Introduction

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

M₂ and M₃ muscarinic receptor subtypes are abundantly expressed in smooth muscle throughout the gastrointestinal tract (Eglen et al., 1996; Ehlert et al., 1997). Radioligand binding (Giraldo et al., 1988; Michel and Whiting, 1990), mRNA hybridization (Maeda et al., 1988), and immunoprecipitation studies (Wall et al., 1991) have shown that the M₂ receptor is the more abundant of the two receptors in the intestine (Ehlert et al., 1997). Interestingly, a large body of pharmacological evidence shows that the less abundant M₃ receptor mediates contraction of gastrointestinal smooth muscle under standard conditions (i.e., no other contractile or relaxant agents present). The M₃ receptor is known to stimulate phospholipase C in a variety of cell types, including smooth muscle. Presumably, this mechanism mobilizes calcium and initiates contraction in smooth muscle.

The M₂ receptor is known to mediate a pertussis toxin-sensitive inhibition of adenylyl cyclase activity in the guinea pig ileum and colon (Candell et al., 1990; Zhang and Buxton, 1991; Thomas and Ehlert, 1994). Muscarinic agonists have been shown in both ileum and colon to oppose the increase in cAMP elicited by forskolin, isoproterenol, and a variety of other compounds that stimulate cAMP accumulation (Griffin and Ehlert, 1992; Ostrom and Ehlert, 1997; Sawyer and Ehlert, 1998). The M₂ receptor also elicits an indirect contraction in both the guinea pig ileum and colon by preventing the relaxant effects of forskolin and isoproterenol on histamine-induced contraction (Thomas et al., 1993; Thomas and Ehlert, 1994; Ostrom and Ehlert, 1997; Sawyer and Ehlert, 1998). The M₂-mediated indirect contraction is pertussis toxin sensitive (Sawyer and Ehlert, 1998), unlike the standard M₃-mediated contraction.

Extensive alkylation of M₃ receptors by the selective irreversible antagonist N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) (Thomas et al., 1993) renders the contractile response of the guinea pig colon sensitive to pertussis toxin treatment (Sawyer and Ehlert, 1998). This sensitivity suggests that the M₂ receptor may directly participate in the contractile response of the colon after most of the M₃ receptors have been inactivated with 4-DAMP mustard. When expressed in high abundance, M₂ receptors have been shown to mediate a weak, pertussis toxin-sensitive, phosphoinositide response by coupling to G_i (Ashkenazi et al., 1987; Lai et al., 1991; Dell'Acqua et al., 1993). Such a mechanism would be difficult to detect in the face of a large M₃ phosphoinositide response via G_q. Thus, it is important to consider whether M₂ receptors in the colon are capable of eliciting contraction through the phosphoinositide pathway after most of the M₃ phosphoinositide response has been inactivated.

In the present report, we investigated this hypothesis and have shown that although the muscarinic contractile response is extremely sensitive to pertussis toxin after 4-DAMP mustard treatment, the phosphoinositide response is not. Thus, our results provide no evidence for the direct coupling of M₂ receptors to phosphoinositide hydrolysis. Instead, our results are consistent with a mathematical model in which the M₂ receptor is capable of eliciting contraction when there is a low level of M₃ receptor activation. This model accounts for the finding that the muscarinic contractile response after 4-DAMP mustard treatment is sensitive to pertussis toxin yet nevertheless relatively insensitive to the M₂-selective antagonist AF-DX 116.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

Contractile Measurements. Male Hartley guinea pigs (300-400 g) were asphyxiated with CO₂ followed immediately by exsanguination, and segments of colon (1-2 cm) were harvested 1 cm from the cecum. Each segment was rapidly cleaned with Krebs-Ringer-bicarbonate (KRB; 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.8 mM CaCl₂, 10 mM glucose) buffer to remove its contents. The segments were connected to a force transducer and mounted longitudinally in an organ bath containing 50 ml of KRB buffer at 37°C gassed with O₂/CO₂ (19:1). The colonic segments were allowed to equilibrate for 40 min at a resting tension equivalent to a load of 1.5g (optimal pretension was determined after constructing a pretension versus force of contraction curve) before measurement of isometric contractions with a force transducer and polygraph. A test dose of the highly efficacious muscarinic agonist oxotremorine-M was then added to each bath. Once each tissue reached a sustained contraction, each bath was washed with KRB buffer and allowed to incubate for 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 Ca²⁺-free KRB buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM KH₂PO₄, 1 mM EGTA, 10 mM glucose). The colon was incubated in Ca²⁺-free medium for 10 min to inhibit myogenic contraction and cause full relaxation. During this period, a resting tension of 1.5g was maintained. Subsequently, the Ca²⁺-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 the addition of the K⁺-deficient KRB buffer, a large contraction was observed that declined to resting tension within 7 to 10 min. Cumulative agonist concentration-response curves were 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. Colonic segments were allowed to incubate for 30 min before any further measurements were made. The above procedure (excluding three test doses) was repeated before each EC₅₀ measurement made with the same tissue.

Contractile Experiments Using 4-DAMP Mustard-Treated Colonic Segments. Some colonic segments were incubated with 40 nM concentration of the aziridinium ion of 4-DAMP mustard for 1 h in the presence of 1.0 µM [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116) to selectively alkylate M₃ muscarinic receptors (Thomas et al., 1993). The colon segments were washed thoroughly to remove AF-DX 116 and any unreacted 4-DAMP mustard before measurement of concentration-response curves as described above. Some tissues were 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 h) before measurement of concentration-response curves. In other experiments, colonic segments were exposed to 4-DAMP mustard for a total of 3 or 4 h using the same washing procedure described above at the end of each hour of exposure. 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 previously described (Thomas et al., 1992).

Phosphoinositide Hydrolysis Assay. Male guinea pigs (300-400 g) were sacrificed as described above, and an approximately 16-cm segment of colon was rapidly harvested 1 cm distal the cecum. The colon was placed in ice-cold KRB gassed with O₂/CO₂ (19:1) and cleaned to remove the contents. The colonic segment was cut longitudinally, and the mucosa was removed using a microscope slide. The segment was cross-chopped at 350 µM using a McIlwain tissue chopper. The resulting colonic slices were immediately suspended in 37°C KRB gassed with O₂/CO₂ (19:1) and then washed with KRB (37°C) three times. The slices were allowed to incubate in KRB at 37°C gassed with O₂/CO₂ (19:1) for 30 min. In some experiments, segments of whole colon were first incubated with AF-DX 116 and 4-DAMP mustard for 2 h before slicing of the tissue. For these incubations, approximately 10 cm of colon was incubated in a final volume of 330 ml of KRB buffer containing AF-DX 116 (1 µM) and 4-DAMP mustard (40 nM). The tissue was washed extensively to remove AF-DX 116 and any unreacted 4-DAMP mustard, and the tissue was sliced as described above. To 10 ml of packed colonic slices, 8 ml of KRB buffer containing 100 µCi of myo-[³H]inositol was added, and the mixture was incubated for 90 min. The slices were gassed with O₂/CO₂ (19:1) every 30 min. After this incubation, the slices were washed with KRB (37°C) three times. The slices were allowed to settle before pipetting 100-µl aliquots of slices into tubes with 0.35 ml of KRB (37°C) gassed with O₂/CO₂ (19:1) containing 5 mM LiCl and various concentrations of oxotremorine-M without or with antagonists in concentrations identified in Results. The slices were incubated for 30 min in these tubes at 37°C. The incubation was stopped by adding 1.13 ml of chloroform/methanol (1:2, v/v). The accumulated inositol phosphates were extracted and separated according to the method of Berridge et al. (1982), as described briefly. Chloroform (0.37 ml) and water (0.37 ml) were added to the tubes to separate the organic and aqueous phases. After centrifugation, 1 ml of the aqueous phase was pipetted into tubes to which 2 ml of water was added. These tubes were centrifuged to sediment any chloroform, and 2.9 ml of the aqueous phase was applied to a Dowex AG 1-X8 (100-200 mesh, 1 ml) anion exchange column. The column was washed three times with 5 ml of water each to remove [³H]inositol and discarded. [³H]Inositol phosphates were eluted from the column with 2.5 ml of 1 M ammonium formate/0.1 M formic acid solution. The elutant was then counted to determine the amount of radioactivity using a scintillation counter. In some experiments and in all experiments in which pertussis toxin-treated colonic slices were used, 200 µl of the organic phase was removed, dried, and then counted to determine the amount of [³H]inositol incorporated into phospholipids. In these experiments, the amount of [³H]inositol phosphates formed is calculated as a percentage of the total amount of radioactivity in the organic phase plus the inositol phosphate fraction. This was done to determine whether there were differences in the uptake of [³H]inositol in tissues treated with pertussis toxin compared with tissues that were not treated with pertussis toxin.

In Vivo Pertussis Toxin Treatment. Male Hartley guinea pigs (300-400 g) were injected i.p. with pertussis toxin (50-100 µg/kg b.wt.) 3 days before being euthanized for the in vitro experiments. For the contractile assays, a dose of 100 µg/kg b.wt. pertussis toxin was used, whereas in the phosphoinositide assays, a dose of 50 µg/kg b.wt. pertussis toxin was used. Initial experiments showed that these two doses yielded similar results in the phosphoinositide assay.

Data Analysis. The concentration of oxotremorine-M eliciting half-maximal contraction (EC₅₀) was estimated by nonlinear regression analysis according to an increasing logistic equation as described previously (Candell et al., 1990).

(1)

Mathematical Modeling. In this investigation, we examined two mathematical models (model I and model II) for an interaction between two receptor subtypes (M₂ and M₃). In model I, activation of the M₃ receptor by itself causes contraction. The M₂ receptor has no effect by itself but can greatly potentiate the contraction elicited by the M₃ receptor when activated. In model II, occupation of the M₃ receptor initiates the activation of two parallel signaling pathways. One pathway causes a direct contraction and is referred to as component A. The other pathway has no effect by itself, but when activated in the presence of occupied M₂ receptor, a contraction ensues (component B). The M₂ receptor is incapable of eliciting contractions by itself. Component B of model II is similar to model I in that it represents an M₂/M₃ interaction. However, the contraction elicited by component B is conditional, occurring only when both M₂ and M₃ receptors are activated. The mathematical derivation of these models is described in Appendix and illustrated in Fig. 1.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Schematic representation of two models for interaction between M2 and M3 receptors. Solid lines indicate contractile signals. Dashed lines indicate silent signals that interact with another signal to elicit contraction.

Materials. Islet-activating protein (pertussis toxin) was obtained from LIST Biological Laboratories (Campbell, CA). myo-[³H]Inositol was from NEN (Boston, MA). AF-DX 116 was from Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT). Pirenzepine, p-F-HHSiD, and oxotremorine-M were from Research Biochemicals Inc. (Natick, MA). 4-DAMP mustard was synthesized as described previously (Thomas et al., 1992). All remaining drugs and chemicals were from Sigma Chemical Co. (St. Louis, MO).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

Effect of 4-DAMP Mustard Treatment on Contraction Elicited by Oxotremorine-M Under Standard Conditions. To investigate the possible contribution of the M₂ receptor to the contractile response of the guinea pig colon, we treated colonic segments with 4-DAMP mustard to inactivate M₃ receptors. The isolated colon was incubated with the aziridinium ion of 4-DAMP mustard (40 nM) in the presence of AF-DX 116 (1 µM) for 1, 2, 3, or 4 h and then washed extensively. AF-DX 116 is used to protect M₂ receptors from alkylation by 4-DAMP mustard. In the presence of AF-DX 116, the aziridinium ion of 4-DAMP mustard has been shown to be an irreversible, muscarinic antagonist that alkylates M₃ receptors selectively over M₂ receptors (Thomas et al., 1993). Incubation of the isolated colon with 4-DAMP mustard for 1, 2, 3, and 4 h caused a progressive shift to the right and decrease in the maximum of the concentration-response curve to oxotremorine-M (Fig. 2 and Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of 4-DAMP mustard treatment on contractile response to increasing concentrations of oxotremorine-M under standard conditions. Contractile measurements were made in tissues before () and following 4-DAMP mustard treatment (40 nM) for 1 h (), 2 h (), 3 h (), and 4 h (). Each data point represents mean ± S.E.M. of four to eight experiments.

Effect of Pertussis Toxin on Contractile Response to Oxotremorine-M. We examined the effect of pertussis toxin on the residual contractile response that persisted after 1- and 4-h treatment with 4-DAMP mustard to determine whether the M₂ receptor is contributing to the remaining contractile response. Pertussis toxin treatment had no inhibitory effect on the contractile response to oxotremorine-M when measured under standard conditions (Fig. 3). In fact, a small potentiation in contraction was observed at high concentrations of oxotremorine-M. After 1- and 4-h 4-DAMP mustard treatment, however, pertussis toxin caused a significant 25.4-fold (p = 3.1 × 10⁴) and 72.5-fold (p = 8.5 × 10⁶) increase in the EC₅₀ value of oxotremorine-M, respectively (Fig. 3, A and B). The sensitivity of the residual contraction to pertussis toxin suggests that the M₂ receptor participates in the contractile response, when a substantial portion of M₃ receptors are alkylated by 4-DAMP mustard. This participation by the M₂ receptor can explain why the effects of 4-DAMP mustard appear to reach a limit (see prior discussion of Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of pertussis toxin treatment (, ) on contractile response to oxotremorine-M in control (, ) and 4-DAMP mustard-treated tissues (, ). Contractile responses were measured in 1-h (A) and 4-h (B) 4-DAMP mustard-treated tissue. Each data point represents mean ± S.E.M. of five to six experiments.

Effect of AF-DX 116 on Contractile Response After 4-DAMP Mustard Treatment. If the M₂ receptor is contributing to the contractile response after 4-DAMP mustard treatment, the remaining response may be antagonized by AF-DX 116 (M₂- and M₄-selective antagonist) in a manner consistent with an M₂-mediated response. Isolated colonic segments were treated with 4-DAMP mustard (40 nM) in the presence of AF-DX 116 (1 µM) for 4 h and then washed extensively as described in Experimental Procedures. Treatment with 4-DAMP mustard caused a 16.3-fold increase in the EC₅₀ value of oxotremorine-M (control EC₅₀ = 94 nM) and a 60% reduction in the maximal contractile response (control E_max = 9.5g). This suppression in maximal response was not as great as that observed in similar experiments shown in Figs. 2 and 3, which we attribute to experimental variation. The contractile response to oxotremorine-M was determined in the absence and presence of 1 µM AF-DX 116 after 4-h 4-DAMP mustard treatment (Fig. 4). AF-DX 116 caused a significant 1.8-fold increase (p = 3.9 × 10³) in the EC₅₀ value of the contractile response elicited to oxotremorine-M. The pK_B value for AF-DX 116 estimated from this rightward shift (see Experimental Procedures) was 5.9 ± 0.11, which is in close agreement with the binding affinity (pK_D = 6.10 ± 0.06) of AF-DX 116 at the human M₃ receptor transfected into Chinese hamster ovary cells (Esqueda et al., 1996). Thus, the contractile response after 4-h 4-DAMP mustard treatment is enigmatic; it is M₂-like in its sensitivity to pertussis toxin, yet it is M₃-like in its weak antagonism by AF-DX 116. AF-DX 116 (1.0 µM) also weakly antagonized the contractile response to oxotremorine-M measured before 4-DAMP mustard treatment (pK_B = 6.07; see Table 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of AF-DX 116 (1 µM) on contractile response to oxotremorine-M in 4-h 4-DAMP mustard-treated tissues. Contractile measurements were made in 4-DAMP mustard-treated colon in the absence () and presence () of AF-DX 116 (1.0 µM). Each data point represents mean ± S.E.M. of three experiments.

Mathematical Modeling. To explain the behavior of the muscarinic contractile response in the guinea pig colon after 4-DAMP mustard treatment, we investigated mathematical models for an interaction between two receptor subtypes (M₂ and M₃). We used a strategy based on the operational model of Black and Leff (1983) to calculate the theoretical concentration-response curves. We also calculated the effects of AF-DX 116 and pertussis toxin treatment on the theoretical responses. Our methods are described in Appendix.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Simulation of a response mediated through interaction of an agonist with two receptors (i.e., M2 and M3 receptors; model I). Response mediated by M2/M3 interaction (filled symbols) was compared with a response mediated solely by M3 receptor (open symbols). Curves are shown before (, ) and after 90% (, ), 99% (, ), and 99.8% (, ) M3 receptor inactivation. A, response of M2 receptor was less sensitive than that of M3 receptor (KM2 = 0.1; KM3 = 0.01). B, response of M2 and M3 receptors were equisensitive (KM2 = 0.01; KM3 = 0.01). C, response of M3 receptor was less sensitive than that of M2 receptor (KM2 = 0.001; KM3 = 0.01). Theoretical points for M3 response and M2/M3 interaction were calculated using eqs. 7 and 8, respectively, as described in the Appendix.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Simulation of effects of AF-DX 116 and M3 receptor inactivation on response to an agonist mediated through interaction of an agonist with two receptors (i.e., M2 and M3 receptors; model I). Curves are shown before (, ) and after 90% (, ), 99% (, ), and 99.8% (, ) M3 receptor inactivation in the absence (filled symbols) and presence (open symbols) of AF-DX 116 1 µM. Response of M2 receptor was less sensitive than that of M3 receptor (KM2 = 0.1; KM3 = 0.01). Each point was calculated using eq. 8 as described in the Appendix.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Simulation of effects of AF-DX 116 and M3 receptor inactivation on response to an agonist mediated through a single receptor site (model II, component A). Curves are shown before (, ) and after 90% (, ), 99% (, ), and 99.8% (, ) receptor inactivation in the absence (filled symbols) and presence (open symbols) of AF-DX 116 (1 µM). Each point was calculated using eq. 7 as described in the Appendix.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Simulation of effects of AF-DX 116 and M3 receptor inactivation on response mediated through interaction of an agonist with two receptors (i.e., M2 and M3; component B, model II). Curves are shown before (, ) and after 90% (, ), 99% (, ), and 99.8% (, ) M3 receptor inactivation in the absence (filled symbols) and presence (open symbols) of AF-DX 116 (1 µM). A, response of M2 receptor was less sensitive than that of M3 receptor (KM2 = 0.03; KM3 = 0.01). B, response of M3 receptor was less sensitive than that of M2 receptor (KM2 = 0.003; KM3 = 0.01). C, response of M2 and M3 receptors were equisensitive (KM2 = 0.01; KM3 = 0.01). Each point was calculated using eq. 10 as described in the Appendix.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Simulation of effects of AF-DX 116 and M3 receptor inactivation on combined response of model II (i.e., combination of components A and B). Curves are shown before (, ) and after 90% (, ), 99% (, ), and 99.8% (, ) M3 receptor inactivation in the absence (filled symbols) and presence (open symbols) of AF-DX 116 (1 µM). A, response of M2 receptor was less sensitive than that of M3 receptor (KM2 = 0.03; KM3 = 0.01). B, response of M3 receptor was less sensitive than that of M2 receptor (KM2 = 0.003; KM3 = 0.01). C, response of M2 and M3 receptors were equisensitive (KM2 = 0.01; KM3 = 0.01). Each point was calculated using eq. 13 as described in the Appendix.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Simulation of effects of pertussis toxin treatment and M3 receptor inactivation on combined response of model II (i.e., combination of components A and B). Curves are shown before (, ) and after 90% (, ), 99% (, ), and 99.8% (, ) M3 receptor inactivation before (filled symbols) and after (open symbols) pertussis toxin treatment. A, response of M2 receptor was less sensitive than that of M3 receptor (KM2 = 0.03; KM3 = 0.01). B, response of M3 receptor was less sensitive than that of M2 receptor (KM2 = 0.003; KM3 = 0.01). C, response of M2 and M3 receptors were equisensitive (KM2 = 0.01; KM3 = 0.01). Theoretical points before and after pertussis toxin treatment were calculated using eqs. 13 and 7 as described in the Appendix.

Phosphoinositide Hydrolysis. When transfected into cells in high abundance, M₂ receptors have been shown to mediate a weak phosphoinositide response through a pertussis toxin-sensitive G protein. Consequently, we investigated whether a pertussis toxin-sensitive phosphoinositide response could be detected in the guinea pig colon after inactivation of most of the M₃ receptors. Such a mechanism could account for a direct, pertussis toxin-sensitive contractile response mediated by the M₂ receptor after inactivation of most of the M₃ receptors with 4-DAMP mustard. However, such a response should be potently antagonized by AF-DX 116, which was not observed in the guinea pig colon (see Fig. 4). Nevertheless, we wished to explore the possible coupling of M₂ receptors to phospholipase C via a pertussis toxin-sensitive G protein. First, we characterized the phosphoinositide response by measuring the ability of the subtype-selective antagonists AF-DX 116, p-F-HHSiD, and pirenzepine to shift the concentration-response curve for oxotremorine-M-induced phosphoinositide hydrolysis to the right (Fig. 11). We estimated the K_B values of the antagonists from these rightward shifts as described in Experimental Procedures. These K_B values are listed in Table 4 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 (Esqueda et al., 1996; Ehlert et al., 1997). Also listed in Table 4 are the pK_B for the antagonists determined in contractile studies in the guinea pig colon (Sawyer and Ehlert, 1998). The binding assays for these K_D estimates were conducted in a HEPES-buffered KRB solution similar to that used in our contractile and phosphoinositide hydrolysis assays. It can be seen that the K_B values of AF-DX 116, p-F-HHSiD, and pirenzepine are in agreement with their respective K_D values for the M₃ receptor but not with those of the M₂ receptor (Table 4). These values also were in close agreement with the values obtained previously from contractile studies conducted in the guinea pig colon (Table 4). There also was a lack of agreement between the antagonist K_B values of AF-DX 116, p-F-HHSiD, and pirenzepine 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). Therefore, we conclude that the M₃ receptor mediates phosphoinositide turnover in the guinea pig colon under standard conditions.

View larger version (12K): [in this window] [in a new window]   Fig. 11.   Competitive antagonism of phosphoinositide response to oxotremorine-M in guinea pig colon. Phosphoinositide response to oxotremorine-M in the absence () and presence () of p-F-HHSiD (0.1 µM) (A), AF-DX 116 (1 µM) (B), and pirenzepine (1 µM) (C).

Effect of 4-DAMP Mustard Treatment and Pertussis Toxin on Phosphoinositide Hydrolysis Elicited by Oxotremorine-M. We measured the pertussis toxin sensitivity of the phosphoinositide response both before and after 4-DAMP mustard treatment to investigate the possible contribution of the M₂ receptor to the response. Colonic slices were incubated with 4-DAMP mustard (40 nM) in the presence of AF-DX 116 (1 µM) for 2 h and washed extensively. Incubation for 2 h with 4-DAMP mustard caused a significant 9.34-fold (p = 1.5 × 10⁵) increase in the EC₅₀ value and a 60% reduction in the maximal response to oxotremorine-M (Fig. 12). As in the functional assays, pertussis toxin did not affect the response to oxotremorine-M; in fact, there was a slight increase in the hydrolysis of phosphoinositides (Fig. 12). Unlike the contractile experiments, however, pertussis toxin had no inhibitory effect on phosphoinositide hydrolysis after 2-h 4-DAMP mustard treatment (Fig. 12). In fact, there was a slight potentiation of the response as well. Consequently, the M₂ receptor does not appear to couple with a phosphoinositide signaling pathway to rescue the contractile response after 4-DAMP mustard treatment.

View larger version (20K): [in this window] [in a new window]   Fig. 12.   Effects of pertussis toxin treatment on phosphoinositide response to oxotremorine-M in control and 2-h 4-DAMP mustard-treated colon. Phosphoinositide hydrolysis was measured in 2-h 4-DAMP mustard-treated (, ) and untreated (, ) colon from pertussis toxin-treated (, ) and untreated (, ) guinea pigs. Each data point represents mean ± S.E.M. of four experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion Appendix References

There is an abundance of data on smooth muscle demonstrating that it is primarily the M₃ receptor that mediates contraction to muscarinic agonists in the absence of other contractile and relaxant agents (Eglen, 1996; Ehlert et al., 1997). This condition occurs in variety of smooth muscle types, including the circular and longitudinal muscles of the colon (Zhang and Buxton, 1991; Sawyer and Ehlert, 1998). The data of the present report are also consistent with this hypothesis; the contractile response was sensitive to 4-DAMP mustard and insensitive to pertussis toxin and the M₂-selective antagonist AF-DX 116.

However, a different picture emerges when we consider the residual contractile response that persisted after selective inactivation of most of the M₃ receptors with 4-DAMP mustard. This response was highly sensitive to pertussis toxin and relatively insensitive to 4-DAMP mustard. Both of these characteristics suggest an M₂ receptor mechanism; but the contractile response was relatively insensitive to the M₂-selective antagonist AF-DX 116. To explain this enigma, we suggest that the response may represent an interaction between M₂ and M₃ receptors. Accordingly, the M₂ receptor may mediate contractions through some mechanism that depends on a low level of signaling through the M₃ receptor. By itself, this low level of M₃ signaling is insufficient to generate a sizable contraction, yet it enables activated M₂ receptors to elicit contraction. The results of our mathematical analysis are not inconsistent with this hypothesis.

We considered two models for the interaction between M₂ and M₃ receptors. In both models, it was assumed that activation of M₂ receptors has no effect by itself, but M₂ receptors can interact with M₃ receptors to trigger a contraction. In the first model (model I), it was assumed that the M₂ receptor simply potentiated the contractile response of the M₃ receptor. This model yielded results similar to the behavior of the colon when it was assumed that oxotremorine-M elicits contraction through the M₃ receptor with greater potency than that with which it activates the potentiating M₂ signal (case 1). Under this condition, oxotremorine-M can elicit a maximal contraction through the M₃ receptor at concentrations that are insufficient to activate the M₂ pathway. Thus, the M₂ receptor does not contribute to the contraction in untreated tissue, and consequently, the response is pertussis toxin insensitive. However, after inactivation of most of the M₃ receptors, the M₂ receptor contributes to the contraction, rendering it sensitive to pertussis toxin. Model I, case 1, also predicts that AF-DX 116 should cause only a 2.3- to 3.6-fold shift to the right in the concentration-response curve both before and after extensive M₃ receptor inactivation. Such results were observed in the colon. After a moderate degree of M₃ receptor inactivation, model I, case 1, predicts that AF-DX 116 (1.0 µM) should cause a 4.2- to 5.5-fold dextral shift in the concentration-response curve. The largest shift that we observed for AF-DX 116 after a moderate degree of receptor inactivation was 4.3-fold (Sawyer and Ehlert, 1998).

We also investigated an additional model (model II) that provided a somewhat better approximation of our data in the colon. Model II is based on the assumption that the M₃ receptor signals through two parallel pathways. The first pathway gives rise to a simple M₃ receptor-mediated contraction (component A), and the second pathway is silent by itself but interacts with a silent M₂ receptor to generate a contraction (component B) (see Fig. 8). Component A is more sensitive, so when contractile responses to oxotremorine-M are measured in untreated tissue, the contractions are due to activation of M₃ receptors. However, after selective inactivation of M₃ receptors, the sensitivity of component A is greatly reduced so component B becomes the more sensitive component.

Our analysis of component B provides an understanding of the competitive antagonism of a response to an agonist that is mediated through an interaction between two receptors. We have shown that there is a tendency for the antagonism to resemble that predicted for the less sensitive receptor mechanism. Consider an example where receptor X interacts with receptor Y to trigger a response. If the signal elicited by receptor X is less sensitive than that elicited by receptor Y, then the nature of the competitive antagonism of the interaction will tend to resemble that predicted for receptor X. If a highly Y-selective competitive antagonist is used, its calculated pK_B value will be less than that of the Y receptor and may approach that of the X receptor. On the other hand, if an X-selective antagonist is used, its calculated pK_B value will be equivalent to that of the X receptor.

Model II agreed best with the contractile data when it was assumed that the M₃ signal of component B was more sensitive than the M₂ signal (i.e., the conditions of model II, B1). Under these conditions, the model predicts that the competitive antagonism of component B1 by AF-DX 116 (1.0 µM) should resemble that expected for the less sensitive receptor signaling pathway: M₂. This antagonism was manifest as a relatively large AF-DX 116-induced shift (19.6-fold) in the concentration-response curve of component B1 (see Fig. 8A). Nevertheless, the complete response obtained by combining components A and B1 exhibited relatively low sensitivity to AF-DX 116 (1 µM) (see Fig. 9A). This behavior can be explained by the greater sensitivity of component A, which is capable of mediating a maximum contraction at agonist concentrations that are insufficient to elicit a response through component B1. It is only after M₃ receptors are inactivated that component B1 dominates the contractile response. However, under these conditions, the M₃ signal of component B1 is less sensitive than the M₂ signal, and consequently, the competitive antagonism of component B1 resembles that expected for an M₃ response, with low sensitivity to AF-DX 116. As a result, the complete response (i.e., the combination of components A and B1) exhibited relatively low sensitivity to AF-DX 116 both before and after varying degrees of M₃ receptor inactivation. However, careful inspection of the theoretical curves shows that the shift in the concentration-response curved caused by AF-DX 116 (1.0 µM) increased from 2.3-fold before receptor inactivation to 3.7-fold after 90% inactivation of M₃ receptors. With further receptor inactivation, the AF-DX 116-induced shift declined to 2.3-fold. Interestingly, we observed a similar phenomenon in the guinea pig colon. We found that the shift in the concentration response curve caused by AF-DX 116 increased from 2.2-fold in untreated tissue to 4.3-fold after 2-h 4-DAMP mustard treatment (Sawyer and Ehlert, 1998). After 4-h treatment, the AF-DX 116-induced shift returned to a low value of 1.8-fold (see Fig. 4).

So far, we have no direct evidence on the possible signaling mechanisms involved in the interaction between M₂ and M₃ receptors. Nevertheless, it is interesting to note that muscarinic agonists trigger a nonselective cation conductance in smooth muscle (Benham et al., 1985; Inoue and Insenberg, 1990a). This conductance is pertussis toxin sensitive, and it is enhanced by calcium, particularly in the colon, where the response is completely dependent on calcium (Inoue and Insenberg, 1990b; Lee et al., 1993). The pertussis toxin sensitivity and the calcium requirement suggest a mechanism involving both M₂ and M₃ receptors. Bolton and Zholos (1997) have shown that the competitive antagonism of the muscarinic-induced cation conductance is consistent with a response mediated by both the M₂ and M₃ receptors. Perhaps this conductance is involved in the interaction between M₂ and M₃ receptors.

The low sensitivity of the postulated interaction between M₂ and M₃ receptors raises the question of its physiological significance. What function could the interaction have if muscarinic agonists are capable of eliciting contraction through a more potent M₃ mechanism? This question is particularly relevant if the nature of the interaction simply involves an M₂ receptor-mediated potentiation of M₃ receptor-mediated contractions (model I). However, if the interaction involves an M₂ potentiation of an alternate M₃ signal that is incapable of eliciting contraction by itself (model II), then this mechanism could function in isolation of the standard M₃ receptor-mediated contraction. Moreover, it could have a pattern of distribution distinct from that of the standard M₃ receptor mechanism, making it the dominant mechanism at some neuroeffector junctions.