The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown. Methods The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca2+]i) was detected by a laser-scanning confocal microscope. Results The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca2+]o (extracellular calcium concentration) or Gd3+ (an agonist of CaSR) induced an increase of [Ca2+]i and PAs constriction in a concentration-dependent manner. In addition, the above-mentioned effects of Ca2+ and Gd3+ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP3 receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase). Conclusions CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca2+]i through G-PLC-IP3 pathway.


Background
Intracellular calcium, a secondary messenger, plays a key role in various physiological processes. Multiple studies have shown that extracellular calcium can act as a first messenger through the calcium-sensing receptor (CaSR) in various cells [1]. The CaSR belongs to the C family of G protein coupled receptors which was first cloned from bovine parathyroid gland by Brown et al [2]. The CaSR is important in maintaining and regulating mineral ion homeostasis. Increasing evidence has indicated that CaSR was functionally expressed in the cardiovascular system. Wang et al showed that CaSR was expressed in cardiac tissues and cardiomyocytes, and the activity of CaSR could be regulated by extracellular calcium and spermine [3]. CaSR is also expressed in vascular smooth muscle cells (SMCs). Wonneberger et al [4] and Ohanian et al [5] demonstrated that CaSR was involved in the regulation of myogenic tone in the gerbil spiral modiolar artery and in rat subcutaneous arteries. Recent study reported that stimulation of CaSR led to up-regulation of VSMC proliferation, and CaSR-mediated PLC activation was important for VSMC survival [6].
Whether the CaSR is expressed in pulmonary artery smooth muscle cells (PASMCs) and its function in PASMCs are unknown. There is marked difference between systemic and pulmonary circulation in physiological and pathophysiological conditions. For example, coronary artery is relaxed but pulmonary artery is contracted under hypoxic condition. Pulmonary vasoconstriction and PASMC proliferation may contribute to hypoxic pulmonary hypertension. Thus, the present study investigated the expression of CaSR in PAMSCs as well as the effect of CaSR activation on pulmonary artery tension in order to provide an experimental basis for the mechanism of pulmonary hypertension involved by CaSR.

Cell preparation and culture
Primary cultures of PASMCs were prepared as previously described [7][8][9]. Briefly, PASMCs were obtained from Wistar rat PAs. The isolated distal arterial rings were incubated in Hanks balanced salt solution containing 1.5 mg/ml of collagenase II (Sigma, USA) for 20 min. After incubation, the connective tissue and a thin layer of the adventitia were carefully stripped off with fine forceps, and the endothelium was removed by gently scratching the intimal surface with a surgical blade. The remaining smooth muscles were then digested with 1.0 mg/ml of collagenase II for 120 min at 37°C. The cells were cultured in DMEM supplemented with 20% FBS, penicillin (100 units/ml), streptomycin (100 units/ ml), and cultured in a humidified incubator with 5% CO 2 for 3-5 d at 37°C. The cells with typical hill-and-valley morphology, were prepared for experiments. Passage 3-8 cells at 80% confluence were used in all reported experiments [10]. This protocol was approved by Harbin Medical University (Harbin 150086, China).

Western blotting analysis
Total proteins of the PASMCs were prepared as previously described [12]. Briefly, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and then incubated in cool protein lysate containing the protease inhibitor phenylmethyl sulfonyl fluoride (PMSF) for 20 min. The cells were centrifuged at 14000 g for 15 min at 4°C to remove nuclei and undisrupted cells. The protein concentration of the supernatant was determined using the Bradford protein assay with BSA as a standard. Pulmonary artery tissues and rat cardiac tissue were homogenized with a polytron homogenizer in cool protein lysate containing the protease inhibitor PMSF for 1 h. Protein samples of 40 μg from different experimental groups were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes by electroblotting (300 mA for 2 h). The membranes were blocked in TBST (137 mM NaCl, 20 mM Tris (pH 7.6), and 0.1% (v/v) Tween 20) containing 5% (w/v) skimmed milk at 37°C for 1 h. The membranes were then incubated overnight at 4°C with antibodies against CaSR and anti-β actin (1:500). The membrane of the negative controls was incubated with the antigen-antibody complex. Primary antibodies (a rabbit polyclonal antibody ) and antigenic peptides were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).The membranes were incubated with secondary antibody AP-IgG(Promega, USA) diluted 1:5000 in TBST for 1 h at room temperature. Antibodyantigen complexes were detected using Western Blue (Promega, USA).

Immunofluorescence study
The isolated PASMCs were placed onto coverslips, which were covered in 24-well culture plates with polylysine. After cultured for 72 h at 37°C, the PASMCs were washed with PBS, fixed with 4% formaldehyde in PBS for 10 min, and blocked in 1% BSA for 30 min. The cells were incubated with antibody against CaSR (1:100) or the antigenantibody complex (Santa Cruz, CA) overnight at 4°C. Then, the cells were incubated with secondary IgG (Santa Cruz, CA) (1:1000) conjugated with fluorescein isothiocyanate (FITC), for 1 h at 37°C and washed in PBS and 0.1% Tween 20. DAPI (4,6-diamidino-2-phenylindole; final concentration of 6 μg/ml, Sigma-Aldrich, USA) was included to label nuclei. Fluorescence images were collected with a fluorescence microscope (Leica, Germany).
The separated pulmonary arteries were submerged in freezing embedding medium (2.5% polyvinyl alcohol) and placed in liquid nitrogen, sliced by a freezing microtome, fixed with acetone for 5 min, washed with PBS for 10 min, and blocked in 1% BSA for 30 min. The pulmonary arteries were stained by immunofluorescence similarly to the isolated PASMCs as described above.

Fluo-3/AM measurements of [Ca 2+ ] i
The isolated PASMCs were placed onto coverslips, which were covered in 6-well culture plates with polylysine. After 72 h at 37°C, the PASMCs were washed with PBS and were then incubated with 5 μM Fluo-3/AM for 30 min at 37°C in the dark. The cells were rinsed three times with Tyrode's solution to remove the remaining dye, and they were further incubated in Tyrode's solution or Ca 2+ -free Tyrode's solution. During the experiment, FI (fluorescence intensity) of fluo-3 in PASMCs was recorded using a laser-scanning confocal microscope (Olympus, Japan) with excitation at 488 nm and emission at 530 nm.

Tension studies of pulmonary artery rings
Adult male Wistar rats (200-250 g) were provided by the Experimental Animal Center of Harbin Medical University, which is fully accredited by the Institutional Animal Care and Use Committee. The experiment was carried out according to the published protocols [21][22][23]. Rats were anesthetized with pentobarbital sodium (50 mg/kg). The chest was opened, and then both the heart and lung were removed and immediately placed in cold Krebs solution (in mM: NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO 4 0.57, KH 2 PO 4 1.2, NaHCO 3 20, EDTA-Na 2 0.02 and Glucose 10, pH 7.4). The pulmonary arteries (PAs) were dissected out, cleaned of connective tissue and cut into rings under a dissecting microscope. Microdissected distal PAs were cut into rings of approximately 0.5 to 1.5 mm in diameter and examined for isometric contractile responses as described [21][22][23]. The rings were attached to tensionmeasuring devices by tungsten wire hooks. Pulmonary arterial rings were treated with CaCl 2 or GdCl 3 (Sigma-Aldrich, USA) at various concentrations, and the ring tensions were recorded. After CaCl 2 or GdCl 3 was washed off, all vessels relaxed to baseline level.

Statistical analysis
Statistical analysis was carried out with SAS version 9.1. A two-sided P < 0.05 was considered significant. Continuous variables were expressed as mean ± standard deviation X SD  . The statistical differences betweengroup were tested with repeated measurement ANOVA.

CaSR mRNA expression in rat PASMCs
A cDNA fragment of 234 bp corresponding to the selected CaSR mRNA sequence was detected in PASMCs ( Figure 1A). In the absence of reverse transcriptase, no PCR-amplified fragments could be detected, indicating the tested RNA samples were free of genomic DNA contamination. Sequencing results were as follows: ttcggcatcagctttgtgctctgtatctcgtgcatcttggtgaagaccaatcgcgtcctcctggtatttgaagccaagatacccaccagcttc caccggaagtggtgggggctcaacct gcagttcctgctggttttcctctgcaccttcatgcagatcctcatctgcatcatctggctctacacggcgcccccctc tagcaccgcaaccatgagctggaagacgaaatcatcttca. The sequence shared 100% identity with the rat CaSR sequence (GenBank/EMBL accession ).

Protein expression of CaSR in rat PASMCs and PAs
Western blotting with monoclonal CaSR-specific antibody revealed signal of apparent molecular seize of 130 kD in the protein lysates of cultured PASMCs and rat pulmonary artery, consistent with the reported band in cardiac tissue, and there were no bands in the specific antigenic peptides groups ( Figure 1B). Immunofluorescence staining showed that CaSR proteins were present in cytoplasm and membrane of the PASMCs ( Figure 1C), as well as in rat PAs ( Figure 1D). The specific antigenic peptide completely abolished CaSR immunostaining ( Figure 1C and 1D).

Increase in [Ca 2+ ] o stimulated an increase in [Ca 2+ ] i via CaSR
An initial FI/FI 0 was regarded as 1.0. As shown in Fig. 2A (n = 20), when [Ca 2+ ] o increased from 5 to 12.5 mM, FI of [Ca 2+ ] i was increased in a concentration-dependent manner. Moreover, we also found that 10 mM Ca 2+ increased the FI of [Ca 2+ ] i to 1.297 ± 0.150 at 30 s, 1.357 ± 0.176 at 60 s, 1.402 ± 0.183 at 90 s, and 1.419 ± 0.176 at 120 s in the absence of NiCl 2 , CdCl 2 and NPS2390 . The FI of [Ca 2+ ] i in both the NiCl 2 + CdCl 2 + CaCl 2 group and the NPS2390 + CaCl 2 group was decreased but higher than that in controls (p < 0.01 versus control), and the FI of [Ca 2+ ] i was decreased significantly in the NiCl 2 + CdCl 2 + NPS2390 + CaCl 2 group (p < 0.01 versus CaCl 2 group) ( Figure 2B, n = 20).

CaSR activation-induced increase in [Ca 2+ ] i is dependent on intracellular Ca 2+ store in PASMCs
Under normal conditions, the increase of intracellular Ca 2 + is from extracellular Ca 2+ entry and release of intracellular Ca 2+ store. To verify that the change in [Ca 2+ ] i induced by activation of CaSR is dependent on the intracellular Ca 2+ store, the PASMCs were incubated with 10 mM caffeine and 10 μM thapsigargin for 30 min, then 10 mM CaCl 2 or 300 μM GdCl 3 were added into the media. It was found that Ca 2+ FI/FI 0 was significantly reduced in the presence of caffeine and thapsigargin (p < 0.01 versus CaCl 2 or GdCl 3 group) ( Figure 3A and 3B, n = 20).

CaSR activation induced an increase in [Ca 2+ ] i in PASMCs via the PLC-IP 3 signal transduction pathway
Compared with the 10 mM Ca 2+ group, FI/FI 0 of [Ca 2+ ] i was decreased in the 2-APB and U73122 pretreated groups. However, U73343 had little effect on [Ca 2+ ] i FI/ FI 0 ( Figure 3A). The treatment with 300 μM Gd 3+ also caused a similar response ( Figure 3B, n = 20).

Calcium-induced constriction of pulmonary artery rings
An isometric tension of 0.3 g (passive force) was regarded as 100% (vehicle). We observed that an increase in the [Ca 2+ ]o from 0.5 to 2.5 mM exerted no effect on tension of the pulmonary artery rings, while increases in [Ca 2+ ]o from 5 to 12.5 mM increased vasoconstriction in a dose-dependent manner. In addition, the vasoconstriction was not completely eliminated by NiCl 2 , CdCl 2 , or NPS2390 (Figure 4, n = 8), indicating that [Ca 2+ ] o -induced vasoconstriction was at least partly mediated via activation of CaSR.

CaSR activation-induced constriction of pulmonary artery rings is dependent on intracellular Ca 2+ store
We observed that preincubation with 10 mM caffeine or 50 μM thapsigargin for 30 min before Ca 2+ and Gd 3+ challenge attenuated the constriction of pulmonary artery rings significantly (p < 0.01 versus the CaCl 2 or GdCl 3 group) ( Figure 5A, B. n = 8).

CaSR activation-induced constriction of pulmonary artery rings via the PLC-IP 3 signal transduction pathway
Both Ca 2+ and Gd 3+ evoked increases in tension of pulmonary artery rings in a concentration-dependent manner. U73122 and 2-APB significantly inhibited the constriction of pulmonary artery rings. However, U73343 did not affect the vasoconstriction induced by Ca 2+ and Gd 3+ (Figure 5A, B. n = 8). Based on these findings, it was speculated that the PLC-IP 3 signal transduction pathway may be involved in CaSR-induced constriction.

Discussion
CaSRs are widely expressed in the vessel system, such as in the mesenteric, basilar, renal, coronary [24,25], spiral modiolar arteries [4], subcutaneous vessels [5]and in the aorta [26]. CaSRs are involved in regulation of vascular tension and cell proliferation in these vessels. Increasing evidence indicates that CaSRs play a potential role in vascular calcification and pathogenesis of atherosclerosis, arteriosclerosis and hypertension [27].
Whether the CaSR is expressed in the pulmonary artery has remained unclear. To confirm the existence of CaSRs and its functional expression in some tissues or cells, the following evidence would be necessary. Firstly, CaSR mRNA and protein would be present in the tissue or cells [4] 20). B. The cells were exposed to 10 mM Ca 2+ , and FI of [Ca 2+ ] i was recorded for 120 s. In some experiments, the cells were pre-exposed to 0.1 mM NiCl 2 , 0.02 mM CdCl 2 , and 10 μM NPS2390 for 30 min before Ca 2+ challenge.  [4,28,29].
In this study, comprehensive experiments were carried out, including RT-PCR with CaSR-specific primers, western blotting, and immunofluorescence staining. A cDNA fragment of 234 bp was found in cultured PASMCs, indicating the presence of CaSR mRNA in rat PASMCs. Western blotting analysis showed that CaSR was clearly expressed in rat PASMCs as well as in whole PAs extracts. Heart tissues were used as positive control, and we detected the same size of band (130 kDa) in the lysates of PAMSCs, PAs and heart. There were no bands in specific antigenic peptide groups. However, Ohanian et al. reported that immunoblotting of rat subcutaneous artery homogenates with monoclonal CaSR antibody revealed a single immunoreactive band at 159 kDa. This antibody also detected another two bands at 145 and 168 kDa in rat kidney homogenate. CaSR protein is present in human aortic smooth muscle cells, and lysate produces a band 160 kDa [30]. It is generally agreed that bands of 130-170 kDa represent a mature, fully glycosylated form of the CaSR [3,23]. Usually, the band size of CaSR detected by western blotting varies considerably depending on the tissue and cell type, cellular fraction analyzed (membrane or cytosolic), and degree of posttranslational modification (glycosylation) of the CaSR protein [31]. Therefore, the CaSR proteins we detected in rat cultured PASMCs and whole pulmonary artery extract may belong to the mature form of CaSR. Immunofluorescence staining showed that CaSR proteins were observed in vessel walls of PAs and were located in the cytoplasm and plasmalemma of the PASMCs, as shown in other cell types [32,33]. Based on these data, we confirmed the expression of CaSR in PASMCs at the mRNA and protein levels.
To  In the NiCl 2 + CdCl 2 pretreated groups, the vasoconstriction of the pulmonary arterys was attenuated, but it was higher than in the vehicle (P < 0.01 versus CaCl 2 groups). In the NPS2390 pretreated groups, the vasoconstriction of the pulmonary arterys was also attenuated, but it was higher than in the vehicle (P < 0.01 versus CaCl 2 groups). In the NPS2390 + NiCl 2 + CdCl 2 treated groups, the vasoconstriction of the pulmonary artery was significantly attenuated.  As we have known, the intracellular Ca 2+ , as an excitation contraction coupling factor, is involved in regulating myocardial contraction and angiotasis. To demonstrate the functional expression of CaSR in PAs, evidence showing that CaSR activation is related to PA tension change needs to be provided. Therefore, we observed the effects of the CaSR agonist, antagonist and other calcium signalrelated factors on PAs tension. The results showed that vasoconstriction appeared in a concentration-dependent manner in PAs when [Ca 2+ ] o was increased from 5 mM to 12.5 mM, and Gd 3+ also induced a similar response. In addition, the vasoconstriction was not reversed by an inhibitor of the Na + -Ca 2+ exchanger and L-type Ca 2+ channels, antagonist of CaSR. These findings suggest that an increased [Ca 2+ ] o or [Gd 3+ ] o evoked vasoconstriction at least in part by the CaSR. In subcutaneous artery a biphasic response was observed. That is increasing [Ca 2+ ] o from 0.5 to 2 mM induced a small vasoconstriction followed by progressive vasodilation from 3 to 10 mM [5]. However, elevation of [Ca 2+ ] o caused a biphasic vasoconstriction in the spiral modiolar artery [4].
The signal transduction mechanism linked to the CaSR is known to involve the release of Ca 2+ from cytosolic stores [35]. Therefore, the PAs were preincubated in caffeine or thapsigargin. We found that caffeine and thapsigargin induced a significant attenuation of the vasoconstriction induced by [

Conclusions
We have demonstrated that functional expression of CaSRs exists in rat PAs and PAMSCs, and that CaSR activation is involved in [Ca 2+ ] i increase and vasoconstriction through the G-PLC-IP 3 signal transduction pathway. Pulmonary artery constriction contributes to pulmonary hypertension, so it is expected that CaSR activation could be involved in the development of pulmonary hypertension.