Introduction

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Muscarinic receptors are expressed abundantly in smooth muscle throughout the gastrointestinal tract in a manner that approximates a three-to-one mixture of the M₂ and M₃ subtypes (see Ehlert et al., 1997). Muscarinic agonists elicit contraction through the M₃ receptor under standard conditions (i.e., no other contractile or relaxant agents present) in smooth muscle ranging from the esophagus to ileum. It is known that the M₃ receptor stimulates phospholipase C- causing inositol-1,4,5-trisphosphate accumulation and calcium mobilization. The extent to which the contractile response depends on this burst of calcium and how this calcium interacts with other transduction mechanisms remains to be determined.

The M₂ muscarinic receptor has been shown to mediate a pertussis toxin-sensitive inhibition of adenylyl cyclase activity in the ileum and colon (Candell et al., 1990; Zhang and Buxton, 1991; Thomas and Ehlert, 1994). This effect opposes the increase in cAMP elicited by forskolin and isoproterenol. In gastrointestinal smooth muscle, forskolin and isoproterenol elicit relaxation through cAMP. The M₂ receptor has been shown to cause an indirect contraction in the ileum by preventing the relaxant effects of forskolin and isoproterenol on histamine-induced contractions (Thomas et al., 1993; Thomas and Ehlert, 1994; Reddy et al., 1995; Ostrom and Ehlert, 1997). In the ileum, therefore, muscarinic agonists are known to have a dual effect on contraction, a direct M₃-mediated contraction and an indirect M₂-mediated inhibition of relaxation.

Muscarinic receptors have also been shown to induce a nonselective cation conductance in the longitudinal smooth muscle of the guinea pig ileum (Inoue, 1991). This conductance is pertussis toxin-sensitive (Inoue and Isenberg, 1990a; Unno et. al., 1995), which suggests that the M₂ receptor may be coupled to the nonselective cation conductance. The nonselective cationic conductance is enhanced by calcium in the guinea pig ileum and jejunum (Inoue and Isenberg, 1990b; Pacaud and Bolton, 1991), and calcium is an absolute requirement in the canine colon (Cole et al., 1989; Lee et al., 1993). This calcium requirement could be provided by M₃ receptor-mediated calcium mobilization. Therefore, it is possible that both the M₂ and M₃ muscarinic receptors mediate the nonselective cation conductance in gastrointestinal smooth muscle. Accordingly, a recent pharmacological analysis suggests that both the M₂ and M₃ receptors cooperate to induce the nonselective cation conductance (Bolton and Zholos, 1997)

In the present study, experiments were conducted to determine the contractile role of muscarinic receptor subtypes in the guinea pig colon. Our results show that the M₃ receptor elicits a pertussis toxin-insensitive contractile response under standard conditions. However, after 4-DAMP mustard treatment the standard contractile response was pertussis toxin-sensitive, which suggests a role for the M₂ receptor. We also show that M₂ receptors can prevent the relaxant effects of forskolin and isoproterenol on histamine-induced contractions.

	Materials and Methods

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In vivo pertussis toxin treatment. In some experiments, male Hartley guinea pigs (300-400 g) were injected i.p. with 100 µg/kg body weight pertussis toxin 3 days before being sacrificed for the experiments.

Cyclic AMP accumulation. Cyclic AMP accumulation was measured in slices of the guinea pig colon by a modification of the procedure described by Daly et al. (1981). Male Hartely guinea pigs (300-400 g) were asphyxiated with CO₂ followed immediately by exsanguination. A 6- to 8-cm segment of colon was quickly dissected 1 cm distal to the cecum and placed in ice-cold KRB buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.8 mM CaCl₂, 10 mM glucose) gassed with O₂/CO₂, (19:1). The segment of colon was cut longitudinally to expose the mucosa, which was removed by a modification of the method described by Diener et al. (1989). The resulting colonic segments (including the longitudinal and circular muscles) were prepared and [³H]cyclic AMP was measured as described previously (Thomas et al., 1993).

Contractile measurements. Male Hartley guinea pigs were sacrificed, and the colon was harvested as described above. The colon was cut into segments 1 to 2 cm in length. Each segment was rapidly cleaned with KRB buffer to remove its contents, connected to a force transducer and mounted longitudinally in a organ bath containing 50 ml of KRB buffer at 37°C gassed with O₂/CO₂ (19:1). The colon segments were allowed to equilibrate for 40 min at a resting tension equivalent to a load of 1.5 g (optimal pretension was determined after constructing a pretension vs. force of contraction curve) before measuring isometric contractions with a force transducer and polygraph. A test dose of either histamine or oxotremorine-M (highly efficacious muscarinic agonist) was then added to each bath. Once each tissue reached a sustained contraction, each bath was washed with KRB buffer and allowed to incubate 5 min before the addition of two more test doses. These three test doses were used to ensure reproducibility of the preparations. Segments of colon that did not contract to at least 60% of that elicited by 100 mM KCl were discarded. After the last 5-min incubation, the KRB buffer was replaced with 50 ml of Ca⁺⁺ free KRB buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1 mM ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 10 mM glucose). The colon was incubated in Ca⁺⁺ free media for 10 min to inhibit myogenic contraction and cause full relaxation. During this period, a resting tension of 1.5 g was maintained. Subsequently, the Ca⁺⁺ free KRB buffer was replaced with 50 ml of K⁺-deficient KRB buffer (124 mM NaCl, 1.3 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.8 mM CaCl₂, 10 mM glucose) to inhibit spontaneous contractions. After addition of the K⁺-deficient KRB buffer, a large contraction was observed which declined to resting tension within 7 to 10 min. A cumulative agonist concentration-response curve was then measured by adding 9 to 18 geometrically spaced (0.33 log unit) concentrations of oxotremorine-M to each of the organ baths. The EC₅₀ value was determined from this curve as described below. The K⁺-deficient KRB buffer was washed from the bath, and 50 ml of KRB buffer was added. Colon segments were allowed to incubate for 30 min before any further measurements were made. The above-mentioned procedure (excluding three test doses) was repeated before each EC₅₀ measurement made with the same tissue. Some colon segments were incubated with 40 nM aziridinium ion of 4-DAMP mustard for 1 hr in the presence of 1.0 µM of AF-DX 116 to alkylate M₃ muscarinic receptors selectively (Thomas et al., 1993). The tissues were then washed with KRB buffer, and the aziridinium ion of 4-DAMP mustard (40 nM) and AF-DX 116 (1.0 µM) were added again for an additional hour (i.e., total incubation time of 2 hr). The colon segments were washed thoroughly to remove AF-DX 116 and any unreacted 4-DAMP mustard. In all experiments, 4-DAMP mustard was first converted to its aziridinium ion by incubation for 30 min at 37°C in 10 mM NaKPO₄, pH 7.4, as described previously (Thomas et al., 1992).

Data analysis. The percent inhibition of agonist-stimulated cAMP accumulation elicited by oxotremorine-M (I_c) was calculated by first subtracting out basal cAMP accumulation (B) before calculating the percent inhibition:

(1)

In this equation, O represents cAMP accumulation in the presence of the agonist and oxotremorine-M, and A represents cAMP accumulation in the presence of the agonist alone.

(2)

(3)

Materials. The drugs and chemicals used in this investigation were obtained from the following sources: islet-activating protein (pertussis toxin), LIST Biological Laboratories, Campbell, CA; [³H]adenine, ICN Biochemicals, Costa Mesa, CA; AF-DX 116, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT; SKF 38393 and Oxotremorine-M, Research Biochemicals Incorporated, Natick, MA; 4-DAMP mustard was synthesized in our laboratory as described previously (Thomas et al., 1992); and all remaining drugs and chemicals were from Sigma Chemical Company, St. Louis, MO.

	Results

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Effect of oxotremorine-M on agonist stimulated cAMP accumulation. Zhang et al. (1991) have shown that the nonselective muscarinic agonist carbachol elicited a concentration-dependent decrease in adenylate cyclase activity stimulated by 10 µM forskolin in homogenates of canine colonic circular smooth muscle cells. This response was antagonized by atropine in a competitive manner and was pertussis toxin-sensitive. To determine which cAMP-stimulating agents the muscarinic receptor opposed in the guinea pig colon, we measured the ability of 10 µM oxotremorine-M to inhibit the cAMP accumulation elicited by 1 µM isoproterenol, 10 µM forskolin, 10 µM SKF 38393 or 10 µM cicaprost. Figure 1, shows that 10 µM oxotremorine-M inhibited the cAMP response to isoproterenol, forskolin, SKF 38393 and cicaprost by 31% (P = .03), 24% (P = .01), 100% (P = .002) and 4.6% (P = .05), respectively. A cursory investigation of the effects of prostaglandin D₂, prostaglandin E₂, serotonin, methoxy tryptamine, dimaprit, dopamine and secretin showed that these agonists caused 1.2- to 1.7-fold increase in cAMP and that oxotremorine-M inhibited these responses by 0% to 13%. These agonists were not investigated further.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of oxotremorine-M on basal and agonist-stimulated cAMP formation in guinea pig colon. Colonic slices were incubated with isoproterenol (1 µM), forskolin (10 µM), SKF 38393 (10 µM) and cicaprost (10 µM) in the absence (closed bars) and presence (open bars) of oxotremorine-M (10 µM). The fold increase in cAMP over basal levels was determined as described under "Materials and Methods." Each bar represents the mean fold increase in cAMP ± S.E.M. of seven (forskolin), four (isoproterenol) and three (SKF 38393, cicaprost) experiments performed in triplicate.

Effect of 4-DAMP mustard treatment and AF-DX 116 on contractions elicited by oxotremorine-M under standard conditions. Previous studies on guinea pig ileum (Candell et al., 1990), gastric fundus (Del Tacca et al., 1990), the longitudinal muscle of the esophagus (Eglen and Whiting, 1988) and the circular muscle of the lower esophageal sphincter (Sohn et al., 1993) have demonstrated that the M₃ receptor mediates contractions in these gastrointestinal smooth muscles under standard conditions. To determine whether the M₃ receptor also elicits the contractile response in the guinea pig colon, we measured the ability of the subtype selective antagonists AF-DX 116, p-F-HHSiD and pirenzipine, to shift the oxotremorine-M-contractile response curve to the right. We estimated the K_B values of the antagonists from these rightward shifts as described under "Materials and Methods." These K_B values are listed in table 1 together with the binding affinities (K_D values) of the same antagonists measured in Chinese hamster ovary cells transfected with the M₂ and M₃ subtypes of the muscarinic receptor (see Esqueda et al., 1996 and Ehlert et al., 1997). These binding experiments were carried out in a HEPES-buffered KRB solution similar to that used in our contractile studies. Keeping the composition of the buffer the same for the binding and functional experiments is important because the binding properties of muscarinic receptors are modulated by ionic strength (Pedder et al., 1991). It can be seen that the K_B values of AF-DX 116, p-F-HHSiD and pirenzipine agree with their respective K_D values for the M₃ receptor, but not with those of the M₂ receptor (see table 1). There was also a lack of congruence between the antagonist K_B values of AF-DX 116, p-F-HHSiD and pirenzipine and their respective K_D values for the M₁ (6.24, 7.08, 7.77), M₄ (6.96, 7.08, 7.23) and M₅ (5.29,6.26, 6.55) subtypes, as reported by Esqueda et al. (1996) and Ehlert et al. (1997). We conclude that the M₃ receptor mediates the contractile response to oxotremorine-M in the guinea pig colon under standard conditions.

5

7

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effects of 4-DAMP mustard treatment on the contractile response to increasing concentrations of oxotremorine-M under standard conditions. All contractile measurements were made in tissues that had been incubated with the aziridinium ion of 4-DAMP mustard (40 nM) for either 1 (A) or 2 (B) hr in the presence of AF-DX 116 (1 µM) at 37°C. Control () and 40 nM 4-DAMP mustard (). Each data point represents the mean ± S.E.M. of four to eight experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of AF-DX 116 (1 µM) on the contractile responses to oxotremorine-M in control and 2-hr 4-DAMP mustard-treated tissues. Contractile measurements were made in untreated (,) and 4-DAMP mustard-treated (,) colon, in the absence (,) and presence (,) of AF-DX 116 (1.0 µM). Each data point represents the mean ± S.E.M. of four experiments.

Contractile studies in 4-DAMP mustard-treated tissue in the presence of histamine and forskolin or isoproterenol. Although the M₃ receptor mediates contractions under standard conditions, the M₂ receptor has been shown to mediate contractions to oxotremorine-M in the ileum and trachea when measured after 4-DAMP mustard treatment and in the presence of histamine and a cAMP stimulating agent, like forskolin (Thomas et al., 1993; Thomas and Ehlert, 1994, 1996). Under these latter conditions, it is likely that the M₂ receptor inhibits the cAMP-mediated, relaxant effects of forskolin and allows histamine to contract the smooth muscle (Thomas et. al., 1993; Thomas and Ehlert, 1994, 1996). In other words, the M₂ receptor mediates a disinhibition of histamine-induced contractions. To investigate whether the M₂ receptor mediates contractile effects in the colon, we used a slightly modified version of the method described by Thomas et al. (1993). After 2 hr 4-DAMP mustard treatment (see "Materials and Methods"), colon segments were contracted with .30 µM histamine, then relaxed back to resting tension with either isoproterenol (0.60 µM) or forskolin (10 µM). The contraction elicited to cumulative addition of oxotremorine-M was measured while histamine and the cAMP stimulating agent remained in the bath. The results of these experiments are shown in figure 4. The dotted lines in this figure indicate the contractile response to histamine (upper line) and the resting level of contraction measured in the presence of histamine and either forskolin or isoproterenol (lower line). In experiments where forskolin was used to cause relaxation, the EC₅₀ value and Hill coefficient of the oxotremorine-M concentration-response curve were 0.27 µM and 1.2, respectively (fig. 4A). AF-DX 116 (1.0 µM) caused a significant 8.0- to 13.2-fold (P = .004) increase in the EC₅₀ value and an increase in the Hill coefficient to 1.84. The shift in the EC₅₀ value corresponds to a pK_B value of 7.09 - 6.85 for AF-DX 116 (table 2). When isoproterenol was used to cause relaxation, the oxotremorine-M concentration-response curve had a EC₅₀ value of 0.97 µM and a Hill coefficient of 1.22 (fig. 4B). AF-DX 116 caused a significant 3.0- to 5.8-fold (P = .05) increase in the EC₅₀ value of oxotremorine-M and an increase in the Hill coefficient to 1.47, with a corresponding pK_B value of 6.30 to 6.68. In the presence of forskolin, the K_B and fold-shift values for AF-DX 116 were in close agreement with those predicted from the estimate of the K_D value of AF-DX 116 measured in binding experiments on the cloned M₂ receptor (pK_D = 7.27 and 19.6-fold; Esqueda et al., 1996). In contrast, when isoproterenol was used as the relaxant agent, the pK_B value (6.30-6.68) of AF-DX 116 at 1.0 µM was intermediate to those expected for either a purely M₂ (7.27) or M₃ (6.1) mediated response (Esqueda et al., 1996).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of AF-DX 116 on the contractile response to oxotremorine-M measured in the presence of histamine and either forskolin or isoproterenol after 4-DAMP mustard treatment. Colon segments were contracted with histamine (0.30 µM) and relaxed with forskolin (10 µM) (A) or isoproterenol (1 µM) (B) before oxotremorine-M-induced contractions were measured. Control () and 1 µM AF-DX 116 (). Each data point represents the mean ± S.E.M. of seven experiments.

Effect of pertussis toxin on the contractile response to oxotremorine-M under standard conditions. The M₂ receptor is known to elicit cellular responses by interacting with pertussis toxin-sensitive G proteins of the G_i family (Kurose and Ui, 1983; Sankary et al., 1988; Zhang and Buxton, 1991). Pertussis toxin catalyzes the ADP-ribosylation of the alpha subunit of G_i and G_o, thereby preventing their coupling to M₂ receptors (Katada and Ui, 1982; Kurose et al., 1983; Brown et al., 1984). Consequently, we were interested in determining the pertussis toxin sensitivity of the contractile response of the colon under different experimental conditions to gain more insight into the muscarinic receptor subtypes that mediate contraction. Pertussis toxin treatment had no inhibitory effects on the contractile response to oxotremorine-M measured under standard conditions (fig. 5). In fact, a small potentiation in contraction by pertussis toxin was noted at high concentrations of oxotremorine-M. After 2-hr 4-DAMP mustard treatment, however, pertussis toxin caused a significant 8.8-fold (P = 2 × 10⁴) increase in the EC₅₀ value of oxotremorine-M under standard conditions. These results suggest that the M₂ receptor may contribute to the standard contractile response after most of the M₃ receptors have been inactivated. The results of these experiments are summarized in table 2.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of pertussis toxin treatment on the contractile response to oxotremorine-M in control and 4-DAMP mustard-treated tissues. Contractile responses were measured in 2-hr 4-DAMP mustard-treated (,) and untreated (,) colon from pertussis toxin-treated (,) and untreated (,) guinea pigs. Each data point represents the mean ± S.E.M. of six experiments.

Effects of pertussis toxin treatment on contractions measured after 4-DAMP mustard treatment and in the presence of histamine and either forskolin or isoproterenol. We also investigated the effects of pertussis toxin treatment on the contractile response to oxotremorine-M measured after 4-DAMP mustard treatment and in the presence of histamine and either forskolin or isoproterenol. In one experiment, pertussis toxin treatment caused a 17.4-fold increase in the EC₅₀ value (fig. 6A and table 3) for those segments relaxed with forskolin. In three other experiments with forskolin, oxotremorine-M was unable to elicit contractions after pertussis toxin treatment (fig. 6A). Nevertheless, in the absence of forskolin, oxotremorine-M was able to elicit contractions in these tissues after pertussis toxin treatment as shown in figure 5. Moreover, pertussis toxin treatment did not affect histamine-induced contractions. Therefore, the lack of responsiveness of the tissue after 4-DAMP mustard treatment and in the presence of histamine and forskolin cannot be attributed to a pertussis toxin-induced depression in contractility. The variation in the effectiveness of pertussis toxin in the experiments described above (fig. 6A) is probably caused by variability in the absorption of pertussis toxin after intraperitoneal injection. In the experiments where isoproterenol was used to relax the colon segments after contraction with histamine, pertussis toxin caused a significant 32.5-fold (P = .02) increase in the EC₅₀ value (fig. 6B). These results strongly indicate that the M₂ receptor mediates contractions under these conditions because the contractile response is pertussis toxin-sensitive. We investigated the effects of AF-DX 116 on the residual contractile response that persisted after pertussis toxin treatment to determine what muscarinic receptor subtype was mediating the response. The EC₅₀ value of oxotremorine-M was 15.1 µM (fig. 6C) when histamine-induced contractions were relaxed with isoproterenol. AF-DX 116 (1 µM) caused a 2.1-fold increase, which yielded a calculated pK_B value of 6.04 (table 3). These data indicate that the receptor mediating the contraction elicited by oxotremorine-M after pertussis toxin treatment is the M₃ because the pK_B value and fold increase in EC₅₀ value were consistent with that expected for a purely M₃-mediated response (pK_D = 6.10 and 2.3-fold increase, respectively).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effects of pertussis toxin treatment and AF-DX 116 on the contractile response to oxotremorine-M measured in the presence of histamine and either forskolin or isoproterenol after 4-DAMP mustard treatment. All contractile responses were measured in colon treated with 4-DAMP mustard for 2-hr as described under "Material and Methods." (A) Contractile responses to oxotremorine-M were measured in the presence of histamine and forskolin in colon from control () and pertussis toxin-treated () guinea pigs. Pertussis toxin treatment completely blocked contractile response to oxotremorine-M under these conditions in three out of four experiments. The number of experiments are denoted by "n." (B) Contractile responses to oxotremorine-M were measured in the presence histamine and isoproterenol in a colon from control () and pertussis toxin-treated () guinea pigs. Each data point represents the mean ± S.E.M. of seven (no pertussis toxin treatment) to two (pertussis toxin treated) experiments. (C) Contractile responses to oxotremorine-M were measured in the presence of histamine and isoproterenol in colon from pertussis toxin-treated guinea pigs. Contractions were measured in the absence () and presence () of AF-DX 116. Each data point represents the mean ± S.E.M. of two experiments.

	Discussion

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In previous experiments with circular smooth muscle of canine colon, the muscarinic agonist carbachol inhibited forskolin-stimulated cAMP accumulation in muscle strips and isoproterenol-stimulated adenylate cyclase activity in broken cell preparations (Zhang and Buxton, 1991). In this investigation, the muscarinic agonist oxotremorine-M inhibited forskolin-, isoproterenol-, SKF 38393- and cicaprost-stimulated cAMP accumulation (fig. 1) in a manner consistent with a M₂-mediated response, as described in previous investigations with circular smooth muscle from canine colon and longitudinal muscle from the guinea pig ileum (Zhang and Buxton, 1991; Thomas et al., 1993; Ostrom and Ehlert, 1997; Kurose and Ui, 1983). The fold increase in cAMP and the percent inhibition elicited in the presence of oxotremorine-M for forskolin, isoproterenol, SKF 38393 and cicaprost were similar to those observed in the guinea pig ileum (Ostrom and Ehlert, 1997).

In this investigation, the standard contractile response of the guinea pig colon to oxotremorine-M was unaffected by pertussis toxin treatment (fig. 5) and antagonized by pirenzipine, p-F-HHSiD and AF-DX 116 (table 1, fig. 3) in a manner consistent with an M₃ receptor-mediated event. This hypothesis is consistent with the well known role of the M₃ receptor in mediating contraction under standard conditions in a variety of other smooth muscles (see Ehlert et al., 1997).

Previously, we showed that treatment with 4-DAMP mustard (40 nM) in the presence of AF-DX 116 (1.0 µM) for 1 hr caused a large inhibition of M₃-mediated responses including oxotremorine-M-stimulated phosphoinositide hydrolysis in the ileum and oxotremorine-M-stimulated contractions in the ileum and trachea. In contrast, the same treatment had no significant effect on oxotremorine-M-mediated inhibition of isoproterenol-stimulated cAMP accumulation in the ileum, an M₂-mediated response. With use of the observed rate constant for alkylation (0.00135 min¹) at M₂ receptors that we previously measured for 4-DAMP mustard (40 nM) in the presence of AF-DX 116, we estimate that 4-DAMP mustard should only inactivate approximately 7.8% and 15% of the M₂ receptors after 1 and 2 hr of 4-DAMP mustard treatment, respectively. These levels of receptor inactivation should only shift an M₂-mediated concentration response curve to the right 1.1- to 1.2-fold. Therefore, the large inhibitory effects of 4-DAMP mustard shown in figure 2 and table 2 are also consistent with the postulate that the M₃ receptor mediates the standard contractile response to oxotremorine-M.

However, after 2-hr 4-DAMP mustard treatment, the standard contractile response to oxotremorine-M was pertussis toxin-sensitive (fig. 5). Under these conditions, pertussis toxin had a great effect on the remaining contraction elicited to oxotremorine-M, making it 8.8-fold less potent. Eglen et al. (1987) and others (Peralta et al., 1988; Kurose et al., 1983) have shown that pertussis toxin uncouples M₂- and M₄-mediated responses without affecting M₁-, M₃- and M₅-mediated responses. Therefore, the M₂ receptor may be contributing to the standard contractile response of the guinea pig colon when most M₃ receptors have been inactivated by 4-DAMP mustard. Data obtained by use of AF-DX 116 to antagonize oxotremorine-M-induced contractions before and after 4-DAMP mustard treatment suggest that both the M₂ and the M₃ receptors contribute to the contractile response (fig. 3). The pK_B value (6.52) estimated after 4-DAMP mustard treatment did not correspond to a purely M₂- or M₃-mediated response, but rather, to an intermediate value, which suggests a role for both the M₂ and M₃ receptors. We have previously shown that antagonism of responses mediated by both the M₂ and M₃ receptors is complex and that the antagonistic profile can resemble an M₃ response even though there is a contribution of the M₂ receptor (Thomas and Ehlert, 1994; Ehlert et al., 1997).

Bolton and Zholos (1997) have used subtype selective antagonists to demonstrate that the M₂ receptor couples to a nonselective cationic channel. Perhaps contractions mediated by this M₂ receptor-activated cation channel can be revealed after most of the M₃ receptors have been inactivated with 4-DAMP mustard.

Analysis of 4-DAMP mustard reaction kinetics also suggests that two receptors are mediating contraction in guinea pig colon after 2-hr treatment. Our laboratory has described the kinetics of alkylation of glandular M₃ muscarinic receptors as being consistent with a first-order decay model having an observed rate constant for alkylation (k_obs) of 0.0321 min¹ when AF-DX 116 (1.0 µM) is present and the concentration of 4-DAMP mustard is 40 nM. Accordingly, 4-DAMP mustard should alkylate 85.4% and 97.8% of the M₃ receptors after 1 and 2 hr of incubation, respectively. This degree of receptor inactivation was observed in pertussis toxin-treated tissue after a 2-hr treatment with 4-DAMP mustard (99.2%, k_obs = 0.040 min¹). However, in control tissue, the calculated level of receptor inactivation after 1 hr (94.6%) was not much different from that estimated at 2 hr (96.7%), even though there was a striking difference in the shapes of the two corresponding oxotremorine-M concentration-response curves (see fig. 2). Thus, the calculations seem inconsistent with our empirical observations; this suggests that the one-site, first-order decay model on which the calculations are based is inaccurate. Furthermore, the one-site, first-order decay model predicts that the estimate of the rate constant for alkylation should be the same for the two periods. However, the k_obs at 1 hr (0.049 min¹) was approximately 73% larger than that estimated at 2 hr (0.0284 min¹). Also, the method for calculating receptor inactivation (see "Material and Methods") provides an estimate of the dissociation constant (K_A) of oxotremorine-M for the receptor mediating the response. This estimate at 1-hr treatment (pK_A = 4.95) was more than 10-fold greater than that measured after 2-hr treatment (pK_A = 6.00). These inconsistencies suggest that the one-site model is inaccurate and that at least two receptors (i.e., M₂ and M₃) showing differential sensitivity to 4-DAMP mustard contribute to the contractile response. A likely interpretation is that the 4-DAMP mustard-insensitive M₂ receptor rescues the contractile response after 2-hr 4-DAMP mustard treatment. By eliminating this M₂ response with pertussis toxin treatment, we should observe data consistent with a single M₃ receptor model. Accordingly, the estimates of pK_A of oxotremorine-M and the k_obs in pertussis toxin-treated tissue after 2-hr 4-DAMP mustard treatment (5.35 and .040 min¹) were similar to those measured after 1-hr 4-DAMP mustard treatment (4.95 and .049 min¹).

Our experiments on 4-DAMP mustard-treated colon show that oxotremorine-M elicits contraction through the M₂ receptor when histamine and either forskolin or isoproterenol are present (fig. 4). Presumably under these conditions, the M₂ receptor inhibits the cAMP-mediated relaxant effects of forskolin and isoproterenol, thereby allowing histamine to cause contraction. Similar results have been reported for the guinea pig ileum (Thomas et al., 1993; Reddy et al., 1995; Ostrom and Ehlert, 1997). The contractile response (disinhibition of contraction) elicited by oxotremorine-M in the presence of histamine and either forskolin or isoproterenol was antagonized by AF-DX 116 in a manner consistent with a M₂ receptor-mediated event. Also, after in-vivo pertussis toxin treatment, three of four colon segments precontracted with histamine (0.30 µM) and relaxed with forskolin (10 µM) did not contract when oxotremorine-M was added even though oxotremorine-M was able to elicit contraction in these tissues under standard conditions (fig. 5). In colon segments precontracted with histamine and relaxed with isoproterenol, pertussis toxin caused a 32.5-fold (fig. 6B) increase in EC₅₀ for contractions elicited to oxotremorine-M. The pertussis toxin sensitivity of the contractile response strongly indicates the role of the M₂ receptor. AF-DX 116 (1 µM) shifted the remaining contractile response in pertussis toxin-treated tissue 2.1-fold (fig. 6C) when isoproterenol was used as the relaxant agent. These data suggest that the remaining contraction elicited to oxotremorine-M is mediated primarily by the M₃ receptor as evidenced by the extremely reduced potency of oxotremorine-M and the inability of AF-DX 116 to shift the remaining contraction more than 2.2-fold.

The guinea pig colon behaves like the ileum in the sense that the colonic M₂ receptor opposes the relaxant effects of both forskolin and isoproterenol, and the role of the M₂ receptor is easier to demonstrate when forskolin is used as the relaxant agent as compared with isoproterenol (see above; also Thomas et al., 1993; Reddy et al., 1995; Ostrom and Ehlert, 1997). We have previously discussed the dependence of the M₂ contractile response in the ileum on the nature of the relaxant agent (see Ostrom and Ehlert, 1997). However, in the trachea, the M₂ receptor only opposes the relaxant effects of forskolin (Ehlert and Thomas, 1996) but not isoproterenol (Watson et al., 1995; Ostrom and Ehlert, unpublished observations).

In summary, our data demonstrate that the M₃ muscarinic receptor mediates the standard contractile response to oxotremorine-M in isolated guinea pig colon. Our data also suggest a role for the M₂ receptor in the standard contraction elicited to oxotremorine-M when the number of M₃ receptors is extremely reduced by 4-DAMP mustard. As observed in other smooth muscle preparations, the M₂ receptor in the guinea pig colon can mediate an indirect contraction by preventing the relaxant effects of forskolin and isoproterenol on histamine-induced contractions.