg mL-1 significantly stimulated CCK secretion in enteroendocrine STC-1 cells (P < 0.05). Furthermore, oral gavage with WPD in mice significantly increased plasma CCK level at 60 min (P < 0.01). Preparative C18 column separation was used to isolate peptide fractions associated with CCK secretion and peptide sequences were identified by liquid chromatography-tandem mass spectrometry. A new CCK-releasing peptide, RYIVPL, that potently stimulated CCK secretion was successfully identified. After pretreatment with a specific calcium-sensing receptor (CaSR) antagonist, NPS 2143, CCK secretion induced by WPD or RYIVPL was greatly suppressed, suggesting that CaSR was involved in WPD- or RYIVPL-induced CCK secretion. CONCLUSION: The present study demonstrated that WPD has an ability to stimulate CCK secretion in vitro and in vivo, and determined that peptide RYIVPL in WPD could stimulate CCK secretion through CaSR. © 2023 Society of Chemical Industry.

Novel Calcium-Binding Peptide from Bovine Bone Collagen Hydrolysates and Its Potential Pro-Osteogenic Activity via Calcium-Sensing Receptor (CaSR).

SCOPE: This paper aims to explore the osteogenic activity and potential mechanism of the peptide-calcium chelate, and provides a theoretical basis for peptide-calcium chelates as functional foods to prevent or improve osteoporosis. METHODS AND RESULTS: In this research, a novel peptide (Phe-Gly-Leu, FGL) with a high calcium-binding capacity is screened from bovine bone collagen hydrolysates (CPs), calcium binding sites of which mainly included carbonyl, amino and carboxyl groups. The FGL-Ca significantly enhances the osteogenic activity of MC3T3-E1 cells (survival rate, differentiation, and mineralization). The results of calcium fluorescence labeling and molecular docking show that FGL-Ca may activate calcium-sensing receptor (CaSR), leading to an increase in intracellular calcium concentration, then enhancing osteogenic activity of MC3T3-E1 cells. Further research found that FGL-Ca significantly promotes the mRNA and protein expression levels of CaSR, transforming growth factor ß (TGF-ß1), TGF-ß-type II receptor (TßRII), Smad2, Smad3, osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegrin (OPG), and collagen type I (COLI). Subsequently, in the signal pathway intervention experiment, the expression levels of genes and proteins related to the TGF-ß1/Smad2/3 signaling pathway that are promoted by FGL-Ca are found to decrease. CONCLUSIONS: These results suggest that FGL-Ca may activate CaSR, increase intracellular calcium concentration, and activate TGF-ß1/Smad2/3 signaling pathway, which may be one of the potential mechanisms for enhancing osteogenic activity.

Nanobodies as negative allosteric modulators for human calcium sensing receptor.

Human calcium sensing receptor (CaSR) senses calcium ion concentrations in vivo and is an important class of drug targets. Mutations in the receptor can lead to disorders of calcium homeostasis, including hypercalcemia and hypocalcemia. Here, 127 CaSR-targeted nanobodies were generated from camels, and four nanobodies with inhibitory function were further identified. Among these nanobodies, NB32 can effectively inhibit the mobilization of intracellular calcium ions (Ca2+i) and suppress the G12/13 and ERK1/2 signaling pathways downstream of CaSR. Moreover, it enhanced the inhibitory effect of the calcilytics as a negative allosteric modulator (NAM). We determined the structure of complex and found NB32 bound to LB2 (Ligand-binding 2) domain of CaSR to prevent the interaction of LB2 domains of two protomers to stabilize the inactive state of CaSR.

Expression of the Calcium-Sensing Receptor on Normal and Abnormal Parathyroid and Thyroid Tissue.

INTRODUCTION: Current imaging techniques have several limitations in detecting parathyroid glands. We have investigated the calcium-sensing receptor (CaSR) as a potential target for specifically labeling parathyroid glands for radiologic detection. For accurate imaging it is vital that a large differential expression exists between the target tissue and adjacent structures. We sought to investigate the relative abundance of the CaSR in normal and abnormal parathyroid tissue, as well as normal and abnormal thyroid. METHODS: Existing clinical specimens were selected that represented a wide variety of pathologically and clinically confirmed malignant and benign thyroid and parathyroid specimens. Sections were stained for the CaSR using immunohistochemistry and scored for intensity and abundance of expression. (H score = intensity scored from 0 to 3 multiplied by the % of cells at each intensity. Range 0-300). RESULTS: All parathyroid specimens expressed the CaSR to a high degree. Normal parathyroid had the highest H score (271, s.d. 25.4). Abnormal parathyroid specimens were slightly lower but still much higher than normal thyroid (H score 38.3, s.d. 23.3). Medullary thyroid cancer also expressed the CaSR significantly higher than normal thyroid (H score 182, s.d. 69.1, P < 0.001) but below parathyroid levels. Hürthle cell carcinoma expressed the CaSR to a lesser degree but higher than normal thyroid (H score 101, s.d. 46.4, P = 0.0037). CONCLUSIONS: The CaSR is differentially expressed on parathyroid tissue making it a feasible target for parathyroid imaging. False positives might be anticipated with medullary and Hürthle cell cancers.

Cinacalcet therapy in a child with novel homozygous CASR p.Glu353Lys mutation causing familial hypocalciuric hypercalcemia type 1: case report and review of the literature.

BACKGROUND: Familial hypocalciuric hypercalcemia (FHH) is one of the conditions that should be considered in the differential diagnosis of hypercalcemia and normo-hypophosphatemia in childhood. Heterozygous Calcium-sensing receptor (CASR) gene mutations cause FHH, and homozygous CASR gene mutations cause neonatal severe primary hyperparathyroidism (NSHPT). Cinacalcet is an allosteric modulator of Calciumsensing receptor (CaSR), and has been used in the treatment of these clinical entities in recent years. CASE: A 26-month-old boy was examined for a recurrent rash. During the evaluation, hypercalcemia (13.3 mg/ dL), hypophosphatemia (2.3 mg/dL) and inappropriately normal PTH level (67 pg/mL) were observed. Neck and renal ultrasonography were normal. The parathyroid scintigraphy was unremarkable. The patient`s family members were also evaluated, and hypocalciuria (fractional excretion of calcium were 0.01%, 0.04% on two separate tests) was detected concurrently with the patient`s hypercalcemia. The mother`s serum calcium was 10.2 mg/dL, the father`s was 10.6 mg/dL, and the brother`s was 12.8 mg/dL. CASR gene sequencing showed a novel homozygous mutation in exon 4 (c.1057G > A), which had generated a substitution of the amino acid glutamate to lysine at codon 353 (p.Glu353Lys). This mutation was homozygous in the children and heterozygous in the parents. Fluid hydration, furosemide, oral phosphorus, prednisolone, pamidronate and cinacalcet treatments were used in the management of hypercalcemia of the proband. A longer and more effective control was achieved with cinacalcet treatment. CONCLUSIONS: FHH can be seen in heterozygous as well as homozygous CASR gene mutations. Different clinical findings may occur in different individuals from the same family. Cinacalcet therapy can be used successfully in the treatment of individuals with FHH.

High-dose remifentanil exacerbates myocardial ischemia-reperfusion injury through activation of calcium-sensing receptor-mediated pyroptosis.

Background: The aim of this study was to investigate whether calcium-sensing receptor (CaSR) was involved in HRF-mediated exacerbation of MI/R injury through NLRP3 inflammasome activation and pyroptosis. Methods: In vivo, a rat MI/R model was established by ligating the left coronary artery, and short-term HRF exposure was induced during reoxygenation. Then, TUNEL, H&E, Masson staining, immunohistochemical (IHC) and serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK), as well as the expression levels of CaSR and pyroptosis-related proteins in heart tissues, were measured. H9c2 cells were cultured to create a hypoxia/reoxygenation (H/R) model and exposed to different concentrations of RF. After pretreatment with the CaSR activator gadolinium chloride (GdCl3) and inhibitor NPS2143 in the H/R model and treatment with HRF, we compared cellular viability, TUNEL, cytosolic [Ca2+]i, the levels of LDH and CK, pyroptosis-related proteins and CaSR in H9c2 cells. We further researched the mechanisms of CaSR-mediated pyroptosis in the H/R+HRF model by CaSR-shRNA, Ac-YVAD-CMK, MCC950 and NAC. Results: We found that HRF significantly increased CaSR expression, rate of cell death, levels of CK and LDH, and exacerbated pyroptosis in MI/R model. In vitro, HRF increased CaSR expression, decreased viability, enhanced cytosolic [Ca2+]i and exacerbated pyroptosis in H/R cells. Pretreated with GdCl3 worsen these changes, and NPS2143, MCC950, Ac-YVAD-CMK, NAC and sh-CaSR can reversed these effects. Conclusion: Exposure to HRF for a short time exacerbates MI/R-induced injury by targeting CaSR to increase cytosolic [Ca2+]i and ROS levels, which mediate the NLRP3 inflammasome and pyroptosis.