Ca2+-sensing Receptor Research Articles

Polyamine stimulation perturbs intracellular Ca2+ homeostasis and decreases viability of breast cancer BT474 cells.

Intracellular polyamines such as spermine and spermidine are essential to cell growth in normal and especially in cancer cells. However, whether extracellular polyamines affect cancer cell survival is unknown. We therefore examined the actions of extracellular polyamines on breast cancer BT474 cells. Our data showed that spermine, spermidine, and putrescine decreased cell viability by apoptosis. These polyamines also elicited Ca2+ signals, but the latter were unlikely triggered via Ca2+-sensing receptor (CaSR) as BT474 cells have been demonstrated previously to lack CaSR expression. Spermine-elicited Ca2+ response composed of both Ca2+ release and Ca2+ influx. Spermine caused a complete discharge of the cyclopiazonic acid (CPA)-sensitive Ca2+ pool and, expectedly, endoplasmic reticulum (ER) stress. The Ca2+ influx pore opened by spermine was Mn2+-impermeable, distinct from the CPA-triggered store-operated Ca2+ channel, which was Mn2+-permeable. Spermine cytotoxic effects were not due to oxidative stress, as spermine did not trigger reactive oxygen species formation. Our results therefore suggest that spermine acted on a putative polyamine receptor in BT474 cells, causing cytotoxicity by Ca2+ overload, Ca2+ store depletion, and ER stress.

The CaSR in Pathogenesis of Breast Cancer: A New Target for Early Stage Bone Metastases.

The Ca2+-sensing receptor (CaSR) is a class-C G protein-coupled receptor which plays a pivotal role in calciotropic processes, primarily in regulating parathyroid hormone secretion to maintain systemic calcium homeostasis. Among its non-calciotropic roles, where the CaSR sits at the intersection of myriad processes, it has steadily garnered attention as an oncogene or tumor suppressor in different organs. In maternal breast tissues the CaSR promotes lactation but in breast cancer it acts as an oncoprotein and has been shown to drive the pathogenesis of skeletal metastases from breast cancer. Even though research has made great strides in treating primary breast cancer, there is an unmet need when it comes to treatment of metastatic breast cancer. This review focuses on how the CaSR leads to the pathogenesis of breast cancer by contrasting its role in healthy tissues and tumorigenesis, and by drawing brief parallels with the tissues where it has been implicated as an oncogene. A class of compounds called calcilytics, which are CaSR antagonists, have also been surveyed in the instances where they have been used to target the receptor in cancerous tissues and constitute a proof of principle for repurposing them. Current clinical therapies for treating bone metastases from breast cancer are limited to targeting osteoclasts and a deeper understanding of the CaSR signaling nexus in this context can bolster them or lead to novel therapeutic interventions.

Calcium sensing receptor exerts a negative regulatory action toward vasopressin-induced aquaporin-2 expression and trafficking in renal collecting duct.

Vasopressin (AVP) plays a major role in the regulation of water homeostasis by its antidiuretic action on the kidney, mediated by V2 receptors. An increase in plasma sodium concentration stimulates AVP release, which in turn promotes water reabsorption. Upon binding to the V2 receptors in the renal collecting duct, AVP induces the expression and apical membrane insertion of the aquaporin-2 (AQP2) water channels and subsequent water reabsorption. AVP regulates two independent mechanisms: the short-term regulation of AQP2 trafficking and long-term regulation of the total abundance of the AQP2 protein in the cells. On the other hand, several hormones, acting through specific receptors, have been reported to antagonize AVP-mediated water transport in kidney. In this respect, we previously described that high luminal Ca2+ in the renal collecting duct attenuates short-term AVP-induced AQP2 trafficking through activation of the Ca2+-sensing receptor (CaSR). This effect is due to reduction of AVP-dependent cAMP generation and possibly hydrolysis. Moreover, CaSR signaling reduces AQP2 abundance both via AQP2-targeting miRNA-137 and the proteasomal degradation pathway. This chapter summarizes recent data elucidating the molecular mechanisms underlying the physiological role of the CaSR-dependent regulation of AQP2 expression and trafficking.

L-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells

Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca2+ ([Ca2+]i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca2+-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca2+ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca2+ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca2+]i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.

Abstract P122: Downregulation and Inhibition of PKCα Causes Increased Ca 2+ -Sensing Receptor-Mediated Vascular Relaxation in Aged Dahl Salt-Sensitive Rats

The G protein-coupled Ca 2+ -Sensing Receptor (CaSR) mediates extracellular calcium (Ca 2+ e ) - induced relaxation of mesenteric arteries under tension and may therefore play a role in blood pressure regulation. This presents CaSR as promising target for therapeutic drug design for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is an important regulator of CaSR signaling and can phosphorylate the receptor leading to desensitization. In this study, we investigated the effects of CaSR mutation and downregulation of PKCα in CaSR-mediated relaxation of phenylephrine (PE)-contracted mesenteric arteries from aged Dahl salt-sensitive rats, fed a low salt (0.1% NaCl) diet, using automated wire myography. We have observed in our laboratory, that there is significant decrease in relaxation response of PE-contracted mesenteric arteries to Ca 2+ e . as animals get older. The results show minimal relaxation responses of arteries to Ca 2+ e and that downregulation of CaSR expression had no significant effect. PKCα downregulation (siRNA) and inhibition of PKC (Gö 6976) showed greater than 80% increase in maximum Ca 2+ -induced relaxation, with significant decreases in EC 50 values compared to untreated controls. The results indicate that PKCα plays an important role in the regulation of CaSR -mediated relaxation of mesenteric arteries. Therefore, inhibition of PKC may lead to increased CaSR sensitivity to Ca 2+ indicating a possible therapeutic mechanism for intervention in age-related vascular dysfunction.

Activation of the Ca2+ sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3.

Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+ in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+ sensing receptor (CaSR) with Gd3+ triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+ triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.

Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells

This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.

Protein Kinase C Downregulation Enhanced Extracellular Ca2+-Induced Relaxation of Isolated Mesenteric Arteries from Aged Dahl Salt-Sensitive Rats.

The Ca2+-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca2+ e) concentration ([Ca2+]e) and transduces the signal into modulation of various signaling pathways. Ca2+-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca2+ e in wild-type (SS) and CaSR mutant (SS-Casrem1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca2+-sensitive Gӧ 6976 resulted in a >80% increase in relaxation. Significant differences in EC50 values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca2+ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.

Activation of the calcium sensing receptor attenuates TRPV6-dependent intestinal calcium absorption.

Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+ sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxy vitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium, however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a non-functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co-expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.

NHE8 attenuates Ca2+ influx into NRK cells and the proximal tubule epithelium.

To garner insights into the renal regulation of Ca2+ homeostasis, we performed an mRNA microarray on kidneys from mice treated with the Ca2+-sensing receptor (CaSR) agonist cinacalcet. This revealed decreased gene expression of Na+/H+ exchanger isoform 8 (NHE8) in response to CaSR activation. These results were confirmed by quantitative real-time PCR. Moreover, administration of vitamin D also decreased NHE8 mRNA expression. In contrast, renal NHE8 protein expression from the same samples was increased. To examine the role of NHE8 in transmembrane Ca2+ fluxes, we used the normal rat kidney (NRK) cell line. Cell surface biotinylation and confocal immunofluorescence microscopy demonstrated NHE8 apical expression. Functional experiments found 5-(N-ethyl-N-isopropyl)amiloride (EIPA)-inhibitable NHE activity in NRK cells at concentrations minimally attenuating NHE1 activity in AP-1 cells. To determine how NHE8 might regulate Ca2+ balance, we measured changes in intracellular Ca2+ uptake by live cell Ca2+ imaging with the fluorophore Fura-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular Na+-enhanced Ca2+ influx into NRK cells. Ca2+ influx was mediated by a voltage-dependent Ca2+ channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil-sensitive Ca2+ influx and NHE8 inhibition-augmented Ca2+ influx via a voltage-dependent Ca2+ channel. Finally, proximal tubules perused ex vivo demonstrated increased Ca2+ influx in the presence of luminal EIPA at a concentration that would inhibit NHE8. The results of the present study are consistent with NHE8 regulating Ca2+ uptake into the proximal tubule epithelium.

Evidence for a regulated Ca2+ entry in proximal tubular cells and its implication in calcium stone formation.

Calcium phosphate (CaP) crystals, which begin to form in the early segments of the loop of Henle (LOH), are known to act as precursors for calcium stone formation. The proximal tubule (PT), which is just upstream of the LOH and is a major site for Ca2+ reabsorption, could be a regulator of such CaP crystal formation. However, PT Ca2+ reabsorption is mostly described as being paracellular. Here, we show the existence of a regulated transcellular Ca2+ entry pathway in luminal membrane PT cells induced by Ca2+-sensing receptor (CSR, also known as CASR)-mediated activation of transient receptor potential canonical 3 (TRPC3) channels. In support of this idea, we found that both CSR and TRPC3 are physically and functionally coupled at the luminal membrane of PT cells. More importantly, TRPC3-deficient mice presented with a deficiency in PT Ca2+ entry/transport, elevated urinary [Ca2+], microcalcifications in LOH and urine microcrystals formations. Taken together, these data suggest that a signaling complex comprising CSR and TRPC3 exists in the PT and can mediate transcellular Ca2+ transport, which could be critical in maintaining the PT luminal [Ca2+] to mitigate formation of the CaP crystals in LOH and subsequent formation of calcium stones.

High Salt Up‐Regulates Ca2+‐Sensing Receptor and Na+/Ca2+ Exchanger Expression in Kidneys of Dahl Salt‐Sensitive Rats

The G protein‐coupled Ca2+‐sensing receptor (CaSR) provides the primary mechanism for Ca2+e‐mediated regulation of the release of parathyroid hormone (PTH) as a result of parathyroid cells sensing small changes in the [Ca2+]e. However, the CaSR is also involved in the fine regulation of serum Ca2+ levels and renal Ca2+ excretion, independent of PTH secretion. The mechanisms underlying these latter effects are not clear. We studied the effect of high salt on urinary ion excretion, blood pressure and expression of the CaSR and NCX1in kidneys of Dahl SR and SS rats. Our results show that four weeks of feeding a high salt diet increased BP in SS, increased urinary Na+/Ca2+ excretion in SR and SS but reduced K+ excretion. The high salt diet caused minimal structural changes in the kidneys, as determined by haematoxylin/eosin (H&amp;E) and periodic acid‐Schiff (PAS) staining. Immunohistochemical analysis of tissue sections showed localization of the CaSR and NCX1 in the thick ascending limb of the loop of Henle, collecting ducts, cortical distal tubules, which was up‐regulated by the high salt in SS animals. NCX1 expression and localization in this study tracks closely with the CaSR. Thus, expression levels and localization patterns of these protein suggest that CaSR modulation of ion transporter and channel proteins could account for the difference in ion excretion observed between SS and SR rats as well as their responses to the high salt diet.Support or Funding InformationNIH Grant SC1 HL136278This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Platelet‐derived growth factor up‐regulates Ca 2+ ‐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.

Calcilytics inhibit the proliferation and migration of human prostate cancer PC-3 cells

The carcinogenesis and development of prostate cancer are mediated by enhanced Ca2+ signaling. In the present study, the pharmacological profile of the Ca2+-sensing receptor (CaSR) antagonists (calcilytics) was examined in human prostate cancer PC-3 cells. NPS2143 and Calhex 231 blocked extracellular Ca2+-induced increases in cytosolic [Ca2+]. NPS2143 and Calhex 231 inhibited cell proliferation (IC50 = 7.4 and 10.3 μM, respectively) and migration. The exposure to NPS2143 or Calhex 231 down-regulated CaSR protein expression. These results demonstrated that calcilytics inhibited cell proliferation/migration and down-regulated CaSR expression in human prostate cancer cells, suggesting their potential as novel therapeutic drugs for prostate cancer.

Calcium-Sensing Receptor Regulates Epidermal Intracellular Ca2+ Signaling and Re-Epithelialization after Wounding

Extracellular Ca2+ (Ca2+o) is a crucial regulator of epidermal homeostasis and its receptor, the Ca2+-sensing receptor (CaSR), conveys the Ca2+o signals to promote keratinocyte adhesion, differentiation, and survival via activation of intracellular Ca2+ (Ca2+i) and E-cadherin-mediated signaling. Here, we took genetic loss-of-function approaches to delineate the functions of CaSR in wound re-epithelialization. Cutaneous injury triggered a robust CaSR expression and a surge of Ca2+i in epidermis. CaSR and E-cadherin were co-expressed at the cell-cell membrane between migratory keratinocytes in the nascent epithelial tongues. Blocking the expression of CaSR or E-cadherin in cultured keratinocytes markedly inhibited the wound-induced Ca2+i propagation and their ability to migrate collectively. Depleting CaSR also suppressed keratinocyte proliferation by downregulating the E-cadherin/epidermal growth factor receptor/mitogen-activated protein kinase signaling axis. Blunted epidermal Ca2+i response to wounding and retarded wound healing were observed in the keratinocyte-specific CaSR knockout (EpidCasr-/-) mice, whose shortened neo-epithelia exhibited declined E-cadherin expression and diminished keratinocyte proliferation and differentiation. Conversely, stimulating endogenous CaSR with calcimimetic NPS-R568 accelerated wound re-epithelialization through enhancing the epidermal Ca2+i signals and E-cadherin membrane expression. These findings demonstrated a critical role for the CaSR in epidermal regeneration and its therapeutic potential for improving skin wound repair.

Hypoxia-Induced Mitogenic Factor Promotes Cardiac Hypertrophy via Calcium-Dependent and Hypoxia-Inducible Factor-1α Mechanisms.

HIMF (hypoxia-induced mitogenic factor/found in inflammatory zone 1/resistin like α) is a secretory and cytokine-like protein and serves as a critical stimulator of hypoxia-induced pulmonary hypertension. With a role for HIMF in heart disease unknown, we explored the possible roles for HIMF in cardiac hypertrophy by overexpressing and knocking down HIMF in cardiomyocytes and characterizing HIMF gene (himf) knockout mice. We found that HIMF mRNA and protein levels were upregulated in phenylephrine-stimulated cardiomyocyte hypertrophy and our mouse model of transverse aortic constriction-induced cardiac hypertrophy, as well as in human hearts with dilated cardiomyopathy. Furthermore, HIMF overexpression could induce cardiomyocyte hypertrophy, as characterized by elevated protein expression of hypertrophic biomarkers (ANP [atrial natriuretic peptide] and β-MHC [myosin heavy chain-β]) and increased cell-surface area compared with controls. Conversely, HIMF knockdown prevented phenylephrine-induced cardiomyocyte hypertrophy and himf ablation in knockout mice significantly attenuated transverse aortic constriction-induced hypertrophic remodeling and cardiac dysfunction. HIMF overexpression increased the cytosolic Ca2+ concentration and activated the CaN-NFAT (calcineurin-nuclear factor of activated T cell) and MAPK (mitogen-activated protein kinase) pathways; this effect could be prevented by reducing cytosolic Ca2+ concentration with L-type Ca2+ channel blocker nifedipine or inhibiting the CaSR (Ca2+ sensing receptor) with Calhex 231. Furthermore, HIMF overexpression increased HIF-1α (hypoxia-inducible factor) expression in neonatal rat ventricular myocytes, and HIMF knockout inhibited HIF-1α upregulation in transverse aortic constriction mice. Knockdown of HIF-1α attenuated HIMF-induced cardiomyocyte hypertrophy. In conclusion, HIMF has a critical role in the development of cardiac hypertrophy, and targeting HIMF may represent a potential therapeutic strategy.

Activation of Ca2+-sensing receptor as a protective pathway to reduce Cadmium-induced cytotoxicity in renal proximal tubular cells

Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca2+ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca2+ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca2+ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.

TRPC1 stimulates calcium‑sensing receptor‑induced store‑operated Ca2+ entry and nitric oxide production in endothelial cells.

Store-operated Ca2+ entry (SOCE) via store-operated Ca2+ channels (SOCC), encoded by transient receptor potential canonical (TRPC) channel proteins, is an important underlying mechanism regulating intracellular Ca2+ concentration ([Ca2+]i) and various intracellular functions in endothelial cells (ECs). TRPC1, the probable candidate for SOCC, is expressed in ECs. Ca2+-sensing receptor (CaSR) is functionally expressed in vascular endothelium and is important in Ca2+ mobilization and cardiovascular functions. To date, there have been no reports demonstrating an association between CaSR and TRPC1 in ECs. The present study investigated the effects of TRPC1 on CaSR-induced Ca2+ influx and nitric oxide (NO) production in human umbilical vein ECs (HUVECs). TRPC1 and CaSR proteins in HUVECs were measured by immunostaining and western blot analysis. [Ca2+]i levels were measured using the Fura-2-acetoxymethyl ester method. The indicator 3-amino, 4-aminomethyl-2, 7-difluorescein diacetate was used to measure NO production in HUVECs. The expression of TRPC1 protein in HUVECs was silenced by transfecting HUVECs with small interfering RNA (siRNA) against TRPC1. Although changes in extracellular Ca2+ failed to alter [Ca2+]i in HUVECs, the CaSR agonist spermine increased [Ca2+]i and NO production in HUVECs. NO production in HUVECs was diminished in Ca2+-free medium or following treatment with a CaSR negative allosteric modulator (Calhex231), SOCC inhibitor (MRS1845) or TRPC inhibitor (SKF96365). The spermine-induced increases in [Ca2+]i and NO production were reduced in HUVECs transfected with TRPC1 siRNA. These results suggested that TRPC1 is a primary candidate in forming SOCC that stimulates CaSR-induced SOCE and NO production in HUVECs and is a potential therapeutic target for vascular diseases.

External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel whose dysfunction is associated with the onset of autosomal dominant polycystic kidney disease (ADPKD). PC2 contributes to Ca2+ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to the external Ca2+ regulation of PC2 channel function. In this study, we explored the effect of external Ca2+ on endogenous PC2 in wild type LLC-PK1 renal epithelial cells. We obtained whole cell currents at different external Ca2+ concentrations, and observed that the basal whole cell conductance in normal Ca2+(1.2mM), decreased by 30.2% in zero (nominal) Ca2+ and conversely, increased by 38% in high external Ca2+(6.2mM). The high Ca2+-increased whole cell currents were completely inhibited by either PC2 gene silencing, or intracellular dialysis with active, but not denatured by boiling, PC2 antibody. Exposure of cells to high Ca2+ was also associated with relocation of PC2 to the plasma membrane. To explore whether a Ca2+ sensing receptor (CaSR) was implicated in the external Ca2+ modulation of PC2 currents, we tested the effect of the CaSR agonists, spermine and the calcimimetic R-568, which largely mimicked the effect of high Ca2+ under Ca2+-free conditions. The CaSR agonist gentamicin also increased the PC2 currents in the presence of normal Ca2+. The presence of CaSR was confirmed by immunocytochemistry, which partially colocalized with the intracellular PC2 protein, in an external Ca2+-dependent manner. The data support a novel Ca2+ sensing mechanism for PC2 expression and functional regulation in renal epithelial cells.

Characterisation of vitamin D-related molecules and calcium-sensing receptor in human Fallopian tube during the menstrual cycle and in ectopic pregnancy

This is a prospective observational study that measures the expression of vitamin D (VD) metabolising and signalling molecules and Ca2+ sensing receptor (CaSR) in human Fallopian tube (FT) during the menstrual cycle and ectopic pregnancy (EP). Fresh FTs were obtained during total abdominal hysterectomy at the follicular (n = 16) and midluteal (n = 16) phases. Specimens from remote and implantation sites as well as trophoblastic tissues were also freshly collected from each FT with EP (n = 10). All women had normal serum VD and ionised Ca2+. The expression of VD synthesising (CYP27B1) and catalysing (CYP24A1) enzymes, binding protein (VDBP), receptor (VDR), retinoid X receptor (RXR) and CaSR was measured by immunohistochemistry and quantitative RT-PCR. All molecules, except VDBP, were significantly increased (P < 0.05) in midluteal compared with follicular samples. Remote EP sites showed significantly (P < 0.05) lower expression of CYP27B1, CYP24A1, VDR and RXR and a higher expression of VDBP and CaSR (P < 0.05) compared with midluteal samples. Significant differences were observed by immunohistochemistry between implantation and remote sites from EP for all molecules, which were also localised in the trophoblastic tissues. In conclusion, VD and calcium are under cycle-dependent regulations within human FT and they appear to play a role in tubal biology through paracrine/autocrine mode of signalling. Furthermore, EP was associated with alterations in the expression of all the studied molecules by the tubal epithelium. Further studies are needed to explore the roles of VD in tubal biology and pathogenesis of EP.