Characterization of Ca2+-Sensing Receptor-Mediated Ca2+ Influx in Microvascular bEND.3 Endothelial Cells

Iat-Lon Leong; Tien-Yao Tsai; Lian-Ru Shiao; Yu-Mei Zhang; Kar-Lok Wong; Paul Chan; Yuk-Man Leung

doi:10.4103/cjp.cjp_93_20

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ORIGINAL ARTICLE

Year : 2021 | Volume : 64 | Issue : 2 | Page : 80-87

Characterization of Ca²⁺-Sensing Receptor-Mediated Ca²⁺ Influx in Microvascular bEND.3 Endothelial Cells

Iat-Lon Leong¹, Tien-Yao Tsai², Lian-Ru Shiao³, Yu-Mei Zhang⁴, Kar-Lok Wong⁵, Paul Chan⁶, Yuk-Man Leung³
¹ Department of Internal Medicine, Division of Cardiology, Kiang Wu Hospital, Macau, China
² Cardiovascular Division, Fu Jen Catholic University Hospital, Fu Jen Catholic University, New Taipei City; Department of Cardiology, Lotung Poh-Ai Hospital, Yilan County, Taiwan
³ Department of Physiology, China Medical University, Taichung, Taiwan
⁴ VIP Department, East Hospital, Tongji University School of Medicine, Shanghai, China
⁵ Department of Anesthesiology, China Medical University Hospital; Department of Anesthesiology, Kuang Tien General Hospital, Taichung, Taiwan
⁶ Division of Cardiology, Department of Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

Date of Submission	16-Nov-2020
Date of Decision	06-Mar-2021
Date of Acceptance	09-Mar-2021
Date of Web Publication	19-Apr-2021

Correspondence Address:
Prof. Yuk-Man Leung
Department of Physiology, China Medical University, Taichung
Taiwan

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjp.cjp_93_20

Abstract

Ca²⁺-sensing receptors (CaSR), activated by elevated concentrations of extracellular Ca²⁺, have been known to regulate functions of thyroid cells, neurons, and endothelial cells (EC). In this report, we studied CaSR-mediated Ca²⁺ influx in mouse cerebral microvascular EC (bEND.3 cells). Cytosolic free Ca²⁺ concentration and Mn²⁺ influx were measured by fura-2 microfluorometry. High (3 mM) Ca²⁺ (CaSR agonist), 3 mM spermine (CaSR agonist), and 10 μM cinacalcet (positive allosteric modulator of CaSR) all triggered Ca²⁺ influx; however, spermine, unlike high Ca²⁺ and cinacalcet, did not promote Mn²⁺ influx and its response was poorly sensitive to SKF 96365, a TRP channel blocker. Consistently, 2-aminoethoxydiphenyl borate and ruthenium red (two other general TRP channel blockers) suppressed Ca²⁺ influx triggered by cinacalcet and high Ca²⁺ but not by spermine. Ca²⁺ influx triggered by high Ca²⁺, spermine, and cinacalcet was similarly suppressed by A784168, a potent and selective TRPV1 antagonist. Our results suggest that CaSR activation triggered Ca²⁺ influx via TRPV1 channels; intriguingly, pharmacological, and permeability properties of such Ca²⁺ influx depended on the stimulating ligands.

Keywords: Ca²⁺, Ca²⁺-sensing receptors, endothelium, spermine, TRP channels

How to cite this article:
Leong IL, Tsai TY, Shiao LR, Zhang YM, Wong KL, Chan P, Leung YM. Characterization of Ca²⁺-Sensing Receptor-Mediated Ca²⁺ Influx in Microvascular bEND.3 Endothelial Cells. Chin J Physiol 2021;64:80-7

How to cite this URL:
Leong IL, Tsai TY, Shiao LR, Zhang YM, Wong KL, Chan P, Leung YM. Characterization of Ca²⁺-Sensing Receptor-Mediated Ca²⁺ Influx in Microvascular bEND.3 Endothelial Cells. Chin J Physiol [serial online] 2021 [cited 2023 Mar 23];64:80-7. Available from: https://www.cjphysiology.org/text.asp?2021/64/2/80/314084

The authors Iat-Lon Leong, Tien-Yao Tsai, Paul Chan and Yuk-Man Leung contribute to the study equally

Introduction

Ca²⁺-sensing receptors (CaSRs) are G-protein-coupled receptors activated by elevated concentrations of extracellular Ca²⁺. One of the biological functions of CaSR is the modulation of parathyroid hormone (PTH) release.^[1] Serum Ca²⁺ elevation stimulates thyroid cell CaSR, the latter then activates G_q/11 and phospholipase C (PLC). As a result, inositol 1, 4, 5-trisphosphate (IP₃) is generated and Ca²⁺ is mobilized from internal stores. PTH release is consequently inhibited by cytosolic elevation of Ca²⁺ concentration.

CaSRs are also expressed in the nervous system and blood vessels. CaSRs, sensing extracellular Ca²⁺, regulate the excitability of nerve cells via opening nonselective cation channels.^[2] CaSRs also promote proliferation and even migration of nerve cells.^[3] Interestingly, isolated blood vessels are dilated by an increase in extracellular Ca²⁺: it was shown that CaSR stimulation in the porcine coronary artery endothelial cell (EC) caused the opening of intermediate conductance Ca²⁺-activated K⁺ channels.^[4] As a consequence, EC hyperpolarized, followed by hyperpolarization of neighboring smooth muscle cells. In a recent work, it was shown that CaSR activation leads to a rise in free cytosolic Ca²⁺ concentration and NO release in human umbilical vein EC.^[5]

In our previous work, we demonstrated protein expression and functions of CaSR in murine brain microvascular EC (bEND.3); the CaSR response (high Ca²⁺-elicited cytosolic [Ca²⁺] elevation) was refractory to suppression of PLC and involved Ca²⁺ influx.^[6] In this work, we further studied the pharmacological properties of CaSR-mediated Ca²⁺ influx. We provided evidence that stimulation of CaSR in bEND.3 cells by three different means, namely high Ca²⁺, spermine, and cinacalcet, triggered Ca²⁺ influx via TRPV1 channels. It was found that, surprisingly, pharmacological and permeability properties of CaSR-triggered Ca²⁺ influx varied with the stimulating ligands.

Materials and Methods

Materials and cell culture

Spermine, cyclopiazonic acid (CPA), and adenosine triphosphate (ATP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 AM was purchased from Calbiochem-Millipore (Darmstadt, Germany). Cinacalcet, 2-aminoethoxydiphenyl borate (2-APB), ruthenium red, CBA, SAR 7334, M084, GSK 2193874, tranilast, Pyr 3, A784168, and SKF 96365 were purchased from Tocris (Bristol, UK). Brain microvascular bEND.3 cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Lung epithelial A549 cells were cultured in RPMI medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Microfluorometric measurement of cytosolic Ca²⁺ concentration and Mn²⁺ influx

Microfluorometric measurement of cytosolic concentration of Ca²⁺ was performed using fura-2 as Ca²⁺-sensitive dye.^[7] Briefly, the cells were grown on small glass coverslips and incubated with 5 μM fura-2 AM (Invitrogen, Carlsbad, CA, USA) for 1 h at 37°C. The cells were then washed in bath solution, containing (mM): 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, and 10 HEPES (pH 7.4 adjusted with NaOH). Ca²⁺-free solution was the same as the bath solution described above except that Ca²⁺ was absent and 100 μM EGTA was supplemented. Cells were alternately excited with 340 nm and 380 nm (1 Hz switching frequency) with an optical filter changer (Lambda 10-2, Sutter Instruments, Novato, CA, USA). The emission wavelength was 500 nm and images were obtained with a CCD camera (CoolSnap HQ2, Photometrics, Tucson, AZ, USA) connected to an inverted Nikon TE2000-U microscope (Tokyo, Japan). When an influx of Mn²⁺ was measured, the excitation wavelength was 360 nm and emission wavelength was 500 nm. Data were analyzed by an MAG Biosystems Software (Sante Fe, MN, USA). Experiments were all conducted at room temperature (25°C). Fluorescence changes were measured and analyzed at a region of interest of single cells within a microscopic view (counted as an individual experiment). Experiments were repeated a few more times to obtain the means of all single cells.

Statistical analysis

Data were means ± standard error of the mean. Unpaired or paired Student's t-test was employed where appropriate to make comparisons of two groups. ANOVA was used to compare multiple groups, followed by the Tukey's HSD post hoc test. P < 0.05 was considered statistically significant.

Results

Spermine-stimulated Ca²⁺ influx was largely unrelated to store-operated Ca²⁺ entry in bEND.3 cells

In our previous report, we have shown that in bEND.3 cells, high Ca²⁺-elicited Ca²⁺ signal was insensitive to PLC inhibition, suggesting that G_q/11 may not be involved.^[6] These data suggest that CaSR-stimulated Ca²⁺ signaling in bEND.3 cells does not depend on Ca²⁺ release but may involve Ca²⁺ influx. To confirm that CaSR response is mainly composed of Ca²⁺ influx but not Ca²⁺ release, we here employed the standard “Ca²⁺-free solution—Ca²⁺ replenishment” protocol [Figure 1]. Ca²⁺ as a CaSR agonist could not be used in this protocol since the addition of this agonist rendered the solution no longer Ca²⁺ free. Cinacalcet [Supplement 1], a positive allosteric modulator of CaSR,^[8] is not suitable in this protocol since its action requires the presence of Ca²⁺ (as agonist). We therefore used spermine, a CaSR agonist, for this purpose. As shown in [Figure 1]a, the addition of spermine caused a substantial rise of Ca²⁺ in Ca²⁺-containing solution. However, in the absence of extracellular Ca²⁺, spermine triggered only a minimal (yet significant, P < 0.05) amount of Ca²⁺ release; subsequent Ca²⁺ replenishment resulted in Ca²⁺ influx [Figure 1]b comparable to the level reached at Ca²⁺-containing solution [Figure 1]a. Such a Ca²⁺ influx was retarded but not suppressed at a steady state in the presence of TRP blocker SKF 96365. Ni²⁺, a nonspecific Ca²⁺ channel blocker, abrogated spermine-stimulated Ca²⁺ influx [Supplement 2].

Figure 1: Ca²⁺ influx stimulated by spermine. (a) [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-containing solution and cells were exposed to 3 mM spermine. (b) [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-free solution. Cells were pretreated with or without 10 μM SKF 96365 for 8 min (red and black lines, respectively); after cells were stimulated by 3 mM spermine, 2 mM Ca²⁺ was replenished. The green line represents the results obtained when cells received no SKF 96365 pre-treatment and spermine stimulation before Ca²⁺ replenishment. There are significant (*P < 0.05) differences between the spermine-SKF group and spermine-no SKF group between 403 and 583 s. (c) [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-containing solution and cells were treated with 30 μM cyclopiazonic acid. Spermine results obtained in Figure 1a are overlapped here for comparison. Significant (*P < 0.05) differences between the spermine and cyclopiazonic acid group began after 278 s. (d) [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-free solution. After cells were treated with 30 μM cyclopiazonic acid, 2 mM Ca²⁺ was replenished. Spermine results obtained in Figure 1b are overlapped here for comparison. Significant (*P < 0.05) differences between the spermine and cyclopiazonic acid group began after 500 s. Results are mean ± SEM, with each group having 23–29 cells obtained from three to four separate experiments.

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The data in [Figure 1]a and [Figure 1]b suggest that spermine stimulated Ca²⁺ influx only with a minimal Ca²⁺ release, casting doubt on the nature of such Ca²⁺ influx as store-operated Ca²⁺ entry. As shown in [Figure 1]c, depletion of endoplasmic reticulum Ca²⁺ store with maximal (CPA, inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺ pump) concentration resulted in a sustained Ca²⁺ signal in the presence of bath Ca²⁺. Also shown in the same figure for comparison is spermine-stimulated Ca²⁺ signal [Figure 1]a, which was significantly stronger than the CPA-stimulated one. [Figure 1]d shows CPA-stimulated Ca²⁺ release (in Ca²⁺-free solution) and Ca²⁺ influx (upon Ca²⁺ replenishment); spermine-stimulated Ca²⁺ release and Ca²⁺ influx [Figure 1]b were also shown for comparison. It is obvious that spermine stimulated less Ca²⁺ release but elicited a much stronger Ca²⁺ influx. Taken together, the results suggest spermine-stimulated Ca²⁺ entry, a major fraction of which was unrelated to store-operated Ca²⁺ entry.

Different sensitivity of high Ca²⁺-, spermine-, and cinacalcet-triggered Ca²⁺ influx to SKF 96365 in bEND.3 cells

We compared SKF 96365 inhibitory effects on Ca²⁺ signals triggered by high Ca²⁺, spermine, and cinacalcet (positive allosteric modulator of CaSR) in Ca²⁺-containing bath solution. As shown in [Figure 2]a and [Figure 2]b, Ca²⁺ signals triggered by high Ca²⁺ and cinacalcet were substantially suppressed by SKF 96365. Ca²⁺ signal triggered by spermine was retarded but only marginally suppressed at steadystate by SKF 96365 [Figure 2]c, an observation consistent with the results shown in [Figure 1]b. The results in [Figure 1] and [Figure 2] therefore suggest the sensitivity of CaSR-stimulated Ca²⁺ influx to SKF 96365 depended on the stimulating ligands.

Figure 2: Suppressive effects of SKF 96365 on Ca²⁺ signals triggered by high Ca²⁺, cinacalcet, and spermine. [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-containing solution; cells were pretreated with or without 10 μM SKF 96365 for 8 min and then treated with (a) 3 mM Ca²⁺, significant (*P < 0.05) differences between the control and SKF group began after 103 s; (b) 10 μM cinacalcet, significant (*P < 0.05) differences between the control and SKF group began after 100 s; or (c) 3 mM spermine, significant (*P < 0.05) differences between the control and SKF group began after 119 s. Results are mean ± SEM, with each group having 16–29 cells obtained from three separate experiments.

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Unlike high Ca²⁺ and cinacalcet, spermine did not promote Mn²⁺ influx in bEND.3 cells

We then examined whether CaSR stimulation triggered Mn²⁺ influx. The results in [Figure 3]a and [Figure 3]b showed that high Ca²⁺ and cinacalcet stimulated Mn²⁺ influx. Unexpectedly, spermine did not stimulate Mn²⁺ influx [Figure 3]c.

Figure 3: Mn²⁺ influx was triggered by extracellular Ca²⁺ and cinacalcet but not spermine. (a) bEND.3 cells were bathed in Ca²⁺-free solution, treated with 1 mM Mn²⁺, followed by the addition of water or 3 mM Ca²⁺. Significant differences (*P < 0.05) between the control and CaCl₂ group began after 462 s. (b) bEND.3 cells were bathed in 1 mM Ca²⁺-containing solution, treated with 1 mM Mn²⁺, followed by the addition of DMSO or 10 μM cinacalcet. Significant differences (*P < 0.05) between the control and cinacalcet group began after 200 s. (c) bEND.3 cells were bathed in Ca²⁺-free solution, treated with 1 mM Mn²⁺, followed by the addition of water or 3 mM spermine. (d) bEND.3 cells were bathed in Ca²⁺-free solution containing 10 μM SKF 96365, treated with 1 mM Mn²⁺, followed by the addition of water or 3 mM Ca²⁺. (e) bEND.3 cells were bathed in 1 mM Ca²⁺-containing solution containing 10 μM SKF 96365, treated with 1 mM Mn²⁺, followed by the addition of DMSO or 10 μM cinacalcet. Results are mean ± SEM, with each group having 26–50 cells obtained from three to five separate experiments.

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Since high Ca²⁺- and cinacalcet-stimulated Ca²⁺ signals were suppressed by SKF 96365, we next investigated whether high Ca²⁺- and cinacalcet-stimulated Mn²⁺ influx was sensitive to SKF 96365. The results in [Figure 3]d and [Figure 3]e show that high Ca²⁺ and cinacalcet did not stimulate Mn²⁺ influx in the presence of SKF 96365.

A784168-suppressed Ca²⁺ influx triggered by cinacalcet, high Ca²⁺, and spermine in bEND.3 cells

We also examined whether Ca²⁺ influx triggered by cinacalcet, high Ca²⁺, and spermine was inhibited by 2-APB and ruthenium red (general TRP channel blockers). As shown in [Figure 4]a and [Figure 4]b, Ca²⁺ signals triggered by high Ca²⁺ and cinacalcet were suppressed by 2-APB and ruthenium red. However, Ca²⁺ signal triggered by spermine was retarded but not suppressed at a steady state by these two agents [Figure 4]c. These results, being consistent with the effects of SKF 96365 shown in [Figure 2], suggest that high Ca²⁺ and cinacalcet differed from spermine in the manner they affected TRP channels.

Figure 4: Suppressive effects of 2-APB and ruthenium red on Ca²⁺ signals triggered by high Ca²⁺, cinacalcet , and spermine. [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-containing solution; cells were pretreated with or without 50 μM 2-APB or 30 μM ruthenium red for 8 min and then treated with (a) 3 mM Ca²⁺; significant (P < 0.05) differences between the control and 2-APB group and ruthenium red group began after 183 (*) and 107 s (**), respectively; (b) 10 μM cinacalcet; significant (P < 0.05) differences between the control and 2-APB group and ruthenium red group began after 115 (*) and 100 s (**), respectively; or (c) 3 mM spermine; there were significant (P < 0.05) differences between the control and 2-APB group from 102 to 318 s (*), and between the control and ruthenium red group from 122 to 340 s (**). Results are mean ± SEM, with each group having 23–41 cells obtained from three to four separate experiments.

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Further experiments were performed to identify which TRP members were involved in CaSR-mediated Ca²⁺ influx. As shown in [Figure 5]a, [Figure 5]b, [Figure 5]c, Ca²⁺ influx triggered by high Ca²⁺, spermine, and cinacalcet was similarly suppressed by A784168 (1 μM), a potent and selective TRPV1 antagonist.^[9] High Ca²⁺-triggered Ca²⁺ influx was not inhibited by CBA (TRPM4 inhibitor; 10 μM), SAR 7334 (TRPC6 inhibitor; 3 μM), Pyr 3 (TRPC3 inhibitor; 10 μM), M084 (TRPC4/5 inhibitor; 10 μM), GSK 2193874 (TRPV4 inhibitor; 300 nM), and tranilast (TRPV2 inhibitor; 10 μM) [Supplement 3].

Figure 5: Suppressive effects of A784168 on Ca²⁺ signals triggered by high Ca²⁺, cinacalcet, and spermine. [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-containing solution; cells were pretreated with or without 1 μM A784168 for 8 min and then treated with (a) 3 mM Ca²⁺; significant (*P < 0.05) differences between the control and A784168 group began after 187 s; (b) 10 μM cinacalcet; significant (*P < 0.05) differences between the control and A784168 group began after 114 s; or (c) 3 mM spermine; significant (*P < 0.05) differences between the control and A784168 group began after 192 s. Results are mean ± SEM, with each group having 25–37 cells obtained from three to four separate experiments.

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Effects of TRP channel blockers on spermine-triggered Ca²⁺ influx in A549 cells

Since spermine-triggered Ca²⁺ influx differed pharmacologically from that triggered by high Ca²⁺ and cinacalcet, we further investigated spermine actions in another cell type, the human alveolar epithelial A549 cell. As shown in [Figure 6]a, the addition of spermine triggered Ca²⁺ signal in Ca²⁺-containing solution. In the absence of extracellular Ca²⁺, spermine did not trigger Ca²⁺ release; subsequent Ca²⁺ replenishment resulted in Ca²⁺ influx [Figure 6]b. SKF 96365 strongly suppressed [Figure 6]a, while A784168 did not inhibit spermine-triggered Ca²⁺ influx [Figure 6]c. These results with SKF 96365 and A784168 as probes in A549 cells appeared to be different from those obtained in bEND.3 cells.

Figure 6: Effects of SKF 96365 and A784168 on Ca²⁺ signals triggered by spermine in A549 cells. (a) [Ca²⁺]i was measured in A549 cells bathed in Ca²⁺-containing solution; cells were pretreated with or without 10 μM SKF 96365 for 8 min and then treated with 3 mM spermine; significant (*P < 0.05) differences between the control and SKF 96365 group began after 138 s; (b) [Ca²⁺]i was measured in A549 cells bathed in Ca²⁺-free solution. Cells were treated with 3 mM spermine and then 2 mM Ca²⁺ was replenished. (c) [Ca²⁺]i was measured in A549 cells bathed in Ca²⁺-containing solution; cells were pretreated with or without 1 μM A784168 for 8 min and then treated with 3 mM spermine. Results are mean ± SEM, with each group having 67–88 cells obtained from four separate experiments.

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Discussion

In a previous report, we have shown protein (Western blot) and functional expression of CaSR in bEND.3 cells.^[6] We have shown that CaSR response (high Ca²⁺-triggered Ca²⁺ signaling) in bEND.3 is unaffected by PLC inhibition caused by two chemically unrelated inhibitors, edelfosine and U73122,^[6] therefore suggesting that the Ca²⁺ signaling is not caused by Ca²⁺ release by IP3 produced via G_q/11-PLC-mediated cleavage of phosphatidyl 4,5-bisphosphate (PIP₂). The “Ca²⁺-free solution—Ca²⁺ replenishment” protocol allows one to dissect Ca²⁺ influx from Ca²⁺ release, but this protocol, as explained, precludes the use of high Ca²⁺ and cinacalcet to stimulate CaSR. We therefore used spermine as a CaSR agonist and found that it stimulated Ca²⁺ influx largely unrelated to store-operated Ca²⁺ entry; this finding is consistent with the insensitivity of high Ca²⁺-triggered Ca²⁺ signaling response to PLC inhibition. CaSR has been demonstrated to be coupled to a variety of G proteins, for instance, G_12/13, G_q/11, Gi, and Gs.^{[10],[11],[12],[13]} CaSR activation has also been shown to stimulate ERK_1/2.^[14],[15] None of the above is directly coupled to Ca²⁺ mobilization except G_q/11, but our data are not supportive of a role of G_q/11 in causing Ca²⁺ influx.

We have excluded the involvement of the reverse mode of Na⁺/Ca²⁺ exchanger in high Ca²⁺-triggered Ca²⁺ signaling.^[6] The suppression of high Ca²⁺- and cinacalcet-triggered Ca²⁺ signals and Mn²⁺ influx by SKF 96365, 2-APB, and ruthenium red suggests that CaSR-mediated Ca²⁺ influx was via TRP channels [Figure 2] and [Figure 3]. As distinguished from voltage-gated Ca²⁺ channels such as L-, T-, and N-type voltage-gated Ca²⁺ channels, TRP channels are a large family of nonvoltage-gated Ca²⁺-permeable channels.^[16] It is noted that bEND.3 cells express multiple TRP channels such as various members of TRPM, TRPC, and TRPV.^[17] Indeed, CaSRs interact with various TRP channels in mesangial cells,^[18] aortic smooth muscle cells,^[19] and mesenteric artery ECs.^[20] The results in [Figure 1], [Figure 2], [Figure 3], [Figure 4] suggest high Ca²⁺-, spermine-, and cinacalcet-triggered Ca²⁺ influx via TRP channels; the Ca²⁺ influx was similarly suppressed by A784168 (a potent and selective TRPV1 antagonist) [Figure 5], suggesting the involvement of TRPV1 channels. This finding is considered novel, as it is noted that, on the contrary, CaSR activation leads to inhibition of TRPV1 in rat vagal bronchopulmonary sensory neurons.^[21] How CaSR is coupled to TRP channels is hitherto undefined.^[22]

CaSR stimulation by high Ca²⁺ and cinacalcet has been shown to activate intermediate conductance Ca²⁺-activated K⁺ channels and thus hyperpolarize porcine coronary artery ECs.^[4] Such hyperpolarization increases the electrochemical driving force for Ca²⁺ influx. Therefore, in our case, it is possible that high extracellular Ca²⁺ stimulated CaSR, TRP channel opening, and Ca²⁺ influx; the latter was enhanced further by hyperpolarization. Spermine-stimulated Ca²⁺ influx via TRP was also expected to be modulated by membrane potential changes since spermine inhibits inward rectifier K⁺ (Kir) channels, whose main role is to set the resting membrane potential.^[23] It has been suggested that a small efflux of K⁺ above equilibrium K⁺ potential is the usual function of Kir channels.^[23] Therefore, spermine inhibition of Kir channels may indeed depolarize bEND.3 cells.

What is rather unexpected is that spermine-stimulated Ca²⁺ influx, in contrast to the responses to high Ca²⁺ and cinacalcet, was much less sensitive to SKF 96365. Consistently, high Ca²⁺- and cinacalcet-triggered Ca²⁺ influx was significantly inhibited by 2-APB and ruthenium red (general TRP channel blockers), but spermine-triggered Ca²⁺ influx could only be slowed down but not suppressed by them. These data suggest that high Ca²⁺ and cinacalcet differed from spermine in the manner they interacted with the CaSR-TRP channel complex. Furthermore, while high Ca²⁺ and cinacalcet stimulated Mn²⁺ influx, spermine failed to do so, although it markedly stimulated an influx of Ca²⁺. It therefore appears that upon activation by different ligands, CaSR-triggered Ca²⁺ influx exhibited different pharmacological and permeability properties. CaSR activation has been known to exhibit biased agonism, in which binding by distinct ligands selectively activates certain signaling pathways such as G_12/13, G_q/11, Gi, Gs, or ERK_1/2.^[14],[22] Ca²⁺, spermine, and cinacalcet may bind to distinct sites of CaSR, causing the latter to adopt different active conformations, eventually leading to biased agonism.^[22] Biased agonism may also explain why CaSR activation could be coupled to different TRP channel members.^{[18],[19],[20],[21]} In line with this notion, we have shown in [Figure 6] that spermine-triggered Ca²⁺ influx may be via TRP channels other than TRPV1 in A549 cells, a result different from what was observed in spermine-stimulated bEND.3 cells. Our work here further reveals that distinct CaSR ligands could differentially affect the pharmacological and permeability properties of TRP channel opening.

Conclusion

CaSR activation in bEND.3 cells led to Ca²⁺ influx largely unrelated to store-operated Ca²⁺ entry; such Ca²⁺ influx might be via TRPV1 channels. Interestingly, different CaSR ligands appeared to affect differently the pharmacological and permeability properties of the elicited Ca²⁺ influx.

Acknowledgements

Y.M.L. and K.L.W. would like to thank China Medical University and China Medical University Hospital, Taiwan, for providing fundings (DMR-108-083; DMR-109-093; CMU108-S-31). This work was also supported by a grant from the Natural Science Foundation of Shanghai (18ZR1430900).

Financial support and sponsorship

Y.M.L. and K.L.W. would like to thank China Medical University and China Medical University Hospital, Taiwan, for providing fundings (DMR-108-083; DMR-109-093; CMU108-S-31). This work was also supported by a grant from the Natural Science Foundation of Shanghai (18ZR1430900).

Conflicts of interest

There are no conflicts of interest.

References

1.	Chen RA, Goodman WG. Role of the calcium-sensing receptor in parathyroid gland physiology. Am J Physiol Renal Physiol 2004;286:F1005-11.
2.	Jones BL, Smith SM. Calcium-sensing receptor: A key target for extracellular calcium signaling in neurons. Front Physiol 2016;7:116.
3.	Ruat M, Traiffort E. Roles of the calcium sensing receptor in the central nervous system. Best Pract Res Clin Endocrinol Metab 2013;27:429-42.
4.	Weston AH, Absi M, Ward DT, Ohanian J, Dodd RH, Dauban P, et al. Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: Studies with calindol and Calhex 231. Circ Res 2005;97:391-8.
5.	Horinouchi T, Mazaki Y, Terada K, Miwa S. Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells. J Pharmacol Sci 2020;143:315-9.
6.	Chen CY, Hour MJ, Lin WC, Wong KL, Shiao LR, Cheng KS, et al. Antagonism of Ca²⁺-sensing receptors by NPS 2143 is transiently masked by p38 activation in mouse brain bEND.3 endothelial cells. Naunyn Schmiedebergs Arch Pharmacol 2019;392:823-32.
7.	Leung YM, Huang CF, Chao CC, Lu DY, Kuo CS, Cheng TH, et al. Voltage-gated K+ channels play a role in cAMP-stimulated neuritogenesis in mouse neuroblastoma N2A cells. J Cell Physiol 2011;226:1090-8.
8.	Messa P, Alfieri C, Brezzi B. Cinacalcet: Pharmacological and clinical aspects. Expert Opin Drug Metab Toxicol 2008;4:1551-60.
9.	Cui M, Honore P, Zhong C, Gauvin D, Mikusa J, Hernandez G, et al. TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists. J Neurosci 2006;26:9385-93.
10.	Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, et al. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 2001;280:F291-302.
11.	Almadén Y, Canalejo A, Ballesteros E, Añón G, Cañadillas S, Rodríguez M. Regulation of arachidonic acid production by intracellular calcium in parathyroid cells: Effect of extracellular phosphate. J Am Soc Nephrol 2002;13:693-8.
12.	Huang C, Hujer KM, Wu Z, Miller RT. The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells. Am J Physiol Cell Physiol 2004;286:C22-30.
13.	Mamillapalli R, VanHouten J, Zawalich W, Wysolmerski J. Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells. J Biol Chem 2008;283:24435-47.
14.	Thomsen AR, Worm J, Jacobsen SE, Stahlhut M, Latta M, Bräuner-Osborne H. Strontium is a biased agonist of the calcium-sensing receptor in rat medullary thyroid carcinoma 6-23 cells. J Pharmacol Exp Ther 2012;343:638-49.
15.	Mizumachi H, Yoshida S, Tomokiyo A, Hasegawa D, Hamano S, Yuda A, et al. Calcium-sensing receptor-ERK signaling promotes odontoblastic differentiation of human dental pulp cells. Bone 2017;101:191-201.
16.	Thakore P, Earley S. Transient receptor potential channels and endothelial cell calcium signaling. Compr Physiol 2019;9:1249-77.
17.	Berrout J, Jin M, O'Neil RG. Critical role of TRPP2 and TRPC1 channels in stretch-induced injury of blood-brain barrier endothelial cells. Brain Res 2012;1436:1-12.
18.	Meng K, Xu J, Zhang C, Zhang R, Yang H, Liao C, et al. Calcium sensing receptor modulates extracellular calcium entry and proliferation via TRPC3/6 channels in cultured human mesangial cells. PLoS One 2014;9:e98777.
19.	Chow JY, Estrema C, Orneles T, Dong X, Barrett KE, Dong H. Calcium-sensing receptor modulates extracellular Ca2+ entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells. Am J Physiol Cell Physiol 2011;301:C461-8.
20.	Greenberg HZE, Carlton-Carew SRE, Khan DM, Zargaran AK, Jahan KS, Vanessa Ho WS, et al. Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced nitric oxide production and vasorelaxation in rabbit mesenteric arteries. Vascul Pharmacol 2017;96-98:53-62.
21.	Gu Q, Vysotskaya ZV, Moss CR II, Kagira MK, Gilbert CA. Calcium-sensing receptor in rat vagal bronchopulmonary sensory neurons regulates the function of the capsaicin receptor TRPV1. Exp Physiol 2013;98:1631-42.
22.	Leach K, Hannan FM, Josephs TM, Keller AN, Møller TC, Ward DT, et al. International union of basic and clinical pharmacology. CVIII. Calcium-sensing receptor nomenclature, pharmacology, and function. Pharmacol Rev 2020;72:558-604.
23.	Hille B. Ion Channels of Excitable Membranes, 3^rd ed. Sunderland, MA: Sinauer Press; 2001: p. 149-52.

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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

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