Effect of Several 5-Hydroxytryptamine1A Receptor Ligands on the Micturition Reflex in Rats: Comparison with WAY

Several novel N-arylpiperazine derivatives were synthesized and tested for their 1) affinity and functional activity on 5-hydroxytryptamine1A (5-HT1A) receptors in vitro; 2) activity in models predictive of antagonism at somatodendritic and postsynaptic 5-HT1A receptors; and 3) effects on the micturition reflex in anesthetized and conscious rats. These studies also included 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN 190), 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378), and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2pyridinyl)cyclohexanecarboxamide (WAY 100635). Almost all compounds were found to be potent and selective for the human recombinant 5-HT1A receptor, with Ki values in the nanomolar range. [S]GTPgS binding in HeLa cells expressing the recombinant human 5-HT1A receptor allowed classification of the compounds into neutral antagonists and partial agonists. Almost all neutral antagonists were active in blocking 8-hydroxy-2dipropylaminotetralin (8-OH-DPAT)-induced forepaw treading in rats (postsynaptic model) and hypothermia in mice (somatodendritic model) with the same potency, whereas compounds showing partial agonistic activity were active in the postsynaptic model but were inactive, or poorly active, in the somatodendritic model. Neutral antagonists potently inhibited volume-induced bladdervoiding contractions in anesthetized rats. Contractions were completely blocked, and the disappearance of bladder contractions lasted 7 to 13 min after the highest doses tested. Furthermore, neutral antagonists increased bladder volume capacity in conscious rats during continuous transvesical cystometry, whereas micturition pressure was only slightly, and not dose-dependently, reduced. Partial agonists were inactive or poorly active, inducing a disappearance time of bladder contractions that did not exceed 6 min in anesthetized rats, and failing to increase bladder volume capacity in conscious rats. These findings indicate that only neutral 5-HT1A receptor antagonists are endowed with inhibitory effects on the bladder. Normal bladder function requires coordinated 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 centers in the spinal cord, pons, and forebrain (de Groat et al., 1993). The descending bulbospinal pathway to the urinary bladder is essentially an inhibitory circuit (de Groat et al., 1993), although bladder contraction may occur through the stimulation of locus coeruleus in the micturition center and consequent release of noradrenaline in the spinal cord (Yoshimura et al., 1988). Several neurotransmitters have been identified as inhibitory transmitters in the micturition reflex pathways at both spinal and supraspinal sites, including 5-hydroxytryptamine (5-HT), g-aminobutyric acid, and glycine, dopamine, acetylcholine, enkephalins, and other peptides (de Groat et al., 1993). Electrical stimulation of 5-HT-containing neurons 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 (Morrison and Spillane, 1986). Multiple 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 (Gerhardt and Heerikhuizen, 1997). Some 5-HT receptor subfamilies have been further subdivided. The largest subclass of 5-HT receptors, 5-HT1 receptors, consists of five subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F; all G protein-coupled receptors), with the 5-HT1A receptor being the most extensively investigated and characterized. The 5-HT1A receptor is located both on the cell body and terminals of nerves (somatodendritic, presynaptic receptor), where it modulates neuronal cell firing, and at the postsynaptic level, where it mediates different functions (Saxena, 1995). Received for publication January 29, 1999. 1 Some of these data have already been presented in abstract form (Testa et al., 1998a; Guarneri et al., 1998). ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); CHO, Chinese hamster ovary; NRM, nucleus raphe magnum; VIVC, volume-induced bladder-voiding contractions; DT, disappearance time; BVC, bladder volume capacity; MP, micturition pressure. 0022-3565/99/2903-1258$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290:1258–1269, 1999 1258 at A PE T Jornals on N ovem er 5, 2016 jpet.asjournals.org D ow nladed from A number of nonselective antagonists of the 5-HT1A receptor have been reported [spiperone and (S)-UH-301], and over the years, other compounds were initially described as selective 5-HT1A antagonists [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) and 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN 190)]. Although these compounds demonstrated antagonist-like activity in several functional measures of postsynaptic 5-HT1A receptor activation, they showed agonist-like activity when examined at the somatodendritic 5-HT1A autoreceptors (Hjorth and Sharp, 1990; Sharp et al., 1990). Recently, a phenylpiperazine derivative, N-[2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635), was shown to be an antagonist at both the preand postsynaptic level (Fletcher et al., 1996). Pharmacological studies have shown that 5-HT1A receptor agonists and antagonists influence central control of lower urinary tract function. The selective agonist 8-hydroxy-2dipropylaminotetralin (8-OH-DPAT), injected i.v., intrathecal, or i.c.v., activates the micturition reflex by inducing an increase in the frequency of volume-induced bladder-voiding contractions (VIVC) in anesthetized rats. On the contrary, the postsynaptic 5-HT1A antagonist NAN 190 blocked the contractions (Lecci et al., 1992), suggesting that 5-HT1A receptors are involved in the control of the micturition reflex, although an a1-adrenergic mechanism cannot be excluded for this compound. To better define the effect of 5-HT1A receptor ligands on the micturition reflex and bladder function, we have synthesized several new, highly selective 5-HT1A compounds and examined their activity on the bladder in comparison to WAY 100635, NAN 190, and BMY 7378. Biochemical and pharmacological experiments to define the nature of the interaction of the compounds with somatodendritic and postsynaptic 5-HT1A receptors are also described. Materials and Methods Male and female Sprague-Dawley rats [Crl:CD(SD)BR, 200–300 g b.wt.) and male mice (Crl:CD-1(ICR)BR, 25–35 g b.wt.) from Charles River, Calco, Italy were used in these 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 1 week before the experiments were carried out. The animals were handled according to internationally accepted principles for care of laboratory animals (European Economic Community Council Directive 86/609, O. J. no. L358, December 18, 1986).