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Am J Physiol Gastrointest Liver Physiol 290: G394-G403, 2006. First published September 15, 2005; doi:10.1152/ajpgi.00321.2005
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NEUROREGULATION AND MOTILITY

Impact of caveolin-1 knockout on NANC relaxation in circular muscles of the mouse small intestine compared with longitudinal muscles

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

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

Submitted 13 July 2005 ; accepted in final form 12 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recently, we showed that caveolin-1 (cav1) knockout mice (Cav1–/– mice) have impaired nitric oxide (NO) function in the longitudinal muscle (LM) layer of the small intestine. The defect was a reduced responsiveness of the muscles to NO compensated by an increase in the function of apamin-sensitive, nonadrenergic, noncholinergic (NANC) mediators. In the present study, we examined similarly the effects of cav1 knockout on the relaxation in circular muscle (CM) of the mouse small intestine. CM of Cav1–/– mice also showed defective NO function, but less than in LM, as well as more activation of apamin-sensitive NANC mediators. CM of Cav1–/– mice, like LM, lacked cav1 but retained small amounts of cav3 and caveolae in the outer CM layer. In addition, we also examined the effects of a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-{alpha}]quinazolin-1-one (ODQ), on electric field stimulation (EFS)-mediated relaxation in both LM and CM. ODQ had an effect similar to the block of NO synthesis. Moreover, we compared the actions of two NO donors in the LM and CM of control and Cav1–/– mice. Similar to LM, CM of Cav1–/– mice showed a reduced responsiveness to the NO donors sodium nitroprusside and S-nitroso-N-acetyl penicillamine. However, both ODQ and apamin blocked the inhibitory effects of the NO donors in LM, whereas apamin had no effect in CM. In conclusion, cav1 knockout affects NO function in both LM and CM, but its effects in CM differ significantly.

caveolin-3; caveolae; nitric oxide donors; gastrointestinal tract pacing; nitric oxide; apamin

NONADRENERGIC, NONCHOLINERGIC NEURONS (NANC neurons) play an important role in the control of gut motility. They mediate most of the inhibitory responses in the gastrointestinal tract (GIT) and control many physiological reflexes, e.g., relaxation of the lower esophageal sphincter after swallowing, receptive relaxation of the proximal stomach during eating, and descending inhibition in response to distention (44). NANC neurotransmitters include nitric oxide (NO) (5, 44), ATP (6, 55), vasoactive intestinal peptide (VIP) (24), pituitary adenylate cyclase-activating peptide (PACAP) (47), and carbon monoxide (37).

NANC inhibitory neurotransmitters induce smooth muscle relaxation, often by evoking inhibitory junction potentials (30, 42, 53). These result from a direct or indirect action on K+ and/or Cl– channels (30, 10). Several intracellular mediators, e.g., cGMP, cAMP, and intracellular calcium, have been reported to be involved in the indirect effect of NANC inhibitory neurotransmitters on K+ and Cl– channels (2, 41, 43). Signaling proteins that are involved in the production and regulation of these mediators such as heterotrimeric G proteins, protein kinase C isoforms, and NO synthase (NOS) isoforms have been reported to bind to caveolin-1 (cav1) (17, 35, 49, 19, 46).

Cav1, -2, and -3 are a family of integral membrane proteins (21–24 kDa) that are the principal components of the caveolar membrane in vivo (8, 39). In smooth muscle and interstitial cells of Cajal (ICC), the most important caveolin is cav1 (12, 14). They form homo- and heterooligomers that insert in the inner leaflet of plasma membrane to form the characteristic flask-shaped caveolae (47). These membrane invaginations serve as membrane-organizing centers. Specialized motifs in the caveolins function to recruit lipids and proteins to caveolae for participation of intracellular trafficking of cellular components and operation in cellular transduction (25). Accordingly, caveolins may play a role in the regulation of the inhibitory responses to NANC neurotransmitters via the control of the function or the formation of the aforementioned intracellular mediators.

The role of cav1 in the regulation of the function of NO has been previously described. Cav1 has been reported to regulate the function of the different NOS isoforms (19, 21, 22, 54). Cav1 interaction with endothelial NOS inhibits NO production in vitro (29) and in vivo (4). In the jejunum and lower esophageal sphincter of the mouse gut, we have shown that cav1 is colocalized with myogenic NOS, a splice variant of neuronal NOS (nNOS) (27) in smooth muscle and ICC. ICC play a crucial role in nerve to muscle signal transmission (40). This suggested a possible role for cav1 in the regulation of NO activity in the murine small intestine.

Cav1 knockout mice (Cav1–/– mice) are reported to lack morphologically identifiable caveolae in the tissues known to express cav1 (38). They show a number of abnormalities: defects in caveolar endocytosis, lung hypercellularity, decreased vascular tone, and atrophic fat pads (38). Moreover, cells and tissues from these mice exhibit behaviors different from wild-type ones, showing defects in the glycosyl phosphatidyl inositol-anchored and lipid-modified proteins (50).

These mice offer a convenient model to study the role of cav1 in the regulation of the function of NANC mediators by examining the changes in their function in Cav1–/– mice. In a recent study (16), we have shown that Cav1–/– mice have a defective NO function in the longitudinal muscles (LM) of the small intestine. N{omega}-nitro-L-arginine (L-NNA) was nearly ineffective to inhibit relaxation, and the exogenous NO donor sodium nitroprusside (SNP) relaxed LM less than in controls. The reduced responsiveness was likely due to a defect in the transduction pathway downstream of the NO and before the level of cGMP-activated effectors. We also showed that an increased activity of the apamin-sensitive mediators could be the reason for the maintenance of a normal response to electrical field stimulation (EFS) in Cav1–/– mice. These results indicated that the cav1 absence impacted not only the function of NO but also other apamin-sensitive NANC mediators. However, this study examined only LM. Whereas LM innervation derives mainly from the myenteric plexus, that of CM derives mainly from the deep muscular plexus. Also, in the absence of NANC conditions, our earlier studies showed that CM, but not LM, was under an inhibitory control provided by release of NO from nerves (13). Thus differences are likely to exist.

The aim of the present study was to examine the effects of cav1 knockout on NO-mediated neurotransmission in circular muscles (CM) and compared these with the changes occurring in the LM. In addition, we examined the possible changes in the function of other NANC mediators that might be imparted by cav1 knockout in CM. Moreover, we also examined the changes imparted by cav1 knockout on the effects of exogenous NO donors in CM. Several reports have pointed out that different NO donors produce different patterns of responses in the GIT (23, 26, 52). However, no data exist to compare the effects of NO donors in CM versus LM. Thus, in the present study, we examined the effect of cav1 knockout on two NO donors, and we also compared their differential responses in CM and LM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 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 Cav1–/– (cav<tm 1 M Is>/J) and control [(B6 129 SF2/J) (Cav+/+)] mice (Jackson Laboratories; Bar Harbor, ME) were killed by cervical dislocation. After the abdominal wall was opened, the digestive tract, beginning from the level of the stomach to the rectum, was removed from the mouse, immediately placed into a beaker of Krebs-Ringer solution containing (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 preequilibrated with carbogen (95% O2-5% CO2). In a dissection dish filled with Krebs-Ringer solution continuously bubbled, small intestinal tissue (ileum and jejunum) was isolated and cut into ~0.5-cm (for CM) and 1- to 1.5-cm (for LM) segments. In the case of LM, the intestinal content, if any, was pushed out by gently rubbing the segments with dissection forceps. To study LM contractions, the tissue segment was placed between two platinum concentric 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). To study CM contraction, the open side of a thin metal triangle hook was slid through the lumen of the tissue segment. The triangle was then hooked together. A stainless steel rod attached to the bottom of the electrode holder was inserted into the lumen of the tissue under the metal triangle. Suture thread, attached to the apex of the triangle opposite to the tissue, was tied to the strain gauge. Two thin platinum rods, situated parallel to and on either side of the tissue, were used to stimulate the tissue electrically. The muscle preparations were placed into muscle baths filled with 10 ml Krebs-Ringer solution, bubbled continuously through the experiment, and maintained at a temperature of 37°C. The tension was increased or decreased slowly until the tension that produced the maximum amplitude of spontaneous phasic activity was reached. Tissue contractile activities were recorded on a Grass Model 7D Polygraph.

Experimental Protocols

In the experiments conducted under NANC conditions, the tissue was equilibrated in atropine (10–7 M), timolol (10–6 M), and prazosin (10–6 M) for 20 min. In some experiments, EFS for a duration of 10 s (parameters: 50 V·cm–1 and 0.5-ms pulse duration) was carried out at 1, 3, 10, and 30 pulses/s with a 10-min interval between each series. In the experiments not run under NANC conditions, the tissue was equilibrated in Krebs-Ringer solution for 20 min followed by EFS at 5 pulses/s to check the nerve activity. Later on in the experiment, TTX (1 µM) was added, and EFS was repeated after 5 min to check the blockade of nerve activity. In case of any residual nerve activity, the amount of TTX was increased until all the activity was blocked. At the end of each experiment, all tissues were washed twice with 10 ml Ca2+-free Krebs with 1.0 mM EGTA. This relaxed the tissue to basal passive tension and abolished spontaneous contractions. This basal passive tension was used as a zero line for measurement of the amplitude.

Experimental Procedures

Experiments on CM. In some experiments, CM segments were set up under NANC conditions, and EFS was carried out following the equilibration period at the four frequencies. The effects of L-NNA (100 µM), apamin (1 µM), 1H-[1,2,4]oxadiazolo-[4,3-{alpha}]quinazolin-1-one (ODQ; 1 µM), and a combination of L-NNA and apamin at the same concentrations on EFS were determined by adding these agents at the beginning of the equilibration period followed by EFS at the four frequencies after the equilibration period. The results were compared with a time control run side by side. In other experiments, S-nitroso-N-acetyl penicillamine (SNAP) and SNP were added to CM segments directly after the equilibration period. In some of these experiments, the effects of the previous agents were examined in tissues treated with ODQ (1 µM), apamin (1 µM), or a combination of both at the same concentrations, which were added at the beginning of the equilibration period. SNP was added at only a single dose of 100 µM to avoid tolerance. SNAP was added cumulatively in three doses that amounted to total concentrations of 1, 10, and 100 µM.

Experiments on LM. In some experiments, LM segments were set up under NANC conditions. ODQ (1 µM) was incubated with the tissue for 20 min following an EFS cycle at the four frequencies. Another cycle of EFS was carried out after the incubation period. The inhibitory responses to EFS after ODQ were compared with those before ODQ. In other experiments, SNP (100 µM), SNAP (100 µM), and 8-bromoguanosine-3',5'-cyclic monophosphate (BCGMP; 100 µM) were added to LM segments set up under NANC conditions directly after the 20-min equilibration period. These doses, based on preliminary experiments, lie in the just submaximal range. The addition of lower concentrations did not result in a clear and consistent response, and the repetitive addition of lower concentrations produced tolerance in the tissue that was sometimes associated with total absence of response to these agents at the 100 µM dose. In these experiments, the effects of the previous agents were examined in tissues treated with ODQ (1 µM) or apamin (1 µM), which were added at the beginning of the equilibration period.

Some of the LM experiments were not conducted under NANC conditions. In these experiments, the nerve activity was tested as mentioned above. SNAP (100 µM) was added to the tissues 10 min later. Ten minutes after the tissue had been washed, recovered, and were nerve blocked, another 100 µM dose of SNAP was added.

Data Analysis

Amplitudes of the spontaneous contractions and the inhibitory phases were measured as the values above the passive tension determined at the end of the experiments. They were measured as the mean of individual contractions over at least 15 contractions. Inhibitions in response to EFS, SNP, SNAP, and BCGMP were calculated by normalizing the amplitude in the inhibitory phase to the amplitude of the muscle activity directly precedent to the inhibitory stimulus (taken as 100%). Frequencies of muscle contractions were measured over a period of at least 20 s. The measurements were entered into GraphPad Instat and statistically tested using ANOVA with the Bonferroni post hoc test, paired t-test, or unpaired t-test, whichever appropriate. A P value of <0.05 was considered to be statistically significant; n values represent the numbers of mice whose intestines provided segments for study.

Ultrastructural Study

The jejunal tissues of Cav1+/+ and Cav1–/– mice were prefixed in a mixed fixative as described previously (14). The tissues were dissected to prepare sample size of 1.0 x 4.0 mm (LM layer x CM layer) and washed in 0.075 M sodium cacodylate buffer (pH 7.4) three times every 5 min at room temperature. The samples were en bloc stained in saturated (1%) uranyl acetate in 70% ethanol overnight at 4°C, postfixed in 1% OsO4 in 0.05 M sodium cacodylate buffer for 2 h at 4°C, dehydrated in graded ethanol, infiltrated in mixture of propylenoxide and TAAB 812 resin, and then embedded in TAAB 812 resin including 1.5% 2,4,6-tri-(dimethylaminomethyl)phenol (DMP-30) overnight at 60°C. Ultra-thin sections were cut, loaded on 200-mesh copper grid or 100-mesh grids coated with 0.25% formvar solution in ethylene dichloride, and stained with 4% uranyl acetate in 50% ethanol and Reynold's lead citrate. The grids were examined in a Philips 410 electron microscope equipped with a charge-coupled device camera (MegView III) at 80 kV.

Immunohistochemical Studies

Tissue preparation and cryosection. The jejunal tissues of Cav1+/+ and Cav1–/– mice were opened along the mesenteric border and pinned on a petri dish of Sylgard silicon rubber filled with oxygenated ice-cold 0.1 M sodium phosphate buffer (0.1 M PB; pH 7.4). The tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M PB for 4 h at room temperature and cryoprotected in 30% sucrose in PB including 0.1 NaN3 overnight at 4°C. Cryosections of 6 µm thickness were obtained by a cryostat (Leica CM 1850). The cryosections were attached to a glass slide coated with 2% 3-aminopropyltriethoxysilane in acetone and dried for 2 h at room temperature. The dried cryosections were used for double immunolabeling.

Double immunolabeling. Cryosections were washed in 0.3% Triton X-100 in PBS (pH 7.0) three times every 10 min. The cryosections were incubated in 10% normal donkey serum to block nonspecific binding proteins for 1 h at room temperature. Primary antibodies, mouse anti-cav3 and guinea pig anti-rat nNOS with COOH-terminal epitope (nNOS-C) in 1% normal donkey serum, were incubated with the tissue for 17–18 h at room temperature. The cryosections were washed in 0.3% Triton X-100 in PBS three times every 15 min. Secondary antibodies, Cy3-conjugated donkey anti-mouse IgG and FITC-conjugated donkey anti-guinea pig IgG, were incubated for 1–1.5 h at room temperature. Cryosections were washed in 0.3% Triton X-100 in PBS twice every 10 min, washed in PBS without Triton X-100 once for 10 min, and then mounted with antifading aquamount medium. To determine the specificity of immunolabeling, primary or secondary antibodies were omitted.

Confocal images. The immunofluorescent-labeled cryosections were observed with a confocal laser scanning microscope (CLSM 1500, Zeiss) with a one-photon laser. The resolution of the confocal images obtained from cryosections was originally 512 x 512 pixels, and the obtained images were edited by Zeiss LSM 5 Image examiner (version 2.80.1123, Zeiss) and Adobe PhotoShop (version 7.0, Adobe).

Solvents

All the drugs used in functional experiments except apamin and ODQ were dissolved in double-distilled water. Apamin was dissolved in 0.05 M acetic acid. ODQ was dissolved in DMSO to prepare a stock solution. Fresh dilutions were prepared before the experiment by 10-fold dilution of the DMSO stock with double-distilled water. Acetic acid (0.05 M) and DMSO, in the amounts added (10 µl in a 10-ml bath and 1 µl in a 10-ml bath, respectively), did not affect the functional activity of the tissue.

Materials

Atropine sulfate, timolol (as maleate salt), prazosin hydrochloride, ODQ, apamin, L-NNA, SNP, SNAP, BCGMP, and VIP were purchased from Sigma (Oakville, Ontario, Canada). TTX was purchased from Alomone Laboratories (Jerusalem, Israel). DMSO and EGTA were purchased from Caledon Laboratories (Georgetown, Ontario, Canada). Uranyl acetate, OsO4, DMP-30, and TAAB 812 resin were purchased from Marivac (St. Laurent, Quebec, Canada). Mesh grids and formvar solution in ethylene dichloride were purchased from Electron Microscopy Sciences (Hatfield, PA). 3-Aminopropyltriethoxysilane was purchased from Sigma (St. Louis, MO). Triton X-100 was purchased from VWR (Edmonton, Alberta, Canada). Normal donkey serum was from Calbiochem (San Diego, CA). Mouse anti-cav3 was from BD Transduction Laboratories (Mississauga, Ontario, Canada). Guinea pig anti-rat nNOS-C was from Eurodiagnostica (Cedarlane Laboratories; Hornby, Ontario, Canada). Cy3-conjugated donkey anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-conjugated donkey anti-guinea pig IgG was from Research Diagnostics (Flanders, NJ). Aquamount medium was purchased from Biomeda (Hayward, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ultrastructural Studies

Ultrastructural examination of the smooth muscles in the CM layer in control and Cav1–/– mice was carried out using an electron microscope. Previous work in our laboratory has demonstrated that ICC and smooth muscles of the mouse intestine (both LM and CM) contain a large number of caveolae that typically appeared in the electron microscope images as flask-shaped invaginations of the plasma membrane (14). Electron microscopic imaging of LM cells in the Cav1–/– mouse intestine revealed a total loss of caveolae structures on the plasma membrane of these cells (16). However, electron microscopic examination of the CM layer in the Cav1–/– mouse intestine revealed that a few caveolae structures remained on the plasma membrane of smooth muscle cells from the outer CM layer (Fig. 1b). Caveolae were absent in the inner CM layer in the Cav1–/– mouse intestine. Control mice showed a normal caveolae distribution on both the outer and inner CM layer (Fig. 1a).



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  		Fig. 1. Electron microscope images of the circular muscle (CM) layer of the mouse small intestine showing smooth muscles from the outer CM layer (OCM) and the inner CM layer (ICM). Numerous caveolar structures appear at the cell membranes from both regions in the caveolin 1 (cav1) control (Cav1-ctr mice; a). Note the total absence of caveolae in the smooth muscles of ICM in cav1 knockout (Cav1–/–-ko; b) mice, whereas a smaller number of caveolae appear in OCM (arrowheads). Thick arrows indicate the endoplasmic reticulum, and thin arrows the indicate sarcoplasmic reticulum. In a, the large arrowhead shows a junction between two OCM cells. ICC, interstitial cells of Cajal of the deep muscular plexus; m, mitochondria; N, nerves; Fib, fibroblasts; lgv, large granular vesicles; sgv, small granular vesicles. Scale bars = 2 µm in a and 1 µm in b.

 
Immunohistochemical Studies

Control experiments in which primary or secondary antibodies were omitted showed no labeling. Double immunolabeling of cryosections from the jejunum of Cav1+/+ and Cav1–/– mice with anti-cav3 antibody and the antibody for nNOS-C, which recognizes the myogenic variant of nNOS (12), showed that cav3 immunoreactivity was found in the periphery of smooth muscle cells in the outer CM layer in both control and Cav1–/– tissue (Fig. 2, b and e). Cav3 immunoreactivity was found to be more intense in Cav1+/+ tissue.



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  		Fig. 2. Immunohistochemical staining of cryosections from control (Cav1+/+; a–c) and Cav1–/– (d–f) jejunum. a and d: cryosections stained for neuronal nitric oxide synthase with COOH-terminal epitope (nNOS-C) show the normal presence of nNOS-C in the myenteric neurons and the cell periphery of smooth muscles of Cav1+/+ jejunum (a) but only residual immunoreactivity in the outer circular muscles of Cav1–/– jejunum (d). b and e: cryosections stained for cav3 show intense immunoreactivity in the outer circular muscles of Cav1+/+ jejunum (b) and lesser immunoreactivity located only in the outer circular muscles of Cav1–/– jejunum (e). The colocalized images (c and f) show very good colocalization between nNOS-C and cav3 in the cell periphery of the outer circular muscles in Cav1+/+ jejunum (c), but no colocalization is seen in Cav1–/– jejunum (f). *Myenteric neurons; lm, longitudinal muscle (LM) layer; mg, myenteric ganglia. Scale bars = 10 µm.

 
In accordance with our previous results (16), nNOS-C immunoreactivity was lost in the LM of Cav1–/– tissue; however, the CM layer showed residual nNOS-C immunoreactivity not fully localized to the plasma membrane (Fig. 2d). nNOS-C immunoreactivity appeared to be clearly present in all smooth muscle layers, localized to the plasma membrane, in Cav+/+ tissue (Fig. 2a). The colocalized images (Fig. 2, c and f) showed a very good colocalization between nNOS-C and cav3 in the periphery of smooth muscle cells in the outer CM layer of the Cav1+/+ intestine but not in the Cav1–/– intestine.

Functional Studies

Spontaneous activity of CM. Under NANC conditions, mouse small intestinal segments set up to record CM activity showed spontaneous uniformly paced rhythmic contractions. The amplitudes of these spontaneous contractions were measured at the end of the equilibration period. The values were as follows: 174.35 ± 32.55 mg tension for Cav1+/+ segments (n = 10) and 119.5 ± 22.45 mg tension for Cav1–/– segments (n = 11). No statistically significant differences were found among the amplitudes of spontaneous contractions.

On the other hand, the spontaneous frequency of pacing was higher in the Cav1–/– jejunum compared with the control strain (Fig. 3A). Because there is a gradient in the frequency of pacing along the mouse small intestine (13), the frequency measurements were all done in the midjejunum in both strains. The frequency of pacing significantly increased in tissues treated with L-NNA (100 µM) or ODQ (1 µM) in Cav1+/+ mice (Fig. 3B) but not in Cav1–/– mice (Fig. 3C). After block of NO production or action, all frequencies became the same.



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  		Fig. 3. The frequency of pacing in jejunal CM. Values shown are means ± SE, and statistical significance was determined using an unpaired t-test and ANOVA followed by Bonferroni's test. A: in Cav1–/– mice, the frequency of pacing was intrinsically higher than in the control mice (**P < 0.01, significant difference from control, n =9 for Cav1+/+ mice and 9 for Cav1–/– mice). B: N{omega}-nitro-L-arginine (L-NNA) and 1H-[1,2,4]oxadiazolo-[4,3-{alpha}]quinazolin-1-one (ODQ) increased the pacing frequency in the Cav1+/+ jejunal CM (*P < 0.5, n = 9, 6, and 7 for control, L-NNA-treated, and ODQ-treated Cav1+/+ mice, respectively). C: L-NNA and ODQ did not affect the frequency of pacing in Cav1–/– jejunal CM (n = 9, 7, and 7 for control, L-NNA-treated, and ODQ-treated Cav1–/– mice, respectively).

 
Effects of L-NNA, apamin, their combination, and ODQ on EFS in CM. Under NANC conditions, EFS of CM induced muscle relaxation that lasted for as long as the stimulus continued in most experiments. The relaxation was maximal at 3 pulses/s. There were no significant differences in the extents of relaxation exhibited by both strains. The relaxation due to EFS stimulation was followed by a rebound contraction, or an "off effect," in Cav1+/+ segments at higher frequencies. Cav1–/– CM segments did not show this off effect at any frequency. This difference, measured at 30 pulses/s, was significant (data not shown).

Inhibiting NOS activity by 100 µM L-NNA significantly reduced the extents of EFS-induced relaxation in the Cav1+/+ control strain at all stimulation frequencies (Fig. 4A). In Cav1–/– segments, the effect of L-NNA was inconsistent and was only significant at 1 and 30 pulses/s (Fig. 5A). ODQ (1 µM) reduced the EFS-evoked relaxation in a manner that was statistically similar to L-NNA in both strains (data not shown).



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  		Fig. 4. Effects of L-NNA, apamin (APA), and their combination on electrical field stimulation (EFS)-induced relaxation under nonadrenergic, noncholergic (NANC) conditions at different stimulation frequencies (0.5 ms and 50 V·cm–1 for 10 s) in Cav1+/+ CM. Extents of EFS-induced responses are indicated as the %inhibition of amplitude (the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the inhibitory stimulus), and values shown are means ± SE. A: L-NNA (100 µM) reduced EFS-evoked relaxation at all stimulation frequencies [in pulses/s (pss)]. B: APA (1 µM) did not have any effect on EFS-induced relaxation. C: the combination of L-NNA (100 µM) and APA (1 µM) almost abolished the EFS-evoked relaxation at all stimulation frequencies. Significance was tested by ANOVA followed by Bonferroni's test (*P < 0.05, **P < 0.01, and ***P < 0.001, n = 9, 6, 7, and 6 for control, L-NNA-treated, APA-treated, and combination-treated tissues, respectively).

 


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  		Fig. 5. Effects of L-NNA, APA, and their combination on EFS-induced relaxation under NANC conditions at different stimulation frequencies (0.5 ms and 50 V·cm–1 for 10 s) in Cav1–/– CM. Extents of EFS-induced responses are indicated as the %inhibition of amplitude (the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the inhibitory stimulus), and values shown are means ± SE. A: L-NNA (100 µM) significantly reduced EFS-evoked relaxation at 1 and 30 pps. B: APA (1 µM) reduced EFS-induced relaxation at all stimulation frequencies. C: the combination of L-NNA (100 µM) and APA (1 µM) almost abolished the EFS-evoked relaxation at all stimulation frequencies. Significance was tested by ANOVA followed by Bonferroni's test (*P < 0.05, **P < 0.01, and ***P < 0.001, n = 9, 6, 7, and 7 for control, L-NNA-treated, APA-treated, and combination-treated tissues, respectively).

 
The addition of 1 µM apamin, which acts by blocking a small-conductance Ca2+-dependant K+-channel (SK3 channels) (33), led to a significant reduction of the extents of EFS-evoked relaxation at all frequencies in Cav1–/– segments (Fig. 5B). In Cav1+/+ segments, apamin did not affect the extents of relaxation (Fig. 4B). A combination of L-NNA (100 µM) and apamin (1 µM) almost abolished the EFS-induced relaxation in Cav1+/+ and Cav1–/– segments (Figs. 4C and 5C).

Effects of ODQ on EFS-induced relaxations in LM. Under NANC conditions, EFS elicited a transient relaxation whose magnitude was proportional to the stimulation frequency to reach a maximum at 10 pulses/s in most experiments. As shown previously (16), there were no significant differences among the extents of relaxation shown by the two mice strains. In Cav1+/+ mice, inhibiting soluble guanylate cyclase (sGC) activity by 1 µM ODQ (52) nearly abolished the relaxation at all stimulation frequencies (Fig. 6A). However, in Cav1–/– mice, ODQ did not affect the magnitudes of relaxation at any frequency (Fig. 6B).



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  		Fig. 6. Effect of ODQ (1 µM) on the EFS-evoked relaxation under NANC conditions at different stimulation frequencies (0.5 ms and 50 V·cm–1 for 10 s) in LM. Extents of EFS-induced responses are indicated as the %inhibition of amplitude (the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the inhibitory stimulus), and values shown are means ± SE. A: ODQ abolished EFS-induced inhibitions at all stimulation frequencies in Cav1+/+ mice. B: ODQ did not have any effect on EFS-mediated relaxation in Cav1–/– mice. Significance was tested by ANOVA followed by Bonferroni's test (**P < 0.01 and ***P < 0.001, n = 6 for all strains).

 
Effects of ODQ and apamin on NO donor-induced relaxation in CM. Under NANC conditions, both SNP (100 µM) and SNAP (1, 10, and 100 µM) produced a reduction in the amplitudes of contraction that was dose dependent in the case of SNAP. At 100 µM, the relaxation induced by both SNP and SNAP was higher in the Cav1+/+ control strain than in Cav1–/– mice (Figs. 7A and 8A). Apamin (1 µM) did not affect the relaxation induced by both NO donors in the CM of both strains. However, ODQ (1 µM) decreased the relaxation due to both NO donors in the control strain and only due to SNAP in Cav1–/– mice (see Figs. 7, B and C, and 8, B and C). The combination of ODQ and apamin had an effect similar to that of ODQ alone in all cases with the exception that it was capable of reducing the relaxation due to SNP in Cav1–/– mice (data not shown). A comparison of the effects of SNP and SNAP (at 100 µM) showed that inhibition of the amplitude was greater for SNP (data not shown).



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  		Fig. 7. Effect of SNP (100 µM) on the amplitude of contraction in CM under NANC conditions. Extents of inhibition are expressed in %inhibition of the amplitude [the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the addition of sodium nitroprusside (SNP)]. Values are shown as means ± SE of 6 experiments. A: SNP produced a greater inhibitory response in the control strain compared with Cav1–/– mice. Significance was tested using the unpaired t-test (***P < 0.001). B: ODQ (1 µM) but not apamin (1 µM) reduced the inhibitory response to SNP in CM of Cav1+/+ mice. C: neither ODQ (1 µM) nor APA (1 µM) reduced the inhibitory response to SNP in Cav1–/– mice. Significance was tested by ANOVA followed by Bonferroni's test (***P < 0.001).

 


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  		Fig. 8. Effect of different doses of S-nitroso-N-acetyl penicillamine (SNAP) on the amplitude of contraction in CM under NANC conditions. Extents of inhibition are expressed in %inhibition of the amplitude (the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the addition of SNP). Values are shown as means ± SE of 6 experiments. A: 100 µM SNAP produced a greater inhibitory response in control mice compared with Cav1–/– mice. Significance was tested using the unpaired t-test (**P < 0.01). B and C: ODQ (1 µM) but not APA (1 µM) reduced the inhibitory responses to 10 and 100 µM SNAP in the two strains. Significance was tested by ANOVA followed by Bonferroni's test (*P < 0.05, **P < 0.01, and ***P < 0.001).

 
Effects of ODQ and apamin on NO donor-induced relaxation in LM. Under NANC conditions, unlike SNP (which produced only inhibition of the amplitudes of contraction), 100 µM SNAP produced a transient contraction followed by a brief relaxation and rapid recovery and sustained excitation in LM segments from the control strain. In Cav1–/– mice, SNAP only inhibited the amplitudes of contractions. Either ODQ (1 µM) or apamin (1 µM) abolished the relaxant phase in the response to SNAP in both strains (Fig. 9, data shown for SNP only). In the control strain, 100 µM SNAP produced a sustained increase in the amplitudes of contractions in the presence of ODQ and apamin (data not shown). In general, the comparison between the inhibitory effects of SNP and SNAP under NANC conditions showed that SNP produced a significantly greater inhibition in both strains (data not shown).



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  		Fig. 9. Effects of ODQ (1 µM) and APA (1 µM) on the inhibitory response to SNP (100 µM) in LM under NANC conditions. Extents of inhibition are expressed in %inhibition of the amplitude (the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the addition of SNP). Values are shown as means ± SE of 6 experiments. ODQ and APA, independently, reduced the SNP-induced inhibition in the two strains, Cav1+/+ (A) and Cav1–/– (B). Significance was tested by ANOVA followed by Bonferroni's test (***P < 0.001).

 
Effects of apamin on BCGMP-induced relaxation in LM. Because of the slow permeability of BCGMP, the inhibitory responses were measured 5 min after its addition. BCGMP (100 µM) produced an inhibition in the amplitudes of contractions that were not statistically different between control and knockout strains. Apamin (1 µM) reduced the BCGMP-induced inhibitions in both strains (data not shown).

Effects of TTX on responses to SNAP in LM. LM segments set up under non-NANC conditions responded to 100 µM SNAP in a manner similar to the response under NANC conditions. After nerve activity was blocked with TTX, the excitatory phases in the response of the segments from the control mice disappeared (Fig. 10A), whereas the magnitude of inhibitions increased (Fig. 10B). In Cav1–/– mice, the inhibitory response to 100 µM SNAP did not change after TTX (Fig. 10C).



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  		Fig. 10. Effects of TTX (1 µM) on the responses to SNAP (100 µM) in LM. A: SNAP produced a response that had excitatory and inhibitory components in the control mice but not in Cav1–/– mice. Excitatory components were abolished after nerve block by TTX, and inhibitory components were enhanced. B: TTX (1 µM) increased the extent of inhibition in response to SNAP (100 µM) in LM of Cav1+/+ mice, whereas it had no effect on the extents of inhibition in LM of Cav1–/– mice. C: extents of inhibition are expressed in %inhibition of the amplitude (the amplitude of the inhibitory phase normalized to the amplitude of muscle activity directly preceding the addition of SNAP). Values are shown as means ± SE of 6 experiments. Significance was tested by paired t-test (*P < 0.05).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Several reports have described a regulatory role for cav1 on the function of different NOS isoforms, which are normally present in the caveolae (19, 20, 22, 54). In a recent study (16), we examined the effects of cav1 absence on NO activity in the LM of the mouse small intestine. Functionally, LM of Cav1–/– mice showed a smaller or no contribution of NO to the EFS-evoked relaxation, despite the fact that immunohistochemical examination of the myenteric plexus neurons showed that they still had a cytoplasmic variant of nNOS. Experiments with SNP and BCGMP suggested that Cav1–/– mice had a reduced responsiveness to NO and that the defect in the NO signal might have occurred before the level of cGMP-controlled effectors. This was confirmed in the present study by experiments examining the effect of the sGC inhibitor ODQ on EFS. ODQ had no effect on EFS-evoked relaxation in LM in the Cav1–/– mice, whereas it reduced the relaxation in control mice. However, a good understanding of the effect of cav1 knockout on the function of NANC mediators in the mouse gut necessitates a study of the CM.

Previous research showed discrepancies between the functional activity of the LM and CM layers in addition to the differences in innervation described in the Introduction. Among these are 1) LM pacing is faster than in the CM and is increased by TTX and L-NNA in CM, not LM; 2) robust pacing is present in the LM of w/wv mice, which lack ICC of the myenteric plexus (32); and 3) differences occur in the effects of agents such as SNP and forskolin (3, 13). In the present study, electron microscopic examination of the CM layer of the Cav1–/– mouse small intestine showed that, unlike LM, they retained some caveolar structures that were in the outer CM layer but not the inner CM layer. These results were consistent with immunohistochemical examination of the same region that showed that only outer CM smooth muscles expressed cav3 in their plasma membrane in Cav1–/– mice. In contrast, control mice had cav3 in outer CM and caveolae in both CM layers. On the basis of protein sequence homology, cav3 and cav1 are about 65% identical and 85% similar (51). Cav3 also has the ability to homooligomerize in vitro and in vivo (51). This self-assembly is thought to drive caveolae formation, and the expression of cav3 is sufficient to cause the formation of caveolae (31). The lower intensity of cav3 immunoreactivity in the Cav1–/– outer CM layer might account for the fact that Cav1–/– mice appear to show fewer caveolae than the outer CM layer in control mice. However, a study that quantifies the differences, if any, will be necessary. The observations that cav3 has a protein binding domain similar to cav1 (31) and that it has very good colocalization with nNOS-C in the control outer CM suggest that it may have a role similar to that of cav1 in the regulation of signal transduction in smooth muscle cells of the outer CM in the mouse small intestine. In fact, the presence of cav3 in the Cav1–/– CM might be the reason for the retention of the residual nNOS-C immunoreactivity, which was not the case in LM. These observations suggest that cav1 knockout affects CM in a manner different from its effect on LM, i.e., in the LM, cav1 is necessary for the expression of nNOS-C and caveolae, whereas in the CM, it is important, but cav3 can partially compensate to allow some expression of nNOS-C and caveolae in the outer CM.

The frequency of pacing in CM in the control mice was apparently decreased by basal NO production, because the addition of either L-NNA or ODQ led to an increase in the frequency. Cav1–/– mice appeared to lack this basal component of NO activity because their pacing frequency was intrinsically higher than the controls and was affected by neither L-NNA nor ODQ. In fact, the addition of L-NNA and ODQ brought the pacing frequencies in control mice to levels similar to the Cav1–/– frequency. In LM, the frequencies of pacing were neither affected by L-NNA nor ODQ in all mice strains, although they were intrinsically higher in Cav1–/– mice (unpublished data).

EFS of CM under NANC conditions led to inhibitory responses that were different in configuration from those induced in LM. EFS-induced inhibitions in CM of control mice were followed by rebound contractions (off effects) that was reported to be due to a depolarization phase brought about by the activation of chloride (11) and nonselective cation conductances (28) reset during the hyperpolarization phase (1). The lack of this off effect in Cav1–/– mice could possibly suggest that they do not have sufficient hyperpolarization to reset these channels. This hypothesis agrees with the observation of reduced NO effects.

The effects of L-NNA on EFS in both Cav1+/+ and Cav1–/– CM showed that the role of NO in the EFS-mediated relaxation (blocked by L-NNA) in Cav1–/– mice was less defective than in LM (16). In Cav1–/– mice, L-NNA reduced the extents of relaxation at 1 and 30 pulses/s. Its effects at 3 and 10 pulses/s were not significant, but it is unclear that this can be considered as no effect. However, it is clear that the overall effect of L-NNA on the EFS-mediated relaxation in Cav1–/– mice was less pronounced than its effect in Cav1+/+ mice. The ODQ effect was similar to L-NNA in Cav1+/+ and Cav1–/– mice, indicating that neurally released NO acts mainly through sGC in these tissues and that the defect in NO response in CM of Cav1–/– is similar to that in LM.

Apamin, an SK3 channel blocker (33), blocks the effects of NANC inhibitory mediators that act through the activation of this channel. In the mouse intestine, apamin-sensitive mediators include ATP (34) and PACAP (36, 56). In LM, apamin reduced the EFS-mediated relaxation in Cav1–/– mice more than in control mice (16). This might be a mechanism to compensate for the reduced NO function. On the other hand, the difference in the effect of apamin on the EFS-evoked relaxation in the CM between Cav1–/– and control mice was much more apparent than in LM. Apamin almost had no effect in control CM, whereas it reduced the EFS-mediated relaxation in Cav1–/– mice at all stimulation frequencies. Preliminary immunohistochemical studies indicate that this might be related to an increased expression of SK3 channels in cells of the CM region in Cav1–/– mice (unpublished data). A combination of L-NNA and apamin almost abolished the EFS-evoked relaxation of CM in Cav1+/+ and Cav1–/– mice.

In our previous study (16), Cav1–/– LM showed reduced responsiveness to SNP compared with control tissues. This observation is confirmed by results from the present study. However, previous studies showed that not all NO donors are equivalent (7, 15, 18), and their induced responses are strongly dependent on the chemical entity of the NO donor (52). In the present study, we examined the differential response to two NO donors, SNP and SNAP, in addition to comparing their effect in LM and CM. In LM, we found that the inhibitory actions of SNP and SNAP involve the activation of both sGC and SK3 channels where ODQ and apamin were independently capable of reducing the induced inhibition in both strains. Our results agree with previous work done on the mouse small intestine where ODQ and apamin reduced inhibitory responses to SNP in LM (48). The ability of apamin to reduce the inhibitory effect of BCGMP in LM of both strains may indicate that NO activates sGC to produce cGMP, which either activates the SK3 channel directly or initiates a series of events that lead to its activation. This, however, was not the case in CM, where apamin did not have any effect on the inhibitions in response to SNP and SNAP. In CM, the inhibitory responses to SNP and SNAP were also found to be higher in the control mice than in Cav1–/– mice, indicating that the same defect in LM is present in CM.

In LM of the control mice, the configuration of the response to SNAP was different from that to SNP. The response of SNP was inhibitory, whereas the response to SNAP had excitatory components (Fig. 10A). These excitatory components were lacking in Cav1–/– mice. They were susceptible to nerve block by TTX, which also increased the extent of inhibition in control mice. On the other hand, the responses of SNAP in CM, similar to SNP, were only inhibitory. This suggests that in LM, SNAP releases an excitatory neural mediator in control mice but not in Cav1–/– mice.

To conclude, the present study shows that cav1 knockout affects NO function in CM and LM of the mouse small intestine differently, and this may be owing to the retention of cav3 in the CM layer of Cav1–/– mice. Also, the increase in the activity of apamin-sensitive NANC mediators in CM of Cav1–/– mice is more pronounced than in LM. On the other hand, although Cav1–/– CM showed reduced responsiveness to NO donors as in LM, there were differences among the actions of these compounds in CM and LM. The reduced NO activity due to cav1 knockout in both LM and CM might implicate a failure of peristaltic function; however, the inhibitory function is largely compensated by apamin-sensitive mediators in the small intestine. Whether this is the case in other regions of the GIT remains to be determined, as are the mechanisms by which cav1 knockout alters innervation patterns.


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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This study was funded by the Canadian Institutes of Health Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. E. Daniel, 9-10 Medical Sciences Bldg., Dept. of Pharmacology, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H (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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 

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