NPS-2143 inhibit glioma progression by suppressing autophagy through mediating AKT-mTOR pathway.

Gliomas are the most common tumours in the central nervous system. In the present study, we aimed to find a promising anti-glioma compound and investigate the underlying molecular mechanism. Glioma cells were subjected to the 50 candidate compounds at a final concentration of 10 µM for 72 h, and CCK-8 was used to evaluate their cytotoxicity. NPS-2143, an antagonist of calcium-sensing receptor (CASR), was selected for further study due to its potent cytotoxicity to glioma cells. Our results showed that NPS-2143 could inhibit the proliferation of glioma cells and induce G1 phase cell cycle arrest. Meanwhile, NPS-2143 could induce glioma cell apoptosis by increasing the caspase-3/6/9 activity. NPS-2143 impaired the immigration and invasion ability of glioma cells by regulating the epithelial-mesenchymal transition process. Mechanically, NPS-2143 could inhibit autophagy by mediating the AKT-mTOR pathway. Bioinformatic analysis showed that the prognosis of glioma patients with low expression of CASR mRNA was better than those with high expression of CASR mRNA. Gene set enrichment analysis showed that CASR was associated with cell adhesion molecules and lysosomes in glioma. The nude mice xenograft model showed NPS-2143 could suppress glioma growth in vivo. In conclusion, NPS-2143 can suppress the glioma progression by inhibiting autophagy.

Ca2+ Signaling and Proliferation via Ca2+-Sensing Receptors in Human Hepatic Stellate LX-2 Cells.

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt). However, the regulatory mechanisms underlying Ca2+ signaling in activated HSCs remain largely unknown. In the present study, functional analyses of Ca2+-sensing receptors (CaSRs) were performed using activated human HSCs, LX-2. Expression analyses revealed that CaSR proteins were expressed in α-smooth muscle actin-positive LX-2 cells. Extracellular Ca2+ restoration (from 0 to 2.2 mM) increased [Ca2+]cyt in these cells. The extracellular Ca2+-induced increase in [Ca2+]cyt was reduced by the CaSR antagonists, NPS2143 and Calhex 231. Furthermore, the growth of LX-2 cells was blocked by NPS2143 and Calhex 231 in concentration-dependent manners (IC50 = 6.0 and 9.5 µM, respectively). LX-2 cell proliferation was also attenuated by NPS2143 and Calhex 231. In conclusion, CaSRs are functionally expressed in activated HSCs and regulate Ca2+ signaling and cell proliferation. The present results provide insights into the molecular mechanisms underlying hepatic fibrosis and will contribute to the development of potential therapeutic targets.

Calcium sensing receptor contribute to early brain injury through the CaMKII/NLRP3 pathway after subarachnoid hemorrhage in mice.

The subversive role of Calcium sensing receptor (CaSR) in cerebral ischemia and traumatic brain injury has been recently reported. Nevertheless, the role of CaSR in early brain injury (EBI) after subarachnoid hemorrhage (SAH) remains unexplored. Using the endovascular perforation model in mice, this study was aimed at investigating the role and potential mechanism of CaSR in EBI after SAH. Gadolinium trichloride (GdCI3), an agonist of CaSR, and NPS-2143, an inhibitor of CaSR, were administered intraperitoneally. The CaMKII inhibitor KN-93 was injected to intracerebroventricular. We found that CaSR expression was increased and widely expressed in neurons, astrocytes, and microglia after SAH. GdCI3 further deteriorated neurological function, brain edema, neurodegeneration, which were alleviated by NPS-2143. Also, GdCI3 increased the level of CaMKII phosphorylation, and upregulated expression of NLRP3, cleaved caspase-1, and IL-1ß, which were attenuated by NPS-2143. Besides, CaMKII inhibitor KN-93 down-regulated the upregulated expression of NLRP3, cleaved caspase-1, and IL-1ß induced by GdCI3. In conclusion, CaSR activation promotes early brain injury, which may be related to the CaMKII/NLRP3 signaling pathway.

Platelet-derived growth factor up-regulates Ca2+-sensing receptors in idiopathic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease associated with remodeling of the pulmonary artery. We previously reported that the Ca2+-sensing receptor (CaSR) is up-regulated in pulmonary arterial smooth muscle cells (PASMCs) from patients with idiopathic PAH (IPAH) and contributes to enhanced Ca2+ responses and excessive cell proliferation. However, the mechanisms underlying the up-regulation of CaSR have not yet been elucidated. We herein examined involvement of platelet-derived growth factor (PDGF) on CaSR expression, Ca2+ responses, and proliferation in PASMCs. The expression of PDGF receptors was higher in PASMCs from patients with IPAH than in PASMCs from normal subjects. In addition, PDGF-induced activation of PDGF receptors and their downstream molecules [ERK1/2, p38, protein kinase B, and signal transducer and activator of transcription (STAT) 1/3] were sustained longer in PASMCs from patients with IPAH. The PDGF-induced CaSR up-regulation was attenuated by small interfering RNA knockdown of PDGF receptors and STAT1/3, and by the treatment with imatinib. In monocrotaline-induced pulmonary hypertensive rats, the up-regulation of CaSR was reduced by imatinib. The combination of NPS2143 and imatinib additively inhibited the development of pulmonary hypertension. These results suggest that enhanced PDGF signaling is involved in CaSR up-regulation, leading to excessive PASMC proliferation and vascular remodeling in patients with IPAH. The linkage between CaSR and PDGF signals is a novel pathophysiological mechanism contributing to the development of PAH.-Yamamura, A., Nayeem, M. J., Al Mamun, A., Takahashi, R., Hayashi, H., Sato, M. Platelet-derived growth factor up-regulates Ca2+-sensing receptors in idiopathic pulmonary arterial hypertension.

The role of calcium sensing receptors in GLP-1 and PYY secretion after acute intraduodenal administration of L-Tryptophan in rats.

Objectives: The calcium-sensing receptor (CaSR), the major sensor of extracellular Ca2+, is expressed in various tissues, including the gastrointestinal tract. Although the essential ligand of CaSR is calcium, its activity can be regulated by aromatic L-amino acids. The expression of CaSR on enteroendocrine cells suggests that CaSR functions as a physiological amino acid sensor for gut hormone release. Here, we investigated the effects of L-tryptophan (L-Trp) on rat glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and insulin secretion, and the role of CaSR in this mechanism in vivo.Methods: The effects of intraduodenal L-Trp on GLP-1, PYY, and insulin secretion were investigated. A CaSR antagonist, NPS 2143, was administered to determine whether CaSR plays a role in L-Trp-mediated gut hormone release. Male Wistar rats were divided into L-Trp, L-Trp+NPS 2143, and L-Trp+vehicle groups. Blood samples were collected, before and after the intraduodenal infusions, for determining plasma glucose, L-Trp, insulin, GLP-1, and PYY levels.Results: Our study showed a significant increase in plasma GLP-1 and insulin levels, but not plasma PYY and glucose levels, following the acute intraduodenal administration of L-Trp. We demonstrated that CaSR plays a role in L-Trp-mediated GLP-1 secretion due to attenuation of GLP-1 release with the CaSR antagonist NPS 2143.Discussion: We demonstrated that GLP-1, but not PYY, secretion following intraduodenal L-Trp administration was mediated through calcium-sensing receptors. This mechanism underlying protein sensing in the gastrointestinal system may be important for the development of new therapeutic strategies without side effects for obesity and diabetes.

Role of Calcium Sensing Receptor in Streptozotocin-Induced Diabetic Rats Exposed to Renal Ischemia Reperfusion Injury.

BACKGROUND/AIMS: Renal ischemia/reperfusion (I/R) injury (RI/RI) is a common complication of diabetes, and it may be involved in altering intracellular calcium concentrations at its onset, which can result in inflammation, abnormal lipid metabolism, the production of reactive oxygen species (ROS), and nitroso-redox imbalance. The calcium-sensing receptor (CaSR) is a G-protein coupled receptor, however, the functional involvement of CaSR in diabetic RI/ RI remains unclear. The present study was intended to investigate the role of CaSR on RI/RI in diabetes mellitus (DM). METHODS: The bilateral renal arteries and veins of streptozotocin (STZ)-induced diabetic rats were subjected to 45-min ischemia followed by 2-h reperfusion with or without R-568 (agonist of CaSR) and NPS-2143 (antagonist of CaSR) at the beginning of I/R procedure. DM without renal I/R rats served as control group. The expressions of CaSR, calmodulin (CaM), and p47phox in the renal tissue were analyzed by qRT-PCR and Western blot. The renal pathomorphology, renal function, oxidative stress, inflammatory response, and calcium disorder were evaluated by detection of a series of indices by hematoxylin-eosin (HE) staining, transmission electron microscope (TEM), commercial kits, enzyme-linked immunosorbent assay (ELISA), and spectrophotofluorometry, respectively. RESULTS: Results showed that the expressions of CaSR, CaM, and p47phox in I/R group were significantly up-regulated as compared with those in DM group, which were accompanied by renal tissue injury, increased calcium, oxidative stress, inflammation, and nitroso-redox imbalance. CONCLUSION: These results suggest that activation of CaSR is involved in the induction of damage of renal tubular epithelial cell during diabetic RI/RI, resulting in lipid peroxidation, inflammatory response, nitroso-redox imbalance, and apoptosis.

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aß42 prompted by exogenous fibrillary or soluble Aß25-35 in human cortical astrocytes and neurons-therapeutic relevance to Alzheimer's disease.

The "amyloid-ß (Aß) hypothesis" posits that accumulating Aß peptides (Aßs) produced by neurons cause Alzheimer's disease (AD). However, the Aßs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aß25-35, an Aß42 proxy, evokes a surplus endogenous Aß42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAß25-35 and soluble (s)Aß25-35 on Aß42 and Aß40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aßs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAß25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aß42, while Aß40 also accrued but its secretion was unchanged. Accordingly, secreted Aß42/Aß40 ratio values rose for astrocytes and neurons. While slightly enhancing Aß40 secretion by fAß25-35-treated astrocytes, NPS 2143 specifically suppressed the fAß25-35-elicited surges of endogenous Aß42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aß42/Aß40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/ß-secretase and γ-secretase activity in fAß25-35-treated astrocytes. Compared to fAß25-35, sAß25-35 also stimulated Aß42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aß/CaSR-induced surplus secretion of endogenous Aß42 and hence further vicious cycles of Aß self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.

Pharmacoperones and the calcium sensing receptor: exogenous and endogenous regulators.

Calcium sensing receptor (CaSR) mutations or altered expression cause disorders of calcium handling. Recent studies suggest that reduced targeting to the plasma membrane is a feature common to many CaSR loss-of-function mutations. Allosteric agonists (calcimimetics) can rescue signaling of a subset of CaSR mutants. This review evaluates our current understanding of the subcellular site(s) for allosteric modulator rescue of CaSR mutants. Studies to date make a strong case for calcimimetic potentiation of signaling not only at plasma membrane-localized CaSR, but at the endoplasmic reticulum, acting as pharmacoperones to assist in navigation of multiple quality control checkpoints. The possible role of endogenous pharmacoperones, calcium and glutathione, in folding and stabilization of the CaSR extracellular and transmembrane domains are considered. Finally, the possibility that dihydropyridines act as unintended pharmacoperones of CaSR is proposed. While our understanding of pharmacoperone rescue of CaSR requires refinement, promising results to date argue that this may be a fruitful avenue for drug discovery.

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.

Synthesis of the calcilytic ligand NPS 2143.

(R)-3 (NPS 2143) is a negative allosteric modulator of the human calcium-sensing receptor (CaSR) and as such represents an important pharmacological tool compound for studying the CaSR. Herein, we disclose for the first time a complete experimental description, detailed characterisation and assessment of enantiomeric purity for (R)-3. An efficient, reproducible and scalable synthesis of (R)-3 that requires a minimum of chromatographic purification steps is presented. (R)-3 was obtained in excellent optical purity (er > 99:1) as demonstrated by chiral HPLC and the pharmacological profile for (R)-3 is in full accordance with that reported in the literature.

The role of calcium-sensitive receptor in ovalbumin-induced airway inflammation and hyperresponsiveness in juvenile mice with asthma.

The role of the calcium-sensitive receptor (CaSR) was assessed in a juvenile mouse model of asthma induced by ovalbumin (OVA). The experiment was divided into normal control, OVA, and OVA +2.5/5 mg/kg NPS2143 (a CaSR antagonist) groups. OVA induction was performed in all groups except the normal control, followed by assessing airway hyperresponsiveness (AHR) and lung pathological changes. Serum OVA-specific IgE and IgG1 were detected with an enzyme-linked immunosorbent assay (ELISA), and inflammatory cells were counted in bronchoalveolar lavage fluid (BALF). Real-time quantitative polymerase chain reaction, ELISA, and western blotting were performed to detect gene and protein expression. NPS2143 improved the OVA-induced AHR in mice, and AHR was higher in the OVA +2.5 mg/kg NPS2143 group than in the OVA +5 mg/kg NPS2143 group. Furthermore, NPS2143 reduced the production of OVA-specific IgE and IgG1 in serum and the number of eosinophils and lymphocytes in BALF in OVA mice with reduced CaSR expression in lung tissues. Besides, OVA-induced mice exhibited peribronchial and perivascular inflammatory cell infiltration, which was accompanied by severe goblet cell hyperplasia/hyperplasia and airway mucus hypersecretion. Furthermore, these mice exhibited increased levels of Interleukin (IL)-5, IL-13, MCP-1, and eotaxin, which were alleviated by NPS2143. The 5 mg/kg NPS2143 showed more effective than the 2.5 mg/kg treatment. CaSR expression was elevated in the lung tissues of OVA-induced asthmatic juvenile mice, whereas the CaSR antagonist NPS2143 reduced AHR and attenuated the inflammatory response in OVA-induced juvenile mice, possibly exerting therapeutic effects on childhood asthma.

Calcium-Sensing Receptor Antagonist NPS-2143 Inhibits Breast Cancer cell Proliferation, Migration and Invasion via Downregulation of p-ERK1/2, Bcl-2 and Integrin ß1 and Induces Caspase 3/7 Activation.

Purpose: Calcium-sensing receptor (CaSR) has been associated with breast cancer metastasis tothe bone. Targeting chemoattractant factors, such as calcium, that are released in response tobone resorption could prevent metastasis and induce apoptosis of cancer cells. In the presentstudy, we investigated the potential caspase 3/7 activation following treatment with a CaSRantagonist, NPS-2143, in breast cancer cells. In addition, the effects of NPS-2143 on breastcancer cell proliferation, migration and invasion were assessed. Methods: Colorimetric MTT assay was used to evaluate cell viability. Apo-one homogeneouscaspase-3/7 assay was used to measure caspase 3/7 activities in breast cancer cells. Cellmigration and invasion were assessed using scratch wound assay and matrigel invasionchambers, respectively. The protein expressions of p-ERK1/2, integrin ß1 and Bcl-2 wereevaluated using western blotting. Results: Our study revealed that NPS-2143 significantly reduced cell proliferation with halfmaximal (50%) inhibitory concentration (IC50) values of 4.08 and 5.71 µM in MDA-MB-231 and MCF-7 cells, respectively. NPS-2143 induced caspase 3/7 activation in MDA-MB-231 breastcancer cells which was accompanied with a remarkable reduction in the expression of Bcl-2antiapoptotic protein. NPS-2143 suppressed migratory and invasive abilities of MDA-MB-231cells with a significant reduction in the expression of p-ERK1/2 and integrin ß1 proteins. Conclusion: Our study confirms the ability NPS-2143 to suppress proliferative, migratory andinvasive effects of breast cancer cells which was accompanied by caspase 3/7 activation andsuggests the potential of NPS-2143 as a promising anti-cancer molecule in breast cancer.

Identification of annotated bioactive molecules that impair motility of the blood fluke Schistosoma mansoni.

Neglected tropical diseases are of growing worldwide concern and schistosomiasis, caused by parasitic flatworms, continues to be a major threat with more than 200 million people requiring preventive treatment. As praziquantel (PZQ) remains the treatment of choice, an urgent need for alternative treatments motivates research to identify new lead compounds that would complement PZQ by filling the therapeutic gaps associated with this treatment. Because impairing parasite neurotransmission remains a core strategy for control of parasitic helminths, we screened a library of 708 compounds with validated biological activity in humans on the blood fluke Schistosoma mansoni, measuring their effect on the motility on schistosomulae and adult worms. The primary phenotypic screen performed on schistosomulae identified 70 compounds that induced changes in viability and/or motility. Screening different concentrations and incubation times identified molecules with fast onset of activity on both life stages at low concentration (1 µM). To complement this study, similar assays were performed with chemical analogs of the cholinomimetic drug arecoline and the calcilytic molecule NPS-2143, two compounds that rapidly inhibited schistosome motility; 17 arecoline and 302 NPS-2143 analogs were tested to enlarge the pool of schistosomicidal molecules. Finally, validated hit compounds were tested on three functionally-validated neuroregulatory S. mansoni G-protein coupled receptors (GPCRs): Sm5HTR (serotonin-sensitive), SmGPR2 (histamine) and SmD2 (dopamine), revealing NPS-2143 and analogs as potent inhibitors of dopamine/epinine responses on both human and S. mansoni GPCRs. This study highlights the potential for repurposing known human therapeutic agents for potential schistosomicidal effects and expands the list of hits for further progression.

Verapamil prevents the effect of calcium-sensing receptor activation on the blood glucose and insulin levels in rats.

BACKGROUND: The Ca2+ triggered insulin exocytosis in ß cells of the pancreatic islets may be the result of Ca2+ influx through L-type voltage dependent calcium channels (VDCC) localized in the plasma membrane, as well as of liberation of Ca2+ from intracellular storages, induced by activation of the calcium receptor (CaR) coupled with the PLC enzyme present in the pancreatic islets. The present study was designated to determine, in in vivo experiments, the effects of CaR activation by R-568 and inhibition of the receptor by NPS 2143 on the plasma glucose and insulin levels in the presence of verapamil, a calcium channel blocker. METHODS: Wistar rats, after fasting for 14 h before the experiment, were anesthetized with inactin and loaded ip with 1 g/kg glucose. RESULTS: In comparison to the control group, the verapamil-induced blockade of the calcium channels in glucose loaded animals increased the blood glucose level and decreased the insulin level, whereas CaR activation with R-568 induced opposite effects. However, in the presence of verapamil, R-568 did not change the concentration of glucose or insulin versus the control animals. Verapamil infusion did not alter elevated glucose concentration in the NPS 2143 animals. At the same time, verapamil reduced the plasma insulin level and potentiated the drop of insulin concentration induced by NPS 2143. CONCLUSION: The observations suggest that under the in vivo conditions, calcium channel blockade may prevent changes in the blood glucose and insulin concentrations induced by the CaR activation.

Antagonism of Ca2+-sensing receptors by NPS 2143 is transiently masked by p38 activation in mouse brain bEND.3 endothelial cells.

Ca2+-sensing receptors (CaSR) are G protein-coupled receptors which are activated by a rise in extracellular Ca2+. CaSR activation has been known to inhibit parathyroid hormone release and stimulate calcitonin release from parathyroid glands and thyroid parafollicular C cells, respectively. The roles of CaSR in other cell types including endothelial cells (EC) are much less understood. In this work, we demonstrated protein and functional expression of CaSR in mouse cerebral EC (bEND.3). Unexpectedly, CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by edelfosine or U73122 but strongly suppressed by SK&F 96365, ruthenium red, and 2-aminoethoxydiphenyl borate (2-APB), suggesting involvement of TRPV and TRPC channels but not Gq-phospholipase C. Acute application of NPS2143, a negative allosteric modulator of CaSR, suppressed CaSR response. However, a 40-min NPS2143 pre-treatment surprisingly enhanced CaSR response. After 4-24 h of application, this enhancement faded away and suppression of CaSR response was observed again. Similar results were obtained when La3+ and Sr2+ were used as CaSR agonists. The transient NPS 2143 enhancement effect was abolished by SB203580, a p38 inhibitor. Consistently, NPS 2143 triggered a transient p38 activation. Taken together, results suggest that in bEND.3 cells, NPS 2143 caused acute suppression of CaSR response, but then elicited a transient enhancement of CaSR response in a p38-dependent manner. NPS 2143 effects on CaSR in bEND.3 cells therefore depended on drug exposure time. These findings warrant cautious use of this agent as a CaSR modulator and potential cardiovascular drug.

NPS 2143, a selective calcium-sensing receptor antagonist inhibits lipopolysaccharide-induced pulmonary inflammation.

NPS 2143, a novel and selective antagonist of calcium-sensing receptor (CaSR) has been reported to possess anti-inflammatory activity. In the present study, we examined the protective effect of NPS 2143 on lipopolysaccharide (LPS)-induced acute lung injury (ALI). NPS 2143 pretreatment significantly inhibited the influx of inflammatory cells and the expression of monocyte chemoattractant protein-1 (MCP-1) in the lung of mice with LPS-induced ALI. NPS 2143 decreased the levels of neutrophil elastase (NE) and protein concentration in the bronchoalveolar lavage fluid (BALF). NPS 2143 also reduced the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the BALF and serum. In addition, NPS 2143 attenuated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and increased the activation of AMP-activated protein kinase (AMPK) in the lung. NPS 2143 also downregulated the activation of nuclear factor-kappa B (NF-κB) in the lung. In LPS-stimulated H292 airway epithelial cells, NPS 2143 attenuated the releases of IL-6 and MCP-1. Furthermore, NPS 2143 upregulated the activation of AMPK and downregulated the activation of NF-κB. These results suggest that NPS 2143 could be potential agent for the treatment of inflammatory diseases including ALI.

Activity of the calcium-sensing receptor influences blood glucose and insulin levels in rats.

BACKGROUND: The calcium-sensing receptor (CaR) has been found not only in parathyroid glands but also in other tissues, e.g. in ß cells of the pancreatic islets. Therefore, CaR might likely mediate the mechanism of insulin secretion. The present study was designed to examine the in vivo effects of R-568, a CaR agonist, and NPS2143, a CaR inhibitor, on plasma insulin and blood glucose concentrations. METHODS: Wistar rats, after fasting for 14h before the experiment, were anesthetized with inactin and loaded ip with 1g/kg glucose. RESULTS: 20, 120 and 180min after iv R-568 administration, plasma insulin increased markedly (by approximately 30%), in glucose-loaded rats, as compared to the control animals. Simultaneously, 180min after R-568 administration, a significant drop by approximately 12% in blood glucose was observed. In contrast, administration of R-568 in rats not given glucose, did not influence the blood glucose or plasma insulin concentrations vs. the control group. Administration of NPS2143 increased the blood glucose level markedly (by about 18% vs. control group) at 180 and 210min of the experiment. Simultaneously, a significant decrease of insulin concentration was observed vs. control group (by about 18 and 23%, respectively). CONCLUSION: We suggest that modulation of the CaR activity may participate in the mechanisms which mediate insulin secretion in rats.

NPS2143 Inhibits MUC5AC and Proinflammatory Mediators in Cigarette Smoke Extract (CSE)-Stimulated Human Airway Epithelial Cells.

Mucus overproduction is a fundamental hallmark of COPD that is caused by exposure to cigarette smoke. MUC5AC is one of the main mucin genes expressed in the respiratory epithelium, and its transcriptional upregulation often correlates with increased mucus secretion. Calcium-sensing receptor (CaSR) antagonists have been reported to possess anti-inflammatory effects. The purpose of the present study was to investigate the protective role of NPS2143, a selective CaSR antagonist on cigarette smoke extract (CSE)-stimulated NCI-H292 mucoepidermoid human lung cells. Treatment of NPS2143 significantly inhibited the expression of MUC5AC in CSE-stimulated H292 cells. NPS2143 reduced the expression of MMP-9 in CSE-stimulated H292 cells. NPS2143 also decreased the release of proinflammatory cytokines such as IL-6 and TNF-α in CSE-stimulated H292 cells. Furthermore, NPS2143 attenuated the activation of MAPKs (JNK, p38, and ERK) and inhibited the nuclear translocation of NF-κB in CSE-stimulated H292 cells. These results indicate that NPS2143 had a therapeutic potential in COPD.

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels.

The present study investigates the effect of commonly used negative and positive allosteric modulators of the calcium-sensing receptor (CaSR) on vascular reactivity. In wire myography studies, increasing [Ca2+]o from 1mM to 6mM induced concentration-dependent relaxations of methoxamine-induced pre-contracted rabbit mesenteric arteries, with 6mM [Ca2+]o producing almost complete relaxation. [Ca2+]o-induced relaxations were attenuated in the presence of the calcilytics Calhex-231 and NPS 2143, and abolished by the removal of the endothelium. In addition to their calcilytic effects, Calhex-231 and NPS 2143 also produced concentration-dependent inhibitions of methoxamine- or KCl-induced precontracted tone, which were unaffected by removal of the endothelium and unopposed in the presence of the calcimimetic Calindol. In vessels with depleted Ca2+ stores, contractions mediated by Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) were inhibited by Calhex231. In freshly isolated single rabbit mesenteric artery smooth muscle cells, Calhex-231 and NPS 2143 inhibited whole-cell VGCC currents. Application of Calindol also inhibited methoxamine- and KCl-induced pre-contracted tone, and inhibited whole-cell VGCC currents. In conclusion, in addition to their CaSR-mediated actions in the vasculature, Calhex-231, NPS 2143 and Calindol reduce vascular contractility via direct inhibition of VGCCs.

Calcilytics enhance sildenafil-induced antiproliferation in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and fatal disease of the pulmonary artery resulting from currently unidentified etiology. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. Phosphodiesterase type 5 (PDE5) inhibitors have been clinically used in the treatment of IPAH. Recently, we have shown that Ca(2+)-sensing receptor (CaSR) antagonists, or calcilytics, inhibit excessive cell proliferation of pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. In this study, the additive or synergistic effect of calcilytics on antiproliferation following PDE5 inhibition was examined in IPAH-PASMCs by MTT assay. Treatment with sildenafil blocked the excessive cell proliferation of IPAH-PASMCs in a concentration-dependent manner with an IC50 value of 16.9µM. However, sildenafil (0.03-100µM) did not affect the cell growth of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Co-treatment with 0.3µM NPS2143, a calcilytic, additively enhanced the antiproliferative effect induced by sildenafil (3 or 30µM) in IPAH-PASMCs. Additionally, the inhibitory effect of calcilytics, NPS2143 or Calhex 231 (1 or 10µM), on excessive cell proliferation of IPAH-PASMCs was synergistic increased in the presence of 1µM sildenafil. Similar results were obtained by BrdU incorporation assay. These findings reveal that calcilytics additively/synergistically enhance the antiproliferative activity mediated by PDE5 inhibition, suggesting that a combination therapy of a PDE5 inhibitor with a calcilytic may be useful as a novel therapeutic approach for IPAH.