| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR |
1 Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK
2 Physiology Department, Crown Street, University of Liverpool, Liverpool L69 3 BX, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
-cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca2+ signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19–21 rats showed that outward K+ current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol-saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K+ channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca2+-sensitive K+ channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca2+ level ([Ca2+]i transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca2+ signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol-rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca2+ signalling and uterine contractility.
(Received 30 January 2007;
accepted after revision 28 February 2007;
first published online 1 March 2007)
Corresponding author S. Wray: Physiology Department, University of Liverpool, Crown Street, Liverpool L69 3BX, UK. Email s.wray{at}liv.ac.uk
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
An additional level of complexity in the regulation of ion channels has emerged in recent years. A growing number of reports suggest that some ion channels and some signal transduction molecules are segregated within the surface membrane into lipid microdomains called rafts (see Brown & London, 2000 and O'Connell et al. 2004 for review). Lipid rafts are regions of the membrane enriched in cholesterol and sphingolipids, which makes them more ordered and less fluid than the bulk plasma membrane. As a consequence of this ‘liquid-ordered’ state, lipid rafts are resistant to solubilization with non-ionic detergents at low temperature (Babiychuk et al. 2002; Dykstra et al. 2003). The level of free cholesterol in the plasma membrane determines the assembling and maintenance of lipid rafts. Many of the membrane proteins involved in signal transduction (e.g. membrane receptors, G-proteins, PI3 kinase, eNOS, integral/structural proteins, etc.) interact with cholesterol either directly or indirectly via other proteins. This provides a platform for compartmentalization of signalling pathways within cellular membrane considered to enhance efficacy of signalling (Simons & Toomre, 2000; Galbiati et al. 2001). Beside other proteins, one family of structural protein components, namely caveolins, is of particular importance, as it drastically changes the morphology and/or function of lipid rafts (Anderson, 1998). Integration of caveolin-1 into lipid rafts causes them to adopt a flask-shaped morphology. These 50–100 nm invaginations of the surface membrane are called caveolae. They are vital for the compartmentalization of signalling systems in many cell types including smooth muscle (Taggart et al. 2000; Gratton et al. 2004). In myometrium, caveolae are involved in the regulation of PKC activity (Turi et al. 2001) and, in turn, are regulated by the sex hormones (Wang et al. 2005).
BK channels are targeted to caveolae and physically associate with cavolin-1 in vascular endothelial cells (Bravo-Zehnder et al. 2000), and this has been also reported for ureteric (Babiychuk et al. 2004) and uterine (Brainard et al. 2005) smooth muscle. Co-immunoprecipitation analysis revealed that BK channels are also associated with and regulated by 
- and 
-actin (Fielding & Fielding, 2000). The maintenance of caveolae depends on the presence of caveolin and free cholesterol (Dreja et al. 2002; Babiychuk et al. 2002; Daniel et al. 2004). Thus, depletion of the cholesterol content of the surface membrane causes changes in caveolin-1 localization and disappearance of caveolae (Babiychuk et al. 2002; Gniadecki, 2004; Babiychuk et al. 2004). Most commonly, the depletion of membrane cholesterol in isolated cells and multicellular strips of smooth muscle is achieved by treatment with methyl--cyclodextrin (MCD) (Kilsdonk et al. 1995; Loyd et al. 1995; Blatter & Niggli, 1998; Babiychuk et al. 2004; Smith et al. 2005; Noble et al. 2006). In our laboratory, recent experiments on rat myometrium found a substantial decrease in cholesterol content and disruption of caveolae after incubation with MCD (Smith et al. 2005). Depletion of membrane cholesterol using either MCD or cholesterol oxidase causes an increase in the amplitude and frequency of spontaneous and oxytocin-induced contractions of myometrium (Smith et al. 2005). In a preliminary study we have also found similar effects in human myometrium (Kendrick et al. 2004).
In the present study, designed to investigate the mechanism underlying the functional effects of cholesterol manipulation in the uterus, we have asked the questions, does cholesterol manipulation affect outward current in the myometrium and can this explain the functional effects, i.e. cholesterol depletion-induced potentiation of uterine contractility? We find that depletion of membrane cholesterol effectively eliminates the large conductance (BK) iberiotoxin-sensitive component of the outward current by triggering internalization of the surface membrane. A decrease in the density of outward current and elevation of [Ca2+]i can account for the previously described increase in spontaneous activity caused by cholesterol depletion. Iberiotoxin or 1 mM TEA pretreatment reduces the effects of MCD in isolated myocytes and intact preparations. Together these data suggest that BK channels are regulated, at least in part, by incorporation or exclusion into membrane caveolae.
A preliminary account of this work has been presented elsewhere in an abstract form (Shmygol & Wray, 2005).
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experiments were performed on small strips of myometrium and acutely isolated uterine smooth muscle cells (see below). Female Wistar rats at the end of gestation (19–21 days) were humanely killed by cervical dislocation under CO2 anaesthesia. All experimental procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. This investigation was approved by the Institutional Animal Care and Use Committee. Small strips of myometrium were dissected from the longitudinal layer of the uterus and mounted horizontally in a small perfusion bath for recording of isometric contractions. Ca2+ was simultaneously measured in strips loaded with indo-1/AM (15 µM, 3 h at room temperature), as described in detail elsewhere (Taggart & Wray, 1993; Taggart et al. 1997). Tissues were superfused with physiological saline, pH 7.4 and 32–35°C. Contraction and Ca2+ transient amplitude, frequency and integral were measured and compared between control (100%) and treatments.
Cells
Smooth muscle cells were enzymatically isolated from the longitudinal layer of the uterus as previously described (Shmigol et al. 1998). Freshly dissociated myocytes were kept in ‘KB’ medium (Klockner & Isenberg, 1985) at 4°C and used for experiments on the same day. During the course of the experiment cells were continuously superfused with warm (35°C) physiological saline solution. Local application of solutions to the cell under study was achieved by the use of pressure-assisted fast application system (Valve Bank 8, Automate Scientific Inc., Berkeley, CA, USA). In a series of experiments designed to investigate the effect of cholesterol depletion on spontaneous activity of isolated uterine myocytes, changes in cytosolic Ca2+ concentration were measured using a Ca2+-sensitive dye, fluo-4, and a confocal microscope (see below). The cells were loaded with fluo-4 by incubation with 7 µm fluo-4/AM for10 min at 35°C in the presence of 0.25% of non-ionic detergent Pluronic F127. In these experiments, we used a x20, 0.7 NA dry objective lens, which allowed us to expand the field of view and record Ca2+ signals from a larger number of cells (typically 10–15) at the same time. Time series of images were collected at 12 frames per second for 3 min before and after cholesterol extraction. Mean fluorescence intensity was measured on-line from regions of interest drawn over individual cells using UltraView software (Perkin Elmer, Boston, MA, USA). The numerical data obtained were saved to an ASCII file for further analysis using Origin 7.0 software (OriginLab Corp., Northampton, MA, USA). The amplitude of [Ca2+]i transients was expressed as normalized fluo-4 fluorescence (
F/F0). Cholesterol extraction caused complex changes in both the amplitude and frequency of spontaneous [Ca2+]i transients (see Fig. 5A for example). These changes were quantified by integrating the normalized fluo-4 signal above the baseline during 3 min time intervals, and integral Ca2+ signals were compared statistically (see Fig. 5B and C). Origin 7.0 software was used for these computations.
|
Membrane cholesterol was extracted using MCD, a cyclic oligosaccharide which sequesters cholesterol as previously described (Smith et al. 2005). In brief, in cells after recording the control I–V curve, 15 mM MCD dissolved in physiological saline (2% MCD solution) was applied to the cell under study for 10 min followed by 5 min washout. After that another I–V curve was recorded. In intact tissues 15 mM MCD was added to the physiological saline and applied for 10–20 min at 35°C. In the experiments designed to test direct effect of MCD on outward currents, cholesterol-saturated MCD was used at 3.75 mM (0.5% solution). All experiments were performed at 32–35°C.
Electrophysiology
Transmembrane ionic currents were studied using the conventional patch clamp technique. Patch electrodes were pulled from thick-walled borosilicate glass capillaries (Harvard Apparatus, Edenbridge, UK) using a PP830 puller (Narishige, Tokyo, Japan). The tips of the electrodes were heat-polished using an MF830 micro forge (Narishige) to achieve 3–5 M
resistance. Tight seals (10–20 G) between the patch electrode and cell membrane were established by pressing the electrode against the cell and applying gentle suction. Whole cell access was established by applying strong pulses of negative pressure. Whole cell currents were recorded using the EPC 9 patch clamp amplifier controlled by the Pulse software (HEKA Elektronik, Lambrecht/Pfalz, Germany). During the experiment, cells were superfused with prewarmed (35°C) physiological saline solution and held at –70 mV holding potential. Recordings started 5 min after establishing the whole cell access to allow for equilibration of ion concentrations between cytosol and the pipette. Current–voltage curves were constructed from the peak outward currents recorded in response to voltage pulses ranging from –60 to +70 mV in 10 mV increments. Cell capacitance and access conductance were measured and corrected automatically before each sweep. Up to 70% of series resistance was routinely compensated.
Confocal microscopy
To visualize the surface membrane of uterine myocytes we used a lipid conjugated dye, Calcium Green C18 (Lloyd et al. 1995). Adherent cells were labelled by perfusing the experimental chamber with physiological saline solution containing 5 µm Calcium Green C18 for 2–3 min. The 18-carbon-long lipophilic chain attached to the Calcium Green fluorophore allows this dye to bind to the surface membrane of the cell but prevents it from entering the cytosol (Blatter & Niggli, 1998). In some experiments another lipophilic dye di-8-ANEPPS was used to visualize the surface membrane, as previously described (Kirk et al. 2003). Upon internalization of the surface membrane, the dye should appear inside the cell. An argon ion laser was used as the 488 nm excitation light source. The cells were imaged through an Olympus UPLAPO60XWPSF-S, 1.2 NA objective lens using the Hamamatsu Orca ER cooled CCD camera. A water- rather than oil-immersion objective lens was used to prevent spherical aberration due to the mismatch between immersion media. To reduce out-of-focus fluorescence, the spinning disk-based confocal scanner (UltraView, Perkin Elmer Optoelectronics, Cambridge, UK) mounted on the Olympus IX-50 inverted microscope was used. Z-stacks of images were collected using the step motor focusing device (OptiScan, Prior Scientific Instruments Ltd, Cambridge, UK) controlled by the UltraView software package. Further reduction in the out-of-focus light was achieved by iterative constrained deconvolution (Huygens Essential version 2.7.2p0, Scientific Volume Imaging B.V., the Netherlands) using a theoretically calculated point spread function. Processed stacks of images were further analysed using the public domain software package Image J (available at http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institute of Health, Bethesda, MD, USA).
Statistics
Where appropriate, the results are presented as means ± S.E.M. Otherwise, the traces shown represent typical results from at least four similar experiments on cells, or myometrial strips obtained from different animals. Statistical differences were tested using ANOVA, and significance was taken at P < 0.05. Where appropriate, Student's t test for paired data was used to compare the cell capacitance, time integrals of spontaneous [Ca2+]i transients or contractile activity before and after cholesterol depletion. Origin 7.0 was used for data analysis and graphing purposes.
Solutions
The solutions used were as follows. Physiological saline solution (mm): NaCl, 130; KCl, 5.8; CaCl2, 2; MgCl2, 1.2; Hepes, 10.0; glucose, 10. KB Solution (mm): KCl, 40; K2HPO4, 10; taurine, 10; TES, 10; glucose, 20; sodium pyruvate, 5; creatine, 5; EGTA, 0.04; potassium glutamate, 105; BSA, 1 mg ml–1. Patch pipette solution (mm): KCl, 140; NaCl, 8; MgATP, 4; Hepes, 10; EGTA, 0.005. Tetraethylammonium (TEA, 1 mm) or iberiotoxin (100 nm) dissolved in physiological solution was used in some experiments to inhibit BK channels and apamin (100 nm) to inhibit SK channels, in intact tissue or cells. All chemicals were from Sigma, apart from fluo-4, indo-1, Calcium Green C18 and di-8-ANEPPS, which were obtained from Molecular Probes.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Voltage clamp pulses above –30 mV applied to cells dialysed with K+-containing solution elicited outward current similar to that described by other authors (Miyoshi et al. 1991; Wang et al. 1998). Figure 1A illustrates typical records obtained from a uterine myocyte bathed in physiological saline solution. Extraction of membrane cholesterol with MCD led to a significant decrease in the outward current density. Figure 1B shows superimposed selected traces recorded from the same cell in response to +50 mV voltage clamp pulses under control conditions (trace a) and after cholesterol extraction (trace b, 15 min later) together with the trace obtained by subtracting trace a from b. Averaged current density–voltage relationships obtained from six different cells before and after cholesterol extraction are shown in Fig. 1C. It can be seen that depletion of membrane cholesterol led to approximately a 30% decrease in the amplitude of the outward current. Control superfusion of cells with physiological saline solution rather than MCD for 15 min produced no change in the density of outward current, and recordings of transmembrane currents without any interventions remained stable for up to 1 h (not shown).
|
|
|
Iberiotoxin inhibits outward current and abolishes the effect of cholesterol depletion
As mentioned earlier BK channels have been localized to caveolae. In order to investigate if BK channels were affected by cholesterol depletion in the rat myometrium, we used iberiotoxin, a highly selective blocker, which inhibits the channels on the external side of the membrane (Candia et al. 1992). Application of iberiotoxin to uterine myocytes caused a substantial decrease in the amplitude of outward current in all cells tested. Typical traces recorded in response to +50 mV voltage pulses in control and in the presence of 100 nM iberiotoxin are shown in Fig. 4A. Averaged I–V curves recorded from seven different cells are presented in Fig. 4C. The extent of reduction in outward current produced by iberiotoxin was similar to that induced by cholesterol depletion (compare Figs 1C and 4C). We hypothesized that a decrease in the outward current density observed upon depletion of membrane cholesterol was due to the abolition of the BK channel current. We tested this by applying iberiotoxin to cells which were treated with MCD. As illustrated in Fig. 4B, 100 nM iberiotoxin was not able to inhibit the outward current in a cell with depleted membrane cholesterol to the same extent as seen in control (Fig. 4A). The I–V curves recorded from the cholesterol depleted cells are not statistically different (n = 5, P > 0.1) in the presence and absence of iberiotoxin (Fig. 4D). In contrast, a blocker of small conductance (SK) potassium channels, apamin (100 nM), was equally effective on both control and MCD treated cells (n = 4, not illustrated) indicating that SK channels were not affected under these conditions. Taken together, these data suggest that the component of outward current eliminated by MCD was indeed flowing through the BK channels.
|
To explore the impact of cholesterol depletion-induced decrease in the outward current on spontaneous activity of uterine myocytes, we took advantage of the fact that many isolated myocytes are capable of generating spontaneous action potentials (Shmygol et al. 2004; Duquette et al. 2005). These action potentials triggered fast [Ca2+]i transients and therefore could be monitored in many cells simultaneously using fluorescence imaging. Figure 5A illustrates typical [Ca2+]i traces recorded from a spontaneously active myocyte before (upper trace) and after (lower trace) cholesterol extraction. It is clear from this record (and 18 others from 7 different digestions) that cholesterol depletion was accompanied by increased spontaneous activity. On average, depletion of membrane cholesterol nearly doubled the integral Ca2+ signal (Fig. 5B). Inhibition of the BK channels using iberiotoxin potentiated spontaneous [Ca2+]i transients to roughly the same extent as cholesterol depletion did. In agreement with our patch clamp data, extraction of membrane cholesterol from cells pretreated with iberiotoxin did not exert any further stimulatory action (Fig. 5C, n = 6). Actually, the integral [Ca2+]i signal recorded from the iberiotoxin treated cells upon cholesterol depletion tended to decrease, although this trend did not reach statistical significance. These data therefore suggest a substantial impact of cholesterol depletion on spontaneous activity of isolated uterine myocytes, due to the abolition of the iberiotoxin-sensitive component of outward current.
The effect of cholesterol extraction on contractile activity and Ca2+ in intact rat myometrium
The above data suggest that BK currents are involved in mediating the effects of MCD in isolated uterine cells. However the question remains of whether these mechanisms would be apparent in intact tissues. To address this question we performed experiments on intact strips of rat myometrium. Figure 6A shows the effect of MCD on spontaneous contractile activity. Consistent with previous data, enhanced activity occurs following MCD treatment. In these pregnant animals there was an increase in amplitude (145 ± 18%) and duration (122 ± 10%) of contractions, resulting in a significant increase in the area under the curve (182 ± 28%; control 100%, n = 6).
|
Previous studies have indicated that changes in [Ca2+] underlie the effects of cholesterol manipulation on rat myometrium. We therefore performed experiments simultaneously measuring Ca2+ and force (Fig. 6B). There was again a good correspondence between Ca2+ and the effects on force (n = 6). As can be seen in Fig. 6Bb, the reduced effects of MCD in the presence of TEA can be seen in the reduced intracellular Ca2+ transients. The baseline Ca2+ remained steady during and after the MCD treatment.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Methyl--cyclodextrin has been used in numerous studies, including recently smooth muscles, and its reversible effects on membrane cholesterol content and caveolae structure are well documented (Kilsdonk et al. 1995; Dreja et al. 2002; Daniel et al. 2004; Babiychuk et al. 2004; Gniadecki, 2004; Smith et al. 2005). Specifically in myometrium we have shown that MCD produces similar functional effect to cholesterol depletion with cholesterol oxidase and in the presence of the agonist oxytocin (Smith et al. 2005). We now additionally show that non-specific effects of MCD do not underlie its action, as after a control period in Krebs solution re-application of MCD to cells produced no effects. Thus it seems reasonable to discuss our data in terms of the effect of MCD on depletion of membrane cholesterol, causing disruption of caveolae, as seen in electron micrographs (Dreja et al. 2002; Babiychuk et al. 2004; Smith et al. 2005).
A clear decrease in outward membrane current occurred in the myometrial cells with cholesterol depletion. This decrease was accompanied by a significant reduction in membrane capacitance. Our finding of internalization of Calcium Green C18 and di-8-ANEPPS suggests membrane internalization underlies the reduction in capacitance. We (Babiychuk et al. 2002, 2004; Draeger et al. 2005) and others (Brainard et al. 2005) have shown associations of caveolin with the actin cytoskeleton; thus as the structure of the caveolae is disrupted by MCD, their cytoskeletal interaction will be disturbed and internalization triggered. The subunit of the BK channel has been shown to be associated with caveolae in smooth muscle (Babiychuk et al. 2004) and it may be speculated that this subunit is undergoing redistribution and internalization. Disruption of the Ca2+ microdomains between caveolae and SR by MCD treatment may also contribute to the effects seen, if coupling between the two membranes is affected.
As discussed earlier, there is evidence for cholesterol manipulation affecting the activity of several ion channels. We found a decrease in outward current in uterine myocytes of 30%, indicating that the effect of MCD was not on all K+ channels, but was selective. For myometrial smooth muscle there is evidence that BK channels are localized to caveolae and that they will therefore be a particular target for MCD (Babiychuk et al. 2004; Brainard et al. 2005). Our data in single cells using iberiotoxin as a blocker of these channels show that BK channels are responsible for the decrease in outward current seen with MCD. Thus, iberiotoxin alone substantially reduces outward current, such that MCD has little further effect. Apamin, a blocker of SK channels, had no effect on the actions of MCD, suggesting specificity to the BK channel. In some previous reports MCD treatment in tissue was associated with a slow rise in basal Ca2+ levels (Babiychuk et al. 2004), suggesting a nonspecific leak of Ca2+ occurs with membrane disruption. Such a rise in basal Ca2+ did not occur in the uterus as seen here and in our previous study (Smith et al. 2005). Thus while not dismissing the possibility that cholesterol manipulation may affect several ion channels, the data here are best explained by a specific effect on BK channels.
Our data on freshly dispersed rat myocytes differ from those of a recent study by Brainard et al. (2005) on human myocytes, where an increase in iberiotoxin-sensitive outward current was found with MCD. We think this is unlikely to be due to a species difference, as MCD has the same functional effects on both rat (Smith et al. 2005) and human myometrium (Kendrick et al. 2004). We consider it more likely that the different findings are from the use of cultured human myocytes, which are known to be phenotypically altered. It is also hard to explain the increased contractility in the myometrium with MCD, if outward current is increased as reported by Brainard et al. (2005).
Our findings provide insight into the previously published results of increased Ca2+ signalling and contractility in intact myometrium with MCD and cholesterol oxidase (Smith et al. 2005). The decrease in outward current will increase the excitability of the tissue, leading to increased firing of action potentials and Ca2+ entry, as shown; indeed spontaneous electrical activity was often observed in single cells following MCD treatment. The effects on Ca2+ entry were inhibited if the cells had been pretreated with iberiotoxin, again consistent with BK channels underlying the effects of MCD.
Our data on single cells are compatible with those found in multicellular preparations. MCD produced a marked increased in contractility and calcium signalling, as shown here and reported previously (Smith et al. 2005). As in the single cell, the stimulatory effect of MCD was significantly reduced after blocking BK channels in multicellular preparations. Thus we conclude that the effects of MCD on uterine Ca2+ signalling and contractility can be explained in part by the reduced outward current consequent to BK channel internalization. It should be noted, however, that in the multicellular preparations, TEA was more effective than iberiotoxin and thus effects on Kv channels may contribute to the functional effects found (Aaronson et al. 2006).
In summary our data support previous evidence for a significant role for BK channels being localized to caveolae in the myometrium. When caveolae are disrupted by cholesterol depletion, the BK channels redistribute and/or become internalized, and thus their contribution to outward current is reduced. In turn the decrease in K+ channel conductance will promote triggering of action potentials and increased Ca2+ entry. This mechanism can explain the effects of cholesterol manipulation in the uterus, and may therefore underlie the increase in contractility found with cholesterol depletion. We also speculate that elevated cholesterol in obese pregnant women may contribute to poor contractility and the increased rate of caesarean sections reported in this group (Crane et al. 1997; Zhang et al. 2007).
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Anderson RG (1998). The caveolae membrane system. Annu Rev Biochem 67, 199–225.[CrossRef][Medline]
Anwer K, Toro L, Oberti C, Stefani E & Sanborn BM (1992). Ca2+-activated K+ channels in pregnant rat myometrium: modulation by -adrenergic agent. Am J Physiol Cell Physiol 263, C1049–C1056.
Babiychuk EB, Monastyrskaya K, Burkhard FC, Wray S & Draeger A (2002). Modulating signaling events in smooth muscle: cleavage of annexin 2 abolishes its binding to lipid rafts. FASEB J 16, 1177–1184.
Babiychuk EB, Smith RD, Burdyga TV, Babiychuk VS, Wray S & Draeger A (2004). Membrane cholesterol selectively regulates smooth muscle phasic contraction. J Membr Biol 198, 95–101.[Medline]
Benkusky NA, Fergus DJ, Zucchero TM & England SK (2000). Regulation of the Ca2+-sensitive domains of the maxi-K channel in the mouse myometrium during gestation. J Biol Chem 275, 27712–27719.
Blatter LA & Niggli E (1998). Confocal near-membrane detection of calcium in cardiac myocytes. Cell Calcium 23, 269–279.[CrossRef][Medline]
Brainard AM, Miller AJ, Martens JR & England SK (2005). Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channel-caveolin complex in the regulation of myometrial smooth muscle K+ current. Am J Physiol Cell Physiol 289, C49–C57.
Bravo-Zehnder M, Orio P, Norambuena A, Wallner M, Meera P, Toro L, Latorre R & Gonzalez A (2000). Apical sorting of a voltage- and Ca2+-activated K+ channel -subunit in Madin-Darby canine kidney cells is independent of N-glycosylation. Proc Natl Acad Sci U S A 97, 13114–13119.
Brown DA & London E (1998). Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14, 111–136.[CrossRef][Medline]
Brown DA & London E (2000). Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275, 17221–17224.
Candia S, Garcia ML & Latorre R (1992). Mode of action of iberiotoxin, a potent blocker of the large conductance Ca2+-activated K+ channel. Biophys J 63, 583–590.
Chanrachakul B, Matharoo-Ball B, Turner A, Robinson G, Broughton-Pipkin F, Arulkumaran S & Khan RN (2003). Reduced expression of immunoreactive 2-adrenergic receptor protein in human myometrium with labor. J Clin Endocrinol Metab 88, 4997–5001.
Chanrachakul B, Pipkin FB & Khan RN (2004). Contribution of coupling between human myometrial 2-adrenoreceptor and the BKCa channel to uterine quiescence. Am J Physiol Cell Physiol 287, C1747–C1752.
Crane SS, Wojtowycz MA, Dye TD, Aubry RH & Artal R (1997). Association between pre-pregnancy obesity and the risk of cesarean delivery. Obstet Gynecol 89, 213–216.[Abstract]
Daniel EE, Bodie G, Mannarino M, Boddy G & Cho WJ (2004). Changes in membrane cholesterol affect caveolin-1 localization and ICC-pacing in mouse jejunum. Am J Physiol Gastrointest Liver Physiol 287, G202–G210.
Draeger A, Wray S & Babiychuk EB (2005). Domain architecture of the smooth-muscle plasma membrane: regulation by annexins. Biochem J 387, 309–314.[CrossRef][Medline]
Dreja K, Voldstedlund M, Vinten J, Tranum-Jensen J, Hellstrand P & Sward K (2002). Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction. Arterioscler Thromb Vasc Biol 22, 1267–1272.
Duquette RA, Shmygol A, Vaillant C, Mobasheri A, Pope M, Burdyga T & Wray S (2005). Vimentin-positive, c-kit-negative interstitial cells in human and rat uterus: a role in pacemaking? Biol Reprod 72, 276–283.
Dykstra M, Cherukuri A, Sohn HW, Tzeng SJ & Pierce SK (2003). Location is everything: lipid rafts and immune cell signaling. Annu Rev Immunol 21, 457–481.[CrossRef][Medline]
Eghbali M, Toro L & Stefani E (2003). Diminished surface clustering and increased perinuclear accumulation of large conductance Ca2+-activated K+ channel in mouse myometrium with pregnancy. J Biol Chem 278, 45311–45317.
Fielding CJ & Fielding PE (2000). Cholesterol and caveolae: structural and functional relationships. Biochim Biophys Acta 1529, 210–222.[Medline]
Galbiati F, Razani B & Lisanti MP (2001). Emerging themes in lipid rafts and caveolae. Cell 106, 403–411.[CrossRef][Medline]
Gniadecki R (2004). Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis. Biochem Biophys Res Commun 320, 165–169.[CrossRef][Medline]
Gratton JP, Bernatchez P & Sessa WC (2004). Caveolae and caveolins in the cardiovascular system. Circ Res 94, 1408–1417.
Heaton RC, Wray S & Eisner DA (1993). Effects of metabolic inhibition and changes of intracellular pH on potassium permeability and contraction of rat uterus. J Physiol 465, 43–56.
Kendrick AJ, Zhang J, Tattersall M, Bricker L, Quenby S & Wray S (2004). Calcium signalling, caveolae and human myometrial contractility. J Physiol 560.P, C50.
Khan R, Mathroo-Ball B, Arilkumaran S & Ashford MLJ (2001). Potassium channels in the human myometrium. Exp Physiol 86, 255–264.[Abstract]
Khan R, Smith SK, Morrison JJ & Ashford MLJ (1993). Properties of large-conductance K+ channels in human myometrium during pregnancy and labour. Proc Roy Soc Lon B Biol Sci 251, 9–15.[CrossRef]
Kilsdonk EPC, Yancey PG, Stoudt GW, Wen Bangerter F, Johnson WJ, Phillips MC & Rothblat GH (1995). Cellular cholesterol efflux mediated by cyclodextrins. J Biol Chem 270, 17250–17256.
Kirk MM, Izu LT, Chen-Izu Y, McCulle SL, Wier WG, Balke CW & Shorofsky SR (2003). Role of the transverse-axial tubule system in generating calcium sparks and calcium transients in rat atrial myocytes. J Physiol 547, 441–451.
Klockner U & Isenberg G (1985). Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig). Pflugers Arch 405, 329–339.[CrossRef][Medline]
Lloyd QP, Kuhn MA & Gay CV (1995). Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophillic calcium-sensitive fluorescent dye, calcium green C18. J Biol Chem 270, 22445–22451.
Matharoo-Ball B, Ashford ML, Arulkumaran S & Khan RN (2003). Down-regulation of the - and -subunits of the calcium-activated potassium channel in human myometrium with parturition. Biol Reprod 68, 2135–2141.
Mershon JL, Mikala G & Schwartz A (1994). Changes in the expression of the L-type voltage-dependent calcium channel during pregnancy and parturition in the rat. Biol Reprod 51, 993–999.[Abstract]
Miyoshi H, Urabe T & Fukiwara A (1991). Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats. Pflugers Arch 419, 386–393.[CrossRef][Medline]
Noble K, Zhang J & Wray S (2006). Lipid rafts, the sarcoplasmic reticulum and uterine calcium signalling: an integrated approach. J Physiol 570, 29–35.
O'Connell KM, Martens JR & Tamkun MM (2004). Localization of ion channels to lipid Raft domains within the cardiovascular system. Trends Cardiovasc Med 14, 37–42.[CrossRef][Medline]
Perez GJ, Toro L, Erulkar SD & Stefani E (1993). Characterization of large-conductance, calcium-activated potassium channels from human myometrium. Am J Obstet Gynecol 168, 652–660.[Medline]
Piedras-Renteria E, Stefani E & Toro L (1991). Potassium currents in freshly dispersed myometrial cells. Am J Physiol Cell Physiol 261, C278–C284.
Shmigol A, Eisner DA & Wray S (1998). Properties of voltage-activated [Ca2+]i transients in single smooth muscle cells isolated from pregnant rat uterus. J Physiol 511, 803–811.
Shmygol A, Burdyga T, Duquette R, Mobasheri A, Vaillant C & Wray S (2004). Spontaneous electrical activity in subpopulation of freshly isolated rat uterine myometrial cells. J Physiol 555.P, C167.
Shmygol A & Wray S (2005). Effect of cholesterol extraction on [Ca2+]i transients and K+ currents in rat uterine myocytes. J Physiol 565.P, C79.
Simons K & Toomre D (2000). Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1, 31–39.[CrossRef][Medline]
Smith RD, Babiychuk EB, Noble K, Draeger A & Wray S (2005). Increased cholesterol decreases uterine activity: functional effects of cholesterol in pregnant rat myometrium. Am J Physiol Cell Physiol 288, C982–C988.
Song M, Zhu N, Olcese R, Barila B, Toro L & Stefani E (1999). Hormonal control of protein expression and mRNA levels o the MaxiK channel subunit in myometrium. FEBS Lett 460, 427–432.[CrossRef][Medline]
Taggart MJ, Leavis P, Feron O & Morgan KG (2000). Inhibition of PKC and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res 258, 72–81.[CrossRef][Medline]
Taggart MJ, Menice CB, Morgan KG & Wray S (1997). Effect of metabolic inhibition on intracellular Ca2+, phosphorylation of myosin regulatory light chain and force in rat smooth muscle. J Physiol 499, 485–496.[CrossRef][Medline]
Taggart MJ & Wray S (1993). Occurrence of intracellular pH transients during spontaneous contractions in rat uterine smooth muscle. J Physiol 472, 23–31.
Turi A, Kiss AL & Mullner N (2001). Estrogen downregulates the number of caveolae and the level of caveolin in uterine smooth muscle. Cell Biol Int 25, 785–794.[CrossRef][Medline]
Wang XL, Ye D, Peterson TE, Cao S, Shah VH, Katusic ZS, Sieck GC & Lee HC (2005). Caveolae targeting and regulation of large conductance Ca2+-activated K+ channels in vascular endothelial cells. J Biol Chem 280, 11656–11664.
Wang SY, Yoshino M, Sui JL, Wakui M, Kao PM & Kao CY (1998). Potassium currents in freshly dissociated uterine myocytes from nonpregnant and late-pregnant rats. J Gen Physiol 112, 737–756.
Wray S, Kupittayanant S, Shmigol A, Smith RD & Burdyga TV (2001). The physiological basis of uterine contractility: a short review. Exp Physiol 86, 239–246.[Abstract]
Yoshino M, Wang SY & Kao CY (1997). Sodium and calcium inward currents in freshly dissociated smooth myocytes of rat uterus. J Gen Physiol 110, 565–577.
Zhang J, Bricker L, Wray S & Quenby S (2007). Poor uterine contractility in obese women. BJOG 114, 343–348.[CrossRef][Medline]
Zhou XB, Wang GX, Ruth P, Huneke B & Korth M (2000). BKCa channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy. Am J Physiol Cell Physiol 279, C1751–C1759.
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. Muller, P. Muller, G. Pincemy, A. Kurz, and C. Labbe Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa Biol Reprod, March 1, 2008; 78(3): 390 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Jie Zhang, A. Kendrick, S. Quenby, and S. Wray Contractility and Calcium Signaling of Human Myometrium Are Profoundly Affected by Cholesterol Manipulation: Implications for Labor? Reproductive Sciences, July 1, 2007; 14(5): 456 - 466. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and after cholesterol extraction (
). The currents were normalized per cell capacitance to decrease cell-to-cell variability. D, MCD itself had no effect on the current–voltage relationship in the uterine myocytes.
