Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023325
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/4/919    most recent
biolreprod.103.023325v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Babich, L. G.
Right arrow Articles by Sanborn, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Babich, L. G.
Right arrow Articles by Sanborn, B. M.
Agricola
Right arrow Articles by Babich, L. G.
Right arrow Articles by Sanborn, B. M.
BIOLOGY OF REPRODUCTION 70, 919–924 (2004)
DOI: 10.1095/biolreprod.103.023325
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Expression of Capacitative Calcium TrpC Proteins in Rat MyometriumDuring Pregnancy1

Lidiya G. Babich3,4, Chun-Ying Ku3,4, Hays W.J. Young3, Helen Huang3, Michael R. Blackburn3, and Barbara M. Sanborn2,3,4

Department of Biochemistry and Molecular Biology,3 University of Texas Medical School at Houston,Houston, Texas 77030 Department of Biomedical Sciences,4 Colorado State University, Fort Collins, Colorado 80523


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
External Ca2+ entry into myometrial smooth-muscle cells is important to uterine contraction and hence to labor progression and parturition. Proteins of the transient receptor potential (Trp) channel family are putative capacitative Ca2+ entry channels that respond to contractant-generated signals and intracellular Ca2+ store depletion. Quantitative reverse transcription-polymerase chain reaction was used to examine the relative expression of TrpC mRNAs in rat myometrium and determine their expression pattern during pregnancy and labor. rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 mRNAs, but not rTrpC3 mRNA, were expressed in nonpregnant rat myometrium. With the exception of rTrpC7, the resulting products were sequenced and found to be identical with published sequences; new rTrpC7 sequence exhibited >88% homology to mouse and human TrpC7 coding regions. Relative to ß-actin mRNA, rTrpC4 mRNA was expressed in the greatest abundance. rTrpC1, 5, and 6 mRNAs were expressed at lower levels, whereas rTrpC2 and 7 mRNAs were barely detectable. This relative expression pattern was also observed throughout the course of gestation. There were no major differences in expression of rTrpC1, 2, 4, or 7 mRNAs between Day 13 and Day 21 of gestation or labor. Rat TrpC5 and TrpC6 mRNA expression decreased in pregnancy but was not altered between Day 13 and Day 21 or in labor. Western blot analysis generally confirmed these observations with respect to protein expression. These data suggest that rTrpC4 may play a major role in regulated Ca2+ entry in myometrial cells and throughout pregnancy but do not rule out contributions from other Trp proteins.

calcium, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The regulation of myometrial contraction is of paramount importance for the maintenance of pregnancy and for parturition [1]. External Ca2+ entry is vitally important to uterine contraction [2]. An increase in intracellular Ca2+ concentration is a major trigger for smooth-muscle contraction [3, 4]. At the same time, activation of smooth-muscle contraction by hormones usually involves a combination of Ca2+ entry and Ca2+ release from intracellular stores [5]. Many contractant receptors use seven-transmembrane domain GTP-binding protein coupled receptors that transmit signals into the cell via interaction with heterotrimeric G-proteins [1]. Stimulation of these proteins results in activation of phospholipase C and the production of diacylglycerol and inositol 1,4,5-trisphosphate (IP3)[6]. IP3 interacts with receptors on the endoplasmic reticulum and releases calcium from these intracellular stores. The release of calcium from intracellular stores is critically important for sustained contractile activity and is an important component of the action of uterine contractants [1, 7, 8].

Ca2+ gating is performed by three classes of ion channels: 1) voltage-gated Ca2+ channels, 2) ligand-gated Ca2+ channels, and 3) voltage-independent Ca2+-permeable channels stimulated by activation of phospholipase C and formation of IP3 or by intracellular Ca2+ release [9]. The suggestion has been made that the voltage-independent calcium channels are formed by proteins of the transient receptor potential channel family [10, 11]. This large family of proteins is divided into three subfamilies: TRPC, TRPV, and TRPM [10, 11]. The TRPC subfamily contains seven major members, termed TrpC1–7. Based on amino acid sequence similarity, the mammalian members of the TrpC subfamily were divided further into four groups [12–14]. All Trp subfamilies may contribute to the regulation of Ca2+ entry [15]. A number of TrpC mRNAs or proteins are expressed in smooth-muscle tissues [10].

Myometrium from pregnant women and human myometrial cell lines expressed mRNAs for TrpC1, TrpC3, TrpC4, TrpC6, and TrpC7 [16, 17]. Western blotting analysis verified expression of hTrpC1, 3, 4, and 6 proteins [16, 17]. A number of lines of evidence point to the importance of capacitative calcium entry in myometrium [1]. We found that human myometrium exhibits capacitative Ca2+ entry in response to oxytocin and the endoplasmic reticulum calcium pump inhibitor thapsigargin [16, 18]. We also showed that overexpression of hTrpC3 in PHM1 myometrial cells enhanced thapsigargin, oxytocin, and 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca2+ entry [19]. The properties of Ca2+ entry mediated by Trp proteins are thought to vary as a result of their relative expression and configuration in heterotetrameric channel complexes [20, 21]. The aims of this study were to determine the relative expression of TrpC mRNAs in rat nonpregnant myometrium and the rTrpC expression pattern during pregnancy and labor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Timed-pregnant Sprague-Dawley rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Animals were maintained at 73°F on a 12L:12D cycle. The day when the vaginal plug was observed was dated Day 0. Experiments were conducted in accordance with institutional practices in an AAALAC-accredited facility. Nonpregnant and pregnant rats on Days 13, 16, 19, and 21 of gestation and in labor (after the first pup was delivered) were killed. The uterus was excised and opened; pups and placenta were discarded. Endometrium was removed by scraping with a scalpel blade.

Total and Messenger RNA Isolation

Total RNA was prepared from rat myometrium using the TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). Messenger RNA was prepared from total RNA using the FastTrack 2.0 Kit mRNA isolation system (Invitrogen, Carlsbad, CA). Three to four rats per group were used for isolation of RNA.

Polymerase Chain Reaction (PCR) Primers

PCR primers for ß-actin and rTrpC1–6 were designed based on published rat sequences in GenBank (Table 1). Because there was no sequence in the databank for rat TrpC7, we used the PCR primers used for rTrpC7 by Jung et al. [22] to generate a clone from which to derive sequence information. The 841-base pair (bp) rTrpC7 cDNA fragment was cloned into the pCR2.1/TOPO vector (Invitrogen) and sequenced. Using this sequence, we designed a new reverse TrpC7 primer to obtain a smaller rTrpC7 cDNA fragment suitable for use in quantitative reverse transcriptase polymerase chain reaction (RT-PCR).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Oligonucleotide sequences of the PCR primers

Reverse Transcription (RT)-PCR

First-strand cDNA was prepared from 300 ng mRNA using AMV reverse transcriptase (Promega, Madison, WI) and 100 ng oligo-dT. The mRNA samples were denaturated at 65°C for 5 min. RT was performed at 42°C for 1.5 h and was stopped by heating samples at 95°C for 5 min. The cDNA was amplified by PCR using the TrpC isoform-specific primers listed in Table 1. The general PCR conditions were 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.1–2.5 mM MgCl2; 0.2 mM dNTP; 0.5 µM forward and reverse primers; and 2.5 U Jump Start Taq DNA polymerase (Sigma, St. Louis, MO) in a total volume of 50 µl. The cDNA samples were initially denaturated at 94°C for 1 min followed by 25–40 cycles of denaturation at 94°C for 30 sec, annealing (56°C for actin and 60°C for all TrpCs) for 30 sec, and extension at 72°C for 1.5 min, with a 10-min final extension. Two negative control reactions (no AMV reverse transcriptase to test for genomic DNA contamination; no template to test for general contamination) were included. The amplified products were separated by electrophoresis on 1.5% agarose gels, and DNA bands were visualized by SYBR Green staining. The PCR products were all of the expected size and were cloned into pCR2.1/TOPO using the TOPO-TA cloning kit (Invitrogen); identity was confirmed by direct sequencing.

Quantitative Real-Time RT-PCR

One-step RT-PCR was performed using the SmartCycler instrument (Cepheid) and QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA). RT-PCR was performed using the kit protocol in a 25-µl reaction volume containing 1x QuantiTect SYBR Green RT-PCR Master Mix, 2.5 mM MgCl2, 1 µM primers (Table 1), QuantiTect RT Mix, and 300 ng template mRNA or 0.1–100 pg standard cDNA. For standard curves, the same protocol was used but without step 1 (reverse transcription). For all experiments, the threshold line was set at fluorescence level 30. The integrity of the RT-PCR products was confirmed by melting curve analysis. Melting curves for rTrpC1, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 and ß-actin cDNA showed one specific signal. Only the melting curve of TrpC2 cDNA showed two peaks, perhaps because of the very low expression of this mRNA in rat myometrium and possible contribution from primer dimers. Therefore, TrpC2 quantitation represents only an approximate estimate. The amount of PCR products calculated in reference to the individual calibration curves were then normalized to that of ß-actin, determined in the same mRNA sample.

Western Blotting

Rat myometrial plasma membrane fractions were isolated according to Ku et al. [23]. A crude membrane fraction was isolated from rat brain using the same method but without sucrose-density gradient centrifugation. Pellets were suspended in sample buffer (10 mM Tris-HCl, pH 7.4; 250 mM sucrose; 1 mM EGTA; 1 µg/ml each leupeptin, pepstatin A, and aprotinin) and stored at -80°C. Twenty micrograms of rat myometrial plasma membranes and 30 µg of crude brain membranes were subjected to 7.5% SDS-PAGE [24] and the resolved proteins were electrotransfered to nitrocellulose membranes (BA85; Protran, Schleicher and Schuell BioScience, Keene, NH). The membranes were blocked for 1 h at room temperature with 5% milk/PBS, briefly washed with two changes of wash buffer (PBS/0.1% Tween20), and incubated in diluted primary antibody in 5% milk/PBS overnight at 4°C. Primary polyclonal antibodies used were anti-TrpC1, anti-TrpC4, anti-TrpC5, and anti-TrpC6 (1:200; Alomone Labs, Jerusalem, Israel) and anti-G{alpha}q/ll (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed with five changes of wash buffer, incubated for 1 h at room temperature with anti-rabbit IgG-horseradish peroxidase antibody (1:5000) (Bio-Rad, Hercules, CA), and detected using ECL+plus (Amersham Biosciences, Little Chalfont, UK).

Statistical Analysis

Data are expressed as mean ± SEM. The mRNA from three to four rats in each group were analyzed by analysis of variance and Duncan new multiple range.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR and Western Blotting

Qualitative RT-PCR was used to determine that rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 mRNAs were expressed in rat myometrium (Fig. 1A). The products were of the predicted size. All products were cloned and sequenced. The rTrpC1, 2, 4, 5, and 6 products exhibited 99–100% identity to the corresponding sequences in GenBank (Table 1). The sequence for rTrpC7 (841 bp) has been entered into GenBank (AY390391) and is located on rat chromosome 17. This sequence exhibited 90% homology with mouse TrpC7 (NM_012035). The coding region (469 bp) exhibited 88% homology with human TrpC7 (NM_020389). PCR failed to amplify rTrpC3 mRNA in rat myometrium, but rTrpC3 fragment was amplified from rat brain mRNA using the same primer set (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 1. A) PCR products obtained using myometrial mRNA from Day 13 pregnant rat. Primers listed in Table 1 were used to amplify cDNA from rTrpC1 (lane1), rTrpC2 (lane 2), rTrpC4 (lane 4), rTrpC5 (lane 5), rTrpC6 (lane 6), rTrpC7 (lane 7). PCR failed to amplify rTrpC3 in rat myometrium (lane 3); M, DNA marker. TrpC1 (B), TrpC4 (C), TrpC5 (D), and TrpC6 (E) protein expression in immunoblots containing myometrial plasma membranes from nonpregnant (NP, lane1), Day 13 pregnant (lane 2), and Day 21 pregnant rats (lane 3) and in rat brain crude membranes (BM, lane 4). Twenty micrograms of myometrial plasma membranes and 30 µg of crude brain membranes were loaded in respective lanes. The G{alpha}q/11 loading control for the TrpC4 blot (C) is shown in (F)

Western blot analysis demonstrated the expression of rTrpC1 (~120 kDa), rTrpC4 (~95 kDa), rTrpC5 (~95 kDa), and rTrpC6 (~110 and 130 kDa) proteins in myometrium from nonpregnant and Day 13 and Day 21 pregnant rats (Fig. 1, B–E). Crude brain membrane protein was used as a positive control and G{alpha}q/11 as loading control in these studies. Purified rat myometrial membranes (20 µg) expressed relatively the same amount of rTrpC4 as 30 µg of crude rat brain membrane protein, slightly more rTrpC1, and significantly less rTrpC5 and rTrpC6.

Quantitative RT-PCR

Quantitative real-time RT-PCR (QRT-PCR) was used to determine the relative expression of all detected myometrial rTrpC mRNAs. This method relies on an inverse linear relationship between the cycle number at which the amplification product is first detected above the threshold and the logarithm of the amount of cDNA. Standard curves for all rTrpCs and ß-actin were constructed using cDNA samples of known concentration and sequence. Each specific PCR product was inserted into the TOPO-TA vector, cut from plasmid, and used as the standard cDNA. The calibration curves were linear over at least a three-log range of cDNAs concentration (0.1–100 pg). One of these standard curves, for TrpC4 cDNA, is presented on Figure 2A. The amounts of PCR products calculated in reference to the individual calibration curves were then normalized to that of ß-actin, determined in the same mRNA sample. It has been shown that ß-actin is constitutively expressed in myometrium from nonpregnant animals and throughout gestation and may be used as internal control [25–27]. This fact was confirmed in the present experiments; expression of ß-actin mRNA was the same in myometrium from Day 13 pregnant rats relative to nonpregnant animals, and there were no significant changes between Day 13 and Day 21 of gestation (Fig. 2B).



View larger version (9K):
[in this window]
[in a new window]
  		FIG. 2. A) The standard curve for TrpC4 cDNA. TGE rTrpC4 was amplified from rat myometrial mRNA and cloned into pCR2.1/TOPO vector; cDNA was cut from the plasmid and used for constructing the standard curve. B) ß-Actin mRNA expression in rat myometrium from nonpregnant, Day 13, and Day 21 pregnant and in-labor rats (n = 3–4). Groups with the same lowercase letters are not significantly different

QRT-PCR confirmed the expression of rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 in rat myometrium. In myometrium from nonpregnant rats, rTrpC4 mRNA was expressed in the greatest relative abundance (Fig. 3). Rat TrpC2 mRNA was expressed at the lowest level relative to other members of this subfamily. Relative expression of rTrpC7 mRNA was significantly higher than that of rTrpC2 mRNA (P < 0.05) but it was also barely detectable. The levels of rTrpC1 and rTrpC5 mRNA relative expression were essentially similar, while rTrpC6 mRNA expression was higher than that of rTrp1 (P < 0.05) and rTrpC5 (P < 0.01) but less than that of rTrpC4 (P < 0.01) (Fig. 3).



View larger version (8K):
[in this window]
[in a new window]
  		FIG. 3. Rat myometrial TrpC mRNA expression. Myometrial mRNA was isolated from nonpregnant rats. Groups with different lowercase letters are significantly different from each other at P < 0.05 (n = 3–4)

TrpC mRNAs in Pregnant Rat Myometrium

Rat TrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 were also expressed in pregnant myometrium between Day 13 and Day 21, and rTrpC4 was again the form expressed in the greatest relative abundance (Fig. 4). Expression of rTrpC1 and rTrpC4 were the same in myometrium from nonpregnant and Day 13 pregnant rats, and no statistically significant changes were seen in their expression between Day 13 and Day 21 of pregnancy or in labor (Fig. 4, A and B). The rTrpC5 and rTrpC6 mRNAs were higher in myometrium from nonpregnant rats than in pregnancy (P < 0.01) but no changes were observed between Day 13 and Day 21 of gestation or in labor (Fig. 4, C and D). There were no pregnancy-related changes in the expression of rTrpC2 and rTrpC7 mRNA, which were barely detectable (data not shown). Figure 1, B–E, shows that the changes in TrpC mRNAs between nonpregnant and pregnant myometrium were reflected in similar relative changes in the respective proteins.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 4. TrpC1 (A), TrpC4 (B), TrpC5 (C), and TrpC6 (D) mRNA expression in myometrium from nonpregnant (NP), Day 13, Day 16, Day 19, and Day 21 pregnant and in-labor rats. Groups with different lowercase letters are significantly different from each other at P < 0.05 (n = 3–4)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A number of TrpC genes have been reported to be present in smooth-muscle tissues [10]. Walker and colleagues found transcripts for TrpC4, TrpC6, and TrpC7 in different murine and canine smooth-muscle cell preparations, TrpC3 was detected only in canine renal artery smooth-muscle cells [28]. TrpC1, TrpC2, TrpC4, TrpC5, and TrpC6 mRNAs were detected in rat pulmonary artery smooth-muscle cells [29]. In human airway smooth muscle, expression of TrpC1, TrpC3, TrpC4, and TrpC6 were observed [30]. We reported expression of hTrpC1, hTrpC3, hTrpC4, hTrpC6, and hTrpC7 mRNAs but not TrpC2 (a pseudogene in humans) or hTrpC5 mRNA in myometrium from pregnant women and in PHM1–41 cells, an immortalized line derived from late pregnant human myometrium [16]. Similar results were reported in another study using myometrial tissue and primary human myometrial cells isolated from term pregnant women [17]. In contrast, in this study, we have found that rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 mRNA are expressed in rat myometrium. No rTrpC3 products were observed, although this product was amplified from rat brain mRNA using the same primer set. This indicates species-related variations in TrpC isoform expression in myometrium.

The rTrpC4 mRNA was expressed in the greatest abundance relative to other TrpC mRNAs in rat myometrium and this pattern did not change over the course of gestation. There were no statistically significant changes in the expression of rTrpC1, rTrpC2, rTrpC4, and rTrpC7 mRNA in rat myometrium during pregnancy or in labor. In contrast, rTrpC5 and rTrpC6 mRNA/ßactin concentrations were decreased in myometrium from Day 13 pregnant relative to nonpregnant rats and were not altered between Day 13 and Day 21 or during labor. The factors that promote the decrease in TrpC5 and TrpC6 during pregnancy are not defined at present. It is unlikely that this is solely attributable to estradiol or progesterone because the ratios of these hormones change markedly before delivery.

Expression of rTrpC1, rTrpC4, rTrpC5, and rTrpC6 proteins in rat myometrium was confirmed using commercially available antibodies. In rat myometrial plasma membranes and crude brain membranes, rTrpC1 was 120 kDa in size, whereas in human myometrial and PHM1–41 cell membranes, hTrpC1 of 95 kDa was detected [16]. A 95-kDa band of rTrpC4 protein was detected in rat myometrial plasma membranes, whereas 100- to 110-kDa bands were reported in human myometrium [17]. The rTrpC5 protein was also detected in rat myometrium as a 95-kDa band. In rat brain membranes, prominent bands of 110 kDa and 130 kDa were detected with the anti-TrpC6 antibody. TrpC6 is highly glycosylated in various cell types [4, 13, 31], deglycosylation of rat pulmonary vascular smooth-muscle cell lysates shifted the 130-kDa rTrpC6 band close to 110 kDa [4]. Consistent with multiple forms of this protein, weak signals were detected at 130 and 110 kDa with anti-Trp6 antibody in rat myometrial plasma membrane samples, whereas only the 130-kDa form was detected in human myometrium in one study [16] and 100 kDa in another [17]. While we cannot quantitate protein expression at present, the relative changes in protein roughly paralleled changes in the respective mRNAs.

In rat myometrium, TrpC4 mRNA was expressed in the greatest abundance relative to ß-actin, with smaller but significant contributions from TrpC1, TrpC5, and TrpC6 mRNA. Schilling [32] has suggested that specific Trp proteins may functionally dominate receptor-mediated responses in specific tissues. In support of this concept, it was shown that bovine adrenal cortex cells expressed TrpC4 abundantly and exhibited endogenous calcium release-activated Ca2+-like currents; expression of TrpC4 antisense cDNA significantly reduced both the amount of native protein and currents [33]. TrpC3 and TrpC4 were more abundant in smooth muscle from human lower esophageal sphincter than in smooth muscle from esophageal body; this correlated with enhanced capacitative calcium entry in the former [34]. In contrast, a study in TrpC4-deficient mice has suggested that TrpC4 is the main subunit involved in store-operated channels in endothelial cells [35]. A number of studies have implicated an important role for activation of store-operated channels in smooth-muscle contraction [36]. When overexpressed under different conditions, TrpC4 and TrpC5 can function as receptor-operated or store-operated channels [37]. It remains to be determined how many of the properties of capacitative calcium entry in myometrium can be explained by the expression of rTrpC4.

TrpC6 is structurally similar to TrpC3 and TrpC7. TrpC3, TrpC6, and TrpC7, but not the others, are activated by diacylglycerol in a protein kinase C (PKC)-independent manner [38, 39]. Both human myometrial cells [19] and pregnant rat myometrial cells (Shlykov and Sanborn, unpublished observations) exhibit Ca2+ entry in response to diacylglycerol. Based on the available data, it is unlikely that TrpC4 is responsible for this activity. TrpC3 mRNA is not expressed in rat myometrium and rTrpC7 mRNA is in very low abundance, suggesting that TrpC6 may account for this activity. TrpC6 is a requisite component of the {alpha}1-adrenoceptor-activated Ca2+-permeable nonselective cation channel in rabbit portal vein smooth-muscle cells [40]. Inhibition of TrpC6 expression using antisense oligomers greatly attenuated arterial smooth-muscle depolarization and pressure-induced constriction in cerebral arteries [41]. This was attributed to a pressure-induced increase in phospholipase C activity, resulting in production of diacylglycerol, which activated TrpC6, leading to membrane depolarization, opening of voltage-dependent calcium channels, increased intracellular Ca2+, and increased tone [41]. Down-regulation of TrpC6 using antisense oligonucleotides also decreased capacitative calcium entry in pulmonary artery smooth muscle [4] and is implicated in receptor-operated calcium channels in A7r5 smooth-muscle cells [22]. Thus, TrpC6 may have an important role in the control of the smooth-muscle contractile response [30, 40, 41].

We have found that human myometrium exhibits capacitative calcium entry in response to oxytocin, the endoplasmic reticulum calcium pump inhibitor thapsigargin, and diacylglycerol [16, 18]. The role of this entry in regulating myometrial contraction is under study in a number of laboratories. Clearly, G-protein-coupled receptor mechanisms lined to the phospholipase C pathway are enhanced near parturition and contribute to contractile activity [1, 42]. Capacitative calcium entry may contribute significantly to this activity. In addition, while TrpC3/6/7 are activated by diacylglycerol, TrpC4/5 are strongly inhibited by diacylglycerol-induced PKC activation, constituting a potential feed-back control of TrpCs [43]. Diacylglycerol produced an increase in intracellular calcium in human myometrial cells expressing hTrpC3, hTrpC6, and hTrpC7 as well as hTrpC1 and hTrpC4 [19], but diacylglycerol produced in response to oxytocin inhibited myometrial contractile activity, possibly by other mechanisms [44]. A balance between opposing mechanisms may account for the apparent differences.

In summary, multiple TrpC mRNAs and proteins are expressed in rat myometrium. There is no change during pregnancy in the rTrpC4 mRNA, the predominant TrpC present, and decreases in rTrpC5 and rTrpC6 mRNAs. TrpC proteins are thought to form homo- and heterotrimeric cation channels. Several studies indicate that, while TrpC4 can interact with TrpC1 and TrpC5, it does not interact with overexpressed TrpC6 [20, 45, 46]. While TrpC5 mRNA/actin decreases during pregnancy, it is already relatively nonabundant in the nonpregnant state. Therefore, changes in TrpC5 or TrpC6 mRNAs are unlikely to have a major effect on channel composition. Because TrpC4 mRNA is present in the greatest abundance, it may play a major role in regulated Ca2+ entry in myometrial cells, but the data do not rule out contributions from other TrpC proteins as well.


   FOOTNOTES
 
1 Supported by NIH HD38970. Back

2 Correspondence: Barbara M. Sanborn, Department of Biomedical Sciences, 101 Physiology Campus Deliveries 1680, Colorado State University, Fort Collins, CO 80523. FAX: 970-491-7569; Barbara.Sanborn{at}colostate.edu Back

Received: 16 September 2003.

First decision: 7 October 2003.

Accepted: 19 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sanborn BM. Hormones and calcium: mechanisms controlling uterine smooth muscle contractile activity. The Litchfield Lecture. Exp Physiol 2001 86:223-237[Abstract]
  2. Wray S, Kupittayanant S, Shmygol A, Smith RD, Burdyga T. The physiological basis of uterine contractility: a short review. Exp Physiol 2001 86:239-246[Abstract]
  3. van Breemen C, Saida K. Cellular mechanisms regulating [Ca2+]i smooth muscle. Annu Rev Physiol 1989 51:315-329[Medline]
  4. Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, Yuan JX. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol 2003 284:C316-330[Abstract/Free Full Text]
  5. Anwer K, Sanborn BM. Changes in intracellular free calcium in isolated myometrial cells: role of extracellular and intracellular calcium and possible involvement of guanine nucleotide-sensitive proteins. Endocrinology 1989 124:17-23[Abstract]
  6. Berridge MJ. Capacitative calcium entry. Biochem J 1995 312:1-11
  7. Sanborn BM, Yue C, Wang W, Dodge KL. G protein signalling pathways in myometrium. Rev Reprod 1998 3:196-205[Abstract]
  8. Burghardt RC, Barhoumi R, Sanborn BM, Andersen J. Oxytocin-induced Ca2+ responses in human myometrial cells. Biol Reprod 1999 60:777-782[Abstract/Free Full Text]
  9. Birnbaumer L. TRPC4 knockout mice: the coming of age of TRP channels as gates of calcium entry responsible for cellular responses. Circ Res 2002 91:1-3[Free Full Text]
  10. McFadzean I, Gibson A. The developing relationship between receptor-operated and store-operated calcium channels in smooth muscle. Br J Pharmacol 2002 135:1-13[CrossRef][Medline]
  11. Minke B, Cook B. TRP channel proteins and signal transduction. Physiol Rev 2002 82:429-472[Abstract/Free Full Text]
  12. Birnbaumer L, Zhu X, Jiang M, Boulay G, Peyton M, Vannier B, Brown D, Platano D, Sadeghi H, Stefani E, Birnbaumer M. On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins. Proc Natl Acad Sci U S A 1996 93:15195-15202[Abstract/Free Full Text]
  13. Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stefani E, Birnbaumer L. Trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 1996 85:661-671[CrossRef][Medline]
  14. Montell C, Birnbaumer L, Flockerzi V, Bindels RJ, Bruford EA, Caterina MJ, Clapham DE, Harteneck C, Heller S, Julius D, Kojima I, Mori Y, Penner R, Prawitt D, Scharenberg AM, Schultz G, Shimizu N, Zhu MX. A unified nomenclature for the superfamily of TRP cation channels. Mol Cell 2002 9:229-231[CrossRef][Medline]
  15. Nilius B, Droogmans G, Wondergem R. Transient receptor potential channels in endothelium: solving the calcium entry puzzle?. Endothelium 2003 10:5-15[CrossRef][Medline]
  16. Yang M, Gupta A, Shlykov SG, Corrigan R, Tsujimoto S, Sanborn BM. Multiple Trp isoforms implicated in capacitative calcium entry are expressed in human pregnant myometrium and myometrial cells. Biol Reprod 2002 67:988-994[Abstract/Free Full Text]
  17. Dalrymple A, Slater DM, Beech D, Poston L, Tribe RM. Molecular identification and localization of Trp homologues, putative calcium channels, in pregnant human uterus. Mol Hum Reprod 2002 8:946-951[Abstract/Free Full Text]
  18. Monga M, Campbell DF, Sanborn BM. Oxytocin-stimulated capacitative calcium entry in human myometrial cells. Am J Obstet Gynecol 1999 181:424-429[CrossRef][Medline]
  19. Shlykov SG, Yang M, Alcorn JL, Sanborn BM. Capacitative cation entry in human myometrial cells and augmentation by hTrpC3 overexpression. Biol Reprod 2003 69:647-655[Abstract/Free Full Text]
  20. Hofmann T, Schaefer M, Schultz G, Gudermann T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci 2002 99:7461-7466[Abstract/Free Full Text]
  21. Trebak M, Vazquez G, Bird GS, Putney JW. The TRPC3/6/7 subfamily of cation channels. Cell Calcium 2003 33:451-461[CrossRef][Medline]
  22. Jung S, Strotmann R, Schultz G, Plant TD. TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells. Am J Physiol Cell Physiol 2002 282:C347-359[Abstract/Free Full Text]
  23. Ku CY, Qian A, Wen Y, Anwer K, Sanborn BM. Oxytocin stimulates myometrial GTPase and phospholipase C activities via coupling to G{alpha}q/11. Endocrinology 1995 136:1509-1515[Abstract]
  24. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
  25. Ou CW, Chen ZQ, Qi S, Lye SJ. Increased expression of the rat myometrial oxytocin receptor messenger ribonucleic acid during labor requires both mechanical and hormonal signals. Biol Reprod 1998 59:1055-1061[Abstract/Free Full Text]
  26. Niiro N, Nishimura J, Hirano K, Nakano H, Kanaide H. Mechanisms of galanin-induced contraction in the rat myometrium. Br J Pharmacol 1998 124:1623-1632[CrossRef][Medline]
  27. Curley M, Cairns MT, Friel AM, McMeel OM, Morrison JJ, Smith TJ. Expression of mRNA transcripts for ATP-sensitive potassium channels in human myometrium. Mol Hum Reprod 2002 8:941-945[Abstract/Free Full Text]
  28. Walker RL, Hume JR, Horowitz B. Differential expression and alternative splicing of TRP channel genes in smooth muscles. Am J Physiol Cell Physiol 2001 280:C1184-C1192[Abstract/Free Full Text]
  29. McDaniel SS, Platoshyn O, Wang J, Yu Y, Sweeney M, Krick S, Rubin LJ, Yuan JX. Capacitative Ca(2+) entry in agonist-induced pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 2001 280:L870-880[Abstract/Free Full Text]
  30. Corteling RL, Li S, Giddings J, Westwick J, Poll C, Hall IP. Expression of TrpC6 and related Trp family members in human airway smooth muscle and lung tissue. Am J Respir Cell Mol Biol 2003 July 18, doi:10.1165/rcmb.2003-0134OC. (e-pub ahead of print)
  31. Zhang L, Saffen D. Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization. J Biol Chem 2001 276:13331-13339[Abstract/Free Full Text]
  32. Schilling WP. TRP proteins: novel therapeutic targets for regional blood pressure control?. Circ Res 2001 88:256-259[Free Full Text]
  33. Philipp S, Trost C, Warnat J, Rautmann J, Himmerkus N, Schroth G, Kretz O, Nastainczyk W, Cavalie A, Hoth M, Flockerzi V. TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells. J Biol Chem 2000 275:23965-23972[Abstract/Free Full Text]
  34. Wang J, Laurier LG, Sims SM, Preiksaitis HG. Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle. Am J Physiol Gastrointest Liver Physiol 2003 284:G1074-1083[Abstract/Free Full Text]
  35. Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weissgerber P, Biel M, Philipp S, Freise D, Droogmans G, Hofmann F, Flockerzi V, Nilius B. Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice. Nat Cell Biol 2001 3:121-127[CrossRef][Medline]
  36. Albert AP, Large WA. Store-operated Ca2+-permeable nonselective cation channels in smooth muscle cells. Cell Calcium 2003 33:345-356[CrossRef][Medline]
  37. Jung S, Muhle A, Schaefer M, Strotmann R, Schultz G, Plant T. Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth. J Biol Chem 2003 278:3562-3571[Abstract/Free Full Text]
  38. Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 1999 397:259-263[CrossRef][Medline]
  39. Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Shimizu S, Ikenaka K, Imoto K, Mori Y. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca2+-permeable cation channel that is constitutively activated and enhanced by stimulation of G-protein-coupled receptor. J Biol Chem 1999 274:27359-27370[Abstract/Free Full Text]
  40. Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, Mori Y. The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel. Circ Res 2001 88:325-332[Abstract/Free Full Text]
  41. Welsh DG, Morielli AD, Nelson MT, Brayden JE. Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res 2002 90:248-250[Abstract/Free Full Text]
  42. Kitazawa T, Maezono Y, Taneike T. The mechanisms of alpha(2)-adrenoceptor agonist-induced contraction in longitudinal muscle of the porcine uterus. Eur J Pharmacol 2000 390:185-195[CrossRef][Medline]
  43. Venkatachalam K, Zheng F, Gill DL. Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 2003 278:29031-29040[Abstract/Free Full Text]
  44. Ascher-Landsberg J, Saunders T, Phillippe M. The role of diacylglycerol as a modulator of oxytocin-stimulated phasic contractions in myometrium from pregnant and nonpregnant rats. Am J Obstet Gynecol 2000 182:943-949[CrossRef][Medline]
  45. Goel M, Sinkins WG, Schilling WP. Selective association of TRPC channel subunits in rat brain synaptosomes. J Biol Chem 2002 277:48303-48310[Abstract/Free Full Text]
  46. Plant TD, Schaefer M. TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels. Cell Calcium 2003 33:441-450[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
H.-T. Ma, Z. Peng, T. Hiragun, S. Iwaki, A. M. Gilfillan, and M. A. Beaven
Canonical Transient Receptor Potential 5 Channel in Conjunction with Orai1 and STIM1 Allows Sr2+ Entry, Optimal Influx of Ca2+, and Degranulation in a Rat Mast Cell Line
J. Immunol., February 15, 2008; 180(4): 2233 - 2239.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
A. Dalrymple, K. Mahn, L. Poston, E. Songu-Mize, and R.M. Tribe
Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells
Mol. Hum. Reprod., March 1, 2007; 13(3): 171 - 179*.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
S. Morales, A. Diez, A. Puyet, P. J. Camello, C. Camello-Almaraz, J. M. Bautista, and M. J. Pozo
Calcium controls smooth muscle TRPC gene transcription via the CaMK/calcineurin-dependent pathways
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C553 - C563.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
C. Y. Ku, L. Babich, R. A. Word, M. Zhong, A. Ulloa, M. Monga, and B. M. Sanborn
Expression of Transient Receptor Channel Proteins in Human Fundal Myometrium in Pregnancy
Reproductive Sciences, April 1, 2006; 13(3): 217 - 225.
[Abstract] [PDF]

Home page
 Reproductive SciencesHome page
B. M. Sanborn, C.-Y. Ku, S. Shlykov, and L. Babich
Molecular Signaling Through G-Protein-Coupled Receptors and the Control of Intracellular Calcium in Myometrium
Reproductive Sciences, October 1, 2005; 12(7): 479 - 487.
[Abstract] [PDF]

Home page
 J. Physiol.Home page
D. J. Beech, K. Muraki, and R. Flemming
Non-selective cationic channels of smooth muscle and the mammalian homologues of Drosophila TRP
J. Physiol., September 15, 2004; 559(3): 685 - 706.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/4/919    most recent
biolreprod.103.023325v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Babich, L. G.
Right arrow Articles by Sanborn, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Babich, L. G.
Right arrow Articles by Sanborn, B. M.
Agricola
Right arrow Articles by Babich, L. G.
Right arrow Articles by Sanborn, B. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS