Department of Physiology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Nonselective cation channels (NSCC) are targets of excitatory agonists in smooth muscle, representing the nonselective cation current Icat. Na+ influx through NSCC causes depolarizations and activates voltage-dependent Ca2+ channels, resulting in contraction. The molecular identity of Icat in smooth muscle has not been elucidated; however, products of the transient receptor potential (TRP) genes have characteristics similar to native Icat. We have determined the levels of TRP transcriptional expression in several murine and canine gastrointestinal and vascular smooth muscles and have analyzed the alternative processing of these transcripts. Of the seven TRP gene family members, transcripts for TRP4, TRP6, and TRP7 were detected in all murine and canine smooth muscle cell preparations. TRP3 was detected only in canine renal artery smooth muscle cells. The full-length cDNAs for TRP4, TRP6, and TRP7, as well as one splice variant of TRP4 and two splice variants of TRP7, were cloned from murine colonic smooth muscle. Quantitative RT-PCR determined the relative amounts of TRP4, TRP6, and TRP7 transcripts, as well as that of the splice variants, in several murine smooth muscles. TRP4 is the most highly expressed, while TRP6 and TRP7 are expressed at a lower level in the same tissues. Splice variants for TRP7, deleted for exons encoding amino acids including transmembrane segment S1, predominated in murine smooth muscles, while the full-length form of the transcript was expressed in canine smooth muscles.
calcium channels; gastrointestinal; vascular; ribonucleic acid expression
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INTRODUCTION |
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IN VISCERAL SMOOTH
MUSCLES, muscarinic stimulation results in excitation and muscle
cell contraction (15). Receptor activation [predominantly
M3 (24)] couples to Gq, which stimulates
phospholipase C (PLC). Inositol 1,4,5-trisphosphate (IP3)
is cleaved from phosphatidylinositol 4,5-bisphosphate
(PIP2), which will result in release of Ca2+
from IP3-dependent stores. In addition, muscarinic receptor
activation will release G, which has been suggested to activate
nonselective cation channels (representing nonselective cation current,
Icat) (11), although other
modes of activation for this current may exist. Influx of
Na+ and Ca2+ through these channels depolarizes
membrane potential, activating voltage-sensitive Ca2+
channels that are the primary source of Ca2+ influx leading
to smooth muscle contraction. The Ca2+ release due to
IP3 augments the activation of smooth muscle
Icat. The properties of
Icat have been determined for several smooth muscle preparations. In physiological solutions, removal of external Na+ reduces Icat by ~90%,
suggesting that Na+ is the primary charge carrier
(10). However, permeability ratios for
Icat from guinea pig jejunum (19),
portal vein (26), guinea pig gastric (14),
and canine pulmonary artery myocytes (13) suggest a high
relative selectivity of Ca2+ over monovalents.
The unitary conductance of Icat recorded from guinea pig jejunum, canine pylorus, and rabbit portal vein has been reported to be ~25-30 pS (11, 25). While the properties of the Icat have been examined, a molecular candidate encoding Icat has not been determined.
Transient receptor potential (TRP) gene products encode nonselective cation channels with properties similar to Icat. First cloned from Drosophila and found to be involved in visual signal transduction (20, 22), TRP channels play a prominent role in store-operated Ca2+ entry (1, 20, 22). To date, seven members of the TRP family have been cloned, and their expression has been detected within a variety of mammalian species and tissues. In addition to contributing to capacitative calcium entry, TRP channels have also been associated with receptor-operated Ca2+ influx stimulated by a Gq-coupled mechanism (9), possibly through direct interaction with diacylglycerol (8, 18). The topological and multimeric structures of TRP channels are similar to those of other cation-selective channels (see Ref. 1). That is, they form from monomeric subunits with six transmembrane segments, a cation-selective pore structure between transmembrane segments 5 and 6, and associate into functional tetramers by either combining four monomers or combining with other TRP family members to form heterotetramers (6, 29).
Because TRP channels are excellent candidates for Icat in smooth muscles, we determined the expression of members of the TRP family in visceral and vascular smooth muscle preparations. We report the identification of TRP4, TRP6, and TRP7 transcripts in isolated smooth muscle cell preparations and the cloning of all three forms from murine smooth muscle. Splice variants were detected for TRP4 and TRP7 expressed in smooth muscle cells from the gastrointestinal (GI) tract and vascular muscles. Quantitative RT-PCR was used to determine the relative expression levels of these TRP channels and splice variants in several smooth muscles.
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MATERIALS AND METHODS |
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Dissection of smooth muscles and smooth muscle cell preparations.
Mongrel dogs of either sex were overdosed with pentobarbital
sodium (100 mg/kg), and incisions were made along the abdomen. Strips
of small bowel and proximal colon were removed and placed into Krebs
solution containing (in mM) 120.35 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 Na2HPO4, and 11.5 glucose. Segments of tissue
were pinned in a dissecting dish with the mucosa facing upward. The
mucosa and submucosa were removed by sharp dissection. Small portions
of the circular smooth muscle tissue were placed into a
Ca2+-free Hanks' solution containing (in mM) 125 NaCl,
5.36 KCl, 15.5 NaOH, 0.336 Na2HCO3, 0.44 KH2PO4, 10 glucose, 2.9 sucrose, and 11 HEPES.
Strips of tissue were incubated in a Ca2+-free Hanks'
solution containing 230 units of collagenase (Worthington Biochemical),
2 mg of fatty acid-free bovine serum albumin (BSA FFA; Sigma), 2 mg of
trypsin inhibitor (Sigma), and 0.11 mg of ATP (Sigma). Incubation in
this enzyme was carried out at 37°C for 8-12 min. The tissues
were washed with Ca2+-free Hanks' solution, and gentle
trituration resulted in the isolation of individual myocytes. Cells
were transferred to the stage of a phase-contrast microscope and
allowed to adhere to the glass coverslip bottom for 5 min. Smooth
muscle cells were differentiated by their characteristic morphology.
Single cells were collected through applied suction by aspirating them
into a wide-bore patch-clamp pipette (borosilicate glass; Sutter
Instruments). Approximately 60 smooth muscle cells were collected,
flash-frozen in liquid nitrogen, and stored at 80°C until use.
BALB/c mice were killed by cervical dislocation, and incisions
were made along the abdomen. Segments of the small bowel and proximal
colon were isolated, and cells were collected as described above.
Pulmonary and renal canine smooth muscle isolation. Mongrel dogs were euthanized as described in Dissection of smooth muscles and smooth muscle cell preparations, and segments of renal and pulmonary arteries were isolated. The main pulmonary and renal arteries were flushed with physiological saline solution (PSS) containing (in mM) 125 NaCl, 5.36 KCl, 0.336 Na2HPO4, 0.44 K2HPO4, 11 HEPES, 1.2 MgCl2, 0.05 CaCl2, 10 glucose, and 2.9 sucrose, with pH 7.4 adjusted with 10 mM Tris and osmolarity of 300 mosM (sucrose). The solution was continuously bubbled with 100% O2 during dissections. Once isolated, arteries were cleaned by removal of bulk connective tissue and then were cut into small squares to prepare for digestion. Pulmonary arteries were digested in a solution containing (in mg/6 ml) 2 mg of collagenase type XI (Sigma), 0.4 mg of elastase type III, and 2 mg of BSA free fatty acids (FFA). Digestion took place for 16-18 h at 4°C. The tissues were then washed in cold Ca2+-free PSS solution and slowly triturated with fire-polished Pasteur pipettes to isolate individual myocytes. Renal arteries were digested in PSS solution containing (in mg/2.5 ml) 5 mg of collagenase type XI, 0.4 mg of elastase type IX, and 2 mg of BSA FFA. Tissues were digested for 18-23 min at 34°C, and cells were isolated. Individual myocytes from renal and pulmonary artery were collected as described above.
Total RNA isolation and RT-PCR. Total RNA was prepared from tissue and isolated smooth muscle cells with the use of a SNAP Total RNA isolation kit (Invitrogen, San Diego, CA) per manufacturer's instructions, including the use of polyinosinic acid (20 µg) as an RNA carrier. Total RNA was also isolated from heart and brain tissue by using this method. First-strand cDNA was prepared from the RNA preparations by using the Superscript II Reverse Transcriptase kit (GIBCO BRL, Gaithersburg, MD); 500 µg/µl oligo(dT) primers were used to reverse transcribe the RNA sample. The cDNA reverse transcription product was amplified with channel-specific primers by PCR. The amplification profile for these primer pairs were as follows: 95°C for 10 min to activate the Amplitaq polymerase (PE Biosystems), 95°C for 15 s, and 60°C for 1 min, each of 40 cycles. The amplified products (5 µl) were separated by electrophoresis on a 4% agarose/1× TAE (Tris, acetic acid, EDTA) gel, and the DNA bands were visualized by ethidium bromide staining. For the RT control, a cDNA reaction was used as template for which the reverse transcriptase was not added, controlling for genomic DNA contamination in the source RNA. The NTC (no-template control) was a PCR amplification for which the template was not added, controlling for nonspecific amplification and spurious primer-dimer fragments. These negative controls were subjected to a second round of amplification to assure specificity of the reactions and the quality of the reagents.
Primer design.
The following PCR primers were used: TRP1 (GenBank accession no.
U73625): sense nt 1794-1816 and antisense nt 2290-2310, amplicon = 516 bp; TRP2 (AF111107): sense nt 1531-1550 and antisense nt 1881-1900, amplicon = 369 bp; TRP3 (U47050):
sense nt 1483-1502 and antisense nt 1795-1814, amplicon = 331; TRP4 (U50922): sense nt 2535-2553 and antisense nt
2781-2800, amplicon = 265; TRP5 (AF029983): sense nt
675-694 and antisense nt 1075-1094, amplicon = 419; TRP6
(U49069): sense nt 738-758 and antisense nt 1128-1148,
amplicon = 410; TRP7 (AF139923): sense nt 2397-2417 and
antisense nt 2637-2657, amplicon = 260; -actin (V01217): sense nt 2384-2402 and antisense nt 3071-3091, amplicon = 498 bp; TRP4 span (U50922): sense nt 2467-2487 and antisense nt 2863-2883, amplicons = 420 or 167 bp depending on splice
variant present; TRP7 span (AF139923): sense nt 747-769 and
antisense nt 1278-1299, amplicons = 550, 385, or 203 bp
depending on splice variant present.
Quantitative RT-PCR.
Real-time quantitative PCR was performed with the use of Syber Green
chemistry on an ABI 5700 sequence detector (PE Biosystems). Regression
analysis of the mean values of eight multiplex RT-PCRs for the
log10 diluted cDNA was used to generate standard curves. Unknown quantities relative to the standard curve for a particular set
of primers were calculated, yielding transcriptional quantitation of
TRP gene products relative to the endogenous standard (-actin). The
reproducibility of the assay was tested by analysis of variance (ANOVA)
comparing repeat runs of samples, and mean values generated at
individual time points were compared by Student's t-test.
Cloning of TRP channels from murine colonic smooth muscle. TRP6 was cloned from murine colonic smooth muscle by performing PCR in the presence of the following gene-specific primers: TRP6 (GenBank accession no. U49069), sense nt 289-310 and antisense 3067-3088. PCR was performed under the following protocol: 94°C for 2 min; 30 cycles of 94°C for 45 s; 63°C for 90 s, and 72°C for 3 min; followed by 72°C for 10 min and holding at 4°C. Full-length fragments were ligated into PCR2.1 vector constructs (Invitrogen) and transformed with the use of a TA cloning kit (Invitrogen). TRP4 was cloned from murine colonic smooth muscle by performing PCR in the presence of the following gene-specific primers: TRP4 (AF019663), sense nt 184-200 and antisense 3101-3118. PCR was performed under the following protocol: 94°C for 5 min; 30 cycles of 94°C for 1 min, 52.4°C for 1 min, and 72°C for 2 min; followed by 72°C for 7 min and holding at 4°C. Wild-type TRP4 fragments, including a splice variant (splice 1), were ligated into pcDNA3.1 mammalian vector (Invitrogen) and transformed as described above. TRP7 was cloned from murine colonic smooth muscle by performing PCR in the presence of the following gene-specific primers: TRP7 (AF139923), sense nt 91-108 and antisense 2673-2692. PCR was performed under the following protocol: 3 cycles of 94°C for 20 s, 54°C for 40 s, and 72°C for 3 min; 3 cycles of 94°C for 20 s, 51°C for 40 s, and 72°C for 3 min; 3 cycles of 94°C for 20 s, 48°C for 40 s, and 72°C for 3 min; and 25 cycles of 94°C for 20 s, 50°C for 40 s, and 72°C for 3 min; followed by 72°C for 7 min and then holding at 4°C. Wild-type TRP7 fragments, as well as two different splice variants, were ligated into pcDNA 3.1 and transformed as described above.
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RESULTS |
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Expression of TRP family members in smooth muscles.
The expression of members of the TRP family in canine and murine smooth
muscle cells was determined by performing RT-PCR on total RNA isolated
from smooth muscle cell preparations. Qualitative RT-PCR was performed
on individually selected smooth muscle cells to avoid the contaminating
effects of other cell types as reported previously (3, 4).
PCR products were generated through the use of gene-specific primers
for TRP 1-7. Canine and murine brain-derived cDNA was used as a
positive control for the various TRP primers to test their ability to
produce the correct amplicon (Fig. 1). -Actin primers were used to confirm that the products generated were
representative of RNA (498-bp band) and not contaminated with genomic
DNA (intron containing 708-bp band) because these primers were designed
to span an intron as well as two exons. PCR reactions were also
performed on aliquots of RNA in which reverse transcriptase was not
added during the cDNA synthesis step. If DNA contamination was noted
with either
-actin or in the RT lane, these samples were discarded.
Detectable amplicons for TRP4 (270 bp), TRP6 (410 bp), and TRP7 (260 bp) were observed in all canine and murine smooth muscle cell
preparations (Fig. 1). TRP3 expression was detected only in canine
renal artery. The presence of other TRP transcripts was not detected
even with 30 additional cycles of PCR amplification.
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TRP channel splice variants expressed in smooth muscle cells.
Full-length cDNA cloning for TRP4 and TRP7 identified several
alternatively spliced transcripts differentially expressed in smooth
muscles. The full-length sequences for TRP4, TRP6, and TRP7
cDNAs were 99% identical to murine sequences
previously reported (GenBank accession no: TRP4, AF019663;
TRP6, U49069; and TRP7, AF139923). Alternatively spliced transcripts
have been reported for TRP7 (18). However, alternatively
spliced transcripts have not been previously published for TRP4. Figure
2A describes the positions on
a linear depiction of the nucleotide sequence for the alternative exons
recovered for these gene transcripts. Primers designed to span the
alternatively spliced exons were used in RT-PCR reactions with RNA from
smooth muscle cells as template. An analysis of the splice variants for
TRP4 and TRP7 from several canine and murine smooth muscles is shown in
Fig. 2B. The expression of specific splice products is
dependent in a qualitative and quantitative manner on the source of
smooth muscle RNA. This is a consistent pattern (n = 3)
and was analyzed further by real-time quantitative PCR procedures (see
Quantitative determination of TRP transcripts in smooth
muscles). TRP7 amplification of canine smooth muscle cells
consistently detected the full-length splice form, while murine smooth
muscle cells did not. However, when murine colonic muscle was used as
the source of RNA, the full-length amplicon was recovered (see Fig.
3C). This is presumably due to non-smooth muscle cell types
in the tissue preparation. Therefore, TRP7 expression in murine smooth
muscle cells is restricted to splice forms 1 and 2.
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Quantitative determination of TRP transcripts in smooth muscles.
The ABI 5700 genetic analyzer (PE Biosystems) was used for accurate
quantitation of steady-state transcript levels by RT-PCR. An analysis
of TRP6 with qualitative methods indicated no alternative splice
variants. Therefore, primers were designed that were specific for TRP6
and used in quantitative analysis. Total RNA was prepared from murine
colon, jejunum, and antrum and from canine pulmonary and renal
arteries. The RNA was prepared from ~25-50 mg of tissue with
mucosa removed from the GI tissues and the endothelium removed from the
vascular muscles, as described in MATERIALS AND METHODS. However, these preparations contain smooth muscle cells and other minor
cell types (e.g., interstitial cells of Cajal, macrophages, and
fibroblasts) that will contribute to the quantitative measurement. RNA
was reverse transcribed to cDNA, and steady-state transcripts were
determined relative to an endogenous control housekeeping gene
(-actin). Therefore, the data are expressed as TRP/
-actin. The
relative transcriptional expression of TRP6 is shown in Fig. 4 for several murine and canine smooth
muscles. Note that the scale for all the TRP expression data is kept
consistent to allow comparison of relative expression levels between
TRP forms, and all expression data are expressed as means ± SE.
TRP6 expression relative to
-actin (arbitrary units) was 0.018 ± 0.0063 for murine colon, 0.0061 ± 0.0018 for murine jejunum,
0.0075 ± 0.0029 for murine antrum, 0.038 ± 0.014 for canine
pulmonary artery, and 0.023 ± 0.0033 for canine renal artery
(n = 3 for all these data).
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DISCUSSION |
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TRP channels have been receiving increasing attention as molecular correlates for capacitative Ca2+ calcium entry (CCE) or store-operated cation channels (1, 7). These channels are activated in response to emptying of intracellular Ca2+ stores and act to replenish those stores (23). This Ca2+ entry mechanism is present ubiquitously in mammalian cells, although the mechanism of channel activation differs depending on cell type (reviewed in Ref. 7). A more specialized form of Ca2+ entry in response to receptor activation occurs in smooth muscles (Icat) and involves the coupling of G protein-linked receptors to isoforms of PLC. This mechanism is independent of store depletion and may involve the direct activation of channels by G proteins (17). TRP channels have been implicated in both store-dependent and store-independent Ca2+ entry mechanisms and may be responsible for the receptor-operated channels (Icat) reported in smooth muscles (2, 27). Given the importance of these channels to receptor-operated mechanisms in smooth muscles, it is important to identify which of these channels are expressed in different types of smooth muscles and their molecular characteristics. In this study we have detected TRP3, TRP4, TRP6, and TRP7 in several canine and murine GI and vascular smooth muscles and have analyzed the molecular pattern of their expression, including a detailed analysis of alternatively spliced transcripts expressed in these tissues.
TRP channels can be functionally organized into two groups, those that are dependent on store depletion for activation and those that are independent. While the mechanism for channel activation is probably multifaceted for both groups, those that are store-depletion dependent are more likely to be CCE channels, and the store-independent TRP channels are more likely to encode receptor-operated nonselective cation channels, responsible for Icat in many types of smooth muscle. Current research suggests that TRP1, TRP3, TRP6, and TRP7 encode channels of the latter type and that TRP2, TRP4, and TRP5 are CCE-type channels (reviewed in Ref. 1). It is striking that, for both canine and murine smooth muscles, the predominant TRP forms expressed are TRP4, TRP6, and TRP7. This suggests that a gene regulatory protein(s) expressed in smooth muscles directs this expression in concert with promoter specificity of these TRPs for smooth muscles. In a study performed on rat tissues including liver, lung, heart, kidney, brain, testis, ovary, and nodose ganglia, TRP1 and TRP3 were detected in all these tissues except liver (5). This finding suggests that these TRP channels might be ubiquitous CCE-type channels. In fact, this study also detected high levels of TRP1 expression in five different mammalian cell lines. However, our results indicate that this is not the case for smooth muscle cells, and TRP4 may carry out this function. Because TRP proteins can form heteromeric channels (16, 28), it is possible that a combination of TRP4, TRP6, and TRP7 may encode CCE in smooth muscles. In bovine adrenal cells, TRP4 was the only TRP channel detected at significant levels (21). A direct connection was made between TRP4 and CCE in these cells by blocking CCE with antisense cDNA directed specifically against TRP4.
In this study we detected specific splice variants of TRP4 and TRP7 expressed in smooth muscles in a species- and tissue-specific expression pattern. Whether these differences are manifest in channel proteins is not known; however, it is striking that in all the murine smooth muscle cells examined, no TRP7 full-length transcripts could be detected. This result was confirmed at the quantitative level by using RNA derived from intact smooth muscles. In addition, the exon-deleted form of TRP4 predominated in all the canine and murine smooth muscles except antrum. It will be important to determine if any differences in channel kinetics or regulation exist between the alternatively spliced forms of these channels.
In conclusion, with the identification of TRP channel expression in several smooth muscles, it now becomes important to critically examine their biophysical and electrophysiological properties. Functional expression of the TRP isoforms will allow investigators to compare channel properties with native receptor-operated and CCE-type channels in smooth muscles and provide information necessary for transgenic studies, delineating their functions in smooth muscle cellular physiology.
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ACKNOWLEDGEMENTS |
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We thank Dr. Sean Wilson for assisting in smooth muscle cell preparation.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-41315 and HL-49254. R. L. Walker is a predoctoral fellow of the American Heart Association-Western States Affiliate.
Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology, Univ. of Nevada School of Medicine, Reno, Nevada 89557 (E-mail: burt{at}physio.unr.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.
Received 26 September 2000; accepted in final form 14 November 2000.
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