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NEUROREGULATION AND MOTILITY
Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Submitted 20 August 2003 ; accepted in final form 7 February 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-clodextrin (CD) to remove cholesterol or with water- soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.
methyl -cyclodexrin; interstitial cells of cajal; water soluble cholesterol; pacing of intestinal contractions
Caveolin 1 knockout mice have been shown to lack caveolae in vascular smooth muscle and endothelial cells and to have deficiencies in signaling through nitric oxide (NO) synthase (NOS) (8, 10, 23, 28, 29). Caveolae are closely associated with lipid rafts containing high concentrations of cholesterol and are involved in cholesterol transport into the membrane (7, 13, 25, 39, 40, 42). Removal of membrane cholesterol from the membrane using molecules such as methyl -cycldextrin (CD) causes loss of caveolin 1 and caveolae and dysfunction of caveolin-associated proteins in various cell types (7, 9, 11, 13, 15, 19, 21, 22, 24, 26, 30, 37, 42).
Previously, we showed that small isolated segments of longitudinal (LM) or circular muscle (CM) from mouse intestine contracted regularly, and the contractions behaved as if they were controlled by ICC pacing: i.e., contractions were independent of nerve function, were reduced but not abolished by block of VDCC, were faster in jejunum than ileum, had high temperature dependence, and were reduced in frequency by block of the sarco(endo)plasmic reticulum Ca2+-ATPase pump.
Our objectives were to determine whether caveolin 1 is present in ICC-MP and other ICC of mouse intestine, to evaluate the effects of removal of cholesterol with CD on ICC-MP pacing on responses to cholinergic stimulation and on the distribution of caveolin 1 and caveolae, and to determine whether any such effects could be reversed by restoration of cholesterol as water-soluble cholesterol (WSC). We also evaluated the effects on pacing of adding WSC first followed by CD and its reversal by treatment with CD.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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BALB/c mice were euthanized by cervical dislocation according to a protocol approved by the Health Sciences Laboratory Animal Services at the University of Alberta. Through a midline incision, the gastrointestinal tissue from the lower esophagus to the rectum was excised and immediately placed into an oxygenated Krebs solution at room temperature, and the mesentery was removed. Tissues were maintained at room temperature in Krebs solution that was constantly bubbled with 95% O2-5% CO2. Segments of the jejunum measuring 
1.5 cm for LM and 1 cm for CM were prepared for study as previously described (34).
In brief, the tissues were suspended in a 10-ml organ bath. To study CM contractions, the tissue was placed in the bath horizontally with an L-shaped holder through the lumen and stimulator electrodes on either side of the tissue. An aluminum wire triangle was inserted through the lumen and attached to a force transducer (Grass FT OC3; Grass Instruments, Quincy, MA) using silk thread. To study LM contractions, the tissue was tied at both ends and suspended vertically in the bath through concentric stimulator electrodes. One end of the tissue was tied to a stationary holder in the bath, and the other end was attached to the force transducer. Muscle contractions were recorded using a Beckman R611 Dynagraph Oscillograph (Beckman Coulter, Fullerton, CA).
For the duration of each experiment, the muscle baths were continuously bubbled with 95% O2-5% CO2 at a constant rate and maintained at 37°C. Before experimental manipulations were performed, the tissues stabilized under slight tension for 5–10 min.
Tissue manipulation.
Before drugs were added, enteric nerves of the tissue preparations were stimulated using electrical field stimulation (EFS) from a Grass S88 stimulator set at 50 V/cm, 5 pulses/s, and 0.5-ms pulse duration to observe whether any inhibition of contractile activity took place. CM contractions were consistently inhibited by nerve stimulation, whereas LM contractions were stimulated. To prevent NO-mediated neural inhibition of muscle contraction, 100 liters of a 10–2 M solution of NG-nitro-L-arginine (L-NNA), an inhibitor of NOS, was added to each bath, giving a final concentration of 10–4 M. EFS was subsequently performed 5 min after the addition of L-NNA to each bath to ensure that inhibitory neural activity was abolished. If needed, an additional 100 µl of L-NNA were added, giving a final concentration of 2 x 10–4 M. The contractions present after L-NNA were abolished by 0.1 µM atropine.
In each experiment either 1 ml of a 10–1 M solution of CD or WSC was added to the test tissues, giving a drug concentration of 10–2 M in the muscle bath. Warmed oxygenated Krebs solution (1 ml) was added to time control tissues in place of WSC or CD. Two experimental protocols are reported involving both LM and CM. First, after at least 60 min or more incubation with CD, the bath was washed out and fresh Krebs and L-NNA were added with WSC to restore membrane cholesterol. The effects of WSC recovery were measured 30 min later. Time control tissues were also washed out and given fresh Krebs and L-NNA. Second, both test and control tissues were incubated for at least 60 min with WS. After the wash plus the addition of fresh Krebs and L-NNA, control tissue was given 1 ml of warmed oxygenated Krebs solution. Test tissues were given 1 ml of 10–1 M CD. The effects of the removal of the WS vs. the addition of CD were compared 30 min after the wash. Third, both test and control tissues were incubated for at least 60 min with WS. After the wash plus the addition of fresh Krebs and L-NNA, test tissues were given CD as above, and other tissues had WSC restored for 30 min.
In some experiments, if a fall in contraction frequency was not observed after 60 min incubation with WSC or CD, the tissues were exposed for an additional 15 min to the respective drug before each bath was washed out.
Additional experiments were carried out in which carbamoyl choline (CCH) was applied at 1 or 10 µM cumulatively before and after CD or WSC to LM segments of ileum and jejunum. Also, the effects of EFS (0.5, 2, 4, and 16 pulses/s) were applied to segments before and after CD and WSC. Changes were evaluated by comparison with responses of time controls. Only LM segments were used after block of neuronal NOS by 10–4 M L-NNA because they produced more reliable cholinergic presponses.
Drugs and solutions.
Krebs-Ringer solution was prepared with (in mM) 115.5 NaCl, 21.9 NaHCO3, 11.1 dextrose, 4.6 KCl, 1.16 MgSO4·H2O, 1.16 NaHPO4·H2O, and 2.5 CaCl2·H2O. The 10–1 M WS, and CD solutions were prepared by dissolving 0.131 g of drug in 1 ml of Krebs solution. A stock solution of 10–2 M L-NNA or CCH was prepared by dissolving the appropriate amount of drug in distilled water. All drugs used were obtained from Sigma-Aldrich (Oakville, ON).
Data analysis.
At each time point (before WSC or CD, 60 min after WSC or CD, and 30 min after wash), contraction frequencies were measured over at least a 20-s interval. Contraction amplitudes were measured and converted to a fraction of the amplitude at time 0 (before WSC or CD), which was set as 1. For contraction frequencies, the statistical program Instat 3 was used to perform mean comparison tests using a one-way ANOVA. Prism 3 software was used to construct graphs using mean frequency values with error bars representing the SE. For the amplitude data, Prism 3 was used to construct graphs comparing the amplitude at each time point and conduct paired t-tests to compare the amplitudes at pairs of time points. Analysis of LM and CM data was done separately.
Immunohistochemistry preparations.
Segments of jejunum, either fresh or after organ bath experiments, were placed in Krebs-Ringer solution, opened along the mesenteric border, and pinned on a petri dish of Sylgard silicon rubber (mucosa side down). For immunofluorescent labeling after cryosection, the segments were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) overnight at 4°C. The fixed tissues were washed in PBS for 30 min x 8 and were cryoprotected in graded sucrose solutions (10, 20% sucrose in PBS) for 2 h each. They were then placed in 30% sucrose in PBS for 24 h at 4°C and stored at –80°C until used. For whole mount preparation, the jejunum was fixed at 4°C overnight in a fixative the same as mentioned earlier. The fixed tissue was washed in PB for 30 min x 8 and was dehydrated and cleared in DMSO for 10 min x 3 and was then rehydrated in PB for 15 min x 4 at room temperature.
Immunofluorescent labeling using cryosections.
Frozen tissues were sectioned by a cryostat (Leitz 1720 digital cryostat) to make sections of 10-µm thickness. The sections were attached on slide glasses coated with 2% (3-aminopropyl)triethoxysilane (cat. no. A3648; Sigma) in acetone and were dried for at least 1 h or overnight at room temperature. The sections were washed in 0.4% Triton X-100 (TX) in PBS for 15 min x 3. The sections were blocked with 10% normal sera that were raised in the host of secondary antibody for 1 or 1.2 h at room temperature. For immunohistochemistry, mouse anti-caveolin mAb (cat. no. 610406; BD Biosciences) was incubated for 18 to 19 h at 4°C. The sections were washed in 0.4% TX in PBS for 15 min x 3. For immunofluorescent-labeling secondary antibodies, immunoglobulin conjugated with Cy3 was used for 1 h at room temperature. The sections were washed in 0.4% TX in PBS for 15 min x 2 and were then washed in PBS for 15 min x 1. The sections mounted with aquamount medium were observed by confocal laser scanning microscope (CLSM; LSM 1500; Zeiss).
Double immunofluorescent labeling using whole mount preparations.
CM and LM layers were separated from the mucosa and the submucosa layer under the dissection microscope. The muscle layers were washed in 0.5% TX in PBS for 15 min x 4 and were then blocked with 10% normal sera that were raised in the host of secondary antibody for 1.5 h at room temperature. For double immunohistochemistry, mouse anti-caveolin mAb and rat anti-cd117 (c-kit; Cedarlane) examined for whole mount were incubated for 48 h at 4°C and were then washed in 0.5% TX in PBS for 15 min x 4. For immunofluorescent-labeling secondary antibodies, immunoglobulin conjugated with Cy3 or FITC were incubated for 2 h at room temperature. The muscle layers were washed in 0.5% TX in PBS for 15 min x 3 and were then washed in PBS for 15 min x 1. The muscle layers mounted with aquamount medium were observed by CLSM.
Preparation of sections for ultrastructural study.
Either fresh or after-organ bath experiment segments of jejunum were opened and pinned in ice-cold oxygenated Krebs solution as described in Immunohistochemistry preparations for immunohistochemistry. The segments were fixed with a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.075 M sodium cacodylate buffer (pH 7.4) containing 3% sucrose and 1 mM CaCl2·2H2O for 2 h at 4°C. The segments were then washed in 0.075 M sodium cacodylate buffer overnight at 4°C. The segments were carefully dissected to a size of 1.0 x 4.0 mm including the mucosa and submucosa, postfixed with 1% OsO4 in 0.05 M sodium cacodylate buffer (pH 7.4) for 2 h at 4°C, dehydrated in graded ethanol and propylenoxide, and embedded in TAAB 812 resin (Marivac, Halifax, Nova Scotia). Ultrathin-sections were cut, mounted on 300-mesh grids, and stained with 13% uranyl acetate in 50% ethanol and lead citrate. The grids were examined in a Philips 410 electron microscope with a charge-coupled device camera (MegaView III) with a resolution of 1,376 x 1,032 x 16 at 80 kV.
Ultrastructural morphometric study: number of caveolae per unit length of ICC-MP and CM membrane.
For the measuring of lengths of ICC-MP and CM membrane, sections (control, CD, CD followed by WSC, WSC alone, and WSC followed by CD) for ultrastructural study were examined. The lengths of each cell membrane were manually drawn and automatically measured by an image analytic program (Laser Scanning Systems LSM510 Image Examiner v.2.80.1123, Carl Zeiss). For counting of caveolae on ICC-MP and CM membranes, open and/or close caveolae on cell membranes, which had membrane length measured, were carefully counted and accepted after agreement by two persons. Uncertain caveolae, which looked similar to sarcoplasmic reticulum or damaged mitochondria and which were located in cytoplasm, were not counted. The number of caveolae per unit length of ICC-MP and CM membrane was shown as means ± SE and was analyzed by one-way ANOVA followed by Tukey-Kramer posttest using the statistical program Instat3.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Segments of both LM and CM exposed to 10 mM CD for 60 min decreased their pacing frequencies. Pacing frequencies were restored by the addition of 10 mM WSC for 30 min (Fig. 1). In time controls, there were no significant changes in frequencies during these time periods. Amplitudes of contractions also significantly decreased in CD compared with initial values as shown but not significantly compared with highly variable time controls. They were not restored by WSC (Fig. 2).
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A possible explanation of our data showing that WSC restored frequencies after reduction by CD was that WSC had some physiological antagonism to CD. However, when 10 mM WSC was added first for 60 min, frequencies in LM or CM did not increase, but they decreased and were restored only in segments to which 10 mM CD was added for 30 min (Fig. 3). Moreover, washout of WSC in half of the tissues after 60 min was followed by the addition of CD or a replacement of WSC with Ringer solution. Only the presence of CD restored contraction frequencies. WSC exposure for 60 min also decreased amplitudes of contractions in LM but not in CM in which it increased. There was no recovery in CD or Ringer solution after 30 min in LM (Fig. 4). Figure 5 shows an example of these findings. In other additional protocols (not shown), WSC reduced frequencies of contractions, and these changes were reversed after washout and replacement of L-NNA Ringer solution with CD but not when the replaced Ringer solution had more WSC.
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Compared with time controls, contractile responses to CCH were unchanged after either 1 or 10 mM CD or WSC (Fig. 6, A and B). Although, responses were reduced after both CD and WSC, the responses of time controls were similarly reduced. Similarly, responses to cholinergic nerve stimulation were unchanged compared with those of time controls after CD or WSC (not shown). These, similar to the responses to CCH, decreased after CD and WSC but no more than time controls.
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Figure 7a shows that caveolin 1 immunoreactivity is located in punctate sites at the periphery of smooth muscle of both LM and CM. This figure also shows that it is also located in what appear to be ICC-MP. Caveolin 1 did not appear to be punctate in distribution in ICC. Figure 7b shows that caveolin 1 immunoreactivity is markedly decreased after CD, and Fig. 7c shows recovery of caveolin 1 immunoreactivity after WSC. One location of caveolin 1 was established to be in ICC membranes, as demonstrated by the colocalization of caveolin 1 and ckit (Fig. 8).
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Compared with control tissues, CD depleted caveolin 1 from plasma membranes of CM, LM, and ICC of mouse intestine, and the distribution was restored to normal after WSC (Fig. 9a). After exposure to WSC, there was enhanced and widespread distribution of caveolin 1, and it appeared no longer to be confined to the plasma membrane. After treatment with CD following WSC, the distribution of caveolin 1 was not restored fully to control conditions (Fig. 9b).
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Caveolae are present in ICC-MP, CM, and ICC-deep MP (DMP) in mouse intestine in control tissues after exposure to muscle bath conditions (Fig. 10). Although the several hours of exposure in the muscle bath affected mitochondrial status and caused other changes, caveolae were present in normal arrays. After exposure to CD, very few caveolae were present in ICC-MP, CM, or ICC-DMP, and those present were often opened in character (Fig. 11). After exposure to WSC, following CD, caveolae were present in normal-appearing arrays in ICC-MP, CM, and ICC-DMP (Fig. 12). After exposure to WSC, caveolae appeared abundant in ICC-MP, CM, and ICC-DMP (Fig. 13) and were restored toward the normal distribution after CD (not shown).
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The numbers of caveolae in ICC and CM were examined quantitatively in ultrastructural profiles from tissues used in these experiments. Figure 14 shows the results of these analyses. CD treatment reduced the number of caveolae per unit membrane length of ICC-MP, and muscle cells of CM and subsequent WSC increased these numbers, which were still reduced but were no longer significantly different from controls. WSC treatment did not increase the number of caveolae per unit membrane length in either ICC-MP or CM cells.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CD clearly altered the distribution of caveolin 1 and caveolae, which we demonstrated in LM, CM, and ICC-MP. It reduced the level of immunoreactivity in all of these cell types and nearly abolished the presence of caveolae. These effects were mostly reversed by subsequent exposure to WSC. Smooth muscle and ICC after these manipulations had abnormal mitochondria and other changes, suggesting that damages had occurred that might account for the failure of contraction amplitudes to recover.
Given first, exposure to WSC increased the level of immunoreactive caveolin 1 and altered its distribution. It was now distributed widely in the cytosol of smooth muscle as well as in the plasma membrane. There were high levels of caveolae present in ICC and smooth muscle, but these were not significantly increased compared with controls. CD exposure after WSC reduced the number of caveolae, but the distribution of immunoreactive caveolin 1 was not fully restored. The actions of WSC on ICC and frequency of pacing may be related to overloading of cholesterol into inappropriate sites or to altered caveolin 1 function with excess cholesterol.
We interpret these changes to mean that caveolae, dependent on caveolin 1 in ICC and affected by cholesterol depletion, participate in the mechanisms controlling pacing. At this point, it is unclear whether ion channels or receptors in ICC are affected. The facts that WSC cholesterol also decreases pacing frequency and this effect is reversed by CD indicate that these interactions are not a kind of physiological antagonism but that there is an optimum level of cholesterol and caveolin 1 in ICC, with either too much or too little leading to decreased pacing frequencies.
Our findings also suggest that the levels of cholesterol in ICC control the level of caveolin 1 as well as its distribution. The literature (7, 13, 25, 39, 40, 42) suggests that caveolin 1 and caveolae contribute to the entrance and exit of cholesterol from smooth muscle cells. So far there has been no study of pacing by ICC in caveolin 1 knockout mouse intestine. Comparison of results from such a study with our results would be of interest. However, studies with cholesterol depletion and restoration or vice versa have some advantages over studies in knockout animals. First, because the effects of cholesterol removal and restoration on structure and function can be studied acutely, there is no possibility that the results reflect a developmental anomaly rather than a change in caveolar function. Second, further analysis is possible to determine what component of caveolar signaling has been affected. Third, comparisons can be made with intestinal segments from the same animals without caveolar disruption, and time controls can be included.
In conclusion, pacing by ICC in mouse intestine may be regulated by the levels of cholesterol in ICC and the effects of this on caveolae and caveolin 1 distribution. Caveolae are sites at which signaling molecules assemble, and one or more of those molecules apparently plays a role in pacing by ICC. Future studies should focus on the nature of that molecule.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J Biol Chem 271: 29182–29190, 1996.-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem 275: 41447–41457, 2000.This article has been cited by other articles:
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  A. Shmygol, K. Noble, and S. Wray Depletion of membrane cholesterol eliminates the Ca2+-activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes J. Physiol., June 1, 2007; 581(2): 445 - 456. [Abstract] [Full Text] [PDF]
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 A. F. El-Yazbi, W. J. Cho, R. Schulz, and E. E. Daniel Caveolin-1 knockout alters beta-adrenoceptors function in mouse small intestine Am J Physiol Gastrointest Liver Physiol, December 1, 2006; 291(6): G1020 - G1030. [Abstract] [Full Text] [PDF]
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A. F. El-Yazbi, W. J. Cho, G. Boddy, R. Schulz, and E. E. Daniel Impact of caveolin-1 knockout on NANC relaxation in circular muscles of the mouse small intestine compared with longitudinal muscles Am J Physiol Gastrointest Liver Physiol, February 1, 2006; 290(2): G394 - G403. [Abstract] [Full Text] [PDF]
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W. J. Cho and E. E. Daniel Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1 Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G571 - G585. [Abstract] [Full Text] [PDF]
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