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Am J Physiol Lung Cell Mol Physiol 290: L1267-L1276, 2006. First published January 6, 2006; doi:10.1152/ajplung.00515.2005
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Functional expression of transient receptor potential melastatin- and vanilloid-related channels in pulmonary arterial and aortic smooth muscle

Xiao-Ru Yang,1 Mo-Jun Lin,1,2 Lionel S. McIntosh,1 and James S. K. Sham1

1Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland; and 2Department of Physiology and Pathophysiology, Fujian Medical University, Fuxhou, Fujian, People's Republic of China

Submitted 7 December 2005 ; accepted in final form 29 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transient receptor potential melastatin- (TRPM) and vanilloid-related (TRPV) channels are nonselective cation channels pertinent to diverse physiological functions. Multiple TRPM and TRPV channel subtypes have been identified and cloned in different tissues. However, their information in vascular tissue is scant. In this study, we sought to identify TRPM and TRPV channel subtypes expressed in rat deendothelialized intralobar pulmonary arteries (PAs) and aorta. With RT-PCR, mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of TRPV family were detected in both PAs and aorta. Quantitative real-time RT-PCR showed that TRPM8 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Moreover, Western blot analysis verified expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in both types of vascular tissue. To examine the functional activities of these channels, we monitored intracellular Ca2+ transients ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) and aortic smooth muscle cells (ASMCs). The TRPM8 agonist menthol (300 µM) and the TRPV4 agonist 4{alpha}-phorbol 12,13-didecanoate (1 µM) evoked significant increases in [Ca2+]i in PASMCs and ASMCs. These Ca2+ responses were abolished in the absence of extracellular Ca2+ or the presence of 300 µM Ni2+ but were unaffected by 1 µM nifedipine, suggesting Ca2+ influx via nonselective cation channels. Hence, for the first time, our results indicate that multiple functional TRPM and TRPV channels are coexpressed in rat intralobar PAs and aorta. These novel Ca2+ entry pathways may play important roles in the regulation of pulmonary and systemic circulation.

transient receptor potential channels; calcium signaling; nonselective cation channels

THE TRANSIENT RECEPTOR POTENTIAL (TRP) channels belong to a large superfamily of cation channels that have diverse physiological functions in both nonexcitable and excitable cells (26, 33). On the basis of sequence homology, the TRP superfamily can be divided into three major subfamilies of canonical (TRPC), melastatin-related (TRPM), and vanilloid-related (TRPV) channels, as well as four more distant subfamilies of polycystin-related (TRPP), mucolipin-related (TRPML), ankyrin-related (TRPA), and no mechanoreceptor potential C, or NOMPC (TRPN), channels and/or proteins.

The classic or canonical TRPC subfamily consists of seven members (TRPC1–7), which have attracted enormous attention because of their putative roles as store-operated and receptor-operated cation channels. In vascular smooth muscle, it is generally accepted that TRPC1 channels are related to store-operated Ca2+ entry, which can be activated by depletion of Ca2+ stores (1, 4, 17, 22, 41, 48), whereas TRPC6 (and TRPC3) channels are involved in receptor-operated Ca2+ entry, which can be activated directly by diacylglycerol in a PKC-independent manner (13, 15, 18, 22). TRPC channels have been shown to play pivotal roles in vasoconstriction induced by {alpha}-adrenoceptor agonists, vasopressin, endothelin-1, and uridine 5'-triphosphate (4, 14, 15, 17, 18, 37, 48); vascular smooth muscle proliferation and remodeling induced by growth factors/mitogens (9, 41, 51, 53); and intravascular pressure-induced depolarization and myogenic tone in small cerebral arteries (47). Moreover, our recent study has provided evidence that chronic hypoxia upregulates TRPC1 and TRPC6 expression in pulmonary arteries and enhances both store- and receptor-operated Ca2+ entries, which contribute to the increased resting intracellular Ca2+ transients ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) and the basal pulmonary arterial tone of hypoxic pulmonary hypertensive rats (22). In addition, idiopathic pulmonary arterial hypertension (IPAH) has been shown to be associated with overexpression of TRPC6 and TRPC3, and inhibition of TRPC6 expression with small interfering RNA (siRNA) markedly attenuated IPAH-PASMC proliferation (50).

Compared with TRPC channels, the physiological functions of TRP channels of other subfamilies are much more elusive in vascular smooth muscle. The melastatin-related TRPM subfamily, which consists of eight mammalian members (TRPM1–8), and vanilloid-related TRPV subfamily, which is composed of six identified members (TRPV1–6), are known to participate in tumor suppression, oxidative stress/reactive oxygen species (ROS)-induced apoptosis, Mg2+ homeostasis, nociception, mechanosensing, osmolarity sensing, and thermosensing (hot and cold) in nonvascular tissues (2, 3, 8, 26, 28, 33). A few recent studies have revealed that some of these TRP channels are expressed and may play different physiological roles in systemic vascular smooth muscles. It has been proposed that TRPM4 contributes to membrane depolarization and vasoconstriction associated with increased intraluminal pressure in cerebral arteries (7). TRPM7 was identified as a functional regulator of Mg2+ homeostasis in mouse and rat mesenteric and aortic smooth muscle cells (11, 42), and TRPV2 has been implicated as an osmotically sensitive cation channel in murine aorta myocytes (27). More interestingly, TRPV4-dependent Ca2+ signals were found to cause membrane hyperpolarization and vasodilation in cerebral arteries through activation of Ca2+ sparks and Ca2+-dependent K+ channels (BKCa) (6).

Besides these several studies, TRPM and TRPV channels have not been characterized systematically in vascular smooth muscle, and information regarding the expression and function of these channels in pulmonary arteries is basically unavailable. In the present study, we hypothesize that some members of the TRPM and TRPV subfamilies operate as functional cation channels in pulmonary and systemic vasculatures. We sought to identify systematically the TRPM and TRPV channels expressed in rat intralobar pulmonary arteries and aorta and compared the mRNA expression pattern using quantitative real-time PCR. Furthermore, we examined their functional activity by measuring Ca2+ transients elicited by agonists specific to TRPM8 and TRPV4 channels in PASMCs and aortic smooth muscle cells (ASMCs). Our results provide the first evidence that multiple TRPM and TRPV channels are coexpressed in rat intralobar pulmonary arteries and aortic smooth muscle and that they are functional Ca2+ entry pathways in vascular arterial myocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of intralobar pulmonary arteries and aorta. Intralobar pulmonary arteries (PAs) and aorta were isolated from male Wistar rats (150–250 g). The procedures involving animals were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee. Rats were injected with heparin and anesthetized with pentobarbital sodium (130 mg/kg ip). They were exsanguinated with the lungs and the thoracic aorta removed and transferred to a petri dish filled with HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Second- and third-generation intrapulmonary arteries (~300–800 µm) were isolated. The PAs and aorta were cleaned free of connective tissue. The endothelium was removed by gently rubbing the luminal surface with a cotton swab.

Total RNA preparation and reverse transcription of RNA. Deendothelialized PAs and aorta were mechanically homogenized. Subsequently, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) with standard procedures. Genomic DNA contamination was removed with TURBO DNA-free DNase (Ambion, Austin, TX). The amounts of RNA were determined by measuring the optical density at 260 nm. Total RNA was also extracted from rat brain, liver, bladder, kidney, and duodenum as positive controls. Total RNA (1 µg) was used for first-strand cDNA synthesis with random hexamer primers and Superscript III RNase H– reverse transcriptase (Invitrogen) according to the manufacturer's protocol.

Conventional RT-PCR. Sense and antisense PCR primers specific to the TRPM and TRPV channels were used (see Table 1). PCR reactions were carried out using Platinum Taq DNA polymerase (Invitrogen) with the following parameters: denaturation at 94°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 90 s. A total of 35 cycles was performed, followed by a final extension at 72°C for 10 min, and the products were then stored at 4°C. PCR products were analyzed by electrophoresis with 1.8% agarose gel and visualized by ethidium bromide staining. Parallel reactions were run for each RNA sample in the absence of Superscript III to ascertain that there was no genomic DNA contamination.


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  		Table 1. Primers for conventional and real-time RT-PCR experiments

 
Quantitative real-time PCR. Gene-specific real-time PCR primers were designed based on the published TRPM or TRPV sequences in GenBank to obtain predicted PCR products of 100–150 bases. At least one primer of each set was designed to span exon-exon junctions to minimize the possibility of amplifying the genomic DNA. PCR reactions were performed with QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA), using 2 µl of cDNA as the template in each 25-µl reaction mixture. PCR assays were performed with a Chrom4 thermal cycler system (MJ Research, Waltham, MA). The PCR protocol consisted of initial enzyme activation at 95°C for 15 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. Using the same protocol, we generated standard curves from serial dilutions of purified PCR products with known copy numbers measured by absorbance at 260 nm. The absolute copy number of mRNA of interest was determined by interpolation of the standard curve with the threshold cycle value of each sample. To confirm the specificity of PCR products, we obtained a melting curve at the end of each run by slow heating with increments of 0.1°C/s from 65 to 95°C, with fluorescence detected at intervals of 0.1°C. Standard gel electrophoresis also was performed to ensure the end product generated a single band with the predicted size (100–150 bases). Data also were normalized with the quantity of 18S rRNA in individual samples to correct for sample variability.

Western blotting. Deendothelialized PAs and aorta were quickly frozen in liquid nitrogen. The frozen tissues were crushed and homogenized using a mortar and pestle and then resuspended in ice-cold cell lysis buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% deoxycholic acid, 0.1% SDS, 0.5% NP-40, and protease inhibitor cocktail (Roche, Mannheim, Germany). The homogenate was centrifuged at 4°C with 1,000 g for 5 min, the supernatant was collected, and the protein concentration was estimated using the bicinchoninic acid assay. The protein sample (30 µg) was resolved in an 8% SDS-PAGE gel and electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked with 5% (wt/vol) nonfat dry milk in PBS containing 0.05% Tween 20 (PBST) for 1 h at room temperature, followed by incubation at 4°C overnight with a specific primary antibody. The primary antibodies were polyclonal rabbit anti-TRPM2 (1:500 dilution), anti-TRPM8 (1:500 dilution), and anti-TRPV1 (1:200 dilution) from Abcam (Cambridge, MA) and anti-TRPV4 (1:400 dilution) from Alomone Labs (Jerusalem, Israel). The nitrocellulose membrane was then washed with PBST. After washing, the membrane was incubated with peroxidase-conjugated goat-anti-rabbit secondary antibody (1:3,000 dilution; Bio-Rad, Hercules, CA) at room temperature for 1 h. Excess secondary antibody was again washed, the bound secondary antibody was detected with enhanced chemiluminescence (Pierce, Rockford, IL), and images were taken using a Gel Logic 200 image system (Kodak, New Haven, CT).

Isolation of PASMCs and ASMCs. PASMCs and ASMCs were enzymatically isolated and transiently cultured as previously described (49). After isolation, deendothelialized arteries were allowed to recover for 30 min in cold (4°C) HBSS, followed by 20 min in reduced-Ca2+ (20 µM) HBSS at room temperature. The tissue was digested at 37°C for 20 min in 20 µM Ca2+ HBSS containing collagenase (type I; 1,750 U/ml), papain (9.5 U/ml), BSA (2 mg/ml), and dithiothreitol (1 mM) and was then removed and washed with Ca2+-free HBSS to stop digestion. Single smooth muscle cells were dispersed gently by trituration with a small-bore pipette in Ca2+-free HBSS at room temperature. The cell suspension was then placed on 25-mm glass coverslips and transiently (16–24 h) cultured in Ham's F-12 medium (with L-glutamine) supplemented with 0.5% FCS, 100 U/ml streptomycin, and 0.1 mg/ml penicillin.

Measurement of [Ca2+]i. [Ca2+]i were monitored using the membrane-permeable Ca2+-sensitive fluorescent dye fluo-3 AM as previously described (22). PASMCs and ASMCs were loaded with 5–10 µM fluo-3 AM (dissolved in DMSO with 20% Pluronic acid) for 30–45 min at room temperature (~22°C) in normal Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Cells were then washed thoroughly with Tyrode solution to remove extracellular fluo-3 AM and rested for 15–30 min in a cell chamber to allow for complete deesterification of cytosolic dye. Fluo-3 was excited at 488 nm, and emission light at >515 nm was detected using a Nikon Diaphot microscope equipped with epifluorescence attachments and a microfluometer (Biomedical Instrument Group, University of Pennsylvania, Philadelphia, PA). Protocols were executed and data collected online with a Digidata analog-to-digital interface (Axon Instruments, Foster City, CA) and the pCLAMP software package (Axon Instruments). [Ca2+]i was calibrated using the equation [Ca2+]i = Kd(F – Fbg)/(Fmax – F), where Fbg is background fluorescence and Fmax is the maximum fluorescence determined in situ in cells superfused with 10 µM 4-bromo-A-23187 and 10 mM Ca2+, or determined via a pseudoratio method (1), using the equation [Ca2+]i = (Kd x R)/{[(Kd/[Ca2+]rest) + 1] – R}, where R is fluorescence/resting fluorescence (F/F0), Kd of fluo-3 is 1.1 µM, and the resting [Ca2+]i ([Ca2+]rest) is assumed to be 100 nM. Data are expressed as means ± SE, and the number of cells is specified. Statistical significance (P < 0.05) was assessed using unpaired or paired Student's t-tests wherever applicable.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of TRPM and TRPV subtype mRNA. Expression of TRPM and TPRV subtypes in PAs and aorta was first identified using conventional RT-PCR. Figures 1 and 2 show the amplified PCR products generated after 35 cycles from deendothelialized PAs and aorta, respectively. Expression profiles were qualitatively identical in the two vascular tissues. PCR products of TRPM2, TRPM3, TRPM4, TRPM7, TRPM8, TRPV1, TRPV2, and TRPV4 were obtained consistently in four separate experiments. Much weaker signals for TRPM5, TRPM6, and TRPV3 also were observed. All these RT-PCR amplified products had sizes corresponding to the predicted values and matched with those of positive controls generated from brain, liver, bladder, kidney, or duodenum in which the specific isoforms have been reported previously. In contrast, TRPM1, TRPV5, and TRPV6 transcripts were not detected in PAs or aorta in three separate preparations, despite the detection of clear signals for these subtypes in positive control samples under identical amplification conditions. Similar results also were obtained in transiently cultured PASMCs (data not shown).


Figure 1
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  		Fig. 1. RT-PCR analysis of transient receptor potential melastatin-related (TRPM) channel subtypes in rat intralobar pulmonary arteries (PA) and aorta. Brain mRNA was used as the positive control for TRPM1, TRPM2, TRPM3, TRPM4, TRPM6, and TRPM7; liver, and bladder mRNAs were used as positive controls for TRPM5 and TRPM8, respectively. Predicted lengths of PCR products are 228, 232, 425, 443, 267, 790, 978, and 489 base pairs (bp) for TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, and TRPM8, respectively.

 

Figure 2
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  		Fig. 2. RT-PCR analysis of transient receptor potential vanilloid-related (TRPV) channel subtypes in rat intralobar PA and aorta. Brain mRNA was used as the positive control for TRPV1, TRPV2, TRPV3, and TRPV4; kidney and duodenum mRNAs were used as positive controls for TRPV5 and TRPV6, respectively. Predicted lengths of PCR products are 964, 1,131, 233, 287, 1,017, and 1,014 bp for TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6, respectively.

 
Quantification of TRPM and TRPV mRNA. The relative expression of TRPM and TRPV mRNA was determined using quantitative real-time RT-PCR. TRPM8 expression was highest among all TRPM subtypes in both PAs and aortic smooth muscle, with levels equivalent to 0.0094 ± 0.0013 and 0.013 ± 0.0018% of 18S rRNA, respectively (Fig. 3). TRPM4 and TRPM7 were moderately expressed, approximately equal to 37 and 31% of TRPM8 in PAs, respectively. In contrast, TRPM2 and TRPM3 transcripts were expressed at <13% of TRPM8, whereas TRPM5 and TRPM6 mRNA levels were exceedingly low even though their products were detectable. Overall, the expression profiles of TRPM channels were similar in PAs and aorta with an order of TRPM8 > TRPM4 > TRPM7 > TRPM3 = TRPM2 > TRPM5 = TRPM6.


Figure 3
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  		Fig. 3. Real-time RT-PCR analysis of the relative expression of TRPM mRNA in rat intralobar PAs and aorta. Data are expressed as percents normalized to 18S rRNA to correct for RNA quantity and integrity. TRPM8 is the most abundant TRPM subtype, and TRPM4 and TRPM7 also are highly expressed in PAs and aorta. Ten animals were used for each channel subtype.

 
For the TRPV subfamily, TRPV4 mRNA was most abundantly expressed in PAs, equivalent to 0.0025 ± 0.0003% of 18S rRNA (Fig. 4). It was followed by TRPV2 and TRPV1, which were ~50 and 13% of TRPV4, respectively. TRPV3 was the least expressed among the detected TRPV channels. The expression profile of TRPV transcripts in PAs had an order of TRPV4 > TRPV2 > TRPV1 > TRPV3. It was similar in aortic smooth muscle, with the exception that TRPV4 was significantly less (0.0011 ± 0.0002% of 18s rRNA, P < 0.05) than that in PAs. Experiments with samples from 10 different animals were performed for the quantification of each channel subtype.


Figure 4
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  		Fig. 4. Real-time RT-PCR analysis of the relative expression of TRPV mRNA in rat intralobar PAs and aorta. Data are expressed as percentages normalized to 18S rRNA to correct for RNA quantity and integrity. TRPV4 is the most abundant TRPV subtype, and TRPV2 also is highly expressed in PAs and aorta. Ten animals were used for each channel subtype.

 
Expression of TRPM and TRPV proteins. Expression of TRPM and TRPV channel proteins in PAs and aorta was examined using Western blot analysis, as shown in Fig. 5. Specific anti-TRPM8, anti-TRPV1, and anti-TRPV4 antibodies detected clear bands at ~250, ~95, and ~105 kDa, respectively, in both PA and aorta samples. Two bands were detected using an anti-TRPM2 antibody at ~250 and ~105 kDa, corresponding to the long and short forms of TRPM2 described previously (54). Positive controls for TRPM2, TRPV1, and TRPV4 were obtained from brain, and for TRPM8 from bladder. Because only a few antibodies for TRPM and TRPV subtypes are commercially available, the expression of other TRPM and TRPV proteins has not been confirmed in PAs and aorta.


Figure 5
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  		Fig. 5. Western blot analysis of TRPM2, TRPM8, TRPV1, and TRPV4 protein expression in rat intralobar PAs and aorta. Brain and bladder proteins were used as positive controls.

 
Functional expressions of TRPM8 and TRPV4 channels in PASMCs. To determine whether the TRPM and TRPV proteins expressed in PAs and aortic smooth muscle are functional Ca2+ entry channels, we examined the effects of TRPM8 and TRPV4 agonists on the activation of Ca2+ responses in PASMCs and ASMCs. TRPM8 and TRPV4 were chosen for examination because they were the most abundantly expressed TRPM and TRPV channels, respectively, in both types of vascular tissues. Activation of TRPM8 channels with 300 µM menthol, a specific TRPM8 agonist (25, 34), elicited prominent Ca2+ transients in both PASMCs and ASMCs. The maximum increase in [Ca2+]i was 308.8 ± 28.1 nM (n = 20) and 342.8 ± 33.8 nM (n = 19) in PASMCs and ASMCs, respectively (Fig. 6). The menthol-induced Ca2+ response was completely abolished by the removal of Ca2+ from extracellular solution and restored after subsequent readmission of extracellular Ca2+ (Fig. 6, A and E), suggesting that the Ca2+ response depended solely on Ca2+ influx. Inhibition of nonselective cation channels with 300 µM Ni2+ completely abolished the menthol-induced Ca2+ response, consistent with the involvement of TRPM8 cation channels. Moreover, inhibition of L-type Ca2+ channels with 1 µM nifedipine had no appreciable effects on the Ca2+ transients, indicating that the dihydropyridine-sensitive voltage-gated Ca2+ channels were not involved.


Figure 6
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  		Fig. 6. Characterization of intracellular Ca2+ response ([Ca2+]i) induced by the TRPM8 channel agonist menthol (300 µM) in pulmonary arterial smooth muscle cells (PASMCs; A–D) and aortic smooth muscle cells (ASMCs; E–H). All traces are ensemble averages of traces from several independent experiments (n = 5–8). A and E: menthol-induced Ca2+ response was obliterated by removal of extracellular Ca2+ and recovered after readmission of extracellular Ca2+. B and F: menthol-induced Ca2+ response was inhibited in the presence of 300 µM Ni2+ and partially recovered after removal of Ni2+. C and G: 1 µM nifedipine had no effect on the menthol-induced Ca2+ response. D and H: summary data show averaged percent changes in intracellular Ca2+ response elicited by menthol after removal of extracellular Ca2+ or application of Ni2+ or nifedipine.

 
TRPV4 channels were activated with the specific agonist 4{alpha}-phorbol 12,13-didecanoate (4{alpha}-PDD) (44). 4{alpha}-PDD (1 µM) caused a progressive increase in [Ca2+]i, reaching 487.5 ± 69.4 nM (n = 17) and 638.1 ± 98.5 nM (n = 19) in PASMCs and ASMCs, respectively, after 4 min of agonist exposure (Fig. 7, A and E). The Ca2+ response induced by 4{alpha}-PDD was irreversible even after prolonged washing with agonist-free solution. Similar to the menthol-induced Ca2+ response, the Ca2+ transients elicited by 4{alpha}-PDD were dependent on extracellular Ca2+. The response was completely abolished after removal of extracellular Ca2+ and activated instantaneously with an overshoot upon reexposure of myocytes to Ca2+-containing solution. The 4{alpha}-PDD-induced Ca2+ response was not blocked by nifedipine but was almost completely inhibited by 300 µM Ni2+ (PASMCs: 78 ± 7.8%, n = 7; ASMCs: 79 ± 4%, n = 6). An increase in [Ca2+]i was observed in ASMCs after nifedipine application because the 4{alpha}-PDD-induced response had not reached a plateau before the blocker application. The results of these physiological experiments clearly suggest that TRPM8 and TRPV4 channels are functional Ca2+ entry pathways in PASMCs and ASMCs.


Figure 7
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  		Fig. 7. Characterization of intracellular Ca2+ response induced by TRPV4 channel agonist 4{alpha}-phorbol 12,13-didecanoate (4{alpha}-PDD; 1 µM) in PASMCs (A–D) and ASMCs (E–H). All traces are ensemble averages of traces from several independent experiments (n = 5–9). A and E: 4{alpha}-PDD-induced Ca2+ response was abolished by removal of extracellular Ca2+, followed by overshoot after reintroduction of extracellular Ca2+. B and F: 4{alpha}-PDD-induced Ca2+ response was inhibited in the presence of 300 µM (for PASMCs) or 1 mM Ni2+ (for ASMCs) and partially returned after removal of Ni2+. C and G: 1 µM nifedipine had no inhibitory effect on the 4{alpha}-PDD-induced Ca2+ response. D and H: summary data show averaged percent changes in intracellular Ca2+ response elicited by 1 µM 4{alpha}-PDD after removal of extracellular Ca2+ or application of Ni2+ or nifedipine.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have systemically identified and quantitatively characterized the relative expression of TRPM and TRPV channels in rat intralobar PAs and aorta. Our results show that 1) multiple TRPM and TRPV channel subtypes are coexpressed and their expression patterns are similar in PAs and aortic smooth muscle; 2) TRPM8, TRPM4, and TRPM7 mRNA are most abundantly expressed among the TRPM members, whereas TRPV4 and TRPV2 mRNA are the predominant TRPV transcripts; 3) TRPM8, TRPM2, TRPV4, and TRPV1 proteins are expressed in both types of vascular smooth muscle; and 4) the TRPM8 agonist menthol and the TRPV4 agonist 4{alpha}-PDD are capable of evoking significant Ca2+ influx in PASMCs and ASMCs. These findings provide the first molecular and physiological evidence that TRPM and TRPV channels operate as Ca2+ entry pathways in pulmonary as well as aortic myocytes.

The complete analysis of TRPM and TRPV expression clearly demonstrates at mRNA and protein levels that multiple TRPM and TRPV channels are coexpressed in PASMCs and ASMCs. It corroborates the limited RT-PCR data of previous studies showing that TRPM4, TRPM6, TRPM7, TRPV2, and TRPV4 channels are expressed in some systemic vascular smooth muscle cells (VSMCs) (6, 7, 11, 27, 42). In addition, the high level of TRPM8 transcript/protein detected in both PAs and aorta has not been reported in other vascular tissues. TRPM8 was originally identified as a prostate-specific gene. Its expression is androgen responsive and upregulated significantly in human prostate carcinoma (43), suggesting possible involvement in cell proliferation/metastasis. TRPM8 is also a menthol- and cold-sensitive ion channel in sensory neurons for the detection of cold temperature (25, 34). Our finding that menthol elicits significant [Ca2+]i increase in PASMCs and ASMCs and that the response could be abolished in the absence of extracellular Ca2+ or in the presence of Ni2+ but not in the presence of nifedipine suggests that TRPM8 proteins are functional Ca2+ influx channels in vascular myocytes. Two recent studies reported independently that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is necessary and sufficient to activate TRPM8 channels in heterologous expression systems (24, 39). Because many receptor-dependent mechanisms and physiological interventions can modulate PtdIns(4,5)P2 synthesis and hydrolysis (12, 29) and the PtdIns(4,5)P2/TRPM8 interactions can be regulated by phosphorylation (23, 52), it is conceivable that TRPM8 in PASMCs and ASMCs may participate in Ca2+ signaling through a PtdIns(4,5)P2-dependent mechanism that does not rely on the cold stimulus.

TRPM4 and TRPM7 are the two other major TRPM transcripts expressed in PAs and aorta, and physiological functions have been implicated for these channels in systemic arteries. TRPM4 is a Ca2+-activated monovalent cationic channel (19, 30, 31) and has been identified in cerebral arteries (7). Even though TRPM4 is impermeable to Ca2+, it can support Ca2+ influx via voltage-gated Ca2+ channels by allowing cells to depolarize in a Ca2+-dependent manner. Knockdown of TRPM4 with antisense oligodeoxynucleotides attenuates the pressure-induced depolarization and vasoconstriction in cerebral arteries, suggesting a critical role in myogenic response (7). However, myogenic vasoconstriction is normally absent in pulmonary circulation. It is likely that TRPM4 in pulmonary myocytes may participate in other processes that involve intracellular Ca2+ mobilization, such as agonist-mediated membrane depolarization.

In contrast, TRPM7 is an endogenous Mg2+ channel, which plays a key modulatory role in Mg2+ homeostasis in VSMCs (11, 42). TRPM7 expression in cultured mesenteric and aortic VSMCs is increased by angiotensin II and aldosterone, and inhibition of TRPM7 by siRNA abolishes the angiotensin II-induced chronic elevation of [Mg2+]i and cell proliferation (11). Moreover, TRPM7 expression is downregulated and [Mg2+]i is reduced in spontaneously hypertensive rats (42). It has been suggested that the low [Mg2+]i leads to increased vascular tone, blunted vasodilation, vascular remodeling, and elevated blood pressure (20, 21). It will be interesting to investigate whether TRPM7 similarly regulates [Mg2+]i in PASMCs and contributes to the vascular dysfunctions observed in pulmonary hypertension.

Another novel finding of the present study is the expression of TRPM2 mRNA and proteins in PAs and aorta. Western blot analysis detected both the full-length (~250 kDa) TRPM2 (TRPM2-L) and the truncated (~105 kDa) TRPM2 (TRPM2-S) in both types of vascular smooth muscle. TRPM2-L is known to be activated by nicotinamide adenine dinucleotide, ADP-ribose, and, more importantly, H2O2 in neurons, immune cells, and other heterologous expression systems (10, 35, 36). It has been proposed that TRPM2 acts as an endogenous redox sensor and mediates oxidative stress/ROS-induced Ca2+ entry and cell death. In contrast, the truncated TRPM2-S is insensitive to H2O2 but is capable of interacting directly with TRPM2-L to suppress H2O2-induced Ca2+ influx (54). Nevertheless, there is no prior report of TRPM2 expression in native vascular tissue. The coexpression of TRPM2-L and TRPM2-S detected in intact PAs and aorta, therefore, raises the intriguing possibility that TRPM2 may operate cooperatively in PASMCs and ASMCs to mediate the diverse ROS-dependent physiological processes.

For the TRPV subfamily, TRPV4 and TRPV2 are the predominant transcripts expressed in PAs and aorta. TRPV4, initially shown to be activated by cell swelling, is activated by very diverse stimuli, including heat, sheer stress, the arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET), and the non-PKC-activating phorbol ester 4{alpha}-PDD (32, 33, 40, 44–46). It is thought to be involved in thermosensing, osmosensing, mechanosensing, and basal Ca2+ homeostasis in different cell types (33). TRPV4 was identified recently in cerebral arteries and was shown to be activated by the putative endothelium-derived hyperpolarizing factor 11,12-EET and by 4{alpha}-PDD (6). More interestingly, antisense-mediated suppression of TRPV4 abolished the 11,12-EET-induced Ca2+ sparks and spontaneous transient outward currents in cerebral arterial myocytes and the membrane hyperpolarization and vasodilatation in cerebral arteries. It was proposed that 11,12-EET stimulates Ca2+ influx through TRPV4, activating ryanodine receptors to generate Ca2+ sparks, which activates the closely coupled Ca2+-activated K+ (KCa) channels to elicit membrane hyperpolarization and vasodilation. However, the function of TRPV4 in PASMCs could be rather different, because 11,12-EET causes vasoconstriction in rabbit pulmonary arteries (55) and Ca2+ sparks causes membrane depolarization instead of hyperpolarization in PASMCs (38).

Similar to TRPV4, TRPV2 is activated by heat, cell-swelling, and mechanical stress (33). It also is activated by growth factors such as insulin growth factor-1 and the neuropeptide head activator (5, 16). In mouse ASMCs, hypotonic solution-induced cell swelling activates a nonselective cation current and elevation in [Ca2+]i, which could be suppressed with TRPV2 antisense oligonucleotides (27). It is proposed that TRPV2 can function as a stretch-activated channel and may contribute to the myogenic response in systemic vessels. However, the physiological significance of mechanical stress in the pulmonary artery is unclear; further studies are necessary to elucidate the functions of TRPV2 and other TRPV channels in PASMCs.

In conclusion, we have identified a large repertoire of TRPM and TRPV channels in PAs and aorta. Our data may serve as the molecular and physiological basis for future explorations of the diverse functions of nonselective ion channels in pulmonary and systemic vasculatures.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-075134 and HL-071835 (to J. S. K. Sham). X. -R. Yang was supported by a postdoctoral fellowship from the American Lung Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. S. K. Sham, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (e-mail: jsks{at}welchlink.welch.jhu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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