The important role of the parasympathetic nervous system in physiologic processes of GI tract motility and in the pathophysiology of motility disorders has been described for various species.1–7 In the cholinergic system, acetylcholine activates G-protein–coupled muscarinic receptors to cause smooth muscle contraction in several organs.1,2 Five mAChR subtypes (M1 to M5, respectively) have been cloned8 and defined pharmacologically.3,9
The M2 and M3 mAChR subtypes are the predominant receptors in the GI tract of several species, with a ratio for M2:M3 of approximately 4:1.1,10 However, the M3 receptor subtype is considered to be of predominant importance in eliciting muscle contractions, and the exact role of the more abundant M2 subtype remains unclear.10,11 A study12 in M3 receptor knockout mice has emphasized the role of M3 mAChRs for smooth muscle contraction because ileal contractile response to carbachol in vitro was reduced by 75% as compared to control animals. In comparison, ileal responses to carbachol were only slightly depressed in smooth muscle tissues from M2 knock-out mice.13 In contrast to these in vitro findings, M3 knock-out mice, as well as M2-M3 knockout mice, were free of apparent GI tract disorders, which suggests redundant regulation mechanisms through other mAChR subtypes or other mediators.12–14
In addition to M2 and M3, other mAChR subtypes are expressed in GI tissues. The M1 to M5 mAChRs have been detected in the digestive tract of several species, such as humans, pigs, dogs, mice, rats, guinea pigs, and rabbits.10,15–22 Immunohistochemical analysis has been used to verify M1, M3, and M5 mAChRs in the bovine GI tract.23 In that study, results for M2 mACHRs were inconclusive, and M4 mAChRs could not be detected.
Loss of intestinal tone and motility resulting in failure of orocaudal movement of GI contents and accumulation of fluid, gas, and ingesta that causes intestinal distention and abdominal pain is a life-threatening condition.24 In contrast to findings in horses, spontaneous paralytic (adynamic) ileus is more common in cattle than is postoperative paralytic ileus.6,24 Although the factors involved in the pathogenesis of postoperative ileus include inflammatory, metabolic, hormonal, neurogenic, and vascular mechanisms,25 spontaneous paralytic ileus in cattle is mainly attributable to metabolic disturbances and electrolyte imbalances, such as hypocalcemia or hypokaliemia,6,26 but sympathetic hyperactivity may also be involved.24
The duodenum also plays a pivotal role in abomasal emptying in ruminants; hence, motility disorders of the duodenum may result in incomplete abomasal emptying. This is believed to be associated with abomasal displacement, a common problem in dairy cattle.27,28 Therefore, prokinetic drugs that act on the small intestines and abomasum in cattle may be useful for clinical management of hypomotility or atony of the intestines and abomasal displacement.
Bethanechol, a methyl derivate of carbachol, induces contraction of smooth muscle cells by direct stimulation of mAChRs.2,29,30 It has been reported that there are no muscarinic receptor agonists with an extremely high selectivity for any particular mAChR subtype8; however, bethanechol was found to act primarily via M2 and M3 mAChR subtypes.31
Several researchers have indicated that bethanechol may be a promising substance for use in the treatment of animals with GI motility disorders. Bethanechol stimulates gastric emptying and intestinal propulsion in rats.30 In healthy horses, bethanechol stimulates gastric emptying32 and myoelectric activity of the ileum, cecum, and right ventral colon.33 In vitro, bethanechol induces a significant concentration-dependent increase in contractility traits in preparations of smooth muscle obtained from the esophageal groove of calves34 as well as the abomasal antrum35 and proximal portion of the colon of healthy cows.36 In contrast, no contractile effect of bethanechol was observed in preparations of smooth muscle obtained from the proximal portion of the duodenum.35 In vivo, bethanechol increases the myoelectric activity of the ileocecocolic area of healthy cows37 and the abomasum and duodenum of healthy yearling cattle.27 Bethanechol (0.07 mg/kg, SC, q 8 h for 2 days) also reportedly can be used successfully for conservative treatment of cows with spontaneous cecal dilatation.38 To our knowledge, in vitro effects of bethanechol on contractility of smooth muscle obtained from the distal portion of the duodenum and jejunum of cows and the various mAChR subtypes involved in mediating contraction have not been investigated.
The purpose of the study reported here was to determine whether bethanechol has an in vitro effect on contractility variables of longitudinal and circular smooth muscle obtained from the duodenum and jejunum of healthy cows and whether mAChR antagonists vary in their effects on the response induced by bethanechol. These results may also indicate the mAChR subtypes that are functionally involved in cholinergic contraction of smooth muscle in the distal portion of the duodenum and jejunum of cattle.
Materials and Methods
Tissue samples—Tissue samples were obtained at an abattoir from dairy cows that did not have a history of GI tract disease. Samples were obtained from 40 dairy cows and randomly allocated to 4 experimental groups. Breed and age distribution of the cows was similar for all experimental groups.
Specimens were collected within 20 minutes after cattle were stunned. Samples from the duodenum were obtained at a point 50 cm aboral to the pylorus, and jejunal samples were obtained at a point 2 m orad to the ileocaecal valve. Tissue samples (approx 8 × 10 cm) were immediately rinsed with cool (4°C) modified Krebs solutiona (118.4mM NaCl, 4.7mM KCl, 1.2mM KH2PO4, 2.5mM MgSO4, 3.3mM CaCl2, 25mM NaHCO3, and 12.2mM glucose monohydrate) that had been oxygenated with carbogen (95% oxygen and 5% carbon dioxide)b for 1 hour before use. Tissue samples were placed into this solution during transport to the laboratory (approx 20 minutes). At the laboratory, rectangular tissue samples were pinned flat in a dissecting dish containing oxygenated modified Krebs solution and cut parallel to the circular and longitudinal muscle fibers, respectively, by use of a custom-designed scalpel with 2 parallel blades. Final size of the resulting preparations was approximately 3 × 8 mm. Finally, the intestinal mucosa was removed from the preparation.
Recording of data—Muscle preparations (DC, DL, JC, and JL) were suspended separately in organ bath chambers,c each of which contained 50 mL of modified Krebs solution (37°C) constantly oxygenated with carbogen. Preparations were randomly allocated to the organ bath chambers. The distal end of each muscle strip was clamped to a hook and the proximal end was attached to an isometric force transducer,d as described elsewhere.35
The mechanical response of the muscle strips was amplifiede and recorded on a personal computer by use of a commercial data acquisition system.f The sampling rate was set at 10 samples/s/channel.
Experimental protocol—Preparations of smooth muscle were allowed to equilibrate for 1 hour, which included a period of 20 minutes of adjustment without tension, followed by a period of 1 g of tension for 10 minutes and a period of 2 g of tension for another 30 minutes. The functional viability of muscle preparations was assessed at the beginning and end of each experiment by the addition of carbacholg (1 × 10−6M) to control for muscle contraction. Only specimens that responded to carbachol with an increase in contractility variables were included in the study.
Carbachol application at the beginning of each experiment was followed by 3 flushes (whereby modified Krebs solution was completely changed 3 times at 15-second intervals in each organ bath) and a 15-minute recovery period. The subsequent 5 minutes were defined as the predrug period. Subsequently, cumulative concentration-response curves for bethanecholh were generated for each tissue location and orientation by increasing the concentration of bethanachol in each organ bath in logarithmic steps from 10−7 to 10−4M at 5-minute intervals. In each experiment, these concentration-response curves were generated with bethanechol, alone or after specimens had been incubated for 20 minutes with antagonists for various mAChR subtypes, which was designed to test the ability of each receptor antagonist to inhibit or enhance contraction generated by the cumulative administration of bethanechol. Each cumulative concentration-response curve was followed by another 3 flushes with modified Krebs solution, a 15-minute recovery period, and a 5-minute predrug period for the subsequent measurement. The order of reversibly binding mAChR antagonists was assigned randomly, but irreversibly binding receptor antagonists and atropine were always tested at the end of the experimental series.
Four experiments were performed. In experiment 1, the specimens (n = 10) were incubated with 11-[[2-[(diethylamino)methyl]-1-piperidinyl=acetyl]-5,11-dihydro-6H-pyrido;2,3-b][1,4]benzodiazepin-6-one (AFDX 116i; 10−6M), a reversible M2 mAChR antagonist, and with 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMPj; 2 × 10−8M), an irreversibly binding M3 mAChR antagonist. In experiment 2, the specimens (n = 10) were incubated with N,Na-bis[6-[[(2-methoxyphenyl)methyl]amino]-1,8-octane diamine tetrahydrochloride (methoctramine tetrahydrochloridek; 3 × 10−7M), a receptor antagonist targeting M2 and M4 mAChR subtypes; cyclohexyl-(4-fluorophenyl)-(3-N-piperidinopropyl)silanol hydrochloride (p-fluorohexahydro-sila-difenidol hydrochloride, p-F-HHSiDl; 8 × 10−7M), an M3 receptor antagonist; and atropine sulfatem (10−6M), a nonselective mAChR antagonist. In experiment 3, the specimens (n = 10) were incubated with 5,11-dihydro-11-;(4-methyl-1-piperazinyl)acetyl=-6H-pyrido;2,3-b=;1,4=benzodiazepin-6-one dihydrochloride (pirenzepine dihydrochloriden; 10−7M), an M1 mAChR antagonist, and with a combination of AF-DX 116 (10−6M) and 4-DAMP (2 × 10−8M). In experiment 4, a cumulative concentration-response curve for bethanechol was recorded for specimens (n = 10) incubated with N-ethyl-3-hydroxy-2-phenyl-N-(pyridinylmethyl) propanamide (tropicamideo; 5 × 10−8M), an M4 mAChR antagonist. Then, the muscle strips were subjected to a protection assay (termed 4-DAMPProtect). Specimens were incubated for 1 hour with 4-DAMP (2 × 10−8M) and AF-DX 116 (10−6M); the reversible receptor antagonist AF-DX 116 was used to protect M2 mAChRs during the alkylation procedure of M3 mAChRs induced by 4-DAMP. Tissue specimens were then flushed 3 times, and the cumulative concentration-response curves for bethanechol were recorded.
At the end of each experiment, muscle specimens were dried between 2 absorbent paper towels, and weight and length of each specimen were measured. All substances were dissolved in distilled water, except for AF-DX 116, 4-DAMP, and tropicamide, which were dissolved in dimethyl sulfoxide.p All concentrations were expressed as final concentrations in the organ baths.
Data analysis and statistical analysis—Several contractility variables were analyzed, which included basal tone (the tension measured between contractions [ie, the minimal amplitude], which was determined for each step of the concentration-response curves), Amax, and AUC. Variables were calculated at 5-minute intervals for each preparation and concentration by use of commercial software.q Calculated values for basal tone, Amax, and AUC were then adjusted by dividing them by the corresponding value of the cross-sectional area of the muscle specimens. Cross-sectional area was calculated for each preparation by assuming a tissue density of 1.056 g/cm3 and by use of the following equation39: area = mass/(density × length).
Concentration-response curves were calculated by use of the Hill equation and estimation by use of a least squares method with commercial simulation software.r
The underlying equation for the Hill function was as follows:
where C is the compound concentration, K is the EC50, and A describes the shape of the function (Hill coefficients with values > 1 describe curves with a flat low-dose region and high curvature, whereas values < 1 correspond to curves that increase rapidly). Significance of comparisons made on the basis of this model was determined by use of the likelihood ratio statistic, which yielded a χ2 test. The SD values for variables in the model were based on the Cramer-Rao statistic.40 Results were expressed as Vmax and EC50 of basal tone, Amax, and AUC, respectively.
Results of the 4 experiments were combined to investigate various treatment effects and were analyzed in 3 separate analyses.
For analysis 1, the effect of cumulative concentrations of bethanechol was tested with the Friedman test for each intestinal location and muscle orientation separately by use of commercial software.s Differences in Vmax and EC50 values for the bethanechol concentration-response curves without prior incubation with antagonists for all experiments (n = 40) were compared among intestinal locations and muscle orientations for basal tone, Amax, and AUC by use of the χ2 test.
For analysis 2, Vmax and EC50 values of the bethanechol concentration-response curves with or without mAChR antagonists were compared for basal tone, Amax, and AUC within each experiment (n = 10) and for each combination of intestinal location and muscle orientation (DC, DL, JC, and JL) separately by use of the χ2 test. Effects of the antagonists were compared between AF-DX 116 and 4-DAMP (experiment 1) and methoctramine and p-F-HHSiD (experiment 2) in accordance with the same principle.
For analysis 3, Vmax and EC50 values of the bethanechol concentration-response curves for incubation with AF-DX 116 (experiment 1; n = 10), 4-DAMP (experiment 1; 10), the combination of AF-DX 116 and 4-DAMP (experiment 3; 10), and 4-DAMPProtect (experiment 4; 10) were compared for Amax and AUC for each intestinal location and muscle orientation separately by use of the χ2 test. Results of Vmax for Amax and AUC were expressed relative to the corresponding predrug period of the concentration-response curves of bethanechol when incubated with the specific receptor antagonist and to the corresponding values of the bethanechol concentration-response curve without prior incubation with antagonists to correct for possible differences among experiments.
For analyses 1 and 2, results of Vmax for basal tone were reported as absolute values, whereas values for Amax and AUC were expressed as proportions relative to the corresponding predrug values. For all comparisons, significant differences were defined as values of P < 0.05.
Results
Comparisons among intestinal locations and muscle orientations for concentration-response curves of bethanechol (analysis 1)—In all preparations, bethanechol induced a significant (P < 0.001) concentration-dependent increase in basal tone. Values for Vmax differed significantly among locations within orientations and were significantly higher in preparations from the jejunum than from the duodenum. The value for JC (20.2 g/cm2) was significantly (P = 0.018) higher than the value for DC (13.5 g/cm2), and the value for JL (31.7 g/cm2) was significantly (P < 0.001) higher than the value for DL (13.7 g/cm2). Within the jejunum, Vmax was significantly (P = 0.016) higher in longitudinal specimens (JL, 31.7 g/cm2), compared with the value for the circular preparations (JC, 20.2 g/cm2); results for the longitudinal and circular samples did not differ significantly within the duodenum. The EC50 values did not differ significantly among locations and orientations.
A significant (P < 0.001) concentration-dependent increase in Amax and AUC was observed after bethanechol application in all preparations (Figure 1). Significant differences among locations and orientations were similar for Amax and AUC. Within locations, Vmax was greater in circular than in longitudinal preparations. Value for Amax in the DC (18.4 g/cm2) was significantly (P = 0.001) higher than the value for DL (6.0 g/cm2), whereas Amax in the JC was 18.3 g/cm2, which was higher but not significantly (P = 0.090) different from Amax in the JL (12.5 g/cm2). The AUC value for DC (13.4 g/cm2) was significantly (P < 0.001) higher than the value for DL (3.1 g/cm2). Similarly, the AUC value for JC (15.7 g/cm2) was significantly (P = 0.003) higher than the value for JL (8.4 g/cm2).
Comparison between locations within longitudinal preparations revealed that Vmax values were significantly higher in preparations from the jejunum than in preparations from the duodenum. The Amax for the JL (12.5 g/cm2) was significantly (P = 0.014) higher than the value for the DL (6.0 g/cm2), and the AUC for the JL (8.4 g/cm2) was significantly (P < 0.001) higher than the AUC for the DL (3.1 g/cm2). No significant differences were found between locations within circular preparations. Significant differences were not found in EC50 values among locations and orientations.
Effects of antagonists specific for various mAChR subtypes on bethanechol-induced contractile responses (analysis 2)—Prior incubation with atropine almost completely abolished the effect of bethanechol for all variables in all preparations. The M1 mAChR antagonist pirenzepine and M4 mAChR antagonist tropicamide had no significant effects on the bethanechol response for any variable. Bethanechol concentration-response curves determined for Amax for circular samples from the duodenum incubated with and without pirenzepine and tropicamide were plotted (Figure 2).
For basal tone, prior incubation with the M3 mAChR antagonist 4-DAMP caused a significant decrease in Vmax in all preparations and a significant increase in EC50 values in circular and longitudinal duodenal samples, compared with results for bethanechol alone(Table 1). Incubation with the M2 mAChR antagonist AF-DX 116 significantly decreased Vmax, and higher EC50 values were apparent in the duodenal samples, compared with results for bethanechol alone. The inhibiting effect of 4-DAMP (M3 receptor antagonist) on Vmax was significantly more pronounced than was the inhibiting effect of AF-DX 116 (M2 receptor antagonist) in all preparations, whereas no significant differences were observed in EC50 values. The combination of AF-DX 116 and 4-DAMP caused a significant decrease in Vmax in all preparations, compared with results for bethanechol alone, whereas significant differences were not found for EC50 values. For basal tone, 4-DAMPProtect significantly inhibited the response to bethanechol for Vmax (JC, P < 0.001; JL, P = 0.006) and EC50 (DC, P < 0.001; JL, P = 0.002).
Mean ± SD values for Vmax and EC50 values for various mAChR antagonists on bethanechol concentration-response curves for basal tone for DL.
Experiment | mAChR agonist or antagonist | Primary selectivity | Vmax(g/cm2) | EC50(M) |
---|---|---|---|---|
1 | Bethanechol | NA | 25.52 ± 28.83a | 2.62 × 10−5 ± 3.34 × 10−5a |
Bethanechol + AF-DX 116 | M2 | 22.01 ± 76.59b | 1.00 × 10−4 ± 7.25 × 10−4b | |
Bethanechol + 4-DAMP | MM3 | 4.45 ± 19.71c | 1.00 × 10−4 ± 1.35 × 10−3b | |
2 | Bethanechol | NA | 12.13 ± 14.47a | 2.59 × 10−5 ± 4.25 × 10−5a |
Bethanechol + methoctramine | M2 and M4 | 12.62 ± 48.47a,c | 1.00 × 10−4 ± 4.87 × 10−4b | |
Bethanechol + p-F-HHSiD | M3 | 1.72 ± 35.32b | 3.40 × 10−5 ± 3.38 × 10−3 | |
Bethanechol + atropine | Nonselective | 0.13 ± 0.31b,c | 1.13 × 10−7 ± 1.59 × 10−6 | |
3 | Bethanechol | NA | 17.96 ± 20.66a | 2.54 × 10−5 ± 1.24 × 10−4 |
Bethanechol + pirenzepine | M1 | 24.52 ± 57.96 | 6.04 × 10−5 ± 2.69 × 10−4 | |
Bethanechol+ AF-DX 116 + 4-DAMP | M2and M3 | 3.82 ± 9.91b | 5.74 × 10−5 ± 2.75 × 10−4 | |
4 | Bethanechol | NA | 13.10 ± 163.68 | 6.00 × 10−5 ± 1.14 × 10−3 |
Bethanechol + tropicamide | M4 | 14.60 ± 52.04 | 1.00 × 10−4 ± 6.81 × 10−4 | |
Bethanechol + 4-DAMPProtect | M3 | 10.95 ± 41.45 | 1.00 × 10−46.53 × 10−4 |
For each experiment, n = 10.
Within a column within an experiment, values with different superscript letters differ significantly (P < 0.05; comparisons among mAChR antagonists were made only for AF-DX 116 vs 4-DAMP and for methoctramine vs p-F-HHSiD [analysis 2]).
NA = Not applicable.4-DAMPProtect = Specimens were incubated for 1 hour with 4-DAMP and the reversible receptor antagonist AF-DX 116, which was used to protect M2 mAChRs during the alkylation procedure of M3 mAChRs induced by 4-DAMP.
The M3 mAChR antagonist p-F-HHSiD caused a significant decrease in Vmax for basal tone in all preparations, compared with results for bethanechol (Figure 3). The EC50 values were significantly increased in circular (P = 0.005) but not in longitudinal (P = 0.089) specimens from the jejunum (Table 1). The M2 mAChR antagonist methoctramine had a significant inhibitory effect on bethanechol for Vmax (DC, P = 0.008) and EC50 values (DL, P = 0.024; JC, P = 0.006). In contrast, methoctramine caused lower but not significantly (P = 0.090) different values in EC50 in circular duodenal samples, compared with results for bethanechol alone. The effect of the M3 mAChR antagonist p-F-HHSiD on Vmax was significantly higher than that of the M2 mAChR antagonist methoctramine (DC, P < 0.001; DL, P < 0.001; JC, P = 0.007; JL, P = 0.011). In contrast, significant differences were not observed in EC50 values.
Prior incubation with 4-DAMP caused a significant decrease in Vmax for Amax and AUC, compared with values for bethanechol alone (Figure 2). The Amax value for DC was significantly (P < 0.001) decreased, compared with the value for bethanechol alone. Similarly, there was a significant decrease for the AUC value for DC (P < 0.001) and JC (P = 0.044), compared with results for bethanechol alone. However, EC50 values did not differ significantly (Amax for JC, P = 0.076; AUC for DL, P = 0.072), compared with results for bethanechol alone. The reduction of Amax and AUC caused by 4-DAMP was more pronounced than that caused by AF-DX 116 for Vmax (Amax for JC, P = 0.049; AUC for DC, P = 0.004; AUC for JC, P = 0.014; and AUC for JL, P = 0.028); no significant differences were observed in EC50 values.
The combination of AF-DX 116 and 4-DAMP caused a significant decrease in Vmax in circular samples from both locations for Amax and AUC (Amax for DC, P = 0.006; Amax for JC, P < 0.001; AUC for DC, P = 0.004; and AUC for JC, P < 0.001). No significant differences were found among EC50 values.
The 4-DAMPProtect resulted in a significant decrease in Vmax for Amax (JC, P = 0.004) but a nonsignificant decrease in Vmax for AUC (JC, P = 0.061), compared with results for bethanechol alone (Table 2). The 4-DAMPProtect significantly inhibited the response to bethanechol for both contractility variables for EC50 (Amax for DC, P = 0.009; Amax for JC, P = 0.011; and AUC for DC, P = 0.010). No significant differences were detected for all other comparisons.
Mean ± SD values for Vmax and EC50 values for the effects of mAChR antagonists on bethanechol concentration-response curves for AUC for JC.
Experiment | mAChR agonist or antagonist | Primary selectivity | Vmax (%)* | EC50(M) |
---|---|---|---|---|
1 | Bethanechol | NA | 25.05 ± 4.09a | 1.45 × 105 ± 3.54 × 106 |
Bethanechol+ AF-DX 116 | M2 | 33.06 ± 19.37a | 3.77 × 10−5 ± 5.12 × 10−5 | |
Bethanechol + 4-DAMP | M3 | 12.38 ± 5,561.5b | 1.00 × 10−4 ± 3.94 × 10−2 | |
2 | Bethanechol | NA | 14.67 + 4 85a | 2.73 × 10−5 + 2.15 × 10−5 |
Bethanechol + methoctramine | M2 and M4 | 15.77 ± 11.29 | 4.49 × 10−5 ± 6.85 × 10−5 | |
Bethanechol + p-F-HHSiD | M3 | 16.15 ± 78.34 | 4.12 × 10−5 ± 4.85 × 10−4 | |
Bethanechol + atropine | Nonselective | 0.5 ± 5.38b | 1.00 × 10−4 ± 3.64 × 10−3 | |
3 | Bethanechol | NA | 11.59 + 2.77a | 1.92 × 10−5 + 1.37 × 10−5 |
Bethanechol + pirenzepine | M1 | 15.97 ± 12.76 | 4.90 × 10−5 ± 8.12 × 105 | |
Bethanechol+ AF-DX 116 + 4-DAMP | M2and M3 | 1.21 ± 1.46b | 1.00 × 10−4 ± 8.12 × 105 | |
4 | Bethanechol | NA | 15.07 ± 2.52 | 1.32 × 10−5 ± 3.55 × 10−6 |
Bethanechol + tropicamide | M4 | 15.03 ± 3.77 | 2.09 × 10−5 ± 1.57 × 10−5 | |
Bethanechol+ 4-DAMPProtect | M3 | 4.21 ± 1,928.01 | 4.84 × 10−5 ± 5.21 × 102 |
For each experiment, n = 10.
Represents results relative to predrug values.
See Table 1 for remainder of key.
The receptor antagonists AF-DX 116, methoctramine, and p-F-HHSiD did not significantly influence the effect of bethanechol on Amax and AUC (Table 2). We did not detect a significant difference for Vmax or EC50 when effects of methoctramine were compared with effects of p-F-HHSiD.
Comparison of the effects of AF-DX 116, 4-DAMP, the combination AF-DX 116 and 4-DAMP, and 4-DAMPProtect(analysis 3)—Because several predrug values for basal tone were extremely low, it was not possible to transform the values relative to the respective bethanechol curves and the predrug values to conduct analysis 3. Consequently, comparisons among receptor antagonists were conducted for Amax and AUC only.
The EC50 values for Amax were significantly lower for 4-DAMPProtect than for 4-DAMP alone (JC, P = 0.047) or after combined incubation with AF-DX 116 and 4-DAMP (DC, P = 0.003; JC, P = 0.025), whereas no significant differences in Vmax were observed for the same comparisons. There was a nonsignificant effect for Amax in 1 location each for EC50 values (JC, P = 0.093), which indicated a less pronounced inhibiting effect for 4-DAMPProtect than for AF-DX 116 alone. Similarly, there was a nonsignificant effect (DL, P = 0.059), which indicated a more pronounced inhibiting effect for AF-DX 116 in combination with 4-DAMP than for AF-DX 116 alone. No significant differences were evident for Vmax for these comparisons. Significant differences in Amax were not observed between the inhibition caused by the combination of AF-DX 116 with 4-DAMP and inhibition for 4-DAMP alone.
The EC50 values for AUC were significantly (JC, P < 0.001) lower for 4-DAMPProtect, compared with values for 4-DAMP alone, whereas no significant differences were observed for the same comparisons for Vmax. Significant differences were not observed for AUC (Vmax and EC50) when comparing the effects of the combination AF-DX 116 and 4-DAMP with effects for 4-DAMPProtect, AF-DX 116, or 4-DAMP alone. Similarly, the effects of 4-DAMPProtect and AF-DX 116 did not differ significantly.
Discussion
Analysis of results of the study reported here revealed that bethanechol has in vitro contractile effects on smooth muscle obtained from the duodenum and jejunum of healthy cows because it induces a significant, concentration-dependent increase in basal tone, Amax, and AUC. Despite the fact that the responsiveness to bethanechol differs among species and anatomic sites,32 our findings are consistent with the effect of bethanechol described for the stomach and small intestines of rats30; descending colon and rectum of humans31; ileum, cecum, and right ventral colon of ponies33; and stomach, duodenum, jejunum, cecum, and pelvic flexure of horses.5,32 In cattle, contractile effects of bethanechol have been reported35–37,t,u for the abomasum, ileum, cecum, ansa proximalis coli, and spiral colon. In healthy yearling cattle, bethanechol significantly increases the myoelectric spike rate of the duodenum at locations 5, 10, and 15 cm aboral to the pylorus.27 In contrast, analysis of results of another study35 on in vitro effects of bethanechol at higher concentrations (1 × 10−10 to 3 × 10−3M) than those used in the study reported here (1 × 10−7 to 1 × 10−4M) indicated no effect of bethanechol on contractility variables in smooth muscle obtained from the proximal portion of the duodenum (10 cm aboral to the pylorus) of healthy cows. These contradictory findings may indicate differing responses to bethanechol caused by differences in the distribution of mAChR subtypes within the duodenum (ie, M2 or M3 mAchR [or both] in the distal but not in the proximal portion of the duodenum).
Another indication of unequal distribution of mAChRs along the GI tract may be evident in the significant differences in contractility among GI locations observed in the study reported here. In general, the effects of bethanechol were more pronounced in jejunal specimens than in duodenal preparations. This result may reflect true differences between locations; however, it may also have been attributable to forces exerted on the duodenum during the slaughtering and evisceration process because the entire intestine was suspended by the proximal portion of the intestines for several minutes during the slaughtering process, which may have contributed to lower responsiveness and contractility of the duodenum.
Finally, the contractile effects of bethanechol were significantly more pronounced in circular than in longitudinal preparations for Amax and AUC, whereas the opposite effects were detected for basal tone (ie, contractile effects were more pronounced in the longitudinal than in the circular preparations of the jejunum and were totally lacking in the duodenum). In another study,35 similar results were found for the abomasal antrum of cattle in which the effects of bethanechol were significantly more pronounced in circular than in longitudinal preparations for mean amplitude and AUC, whereas opposite effects were observed for basal tone.
To determine the mAChR subtypes functionally involved in mediation of the cholinergic contraction induced by bethanechol, experiments were designed that included prior incubation with mAChR antagonists. Concentration of the anticholinergic agent atropine (10−6M) was selected on the basis of results of other studies.41–43 The fact that this concentration of atropine almost completely abolished the effects of bethanechol confirmed that the mAChRs were blocked and corroborated that the effect of bethanechol is mediated via mAChRs. Concentrations of specific mAChR antagonists used in the study were determined from antagonist affinity values reported in other studies,8,15,44–46 which allowed maximal occupancy of the receptor subtype of interest with minimal occupation of other subtypes. The mAChR antagonists used in the study reported here possess differing selectivity for the various mAChR subtypes, depending on their affinity patterns. Thus, the affinity pattern for pirenzepine47 is M1 > M4 > M3 > M2, whereas the pattern for AF-DX 11615,48,49 is M2 > M1 ≥ M4 > M3, and the pattern for methoctramine15,41,45 is M2 ≥ M4 > M1 > M3. The antagonist patterns for 4-DAMP and p-F-HHSiD are M3 > M1 ≥ M4 > M2.15,45,50 Affinity data for tropicamide are sparse, but this receptor antagonist appears to bind primarily to M4.51,52 We are not aware of any receptor antagonists with high affinity for M5 receptors.
Studies11,44,53–55 have revealed that > 1 mAChR sub-type may be responsible for mediation of cholinergic contraction in tissues. Analysis of results of the study reported here indicated that the effect of bethanechol in the intestinal segments of interest was mediated by both M2 and M3 mAChRs, with activation of M3 mAChRs causing stronger increases in contractility traits than for activation of M2 mAChRs, as indicated by use of both sets of receptor antagonists. This finding is consistent with findings in rodents that have revealed a predominant role of M3 mAChRs in the cholinergic mediation of smooth muscle contraction,2,3,10 despite the fact that the density of M2 receptors is higher than that of M3 receptors.1,3,10 The M2 mAChRs appear to play a role mainly under pathologic conditions (eg, increased sympathetic activity; in knockout mice deficient in M3 receptors; in animals with dysfunctional M3 receptors; and in some disease states, such as rats with experimentally induced diabetes mellitus, intestinal denervation, or bladder denervation).3,12,45
The predominant role of M3 mAChRs in smooth muscle contraction, compared with that for M2 mAChRs, may be partly explained by differences in the pathways to mediate these effects. Both receptors are linked to G-protein–coupled second messenger systems15; however, in contrast to M3 mAChRs that act directly through activation of phospholipase C, M2 mAChRsindirectly mediate contraction via inhibition of the relaxation caused by β-adrenoceptor stimulation, which causes activation of adenylate cyclase.3,10,56
Binding characteristics of competitive and irreversibly binding receptor antagonists were confirmed in our study because the competitive M2 mAChR antagonist AF-DX 116 caused a visible rightward shift of concentration-response curves, whereas Vmax values for Amax and AUC did not differ from those for concentration-response curves without receptor antagonists. In contrast, the alkylating M3 mAChR antagonist 4-DAMP caused a significant decrease in Vmax for the examined contractility variables, which could not be counteracted by increases in bethanechol concentrations.
Data analysis to compare the effects of M2 and M3 mAChR antagonists alone or in combination was complicated by the fact that it was impossible to test all mAChR antagonists in 1 experimental series. The effects of the irreversibly binding M3 receptor antagonist 4-DAMP alone or in combination could not be investigated consecutively during the same experiment. It has been reported57 that repeated stimulation of mAChRs up to 6 consecutive times does not affect the responsiveness of smooth muscle preparations. Limiting the duration of experiments to 5 hours and a maximum of 4 concentration-response curves of bethanechol conducted in 1 experimental series allowed us to exclude bias caused by exhaustion of the muscle preparations. However, the experiments had to be conducted in 4 series, which complicated data analysis. Expression of the results for Amax and AUC relative to the corresponding predrug periods and the corresponding bethanechol concentration-response curves allowed us to conduct valid comparisons among receptor antagonists in the various experiments, but this transformation was not possible for basal tone because of the numerous extremely low values at the beginning of the experiments (predrug values).
Use of M2 and M3 mAChR antagonists alone or in combination resulted in significantly smaller EC50 values for the bethanechol concentration-response curves for 4-DAMPProtect than after prior incubation of muscle preparations with 4-DAMP alone or with a combination of AF-DX 116 and 4-DAMP. The protection assay was designed to eliminate potential unspecific effects of the M3 mAChR antagonist 4-DAMP on M2 mAChRs during incubation with 4-DAMP alone.58 Protection of M2 mAChRs by use of AF-DX 116 during the incubation period was followed by thorough flushing; thus, the observed effect of 4-DAMP must have been caused through binding of bethanechol to M3 mAChRs. The fact that the inhibitory effect of the M3 receptor antagonist 4-DAMP (2 × 10−8M) without AF-DX 116 to protect the M2 receptors was significantly greater than in the protection assay indicated that there is unspecific binding of 4-DAMP to the M2 mAChRs. This observation corroborates the fact that commercially available mAChR antagonists possess limited selectivity for specific receptor subtypes and confirms that their effects cannot be assigned exclusively to 1 mAChR subtype.46,59
Analysis of results of the study reported here suggests that the M1 and M4 mAChR subtypes are not involved in mediation of the contractile effect of bethanechol in the locations examined, although M1 mAChRs have been found in the plexus submucosus and myenteric plexus of the bovine GI tract (from the abomasum to the colon) by use of immunohistochemical analysis.23 In the ileum of guinea pigs, blot hybridization techniques60 revealed large amounts of mRNA coding for M2, a small amount of mRNA coding for M3, and only traces of mRNA for M1. In a study4 of the colon of dogs, M2 and M3 mAChR subtypes, but not M1 and M4 subtypes, were detected by use of radioligand binding and cDNA hybridization. The choice for the pirenzepine (an M1 receptor antagonist) concentration used here (10−7M) was determined on the basis of protocols from other studies42,61–63 in which it was judged that this concentration achieved adequate inhibition of M1 mAChRs. Therefore, it does not appear that M1 receptors are involved in mediation of smooth muscle contraction induced by bethanechol. This observation does not preclude a possible role of M1 mAChRs in the regulation of intestinal motility, but such effects of M1 receptors would be mediated by other receptor agonists. Our results concerning the role of M4 receptors in cholinergic mediation of contraction is in accordance with the fact that M4 receptors were not detected by use of immunohistochemical analysis in the bovine GI tract.23
Bethanechol increased contractility variables of smooth muscle specimens obtained from the duodenum and jejunum of healthy cows in a concentrationdependent manner. In general, the contractile effects of bethanechol were more pronounced in jejunal than in duodenal specimens and in circular than in longitudinal preparations. Furthermore, analysis of the results of experiments involving mAChR antagonists indicated that the effect of bethanechol was primarily mediated via M3 mAChRs and, with a minor role, via M2 mAChRs. A similar effect of bethanechol has been reported for the abomasum,35,t ileum, and large intestineu of cattle in vitro. Thus, analysis of results of the study reported here and other studies suggests that there may be potential beneficial clinical effects from the use of bethanechol as a prokinetic drug for GI motility disorders, such as paralytic ileus, displacement of the abomasum, or cecal dilatation in cattle. Additional studies are warranted to investigate the in vivo effects of bethanechol in cows with the aforementioned diseases.
ABBREVIATIONS
GI | Gastrointestinal |
mAChR | Muscarinic acetylcholine receptor |
DC | Duodenum circular |
DL | Duodenum longitudinal |
JC | Jejunum circular |
JL | Jejunum longitudinal |
Amax | Maximal amplitude of contractions |
AUC | Area under the concentration-response curve |
Vmax | Maximal attainable response |
EC50 | Concentration that causes 50% of the maximal effect |
Modified Krebs solution, Dr. E. Graeub AG, Berne, Switzerland.
Carbogen, Carbagas, Liebefeld-Bern, Switzerland.
Organ baths, ML0186, LSi LETICA, Panlab s.l., Barcelona, Spain.
MLT0201, LSi LETICA, Panlab s.l., Barcelona, Spain.
ML119, ADInstruments GmbH, Spechbach, Germany.
Power Lab, ADInstruments GmbH, Spechbach, Germany.
Carbachol, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland.
Carbamyl-β-methylcholine chloride, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland.
AF-DX 116, Tocris, Bristol, UK.
4-DAMP, Tocris, Bristol, UK.
Methoctramine tetrahydrochloride, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland.
p-F-HHSiD, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland.
Atropine sulfate salt hydrate, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland.
Pirenzepine dihydrochloride, Tocris, Bristol, UK.
Tropicamide, Tocris, Bristol, UK.
Dimethyl sulfoxide, Dr. Grogg Chemie AG, Stettlen-Deisswil, Switzerland.
ChartTM, Power Lab, ADInstruments GmbH, Spechbach, Germany.
MatLab Simulation software, release 14, The MathWorks Inc, Cambridge, Mass.
SYSTAT, SPSS Inc, Chicago, Ill.
Bühler M. In vitro effects of bethanechol on smooth muscle preparations from abomasal fundus, corpus, and antrum of healthy dairy cows. Doctoral thesis, Vetsuisse Faculty, University of Berne, Berne, Switzerland, 2005.
Zanolari P, Marti M, Meylan M, et al. In vitro Effekte von Bethanechol auf Glattmuskelpräparate von Dünndarm, Blinddarm und Kolon bei der laktierenden Kuh (abstr), in Proceedings. Buiatrissima 1st Swiss Buiatrics Cong 2005;111.
References
- 1
Eglen RM, Hegde SS, Watson N. Muscarinic receptor subtypes and smooth muscle function. Pharmacol Rev 1996;48:531–565.
- 2↑
Wrzos HF, Tandon T, Ouyang A. Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats. World J Gastroenterol 2004;10:3292–3298.
- 3↑
Uchiyama T, Chess-Williams R. Muscarinic receptor subtypes of the bladder and gastrointestinal tract. J Smooth Muscle Res 2004;40:237–247.
- 4↑
Zhang LB, Horowitz B, Buxton IL. Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes. Mol Pharmacol 1991;40:943–951.
- 5
Marti M, Mevissen M, Althaus H, et al. In vitro effects of bethanechol on equine gastrointestinal contractility and functional characterization of involved muscarinic receptor subtypes. J Vet Pharmacol Ther 2005;28:565–574.
- 6
Steiner A. Modifiers of gastrointestinal motility of cattle. Vet Clin North Am Food Anim Pract 2003;19:647–660.
- 7
Oyachi N, Lakshmanan J, Ahanya SN, et al. Development of ovine fetal ileal motility: role of muscarinic receptor subtypes. Am J Obstet Gynecol 2003;189:953–957.
- 8↑
Caulfield MP, Birdsall NJ. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 1998;50:279–290.
- 9
Eglen RM. Muscarinic receptor subtype pharmacology and physiology. Prog Med Chem 2005;43:105–136.
- 10
Eglen RM. Muscarinic receptors and gastrointestinal tract smooth muscle function. Life Sci 2001;68:2573–2578.
- 11
Ehlert FJ, Sawyer GW, Esqueda EE. Contractile role of M2 and M3 muscarinic receptors in gastrointestinal smooth muscle. Life Sci 1999;64:387–394.
- 12↑
Matsui M, Motomura D, Karasawa H, et al. Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci U S A 2000;97:9579–9584.
- 13↑
Stengel PW, Gomeza J, Wess J, et al. M(2) and M(4) receptor knockout mice: muscarinic receptor function in cardiac and smooth muscle in vitro. J Pharmacol Exp Ther 2000;292:877–885.
- 14
Matsui M, Motomura D, Fujikawa T, et al. Mice lacking M2 and M3 muscarinic acetylcholine receptors are devoid of cholinergic smooth muscle contractions but still viable. J Neurosci 2002;22:10627–10632.
- 15↑
Caulfield MP. Muscarinic receptors—characterization, coupling and function. Pharmacol Ther 1993;58:319–379.
- 16
Ehlert FJ, Ostrom RS, Sawyer GW. Subtypes of the muscarinic receptor in smooth muscle. Life Sci 1997;61:1729–1740.
- 17
Nelson DK, Pieramico O, Dahmen G, et al. M1-muscarinic mechanisms regulate interdigestive cycling of motor and secretory activity in human upper gut. Dig Dis Sci 1996;41:2006–2015.
- 18
Akbulut H, Goren Z, Iskender E, et al. Subtypes of muscarinic receptors in rat duodenum: a comparison with rabbit vas deferens, rat atria, guinea-pig ileum and gallbladder by using imperialine. Gen Pharmacol 1999;32:505–511.
- 19
Stadelmann AM, Walgenbach-Telford S, Telford GL, et al. Distribution of muscarinic receptor subtypes in rat small intestine. J Surg Res 1998;80:320–325.
- 20
Wess J. Muscarinic acetylcholine receptor knockout mice: novel phenotypes and clinical implications. Annu Rev Pharmacol Toxicol 2004;44:423–450.
- 21
So I, Yang DK, Kim HJ, et al. Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig. Exp Mol Med 2003;35:46–52.
- 22
Dorje F, Levey AI, Brann MR. Immunological detection of muscarinic receptor subtype proteins (m1–m5) in rabbit peripheral tissues. Mol Pharmacol 1991;40:459–462.
- 23↑
Stoffel MH, Wicki Monnard C, Steiner A, et al. Distribution of muscarinic receptor subtypes and interstitial cells of Cajal in the gastrointestinal tract of healthy dairy cows. Am J Vet Res 2006;67:1992–1997.
- 24↑
Radostits OM, Blood DC, et al. Manifestation of alimentary tract dysfunction. In: Radostits OM, Blood DC, Hinchcliff KW, ed.Veterinary medicine. 9th ed.London: WB Saunders Co, 2000;171–176.
- 25↑
Bauer AJ, Boeckxstaens GE. Mechanisms of postoperative ileus. Neurogastroenterol Motil 2004;16:54–60.
- 26
Sattler D, Fecteau G, Girard C, et al. Description of 14 cases of bovine hypokalemia syndrome. Vet Rec 1998;143:503–507.
- 27↑
Roussel AJ, Brumbaugh GW, Waldron RC, et al. Abomasal and duodenal motility in yearling cattle after administration of prokinetic drugs. Am J Vet Res 1994;55:111–115.
- 28
Eicher R, Audige L, Braun U, et al. Epidemiology and risk factors of cecal dilatation/dislocation and abomasal displacement in dairy cows. Schweiz Arch Tierheilkd 1999;141:423–429.
- 29
Kilbinger H, Weihrauch TR. Drugs increasing gastrointestinal motility. Pharmacology 1982;25:61–72.
- 30↑
Megens AA, Awouters FH, Niemegeers CJ. General pharmacology of the four gastrointestinal motility stimulants bethanechol, metoclopramide, trimebutine, and cisapride. Arzneimittelforschung 1991;41:631–634.
- 31↑
Law NM, Bharucha AE, Undale AS, et al. Cholinergic stimulation enhances colonic motor activity, transit, and sensation in humans. Am J Physiol Gastrointest Liver Physiol 2001;281: G1228–G1237.
- 32↑
Ringger NC, Lester GD, Neuwirth L, et al. Effect of bethanechol or erythromycin on gastric emptying in horses. Am J Vet Res 1996;57:1771–1775.
- 33↑
Lester GD, Merritt AM, Neuwirth L, et al. Effect of alpha 2-adrenergic, cholinergic, and nonsteroidal anti-inflammatory drugs on myoelectric activity of ileum, cecum, and right ventral colon and on cecal emptying of radiolabeled markers in clinically normal ponies. Am J Vet Res 1998;59:320–327.
- 34↑
Barahona MV, Sanchez-Fortun S, San Andres MD, et al. Acetylcholinesterase histochemistry and functional characterization of the muscarinic receptor mediating the contraction of the bovine oesophageal groove. J Auton Pharmacol 1997;17:77–86.
- 35↑
Michel A, Mevissen M, Burkhardt HW, et al. In vitro effects of cisapride, metoclopramide and bethanechol on smooth muscle preparations from abomasal antrum and duodenum of dairy cows. J Vet Pharmacol Ther 2003;26:413–420.
- 36↑
Steiner A, Denac M, Ballinari U. Effects of adrenaline, dopamine, serotonin, and different cholinergic agents on smooth muscle preparations from the ansa proximalis coli in cattle: studies in vitro. Zentralbl Veterinarmed [A] 1992;39:541–547.
- 37↑
Steiner A, Roussel AJ, Martig J. Effect of bethanechol, neostigmine, metoclopramide, and propranolol on myoelectric activity of the ileocecocolic area in cows. Am J Vet Res 1995;56:1081–1086.
- 38↑
Steiner A, Meylan M, Eicher R. New aspects on the etiopathogenesis and treatment of cecal dilatation/dislocation in cows—a review. Schweiz Arch Tierheilkd 1999;141:419–422.
- 39↑
Hirsbrunner G, Knutti B, Liu I, et al. An in vitro study on spontaneous myometrial contractility in the cow during estrus and diestrus. Anim Reprod Sci 2002;70:171–180.
- 40↑
Portier C, Tritscher A, Kohn M, et al. Ligand/receptor binding for 2,3,7,8-TCDD: implications for risk assessment. Fundam Appl Toxicol 1993;20:48–56.
- 41
Dorje F, Wess J, Lambrecht G, et al. Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Ther 1991;256:727–733.
- 42
Stengel PW, Cohen ML. Muscarinic receptor knockout mice: role of muscarinic acetylcholine receptors M(2), M(3), and M(4) in carbamylcholine-induced gallbladder contractility. J Pharmacol Exp Ther 2002;301:643–650.
- 43
Shi H, Wang H, Wang Z. Identification and characterization of multiple subtypes of muscarinic acetylcholine receptors and their physiological functions in canine hearts. Mol Pharmacol 1999;55:497–507.
- 44
Eglen RM, Harris GC. Selective inactivation of muscarinic M2 and M3 receptors in guinea-pig ileum and atria in vitro. Br J Pharmacol 1993;109:946–952.
- 45
Eglen RM, Reddy H, Watson N, et al. Muscarinic acetylcholine receptor subtypes in smooth muscle. Trends Pharmacol Sci 1994;15:114–119.
- 46
Mansfield KJ, Mitchelson FJ, Moore KH, et al. Muscarinic receptor subtypes in the human colon: lack of evidence for atypical subtypes. Eur J Pharmacol 2003;482:101–109.
- 47↑
Grimm U, Fuder H, Moser U, et al. Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens. Naunyn Schmiedebergs Arch Pharmacol 1994;349:1–10.
- 48
Hammer R, Giraldo E, Schiavi GB, et al. Binding profile of a novel cardioselective muscarine receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat. Life Sci 1986;38:1653–1662.
- 49
Giraldo E, Monferini E, Ladinsky H, et al. Muscarinic receptor heterogeneity in guinea pig intestinal smooth muscle: binding studies with AF-DX 116. Eur J Pharmacol 1987;141:475–477.
- 50
Eltze M, Ullrich B, Mutschler E, et al. Characterization of muscarinic receptors mediating vasodilation in rat perfused kidney. Eur J Pharmacol 1993;238:343–355.
- 51
Hernandez M, Simonsen U, Prieto D, et al. Different muscarinic receptor subtypes mediating the phasic activity and basal tone of pig isolated intravesical ureter. Br J Pharmacol 1993;110:1413–1420.
- 52
Lazareno S, Buckley NJ, Roberts FF. Characterization of muscarinic M4 binding sites in rabbit lung, chicken heart, and NG108-15 cells. Mol Pharmacol 1990;38:805–815.
- 53
Stengel PW, Yamada M, Wess J, et al. M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea. Am J Physiol Regul Integr Comp Physiol 2002;282:R1443–R1449.
- 54
Dietrich C, Kilbinger H. Prejunctional M1 and postjunctional M3 muscarinic receptors in the circular muscle of the guinea-pig ileum. Naunyn Schmiedebergs Arch Pharmacol 1995;351:237–243.
- 55
Sawyer GW, Ehlert FJ. Muscarinic M3 receptor inactivation reveals a pertussis toxin-sensitive contractile response in the guinea pig colon: evidence for M2/M3 receptor interactions. J Pharmacol Exp Ther 1999;289:464–476.
- 56
Peralta EG, Ashkenazi A, Winslow JW, et al. Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature 1988;334:434–437.
- 57↑
Kaze C, Mevissen M, Hirsbrunner G, et al. Effect of endotoxins on contractility of smooth muscle preparations from the bovine abomasal antrum. Dtsch Tierarztl Wochenschr 2004;111:28–35.
- 58↑
Thomas EA, Baker SA, Ehlert FJ. Functional role for the M2 muscarinic receptor in smooth muscle of guinea pig ileum. Mol Pharmacol 1993;44:102–110.
- 59
Wess J. Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 1996;10:69–99.
- 60↑
Maeda A, Kubo T, Mishina M, et al. Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes. FEBS Lett 1988;239:339–342.
- 61
Eglen RM, Cornett CM, Whiting RL. Interaction of p-F-HHSiD (p-fluoro-hexahydrosila-difenidol) at muscarinic receptors in guinea-pig trachea. Naunyn Schmiedebergs Arch Pharmacol 1990;342:394–399.
- 62
Eglen RM, Michel AD, Montgomery WW, et al. The interaction of parafluorohexahydrosiladiphenidol at muscarinic receptors in vitro. Br J Pharmacol 1990;99:637–642.
- 63
Shi H, Wang H, Wang Z. M3 muscarinic receptor activation of a delayed rectifier potassium current in canine atrial myocytes. Life Sci 1999;64:PL251–PL257.