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Keywords:

  • rat bladder function;
  • urodynamics;
  • serotoninergic antagonists

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Objective To evaluate the effects of antagonists of different subfamilies of 5-hydroxytryptamine (5-HT) receptors on bladder function in anaesthetized and conscious rats.

Materials and methods The urinary bladder of female anaesthetized rats was catheterized urethrally and filled with physiological saline until spontaneous bladder contractions occurred. Infravesical pressure was measured by a pressure transducer and displayed continuously on a chart recorder. The time of bladder quiescence (to the disappearance of rhythmic contractions) after injection with different compounds tested was recorded. Conscious rats underwent cystometry with chronically (infravesical) implanted catheters to continuously record bladder capacity (evaluated as amount of saline infused between voiding cycles) and maximal voiding pressure. The affinity for the human recombinant serotoninergic 5-HT1A subtype (inhibition of specific binding of [3H]8-hydroxy-2-(di-n-propylamino) tetralin) and the effects on the [35S]guanosine 5′-(γ-thio) triphosphate (GTPγS) binding in HeLa cells was also evaluated.

Results Among the compounds tested, only 4-(2′-methoxy-phenyl)-1-[2′-(n-2′′-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI) and methiothepin showed nanomolar affinity for the 5-HT1A receptors, the former being a neutral antagonist and the latter an inverse agonist in the [35S]GTPγS binding model. Intravenous injection of low doses of p-MPPI and methiothepin induced a dose-dependent disappearance of isovolumic bladder contractions in anaesthetized rats (> 10 min). At the highest doses, the dose-response curves were bell-shaped. The amplitude of bladder contractions was not markedly altered. The tested antagonists of 5-HT2, 5-HT3, 5-HT4, and 5-HT6 serotoninergic subtypes were poorly active or inactive in the model. Similarly, these compounds were inactive on cystometry in conscious rats, whereas p-MPPI and methiothepin induced a consistent increase in bladder capacity. Methiothepin also decreased the voiding pressure, whereas p-MPPI did not affect this variable.

Conclusions These findings confirm that only selective 5-HT1A receptor antagonists have favourable effects on the bladder, inducing an increase in bladder capacity with no derangement of bladder contractility.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Bladder overactivity and urinary incontinence caused by bladder overactivity are common disorders; an incidence of 33–61% has been reported in the elderly (> 65 years old [1,2]). Bladder overactivity may have different pathogeneses; sensitization of peripheral afferent nerve terminals in the bladder or damage to central inhibitory pathways [3], and increased sensitivity to efferent stimulation in the detrusor muscle [4], or a combination of these factors. Therefore, spinal and supraspinal structures of the CNS, and peripheral structures (bladder, urethra, ganglia or peripheral nerves) may be drug targets for treating bladder overactivity. Traditionally, drugs used to treat bladder overactivity and incontinence have had a peripheral site of action [5], but drugs acting in the CNS could be developed.

Several neurotransmitters have been identified as inhibitory transmitters in the micturition reflex pathways at both spinal and supraspinal sites, including 5-HT, γ aminobutyric acid, glycine, dopamine, acetylcholine, enkephalins and other peptides [6]. The electrical stimulation of 5-HT-containing neurones in the caudal raphe and activation of postsynaptic 5-HT receptors in the spinal cord of cats, via the release of 5-HT, inhibits bladder contractions [7].

Many 5-HT receptors have been characterized in mammalian species and divided into seven subfamilies (from 5-HT1 to 5-HT7) based on their affinity for different 5-HT agonists and antagonists and/or gene structure [8]. Some 5-HT receptor subfamilies have been further subdivided, e.g., 5-HT1 receptors, the largest subclass of 5-HT receptors, consist of five subtypes, with the 5-HT1A receptor being the most extensively investigated and characterized.

Our previous studies [9] showed that 5-HT1A receptor neutral antagonists influence the central control of lower urinary tract function, decreasing the frequency of bladder voiding contractions and increasing bladder capacity. To further extend knowledge of the role of 5-HT receptor antagonists on the micturition reflex, we assessed the effects of available antagonists of the different 5-HT families in the anaesthetized rat bladder isovolumic-contraction model, and on cystometrographic variables in conscious rats. The affinity of the compounds tested for the human recombinant 5-HT1A receptors, and their effects on [35S]guanosine 5′-(γ-thio)triphosphate (GTPγS) binding in HeLa cells (a functional model of receptor-mediated G-protein activation) were also evaluated. The following antagonists were evaluated: 4-(2′-methoxy-phenyl)-1-[2′-(n-2′′-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI, 5-HT1A selective [10]), GR 55562 (5-HT1B selective [11]), ketanserin (5-HT2A partially selective [12]), SB 242084 (5-HT2C selective [13]), mesulergine (5-HT2 nonselective [12]), Y 25130 and zatosetron (5-HT3 selective [14,15]), RS 39604 (5-HT4 selective [16]), Ro 04–6790 (5-HT6 selective [17]), and methiothepin, a compound showing high affinity for most of 5-HT subfamilies, including 5-HT2A,6,7[18]. Some of the present data are published in abstract form [19].

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Male and female Sprague Dawley rats (Crl:CD (SD) BR, 200–300 g body weight; Charles River, Italy) were used in the experiments. Animals were housed with free access to food and water and maintained on a forced 12 h light-dark cycle at 22–24 °C for at least one week before the experiments were carried out. The animals were handled according to internationally accepted principles for the care of laboratory animals (EC Council Directive 86/609, O. J. no L358, 18/12/86).

Binding studies

The affinity of tested compounds for the human recombinant 5-HT1A serotoninergic subtype in HeLa cells was evaluated using [3H]8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) as the labelled ligand. Briefly, HeLa cells were detached from the growth flask at 95% confluence by a cell scraper and lysed in ice-cold Tris (5 mmol/L) and EDTA (5 mmol/L) buffer (pH 7.4). Homogenates were centrifuged for 20 min at 40 000 g, the pellets resuspended in a small volume of ice-cold Tris/EDTA buffer, immediately frozen and stored at −70 °C until use. On the day of the experiment, cell membranes were resuspended in binding buffer (50 mmol/L Tris, 2.5 mmol/L MgCl2, 10 µmol/L pargiline, pH 7.4) and incubated in a final volume of 1 mL for 30 min at 30 °C with 1 nmol/L [3H]8-OH-DPAT, in the absence or presence of various concentrations of the competing drugs (1 pmol/L to 10 µmol/L); each estimate was triplicated. Nonspecific binding was determined in the presence of 10 µmol/L 5-HT.

To evaluate the effects of the compounds tested on [35S]GTPγS binding in HeLa cells, cell membranes were resuspended in buffer containing 20 mmol/L HEPES, 3 mmol/L MgCl2 and 120 mmol/L NaCl (pH 7.4). The membranes were incubated with 30 µmol/L GDP and different concentrations (0.1 nmol/L to 100 µmol/L) of test drugs or 5-HT (reference curve) for 20 min at 30 °C in a final volume of 0.5 mL. Samples were transferred to ice, [35S]GTPγS (200 pmol/L) added and samples incubated for a further 30 min at 30 °C. Antagonism to 5-HT-induced stimulation of [35S]GTPγS binding was evaluated on cell membranes resuspended as described above, and incubated with 30 µmol/L GDP and various concentrations of 5-HT (from 0.1 nmol/L to 100 µmol/L) in the absence or presence of a fixed concentration of antagonist, for 20 min at 30 °C in a final volume of 0.5 mL. Samples were transferred to ice, [35S]GTPγS (200 pmol/L) added and samples incubated for a further 30 min at 30 °C. For both procedures, nonspecific binding was determined in the presence of 10 µmol/L GTPγS. Incubation was stopped by adding ice-cold HEPES buffer and rapid filtration on filters using a cell harvester. The filters were washed with ice-cold buffer and the radioactivity retained on the filters counted by liquid scintillation spectrometry at an efficiency of > 90%.

Activity on isovolumic bladder contractions in anaesthetized rats

Female rats were anaesthetized with a subcutaneous injection of urethane 1.25 g/kg (5 mL/kg), and the urinary bladder catheterized urethrally with polyethylene tubing (0.58 mm internal diameter, ID, 0.96 mm outer, OD) filled with physiological saline. The catheter was tied in place with a ligature around the external urethral orifice, and the intravesical pressure measured using a pressure transducer connected to the external end of the bladder cannula, displayed continuously on a chart recorder. The bladder was filled through the recording catheter by incremental volumes of warmed (37 °C) saline until spontaneous bladder contractions occurred (usually 0.8–1.5 mL) as a result of central activity. The activity of the tested compounds was assessed after the intravenous administration (through a polyethylene cannula inserted into the jugular vein) in individual animals (at least 4–6 rats per group per dose), by measuring the duration of bladder quiescence (the time for the disappearance of contractions). Each rat was challenged with vehicle before injecting with the active substances and the maximal duration of bladder quiescence during a 15-min period after each injection recorded. The effects of the tested compounds on the amplitude of bladder contraction were estimated comparing them (when contractions re-started) with those previously recorded for 15 min after the intravenous administration in the same animals of vehicle alone.

Cystometrography in conscious rats

Male rats, anaesthetized with an intraperitoneal administration of 3 mL/kg of Equithensin solution, were placed supine and a ≈ 10 mm long midline incision made in the shaved and cleaned abdominal wall. The urinary bladder was gently freed from adhering tissues, emptied and then cannulated, via an incision in the dome, with a polyethylene cannula (0.58 mm ID, 0.96 mm OD), which was permanently sutured with silk thread. For intravenous bolus injection a similar polyethylene tube filled with physiological saline was inserted into the jugular vein. The cannulae were exteriorized through a subcutaneous tunnel in the retroscapular area, where they were connected with a plastic adapter, to avoid the risk of removal by the animal. For drug testing, the rats were used one day after implantation. On the day of the experiment, the rats were placed in Bollman's cages; after a stabilization period of ≈ 20 min, the free tip of the cannula was connected through a T-shaped tube to a pressure transducer and to a peristaltic pump for the continuous infusion of warmed saline solution (37 °C) into the urinary bladder, at a constant 0.1 mL/min. The intraluminal pressure signal during the infusion of saline into the bladder was recorded continuously on a polygraph and two urodynamic variables of the cystometrogram evaluated, i.e. the volume of saline infused in the bladder necessary to induce detrusor contractions followed by micturition (BVC) and the maximal intravesical pressure determined by the contraction of the detrusor during micturition (MP). The basal BVC and MP were evaluated as the mean of the values in the first two complete and reproducible recorded cystometrograms. At this point, drugs were administered intravenously by bolus injection under continuous filling of the bladder. Changes in BVC and MP were evaluated from the cystometrograms at ≈ 15, 30, 45 and 60 min after treatment.

p-MPPI HCl, ketanserin(+) tartrate, methiothepin mesylate, Ro 04–6790 2HCl and mesulergine HCl were from Research Biochemicals International, Natick, MA, USA. GR 55562, Y25130 HCl and RS 39604 HCl were from Tocris Cookson Ltd., Avonmouth Bristol, UK. Zatosetron maleate and SB 242084 were a gift from K.B. Thor (Eli Lilly and Co., Indiana, USA) and J.P. Hieble (SmithKline Beecham Pharmaceuticals, Pennsylvania, USA), respectively. [3H]8-OH-DPAT and [35S]GTPγS) were obtained from NEN Life Science Products, Milan, Italy. All the other substances were from commercial sources. For receptor-binding studies, the compounds were dissolved in absolute alcohol or demineralized water according to their solubility. For intravenous administration, methiotepin and Ro 04–6790 were dissolved in saline solution; p-MPPI, GR 55562, Y25130, mesulergine and ketanserin were dissolved in demineralized water; RS 39604, zatosetron and SB 242084 were dissolved using N,N-dimethylformamide (4% v/v) and TWEEN 80 (8% v/v) in demineralized water. All the reported doses were those of the corresponding salts or bases. The Equithensin solution for animal anaesthesia had the following composition: pentobarbital 1.215 g, chloral hydrate 5.312 g, magnesium sulphate 2.657 g, ethanol 12.5 mL, propylene glycol 49.5 mL, distilled water to 125 mL final volume.

Statistical analysis

The inhibition of specific binding of [3H]8-OH-DPAT by the tested drugs was analysed to estimate the concentration required for 50% inhibition (IC50), using the nonlinear curve-fitting program ALLFIT [20]. The IC50 value was converted to an affinity constant (Ki) by the equation of Cheng and Prusoff [21]. Stimulation or inhibition of [35S]GTPγS binding in HeLa cells expressing the recombinant human 5-HT1A receptor induced by the compounds tested was expressed as a percentage of the basal value, the maximum stimulation with 5-HT taken as 100%. The antagonism toward [35S]GTPγS binding was quantified by evaluating the shift to the right of the 5-HT concentration-response curve (CRC) in presence of the concentration of tested compounds. The apparent pKB value was evaluated according as log10 ([B]/(Dr-1)), where [B] is the antagonist concentration and the dose-ratio (Dr) is the ratio between the concentrations of agonist required to produce a half-maximal response in the presence and the absence of the antagonist.

In the isovolumic bladder contractions model, the maximum time between consecutive contractions during the 15-min period after vehicle injection, and the time of disappearance of bladder contractions after injecting different doses of the compounds tested, were expressed as the mean (sem). The statistical significance of the difference of bladder quiescence recorded after vehicle vs compound injection was assessed using Student's t-test for paired data.

The BVC and MP values were assessed as the difference between vehicle and active treatments effect, evaluated by the general linear model procedure, repeated measures anova, a univariate test of hypotheses for within subject effects and anova of contrast variables. The difference between the values at different times from the basal values was evaluated by the least squares means procedure.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Binding studies

In our previous report [9] we showed that only compounds with a high affinity for the 5-HT1A receptors and behaving as neutral antagonists had favourable effects on the bladder. Therefore, the affinity of all the compounds was evaluated for this serotoninergic subtype.

p-MPPI was the most potent compound, with a pKi of 8.6 (0.02); methiothepin also showed high affinity, with a pKi of 8.4 (0.11), only slightly lower than that of p-MPPI. Ketanserin, SB 242084, Y 25130, zatosetron, RS 39604, and Ro 04–6790 had pKi values of < 6.0, whereas GR 55562, with a pKi of 6.2 (0.04) and mesulergine of 6.0 (0.16), showed micromolar affinity for the 5-HT1A receptors.

The two compounds with high affinity for the 5-HT1A receptors (p-MPPI and methiothepin) were also tested in the functional model of receptor-mediated G-protein activation (stimulation of [35S]GTPγS binding in HeLa cells expressing the cloned human 5-HT1A receptor). As shown in Fig. 1 (upper panel), p-MPPI behaved as a neutral antagonist, whereas methiothepin was an inverse agonist, inducing a decrease of the basal binding of [35S]GTPγS, in agreement with previous findings [22]. The antagonistic activity of these compounds was evaluated as the inhibition of the 5-HT-induced increase of [35S]GTPγS binding, as shown in Fig. 1 (lower panel).

image

Figure 1. Stimulation of [35S]GTPγS binding at recombinant human 5HT1A receptors stably expressed in HeLa cells by different concentrations of: upper panel, 5-HT (green), p-MPPI (light red) and methiothepin (red); lower panel, 5-HT in the absence (green) and presence (light red) of 30 nmol/L p-MPPI or 100 nmol methiothepin (red). The values are the percentage of the maximum binding by 5-HT, the points being single replicates from a representative experiment.

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Both p-MPPI and methiothepin (30 and 100 nmol/L, respectively) produced a dose-dependent parallel rightward shift of the 5-HT CRC, with no apparent reduction in Emax, indicative of competitive antagonism. The pKB values were 9.3 (0.28) and 8.7 (0.25), respectively.

Activity on isovolumic bladder contractions in anaesthetized rats

In urethane-anaesthetized rats, distension of the urinary bladder by filling with saline through a transurethral catheter produced a series of rhythmic bladder contractions which were fairly reproducible and repetitive. The frequency of these contractions in untreated rats was 0.4–1.3 contractions/min, with a peak amplitude of 25–50 cmH2O. In general, the time between consecutive contractions was < 1–1.5 min (Table 1).

Table 1.  Effects on isovolumic bladder contractions after intravenous administration of drugs in anaesthetized rats. The values are the time of bladder quiescence (disappearance of contractions) after injection with vehicles (before) and indicated doses (after) of the tested compounds
AntagonistDose (µg/kg), before/after
310301003001000
  • *

    P  < 0.01;

  • P  < 0.001 vs before, Student's t-test for paired data.

Mean (sem) time, min
5-HT1
p-MPPI-0.78 (0.11)1.18 (0.34)1.03 (0.14)1.72 (0.37)1.90 (0.35)
-1.08 (0.15)7.52 (0.99)15.98 (2.40)11.75 (0.87)7.97 (1.05)*
GR 55562---0.93 (0.11)-0.97 (0.26)
---1.20 (0.27)-1.30 (0.57)
5-HT2
Mesulergine-1.05 (0.11)0.78 (0.09)1.00 (0.09)1.30 (0.19)1.55 (0.17)
-1.57 (0.18)1.18 (0.12)1.05 (0.11)1.33 (0.23)1.07 (0.15)
Ketanserin---1.21 (0.11)0.98 (0.07)1.29 (0.11)
---2.55 (0.51)*2.76 (1.09)2.57 (0.77)
SB 242084---0.83 (0.06)0.75 (0.32)0.85 (0.20)
---1.58 (0.64)0.68 (0.06)1.58 (0.39)
5-HT3
Zatosetron0.95 (0.22)1.20 (0.30)1.00 (0.04)0.89 (0.18)-0.92 (0.20)
2.20 (0.49)5.35 (1.97)3.39 (1.43)4.49 (2.27)-4.77 (1.51)
Y 25130---1.07 (0.13)-1.24 (0.16)
---3.48 (0.89)*-5.50 (2.50)
5-HT4
RS 39604---1.03 (0.20)1.28 (0.18)1.20 (0.09)
---2.65 (1.52)3.63 (0.77)5.54 (1.89)
5-HT6
Ro 04–6790---0.80 (0.18)-1.08 (0.29)
---1.00 (0.50)-1.30 (0.68)
Not selective
Methiothepin1.24 (0.34)1.30 (0.20)2.03 (0.49)1.38 (0.24)1.37 (0.18)1.22 (0.24)
3.58 (1.30)5.10 (1.68)9.32 (2.75)10.92 (1.84)9.23 (0.94)4.60 (0.91)

Injection with the different vehicles used for the compounds only sporadically induced a block of bladder contractions of > 2–2.5 min and did not affect their amplitude. Intravenous injection with low doses (10 to 100 µg/kg) of the 5-HT1A antagonist p-MPPI induced a dose-dependent disappearance of bladder contractions, to ≈ 16 min of bladder inactivity (Fig. 2). At the highest doses tested (300–1000 µg/kg) the inhibition induced by the compound showed a bell-shaped effect. The amplitude of bladder contractions was not markedly altered (< 20% and not dose-dependent), indicating no direct effect upon bladder contractility (Fig. 2).

image

Figure 2. Representative cystometrograms showing the effects of p-MPPI (upper tracing 30 µg/kg; lower tracing 100 µg/kg) on isovolumic bladder contractions in anaesthetized rats. Open and closed arrows indicate vehicle and drug injection, respectively. Note (a) the short disappearance time of bladder contractions after vehicle injection compared with the effect of p-MPPI (lower tracing); (b) the amplitude of contractions was unaffected by vehicle and compound. Vertical bars 25 cmH2O; horizontal bars 5 min.

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The 5-HT1B antagonist GR 55562, and all the 5-HT2 (mesulergine, ketanserin, and SB 242084) and 5-HT6 (Ro 04–6790) antagonists tested had no blocking effects on bladder contractions at doses up to 1.0 mg/kg. After injecting the lowest doses of mesulergine and ketanserin, there was a statistically significant difference from basal values. However, the disappearance time recorded after treating with the lowest doses of these two compounds was in the range of that generally recorded in the different groups of rats before treatment, and may be considered insignificant. The lack of effect of GR 55562 and Ro 04–6790 was confirmed at the highest doses (10 and 3 mg/kg, respectively; data not shown). In some animals, mesulergine and SB 242084 showed a tendency to increase the frequency of bladder contractions.

The 5-HT3 (Y 25130 and zatosetron) and 5-HT4 (RS 39604) antagonists induced no dose-dependent disappearance of bladder contractions not exceeding 6 min. Methiothepin showed a bell-shaped dose–response curve, with the maximal inhibitory effect at 30–300 µg/kg.

Cystometrography in conscious rats

Only the compounds showing activity in the isovolumic contractions model (blocking contractions for >4 min) were also evaluated in conscious rats. Generally, the doses eliciting the maximum effect on the isovolumic contractions were tested in the cystometry model. Injection with p-MPPI at 0.3 mg/kg induced a consistent (> 40%) and significant increase in BVC, compared with both basal values and saline-injected animals, through the observation period. In the same animals, bladder contractility was not substantially changed and did not differ from changes in the controls, except for the first recording (Fig. 3). After injecting with the higher dose (1.0 mg/kg), the effects on BVC were generally lower than those recorded with 0.3 mg/kg, confirming the bell-shaped dose–response curve previously observed in the contraction model. The MP was not changed at this dose (Fig. 3).

image

Figure 3. The time-course of the effects of an intravenous injection with saline (green) and p-MPPI (0.3 mg/kg, red; 1 mg/kg, light red) on BVC (left) and MP (right) in conscious rats. The values are the percentage change from basal values at different times during infusion. The significance (< …) shown is between treatments (anova of contrast variables), indicating the difference between the trend observed in the control (saline) and treated groups at each time. **P < 0.01, ***P < 0.001 vs basal values (within treatment).

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Methiothepin (0.1 mg/kg) induced increases in BVC statistically different from basal values through the observation period, and different from control animals at 60 min. After 1.0 mg/kg, the increase in BVC was significantly different from basal values and saline-treated rats only 15 min after administration (Fig. 4). This compound induced a dose-dependent and statistically significant decrease in MP, reaching about half after giving the higher dose.

image

Figure 4. The time-course of the effects of injection with saline (green) and methiothepin (0.1 mg/kg, red; 1.0 mg/kg, light red) on BVC (left) and MP (right) in conscious rats. The values are the percentage change from basal values at different times during infusion. The significance (< …) shown is between treatments (anova of contrast variables), indicating the difference between the trend observed in the control (saline) and treated groups at each time. *P < 0.05, **P < 0.01 and ***P < 0.001 vs basal values (within treatment).

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Zatosetron (1 mg/kg) was chosen as an example of the 5-HT3 antagonists. The BVC in rats treated with the compound tended to increase with time and was significantly different from baseline 1 h after injection (31% increase). As a similar (and significant) increase in BVC also occurred in the control group, the time-course of the changes induced by zatosetron was not significantly different from that in saline-injected rats. The MP was sporadically increased (compared with basal values) in the control group, and decreased in treated animals. The time-course of MP changes in treated rats was significantly different from that of controls 15 and 60 min after zatosetron injection (data not shown). However, the decrease in MP induced by the compound was leqslant R: less-than-or-eq, slant 15% from the basal value and may be considered insignificant. The administration of the 5-HT4 antagonist RS 39604 (1 mg/kg) caused no significant effects on either BVC or MP (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

Normal bladder function requires co-ordinated detrusor relaxation and urethral sphincter contraction during the filling phase, and the converse during micturition. This is achieved by the integration of excitatory, inhibitory and sensory nerve activity in control centres in the spinal cord, pons and forebrain. The descending bulbospinal pathway to the urinary bladder is essentially an inhibitory circuit involving the release of 5-HT at the spinal level [6,7]. Serotonin within the dorsal horn of the spinal cord arises primarily from neurones in the pontomedullary raphe nuclei [23]. Electrical stimulation of 5-HT-containing neurones of nucleus raphe magnum and activation of postsynaptic 5-HT receptors in the spinal cord of cats inhibit bladder contractions and reflex firing in the sacral efferent pathways to the bladder [7,24]. Stimulating the nucleus raphe magnum also inhibits the firing of spinal dorsal horn neurones activated by afferents in the pelvic nerve [25], and raphe neurones are activated by bladder distension [25,26]. This raises the possibility that afferent input from the bladder may trigger a reflex loop, resulting in activation of a raphe-spinal inhibitory pathway which could suppress the micturition reflex.

On the cell bodies and dendrites of serotoninergic neurones within the raphe nuclei, 5-HT receptors are of the 5-HT1A subtype [27]. This evidence prompted an evaluation of the effects of several 5-HT1A ligands on bladder function [9]. Our findings showed that neutral 5-HT1A antagonists have favourable effects on the bladder. These compounds induced a marked block in voiding contractions in anaesthetized rats, and increased bladder capacity without consistently impairing bladder contractility in conscious animals. On the contrary, agonists or partial agonists of this subtype generally behaved in the opposite way [9,28].

The effects of 5-HT1A ligands on the micturition reflex in rats were clearly exerted at a central level, as both the selective agonist 8-OH-DPAT [28] and the neutral antagonist WAY 100635 [29] were inactive on the peripheral control of the bladder.

Because there are several families of central 5-HT receptors [30] there is a need to study the effects on the lower urinary tract of compounds with different antagonistic profiles for the 5-HT receptor subtypes. From receptor-binding profiles, common secondary messenger coupling and the functional activity of ligands, four main subgroups of 5-HT receptors, termed 5-HT1, 5-HT2, 5-HT3, and 5-HT4, were identified. Molecular biological techniques have both confirmed this classification and led to the identification of novel 5-HT receptors (5-HT1F, 5-HT5, 5-HT6, and 5-HT7), that have been cloned and characterized [8,18]. Besides the 5-HT1A and the 5-HT1F, at least three other 5-HT1 receptor subtypes (5-HT1B, 5-HT1D, and 5-HT1E) have been recognized, whereas the 5-HT2 group has been divided into three subgroups (5-HT2A, 5-HT2B, and 5-HT2C).

Among the compounds tested in the present study, p-MPPI and methiothepin only showed relevant affinity for the 5-HT1A subtype (pKi values of 8.6 and 8.4, respectively) and were potent in blocking isovolumic voiding contractions in anaesthetized rats after the administration of 30–300 µg/kg. All the other compounds were poorly active (5-HT3 and 5-HT4 antagonists) or inactive (5-HT1B, 5-HT2 and 5-HT6 antagonists) after the administration of doses up to 1000 µg/kg. Furthermore, p-MPPI and methiothepin significantly increased the BVC in conscious rats, whereas 5-HT3 and 5-HT4 antagonists were inactive.

Espey and Downey [31] showed that intrathecal methysergide (a partially selective 5-HT2A/2C antagonist) induced a reduction in the volume at which micturition occurs in conscious, spinally intact cats. Accordingly, in the present study, mesulergine, at 0.1 and 1 mg/kg (data not shown), induced a decrease (20–37%) in BVC and MP that was not dose-dependent. Furthermore, the partially selective 5-HT2C agonist mCPP [32], was active on the micturition reflex [33], and its effects were blocked by mesulergine [34]. All these data together indicate that the 5-HT2C subtype is involved in the micturition reflex, but argue against the use of 5-HT2C antagonists to improve bladder control.

Espey et al.[35] showed that intrathecal zatosetron decreased the BVC in spinally intact cats, and suggested that endogenous 5-HT, through activation of 5-HT3 receptors, inhibits micturition. The present data in rats are partly in agreement with these previous findings, as zatosetron had no effects on the BVC in conscious rats, but both the 5-HT3 antagonists weakly and not dose-dependently inhibited bladder voiding contractions in anaesthetized rats.

Evidence for a peripheral inhibitory effect in the urinary bladder of monkeys [36] and an excitatory effect in man exerted through the 5-HT4 receptor [37] has been reported. However, after administering the 5-HT4 antagonist tested there was only a slight inhibitory effect on the bladder contractions in anaesthetized rats and no effects on bladder capacity in conscious rats. The 5-HT3 and 5-HT4 subtypes seem to be involved in the micturition reflex, but selective antagonists of these subtypes did not affect bladder voiding in the desired way.

In conclusion, the present data confirm our previous findings [9] that 5-HT1A antagonists have favourable effects on the bladder and exclude the possibility that any antagonism at the level of the 5-HT1B, 5-HT2, 5-HT3, 5-HT4, and 5-HT6 subtypes can positively influence the central control of bladder function. Methiothepin also shows affinity for the 5-HT7 subtype [18], so that any relevance to micturition of blocking this subtype cannot be excluded.

Although the receptor profile of the antagonists used in the present study support the preferential involvement of the 5-HT1A subtype, we cannot exclude a degree of nonselectivity. Therefore, the availability of more selective 5-HT antagonists is crucial to better understand the physiological relevance for bladder control of blocking other serotonergic subtypes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

The skilful technical assistance of R. Cova and I. Simonazzi is gratefully acknowledged.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors
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Authors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Authors

R. Testa.

L. Guarneri.

P. Angelico.

C. Velasco.

E. Poggesi.

A. Cilia.

A. Leonardi.