In human isolated detrusor strips, submaximal contractile responses evoked by electrical stimulation were resistant to hexamethonium (30 tiM) and abolished by tetrodotoxin (0.6 ELM) and hyoscine (1 fM), indicating the activation of postganglionic cholinergic nerves. In methysergide (1 tM) and ondansetron (3 ,iM) pretreated tissues, 5-hydroxytryptamine (5-HT) (0.3 nM-1ILM) caused a concentration-dependent increase in the amplitude of contractions (pEC_ = 8.1), which was antagonized by the selective 5-HT4 receptor antagonist GR 113808 (3, 10 and 30 nM) in a competitive manner. Schild analysis yielded a pA2 estimate of 8.9, a value comparable to that reported for GR 113808 in other animal and human peripheral tissues (8.8-9.7). Our findings indicate that neuromuscular cholinergic transmission in human isolated detrusor muscle is facilitated by neural 5-HT receptors belonging to the 5-HT4 subtype. The human urinary bladder can thus be regarded as an additional site in which 5-HT4 receptors are distributed.

1 In strips of human isolated detrusor muscle, the 5-hydroxytryptamine (5-HT) receptor (5-HT4) that mediates facilitation of neuromuscular cholinergic transmission was further characterized by using 5-HT and a series of ligands known for their 5-HT4 agonist (5-methoxytryptamine: 5-MeOT, cisapride, (R,S)-zacopride, BIMU 8) or antagonist (RS 23597, GR 125487, DAU 6285) properties. 4 Thus, in the human isolated detrusor muscle, the 5-HT4 receptors mediating facilitation of cholinergic neuromuscular transmission are activated by indoleamines (5-HT, 5-MeOT), substituted benzamide (cisapride, (R,S)-zacopride), benzoate (RS 23597) and benzimidazolone (BIMU 8) derivatives. The activities (in terms of both potency and efficacy) of most agonists, as well as the affinity estimates of the antagonists GR 125487 and DAU 6285, are comparable to those found in other peripheral tissues. Exceptions are RS 23597, which acted either as a partial agonist or as an antagonist of the response to 5-HT, and 5-MeOT that showed an unusually low potency. The latter findings may be ascribed to differences in the efficiency of receptor coupling mechanisms and/or in the molecular structure (i.e. splice variants) of the 5-HT4 receptor.

Bladder overactivity (OAB) is a chronic and debilitating lower urinary tract (LUT) disorder that affects millions of individuals worldwide. LUT symptoms associated with OAB, such as urgency and urinary incontinence, cause a hygienic and social concern to patients, but their current pharmacological treatment is largely inadequate due to the lack of uroselectivity. Although OAB etiology remains multifactorial and poorly understood, increasing evidence indicates that serotonin [5-hydroxytryptamine (5-HT)] is an endogenous substance involved in the control of micturition at central and peripheral sites. In this study, we demonstrated the presence of three distinct 5-HT receptors localized at parasympathetic nerve terminals of the human bladder by measuring electrically evoked tritiated acetylcholine release in isolated detrusor strips. These prejunctional receptors, involved in both positive and negative feedback mechanisms regulating cholinergic transmission, have been characterized by means of three highly selective 5-HT antagonists for 5-HT 4 , 5-HT 7 , and 5-HT 1A receptors,. Under these conditions, we confirmed the facilitatory role of 5-HT 4 heteroreceptors on acetylcholine release and revealed for the first time the occurrence of 5-HT 7 and 5-HT 1A heteroreceptors with a facilitatory and an inhibitory action, respectively. Our findings strengthen the novel concept for the use of recently patented selective 5-HT agonists and antagonists for the control of OAB dysfunctions associated with inflammatory conditions, although their therapeutic efficacy needs to be explored in the clinical setting.

We investigated whether in human isolated detrusor strips the atropine-resistant contractile response to electrical field stimulation was mediated by ATP (or a related purine), as previously shown in the urinary bladder of other mammalian species. Electrical stimulation (1-50 Hz for 5 s at 1 min intervals, 0.1 ms pulse width, 60 V) elicited reproducible, frequency-dependent twitch contractions, which were markedly reduced by atropine (10 microM). Tetrodotoxin (TTX: 1 microM) inhibited contractile responses to a similar degree. When applied together, atropine and TTX caused an inhibition which was superimposable to that caused by either drug alone. The TTX-resistant contractions were totally unaffected by omega-conotoxin GVIA (omega-CTX: 0.1 microM). The atropine-resistant contractions were unaffected by the P2-purinoceptor antagonists suramin (300 microM) and PPADS (30 microM), at concentrations which virtually suppressed the contractile response induced by applied ATP (10 microM(-1) mM). As previously described, antagonism of the ATP-induced contractions by suramin (30, 100, 300 microM) and PPADS (3, 10, 30 microM) was insurmountable, with apparent 'pA2' values (calculated at the lowest antagonist concentrations) of 4.9 and 5.2, respectively. It is concluded that, under our experimental conditions, the non-cholinergic (atropine-resistant) component of the excitatory transmission in the human detrusor is not mediated by neural release of ATP, in spite of the presence of excitatory P2-purinoceptors on the effector cells. The TTX- and omega-CTX-resistant, non-cholinergic component might be related to the release of unknown transmitter(s) through a mechanism independent of both Na+- and N-type Ca2+-channels. More likely, the atropine-resistant component may reflect direct smooth muscle excitation since the human detrusor has a very short chronaxie (Sibley 1984).

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