Abstract
The muscarinic receptor subtype-activated signal transduction mechanisms mediating rat urinary bladder contraction are incompletely understood. M3 mediates normal rat bladder contractions; however, the M2 receptor subtype has a more dominant role in contractions of the hypertrophied bladder. Normal bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a single cumulative concentration-response curve to the muscarinic receptor agonist carbachol. The outcome measures were the maximal contraction, the potency of carbachol, and the affinity of the M3 -selective antimuscarinic agent darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) reduces carbachol potency and reduces darifenacin affinity, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximal contraction. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol potency and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine·HCl (HA-1077) reduces the carbachol maximal contraction, carbachol potency, and darifenacin affinity. Inhibition of protein kinase C (PKC) with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl (H7) reduces the carbachol maximum and carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl (H89) reduces carbachol maximum and carbachol potency. Both the M2 and the M3 receptor subtype are involved in normal rat bladder contractions. The M3subtype seems to mediate contraction by activation of PI-PLC, PC-PLC, and PKA, whereas the M2 signal transduction cascade may include activation of rho kinase, PKC, and an additional contractile signal transduction mechanism independent of rho kinase or PKC.
Pharmacological data, based on the actions of subtypeselective antimuscarinic agents, can distinguish four subtypes of muscarinic acetylcholine receptors (M1-M4). Molecular techniques have identified five muscarinic receptor subtypes (M1-M5) arising from five separate genes (Caulfield, 1993). Both M2 and M3 muscarinic receptor subtypes are found in most smooth muscles. The M2 receptor preferentially couples to the inhibition of adenylyl cyclase through the Gi family of proteins, whereas the M3 receptor preferentially couples to IP3 generation and calcium mobilization through the Gq family of proteins (Caulfield, 1993). Pertussis toxin, which ADP ribosylates and therefore inactivates the Gi family of proteins, has no apparent effect on contraction (Sawyer and Ehlert, 1999). Even though the M2 muscarinic receptor density is greater than the M3 receptor density in bladder and other smooth muscles, the affinity of subtypeselective muscarinic receptor antagonist drugs indicates that contraction is mediated by the M3 receptor in most smooth muscles under normal conditions (Caulfield, 1993; Sawyer and Ehlert, 1999). Although not directly involved in mediating smooth muscle contraction, we and others have shown that prejunctional M1 receptors mediate augmentation of neuronal acetylcholine release and that M2 receptors inhibit acetylcholine release (Somogyi and de Groat, 1992; Braverman et al., 1998a).
Subsequent steps in the contractile signal transduction pathway are not known for certain in bladder smooth muscle, but one possibility involves Gq/11 activation of phosphatidylinositol-specific phospholipase C (PI-PLC), which generates IP3 and diacylglycerol. IP3 is thought to act on sarcoplasmic reticulum IP3 receptors inducing release of internal stores of calcium that sequentially activate calmodulin and myosin light chain kinase. Recent studies indicate that this pathway is not involved in mediating contraction because complete suppression of IP3 production with the PI-PLC inhibitor U73122 has no effect on cholinergic contractions of the rat (Schneider et al., 2004b) or human (Schneider et al., 2004a) bladder.
Results from many studies in gastrointestinal (Murthy and Makhlouf, 1997; Murthy et al., 2003a; Zhou et al., 2003), airway (Yang et al., 1996; Sohn et al., 2000), vascular (Horowitz et al. 1996; Woodrum et al. 1999; Rembold et al. 2000; Komalavilas et al., 2001), bladder and uterine smooth muscle (Taggart et al., 1999), and cells transfected with muscarinic receptor subtypes (May et al., 1999; Strassheim et al., 1999; Wang et al., 1999; Rumenapp et al., 2001; Ruiz-Velasco et al., 2002; Murthy et al., 2003b) indicate that many signal transduction enzymes are involved in muscarinic receptormediated contraction, and they are summarized in Fig. 1. Few if any of these previously published studies were designed to determine which muscarinic receptor subtype mediates which of these pathways or the interaction between the pathways activated by the individual receptor subtypes.
Materials and Methods
Materials. The following drugs or chemicals were obtained from the sources indicated: carbachol (Sigma-Aldrich, St. Louis, MO) and ET-18-OCH3, D609, Y-27632, HA-1077, chelerythrine [1,2-dimethoxy-N-methyl(1,3)benzodioxolo(5,6-c)phenanthridinium chloride], H7, and H89 (BIOMOL Research Laboratories, Plymouth Meeting, PA). Darifenacin was a generous gift from Pfizer Central Research (Sandwich, Kent).
Muscle Strips. Urinary bladders were removed from rats euthanized by CO2 asphyxiation. The urinary bladder body (tissue above the ureteral orifices) was dissected free of the serosa and surrounding fat. The bladder was divided in the mid-sagittal plane and cut into longitudinal smooth muscle strips (approximately 3 × 8 mm). The muscle strips were then suspended with 1 g of tension in tissue baths containing 15 ml of modified Tyrode's solution (125 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.8 mM CaCl2, 0.5 mM MgCl2, 23.8 mM NaHCO3, and 5.6 mM glucose) and equilibrated with 95% O2, 5% CO2 at 37°C.
Carbachol Concentration Response. After equilibration to the bath solution for 30 min, a maximal contraction induced by a 3-min exposure to 120 mM potassium was recorded. The strips were ranked based on their contractile response to KCl and sorted so that the average response in each treatment group was equal. The strips were incubated for 30 min in the presence or absence of an enzyme inhibitor and in the presence or absence of 30 nM darifenacin. Because higher doses of darifenacin seemed insurmountable and lower doses did not produce a significant shift in the concentration-response curve, a single dose of 30 nM darifenacin was used. Doseresponse curves were derived from the peak tension developed after cumulative addition of carbachol (10 nM to 300 μM final bath concentration) and normalized to the response to 120 mM KCl. Only one concentration of enzyme inhibitor and/or darifenacin was used for each muscle strip.
The target enzymes and Ki for the enzyme inhibitors are listed in Tables 1 and 2. The three different enzyme inhibitor concentrations used were based on the concentrations reported for isolated, purified enzymes starting at just above the published Ki and increasing at half-log intervals. Dose ratios were determined based on the average responses of antagonist free strips. Because some of the enzyme inhibitors decreased the maximal contraction to less than 50%, darifenacin affinity is based on EC25 values. The EC25 and EC50 values were determined for each strip using a sigmoidal curve fit of the data (Origin; OriginLab Corp., Northampton, MA). The EC25 values determined in the presence of darifenacin were used to estimate the pKb, and the dose ratios were determined using the same concentration of inhibitor with and without darifenacin. The estimated pKb for darifenacin was calculated using the formula pKb = -[log[darifenacin concentration] -log(dose ratio - 1)].
Because there were statistically significant differences in the variance between groups, statistical analysis of multiple group comparisons was performed by nonparametric Kruskal-Wallis analysis of variance followed by a post hoc Mann-Whitney U test for pairwise comparisons (GB-STAT; Dynamic Microsystems, Silver Spring, MD). Because there was no statistical difference between the outcome measures for the different vehicle control groups, these data were pooled. For maximal contraction and carbachol potency, groups with enzyme inhibitors were compared with these pooled, no inhibitor added, vehicle controls.
Results
Inhibition of Phospholipases. Inhibition of PI-PLC with ET-18-OCH3 had no effect on the carbachol-stimulated maximal contraction response. A representative graph of the data for ET-18-OCH3 is shown in Fig. 2, and Table 3 includes this information and data for all of the enzyme inhibitors used. Inhibition of PI-PLC with both 30 μM and 100 μM ET-18-OCH3 decreased carbachol potency. Furthermore, when the strips were exposed to the highest concentration of ET-18-OCH3 (100 μM), the affinity of darifenacin for inhibiting contraction was significantly lower than in strips exposed to vehicle only. Inhibition of PC-PLC with 100 μM D609 reduced the maximal contraction with no effect on carbachol potency or darifenacin affinity. Lower concentrations of D609 had no significant effects. The nonspecific phospholipase C inhibitor neomycin had only minor effects: a statistically significant reduction in the maximal contraction at the lowest concentration used with no effect at higher concentrations.
Inhibition of Protein Kinases. Inhibition of ROCK with 3 and 10 μM Y-27632 reduced carbachol potency with no effect on the maximal contraction. In addition, darifenacin affinity was significantly increased after ROCK inhibition with both 1 and 10 μM Y-27632. Inhibition of ROCK along with PKG and PKA by all concentrations of HA-1077 decreased the carbachol maximal contraction and carbachol potency and at the same time decreased darifenacin affinity. Inhibition of PKC with chelerythrine had no effect on the maximal contraction response or carbachol potency. Inhibition of PKC did significantly increase darifenacin affinity. Inhibition of ROCK, PKA, PKC, and PKG by all concentrations of H7 reduced the maximal carbachol contraction and carbachol potency. The two highest concentrations of H7 (30 and 100 μM) increased darifenacin affinity. Inhibition of PKA, ROCK, and PKG with both 3 and 10 μM H89 reduced the maximal carbachol contraction, whereas all concentrations used reduced carbachol potency. H89 had no effect on darifenacin affinity.
Discussion
Our previous studies demonstrated that the M2 receptor subtype participates in mediating contraction from several experimental pathological conditions that result in hypertrophy (Braverman et al., 1998b, 2000, 2002; Braverman and Ruggieri, 2003). Because the M2 subtype preferentially couples to Gαi, whereas the M3 subtype preferentially couples to Gαq, this led to the hypothesis that the signal transduction pathways activated by the two receptor subtypes are different and may act in parallel to mediate contraction. Darifenacin is an M3-selective muscarinic antagonist with approximately a 30-fold selectivity for M3 over M2 receptors. The affinity of darifenacin for inhibiting carbachol-stimulated contraction is high in normal bladders consistent with the M3 receptor subtype mediating contraction (Braverman and Ruggieri, 2003). A change in affinity of darifenacin to lower values induced by a given enzyme inhibitor suggests involvement of that enzyme in a signal transduction pathway mediated by the M3 receptor. Alternatively, if an enzyme inhibitor increases the affinity of darifenacin, this may suggest the involvement of the inhibited enzyme in a parallel signal transduction pathway mediated by the M2 receptor. Decreasing the maximal agonist-induced contraction or the agonist potency with no change in darifenacin affinity could suggest the involvement of that enzyme in both M2- and M3-mediated pathways. A summary of our findings implicating the different possible signal transduction enzymes in M2- and M3-mediated contractile pathways is shown in Table 4.
The PI-PLC inhibitor ET-18-OCH3 reduces carbachol potency and darifenacin affinity, indicating the possible involvement of PI-PLC in mediating the M3 contractile signal. This suggests that the M2 receptor subtype has a greater role in mediating contraction after PI-PLC inhibition. Because D609 inhibits maximal contraction with no effect on darifenacin affinity, this suggests that PC-PLC is activated by both M2 and M3 receptors. Because the contraction after PC-PLC inhibition is mediated by the M3 receptor subtype, this suggests that PC-PLC is essential for maximal force generation. Similar findings have been reported in the cat lower esophageal sphincter (Biancani et al., 1994). In permeabilized, isolated cat detrusor smooth muscle cells, inhibition of contraction with antibodies to G proteins and PI-PLC isoforms indicates that cholinergic contraction occurs by M3 activation of Gαq/11, PI-PLC-1 and IP3-dependent calcium release (An et al., 2002). Studies using M2 and M3 knockout mice indicate that M2 receptors mediate bladder contraction indirectly by inhibition of adenylyl cyclase (Ehlert et al., 2005); however, findings in isolated rabbit intestine circular smooth muscle cells indicate that M2 receptors can activate contraction directly through Gβγi3-dependent activation of PI-PLC-β3 (Murthy et al., 2003a).
Our data differ from other reports of PI-PLC mediating rat and human bladder contraction (Fleichman et al., 2004; Schneider et al., 2004a,b). These reports concluded that bladder contraction via M3 receptors largely depends on calcium entry through nifedipine-sensitive channels and activation of ROCK, whereas phospholipase D and store-operated calcium channels contribute in a minor way, and phospholipase C or PKC do not seem to be involved. This is likely because of differences in experimental paradigms. In our paradigm, each muscle strip is exposed to one single agonist concentration-response curve (CRC) in separate smooth muscle strips with and without the enzyme inhibitor. The previously published studies performed five repetitive CRCs in the same smooth muscle strip with increasing concentrations of enzyme inhibitor. We found that after five successive carbachol CRCs, the affinity of the M3-selective antagonist para-fluoro hexahydro siladifenadol was shifted to a lower value, consistent with M2 receptors participating in the contraction (data not shown). Others have shown that the M3 receptor subtype is subject to agonist-induced desensitization (Tobin et al., 1992; Willets et al., 2001, 2002; Griffin et al., 2004). Therefore, M3 receptor desensitization may occur during the repeated agonist CRCs, which could explain these differences.
Recently, much attention has been focused on calcium sensitization induced by the small GTP binding protein rho (Taggart et al., 1999; Murthy et al., 2003a; Fleichman et al., 2004; Schneider et al., 2004a). Calcium sensitization refers to the ability of the muscle to generate the same contractile force with lower levels of intracellular calcium and is likely because of inhibition of MLC phosphatase. Smooth muscle contractile force is largely a function of the phosphorylation state of myosin light chain. This phosphorylation state is in a balance between the activity of myosin light chain kinase and myosin light chain phosphatase. Rho kinase has been shown to phosphorylate CPI-17 which dramatically increases the inhibitory activity of CPI-17 on MLC phosphatase activity (Koyama et al., 2000). Y-27632, a specific inhibitor of rho kinase (Uehata et al., 1997), reduces carbachol potency and increases darifenacin affinity. This suggests that the M3 receptor subtype mediates contraction after inhibition of the rho pathway and that the rho pathway is activated by M2 receptor activation. The finding that inhibition of the rho pathway in normal bladders results in decreased carbachol potency with no decrease in the carbachol maximal contraction, suggests that this pathway is active in normal bladders, but maximal contraction is not critically dependent on inhibition of MLC phosphatase.
HA-1077 has approximately a 5-fold higher affinity for inhibition of ROCK than PKA and PKG (Nagumo et al., 2000). When PKA and PKG along with ROCK are inhibited by HA-1077, there is a decrease in carbachol potency, a decrease in the carbachol maximum, and a decrease in darifenacinaffinity. The reduction in carbachol potency is likely the result of ROCK inhibition, because the specific ROCK inhibitor Y-27632 reduces carbachol potency. However, ROCK inhibition alone has no effect on the carbachol maximum, thus inactivation of either PKA or PKG blocks the carbachol maximum. The darifenacin affinity is decreased after inhibition of PKA, PKG, and ROCK by HA-1077, but it is increased after inhibition of only ROCK with Y-27632. This suggests that when ROCK is inhibited, PKA or PKG becomes involved in the pathway activated by the M3 receptor and mediates contraction with only a decrease in carbachol potency. Once all three of these enzymes are inhibited, contraction occurs via an M2-mediated pathway independently of ROCK, PKA, and PKG.
Inhibition of PKC by chelerythrine has no effect on the carbachol maximum or carbachol potency. However, the darifenacin affinity is increased after PKC inhibition, which suggests that PKC is involved in the M2 receptor contractile signal transduction pathway. There are at least 11 different isoforms of PKC (α, βI, βII, γ, δ, ϵ, η, θ, μ, ζ, and λ), and the isoform mediating this M2 contractile pathway in rat bladder is not known. Because PKCα has been reported to be relatively insensitive to inhibition by chelerythrine (Lee et al., 1998; Davies et al., 2000), it is likely that isoforms other than PKCα mediate this M2 receptor-activated contractile signal transduction pathway in rat bladder.
H89 has approximately a 5-fold higher affinity for inhibiting PKA than ROCK and approximately a 10-fold higher affinity for inhibiting PKA than PKG (Leemhuis et al., 2002). H89 inhibits the carbachol maximum and carbachol potency with no effect on darifenacin affinity. Because inhibition of ROCK alone only results in a decrease in carbachol potency, the decrease in carbachol maximum is likely the result of inhibition of PKA after H89. This suggests that PKA may be involved in mediating the M3 contractile signal.
H7 has approximately a 6-fold higher affinity for inhibition of ROCK than PKA and approximately a 10-fold higher affinity for inhibiting ROCK than PKC and PKG (Uehata et al., 1997). H7 inhibits the maximum contraction while increasing darifenacin affinity, suggesting that after inhibition of these enzymes, the M3 receptor mediates contraction, but the remaining contractile signal is insufficient to stimulate maximum force. These results as well as the results with HA-1077 suggest that PKA is involved in mediating the M3 contractile signal. Because the darifenacin affinity is increased after H7, the M3 receptor predominately mediates contraction in the presence of H7. However, when ROCK, PKA, and PKG are inhibited with HA-1077, the residual contraction is mediated by the M2 receptor. This difference is likely because HA-1077 does not inhibit PKC, whereas H7 does inhibit PKC. This result, as with the chelerythrine result described above, suggests that PKC is involved in mediating the M2 contractile pathway.
Our results suggest that in normal bladders, where the M3 receptor subtype predominantly mediates contraction, PI-PLC, PC-PLC, ROCK, PKC, PKA, and/or PKG are all involved in transducing the contractile signal. The M2 receptor may activate ROCK in the normal rat bladder because darifenacin affinity is increased after ROCK inhibition. This demonstrates that although the M3 receptor seems to predominate in mediating contraction, the M2 receptor does participate in the contractile signal. The contractile pathways activated by muscarinic receptors in the rat urinary bladder are complex, with redundant signaling pathways that become active when other signaling systems are inhibited. This redundant mechanism includes a more dominant role for the M2 receptor after M3 receptor desensitization, which probably occurs after multiple exposures to agonist.
Acknowledgments
We acknowledge the expert technical assistance provided by Neil Lamarre, Kimberly Buck, and Bernadette Simpkiss.
Footnotes
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This study was supported by Public Health Service Grant R01 DK43333 (to M.R.R.).
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doi:10.1124/jpet.105.097303.
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ABBREVIATIONS: IP3, inositol 1,4,5-trisphosphate; PI-PLC, phosphoinositide specific phospholipase C; U73122, 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; ET-18-OCH3, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine; D609, O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt; Y-27632, (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride; HA-1077, 1-(5-isoquinolinesulfonyl)-homopiperazine·HCl; H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl; ROCK, rho kinase; PKG, protein kinase G; PKA, protein kinase A; PKC, protein kinase C; PC-PLC, phosphatidyl choline specific phospholipase C; CRC, concentration-response curve; CPI-17, protein kinase C-potentiated phosphatase inhibitor of 17 kDa; MLC, myosin light chain.
- Received October 17, 2005.
- Accepted October 20, 2005.
- The American Society for Pharmacology and Experimental Therapeutics