Introduction

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In the urinary bladder of rodent (e.g., rat) and nonrodent mammal species, the excitatory neuromuscular transmission depends primarily on the activation of parasympathetic pathways, which possess both a cholinergic and noncholinergic component (Andersson, 1993). The latter is apparently mediated by ATP or a related purine compound acting at P₂-purinoceptors (Hashimoto and Kokubun, 1995), whereas the cholinergic component leads to contractile responses mediated by postjunctional muscarinic receptors (Andersson, 1993; Wein et al., 1987).

Based on differential potencies of selective antagonists, central and peripheral muscarinic receptors can be subdivided into a population of four subtypes denoted as M₁-M₄ (Caulfield, 1993; Eglen et al., 1994; Eglen and Watson, 1996), whereas muscarinic receptor genes encode five receptor proteins (m₁-m₅), which share the same overall structure and a large degree of protein sequence homology (Bonner, 1989). With regard to the characterization of muscarinic receptor subtypes located on the effector cells of the rat urinary bladder, radioligand binding studies revealed the presence of M₂ and M₃ sites, corresponding to the m₂ and m₃ subtypes as demonstrated by subtype-sensitive antisera (Wall et al., 1991; Wang et al., 1995). Nevertheless, the functional receptors that mediate contractile responses in the detrusor, as well as in intestinal and tracheal preparations (Eglen et al., 1996), belong to the M₃ subtype (D'Agostino et al., 1993; Longhurst et al., 1995). In addition to postjunctional muscarinic receptors, the rat urinary bladder is endowed with muscarinic autoreceptors regulating, via a negative (D'Agostino et al., 1986, 1989; Somogyi and de Groat, 1992) and a positive feedback mechanism mediated by M₁ receptors (Somogyi et al., 1994), the release of [³H]ACh from nerve terminals. Based on a study with selective muscarinic receptor antagonists, it was suggested that inhibitory autoreceptors could not be probably regarded as M₁/M₃ or M₂ subtypes, but the lack of antagonists with a high selectivity ratio for M₂/M₄ hindered their final recognition (D'Agostino et al., 1993). Recently, tripitramine, a novel muscarinic receptor antagonist with an improved M₂/M₄ affinity ratio (~10) compared with methoctramine (~2), emerged as a suitable tool for the pharmacological characterization of these muscarinic receptor subtypes (Angeli et al., 1995; Chiarini et al., 1995; Maggio et al., 1994; Melchiorre et al., 1995).

The present study was therefore designed to assess whether in the urinary bladder of the rat, the muscarinic autoreceptor inhibiting the electrically induced [³H]ACh release from cholinergic nerve terminals can belong to the M₄ receptor subtype.

	Materials and Methods

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Preparation of rat bladder strips. Male albino rats (average body weight, 350 g) of the Wistar Morini strain (San Polo d'Enza, Parma, Italy) were used. Procedures involving animals and their care were conducted in conformity with institutional guidelines, which are in compliance with Italian and international laws and policies. The whole urinary bladder was dissected out and placed in Krebs-Henseleit solution (composition in mM: NaCl 118, KCl 5.6, CaCl₂ 2.5, MgSO₄ 1.19, NaHCO₃ 25, NaH₂PO₄ 1.3, glucose 10; pH 7.4). Longitudinal muscle strips were isolated from the extratrigonal area. Four preparations (10 mm long, 1.5 mm wide, 12-18 mg weight) were obtained from each urinary bladder and suspended isometrically (tension, 10 mN) in organ baths containing 2 ml of Krebs-Henseleit solution maintained at 37°C and gassed with 95% O₂/5% CO₂. Each preparation was used for different protocols. Tritiated acetylcholine ([³H]ACh) release and the concomitant smooth muscle contraction due to activation of postjunctional muscarinic receptors were evoked by EFS and recorded simultaneously. Any antagonist-induced change in both [³H]ACh overflow (see below) and neurogenic smooth muscle contractions was considered as an interaction with muscarinic receptors located at prejunctional and postjunctional level, respectively. In fact, to minimize the presence of excitatory purinergic transmission to smooth muscle cells, all experiments were carried out in the presence of 3 µM indomethacin to prevent ATP-mediated biosynthesis of prostaglandins (Kasakov and Vlaskovska, 1985), which are known to participate in the contractile response to ATP. In the rat bladder, single-pulse EFS was found to evoke an "early" phasic (mainly ATP-mediated) contraction and a "late" tonic cholinergic component (Maggi et al., 1985). This motor pattern is less clear-cut when a stimulus train is used, due to the overlapping of the atropine-sensitive and -resistant components (Maggi et al., 1985). In untreated bladder from the rat, the noncholinergic component caused by a stimulus train (360 pulses) was previously found to account for ~30% (in terms of area) of the total mechanical response (D'Agostino et al., 1986). In the present experiments with indomethacin, contractile responses induced by EFS (540 pulses) were reduced by muscarinic receptor antagonists by 90% to 95% (in terms of area) (see Results), indicating that under our experimental conditions, they were mainly mediated by activation of cholinergic nerves. Indomethacin (3 µM) did not affect tritiated ACh release (data not shown).

Labeling and release experiments. Neuronal ACh stores were labeled according to the procedure described in detail previously (D'Agostino et al., 1988, 1989). Briefly, the preparation was incubated for 30 min with [³H]choline (92.5 KBq/ml; 32 nM) and stimulated continuously during this period by means of two parallel platinum electrodes (0.8 Hz, 1-msec pulse duration at 8 V/cm). At the end of the labeling period, the preparation was washed out by superfusion at a constant rate of 2 ml/min for 120 min (Minipulse 2HP8 flow inducer, Gilson Medical Electronics, Villiers le Bel, France). Hemicholinium-3 (10 µM) was present in the washout solution throughout the experiment to prevent the uptake of [³H]choline. Starting from the 121th min (zero time), superfusion fluid was collected at 3-min intervals (6-ml samples). The strip was stimulated three times (S₁, S₂ and S₃), beginning at 9 (S₁), 39 (S₂) and 69 (S₃) min after zero time. The release was evoked with square-wave pulses (1-msec duration, 8 V/cm) at the frequency of 3 Hz (540 pulses). Aliquots (1 ml) of the superfusate were added to 5 ml of Ultima Gold (Packard) and the tritium content was measured by liquid scintillation spectrometry (Packard 1900). Quench correction curves were established, and external standardization was used for counting efficiency. Both resting and stimulated outflow of radioactivity were expressed as disintegrations/sec (Bq)/g of tissue (Bq/g). The increase in the release caused by electrical stimulation was obtained from the difference between the total tritium outflow during 3-min stimulation plus the following 12 min (stimulation outflow period) and the calculated spontaneous outflow. The decline of the spontaneous outflow was calculated by fitting a linear regression line to the values (expressed in Bq/g) of three 3-min samples before and after the stimulation period.

Prejunctional and postjunctional effects of muscarinic receptor antagonists. Increasing concentrations of a series of muscarinic receptor antagonists (atropine, pirenzepine, 4-DAMP, HHSiD, AF-DX 116 [(±)-11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one, methoctramine and tripitramine) were added 27 min before the onset of S₂ and S₃. In preliminary trials, to study the repeatibility of the effects of a given antagonist at prejunctional and postjunctional level, the same concentration of the tested drug was administered twice before S₂ and before S₃. The prejunctional effects of muscarinic antagonists were expressed as ratios S₂/S₁ or S₃/S₁. Electrically evoked contractions of the smooth muscle (postjunctional effects) were recorded by a force-displacement transducer and displayed on a chart polygraph. The area underlying the peak tension (C₁) developed during S₁ stimulation was calculated and compared with that obtained during S₂ (C₂) and S₃ (C₃), so prejunctional and postjunctional effects were estimated simultaneously in each experiment.

Postjunctional antagonist pA₂ values against muscarone. Urinary bladder strips were suspended isometrically under a tension of 10 mN in Krebs-Henseleit solution at 37°C. After an equilibration period of 60 min, the preparation was primed with a submaximal concentration of muscarone, a potent muscarinic receptor agonist in the rat bladder (Grana et al., 1987). After a washout of 30 min, cumulative concentration-response curves to muscarone were obtained using half-logarithmic dosing increments. At the end of the first concentration-response curve, the preparation was washed for 40 min, during which time the tension returned to the base-line level. The preparation was then exposed for 30 min to a given muscarinic receptor antagonist, and a second curve to muscarone was constructed. Only one antagonist concentration was tested in each preparation. Changes in sensitivity to muscarone were evaluated in parallel time control experiments without antagonist.

Data analysis. Drug potency estimates were evaluated as log EC₅₀ (negative log of the molar concentration producing half-maximal effect) by nonlinear curve fitting (GraphPAD Prism, Version 1.3, GraphPAD Software, San Diego, CA). Values from individual experiments were averaged, and the S.E.M. values were calculated. Statistical significance between the mean potency of a given antagonist at prejunctional and postjunctional levels was assessed by Student's unpaired t test. Antagonist affinity estimates (pA₂ values) were calculated following Schild regression analysis (Arunlakshana and Schild, 1959) using (±)-muscarone concentration ratios determined at EC₅₀ levels in control and test curves. Confidence limits at 95% probability for the slope of the regression were evaluated by using a computer program based on a manual for pharmacological calculations (PHARM/PCS, Version 4.1, Tallarida and Murray, 1986). The subtype classification of prejunctional and postjunctional muscarinic receptors was carried out by comparing the potency or affinity estimates for antagonists obtained in the bladder with the constants present in the literature for the same compounds for cloned and native muscarinic receptors (correlation analysis). The statistic t for any relationship was also calculated (Kenakin, 1993).

Drugs. We purchased [methyl-³H]choline chloride (78 Ci/mM; 2.89 TBg/mM) from Amersham (Arlington Heights, IL); hemicholinium-3, indomethacin, atropine sulfate and pirenzepine dihydrochloride monohydrate from Sigma Chemical (St. Louis, MO); and HHSiD, 4-DAMP and AF-DX 116, (±)-11-[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5-11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one from Research Biochemicals (Natick, MA). Methoctramine hydrochloride and tripitramine sesquifumarate were kindly donated by Prof. C. Melchiorre (University of Bologna, Italy), and (±)-muscarone was kindly donated by Prof. C. De Micheli (University of Milan, Italy).

	Results

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Responses to EFS. The measurements of electrically evoked tritium outflow as a direct evaluation of neuronal [³H]ACh release in the rat urinary bladder have been established previously (D'Agostino et al., 1986, 1988). When EFSs (S₁, S₂ and S₃) were applied at 3 Hz (540 pulses) 30 min apart, the preparation showed a consistent release of labeled ACh (fig. 1A) and reproducible contractile responses (fig. 1B). In control experiments, S₂/S₁ and S₃/S₁ overflow ratios were 0.70 ± 0.03 and 0.53 ± 0.08, respectively, with an S₁ overflow value of 12,290 ± 1,497 Bq/g (n = 9). On the contrary, smooth muscle contractions (C₁, C₂ and C₃) were not significantly different (C₁ = 26.6 ± 0.9 mN, n = 9). All these effects were prevented by 300 nM tetrodotoxin (n = 3, data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Prejunctional (transmitter release) and postjunctional (smooth muscle contraction) effects caused by EFS in the rat urinary bladder strips. A, Time course of [3H]ACh release evoked by EFS delivered at 30-min intervals (S1, S2 and S3: 3 Hz, 540 pulses each). Each point represents the radioactivity per gram of tissue in the superfusate collected in 3-min samples. Given are the mean ± S.E.M. of 9 experiments. B, Example of isometric contractions (C1,C2 and C3) evoked by EFS (S1, S2 and S3) in a single bladder strip.

Prejunctional and postjunctional potencies of muscarinic receptor antagonists. A series of muscarinic receptor antagonists, including atropine (1-100 nM), pirenzepine (100 nM to 10 µM), 4-DAMP (1 nM to 1 µM), HHSiD (3 nM to 3 µM), AF-DX 116 (300 nM to 10 µM), methoctramine (30 nM to 10 µM) and tripitramine (3 nM to 1 µM) (see table 1 for their receptor selectivity profile) increased the evoked [³H]ACh release and inhibited the electrically induced contractions in a concentration-dependent manner (as an example, see fig. 2 for tripitramine). Conversely, the basal overflow of tritium and the resting tension of the preparation were not affected during the exposure to all the antagonists. Regardless of the antagonist used, the maximal prejunctional facilitation was ~45%, whereas the maximal postjunctional inhibition was ~90% (fig. 3). Therefore, with 3 µM indomethacin in the medium, the noncholinergic component of the excitatory neuromuscular transmission accounted for 5% to 10% of the total response, indicating that the response under investigation was mainly cholinergic in nature. The potencies of the antagonists at prejunctional and postjunctional level are shown in table 1. The rank orders of the potency of antagonists at prejunctional and postjunctional level were: atropine > 4-DAMP > tripitramine > HHSiD > methoctramine > AF-DX 116 > pirenzepine, and atropine > 4-DAMP > HHSiD > tripitramine > pirenzepine > AF-DX 116 > methoctramine. For comparison purposes (table 1), the postjunctional affinity estimates (pA₂ values) of all the antagonists are also reported (see table 4). The correlation between the potencies of antagonists at prejunctional and postjunctional levels and the published constants for cloned and native muscarinic receptors is shown in tables 2 and 3, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A, Time course of [3H]ACh release evoked by EFS (3 Hz, 540 pulses) in the rat bladder strips in the absence (S1) and in the presence (S2, S3) of increasing concentrations of tripitramine (10 and 1000 nM: horizontal bars). Each point represents the radioactivity per gram of tissue in the superfusate collected in 3-min samples. Given are the mean ± S.E.M. of 5 experiments. B, Example of isometric contractions (C1,C2 and C3) evoked by EFS in a single bladder strip in the absence (C1) and in the presence of 10 nM (C2) and 1000 nM tripitramine (C3). Note the opposite effects of tripitramine at prejunctional (enhancement of [3H]ACh release) and postjunctional (depression of contractile responses) levels.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effects of muscarinic receptor antagonists on [3H]ACh outflow () and smooth muscle contraction () evoked by EFS (3 Hz, 540 pulses) in bladder strips from the rat. Control experiments were carried out in parallel, and the effects of the antagonists are given as a percentage of the corresponding control value. Given are the mean ± S.E.M. of 4-10 experiments.

View this table: [in this window] [in a new window]   TABLE 2 Correlation of log EC50 values for the facilitation of [3H]ACh outflow in the rat urinary bladder with constants for cloned (m) and native (M) muscarinic receptors

View this table: [in this window] [in a new window]   TABLE 3 Correlation of log EC50 values for the inhibition of contractile responses in the rat urinary bladder with constants for cloned (m) and native (M) muscarinic receptors

Antagonist postjunctional affinities. Muscarone caused concentration-dependent contractions with a log EC₅₀ value of 7.26 ± 0.03 (n = 19). All antagonists produced parallel shifts of the concentration-response curves to muscarone, without depression of the maximum response. Schild regression analysis was linear with a slope not significantly different from unity (fig. 4, table 4). The rank order of antagonist affinities (pA₂ values) was atropine > 4-DAMP > HHSiD > tripitramine > pirenzepine > AF-DX 116 > methoctramine (table 4).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Schild plots for atropine (), 4-DAMP (), HHSiD (), pirenzepine (), tripitramine (), methoctramine () and AF-DX 116 (). Each point represents the mean ± S.E.M. of 4-7 individual observations. The pA2 values and slopes for the regression lines are given in table 4.

	Discussion

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The main purpose of this study was the pharmacological characterization of muscarinic autoreceptors that inhibit the electrically evoked [³H]ACh release in urinary bladder strips from the rat. In strips preincubated with [³H]choline, EFS caused two effects: (1) an increase in overflow of radioactivity that can be assumed as a reliable marker of neuronal [³H]ACh release (D'Agostino et al., 1986, 1988) associated with (2) a contractile response of the smooth muscle. Therefore, in this study, antagonist-induced changes in both [³H] overflow and contraction were considered as due to blockade of muscarinic receptors located at prejunctional and postjunctional level, respectively.

In the urinary bladder from the rat, previous biochemical studies revealed the presence of M₂ and M₃ receptor subtypes, with the former site being predominant (Kamai et al., 1994; Monferini et al., 1988; Wall et al., 1991; Wang et al., 1995). The role of M₃ receptors was established in previous functional studies (D'Agostino et al., 1993; Longhurst et al., 1995) and related to postjunctional muscarinic receptors mediating smooth muscle contractility. The characterization of postjunctional receptors as an M₃ receptor was further corroborated in the present investigation with the use of additional antagonists, such as AF-DX 116 and tripitramine, which were included in this study to characterize the nature of prejunctional autoreceptors. In fact, the rank order of both potencies and affinities at postjunctional level of six subtype-preferring antagonists (atropine > 4-DAMP > HHSiD > tripitramine > pirenzepine > AF-DX 116 > methoctramine) was found to be similar and consistent with the activation of M₃ receptors (Caulfield, 1993; Lambrecht et al., 1995), thus excluding the participation of other muscarinic receptor subtypes in the contractile response (see selectivity profile in table 1). Additionally, potency relationship calculated as correlation coefficient between the potency of antagonists at postjunctional level and published affinity constants for the same antagonists was significant (P < .001) for the M₃ subtype only (table 3).

The comparison of prejunctional and postjunctional potencies of a series of muscarinic antagonists revealed that muscarinic receptors located at the two sides of the cholinergic cleft are pharmacologically different, as suggested in a preliminary study (D'Agostino et al., 1993). Analysis of antagonist effects showed that atropine was equipotent in suppressing the contractile response and facilitating the release of labeled neurotransmitter evoked by EFS (see table 1). Therefore, as expected for a nonselective antagonist, atropine was not able to discriminate between prejunctional and postjunctional levels. Pirenzepine, an antagonist possessing a 10-fold higher affinity at M₁ receptors than at M₃ receptors, in our study discriminated between prejunctional and postjunctional sites by 2-fold only (P < .05), suggesting as unlikely the participation of M₁ receptors.

HHSiD and 4-DAMP, which are known to possess an M₃/M₂ affinity ratio close to 10 but to have similar affinity at M₃ and M₄ sites (table 1), in our hands did not discriminate between prejunctional and postjunctional effects. This indicates that prejunctional muscarinic autoreceptors do not belong to the M₂ subtype. AF-DX 116 and methoctramine, selective M₂/M₄ antagonists, were more potent at the prejunctional than at postjunctional level (P < .01), showing a potency ratio of 2.5 and 30, respectively. These potency ratios correspond more closely to the M₄/M₃ affinity ratio than to the M₂/M₃ ratio, with the latter being close to 10 and 100 for AF-DX 116 and methoctramine, respectively (Caulfield, 1993). These data suggest that the M₃ receptors are not involved in the inhibition of [³H]ACh release in the bladder and further exclude the participation of M₂ subtypes in this mechanism. Furthermore, tripitramine, which is a potent and selective M₂ antagonist with an M₂/M₃ ratio of ~300 (Chiarini et al., 1995; Melchiorre et al., 1993), and an M₄/M₃ ratio of 8-10 times (Maggio et al., 1994), showed in our study a ratio of 9 (P < .01). This clearly indicates the involvement of M₄, and it does exclude involvement of M₂ receptor subtypes. Finally, when potency relationship was determined by correlating prejunctional log EC₅₀ values with published constants for cloned and native muscarinic receptor subtypes, the correlations were significant for both M₄ and M₅ subtypes, with the best correlation found (P < .001) for the M₄ subtype (see table 2). Although this evidence may result from a lack of antagonist selectivity between M₄ and M₅ subtypes, it is also compatible with the presence of M₅ receptors in the rat bladder. However, because M₅ receptors (like M₁ and M₃ receptors) preferentially couple to mobilization of intracellular calcium, by augmentation of phosphoinositide hydrolysis (Eglen et al., 1996; Felder, 1995), it is unlikely that they may participate in the inhibition of ACh release through this mechanism.

In conclusion, our results are compatible with the notion that the prejunctional muscarinic autoreceptors inhibiting depolarization-evoked ACh release from postganglionic cholinergic nerve endings in the rat urinary bladder belong to the M₄ subtype. In early studies, the presence of M₄ muscarinic receptors in the rat bladder was not detected immunologically using subtype-selective antibodies (Wall et al., 1991; Wang et al., 1995). Recently, however, using the reverse transcriptase-polymerase chain reaction, M₄ transcript has been identified (Braverman et al., 1997). This new molecular finding corroborates further our functional analysis, in which, unlike other studies (Alberts, 1995), prejunctional feedback mechanisms are operating in the physiological range because experiments were carried out in the absence of cholinesterase inhibitors. In fact, the eserine-induced "excess" of ACh in the synaptic cleft while is at work simultaneously with antagonists in regulating its own release may lead to an inadequate evaluation of the potencies of antagonists at prejunctional level (Starke et al., 1989). This would make uncertain the characterization of muscarinic autoreceptors, especially with antagonists possessing a narrow "selectivity window".

The finding that inhibitory M₄ autoreceptors are also operative in the bladder (Alberts, 1995) and the trachea of the guinea pig (Kilbinger et al., 1995) can be taken as evidence that these receptors, as well as the M₂ autoreceptors in the intestine (Eglen and Watson, 1996; Starke et al., 1989), may be involved, via inhibition of adenylyl cyclase activity (Eglen et al., 1996; Felder, 1995), in a general negative feedback mechanism regulating ACh release in peripheral cholinergic nerves. In the urinary bladder, negative feedback mechanisms are operating when the frequency of stimulation is in the range of 0.5 to 5 Hz (Alberts, 1995; D'Agostino et al., 1986, 1989; Somogyi and de Groat, 1992; present study). Conversely, M₁ autoreceptors that enhance [³H]ACh release from postganglionic cholinergic nerves are activated by higher frequencies (10 Hz) of stimulation (Somogyi et al., 1994). With regard to the final mode of action through which M₄ receptors inhibit transmitter release, these receptors in DNA-transfected NG 108-15 cells have been shown to inhibit a voltage-gated calcium current (Higashida et al., 1990), an effect that could be involved in the reduction of neurotransmitter release through inhibition of exocytosis.