Ca2+-sensing Receptor Research Articles

Molecular Mechanism of Dihydropyridine Ca2+ Channel Blockers in Pulmonary Hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and fatal disease of unidentified pathogenesis. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. In pulmonary arterial smooth muscle cells (PASMCs), an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) triggers vasoconstriction and stimulates cell proliferation leading to vascular remodeling. However, dihydropyridine-type voltage-dependent Ca2+ channel blockers are only effective in very few patients with IPAH (<10%). It is unclear why dihydropyridine Ca2+ channel blockers are not therapeutically effective in a majority of IPAH patients. We have previously shown that extracellular Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, and it contributes to enhanced [Ca2+]cyt responses and augmented cell proliferation. In this study, the effects of dihydropyridine Ca2+ channel blockers on [Ca2+]cyt responses mediated by CaSR were examined in IPAH-PASMCs. Nifedipine (dihydropyridines) enhanced the CaSR-mediated increase in [Ca2+]cyt in IPAH-PASMCs, but not in PASMCs from normal subjects. Nicardipine (dihydropyridines) and Bay K 8644 (a dihydropyridine Ca2+ channel activator) also augmented the CaSR-mediated [Ca2+]cyt increase in IPAH-PASMCs. In contrast, non-dihydropyridine Ca2+ channel blockers such as diltiazem (benzothiazepines) and verapamil (phenylalkylamines) had no effect on the [Ca2+]cyt response in IPAH-PASMCs. Finally, in monocrotaline-induced pulmonary hypertensive rats, nifedipine caused further increase in right ventricular systolic pressure and thus right ventricular hypertrophy. In conclusion, dihydropyridine Ca2+ channel blockers could exacerbate symptoms of pulmonary hypertension in IPAH patients with upregulated CaSR in PASMCs.

Notch activation of Ca2+-sensing receptor mediates hypoxia-induced pulmonary hypertension.

A recent study from our group demonstrated that the Ca2+-sensing receptor (CaSR) was upregulated and that the extracellular Ca2+-induced increase in the cytosolic Ca2+ concentration [Ca2+]cyt was enhanced in pulmonary arterial smooth muscle cells (PASMCs) from patients with idiopathic pulmonary arterial hypertension. Here, we examined whether hypoxia-induced activation of Notch signaling leads to the activation and upregulation of CaSR in hypoxia-induced pulmonary hypertension (HPH). The activation of Notch signaling with Jag-1, a Notch ligand, can activate the function and increase the expression of CaSR in acute and chronic hypoxic PASMCs. Downregulation of Notch3 with a siRNA attenuates the extracellular Ca2+-induced increase in [Ca2+]cyt and the increase in hypoxia-induced PASMC proliferation in acute hypoxic rat PASMCs. Furthermore, we tested the prevention and rescue effects of a γ-secretase inhibitor (DAPT) in HPH rats. For the Jag-1-treated group, right ventricular systolic pressure (RVSP), right heart hypertrophy (RV/LV+S ratio), and the level of right ventricular myocardial fibrosis were higher than the hypoxia alone group. Meanwhile, DAPT treatment prevented and rescued pulmonary hypertension in HPH rats. The Notch activation of CaSR mediates hypoxia-induced pulmonary hypertension. Understanding the new molecular mechanisms that regulate [Ca2+]cyt and PASMC proliferation is critical to elucidating the pathogenesis of HPH and the development of novel therapies for pulmonary hypertension.

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 Ca2+-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.

Regulation of Polycystin-2 TRPP2 Associated Cation Currents in the Renal Epithelial Cell Line LLC-PK1 by the Calcium-Sensing Receptor

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel that contributes to Ca2+ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to how Ca2+ triggers regulatory mechanisms that control PC2 channel function. In this study, we explored the effect external Ca2+ has on endogenous PC2 channel function in wild type LLC-PK1 renal epithelial cells. Whole cell currents were obtained with the voltage clamping technique at different external Ca2+ concentrations, and observed that the basal whole cell conductance in normal Ca2+ (1.2 mM), decreased by 30.2% in zero (nominal) Ca2+, while it increased by 38% in the presence of high external Ca2+ (6.2 mM). Exposure to high Ca2+ also was associated with PC2 relocation to the plasma membrane. The high Ca2+-induced whole cell currents were inhibited by intracellular dialysis with active, but not denatured, anti-PC2 antibody. To confirm the possible role of a Ca2+ sensing receptor (CaR) on the external Ca2+ modulation of PC2 currents, other maneuvers were also tested, including the CaR agonist spermine, which stimulated the whole cell currents under Ca2+-free conditions, and gentamicin, which also increased the PC2-associated whole-cell currents in normal external Ca2+. Conversely, intracellular dialysis with ketamine used to inhibit the CaR, eliminated both the high Ca2+, and gentamicin-induced whole cell currents of LLC-PK1 cells. The presence of CaR was confirmed by immunocytochemistry as well as its external Ca2+ dependent colocalization with PC2. The data support a novel mechanism for the regulation of PC2 by external Ca2+, involving the Ca2+-sensing receptor. This regulation by Ca2+ may have important implications to the physiology of renal epithelial cells.

Intracellular Ca2+ oscillations generated via the extracellular Ca2+-sensing receptor (CaSR) in response to extracellular Ca2+ or l-phenylalanine: Impact of the highly conservative mutation Ser170Thr

The extracellular Ca2+-sensing receptor (CaSR) is an allosteric protein that responds to changes in the extracellular concentration of Ca2+ ([Ca2+]e) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]e stimulates sinusoidal oscillations in [Ca2+]i whereas aromatic amino acid-induced CaR activation in the presence of a threshold [Ca2+]e promotes transient oscillations in [Ca2+]i. Here, we examined spontaneous and ligand-evoked [Ca2+]i oscillations in single HEK-293 cells transfected with the wild type CaSR or with a mutant CaSR in which Ser170 was converted to Thr (CaSRS170T). Our analysis demonstrates that cells expressing CaSRS170T display [Ca2+]i oscillations in the presence of low concentrations of extracellular Ca2+ and respond to L-Phe with robust transient [Ca2+]i oscillations. Our results indicate that the S170T mutation induces a marked increase in CaSR sensitivity to [Ca2+]e and imply that the allosteric regulation of the CaSR by aromatic amino acids is not only mediated by an heterotropic positive effect on Ca2+ binding cooperativity but, as biased agonists, aromatic amino acids stabilize a CaSR conformation that couples to a different signaling pathway leading to transient [Ca2+]i oscillations.

Effect of extracellular calcium on regucalcin expression and cell viability in neoplastic and non-neoplastic human prostate cells

Extracellular calcium (Ca2+o) and its receptor, the Ca2+-sensing receptor (CaSR), play an important role in prostate physiology, and it has been shown that the deregulation of Ca2+ homeostasis and the overexpression of CaSR are involved in prostate cancer (PCa). Regucalcin (RGN), a Ca2+-binding protein that plays a relevant role in intracellular Ca2+ homeostasis, was identified as an under-expressed protein in human PCa. Moreover, RGN was associated with suppression of cell proliferation, suggesting that the loss of RGN may favor development and progression of PCa. This work aims to unveil the role of Ca2+o on RGN expression and viability of non-neoplastic (PNT1A) and neoplastic (LNCaP) prostate cell lines. It was demonstrated that Ca2+o up-regulates RGN expression in both cell lines, but important differences were found between cells for dose- and time-responses to Ca2+o treatment. It was also shown that high [Ca2+]o triggers different effects on cell proliferation of neoplastic and non-neoplastic PCa cells, which seems to be related with RGN expression levels. This suggests the involvement of RGN in the regulation of cell proliferation in response to Ca2+o treatment. Also, the effect of Ca2+o on CaSR expression seems to be dependent of RGN expression, which is strengthened by the fact that RGN-knockdown in PNT1A cells increases the CaSR expression, whereas transgenic rats overexpressing RGN exhibit low levels of CaSR. Overall, our results highlighted the importance of RGN as a regulatory protein in Ca2+-dependent signaling pathways and its deregulation of RGN expression by Ca2+o may contribute for onset and progression of PCa.

The Functional Coupling of CaSR and TRPC6 Contributes to Enhanced Proliferation in Pulmonary Arterial Smooth Muscle Cells

An increase in cytosolic free Ca2+ ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for PASMC proliferation.We have previously shown both extracellular Ca2+‐sensing receptor (CaSR), a G protein‐coupled receptor, and TRPC6, a transient receptor potential cation channel, are upregulated in PASMC from idiopathic pulmonary arterial hypertension (IPAH) patients. Furthermore, extracellular Ca2+‐induced increase in [Ca2+]cyt was enhanced in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) compared to normal patients. However, the implication of increased CaSR and TRPC6 in pulmonary hypertension remains unknown. In this study, our data show the inhibition of CaSR by siRNA significantly inhibited hypoxia‐induced proliferation in both normal and IPAH PASMC. Furthermore, IPAH‐PASMC treated with siRNA targeting CaSR or TRPC6 inhibited Ca2+‐induced Ca2+ entry, suggesting functional coupling of CaSR and TRPC6. In addition, PDGF‐induced proliferation in IPAH‐PASMC was inhibited by blocking either CaSR or TRCP6 via inhibitors NPS2143 and 2‐APB, respectfully, while NPS2143 was unable to block PDGF‐induced proliferation in normal PASMC. The data from this study indicate that upregulation of CaSR is functionally coupled to TRPC6 channels to increase [Ca2+]cyt; the enhanced extracellular Ca2+‐induced increase in [Ca2+]cyt due to upregulation of CaSR and TRPC6 plays an important role in PASMC proliferation. This suggests that CaSR could be a novel therapeutic candidate for the treatment of PH.

Acetylcholine protects mesenteric arteries against hypoxia/reoxygenation injury via inhibiting calcium-sensing receptor

The Ca2+-sensing receptor (CaSR) plays an important role in regulating vascular tone. In the present study, we investigated the positive effects of the vagal neurotransmitter acetylcholine by suppressing CaSR activation in mesenteric arteries exposed to hypoxia/reoxygenation (H/R). The artery rings were exposed to a modified ‘ischemia mimetic’ solution and an anaerobic environment to simulate an H/R model. Our results showed that acetylcholine (10−6 mol/L) significantly reduced the contractions induced by KCl and phenylephrine and enhanced the endothelium-dependent relaxation induced by acetylcholine. Additionally, acetylcholine reduced CaSR mRNA expression and activity when the rings were subjected to 4 h of hypoxia and 12 h of reoxygenation. Notably, the CaSR antagonist NPS2143 significantly reduced the contractions but did not improve the endothelium-dependent relaxation. When a contractile response was achieved with extracellular Ca2+, both acetylcholine and NPS2143 reversed the H/R-induced abnormal vascular vasoconstriction, and acetylcholine reversed the calcimimetic R568-induced abnormal vascular vasoconstriction in the artery rings. In conclusion, this study suggests that acetylcholine ameliorates the dysfunctional vasoconstriction of the arteries after H/R, most likely by decreasing CaSR expression and activity, thereby inhibiting the increase in intracellular calcium concentration. Our findings may be indicative of a novel mechanism underlying ACh-induced vascular protection.

Computational analysis of the extracellular domain of the Ca2+-sensing receptor: An alternate model for the Ca2+ sensing region

The extracellular Ca2+ sensing receptor (CaSR) belongs to Class C G-protein-coupled receptors (GPCRs) which include receptors for amino acids, γ-aminobutyric acid and glutamate neurotransmitters. CaSR has been described as having an extended sequence containing a Ca2+ binding pocket within an extracellular amino (N)-terminal domain, called a Venus Fly Trap (VFT) module. CaSR is thought to consist of three domains: 1) a Ca2+−sensory domain, 2) a region containing 7 transmembrane (TM) helices, and 3) a carboxy (C)-terminal tail. We find that SPOCTOPUS (a combination of hidden Markov models and artificial neural networks) predicts that Homo sapiens CaSR contains two additional TM helices (190D – G210; 262S–E282), with the second TM helix containing a pore-lining region (265K – I280). This predicts that the putative Ca2+ sensory domain is within an extracellular loop, N-terminal to the highly conserved heptahelical bundle. This loop contains both the cysteine-rich domain (537V – C598) and a 14 residue “linker” sequence (599I – F612) thought to support signal transmission to the heptahelical bundle. Thus domain 1 may contain a 189 residue N-terminal extracellular region followed successively by TM-1, a short intracellular loop, TM-2 and a 329 residue extracellular loop; rather than the proposed 620 residue VFT module based on crystallography of the N-terminal region of mGluR1. Since the topologies of the two proteins differ, the published CaSR VFT model is questionable. CaSR also contains multiple caveolin-binding motifs and cholesterol-binding (CRAC/CARC) domains, facilitating localization to plasma membrane lipid rafts. Ion sensing may involve combination of pore-lining regions from CaSR dimers and CaSR-bound caveolins to form ion channels capable of monitoring ionized Ca2+ levels.

Negative feedback from CaSR signaling to aquaporin-2 sensitizes vasopressin to extracellular Ca2.

We previously described that high luminal Ca(2+) in the renal collecting duct attenuates short-term vasopressin-induced aquaporin-2 (AQP2) trafficking through activation of the Ca(2+)-sensing receptor (CaSR). Here, we evaluated AQP2 phosphorylation and permeability, in both renal HEK-293 cells and in the dissected inner medullary collecting duct, in response to specific activation of CaSR with NPS-R568. In CaSR-transfected cells, CaSR activation drastically reduced the basal levels of AQP2 phosphorylation at S256 (AQP2-pS256), thus having an opposite effect to vasopressin action. When forskolin stimulation was performed in the presence of NPS-R568, the increase in AQP2-pS256 and in the osmotic water permeability were prevented. In the freshly isolated inner mouse medullar collecting duct, stimulation with forskolin in the presence of NPS-R568 prevented the increase in AQP2-pS256 and osmotic water permeability. Our data demonstrate that the activation of CaSR in the collecting duct prevents the cAMP-dependent increase in AQP2-pS256 and water permeability, counteracting the short-term vasopressin response. By extension, our results suggest the attractive concept that CaSR expressed in distinct nephron segments exerts a negative feedback on hormones acting through cAMP, conferring high sensitivity of hormone to extracellular Ca(2+).

Do Astrocytes Collaborate with Neurons in Spreading the “Infectious” Aβ and Tau Drivers of Alzheimer’s Disease?

Evidence has begun emerging for the “contagious” and destructive Aβ42 (amyloid-beta42) oligomers and phosphorylated Tau oligomers as drivers of sporadic Alzheimer’s disease (AD), which advances along a pathway starting from the brainstem or entorhinal cortex and leading to cognition-related upper cerebral cortex regions. Seemingly, Aβ42 oligomers trigger the events generating the neurotoxic Tau oligomers, which may even by themselves spread the characteristic AD neuropathology. It has been assumed that only neurons make and spread these toxic drivers, whereas their associated astrocytes are just janitorial bystanders/scavengers. But this view is likely to radically change since normal human astrocytes freshly isolated from adult cerebral cortex can be induced by exogenous Aβ25-35, an Aβ42 proxy, to make and secrete increased amounts of endogenous Aβ42. Thus, it would seem that the steady slow progression of AD neuropathology along specific cognition-relevant brain networks is driven by both Aβ42 and phosphorylated Tau oligomers that are variously released from increasing numbers of “contagion-stricken” members of tightly coupled neuron–astrocyte teams. Hence, we surmise that stopping the oversecretion and spread of the two kinds of “contagious” oligomers by such team members, perhaps via a specific CaSR (Ca2+-sensing receptor) antagonist like NPS 2143, might effectively treat AD.

The ZnR/GPR39 Interacts With the CaSR to Enhance Signaling in Prostate and Salivary Epithelia

Zinc signaling is mediated by the zinc sensing receptor, ZnR, recently suggested to be the same receptor as G-protein coupled receptor 39, GPR39. However, it is unknown if GPR39 is mediating Zn(2+) -dependent signaling in prostate and salivary tissue where changes in zinc concentrations are frequent and of physiological significance. Here, we show that GPR39 is mediating Zn(2+) -dependent Ca(2+) responses and is regulating activity of MAP and PI3 pathways in prostate cancer cells, PC3, and ductal salivary gland cells, HSY. We next ask whether ZnR/GPR39 interacts with other GPCR family members. We find that endogenous ZnR/GPR39 activity is regulated by the expression and activity of another cation sensing GPCR, the Ca(2+) -sensing receptor (CaSR). Although CaSR is not activated by Zn(2+), co-expression of CaSR and ZnR/GPR39 synergistically enhances Ca(2+) responses in PC3 and HSY cells. Silencing of the CaSR using siRNA or a dominant negative construct reduces the Zn(2+) -dependent signaling. Importantly, overexpression of GPR39 in HEK293 cells is sufficient to trigger Zn(2+) -dependent responses. Nevertheless, application of the CaSR agonist spermine, at concentration below its threshold, enhanced Zn(2+) -dependent Ca(2+) response. Our results suggest that the CaSR interacts with ZnR/GPR39 and thereby regulates its activity. Finally, we show that in PC3 cells ZnR/GPR39 is required for mediating the Zn(2+) -dependent activation of MAPK and PI3K, pathways leading to enhanced cell growth. Importantly, Zn(2+) -dependent activation of ZnR/GPR39 also enhances the expression of the Ca(2+) -binding protein S100A4 that is linked to invasion of prostate cancer cells.

Functional expression of the multimodal extracellular calcium-sensing receptor in the complex pulmonary neuroepithelial body microenvironment

The Ca2+-sensing receptor (CaSR) is the master regulator of whole body extracellular free ionized Ca2+ (Ca2+o). In addition to sensing Ca2+o, the CaSR integrates inputs from a variety of different physiological stimuli such as nutrient availability, redox status, pH and ionic strength. Owing to this multimodal ability, the CaSR is expressed in many regions outside of the Ca2+ homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR.

Abstract 474: High Salt Diet Modulates Ca 2 +-Sensing Receptor and Cytochrome P450 Isoform Expression in Mesenteric Arteries in Dahl Salt-Sensitive Rats

The Ca 2+ -sensing receptor (CaSR) mediates relaxation of isolated, phenylephrine-contracted mesenteric arteries, which express the receptor in the sensory network. We tested the hypothesis that high salt alters the expression of CaSR and its CYP enzymes downstream of the receptor in salt-sensitive hypertension. Dahl salt−sensitive (DS) and Dahl salt-resistant (DR) rats were placed on an 8% NaCl diet (HS) for 1, 2, and 4 weeks. Using tail cuff plethysmyography, blood pressures were measured before and after HS diet treatment. Our results show a significant increase in blood pressure in DS animals on HS diet (Control : 116 ± 4.8 mmHg; 1 week HS: 164.4 ± 8.7 mmHg; 2 weeks HS: 175.1 ± 7.2 mmHg; p &lt; 0.05 , ANOVA). Western blot analysis of proteins extracted from mesenteric arteries showed an increase in CaSR, CYP1A1, and CYP2J2 expression in DS rats on HS diet, but not in arteries from DR rats on the same diet. The data show that HS increased mean arterial blood pressure, as expected, and increased the expression of the mature form of the CaSR while decreasing expression of the immature form. Increased CYP isoform expression was observed as well, however, these changes did not reduce blood pressure. These new findings suggest an abnormality in CaSR activity in salt −sensitive hypertension, which may provide a molecular target for anti-hypertensive therapies.

Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release

Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 μm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 μm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10 μm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 μm) and attenuated by the protein kinase C inhibitor Gö6983 (3 μm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 μm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage-gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.

The key role of correlation between MR spectroscopy choline peak and calcium sensing receptor for breast cancer: Diagnosis by a 3T MR scanner.

e22132 Background: The Ca2+-sensing receptor (CaSR) regulates the Ca2+ signaling and its expression is present in both normal and malignant breast tissues. Recently, we demonstrated in a pre-clinical setting the CaSR expression in breast cancer mouse model by manganese enhanced MRI (Baio G et al. Eur Rad 2011). Alteration in choline phospholipid metabolism as detected by MRI, is a common feature of breast and many other cancer cells or tumors. Since CaSR-ChoK signaling plays an important role in breast cancer, we compared the MRS (MR spectroscopy) choline peak by a 3T MR scanner between calcium sensing receptor positive and negative breast cancers. Methods: Breast MRI of 16 consecutive patients with breast lesions were performed by a 3T MR and MRS findings were defined as positive by the visual inspection for the presence or the absence of choline peak. MRS imaging were compared with histologic findings and the pattern of CaSR by immunohistochemistry. The immunohistochemical results were qualitatively classified according to intensity and pattern of the staining, using a 6-point scale to score the intensity of the CaSR expression (0, absent; 5, intense, widespread expression). Results: A choline peak was present in 10 of 15 malignant lesions and the CaSR expression was between 3 to 5 score at immunohistochemestry analysis (p&lt;.0006). In 4 of 15 malignant lesions the choline peak was absent or not consistent at the visual analysis with a CaSR patterns of 1-2. In one benign lesion (papillary hyperplasia), the choline peak was consistent and the CaSR pattern was with score 3. Conclusions: In this study we evaluated if MRS presence or absence of choline peak well correlated with CaSR positive and negative breast cancers. We observed a choline peak with an high level expression of CaSR (score 3-5) in 67% of the breast lesions, while in 27% of the lesions the choline peak was absent with a low CaSR expression (score 1-2). These preliminary results support the hypothesis that CaSR represents an important role in the production of choline in breast cancer, determining an increase of his production when CaSR is expressed at high level and implicating and absence of choline peak when CaSR is not expressed.

Nitric-Oxide Synthase Knockout Modulates Ca2+-Sensing Receptor Expression and Signaling in Mouse Mesenteric Arteries

Extracellular calcium (Ca²⁺(e))-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries is dependent on an intact perivascular sensory nerve network that expresses the Ca²⁺-sensing receptor (CaSR). Activation of the receptor stimulates an endocannabinoid vasodilator pathway, which is dependent on cytochrome P450 and phospholipase A₂ but largely independent of the endothelium. In the present study, we determined the role of nitric oxide (NO) in perivascular nerve CaSR-mediated relaxation of PE-contracted mesenteric resistance arteries isolated from mice. Using automated wire myography, we studied the effects of NO synthase (NOS) gene knockout (NOS(-/-)) and pharmacologic inhibition of NOS on Ca²⁺(e)-induced relaxation of PE-contracted arteries. Endothelial NOS knockout (eNOS(-/-)) upregulates but neuronal NOS knockout (nNOS(-/-)) downregulates CaSR expression. NOS(-/-) reduced maximum Ca²⁺(e)-induced relaxation with no change in EC₅₀ values, with eNOS(-/-) having the largest effect. The responses of vessels to calindol and Calhex 231 indicate that the CaSR mediates relaxation. L-N⁵-(1-iminoethyl)-ornithine reduced Ca²⁺(e)-induced relaxation of PE-contracted arteries from C57BL/6 control mice by ≈38% but had a smaller effect in vessels from eNOS(-/-) mice. 7-Nitroindazole had no significant effect on relaxation of arteries from NOS(-/-) mice, but both N(G)-nitro-L-arginine methylester and N(G)-monomethyl-L-arginine significantly reduced the relaxation maxima in all groups. Interestingly, the nNOS-selective inhibitor S-methyl-L-thiocitrulline significantly increased the EC₅₀ value by ≈60% in tissues from C57BL/6 mice but reduced the maximum response by ≈80% in those from nNOS(-/-) mice. Ca²⁺-activated big potassium channels play a major role in the process, as demonstrated by the effect of iberiotoxin. We conclude that CaSR signaling in mesenteric arteries stimulates eNOS and NO production that regulates Ca²⁺(e)-induced relaxation.

Key role for the multimodal CaSR in pulmonary neuroepithelial body microenvironment physiology

The neuroepithelial body (NEB) microenvironment consists of densely innervated groups of neuroendocrine cells, representing complex sensory receptors in the airways, and a stem cell population. The extracellular Ca2+‐sensing receptor (CaSR) is a master regulator of extracellular free ionized Ca2+ ([Ca2+]o), and a multimodal sensor able to integrate inputs from a variety of physiological stimuli. Immunostaining and (q)RT‐PCR recently revealed a selective CaSR expression on the plasmamembrane of NEB cells in postnatal mouse lungs. We further aimed at exploring potential roles for the CaSR in pulmonary NEB physiology. Ca2+ imaging of mouse lung slices demonstrated a CaSR‐mediated rise in intracellular Ca2+ concentration ([Ca2+]i) following an increase of [Ca2+]o or application of established CaSR agonists. The CaSR was shown to be involved in regulating baseline [Ca2+]i in NEBs, and in mediating intercellular communication in the NEB microenvironment. By integrating multiple physio(patho)logical signals and coordinating intra‐ and extracellular Ca2+ signaling, the CaSR appears to serve key roles for the NEB microenvironment as a complex chemosensory receptor and airway stem cell niche.

Dihydropyridine Ca 2+ Channel Blockers Increase Cytosolic [Ca 2+ ] by Activating Ca 2+ -sensing Receptors in Pulmonary Arterial Smooth Muscle Cells

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC. The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt). The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

Activation of the Ca2+-sensing receptor increases renal claudin-14 expression and urinary Ca2+ excretion

Kidney stones are a prevalent clinical condition imposing a large economic burden on the healthcare system. Hypercalciuria remains the major risk factor for development of a Ca(2+)-containing stone. The kidney's ability to alter Ca(2+) excretion in response to changes in serum Ca(2+) is in part mediated by the Ca(2+)-sensing receptor (CaSR). Recent studies revealed renal claudin-14 (Cldn14) expression localized to the thick ascending limb (TAL) and its expression to be regulated via the CaSR. We find that Cldn14 expression is increased by high dietary Ca(2+) intake and by elevated serum Ca(2+) levels induced by prolonged 1,25-dihydroxyvitamin D3 administration. Consistent with this, activation of the CaSR in vivo via administration of the calcimimetic cinacalcet hydrochloride led to a 40-fold increase in Cldn14 mRNA. Moreover, overexpression of Cldn14 in two separate cell culture models decreased paracellular Ca(2+) flux by preferentially decreasing cation permeability, thereby increasing transepithelial resistance. These data support the existence of a mechanism whereby activation of the CaSR in the TAL increases Cldn14 expression, which in turn blocks the paracellular reabsorption of Ca(2+). This molecular mechanism likely facilitates renal Ca(2+) losses in response to elevated serum Ca(2+). Moreover, dysregulation of the newly described CaSR-Cldn14 axis likely contributes to the development of hypercalciuria and kidney stones.