Calcium-sensing Receptor Agonist Research Articles

The calcium‐sensing receptor alleviates endothelial inflammation in atherosclerosis through regulation of integrin β1‐NLRP3 inflammasome

Atherosclerosis (AS) is a chronic inflammatory disease of arteries. Endothelial inflammation is key to the initiation and development of AS. The calcium‐sensing receptor (CaSR) is expressed in endothelial cells (ECs) but its role in endothelial inflammation during AS remains unclear. This study focused on the involvement of CaSR in regulating endothelial inflammation and its underlying mechanisms, providing novel insights for AS therapy. Here, we observed that CaSR agonist NPS‐R568 significantly reduced atherosclerotic lesions and aortic inflammation in high‐fat diet (HFD)‐fed ApoE−/− mice, while enhancing the expression of CaSR in aortic tissues. In vitro, human umbilical vein endothelial cells (HUVECs) exposed to oxidized low‐density lipoprotein (oxLDL) at 20 μg·mL−1 triggered inflammation, as indicated by the upregulation of vascular cell adhesion molecule‐1 (VCAM‐1), interleukin (IL)‐6, and IL‐1β expression, along with increased adherence of THP‐1 or U937 cells to the HUVECs. Additionally, treatment with 20 μg·mL−1 oxLDL led to downregulation of CaSR expression in HUVECs. The administration of CaSR agonist NPS‐R568 or overexpression of CaSR in HUVECs resulted in a significant reversal of inflammation induced by oxLDL. Mechanistically, CaSR was found to mitigate NLRP3 inflammasome activation by downregulating the protein level of integrin β1. In conclusion, our study elucidates the beneficial role of CaSR in reducing endothelial inflammation in AS through the regulation of integrin β1 and the subsequent NLRP3 inflammasome. CaSR emerges as a promising target for potential therapeutic interventions in AS.

Calcium-sensing receptor regulates the angiogenic differentiation of LPS-treated human dental pulp cells via the phosphoinositide 3-kinase/Akt pathway invitro.

The purpose of this study was to investigate the role of calcium-sensing receptor (CaSR) in the angiogenic differentiation of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The LPS-induced hDPCs were cultured in the medium with different combinations of CaSR agonist R568 and antagonist Calhex231. The cell proliferation, migration, and angiogenic capacity were measured by Cell Counting Kit-8 (CCK-8), scratch wound healing, and tube formation assays, respectively. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were conducted to determine the gene/protein expression of CaSR, inflammatory mediators, and angiogenic-associated markers. The activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) was assessed by western blot analysis. The cell proliferation was elevated in response to R568 or Calhex231 exposure, but an enhanced cell migration was only found in cultures supplemented with Calhex231. Furthermore, R568 was found to potentiate the formation of vessel-like structure, up-regulated the protein expression of tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1; comparable influences were also observed in R568-stimulated cells in the presence of PI3K inhibitor LY294002. In contrast, Calhex231 obviously inhibited the tube formation and VEGF protein level, whereas promoted the production of IL-6, TNF-α, and eNOS; however, in the presence of LY294002, Calhex231 showed a significant promotion on the protein expression of CaSR, VEGF, and SDF-1. In addition, R568 exhibited a promotive action on the Akt phosphorylation, which can be reversed by LY294002. Our results demonstrated that CaSR can regulate the angiogenic differentiation of LPS-treated hDPCs with an involvement of the PI3K/Akt signalling pathway.

Comparative analysis of calcium-sensing receptor (CaSR) expression and function in normal and abnormal human sperm and spermatogenic cells.

The calcium-sensing receptor (CaSR) is a critical mediator of calcium homeostasis in various tissues. Its role in human reproduction, especially in sperm function and male fertility, remains not fully elucidated. This study investigates the expression patterns of CaSR in normal and abnormal sperm and spermatogenic cells and evaluates its potential effect on sperm motility and morphology. Using immunohistochemistry (IHC), quantitative PCR (qPCR), we assessed the expression levels of CaSR in normal sperm, spermatogonia, and cases of asthenozoospermia, oligozoospermia, and teratozoospermia. In vitro functional assays were performed to analyze the effects of CaSR modulation on sperm motility under varying conditions, including the presence of specific CaSR agonists and antagonists. Our study revealed distinct patterns of CaSR expression in normal sperm and spermatogonia compared with those in abnormal sperm samples, particularly in cases of asthenozoospermia, oligozoospermia, and teratozoospermia. A marked decrease in CaSR expression was evident in these abnormal samples, highlighting its significance in normal sperm functionality. Functional assays further elucidated the role of CaSR in sperm motility. Activation of CaSR through specific agonists enhanced sperm motility, while inhibition by antagonists led to reduced motility. Our findings suggest that CaSR plays a significant role in maintaining sperm functionality and that changes in its expression may be associated with male infertility. These insights into the molecular underpinnings of sperm physiology highlight CaSR as a potential therapeutic target for treating certain forms of male infertility.

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

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

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. To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin (CTX) in mice. 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. 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). 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.

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.

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.

Distinct and Targetable Role of Calcium and Calcium Sensing Receptor in Leukaemia

Calcium, the most abundant mineral in the body, is a key component of bones and is released by bone remodelling. Calcium ions play a role in the localisation, engraftment and the adhesion of normal haematopoietic stem cells (HSC) to extracellular matrix (ECM) proteins in the bone marrow microenvironment (BMM) via the calcium sensing receptor (CaSR), thereby maintaining normal haematopoiesis. However, the role of this heterotrimeric G-protein-coupled receptor and its associated pathways in the local BMM for the development of leukaemia is poorly understood. We hypothesized that calcium ions, subject to a fine balance between osteoblasts and osteoclasts and released from bone, and/or CaSR contribute to development, progression and response to therapy in leukaemia and might be targetable via CaSR. By imaging of a genetically-encoded calcium indicator using intravital microscopy (IVM), we have shown that the local calcium concentration forms a gradient in the BMM with the highest concentrations close to the endosteal area. Furthermore, calcium levels differ between different leukaemias, with the calcium concentration of mice and human patients being highest in acute myeloid leukaemia (AML). Additionally, CaSR was expressed at a higher level on AML compared to chronic myeloid leukaemia (CML) cells. Using genetic deletion or overexpression or pharmacological inhibition of CaSR, we revealed that this receptor influences the localization of CML and AML cells in the BMM. Furthermore, we showed that the secondary messenger intracellular calcium (iCa 2+) can be regulated by CaSR in different leukaemia cell lines which have differential sensitivity to extracellular calcium (eCa 2+). Exposure of the human cell lines K562 (BCR-ABL1 +) and THP1 (MLL-AF9 +) to increasing calcium concentrations revealed that CaSR expression, migration and adhesion to the extracellular matrix (ECM) protein fibronectin were not altered in CML, but significantly increased in AML. Genetic deletion or overexpression of Casr in leukaemia-initiating cells in murine models revealed that CaSR acts as tumour suppressor in BCR-ABL1-driven CML and B-cell acute lymphoblastic leukaemia (B-ALL) and as oncogene in AML. Conversely, overexpression of CaSR in CaSR-deficient AML-initiating cells ‘rescued’ the disease. Limiting dilution transplantation of CaSR-deficient AML-initiating cells to assess leukaemic stem cell number and/or function revealed a 6.5-fold reduction of leukemic stem cells. Treatment of mice with the CaSR agonist cinacalcet and imatinib prolonged survival of mice with CML, while treatment with a CaSR antagonist NPS-2143 significantly reduced tumour burden and prolonged survival of mice with AML in syngeneic and xenogeneic transplantation experiments. In summary, our results suggest that calcium ions, likely originating from the BMM and/or CaSR strongly and differentially influence leukaemia localisation and progression. As an adjunct to existing treatment strategies, targeting of CaSR with specific pharmacologic agonists may be beneficial in CML and B-ALL, while CaSR antagonists may prolong survival of patients with AML.

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

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

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.

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

Asthma is a heterogeneous chronic airways disease with an unmet need for improved therapeutics in uncontrolled severe disease. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor upregulated in asthma. The CaSR agonist, spermine, is also increased in asthmatic airways and contributes to bronchoconstriction. CaSR negative allosteric modulators (NAMs) oppose chronic airway inflammation, remodelling and hyperresponsiveness in murine and guinea pig asthma models, but whether CaSR NAMs are effective acute bronchodilators compared to standard-of-care has not yet been established. Further, the ability of different classes of NAMs to inhibit spermine-induced CaSR signaling or MCh-induced airway contraction has not been quantified. Here, we show CaSR NAMs differentially inhibit spermine-induced intracellular calcium mobilisation and inositol monophosphate accumulation in HEK293 cells stably expressing the CaSR. NAMs reverse methacholine (MCh)-mediated airway contraction in mouse precision cut lung slices with similar maximal relaxation compared to the standard treatment, salbutamol. Of note, the bronchodilator effects of CaSR NAMs are maintained under conditions of β2 adrenergic receptor desensitization when salbutamol efficacy is abolished. Furthermore, overnight treatment with some, but not all, CaSR NAMs prevents MCh-mediated bronchoconstriction. These findings further support the CaSR as a putative drug target, and NAMs as alternative or adjunct bronchodilators, in asthma.

The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

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.

Role of calcium-sensing receptor in regulating activation susceptibility of postovulatory aging mouse oocytes.

The mechanisms underlying postovulatory oocyte aging (POA) remain largely unknown. The expression of the calcium-sensing receptor (CaSR) in mouse oocytes and its role in POA need to be explored. Our objective was to observe CaSR expression and its role in the susceptibility to activating stimuli (STAS) in POA mouse oocytes. The results showed that, although none of the newly ovulated oocytes were activated, 40% and 94% of the oocytes recovered 19 and 25 h after human chorionic gonadotropin (hCG) injection were activated, respectively, after ethanol treatment. The level of the CaSR functional dimer protein in oocytes increased significantly from 13 to 25 h post hCG. Thus, the CaSR functional dimer level was positively correlated with the STAS of POA oocytes. Aging in vitro with a CaSR antagonist suppressed the elevation of STAS, and cytoplasmic calcium in oocytes recovered 19 h post hCG, whereas aging with a CaSR agonist increased STAS, and cytoplasmic calcium of oocytes recovered 13 h post hCG. Furthermore, the CaSR was more important than the Na-Ca2+ exchanger in regulating oocyte STAS, and T- and L-type calcium channels were inactive in aging oocytes. We conclude that the CaSR is involved in regulating STAS in POA mouse oocytes, and that it is more important than the other calcium channels tested in this connection.

Cinacalcet inhibition of neuronal action potentials preferentially targets the fast inactivated state of voltage-gated sodium channels.

Voltage-gated sodium channel (VGSC) activation is essential for action potential generation in the brain. Allosteric calcium-sensing receptor (CaSR) agonist, cinacalcet, strongly and ubiquitously inhibits VGSC currents in neocortical neurons via an unidentified, G-protein-dependent inhibitory molecule. Here, using whole-cell patch VGSC clamp methods, we investigated the voltage-dependence of cinacalcet-mediated inhibition of VGSCs and the channel state preference of cinacalcet. The rate of inhibition of VGSC currents was accelerated at more depolarized holding potentials. Cinacalcet shifted the voltage-dependence of both fast and slow inactivation of VGSC currents in the hyperpolarizing direction. Utilizing a simple model, the voltage-dependence of VGSC current inhibition may be explained if the affinity of the inhibitory molecule to the channel states follows the sequence: fast-inactivated > slow-inactivated > resting. The state dependence of VGSC current inhibition contributes to the non-linearity of action potential block by cinacalcet. This dynamic and abundant signaling pathway by which cinacalcet regulates VGSC currents provides an important voltage-dependent mechanism for modulating central neuronal excitability.

Activation of Calcium-Sensing Receptor in the Area Postrema Inhibits Food Intake via Glutamatergic and GABAergic Signaling Pathways.

A high-protein diet has become a popular way to lose weight. Calcium-sensing receptor (CaSR) is activated by amino acids in addition to calcium ions. CaSR shows dense expression in the area postrema (AP), which participates in feeding regulation. The effect of CaSR in the AP on food intake and the potential mechanism involved is investigated. Male C57BL/6 mice are used to observe the effect of R568 (agonist of CaSR) on food intake. Enzyme-linked immunosorbent assay, immunofluorescence staining, and chemogenetics are used to explore the neural signaling involved. CaSR activation in the AP inhibited acute feeding; R568 increases the content of glutamate and γ-aminobutyric acid (GABA) in the AP, whereas only glutamatergic neurons mediate the effect of R568. GABA-A receptor and ionic glutamate receptor (N-methyl-D-aspartate receptor [NMDAR]) in the paraventricular nucleus of hypothalamus (PVN) are involved in the effect of R568. Promotion of oxytocin (OT) synthesis in the PVN also participates in the effect of R568, and this mechanism is mediated by NMDAR in the PVN. CaSR activation in the AP suppresses feeding, and AP-PVN glutamatergic and GABAergic signaling pathways are involved.

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

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

Drug-Induced QT Prolongation and Torsade de Pointes in Spontaneous Adverse Event Reporting: A Retrospective Analysis Using the Japanese Adverse Drug Event Report Database (2004-2021).

Drugs with new mechanisms of action are continually being developed, but it is difficult to capture whether a drug induces QT prolongation/torsade de pointes (TdP) in preclinical and preapproval clinical trials. To evaluate drugs associated with drug-induced QT prolongation/TdP using a real-world database in Japan. A search was performed in the Japanese Adverse Drug Event Report (JADER) database for QT prolongation and TdP. The reporting odds ratio (ROR) was calculated to identify potential drug-induced QT prolongation/TdP association. Among the reported 4,326,484 data entries, 3410 patients exhibited QT prolongation/TdP (2707 with QT prolongation, 703 with TdP) with the suspected drugs. Of these patients, 53.9% were females. The highest occurrence was in the 70- to 79-year-old age group (24.7%). The most common types of drugs involved were cardiovascular drugs, central nervous system (CNS) drugs, anticancer drugs, and anti-infective drugs; the rate of overdose was reportedly very low at 1.6%. The highest adjusted RORs were observed for nifekalant (351.41, 95% confidence interval (CI) 235.85-523.59), followed by vandetanib (182.55, 95% CI 108.11-308.24), evocalcet (181.59, 95% CI 132.96-248.01), bepridil (160.37, 95% CI 138.17-186.13), diarsenic trioxide (79.43, 95% CI 63.98-98.62), and guanfacine (78.29, 95% CI 58.51-104.74). Among the drugs launched in Japan during the last decade, vandetanib had the highest adjusted RORs. This study using the JADER database showed that antiarrhythmic drugs, calcium-sensing receptor agonists, small-molecule targeted anticancer drugs, and CNS drugs are associated with QT prolongation/TdP. Further pharmacoepidemiological studies, such as cohort studies using large databases, are needed to prove these causal relationships.

Label-Free Mass Spectrometry-Based Quantitative Proteomics to Evaluate the Effects of the Calcium-Sensing Receptor Agonist Cinacalcet on Protein Expression in Rat Brains and Livers.

BackgroundCinacalcet is a calcium-sensing receptor agonist that is clinically approved for the treatment of secondary hyperparathyroidism in chronic kidney disease and hypercalcemia in patients with parathyroid carcinoma. This study aimed to use quantitative mass spectrometry-based label-free proteomics to evaluate the effects of cinacalcet on protein expression in rat brains and livers.Material/MethodsWe randomly assigned 18 Wistar rats to 2 groups: an untreated control group (n=6) and a group treated with cinacalcet at a dose corresponding to the maximum dose used in humans (2 mg/kg/body weight, 5 days/week) divided into 7-day (n=6) and 21-day (n=6) treatment subgroups. A mass-spectrometry-based label-free quantitative proteomics approach using peptides peak area calculation was used to evaluate the changes in protein expression in examined tissues. Bioinformatics analysis of quantitative proteomics data was done using MaxQuant and Perseus environment.ResultsNo changes in protein expression were revealed in the 7-day treatment subgroup. We detected 10 upregulated and 3 downregulated proteins in the liver and 1 upregulated protein in the brain in the 21-day treatment subgroup compared to the control group. Based on Gene Ontology classification, all identified differentially expressed proteins were indicated as molecular functions involved in the enzyme regulator activity (36%), binding (31%), and catalytic activity (19%).ConclusionsThese findings indicate that long-term cinacalcet therapy can impair phase II of enzymatic detoxication and can cause disturbances in blood hemostasis, lipid metabolism, and inflammatory mediators or contribute to the acceleration of cognitive dysfunction; therefore, appropriate patient monitoring should be considered.

A role for the calcium-sensing receptor in the expression of inflammatory mediators in LPS-treated human dental pulp cells.

The aim of this study is to investigate the role of calcium-sensing receptor (CaSR) in the expression of inflammatory mediators of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The expression profile of CaSR in LPS-simulated hDPCs was detected using immunofluorescence, real time quantitative PCR (RT-qPCR), and Western blot analyses. Then, its regulatory effects on the expression of specific inflammatory mediators such as interleukin (IL)-1β, IL-6, cyclooxygenase 2 (COX2)-derived prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and IL-10 were determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA). LPS significantly downregulated the gene expression of CaSR, but upregulated its protein expression level in hDPCs. Treatments by CaSR agonist R568 or its antagonist Calhex231, and their combinations with protein kinase B (AKT) inhibitor LY294002 showed obvious effects on the expression of selected inflammatory mediators in a time-dependent manner. Meanwhile, an opposite direction was found between the action of R568 and Calhex231, as well as the expression of the pro- (IL-1β, IL-6, COX2-derived PGE2, and TNF-α) and anti-inflammatory (IL-10) mediators. The results provide the first evidence that CaSR-phosphatidylinositol-3 kinase (PI3K)-AKT-signaling pathway is involved in the release of inflammatory mediators in LPS-treated hDPCs, suggesting that the activation or blockade of CaSR may provide a novel therapeutic strategy for the treatment of pulp inflammatory diseases.