Calcium-sensing receptor modulates extracellular Ca²⁺ entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Abstract

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca²⁺ concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca²⁺ ([Ca²⁺]_o) dose-dependently increased free cytosolic Ca²⁺ ([Ca²⁺]_cyt) in HASMC, with a half-maximal response (EC₅₀) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca²⁺]_o-induced [Ca²⁺]_cyt rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca²⁺]_cyt was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, Gö6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca²⁺]_cyt. Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La³ significantly inhibited [Ca²⁺]_o entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca²⁺]_o entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca²⁺]_cyt in [Ca²⁺]_o-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca²⁺]_o induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca²⁺]_o entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca²⁺]_o entry in HASMC.

cytosolic free Ca²⁺ ([Ca²⁺]_cyt) is known to play an essential role in various mammalian cells, regulating physiological functions such as neurotransmitter release, muscle contraction, fertilization, gene regulation, proliferation, and apoptosis (4, 10, 17, 28). Therefore, dysregulation of Ca²⁺ homeostasis in the body results in pathological changes in different systems (4, 22, 25). One of the regulators of Ca²⁺ homeostasis is the plasma membrane Ca-sensing receptor (CaSR), which is a plasma membrane protein with 1,078 amino acids that was initially cloned from bovine parathyroid cells (5–7). CaSR is a member of the G protein-coupled receptor (GPCR) family and regulates the synthesis of parathyroid hormone (6, 8, 15) in response to changes in serum Ca²⁺ concentrations. In addition, CaSR activation elicits complex intracellular signaling events through modulation of a wide range of intracellular mediators, including G proteins and phospholipase C (PLC). These, in turn, stimulate both protein kinase C (PKC) activation and inositol 1,4,5-trisphosphate (IP₃) production, the latter inducing intracellular Ca²⁺ release (6, 15). Therefore, CaSR plays an important role in the regulation of serum Ca²⁺ concentrations in the body and [Ca²⁺]_cyt homeostasis in mammalian cells. The plasma membrane CaSR can be directly activated by several CaSR agonists, such as extracellular Ca²⁺ ([Ca²⁺]_o) and spermine (6–8, 14).

Although CaSR has been well documented to play important physiological roles in various types of mammalian cells, especially in parathyroid cells (6, 8, 15), the knowledge of its expression and function in the human cardiovascular system is limited. Recently, growing evidence suggests that CaSR is expressed in vascular smooth muscle cells (VSMC), likely modulates arterial blood pressure, and mediates VSMC proliferation through the MEK1/ERK1/2 signaling pathway (21, 32). However, the mechanism(s) whereby CaSR mediates physiological events in VSMC are not fully understood. Here, we sought to investigate the mechanisms underlying CaSR-mediated [Ca²⁺]_cyt signaling pathway in human aortic smooth muscle cells (HASMC) as a model of VSMC because CaSR is known to be functionally expressed in this cell type (21).

MATERIALS AND METHODS

Cell culture.

HASMC (ScienceCell Research Laboratories, San Diego, CA) were cultured in smooth muscle growth medium (SmGM, ScienceCell) at 37°C in a humidified 5% CO₂ plus 95% air atmosphere. SmGM was supplemented with 5% fetal bovine serum, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 μg/ml insulin. The medium was changed every 48 h thereafter and cells of passages 1-10 were used for experiments (21).

Transfection of Flag-tagged dominant negative-CaSR constructs into HASMC.

We used two CaSR dominant negative mutants that are mutated at different locations of the wild-type CaSR, R185Q and R795W (gifts of Dr. Edward Brown, Harvard University). Flag protein was also cloned into these mutants. R185Q is characterized by mutation of the ligand binding region of the wild-type CaSR, while R795W is characterized by mutation of the transmembrane region. Both R185Q and R795W had substantially attenuated responses to [Ca²⁺]_o although they exhibited normal patterns of receptor protein expression (1). R185Q showed a prominent “dominant negative” effect on the coexpressed wild-type receptor, while R795W mutation could potentially still bind to its respective G protein(s) but fail to activate them. HASMC were transfected with the plasmid DNA constructs with Fugene 6. Six-well plates containing 80–90% confluent cells in each well were transfected with a total of 2 μg/well plasmid DNA. [Ca²⁺]_cyt measurement and membrane protein extraction for Western blots were performed 48 h after transfection.

[Ca²⁺]_cyt measurement.

[Ca²⁺]_cyt levels in HASMC were measured by fura-2 fluorescence ratio digital imaging as described previously (36). Briefly, HASMC were trypsinized and replated onto 10-mm round glass coverslips that had been precoated with 1 mg/ml poly-d-lysine (Sigma) at a density such that 70–90% confluence was achieved within 24 h. They were then loaded with 5 μM fura-2 acetoxymethyl ester (AM) [dissolved in 0.01% Pluronic F-127 plus 0.1% DMSO in normal physiological salt solution (PSS), described below] at room temperature for 50 min, then washed in PSS for at least 20 min. Thereafter, the coverslips with HASMC were mounted in a perfusion chamber on a Nikon microscope stage. Cells were initially superfused with PSS for 5 min and then switched to Ca²⁺-free or Ca²⁺ solutions containing drugs of interest. Fura-2 fluorescence ratio (510-nm light emission excited by 340- or 380-nm illuminations), as well as background fluorescence, was collected at room temperature (22°C) with the use of a ×40 Nikon UV-Fluor objective and an intensified CCD camera (ICCD200). The fluorescence signals emitted from the cells were monitored continuously using a MetaFluor Imaging System (Universal Imaging, Downingtown, PA) and recorded for later analysis. PSS used in digital Ca²⁺ measurement contained the following (in mM): 140 Na⁺, 5.0 K⁺, 2 Ca²⁺, 147 Cl⁻, 10 HEPES, and 10 glucose, pH 7.4. For the Ca²⁺-free PSS solution, Ca²⁺ was omitted, and 0.5 mM EGTA was added to prevent possible Ca²⁺ contamination. The osmolalities for all solutions were ∼300 mosM/l.

RT-PCR analysis of TRPC.

Briefly, total RNA (5 μg) from HASMC was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and our previous publication (2). RNA were converted into cDNA with reverse transcriptase and PCR was performed using the following primers: TRPC1 (accession no. NM_003304) forward strand, 5′-TGCTTACCAAACTGCTGGTG-3′ and reverse strand, 5′-AACTGTTTTGCCGTTTGACC-3′; TRPC3 (accession no. NM_003305) forward strand, 5′-CTTTCCTTCAGGCAACGAAG-3′ and reverse strand, 5′-GTACGCAATCCGAGAGAAGC-3′; TRPC4 (accession no. NM_016179) forward strand, 5′-GCTGGAGGAGAAGACACTGG-3′ and reverse strand, 5′-GACCTGTCGATGTGCTGAGA-3′; and TRPC6 (accession no. NM_004621) forward strand, 5′-GCCAATGAGCATCTGGAAAT-3′ and reverse strand, 5′-AACCTCTTGCCTTCAGCAAA-3′. The conditions for PCR reactions for these genes were similar to those previously described (9). Primers for GAPDH were used as a control (forward strand, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse strand, 5′-TCCACCACCCT-GTTGCTGTA-3′). The samples were amplified in an automated thermal cycler (GeneAmp 2400; Applied Biosystems). DNA amplification conditions included an initial 3-min denaturation step at 94°C, 35 cycles of 30 s at 94°C, 30 s at 57°C, 40 s at 72°C, and a final elongation step of 10 min at 72°C. The products were separated on a 1.5% agarose gel, stained with ethidium bromide, and then photographed under UV light.

Western blot analysis.

HASMC cells were washed three times with ice-cold PBS. Cells were then lysed with total lysis buffer [150 mM NaCl, 10 mM Tris·HCl (pH 7.8), 1 mM EDTA, 0.5% Triton X-100, and 1 mM sodium orthovanadate] containing protease inhibitors (1 μg/ml leupeptin and 100 μg/ml PMSF) and incubated at 4°C for 30 min with constant shaking. The cells were then scraped into microcentrifuge tubes and centrifuged at 12,000 g for 15 min to remove insoluble material. The protein content in each sample was determined and normalized. Cell lysates were then resuspended in 2× gel loading buffer, boiled for 5 min, and then separated by SDS-polyacrylamide gel electrophoresis (9% polyacrylamide). Resolved proteins were transferred overnight at 4°C onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membranes were then blocked with a 5% solution of skim milk for 30 min at room temperature, followed by further incubation with monoclonal antibodies specific for CaSR (1:1,000, Abcam, Cambridge, MA), TRPC1, -4, and -6 (1:500, Alomone Lab, Israel), phospho-PKCε 1:1,000 (Upstate Biotechnology, Lake Placid, NY), PKCα 1:1,000 (BD Biosciences, San Jose, CA), or GAPDH 1:5,000 (Ambion, Austin, TX). After being washed with PBS with 1% Tween (PBST), the rabbit anti-mouse secondary antibody was applied to the membrane. After washing with PBST, the membrane was treated with a chemiluminescent solution (Fivephoton Biochemicals, San Diego, CA) according to manufacturer's instructions and exposed to X-ray film. Densitometric analysis of the blots was performed with the use of an AlphaImager digital imaging system (Alpha Innotech, San Leandro, CA).

Chemicals and solutions.

SKF-96365, U-73122, nifedipine, chelerythrine, and Gö6976 were purchased from Sigma. 2-Aminoethoxydiphenyl borate (2-APB) was purchased from Tocris Bioscience (Ellisville, MO). Chiral enantiomers of BEL (S- and R-BEL) were from Cayman Chemical (Ann Arbor, MI). Fura 2-AM was from Molecular Probes (Eugene, OR). The other chemicals were obtained from Fisher Scientific (Santa Clara, CA).

Statistical analysis.

Results are expressed as means ± SE. Differences between means were considered to be statistically significant at P < 0.05 using Student's t-test or one-way ANOVA followed by Newman-Keuls post hoc test, as appropriate.

RESULTS

CaSR activation induces Ca²⁺ signaling in HASMC.

Although activation of CaSR resulted in ERK1/2 phosphorylation in HASMC (6, 21), little is known about Ca²⁺ signaling downstream of CaSR activation in this cell type. Therefore, [Ca²⁺]_cyt levels in HASMC were measured, examining the dose-response of [Ca²⁺]_cyt changes in HASMC stimulated with various levels of [Ca²⁺]_o. Following a short exposure to Ca-free solutions (2–3 min), cells were superfused with different concentrations of [Ca²⁺]_o (0.2–2.0 mM) (Fig. 1). While [Ca²⁺]_o at 0.2 mM did not affect the basal [Ca²⁺]_cyt, obvious increases were seen at higher levels of [Ca²⁺]_o (Fig. 1A). Elevated [Ca²⁺]_cyt was dependent on [Ca²⁺]_o because it was promptly reversed when [Ca²⁺]_o was removed (Fig. 1A). CaSR activation appeared to be cooperative, with a steep dose-response relationship (Fig. 1B). The half-maximal response (EC₅₀) was seen when [Ca²⁺]_o was 0.52 mM and the Hill coefficient for [Ca²⁺]_o stimulation was 5.5 (Fig. 1B).

Fig. 1.

Extracellular Ca²⁺ ([Ca²⁺]_o) and spermine stimulate an increase in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) in human aortic smooth muscle cells (HASMC). After HASMC were loaded with Fura-2 AM, [Ca²⁺]_cyt in the cells was measured by a digital Ca²⁺ imaging system. A: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o. B: summary data showing a dose-dependent relationship for the effect of [Ca²⁺]_o stimulation or peak [Ca²⁺]_cyt responses. C: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of spermine in normal Ca²⁺ physiological salt solution (PSS)_. D: time course of [Ca²⁺]_cyt changes in HASMC induced by same concentration of spermine (3 mM) in Ca²⁺-free or normal Ca²⁺ PSS. Data are shown as means ± SE of 40–50 cells.

Although [Ca²⁺]_o stimulates CaSR, it may enter healthy cells through store-operated Ca²⁺ entry pathway (26), or may even directly leak into unhealthy cells through nonspecific pathways. We therefore tested these possibilities using spermine, another direct activator of CaSR (6, 21). While spermine at 1 mM did not affect the basal [Ca²⁺]_cyt in normal PSS, obvious increases were seen at 3 mM (Fig. 1C). In addition, spermine (3 mM) did not affect the basal [Ca²⁺]_cyt in Ca²⁺-free PSS, but significantly increased [Ca²⁺]_cyt in normal Ca²⁺ PSS (Fig. 1D). These results provide further evidence for a functional role of CaSR in the regulation of [Ca²⁺]_cyt in HASMC.

To demonstrate the presence of CaSR in HASMC, we performed Western blot analysis of lysates from HASMC and HT29, a human colonic cancer cell line used as a positive control. HASMC lysate produced a band of 160 kDa (Fig. 2A), consistent with the mature, full-size CaSR (21). To confirm the functional role of CaSR in the regulation of [Ca²⁺]_cyt in HASMC, we used the CaSR mutants R185Q and R795W. The former showed a prominent dominant negative effect on the coexpressed wild-type receptor, while the latter could potentially still bind to its respective G protein(s) but fail to activate them (1). After transfection of dominant negative-CaSR constructs in HASMC for 48 h, expression of Flag-R185Q and Flag-R795W, but not empty (MT) vector, was seen (Fig. 2B). [Ca²⁺]_o (0.3–1.0 mM) increased [Ca²⁺]_cyt in MT-transfected HASMC (Fig. 2C) but not in R185Q- or R795W-transfected HASMC (Fig. 2, D and E). Together, these results confirm the functional role of CaSR in the regulation of [Ca²⁺]_cyt in HASMC.

Fig. 2.

Protein expression and function of Ca-sensing receptor (CaSR) wild type and mutants in HASMC. A: HT29 (a human colonic cancer cell line used as a positive control) and HASMC were lysed and then Western blot analysis was performed to detect protein expression of CaSR wild type. B–E: after transfection of Flag-tagged empty (MT) vector, R185Q, or R795W construct in HASMC for 48 h, Western blot analysis was performed to detect protein expression of CaSR mutants (B), and digital Ca²⁺ imaging was performed to determine [Ca²⁺]_o-induced [Ca²⁺]_cyt signaling in HASMC transfected with MT (C), R185Q (D), or R795W construct (E). Data are shown as means ± SE of 30–40 cells.

CaSR functions as a GPCR.

CaSR activation elicits intracellular signaling events, including those mediated by G proteins, PLC and PKC, that in turn modulate [Ca²⁺]_cyt (6, 15). Therefore, we sought to test the involvement of these pathways following CaSR activation in HASMC, using [Ca²⁺]_o at both the threshold concentration (0.5 mM) and maximal concentration (1.0 mM). We first examined whether CaSR activation stimulates G proteins. HASMC were superfused with [Ca²⁺]_o with or without pertussis toxin (PTX, 100 ng/ml) (Fig. 3A), a G_i/0 protein inhibitor (37). PTX significantly reduced the [Ca²⁺]_o-induced Ca²⁺ signal (Fig. 3B). We next investigated the role of PLC using a selective PLC inhibitor (20), U-73122 (30 μM) (Fig. 3C). As shown in Fig. 3, C and D, the [Ca²⁺]_o-induced Ca²⁺ signal was also significantly reduced by U-73122, at least at the lower level of [Ca²⁺]_o. Taken together, both G proteins and PLC are involved in CaSR-mediated Ca²⁺ signaling in HASMC, which is consistent with other reports (5, 11).

Fig. 3.

Inhibition of G proteins or phospholipase C (PLC) attenuates [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt in HASMC. A: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in control cells. B and C: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in the presence of pertussis toxin (PTX, 100 ng/ml) or U-73122 (30 μM). D: summary data showing inhibition of [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt by PTX or U-73122. Data are shown as means ± SE of 40–50 cells for each group. **P < 0.01, ***P < 0.001 vs. control in the absence of inhibitors.

To test whether CaSR activation stimulates PKC, HASMC were superfused with [Ca²⁺]_o in the absence (Fig. 4A) or the presence of chelerythrine (10 μM) (Fig. 4B), an inhibitor of the majority of PKC isoforms (39), or Gö6976 (10 μM) (Fig. 4C), a relatively selective inhibitor for conventional isoforms of PKC (39). As shown in Fig. 4D, the [Ca²⁺]_o-induced Ca²⁺ signal in HASMC was prevented by chelerythrine, but not by Gö6976, suggesting that PKC isoforms other than conventional isoforms are involved in CaSR-mediated Ca²⁺ signaling in HASMC (35).

Fig. 4.

[Ca²⁺]_o-induced increase in [Ca²⁺]_cyt in HASMC is inhibited by chelerythrine (CHE) but not by Gö6976. A: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in control cells. B and C: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in the presence of chelerythrine (10 μM) or Gö6976 (10 μM). D: summary data showing effect of chelerythrine or Gö6976 on [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt. Data are shown as means ± SE of 40–50 cells for each group. ***P < 0.001 vs. control in the absence of inhibitors.

PKCε activation plays a role in CaSR-mediated Ca²⁺ signaling.

More than 11 isoforms of PKC have been identified (23). To further our understanding of the expression of different isoforms of PKC in HASMC, we screened for expression of PKC isoforms using Western blot analysis of whole cell lysates. Like BxPc3 cells, pancreatic ductal cells that were used as positive controls (9), HASMC contained proteins immunoreactive with antibodies to PKCα, -β, and -γ in the conventional family, PKCε, -μ, and -δ in the novel family, and PKCι in the atypical family (Fig. 5).

Fig. 5.

Expression profiles of PKC isoforms in HASMC. HASMC and BxPc3 (a pancreatic ductal cell line used as a positive control) were lysed with lysis buffer and then Western blot analysis was performed. These data are representative of 3 experiments with similar results.

Since PKCε was previously implicated in the regulation of Ca²⁺ signaling in rabbit aortic smooth muscle cells (23), we focused on PKCε phosphorylation in HASMC in response to CaSR activation. As shown in Fig. 6, PMA (1 μM)-induced serine phosphorylation of PKCε in HASMC was reversed by chelerythrine (10 μM). Similarly, [Ca²⁺]_o-induced (0.5 mM) PKCε serine phosphorylation was also prevented by chelerythrine (10 μM) (Fig. 6), suggesting that CaSR activation results in PKCε phosphorylation in HASMC (35).

Fig. 6.

[Ca²⁺]_o-induced PKCε serine phosphorylation in HASMC. HASMC were pretreated with chelerythrine (10 μM) followed by treatment with or without [Ca²⁺]_o (0.5 mM) for 30 min. PMA treatment was used as a positive control. Blots were then probed with an antibody to phosphorylated PKCε or total PKCε. GAPDH was used as a loading control. [Ca²⁺]_o or PMA (1 μM) alone significantly induced PKCε serine phosphorylation, but the combination had no further effect. Chelerythrine alone did not affect PKCε serine phosphorylation but was able to suppress PKCε serine phosphorylation induced by [Ca²⁺]_o or PMA. These data are representative of 3 experiments with similar results.

Source of Ca²⁺ mobilized by CaSR activation.

Since GPCR activation may mobilize different Ca²⁺ sources in different cell types, we investigated whether CaSR activation induces intracellular Ca²⁺ release, [Ca²⁺]_o entry, or both in HASMC. To test whether intracellular Ca²⁺ release from the sarcoplasmic/endoplasmic reticulum (S/ER) plays a role, we used cyclopiazonic acid (CPA, 30 μM) to deplete intracellular Ca²⁺ stores (41), or ionomycin (10 μM) and caffeine (10 mM) to release total intracellular Ca²⁺ stores (18) in Ca²⁺-free solutions. None of these reagents induced any change in the basal [Ca²⁺]_cyt (Fig. 7, B and C), but 1 mM [Ca²⁺]_o induced significant [Ca²⁺]_cyt signaling in HASMC (Fig. 7, A, C, and D). Interestingly, re-addition of [Ca²⁺]_o after application of CPA plus caffeine did not induce a significant [Ca²⁺]_cyt increase in HASMC (Fig. 7, B vs. A). Together, these results suggest that neither intracellular Ca²⁺ release mechanisms nor store-operated Ca²⁺ entry plays a significant role in regulating [Ca²⁺]_cyt in HASMC.

Fig. 7.

[Ca²⁺]_o mediates Ca²⁺ entry rather than intracellular Ca²⁺ release to raise [Ca²⁺]_cyt in HASMC. A: time course of [Ca²⁺]_cyt changes induced by 1 mM [Ca²⁺]_o in control cells. B and C: time course of [Ca²⁺]_cyt changes after addition of cyclopiazonic acid (CPA, 30 μM) and caffeine (Caff, 10 mM), ionomycin (10 μM), or [Ca²⁺]_o in the Ca²⁺-free solutions. D: summary data showing that, although [Ca²⁺]_o induces a marked increase in [Ca²⁺]_cyt, CPA, caffeine, and ionomycin did not significantly affect basal [Ca²⁺]_cyt in HASMC. Data are shown as means ± SE of 40–50 cells for each group. ***P < 0.001 vs. basal [Ca²⁺]_cyt in HASMC.

Although voltage-gated Ca²⁺ channels (VGCC) in plasma membrane play an important role in regulating Ca²⁺ entry into VSMC, nifedipine (10 μM), which blocks VGCC, did not significantly affect [Ca²⁺]_o-induced [Ca²⁺]_cyt signaling in HASMC (data not shown), suggesting a minor role for VGCC in CaSR-mediated [Ca²⁺]_cyt signaling.

TRPC-encoded nonselective cation channels are involved in CaSR-mediated Ca²⁺ entry.

Since neither intracellular Ca²⁺ release from the S/ER nor VGCC appeared to be involved in CaSR-mediated [Ca²⁺]_cyt signaling in HASMC, we further investigated whether other Ca²⁺ pathways, such as plasma membrane nonselective cation channels (NSCC), are involved. All of the TRPC-encoded NSCC blockers, including La³⁺ (10 μM), SKF-96365 (50 μM), or 2-APB (100 μM) (16), significantly inhibited CaSR-mediated [Ca²⁺]_cyt signaling in HASMC (Fig. 8), While flufenamic acid (100 μM), an activator of TRPC6 channels (16), did not alter basal [Ca²⁺]_cyt in Ca²⁺-free solutions, it significantly elevated [Ca²⁺]_cyt in a [Ca²⁺]_o-dependent manner, suggesting the involvement of TRPC6-encoded NSCC in CaSR-mediated Ca²⁺ entry into HASMC (Fig. 9).

Fig. 8.

[Ca²⁺]_o-mediated Ca²⁺ entry is likely through receptor-operated Ca²⁺ channels in HASMC. A: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in control cells. B–D: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in the presence of La³⁺ (10 μM), SKF-96365 (50 μM), or 2-aminoethoxydiphenyl borate (2-APB; 100 μM). E and F: summary data showing effect of La³⁺, SKF-96365, or 2-APB [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt. Data are shown as means ± SE of 40–50 cells for each group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control in the absence of inhibitors.

Fig. 9.

[Ca²⁺]_o-mediated Ca²⁺ entry through TRPC-encoded receptor-operated channels (ROC) and expression of TRPC isoforms in HASMC. A: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in control cells. B and C: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in the presence of flufenamic acid (FFA, 100 μM). D: summary data showing effect of flufenamic acid on different concentrations of [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt. Please note that ΔF (340/380) in the 0.2 mM [Ca²⁺]_o control group is close to zero. Data are shown as means ± SE of 40–50 cells for each group. ***P < 0.001 vs. the control in the absence of flufenamic acid. E and F: murine brains (used as a positive control) or HASMC were lysed with either TRIzol reagent or lysis buffer and then mRNA or protein expression of TRPC isoforms was detected by RT-PCR (E) or by Western blot analysis (F), respectively. GAPDH (GAP) was used as an internal standard. M, molecular weight marker. These data are representative of 3 experiments with similar results.

We then examined the expression of TRPC channels in HASMC. Our RT-PCR data revealed transcripts for TRPC1, -3, -4, and -6 in HASMC (Fig. 9E), and Western blotting identified protein expression of TRPC1 and -6 (Fig. 9F). These data further support that TRPC-encoded NSCC may contribute to CaSR-mediated Ca²⁺ entry in HASMC.

Caveolae are involved in CaSR-mediated Ca²⁺ signaling.

Spatially organized complexes of signaling molecules, such as caveolae, were found recently in microdomains of the plasma membrane in VSMC (13). These are important microstructures within the plasma membrane that contain signaling molecules such as caveolins, GPCR, and TRPC, all of which are involved in multiple cellular processes including Ca²⁺ homeostasis and signal transduction (27). Since caveolae are enriched in cholesterol, we tested whether caveolae were involved in CaSR-mediated Ca²⁺ entry by pretreating HASMC with MβCD (5 mM) for 1 h to deplete membrane cholesterol (27). Indeed, MβCD decreased CaSR-mediated Ca²⁺ entry in HASMC (Fig. 10), suggesting that CaSR in caveolae plays a role in [Ca²⁺]_o-mediated [Ca²⁺]_cyt signaling in HASMC.

Fig. 10.

Impairment of caveolae in the plasma membrane attenuates [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt in HASMC. A: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in control cells. B: time course of [Ca²⁺]_cyt changes in HASMC induced by different concentrations of [Ca²⁺]_o in the presence of MβCD (5 mM) for 1 h to deplete membrane cholesterol. C: summary data showing effect of MβCD on [Ca²⁺]_o-induced increases in [Ca²⁺]_cyt. Data are shown as means ± SE of 40–50 cells for each group. **P < 0.01, ***P < 0.001 vs. the control in the absence of MβCD.

DISCUSSION

Here we found that 1) [Ca²⁺]_o induces Ca²⁺ entry into HASMC likely via activation of CaSR; 2) CaSR activation mediates Ca²⁺ entry through receptor-operated channels (ROC); and 3) the PLC/PKCε pathway is involved in the CaSR-mediated [Ca²⁺]_cyt rise in HASMC. Our study demonstrates a role for CaSR in regulating [Ca²⁺]_o entry via ROC, and advances our understanding of the molecular mechanisms underlying CaSR-mediated [Ca²⁺]_cyt rise in human VSMC.

Although CaSR was cloned from and characterized in bovine parathyroid cells in 1993 (5), it was recently shown also to be expressed in human VSMC and to function via the ERK signaling pathway (21). However, the proximal mechanism(s) by which CaSR transduces calcium-dependent signaling in VSMC are largely unknown. CaSR has physiological roles in the cardiovascular system by modulating myogenic tone in small arteries (24), arterial blood pressure (12, 34), and VSMC proliferation (32), through Ca²⁺ and/or ERK1/2 pathways (21, 32). In the present study, we found that both [Ca²⁺]_o and spermine, two direct CaSR agonists with different molecular structures (33, 34), raised [Ca²⁺]_cyt through CaSR activation in HASMC. We have also demonstrated that 1) CaSR proteins are expressed in HASMC, 2) [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt was abolished in HASMC transfected with two CaSR mutant constructs that are mutated at different locations of the wild-type CaSR, and 3) [Ca²⁺]_o-induced increase in [Ca²⁺]_cyt was attenuated by inhibition of G_i/0 protein, PLC or PKC. Together, our results confirm the functional role of CaSR in the regulation of [Ca²⁺]_cyt in HASMC. Similar to other GPCR, CaSR activation stimulates PLC, which cleaves phosphatidylinositol 4,5-bisphosphate into 1,2-diacylglycerol (DAG) and IP₃ (6, 8, 33, 34). While DAG activates PKCε, and Ca²⁺ entry via receptor-operated calcium channels (ROC) (19, 29, 35, 38), IP₃ causes intracellular Ca²⁺ release from S/ER stores and depletion of intracellular Ca²⁺ stores leads to Ca²⁺ entry via store-operated calcium channels (SOC) in different cell types (26, 30).

Both [Ca²⁺]_o and [Ca²⁺]_cyt play essential roles in numerous physiological process (10). [Ca²⁺]_o is controlled by a complex homeostatic system, including the parathyroid glands and calcitonin-secreting C cells of various organs (6). In resting conditions, little or no Ca²⁺ crosses the plasma membrane passively, so as to maintain an almost 20,000-fold concentration gradient between [Ca²⁺]_cyt (∼100 nM) and [Ca²⁺]_o (∼1.2 mM) (6, 10). This is coordinated by activity of membrane ion channels, Ca²⁺ pumps, Na⁺/Ca²⁺ exchangers, and Ca²⁺ sequestration into the ER (10, 17). Our data suggest that [Ca²⁺]_o specifically activates CaSR to induce Ca²⁺ entry via a specific PLC/PKCε pathway, rather than nonspecifically causing [Ca²⁺]_o to leak into HASMC because 1) either [Ca²⁺]_o or spermine dose-dependently raised [Ca²⁺]_cyt in HASMC, which was inhibited by a G protein inhibitor or a PLC inhibitor; 2) a PKC inhibitor reduced CaSR-mediated [Ca²⁺]_o entry; 3) [Ca²⁺]_ob induced PKCε phosphorylation in HASMC, which was prevented by chelerythrine; 4) ROC blockers reduced, but a TRPC activator potentiated, CaSR-mediated [Ca²⁺]_o entry; and 5) mRNA and protein for TRPC were identified in HASMC.

Our data show a Hill coefficient for CaSR activation by [Ca²⁺]_o of 5.50 in HASMC, which is close to that of 4.73 in HEK-293 cells transfected with the human CaSR as reported by Quinn et al. (31), suggesting that multiple CaSR binding sites for [Ca²⁺]_o in HASMC (1, 31). However, the EC₅₀ for [Ca²⁺]_o to raise [Ca²⁺]_cyt in HASMC (0.52 mM) is about fourfold lower than seen in CaSR-transfected human embryonic kidney (HEK)-293 cells (∼2.0 mM) (31), suggesting that CaSR endogenously expressed in native HASMC might be more sensitive to [Ca²⁺]_o than that heterologously overexpressed in HEK-293 cells. Our findings in HASMC also support an important role for the PLC/PKCε cascade during CaSR activation in different types of VSMC (34, 35, 38).

CaSR-mediated [Ca²⁺]_o entry has previously been studied mostly in vascular endothelial cells (3, 42), but not in vascular smooth muscle cells. Moreover, little was known about [Ca²⁺]_o entry mechanisms upon activation of CaSR. Although CaSR activation was found to enhance the activity of permeable NSCC in HEK-293 cells stably transfected with CaSR (40), the precise mechanisms underlying CaSR-mediated [Ca²⁺]_cyt homeostasis in VSMC were largely unknown. After excluding the involvement of VGCC, we focused on SOC and ROC, both of which exist in VSMC. The major differences between SOC and ROC are 1) SOC is known to be functionally activated by depletion of intracellular Ca²⁺ stores, but ROC is activated via a PLC/PKCε cascade (19, 29, 35, 38); and 2) TRPC1 is a molecular candidate for SOC in VSMC, compared with TRPC6 for ROC (16, 19, 29, 38). SOC is unlikely involved in the [Ca²⁺]_o-induced [Ca²⁺]_cyt increase we observed in the present study because 1) the [Ca²⁺]_cyt signal was not only induced by [Ca²⁺]_o in Ca²⁺-free solutions, but also induced by spermine in Ca²⁺-containing solutions; 2) [Ca²⁺]_o-induced [Ca²⁺]_cyt increase was abolished in HASMC transfected with two different CaSR dominant negative mutants; and 3) [Ca²⁺]_o re-addition did not induce a significant [Ca²⁺]_cyt signal in HASMC after application of CPA plus caffeine. Together, our data suggest that TRPC1- or Orai1-mediated SOC does not play a major role in [Ca²⁺]_o-induced [Ca²⁺]_cyt increase in HASMC although Orai1 is another molecular candidate of SOC (2).

We therefore tested whether ROC participate in [Ca²⁺]_o entry because they are NSCC that have been characterized in VSMC. Here, we showed that ROC blockers with different chemical structures significantly inhibited CaSR-mediated Ca²⁺ entry in HASMC, suggesting an involvement of ROC in this process. Moreover, we also found that TRPC6 is expressed in HASMC and that CaSR-mediated [Ca²⁺]_o entry into HASMC was significantly enhanced by flufenamic acid, an activator of TRPC6 channels (16). Since TRPC6 satisfies many of the functional criteria of ROC and thus has been considered a ROC candidate in VSMC (16, 19, 29, 38), our findings support that TRPC6-encoded ROC may play a key role in CaSR-mediated [Ca²⁺]_o entry into HASMC.

In summary, we demonstrate that [Ca²⁺]_o activates CaSR in HASMC, and mediates [Ca²⁺]_o entry into the cell interior likely through TRPC6-encoded ROC. We have also provided evidence that CaSR-mediated Ca²⁺ entry is regulated by the PLC/PKCε signaling cascade. Further studies are needed to assess the physiological role of CaSR-mediated [Ca²⁺]_cyt in modulating cardiovascular function, and eventually to discover a novel therapeutic target for the treatment of cardiovascular diseases.

GRANTS

This work was supported by American Heart Association Beginning Grant-in-Aid Award (0565025Y) and American Lung Association research grant to H. Dong and by the University of California San Diego Digestive Diseases Research Development Center Grant DK-080506, in which H. Dong serves as the director of cell imaging core. It was also partially supported by National Institutes of Health Grant DK-073090 and the University of California San Diego Academic Senate Research Grant RH154H to J. Y. C. Chow.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).