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The Role of M2 Muscarinic Receptor Subtypes Mediating Contraction of the Circular and Longitudinal Smooth Muscle of the Pig Proximal Urethra
0022-5347/02/1681-0308/0 THE JOURNAL OF UROLOGY® Copyright © 2002 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 168, 308 –314, July 2002 Printed in U.S.A.
THE ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES MEDIATING CONTRACTION OF THE CIRCULAR AND LONGITUDINAL SMOOTH MUSCLE OF THE PIG PROXIMAL URETHRA TOMONORI YAMANISHI, CHRISTOPHER R. CHAPPLE, KOSAKU YASUDA, KEN-ICHIRO YOSHIDA AND RUSSELL CHESS-WILLIAMS From the Department of Biomedical Science, University of Sheffield and Department of Urology, Royal Hallamshire Hospital, Sheffield, United Kingdom, and Department of Urology, Dokkyo University, Tochigi, Japan
ABSTRACT
Purpose: We investigated the characterization of muscarinic receptor subtypes in the female pig urethra. Materials and Methods: The affinities of muscarinic antagonists against carbachol responses were calculated in normal and cyclic adenosine monophosphate (cAMP) elevated tissues (contraction with KCl and relaxation with isoprenaline) using longitudinal and transverse strips of urethra. Results: In displacement experiments with 1-quinuclidinyl [phenyl-4-3H] benzilate inhibitory constant (pKi) values of 4-diphenylacetoxyl-N-methyl-piperidine methiodide (DAMP) M3 selective antagonist) and methoctramine (M2 antagonist) indicated the presence of the M2 receptor. In functional studies contraction responses to carbachol were greater in longitudinal than in circular muscle. After cAMP elevation the contraction response increased in circular muscles to the level close to that of longitudinal muscles but did not change significantly in cAMP elevated longitudinal muscle. On normal circular tissues 4-DAMP had high mean affinity (mean apparent pKB value 9.3) but with a Schild slope of less than unity (0.76). Methoctramine competitively antagonized carbachol responses (mean affinity [pA2] value 6.9). On normal longitudinal tissues 4-DAMP and methoctramine competitively antagonized carbachol responses (mean pA2 9.0 and 6.2, respectively). After cAMP elevation in circular tissues mean pA2 values for 4-DAMP (8.7) were significantly lower (p ⫽ 0.0015), and those for methoctramine (7.3) were significantly higher (p ⫽ 0.0193) than in normal tissues. In longitudinal tissues the mean pA2 value for methoctramine (6.9) was significantly greater than in normal tissues (p ⬍0.0001) but the value for 4-DAMP (8.8) did not alter. Conclusions: Pig urethra appears to have predominantly M2 muscarinic receptors. Contraction of the normal urethra appears to be predominantly mediated by M2 and M3 receptors in circular muscle but by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors appeared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle. KEY WORDS: urethra; muscle, smooth; swine; receptors, muscarinic; carbachol
It has been reported that urethral smooth muscle receives a rich cholinergic innervation, of which the role is largely unknown.1, 2 The bladder shows a heterogeneous populations of muscarinic receptors.3 The presence of messenger RNA encoding only M2 and M3 subtypes has been detected in human bladder4 but M1, M2, M3 and M4 subtypes in rat bladder have been found.5 Predominance of the M2 muscarinic receptor subtype with a minor population of M3 receptors has been reported for bladder smooth muscle in several species with an M2-to-M3 ratio of 3:1 in pig and human bladders.6, 7 However, pharmacological characterization of muscarinic receptors mediating contraction of detrusor muscle in pig and human bladder suggest the singular involvement of M3 receptors.8, 9 The predominance of the M3 receptor subtype for mediating bladder contraction has also been demonstrated in experiments in mutant mice lacking the receptor gene for the M3 subtype.10 Although no direct contractile response to M2 receptor activation can be demonstrated, an indirect influence on contraction via the inhibition of cyclic adenosine monophosphate (cAMP) mediated smooth muscle relaxation by Accepted for publication January 11, 2002.
-adrenoceptors has been reported. The involvement of M2 receptors in contraction of the bladder has been observed in tissues after the inactivation of M3 receptors and the elevation of cAMP levels in vitro.3, 6, 11, 12 Recently the involvement of M2 (and not M4) receptors for mediating contraction of the bladder has also been demonstrated in experiments in M2 and M4 muscarinic receptor subtype knockout mice.13 M2 receptors have also been suggested to be able to activate smooth muscle through an action on ion channels, the opening of nonspecific cation channels resulting in depolarization and calcium influx.14, 15 It has also been reported that M2 receptors participate in the contractile response under pathological conditions in rat and human detrusor smooth muscle.3, 11, 16 –18 Although others have investigated responses to muscarinic receptor stimulation in the urethra,19 –21 the physiological significance of muscarinic receptors in the urethra remains unclear. It has been reported that M1, M2 and M3 receptors mediate contraction to carbachol stimulation in the circular muscle of the rabbit urethra.20, 21 However, to our knowledge there have been no reports characterizing muscarinic receptor subtypes in the longitudinal muscles of the urethra or the
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functional role of M2 subtypes in circular and longitudinal urethral tissues after cAMP elevation. We investigated the characterization of muscarinic receptor subtypes in circular and longitudinal isolated strips of the female pig in normal tissue and after cAMP elevation. MATERIALS AND METHODS
Female pig bladders were obtained from the abattoir and immediately placed in cold (4C) Krebs solution (118.4 mM. NaCl, 4.7 mM. KCl, 1.9 mM. CaCl2, 24.9 mM. NaHCO3, 1.15 mM. MgSO4, 1.15 mM. KH2PO4 and 11.7 mM. glucose). The urethra was opened longitudinally at the dorsal portion and the proximal urethra was separated from the bladder at the level of the bladder neck. Longitudinal and transverse strips of the proximal urethra (3 ⫻ 10 mm.) were cut, and the mucosa and serosa were removed. In functional studies in vitro tissues were mounted in 30 ml. organ baths containing Krebs solution maintained at 37C and continuously gassed with 95% O2 and 5% CO2. Tissues for binding studies were freshly used or frozen in liquid nitrogen and stored at ⫺80C until used. Radioligand binding studies. The methods for receptor binding studies have been reported previously.6 Briefly, tissues were homogenized in 50 mM. tris-HCl buffer (pH 7.4) and filtered through muslin. Homogenates were centrifuged at 45,000 ⫻ gravity for 10 minutes at 4C. The pellets were re-suspended in 5 ml. tris-HCl buffer. Homogenates were again centrifuged and the pellets were re-suspended in 5 ml. tris-HCl buffer. Saturation experiments were conducted using 7 concentrations (0.0625 to 4 nM.) 1-quinuclidinyl[phenyl-4-3H]benzilate ([3H]QNB). Binding was determined in a final volume of 0.25 ml. and nonspecific binding was determined with 10 M. atropine. The assay tubes were incubated at 37C for 60 minutes before filtration through an M30 cell harvester (Brandel, Gaithersburg, Maryland). Radioactivity on the filters was determined by liquid scintillation spectrometry. Displacement experiments using 0.3 nM. [3H]QNB with the unlabeled M3 selective antagonist 4-diphenylacetoxyl-Nmethyl-piperidine methiodide (4-DAMP) (0.1 nM. to 1 M.) and the M2 selective antagonist methoctramine (25 nM. to 0.25 mM.) was used to determine the presence of M2 and M3 receptors in pig urethra membranes. In competition binding studies the inhibitory constant (pKi) values were calculated and analyzed for 1 or 2-site binding using Prism software (GraphPad, San Diego, California). A 2-site fit was accepted only if it is resulted in a significant improvement of the fit as judged by the F test.6, 12, 22 In vitro functional studies. Isolated tissues were subjected to a resting tension of 1 gm. and allowed to equilibrate for 60 minutes before the first cumulative concentration-response curves to carbachol were obtained. Tissues were washed and equilibrated for 30 minutes with Krebs solution containing the appropriate concentration of antagonist or vehicle (time
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control). The second concentration-response curves to carbachol were obtained in the presence of 4-DAMP, methoctramine or pirenzepine (M1 selective antagonist), or vehicle. In control experiments the 2 concentration-response curves to carbachol were reproducible but the third concentrationresponse curve varied, probably because of tachyphylaxis in some experiments. Thus, 2 concentration-response curves per tissue were used to calculate affinity values for each muscarinic antagonist. The affinity of these antagonists against carbachol induced contractions of tissue strips were calculated in normal and cAMP elevated tissues. cAMP elevation was performed by precontraction with 50 mM. KCl and relaxation with isoprenaline (30 M.).6 In functional studies, responses are expressed as the mean percent plus or minus standard error of mean (SEM) of the response to 50 mM. KCl. Agonist potencies are expressed as the mean pEC50 plus or minus SEM (-logarithm of the molar concentration of agonist resulting in EC50). Antagonist affinities (apparent affinity [KB] values) were calculated from the equation, KB-antagonist concentration in moles/(dose ratio-1). Schild analysis was performed with the intercept considered the pA2 value. Radioligand binding data and in vitro functional data were analyzed using Prism software. The nonpaired Student t test was used for statistical analysis with p ⬍0.05 considered significant. [3H]QNB (specific activity 42 Ci./mmol.) was stored at ⫺20C. RESULTS
Radioligand binding studies. In saturation binding studies in 4 animals binding of [3H]QNB was saturable and displaceable by atropine. The mean dissociation constant for [3H]QNB was 0.26 ⫾ 0.07 nM. and mean receptor density was 44.1 ⫾ 13.2 fmol./mg. protein. In competition binding studies displacement of [3H]QNB by 4-DAMP in 8 animals and methoctramine in 8 best fitted a 1-site model with mean Hill’s slopes close to unity (0.97 ⫾ 0.13 and 1.05 ⫾ 0.09, respectively). Mean inhibitory constant (pKi) values for 4-DAMP and methoctramine were 7.67 ⫾ 0.11 and 7.56 ⫾ 0.18, respectively, which were comparable with M2 receptors by reported antagonist affinity estimates derived from radioligand binding studies in human recombinant receptors.3, 23, 24 In vitro functional studies. Circular strips from the proximal third of the urethra responded to carbachol but circular muscle from the mid to distal part of the urethra had little or no response to carbachol. Carbachol also induced contractions of longitudinal smooth muscle of the proximal to mid part of the urethra in a concentration dependent manner. In normal strips mean pEC50 values were significantly greater in circular muscles than in longitudinal muscles in 14 animals each (6.78 ⫾ 0.004 versus 6.13 ⫾ 0.06, p ⬍0.0001). The maximum contraction to carbachol relative to that to 50 mM. KCl was significantly larger in longitudinal muscle than in circular muscle (mean 99.8% ⫾ 9.5% versus 55.3% ⫾ 5.8%, p ⫽ 0.0005, fig. 1). After cAMP elevation in circular (16
FIG. 1. Comparison of concentration-response curves to carbachol in normal circular (open circles) and longitudinal (filled circles) muscles (A) and in muscles after cAMP elevation (B).
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animals) and longitudinal (29 animals) urethral muscle mean pEC50 values for carbachol were significantly reduced (5.78 ⫾ 0.03, p ⬍0.0001 and 5.46 ⫾ 0.04, p ⬍0.0001, respectively). The mean maximum contractions to carbachol relative to those to 50 mM. KCl were increased (marginally significantly because of large variations p ⫽ 0.0956) in circular muscle (84.7% ⫾ 15.2%) to a level not significantly different from those in longitudinal muscle (119.4% ⫾ 15.7%). In cAMP elevated longitudinal muscle the maximum contraction did not change significantly compared to that in normal longitudinal muscle (p ⫽ 0.4119, fig. 1). On normal circular muscle strips in vitro 4-DAMP antagonized responses to carbachol with a high affinity (mean apparent pKB 9.30 ⫾ 0.12) without affecting maximum responses, although the mean slope of the Schild plot was significantly less than unity (0.76 ⫾ 0.10, p ⬍0.0001, fig. 2). Methoctramine demonstrated competitive antagonism (mean pA2 6.90 ⫾ 0.14) without affecting maximum responses and with a Schild slope close to unity (fig. 3). Pirenzepine antagonized contractile responses to carbachol with a mean apparent pKB value of 6.73 ⫾ 0.08 without affecting maximum responses, although the mean Schild slope was significantly less than unity (0.77 ⫾ 0.04, p ⬍0.01, see table). In longitudinal muscle 4-DAMP and methoctramine competitively antagonized responses to carbachol without affecting maximum responses and with Schild plots with slopes of unity (see table, and figs. 4 and 5). Mean pA2 values of 8.98 ⫾ 0.08 and 6.22 ⫾ 0.11 were obtained for 4-DAMP and methoctramine, respectively. Pirenzepine antagonized contractile responses to carbachol with low affinity (mean pA2 6.88 ⫾ 0.09) without affecting maximum response and with a mean Schild slope not significantly different from unity (1.02 ⫾ 0.39) (see table). After cAMP elevation in circular muscle of the urethra 4-DAMP produced surmountable competitive antagonism against response to carbachol with a pA2 value of 8.7 ⫾ 0.08, which was significantly lower than those in the normal tissues but with a Schild slope close to unity (p ⫽ 0.0015, see
table and fig. 2). Methoctramine also antagonized responses without affecting maximum responses, yielding a mean apparent pKB value of 7.32 ⫾ 0.09, significantly greater than that for normal tissues (p ⫽ 0.0193). However, the Schild slope was significantly less than unity (0.68 ⫾ 0.05, p ⬍0.0001, fig. 3). In longitudinal tissues after cAMP elevation 4-DAMP and methoctramine antagonized responses without affecting maximum responses and with mean apparent pKB values of 8.78 ⫾ 0.09 and 6.92 ⫾ 0.09, respectively. In cAMP elevated longitudinal tissues apparent pKB values for 4-DAMP were not significantly different from normal tissues but those for methoctramine were significantly increased compared with normal tissues (p ⬍0.0001). The Schild slopes for 4-DAMP and methoctramine were low but not significantly less than unity (see table, and figs. 4 and 5). DISCUSSION
Using computer assisted 3-dimensional reconstruction Dass et al noted that the female pig urethra is arranged in 3 smooth muscle layers, namely an inner longitudinal, a mid circular and an outer longitudinal layer, but the female human urethra has a smooth muscle bilayer consisting of an inner longitudinal and an outer circular layer.25 Considering this anatomical difference in pig and human urethras the pig lower urinary tract has similar characteristics physiologically and pharmacologically to the human lower urinary tract and is large enough to provide tissue samples from the proximal urethra.6, 12, 26 Thus, in the current study we used isolated strips of female pig urethra. In saturation binding studies the dissociation constant for [3H]QNB in the urethra in the current study (0.26 nM.) was similar to the value that we have previously obtained in the pig bladder dome (0.30 nM.) but receptor density in the urethra was significantly lower than that in the bladder dome (44.1 versus 89.3 fmol./mg. protein, p ⬍0.05).6 This result was consistent with that of Mutoh et al,20 in that the
FIG. 2. Effect of 4-DAMP on concentration-response curves to carbachol in normal circular muscle and in circular muscle after cAMP elevation. Schild plots show antagonism of responses to carbachol by 4-DAMP.
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ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES IN PROXIMAL URETHRA
FIG. 3. Effect of methoctramine on concentration-response curves to carbachol in normal circular muscle and in tissues after cAMP elevation. Schild plot shows antagonism of responses to carbachol by methoctramine.
Affinity values of antagonists in pig proximal urethra in vitro Tissues/Antagonist (concentration)
Mean pA2 ⫾ SEM
No. Subjects
Mean Schild Slope ⫾ SEM
Circular Normal: 4-DAMP (3–30 nM.) Methoctramine (300 nM.–3 M.) Pirenzepine (1–10 M.) cAMP elevation: 4-DAMP (3–30 nM.) Methoctramine (300 nM.–3 M.)
18 15 14
9.30 ⫾ 0.12* 6.90 ⫾ 0.14 6.73 ⫾ 0.08*
0.76 ⫾ 0.10† 1.03 ⫾ 0.02 0.77 ⫾ 0.04†
12 12
8.73 ⫾ 0.08‡ 7.32 ⫾ 0.09*, §
1.01 ⫾ 0.06 0.68 ⫾ 0.05†
8.98 ⫾ 0.08 6.22 ⫾ 0.11 6.88 ⫾ 0.09
0.82 ⫾ 0.3 1.29 ⫾ 0.03 1.02 ⫾ 0.39
8.78 ⫾ 0.09* 6.92 ⫾ 0.09*, 㛳
0.78 ⫾ 0.19 0.79 ⫾ 0.20
Longitudinal Normal: 4-DAMP (1–30 nM.) 20 12 Methoctramine (1–10 M.) 17 Pirenzepine (1–10 M.) cAMP elevation: 4-DAMP (3–100 nM.) 25 12 Methoctramine (300 nM.–3 M.) * Apparent pKB values were calculated because of noncompetitive antagonism. † Significantly less than unity (p ⬍0.05). ‡ Significantly lower than normal tissues (p ⫽ 0.0015). § Significantly higher than normal tissues (p ⫽ 0.0193). 㛳 Significantly higher than normal tissues (p ⬍0.0001).
density of muscarinic receptors was greatest in the rabbit bladder dome and lowest in the rabbit urethra. Interestingly receptor densities of the pig urethra in our study were similar to those identified after selective M3 inactivation in the pig bladder dome (61.1 fmol./mg. protein).6 In competition binding studies displacement of [3H]QNB by 4-DAMP and methoctramine in the urethra indicated the sole presence of M2 receptors or at least a small population of M3 receptors that were masked by the large population of M2 receptors. Mutoh et al reported that binding data in rabbit bladder indicated the predominance of the M2 receptor subtype in the whole bladder dome, bladder base and urethra.20 Although our previous studies in the dome and base of the pig bladder have shown mixed populations of M2 and M3 receptors with a ratio of 3:1,6, 12 displacement experiments in the current study in the pig urethra were comparable to those of
Mutoh et al in the rabbit urethra.20 In our functional studies in vitro the contraction responses to carbachol were greater in the longitudinal muscle of the urethra than in the circular muscle. This result was comparable to that in previous reports.2 It has been reported that the longitudinal smooth muscle layer of the female pig urethra is continuous with the longitudinal layer of the detrusor, implying a possible role in bladder neck opening and urethral shortening at the onset of voiding.25, 27 The circular layer of the urethra is not continuous with the detrusor. It may be important for maintaining urethral closure and may be more sensitive to noradrenaline than to acetylcholine. Thus, the longitudinal muscle of the urethra may show greater responses to carbachol than the circular muscle of the urethra. The circular muscles of the urethra have been reported to
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ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES IN PROXIMAL URETHRA
FIG. 4. Effect of 4-DAMP on concentration-response curves to carbachol in normal longitudinal muscle and in longitudinal muscle after cAMP elevation. Schild plot shows antagonism of responses to carbachol by 4-DAMP.
FIG. 5. Effect of methoctramine on concentration-response curves to carbachol in normal longitudinal muscle and in tissues after cAMP elevation. Schild plot shows antagonism of responses to carbachol by methoctramine.
show a minimum or no response to muscarinic stimulation in vitro.19 Mutoh et al reported that in the rabbit maximum contractions to carbachol are greatest in the bladder dome and bladder base responses are 64% of that of the bladder dome but only 2% to 3% in the urethra (transverse strips).20 Nagahama et al also reported that in transverse strips of male rabbit urethra carbachol produced a concentration dependent contraction of the proximal but not of the distal
portion.21 They speculated that the reason for these differences would be differences in embryological origin (the proximal urethra is from the mesoderm and endoderm, whereas the distal urethra is from the endoderm and ectoderm). The results of the current study in circular muscle of the female pig urethra appear comparable to these in previous studies because only the proximal third of the circular urethral muscles responded to carbachol. The smooth muscle of the ure-
ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES IN PROXIMAL URETHRA
thra is responsible for closing and opening the bladder outlet. Contraction of the circular urethral muscle during muscarinic stimulation may increase bladder outlet resistance during voiding. However, this effect may be negligible considering the small contraction responses to muscarinic agonists in vitro.2, 19 After cAMP elevation maximum responses to carbachol were increased in circular muscles to almost those of longitudinal muscles. However, in cAMP elevated longitudinal muscle the maximum contraction did not change significantly compared to that in normal longitudinal muscle (p ⫽ 0.4119). It has been reported that muscarinic M3 receptor stimulates phosphoinositide hydrolysis, causing the release of intracellular calcium, and it is responsible for the direct contractile responses, while M2 receptors couple to the pertussis toxin sensitive guanine nucleotide regulatory protein Gi and inhibit adenylyl cyclase activation.3 Thus, the greater contractile responses to carbachol in cAMP elevated circular muscle may suggest that the involvement of M2 muscarinic receptors is greater in the circular than in the longitudinal muscles. Antagonist affinities in normal circular muscle of the urethra may suggest the involvement of M3 and M2 receptors. In these tissues 4-DAMP had high affinity but the Schild slope was less than unity, suggesting involvement of more than 1 receptor subtype. The mean affinity value for methoctramine in these tissues (pA2 6.9) was relatively higher than those at M3 receptors in the pig bladder dome (6.05).6 Although Mutoh20 and Nagahama21 et al reported the involvement of M1, M2 and M3 subtypes in contraction of circular muscle of the female and male rabbit urethra, we were unable to demonstrate the involvement of M1 receptors in the circular muscle of the pig urethra. The affinity of pirenzepine (apparent pKB 6.73) in our study was significantly lower than that reported by these other groups for rabbit urethra (7.7 and 7.5, respectively), suggesting little involvement of M1 receptors in mediating urethral contraction in the pig. After cAMP elevation in circular muscles affinity values for 4-DAMP (pA2 8.7) were significantly lower (p ⫽ 0.0015), and those for methoctramine (apparent pKB 7.3) were significantly higher (p ⫽ 0.0193) than in normal tissues. The Schild slopes for 4-DAMP were not different from unity but those for methoctramine (0.68) were significantly less than unity (p ⬍0.0001). The changes in maximal tension development and in antagonist affinity values in circular muscles may suggest the involvement of M2 receptors after cAMP elevation. In contrast, in the longitudinal muscles M3 receptors appeared to mediate the contractile response to carbachol in normal longitudinal muscles because the affinity values for 4-DAMP, methoctramine and pirenzepine correlated closely with the affinities at human recombinant M3 receptors.3, 23, 24 After cAMP elevation affinity values for 4-DAMP did not change compared with normal tissues, although those for methoctramine were significantly increased (p ⬍0.0001). These changes of muscarinic antagonist affinity after cAMP elevation suggest that M2 and M3 receptors appear to mediate longitudinal muscle contraction after cAMP elevation. Thus, in our current study contraction of the longitudinal muscles of the urethra, like detrusor muscle, appeared to be mediated mainly by M3 receptors. These results seem to be reasonable considering that the longitudinal urethral muscle is continuous with detrusor muscle.
CONCLUSIONS
Pig urethra appears to have a population of predominantly M2 receptors. Contraction of the normal urethra appears to be mediated via M2 and M3 receptors in circular muscle but only by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors ap-
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peared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle. [3H]QNB was obtained from NEN Life Science Products, Inc., Boston, Massachusetts. Atropine, carbachol, isoprenaline, 4-DAMP, methoctramine and pirenzepine were obtained from Sigma Chemical Co., St. Louis, Missouri. REFERENCES
1. Andersson, K. E.: New roles for muscarinic receptors in the pathophysiology of lower urinary tract symptoms. BJU Int, suppl., 86: 36, 2000 2. Mattiasson, A., Andersson, K.-E., Andersson, P.-O., Larsson, B., Sjo¨ gren, C. and Uvelius, B.: Nerve-mediated functions in the circular and longitudinal muscle layers of the proximal female rabbit urethra. J Urol, 143: 155, 1990 3. Hegde, S. S., Choppin, A., Bonhaus, D., Briaud, S., Loeb, M., Moy, T. M. et al: Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo. Br J Pharmacol, 120: 1409, 1997 4. Yamaguchi, O., Shishido, K., Tamura, K., Ogawa, T., Fujimura, T. and Ohtsuka, M.: Evaluation of mRNAs encoding muscarinic receptor subtypes in human detrusor muscle. J Urol, 156: 1208, 1996 5. Braverman, A. S., Kohn, I. J., Luthin, G. R. and Ruggieri, M. R.: Prejunctional M1 facilitatory and M2 inhibitory muscarinic receptors mediate rat urinary bladder contractility. Am J Physiol, 274: R517, 1998 6. Yamanishi, T., Chapple, C. R., Yasuda, K. and Chess-Williams, R.: The role of M(2)-muscarinic receptors in mediating contraction of the pig urinary bladder in vitro. Br J Pharmacol, 131: 1482, 2000 7. Wang, P., Luthin, G. R. and Ruggieri, M. R.: Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J Pharmacol Exp Ther, 273: 959, 1995 8. D’Agostino, G., Bolognesi, M. L., Lucchelli, A., Vicini, D., Balestra, B., Spelta, V. et al: Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype. Br J Pharmacol, 129: 493, 2000 9. Sellers, D. J., Yamanishi, T., Chapple, C. R., Couldwell, C., Yasuda, K. and Chess-Williams, R.: M3 muscarinic receptors but not M2 mediate contraction of the porcine detrusor muscle in vitro. J Auton Pharmacol, 20: 171, 2000 10. Matsui, M., Motomura, D., Karasawa, H., Fujikawa, T., Jiang, J., Komiya, Y. et al: Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci USA, 97: 9579, 2000 11. Braverman, A. S. and Ruggieri, M. R.: Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for M2 muscarinic receptor. J Recept Signal Transduct Res, 19: 819, 1999 12. Yamanishi, T., Chapple, C. R., Yasuda, K. and Chess-Williams, R.: The role of M2 muscarinic receptor subtypes in mediating contraction of the pig bladder base after cyclic adenosine monophosphate elevation and/or selective M3 inactivation. J Urol, 167: 397, 2002 13. Stengel, P. W., Gomeza, J., Wess, J. and Cohen, M. L.: M2 and M4 receptor knockout mice: muscarinic receptor function in cardiac and smooth muscle in vitro. J Pharmacol Exp Ther, 292: 877, 2000 14. Nakamura, T., Yagaguchi, O. and Kimura, J.: Muscarinic M2 receptors regulate Ca2⫹-activated K channels in bladder smooth muscle cells. Neurourol Urodyn, 17: 400, 1998 15. Hegde, S. S. and Eglen, R. M.: Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder. Life Sci, 64: 419, 1999 16. Braverman, A. S., Luthin, G. R. and Ruggieri, M. R.: M2 muscarinic receptor contributes to contraction of the denervated rat urinary bladder. Am J Physiol, 275: R1654, 1998 17. Braverman, A. S., Legos, J., Young, W., Luthin, G. and Ruggieri, M.: M2 receptors in genito-urinary smooth muscle pathology. Life Sci, 64: 429, 1999 18. Braverman, A. S., Ruggieri, M. R. and Pontari, M. A.: The M2 muscarinic receptor subtype mediates cholinergic bladder contractions in patients with neurogenic bladder dysfunction.
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Vol. 168, 308 –314, July 2002 Printed in U.S.A.
THE ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES MEDIATING CONTRACTION OF THE CIRCULAR AND LONGITUDINAL SMOOTH MUSCLE OF THE PIG PROXIMAL URETHRA TOMONORI YAMANISHI, CHRISTOPHER R. CHAPPLE, KOSAKU YASUDA, KEN-ICHIRO YOSHIDA AND RUSSELL CHESS-WILLIAMS From the Department of Biomedical Science, University of Sheffield and Department of Urology, Royal Hallamshire Hospital, Sheffield, United Kingdom, and Department of Urology, Dokkyo University, Tochigi, Japan
ABSTRACT
Purpose: We investigated the characterization of muscarinic receptor subtypes in the female pig urethra. Materials and Methods: The affinities of muscarinic antagonists against carbachol responses were calculated in normal and cyclic adenosine monophosphate (cAMP) elevated tissues (contraction with KCl and relaxation with isoprenaline) using longitudinal and transverse strips of urethra. Results: In displacement experiments with 1-quinuclidinyl [phenyl-4-3H] benzilate inhibitory constant (pKi) values of 4-diphenylacetoxyl-N-methyl-piperidine methiodide (DAMP) M3 selective antagonist) and methoctramine (M2 antagonist) indicated the presence of the M2 receptor. In functional studies contraction responses to carbachol were greater in longitudinal than in circular muscle. After cAMP elevation the contraction response increased in circular muscles to the level close to that of longitudinal muscles but did not change significantly in cAMP elevated longitudinal muscle. On normal circular tissues 4-DAMP had high mean affinity (mean apparent pKB value 9.3) but with a Schild slope of less than unity (0.76). Methoctramine competitively antagonized carbachol responses (mean affinity [pA2] value 6.9). On normal longitudinal tissues 4-DAMP and methoctramine competitively antagonized carbachol responses (mean pA2 9.0 and 6.2, respectively). After cAMP elevation in circular tissues mean pA2 values for 4-DAMP (8.7) were significantly lower (p ⫽ 0.0015), and those for methoctramine (7.3) were significantly higher (p ⫽ 0.0193) than in normal tissues. In longitudinal tissues the mean pA2 value for methoctramine (6.9) was significantly greater than in normal tissues (p ⬍0.0001) but the value for 4-DAMP (8.8) did not alter. Conclusions: Pig urethra appears to have predominantly M2 muscarinic receptors. Contraction of the normal urethra appears to be predominantly mediated by M2 and M3 receptors in circular muscle but by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors appeared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle. KEY WORDS: urethra; muscle, smooth; swine; receptors, muscarinic; carbachol
It has been reported that urethral smooth muscle receives a rich cholinergic innervation, of which the role is largely unknown.1, 2 The bladder shows a heterogeneous populations of muscarinic receptors.3 The presence of messenger RNA encoding only M2 and M3 subtypes has been detected in human bladder4 but M1, M2, M3 and M4 subtypes in rat bladder have been found.5 Predominance of the M2 muscarinic receptor subtype with a minor population of M3 receptors has been reported for bladder smooth muscle in several species with an M2-to-M3 ratio of 3:1 in pig and human bladders.6, 7 However, pharmacological characterization of muscarinic receptors mediating contraction of detrusor muscle in pig and human bladder suggest the singular involvement of M3 receptors.8, 9 The predominance of the M3 receptor subtype for mediating bladder contraction has also been demonstrated in experiments in mutant mice lacking the receptor gene for the M3 subtype.10 Although no direct contractile response to M2 receptor activation can be demonstrated, an indirect influence on contraction via the inhibition of cyclic adenosine monophosphate (cAMP) mediated smooth muscle relaxation by Accepted for publication January 11, 2002.
-adrenoceptors has been reported. The involvement of M2 receptors in contraction of the bladder has been observed in tissues after the inactivation of M3 receptors and the elevation of cAMP levels in vitro.3, 6, 11, 12 Recently the involvement of M2 (and not M4) receptors for mediating contraction of the bladder has also been demonstrated in experiments in M2 and M4 muscarinic receptor subtype knockout mice.13 M2 receptors have also been suggested to be able to activate smooth muscle through an action on ion channels, the opening of nonspecific cation channels resulting in depolarization and calcium influx.14, 15 It has also been reported that M2 receptors participate in the contractile response under pathological conditions in rat and human detrusor smooth muscle.3, 11, 16 –18 Although others have investigated responses to muscarinic receptor stimulation in the urethra,19 –21 the physiological significance of muscarinic receptors in the urethra remains unclear. It has been reported that M1, M2 and M3 receptors mediate contraction to carbachol stimulation in the circular muscle of the rabbit urethra.20, 21 However, to our knowledge there have been no reports characterizing muscarinic receptor subtypes in the longitudinal muscles of the urethra or the
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functional role of M2 subtypes in circular and longitudinal urethral tissues after cAMP elevation. We investigated the characterization of muscarinic receptor subtypes in circular and longitudinal isolated strips of the female pig in normal tissue and after cAMP elevation. MATERIALS AND METHODS
Female pig bladders were obtained from the abattoir and immediately placed in cold (4C) Krebs solution (118.4 mM. NaCl, 4.7 mM. KCl, 1.9 mM. CaCl2, 24.9 mM. NaHCO3, 1.15 mM. MgSO4, 1.15 mM. KH2PO4 and 11.7 mM. glucose). The urethra was opened longitudinally at the dorsal portion and the proximal urethra was separated from the bladder at the level of the bladder neck. Longitudinal and transverse strips of the proximal urethra (3 ⫻ 10 mm.) were cut, and the mucosa and serosa were removed. In functional studies in vitro tissues were mounted in 30 ml. organ baths containing Krebs solution maintained at 37C and continuously gassed with 95% O2 and 5% CO2. Tissues for binding studies were freshly used or frozen in liquid nitrogen and stored at ⫺80C until used. Radioligand binding studies. The methods for receptor binding studies have been reported previously.6 Briefly, tissues were homogenized in 50 mM. tris-HCl buffer (pH 7.4) and filtered through muslin. Homogenates were centrifuged at 45,000 ⫻ gravity for 10 minutes at 4C. The pellets were re-suspended in 5 ml. tris-HCl buffer. Homogenates were again centrifuged and the pellets were re-suspended in 5 ml. tris-HCl buffer. Saturation experiments were conducted using 7 concentrations (0.0625 to 4 nM.) 1-quinuclidinyl[phenyl-4-3H]benzilate ([3H]QNB). Binding was determined in a final volume of 0.25 ml. and nonspecific binding was determined with 10 M. atropine. The assay tubes were incubated at 37C for 60 minutes before filtration through an M30 cell harvester (Brandel, Gaithersburg, Maryland). Radioactivity on the filters was determined by liquid scintillation spectrometry. Displacement experiments using 0.3 nM. [3H]QNB with the unlabeled M3 selective antagonist 4-diphenylacetoxyl-Nmethyl-piperidine methiodide (4-DAMP) (0.1 nM. to 1 M.) and the M2 selective antagonist methoctramine (25 nM. to 0.25 mM.) was used to determine the presence of M2 and M3 receptors in pig urethra membranes. In competition binding studies the inhibitory constant (pKi) values were calculated and analyzed for 1 or 2-site binding using Prism software (GraphPad, San Diego, California). A 2-site fit was accepted only if it is resulted in a significant improvement of the fit as judged by the F test.6, 12, 22 In vitro functional studies. Isolated tissues were subjected to a resting tension of 1 gm. and allowed to equilibrate for 60 minutes before the first cumulative concentration-response curves to carbachol were obtained. Tissues were washed and equilibrated for 30 minutes with Krebs solution containing the appropriate concentration of antagonist or vehicle (time
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control). The second concentration-response curves to carbachol were obtained in the presence of 4-DAMP, methoctramine or pirenzepine (M1 selective antagonist), or vehicle. In control experiments the 2 concentration-response curves to carbachol were reproducible but the third concentrationresponse curve varied, probably because of tachyphylaxis in some experiments. Thus, 2 concentration-response curves per tissue were used to calculate affinity values for each muscarinic antagonist. The affinity of these antagonists against carbachol induced contractions of tissue strips were calculated in normal and cAMP elevated tissues. cAMP elevation was performed by precontraction with 50 mM. KCl and relaxation with isoprenaline (30 M.).6 In functional studies, responses are expressed as the mean percent plus or minus standard error of mean (SEM) of the response to 50 mM. KCl. Agonist potencies are expressed as the mean pEC50 plus or minus SEM (-logarithm of the molar concentration of agonist resulting in EC50). Antagonist affinities (apparent affinity [KB] values) were calculated from the equation, KB-antagonist concentration in moles/(dose ratio-1). Schild analysis was performed with the intercept considered the pA2 value. Radioligand binding data and in vitro functional data were analyzed using Prism software. The nonpaired Student t test was used for statistical analysis with p ⬍0.05 considered significant. [3H]QNB (specific activity 42 Ci./mmol.) was stored at ⫺20C. RESULTS
Radioligand binding studies. In saturation binding studies in 4 animals binding of [3H]QNB was saturable and displaceable by atropine. The mean dissociation constant for [3H]QNB was 0.26 ⫾ 0.07 nM. and mean receptor density was 44.1 ⫾ 13.2 fmol./mg. protein. In competition binding studies displacement of [3H]QNB by 4-DAMP in 8 animals and methoctramine in 8 best fitted a 1-site model with mean Hill’s slopes close to unity (0.97 ⫾ 0.13 and 1.05 ⫾ 0.09, respectively). Mean inhibitory constant (pKi) values for 4-DAMP and methoctramine were 7.67 ⫾ 0.11 and 7.56 ⫾ 0.18, respectively, which were comparable with M2 receptors by reported antagonist affinity estimates derived from radioligand binding studies in human recombinant receptors.3, 23, 24 In vitro functional studies. Circular strips from the proximal third of the urethra responded to carbachol but circular muscle from the mid to distal part of the urethra had little or no response to carbachol. Carbachol also induced contractions of longitudinal smooth muscle of the proximal to mid part of the urethra in a concentration dependent manner. In normal strips mean pEC50 values were significantly greater in circular muscles than in longitudinal muscles in 14 animals each (6.78 ⫾ 0.004 versus 6.13 ⫾ 0.06, p ⬍0.0001). The maximum contraction to carbachol relative to that to 50 mM. KCl was significantly larger in longitudinal muscle than in circular muscle (mean 99.8% ⫾ 9.5% versus 55.3% ⫾ 5.8%, p ⫽ 0.0005, fig. 1). After cAMP elevation in circular (16
FIG. 1. Comparison of concentration-response curves to carbachol in normal circular (open circles) and longitudinal (filled circles) muscles (A) and in muscles after cAMP elevation (B).
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animals) and longitudinal (29 animals) urethral muscle mean pEC50 values for carbachol were significantly reduced (5.78 ⫾ 0.03, p ⬍0.0001 and 5.46 ⫾ 0.04, p ⬍0.0001, respectively). The mean maximum contractions to carbachol relative to those to 50 mM. KCl were increased (marginally significantly because of large variations p ⫽ 0.0956) in circular muscle (84.7% ⫾ 15.2%) to a level not significantly different from those in longitudinal muscle (119.4% ⫾ 15.7%). In cAMP elevated longitudinal muscle the maximum contraction did not change significantly compared to that in normal longitudinal muscle (p ⫽ 0.4119, fig. 1). On normal circular muscle strips in vitro 4-DAMP antagonized responses to carbachol with a high affinity (mean apparent pKB 9.30 ⫾ 0.12) without affecting maximum responses, although the mean slope of the Schild plot was significantly less than unity (0.76 ⫾ 0.10, p ⬍0.0001, fig. 2). Methoctramine demonstrated competitive antagonism (mean pA2 6.90 ⫾ 0.14) without affecting maximum responses and with a Schild slope close to unity (fig. 3). Pirenzepine antagonized contractile responses to carbachol with a mean apparent pKB value of 6.73 ⫾ 0.08 without affecting maximum responses, although the mean Schild slope was significantly less than unity (0.77 ⫾ 0.04, p ⬍0.01, see table). In longitudinal muscle 4-DAMP and methoctramine competitively antagonized responses to carbachol without affecting maximum responses and with Schild plots with slopes of unity (see table, and figs. 4 and 5). Mean pA2 values of 8.98 ⫾ 0.08 and 6.22 ⫾ 0.11 were obtained for 4-DAMP and methoctramine, respectively. Pirenzepine antagonized contractile responses to carbachol with low affinity (mean pA2 6.88 ⫾ 0.09) without affecting maximum response and with a mean Schild slope not significantly different from unity (1.02 ⫾ 0.39) (see table). After cAMP elevation in circular muscle of the urethra 4-DAMP produced surmountable competitive antagonism against response to carbachol with a pA2 value of 8.7 ⫾ 0.08, which was significantly lower than those in the normal tissues but with a Schild slope close to unity (p ⫽ 0.0015, see
table and fig. 2). Methoctramine also antagonized responses without affecting maximum responses, yielding a mean apparent pKB value of 7.32 ⫾ 0.09, significantly greater than that for normal tissues (p ⫽ 0.0193). However, the Schild slope was significantly less than unity (0.68 ⫾ 0.05, p ⬍0.0001, fig. 3). In longitudinal tissues after cAMP elevation 4-DAMP and methoctramine antagonized responses without affecting maximum responses and with mean apparent pKB values of 8.78 ⫾ 0.09 and 6.92 ⫾ 0.09, respectively. In cAMP elevated longitudinal tissues apparent pKB values for 4-DAMP were not significantly different from normal tissues but those for methoctramine were significantly increased compared with normal tissues (p ⬍0.0001). The Schild slopes for 4-DAMP and methoctramine were low but not significantly less than unity (see table, and figs. 4 and 5). DISCUSSION
Using computer assisted 3-dimensional reconstruction Dass et al noted that the female pig urethra is arranged in 3 smooth muscle layers, namely an inner longitudinal, a mid circular and an outer longitudinal layer, but the female human urethra has a smooth muscle bilayer consisting of an inner longitudinal and an outer circular layer.25 Considering this anatomical difference in pig and human urethras the pig lower urinary tract has similar characteristics physiologically and pharmacologically to the human lower urinary tract and is large enough to provide tissue samples from the proximal urethra.6, 12, 26 Thus, in the current study we used isolated strips of female pig urethra. In saturation binding studies the dissociation constant for [3H]QNB in the urethra in the current study (0.26 nM.) was similar to the value that we have previously obtained in the pig bladder dome (0.30 nM.) but receptor density in the urethra was significantly lower than that in the bladder dome (44.1 versus 89.3 fmol./mg. protein, p ⬍0.05).6 This result was consistent with that of Mutoh et al,20 in that the
FIG. 2. Effect of 4-DAMP on concentration-response curves to carbachol in normal circular muscle and in circular muscle after cAMP elevation. Schild plots show antagonism of responses to carbachol by 4-DAMP.
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FIG. 3. Effect of methoctramine on concentration-response curves to carbachol in normal circular muscle and in tissues after cAMP elevation. Schild plot shows antagonism of responses to carbachol by methoctramine.
Affinity values of antagonists in pig proximal urethra in vitro Tissues/Antagonist (concentration)
Mean pA2 ⫾ SEM
No. Subjects
Mean Schild Slope ⫾ SEM
Circular Normal: 4-DAMP (3–30 nM.) Methoctramine (300 nM.–3 M.) Pirenzepine (1–10 M.) cAMP elevation: 4-DAMP (3–30 nM.) Methoctramine (300 nM.–3 M.)
18 15 14
9.30 ⫾ 0.12* 6.90 ⫾ 0.14 6.73 ⫾ 0.08*
0.76 ⫾ 0.10† 1.03 ⫾ 0.02 0.77 ⫾ 0.04†
12 12
8.73 ⫾ 0.08‡ 7.32 ⫾ 0.09*, §
1.01 ⫾ 0.06 0.68 ⫾ 0.05†
8.98 ⫾ 0.08 6.22 ⫾ 0.11 6.88 ⫾ 0.09
0.82 ⫾ 0.3 1.29 ⫾ 0.03 1.02 ⫾ 0.39
8.78 ⫾ 0.09* 6.92 ⫾ 0.09*, 㛳
0.78 ⫾ 0.19 0.79 ⫾ 0.20
Longitudinal Normal: 4-DAMP (1–30 nM.) 20 12 Methoctramine (1–10 M.) 17 Pirenzepine (1–10 M.) cAMP elevation: 4-DAMP (3–100 nM.) 25 12 Methoctramine (300 nM.–3 M.) * Apparent pKB values were calculated because of noncompetitive antagonism. † Significantly less than unity (p ⬍0.05). ‡ Significantly lower than normal tissues (p ⫽ 0.0015). § Significantly higher than normal tissues (p ⫽ 0.0193). 㛳 Significantly higher than normal tissues (p ⬍0.0001).
density of muscarinic receptors was greatest in the rabbit bladder dome and lowest in the rabbit urethra. Interestingly receptor densities of the pig urethra in our study were similar to those identified after selective M3 inactivation in the pig bladder dome (61.1 fmol./mg. protein).6 In competition binding studies displacement of [3H]QNB by 4-DAMP and methoctramine in the urethra indicated the sole presence of M2 receptors or at least a small population of M3 receptors that were masked by the large population of M2 receptors. Mutoh et al reported that binding data in rabbit bladder indicated the predominance of the M2 receptor subtype in the whole bladder dome, bladder base and urethra.20 Although our previous studies in the dome and base of the pig bladder have shown mixed populations of M2 and M3 receptors with a ratio of 3:1,6, 12 displacement experiments in the current study in the pig urethra were comparable to those of
Mutoh et al in the rabbit urethra.20 In our functional studies in vitro the contraction responses to carbachol were greater in the longitudinal muscle of the urethra than in the circular muscle. This result was comparable to that in previous reports.2 It has been reported that the longitudinal smooth muscle layer of the female pig urethra is continuous with the longitudinal layer of the detrusor, implying a possible role in bladder neck opening and urethral shortening at the onset of voiding.25, 27 The circular layer of the urethra is not continuous with the detrusor. It may be important for maintaining urethral closure and may be more sensitive to noradrenaline than to acetylcholine. Thus, the longitudinal muscle of the urethra may show greater responses to carbachol than the circular muscle of the urethra. The circular muscles of the urethra have been reported to
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FIG. 4. Effect of 4-DAMP on concentration-response curves to carbachol in normal longitudinal muscle and in longitudinal muscle after cAMP elevation. Schild plot shows antagonism of responses to carbachol by 4-DAMP.
FIG. 5. Effect of methoctramine on concentration-response curves to carbachol in normal longitudinal muscle and in tissues after cAMP elevation. Schild plot shows antagonism of responses to carbachol by methoctramine.
show a minimum or no response to muscarinic stimulation in vitro.19 Mutoh et al reported that in the rabbit maximum contractions to carbachol are greatest in the bladder dome and bladder base responses are 64% of that of the bladder dome but only 2% to 3% in the urethra (transverse strips).20 Nagahama et al also reported that in transverse strips of male rabbit urethra carbachol produced a concentration dependent contraction of the proximal but not of the distal
portion.21 They speculated that the reason for these differences would be differences in embryological origin (the proximal urethra is from the mesoderm and endoderm, whereas the distal urethra is from the endoderm and ectoderm). The results of the current study in circular muscle of the female pig urethra appear comparable to these in previous studies because only the proximal third of the circular urethral muscles responded to carbachol. The smooth muscle of the ure-
ROLE OF M2 MUSCARINIC RECEPTOR SUBTYPES IN PROXIMAL URETHRA
thra is responsible for closing and opening the bladder outlet. Contraction of the circular urethral muscle during muscarinic stimulation may increase bladder outlet resistance during voiding. However, this effect may be negligible considering the small contraction responses to muscarinic agonists in vitro.2, 19 After cAMP elevation maximum responses to carbachol were increased in circular muscles to almost those of longitudinal muscles. However, in cAMP elevated longitudinal muscle the maximum contraction did not change significantly compared to that in normal longitudinal muscle (p ⫽ 0.4119). It has been reported that muscarinic M3 receptor stimulates phosphoinositide hydrolysis, causing the release of intracellular calcium, and it is responsible for the direct contractile responses, while M2 receptors couple to the pertussis toxin sensitive guanine nucleotide regulatory protein Gi and inhibit adenylyl cyclase activation.3 Thus, the greater contractile responses to carbachol in cAMP elevated circular muscle may suggest that the involvement of M2 muscarinic receptors is greater in the circular than in the longitudinal muscles. Antagonist affinities in normal circular muscle of the urethra may suggest the involvement of M3 and M2 receptors. In these tissues 4-DAMP had high affinity but the Schild slope was less than unity, suggesting involvement of more than 1 receptor subtype. The mean affinity value for methoctramine in these tissues (pA2 6.9) was relatively higher than those at M3 receptors in the pig bladder dome (6.05).6 Although Mutoh20 and Nagahama21 et al reported the involvement of M1, M2 and M3 subtypes in contraction of circular muscle of the female and male rabbit urethra, we were unable to demonstrate the involvement of M1 receptors in the circular muscle of the pig urethra. The affinity of pirenzepine (apparent pKB 6.73) in our study was significantly lower than that reported by these other groups for rabbit urethra (7.7 and 7.5, respectively), suggesting little involvement of M1 receptors in mediating urethral contraction in the pig. After cAMP elevation in circular muscles affinity values for 4-DAMP (pA2 8.7) were significantly lower (p ⫽ 0.0015), and those for methoctramine (apparent pKB 7.3) were significantly higher (p ⫽ 0.0193) than in normal tissues. The Schild slopes for 4-DAMP were not different from unity but those for methoctramine (0.68) were significantly less than unity (p ⬍0.0001). The changes in maximal tension development and in antagonist affinity values in circular muscles may suggest the involvement of M2 receptors after cAMP elevation. In contrast, in the longitudinal muscles M3 receptors appeared to mediate the contractile response to carbachol in normal longitudinal muscles because the affinity values for 4-DAMP, methoctramine and pirenzepine correlated closely with the affinities at human recombinant M3 receptors.3, 23, 24 After cAMP elevation affinity values for 4-DAMP did not change compared with normal tissues, although those for methoctramine were significantly increased (p ⬍0.0001). These changes of muscarinic antagonist affinity after cAMP elevation suggest that M2 and M3 receptors appear to mediate longitudinal muscle contraction after cAMP elevation. Thus, in our current study contraction of the longitudinal muscles of the urethra, like detrusor muscle, appeared to be mediated mainly by M3 receptors. These results seem to be reasonable considering that the longitudinal urethral muscle is continuous with detrusor muscle.
CONCLUSIONS
Pig urethra appears to have a population of predominantly M2 receptors. Contraction of the normal urethra appears to be mediated via M2 and M3 receptors in circular muscle but only by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors ap-
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peared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle. [3H]QNB was obtained from NEN Life Science Products, Inc., Boston, Massachusetts. Atropine, carbachol, isoprenaline, 4-DAMP, methoctramine and pirenzepine were obtained from Sigma Chemical Co., St. Louis, Missouri. REFERENCES
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23. Eglen, R. M. and Nahorski, S. R.: The muscarinic M(5) receptor: a silent or emerging subtype? Br J Pharmacol, 130: 13, 2000 24. Watson, N., Daniels, D. V., Ford, A. P., Eglen, R. M. and Hegde, S. S.: Comparative pharmacology of recombinant human M3 and M5 muscarinic receptors expressed in CHO-K1 cells. Br J Pharmacol, 127: 590, 1999 25. Dass, N., McMurray, G., Greenland, J. E. and Brading, A. F.: Morphological aspects of the female pig bladder neck and urethra: quantitative analysis using computer assisted 3-dimensional reconstructions. J Urol, 165: 1294, 2001 26. Mills, I. W., Noble, J. G. and Brading, A. F.: Radiotelemetered cystometry in pigs: validation and comparison of natural filling versus diuresis cystometry. J Urol, 164: 1745, 2000 27. Hutch, J. A.: The internal urinary sphincter: a double-loop system. J Urol, 105: 375, 1971