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MUSCARINIC M2RECEPTORS ARE NOT PRIMARILY INVOLVED IN THE CONTRACTION OF GUINEA-PIG GALLBLADDER SMOOTH MUSCLE
Pharmacological Research, Vol. 40, No. 5, 1999 Article No. phrs.1999.0536, available online at http:rrwww.idealibrary.com on
MUSCARINIC M 2 RECEPTORS ARE NOT PRIMARILY INVOLVED IN THE CONTRACTION OF GUINEA-PIG GALLBLADDER SMOOTH MUSCLE AHMET AKICI a , ATILA KARAALP a , ECE ˙ISKENDERa , ESAM E. EL-FAKAHANY b and ¸ S ULE OKTAY a,U a
Department of Pharmacology and Clinical Pharmacology, Marmara Uni¨ ersity School of ˙ Medicine, Haydarpas¸a, 81326 Istanbul, Turkey and b Department of Psychiatry, the Uni¨ ersity of Minnesota, Minneapolis, MN, USA Accepted 8 May 1999
The presence of M 1 ]M 4 receptors in guinea-pig gallbladder smooth muscle cells has been reported recently. The majority of these receptors are said to be of M 2 subtype. However, there are controversial reports about the functional muscarinic receptors that mediate contraction in this tissue. Similar to gallbladder, it was claimed that M 4 receptors mediate guinea-pig uterine contractions, but these receptors have appeared to be of M 2 subtypes later. Therefore, the antagonistic affinities of three M 2-selective muscarinic antagonists were determined in contraction and radioligand binding experiments in guinea-pig gallbladder in the present study. The antagonistic affinity values Žp K i . of gallamine, tripitramine and imperialine were as follows, respectively: 6.28" 0.15, 8.65" 0.10 and 6.55" 0.07 against 0.250 nM w 3 HxQNB binding. All three antagonists displaced the concentration] response curves to carbachol to the right in parallel without affecting the maximum responses. The p A 2 values obtained from constrained Schild plots Žylog K B . were 4.14" 0.18 for gallamine, 6.79" 0.09 for tripitramine, and 7.02" 0.09 for imperialine. The antagonistic affinity values of gallamine, tripitramine and imperialine for M 2 receptors are reported to be 6.3, 9.6, 7.7, respectively. The p A 2 values obtained in this study clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M 2 subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand binding Žp K i . and contraction Žp A 2 . experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M 2 do not mediate the contractile responses.Q 1999 Academic Press KEY WORDS: muscarinic, guinea-pig gallbladder, receptor binding, contraction, gallamine, imperialine, tripitramine.
INTRODUCTION Five different muscarinic receptor subtypes have been defined based on their molecular structure and on in ¨ itro binding or second messenger studies w1]3x. Most of the smooth muscle preparations have been demonstrated to have functional muscarinic M 3 receptors w4x. The contractile responses have been reported to be mediated by muscarinic M 2 receptors in a few smooth muscle preparations such as porcine basilar artery w5x and guinea-pig uterus w6]8x, and by muscarinic M 1 receptors in canine U
Corresponding author.
1043]6618r99r110443]07r$30.00r0
saphenous and femoral veins w9x. Recently, the contractile responses of the guinea-pig gallbladder smooth muscle cells have been suggested to be me¨ diated via M 4 receptors by Ozkutlu et al. w10x whereas others w11]14x have claimed the presence of functional M 3 receptors in guinea-pig gallbladder smooth muscle which are coupled to both phosphoinositide hydrolysis and inhibition of adenylate cyclase activity, depending on the affinities of a series of selective antagonists. On the other hand, radioligand binding studies and Northern blot analysis have revealed the existence of a mixed population of muscarinic binding sites in the majority of smooth muscles from many species w15]19x. Chen et al. w20x have reported that Q 1999 Academic Press
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M 2 and M 3 muscarinic receptor subtypes may be involved in the contractile responses of cat gallbladder. These investigators have suggested that M 2 receptors stimulate calcium influx, whereas M 3 receptors account for phosphoinositide breakdown via activation of phospholipase C. Similarly, Von Schrenck et al. w12x have reported that carbacholinduced inhibition in adenylate cyclase activity, but not phosphoinositide hydrolysis, is pertussis toxin sensitive in guinea-pig gallbladder indicating the presence of more than one subtype of muscarinic receptors. Radioligand binding experiments, Western blot analysis, electrical field stimulation and second messenger studies have revealed the presence of M 1 , M 2 , M 3 and M 4 muscarinic receptor subtypes in this tissue w21, 22x. M 2 receptors have been reported to be the predominant subtype Žapprox. 80%. w21x. Similar to gallbladder, M 4 receptors have been claimed to be involved in guinea-pig uterine contractions w23x, however, later reports revealed that M 2 receptors mediate contractile responses w6x. Therefore, it was planned to investigate the possible involvement of M 2 muscarinic receptors in the guinea-pig gallbladder contraction by comparing the antagonistic affinities of three M 2-selective muscarinic antagonists, gallamine, imperialine and tripitramine in binding and functional experiments.
MATERIALS AND METHODS
Radioligand binding studies Gallbladders were removed from guinea-pigs of both sexes weighing 250]300 g, and homogenized in sodium-potassium phosphate buffer ŽSPPB. Ž50 mM. with an Ultra Turrax homogenizer at maximal speed. Saturation binding assay was performed by the method previously reported w24x. Briefly, the membrane suspension of gallbladder containing approximately 0.1]0.2 mg protein in 2 ml of SPPB Ž50 mM., was incubated at 378C for 90 min with 0.01]5 nM w 3 Hxquinuclydinyl benzilate Žspec. act. 47 Ci mmoly1 ., with or without 1 m M atropine sulphate. The reaction was stopped by filtration through glass fiber filters ŽSigma, F-6019., washed with 3 ml of ice-cold SPPB twice, air-dried, kept in 3 ml of scintillation fluid overnight and counted in a liquid scintillation analyser ŽPackard Tri-Carb 1500. with an efficiency of 58]60%. The filters were pretreated with 0.1% polyethylenimine to reduce the amount of radioactivity retained by the filter. Specific binding is defined as the difference between the radioactivity bound with and without atropine. Protein concentration was determined according to Lowry et al. w25x using bovine serum albumin as standard. For competition binding assay, 0.250 nM w 3 Hxquinuclydinyl benzilate was incubated at 378C for 90 min with guineapig gallbladder smooth muscle membranes Ž0.1]0.2 mg protein. and increasing concentrations of the
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antagonist in a total volume of 2 ml. At least ten concentrations of each of the competitors were chosen to bracket the expected IC 50 and to cover five orders of magnitude. Statistical analysis. Saturation binding curves were analysed using the computer program GraphPAD InPlot. Non-linear curve fitting was used to generate the equilibrium dissociation constant Ž K D . and capacity Ž Bmax . values. The competition curves were also analysed by a computer-assisted nonlinear, least squares fit of the binding data which determines the IC 50 values and slopes ŽHill coefficients, n H .. This approach fits the data to either one or two classes of binding sites and assists in determining whether the two-site model is significantly better than the one-site fit of the data ŽF test.. Apparent dissociation constants Ž K i . were calculated from IC 50 values according to Cheng and Prusoff w26x. The values were expressed as p K i Žylog K i .. All determinations were performed in duplicate. Data were expressed as mean " SEM of four to eight independent experiments.
Contraction experiments Longitudinal strips of gallbladders of guinea-pigs Ž300]350 g. of both sexes were prepared and mounted in an organ bath containing Krebs solution Žcomposition in mmol ly1 : NaCl, 118.4; KCl, 4.7; CaCl 2 , 2.5; MgSO4 , 1.2; NaHCO3 , 25.0; KH 2 PO4 , 1.2; glucose, 11.1. at 378C administered with a mixture of 95% O 2 and 5% CO 2 under a resting tension of 1.0 g and were allowed to equilibrate for 3 h. Isometric contractions were recorded on a polygraph ŽGrass Model 7. via a force-displacement transducer ŽGrass FT03.. Cumulative concentration]response curves were constructed to carbachol, using incremental increases in concentration spaced at 0.5 log intervals. Concentrations were added once a sustained response to the previous concentration was reached. An interval of 45 min was then allowed during which the tissues were washed with Krebs solution. The gallbladder strips were incubated with a selective muscarinic receptor antagonist Žgallamine, tripitramine or imperialine. for the last 15 min and a second concentration]response curve to carbachol was constructed in the presence of the antagonist. One or two concentrations of only one antagonist was used for each strip. Statistical analysis. EC 50 values and the slopes of concentration]response curves were calculated by means of the logistic transformation with individual maximal responses accepted as 100%, and linear regression analysis. Schild plots for competitive antagonism were constructed from the dose ratios of the agonist, obtained from at least four different antagonist concentrations w B x to estimate the p A 2 values. A second approach related to a theoretical competitive antagonism was used in which the slopes
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of the regression lines in the Schild plots were constrained to 1 to assess the p A 2 values Žylog K B . better, since the slopes of Schild plots for the antagonists were not significantly different from unity. Data were expressed as mean " SEM.
binding experiments were as follows: 6.28" 0.15 for gallamine, 8.65" 0.10 for tripitramine and 6.55" 0.07 for imperialine. All displacement curves were monophasic and the Hill coefficients were not significantly different from unity ŽFig. 1..
Drugs used in this study
Contraction experiments
Carbachol, atropine sulphate and gallamine ŽSigma., w 3 Hxquinuclydinyl benzilate ŽAmersham., tripitramine Žgift from Prof. C. Melchiorre, Italy., and imperialine Žgift from Dr Yu R. Mirzaev, Tashkent, Republic of Uzbekistan and Latoxan, France.. RESULTS
Radioligand binding studies
The specific binding of w 3 Hxquinuclydinyl benzilate to guinea-pig gallbladder smooth muscle membranes was a saturable process and shown to be dependent on temperature and protein concentration. Scatchard transformation of the saturation curves at 378C indicated a linear plot. Non-specific binding represented approximately 10% of the total binding at concentrations of w 3 Hxquinuclydinyl benzilate near the K D . The K D and Bmax values were 0.321" 0.048 nM and 189.2" 28.5 fmol mgy1 protein, respectively. The antagonistic affinity values Žp K i . of the antagonists obtained in competition
Gallamine Ž1 = 10y6 ]1 = 10y3 M., tripitramine Ž1 = 10y8 ]3 = 10y5 M. and imperialine Ž1 = 10y7 ]3 = 10y5 M. inhibited the contractile responses of guinea-pig gallbladder smooth muscle strips to carbachol in a concentration-dependent manner. All antagonists displaced the concentration]response curves to carbachol to the right in parallel without affecting the maximum responses ŽFigs 2]4.. Schild analysis of data was consistent with competitive antagonism, since the slopes of the Schild plots were not significantly different from unity. The p A 2 values obtained from constrained plots Žylog K B . were 4.14 " 0.18 for gallamine, 6.79 " 0.09 for tripitramine, and 7.02" 0.09 for imperialine ŽFigs 2]4.. A correlation analysis between the p A 2 and p K i values revealed a correlation constant of 0.5277 Ž P ) 0.05.. DISCUSSION The presence of M 1 ]M 4 receptors in guinea-pig gallbladder smooth muscle with the majority of M 2
Fig. 1. Displacement curves for gallamine, imperialine and tripitramine for guinea-pig gallbladder muscarinic binding sites. Each data point represents the mean of four to six independent experiments performed in duplicate. Standard errors were - 10%.
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Fig. 2. Concentration-contractile response curves of carbachol ŽCCh. in the absence Žcontrol. and presence of gallamine in guinea-pig gallbladder. Data points represent the means of four to eight experiments. Standard errors were - 10%.
subtypes have been recently suggested depending on radioligand binding experiments, Western blot analysis, second messenger and electrical stimulation studies w21, 22x. The finding that gallbladder smooth muscle contains multiple muscarinic binding sites is not surprising, since the majority of smooth muscles from many species exhibit heterogeneous popu-
lations of muscarinic receptors w15]19, 27x. Many smooth muscles exhibit a preponderance of muscarinic M 2 receptors with a minor population of muscarinic M 3 receptors which mediate the contractile responses w4, 28]32x. Despite being the predominant muscarinic receptor subtype in most smooth muscles, the contractile responses have been
Fig. 3. Concentration-contractile response curves of carbachol ŽCCh. in the absence Žcontrol. and presence of imperialine in guinea-pig gallbladder. Data points represent the means of four to eight experiments. Standard errors were - 10%.
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Fig. 4. Concentration-contractile response curves of carbachol ŽCCh. in the absence Žcontrol. and presence of tripitramine in guinea-pig gallbladder. Data points represent the means of four to eight experiments. Standard errors were - 10%.
reported to be mediated by muscarinic M 2 receptors in only a few smooth muscle preparations such as porcine basilar artery w5x and guinea-pig uterus w6]8x. We have reported evidence showing that the functional muscarinic receptors in guinea-pig gallbladder could be distinct from those of ileum ŽM 3 . w33, 34x and are more likely to be of M 4 subtype w10, 21, 34x. Similarly, Barocelli et al. w35x have also concluded that gallbladder muscarinic receptors may be distinct from ileum whereas the involvement of muscarinic M 3 receptors in guinea-pig gallbladder contraction has been postulated by Von Schrenck et al. w11, 12x and Eltze et al. w14x. A similar discussion had appeared in the literature for guinea-pig uterus during the last decade. It was reported that guinea-pig uterus expresses both M 4 and predominantly M 2 receptors w36x. Dorje ¨ et al. w23x have suggested that either M 3 or M 4 receptors mediate contraction, but it was then concluded that guinea-pig uterus contracts via M 2 receptor w6x. Therefore, in the present study, the possible involvement of M 2 receptors has been planned to be further investigated via three M 2-selective muscarinic receptor antagonists. The antagonistic affinity values of gallamine, tripitramine and imperialine for M 2 receptors are as follows, respectively: 6.3, 9.6, 7.7 w6, 37, 38x. The p A 2 values obtained in this study Ž4.14 for gallamine, 6.99 for tripitramine, 7.15 for imperialine. clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M 2 subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand
binding Žp K i . and contraction Žp A 2 . experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M 2 , do not mediate the contractile responses. Since all of these three antagonists do not significantly distinguish M 3 and M 4 receptors w6, 14, 37, 38x, the results of this study may not provide any evidence of the involvement of either of these subtypes in guinea-pig gallbladder contraction. On the other hand, the p K i values of both imperialine and tripitramine obtained at guinea-pig gallbladder homogenates were interestingly very close to the reported antagonistic affinity values of these antagonists at M 1rM 4 receptors w37]39x. However, both agents are not highly selective to distinguish M 4 receptors from M 2 receptors. Therefore, these values may not support the presence of M 4 receptors in this tissue. Muscarinic receptors regulate multiple effector systems by activating G proteins. The genes for five muscarinic receptor subtypes, M 1 ]M 5 , have been cloned and expressed in mammalian cells w40]42x. A number of studies have revealed that M 1 , M 3 and M 5 receptors preferentially couple to phosphatidylinositol 4,5-biphosphate hydrolysis via a pertussis toxin independent G protein ŽGq ., whereas M 2 and M 4 receptor subtypes inhibit adenylate cyclase through Gi w40, 43, 44x and possibly through Gz w45x. Carbachol was found to inhibit cAMP formation in the guinea-pig gallbladder smooth muscle homogenates concentration-dependently w13, 21x supporting the presence of functional muscarinic M 2 andror M 4 receptor subtypes. Carbachol also stimulated
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phosphoinositide hydrolysis but it appeared to be less efficient than the inhibition of adenylate cyclase based on the respective EC 50 values w13, 21x. In conclusion, our results provide further evidence for the heterogeneity of muscarinic receptors in guinea-pig gallbladder smooth muscle and unlike guinea-pig uterus, gallbladder contractions are not mediated via M 2 receptors. It remains to be seen whether M 3 andror M 4 receptors are responsible for eliciting the second messenger ŽPI hydrolysis and adenylate cyclase inhibition. responses and the consequent contraction.
ACKNOWLEDGEMENTS The authors wish to thank Prof. C. Melchiorre ŽItaly. for tripitramine and Dr Yu R. Mirzaev ŽTashkent, Republic of Uzbekistan. and Latoxan ŽFrance. for imperialine. This work was supported by National Institutes of Health, USA ŽFIRCA grant R03 TW00615 and NS 30454..
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Mol Pharmacol 1990; 38: 689]97. 29. Entzeroth M, Doods HN, Mayer N. Characterization of porcine coronary muscarine receptors. NaunynSchmied Arch Pharmacol 1990; 341: 432]8. 30. Hearwi MG, Lambrecht G, Mutschler E, Moser U, Pfeiffer A. Different binding properties of muscarinic M 2-receptor subtypes for agonists and antagonists in porcine gastric smooth muscle and mucosa. Gastroenterology 1988; 94: 630]7. 31. Lucarbacholesi PA, Scheid CR, Romano FD, Kargacin ME, Mullikin-Kilpatrick D, Yamaguchi H, Honeyman TW. Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle. Am J Physiol 1990; 258: C730]8. 32. Zhang L, Horowitz B, Buxton LO. Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M 2 and M 3 subtypes. Mol Pharmacol 1991; 40: 943]51. ¸, Dedeoglu 33. Kurtel H, Yegen ˘ BC ˘ A, Ulusoy NB, Oktay ¸ S. Muscarinic receptor subtypes of guinea-pig gallbladder smooth muscle. Arch Int Pharmacodyn 1990; 308: 39]46. 34. Karaalp A, Akıcı A, Akbulut H, Ulusoy NB, Oktay ¸ S. Distinct functional muscarinic receptors in guinea-pig gallbladder, ileum and atria. Pharmacol Res 1999: in press. 35. Barocelli E, Ballabeni V, Chiavarini M, Molina E, Impicciatore M. Functional comparison between nuvenzepine and pirenzepine on different guinea-pig isolated smooth muscle preparations. Pharmacol Res 1994; 30: 161]70. 36. Eglen RM, Ford APDW, Levine WB, Harris GC, Michel AD, Whiting RL. Multidisciplinary analysis of muscarinic receptors in guinea-pig isolated ileum, atria and uterus in ¨ itro. In: Trends in receptor research. Angeli P, Gulini U, Guaglia W, eds. 1992.
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37. Eglen RM, Harris GC, Cox H, Sullivan AO, Stefanich E, Whiting RL. Characterization of the interaction of the cervane alkaloid, imperialine, at muscarinic receptors in ¨ itro. Naunyn-Schmied Arch Pharmacol 1992; 346: 144]51. 38. Maggio R, Barbier P, Bolognesi ML, Minarini A, Tedeschi D, Melchiorre C. Binding profile of the selective muscarinic receptor antagonist tripitramine. Eur J Pharmacol 1994; 268: 459]62. 39. Caulfield MP, Birdsall NJ. International union of pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Re¨ 1998; 50: 279]90. 40. Bonner TI, Buckley NJ, Young AC, Brann MR. Identification of a family of muscarinic acetylcholine receptor genes. Science 1987; 237: 527]32. 41. Bonner TI, Young AC, Brann MR, Buckley NJ. Cloning and expression of the human and rat m 5 muscarinic acetylcholine receptor genes. Neuron 1988; 1: 403]10. 42. Peralta EG, Ashkenazi A, Winslow JW, Smith DH, Ramachandran J, Capon DJ. Distinct primary structures, ligand binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J 1987; 6: 3923]9. 43. Peralta EG, Ashkenazi A, Winslow JW, Ramachandran J, Capon DJ. Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes. Nature 1988; 334: 434]7. 44. Bonner TI. New subtypes of muscarinic acetylcholine receptors. Trends Pharmacol Sci 1989; (Suppl): 11]15. 45. Parker EM, Kameyama K, Higashijima T, Ross EM. Reconstitutively activity G-protein-coupled receptors purified from baculovirus-infected insect cells. J Biol Chen 1991; 266: 519]27.
MUSCARINIC M 2 RECEPTORS ARE NOT PRIMARILY INVOLVED IN THE CONTRACTION OF GUINEA-PIG GALLBLADDER SMOOTH MUSCLE AHMET AKICI a , ATILA KARAALP a , ECE ˙ISKENDERa , ESAM E. EL-FAKAHANY b and ¸ S ULE OKTAY a,U a
Department of Pharmacology and Clinical Pharmacology, Marmara Uni¨ ersity School of ˙ Medicine, Haydarpas¸a, 81326 Istanbul, Turkey and b Department of Psychiatry, the Uni¨ ersity of Minnesota, Minneapolis, MN, USA Accepted 8 May 1999
The presence of M 1 ]M 4 receptors in guinea-pig gallbladder smooth muscle cells has been reported recently. The majority of these receptors are said to be of M 2 subtype. However, there are controversial reports about the functional muscarinic receptors that mediate contraction in this tissue. Similar to gallbladder, it was claimed that M 4 receptors mediate guinea-pig uterine contractions, but these receptors have appeared to be of M 2 subtypes later. Therefore, the antagonistic affinities of three M 2-selective muscarinic antagonists were determined in contraction and radioligand binding experiments in guinea-pig gallbladder in the present study. The antagonistic affinity values Žp K i . of gallamine, tripitramine and imperialine were as follows, respectively: 6.28" 0.15, 8.65" 0.10 and 6.55" 0.07 against 0.250 nM w 3 HxQNB binding. All three antagonists displaced the concentration] response curves to carbachol to the right in parallel without affecting the maximum responses. The p A 2 values obtained from constrained Schild plots Žylog K B . were 4.14" 0.18 for gallamine, 6.79" 0.09 for tripitramine, and 7.02" 0.09 for imperialine. The antagonistic affinity values of gallamine, tripitramine and imperialine for M 2 receptors are reported to be 6.3, 9.6, 7.7, respectively. The p A 2 values obtained in this study clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M 2 subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand binding Žp K i . and contraction Žp A 2 . experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M 2 do not mediate the contractile responses.Q 1999 Academic Press KEY WORDS: muscarinic, guinea-pig gallbladder, receptor binding, contraction, gallamine, imperialine, tripitramine.
INTRODUCTION Five different muscarinic receptor subtypes have been defined based on their molecular structure and on in ¨ itro binding or second messenger studies w1]3x. Most of the smooth muscle preparations have been demonstrated to have functional muscarinic M 3 receptors w4x. The contractile responses have been reported to be mediated by muscarinic M 2 receptors in a few smooth muscle preparations such as porcine basilar artery w5x and guinea-pig uterus w6]8x, and by muscarinic M 1 receptors in canine U
Corresponding author.
1043]6618r99r110443]07r$30.00r0
saphenous and femoral veins w9x. Recently, the contractile responses of the guinea-pig gallbladder smooth muscle cells have been suggested to be me¨ diated via M 4 receptors by Ozkutlu et al. w10x whereas others w11]14x have claimed the presence of functional M 3 receptors in guinea-pig gallbladder smooth muscle which are coupled to both phosphoinositide hydrolysis and inhibition of adenylate cyclase activity, depending on the affinities of a series of selective antagonists. On the other hand, radioligand binding studies and Northern blot analysis have revealed the existence of a mixed population of muscarinic binding sites in the majority of smooth muscles from many species w15]19x. Chen et al. w20x have reported that Q 1999 Academic Press
444
M 2 and M 3 muscarinic receptor subtypes may be involved in the contractile responses of cat gallbladder. These investigators have suggested that M 2 receptors stimulate calcium influx, whereas M 3 receptors account for phosphoinositide breakdown via activation of phospholipase C. Similarly, Von Schrenck et al. w12x have reported that carbacholinduced inhibition in adenylate cyclase activity, but not phosphoinositide hydrolysis, is pertussis toxin sensitive in guinea-pig gallbladder indicating the presence of more than one subtype of muscarinic receptors. Radioligand binding experiments, Western blot analysis, electrical field stimulation and second messenger studies have revealed the presence of M 1 , M 2 , M 3 and M 4 muscarinic receptor subtypes in this tissue w21, 22x. M 2 receptors have been reported to be the predominant subtype Žapprox. 80%. w21x. Similar to gallbladder, M 4 receptors have been claimed to be involved in guinea-pig uterine contractions w23x, however, later reports revealed that M 2 receptors mediate contractile responses w6x. Therefore, it was planned to investigate the possible involvement of M 2 muscarinic receptors in the guinea-pig gallbladder contraction by comparing the antagonistic affinities of three M 2-selective muscarinic antagonists, gallamine, imperialine and tripitramine in binding and functional experiments.
MATERIALS AND METHODS
Radioligand binding studies Gallbladders were removed from guinea-pigs of both sexes weighing 250]300 g, and homogenized in sodium-potassium phosphate buffer ŽSPPB. Ž50 mM. with an Ultra Turrax homogenizer at maximal speed. Saturation binding assay was performed by the method previously reported w24x. Briefly, the membrane suspension of gallbladder containing approximately 0.1]0.2 mg protein in 2 ml of SPPB Ž50 mM., was incubated at 378C for 90 min with 0.01]5 nM w 3 Hxquinuclydinyl benzilate Žspec. act. 47 Ci mmoly1 ., with or without 1 m M atropine sulphate. The reaction was stopped by filtration through glass fiber filters ŽSigma, F-6019., washed with 3 ml of ice-cold SPPB twice, air-dried, kept in 3 ml of scintillation fluid overnight and counted in a liquid scintillation analyser ŽPackard Tri-Carb 1500. with an efficiency of 58]60%. The filters were pretreated with 0.1% polyethylenimine to reduce the amount of radioactivity retained by the filter. Specific binding is defined as the difference between the radioactivity bound with and without atropine. Protein concentration was determined according to Lowry et al. w25x using bovine serum albumin as standard. For competition binding assay, 0.250 nM w 3 Hxquinuclydinyl benzilate was incubated at 378C for 90 min with guineapig gallbladder smooth muscle membranes Ž0.1]0.2 mg protein. and increasing concentrations of the
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antagonist in a total volume of 2 ml. At least ten concentrations of each of the competitors were chosen to bracket the expected IC 50 and to cover five orders of magnitude. Statistical analysis. Saturation binding curves were analysed using the computer program GraphPAD InPlot. Non-linear curve fitting was used to generate the equilibrium dissociation constant Ž K D . and capacity Ž Bmax . values. The competition curves were also analysed by a computer-assisted nonlinear, least squares fit of the binding data which determines the IC 50 values and slopes ŽHill coefficients, n H .. This approach fits the data to either one or two classes of binding sites and assists in determining whether the two-site model is significantly better than the one-site fit of the data ŽF test.. Apparent dissociation constants Ž K i . were calculated from IC 50 values according to Cheng and Prusoff w26x. The values were expressed as p K i Žylog K i .. All determinations were performed in duplicate. Data were expressed as mean " SEM of four to eight independent experiments.
Contraction experiments Longitudinal strips of gallbladders of guinea-pigs Ž300]350 g. of both sexes were prepared and mounted in an organ bath containing Krebs solution Žcomposition in mmol ly1 : NaCl, 118.4; KCl, 4.7; CaCl 2 , 2.5; MgSO4 , 1.2; NaHCO3 , 25.0; KH 2 PO4 , 1.2; glucose, 11.1. at 378C administered with a mixture of 95% O 2 and 5% CO 2 under a resting tension of 1.0 g and were allowed to equilibrate for 3 h. Isometric contractions were recorded on a polygraph ŽGrass Model 7. via a force-displacement transducer ŽGrass FT03.. Cumulative concentration]response curves were constructed to carbachol, using incremental increases in concentration spaced at 0.5 log intervals. Concentrations were added once a sustained response to the previous concentration was reached. An interval of 45 min was then allowed during which the tissues were washed with Krebs solution. The gallbladder strips were incubated with a selective muscarinic receptor antagonist Žgallamine, tripitramine or imperialine. for the last 15 min and a second concentration]response curve to carbachol was constructed in the presence of the antagonist. One or two concentrations of only one antagonist was used for each strip. Statistical analysis. EC 50 values and the slopes of concentration]response curves were calculated by means of the logistic transformation with individual maximal responses accepted as 100%, and linear regression analysis. Schild plots for competitive antagonism were constructed from the dose ratios of the agonist, obtained from at least four different antagonist concentrations w B x to estimate the p A 2 values. A second approach related to a theoretical competitive antagonism was used in which the slopes
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of the regression lines in the Schild plots were constrained to 1 to assess the p A 2 values Žylog K B . better, since the slopes of Schild plots for the antagonists were not significantly different from unity. Data were expressed as mean " SEM.
binding experiments were as follows: 6.28" 0.15 for gallamine, 8.65" 0.10 for tripitramine and 6.55" 0.07 for imperialine. All displacement curves were monophasic and the Hill coefficients were not significantly different from unity ŽFig. 1..
Drugs used in this study
Contraction experiments
Carbachol, atropine sulphate and gallamine ŽSigma., w 3 Hxquinuclydinyl benzilate ŽAmersham., tripitramine Žgift from Prof. C. Melchiorre, Italy., and imperialine Žgift from Dr Yu R. Mirzaev, Tashkent, Republic of Uzbekistan and Latoxan, France.. RESULTS
Radioligand binding studies
The specific binding of w 3 Hxquinuclydinyl benzilate to guinea-pig gallbladder smooth muscle membranes was a saturable process and shown to be dependent on temperature and protein concentration. Scatchard transformation of the saturation curves at 378C indicated a linear plot. Non-specific binding represented approximately 10% of the total binding at concentrations of w 3 Hxquinuclydinyl benzilate near the K D . The K D and Bmax values were 0.321" 0.048 nM and 189.2" 28.5 fmol mgy1 protein, respectively. The antagonistic affinity values Žp K i . of the antagonists obtained in competition
Gallamine Ž1 = 10y6 ]1 = 10y3 M., tripitramine Ž1 = 10y8 ]3 = 10y5 M. and imperialine Ž1 = 10y7 ]3 = 10y5 M. inhibited the contractile responses of guinea-pig gallbladder smooth muscle strips to carbachol in a concentration-dependent manner. All antagonists displaced the concentration]response curves to carbachol to the right in parallel without affecting the maximum responses ŽFigs 2]4.. Schild analysis of data was consistent with competitive antagonism, since the slopes of the Schild plots were not significantly different from unity. The p A 2 values obtained from constrained plots Žylog K B . were 4.14 " 0.18 for gallamine, 6.79 " 0.09 for tripitramine, and 7.02" 0.09 for imperialine ŽFigs 2]4.. A correlation analysis between the p A 2 and p K i values revealed a correlation constant of 0.5277 Ž P ) 0.05.. DISCUSSION The presence of M 1 ]M 4 receptors in guinea-pig gallbladder smooth muscle with the majority of M 2
Fig. 1. Displacement curves for gallamine, imperialine and tripitramine for guinea-pig gallbladder muscarinic binding sites. Each data point represents the mean of four to six independent experiments performed in duplicate. Standard errors were - 10%.
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Fig. 2. Concentration-contractile response curves of carbachol ŽCCh. in the absence Žcontrol. and presence of gallamine in guinea-pig gallbladder. Data points represent the means of four to eight experiments. Standard errors were - 10%.
subtypes have been recently suggested depending on radioligand binding experiments, Western blot analysis, second messenger and electrical stimulation studies w21, 22x. The finding that gallbladder smooth muscle contains multiple muscarinic binding sites is not surprising, since the majority of smooth muscles from many species exhibit heterogeneous popu-
lations of muscarinic receptors w15]19, 27x. Many smooth muscles exhibit a preponderance of muscarinic M 2 receptors with a minor population of muscarinic M 3 receptors which mediate the contractile responses w4, 28]32x. Despite being the predominant muscarinic receptor subtype in most smooth muscles, the contractile responses have been
Fig. 3. Concentration-contractile response curves of carbachol ŽCCh. in the absence Žcontrol. and presence of imperialine in guinea-pig gallbladder. Data points represent the means of four to eight experiments. Standard errors were - 10%.
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Fig. 4. Concentration-contractile response curves of carbachol ŽCCh. in the absence Žcontrol. and presence of tripitramine in guinea-pig gallbladder. Data points represent the means of four to eight experiments. Standard errors were - 10%.
reported to be mediated by muscarinic M 2 receptors in only a few smooth muscle preparations such as porcine basilar artery w5x and guinea-pig uterus w6]8x. We have reported evidence showing that the functional muscarinic receptors in guinea-pig gallbladder could be distinct from those of ileum ŽM 3 . w33, 34x and are more likely to be of M 4 subtype w10, 21, 34x. Similarly, Barocelli et al. w35x have also concluded that gallbladder muscarinic receptors may be distinct from ileum whereas the involvement of muscarinic M 3 receptors in guinea-pig gallbladder contraction has been postulated by Von Schrenck et al. w11, 12x and Eltze et al. w14x. A similar discussion had appeared in the literature for guinea-pig uterus during the last decade. It was reported that guinea-pig uterus expresses both M 4 and predominantly M 2 receptors w36x. Dorje ¨ et al. w23x have suggested that either M 3 or M 4 receptors mediate contraction, but it was then concluded that guinea-pig uterus contracts via M 2 receptor w6x. Therefore, in the present study, the possible involvement of M 2 receptors has been planned to be further investigated via three M 2-selective muscarinic receptor antagonists. The antagonistic affinity values of gallamine, tripitramine and imperialine for M 2 receptors are as follows, respectively: 6.3, 9.6, 7.7 w6, 37, 38x. The p A 2 values obtained in this study Ž4.14 for gallamine, 6.99 for tripitramine, 7.15 for imperialine. clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M 2 subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand
binding Žp K i . and contraction Žp A 2 . experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M 2 , do not mediate the contractile responses. Since all of these three antagonists do not significantly distinguish M 3 and M 4 receptors w6, 14, 37, 38x, the results of this study may not provide any evidence of the involvement of either of these subtypes in guinea-pig gallbladder contraction. On the other hand, the p K i values of both imperialine and tripitramine obtained at guinea-pig gallbladder homogenates were interestingly very close to the reported antagonistic affinity values of these antagonists at M 1rM 4 receptors w37]39x. However, both agents are not highly selective to distinguish M 4 receptors from M 2 receptors. Therefore, these values may not support the presence of M 4 receptors in this tissue. Muscarinic receptors regulate multiple effector systems by activating G proteins. The genes for five muscarinic receptor subtypes, M 1 ]M 5 , have been cloned and expressed in mammalian cells w40]42x. A number of studies have revealed that M 1 , M 3 and M 5 receptors preferentially couple to phosphatidylinositol 4,5-biphosphate hydrolysis via a pertussis toxin independent G protein ŽGq ., whereas M 2 and M 4 receptor subtypes inhibit adenylate cyclase through Gi w40, 43, 44x and possibly through Gz w45x. Carbachol was found to inhibit cAMP formation in the guinea-pig gallbladder smooth muscle homogenates concentration-dependently w13, 21x supporting the presence of functional muscarinic M 2 andror M 4 receptor subtypes. Carbachol also stimulated
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phosphoinositide hydrolysis but it appeared to be less efficient than the inhibition of adenylate cyclase based on the respective EC 50 values w13, 21x. In conclusion, our results provide further evidence for the heterogeneity of muscarinic receptors in guinea-pig gallbladder smooth muscle and unlike guinea-pig uterus, gallbladder contractions are not mediated via M 2 receptors. It remains to be seen whether M 3 andror M 4 receptors are responsible for eliciting the second messenger ŽPI hydrolysis and adenylate cyclase inhibition. responses and the consequent contraction.
ACKNOWLEDGEMENTS The authors wish to thank Prof. C. Melchiorre ŽItaly. for tripitramine and Dr Yu R. Mirzaev ŽTashkent, Republic of Uzbekistan. and Latoxan ŽFrance. for imperialine. This work was supported by National Institutes of Health, USA ŽFIRCA grant R03 TW00615 and NS 30454..
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