Introduction

Top Abstract Introduction Methods Results Discussion References

Alcuronium, C₇/3-phth and gallamine have all been demonstrated to act as allosteric modulators at cardiac muscarinic acetylcholine M₂ receptors in functional studies (Christopoulos and Mitchelson, 1994; Clark and Mitchelson, 1976; Maaß et al., 1995). Although some recent binding studies (Ellis and Seidenberg, 1992; Jakubík et al., 1995; Waelbroeck, 1994) have suggested that gallamine and other allosteric modulators recognize a common modulatory site, such a mode of interaction has yet to be demonstrated in functional studies.

One method for detecting whether an inhibitor is acting at the same site as another inhibitor, in functional studies, is to use them in combination against an agonist. In such studies, combinations of competitive antagonists produce additive dose ratios, based on the expected shifts of the agonist C-R curve that each antagonist produces alone (Paton and Rang, 1965). Theoretical considerations (Ehlert, 1988a) predict that combination of an allosteric modulator with a competitive antagonist will lead to a dose ratio that is less than, equal to or greater than that expected for combination of two competitive antagonists, depending on the relative effects of the allosteric modulator on the affinity of the agonist and the competitive antagonist and providing that the intrinsic efficacy of the agonist is not affected by the allosteric modulator. For example, using acetylcholine as agonist, gallamine in combination with atropine produced underadditive dose ratios, whereas with carbachol as agonist, the dose ratios were no different to those expected for combination of two competitive antagonists (Clark and Mitchelson, 1976). Both C₇/3-phth and alcuronium, in combination with NMS produced dose ratios that were supra-additive (Christopoulos and Mitchelson, 1994; Maaß et al., 1995).

To obtain evidence of whether the three allosteric modulators alcuronium, C₇/3-phth and gallamine acted at a similar or different site, combinations of these compounds were investigated using carbachol as agonist. Some experiments were also conducted using either atropine or NMS, as the competitive antagonist, in combination with one of the allosteric modulators, for comparison with the data obtained when the modulators were combined. In a few experiments, acetylcholine was used in place of carbachol. The results suggested that the combination dose ratios were those expected if the allosteric modulators acted at a common site, different from that for agonists and competitive antagonists.

	Methods

Top Abstract Introduction Methods Results Discussion References

Isolated atria preparations. Guinea pigs of either sex were killed by cervical dislocation followed by exsanguination, and their hearts were rapidly removed and placed in ice-cold Krebs' solution of the following composition (mM): NaCl 118.4, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0, glucose 11.7 and CaCl₂ 2.2. The left atrium was dissected, attached to a tissue hook on the end of an electrode assembly and placed in a 20-ml organ bath containing Krebs' buffer at 37°C, bubbled with a mixture of 95% O₂ and 5% CO₂. A Grass force-displacement transducer (FT.03C), connected to a Grass polygraph (Model 79D), was used to record the responses. The atrium was electrically driven by a Grass S48 stimulator (3 Hz, 10 msec, 15 V). The tissue was allowed to equilibrate for 20 min under a resting tension of 1 g before exposure to an agonist.

Experiments using competitive antagonists or allosteric modulators. A contact time of 1 min with the tissue was used for each of the agonists (acetylcholine or carbachol). The tissue was washed twice with fresh Krebs' solution after each dose of agonist, with 5-min periods allowed between additions of agonist. A minimum of three concentrations of the agonist were used to construct a reproducible, control C-R curve, ranging from 20% to 80% of maximal inhibition of atrial contraction. The negative inotropic responses to any one concentration of agonist were obtained, at least in duplicate, with a 20- to 30-min period elapsing between the determination of the initial and duplicate response.

Experiments with combinations of inhibitors. After establishment of a mean C-R curve in the presence of one concentration of inhibitor following washing of the tissue, a second inhibitor was added together with the initial inhibitor and an additional 40-min incubation period with the tissue was undertaken, after which a third mean C-R curve for the agonist was determined. In some experiments, the incubation period with both inhibitors present in the tissue bath was extended up to 180 min, with washing and replacement every 30 min. In the same fashion, as for the other curves, responses of the agonist were duplicated over a period of 20 to 30 min.

Data analysis. Using the program PRISM 2.01 (GraphPAD Software, San Diego, CA) the EC₅₀ values for agonists (concentration producing 50% inhibition of atrial contractility) from the C-R curves were determined by fitting the data to an equation of the following form: where E is the effect, expressed as percent inhibition of atrial contractility, and [A] is the concentration of agonist. Dose ratios were calculated as the ratio of the EC₅₀ obtained in the presence of inhibitor(s) to that obtained in the absence of inhibitor(s) and are expressed as geometric means together with 95% confidence limits. Statistical significance was determined using Student's t test; values of P < .05 were considered significant.

Drugs. Acetylcholine chloride, atropine sulphate, carbamylcholine chloride (carbachol), gallamine triethiodide and NMS were obtained from Sigma Chemical (St. Louis, MO). C₇/3-phth was from the Institute of Drug Technology (Boronia, Australia), and alcuronium chloride was a gift from Hoffman-La Roche (Basle, Switzerland).

	Results

Top Abstract Introduction Methods Results Discussion References

Effects of NMS and atropine. Both NMS and atropine behaved as competitive antagonists. The Schild plots were linear with slopes not significantly different from unity (P > .05); for NMS (3-30 nM), the slope of the plot of log (DR  1) vs. log [antagonist] was 1.18 ± 0.11, and the estimated K_B value, with the regression constrained to unity, was 0.24 nM (95% confidence limits, 0.12-0.45, 8 experiments, 3 concentrations). For atropine, the slope was 1.07 ± 0.06 and the K_B, estimated as above, was 0.97 nM (0.83-1.16, 4 experiments, 4 concentrations).

Effect of gallamine in combination with a competitive antagonist. Combination of NMS and gallamine produced a small degree of supra-additivity with either carbachol or acetylcholine as agonist (table 1), but the level of supra-additivity (1.3-1.4-fold) did not reach statistical significance.

Effect of C₇/3-phth in combination with atropine. Experiments conducted with a combination of atropine (200 nM) and C₇/3-phth (10 µM) produced a ~5-fold supra-additive effect (table 1), which was statistically significant (P < .01).

Effect of alcuronium in combination with a competitive antagonist. Combination of NMS and alcuronium showed supra-additive dose ratios, the shifts of the C-R curves for carbachol being  33-fold greater than additive (table 1). Increasing the incubation time with the second inhibitor, alcuronium, from 40 to 180 min did not alter the degree of supra-additivity (table 3). However, this was not the situation when the order of addition was alcuronium (10 µM) followed by NMS (3 nM). After a 40-min incubation with the competitive antagonist, there was only a 2- to 3-fold degree of supra-additivity (table 2), but this was increased considerably with a 180-min incubation (table 3). For the higher concentration of NMS (30 nM), the order of addition of inhibitor was not important; the dose ratio for the combination obtained after a 40-min incubation was of a similar order to that obtained after a 180-min incubation (tables 1-3).

Effect of combinations of allosteric modulators. The combination of C₇/3-phth with gallamine (table 4) or alcuronium (table 5) gave dose ratios that were generally less than additive. Extending the time of incubation with the second antagonist, alcuronium or gallamine, from 40 to 180 min did not greatly alter the magnitude of the dose ratios obtained (table 6). Gallamine and alcuronium in combination gave a small degree of supra-additivity (<1.5 fold), which is in contrast to the combination of NMS and alcuronium (compare tables 1 and 5). Extending the incubation time with the combination of the two modulators to 180 min increased the supra-additivity to ~4-fold (table 6).

View this table: [in this window] [in a new window]   TABLE 4 Dose ratios obtained with either C7/3-phth or gallamine alone or in combination using carbachol as agonist

View this table: [in this window] [in a new window]   TABLE 5 Dose ratios obtained with alcuronium and either C7/3-phth or gallamine alone and in combination using carbachol as agonist

Comparison of data with predicted values. Using heterotropic cooperativity factors ( values) previously determined in this laboratory for the interaction of carbachol with C₇/3-phth ( = 1064; Lanzafame et al., 1996) and gallamine ( = 191; calculated from Clark and Mitchelson, 1976), the dose ratios expected from a combination of the two allosteric modulators were estimated using equation 15 (Appendix); these are also shown in table 4. Because the experimental protocol for the combination experiments did not allow the determination of the actual dose ratio produced by the second modulator in the same preparation, the predicted dose ratios were calculated as a range based on the 95% confidence limits of the dose ratios produced by the second modulator when used alone with carbachol in separate experiments. That is, the dose ratio obtained experimentally for the first inhibitor (DR₁) together with the upper and lower 95% confidence limits for DR₂ was used for the calculation in equation 15 to provide the corresponding predicted range of combination dose ratios. The mean dose ratios obtained experimentally (DR₃) in all cases did not differ significantly (P > .05) from the lower range value for the predicted dose ratio.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Allosteric modulation of the guinea pig atrial muscarinic receptor was evident in the present study with the three compounds alcuronium, C₇/3-phth and gallamine. Previously, in functional experiments, C₇/3-phth (Lanzafame et al., 1996; Lüllmann et al., 1969) and gallamine (Clark and Mitchelson, 1976) were reported to produce curvilinear Schild plots that trended to a limiting maximal dose ratio. Ehlert (1988a) has shown that this maximal dose ratio is an estimate of the heterotropic cooperativity factor () for the interaction between the binding of an agonist at the orthosteric site and an allosteric modulator at the secondary site. Thus far, alcuronium has only been investigated against oxotremorine-M in concentrations 100 µM in functional experiments and exhibited a linear Schild plot over the concentration range studied (Maaß et al., 1995).

However, the allosteric nature of the interaction of alcuronium with muscarinic receptors at a functional level was clearly evident, along with that of C₇/3-phth and gallamine, when each was combined with a competitive antagonist, such as NMS or atropine. The dose ratio produced differed from that predicted for combination of two competitive antagonists. Alcuronium, in combination with NMS, produced a supra-additive dose ratio which was ~10- to 30-fold greater than predicted if both alcuronium and NMS had been acting competitively. Maaß et al. (1995) found a similar degree of supra-additivity, using oxotremorine-M as the agonist, with the two inhibitors.

In the case of gallamine, the combination dose ratio with NMS was only ~1.5-fold greater than expected for two competitive antagonists. However, when combined with atropine, gallamine produced underadditive dose ratios when acetylcholine was the agonist, indicating that gallamine was not acting competitively. Similar findings with gallamine have been reported previously (Clark and Mitchelson, 1976), and this effect could not be attributed to any anticholinesterase activity of gallamine because the same result was obtained after pretreatment of the tissue with dyflos. The third allosteric modulator, C₇/3-phth, produced a supra-additive effect with atropine similar to that observed previously with NMS (Christopoulos and Mitchelson, 1994). It should be noted that the nature of the cooperativity for the three allosteric modulators differs. In binding studies, alcuronium has been shown to act as a positive allosteric modulator because it increased the binding of [³H]NMS (Tucek et al., 1990), whereas C₇/3-phth (Christopoulos et al., 1993) and gallamine (Stockton et al., 1983) are negative allosteric modulators; they were found to inhibit binding of the [³H] ligand.

The estimates of heterotropic cooperativity factors () obtained in the present study (tables 1 and 2) are of the same order as previously published values for the interaction of these allosteric modulators with antagonists. Proka and Tucek (1995) and Jakubík et al. (1995) reported values of 0.3 to 0.4 for alcuronium vs. NMS, and a value of 33 has been reported for C₇/3-phth with atropine (Mitchelson, 1975). For gallamine, a value of ~30 was reported for interaction with atropine in binding studies (Stockton et al., 1983), and a value of ~55 was reported for the same interaction in functional experiments (Clark and Mitchelson, 1976). The latter value was independent of the agonist used, as in the present experiments. The interaction of gallamine with [³H]NMS in binding studies has provided wider estimates of ranging from 13 to 74.5 (Ehlert, 1988b; Lee et al., 1992; Proka and Tucek, 1995; Stockton et al., 1983). This may be due to the fact that the degree of negative cooperativity existing between gallamine and [³H]NMS is very sensitive to the ionic composition of the various buffers used, as has been noted previously (Tränkle et al., 1996; Waelbroeck, 1994).

From the present studies, it is obvious that allosteric interactions may give rise to either supra-additivity, underadditivity or pseudocompetitive behavior. The basis underlying these phenomena may be appreciated from a consideration of the difference in the magnitude of the cooperativity factors between modulator/agonist and modulator/antagonist pairs. For interactions characterized by negative cooperativity, a large  value between a modulator/agonist pair coupled with a small value for the modulator/antagonist pair will give rise to supra-additive inhibition when the three drugs are combined. On the other hand, a small  value and large value will lead to underadditive inhibition. If the  and values are of a similar magnitude, combination of the agonist with the modulator and antagonist will produce inhibition that appears indistinguishable from competitive behavior. Similar considerations apply to positive allosteric interactions.

Estimates of cooperativity factors for the interaction of the modulators with agonists range from >1000 for C₇/3-phth with carbachol (Lanzafame et al., 1996) to 78 to 191 for gallamine with carbachol and 39 to 100 for gallamine with acetylcholine (Clark and Mitchelson, 1976; Stockton et al., 1983). Thus, these values suggest that C₇/3-phth will produce supra-additive inhibition of responses to an agonist when combined with a competitive antagonist because the higher  value obtained with the agonist, compared with the value obtained with the competitive antagonist, indicates that C₇/3-phth will inhibit the agonist selectively. In the case of gallamine, with NMS, the cooperativity factor was only slightly smaller (~10-fold) than the value of ~200 obtained with carbachol (Clark and Mitchelson, 1976), so the observed supra-additivity was small, as expected. The interaction of gallamine with acetylcholine has provided an  value of ~40 in functional studies (Clark and Mitchelson, 1976), whereas the value for atropine was ~100, leading to a prediction of underadditive dose ratios when acetylcholine, gallamine and atropine were combined. This was in agreement with the experimental findings. In the case of alcuronium, a linear Schild plot with oxotremorine-M for dose ratios up to ~100 (Maaß et al., 1995) suggests a cooperativity factor 100 with agonists, so the degree of supra-additivity in combination with NMS should be large with this modulator because of the very low values found for its interaction with the competitive antagonist.

In contrast, the combination of gallamine (100 µM) with C₇/3-phth gave dose ratios that were less than additive and comparable to those predicted for a ternary complex in which C₇/3-phth and gallamine were competing for a common site. In this model, each allosteric modulator would influence the binding of the agonist at the orthosteric site depending on the amount bound at the allosteric site and on its cooperativity factor for interaction with the agonist. Results with the lower concentration of gallamine (30 µM) were more divergent from the model. This finding is puzzling and points to some other possible action of gallamine complicating the data. One explanation is the recent finding (Jakubík et al., 1996) that gallamine and alcuronium, binding at the allosteric site on cloned m2 receptors, produced agonist-like effects and inhibited adenylyl cyclase; this effect was not influenced by competitive antagonists. For gallamine, the effect peaked at ~1 µM and was absent at 100 µM. Thus, it would be expected that a 30 µM concentration of gallamine would have two opposing actions: an inhibitory action on responses to agonists and some ability to activate the receptor, the latter effect being absent at a concentration of 100 µM. Consequently, the combination dose ratio with the lower concentration of gallamine would be less than that predicted from the ternary complex model. Other possible factors considered were an effect on atrial contractility, possible nonequilibrium conditions and the ability of gallamine to bind to more than one site on the receptor. The level of contractility of the atria did not change significantly at the end of the incubation periods with the modulators, so the low combination dose ratio did not involve some additional effect of the modulators on the contractility of the tissue, although gallamine has been shown to inhibit K⁺ channels (Cook and Haylett, 1985; Dunn et al., 1996). Incubation time did not appear to be a factor, as there was little change in dose ratios when the incubation with the two inhibitors was extended to 180 min (table 6). Ellis and Seidenberg (1989) reported that gallamine bound to an allosteric site that facilitated [³H]quinuclidinyl benzilate dissociation, as well as to a site that inhibited dissociation of both [³H]quinuclidinyl benzilate and [³H]NMS. Under this scheme, binding of gallamine at an additional site to enhance dissociation of C₇/3-phth over that predicted from competition for a common site could occur. However, the phenomenon reported by Ellis and Seidenberg (1989) appears to occur only in a low ionic strength medium (5 mM phosphate buffer), as others have not observed this in dissociation rate studies using a high ionic strength medium (Jakubík et al., 1995); therefore, it would appear to be an unlikely possibility in the present experiments with a physiological buffer.

Combination of C₇/3-phth with alcuronium also gave a dose ratio that was additive or underadditive. No supra-additivity was observed, even after a 180-min incubation with the two modulators, which is in contrast to the experiments in which either modulator was combined with a competitive antagonist. Because a cooperativity factor for the interaction of alcuronium with agonists at the muscarinic receptor has not been established, a value of 200 or 1000 was used to estimate the predicted dose ratio, as outlined in Results. The use of either value produced similar estimates. On the basis of findings in binding studies, it has been suggested that alcuronium has two facets to its interaction with competitive antagonists at the muscarinic receptor: an interaction at the allosteric site accompanied by some physical occlusion of the orthosteric site, so that a competitive antagonist is unable to gain access to the site or to leave the site readily if the allosteric modulator is present (Tucek and Proka, 1995). Accordingly, the effect of combinations of alcuronium with NMS has been shown to be dependent on the order in which the inhibitors were added in binding experiments (Proka and Tucek, 1994). In the present functional experiments, it was found that when C₇/3-phth was added as first modulator followed by alcuronium, there did not appear to be any difference in the dose ratios obtained after a 40-min incubation with alcuronium compared to that obtained after 180 min. Adding alcuronium first and then C₇/3-phth gave similar dose ratios to that observed with the reverse order, although the combination dose ratio was slightly smaller. These findings contrast with the results obtained when alcuronium was combined with NMS (3 nM). In these experiments, the order of addition was important and the degree of supra-additivity continued to increase over 180 min when alcuronium was the first of the two drugs, supporting the suggestion of Proka and Tucek (1994) that access of NMS to the receptor was inhibited by alcuronium. However, there are two points that should be noted in connection with the findings. First, the order of addition did not appear important when a higher concentration of N-methylscopolamine (30 nM) was used, and second, no delay in the rate of onset of action of carbachol was noted in these experiments, suggesting that any steric effect of alcuronium must be limited to the binding groups for competitive antagonists and not those for agonists at the orthosteric site. Maaß et al. (1995) also did not report any delayed negative inotropic response with oxotremorine-M as agonist in the presence of alcuronium.

In binding experiments, Proka and Tucek (1995) found concentrations of gallamine or of alcuronium >3 µM slowed the binding of [³H]NMS to the extent that equilibration was not reached over a 5-hr incubation period in HEPES buffer at 25°C. In the functional experiments reported here, the concentrations of alcuronium and gallamine used exceeded 3 µM, but, as noted, the dose ratios did not change markedly on extending incubations from 40 to 180 min.

Proka and Tucek (1995) also found that gallamine and alcuronium competed for a common site on the muscarinic receptor when modulating the binding of [³H]NMS. The present finding, with the use of the two allosteric modulators in combination as inhibitors of the response to carbachol, appears to be in substantial agreement with their findings. However, there was a small degree of supra-additivity that increased further on prolonged incubation, and this appeared unlikely, on the basis of the predicted dose ratios, even allowing for the uncertainty surrounding the value of the cooperativity factor for alcuronium with carbachol. Whether the ability of both gallamine and alcuronium to influence G protein interactions (Jakubík et al., 1996) contributed to this small effect remains to be determined. Thus, it is possible that the small differences, from theoretical predictions, occurring with some combinations may be due to effects of the modulators on agonist efficacy; this may be resolved by further experiments with the technique of resultant analysis (Black et al., 1986). Indeed, Kenakin and Boselli (1989) used resultant analysis to demonstrate that gallamine recognized an allosteric site on muscarinic receptors in rat trachea.

In conclusion, combination of C₇/3-phth with either gallamine or alcuronium gave dose ratios in general accordance with that predicted using an allosteric ternary complex model for binding at the muscarinic receptor. Overall, the results suggested that the three allosteric modulators act at a common accessory site.