Introduction

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Some local anesthetics have been reported to inhibit agonist-induced desensitization in various tissues. For example, tetracaine inhibits desensitization of the beta adrenoceptor system (Mallorga et al., 1980), muscarinic receptor system (Hishinuma and Uchida, 1991) and histamine H₁ receptor system (Hishinuma and Uchida, 1987; Horio et al., 1997). Similarly, procaine inhibits desensitization of the muscarinic and histamine H₁ receptor system (Magaribuchi et al., 1973; Hishinuma and Uchida, 1987; Horio et al., 1997), and some related compounds like quinacrine and chloroquine, with a similar chemical structure to amine local anesthetics, also inhibit desensitization of various systems (Mallorga et al., 1980; Higuchi et al., 1982; Siegel et al., 1984).

These drugs have several common actions on the receptor-mediated cellular processes in approximately the same concentration range. First, all of them are phospholipase A₂ inhibitors. Second, tetracaine and procaine have inhibitory effects on voltage-dependent Ca⁺⁺ channels in smooth muscle cells (Ishii and Shimo, 1984; Spedding and Berg, 1985; Ahn and Karaki, 1988). Third, some of them interact with muscarinic and histamine H₁ receptors (Burgermeister et al., 1978; Aguilar et al., 1980; Fairhurst et al., 1980; Taylor et al., 1980; Horio et al., 1997). At present, however, it is not clear whether these local anesthetics inhibit various types of desensitization through a common mechanism or their respective mechanisms, although their inhibitory actions have been, in some cases, attributed to the inhibition of phospholipase A₂ (Mallorga et al., 1980) or to receptor blockade (Horio et al., 1997).

In the present study, we examined the effects of these local anesthetics on acetylcholine-induced desensitization of guinea pig ileal smooth muscle to clarify the mechanism with which these drugs inhibit desensitization of muscarinic receptor system. Especially, we focused on their interaction with the receptors because our previous study showed that these drugs inhibited histamine-induced desensitization mainly through their blocking action on histamine H₁ receptors (Horio et al., 1997).

	Methods

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Measurement of contractile responses. Guinea pigs of either sex, weighing 250 to 500 g, were killed by a blow on the head and cutting of the throat (this study was approved by the Institutional Animal Care and Use Committee of the University of Tokushima). The ileum was removed, and strips of longitudinal muscle were obtained according to the method of Rang (1964). The strips were suspended in Tyrode's solution at 31°C and bubbled with air under a resting tension of ~0.5 g. The Tyrode's solution had the following composition (in mM): NaCl 136.9, KCl 2.7, CaCl₂ 1.8, MgCl₂ 1.05, NaH₂PO₄ 0.42, NaHCO₃ 11.9 and glucose 5.6. Isotonic contractions were recorded with a lever on a smoked drum.

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Measurement of desensitization. Cumulative dose-response curves to acetylcholine were measured on a longitudinal muscle strip at intervals of ~1 hr. The muscle strip was then treated with a desensitizing agent (10⁴ M acetylcholine) for 30 min. After washing of the muscle with Tyrode's solution for 10 min, the dose-response curve for acetylcholine was reexamined. The curve shifted almost in parallel to the right after this desensitizing treatment. The dose-ratio for shifted dose-response curves was determined to assess the extent of desensitization (Paton, 1961; Horio et al., 1990b). Here, the dose-ratio is the ratio of the concentration of acetylcholine required to elicite 50% of maximal response after the desensitizing treatment to the concentration needed to elicite the same response in the control experiment.

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Measurements of binding of [³H]NMS and [³H]nitrendipine. For experiments on [³H]NMS binding, strips of guinea pig ileal longitudinal muscle were cut into small pieces with scissors and homogenized in 10 volumes of 50 mM sodium-potassium phosphate buffer (pH 7.4) with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) (setting 6) for three periods of 15 sec at 1-min intervals. The homogenate was centrifuged at 50,000 × g for 30 min, and the pellet was resuspended in 5 mM phosphate buffer. Digitonin (1%) was added to the suspension, and the mixture was stirred for 60 min at 4°C and then centrifuged at 90,000 × g for 60 min. The supernatant was used for binding assay immediately. Incubations in 20 mM Tris·HCl (pH 7.4) contained 500 pM [³H]NMS, the drugs (quinacrine, chloroquine, tetracaine and procaine) and the supernatant membrane fraction (0.08 mg protein) in a total volume of 0.5 ml. Equilibration was for 30 min at 25°C. Incubations were then cooled to 0°C, and a 0.2-ml sample was applied in duplicate to a column of Sephadex G-50 (preequilibrated with 20 mM Tris·HCl, pH 7.4) and then eluted with 1.1 ml buffer (Haga and Haga, 1983). The whole elute was collected in a vial, and the radioactivity was measured by liquid scintillation spectrophotometry in a toluene-Triton X-100 base scintillation cocktail. The level of nonspecific binding was defined as that insensitive to inhibition by 1 µM atropine. It represented <1% of the total binding.

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Calculation of receptor occupancy. Receptor occupancy in the presence of the inhibitor drug was calculated by use of a one-site model with the equation: where Y is the percentage of receptors occupied by agonist, A is the concentration of agonist, K_A is its dissociation constant, B is the concentration of the inhibitor drug and K_B is its dissociation constant. In the calculation, we used K_A = 1.0 × 10⁶ M (Yamamura and Snyder, 1974). The values of K_B were obtained from functional studies as described in the section Measurement of contractile responses.

Statistics. Statistical evaluation of significant differences was performed with Student's t test. Differences with P values of < .05 were considered statistically significant.

Drugs. [³H]NMS (80 Ci/mmol) and [³H]nitrendipine (74 Ci/mmol) were obtained from New England Nuclear Research Products (Boston, MA). Quinacrine dihydrochloride, chloroquine diphosphate, nifedipine and p-bromophenacyl bromide were purchased from Sigma Chemical (St. Louis, MO). Acetylcholine chloride and procaine hydrochloride were from Daiichi Pharmaceutical (Tokyo, Japan). Tetracaine hydrochloride was from Kyorin Pharmaceutical (Tokyo, Japan). Atropine sulfate, manoalide and digitonin were from Wako Pure Chemicals (Tokyo, Japan).

	Results

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Effect of local anesthetics on desensitization. Local anesthetics and related compounds, quinacrine, chloroquine, tetracaine and procaine inhibited acetylcholine-induced desensitization in guinea pig ileal longitudinal muscle (fig. 1). Here the extent of desensitization was assessed by dose-ratios as described previously (Paton, 1961; Horio et al., 1990b). The order of potency was quinacrine > chloroquine > tetracaine > procaine. Values of pIC₅₀, which denotes negative logarithm of IC₅₀ for desensitization, are shown in table 1.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Inhibition by four drugs, quinacrine (), chloroquine (), tetracaine () and procaine (), of desensitization in guinea pig ileal longitudinal muscle. Desensitization was induced by pretreatment with 10 M acetylcholine for 30 min. Dose-ratios on the ordinate scale were determined as described in Methods to express the extent of desensitization. Control desensitization was measured in the absence of any drug and is represented by a dotted line (dose-ratio = 45.0 ± 1.0). Desensitization in the presence of the drugs was measured by treating the tissue with the drugs for 10 min before the desensitizing treatment with acetylcholine in the continued presence of the drugs. Each point is the mean of three experiments, and S.E.M. values are indicated by vertical bars.

View this table: [in this window] [in a new window]   TABLE 1 An index of inhibitory potency of local anesthetics on desensitization and muscarinic receptors in guinea pig ileal longitudinal muscle Values of pIC50 for desensitization were obtained from the results shown in figure 1. The IC50 of procaine could not be determined because this drug did not inhibit desensitization over 50% in this experiment. Hill coefficients were obtained by a nonlinear least-squares regression. Dissociation constants (pKi) were calculated from concentrations giving 50% inhibition of the binding. Dissociation constants (pKB) were calculated from the parallel shift of the dose-response curves for acetylcholine obtained in the presence of 5 × 106 M quinacrine, 105 M chloroquine, 3 × 105 M tetracaine or 104 M procaine after preexposure for 30 min, under which condition maximal response was not suppressed (>95%). Values are mean ± S.E.M. (n = 3-5).

Effect of phospholipase A₂ inhibitors on desensitization. At the concentration of IC₅₀ for desensitization (4.79 × 10⁶ M), quinacrine was fully effective as a phospholipase A₂ inhibitor (e.g., Nishimura et al., 1995). Thus, to determine whether the effect of local anesthetics on desensitization was due to their inhibition of phospholipase A₂ activity, the effect of two specific inhibitors of phospholipase A₂, manoalide and p-bromophenacyl bromide on desensitization was examined. Both drugs had no significant effect on desensitization (fig. 2).

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Effects of manoalide and p-bromophenacyl bromide on desensitization in guinea pig ileal longitudinal muscle. Desensitization was induced by pretreatment with 10 M acetylcholine for 30 min, in the absence (control) or presence of 10 M manoalide (Mano) or 5 × 10 M p-bromophenacyl bromide (p-BPB). Dose-ratios were determined as described in Methods to express the extent of desensitization. Vertical bars indicate mean ± S.E.M. (n = 3). Both manoalide and p-bromophenacyl bromide were without significant effect on desensitization (P > .05).

Interaction of local anesthetics with Ca⁺⁺ channels. These local anesthetics inhibited Ca⁺⁺-induced contractions in K⁺-depolarized muscle (fig. 3), indicating that these drugs interacted with Ca⁺⁺ channels. Values of pIC₅₀ for Ca⁺⁺-induced contractions are shown in table 2. Local anesthetics also inhibited [³H]nitrendipine binding to membrane preparations from guinea pig ileal longitudinal muscle. The Hill coefficients and pIC₅₀ values are summarized in table 2. The values of Hill coefficient were far apart from unity and thus we did not further analyze these data. The rank order of the pIC₅₀ values for both Ca⁺⁺-induced contractions and [³H]nitrendipine binding was quinacrine > chloroquine = tetracaine  procaine and was not in agreement with the potency order of inhibiting desensitization.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition by quinacrine (), chloroquine (), tetracaine () and procaine () of Ca++-induced contraction in K+-depolarized guinea pig ileal longitudinal muscle. The contractile response obtained at 3 mM CaCl2 was used as a control. The response examined in the presence of each inhibitor drug was expressed as a percentage of the control response. Each point is the mean of four experiments, and S.E.M. values are indicated by vertical bars.

View this table: [in this window] [in a new window]   TABLE 2 An index of inhibitory potency of local anesthetics on Ca++ channels in guinea pig ileal longitudinal muscle Values of pIC50 for Ca++-induced contractions were obtained from the results shown in figure 3. Values of pIC50 for [3H]nitrendipine binding (0.3 nM) were obtained from the curves of inhibition of the binding, and Hill coefficients were obtained by a nonlinear least-squares regression. Procaine did not inhibit [3H]nitrendipine binding at 102 M. Values are mean ± S.E.M. (n = 4).

Interaction of local anesthetics with muscarinic receptors. Local anesthetics inhibited the binding of [³H]NMS to solubilized membranes from guinea pig ileal longitudinal muscle (fig. 4). The values of the Hill coefficient obtained from the inhibition curves are shown in table 1. These values were all close to unity, indicating that these drugs bound to a single site on the receptors. The dissociation constants (pK_i) are shown in table 1. In this study, we used solubilized receptors instead of membrane preparations to eliminate the indirect effects of the local anesthetics through their action on membrane lipids (Seeman, 1972). Essentially the same results were obtained by the binding experiments using membrane preparations (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Inhibition by quinacrine (), chloroquine (), tetracaine () and procaine () of [3H]NMS binding to solubilized membranes prepared from guinea pig ileal longitudinal muscle. Measurement of the inhibition of the binding of 0.5 nM [3H]NMS was performed in 20 mM Tris·HCl, pH 7.4. Each point is the mean of three or four experiments, each performed in duplicate, and S.E.M. values are indicated by vertical bars.

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View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effects of four drugs, quinacrine (A), chloroquine (B), tetracaine (C) and procaine (D), on the dose-response curves for acetylcholine in guinea pig ileal longitudinal muscle, showing control dose-response curves () and dose-response curves in the presence of each drug at 3 µM (), 10 µM (), 30 µM () and 100 µM (). Each point is the mean of five experiments, and S.E.M. values are indicated by vertical bars.

View this table: [in this window] [in a new window]   TABLE 3 Dose-ratio test for competitive antagonism of local anesthetics at muscarinic receptors Drugs used were 5 × 106 M quinacrine, 105 M chloroquine, 3 × 105 M tetracaine, 104 M procaine and 108 M atropine. The guinea pig ileal longitudinal muscle strips were pretreated with each drug or a combination of the drugs for 30 min, and the dose-response curves for acetylcholine were examined in the presence of the drugs. Dose-ratios were determined from the parallel shift of the curves. Values are mean ± S.E.M. (n = 3-5).

Blockade of muscarinic receptors by local anesthetics. The rank order of pK_i and pK_B values agreed well with the order of pIC₅₀ values for desensitization, suggesting that blockade of the receptors by the local anesthetics was responsible for the inhibition of desensitization. To clarify this point, we calculated the receptor occupancy by agonist in the presence of these inhibitor drugs. For simplicity, we used a one-binding-site model as described in Methods, although muscarinic receptors of guinea pig ileal muscle have been shown to be best fitted by a two-binding-site model (Eglen et al., 1992). Here, we must note that quinacrine probably interacted at an allosteric site. The Schild plot for such negative allosteric ligand is shown to be curvilinear, and the inhibiting effect of the ligand reaches a plateau at high concentrations (Stockton et al.,1983; Ehlert, 1988). Therefore, when we calculated receptor occupancy by using the above equation, we might overestimate the blocking action of quinacrine. The results are summarized in table 4. Receptor occupancy was calculated for the conditions when the desensitizing treatment (coexistence of agonist and inhibitor) induced 50% desensitization. For comparison, receptor occupancy was also calculated for the following two conditions in which desensitizing treatment was performed: (1) at a low concentration of acetylcholine (0.93 ± 0.21 µM, n = 5) in the absence of any inhibitor, or (2) in the presence of atropine (28.0 ± 3.2 nM, n = 4); both treatments gave 50% desensitization. In the latter experiment, we examined acetylcholine-induced desensitization of histamine response (see Methods). Quinacrine, chloroquine and tetracaine had little blocking effect on muscarinic receptors. Procaine effectively inhibited agonist binding, to ~50%. The treatment at a low concentration of acetylcholine (0.93 µM) or in the presence of atropine (28.0 nM) also inhibited it to a similar extent.

View this table: [in this window] [in a new window]   TABLE 4 Receptor occupancy by agonist under the various desensitizing conditions at muscarinic receptors Receptor occupancy was calculated by use of a one-site model. Desensitization induced by 104 M acetylcholine was used as control. Receptor occupancy was calculated for the conditions that induced 50% desensitization, which was obtained either by adding inhibitor drug (local anesthetic or atropine at a concentration of IC50 for desensitization) or by reducing the concentration of desensitizing agent (9.3 × 107 M acetylcholine).

	Discussion

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Agonist-induced desensitization, the loss of sensitivity subsequent to agonist treatment, has long been observed in a wide variety of cellular systems and is generally considered to be due to events at the signaling pathway. In the present study, we focused on acetylcholine-induced desensitization and the effect of local anesthetics on this type of desensitization, especially on the signaling pathway, although there remained the possibility that local anesthetics acted through other mechanism such as membrane-stabilizing actions or hydrophobic interactions. Here, we determined which of the main actions of local anesthetics (i.e., blockade of receptors, inhibition of phospholipase A₂ or interaction with Ca⁺⁺ channels) was responsible for the inhibition of acetylcholine-induced desensitization in guinea pig ileal smooth muscle.

The potency order of these drugs as a phospholipase A₂ inhibitor is quinacrine > chloroquine > tetracaine > procaine, according to the literature (Kunze et al., 1976; Higuchi et al., 1983; Loffler et al., 1985; Abubakar et al., 1990) and agreed with the order of inhibition of desensitization (table 1), suggesting that this action was responsible for the inhibition of desensitization. However, this view was negated because two selective inhibitors of phospholipase A₂, manoalide and p-bromophenacyl bromide, had no significant effect on desensitization (fig. 2). Manoalide at the concentration tested was fully effective as a phospholipase A₂ inhibitor, to the same extent as quinacrine (Nishimura et al., 1995). Previously, Siegel et al. (1984) showed a slight inhibition of desensitization by p-bromophenacyl bromide in guinea pig ileum. However, higher concentration (5 × 10⁶ M) of this drug than that used by them (10⁷ M) had no inhibitory effect. These results indicated that local anesthetics did not inhibit desensitization through their inhibitory action on phospholipase A₂.

Muscarinic stimulation inhibits voltage-gated Ca⁺⁺ channel currents in smooth muscle cells (Mitsui and Karaki, 1990; Russell and Aaronson, 1990; Unno et al., 1995), suggesting that an inactivation of voltage-gated Ca⁺⁺ channels is responsible for desensitization (Himpens et al., 1991). Because most local anesthetics interact with Ca⁺⁺ channels (Spedding and Berg, 1985), their inhibitory effects during desensitization might protect Ca⁺⁺ channels from being desensitized. Our test, however, showed that the potency order of these local anesthetics in inhibiting Ca⁺⁺ channels did not agree with that of inhibiting desensitization. Especially, chloroquine had a 10-fold stronger effect than tetracaine on desensitization (table 1), whereas both drugs had the same effect on Ca⁺⁺ channels (table 2). Moreover, quinacrine and chloroquine, both potent inhibitors of desensitization, did not inhibit Ca⁺⁺ channel currents at the concentration of IC₅₀ for desensitization (fig. 3). These results indicated that local anesthetics did not inhibit desensitization through their action on Ca⁺⁺ channels. This conclusion is in accord with that of Hishinuma and Uchida (1987) showing that dibucaine, with stronger inhibitory effect than tetracaine on Ca⁺⁺ channels, did not inhibit agonist-induced desensitization of guinea pig taenia.

The present study clearly demonstrated that quinacrine, chloroquine, tetracaine and procaine interacted with muscarinic receptors in guinea pig ileal longitudinal muscle. However, there were some differences in the manner each drug interacted with the receptor. That is, tetracaine and procaine were competitive at this receptor, as shown by the good agreement of the pK_i values of each drug with the corresponding pK_B values and by the results from the combined dose-ratio test of Paton and Rang (1965). On the other hand, chloroquine was only partially competitive, and quinacrine was noncompetitive at muscarinic receptors according to the results of the combined dose-ratio test. The disagreement of the pK_i values of these drugs with the corresponding pK_B values also supported this view. Our result on the competitive interaction of tetracaine and procaine with muscarinic receptors was in accord with that of previous studies (Richelson et al., 1978; Fairhurst et al., 1980; Taylor et al., 1980; Aguilar et al., 1980; Hisayama et al., 1989), although some of these studies indicated that the interaction became noncompetitive (or allosteric) at higher drug concentrations. On the other hand, the present study showed that quinacrine bound to this receptor in a noncompetitive manner, indicating that it bound to a site different from the receptor site (i.e., an allosteric site) and led to the inhibition of the binding of agonist and antagonist to the receptor site.

Each local anesthetic, whether its action on muscarinic receptors is competitive or not, would inhibit agonist binding to the receptor site. Therefore, it is probable that these drugs inhibited desensitization through their blocking action on the receptor. The result that the order of potency of these drugs in inhibiting desensitization agreed well with the rank order of affinity for the receptors (table 1) supported this view. To check this point, we examined the effects of these drugs on receptor occupancy by agonist. Quinacrine, chloroquine and tetracaine reduced receptor occupancy only slightly, that is, from 99.0% (control) to 96.8%, 95.1% and 89.4%, respectively, under conditions when the desensitizing treatment (coexistence of agonist and the inhibitor) induced half-maximum inhibition of desensitization, whereas procaine reduced it to 49.8%. These results indicated that quinacrine, chloroquine and tetracaine exerted only slight blocking action on the receptor, which was insufficient for inhibiting desensitization, whereas procaine could have inhibited desensitization through its antagonizing action.

Nevertheless, there remained the possibility that these local anesthetics inhibited desensitization through their action on the receptor. It should be noted that the K_i values of quinacrine and chloroquine were smaller than the corresponding K_B values by factors of 10 to 20, indicating that these drugs interacted with the receptor under such conditions that they did not inhibit agonist-binding (more correctly, agonist-induced contraction, because the pK_B values were obtained from functional studies). These drugs probably acted on the allosteric site, altered the conformation of the receptor site and thus modified ligand receptor interaction. Therefore, it was possible that muscarinic agonist stimulated two cellular processes in parallel; the contractile process and desensitization process, and that the local anesthetics inhibited desensitization process specifically at the concentrations whereby contractile process was not inhibited. This idea is supported by the findings that muscarinic stimulation is mediated by two types of G protein: pertussis toxin-sensitive G protein and pertussis toxin-insensitive G protein (Inoue and Isenberg, 1990; Unno et al., 1995); the former leads to opening of nonselective cationic channels and contraction of smooth muscle, and the latter leads to inactivation of voltage-gated Ca⁺⁺ channel currents and possibly to desensitization. Tetracaine could have inhibited desensitization by a similar mechanism because this drug acted allosterically at higher concentrations (Taylor et al., 1980; Aguilar et al., 1980).

In conclusion, our results showed that although local anesthetics interacted with muscarinic receptors, they did not inhibit desensitization through their simple blocking action on the receptors (except for procaine). However, there remained the possibility that these local anesthetics bound to an allosteric site on the receptor, modified agonist receptor interaction (e.g., the coupling between the receptor and a G protein) and thus inhibited the pathway specific to the desensitization process. To elucidate this point further requires additional work.