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

Cing-Yu Chen • Mann-Jen Hour • Wen-Chuan Lin • Kar-Lok Wong • Lian-Ru Shiao • Ka-Shun Cheng • Paul Chan • Yuk-Man Leung
1 School of Pharmacy, China Medical University, Taichung 40402, Taiwan
2 Department of Anesthesiology, China Medical University Hospital, Taichung, Taiwan
3 Department of Physiology, China Medical University, Taichung 40402, Taiwan
4 Department of Anesthesiology, The Qingdao University Yuhuangding Hospital, Yantai, Shandong, China
5 Division of Cardiology, Department of Medicine, Taipei Medical University Wan Fang Hospital, Taipei, Taiwan

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.

The most well-known physiological function of Ca2+-sensing receptors (CaSR) is the regulation of parathyroid hormone (PTH) release from parathyroid glands (Chen and Goodman2004). A rise in serum Ca2+ activates thyroid cell CaSR, which in turn activates Gq and subsequently phospholipase C (PLC), generating inositol 1,4,5-trisphophate (IP3) and Ca2+ release. A rise in intracellular Ca2+ inhibits PTH release; the drop in serum PTH will provide less stimulation of Ca2+ re-lease from bones. Stimulation of thyroid parafollicular C cellCaSR promotes secretion of calcitonin, which lowers serum Ca2+ by suppressing osteoclasts and inhibiting renal tubular Ca2+ reabsorption (Brown 2013). These dual actions of CaSR serve as a major mechanism regulating serum Ca2+.
CaSR are present in other tissues as well, such as in the kidney and the brain. In the brain, CaSR regulates neuronal cell proliferation and migration (Ruat and Traiffort 2013). Further, CaSR also regulates neuronal excitability via an acti- vation of non-selective cation channel by decreased extracel- lular Ca2+ (see Jones and Smith 2016 for a review). Non- selective cation channel activation by decreased extracellular Ca2+ (hence decreased CaSR stimulation) also leads to action potential broadening, prolonged Ca2+ entry, and augmented synaptic transmission (Jones and Smith 2016). However, in addition to the neurophysiological roles, CaSR activation in asubpopulation of neurons and astrocytes appears to be in- volved in the pathological relation between hypoxia and Alzheimer’s disease. For instance, hypoxia has been shown to upregulate CaSR expression, raise cytosolic Ca2+, and cause overproduction of beta-amyloid in hippocampal neu- rons; Calhex 231, a negative allosteric modulator of CaSR, could suppress cytosolic Ca2+ elevation and hypoxia-induced beta-amyloid overproduction (Bai and Mao 2015). Thus, CaSR antagonists are potentially promising therapeutic agents for Alzheimer’s disease.
A small increase in extracellular Ca2+ has been known to dilate isolated blood vessels (Weston et al. 2005). In this work, it was shown that CaSR activation in rat mesenteric arteries and in porcine coronary artery EC was coupled to EC intermediate conductance Ca2+- activated K+ channel opening, EC hyperpolarization, and eventually leading to smooth muscle hyperpolariza- tion. In addition to the physiological role of CaSR in vascular reactivity, CaSR (overactivation) appears to be involved in EC pathophysiology. Inhibition of CaSR protects rat mesenteric arteries (thus, preserving EC- dependent vasorelaxation) from hypoxia/reoxygenation insult (Zhao et al. 2015). Suppression of CaSR expres- sion accounts for tetrahydropalmatine protection of rat pulmonary EC from irradiation-induced apoptosis (Yu et al. 2016).
NPS 2143, a negative allosteric modulator of CaSR (Leach et al. 2013), has been shown to raise PTH levels (Gowen et al. 2000; Nemeth et al. 2001). Later, NPS 2143 was found to sup- press fibrillary Aβ25–35-elicited elevation of endogenous Aβ42 secretion by astrocytes and neurons, revealing its thera- peutic potential of Alzheimer’s disease (Armato et al. 2013; also see above). In this work, we examined the effects of NPS 2143 on high Ca2+-triggered Ca2+ signaling in brain endothelial cells. Acute NPS 2143 addition caused suppression of Ca2+-triggered Ca2+ signaling; however, longer exposure to NPS 2143 surpris- ingly enhanced it. We investigated the mechanisms.

Materials and methods
Materials and cell culture
NPS 2143, 2-aminoethoxydiphenyl borate (2-APB), edelfosine, U73122, and SB203580 were from Sigma- Aldrich (St. Louis, MO, USA). SK&F 96365 and ruthenium red were from Tocris Bioscience (UK). Fura-2 AM was pur- chased from Calbiochem-Millipore. Brain microvascular bEND.3 cells and breast cancer BT474 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Invitrogen).

Microfluorimetric measurement of cytosolic Ca2+
Microfluorimetric measurement of cytosolic Ca2+ con- centration was performed using fura-2 as the Ca2+-sen- sitive fluorescent dye as described previously (Leung et al. 2011). Briefly, the cells were grown on tiny glass cover slips and incubated with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA) for 1 h at 37 °C and then washed in extracellular bath solution which contains the following (mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES (pH 7.4 adjusted with NaOH). When intra- cellular Ca2+ release was assayed, Ca2+-free solution was used. This Ca2+-free solution was the same as the extracellular bath solution mentioned above except that Ca2+ was omitted and 100 μM EGTA was supplement- ed. Cells were alternately excited with 340 nm and 380 nm (switching frequency at 1 Hz) using an optical filter changer (Lambda 10-2, Sutter Instruments). Emission was collected at 500 nm and images were captured using a CCD camera (CoolSnap HQ2, Photometrics, Tucson, AZ) linked to an inverted Nikon TE2000-U microscope. Images were analyzed with the MAG Biosystems Software (Sante Fe, MN). All imag- ing experiments were performed at room temperature (25 °C). We measured and analyzed the 340/380 ratio changes at a region of interest of single cells within the microscopic views (regarded as one experiment) and then repeated this experiment a few more times to get the mean of all single cells examined. The cells exam- ined had about 30% confluence and in general there were about 8–10 cells in each microscopic view. The solutions were applied by gravity-fed perfusion.

Western blot
Western blotting was performed as described previously (Lu et al. 2009). Briefly, cells were washed in cold PBS and lysed for 30 min on ice with radioimmunoprecipitation assay (RIPA) buffer. Protein samples containing 30 μg pro- tein were separated on 10% sodium dodecyl sulfate- polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h with 5% nonfat milk in TBST buffer to block nonspe- cific binding. The membranes were incubated with various primary antibodies such as anti-CaSR, anti-p38, and anti- phospho-p38 (all 1:1000; Cell Signaling Technology, Beverly, MA, USA; cat. # 73303, 9212, and 9211, respec- tively). Anti-actin was from Gene Tex (cat. # GTX109639; 1:1000). Subsequently, the membranes were incubated with goat anti-rabbit or goat anti-mouse peroxidase-conjugated secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) for 1 h.

Functional and protein expression of CaSR in bEND.3 cells
Addition of increasing bath concentrations of Ca2+ caused an elevation in [Ca2+]i (Fig. 1), suggesting the presence of CaSR. Likewise, addition of a known CaSR agonist, La3+ (Díaz-Soto et al. 2016), also stimulated elevation in [Ca2+]i (Fig. 2a). Addition of another CaSR agonist, Sr2+ (Díaz-Soto et al. 2016), also stimulated [Ca2+]i elevation; however, such rise

measured in bEND.3 cells, which were stimulated by different concentrations of extracellularly added Ca2+. Results are mean ± SEM, each group having 24–35 cells from four separate experimentswere visualized by enhanced chemiluminescence (ECL; Millipore) using Kodak X-OMAT LS film (Eastman Kodak, Rochester).

Statistical analysis was much weaker (Fig. 2b) compared to those stimulated by Ca2+ and La3+, suggesting weak agonism towards Ca2+ sig- naling. This is consistent with the weak agonistic action on Ca2+ signaling of Sr2+ in rat medullary thyroid carcinoma 6– 23 cells (Thomsen et al. 2012). We performed Western blot to confirm CaSR protein expression in bEND.3 cells (Fig. 3a). However, BT474 cells did not express CaSR, and consistent- ly, was not responsive to high Ca2+ (Fig. 3a, b). Response to cyclopiazonic acid (CPA, an inhibitor of endoplasmic

Data are presented as means ± SEM. The unpaired or paired Student’s t test was used where appropriate to compare two groups. ANOVA was used to compare multiple groups. A value of p < 0.05 was considered to represent a significant difference. CaSR response was inhibited by TRP channel blockers One of the signaling pathways that CaSR has been shown to activate is the Gq-PLC pathway. We therefore examined if edelfosine, a PLC inhibitor, would inhibit Ca2+-triggered Ca2+ signals. Rather unexpectedly, edelfosine had no effect (Fig. 4a). Another structurally unrelated PLC inhibitor, U73122, also showed no effect on Ca2+-triggered Ca2+ signals (Fig. 4b). Previous works have shown CaSR stimulation activates TRPC and TRPV channels in various cells (Chow et al. 2011; Meng et al. 2014; Greenberg et al. 2017). We then ex- amined the effects of TRP channel blockers. We showed that SK&F 96365 and 2-APB (TRPC channel inhibitors) and ruthe- nium red (TRPV channel inhibitors) could strongly suppress Ca2+-triggered Ca2+ signals, suggesting involvement of TRP channels (Fig. 4c). We then examined if the reverse mode of Na+/Ca2+ exchanger (NCX) played a role in the Ca2+-triggered Ca2+ influx. Experiments were conducted in normal Na+-con- taining extracellular bath solution or in an extracellular bath solution with NaCl completely substituted by choline chloride. The Na+-free solution was expected to favor the reverse modeof NCX. Results in Fig. 4d show that Ca2+-triggered Ca2+ signal was not significantly affected in the absence of extracel- lular Na+, arguing against NCX involvement. The pathway by which Ca2+ entered the cell upon high Ca2+ stimulation was not attributable to voltage-gated Ca2+ channel (VGCC), since high (60 mM) K+ depolarization did not stimulate any Ca2+ signal (data not shown), ruling out involvement of VGCC. NPS 2143 effects on CaSR was time-dependent When we examined the effects of NPS 2143 on Ca2+- triggered Ca2+ signals, we noted that after NPS 2143 (1 μM) was added for 2 min, it suppressed Ca2+ signal (Fig. 5a). No further suppression of Ca2+ signal was observed with 10 μM NPS 2143 (data not shown). When cells were pre-treated with NPS 2143 longer (40 min), we surprisingly noted that the Ca2+-triggered Ca2+ response was faster than that in the control (Fig. 5b). After cells were pre-treated with NPS 2143 for 4 h, there was neither enhancement nor inhibition of Ca2+- triggered Ca2+ signals (Fig. 5c). If cells were pre-treated with NPS 2143 for 24 h, inhibition of Ca2+-triggered Ca2+ signals reappeared (Fig. 5d). Using this protocol, we repeated the experiments using La3+: that is, La3+- triggered Ca2+ signals (Fig. 6). Almost the same results We speculated that whilst NPS 2143 expectedly exerted an acute inhibition of CaSR (2 min), it produced an unreported time-dependent enhancement of CaSR (man- ifested after 40 min), then fading away gradually after a few hours and overnight so that after 24 h the NPS 2143 inhibition of CaSR would reappear. We then ex- amined if this NPS 2143 activation was mediated by some kinases. An experiment was performed in which SB203580, an inhibitor of p38, was co-added with NPS 2143. After 40 min incubation, NPS 2143 alone pro- duced an enhancement of Ca2+-triggered Ca2+ signals (Fig. 8a); with SB203580 co-addition, such enhance- ment was lost and in fact an inhibition of Ca2+-triggered Ca2+ signals was observed (Fig. 8b). This result sug- gests NPS 2143 produced the enhancement effect via activating p38. A Western blot analysis was performed to show whether NPS 2143 activated p38 (Fig. 9a). Results revealed that there was a moderate enhancement of p-p38 protein levels after 2 and 40 min of NPS 2143 addition. CaSR activation has been shown before to activate p38 in human kidney HK-2G epithelial cells (Maiti et al. 2008). However, we did not observe any p38 activation upon CaSR stimulation in bEND.3 cells (Fig. 9b). Discussion In this work, we showed in murine bEND.3 EC protein ex- pression of CaSR (Western blot) and its functional presence (activation by Ca2+, Sr2+, and La3+). The Ca2+-sensing recep- tor (CaSR) is a member of the G protein-coupled receptor (GPCR) superfamily. Its pleiotropicity is reflected in its cou- pling to various G proteins, namely, Gi (inhibition of adenyl- ate cyclase), Gq/11 (IP3/Ca2+/diacylglycerol pathway via PLC hydrolysis), and G12/13 (via use of guanine nucleotide ex- change factors) and even Gs (stimulating adenylate cyclase) (Kifor et al. 2001; Almaden et al. 2002; Huang et al. 2004; Mamillapalli et al. 2008). CaSR signaling also leads to ERK1/2 activation in rat medullary thyroid carcinoma 6–23 cells. (Thomsen et al. 2012) and human dental pulp cells (Mizumachi et al. 2017). CaSR also interacts with several intracellular proteins, including β-arrestins and filamin A (Awata et al. 2001; Hjalm et al. 2001; Bouschet et al. 2007;Gorvin et al. 2018). We here reported, however, CaSR re- sponse in bEND.3 cells was not inhibited by edelfosine and U73122, suggesting high Ca2+-triggered Ca2+ response was not mediated through Gq-PLC pathway. The possibility that high Ca2+-triggered Ca2+ influx was via VGCC could be ruled out by the absence of functional VGCC in bEND.3 cells (no high KCl response, not shown). There was also no indication of any NCX participation (Fig. 4d). Suppression by TRPC and TRPV channel blockers sug- gests the involvement of TRPC and TRPV channels. Given the pleitropicity explained above, this would not appear entirely surprising. Further, previous works have shown CaSR stim- ulation activates TRPC channels in human aortic smooth muscle cells (Chow et al. 2011), human mesangial cells (Meng et al. 2014), and TRPV/TRPC heteromeric channels in rabbit mesenteric artery endothelial cells (Greenberg et al. 2017). Of note, TRP channel heteromerization across fami- lies has been observed (Du et al. 2014). CaSR stimulation also activates TRPP2 channels in LLC-PK1 renal epithelial cells (Dai et al. 2017). Given CaSR is a GPCR and TRP channels are G protein-regulated (Veldhuis et al. 2015), cou- pling between CaSR and TRP channels could be via a G protein. However, there is hitherto no report to suggest any G protein to play this role. Alternatively, as CaSR has been known to interact with arrestins, and that arrestins have been recently shown to bridge GPCR to activate TRP channels (Liu et al. 2017; Chai et al. 2017), a CaSR–arrestin–TRP complex is a possibility. Recent works have also shown a role of CaSR in ischemic brain injury. In rats undergoing transient focal cerebral ischemia, CaSR protein expression was upregulated in neurons and vascu- lar cells in ischemic and border zones of lesions and in reactive astrocytes in peri-infarct areas (Noh et al. 2015). Kim et al. (2014) showed that genetic ablation of CaSR genes or pharma- cological inhibition of CaSR activities could protect neurons and rescue learning and memory abilities in mice suffering from global and local brain ischemia. CaSR modulators are therefore important in elucidating the roles of CaSR. NPS2143 is a nega- tive allosteric modulator of CaSR. This drug suppresses pulmo- nary arterial smooth muscle cell proliferation in idiopathic pul- monary arterial hypertension patients (Yamamura et al. 2015). NPS2143 has also been reported to decrease the release of proinflammatory cytokines such as IL-6 and tumor ne- crosis factor-α in cigarette smoker extract-stimulated hu- man airway epithelial cells (Lee et al. 2017). NPS 2143 could suppress inflammation in lipopolysaccharide-induced acute lung injury in mice: inhibition of influx of inflammatory cells, reduce inflammatory cytokine production, and down- regulation of NF-κB activation (Lee et al. 2017). Remarkably, in this work, we found that NPS2143 only caused weak suppression of CaSR. The reason may be that the putative CaSR–TRP complex (perhaps coupled via a G protein or arrestin as described above) may not be very sensitive to NPS2143. This novel find- ing is important in showing that NPS 2143 may not be equipotent to all CaSRs (due to diversity in effector coupling). Another explanation could be that CaSR only mediated a fraction of the response to raised extracellu- lar Ca2+; the latter also triggered Ca2+ signals via non- selective cation channels. In this regard, it is important to note that SK&F 96365 and 2-APB, two non-selective cation channel blockers, caused very substantial inhibi- tion of high Ca2+-triggered Ca2+ response (Fig. 4c). NPS2143 caused an unexpected transient activation or sen- sitization of CaSR, an enhanced response in the 40-min time range. A possible explanation could be that NPS2143, via a negative allosteric modulation of the CaSR, uncoupled a con- stitutive recycling of this receptor, thus increasing the CaSR density at the plasma membrane momentarily. In addition, NPS enhancement of CaSR was p38-dependent, as NPS2143 caused a moderate p38 activation, and the fact that the response was abolished by SB203580, an inhibitor of p38. Therefore, NPS2143 might cause the enhancement by p38-dependent transcriptional or post-transcriptional activities, hence increas- ing the amounts of CaSR, TRP channels, or CaSR–TRP com- plexes at the plasma membrane. In support for this, previous works have shown p38 activities enhance TRPA and TRPV channels in odontoblast-like cells (El Karim et al. 2015). p38 also enhances TRPC expression in pulmonary artery smooth muscle cells (Li et al. 2013). Further, TRPV6 transcription was enhanced by p38 (Ishizawa et al. 2017). The role of CaSR in EC appears to be complex, involving both promotion of vascular smooth muscle relaxation (Weston et al. 2005) and cytotoxicity (Yu et al. 2016). The transient enhancement of CaSR by NPS 2143 may add another layer of complexity to interpreting the role of CaSR in EC if this drug is deployed. Whether such transient enhancement of CaSR byNPS 2143 happens in other EC types or other cell types re- mains unknown, and this certainly warrants future investiga- tion. What is also worth investigating is the development of NPS 2143 derivatives devoid of such transient enhancement. Conclusion CaSR in bEND.3 cells appeared to be coupled to TRP chan- nels. NPS 2143 caused in bEND.3 cells an immediate sup- pression of CaSR response, followed by a transient enhance- ment in the medium time frame; this may prevent NPS 2143 from being an effective negative allosteric modulator of CaSR. This novel finding draws attention to the cautious us- age of NPS 2143 as a potential cardiovascular drug. References Almaden Y, Canalejo A, Ballesteros E, Anon G, Canadillas S, Rodriguez M (2002) Regulation of arachidonic acid production by intracellular calcium in parathyroid cells: effect of extracellular phosphate. J Am Soc Nephrol 13:693–698 Armato U, Chiarini A, Chakravarthy B, Chioffi F, Pacchiana R, Colarusso E, Whitfield JF, Dal PI (2013) Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Abeta42 prompted by exogenous fibrillary or soluble Abeta25-35 in human cortical astrocytes and neurons- therapeutic relevance to Alzheimer’s disease. Biochim Biophys Acta 1832:1634–1652 Awata H, Huang C, Handlogten ME, Miller RT (2001) Interaction of the calcium-sensing receptor and filamin, a potential scaffolding pro- tein. J Biol Chem 276:34871–34879 Bai S, Mao M, Tian L. Yu Y, Zeng J, Ouyang K, Yu L, Li L, Wang D, Deng X, Wei C and Luo Y. (2015) Calcium sensing receptormediated the excessive generation of beta-amyloid peptide induced by hypoxia in vivo and in vitro. Biochem Biophys Res Commun 459: 568–573 Bouschet T, Martin S, Kanamarlapudi V, Mundell S, Henley JM (2007) The calcium-sensing receptor changes cell shape via a beta-arrestin- 1 ARNO ARF6 ELMO protein network. J Cell Sci 120:2489–2497 Brown EM (2013) Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best Pract Res Clin Endocrinol Metab 27: 333–343 Chai Z, Chen Y, Wang C (2017) β-Arrestin-1: bridging GPCRs to active TRP channels. Channels (Austin) 11(5):357–359 Chen RA, Goodman WG (2004) Role of the calcium-sensing receptor in parathyroid gland physiology. Am J Physiol Ren Physiol 286: F1005–F1011 Chow JY, Estrema C, Orneles T, Dong X, Barrett KE, Dong H (2011) Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells. Am J Phys Cell Phys 301(2):C461–C468 Dai XQ, Perez PL, Soria G, Scarinci N, Smoler M, Morsucci DC, Suzuki K, Cantero MDR, Cantiello HF (2017) External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells. Exp Cell Res 350(1):50–61 Díaz-Soto G, Rocher A, García-Rodríguez C, Núñez L, Villalobos C (2016) The calcium-sensing receptor in health and disease. Int Rev Cell Mol Biol 327:321–369 Du J, Ma X, Shen B, Huang Y, Birnbaumer L, Yao X (2014) TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel. FASEB J 28(11):4677–4685 El Karim I, McCrudden MT, Linden GJ, Abdullah H, Curtis TM, McGahon M, About I, Irwin C, Lundy FT (2015) TNF-α-induced p38MAPK activation regulates TRPA1 and TRPV4 activity in odontoblast-like cells. Am J Pathol 185(11):2994–3002 Gorvin CM, Babinsky VN, Malinauskas T, Nissen PH, Schou AJ, Hanyaloglu AC, Siebold C, Jones EY, Hannan FM, Thakker RV (2018) A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling. Sci Signal 20:11(518) Gowen M, Stroup GB, Dodds RA, James IE, Votta BJ, Smith BR, Bhatnagar PK, Lago AM, Callahan JF, DelMar EG, Miller MA, Nemeth EF, Fox J (2000) Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone forma- tion in osteopenic rats. J Clin Invest 105(11):1595–1604 Greenberg HZE, Carlton-Carew SRE, Khan D, Zargaran AK, Jahan KS, Vanessa Ho WS, Albert AP (2017) Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced nitric oxide pro- duction and vasorelaxation in rabbit mesenteric arteries. Vasc Pharmacol 96-98:53–62 Hjalm G, MacLeod RJ, Kifor O, Chattopadhyay N, Brown EM (2001) Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase. J Biol Chem 276:34880–34887 Huang C, Hujer KM, Wu Z, Miller RT (2004) The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin- Darby canine kidney cells. Am J Phys Cell Phys 286:C22–C30 Ishizawa M, Akagi D, Yamamoto J, Makishima M (2017) 1α,25- Dihydroxyvitamin D3 enhances TRPV6 transcription through p38 MAPK activation and GADD45 expression. J Steroid Biochem Mol Biol 172:55–61 Jones BL, Smith SM (2016) Calcium-sensing receptor: a key target for extracellular calcium signaling in neurons. Front Physiol 7:116 Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, Brown EM (2001) Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Ren Physiol 280:F291–F302 Kim JY, Ho H, Kim N, Liu J, Tu CL, Yenari MA, Chang W (2014) Calcium-sensing receptor (CaSR) as a novel target for ischemic neuroprotection. Ann Clin Transl Neurol 1:851–866 Leach K, Wen A, Cook AE, Sexton PM, Conigrave AD, Christopoulos A (2013) Impact of clinically relevant mutations on the pharmacoregulation and signaling bias of the calcium-sensing re- ceptor by positive and negative allosteric modulators. Endocrinology 154(3):1105–1116 Lee JW, Park JW, Kwon OK, Lee HJ, Jeong HG, Kim JH, Oh SR, Ahn KS (2017) NPS2143 inhibits MUC5AC and proinflammatory me- diators in cigarette smoke extract (CSE)-stimulated human airway epithelial cells. Inflammation. 40(1):184–194 Leung YM, Huang CF, Chao CC, Lu DY, Kuo CS, Cheng TH, Chang LY, Chou CH (2011) Voltage-gated K+ channels play a role in cAMP- stimulated neuritogenesis in mouse neuroblastoma N2A cells. J Cell Physiol 226:1090–1098 Li X, Lu W, Fu X, Zhang Y, Yang K, Zhong N, Ran P, Wang J (2013) BMP4 increases canonical transient receptor potential protein ex- pression by activating p38 MAPK and ERK1/2 signaling pathways in pulmonary arterial smooth muscle cells. Am J Respir Cell Mol Biol 49(2):212–220 Liu CH, Gong Z, Liang ZL, Liu ZX, Yang F, Sun YJ, Ma ML, Wang YJ, Ji CR, Wang YH, Wang MJ, Cui FA, Lin A, Zheng WS, He DF, Qu CX, Xiao P, Liu CY, Thomsen ARB, Joseph Cahill T, Kahsai AW, Yi F, Xiao KH, Xue T, Zhou Z, Yu X, Sun JP (2017) Arrestin-biased AT1R agonism induces acute catecholamine secretion through TRPC3 coupling. Nat Commun 8:14335 Lu DY, Tang CH, Yeh WL, Wong KL, Lin CP, Chen YH, Lai CH, Chen YF, Leung YM, Fu WM (2009) SDF-1alpha up-regulates interleukin-6 through CXCR4, PI3K/Akt, ERK, and NF-kappaB- dependent pathway in microglia. Eur J Pharmacol 613:146–154 Maiti A, Hait NC, Beckman MJ (2008) Extracellular calcium-sensing receptor activation induces vitamin D receptor levels in proximal kidney HK-2G cells by a mechanism that requires phosphorylation of p38alpha MAPK. J Biol Chem 4283(1):175–183 Mamillapalli R, VanHouten J, Zawalich W, Wysolmerski J (2008) Switching of G-protein usage by the calcium-sensing receptor re- verses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells. J Biol Chem 283:24435– 24447 Meng K, Xu J, Zhang C, Zhang R, Yang H, Liao C, Jiao J (2014) Calcium sensing receptor modulates extracellular calcium entry and prolifer- ation via TRPC3/6 channels in cultured human mesangial cells. PLoS One 9(6):e98777 Mizumachi H, Yoshida S, Tomokiyo A, Hasegawa D, Hamano S, Yuda A, Sugii H, Serita S, Mitarai H, Koori K, Wada N, Maeda H (2017) Calcium-sensing receptor-ERK signaling promotes odontoblastic differentiation of human dental pulp cells. Bone. 101:191–201 Nemeth EF, Delmar EG, Heaton WL, Miller MA, Lambert LD, Conklin RL, Gowen M, Gleason JG, Bhatnagar PK, Fox J (2001) Calcilytic compounds: potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone. J Pharmacol Exp Ther 299(1):323–331 Noh JS, Pak HJ, Shin YJ, Riew TR, Park JH, Moon YW, Lee MY (2015) Differential expression of the calcium-sensing receptor in the ische- mic and border zones after transient focal cerebral ischemia in rats. J Chem Neuroanat 66-67:40–51 Ruat M, Traiffort E (2013) Roles of the calcium sensing receptor in the central nervous system. Best Pract Res Clin Endocrinol Metab 27: 429–442 Thomsen AR, Worm J, Jacobsen SE, Stahlhut M, Latta M, Brauner- Osborne H (2012) Strontium is a biased agonist of the calcium- sensing receptor in rat medullary thyroid carcinoma 6-23 cells. J Pharmacol Exp Ther 343:638–649 Veldhuis NA, Poole DP, Grace M, McIntyre P, Bunnett NW (2015) The G protein-coupled receptor-transient receptor po- tential channel axis: molecular insights for targeting disorders of sensation and inflammation. Pharmacol Rev 67(1):36–73 Weston AH, Absi M, Ward DT, Ohanian J, Dodd RH, Dauban P, Petrel C, Ruat M, Edwards G (2005) Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231. Circ Res 97:391–398 Yamamura A, Ohara N, Tsukamoto K (2015) Inhibition of excessive cell proliferation by calcilytics in idiopathic pulmonary arterial hyper- tension. PLoS One 10(9):e0138384 Yu J, Zhao L, Liu L, Yang F, Zhu X, Cao B (2016) Tetrahydropalmatine protects rat pulmonary endothelial cells from irradiation-induced apoptosis by inhibiting oxidative stress and the calcium sensing receptor/phospholipase C-gamma1 pathway. Free Radic Res 50: 611–626 Zhao M, He X, Yang YH, Yu XJ, Bi XY, Yang Y, Xu M, Lu XZ, Sun Q, Zang WJ (2015) Acetylcholine SB203580 protects mesenteric arteries against hypoxia/reoxygenation injury via inhibiting calcium-sensing recep- tor. J Pharmacol Sci 127:481–488