The role of central 5-hydroxytryptamine (5-HT, serotonin) receptors in the control of micturition

Abstract

At present the most investigated 5-HT receptor that has been shown to play a role in the control of micturition is the 5-HT_1A receptor followed by 5-HT₇, 5-HT₂ and 5-HT₃ receptors. Most experiments focus on the control these receptors have on the parasympathetic outflow to the bladder and the somatic outflow to the external urethral sphincter (EUS) in the rat. Furthermore, 5-HT_1A and 5-HT₇ receptors have been identified as having an excitatory physiological role in the control of bladder function. 5-HT_1A receptors act, at least in the rat, at both a spinal (probably a heteroreceptor) and supraspinal (probably an autoreceptor) level, while 5-HT₇ receptors only act at a supraspinal level. Additionally, in the rat, 5-HT administered at a spinal or supraspinal site has an excitatory action, although earlier experiments have shown that activating 5-HT-containing brain areas causes inhibition of the bladder. Recent experiments have also indicated that blockade of the 5-HT_1A receptor pathway shows rapid tolerance. However, no data exist for the development of tolerance for the 5-HT₇ receptor pathway. Neither receptor seems to play a role in the control of the urethra. Regarding 5-HT₂ receptors, activation of this receptor subtype inhibits micturition, and this inhibitory action may occur at a spinal, supraspinal or both levels. Although no physiological role for 5-HT_2C receptors can yet be identified, 5-HT_2C receptors have been implicated in the proposed supraspinal tonically active 5-HT_1A autoreceptor (negative feedback) pathway. This proposition reconciles the data that central 5-HT-containing pathways are inhibitory to micturition, while 5-HT_1A receptors, although inhibitory to adenylyl cyclase, have an excitatory function. This is because activation of 5-HT_1A autoreceptors reduces the release of 5-HT thus reducing the activation of the 5-HT_2C receptors, which are inhibitory in the control of micturition (disinhibition). Furthermore, 5-HT_2A receptors in the rat and 5-HT_2C receptors in the guinea pig cause activation of the EUS. In this respect, 5-ht_5A receptors have also been identified in Onuf's nucleus, the site of somatic motoneurones controlling this sphincter. In the cat there is very little evidence to indicate that 5-HT receptors are involved in micturition except under pathological conditions in which activation of 5-HT_1A receptors causes inhibition of micturition. Interestingly, under such conditions 5-HT_1A receptors cause excitation of the EUS. Nevertheless, spinal 5HT₃ receptors have been implicated in the physiological control of micturition in the cat, but not yet in the rat. Overall, the data support the view that 5-HT receptors are important in the control of micturition. However, many more studies are required to fully understand these roles and why there are such species differences.

Introduction

Along with other parts of the autonomic nervous system, for example heart, vasculature and airways (see Ramage, 2001), central 5-HT receptors have been implicated in the control of micturition (see De Groat et al., 1993). There are 14 different, structurally distinct 5-HT receptors, which are divided up into seven families (5-HT_1–7), and those receptors that have already been implicated in the control of micturition are the 5-HT₁, 5-HT₂ and 5-HT₃ receptors. These receptors seem to modulate all the pathways involved in the control of micturition, the parasympathetic, the sympathetic and the somatic (see De Groat et al., 1993), although there is little experimental data on the sympathetic pathway. These conclusions are based on the use of such 5-HT receptor ligands as 5-methoxydimethyl tryptamine, lysergic acid diethylamide (LSD), methysergide and 8-OH-DPAT, the archetypical 5-HT_1A receptor agonist. Although 8-OH-DPAT can be considered much more selective than the other compounds for a particular 5-HT receptor subtype, it is still not specific (see Ramage, 2004). More recently, the 5-HT₇ receptor (Read et al., 2003) and, interestingly, the least well-understood 5-HT receptor, the 5-ht_5A receptor, (Doly et al., 2004), have been added to this list. As with all areas of pharmacology, our understanding is always dependent on the known selectivity of the particular ligands used plus the development of novel ligands for these 5-HT receptors. In addition, it is becoming clear that different species may give a different interpretation to the function of these receptor subtypes. In this respect, a great deal of our original understanding of the mechanisms involved in the control of micturition comes from the cat. However, the rat has now taken over as the main species for the investigation of urine storage and micturition reflexes. Importantly, the guinea pig may begin to displace the rat as species of choice for micturition studies, since this species has a guarding reflex, something that is not observed in the rat. That is, in the guinea pig, before voiding, the external urethral sphincter (EUS) has an increase in activity, which shuts off during expulsion of urine. Once expulsion has finished, activity increases again before finally switching off. On the other hand, in the rat, the EUS is initially activated in bursts during voiding, and this probably aids in micturition.

In addition to the recognition that certain 5-HT receptor subtypes are involved in the control of micturition, central 5-HT-containing areas, for example, the brainstem raphé, when activated in both cat and rat, have been shown to cause inhibition of micturition (McMahon & Spillane, 1982; Chen et al., 1993; Sugaya et al., 1998). These areas also inhibit the firing of spinal dorsal horn neurones, which are activated by afferents in the pelvic nerve (Lumb, 1986a). Further, neurones in the raphé are activated by bladder afferents (McMahon & Spillane, 1982; Sugaya et al., 1998) as well as distension of the bladder (Oh et al., 1986; Lumb, 1986b). Interestingly, in the cat this inhibition of bladder reflexes can be blocked by i.v. or topical application of LSD to the sacral spinal cord (Morrison & Spillane, 1986). The problem here is knowing whether this is due to an agonist or antagonist action of LSD. Thus, from these stimulation experiments the view has evolved that 5-HT-containing pathways play an inhibitory role in the control of micturition. Nevertheless, more recent data using new pharmacological tools suggest, at least in the rat, that 5-HT pathways may be excitatory as well (see below). Thus, it is the purpose of this review to reconsider some of the older concepts and integrate the newer experimental evidence that implicates the involvement of different 5-HT receptor subtypes in micturition. This will also be carried out in the light of the newer developments in our understanding of the pharmacology of these various 5-HT receptor subtypes.

5-HT_1A receptors

Rats – agonist studies

Giving either 8-OH-DPAT or buspirone i.v., Lecci et al. (1992) showed that, in the rat, these drugs could induce rhythmic bladder contractions in bladders 8% filled (approx. 100 μl saline) but not in empty bladders. For 8-OH-DPAT this effect was not dose related and for buspirone it only occurred at the lowest dose (0.1 mg kg⁻¹). This action also occurred in spinalized rats and was inhibited by i.v. spiroxatrine. These responses did not show desensitization. Similar data were obtained with i.t. (intrathecally – lumbar level) and i.c.v. administration of 8-OH-DPAT (see Figure 1a). Depletion of 5-HT by 5,7-dihydroxtryptamine (5,7-DHT) increased the effective dose of i.c.v. 8-OH-DPAT but did not affect i.t. 8-OH-DPAT. This indicates that 5-HT_1A receptors in the brainstem are probably autoreceptors located on 5-HT terminals and/or on 5-HT-containing neurones (somatodendetric autoreceptors), while those at the level of the sacral spinal cord are heteroreceptors, controlling the release of another transmitter. Furthermore, distension-induced bladder contraction (isovolumetric contractions) could be blocked by i.v. buspirone (high doses), spiroxatrine and NAN-190 and a very high dose of methysergide. Further, evidence that 5-HT_1A receptor activation was excitatory came from anaesthetized rats (Conley et al., 2001) showing that 8-OH-DPAT and BMY-7378 (a partial 5-HT_1A receptor agonist and α_1D-adrenoceptor antagonist) decreased the pressure and volume threshold to evoke a micturition reflex (these values would be expected change in parallel but as volume threshold also gives an indication of any changes in compliance of the bladder i.e. if the sympathetic has been switched in to aid storage, discrepancy between these two values would indicate a change in bladder compliance); again these effects were not dose related. However, activation of 5-HT_1A receptors did not affect the associated urethral reflexes. Using cystometry in conscious rats (Testa et al., 1999; Pehrson et al., 2002) showed that 8-OH-DPAT i.v. decreased bladder capacity, micturition volume and pressure and this was also observed (Pehrson et al., 2002) for i.c.v. and i.t. administration (bladder capacity is the sum of the residual volume plus the micturition volume). Interestingly, for i.t. administration there was an associated increase in bladder pressure (this measurement indicates the level of parasympathetic drive to the bladder). Similar effects were observed for 5-HT given i.t. (Pehrson et al., 2002) and i.c.v. (Ishizuka et al., 2002; see Figure 1b). The effect of 5-HT i.c.v. lasted for 60 min and was repeatable and for higher doses 50% of the rats had urinary incontinence, presumably due to this hyperactivity of the bladder. However, BMY-7378 was with little effect when given i.v. (Testa et al., 1999). Nevertheless, BMY-7378 although reported not to affect cystometric variables in conscious, bladder obstructed rats, inhibited nonvoiding contractions (Velasco et al., 2003). These variable results with BMY-7378 may just simply reflect its lack of pharmacological selectivity. Thus, data indicate that 5-HT_1A receptors along with 5-HT at supraspinal and spinal sites have an excitatory effect on micturition in the rat.

Figure 1.

Rats: experimental traces showing the effects of: – (a) the 5-HT_1A receptor agonist 8-OH-DPAT on baseline bladder pressure given intrathecally (i.t.) and intracerebroventricular (i.c.v.), (b) 5-HT (A) and 8-OH-DPAT (B) given i.c.v. in conscious rats on bladder pressure and volume changes evoked by bladder distension induced by infusing saline into the bladder, (c) the 5-HT_1A receptor antagonists Rec 0/0259 and WAY-100635 given i.v. on rhythmic isovolumetric bladder contractions and (d) effect of WAY-100635 i.v. on changes evoked in bladder and urethral pressure caused by dissention induced by infusion of saline into the bladder.

Antagonist studies

In support of the view above that 5-HT_1A receptors are part of the mechanism by which the rat controls micturition, Lecci et al. (1992) showed that i.v. high doses of buspirone (partial agonist), spiroxatrine and NAN-190 could inhibit isovolumetric contractions in a dose-dependent manner. Further, the selective and archetypical 5-HT_1A receptor antagonist, WAY-100635, given i.v. (Testa et al., 1999) in conscious rats undergoing continuous cystometry, increased bladder capacity with a very steep dose–response curve and decreased micturition pressure. The latter effect was not dose related and minor. Further, in anaesthetized rats, WAY-100635 i.v. (Testa et al., 1999) caused a transient disappearance of regular isovolumetric bladder contractions (Figure 1c) and this action could be potentiated by pretreatment with the SSRI citalopram, which had no effect on its own. Interestingly and surprisingly this action of WAY-100635 could be blocked by pretreatment i.v. with the nonselective 5-HT_2C receptor antagonist mesulergine (100 μg kg⁻¹), which also had no effect on its own (Testa et al., 1999; see perspectives for more details). Neither of these pretreatments affected the ‘partial agonist' NAN-190. Similar results for WAY-100635 were observed in anaesthetized rats (Conley et al., 2001) in which it caused a dose-related increase in pressure threshold for initiation of the micturition reflex (Figure 1d) and only at high doses did it affect reflex changes evoked in the urethra and this was attributed to α₁-adrenoceptor blockade. Using a structurally similar antagonist in anaesthetized and conscious rats and guinea pigs (Leonardi et al., 2001) undergoing cystometry, Rec 15/3079 i.v. also caused an increase in bladder volume capacity without affecting residual volume (changes in residual volume would indicate interference in the urethra and/or reduction in the force of bladder contraction). Again, in the anaesthetized rats, citalopram potentiated the ability of neutral 5-HT_1A receptor antagonists to inhibit these regular isovolumetric bladder contractions, supporting the view, that 5-HT release is important in the action of these 5-HT_1A receptor antagonists. Further, both WAY-100635 and Rec 15/3079 could reverse the decrease in bladder volume capacity caused by irritating the bladder with acetic acid in conscious rats. Testa et al. (2001) also reported that another structurally related antagonist to WAY-100635, p-MPPI given i.v. had the same effect, inhibiting the regular isovolumetric bladder contractions.

Administration of WAY-100635 i.t. (Kakizaki et al., 2001) and i.c.v. (Yoshiyama et al., 2003) in anaesthetized rats also inhibited regular isovolumetric bladder contractions, and the effect was dose related (Figure 2a and b). Thus, the regulation of the bladder in micturition involves 5-HT_1A receptors at spinal and supraspinal sites, which overall have an excitatory action on parasympathetic supply to the bladder. In this respect, 5-HT_1A receptors also play an excitatory role in the reflex control of parasympathetic outflow to the heart and airways (see Ramage, 2001). Further it was suggested that supraspinal 5-HT_1A receptors are involved in modulating afferent input involved in the timing of the reflex while the spinal (sacral) 5-HT_1A receptors are involved in the activation of parasympathetic preganglionic neurones. In this respect, bladder contractions evoked by stimulation of the pontine micturition centre (PMC) were attenuated by i.t. WAY-100635 (Kakizaki et al., 2001). Nevertheless, the ability of WAY-100635 (Figure 2a and b) given at both central sites to inhibit the appearance of regular isovolumetric bladder contractions has been considered to intuitively favour the interruption of afferent input. However, field potentials evoked in the rostral pons (micturition centre) by stimulation of afferents running in the pelvic nerve were not affected by i.v. or i.t. WAY-100635 (Kakizaki et al., 2001). Interestingly, the return of these regular isovolumetric bladder contractions was reported to be only of a similar height for i.t. but not for i.c.v. WAY-100635. This may favour the reverse interpretation, that is, the sacral spinal 5-HT_1A receptor interferes with afferent input while the supraspinal 5-HT_1A receptor interferes with efferent outflow to the bladder. However, such a phenomenon with this technique, regular isovolumetric bladder contractions, was not observed (compare Figure 2a and c, also see Sercker, 2004), and thus probably represents experimental variation with this particular technique. However, the ability of WAY-100635 to increase the pressure threshold for the micturition reflex (Conley et al., 2001) still intuitively favours an effect on afferent input, although this could be due to inhibition of the central ‘switching' mechanism from storage to expulsion involving the raphé (dorsal?), the locus coeruleus and the PMC. Interestingly, there is evidence to suggest that there is dorsal raphé feedback on locus coeruleus to control neuronal activity within this nucleus (Kaehler et al., 1999). Additionally, an effect on the switching mechanism may explain the steep dose–response curve for the effects of WAY-100635. Thus, at present the precise function of these two different populations of 5-HT_1A receptors involved in the control of the micturition reflex remains to be determined.

Figure 2.

Anaesthetized female rats: experimental traces comparing the effects on rhythmic isovolumetric bladder of WAY-100635 given in (a) i.c.v., (b) .i.t and (c) i.c.v. and (d) histograms comparing the effects of sequential doses of WAY-100635 given i.c.v. or i.t.12 min apart on the duration (s) of suppression of rhythmic isovolumetric bladder.

More support for the central excitatory physiological role of 5-HT_1A receptors comes from the study of the structurally distinct 5-HT_1A receptor antagonist robalzotan (NAD-299) in the control of micturition in conscious rats (Pehrson et al., 2002). This antagonist has very little affinity for α₁ adrenoceptors compared with WAY-100635 (approx pA₂ 7.5 on all subtypes). Both robalzotan and WAY-100635 i.v. increased bladder capacity and volume. Interestingly, at higher doses robalzotan was reported to decrease micturition pressure. Further, repeated doses of robalzotan at high concentrations also showed some desensitization. Both drugs given i.c.v. also increased bladder capacity while robalzotan increased micturition pressure and WAY-100635 decreased micturition pressure. Robalzotan and WAY-100635 i.t. at the doses chosen were found not have any effect although 8-OH-DPAT had the expected action. 5-HT i.t. increased micturition pressure and decreased bladder capacity and micturition volume. However, in anaesthetized rats WAY-100635 given i.c.v. or i.t (Secker et al., 2003) has been shown to inhibit micturition, but for both routes of administration, in this study, this action showed the development of rapid tolerance (Figure 2b). This was further extended in conscious rats to demonstrate that this effect was not compound specific, as it also occurred with robalzotan (Glew et al., 2004a) and was due to an upregulation of 5-HT_1A receptors (Glew et al., 2004b). More recently, another 5-HT_1A receptor antagonist has been developed to improve cognitive ability in Alzheimer's disease and has been reported not to cause tolerance (Schechter et al., 2005). It would be interesting to test this compound on micturition.

Perspective – rat 5-HT_1A receptors

The overall data indicate that 5-HT_1A receptors, although inhibitory to adenylyl cyclase, have an excitatory physiological role in the control of micturition in the rat and, it would seem, in the guinea pig, at both a supraspinal and sacral spinal level. The supraspinal receptor is believed to have an autoreceptor function and thus blockade of this 5-HT_1A autoreceptor would cause potentiation of the supraspinal 5-HT inhibitory pathways by increasing 5-HT release, which is consistent with the citalopram data. This putative pathway would also be expected to be tonically active at rest. (It should be noted that Lecci et al. (1992) also favoured the presence of supraspinal 5-HT_1A heteroreceptor as well.) Furthermore this putative supraspinal 5-HT_1A pathway would have to activate another 5-HT receptor, possibly 5-HT_2C, to mediate its overall inhibitory action (see De Groat, 2002, Figure 3). This could explain the reported ability of mesulergine (Testa et al., 1999) to block the effects of WAY-100635. As this pathway is hypothesized to be a tonic inhibitory pathway mesulergine would also be expected to cause spontaneous activity in the bladder. This has not been observed. In addition, this proposed effect of mesulergine should be more overt against i.c.v. than i.v. WAY-100635. However, Yoshiyama et al. (2001) obtained very variable results with mesulergine i.v. against i.c.v. WAY-100635 and thus no conclusion can be drawn at present. A probable reason for the large variability may be due to the lack of selectivity of mesulergine for 5-HT_2C receptors. For instance mesulergine is a potent 5-HT₇ receptor antagonist (Wood et al., 2000) and this receptor also plays an excitatory role in the control of micturition (see later). Interestingly, in this context Testa et al. (2001) reported that a low dose (10 μg kg⁻¹, i.v.) of mesulergine did inhibit regular isovolumetric contractions, although increasing doses were ineffective. Thus, further studies are still required to identify the putative 5-HT receptor mediating this tonic inhibitory pathway and which raphé nucleus/nuclei are involved. However, the above data indicate the importance of 5-HT, via activation of 5-HT_1A receptors, in micturition control. In this respect, it would be expected that depletion of 5-HT with either p-chlorophenyalanine (Yoshiyama et al., 1994) or destruction with 5,7-DHT (Lecci et al., 1992) would interfere with micturition. This was not observed in these studies, although 5,6-DHT i.t., reducing spinal content by around 75%, was reported to increase micturition volume (Durant & Yaksh, 1988) favouring an excitatory action of 5-HT at least at the level of sacral spinal cord. These discrepancies may mean that the pretreatments in the first two studies may not have reduced the level of 5-HT to below that which is critically required for operation of the reflex.

Figure 3.

Diagrammatic representation of the putative supraspinal 5-HT_1A autoreceptor pathway involving a 5-HT_2C receptor probably located in the sacral region of the spinal cord which activates an inhibitory interneurone to cause inhibition of bladder contractions, adapted from De Groat (2002). 5-HT_1A autoreceptors are involved in negative feedback control of 5-HT release from raphé neurones. Blockade of these autoreceptors will cause an increase in firing of the raphé neurones and thus greater release of 5-HT onto the 5-HT_2C receptors which activate an inhibitory interneurone to ‘switch off' the parasympathetic drive to the bladder. This pathway is postulated to be tonically activated. However, how the spinal 5-HT_1A receptor pathway integrates into this postulated pathway remains unknown.

Cats-5-HT_1A receptor agonists/antagonists and uptake inhibition

Although 5-HT_1A receptors play an excitatory role in the reflex control of parasympathetic outflow to heart and airways in cats (see Ramage, 2001) the limited data in cats using 8-OH-DPAT and WAY-100635 suggest the opposite, in that activation of these receptors is inhibitory to the bladder, although excitatory to the EUS. Further, these effects (Figure 4a) are only observed under conditions in which the bladder had been irritated by intravesical acetic acid (Thor et al., 2002). In addition, the absence of effects of WAY-100635 and LY206130 indicates that 5-HT_1A receptors are not physiologically activated in the cat during micturition. More recently, it was reported (Gu et al., 2004) that 8-OH-DPAT decreased volume threshold in cats with chronic spinal injury and this could be reversed by WAY-100635, again the effects of these ligands in intact cats were very minor. These authors concluded that 5-HT_1A receptors facilitate the nociceptive bladder afferent reflex pathway to the sphincter muscle and inhibit nociceptive-driven reflex micturition. Interestingly some of this group had previously shown that the 5-HT and noradrenaline uptake inhibitors duloxetine (Thor & Katofiasc, 1995) and venlafaxine (Katofiasc et al., 2002) had very similar effects to 8-OH-DPAT, again only in cats whose bladder had been irritated by intravesical acetic acid. These effects could be blocked by the nonselective 5-HT receptor antagonist methiothepin. Further, the selective SSRI s-norfluoxetine had a similar effect to duloxetine, but the selective noradrenaline uptake inhibitor thionisoxetine was ineffective (Katofiasc et al., 2002). These results suggest that central 5-HT may be involved in nociceptive effects on the bladder but at present there is no indication that, in the cat, the micturition reflex involves the activation of 5-HT_1A receptors. Interestingly, when 5-HT was given i.t. to conscious cats it caused a 200% increase in volume threshold for micturition, while in spinal cats 5-HT, as 8-OH-DPAT, reduced the volume threshold (Espey & Downie, 1995; Figure 4b). The spinal receptor/s at which 5-HT in intact cats causes inhibition of micturition remains to be determined.

Figure 4.

Cats: experimental traces showing the effects of: – (a) 8-OH-DPAT i.v. on baseline external urethral sphincter activity and bladder pressure in hyperactive bladder induced by acetic acid and the effect of WAY-1006235 i.v. on these changes, (b) 5-HT given intrathecally (i.t.) in conscious intact (A) and spinalized (B) cats and c) the effect of the 5-HT₃ receptor antagonist, zatosetron given i.t. in an intact cat and the 5-HT₃ receptor agonist 2-methyl-5-HT given i.t. in a spinalized cat.

5-HT₇ receptors

Another receptor subtype that needs to be considered when investigating 5-HT_1A receptors is 5-HT₇ receptors as the ligands used to study 5-HT_1A receptors also have affinity for 5-HT₇ receptors; 8-OH-DPAT has pK_i of 6.9 and WAY-100635 has a pK_i of 7 (100 × lower than at 5-HT_1A). Other 5-HT receptor ligands that have been used to study micturition which also have significant affinity for 5-HT₇ receptors are methysergide (Krobert & Levy, 2002), methiothepin, mesulergine (Hagan et al., 2000), LSD (To et al., 1995) and meta-chlorophenylpiperazine (mCPP; see Hoyer et al., 2002). However, these receptors differ from 5-HT_1A in that they activate adenylyl cyclase and thus would be considered excitatory. Further, it is now recognized that 8-OH-DPAT-induced hypothermia in mice, a screen sometimes used to determine if a drug has activity at 5-HT_1A autoreceptors (presynaptic) is mediated by 5-HT₇ receptors as for guinea pig and rat (see Hedlund et al., 2004).

With the development of the selective 5-HT₇ receptor antagonist SB-266970 (10–30 μg kg⁻¹) the role of these receptors could be investigated. SB-269970 given i.c.v. (Figure 5a) caused an increase in bladder pressure and volume threshold and at higher doses abolished the micturition reflex in anaesthetized rats but not when given i.t. although WAY-100635 was effective (Figure 5b; Read et al., 2003). Further, SB-656104 a 5-HT₇ receptor antagonist from the same chemical series but structurally distinct and with a different selectivity profile to SB-269970 had a similar effect (Figure 5a) with the additional action of causing an increase in residual volume. In this respect, it has recently been reported that 5-HT₇ receptors are located in Onuf's nucleus in the rat (Doly et al., 2005) however, why SB-269970 has no effect on the urethra (Figure 5c) remains to be determined but it could be related to its ability to interfere with 5-ht_5A receptors also found in this nucleus, see below. Thus, the effects of SB-269970 are receptor specific rather compound specific. Interestingly, central 5-HT₇ receptors also control reflex activation of parasympathetic outflow to the heart (Kellett et al., 2005).

Figure 5.

Anaesthetized rats showing the effects of: (a) 5-HT₇ receptor antagonists on bladder contractions evoked by saline infusion into the bladder, (b) a comparison of i.t. SB-269970 with that of WAY-100635 on the same variables and (c) SB-269970 on bladder and urethral-evoked changes caused by infusion of saline into the bladder (Read et al., 2003).

5-carboxamidotryptamine (5-CT) – 5-HT₇ receptor agonist plus 5-HT_1B receptors

At present there are no selective agonists available to study 5-HT₇ receptors. The agonist that has been used in a large number of in vitro studies is 5-CT; however, this compound activates most of the 5-HT₁ receptor subtypes. As may be expected it has variable effects on micturition causing inhibition and excitation (Figure 6a) when given i.c.v. at the same dose (Read et al., 2004). In the presence of the 5-HT_1B/1D receptor antagonist GR127935, which has a pK_i of 8.7 and 8.3, respectively, at these receptors (Roberts et al., 2001), while at rat 5-HT_1A it has a pK_i 6.5–6.9 (see Pauwels, 1997), 5-CT i.c.v caused a reduction in the volume threshold and evoked spontaneous bladder contractions (Figure 6b). Interestingly, in preliminary experiments GR127935 i.c.v. alone caused a large increase in the volume threshold by 94±35%. In this respect, the 5-HT_1B receptor agonist CP-93,129 (i.c.v.) abolished (Figure 6c) the micturition reflex in five out of six rats while the 5-HT_1B/1D receptor agonist sumatriptan caused attenuation of micturition. Thus, these data support the view that 5-HT₇ receptors are excitatory but are complicated by the involvement of 5-HT_1B and 5-HT_1D receptors. Overall these data suggest that other 5-HT₁ receptor subtypes may be involved in micturition, however, further studies are required.

Figure 6.

Anaesthetized rats comparisons of the effects of the nonselective 5-HT₇ receptor agonist 5-CT given i.c.v. (a) alone and (b) in the presence of 5HT_1B/1D receptor antagonist GR127935 on bladder contractions induced by saline infusion into the bladder; (c) shows the effect of the 5-HT_1B receptor agonist CP-93,129 given i.c.v. again bladder contractions induced by saline infusion into the bladder (Read et al., 2004).

5-ht_5A receptors

These receptors, like 5-HT_1A receptors, are coupled negatively to adenylyl cyclase and would seem to have a similar location in the spinal cord and brain. Nevertheless, in respect to micturition it is the high density of these receptors in Onuf's nucleus (Doly et al., 2004) that is of interest. At present, there are no available selective ligands for these receptors. Nevertheless, they have a similar pharmacology to 5-HT₇ receptors in that the selective 5-HT₇ receptor antagonist SB-269970 has as pK_i of 7.8 at the rat 5-ht_5A receptor (50 × less potent than on 5-HT₇) and the rank order of agonist potency (5-CT>5-HT>8-OH-DPAT) is also similar (see Ramage, 2004).

5-HT₂ receptors

This receptor has three subtypes A, B and C and the subtypes that are of interest in micturition are 5-HT_2A and 5-HT_2C receptors. It should be noted that activation of 5-HT_2A receptors causes smooth muscle contraction, thus causing contraction of the bladder, a rise in blood pressure and bronchoconstriction and centrally it is believed to cause hallucinations, while 5-HT_2C receptors are involved in the control of appetite as well as other functions. These receptors are considered to have an excitatory action as they couple preferentially to G_q/11 to increase the hydrolysis of inositol phosphates and elevate cytosolic [Ca²⁺].

The indication that 5-HT₂ receptors are involved in micturition came from the study of the very unselective 5-HT_2C receptor agonist mCPP (Steers & De Groat, 1989). This agonist i.v. (100 μg kg⁻¹) caused inhibition of rhythmic isovolumetric bladder contraction caused ‘by infusion 0.6 ml of saline into the bladder after the distal urethra was completely obstructed'. This was surprisingly blocked by the neuromuscular blocker pancuronium implying that this inhibition of rhythmic isovolumetric bladder contractions was related to ‘somatic muscle contractions'. The ability of mCPP to block micturition was confirmed by Guarneri et al. (1996) and, interestingly, it was reported that these effects could by blocked by mesulergine, supposedly by blocking 5-HT_2C receptors, and potentiated by ketanserin a 5-HT₂ receptor antagonist, which is considered to be selective for 5-HT_2A. However, ketanserin has been reported to inhibit isovolumetric bladder contractions (Testa et al., 2001). Nevertheless, the role of 5-HT_2C receptors in the control of the EUS (striated muscle) has been confirmed using the selective agonist Ro 60-60175 in anaesthetized guinea pigs where the agonist activated the EUS at rest and increased bladder capacity and volume threshold although voiding occurred normally (McMurray & Miner, 2005). The selective 5-HT_2C receptor antagonist SB-242084 could reverse these effects. Recently, studies (Mbaki et al., 2006) in anesthetized rats have also confirmed that mCPP i.v. can inhibit the micturition reflex, activate the EUS and cause contraction of the urethra. However, the ability of the so-called 5-HT_2C receptor agonists to activate (see Figure 7a) the EUS in the rat is also mediated by 5-HT_2A (Mbaki et al., 2006), although inhibition of micturition is mediated by 5-HT_2C receptors. Interestingly, ketanserin was found to potentiate this inhibitory effect of 5-HT_2C receptor agonists on micturition (Figure 7b). Furthermore, these data are consistent with the view that the supraspinal 5-HT pathways using 5-HT_1A receptors mediate their actions via 5-HT_2C receptors (see above; Figure 3). However, the selective 5-HT_2C receptor antagonist SB 242084 alone i.v. failed to reduce bladder capacity and volume threshold. The mechanism by which ketanserin poteniates this inhibitory action of 5-HT_2C receptors is difficult to explain but may be due to involvement of excitatory 5-HT_1D receptors in micturition as ketanserin also blocks these receptors. Interestingly, a high dose (100 μg kg⁻¹, i.v.) of ketanserin can also inhibit micturition (Testa et al., 2001 and Mbaki; personal communication, Figure 7b).

Figure 7.

Anaesthetized rats: (a) experimental traces showing the effect of the 5-HT_2C receptor agonist WAY 161503 on external urethral sphincter EMG and urethral pressure and (b) histograms of changes caused by WAY 161503 on bladder variables during micturition evoked by a saline-induced distension of the bladder in the absence and presence of the 5-HT_2A receptor antagonist ketanserin.

The species differences between which 5-HT₂ receptor is involved in activation of the EUS could be related to a difference in the physiological role of the sphincter in these species. In the rat it is used to expel urine while in the guinea pig it is used to prevent expulsion as the bladder fills, a guarding function. A possible explanation for this difference is that if 5-HT_2C receptors are considered inhibitory to micturition and that activation of the EUS in guinea pig is part of this inhibitory mechanism thus 5-HT_2C receptor activation of the EUS would be consistent with this function. However, in the rat, EUS activity is associated with the activation of micturition and thus if 5-HT_2A receptors are considered excitatory to micturition this activation of EUS by 5-HT_2A would also be consistent with that function. This would suggest that in the guinea pig 5-HT_2A receptor agonists might switch off EUS activity. Interestingly, in the cat, which has a guarding reflex, central 5-HT₂ receptors have also been implicated in the excitatory control of the EUS (Thor et al., 1990; Danuser & Thor, 1996) but this is only observed in spinalized cats. However, the role of different 5-HT₂ receptor subtypes remains to be determined, although DOI was used (Danuser & Thor, 1996) which is considered to be selective for 5-HT_2A over 5-HT_2C receptors. Interestingly, Danuser & Thor (1996) suggested that the reason for not observing excitation in the intact cat is that supraspinal 5-HT₂ receptors (5-HT_2C?) are inhibitory while spinally such receptors (5-HT_2A?) have an excitatory function.

Perspective

These data indicate that 5-HT₂ receptor stimulation activates the pudendal nerve causing an increase in EUS activity as well as causing inhibition of micturition. In the guinea pig, it is the 5-HT_2C receptor that has this action on the pudendal nerve while in rat it is the 5-HT_2A receptor, however, the involvement of both receptors in both species cannot be ruled out. Nevertheless, in both species 5-HT_2C receptors have an inhibitory action on the micturition reflex. However, the physiological role of these receptors remains to be determined.

5-HT₃ receptors

The 5-HT₃ receptor differs from all other 5-HT receptors in that it is a ligand-gated ion channel receptor which has been demonstrated to play an important excitatory role in the processing of cardiovascular and gut afferent input at the level of the NTS (see Jeggo et al., 2005). Zatosetron, a 5-HT₃ receptor antagonist given intrathecally to conscious cats decreased the volume threshold for micturition (Figure 4c). In spinalized conscious cats the 5-HT₃ receptor agonist, 2-methyl-5-HT given intrathecally (Figure 4c) increased the volume threshold for micturition (Espey & Downie, 1995). This implies that spinal 5-HT₃ receptors are inhibitory to the reflex, at least when there is descending drive, and physiologically involved in the micturition reflex. Further along with two other 5-HT₃ receptor antagonists tropisetron (ICS 205-930) and MDL 72222, again given intrathecally, pelvic nerve-evoked neuronal activity in the lower thoracic spinal cord was found to be potentiated (Espey et al., 1998). This suggests that during bladder filling, 5-HT is released to activate 5-HT₃ receptors at the level of the spinal cord to inhibit afferent input. Interestingly, 5-HT₃ receptors may also have a role in the control of the EUS as the pelvic – pudendal nerve reflex was attenuated by zatosetron and potentiated by 2-methyl-5-HT also given intrathecally (Espey et al., 1998) implying that, at least in the cat, 5-HT₃ receptors are also involved in the control of the EUS.

In anaesthetized rats i.v. zatosetron and Y 25130 showed no overall effect on isovolumetric bladder contractions (Testa et al., 2001) while in conscious rats, 2-methyl-5-HT given i.c.v. also failed to have any effect on cystometric parameters (Ishizuka et al., 2002).

Overall, further studies are required to determine the importance of central 5-HT₃ receptors in micturition.

5-HT₄ and 5-HT₆ receptors

Drugs selective for these receptors have been tested in only two studies in rats. The 5-HT₄ receptor antagonist RS 39604 given i.v. to anaesthetized rats did not affect regular isovolumetric bladder contraction as did the 5-HT₆ receptor antagonist Ro 04-6790 (Testa et al., 2001). Nevertheless the 5-HT₄ receptor agonist RS 67506 given i.c.v. to conscious rats decreased bladder capacity and micturition volume (Ishizuka et al., 2002).

Conclusion

Traditionally, central 5-HT-pathways are considered to be inhibitory in the control of micturition. However, at least in the rat, 5-HT_1A and 5-HT₇ receptors have excitatory actions. Further, the use of antagonists for these two receptors indicates that both play an essential role in micturition in the rat and probably in the guinea pig. Interestingly, both receptors seem to have a similar role supraspinally, although they have opposing effects on adenylyl cyclase. At a spinal level, however, only 5-HT_1A receptors have a role in micturition. Whether these receptors are acting as autoreceptors, heteroreceptors or both needs to be determined. In this respect, 5-HT_1A receptor data suggest that supraspinal 5-HT_1A receptors may be predominantly acting as autoreceptors, while the spinally located receptors may be heteroreceptors. Further, the spinally located receptors seem to modulate efferent activity to the bladder parasympathetic preganglionic neurones. Nevertheless, the precise function of these receptors in the control of micturition remains to be determined. However, surprisingly, in the cat, 5-HT_1A receptors do not seem to play a physiological role in micturition, and may only have a pharmacological role in pathological situations. Further, in the cat, 5-HT_1A receptors have the opposite effect to that expected in that they are inhibitory but do cause excitation of the EUS muscle, whereas in rats and guinea pigs this is caused by activation of 5-HT_2A and 5-HT_2C receptors, respectively. There is, however, no evidence that these receptors are physiologically involved in the control of this sphincter or in bladder function except that activation of 5-HT_2C receptors has an inhibitory action on micturition and somewhat surprisingly, antagonists to this receptor are without effect. In this respect, only 5-HT₃ receptors have been suggested to be physiologically involved in the control of micturition in the cat, at least at a spinal level. The paucity of evidence compared with rat indicating that 5-HT plays an important role in the control of micturition may just reflect the lack of experiments carried out in this species. Overall the data indicate that 5-HT is an important transmitter involved in the control of micturition. However, further experiments are required to elucidate its precise role and the seeming difference in importance it has in this function between species.

Glossary

5,6-DHT

5,6-dihydroxytryptamine

5,7-DHT

5,7-dihydroxtryptamine

DOI

1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane

EUS

external urethral sphincter

5-HT

5-hydroxytryptamine

i.c.v.

intracerebroventricular

i.t.

intrathecal

i.v.

intravenous

LSD

lysergic acid diethylamide

mCPP

meta-chlorophenylpiperazine

5-MeODMT

5-methoxydimethyl tryptamine

8-OH-DPAT

8-hydroxy-2-(di-n-propylamino)tetralin

SSRI

selective serotonin reuptake inhibitor