Skip Navigation


Mol. Hum. Reprod. Advance Access originally published online on January 5, 2007
Molecular Human Reproduction 2007 13(3):171-179*; doi:10.1093/molehr/gal110
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/3/171    most recent
gal110v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dalrymple, A.
Right arrow Articles by Tribe, R.M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dalrymple, A.
Right arrow Articles by Tribe, R.M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells

A. Dalrymple1, K. Mahn2, L. Poston1, E. Songu-Mize3 and R.M. Tribe1,4

1 Maternal and Fetal Research Unit, Division of Reproduction and Endocrinology, King's College London, St Thomas' Hospital Campus, London 2 Division of Allergy, Asthma and Respiratory Science, King's College, Guy's Hospital Campus, London, UK 3 Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center, New Orleans, LA, USA

4 To whom correspondence should be addressed at: Maternal and Fetal Research Unit, Division of Reproduction and Endocrinology, King's College London, St Thomas' Hospital Campus, Lambeth Palace Road, London, SE1 7EH, UK. E-mail: rachel.tribe{at}kcl.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Stretch is known to stimulate myometrial hyperplasia and hypertrophy in early pregnancy and uterine contraction at term. We propose that transduction of the stretch signal involves alteration of intracellular calcium signalling, including changes in transient receptor potential canonical (TRPC) isoform expression. The aim of the present study was to investigate the effect of prolonged mechanical (tonic) stretch in vitro on human myometrial smooth muscle cell calcium signalling and TRPC expression. Cells were cultured from myometrial biopsies, obtained from women undergoing elective Caesarean section at term, grown on FlexiplatesTM and subjected to 25% tonic mechanical stretch for 1, 4 and 14 h. Time-matched control cells were not stretched. Mechanical stretch (14 h) increased basal calcium entry and cyclopiazonic acid (CPA)-induced calcium/Mn2+ entry (P < 0.05) in Fura-2 loaded cells. The calcium selectivity of CPA-thapsigarin induced inward currents, measured by patch clamp electrophysiology, was also increased in stretched cells compared with control cells (P < 0.05). Real time PCR and Western blot data demonstrated that TRPC3 and TRPC4 mRNA and TRPC3 protein expression were increased by stretch (P < 0.05), respectively. These data support the hypothesis that uterine stretch modulates uterine growth and contractility in pregnancy via alterations in calcium signalling.

Key words: cell signalling/ion channels/pregnancy/smooth muscle/uterus


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
To ensure successful reproduction, the uterus has to rapidly adapt in response to the needs of the developing fetus(es). In the early stages of pregnancy, uterine enlargement is mediated by asymmetric uterine smooth muscle cellular hypertrophy and, subsequently, minor hyperplasia (Laguens and Lagrutta, 1964). Conversely, at the end of pregnancy, a period of rapid atrophy must occur for the uterus to return to a non-pregnant state. While a number of these adaptive processes have been examined in the rat, relatively little is known in human pregnancy. In the rat, there appear to be two modulatory influences on uterine growth; hormonal through progesterone (Csapo and Wiest, 1969; Michael and Schofield, 1969, Halme and Woessner 1975;) and mechanical through uterine stretch (Csapo et al., 1965; Cullen and Harkness, 1968; Alexandrova and Soloff, 1980; Douglas et al., 1988). As pregnancy progresses and as the fetus grows, it is likely that the contribution of prolonged (tonic) mechanical stretch becomes increasingly important (Csapo et al., 1965).

Stretch of myometrial tissue, in vivo and in vitro, stimulates expression of a range of labour-associated proteins, such as COX-2 and prostaglandin as well as oxytocin receptors (Manabe et al., 1992; Ou et al., 1997, 1998; Wu et al., 1999; Korita et al., 2002; Loudon et al., 2004; Sooranna et al., 2004; Terzidou et al., 2005). Myometrial stretch is also associated with activation of AP-1 transcription factors, MAP kinase cascades, ERK1/2 phosphorylation and increased expression of c-fos and cytoskeleton-related genes (Mitchell and Lye, 2002; Oldenhof et al., 2002; Shynlova et al., 2002; Mitchell et al., 2004; Sooranna et al, 2004, 2005).

The transduction mechanisms linking myometrial stretch and downstream events have not been fully explored, but in most cell types mechanotransduction is mediated by integrin signalling and stretch-activated calcium entry (Shaw and Xu, 2003; Iqbal and Zaidi, 2005). A rise in intracellular calcium mediated via stretch-activated channels and release from intracellular calcium stores has been described in cardiac myocytes and vascular smooth muscle cells (McCarron et al., 1997; Zou et al., 2002; Calaghan et al., 2003; Liao et al., 2003). Recent reports suggest that proteins of the TRP superfamily form mechanosensitive calcium channels (Corey et al., 2004; Lin and Corey, 2005; Maroto et al., 2005),which could theoretically play a role, as we and others have shown that transient receptor potential canonial (TRPC) proteins, putative components of store-operated calcium entry, are expressed in human myometrial tissue and cells and regulated during pregnancy (Dalrymple et al., 2002, 2004; Yang et al., 2002; Babich et al., 2004). The aim of this study was to examine the effect of prolonged tonic stretch on TRPC channel gene and protein expression and store-operated calcium entry in human myometrial smooth muscle cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects
Human myometrial biopsies were obtained at Caesarean section with informed written consent and institutional Ethics Committee approval (Guy's and St Thomas' Hospital Trusts, London, UK) in accordance with the principles set out in the Declaration of Helsinki. Biopsies were collected from women without underlying disease at term prior to labour (n = 20, 38–41 weeks’ indications: previous section, breech presentation or maternal request) and used for cell culture.

Cell culture and tonic mechanical stretch protocol
Primary uterine myocytes were dispersed enzymatically from tissue biopsies as described previously from myometrial biopsies from women at term (not in labour) (Tribe et al., 2000; Dalrymple et al., 2002). Cells were incubated (37°C, 95% air, 5% CO2) in 25 cm3 culture flasks in Dulbecco's modified Eagle's medium (DMEM) (Sigma, Poole, UK) plus 5% fetal calf serum (FCS) (Invitrogen, Paisley, UK) and maintained in culture until Passage 1. Cells were then detached using trypsin-EDTA (x1, Sigma) for 5 min at room temperature and cultured (passage 2) in six well-flexible bottom culture plates precoated with collagen type I in 3 ml DMEM plus 5% FCS (Flexcell International Corp., PA) until 85–95% confluent. Following replacement of media (10–12 h prior to experiment), cells were subjected to 25% tonic mechanical stretch for 1, 4 or 14 h using a strain unit (Flexcell) housed in the cell culture incubator. Time matched control cells (0, 1, 4 and 14 h) were grown on the same flexible-bottomed culture plates, but were not stretched. Cells were then washed with phosphate buffered saline (Sigma). For imaging and patch clamp protocols, cells exposed to 14 h stretch (and time matched controls) were detached using trypsin-EDTA, pelleted by centrifugation (450g, 5 min) and resuspended in 1 ml of DMEM plus 5% FCS. The cell solution (100 µl) was placed on glass cover-slips and cells allowed time (1 h) to adhere. Whole cell lysates (taken at t = 14 h) for Western blot analysis were obtained by adding 100 µl of homogenization buffer [10 mmol/l Hepes-KOH, pH 7, 1 mmol/l DTT, 1% (v/v) nonidet-P40 (Sigma) and protease inhibitor cocktail (COMPLETETM tablets, Boehringer Mannheim Biochemical Lewes, UK)] to each well. For RNA studies, total RNA (tRNA) was extracted from stretched and control cells (at t = 1, 4 and 14 h) and purified using Trizol (Invitrogen, Paisley, UK, as recommended by the manufacturer).

Digital Ca2+ imaging
Cells exposed to mechanical stretch for 14 h and paired non-stretched time controls were loaded with the fluorescent Ca2+ indicator Fura-2-AM (1 µM, 1 h RT). This time point was chosen to address the functional impact of a period of prolonged tonic stretch (to mimic in vivo events at the end of pregnancy) on intracellular calcium responses. Cells were perfused (15 min) with physiological salt solution (PSS) (140 mM NaCl, 5.9 mM KCl, 1.2 mM NaH2PO4, 5 mM NaHCO3, 1.4 mM MgCl2, 1.8 mM CaCl2, 11.5 mM glucose, and 10 mM Hepes titrated to pH 7.4 with NaOH). Following excitation at 360 and 380 nm, emission was measured at 510 nm. Human myometrial cells were imaged every 4–6 s using a digital camera (Pentamax CCD, Princeton Instruments, Trenton, NJ, USA) controlled by Universal Imaging Metafluor computer software (Downington, PA, USA) and the signal normalized for background fluorescence. Images were captured and background corrected 360/380 ratios calculated for all cells within the field of view. Store-operated calcium entry was examined by initiating Ca2+ release from intracellular stores using cyclopiazonic acid (CPA) (5 µM) in the presence or absence of extracellular Ca2+. The peak change in Fura-2 360/380 nm ratio (F360/380) from baseline is reported. Store-operated calcium entry was also examined by determining the rate of Ca2+ re-entry ({Delta}F360/380/s; assessed using linear regression) on readdition of Ca2+ to Ca2+ free PSS containing CPA. The protocol was then repeated in the presence of the L-type Ca2+ channel inhibitor, diltiazem (20 µM). Experiments were also performed to determine the La3+-sensitivity (50 µM) and Gd3+-sensitivity (10 µM) of basal and store-operated calcium entry in stretch and control cells. Store-operated calcium entry was further investigated by the application of extracellular Mn2+ (200 µM, MnCl2 in nominally Ca2+ free PSS, no EGTA), a surrogate ion for Ca2+ that is not extruded by the plasma membrane calcium ATPase or by Na/Ca exchange. The initial rate of Mn2+ influx [quench of Fura-2 360 nm fluorescence, {Delta}F360/s] was measured. Modified PSS (sodium phosphate and sodium bicarbonate removed) was used for La3+, Gd3+ and Mn2+ experiments.

Whole-cell current recording
Recordings were made using the tight-seal whole-cell configuration of the patch clamp technique (Hamill et al., 1981). Experiments were performed in single cells, dispersed on a glass coverslip, using the standard patch-clamp technique with Axopatch 200A amplifier and pCLAMP-6 software (Axon Instruments, Union City, CA, USA). The bath solution was either a standard NaCl-PSS (130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, 10 mM glucose) or a N-methyl-D-glucamine-PSS (0 Na) and contained TEA and 4-AP, the pipette solution was: 135 mM CsCl, 2.5 mM MgCl2, 10 mM EGTA, 10 mM HEPES, 5 mM NaATP. Neither K+ nor L-type Ca2+channels were active under these conditions. The membrane potential was clamped at –70 mV and a combination of CPA (10 µM) and thapsigargin (100 nM) used to activate store-operated currents that were continuously recorded.

Quantitative real-time PCR
After quantification, 1.0 µg of tRNA was reverse transcribed with oligo dT random primers using MuLV reverse transcriptase (Promega, Southampton, UK). Validated primer and probe sets for TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7 and 18s rRNA were obtained from Applied Biosystems Ltd, Warrington, UK. Quantitative PCR was performed in the presence of either VIC (for 18s) or FAM (for TRPCs) (Applied Biosystems Ltd) and amplicon yield monitored during cycling in an ABI Prism® 7000 Sequence Detection System (Applied Biosystems Ltd). Pre-PCR cycle was 2 min at 50°C and 10 min at 95°C for 1 cycle followed by 40 cycles of 95°C for 15 s and annealing/final extension at 60°C for 1 min. The cycle at which the fluorescence reached a preset threshold (cycle threshold) was used for quantitative analyses. The cycle threshold (CT) in each assay was set so that the exponential increase in amplicon abundance was approximately parallel between all samples. All mRNA abundance data were expressed relative to the amount of the constitutively expressed 18s rRNA using the 2–{Delta}{Delta}CT method of analysis (Livak and Schmittgen, 2001) and, subsequently, normalized to control samples taken at t = 0 h (assigned a value of one).

Western blot analysis
Proteins were separated from cell debris by centrifugation (13 000g, 10 min, 4°C) and the protein concentration determined using BSA as a standard and the DC protein assay kit (Bio-Rad Laboratories Ltd, Herts, UK). Control and stretched HMSM cellular proteins (10 µg) were denatured at 95°C for 10 min in Laemmli sample buffer (Sigma), loaded onto Novex® 10% Tris–glycine gels (Invitrogen) and subsequently transferred to polyvinylidene difluoride membrane (Amersham Pharmacia Biotech Ltd, Buckinghamshire, UK). The transfer efficiency and equal loading of protein samples was assessed by incubating membranes with Ponceau red solution (Sigma) and then incubated (overnight, 4°C) in blocking buffer (PBS-T, 5% goat serum, Chemicon, Harrow, UK). Membranes were incubated (3 h, room temperature) with TRPC1 (Xu and Beech, 2001; 1:1000), TRPC3, TRPC4 or TRPC6 antibodies (Alomone Labs, Jerusalem, Israel, 1:200) which were diluted in blocking buffer and subsequently incubated (1 h, room temperature) with a horseradish peroxidase conjugated goat anti-rabbit secondary antibody (Bio-Rad, diluted 1:2500 in blocking buffer). Protein bands were visualized with ECL solution on hyperfilm (Amersham Pharmacia Biotech Ltd). For the negative controls, membranes were incubated with a 1:1000 dilution of rabbit pre-immune serum (TRPC1) or incubated with primary antibodies that were pre-absorbed (1 h, room temperature) with the respective peptides (TRPC3, TRPC4 or TRPC6, Alomone Labs, as recommended by the supplier). TRPC5 and TRPC7 protein expression was not investigated.

Statistical analysis
Data are presented as mean ± SEM; n refers to the total number of cell cultures (each originating from a biopsy from a different subject) except for calcium imaging and patch clamp data when n refers to number of cells (cells were obtained from myometrial biopsies originating from at least three subjects). Data from calcium imaging and patch clamp studies were analysed in Microsoft® Excel 97 (Bellvue, WA, USA) and two-way ANOVA or Student's t-test as appropriate. Western blot hyperfilms were analysed using Total Lab software (Nonlinear Dynamics, Newcastle upon Tyne, UK) which allows the calculation of the density of each band and corresponding background values. Final intensity values (arbitrary units, a.u.) were background subtracted. Western blot data were analysed using Student's t-test. Real time PCR data were analysed using the Box–Cox method, and normal distribution plots were used to confirm that data transformations were not needed. Analysis was by repeated measures ANOVA of treatment by myometrial sample and regression with robust standard errors (Huber, 1967). Planned comparisons gave estimates of the ratios between stretch and control samples for each time point. Smaller P-values in the range 0.05–0.001 are taken as increasing evidence against the null hypothesis, but the totality of evidence is regarded as more important than any single test (Sterne and Davey-Smith, 2001). Marginally significant results (0.05 > P > 0.01) are mentioned, but not discussed in detail. Accordingly, no formal adjustment is made for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Basal calcium entry is enhanced in stretched cells
Stretch did not alter the resting calcium baseline in the presence of calcium (Figure 1A, F360/380, 0.965 ± 0.010, n = 84 cells exposed to 14 h stretch versus F360/380, 0.962 ± 0.015, n = 96 control cells, not significant (NS). However, in the absence of extracellular calcium (Ca2+-free PSS), the resting baseline in stretched cells was significantly reduced to 0.921 ± 0.010 (F360/380, n = 84, P ≤ 0.001) in stretched cells, but not controls (F360/380, 0.936 ± 0.015, n = 96 cells, NS).


Figure 1
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Calcium entry, following a period of prolonged calcium store depletion, is enhanced in tonically (14 h) stretched human myometrial cells. (A) Representative trace of resting calcium responses and cyclopiazonic (CPA) (10 µM) induced Ca2+ mobilization in control and stretched cells in physiological salt solution (PSS); arrow indicates initiation of Ca2+ re-entry following a period of prolonged store depletion in Ca2+ free PSS containing CPA. (B) Mean ± SEM data demonstrating an increase in the calcium re-entry slope in stretched cells (n = 147) versus control cells (n = 151), ***P = 0.0002.

 
CPA-induced calcium responses
CPA-induced peak calcium responses in PSS were unaltered by 14 h stretch (change from baseline F360/380, 0.599 ± 0.055, n = 95 cells) versus controls (0.633 ± 0.079, n = 114 cells, NS, (Figures 1A and 2). In Ca2+-free PSS, the mean CPA-evoked Ca2+ peak was of similar magnitude in both stretch (change from Ca2+-free PSS baseline F360/380, 0.577 ± 0.059, n = 84 cells) versus controls (0.535 ± 0.077, n = 96 cells, NS) (Figure 2). The re-addition of Ca2+ in the presence of CPA elicited a rapid rise in cytoplasmic Ca2+ in control and stretched cells (Figure 1). The initial rate of Ca2+ entry in stretched cells ({Delta}F360/380/s, 0.0075 ± 0.0006, n = 147 cells) was significantly greater than controls (0.0049 ± 0.0003, n = 151, P ≤ 0.0002) (Figure 1B).


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. In vitro tonic 14 h stretch does not alter CPA-induced Ca2+ transient peaks in cultured human myometrial smooth muscle cells. Mean ± SEM data show that CPA-induced Ca2+ peaks (change from baseline F360/380) are of similar magnitude in control and stretched cells when measured in PSS (not significant, NS; n = 96–114 cells) and Ca2+-free PSS (NS, n = 84–96 cells).

 
The CPA-induced Ca2+ peak observed in stretched and control cells was not affected by diltiazem (stretched cells, 0.602 ± 0.066, n = 41 cells; control cells, 0.722 ± 0.099, n = 46 cells, NS). The difference in the initial rate of Ca2+ re-entry between stretched and controls cells remained in the presence of diltiazem (P ≤ 0.024).

Basal and CPA-induced Mn2+ influx in control and stretched cells
The basal rate of Mn2+ entry was significantly greater in stretched cells ({Delta}F360/380/s, –0.154 ± 0.013, n = 53 cells) compared with controls (–0.122 ± 0.005, n = 56 cells, P ≤ 0.022) (Figure 3A and C). CPA-stimulated Mn2+ entry in Ca2+-free PSS was also greater in stretched cells ({Delta}F360/380/s, –0.218 ± 0.017, n = 60 cells) compared with controls (–0.147 ± 0.008, n = 76 cells, P ≤ 0.0001) (Figure 3B and C). CPA-stimulated Mn2+ entry was also significantly greater than basal entry in stretched (#, P ≤ 0.005) and control cells ({dagger}, P ≤ 0.02, Figure 3C).


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Basal and CPA-induced Mn2+ influx is enhanced in tonically (14 h) stretched human myometrial cells. Representative plots to show (A) Basal Mn2+ quench of Fura-2 fluorescence at 360 nM ({Delta}F360) and (B) CPA-induced Mn2+ entry in control and stretched cells. (C) Mean ± SEM data for basal Mn2+ entry in stretched cells (n = 53 cells) versus controls (n = 56 cells; *P < 0.02) and CPA-induced Mn2+ entry in stretched (n = 60 cells) versus controls (n = 76 cells; ***P < 0.00011). CPA (5 µM) induced Mn2+ entry was greater than basal entry in both stretched (#, P < 0.005) and control ({dagger}, P < 0.020) groups.

 
The effect of Ca2+-free PSS,La3+, Gd3+ to CPA-induced Ca2+ entry in control and stretched cells
The effect of calcium removal on the CPA-induced plateau was compared with the addition of La3+ and Gd3+ in stretched and control cells (Figure 4). There was no significant reduction in the CPA-induced plateau in the presence of extracellular calcium (Figure 4A) over 3.5 min in either stretched or control cells (% reduction F360/380 in CPA-PSS plateau, 6.08 ± 0.39, n = 117 stretched cells versus 6.25 ± 0.89, n = 102 control cells, NS) (Figure 4E). However, Ca2+ removal significantly reduced the CPA-induced plateau in stretched cells (percentage reduction F360/380 from CPA plateau, 16.10 ± 0.65, n = 71 cells) versus controls (12.21 ± 0.94, n = 72 cells, P < 0.001) (Figure 4B and 4E). La3+ addition had a minimal affect on the CPA-induced PSS plateau (Figure 4C), and there was no significant difference between the stretched (2.56 ± 0.66, n = 43 cells) and control cells (1.04 ± 0.47, n = 43 cells, NS) (Figure 4E). Gd3+ also had a minimal effect on the CPA-induced PSS plateau (Figure 4D), but in stretched cells Gd3+ evoked a small but significant reduction in the plateau compared with controls (7.64 ± 0.34, n = 83 cells) (5.75 ± 0.36, n = 96 cells, P < 0.0002) (Figure 4E).


Figure 4
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. CPA-induced Ca2+ plateau is dependent on calcium entry, a pathway that is enhanced in tonically stretched cells. Representative plots to show (A) the CPA (5 µM) plateau in the presence of PSS and the effect of (B) extracellular Ca2+ removal, (C) addition of La3+(100 µM) and (D) addition of Gd3+ (10 µM) on the CPA-induced Ca2+ plateau. (E) Mean ± SEM data to show the % reduction in the CPA induced Ca2+ plateau following extracellular Ca2+ removal and addition of La3+(50 µM) and Gd3+(10 µM) (n = 43–117 cells).

 
Comparison of CPA and thapsigargin induced currents in stretched and control human myometrial cells
Application of CPA (10 µM) and thapsigargin (100 nM) induced an inward rectifying current in control (n = 5) and stretched cells (n = 4) (Figure 5A). The currents were dependent on extracellular Na+ and extracellular Ca2+. The Na+ component of the current was greater in control cells than stretched cells as removal of extracellular Na+ reduced the current amplitude by 68.7 ± 2.3% in controls versus 29.4 ± 7.5% in stretched cells (P = 0.0017, Figure 5B). The residual current, which was carried by calcium, was relatively insensitive to lanthanum in stretched cells (61.7 ± 9.9% inhibition) compared with controls 95.9 ± 3.4 (P = 0.0109, Figure 5B).


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Current clamp recordings in human myometrial smooth muscle cells. (A) Representative trace of CPA (10 µM) & thapsigargin (Thg) (100 nM) induced inward rectifying current in cultured control and stretched (14 h) cells in the presence and absence of Na+ and La3+. Holding potential was –70 mV. (B) Mean ± SEM of CPA and Thg-induced inward rectifying current in cultured control (n = 5) and stretched (n = 4) cells in PSS and in the absence of extracellular Na+ as well as the presence of La3+. *P < 0.05 compared with PSS within experimental group; #P < 0.05 stretched cells versus control cells for current measured in 0 Na+ or presence of La3+.

 
TRPC mRNA expression in control and stretched HMSM cells
TRPC5 or 7 mRNA expression was not detected in either control or stretched HMSM cells (n = 8, data not shown). TRPC1, 3, 4 and 6 mRNA were expressed in both control and stretched HMSM cells (n = 8) (Figure 6). TRPC1 mRNA expression was transiently increased after 1 h in stretched cells versus the paired time control (P = 0.03, Figure 6A) and TRPC6 mRNA expression was slightly reduced at 14 h (P = 0.01, Figure 6D) compared with the 14 h control. There was a significant up-regulation of TRPC3 mRNA expression in stretched cells 1 h (P ≤ 0.0001) and 4 h (P ≤ 0.0001) versus control cells, which returned to basal levels by 14 h (Figure 6B). TRPC4 mRNA expression was enhanced at 1 h (P = 0.01), 4 h (P ≤ 0.001) and 14 h (P ≤ 0.001) in stretched versus controls cells (Figure 6C).


Figure 6
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. In vitro stretch modulates TRPC mRNA expression in cultured human myometrial cells. Graphs depicting mean ± SEM real time PCR mRNA expression data for (A) TRPC1, (B) TRPC3, (C) TRPC4 and (D) TRPC6 in control cells and cells that were stretched for 1, 4 or 14 h. All target TRPC isofom mRNA abundance data was normalized to the housekeeping gene 18s rRNA using the 2–{Delta}{Delta}CT method. Subsequently, changes in 2–{Delta}{Delta}CT values for each TRPC isoform were expressed relative to controls (taken at t = 0; assigned a value of 1). Statistical comparisons were made between stretched (1, 4 and 14 h) and non-stretched control cells (1 4 and 14 h).

 
TRPC protein expression in control and stretched human myometrial cells
TRPC1 (90 kDa), TRPC3 (90 kDa), TRPC4 (100 kDa) and TRPC6 (100 kDa) proteins were detected by Western blot analysis in control and stretched cells (Figure 7). No bands were observed when the primary antibody was omitted or when primary antibodies were pre-absorbed with the appropriate peptide (data not shown). TRPC1 (control, 9104 ± 759 a.u. versus stretch 11890 ± 1296 a.u., NS, Figure 7A), TRPC4 (control, 5082 ± 1656 a.u. versus stretch, 7971 ± 1757 a.u., NS, Figure 7C) and TRPC6 (control, 20361 ± 1331 versus stretch, 17631 ± 673, NS, Figure 7D) proteins were expressed at a similar level in control and stretched cells. TRPC3 protein expression was significantly increased following 14 h tonic mechanical strain (control, 4149 ± 1314 versus stretch 11692 ± 2241, P ≤ 0.05, Figure 7B).


Figure 7
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7. In vitro stretch enhances TRPC3 protein expression in cultured human myometrial cells. Representative western blots and graphs depicting mean ± SEM (n = 7) of western blot densitometry of (A) TRPC1 (90 Mr x 10–3), (B) TRPC3 (90 Mr x 10–3), (C) TRPC4 (100 Mr x 10–3) and TRPC6 (100 Mr x 10–3) protein expression in control and stretched cells analysis for (A) TRPC1, (B) TRPC3, (C) TRPC4 and (D) TRPC6 protein expression. *P < 0.05; NS, P > 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Mechanical stretch is hypothesized to be an important regulator of uterine growth and contractility during pregnancy. Several studies report stretch-induced regulation of contraction-associated genes and proteins in human myometrial smooth muscle (Manabe et al., 1992; Ou et al., 1997, 1998; Wu et al., 1999; Korita et al., 2002; Loudon et al., 2004; Sooranna et al., 2004; Terzidou et al., 2005), but functional correlates have not been fully explored. This study has determined the effect of prolonged tonic mechanical strain on intracellular calcium responses and calcium currents in parallel with expression of TRPC isoforms. Tonic mechanical stretch increased basal and store-operated calcium entry in association with an increase in TRPC3 and 4 gene expression. This effect was translated into an increase in TRPC3 protein expression.

Acute stretch of vascular smooth muscle and cardiac muscle in vitro increases intracellular calcium levels in muscle (McCarron et al., 1997; Calaghan et al., 2003; Zou et al., 2002; Liao et al., 2003). Uterine distension, whether in the early or late stages of pregnancy, is more likely to be a tonic stretch, but the influence of prolonged stretch on smooth muscle calcium dynamics is unclear. In our in vitro model, 14 h of tonic stretch induced a rise in basal calcium as evidenced by a drop in resting intracellular calcium on removal of extracellular calcium and stretch-enhanced Mn2+ entry. Despite increased basal calcium entry in stretched cells, resting calcium was unaltered in stretched cells compared with controls. This implies that stretch may also facilitate Ca2+ sequestration and/or removal. The amplitude of CPA-induced calcium transients (in the presence and absence of extracellular calcium) were similar in control and stretched cells which suggests that the CPA-releasable SR calcium store is unaffected by stretch. However, if calcium removal mechanisms are co-regulated by stretch, then it is possible that a stretch-induced change in the ‘peak CPA’ response is masked.

In contrast, our data clearly support a role for tonic in vitro mechanical stretch in mediating enhanced store-operated calcium entry in cultured human myometrial cells. This to our knowledge has not been reported in other smooth muscle. The plateau phase of the CPA response in both control and stretched cells is dependent on a contribution from store-operated calcium entry. In stretched cells, the reduction in the plateau following calcium removal and the rate of CPA-induced Mn2+ entry were more rapid and under conditions of prolonged store depletion, the re-addition of Ca2+ also elicited a more rapid rise in intracellular calcium in the stretched cells. A contribution of L-type calcium channels to the calcium response is unlikely, as the addition of diltiazem had no effect. Patch clamp data also demonstrated that in vitro mechanical stretch modulated the characteristics of the inwardly rectifying current activated by sarcoplasmic store depletion. In stretched cells, the current was less dependent on extracellular sodium and more dependent on Ca2+, compared with controls. Overall, these data indicate that store-operated calcium entry is enhanced by tonic stretch and imply that Ca2+ removal/sequestration mechanisms may increase in parallel.

Further characterization of CPA-induced calcium response showed that the plateau phase was insensitive to La3+ and only minimally inhibited by Gd3+ at concentrations known to inhibit store-operated calcium entry (Shlykov et al., 2003; Bradley et al., 2005). The current activated in stretched cells by store depletion (in the absence of extracellular sodium) was also relatively insensitive to La3+. These results differ from our data in primary human myometrial cells (Tribe et al., 2003) in which the CPA-induced calcium response was La3+ sensitive. It is possible, that the baseline molecular characteristics of store-operated calcium channels in the cultured cells used in the present study were altered due to different culture conditions (Golovina et al., 2001).

The stretch-induced effects on TRPC isoform protein expression were varied. TRPC1, which is reported to form a mechanosensitive channel that responds to acute stretch (Maroto et al., 2005), was unaffected in the present study by chronic stretch, despite a transient rise in TRPC1 mRNA at 1 h. TRPC6 protein and mRNA expression was unchanged by in vitro mechanical stretch, except for a slight down-regulation in mRNA expression at 4 h. In contrast, our data clearly show a significant increase in TRPC3 and TRPC4 mRNA expression following 1 and 4 h of tonic stretch. This was translated into a significant increase in TRPC3 protein expression at 14 h and a non-significant increase in TRPC4 protein expression. The discrepancy between changes in TRPC4 mRNA compared with protein expression may indicate that TRPC4 expression is regulated post-transcriptionally; indeed we have previously reported a similar lack of correlation between TRPC4 mRNA and protein expression in human myometrial tissue (Dalrymple et al., 2004). Alternatively, the non-significant change in TRPC4 protein expression may reflect the inability of western blot to accurately resolve small changes in protein expression. Interestingly, TRPC4 mRNA expression, unlike TRPC3, remained elevated at the 14 h time point which could suggest that a significant rise in TRPC4 protein might have been detected at a later time point; this observation requires further examination. The impact of TRPC3 and 4 gene silencing on stretch-induced changes in calcium signalling could also provide further insight into the functional role of these TRPC isoforms.

In previous studies, we reported that a similar increase in TRPC3 protein expression was associated with enhanced basal entry and store-operated calcium entry in interleukin (IL)-1ß treated HMSM cells (Tribe et al., 2003; Dalrymple et al., 2004). However, there are marked differences in calcium responses activated by the two stimuli. IL-1ß treated cells exhibited spontaneous generation of calcium transients and the enhanced store-operated calcium entry was La3+ sensitive. There was also no indication that IL-1ß altered TRPC4 gene expression. This may be explained by (i) differential effects of stretch on TRPC4 isoform expression and (ii) divergence in the activation of other signalling cascades during the period of exposure to stretch or IL-1ß (Sooranna et al., 2005). The present study may also reflect differences between primary human myometrial cells and those maintained in longer-term culture. Interestingly, application of stretch or IL-1ß results in alterations in TRPC isoforms which are up-regulated in human myometrial tissue taken from women in labour (Dalrymple et al., 2004), providing additional support for a contribution of TRPC3/4 to myometrial function.

Overall, our data suggests that tonic stretch results in co-regulation of calcium entry pathways and TRPC3 and TRPC4 expression in cultured human myometrial cells. Since an up-regulation of calcium entry may lead to increased contractility of smooth muscle, this provides a possible pathway by which uterine function can be altered in response to a growing fetus during pregnancy and at the end of gestation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank all the women who kindly participated in this study, Julie Adams and Guy's and St Thomas' Hospital Trusts labor ward staff for myometrial sample collection. We are indebted to Jeanette Judah for technical assistance in preparing cells, Professor David Beech (University of Leeds) for kindly supplying the TIE3 antibody, Mr Paul Seed for statistical assistance, and Drs Neil Levinson and Jason Tennant for the real time PCR data. This study was funded by Tommy's, the baby charity (Reg. Charity No: 1060508).


   Footnotes
 
* N.B. An error was made in the initial online pagination of Molecular Human Reproduction 13/3. The page span of this article was originally shown as 31–39. The publisher wishes to apologise for this error.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Alexondrova M and Soloff MS. (1980) Oxytocin receptors and parturition II: Concentrations of receptors for oxytocin and estrogen in the gravid and non gravid uterus at term. Endocrinol 106:736–738.[Abstract/Free Full Text]

Babich LG, Ku CY, Young HW, Huang H, Blackburn MR, Sanborn BM. (2004) Expression of capacitative calcium TrpC proteins in rat myometrium during pregnancy. Biol Reprod 70:919–924.[Abstract/Free Full Text]

Bradley E, Hollywood MA, McHale NG, Thornbury KD, Sergeant GP. (2005) Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry. Am J Physiol Cell Physiol 289:C625–C632.[Abstract/Free Full Text]

Calaghan SC, Belus A, White E. (2003) Do stretch-induced changes in intracellular calcium modify the electrical activity of cardiac muscle? Prog Biophys Mol Biol 82:81–95.[CrossRef][Web of Science][Medline]

Corey DP, Garcia-Anoveros J, Holt JR, Kwan KY, Lin SY, Vollrath MA, Amalfitano A, Cheung EL, Derfler BH, Duggan A, et al. (2004) TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells. Nature 432:723–730.[CrossRef][Medline]

Csapo AI and Wiest WG. (1969) An examination of the quantitative relationship between progesterone and the maintenance of pregnancy. Endocrinol 85:735–746.[Abstract/Free Full Text]

Csapo A, Erdos T, De Mattos CR, Gramss E, Moscowitz C. (1965) Stretch-induced uterine growth, protein synthesis and function. Nature 207:1378–1379.[CrossRef][Medline]

Cullen BM and Harkness RD. (1968) Collagen formation and changes in cell population in the rat's uterus after distension with wax. Q J Exp Physiol Cogn Med Sci 53:33–42.[Abstract/Free Full Text]

Dalrymple A, Slater DM, Beech D, Poston L, Tribe RM. (2002) Molecular identification and localization of Trp homologues, putative calcium channels, in pregnant human uterus. Mol Hum Reprod 8:946–951.[Abstract/Free Full Text]

Dalrymple A, Slater DM, Poston L, Tribe RM. (2004) Physiological induction of transient receptor potential canonical proteins, calcium entry channels, in human myometrium: influence of pregnancy, labor, and interleukin-1 beta. J Clin Endocrinol Metab 89:1291–1300.[Abstract/Free Full Text]

Douglas AJ, Clarke EW, Goldspink DF. (1988) Influence of mechanical stretch on growth and protein turnover of rat uterus. Am J Physiol 254:E543–E548.[Web of Science][Medline]

Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, Yuan JX. (2001) Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 280:H746–H755.[Abstract/Free Full Text]

Halme J and Woessner JF Jr. (1975) Effect of progesterone on collagen breakdown and tissue collagenolytic activity in the involuting rat uterus. J Endocrinol 66:357–362.[Abstract/Free Full Text]

Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. (1981) Improved patch-clamp techniques for high resolution current recording from cells and cell free membrane patches. Pflugers Arch 391:85–100.[CrossRef][Web of Science][Medline]

Huber PJ. (1967) In LeCam LM and Neyman J (Eds.). The behavior of maximum likelihood estimates under nonstandard conditions. Proceedings of the Fifth Annual Berkeley Symposium on Mathematical Statistics and ProbabilityUniversity of California Press pp. 221–233.

Iqbal J and Zaidi M. (2005) Molecular regulation of mechanotransduction. Biochem Biophys Res Commun 328:751–755.[CrossRef][Web of Science][Medline]

Korita D, Sagawa N, Itoh H, Yura S, Yoshida M, Kakui K, Takemura M, Yokoyama C, Tanabe T, Fujii S. (2002) Cyclic mechanical stretch augments prostacyclin production in cultured human uterine myometrial cells from pregnant women: possible involvement of up-regulation of prostacyclin synthase expression. J Clin Endocrinol Metab 87:5209–5219.[Abstract/Free Full Text]

Laguens R and Lagrutta J. (1964) Fine structure of human uterine muscle in pregnancy. Am J Obstet Gynecol 89:1040–1048.[Web of Science][Medline]

Liao XD, Tang AH, Chen Q, Jin HJ, Wu CH, Chen LY, Wang SQ. (2003) Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells. Biochem Biophys Res Commun 310:405–411.[CrossRef][Web of Science][Medline]

Lin SY and Corey DP. (2005) TRP channels in mechanosensation. Curr Opin Neurobiol 15:350–357.[CrossRef][Web of Science][Medline]

Livak KJ and Schmittgen TD. (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 25:402–408.[CrossRef][Web of Science][Medline]

Loudon JA, Sooranna SR, Bennett PR, Johnson MR. (2004) Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis. Mol Hum Reprod 10:895–899.[Abstract/Free Full Text]

Manabe Y, Sagawa N, Mori T. (1992) Fetal viability does not affect the onset of stretch-induced labor and the increase in amniotic fluid prostaglandin F2 alpha and plasma prostaglandin F2 alpha metabolite levels. Prostaglandins 44:119–128.[CrossRef][Web of Science][Medline]

Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP. (2005) TrpC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol 7:105–107.[CrossRef][Web of Science][Medline]

McCarron JG, Crichton CA, Langton PD, MacKenzie A, Smith GL. (1997) Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries. J Physiol 498:371–379.[Abstract/Free Full Text]

Michael CA and Schofield BM. (1969) The influence of the ovarian hormones on the actomyosin content and the development of tension in uterine muscle. J Endocrinol 44:501–511.[Web of Science][Medline]

Mitchell JA and Lye SJ. (2002) Differential expression of activator protein-1 transcription factors in pregnant rat myometrium. Biol Reprod 67:240–246.[Abstract/Free Full Text]

Mitchell JA, Shynlova O, Langille BL, Lye SJ. (2004) Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells. Am J Physiol Endocrinol Metab 287:E439–E445.[Abstract/Free Full Text]

Oldenhof AD, Shynlova OP, Liu M, Langille BL, Lye SJ. (2002) Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells. Am J Physiol Cell Physiol 283:C1530–C1539.[Abstract/Free Full Text]

Ou CW, Orsino A, Lye SJ. (1997) Expression of connexin-43 and connexin-26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and horomonal signals. Endocrinol 138:5398–5407.[Abstract/Free Full Text]

Ou CW, Chen ZQ, Qi S, Lye SJ. (1998) Increased expression of the rat myometrial oxytocin receptor messenger ribonucleic acid during labor requires both mechanical and hormonal signals. Biol Reprod 59:1055–1061.[Abstract/Free Full Text]

Shaw A and Xu Q. (2003) Biomechanical stress-induced signaling in smooth muscle cells: an update. Curr Vasc Pharmacl 1:41–58.[CrossRef]

Shlykov SG, Yang M, Alcorn JL, Sanborn BM. (2003) Capacitative cation entry in human myometrial cells and augmentation by hTrpC3 overexpression. Biol Reprod 69:647–655.[Abstract/Free Full Text]

Shynlova OP, Oldenhof AD, Mingyao L, Lowell Langille L, Lye SJ. (2002) Regulation of c-fos expression by static stretch in rat myometrial smooth muscle cells. Am J Obstet Gynecol 186:1358–1365.[CrossRef][Web of Science][Medline]

Sooranna SR, Lee Y, Kim LU, Mohan AR, Bennett PR, Johnson MR. (2004) Mechanical stretch activates type 2 cyclooxygenase via activator protein-1 transcription factor in human myometrial cells. Mol Hum Reprod 10:109–113.[Abstract/Free Full Text]

Sooranna SR, Engineer N, Loudon JA, Terzidou V, Bennett PR, Johnson MR. (2005) The mitogen-activated portein kinase dependent expression of prostaglandin H synthase-2 and interleukin messenger ribonucleic acid by myometrial cells: the differential effect of stretch and interleukin-1ß. J Clin Endocrinol Metab 90:517–527.

Sterne JA and Davey Smith G. (2001) Sifting the evidence – what's wrong with significance tests? Br Med J 322:226–231.[Free Full Text]

Terzidou V, Sooranna SR, Kim LU, Thornton S, Bennett PR, Johnson MR. (2005) Mechanical stretch up-regulates the human oxytocin receptor in primary human uterine myocytes. J Clin Endocrinol Metab 90:237–246.[Abstract/Free Full Text]

Tribe RM, Moriarty P, Poston L. (2000) Calcium homeostatic pathways change with gestation in human myometrium. Biol Reprod 63:748–755.[Abstract/Free Full Text]

Tribe RM, Moriarty P, Dalrymple A, Hassoni AA, Poston L. (2003) Interleukin-1ß induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle. Biol Reprod 68:1842–1849.[Abstract/Free Full Text]

Wu WX, Ma XH, Yoshizato T, Shinozuka N, Nathanielsz PW. (1999) Differential expression of myometrial oxytocin receptor and prostaglandin H synthase 2, but not estrogen receptor alpha and heat shock protein 90 messenger ribonucleic acid in the gravid horn and nongravid horn in sheep during betamethasone-induced labor. Endocrinol 140:5712–5718.[Abstract/Free Full Text]

Xu SZ and Beech DJ. (2001) TrpC1 is a membrane-spanning subunit of store-operated Ca(2+) channels in native vascular smooth muscle cells. Circ Res 88:84–87.[Abstract/Free Full Text]

Yang M, Gupta A, Shlykov SG, Corrigan R, Tsujimoto S, Sanborn BM. (2002) Multiple Trp isoforms implicated in capacitative calcium entry are expressed in human pregnant myometrium and myometrial cells. Biol Reprod 67:988–994.[Abstract/Free Full Text]

Zou H, Lifshitz LM, Tuft RA, Fogarty KE, Singer JJ. (2002) Visualization of Ca2+ entry through single stretch-activated cation channels. Proc Natl Acad Sci USA 99:6404–6409.[Abstract/Free Full Text]

Submitted on August 25, 2006; resubmitted on November 13, 2006; accepted on November 14, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Vet Rec.Home page
A. P. McNaughton and R. D. Murray
Structure and function of the bovine fetomaternal unit in relation to the causes of retained fetal membranes
Vet Rec., November 21, 2009; 165(21): 615 - 622.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
J. Abramowitz and L. Birnbaumer
Physiology and pathophysiology of canonical transient receptor potential channels
FASEB J, February 1, 2009; 23(2): 297 - 328.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/3/171    most recent
gal110v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dalrymple, A.
Right arrow Articles by Tribe, R.M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dalrymple, A.
Right arrow Articles by Tribe, R.M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?