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Am J Physiol Gastrointest Liver Physiol 291: G1020-G1030, 2006. First published June 15, 2006; doi:10.1152/ajpgi.00159.2006
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MUCOSAL BIOLOGY

Caveolin-1 knockout alters beta-adrenoceptors function in mouse small intestine

Ahmed F. El-Yazbi,1 Woo Jung Cho,1 Richard Schulz,1,2 and Edwin E. Daniel1

Departments of 1Pharmacology and 2Pediatrics, University of Alberta, Edmonton, Alberta, Canada

Submitted 13 April 2006 ; accepted in final form 11 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 Immunohistochemistry
 RESULTS
 DISCUSSION
 REFERENCES
 
beta-Adrenoceptors are G protein-coupled receptors whose functions are closely associated with caveolae in the heart and cultured cell lines. In the gut, they are responsible, at least in part, for the mediation of the sympathetic stimulation that might lead to intestinal paralysis postoperatively. We examined the effect of caveolin-1 knockout on the beta-adrenoceptor response in mouse small intestine. The relaxation response to (–)-isoprenaline in carbachol-contracted small intestinal tissue segments was reduced in caveolin-1 knockout mice (cav1–/–) compared with their genetic controls (cav1+/+). Immunohistochemical staining showed that beta-adrenoceptor expression was similar in both strains in gut smooth muscle. Selective beta-adrenoceptor blockers shifted the concentration response curve (CRC) of (–)-isoprenaline to the right in cav1+/+ intestine, but not in cav1–/–, with greatest shift in case of the beta3-blocker, SR59230A. The CRC of the selective beta3-agonist BRL 37344 was also shifted to the right in cav1–/– compared with cav1+/+. The cAMP-dependent protein kinase (PKA) inhibitor H-89 shifted the CRC of (–)-isoprenaline to the right in cav1+/+ but not in cav1–/–. H-89 reduced the relaxation due to forskolin and dibutyryl cAMP in cav1+/+ but not in cav1–/–, suggesting a reduction in PKA activity in cav1–/–. In cav1+/+, PKA was colocalized with caveolin-1 in the cell membrane, but PKA immunoreactivity persisted in cav1–/–. Examination of PKA expression in the lipid raft-rich membrane fraction of the jejunum revealed reduced PKA expression in cav1–/– compared with cav1+/+. The results of the present study show that the function of beta-adrenoceptors is reduced in cav1–/– small intestine likely owing to reduced PKA activity.

protein kinase A; isoprenaline; forskolin; adenosine 3',5'cyclic-monophosphate

FOLLOWING AN ABDOMINAL SURGERY or injury, the motility of the gastrointestinal tract (GIT) is transiently impaired. Among the pathogenic mechanisms underlying the paralytic state is the sympathetic inhibitory reflex (19). The sympathetic hyperactivity in the postoperative period is the result of increased adrenergic neuronal hyperactivity or high circulating epinephrine released from the adrenal gland in response to surgical trauma (39, 34). Thus adrenergic receptors play a role in gut pathophysiology, and modification of their function may alter these responses.

The inhibitory sympathetic effects in the GIT are mediated by {alpha}- (48) and beta-adrenoceptors (51). Sympathetic innervation to the GIT comes from extrinsic nerves, which mostly end on and modulate enteric nerves (17). However, some adrenergically mediated inhibitory actions occur at the level of receptors expressed on smooth muscle cells (47, 30). A recent study (44) showed that most of the beta-adrenoceptor-mediated inhibition, in response to beta-adrenergic agonists, was independent of the enteric nervous system. Among the beta-adrenoceptors, the beta3 subtype is of a special interest in gastrointestinal motility because it is abundantly present in the GIT (3).

beta-Adrenoceptors are G-protein-coupled receptors, which upon activation by an agonist, stimulate adenylyl cyclase to produce the second messenger cAMP. The end effect of this process is the activation of cAMP-dependent protein kinase (PKA) (43). The receptor/G-protein/PKA interaction is not due to a random collision of freely moving proteins in the plasma membrane but is rather due to a compartmentalized membrane process (29). For beta-adrenoceptors, this compartmentalization has been shown to occur in caveolae in cardiac myocytes (13, 38) and cultured cell lines (42). In addition, beta2-adrenoceptor stimulation triggered the transfer of Gs{alpha} to caveolae (2, 49). Furthermore, disruption of caveolae by fillipin altered beta2-adrenoceptor function in cardiac myocytes (56).

Caveolae are non-clathrin-coated plasma membrane invaginations that are abundant in terminally differentiated cells such as endothelial cells, adipocytes, fibroblasts, and myocytes (8). They are present in cholesterol- and sphingolipid-rich domains of the plasma membrane and are coated on their cytoplasmic face by caveolins (8). Caveolins (1, 2, and 3) are a family of integral membrane proteins that are the principal components of caveolae in vivo (37). They form homo- and hetero-oligomers that insert in the inner leaflet of the plasma membrane to form the characteristic flask-shaped caveolae (40). Caveolae and caveolins are thought to be involved in signal transduction. Caveolin-1, through the caveolin scaffolding domain, binds and regulates the activity of several distinct classes of signaling molecules (21). Among these are the heterotrimeric G-proteins (26) and adenylyl cyclase (52) that are essential for signal transduction downstream of beta-adrenoceptors.

In the mouse small intestine, we showed that caveolin-1 is present in smooth muscle cells and interstitial cells of Cajal and is required for the normal pacing activity of the small intestine (9). We also found that caveolin-1 knockout (cav1–/–) mice, which lack caveolin-1 and most caveolae, have an altered response to nitric oxide and other nonadrenergic, noncholinergic mediators in the small intestine (11, 12). In the present study, we investigated alterations of GIT motility in the cav1–/– mice small intestine associated with beta-adrenoceptor function. We hypothesized that their function would be altered in the cav1–/– small intestine owing to the close relationship between caveolin-1 and beta-adrenoceptor signaling. We evaluated this hypothesis by comparing the structural and functional changes related to beta-adrenoceptors in the small intestine of cav1–/– mice to those in small intestine from control mice. We also examined the underlying mechanism of alteration in the signaling cascade downstream of beta-adrenoceptors.


   METHODS
TOP
 ABSTRACT
 METHODS
Immunohistochemistry
 RESULTS
 DISCUSSION
 REFERENCES
 
All animal experiments were conducted according to a laboratory animal protocol approved by our institutional Animal Policy and Welfare Committee.

Functional Studies

Preparation of the tissue. Male 6- to 8-wk-old caveolin-1 knockout (cav1–/–) and control [(B6 129 SF2/J) (cav1+/+)] mice (Jackson Laboratory, Bar Harbor, ME) were killed by cervical dislocation. After the abdominal wall was opened, the digestive tract, from the stomach to the rectum, was removed from the mouse and placed in a beaker of Krebs-Ringer solution. This contained (in mM): 115.5 NaCl, 21.9 NaHCO3, 11.1 D-glucose, 4.6 KCl, 1.16 MgSO4, 1.16 NaH2PO4, and 2.5 CaCl2, at room temperature (21–22°C), and had been preequilibrated with carbogen (95% O2 and 5% CO2). In a dissection dish, filled with Krebs-Ringer solution and continuously bubbled with carbogen, small intestinal tissue (jejunum) was isolated and cut into segments of 1–1.5 cm. The intestinal content, if any, was removed by gently rubbing the tissue segments with dissection forceps. The tissue segments were suspended longitudinally between two concentric platinum electrodes and tied to a hook at the bottom of the electrode holder with silk suture thread. The top of the tissue was also tied with thread and attached to a strain gauge (Grass FT-03). The tissue preparations were placed into jacketed tissue baths filled with 10 ml of Krebs-Ringer solution, bubbled continuously throughout the experiment with carbogen. The tissue baths were maintained at 37°C by water circulating through the glass jacket during the length of the experiment. The tissues were subjected to slight tension, adjusted to obtain the maximum amplitude of spontaneous phasic activity. Tissue contractile activities were recorded on a Grass model 7D polygraph.

Experimental protocols. Tissue segments were equilibrated in Krebs-Ringer solution for 15 min at the beginning of the experiments. Tetrodotoxin (10–6 M) was added to the solution at the beginning of the equilibration period to block enteric nerve activity. In preliminary experiments, this concentration was found to be sufficient to block responses to nerve activity elicited by electric field stimulation. In experiments to study the effects of beta-adrenoceptor antagonists and H-89 on the (–)-isoprenaline-induced relaxation, the compounds were added at the beginning of the equilibration period and left in contact with the tissue throughout the experiment. CGP20712A (10–7 M) was used to block beta1-adrenoceptors, ICI118551 (10–7 M) was used to block beta2-adrenoceptors, and SR59230A (10–7 M) was used to block beta3-adrenoceptors. H-89 (10–6 M) was used to study the effect of PKA block on the response to (–)-isoprenaline. After the equilibration period, the tissues were contracted by carbachol (10–6 M). A period of 15 min was allowed after the addition of carbachol for the stabilization of the induced tone. In control experiments, the carbachol-induced tone came to plateau after 10 min. Cumulative concentration-response curves (CRC) for (–)-isoprenaline were constructed by using nine doses separated by 0.5 log units (10–9–10–5 M). Similar CRCs were constructed for the selective beta3-agonist BRL37344. At the end of a CRC, the tissue was brought to a state of maximum relaxation by washing and incubation in calcium-free Krebs-Ringer solution containing 1 mM EGTA. The relaxing effect of each dose was calculated as a percentage of the decrease from the carbachol-induced tone before the addition of (–)-isoprenaline to the passive tone in calcium-free solution. Sigmoid dose-response curves with constant Hill slope were estimated with GraphPad Prism 4.0. The negative logarithms of the EC50 (pEC50) values were calculated and expressed as means ± SE. Statistical significance among pEC50 values was determined by unpaired t-test. A P value <0.05 was considered significant. In case of comparison of more than 2 pEC50 values, ANOVA was used followed by Bonferroni post hoc test.

In experiments in which H-89 (5 x 10–7 M) was added to some tissues at the beginning of the equilibration period, all tissues were contracted with 10–6 M carbachol after nerve blockade with tetrodotoxin. After 15 min, a single dose of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (di-bu cAMP, 10–4 M) or forskolin (10–6 M) was added. The relaxing effect was measured after 5 min following the addition of the drug and represented as a percentage of the relaxation brought about by calcium-free Krebs-Ringer solution with 1 mM EGTA added at the end of the experiment. The results are expressed as means ± SE. In all cases n represents the number of animals whose small intestine provided segments for the study. The statistical significance of the H-89 effect was determined by comparison to controls run side by side using unpaired t-test. A P value <0.05 was considered significant.

In a third type of experiments to examine the role of the different subtypes of beta-adrenoceptors in the regulation of the spontaneous pacing frequency in mouse small intestine, the tissues were incubated with tetrodotoxin or tetrodotoxin and one of the selective beta-adrenoceptor blockers for 15 min. (–)-Isoprenaline (10–6 M) was added, and the number of contractions per minute was determined before and after the addition of (–)-isoprenaline and compared by paired t-test.


   Immunohistochemistry
TOP
 ABSTRACT
 METHODS
 Immunohistochemistry
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue preparation and cryosections. Intestinal tissue was put in ice-cold oxygenated (95% O2 and 5% CO2) Krebs-Ringer solution. The jejunum was separated, pinned on a petri dish, and fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. The fixed jejunum was rinsed with 0.1 M sodium phosphate buffer and cryoprotected in 30% sucrose in phosphate-buffered saline (PBS). The cryoprotected jejunum was trimmed, embedded, and frozen for cryosection. Cryosections of 6-µm thickness were obtained by a cryostat (Leica CM 1850; Leica Microsystems, Richmond Hill, ON, Canada) and attached on a slide glass coated with 3-aminopropyltriethoxysilane (6).

Double-immunofluorescent labeling. For colocalization studies of two different proteins, double immunolabeling was accomplished by two different methods: 1) double labeling of primary antibodies from different host species and 2) double labeling of primary antibodies from the same host species as described previously (6).


DOUBLE-LABELING OF CAV1 AND B1-, B2, or B3-ADRENOCEPTORS. The cryosections were blocked with mixture of 10% normal donkey serum and 10% normal goat serum in PBS for 30 min to reduce nonspecific binding. Primary antibodies (mouse anti-Cav1 and rabbit anti-beta1-, rabbit anti-beta2-, or rabbit anti-beta3-adrenoceptor) from the different host species were mixed together and incubated at 4°C for 17 or 18 h. The cryosections were rinsed with 0.3% Triton X-100 in PBS twice every 10 min, followed by with PBS once for 10 min. Two different secondary antibodies conjugated with Cy3 and Alexa488 were mixed together and incubated for 1 h. The cryosections were washed with 0.3% Triton X-100 in PBS twice every 10 min, followed by a wash with PBS for 10 min.


DOUBLE-LABELING OF CAV1 AND PKAc. The cryosections were blocked with 10% normal donkey serum for 30 min. Cav1 and the catalytic subunit of PKA (PKAc) were from the same host animal (mouse). Therefore, Cav1 was designated as a first, primary antibody and then PKAc was designated as a second, primary antibody. Cav1 as the first, primary antibody was incubated for 17 or 18 h. The cryosections were washed with 0.3% Triton X-100 in PBS twice every 10 min, followed by wash with PBS once for 10 min. The secondary antibody, Fab fragment rabbit anti-mouse IgG, was applied for 17 to 18 h to convert the first, primary antibody into one recognized as rabbit. The cryosections were rinsed with 0.3% Triton X-100 twice every 10 min, followed by with PBS once for 10 min. Tertiary antibody, Alexa488-conjugated goat anti-rabbit IgG, was incubated for 1 h. The cryosection was rinsed with 0.3% Triton X-100 twice every 10 min, followed by with PBS once for 10 min. PKAc as the second, primary antibody was incubated for 17 or 18 h. The cryosection was rinsed with 0.3% Triton X-100 in PBS twice every 10 min, followed by PBS once for 10 min. Secondary antibody, Cy3-conjugated donkey anti-mouse IgG, was incubated for 1 h. The cryosection was rinsed with 0.3% Triton X-100 in PBS twice every 10 min, followed by with PBS once for 10 min.

The cryosections from methods 1 and 2 were mounted with aqueous mounting medium with anti-fading agent and covered with cover glass. The immunolabeled cryosections were observed by confocal laser scanning microscope (LSM 510, Carl Zeiss, Jena, Germany).

All antibodies, antigens, and normal sera used are summarized in Table 1. During the incubation with all primary antibodies, 1% normal donkey serum and 1% normal goat serum of total incubation volume was added. All incubations for antibodies and normal sera were performed at room temperature. To determine specificity of immunolabeling, primary antibody was omitted, or when the antigen was available, it was used to saturate the primary antibody.


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  		Table 1. Antibodies and normal sera

 
In the images obtained from confocal laser scanning microscope, mucosa and submucosa were eliminated and muscularis was extracted by Graphire 4 pen with pen tablet (Wacom, WA) and LSM 5 image (Carl Zeiss, Jena, Germany). All final images were enhanced by brightness, contrast, and gamma tool of LSM 5 image and edited by Adobe PhotoShop (Version 7.0, Adobe).

Western Blotting of Membrane Fractions

Tissue collection and preparation of fractions. Mouse jejunum was collected on ice-cold oxygenated Krebs-Ringer solution. The tissue was opened along the mesenteric border and the mucosa was scraped off. The remaining smooth muscle tissue was immediately frozen in liquid nitrogen and stored at –80°C. Tissues from 3 Cav1+/+ and 3 Cav1–/– mice were pooled. A lipid raft-rich membrane fraction was prepared as described previously (50). Briefly, tissues were homogenized on ice in 150 mM Na2CO3 containing a protease inhibitor cocktail using a Polytron homogenizer. Three 20-s strokes separated by 1 min cooling periods were used. Samples were fractionated by ultracentrifugation on a sucrose gradient at 180,000 g for 18 h at 4°C. Lipid raft-enriched fractions were collected from the interface of the 5 and 35% sucrose layers while heavier membrane fractions were collected from the lower half of the 45% sucrose layer.

Immunoblotting. Sample aliquots containing 40-µg proteins were blotted as described previously (24). Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane using Towbin's transfer buffer. The membranes were blocked by 5% skim milk in Tris-buffered saline for 2 h at room temperature, incubated with primary antibody (1:1,000 anti caveolin-1, and 1:1,000 anti-PKAc) in 5% skim milk for 2 h at room temperature. Horseradish peroxidase-conjugated secondary antibody was incubated with the membranes for 1 h at room temperature and protein expression was visualized by using an ECL chemiluminescence kit.

Materials. (–)-Isoprenaline, ICI118551, CGP20712A, SR59230A, BRL37344, forskolin, and di-bu cAMP were purchased from Sigma Canada (Oakville, ON). H-89 was purchased from Calbiochem (San Diego, CA). TTX was purchased from the Alomone Laboratories (Jerusalem, Israel). Antifading agent was from Biomeda (Foster, CA). Glass coverslips were from Electron Microscopy Sciences (Washington, PA). ECL kit was from GE Health Care (Baie d'Urfe, QC).

All the drugs used for functional experiments except SR59230A, forskolin, and tetrodotoxin were dissolved as stock solutions in distilled water and frozen at –20°C till used. Fresh dilutions were prepared in distilled water on the day of the experiment. SR59230A and forskolin were dissolved in dimethylsulfoxide, and tetrodotoxin was dissolved in 0.1 M acetic acid. These solvents in the quantities added (10 µl in 10 ml) did not alter the tissue function in preliminary experiments.


   RESULTS
TOP
 ABSTRACT
 METHODS
 Immunohistochemistry
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemistry

Double immunostaining for caveolin-1 and beta-adrenoceptors. We examined the distribution of the different subtypes of beta-adrenoceptors with respect to caveolin-1 in cryosections of cav1+/+ and cav1–/– mice small intestine. When the primary antibodies were omitted or a blocking antigen peptide was used to saturate the primary antibody, no immunoreactivity was detected. Cav1–/– cryosections lacked caveolin-1 immunoreactivity and thus showed immunoreactivity only for beta-adrenoceptors (Fig. 1). In cav1+/+, all three subtypes of beta-adrenoceptors showed a similar distribution. Their immunoreactivities were diffusely distributed in smooth muscles (circular and longitudinal) and myenteric plexus cells with only a limited colocalization with caveolin-1 in possible interstitial cells of Cajal in the deep muscular plexus, interstitial cells of Cajal in the myenteric plexus, and some epithelial cells in the serosal layer (Fig. 1, c, i, and o). In cav1–/–, the receptors had a distribution similar to that in cav1+/+ and the immunofluorescence showed a comparable intensity (Fig. 1, e, k, and q).


Figure 1
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  		Fig. 1. Double immunohistochemical staining of caveolin-1 (red) and beta-adrenoceptors (green). a–f: Immunohistochemical staining for caveolin-1 and beta1-adrenoceptors in cav1+/+ (a–c) and cav1–/– (d–f). g–l: Immunohistochemical staining for caveolin-1 and beta2-adrenoceptors in cav1+/+ (g–i) and cav1–/– (j–l). m–r: Immunohistochemical staining for caveolin-1 and beta3-adrenoceptors in cav1+/+ (m–o) and cav1–/– (p–r). Colocalization is indicated in c, f, i, l, o, and r by yellow color. Arrowheads indicate colocalization in possible interstitial cells of Cajal in the deep muscular plexus, thin arrows indicate colocalization in the myenteric plexus interstitial cells of Cajal, and thick arrows indicate colocalization in epithelial cells in the serosal layer. The scale bar is 20 µm. icm, inner circular muscle; ocm, outer circular muscle; lm, longitudinal muscle; mp, myenteric plexus; bv, blood vessel.

 
Double immunostaining for caveolin-1 and PKA. We examined the distribution of the catalytic subunit of PKA immunoreactivity in cryosections from both cav1+/+ and cav1–/– with respect to caveolin-1. When the primary antibodies were omitted or a blocking antigen peptide was used to saturate the primary antibody, no immunoreactivity was detected. In cav1+/+, the PKA catalytic subunit immunoreactivity was mainly localized to the cell membrane of smooth muscles (Fig. 2b). It showed a close colocalization with caveolin-1 in the cell membrane (Fig. 2c). In cav1–/–, no immunoreactivity to caveolin-1 was detected (Fig. 2d); however, the immunoreactivity to PKA catalytic subunit persisted in the cell membrane (Fig. 2e). The immunofluorescence due to PKA catalytic subunit immunoreactivity was of a comparable intensity in cav1–/– and cav1+/+.


Figure 2
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  		Fig. 2. Double immunohistochemical staining for caveolin-1 and the catalytic subunit of PKA (PKAc). a–c: Immunostaining for caveolin-1 and PKAc in cav1+/+ tissue. c: Colocalization as a yellow color in the membrane of smooth muscle cells, possible interstitial cells of Cajal in the deep muscular plexus (indicated by arrow heads), interstitial cells of Cajal in the myenteric plexus (indicated by thin arrows), and epithelial cells in the serosal layer (indicated by thick arrows). d–f: Immunostaining for caveolin-1 and PKAc in cav1–/– tissue. The scale bar is 10 µm.

 
Functional Experiments

Contractile response to carbachol. Upon treatment with carbachol (1 µM), mouse small intestinal tissue segments responded by a sustained tonic contraction, i.e., the phasic contractions persisted but at a higher tone. The tonic contraction level (after relaxation of a phasic contraction) came to a plateau within 10 min. The magnitudes of the carbachol-induced tone measured at 15 min were 37.21 ± 4.66 in cav1+/+ (n = 11) and 33.63 ± 5.17 in cav1–/– (n = 13) and were not significantly different.

Effect of (–)-isoprenaline. The nonselective beta-agonist (–)-isoprenaline produced a dose-dependent relaxation of the tissue segments contracted with carbachol in both cav1+/+ and cav1–/–. However, the response to (–)-isoprenaline in cav1+/+ segments started at lower concentration than in cav1–/– segments (Fig. 3A). The CRC of (–)-isoprenaline in the cav1–/– (pEC50: 6.94 ± 0.09) was shifted significantly (P < 0.01) to the right with respect to the cav1+/+ CRC (pEC50: 7.60 ± 0.15) (Fig. 3B).


Figure 3
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  		Fig. 3. The relaxing effect of (–)-isoprenaline in small intestinal tissue segments from cav1+/+ and cav1–/– mice contracted with 10–6 M carbachol. A: representative tracings of the effect of (–)-isoprenaline in cav1+/+ and cav1–/–. Note that relaxation started at a lower concentration in cav1+/+ compared with cav1–/–. B: concentration response curve of (–)-isoprenaline in cav1+/+ compared with cav1–/– (n = 6). The relaxing effect was measured as the tonic amplitude (the relaxed phase of phasic contractions).

 
Effects of selective beta-antagonists on the relaxation due to (–)-isoprenaline. To evaluate the relative roles of the different beta-receptor subtypes in the (–)-isoprenaline-induced relaxation in the mouse small intestine in the presence or absence of caveolin-1, we examined the effects of selective beta-adrenoceptor antagonists on the (–)-isoprenaline-induced relaxation. The concentrations of the antagonists used were based on those reported in previous work on mouse intestine (16). In cav1+/+, 100 nM of CGP20712A, a selective beta1-antagonist, significantly shifted the CRC of isoprenaline to the right (pEC50: control, 7.60 ± 0.15; CGP20712A, 7.08 ± 0.14; P < 0.05) (Fig. 4A). However, in cav1–/–, CGP20712A did not significantly shift the CRC of (–)-isoprenaline (pEC50: control, 6.94 ± 0.09; CGP20712A, 7.27 ± 0.09) (Fig. 4A). The selective beta2-adrenoceptor antagonist ICI118551 (100 nM) had the same effect as the beta1-antagonist. It slightly shifted the CRC of (–)-isoprenaline in cav1+/+ (pEC50: control, 7.60 ± 0.15; ICI118551: 6.95 ± 0.15, P < 0.05) and had no significant effect on the CRC in cav1–/– (pEC50: control, 6.94 ± 0.09; ICI118551: 6.72 ± 0.13) (Fig. 4B). The greatest shift in the CRC of (–)-isoprenaline in cav1+/+ small intestinal tissue was obtained with the selective beta3-adrenoceptor antagonist, SR59230A (100 nM) (pEC50: control, 7.60 ± 0.15, SR59230A: 6.39 ± 0.13, P < 0.001) (Fig. 4C). SR59230A (100 nM) did not affect the CRC in cav1–/– (pEC50: control, 6.94 ± 0.09, SR59230A: 6.71 ± 0.09) (Fig. 4C). The shift in the CRC in cav1+/+ imparted by SR59230A was higher than that imparted by CGP20712A (P < 0.01) and ICI118551 (P < 0.05) when measured in terms of pEC50.


Figure 4
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  		Fig. 4. Effect of selective beta-adrenoceptor antagonists on the (–)-isoprenaline-induced relaxation in small intestinal tissue segments from cav1+/+ and cav1–/– mice contracted with 10–6 M carbachol. A: effect of the selective beta1-adrenoceptor antagonist CGP20712A. B: effect of the selective beta2-adrenoceptor antagonist ICI118551. C: effect of the selective beta3-adrenoceptor antagonist SR59230A (n = 6 for all experiments).

 
Responses to BRL37344. We examined the effects of the selective beta3-agonist, BRL37344, on the carbachol-induced tone in mouse small intestine. It caused a dose-dependent relaxation of the carbachol-induced tone in cav1+/+ (pEC50: 6.49 ± 0.12) (Fig. 5). In the cav1–/– tissue, the CRC of BRL37344 was shifted to the right (pEC50: 5.55 ± 0.19, P < 0.01) (Fig. 5) with respect to the CRC in cav1+/+.


Figure 5
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  		Fig. 5. Concentration response curves of the selective beta3-agonist BRL37344 in small intestinal tissue segments from cav1+/+ and cav1–/– mice precontracted with carbachol (n = 6).

 
Effect of H-89 on (–)-isoprenaline-induced relaxation. We used H-89 to examine the role of PKA in the (–)-isoprenaline-induced relaxation in mouse small intestine. In cav1+/+ tissue, H-89 drastically shifted the CRC of (–)-isoprenaline to the right (pEC50: control, 7.60 ± 0.15; H-89: 6.02 ± 0.22, P < 0.001) (Fig. 6). However, in cav1–/– treatment with H-89 did not affect the CRC of (–)-isoprenaline significantly (pEC50: control, 6.94 ± 0.09; H-89: 6.75 ± 0.19) (Fig. 6).


Figure 6
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  		Fig. 6. Effect of the PKA inhibitor H-89 (1 µM) on the (–)-isoprenaline-induced relaxation in small intestinal tissue segments from cav1+/+ (A) and cav1–/– (B) mice contracted with 10–6 M carbachol (n = 6).

 
Effect of H-89 on relaxation due to forskolin. The direct activator of adenylyl cyclase, forskolin, produced a relaxation of the carbachol-induced tone in both cav1+/+ and cav1–/– tissue (Fig. 7A). In cav1+/+, the relaxation was reduced when the tissue was pretreated with H-89. On the other hand, H-89 pretreatment did not affect the relaxation due to forskolin in cav1–/– tissue.


Figure 7
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  		Fig. 7. Effect of the PKA inhibitor H-89 (0.5 µM) on the forskolin- (A) and N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (di-bu cAMP)-induced (B) relaxation in small intestinal tissue segments from cav1+/+ and cav1–/– mice contracted with 10–6 M carbachol (n = 6). Statistical significance was measured by the unpaired t-test and denoted by **P < 0.01 and ***P < 0.001.

 
Effect of H-89 on relaxation due to di-bu cAMP. We studied the effect of di-bu cAMP, a membrane permeable analog of cAMP, on the carbachol-induced tone in intestinal tissue. Di-bu cAMP produced a relaxation in cav1+/+ tissue, which was greater than its effect in cav1–/– (P < 0.05). H-89 almost abolished the relaxation due to di-bu cAMP in cav1+/+ but had no significant effect in the cav1–/–. In addition, the response of the cav1+/+ tissue pretreated with H-89 to di-bu cAMP became similar to the control response in cav1–/– (Fig. 7B).

Effect of (–)-isoprenaline on spontaneous frequency of pacing. To examine the effect the role of beta-adrenoceptors in the regulation of the frequency of spontaneous pacing by interstitial cells of Cajal in mouse small intestine, we treated the spontaneously contracting tissue with (–)-isoprenaline in presence and absence of selective beta-adrenoceptor blockers. The frequencies of pacing, measured as the number of contractions per minute, were not affected by the addition of (–)-isoprenaline in control tissues and in those treated with the selective beta-blockers in both cav1+/+ and cav1–/–. In addition, treatment with beta-blockers did not produce any change in the spontaneous frequency of pacing (data not shown).

Western Blotting

Probing for the PKA catalytic subunit showed that it was expressed in the lipid raft-rich membrane fractions in cav1+/+ jejunal smooth muscles and in the heavier membrane fraction of the two strains. In cav1–/–, PKA catalytic subunit expression was very much reduced in the lipid raft fraction compared with cav1+/+. Caveolin-1 expression was observed only in the lipid raft-rich membrane fraction of cav1+/+. Figure 8 shows a representative blot.


Figure 8
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  		Fig. 8. A representative blot showing the expression of caveolin-1 (22–24 kDa) and PKA catalytic subunit (40 kDa) in the lipid raft-rich (LRF) and the lower heavier (LF) membrane fraction in cav1+/+ and cav1–/– mice jejunal smooth muscles. A 40-µg protein aliquot was loaded in each lane, which was the lowest amount necessary to show PKA in cav1–/– LRF. Note the reduced expression of PKA catalytic subunit in the cav1–/– LRF compared with cav1–/–.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 Immunohistochemistry
 RESULTS
 DISCUSSION
REFERENCES
 
Cav1–/– mice offer a convenient model to study the alterations in the functions of cellular components that are associated with caveolin-1. Previous evidence suggests that the function of beta-adrenoceptors is related to caveolae and caveolins in the heart (13, 31, 38). Some reports also showed that such interactions occurred in cultured cell lines (2, 42). Although, beta-adrenoceptors have an important modulatory role on GIT movement, the relationship between their function in the GIT and caveolae and caveolin-1 has not been examined to date. In this study, we used cav1–/– mice tissue for that purpose.

Initially, we found that the CRC representing the relaxation induced by (–)-isoprenaline in tissues contracted with carbachol was shifted to the right in cav1–/– tissues compared with cav1+/+. We considered this result to be related to the function of beta-adrenoceptors expressed on smooth muscle cells since our experiments were conducted after nerve blockade with tetrodotoxin. Considering that beta-adrenoceptors were reported to be localized in caveolae (33, 38, 42), a possible explanation could have been a reduction in the expression of beta-adrenoceptors on smooth muscle cells in cav1–/– due to the absence of caveolae. However, using immunohistochemistry, we found that the different subtypes of beta-adrenoceptors are similarly present in cav1–/– and cav1+/+ tissues. Both the distribution and immunofluorescence intensity were similar to those in cav1+/+ tissue. The persistence of these receptors in the small intestinal tissues from cav1–/– mice could be related to our observation that they were not colocalized with caveolin-1 in the small intestinal tissue of cav1+/+ control mice and that localization of a particular G-protein-coupled receptor to caveolae is cell type dependent (32). A previous study on cav1–/– mice (7) showed that they retained the beta-adrenoceptors in adipocytes.

Consequently, we concluded that the defect in the beta-adrenoceptor function in cav1–/– small intestine lay in the signal transduction pathway downstream of these receptors. We examined the roles of the different beta-adrenoceptor subtypes in the (–)-isoprenaline-induced relaxation and whether the functional defect was in all or some of them. Selective beta-adrenoceptor antagonists were used to block the relaxation of carbachol-contracted tissue by (–)-isoprenaline. In cav1+/+, both beta1- and beta2-antagonists shifted the CRC of (–)-isoprenaline to the right, slightly but significantly suggesting that they might have a role in the relaxation due to (–)-isoprenaline. However, similar to previous observations in mouse intestine (16), beta3-adrenoceptors play the major role in the relaxation due to (–)-isoprenaline. On the other hand, none of the selective beta-adrenoceptor blockers appeared to have a significant effect on the relaxation due to (–)-isoprenaline in cav1–/– small intestine. This suggests that the lower relaxing effect of (–)-isoprenaline observed in cav1–/– might be due to an altered response of all three beta-adrenoceptor subtypes. A failure of the three beta-adrenoceptor antagonists to shift the CRC of (–)-isoprenaline in cav1–/– muscle at the concentration used (100 nM), despite the persistence of a response to (–)-isoprenaline, might be due to the requirement for a higher concentration of antagonist in the cav1–/– muscle. Note that higher concentrations of (–)-isoprenaline were required to show responses in the cav1–/– tissues. This suggestion is supported by the observation that in the cav1+/+ tissues in presence of the selective antagonists, the same high concentrations of (–)-isoprenaline produced responses similar to those of cav1–/–. Moreover, the (–)-isoprenaline responses in cav1–/– were still sensitive to higher concentrations of the selective antagonists. A 10-fold higher concentration of any of these antagonists shifted the CRC of (–)-isoprenaline significantly in both cav1+/+ and cav1–/–. However, as the use of such high concentrations of antagonists interferes with their selectivity, their effects were not mutually distinguishable and were similar to the effects of a similar concentration (1 µM) of the nonselective beta-adrenoceptor antagonist timolol in both strains (data not shown).

Another possible explanation could be a change in the beta-adrenoceptors conformational properties in the absence of caveolin-1 rendering them relatively insensitive to the antagonists at the concentration used (100 nM). However, a future, detailed study of the changes of receptor affinity and conformation in cav1–/– is necessary to test this assumption. A third possibility might be that the activation of {alpha}-adrenoceptors as well as altered beta-adrenoceptors occurred at the higher concentrations of (–)-isoprenaline, but this is unlikely, as the CRCs were best fit by a Hill slope of 1. So far, {alpha}-adrenoceptor antagonists have not been tested.

Furthermore, to specifically examine the function of beta3-adrenoceptor, the subtype that is mainly responsible for relaxation due to (–)-isoprenaline in mouse small intestine, we used a selective beta3-agonist, BRL37344. Relaxation to BRL37344 was reduced in cav1–/– segments compared with cav1+/+, again indicating a reduction in the beta3-adrenoceptor activity.

To examine further the defect in the beta-adrenoceptors signaling in the cav1–/– small intestinal tissue, we studied the effect of H-89, the PKA inhibitor, on the (–)-isoprenaline-induced relaxation. beta-Adrenoceptors function through the activation of PKA (41). In the GIT, beta3-adrenoceptors were shown to effect relaxation by a mechanism involving an increase in cAMP (20). In cav1+/+ tissues, H-89 caused about a 40-fold increase in the EC50 of (–)-isoprenaline, indicating that the relaxation observed is due to the activation of PKA downstream of beta-adrenoceptors. Conversely, in cav1–/– H-89 did not have a significant effect on the CRC of (–)-isoprenaline, indicating that the reduced response to (–)-isoprenaline might be due to a decreased PKA function. To investigate this possibility further, we examined the effect of H-89 on the relaxation of carbachol-contracted tissues due to forskolin, a direct adenylyl cyclase activator, and di-bu cAMP, a membrane-permeable analog of cAMP. Only in cav1+/+ tissue did H-89 reduce the relaxation due to forskolin and di-bu cAMP. The relaxation due to di-bu cAMP in cav1–/– tissues was very small in tissues not treated with H-89; in fact, it was similar to the response of cav1+/+ control tissues treated with H-89. Taken together, these results suggest a reduced PKA function in the small intestinal tissue of cav1–/– mice. Although activation of PKC and MAP kinases and mobilization of intracellular calcium have been reported to mediate some of the beta-adrenoceptors responses (54, 46, 41), this has not been reported to occur in mouse intestinal smooth muscles. Neither has it been shown to happen downstream of beta3-adrenoceptor (53). In any case, the concentrations of H-89 used in our experiments (0.5 and 1 µM) are highly selective for inhibition of PKA and show no appreciable activity against PKC, calcium/calmodulin-dependent kinases, and MAP kinases (14, 10). In addition, blockade by H89 of the relaxations to mediators of different stages in the signal transduction sequence (beta-adrenoceptor activation, adenylyl cyclase activation, and PKA activation) further strengthens our hypothesis that PKA function is reduced in cav1–/– small intestine.

The relaxation elicited by forskolin and di-bu cAMP in cav1–/– could be attributed to PKA-independent effects. These have also been shown to exist in cav1+/+ mice as a relaxation equivalent to that seen in the knockout mice persisted in cav1+/+ tissues treated with H89. cAMP production by nonselective activation of adenylyl cyclase, i.e., through the use of forskolin is not expected to be compromised in cav1–/– because many adenylyl cyclase isoforms are known to exist mainly outside of caveolae (32). Forskolin and cAMP were shown to elicit smooth muscle relaxation by opening of calcium-activated potassium channels through mechanisms that are not dependent on PKA activation (57, 4). In addition, cAMP was postulated to produce relaxation in the intestinal smooth muscles by PKA-independent mechanisms such as the activation of uncoupling protein 1 (45) or cross activation of PKG (28). In previous studies, we showed that smooth muscle relaxation downstream of nitric oxide is reduced in cav1–/– as a result of a defective soluble guanylate cyclase function (11, 12), but the function of PKG was not tested.

Immunohistochemical staining of the catalytic unit of PKA showed that it was colocalized with caveolin-1 in the cell membrane of smooth muscles in cav1+/+ small intestine. This was similar to other results (27) in which PKA was shown to be colocalized with caveolin-1 in aortic rings. Caveolin-1 interacted directly with PKA (25, 35, 36), and this interaction was necessary for the modulation of cAMP-mediated signal transduction (36). In cav1–/–, PKA immunoreactivity persisted despite the absence of caveolae. However, further examination of the expression of PKA showed that the distribution of PKA within the membrane domains is not identical in cav1+/+ and cav1–/–. In the presence of caveolin-1, PKA is concentrated in the lipid raft membrane fraction, i.e., in close association with caveolin-1 and caveolae. In contrast, in the absence of caveolin-1 very little PKA is expressed in these domains, providing more evidence to support a role of caveolin-1 in the regulation of PKA in small intestinal tissue. The persistence of PKA in cav1–/– in general is in agreement with previous findings in these mice (7), in which PKA expression persisted in adipocytes. In that study, it was shown that the main role of caveolin-1 was to recruit the target proteins to be phosphorylated by PKA downstream of beta3-adrenoceptor activation. This might be the case in the present study, in which the absence of caveolin-1 did not alter the expression of PKA but clearly reduced its activity in relaxing smooth muscle as shown in functional experiments. Potential target proteins that may be recruited by caveolin-1 for phosphorylation by PKA to produce smooth muscle relaxation include inositol triphosphate receptor, plasma membrane calcium pump, myosin light chain kinase, phospholamban (54), and ATP-dependent K+ channels (22).

Nevertheless, the PKA phosphorylation of intracellular targets is not the only signal transduction mechanism downstream of beta-adrenoceptors. In addition to the previously mentioned mechanisms by which cAMP can elicit PKA-independent relaxation, several other mechanisms have been suggested to be associated with beta-adrenoceptor activation. Among these mechanisms are activation of extracellular signal-related kinases 1 and 2 (ERK 1 and 2) (15), activation of cytosolic phospholipase A2 (1), and the activation of calcium-activated potassium channels by Gs{alpha} that occurs independent of cAMP and PKA in a membrane delimited manner (23). Any of these mechanisms could be active in cav1–/– tissue to cause relaxation despite the reduced PKA activity and account for the residual response to beta-adrenoceptor agonists. These assumptions must be tested by future studies of the nature of (–)-isoprenaline-induced relaxation in cav1–/–.

Finally, beta-adrenoceptors were reported to have a role in the regulation of pacemaker currents generated by cultured interstitial cells of Cajal from mouse small intestine (18). However, our experiments show that the different beta-adrenoceptor subtypes did not have any effect on the frequency of pacing in intact tissue segments. Indeed, the properties of pacing differ considerably between intact tissue and cultured interstitial cells of Cajal as we reported earlier (5).

To conclude, our present results suggest that the beta-adrenoceptor function in the mouse small intestine is reduced due to caveolin-1 knockout. The absence of caveolae might have reduced the relaxation response seen as a result of beta3-adrenoceptor activation. This could be due to the reduced activity of PKA that requires the recruitment of target proteins by caveolin-1 to elicit full activity.


   ACKNOWLEDGMENTS
 
Preliminary report of this work was presented in Experimental Biology 06, San Francisco, California. A. F. El-Yazbi is a Killam Trusts Scholar.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. E. Daniel, 9–10 Medical Sciences Bldg., Univ. of Alberta, Edmonton, AB T6G 2H7, Canada (e-mail: edaniel{at}ualberta.ca)

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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