QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.097303v1 316/2/869    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Braverman, A. S. Articles by Ruggieri, M. R. Search for Related Content PubMed PubMed Citation Articles by Braverman, A. S. Articles by Ruggieri, M. R., Sr Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CARBACHOL CHLORIDE

GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

M₂ and M₃ Muscarinic Receptor Activation of Urinary Bladder Contractile Signal Transduction. I. Normal Rat Bladder

Departments of Urology (A.S.B., A.S.T., M.R.R.) and Pharmacology (M.R.R.), Temple University School of Medicine, Philadelphia, Pennsylvania

Received October 17, 2005; accepted October 20, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
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.

Subsequent steps in the contractile signal transduction pathway are not known for certain in bladder smooth muscle, but one possibility involves G_q/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 [GenBank] 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.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Diagram of potential muscarinic receptor stimulated signal transduction mechanisms. DAG, diacylglycerol; MYPT-1, regulatory myosin phosphatase targeting subunit 1; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PLA2 phospholipase A2 PLD, phospholipase D; RGS, regulator of G protein signaling; SR, sarcoplasmic reticulum.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

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 x 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 K_i 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 K_i 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 pK_b, and the dose ratios were determined using the same concentration of inhibitor with and without darifenacin. The estimated pK_b for darifenacin was calculated using the formula pK_b = -[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

Top Abstract Materials and Methods Results Discussion References

 
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.

View larger version (13K): [in this window] [in a new window]   Fig. 2. A, cumulative carbachol concentration-response curves of rat bladder contraction with and without the PI-PLC inhibitor ET-18-OCH3 The maximal carbachol response in the absence of any inhibitor is 121 ± 5% of the response to 120 mM KCl (2.41 ± 0.14 g), and the carbachol EC50 is 1.9 ± 0.6 µM. B, effect of ET-18-OCH3 on the maximal carbachol response. C, effect of ET-18-OCH3 on carbachol EC50. No inhibitor (n = 41), 10 µM (n = 18), 30 µM (n = 4), and 100 µM (n = 4). **, p < 0.01 compared with no inhibitor.

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

Top Abstract Materials and Methods Results Discussion References

 
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 G_i, whereas the M₃ subtype preferentially couples to G_q, 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 G_q/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 G_i3-dependent activation of PI-PLC-3 (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 (, I, II, , , , , , µ, , and ), and the isoform mediating this M₂ contractile pathway in rat bladder is not known. Because PKC 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 PKC 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.

	Acknowledgements

 
We acknowledge the expert technical assistance provided by Neil Lamarre, Kimberly Buck, and Bernadette Simpkiss.

	Footnotes

 
This study was supported by Public Health Service Grant R01 DK43333 (to M.R.R.).

doi:10.1124/jpet.105.097303.

ABBREVIATIONS: IP₃, inositol 1,4,5-trisphosphate; PI-PLC, phosphoinositide specific phospholipase C; U73122 [GenBank] , 1-[6-[[17-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.

Address correspondence to: Dr. Michael R. Ruggieri, Sr., 715 OMS, 3400 N. Broad St., Philadelphia, PA 19140. E-mail: rugg1{at}msn.com

	References

Top Abstract Materials and Methods Results Discussion References

 

An JY, Yun HS, Lee YP, Yang SJ, Shim JO, Jeong JH, Shin CY, Kim JH, Kim DS, and Sohn UD (2002) The intracellular pathway of the acetylcholine-induced contraction in cat detrusor muscle cells. Br J Pharmacol 137: 1001-1010.[CrossRef][Medline]

Biancani P, Harnett KM, Sohn UD, Rhim BY, Behar J, Hillemeier C, and Bitar KN (1994) Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh. Am J Physiol 266: G767-G774.[Medline]

Braverman AS, Bartula LL, Myers SI, Parkman HP, and Ruggieri MR (2000) Inflammation changes the muscarinic receptor subtype and signal transduction pathway that mediates gallbladder contraction. Gastroenterology 118: A197.[CrossRef]

Braverman AS, Kohn IJ, Luthin GR, and Ruggieri MR (1998a) Prejunctional M1 facilitory and M2 inhibitory muscarinic receptors mediate rat bladder contractility. Am J Physiol 274: R517-R523.[Medline]

Braverman AS, Luthin GR, and Ruggieri MR (1998b) M2 muscarinic receptor contributes to contraction of the denervated rat urinary bladder. Am J Physiol 275: R1654-R1660.[Medline]

Braverman AS and Ruggieri MR Sr (2003) Hypertrophy changes the muscarinic receptor subtype mediating bladder contraction from M3 toward M2. Am J Physiol 285: R701-R708.

Braverman AS, Tallarida RJ, and Ruggieri MR Sr (2002) Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction. Am J Physiol 283: R663-R668.

Caulfield MP (1993) Muscarinic receptors-characterization, coupling and function. Pharmacol Ther 58: 319-379.[CrossRef][Medline]

Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105.[CrossRef][Medline]

Ehlert FJ, Griffin MT, Abe DM, Vo TH, Taketo MM, Manabe T, and Matsui M (2005) The M₂ muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder. J Pharmacol Exp Ther 313: 368-378.[Abstract/Free Full Text]

Fleichman M, Schneider T, Fetscher C, and Michel MC (2004) Signal transduction underlying carbachol-induced contraction of rat urinary bladder. II. Protein kinases. J Pharmacol Exp Ther 308: 54-58.[Abstract/Free Full Text]

Griffin MT, Matsui M, Shehnaz D, Ansari KZ, Taketo MM, Manabe T, and Ehlert FJ (2004) Muscarinic agonist-mediated heterologous desensitization in isolated ileum requires activation of both muscarinic M₂ and M₃ receptors. J Pharmacol Exp Ther 308: 339-349.[Abstract/Free Full Text]

Horowitz A, Clement-Chomienne O, Walsh MP, and Morgan KG (1996) Epsilonisoenzyme of protein kinase C induces a Ca(2+)-independent contraction in vascular smooth muscle. Am J Physiol 271: C589-C594.[Medline]

Komalavilas P, Mehta S, Wingard CJ, Dransfield DT, Bhalla J, Woodrum JE, Molinaro JR, and Brophy CM (2001) PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. J Appl Physiol 91: 1819-1827.[Abstract/Free Full Text]

Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, and Nakano T (2000) Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett 475: 197-200.[CrossRef][Medline]

Lee SK, Qing WG, Mar W, Luyengi L, Mehta RG, Kawanishi K, Fong HH, Beecher CW, Kinghorn AD, and Pezzuto JM (1998) Angoline and chelerythrine, benzophenanthridine alkaloids that do not inhibit protein kinase C. J Biol Chem 273: 19829-19833.[Abstract/Free Full Text]

Leemhuis J, Boutillier S, Schmidt G, and Meyer DK (2002) The protein kinase A inhibitor H89 acts on cell morphology by inhibiting Rho kinase. J Pharmacol Exp Ther 300: 1000-1007.[Abstract/Free Full Text]

May LG, Johnson S, Krebs S, Newman A, and Aronstam RS (1999) Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation. Cell Signal 11: 179-187.[CrossRef][Medline]

Murthy KS and Makhlouf GM (1997) Differential coupling of muscarinic m₂ and m₃ receptors to adenylyl cyclases V/VI in smooth muscle. Concurrent M₂-mediated inhibition via G_i3 and m₃-mediated stimulation via G_q. J Biol Chem 272: 21317-21324.[Abstract/Free Full Text]

Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, and Makhlouf GM (2003a) Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145-155.[CrossRef][Medline]

Murthy KS, Zhou H, Grider JR, and Makhlouf GM (2003b) Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA. Am J Physiol 284: G1006-G1016.

Nagumo H, Sasaki Y, Ono Y, Okamoto H, Seto M, and Takuwa Y (2000) Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells. Am J Physiol 278: C57-C65.

Rembold CM, Foster DB, Strauss JD, Wingard CJ, and Eyk JE (2000) cGMPmediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation in swine carotid artery. J Physiol (Lond) 524: 865-878.[Abstract/Free Full Text]

Ruiz-Velasco R, Lanning CC, and Williams CL (2002) The activation of Rac1 by M₃ muscarinic acetylcholine receptors involves the translocation of Rac1 and IQGAP1 to cell junctions and changes in the composition of protein complexes containing Rac1, IQGAP1, and actin. J Biol Chem 277: 33081-33091.[Abstract/Free Full Text]

Rumenapp U, Asmus M, Schablowski H, Woznicki M, Han L, Jakobs KH, Fahimi-Vahid M, Michalek C, Wieland T, and Schmidt M (2001) The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not G_q-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptor-effector coupling. J Biol Chem 276: 2474-2479.[Abstract/Free Full Text]

Sawyer GW and Ehlert FJ (1999) Muscarinic M₃ receptor inactivation reveals a pertussis toxin-sensitive contractile response in the guinea pig colon: evidence for M₂/M₃ receptor interactions. J Pharmacol Exp Ther 289: 464-476.[Abstract/Free Full Text]

Schneider T, Fetscher C, Krege S, and Michel MC (2004a) Signal transduction underlying carbachol-induced contraction of human urinary bladder. J Pharmacol Exp Ther 309: 1148-1153.[Abstract/Free Full Text]

Schneider T, Hein P, and Michel MC (2004b) Signal transduction underlying carbachol-induced contraction of rat urinary bladder. I. Phospholipases and Ca²⁺ sources. J Pharmacol Exp Ther 308: 47-53.[Abstract/Free Full Text]

Sohn UD, Hong YW, Choi HC, Ha JH, Lee KY, Kim WJ, Biancani P, Jeong JH, and Huh IH (2000) Increase of [Ca(2+)]_i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle. Cell Signal 12: 215-222.[CrossRef][Medline]

Somogyi GT and de Groat WC (1992) Evidence for inhibitory nicotinic and facilitatory muscarinic receptors in cholinergic nerve terminals of the rat urinary bladder. J Auton Nerv Syst 37: 89-97.[CrossRef][Medline]

Strassheim D, May LG, Varker KA, Puhl HL, Phelps SH, Porter RA, Aronstam RS, Noti JD, and Williams CL (1999) M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms. J Biol Chem 274: 18675-18685.[Abstract/Free Full Text]

Taggart MJ, Lee YH, and Morgan KG (1999) Cellular redistribution of PKCalpha, rhoA and ROKalpha following smooth muscle agonist stimulation. Exp Cell Res 251: 92-101.[CrossRef][Medline]

Tobin AB, Lambert DG, and Nahorski SR (1992) Rapid desensitization of muscarinic m3 receptor-stimulated polyphosphoinositide responses. Mol Pharmacol 42: 1042-1048.[Abstract]

Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, et al. (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature (Lond) 389: 990-994.[CrossRef][Medline]

Wang YX, Dhulipala PD, Li L, Benovic JL, and Kotlikoff MI (1999) Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C. J Biol Chem 274: 13859-13864.[Abstract/Free Full Text]

Willets JM, Challiss RA, Kelly E, and Nahorski SR (2001) G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells. Mol Pharmacol 60: 321-330.[Abstract/Free Full Text]

Willets JM, Challiss RA, and Nahorski SR (2002) Endogenous G protein-coupled receptor kinase 6 Regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. J Biol Chem 277: 15523-15529.[Abstract/Free Full Text]

Woodrum DA, Brophy CM, Wingard CJ, Beall A, and Rasmussen H (1999) Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol 277: H931-H939.[Medline]

Yang CM, Hsu MC, Tsao HL, Chiu CT, Ong R, Hsieh JT, and Fan LW (1996) Effect of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells. Cell Calcium 19: 243-254.[CrossRef][Medline]

Zhou H, Das S, and Murthy KS (2003) Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA₂ by m3 and m2 receptors. Am J Physiol 284: G472-G480.

This article has been cited by other articles:

				E. P. Frazier, A. S. Braverman, S. L. M. Peters, M. C. Michel, and M. R. Ruggieri Sr. Does Phospholipase C Mediate Muscarinic Receptor-Induced Rat Urinary Bladder Contraction? J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 998 - 1002. [Abstract] [Full Text] [PDF]

				A. S. Braverman, L. R. Doumanian, and M. R. Ruggieri Sr M2 and M3 Muscarinic Receptor Activation of Urinary Bladder Contractile Signal Transduction. II. Denervated Rat Bladder J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 875 - 880. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.097303v1 316/2/869    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Braverman, A. S. Articles by Ruggieri, M. R. Search for Related Content PubMed PubMed Citation Articles by Braverman, A. S. Articles by Ruggieri, M. R., Sr Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CARBACHOL CHLORIDE