Activation Of Calcium-sensing Receptor Research Articles

#1447 Calcimimetics improve impaired cardiac function and contractility in chronic phosphate loading by activating the CaSR/cAMP signaling cascade

Abstract Background and Aims High phosphate loading stimulate the synthesis of PTH and FGF23 and is associated with increased cardiovascular (CV) mortality. In hemodialysis patients, the administration of calcimimetics led to reductions in PTH and FGF23 and the latter was associated with a lower rate of CV events. We have previously shown that mice with a high dietary phosphate load show elevated PTH and FGF23 levels and develop impaired systolic cardiac function, which could be improved by calcimimetics. Calcimimetics are allosteric activators of the calcium-sensing receptor (CaSR). Phosphate (Pi) was shown to inactivate the parathyroid CaSR at concentrations >1.4 mM Pi. Whether calcimimetics contribute to CV event reduction indirectly via lowering PTH and FGF23 or directly via activation of the cardiac CaSR is unknown. Method Wild-type mice were fed a high phosphate diet (HPD) ± etelcalcetide (Etl) or a normal phosphate diet (NPD), cardiac function was assessed by echocardiography and Millar catheterization, and cardiac tissue was harvested for histology and RNAseq analyses. In addition, isolated adult mouse cardiomyocytes (AMCM) were stimulated ex vivo with PTH, FGF23 or Pi, contractility and calcium handling were measured with the addition of Etl and activity analyses were performed. Results In addition to the positive effect on HPD-induced systolic cardiac dysfunction, Etl improved impaired LV contractility and reduced fibrosis. RNAseq analyses revealed reduced Ca, PKA and cAMP signaling by HPD. Ex vivo stimulation of AMCM with PTH and FGF23 had no functional effects. In contrast, Pi significantly worsened contractility and Ca handling, which was blunted by Etl co-stimulation. Downstream of cardiac CaSR, Pi reduced the formation of cAMP, which led to PKA-dependent dephosphorylation of PLN and subsequently to lower Serca2a activity, which was ultimately responsible for the reduced contractility. Etl led to improved cardiomyocyte contractility via increased cAMP synthesis and increased PLN phosphorylation and Serca2a activity. Conclusion Our data suggest that Etl ameliorates the Pi-induced pathologic cardiac phenotype associated with high phosphate load via direct activation of CaSR/cAMP signaling cascade, resulting in improvement of cardiomyocyte contractility.

New insights into renal calcium-sensing receptor activation.

Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood. Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation. These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms.

Molecular regulation of calcium‐sensing receptor (CaSR)‐mediated signaling

AbstractCalcium‐sensing receptor (CaSR), a family C G‐protein‐coupled receptor, plays a crucial role in regulating calcium homeostasis by sensing small concentration changes of extracellular Ca2+, Mg2+, amino acids (e.g., L‐Trp and L‐Phe), small peptides, anions (e.g., HCO3− and PO43−), and pH. CaSR‐mediated intracellular Ca2+ signaling regulates a diverse set of cellular processes including gene transcription, cell proliferation, differentiation, apoptosis, muscle contraction, and neuronal transmission. Dysfunction of CaSR with mutations results in diseases such as autosomal dominant hypocalcemia, familial hypocalciuric hypercalcemia, and neonatal severe hyperparathyroidism. CaSR also influences calciotropic disorders, such as osteoporosis, and noncalciotropic disorders, such as cancer, Alzheimer's disease, and pulmonary arterial hypertension. This study first reviews recent advances in biochemical and structural determination of the framework of CaSR and its interaction sites with natural ligands, as well as exogenous positive allosteric modulators and negative allosteric modulators. The establishment of the first CaSR protein–protein interactome network revealed 94 novel players involved in protein processing in endoplasmic reticulum, trafficking, cell surface expression, endocytosis, degradation, and signaling pathways. The roles of these proteins in Ca2+‐dependent cellular physiological processes and in CaSR‐dependent cellular signaling provide new insights into the molecular basis of diseases caused by CaSR mutations and dysregulated CaSR activity caused by its protein interactors and facilitate the design of therapeutic agents that target CaSR and other family C G‐protein‐coupled receptors.

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.

Calcium-sensing Receptor, a Potential Biomarker Revealed by Large-scale Public Databases and Experimental Verification in Metastatic Breast Cancer.

Breast cancer (BC) is a common cancer characterized by a high molecular heterogeneity. Therefore, understanding its biological properties and developing effective treatments for patients with different molecular features is imperative. Calcium-sensing receptor (CaSR) has been implicated in several regulatory functions in various types of human cancers. However, its underlying pathological mechanism in BC progression remains elusive. We utilized The Cancer Genome Atlas and Gene Expression Omnibus databases to explore the function of CaSR in the metastasis of BC. Gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Gene Set Enrichment Analysis of biological processes and cell signaling pathways revealed that CaSR could be activated or inhibited. Importantly, quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to verify the gene expression of the CaSR. Wound healing and transwell assays were conducted to assess the effect of CaSR on the migration of BC cells. We demonstrated that CaSR expression in metastatic BC was higher than that in non-metastatic BC. It is the first time that database information has been used to reveal the biological process and molecular mechanism of CaSR in BC. Moreover, the CaSR expression in normal breast epithelial cells was notably less compared to that in BC cells. The activation of CaSR by Cinacalcet (a CaSR agonist) significantly enhanced the migration of BC cells, whereas NPS-2143 (a CaSR antagonist) treatment dramatically inhibited these effects. Bioinformatics techniques and experiments demonstrated the involvement of CaSR in BC metastasis. Our findings shed new light on the receptor therapy and molecular pathogenesis of BC, and emphasize the crucial function of CaSR, facilitating the metastasis of BC.

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 alleviates gut damage caused by endotoxemia by regulating gut microbiota.

Growing evidence points to an association between the gut microbiota and neonatal diseases. Calcium-sensing receptor (CaSR) is a major modulator of tissue responses associated with calcium homeostasis and is highly expressed in the mammalian gut. CaSR may affect the composition and balance of the intestinal microenvironment. Neonatal rats were randomized to the control, lipopolysaccharide (LPS), CaSR agonist, and CaSR inhibitor groups. The intestinal contents of neonatal rats were collected within 24 hours or 7 days after intervention. Then, 16S rRNA short amplicon sequencing was used to analyze biological information and the richness and diversity of individual taxa. LPS aggravated intestinal injury. The CaSR agonist alleviated injury, and the inhibitor further enhance intestinal injury. Activation of CaSR enhanced the diversity of the gut microbiota and the abundance of Lactobacillus. The lowest abundance of Firmicutes and the highest abundance of Bacteroidetes were found in the agonist group. CaSR impacted the bacterial species in rats with endotoxemia, and Akkermansia had the greatest effect on the differences among groups. Activation of CaSRs could enhance the species richness and β-diversity of the gut microbiota and alter the abundance of many taxa. This could attenuate LPS-induced gut injury by modulating the gut microbiota.

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.

THU396 Digital Spatial Profiling Of Human Parathyroid Tumors Reveals Distinct Cellular And Molecular Alterations Linked To Vitamin D Deficiency

Abstract Disclosure: C. Tu: None. W. Chang: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly & Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly & Company. J. Koh: None. Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population, but the pathophysiological basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from 9 vitamin D-deficient or 34 vitamin D-replete PHPT patients with 12 eucalcemic cadaveric donor parathyroid glands as normal tissue controls. Parathyroid oxyphil cell content is markedly higher in tumors from vitamin D-deficient PHPT patients (Def-Ts) relative to tumors from vitamin D-replete PHPT patients (Rep-Ts) (47.8% vs 17.8%, respectively; p <0.0001) and normal donor parathyroid glands (7.7%). Def-Ts display increased expression of genes encoding electron transport chain (corr. coefficient=0.546), proteasomal catabolism (corr. coefficient=0.587), and oxidative phosphorylation (corr. coefficient=0.982) pathway components. In contrast, Rep-Ts are characterized by upregulation of the ras (corr. coefficient=0.632) and myc (corr. coefficient=0.811) signaling pathways, suggestive of an oncogene-driven etiology. Parathyroid oxyphil cells are comparable to chief cells at the transcriptional level, and vitamin D-status is reflected in the transcriptional profiles of both cell types in a similar manner. Among PHPT patients who did not present as vitamin D deficient, three distinct tumor transcriptional profile clusters showed a dose-dependent relationship to pre-operative 25OHD levels (p <0.0001 by ANOVA). Relative to normal tissue, the abundance of calcium sensing receptor (CaSR) protein is reduced in Def-Ts (mean protein count 253.6 vs 492.3, respectively; p=0.039 for difference), while the type B γ-aminobutyric acid receptor 1 (GABAB1R) is increased (mean protein count 114.5 vs 64.65, respectively; p=0.0099 for difference). These expression changes resulted in a vitamin D dose-dependent reduction in the ratio of CaSR to GABAB1R receptors in Def-T tumor cells (r2 = 0.218, p<0.0001), favoring the formation of calcium sensing-attenuated CaSR:GABAB1R heteromers. The abundance of the amyloid precursor protein (APP) peptide derivative Aβ1-42, a putative GABAB1R ligand, is preferentially increased in Def-Ts compared to normal tissue (mean protein count 5797 vs 4500, respectively; p=0.0318). Collectively, these findings suggest a novel mechanism where chronic vitamin D deficiency can contribute to tonic PTH hypersecretion in PHPT patients by increasing GABAB1R-mediated antagonism of CaSR activity in parathyroid adenomas. Presentation: Thursday, June 15, 2023

THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells

Abstract Disclosure: K.A. Alghamdi: None. D. Ward: None. Calcium-sensing receptor (CaR) is the key controller of parathyroid hormone (PTH) secretion and extracellular calcium homeostasis. Hyperphosphataemia increases PTH secretion and we reported recently that pathophysiologic phosphate (Pi) concentrations can attenuate CaR activity directly in transfected HEK-293 cells and can increase PTH secretion from human and murine parathyroid cells (1). This was investigated further using thyroidal C cell-derived TT cells (ATCC), which express CaR endogenously. Intracellular Ca2+ (Ca2+i) mobilisation was assayed by epifluorescence microscopy, calcitonin secretion by ELISA, protein expression by immunoblotting and CaR mRNA expression by qRT-PCR. Co-stimulation of TT cells with the (CaR-activating) calcimimetic R568 (1µM) and spermine (1mM) elicited robust Ca2+i mobilisation, that was inhibited (33±4%; P<0.001; N=4) by 2mM (pathophysiologic) Pi-containing buffer vs 0.8mM Pi control. In contrast, raising Pi concentration was without effect on carbachol-induced Ca2+i mobilisation (acting via muscarinic receptors) (P=0.89; N=3). Also, 1.2mM (high) sulphate elicited a similar CaR inhibition as for Pi vs 0.3mM control (-28±16%; P<0.05; N=4). Next, it was found that CaR-mediated stimulation of the TT cells with 1µM R568 & 1mM spermine (in 2mM Ca2+ and 0.8mM Pi) increased calcitonin secretion, as expected, whereas the same stimulation in 2mM Pi (in the presence of 2.2mM Ca2+ to correct for any reduction in free Ca2+) significantly blunted this response (-59.7±15%; P<0.05;N=4). Interestingly, CaR mRNA expression was significantly downregulated 45±13%; P<0.05;N=6) after 48 hours treatment with media containing 2mM Pi (vs 0.8mM control). However, while CaR protein expression in the TT cells was confirmed by immunoblotting, its abundance was unaffected by the same 2mM Pi cotreatment (P=0.79; N=8). Therefore, pathophysiologic Pi treatment inhibits endogenous CaR-induced signalling, calcitonin secretion and CaR mRNA expression in thyroidal TT cells but without affecting CaR abundance. These results further support the idea that the CaR represents a mineral sensor, at which Pi acts directly as a non-competitive antagonist to limit CaR-induced suppression of PTH secretion. Reference: (1) Centeno et al. Nat Commun. 2019;10:4693. Presentation: Thursday, June 15, 2023

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.

Alcohol Intake Provoked Cardiomyocyte Apoptosis Via Activating Calcium-Sensing Receptor and Increasing Endoplasmic Reticulum Stress and Cytosolic [Ca2+]i.

Cardiomyocyte apoptosis plays an important role in alcoholic cardiac injury. However, the association between calcium-sensing receptor (CaSR) and alcohol-induced cardiomyocyte apoptosis remain unclear. Therefore, we investigated the role and its moleculer mechanism of CaSR in rat cardiomyocyte apoptosis induced by alcohol. Alcohol-induced cardiomyocyte apoptosis in vivo and in vitro model of rats were applied in this study. The expression of CaSR, endoplasmic reticulum stress markers and apoptosis were tested by immunohistological staining, western blot, TUNEL and flow cytometry, respectively. [Ca2+]i were detected by confocal laser scanning microscopy. Compared with the control group, alcohol intake (AI) led to abnormal arrangements of cardiomyocytes and obvious increase of myocardial apoptosis. Moreover, AI also significantly upregulated protein expression of CaSR, GRP94, caspase-12 and CHOP. Alcohol induced apoptosis of cultured cardiomyocytes of rats in a dose-dependent way. Activation of CaSR markedly enhanced cardiomyocyte apoptosis and ERS induced by alcohol, ERS inducer also significantly increased cardiomyocyte apoptosis without activating CaSR. Furthermore, GdCl3 augmented alcohol-induced increase of [Ca2+]i in cardiomyocytes, which was attenuated by NPS2390 but not 4-PBA pre-treatment. Alcohol could induce cardiomyocyte apoptosis in rats in vivo and in vitro, which was mediated probably via activating CaSR, and then ERS and the increase of the cytosolic [Ca2+]i. This provides a potential target for preventing cardiomyocyte apoptosis and cardiomyopathy induced by alochol.

Phenylalanine promotes alveolar macrophage pyroptosis via the activation of CaSR in ARDS.

Acute respiratory distress syndrome (ARDS) is associated with high mortality rates in patients admitted to the intensive care unit (ICU) patients with overwhelming inflammation considered to be an internal cause. The authors' previous study indicated a potential correlation between phenylalanine levels and lung injury. Phenylalanine induces inflammation by enhancing the innate immune response and the release of pro-inflammatory cytokines. Alveolar macrophages (AMs) can respond to stimuli via synthesis and release of inflammatory mediators through pyroptosis, one form of programmed cell death acting through the nucleotide-binging oligomerization domain-like receptors protein 3 (NLRP3) signaling pathway, resulting in the cleavage of caspase-1 and gasdermin D (GSDMD) and the release of interleukin (IL) -1β and IL-18, aggravating lung inflammation and injury in ARDS. In this study, phenylalanine promoted pyroptosis of AMs, which exacerbated lung inflammation and ARDS lethality in mice. Furthermore, phenylalanine initiated the NLRP3 pathway by activating the calcium-sensing receptor (CaSR). These findings uncovered a critical mechanism of action of phenylalanine in the context of ARDS and may be a new treatment target for ARDS.

Expression of Concern: Crucial Role of Calcium-Sensing Receptor Activation in Cardiac Injury of Diabetic Rats.

Expression of Concern: Crucial Role of Calcium-Sensing Receptor Activation in Cardiac Injury of Diabetic Rats.

Asymmetric dimethylarginine positively modulates calcium-sensing receptor signalling to promote lipid accumulation

Asymmetric dimethylarginine (ADMA) is generated through the irreversible methylation of arginine residues. It is an independent risk factor for cardiovascular disease, currently thought to be due to its ability to act as a competitive inhibitor of the nitric oxide (NO) synthase enzymes. Plasma ADMA concentrations increase with obesity and fall following weight loss; however, it is unknown whether they play an active role in adipose pathology. Here, we demonstrate that ADMA drives lipid accumulation through a newly identified NO-independent pathway via the amino-acid sensitive calcium-sensing receptor (CaSR). ADMA treatment of 3T3-L1 and HepG2 cells upregulates a suite of lipogenic genes with an associated increase in triglyceride content. Pharmacological activation of CaSR mimics ADMA while negative modulation of CaSR inhibits ADMA driven lipid accumulation. Further investigation using CaSR overexpressing HEK293 cells demonstrated that ADMA potentiates CaSR signalling via Gq intracellular Ca2+ mobilisation. This study identifies a signalling mechanism for ADMA as an endogenous ligand of the G protein-coupled receptor CaSR that potentially contributes to the impact of ADMA in cardiometabolic disease.

Inhibition of the calcium-sensing receptor by extracellular phosphate ions and by intracellular phosphorylation.

As both a sensor of extracellular calcium (Ca2+ o) concentration and a key controller of Ca2+ o homeostasis, one of the most interesting properties of the calcium-sensing receptor (CaR) is its sensitivity to, and modulation by, ions and small ligands other than Ca2+. There is emerging evidence that extracellular phosphate can act as a partial, non-competitive CaR antagonist to modulate parathyroid hormone (PTH) secretion, thus permitting the CaR to integrate mineral homeostasis more broadly. Interestingly, phosphorylation of certain intracellular CaR residues can also inhibit CaR responsiveness. Thus, negatively charged phosphate can decrease CaR activity both extracellularly (via association with arginine) and intracellularly (via covalent phosphorylation).

Cadmium Disrupted ER Ca2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells

Cadmium (Cd2+) exposure induces chronic kidney disease and renal cancers, which originate from injury and cancerization of renal tubular cells. Previous studies have shown that Cd2+ induced cytotoxicity by disrupting the intracellular Ca2+ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca2+ store. However, the molecular mechanism of ER Ca2+ homeostasis in Cd2+-induced nephrotoxicity remains unclear. In this study, our results firstly revealed that the activation of calcium-sensing receptor (CaSR) by NPS R-467 could protect against Cd2+ exposure-induced cytotoxicity of mouse renal tubular cells (mRTEC) by restoring ER Ca2+ homeostasis through the ER Ca2+ reuptake channel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Cd2+-induced ER stress and cell apoptosis were effectively abrogated by SERCA agonist CDN1163 and SERCA2 overexpression. In addition, in vivo, and in vitro results proved that Cd2+ reduced the expressions of SERCA2 and its activity regulator phosphorylation phospholamban (p-PLB) in renal tubular cells. Cd2+-induced SERCA2 degradation was suppressed by the treatment of proteasome inhibitor MG132, which suggested that Cd2+ reduced SERCA2 protein stability by promoting the proteasomal protein degradation pathway. These results suggested that SERCA2 played pivotal roles in Cd2+-induced ER Ca2+ imbalance and stress to contribute to apoptosis of renal tubular cells, and the proteasomal pathway was involved in regulating SERCA2 stability. Our results proposed a new therapeutic approach targeting SERCA2 and associated proteasome that might protect against Cd2+-induced cytotoxicity and renal injury.

Secretory Factors from Calcium-Sensing Receptor-Activated SW872 Pre-Adipocytes Induce Cellular Senescence and A Mitochondrial Fragmentation-Mediated Inflammatory Response in HepG2 Cells

Adipose tissue inflammation in obesity has a deleterious impact on organs such as the liver, ultimately leading to their dysfunction. We have previously shown that activation of the calcium-sensing receptor (CaSR) in pre-adipocytes induces TNF-α and IL-1β expression and secretion; however, it is unknown whether these factors promote hepatocyte alterations, particularly promoting cell senescence and/or mitochondrial dysfunction. We generated conditioned medium (CM) from the pre-adipocyte cell line SW872 treated with either vehicle (CMveh) or the CaSR activator cinacalcet 2 µM (CMcin), in the absence or presence of the CaSR inhibitor calhex 231 10 µM (CMcin+cal). HepG2 cells were cultured with these CM for 120 h and then assessed for cell senescence and mitochondrial dysfunction. CMcin-treated cells showed increased SA-β-GAL staining, which was absent in TNF-α- and IL-1β-depleted CM. Compared to CMveh, CMcin arrested cell cycle, increased IL-1β and CCL2 mRNA, and induced p16 and p53 senescence markers, which was prevented by CMcin+cal. Crucial proteins for mitochondrial function, PGC-1α and OPA1, were decreased with CMcin treatment, concomitant with fragmentation of the mitochondrial network and decreased mitochondrial transmembrane potential. We conclude that pro-inflammatory cytokines TNF-α and IL-1β secreted by SW872 cells after CaSR activation promote cell senescence and mitochondrial dysfunction, which is mediated by mitochondrial fragmentation in HepG2 cells and whose effects were reversed with Mdivi-1. This investigation provides new evidence about the deleterious CaSR-induced communication between pre-adipocytes and liver cells, incorporating the mechanisms involved in cellular senescence.

Activation of calcium-sensing receptors in the basolateral nucleus of the amygdala inhibits food intake and induces anxiety-depressive-like emotions via dopamine system

The calcium-sensing receptor (CaSR) is abundantly expressed in gastrointestinal mucosa and participates in the regulation of feeding by affecting hormone secretion. Studies have demonstrated that the CaSR is also expressed in feeding-related brain areas, such as the hypothalamus and limbic system, but the effect of the central CaSR on feeding has not been reported. Therefore, the aim of this study was to explore the effect of the CaSR in the basolateral amygdala (BLA) on feeding, and the potential mechanism was also studied. CaSR agonist R568 was microinjected into the BLA of male Kunming mice to investigate the effects of the CaSR on food intake and anxiety-depression-like behaviours. The enzyme-linked immunosorbent assay (ELISA) and fluorescence immunohistochemistry were used to explore the underlying mechanism. Our results showed that microinjection of R568 into the BLA could inhibit both standard and palatable food intake in mice for 0–2 h, induce anxiety-depression-like behaviours, increase glutamate levels in the BLA, and activate dynorphin and gamma-aminobutyric acid neurons through the N-methyl-D-aspartate receptor and thus reduce the content of dopamine in the arcuate nucleus of the hypothalamus (ARC) and ventral tegmental area (VTA), respectively. Our findings suggest that activation of the CaSR in the BLA inhibited food intake and caused anxiety-depression-like emotions. The reduced dopamine levels in the VTA and ARC via glutamatergic signals are involved in these functions of CaSR.

Low molecular weight hyaluronan inhibits lung epithelial ion channels by activating the calcium-sensing receptor

Herein, we tested the hypothesis that low molecular weight hyaluronan (LMW-HA) inhibits lung epithelial ions transport in-vivo, ex-vivo, and in-vitro by activating the calcium-sensing receptor (CaSR). Twenty-four hours post intranasal instillation of 50–150 µg/ml LMW-HA to C57BL/6 mice, there was a 75% inhibition of alveolar fluid clearance (AFC), a threefold increase in the epithelial lining fluid (ELF) depth, and a 20% increase in lung wet/dry (W/D) ratio. Incubation of human and mouse precision cut lung slices with 150 µg/ml LMW-HA reduced the activity and the open probability (Po) of epithelial sodium channel (ENaC) in alveolar epithelial type 2 (ATII) cells, and in mouse tracheal epithelial cells (MTEC) monolayers as early as 4 h. The Cl− current through cystic fibrosis transmembrane conductance regulator (CFTR) and the activity of Na,K-ATPase were both inhibited by more than 66% at 24 h. The inhibitory effects of LMW-HA on ion channels were reversed by 1 µM NPS-2143, or 150 µg/ml high molecular weight hyaluronan (HMW-HA). In HEK-293 cells expressing the calcium-sensitive Cl− channel TMEM16-A, CaSR was required for the activation of the Cl− current by LMW-HA. This is the first demonstration of lung ions and water transport inhibition by LMW-HA, and its mediation through the activation of CaSR.