Sensitivity of Ca2+-sensing receptor-transient receptor potential-mediated Ca2+ influx to extracellular acidity in bEND.3 endothelial cells

Iat-Lon Leong; Chung-Ming Yu; Lian-Ru Shiao; Paul Chan; King-Chuen Wu; Yuk-Man Leung

doi:10.4103/0304-4920.365460

ORIGINAL ARTICLE

Year : 2022 | Volume : 65 | Issue : 6 | Page : 277-281

Sensitivity of Ca²⁺-sensing receptor-transient receptor potential-mediated Ca²⁺ influx to extracellular acidity in bEND.3 endothelial cells

Iat-Lon Leong¹, Chung-Ming Yu², Lian-Ru Shiao³, Paul Chan⁴, King-Chuen Wu⁵, Yuk-Man Leung³
¹ Division of Cardiology, Department of Internal Medicine, Kiang Wu Hospital, Macau, China
² Department of Anesthesiology, Chang Gung Memorial Hospital, Chiayi, Taiwan
³ Department of Physiology, China Medical University, Taichung, Taiwan
⁴ Division of Cardiology, Department of Medicine, Taipei Medical University Wan Fang Hospital, Taipei, Taiwan
⁵ Department of Anesthesiology, Chang Gung Memorial Hospital, Chiayi; Department of Nursing, Chang Gung University of Science and Technology, Chiayi; Department of Information Management, Shu-Zen Junior College of Medicine and Management, Kaohsiung, Taiwan

Date of Submission	16-Jul-2022
Date of Decision	04-Sep-2022
Date of Acceptance	08-Oct-2022
Date of Web Publication	26-Dec-2022

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

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0304-4920.365460

Abstract

Ca²⁺-sensing receptors (CaSRs) are G protein-coupled receptors activated by elevated concentrations of extracellular Ca²⁺. In our previous works, we showed protein and functional expression of CaSR in mouse cerebral endothelial cell (EC) (bEND.3); the CaSR response (high Ca²⁺-elicited cytosolic [Ca²⁺] elevation) was unaffected by suppression of phospholipase C but in part involved Ca²⁺ influx through transient receptor potential V1 (TRPV1) channels. In this work, we investigated if extracellular acidity affected CaSR-mediated Ca²⁺ influx triggered by high (3 mM) Ca²⁺ (CaSR agonist), 3 mM spermine (CaSR agonist), and 10 mM cinacalcet (positive allosteric modulator of CaSR). Extracellular acidosis (pH 6.8 and pH 6.0) strongly suppressed cytosolic [Ca²⁺] elevation triggered by high Ca²⁺, spermine, and cinacalcet; acidosis also inhibited Mn²⁺ influx stimulated by high Ca²⁺ and cinacalcet. Purinoceptor-triggered Ca²⁺ response, however, was not suppressed by acidosis. Extracellular acidity also did not affect membrane potential, suggesting suppressed CaSR-mediated Ca²⁺ influx in acidity did not result from the reduced electrical driving force for Ca²⁺. Our results suggest Ca²⁺ influx through a putative CaSR-TRP complex in bEND.3 EC was sensitive to extracellular pH.

Keywords: Ca²⁺, Ca²⁺-sensing receptors, cinacalcet, endothelium, spermine, transient receptor potential channels

How to cite this article:
Leong IL, Yu CM, Shiao LR, Chan P, Wu KC, Leung YM. Sensitivity of Ca²⁺-sensing receptor-transient receptor potential-mediated Ca²⁺ influx to extracellular acidity in bEND.3 endothelial cells. Chin J Physiol 2022;65:277-81

How to cite this URL:
Leong IL, Yu CM, Shiao LR, Chan P, Wu KC, Leung YM. Sensitivity of Ca²⁺-sensing receptor-transient receptor potential-mediated Ca²⁺ influx to extracellular acidity in bEND.3 endothelial cells. Chin J Physiol [serial online] 2022 [cited 2023 Sep 28];65:277-81. Available from: https://www.cjphysiology.org/text.asp?2022/65/6/277/365460

^#Iat-Lon Leong and Chung-Ming Yu contributed equally as first authors. ^##Paul Chan, King-Chuen Wu and Yuk-Man Leung contributed equally as corresponding authors.

Introduction

Ca²⁺-sensing receptors (CaSRs) are G protein-coupled receptors activated by elevated extracellular Ca²⁺ concentration. A rise in serum Ca²⁺ concentration stimulates parathyroid cell CaSR, which then activates G_q/11 and phospholipase C (PLC); as a result, inositol 1, 4, 5-trisphosphate (IP₃) is generated which mobilizes Ca²⁺ from internal stores.^[1] Parathyroid hormone release is consequently inhibited by cytosolic elevation of Ca²⁺ concentration.^[1] Pleiotropy of CaSR has been demonstrated: in addition to G_q/11, CaSR is coupled to, for instance, G_12/13, Gi, and Gs.^{[2],[3],[4],[5]} CaSR stimulation also activates the extracellular signal-regulated kinase (ERK)_1/2 pathway.^[6],[7] A number of studies have also shown that CaSR stimulation leads to the opening of various transient receptor potential (TRP) channels.^[8],[9],[10]

In our previous work, we demonstrated protein expression and functions of CaSR in murine bEND.3 brain microvascular endothelial cell (EC); the CaSR response (high Ca²⁺-elicited cytosolic [Ca²⁺] elevation) was refractory to suppression of PLC, but appeared to stimulate Ca²⁺ influx through TRP channels.^[11] In our most recent work, we provided evidence that stimulation of CaSR in bEND.3 cells by three different means, namely, high Ca²⁺, spermine, and cinacalcet, triggered Ca²⁺ influx in part through TRPV1 channels.^[12] Remarkably, spermine-triggered Ca²⁺ influx was much less sensitive to SKF96365, ruthenium red, and 2-aminoethoxydiphenyl borate (2-APB) than Ca²⁺ influx triggered by high Ca²⁺ and cinacalcet.^[12] High Ca²⁺ and cinacalcet, but not spermine, triggered Mn²⁺ influx. Therefore, pharmacological and permeability properties of CaSR-triggered Ca²⁺ influx varied with the stimulating ligands.

Cells switch to anaerobic glycolysis in case of oxygen shortage; the consequence is acidosis caused by lactic acid and proton accumulation.^[13],[14] Extracellular pH could reach 6.0 under ischemia.^{[13],[14],[15]} Whether extracellular acidosis affects CaSR responses is not much studied. In one report, it was shown that in human embryonic kidney (HEK) cells expressing CaSR and in bovine parathyroid cells, extracellular acidosis suppresses high Ca²⁺-stimulated Ca²⁺ elevation and ERK signaling.^[16] It is hitherto unknown if acidosis affects CaSR response in other cell types or CaSR coupled to other signaling pathways. Given that pharmacological and permeability properties of CaSR-triggered Ca²⁺ influx through TRP varied with stimulating ligands,^[12] it will be of interest in our present study to examine if extracellular acidity affects CaSR-TRP activities triggered by all these stimulating ligands.

Materials and Methods

Materials and cell culture

Spermine, cyclopiazonic acid, and ATP were from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 AM was purchased from Calbiochem Millipore. Cinacalcet, 2-APB, ruthenium red, and SKF 96365 were from Tocris (Bristol, UK). Brain microvascular bEND.3 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (Invitrogen).

Microfluorimetric measurement of cytosolic Ca²⁺ concentration and Mn²⁺ influx

Microfluorimetric measurement of the cytosolic concentration of Ca²⁺ was conducted using fura-2 as a Ca²⁺-sensitive probe.^[17] In brief, the cells were grown on small glass coverslips and incubated with 5 mM 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). The Ca²⁺-free solution was the same as the bath solution described above except that Ca²⁺ was absent and 100 μM EGTA was added. Cells were alternately excited with 380 nm and 340 nm (switching frequency 1 Hz) with the aid of an optical filter changer (Lambda 10-2, Sutter Instruments). The emission wavelength was set at 500 nm and images were acquired with a charge-coupled device (CCD) camera (CoolSnap HQ2, Photometrics, Tucson, AZ, USA) connected to an inverted Nikon TE2000-U microscope. When Mn²⁺ influx was measured, the excitation wavelength was 360 nm and the emission wavelength was 500 nm. Data were analyzed by MAG Biosystems Software (Santa Fe, NM, USA). Experiments were performed at room temperature (~25°C).

Electrophysiology

Electrophysiological experiments were performed as previously reported.^[17] Cells were voltage-clamped in the whole-cell configuration. Thin-walled borosilicate glass tubes (O.D. 1.5 mm, I.D. 1.10 mm, Sutter Instrument, Novato, CA, USA) were pulled with a micropipette puller (P-87, Sutter Instrument), and then heat polished by a microforge (Narishige Instruments, Inc., Sarasota, FL, USA). The pipettes, filled with intracellular solution, containing (mM): 140 KCl, 1 MgCl₂, 1 EGTA, 10 HEPES, and 5 Mg ATP (pH 7.25 adjusted with KOH), had typical resistance of 4-7 MΩ. The bath solution contained (mM): 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, and 10 HEPES (pH 7.4 adjusted with NaOH). The currents were recorded using an EPC-10 amplifier with Pulse 8.60 acquisition software and analyzed by Pulsefit 8.60 software (HEKA Elektronik, Lambrecht, Germany). Data were filtered at 2 kHz and sampled at 10 kHz. After a whole-cell configuration was established, the current-clamp mode was used to measure resting membrane potential. All experiments were performed at room temperature (~25°C).

Statistical Analysis

Data are means ± standard error of mean. Unpaired or paired Student's t-test was used where appropriate to compare the two groups. Multiple groups were analyzed by analysis of variance. P < 0.05 was considered statistically significant.

Results

As shown in [Figure 1]a, switching the bath pH from 7.4 to 6.8 and then 6.0 did not significantly alter Ca²⁺ level. However, pH 6.8 and pH 6.0 significantly suppressed Ca²⁺ signals triggered by 3 mM Ca²⁺ [Figure 1]b. Similarly, pH 6.8 and pH 6.0 significantly repressed Ca²⁺ signals triggered by spermine and cinacalcet [Figure 1]c and [Figure 1]d.

Figure 1: Extracellular acidity did not affect basal cytosolic Ca²⁺ level but suppressed CaSR-triggered Ca²⁺ signaling. [Ca²⁺]i was measured in bEND.3 cells bathed in Ca²⁺-containing solution. Cells were exposed to extracellular acidity (a). Cells bathed in Ca²⁺-containing solution of different pH were stimulated by (b) 3 mM Ca²⁺, significant (P < 0.05) differences between the pH 7.4 group and the pH 6.8 and 6.0 groups began after 171 and 116 s, respectively; (c) 3 mM spermine, significant (P < 0.05) differences between the pH 7.4 group and the pH 6.8 and 6.0 groups began after 132 s, or (d) 10 mM cinacalcet, significant (P < 0.05) differences between the pH 7.4 group and the pH 6.8 and 6.0 groups began after 233 and 132 s, respectively. Results are mean ± SEM, each group had 22-28 cells obtained from three to four separate experiments. SEM: Standard error of the mean.

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As Mn²⁺ is a surrogate ion for Ca²⁺, we studied whether extracellular acidity would affect CaSR-triggered Mn²⁺ influx. Results in [Figure 2] showed that high Ca²⁺ stimulated Mn²⁺ influx at pH 7.4 but not pH 6.8 and pH 6.0. Similarly, cinacalcet stimulated Mn²⁺ influx at pH 7.4 but not at acidic pH [Figure 3]. We reported earlier that spermine did not stimulate Mn²⁺ influx at pH 7.4.^[12]

Figure 2: Acidity-suppressed Mn²⁺ influx triggered by extracellular Ca²⁺. bEND.3 cells were bathed in Ca²⁺-free solution of different pH, treated with 1 mM Mn²⁺, followed by the addition of water or 3 mM Ca²⁺. At pH 7.4, significant differences (P < 0.05) between the control and CaCl₂ groups began after 462 s. Results are mean ± SEM, each group had 40-50 cells obtained from three to five separate experiments. SEM: Standard error of the mean.

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Figure 3: Acidity-suppressed Mn²⁺ influx triggered by cinacalcet. bEND.3 cells were bathed in 1 mM Ca²⁺-containing solution of different pH, treated with 1 mM Mn²⁺, followed by the addition of DMSO or 10 mM cinacalcet. At pH 7.4, significant differences (P < 0.05) between the control and cinacalcet groups began after 200 s. Results are mean ± SEM; each group had 29-108 cells obtained from three to nine separate experiments. SEM: Standard error of the mean, DMSO: dimethyl sulfoxide.

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Resting membrane potential is one of the factors controlling Ca²⁺ influx. We performed a current clamp to examine whether extracellular acidity would affect resting membrane potential. As shown in [Figure 4], resting membrane potential was not affected by acidic pH, suggesting suppression of Ca²⁺ influx triggered by high Ca²⁺, cinacalcet, and spermine under acidic conditions did not result from a reduced electrical driving force for Ca²⁺.

Figure 4: Extracellular acidity did not affect membrane potential. bEND.3 cells, bathed in Ca²⁺-containing bath solution of different pH, were subject to current-clamp measurements. Results are mean ± SEM, each group having four cells. SEM: Standard error of the mean.

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Whether extracellular acidity produced a general untoward effect on Ca²⁺, signaling was investigated. We examined if extracellular acidity would affect ATP-triggered Ca²⁺ response [Figure 5]. It was shown that ATP-triggered Ca²⁺ responses were not suppressed by pH 6.8 and pH 6.0.

Figure 5: ATP-triggered Ca²⁺ signaling was not diminished by acidosis. [Ca²⁺]i was measured in bEND.3 cells in Ca²⁺-containing bath solutions of different pH. Cells were stimulated by 10 mM ATP. Results are mean ± SEM, each group having 29–39 cells from three to four separate experiments. SEM: Standard error of the mean.

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Discussion

CaSR exhibits pleiotropy in that in addition to G_q/11, CaSR is coupled to, for instance, G_12/13, Gi, and Gs.^{[2],[3],[4],[5]} CaSR activation has also been shown to stimulate ERK_1/2.^[6],[7] A number of studies have also shown that CaSR stimulation leads to the modulation of various TRP channels in mesangial cells,^[8] aortic smooth muscle cells,^[9] and mesenteric artery ECs.^[10] We found that in bEND.3 cells, CaSR-mediated Ca²⁺ influx is independent of PLC activity (thus, not related to G_q/11) and via TRP channels (in part TRPV1).^[11],[12] How CaSR is coupled to TRP channels is hitherto unknown. Given that CaSR is a GPCR and TRP channels are G protein-regulated,^[18] coupling between CaSR and TRP channels could be through 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 arrestins have been recently shown to bridge GPCR to activate TRP channels,^[19],[20] a CaSR-arrestin-TRP complex is a possibility.

Reports on whether and how extracellular acidosis affects CaSR responses have been rare. Campion et al.^[16] have shown that in HEK cells expressing CaSR and in bovine parathyroid cells, extracellular acidosis suppressed high Ca²⁺-stimulated Ca²⁺ elevation and ERK signaling. How acidosis affects CaSR-TRP responses is unknown. This is further complicated by the fact that some TRP channels are pH-sensitive (see below). In addition, pharmacological and permeability properties of CaSR-triggered Ca²⁺ influx through TRP varied with the stimulating ligands (high Ca²⁺, cinacalcet, and spermine).^[12] Does extracellular acidity affect Ca²⁺ influx triggered by all these stimulating ligands? Here, we observed that extracellular acidosis similarly inhibited Ca²⁺ signaling triggered by high Ca²⁺, spermine, and cinacalcet in mouse brain bEND.3 EC. Based on our data, acidosis did not suppress ATP-elicited Ca²⁺ signaling which ruled out the possibility that acidity had a generally negative effect on Ca²⁺ transport. In addition, acidosis did not cause membrane depolarization; hence, it is unlikely that suppressed Ca²⁺ influx was due to reduced electrical driving force for Ca²⁺. How acidosis modulates CaSR is so far unclear. Mutating extracellular domain histidine and cysteine residues did not affect sensitivity of CaSR to extracellular pH.^[16] The latter work also shows that extracellular pH changes do not significantly affect cytosolic pH values.

bEND.3 cells express multiple TRP channels such as TRPM2, TRPM4, TRPM7, TRPP2, TRPC1, TRPC4, TRPC6, TRPV2, and TRPV4.^[21] Expression of TRPV1 was not probed in the latter study. Our previous study showed that Ca²⁺ influx elicited by high Ca²⁺, cinacalcet, and spermine was suppressed by SKF96365, ruthenium red, and 2-APB (general TRP blockers) and inhibited by A784168 (a potent and selective TRPV1 antagonist), suggesting CaSR activation triggered Ca²⁺ influx in part through TRPV1 channels.^[12] It is possible that the pH sensitivity of CaSR responses may in part be attributable to the sensitivity of TRP channels to extracellular pH. Indeed, TRPC5 and TRPM2 are inhibited by extracellular acidic pH.^[22],[23] Thus, extracellular acidosis may exert dual inhibition, one on CaSR and another on TRP channels in bEND.3 cells.

Conclusion

Our data suggest that CaSR-mediated Ca²⁺ influx, through a putative CaSR-TRP complex activated by high Ca²⁺, cinacalcet, and spermine, was sensitive to extracellular pH.

Ethical statement

No humans or animals were used in this study; only cell lines were used in this work and therefore ethical approval is not required.

Acknowledgments

Y.M.L. would like to thank China Medical University, Taiwan for support (CMU110-S-30). K.C.W. would like to thank Chang Gung Memorial Hospital, Chiayi, Taiwan, for support (CMRPG6J0371).

Financial support and sponsorship

Y.M.L. would like to thank China Medical University, Taiwan for support (CMU110-S-30). K.C.W. would like to thank Chang Gung Memorial Hospital, Chiayi, Taiwan, for support (CMRPG6J0371).

Conflicts of interest

There are no conflicts of interest.

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