Signal Transduction Underlying Carbachol-Induced Contraction of Rat Urinary Bladder. I. Phospholipases and Ca²⁺ Sources

Muscarinic acetylcholine receptors are the physiologically most important mechanisms to elicit contraction of the urinary bladder (Andersson, 1993). In the bladder of various mammalian species, including humans, M₂ and M₃ muscarinic receptors coexist, but the expression of M₂ receptors is much greater than that of the M₃ receptors (Wang et al., 1995; Goepel et al., 1998; Yamanishi et al., 2000; Kories et al., 2003). Nevertheless, the contractile response to the exogenous agonist carbachol and to endogenous agonists released by field stimulation have been attributed predominantly, if not exclusively, to M₃ receptors in rats (Longhurst et al., 1995; Hegde et al., 1997; Tong et al., 1997; Braverman et al., 1998; Choppin et al., 1998; Longhurst and Levendusky, 2000; Kories et al., 2003), mice (Choppin and Eglen, 2001b), pigs (Yamanishi et al., 2000), dogs (Choppin and Eglen, 2001a), and humans (Chess-Williams et al., 2001; Fetscher et al., 2002). Moreover, at least male M₃ (but not M₂) receptor-knockout mice exhibit bladder distension and develop urinary retention (Matsui et al., 2000). On the other hand, it should be considered that hitherto available antagonists have only modest subtype selectivity and/or do not act in a purely competitive manner; hence, they were not well suited for detecting a potential minor component of M₂ receptors in bladder contraction. The present study was primarily designed to determine the proximal signaling mechanisms underlying M₃ receptor-mediated contraction of rat urinary bladder, but we have also reinvestigated the role of muscarinic receptor subtypes using two novel and highly M₂- and M₃-selective antagonists, i.e., (R)-4-{2-[3-(4-methoxy-benzoylamino)-benzyl]piperidin-1-ylmethyl}-piperidine-1-carboxylic acid amide (Ro-320-6206) (Zhao et al., 2001) and [3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine] APP (MacKenzie and Cross, 1991), respectively.

The prototypical signal transduction mechanism of M₃ receptors is stimulation of a phospholipase (PL)C to generate inositol phosphates and diacylglycerol (Caulfield and Birdsall, 1998). Muscarinic stimulation of PLC has also been demonstrated in cultured smooth muscle cells from human bladder (Marsh et al., 1996) and in rat bladder slices (Kories et al., 2003), and the latter response was shown to be M₃ receptor mediated. However, muscarinic receptors can also activate a PLD or PLA₂ in a variety of cell types (Felder, 1995), the latter possibly leading to cyclooxygenase activation (Nishimura et al., 1995). Several cyclooxygenase products can contract isolated detrusor muscle (Andersson, 2000), and cyclooxygenase activation was shown to at least partly mediate rat urinary bladder contraction induced by protease-activated receptor-2 (Nakahara et al., 2003). Therefore, we have determined the possible roles of PLC, PLD, PLA₂, and cyclooxygenase in muscarinic M₃ receptor-mediated contraction of rat urinary bladder.

Similar to all other types of smooth muscle, urinary bladder contraction evoked by muscarinic receptor stimulation involves elevations of intracellular Ca²⁺ concentrations in rat and guinea pig bladder smooth muscle cells (Ikeda et al., 2002; Ma et al., 2002). Accordingly, L-type Ca²⁺ entry blockers can inhibit muscarinic receptor-mediated bladder contraction in guinea pigs and humans (Sjögren et al., 1982; Masters et al., 1999; Ikeda et al., 2002). However, Ca²⁺ sources apart from L-type channels may also contribute in human bladder smooth muscle cells (Masters et al., 1999; Visser and van Mastrigt, 2000). Therefore, we have also determined the roles of nifedipine-sensitive and receptor-operated Ca²⁺ channels in M₃ receptor-mediated rat bladder contraction.

Materials and Methods

Force of Contraction. Urinary bladder strips were prepared from female Wistar rats (body weight 231 ± 9 g, bladder weight 65 ± 2 mg) obtained from the central animal breeding facility at the University of Essen. Experiments were performed as previously described (Kories et al., 2003). Briefly, longitudinal bladder strips (approximately 1 mm diameter, 18 ± 1 mm length, and 9.6 ± 0.5 mg weight, n = 95) were mounted under a tension of 10 mN in 10-ml organ baths containing Krebs-Henseleit solution (119 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄, 2.5 mM CaCl₂, 0.027 mM EDTA, 5.5 mM glucose, and 10 mM HEPES), which was aerated with 95% O₂ and 5% CO₂ to yield a pH of 7.4 at 37°C. After 60 min of equilibration, including washes with fresh buffer every 15 min, the bladder strips were challenged three times with a combination of 50 mM KCl and 0.1 mM carbachol with 5-min rest and washes between each challenge. After washout and an additional 30 min of equilibration, cumulative concentration-response curves were constructed for carbachol in the absence of any inhibitor or vehicle. Using 15-min washouts and then 15-min equilibration periods in between, up to four additional curves were then generated in the presence of increasing concentrations of the indicated antagonists and inhibitors, their negative controls, or their vehicles. Previous work had shown that carbachol-induced rat bladder contraction remains fairly stable under these conditions (Kories et al., 2003).

Carbachol concentration-response curves were analyzed by fitting sigmoidal curves to the experimental data, in which the bottom of the curve was fixed at 0. The force of contraction in the absence and presence of inhibitors were expressed as the percentage of maximum carbachol effects observed within the same bladder strip in the first concentration-response curve, i.e., before addition of any inhibitor or vehicle. To assess inhibitor effects, alterations in E_max or pEC₅₀ in its presence relative to the first curve were compared with those in the presence of a matching vehicle time control using two-way analysis of variance testing for main treatment effect and concentration dependence; if this indicated statistical significance, the effect of individual inhibitor concentrations relative to time-matched controls was assessed by Bonferroni post tests. p < 0.05 was considered to be significant in all statistical analyses. To assess antagonist effects, analysis according to Arunlakshana and Schild (1959) was performed. All curve fitting and statistical calculations were performed with the Prism program (version 4.0; GraphPad Software Inc., San Diego, CA).

Phospholipase C activation was assessed as [³H]inositol phosphate formation in 350 × 350-μm bladder slices as previously described (Kories et al., 2003). Briefly, slices were suspended in 10 ml of Ringer solution (147.2 mM NaCl, 4.0 mM KCl, 2.25 mM CaCl₂, 10 mM glucose, and 20 mM HEPES at pH 7.4) supplemented with 10 mM LiCl to block inositol phosphate degradation and 2 U/ml adenosine deaminase. They were incubated for 60 min at 37°C in the presence of 100 μCi of [³H]myoinositol/12 ml. Thereafter, 300 μl of the slice suspension (corresponding to 6–8-mg slice wet weight) were pipetted into flat-bottom polystyrene tubes under gentle swirling, and agonists and antagonists were added in the indicated concentrations to yield a final volume of 330 μl. After incubation for 45 min, the reaction was stopped by addition of 400 μl of ice-cold methanol and 700 μl of chloroform. The mixture was vigorously vortexed twice, and thereafter, the phases were separated by centrifugation at 820g for 10 min at 4°C. Aliquots (450 μl) of the upper phase were placed on Dowex AG1-X8 columns (200 mg per column). Free inositol was eluted twice each with 5 ml of H₂O and 5 ml of 60 mM ammonium formate. Total inositol phosphates were eluted twice by addition of 1 ml of 1 M ammonium formate dissolved in 100 mM formic acid. Each data point was measured in quadruplicate within each experiment. Statistical significance of differences was determined by one-way analysis of variance; if this indicated significant differences among group means, individual groups were compared by Dunnett's multiple comparison tests.

Phospholipase D was assessed as [³H]phosphatidylethanol ([³H]PEtOH) formation. The bladder was quickly removed and stored in buffer containing 147.2 mM NaCl, 4 mM KCl, 2.25 mM CaCl₂, 20 mM HEPES, and 1 mg/ml glucose at 37°C and a pH of 7.4. Bladder slices of 200 × 200 μm were prepared as described above. The suspension was resuspended and incubated twice with adenosine deaminase (2 U/ml) for 15 min each. Next, the slices were resuspended in 7 ml of fresh buffer containing 40 μl of [³H]oleic acid (specific activity, 5 mCi/ml) and again incubated at 37°C for 60 min. After resuspending the slices in fresh buffer containing 5% (v/v) ethanol, they were incubated in a total volume of 400 μl with the indicated drugs for 45 min at 37°C. Afterward, the incubation was stopped by adding 0.5 ml each of ice-cold methanol, trichloromethane, and H₂O. The mixture was vortexed vigorously twice and centrifuged for 10 min at 2000g and 4°C. Four hundred microliters of the lower phase were put into small reaction tubes, and the solvent was evacuated using a SpeedVac centrifuge. The pellet was then resuspended with 25 μl of a 1:1 mixture of chloroform and methanol, and 20 μl were placed on silica gel 60 thin-layer chromatography plates (Whatman, Clifton, NJ). The lipids were separated using the organic phase of a mixture of ethyl acetate/isooctane/acetic acid/water (91: 14:21:70 by volume), migrated with authentic standards, and localized by iodine staining. Areas corresponding to the PEtOH standard were scraped into scintillation vials, as were the areas below containing other labeled phospholipids. The formation of [³H]PEtOH was assessed as the ratio of total labeled phospholipids and is given as the percentage of basal.

Chemicals. Carbachol HCl, nifedipine, SKF 96,365 [1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl], U 73,122 [1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione], and U 73,343 [1-(6-[-([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-2,5-pyrrolidinedione] were obtained from Sigma-Aldrich Laborchemikalien (Seelze, Germany). AACOCF₃ (arachidonyltrifluoromethyl ketone), indomethacin, and phorbol myristyl acetate were from Calbiochem (Bad Soden, Germany). [³H]myoinositol (specific activity, 115 Ci/mmol) was from Amersham Biosciences Inc. (Piscataway, NJ), and [³H]oleic acid (specific activity, 23 Ci/mmol) was from PerkinElmer Life and Analytical Sciences (Boston, MA). Darifenacin and tolterodine were provided by Pfizer (New York, NY), and Ro-320-6206 and APP were synthesized as previously described (MacKenzie and Cross, 1991; Zhao et al., 2001).

AACOCF₃ (at 10 mM), APP (at 10 mM), darifenacin (at 10 mM), tolterodine (at 10 mM), Ro-320-6206 (at 10 mM), U 73,122 (at 3 mM), U 73,343 (at 3 mM), and phorbol myristate acetate (at 1 mM) were dissolved in dimethylsulfoxide. Indomethacin (at 10 mM) and nifedipine (at 1 mM) were dissolved in ethanol. SKF 96,365 was dissolved at 1 mM in distilled water. The experiments involving nifedipine were performed in light-shielded organ baths.

Results

Before addition of antagonist or inhibitor, i.e., in the first curve generated within each bladder strip, carbachol concentration-dependently increased force of contraction with a pEC₅₀ of 5.65 ± 0.03 and maximum effects of 35.4 ± 1.4 mN (n = 127 muscle strips). All further contraction data are expressed as percentage of the maximum carbachol effect within the same preparation before addition of any inhibitor.

Antagonist Experiments. Relative to the first curve in the absence of any antagonist, the second to fifth consecutive curve within a preparation exhibited a pEC₅₀ that was -0.06, 0.05, 0.16, and 0.23 log units smaller, respectively; concomitantly, maximum effects were reduced by -2 ± 5%, 8 ± 9%, 18 ± 11%, and 26 ± 13%, respectively (n = 6). These alterations were taken into account when analyzing the effects of the antagonists. Within the tested concentration range, tolterodine, Ro-320-6206, and APP did not affect maximum carbachol responses in a manner that was significantly different from vehicle (data not shown), whereas 10, 30, 100, and 300 nM darifenacin reduced it by 21 ± 5%, 38 ± 9%, 52 ± 11%, and 60 ± 10%, respectively (except for the highest concentration, all p < 0.05 versus vehicle). All four antagonists concentration-dependently right-shifted the carbachol concentration-response curve (Fig. 1). The Schild regression for the nonselective tolterodine (30–1000 nM) had a slope of slightly less than unity (0.80 ± 0.06) and an x-axis intercept (apparent pA₂ value) of 8.93 (95% confidence interval, 8.57–9.42). The Schild regression for the M₃-selective darifenacin (10–300 nM) had a slope close to unity (1.11 ± 0.15) and an x-axis intercept (apparent pA₂ value) of 8.67 (95% confidence interval, 8.20–9.38). The Schild regression for the M₃-selective APP (10–300 nM) had a slope close to unity (1.08 ± 0.15) and an x-axis intercept (apparent pA₂ value) of 8.73 (95% confidence interval, 8.24–9.49). The M₂-selective Ro-320-6206 had only little effect on the carbachol concentration-response curve. Thus, at concentrations of 0.3, 1, 3, and 10 μM, it right-shifted the carbachol concentration-response curve by only 0.14 ± 0.10, 0.25 ± 0.11, 0.42 ± 0.14, and 0.75 ± 0.16 log units, respectively; accordingly, a Schild slope of only 0.40 ± 0.11 was obtained, and the x-axis intercept of this shallow regression line was 6.72 (95% confidence interval, 5.92–8.53).

Role of Phospholipase C. In confirmation of previous findings from our laboratory (Kories et al., 2003), 1 mM carbachol enhanced IP formation by approximately 60% over basal (Fig. 2). This concentration of carbachol had been chosen based on our previously published concentration-response curves to obtain a good signal/noise ratio. Although U 73,122 (10 μM) alone had no effect on basal IP formation, it abolished carbachol-stimulated IP formation (Fig. 2).

Nevertheless, U 73,122 (1–10 μM) did not significantly alter the potency or maximum effects of carbachol-induced bladder contraction relative to its vehicle (Fig. 3). In light of the unexpectedness of this finding, it was confirmed for 10 μM U 73,122 in a second series of experiments performed by a different investigator; in that series, U 73,122 failed to significantly affect carbachol-induced bladder contraction not only relative to its vehicle but also relative to 10 μM of its negative control, U 73,343 (n = 11–12; data not shown). Moreover, we confirmed the effectiveness of U 73,122 in organ bath experiments by demonstrating that it markedly inhibited α₁-adrenoceptor-induced contraction of rat mesenteric microvessels (Altmann et al., 2003).

Role of Phospholipase D. PLD activity was markedly stimulated by 1 μM of the positive control phorbol myristyl acetate (210 ± 19% over basal, n = 13). In contrast, 1 mM carbachol had only little effect on PLD activity, i.e., enhanced [³H]PEtOH accumulation nonsignificantly by only 13 ± 10% over basal (Fig. 4).

The PLD inhibitor butan-1-ol did not significantly alter carbachol-induced contraction relative to its negative control butan-2-ol when tested at concentrations of 0.03 or 0.1%, but a statistically significant reduction of potency and maximum effects of carbachol was obtained at a butan-1-ol concentration of 0.3% (Fig. 5).

Role of Phospholipase A₂ and Cyclooxygenase. The PLA₂ inhibitor AACOCF₃ did not significantly alter carbachol-induced contraction relative to its vehicle when tested at concentrations of 30 and 100 μM, whereas a statistically significant reduction of maximum effects of carbachol (but not of its potency) was observed at an inhibitor concentration of 300 μM (Fig. 6). The cyclooxygenase inhibitor indomethacin (1–10 μM) did not significantly alter the potency or maximum effects of carbachol-induced bladder contraction relative to its vehicle (Fig. 7).

Role of Ca²⁺ Sources. The Ca²⁺ entry blocker nifedipine (10–100 nM) markedly inhibited carbachol-induced bladder contraction relative to its vehicle, ethanol (Fig. 8). This inhibition was due to reductions of maximum carbachol responses reaching up to 90% at 100 nM, which were not accompanied by statistically significant alterations of the agonist potency for the remaining response.

SKF 96,365, an inhibitor of receptor-operated Ca²⁺ channels, did not significantly affect carbachol-induced bladder contractions at concentrations of 1 or 3 μM, whereas a significant reduction of maximum responses (-47%) but not of carbachol potency was seen with 10 μM SKF 96,365 (Fig. 9).

Discussion

The present study was primarily designed to investigate proximal signaling mechanisms potentially involved in carbachol-induced muscarinic receptor-mediated contraction of rat urinary bladder. Although M₂ receptors are more numerous in rat bladder than M₃ receptors (Wang et al., 1995; Kories et al., 2003), numerous studies have proposed that rat bladder contraction is mediated predominantly if not exclusively by the minor population of M₃ receptors (Longhurst et al., 1995; Hegde et al., 1997; Tong et al., 1997; Braverman et al., 1998; Choppin et al., 1998; Longhurst and Levendusky, 2000; Kories et al., 2003). However, all of these studies were based on antagonists with only moderate subtype selectivity or on darifenacin, which has considerable selectivity for M₃ receptors but does not act purely competitively (as confirmed in the present study). Therefore, we reinvestigated the muscarinic receptor subtype mediating rat bladder contraction using APP (MacKenzie and Cross, 1991), a compound that, similar to darifenacin, is about 40-fold selective for M₃ receptors (K_i, 2.6 versus 111 nM; S. Hegde, personal communication) but does not reduce maximum responses, and Ro-320-6206, an approximately 100-fold M₂-selective antagonist APP (5.0 versus 500 nM; Zhao et al., 2001); the nonselective tolterodine and the M₃-selective darifenacin were studied in comparison. Using more selective and apparently purely competitive tools, our present experiments confirm that carbachol-induced contraction of rat bladder occurs via M₃ receptors.

Coimmunoprecipitation studies demonstrate that the M₃ receptors in rat bladder couple predominantly to G-proteins of the G_q/11 and, surprisingly, also the G_i1 type (Wang et al., 1995). Activation of a PLC is the prototypical signaling response of G_q-coupled receptors in general and of M₃ muscarinic receptors in particular (Caulfield and Birdsall, 1998). Muscarinic receptor stimulation also activates PLC in cultured smooth muscle cells from human bladder (Marsh et al., 1996) and in rat bladder slices (Kories et al., 2003), and at least the latter response is mediated by M₃ receptors, i.e., the same subtype mediating the contraction. Studies in feline isolated bladder smooth muscle cells using neomycin as the PLC inhibitor have proposed that PLC activation is important for carbachol-induced bladder contraction (An et al., 2002). The present data based on rat bladder strips and U 73,122 as the PLC inhibitor surprisingly do not support this proposal. Although we do not know whether these discrepancies are due to differences in species (rat versus cat), type of preparation (strip versus cultured cell), or PLC inhibitor (U 73,122 versus neomycin), it should be noted that the present study did not detect inhibition of contraction under conditions where PLC activation was clearly abolished within the same study. Thus, at least in rats, M₃ receptor-mediated PLC activation and contraction occur concomitantly, but contraction is not dependent on PLC activation.

Activation of PLD is another potential signaling mechanism of M₃ muscarinic receptors (Zhou et al., 1994; Schmidt et al., 1995). In the present study, the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol, caused some inhibition of carbachol-induced bladder contraction, but the effect was weak and reached statistical significance only at the highest inhibitor concentration. However, butan-1-ol concentrations up to 0.5% can still be considered to be selectively inhibiting PLD (Banno et al., 2001; Bechoua and Daniel, 2001), and hence, the inhibition by 0.3% butan-1-ol in our study is unlikely to be nonspecific. The small extent of the inhibition is not surprising, since carbachol caused only little if any PLD activation in rat bladder slices, i.e., an effect of less than 10% of the positive control phorbol myristate acetate. Thus, PLD activation appears to play only a minor role in M₃ receptor-mediated rat bladder contraction.

Activation of a cytosolic PLA₂, possibly followed by that of a cyclooxygenase, is another potential signaling mechanism of muscarinic receptors (Hunt et al., 1994; Felder, 1995; Nishimura et al., 1995). This could potentially also be involved in muscarinic receptor-mediated bladder contraction, since several cyclooxygenase products are known to contract the bladder (Andersson, 2000), and since it has recently been shown that rat bladder contraction elicited by protease-activated receptor-2 involves activation of a cyclooxygenase (Kubota et al., 2003; Nakahara et al., 2003). In the present study, AACOCF₃, an inhibitor of cytosolic PLA₂, caused only minor, if any, inhibition of rat bladder contraction. Moreover, the cyclooxygenase inhibitor indomethacin, when applied in concentrations inhibiting protease-activated receptor-2-mediated rat bladder contraction (Nakahara et al., 2003), was completely ineffective. Thus, cytosolic PLA₂ and cyclooxygenase do not appear to play a role for M₃ receptor-mediated rat bladder contraction.

Elevations of intracellular Ca²⁺ concentrations play a central role in smooth muscle contraction. Muscarinic receptor-induced Ca²⁺ elevations have been demonstrated in rat and guinea pig bladder smooth muscle cells (Ikeda et al., 2002; Ma et al., 2002). They could come from intracellular stores, e.g., inositol phosphate or ryanodine receptor-sensitive stores, or from the extracellular space via a variety of ion channels. In light of our negative data regarding an involvement of PLC, we have not further investigated a possible role of inositol phosphate-sensitive Ca²⁺ stores. A role for ryanodine-sensitive Ca²⁺ stores has previously been demonstrated in muscarinic receptor-stimulated contraction of human detrusor isolated smooth muscle cells (Visser and van Mastrigt, 2000). In guinea pig and human bladder smooth muscle cells, L-type Ca²⁺ channels also appear to contribute to the muscarinic receptor-mediated bladder contractions (Sjögren et al., 1982; Masters et al., 1999; Visser and van Mastrigt, 2000; Ikeda et al., 2002). In the present study, the L-type Ca²⁺ channel inhibitor nifedipine potently and effectively inhibited carbachol-induced bladder contraction. Indeed, this response was much more sensitive to nifedipine- than noradrenaline- or sphingosylphosphorylcholine-induced blood vessel contraction (Chen et al., 1996; Bischoff et al., 2001). Moreover, knockout mice lacking the Ca_v1.2 gene, which encodes for a subunit of voltage-operated Ca²⁺ channels, exhibit a markedly reduced bladder contraction in response to muscarinic stimulation (Wegener et al., 2003). In contrast, SKF 96,365, an inhibitor of receptor-operated Ca²⁺ channels, caused only minor, if any, inhibition of carbachol-induced bladder contraction in the present study. Thus, influx of extracellular Ca²⁺ through L-type voltage-dependent channels but not through receptor-operated channels appears to play a pivotal role for rat bladder contraction.

In conclusion, carbachol-induced, M₃ muscarinic receptor-mediated contraction of rat bladder is largely mediated by Ca²⁺ influx through L-type, voltage-dependent channels. Surprisingly, PLC activation is not involved, although it is concomitantly activated. Moreover, PLD, PLA₂, cyclooxygenase, and receptor-operated Ca²⁺ channels also play only a minor, if any, role in muscarinic receptor-mediated contraction of rat bladder. The role of various protein kinases, which may be activated secondary to these proximal signaling mechanisms, was determined in the accompanying paper (Fleichman et al., 2004).