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Am J Physiol Gastrointest Liver Physiol 286: G899-G905, 2004; doi:10.1152/ajpgi.00408.2003
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NEUROREGULATION AND MOTILITY

G protein-mediated dysfunction of excitation-contraction coupling in ileal inflammation

Xuan-Zheng Shi and Sushil K. Sarna

Departments of Internal Medicine, Physiology and Biophysics, Enteric Neuromuscular Disorders and Visceral Pain Center, Division of Gastroenterology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1064

Submitted 17 September 2003 ; accepted in final form 12 December 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inflammation impairs the circular muscle contractile response to muscarinic (M) receptor activation. The aim of this study was to investigate whether the expression of muscarinic receptors, their binding affinity, and the expression and activation of receptor-coupled G proteins contribute to the suppression of contractility in inflammation. The studies were performed on freshly dissociated single smooth muscle cells from normal and inflamed canine ileum. Northern blotting indicated the presence of only M2 and M3 receptors on canine ileal circular muscle cells. Inflammation did not alter the mRNA or protein expression of M2 and M3 receptors. The maximal binding and Kd values also did not differ between normal and inflamed cells. However, the contractile response to ACh in M3 receptor-protected cells was suppressed, whereas that in M2 receptor-protected cells was enhanced. Further experiments indicated that the expression and binding activity of G{alpha}q/11 protein, which couples to M3 receptors, were downregulated, whereas those of G{alpha}i3, which couples to M2 receptors, were upregulated in inflamed cells. We concluded that inflammation depresses M3 receptor function, but it enhances M2 receptor function in ileum. These effects are mediated by the differentially altered expression and binding activity of their respective coupled G{alpha}q/11 and G{alpha}i3 proteins.

smooth muscle; motility; inflammatory bowel disease; signalopathy; acetylcholine

ACTIVATION OF MUSCARINIC (M/m) receptors on circular smooth muscle cells by release of ACh from excitatory cholinergic motor neurons plays an important role in the stimulation of spontaneous ileal contractions (8, 19). The ligand receptor binding initiates cascades of signaling pathways, which result in the phosphorylation/dephosphorylation of contractile proteins. The in vivo and in vitro contractions stimulated by ACh are suppressed in the inflamed ileum (1, 9, 11, 20–22), which suggests alterations in the expression or activation of some of the signaling molecules responsible for excitation-contraction coupling. Previous studies (1) have shown that the expression of PKC isoforms {alpha}, {beta}, and {epsilon} is downregulated and that of {lambda} and {alpha} is upregulated, whereas the expression of the rest of the PKC isozymes is not altered by inflammation. The expression of L-type Ca2+ channels is downregulated by inflammation, resulting in a decrease of Ca2+ influx, whereas the release of Ca2+ from the intracellular stores is not affected (11, 22). These findings suggest that inflammation induces specific alterations in cell signaling that produce abnormal motility in the inflamed ileum.

Five molecular types of muscarinic receptors (m1, m2, m3, m4, and m5) have been cloned and characterized, and four pharmacological counterparts of these receptors (M1, M2, M3, and M4) have been identified (2, 3, 6, 8). Functionally, M1, M3, and M5 receptors couple to the activation of phospholipases A2, C, and D, tyrosine kinase, and PKC, and calcium influx, and intracellular Ca2+ release (7). On the other hand, M2 and M4 receptors couple to adenylyl cyclase, whose activation is inhibited by the stimulation of these receptors, resulting in an indirect increase of cell contraction (6, 7). The effect of inflammation on the expression of muscarinic receptors, their affinity for ACh, or the G proteins to which they couple for the activation of the above signaling molecules are not known. The first aim of this study was to test the hypothesis that inflammation differentially alters the expression of muscarinic receptors and their associated G proteins.

Although the M2 receptors are present in abundance on the membranes of gastrointestinal smooth muscle cells, most studies (20, 21) indicate that they do not mediate contractions in the normal state of these cells. By contrast, in the ileum or the lower esophageal sphincter, inflammation seems to turn on the M2 receptors to mediate a part of the contractile response (20, 21, 23). The identity of this "molecular switch" is unknown. The second aim of this study was to test the hypothesis that an increased expression of G{alpha}i3, which couples M2 receptors to adenylyl cyclase, may be the molecular switch that turns on the M2 receptor function in inflammation. The studies were performed on freshly dissociated single circular muscle cells of the canine ileum.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of canine ileal inflammation. An intraluminal Silastic catheter with side holes was implanted surgically under general anesthesia (30 mg/kg iv pentobarbital sodium; Abbott Laboratories, Chicago, IL) in the ileum with its tip resting ~160 cm from the ileocolonic junction (9). A stainless steel cannula was implanted 20 cm orad to the ileocolonic junction. The dogs were allowed to recover for 7 days from surgery. The intraluminal catheter was used to infuse ethanol and acetic acid to induce ileal inflammation. The luminal contents were drained during the induction of inflammation from the ileal cannula so that they did not reach the colon. This study was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch.

Ileal inflammation was induced by three mucosal exposures to ethanol and acetic acid, as reported previously (9, 18, 20, 21). Briefly, 75 ml of 95% ethanol were infused intraluminally on day 1. The same amount of ethanol was infused on days 3 and 5, followed 1 h later by infusions of 50 ml of 20% acetic acid. Ileal inflammation, induced as above, lasts for ~10 days and is marked by suppression of individual phasic contractions and migrating motor complex cycling but stimulation of giant migrating contractions (9). MPO activity, as well as neutrophil count, is increased in the muscularis externa and the lamina propria during inflammation induced by this method (9). Dogs were killed on day 6 after induction of inflammation to obtain tissue for experiments.

Enzymatic dispersion of ileal circular smooth muscle cells and measurement of cell contraction. Single smooth muscle cells were isolated by two consecutive digestions with papain and collagenase as reported previously (21, 22). A 10-cm segment of ileum was removed from dogs anesthetized with pentobarbital sodium (Abbott Laboratories). The longitudinal muscle layer and myenteric plexus were peeled off after lightly scoring the segment with a blunt scalpel blade along the longitudinal axis and then discarded. The remaining tissue was scored deeper along the circular muscle axis, and the circular muscle layer was peeled off and collected in ice-cold HEPES buffer (pH 7.4). The circular muscle sheets were cut into 0.5 x 0.5 cm2 pieces and incubated at 37°C in 20 ml of Ca2+-free Hanks' solution (pH 7.2) for 15 min, and then in Hanks' solution containing 0.4 mg/ml of papain and 0.3 mg/ml of 1,4-dithiothreitol until the tissue appeared loose and sticky (~10 min). The tissue was washed with HEPES buffer and further digested at 31°C with 0.5 mg/ml collagenase (type II, 319 U/mg) and 0.1 mg/ml soybean trypsin inhibitor for 40 min. The digested tissue was washed three times with enzyme-free HEPES buffer, and the muscle cells were allowed to disperse spontaneously under gentle to-and-fro motion. Circular muscle cells were harvested by filtration through a 500-µm Nitex mesh and collected by centrifugation at 350 g for 5 min. Dispersed cells were relaxed at rest, and they responded with cell length shortening in the presence of ACh.

To quantitate muscle cell contraction, cells were resuspended in HEPES buffer to reach the concentration of 5 x 104 cells/ml. Cell length was measured by scanning micrometry, as described previously (21, 22). An aliquot (0.45 ml) of cells was exposed to 50 µl of ACh or vehicle control for 40 s at 31°C and fixed with 1% acrolein. The lengths of 30 consecutive intact healthy cells were measured through a phase-contrast microscope (Nikon TMS) fitted with a video camera (Javelin CCD), and connected to a Macintosh computer. NIH Image 1.61 was used to measure the cell length. The contractile response was expressed as percent cell shortening from the vehicle control.

Preparation of permeable smooth muscle cells. In the experiments in which G protein antibodies (1:400) were used, the smooth muscle cells were permeabilized as previously described (22). Briefly, the smooth muscle cells were incubated with 35 µg/ml of saponin for 10 min in a medium with the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 0.96 NaH2PO4, 0.48 CaCl2, 1 EGTA, and 2% bovine serum albumin. The cells were then centrifuged at 350 g for 5 min, washed free of saponin, and resuspended in fresh medium.

M2 and M3 receptor protection and measurement of cell contraction. Receptor-protection assay was used in single smooth muscle cells to selectively preserve M2 or M3 receptors, as described previously by Murthy and Makhlouf (13). M2 receptors were protected by exposing them initially to 10 nM methoctramine for 2 min and then adding 5 µM N-ethylmaleamide for 20 min to block irreversibly all the remaining unprotected receptors. The cell suspension was washed two times and allowed to equilibrate at 31°C for 30 min before proceeding with cell contraction experiments. M3 receptors were protected by exposing them first to 5 nM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP) and then to N-ethylmaleamide. Preliminary [3H]quinuclidinyl benzilate ([3H]QNB) binding experiments confirmed that in M2-protected cells the muscarinic receptor binding was blocked 87% by 10 nM methoctramine, but not significantly affected by 5 nM 4-DAMP. The binding activity in M3-protected cells was blocked 91% by 5 nM 4-DAMP, but not affected by 10 nM methoctramine.

[3H]QNB binding. Ileal circular muscle cells at 200,000 cells/ml in HEPES buffer were incubated with different concentrations of [3H]QNB in the presence and absence of 5 µM atropine at 31°C for 30 min. After the addition of 1 volume of ice-cold HEPES buffer, the cell suspension was centrifuged at 14,000 g at 4°C for 10 min. The cells were washed and centrifuged one more time. The resultant cell pellet was lysed with 200 µl of tissue solubilizer at 50°C for 20 to 30 min until the cell pellet was dissolved completely. The radioactivity of the solubilized suspension was measured in 8 ml of scintillation cocktail with a liquid scintillation counter (Packard Instruments, Downers Grove, IL). Specific muscarinic receptor binding was defined as the difference in bindings in the absence and presence of atropine. All binding assays were performed in duplicate. The maximal binding (Bmax) and Kd were calculated by using Graphpad software.

RNA preparation, Northern blot, and RNase protection assay. Total RNA was extracted from ileal circular muscle tissue with a TRIzol RNA isolation kit (Life Technologies, Rockville, MD) by following the manufacturer's protocol. Total RNAs from dog cerebral cortex, heart, and rat pancreas were also processed as positive controls for m1, m2, m3, and m4 mRNAs. RNA samples were separated on 1% agarose/0.66 M formaldehyde gel, and transferred to nylon membrane. RNA blots were hybridized for 16 to 24 h at 42°C with [32P]dCTP-labeled rat m1, m3, m4 and m5, and human m2 cDNA probes, separately. All cDNA probes (gift from Dr. T. I. Bonner, National Institutes of Health) were fragments corresponding to the third intracellular loop of their protein sequences (2). The hybridized blots were washed two times for 20 min each in 1x SSC/0.1% SDS at room temperature followed by two washes for 20 min each in 0.1x SSC/0.1% SDS at 42°C. The blots were exposed to X-ray films at –80°C for 24 to 48 h.

The m2 and m3 mRNA levels were determined by RNase protection assay using 5 µg of total RNA samples extracted from the circular muscle of normal and inflamed ileums. The cRNA probes for m2 and m3 were generated with SP6 RNA polymerase in the presence of [{alpha}-32P]UTP. After 16 h of hybridization at 45°C, excess, nonprotected RNA was digested with RNAse A (40 µg/ml, ~1 U/sample) and RNase T1 (2 µg/ml). The protected hybridization products were purified by extraction in phenol/chloroform/isoamyl alcohol mixture (25:24:1). Protected fragments were separated on 8% polyacrylamide/8 M urea sequencing gels. The gels were dried and subjected to autoradiography at –80°C with an intensifying screen.

Membrane protein extraction and Western blot analysis. Smooth muscle membrane extracts were prepared as described previously (4). Ileal circular muscle cells from dogs with normal and inflamed ileums were homogenized in 20 mM HEPES medium (pH 7.4) containing 2 mM MgCl2, 1 mM EDTA, and 2 mM DTT. The homogenates were centrifuged at 600 g for 5 min. The supernatants were ultracentrifuged at 25,000 g for 30 min. The membrane protein extracts were collected for Western blotting and GTP{gamma}S binding.

For Western blotting, 10 µg of membrane extracts were electrophoresed though 10% SDS-polyacrylamide gel. After blotting, the nitrocellulose membrane was blocked with 5% nonfat dry milk for 30 min. The blot was then incubated overnight with primary antibody in 5% milk at 4°C. After four 10-min washings, the blot was incubated with secondary antibody for 1 h at room temperature. The blot was washed again, and the protein bands were visualized with an enhanced chemiluminescence assay kit (Amersham Pharmacia Biotech, Piscataway, NJ).

[35S]GTP{gamma}S binding. [35S]GTP{gamma}S binding was assayed by the method of Okamoto et al. (16) with slight modifications (4, 13). Membrane extracts were first solubilized for 60 min at 4°C in HEPES medium (pH 7.4) containing 20 mM HEPES, 2 mM EDTA, 240 mM NaCl, and 1% CHAPS. The membrane extracts (2.5 µg/µl) were incubated for various periods at 37°C with 50 nM [35S]GTP{gamma}S in a solution containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM MgCl2. The reaction was stopped with 10 volumes of 100 mM Tris·HCl medium (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP, and the mixture (200 µl) was placed in wells precoated with specific G protein antibodies. After incubation for 2 h on ice, the wells were washed three times with phosphate buffer solution containing 0.1% Tween 20, and the radioactivity from each well was counted. Coating with G protein antibodies (1:1,000) was done after the wells were first coated with anti-rabbit IgG (1:1,000) for 2 h on ice. The selective M2 receptor antagonist methoctramine and M3 receptor antagonist 4-DAMP were used to identify the receptor subtype coupled to a given G protein.

Materials and solutions. Collagenase type II and soybean trypsin inhibitor were obtained from Worthington (Freehold, NJ). Papain, 1,4-dithiothreitol, ACh chloride, essential amino acid mixture, and N-ethylmaleamide were purchased from Sigma (St. Louis, MO); methoctramine and 4-DAMP were from RBI (Natick, MA); and [{alpha}-32P]UTP, [32P]dCTP, [35S]GTP{gamma}S, and [3H]QNB were from New England Nuclear Life Science (Boston, MA).

The composition of HEPES buffer (pH 7.4) was (in mM) 120 NaCl, 2.6 KH2PO4, 4 KCl, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% essential amino acid mixture. Krebs solution consisted of (in mM) 120 NaCl, 6 KCl, 14 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, and 11 glucose (bubbled with 95% O2-5% CO2 to maintain pH 7.4). The composition of Hanks' solution was (in mM) 135 NaCl, 5.5 KCl, 0.5 KH2PO4, 4 NaHCO3, 0.4 Na2HPO4, 0.5 MgCl2, and 5.5 +glucose (pH 7.3).

Statistical analysis. All values are expressed as means ± SE; n represents the number of animals studied. Statistical analysis was performed by ANOVA with nonrepeated measures or unpaired t-test. Multiple comparisons were performed by Student-Newman-Keuls test, and P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of inflammation on contractile response of ileal circular muscle cells to ACh. Freshly dispersed circular smooth muscle cells from the normal ileum responded to ACh concentration dependently with an EC50 of 0.8 ± 0.3 nM (n = 4) and maximal contraction of 24 ± .1% (Fig. 1). The contractile response was significantly reduced in cells from the inflamed ileum with an EC50 of 21 ± 3 nM and maximal contraction of 15.3 ± 1.0% (n = 4).



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  		Fig. 1. Contraction of freshly dispersed ileal circular smooth muscle cells in response to ACh (10–12-10–4 M). The contractile response was suppressed in the inflamed cells compared with that in normal cells (*P < 0.05 vs. normal; n = 4 each). Basal cell lengths of normal (99.3 ± 2.2 µm) and inflamed cells (97.7 ± 2.1) were not different.

 
Effects of inflammation on mRNA and protein expression of muscarinic receptors. We examined first whether the mRNA and protein expression of muscarinic receptor subtypes were altered by inflammation. Northern blot analysis with m1, m2, m3, m4, and m5 cDNAs detected only m2 and m3 mRNAs in canine ileal circular muscle cells (Fig. 2A). Positive controls indicated the presence of m1 and m4 receptors mRNAs in brain, m2 receptors in cardiac, and m3 receptors in pancreatic tissue. Quantification of m2 and m3 mRNAs with RNase protection assay showed that m2 and m3 mRNA levels were not significantly different between cells obtained from the normal and inflamed ileums (Fig. 2B). Western immunoblotting (Fig. 3) indicated that the protein expression of M2 and M3 receptors was also not different between cells from the normal and inflamed ileums.



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  		Fig. 2. A: Northern blot analysis was performed to determine the subtypes of muscarinic receptor transcripts (m) in canine ileal circular muscle cells. Only m2 (7 kb) and m3 (8 kb) mRNA messages were detected on these cells. Total RNA (10 µg) was loaded in each lane. B: expression level of m2 and m3 mRNA was determined by RNAse protection assay. Total RNA (5 µg) was loaded in each lane. Blots are representative of 4 experiments.

 


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  		Fig. 3. Western blot analysis of the expression of M2 (A) and M3 (B) receptor proteins on the canine ileal circular muscle cells in normal and inflamed states. Subsequent stripping of blots followed by incubation with {beta}-actin antibody was performed to monitor protein loading. The results are representative of 4 experiments.

 
Differential effects of inflammation on M3 and M2 receptor-mediated contractions. We then examined whether inflammation modulates M3 or M2 receptor-induced excitation-contraction coupling. The contractile response to ACh in cells in which M3 receptors were preserved selectively was not different from that in cells in which all receptors were present (Figs. 1 and 4A); the maximal contraction amplitudes were 22.5 ± 0.8 and 24 ± 1%, respectively. The suppression of contractile response to ACh in M3 receptor-protected cells from the inflamed ileum was also not significantly different from that in cells without receptor protection (Figs. 1 and 4A). By contrast, the M2 receptor-protected cells from the normal ileum contracted to ACh much less potently compared with M3 receptor-protected cells or receptor-unprotected cells (Fig. 4, A and B). The maximal contraction to 100 µM ACh was only 8.2 ± 0.7% in M2-protected cells from the normal ileum. There was little or no contractile response to doses of ACh <10–8 M in these cells. Inflammation shifted the ACh dose-response curve to the left in M2 receptor-protected cells (Fig. 4B) in contrast to the right shift of concentration response curve to ACh in M3 receptor-protected cells (Fig. 4A). The maximal response to ACh in M2 receptor-protected cells was significantly greater in cells from the inflamed ileum (14.6 ± 1.2%) than in those from the normal ileum (10.4 ± 1.0%).



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  		Fig. 4. Contractile response of ileal circular smooth muscle cells in which M3 (A) or M2 (B) muscarinic receptors were selectively protected. The contractile response of M3 receptor alone was significantly decreased in inflammation, whereas that of M2 receptor was increased (*P < 0.05 vs. normal ileum; n = 4 each). The basal cell length of M3-protected cells from normal ileum was 96.6 ± 1.9 µm and was 96.4 ± 2.2 µm in those from the inflamed ileum. The basal cell lengths of M2-protected cells were 97.1 ± 2.0 and 96.5 ± 1.8 µm in cells from the normal and inflamed ileums, respectively.

 
[3H]QNB binding activity of muscarinic receptors. To determine whether the altered muscarinic receptor activation in inflammation is due to the impairment of receptor binding, the ileal circular muscle cells were incubated with [3H]QNB, and their binding affinity and capacity were quantitated. In normal cells, Kd was 0.13 + 0.05 nM, and Bmax was 21.1 + 1.5 fmol. The corresponding Kd and Bmax values of 0.17 + 0.09 nM and 23.6 + 2.5 fmol, respectively, were not significantly different in inflamed cells (Fig. 5).



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  		Fig. 5. Specific [3H]quinuclidinyl benzilate ([3H]QNB) binding to muscarinic receptors on circular smooth muscle cells from normal and inflamed ileums. Values are means ± SE of 4 separate experiments performed in duplicate. Bmax, maximal binding.

 
Effects of inflammation on the expression of GTP-binding proteins. The above data indicated that the expression of M2 and M3 receptors and their affinity to ACh may not be altered in inflammation, and yet cell contractility associated with the two subtypes of muscarinic receptors is altered differentially. We therefore examined whether the alteration of M2 and M3 receptor-associated excitation-contraction coupling was due to changes in the next downstream signaling step, i.e., the expression and/or activation of G proteins coupled to these receptors. Freshly dispersed smooth muscle cells were permeabilized, and then incubated with anti-G{alpha}i1–2, -G{alpha}i3, -G{alpha}q/11, and -G{alpha}s antibodies. In M3 receptor-protected cells, only the antibody to G{alpha}q/11 inhibited the response to ACh (Fig. 6B). By contrast, only the antibody to G{alpha}i3 protein inhibited the contractile response to ACh in M2 receptor-protected cells (Fig. 6B). The antibodies to G{alpha}i1–2 or G{alpha}s had no effect on the contractile response to ACh in M3 or M2 receptor-protected cells. Pertussis toxin (PTx) treatment of the cells from the normal ileum had no effect on their contractile response to ACh. On the contrary, PTx significantly reduced the contractile response to ACh in inflamed cells (Fig. 7). These data confirmed that M2 receptors were coupled to G{alpha}i3 and M3 receptors to G{alpha}q/11.



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  		Fig. 6. A: in M3 receptor-protected cells, only the antibody to G{alpha}q/11 protein inhibited ACh-induced cell shortening. B: in the M2 receptor-protected cells, only the antibody to G{alpha}i3 protein blocked ACh effect. In all the experiments, cells were permeabilized before preincubation with G protein antibodies at 1:400 dilution for 1 h. Values are means + SE of 3 experiments. *P < 0.05 vs. non-antibody control.

 


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  		Fig. 7. Effect of pertussis toxin (PTX; 200 ng/ml, 1 h incubation) on ACh (10–6 M)-stimulated contraction in normal (n = 4) and inflamed (n = 4) cells. *P < 0.05 vs. normal ileum ACh control. #P < 0.05 vs. inflamed ileum ACh control.

 
Western blot analysis indicated that the expressions of G{alpha}i1–2 and G{alpha}s in circular muscle cells were not affected by ileal inflammation (Figs. 8, A and D). However, the expression of G{alpha}q/11 was significantly decreased in inflamed cells (54 ± 7.5% of that normal cells; Fig. 8C), whereas that of G{alpha}i3 was significantly increased (156 ± 19% of that normal cells; Fig. 8B).



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  		Fig. 8. Western blot analysis of the expression level of G proteins in circular muscle cells from the normal and inflamed canine ileums. Ten micrograms of protein were loaded in each lane. Shown on the blots are 4 representative samples. On each blot, the left 2 lanes are from 2 normal ileums, whereas the right 2 lanes are 2 samples of inflamed ileums. Values of the bar graphs are means ± SE of 4 separate experiments. *P < 0.05 vs. normal ileum.

 
Effects of inflammation on [35S]GTP{gamma}S binding to G proteins. The activation of G proteins was examined by measuring the magnitude of [35S]GTP{gamma}S binding with Gs, Gi1–2, Gq/11, and Gi3 proteins in response to ACh (Fig. 9). There was only minimal GTP{gamma}S binding to Gi1–2 and Gs proteins in response to ACh treatment in normal or inflamed cells. The binding to Gq/11 protein was 28 ± 6.2% in normal cells, and it decreased to 7.8 + 6.7% (P < 0.05) in inflamed cells (Fig. 9). On the other hand, the GTP{gamma}S binding to G{alpha}i3 protein was only 4 + 3.2% in cells from the normal ileum, and it increased to 14 + 2% (P < 0.05) in cells from the inflamed ileum (Fig. 9).



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  		Fig. 9. ACh (10–6 M)-stimulated [35S]GTP{gamma}S binding in ileal circular muscle cell membranes from normal (left bars, n = 4) and inflamed (right bars, n = 4) ileums. Data are expressed as %increase of binding from basal level in the absence of ACh. Basal levels were counts per minute per milligram of protein in cells from the normal and inflamed ileums. Basal levels for G{alpha}i1–2 binding (A) were 3,337 ± 167 and 3,124 ± 112; for G{alpha}i3 binding (B) were 3,914 ± 450 and 6,022 ± 528; for G{alpha}q/11 binding (C) were 3,859 ± 312 and 3,364 ± 302; and for G{alpha}s binding (D) were 2,686 ± 326 and 2,616 ± 238 for cells from the normal (left) and inflamed (right) ileum, respectively. *P < 0.05 inflamed vs. normal ileum.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The binding of ACh to muscarinic receptors and activation of their coupled G proteins are early steps in excitation-contraction coupling in smooth muscle cells. Any alterations in the expression or activation of these proteins would affect the entire downstream signaling cascades coupled to them. Our findings show that ileal inflammation does not alter the expression of M2 and M3 receptors or their affinity to ACh, but it differentially modulates the expression and activation of G proteins coupled to them. It is interesting that the expression and activation of other G proteins, such as G{alpha}s and G{alpha}i1–2, that are not coupled to muscarinic receptors are not affected by inflammation.

We found that the canine ileal circular muscle cells express only the M2 and M3 receptors, and of these, M3 receptor mediates most of the excitation-contraction coupling in the normal state. The concentration-response to ACh in M3 receptor-protected cells did not differ from that in normal cells that had all receptors intact. By contrast, the contractile response of M2 receptor-protected cells was significantly smaller and seen only at supramaximal doses of ACh. These findings are consistent with a previous report from our lab that, in canine normal ileal circular muscle cells, ACh does not decrease forskolin-induced production of cAMP, indicating near absence of M2 receptor activation on normal ileum. Using antibodies to G proteins, Murthy and Makhlouf (13) also found that M3, rather than M2, receptors mediate the contraction of normal circular muscle cells of the rabbit stomach.

The lack of mediation of normal circular muscle contraction by M2 receptors is despite the fact that these receptors are present in abundance on these cells and have a well-defined signaling pathway independent of that used by M3 receptors (6, 7, 13, 14, 15). Our findings suggest that the lack of a role of M2 receptors in mediating contractions in normal cells may be due to the low level of activation of G{alpha}i3. GTP{gamma}S binding to G{alpha}i3 was only 4 ± 3.2% after ACh stimulation compared with the 28 ± 6% binding for G{alpha}q/11, which is coupled to M3 receptors.

The expression and activation of G{alpha}q/11 were significantly decreased in inflamed cells. These alterations would contribute to the suppression of contractility seen in these cells. On the other hand, the expression and activation of G{alpha}i3 were upregulated, which may partly reverse the suppression of contractility in inflammation through the activation of M2 receptors. The contractile response to ACh in M2 receptor-protected inflamed cells increased significantly over that seen in M2 receptor-protected cells from the normal ileum. Shi and Sarna (21) noted previously that M2 receptor activation decreases forskolin-induced synthesis of cAMP in inflamed cells, but not in normal cells. In this study, we found that PTx, which blocks the activation of G{alpha}i3, had no effect on ACh-induced contraction in normal cells, but it reduced it significantly in inflamed cells. These findings indicate a contribution of M2 receptors to the overall muscarinic response in the inflamed state, but not in the normal state. Sohn et al. (23) also noted the activation of M2 receptor-linked signaling pathway in circular muscle cells from the inflamed lower esophageal sphincter of cats. However, they did not identify the increased expression and activation of G{alpha}i3 as the molecular switch for this phenomenon.

It seems that different pathological conditions may alter the activation and expression of G proteins differently in gut smooth muscle cells. In gastric smooth muscle cells of streptozotocin-treated diabetic rats, G{alpha}s is overexpressed without any change in the expression of G{alpha}i3 or G{alpha}q/11 (10). On the other hand, the expression of G{alpha}q/11 is decreased in spontaneously diabetic WBN/Kob rats without a change in the expression G{alpha}s, G{alpha}i, or G{alpha}i3 (10). By contrast, our data show that, in inflammation, G{alpha}q/11 is downregulated and G{alpha}i3 is upregulated, whereas G{alpha}s and G{alpha}i1–2 are not affected. The expression and affinity of muscarinic receptors to ACh are also not altered in the above two models of diabetic rats, similar to that seen in inflammation. Chen et al. (4) found that progesterone treatment of guinea pigs significantly decreases [35S]GTP{gamma}S binding to G{alpha}i3, resulting in hypomotility of gallbladder smooth muscle cells in response to CCK-8. The contraction in guinea pig gallbladder seems to be mediated primarily by M2 receptor-induced activation of G{alpha}i3. On the other hand, G{alpha}q/11 is decreased and G{alpha}s increased during pregnancy in both antral and colonic smooth muscle cells of the guinea pig (5). This diversity of alterations in G proteins in different motility disorders may offer an opportunity to target specific early signaling molecules to normalize abnormal contractility.

In conclusion, inflammation downregulates the expression and activation of G{alpha}q/11 and upregulates those of G{alpha}i3 without affecting any of these parameters for G{alpha}s and G{alpha}i1–2. The density of muscarinic receptors and their affinity to ACh are not affected in inflammation. The downregulation of G{alpha}q/11 protein may account, in part, for the suppression of contractility in circular muscle cells of the inflamed ileum (9, 20). The upregulation of G{alpha}i3 would partly counter the suppression of contractility due to the downregulation of G{alpha}q/11 in inflammation. Because G protein activation is an early step in excitation-contraction coupling, its modulation would have significant effects on all signaling cascades coupled to them. Differential modulation of G proteins in different pathological conditions may lead to selective alterations of motility in different organs of the gastroinstestinal tract.


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The study was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32346.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. K. Sarna, Div. of Gastroenterology, Dept. of Internal Medicine, The Univ. of Texas Medical Branch at Galveston, Galveston, TX 77555-0632 (E-mail: sksarna{at}utmb.edu).

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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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Ali I and Sarna SK. Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells. Gastroenterology 122: 483–494, 2002.[CrossRef][Web of Science][Medline]
  2. Bonner TI, Buckley NJ, Young AC, and Brann MR. Identification of a family of muscarinic acetylcholine receptor genes. Science 237: 527–532, 1987.[Abstract/Free Full Text]
  3. Buckley NJ, Bonner TI, Young AC, and Brann MR. Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Mol Pharmacol 35: 469–476, 1989.[Abstract]
  4. Chen Q, Chitinavis V, Xiao Z, Yu P, Oh S, Biancani P, and Behar J. Impaired G protein function in gallbladder muscle from progesterone-treated guinea pigs. Am J Physiol Gastrointest Liver Physiol 274: G283–G289, 1998.[Abstract/Free Full Text]
  5. Chen Q, Xiao ZL, Biancani P, and Behar J. Downregulation of G{alpha}q-11 protein expression in guinea pig antral and colonic circular muscle during pregnancy. Am J Physiol Gastrointest Liver Physiol 276: G895–G900, 1999.[Abstract/Free Full Text]
  6. Eglen RM, Hegde SS, and Watson N. Muscarinic receptor subtypes and smooth muscle function. Pharmacol Rev 48: 531–565, 1996.[Web of Science][Medline]
  7. Felder CC. Muscarinic acetylcholine receptors: signal transduction through multiple effectors. FASEB J 9: 619–25, 1995.[Abstract]
  8. Goyal RK. Muscarinic receptor subtypes: physiology and clinical implications. N Engl J Med 321: 1022–1029, 1989.[Web of Science][Medline]
  9. Jouet P, Sarna SK, Singaram C, Ryan RP, Hillard CJ, Telford GL, Fink J, and Henderson JD. Immunocytes and abnormal gastrointestinal motor activity during ileitis in dogs. Am J Physiol Gastrointest Liver Physiol 269: G913–G924, 1995.[Abstract/Free Full Text]
  10. Lin S, Kajimura M, Takeuchi K, Kodaira M, Hanai H, Nishimura M, and Kaneko E. Alterations of GTP-binding proteins (Gs{alpha} and Gq/11{alpha}) in gastric smooth muscle cells from streptozotocin-induced and WBN/Kob diabetic rats. Dig Dis Sci 45: 1517–1524, 2000.[CrossRef][Web of Science][Medline]
  11. Liu XR, Rusch NJ, Striessnig J, and Sarna SK. Down-regulation of L-type calcium channels in inflamed circular smooth muscle cells of the canine colon. Gastroenterology 120: 480–489, 2001.[CrossRef][Web of Science][Medline]
  12. Marzio L, Blennerhassett P, Chiverton S, Vermillion DL, Langer J, and Collins SM. Altered smooth muscle function in worm-free gut regions of Trichinella-infected rats. Am J Physiol Gastrointest Liver Physiol 259: G306–G313, 1990.[Abstract/Free Full Text]
  13. Murthy KS and Makhlouf GM. Differential coupling of muscarinic m2 and m3 receptors to adenylyl cyclases V/VI in smooth muscle. Concurrent M2-mediated inhibition via G{alpha}i3 and m3-mediated stimulation via G{alpha}{gamma}q. J Biol Chem 272: 21317–21324, 1997.[Abstract/Free Full Text]
  14. Murthy KS. cAMP inhibits IP3-dependent Ca2+ release by preferential activation of cGMP-primed PKG. Am J Physiol Gastrointest Liver Physiol 281: G1238–G1245, 2001.[Abstract/Free Full Text]
  15. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, and Makhlouf GM. Differential signaling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145–155, 2003.[CrossRef][Web of Science][Medline]
  16. Okamoto T, Ikezu T, Murayama Y, Ogata E, and Nishimato I. Measurement of GTP{gamma}S binding to specific G proteins in membranes using G protein antibodies. FEBS Lett 305: 125–128, 1992.[CrossRef][Web of Science][Medline]
  17. Sarna SK. Giant migrating contractions and their myoelectric correlates in the small intestine. Am J Physiol Gastrointest Liver Physiol 253: G697–G705, 1987.[Abstract/Free Full Text]
  18. Sarna SK. Neuronal locus and cellular signaling for stimulation of ileum giant migrating and phasic contractions. Am J Physiol Gastrointest Liver Physiol 284: G789–G797, 2003.[Abstract/Free Full Text]
  19. Sarna SK, Stoddard C, Belbeck L, and McWade D. Intrinsic neural control of migrating myoelectric complexes. Am J Physiol Gastrointest Liver Physiol 241: G16–G23, 1981.[Abstract/Free Full Text]
  20. Shi XZ and Sarna SK. Inflammatory modulation of muscarinic receptor activation in canine ileal circular muscle cells. Gastroenterology 112: 864–874, 1997.[CrossRef][Web of Science][Medline]
  21. Shi XZ and Sarna SK. Differential inflammatory modulation of canine ileal longitudinal and circular muscle cells. Am J Physiol Gastrointest Liver Physiol 277: G341–G350, 1999.[Abstract/Free Full Text]
  22. Shi XZ and Sarna SK. Impairment of Ca2+ mobilization in circular muscle cells of the inflamed colon. Am J Physiol Gastrointest Liver Physiol 278: G234–G242, 2000.[Abstract/Free Full Text]
  23. Sohn UD, Harnett KM, Cao W, Rich H, Kim N, Behar J, and Biancani P. Acute experimental esophagitis activates a second signal transduction pathway in cat smooth muscle from the lower esophageal sphincter. J Pharmacol Exp Ther 283: 1293–1304, 1997.[Abstract/Free Full Text]



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