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The GABAB receptor agonist baclofen reduces urethral resistance and detrusor overactivity in patients with spasticity. However, baclofen's side effects limit its use for the treatment of overactive bladder (OAB). Here, we tested a novel GABAB positive allosteric modulator (PAM) ADX71441 in models of OAB in mice and guinea pigs.Mice were left untreated or given (p.o.) vehicle (1% CMC), ADX71441 (1, 3, 10 mg kg(-1) ) or oxybutynin (100 mg kg(-1) ; Experiment 1) or vehicle (1% CMC), baclofen (1, 3, 6 mg kg(-1) ) or oxybutynin (Experiment 2). Treated mice were then overhydrated with water, challenged with furosemide, before being placed into micturition chambers and monitored for urinary parameters. In anaesthetized guinea pigs, intravesical infusion of acetic acid was used to induce OAB and the effects of ADX71441 (1, 3 mg kg(-1) ) or baclofen (1 mg kg(-1) ), administered i.v., on cystometric parameters were monitored.In mice, 10 mg kg(-1) ADX71441 increased urinary latencies, reduced the number of urinary events and the total and average urinary volumes. In guinea pigs, ADX71441 (1 and 3 mg kg(-1) ) increased the intercontraction interval (ICI) and bladder capacity (BC), and reduced micturition frequency (MF) compared to vehicle. At 3 mg kg(-1) ADX71441 completely inhibited the micturition reflex and induced overflow incontinence in five out of 10 animals. Baclofen slightly increased ICI and BC and reduced MF.Our findings demonstrate, for the first time, that a GABAB PAM has potential as a novel approach for the treatment of OAB.

To compare the direct relaxant effects of alfuzosin, phentolamine and sildenafil in rabbit isolated corpus cavernosum (CC) pre-contracted with phenylephrine or KCl.Penile erectile tissue was obtained from male New Zealand White rabbits (22-26 weeks old). The CC was cut into longitudinal strips and mounted under 2 g resting tension in 5-mL jacketed organ baths containing a modified Krebs solution bubbled with 95% O2, 5% CO2 and maintained at 37 degrees C. Tissue strips were pre-contracted by 60 mmol/L KCl or 10 micro mol/L phenylephrine. After obtaining a stable plateau of contractions, test compounds were added to the organ bath. The relaxant potencies were expressed as the percentage of inhibition of the plateau of contraction induced by 10 micro mol/L phenylephrine.Alfuzosin showed a concentration-dependent relaxing effect on rabbit CC pre-contracted by 10 micro mol/L phenylephrine, with a mean (sd) pIC50 of 7.64 (0.06). The relaxant effect was unaffected by pre-incubation with 100 micro mol/L Nomega-nitro-l-arginine methyl ester (L-NAME). Phentolamine had a potency similar to alfuzosin, with a pIC50 of 7.44 (0.08). Both alfuzosin and phentolamine were completely ineffective on the plateau of contraction induced by 60 mmol/L KCl. In contrast to alfuzosin, sildenafil was equipotent in relaxing the rabbit CC against each contractile agent, with pIC50 values of 7.25 (0.09) and 7.23 (0.22) with 10 micro mol/L phenylephrine and 60 mmol/L KCl, respectively. The relaxant response to sildenafil was partly blocked by pretreatment with 100 micro mol/L L-NAME, with pIC50 values of 7.94 (0.09) and 6.63 (0.32) without and with L-NAME, respectively. Sildenafil, incubated for 45 min at 10 micro mol/L, had no relaxant effect on the resting tension of the preparation or on the concentration-response curve to phenylephrine.The direct relaxant effect of alfuzosin is mediated through alpha1-adrenoceptor blockade. The relaxations induced by phentolamine and alfuzosin are independent of nitric oxide, whereas those induced by sildenafil are, at least partly, sensitive to L-NAME and a selective soluble guanylate cyclase inhibitor, indicating the involvement of nitric oxide and soluble guanylate cyclase. Alfuzosin and phentolamine effectively counteract alpha1-adrenoceptor-mediated contractions of rabbit CC. If valid for human CC, such an effect may contribute to an improved erectile function in patients treated for benign prostatic hyperplasia.

The aim of this work was to study the role of capsaicin-sensitive afferent fibers in modulating the micturition reflex at spinal level in urethane-anesthetized guinea pigs after spinal cord transection at level T3-T4.The intravesical effect of capsaicin was investigated in a series of cystometrograms performed in intact and spinalized animals.In both intact and spinalized animals capsaicin, at 30 microM, induced a significant increase of volume threshold only, whereas at 100 microM it induced a complete inhibition of the spinal micturition reflex in 60% and 85% of the animals tested, respectively. This capsaicin inhibitory effect (CIE) was unaffected by intravenous phentolamine and propranolol (0.5 and 1 mg./kg., respectively), indomethacin at 100 nmoles intrathecally (i.t.), the CGRP receptor antagonist hCGRP8-37 (3 nmoles i.t.) and the NK2 receptor selective antagonist GR 94800 (1 nmol. i.t.). However, both naloxone (30 microg. i.t.) and the NK1 antagonist GR 82334 (10 to 20 nmoles i.t.) prevented CIE in the majority of spinalized animals.These results suggest that CIE could be mediated by enkephalines released by dorsal root ganglion neurons through substance P release and subsequent activation of NK1 receptors in acutely spinalized guinea pigs.

The contractile effect of two highly potent, selective and peptidase-resistant neurokinin (NK) 1 and NK2 receptor agonists, namely delta-Aminovaleryl-[L-Pro9, N-MeLeu10]substance P-(7-11) (GR 73632) and [Lys3, Gly8-R-gamma-lactam-Leu9]NKA-(3-10) (GR 64349), respectively, was investigated on smooth muscle strips dissected from specimens of human detrusor, prostatic urethra and prostate. Furthermore, the potencies of two peptidic NK2 receptor antagonists, GR 87389 L 659,837, in antagonizing GR 64349-induced contractions were compared in these three tissues. In human detrusor muscle the rank order of agonist potency was: [beta Ala8 (NKA-(4-10)]GR 64349NKA-(4-10)SP = GR 73632SP-methylester. The NK2 receptor antagonist, GR 87389, antagonized GR 64349-induced contractions in a competitive manner, whereas L 659,837 was a noncompetitive antagonist. In the prostatic urethra the rank order of agonist potency was GR 64349NKA-(4-10)SPGR 73632, whereas in the prostate it was: GR 64349[beta Ala8 (NKA-(4-10)]NKA-(4-10)SP; GR 73632 was ineffective up to 30 microM. In the prostatic urethra and in the prostate GR 87389 was a noncompetitive antagonist with a potency similar to that exhibited in the detrusor. On the contrary, L 659,837 appeared to be a competitive antagonist in the prostate and in the prostatic urethra, having approximately the similar potency in these two tissues. The selective NK3 agonist senktide was ineffective up to 30 microM in all three tissues. These results are discussed in the view of the proposed NK2 receptor subtypes and considering possible therapeutic implications in the treatment of urinary bladder disorders.

The activity of a selective cholecystokinin (CCK)-A receptor agonist, N-acetyl derivative of A71623 (Ac-Trp-Lys(epsilon-N-[2-methylphenylamino-carbonyl]) -Asp-(NMe)Phe-NH2) was investigated in the guinea pig isolated ileum longitudinal muscle myenteric plexus. NAA caused both a phasic and tonic contraction at all concentrations tested (1-1000 nM). The selective CCK-A antagonist L-364,718 (Devazepide) antagonized both types of contraction with a pKB of 10.10 and 9.95, respectively. The CCK-B selective antagonist L-365,260 ((3R(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1, 4-benzodiazepine-3yl)-N-(3-methylphenyl)-urea) was inactive up to a concentration of 30 nM. Atropine at 300 nM and 1000 nM reduced the maximal response of NAA by only 17% and 50%, respectively. The selective neurokinin (NK)-1 antagonists GR 82334 ([D-pro9[Spiro-gamma-Lactam] Leu10, Trp11]-Phys (1-11)9) at 300 and 1000 nM and (+-) CP-96,345 [(2S, 3S)-cis- 2-(diphenylmethyl)-N- [(2-methoxyphenyl)-methyl] -1-azabici-clo [2.2.2]octan-3-amine] at 10 nM were inactive or partially active. When atropine and GR 82334 or (+/-) CP-96,345 were combined, they produced a dose-dependent synergistic inhibition of both phasic and tonic contractions induced by NAA. The selective NK-3 receptor agonist senktide induced both phasic and tonic contractions that were blocked by tetrodotoxin. In the presence of atropine and GR 82334, both 300 nM, a synergistic depression of the response to senktide similar to that observed for the agonist NAA was disclosed.(ABSTRACT TRUNCATED AT 250 WORDS)

The classification of purinergic receptors is seriously hampered by the lack of specific antagonists. Furthermore, there is increasing evidence that other purinoceptor subtypes may exist that are different than the relatively well characterized P2X, P2Y, P2Z and P2T. Human isolated urinary bladder was reported to contract in response to challenge with alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) and adenosine 5'-triphosphate (ATP), probably through activation of P2X purinoceptors. In this work, we tried to classify the purinoceptors subtypes present in human detrusor muscle by using adenosine 5'-[beta-thio]diphosphate (ADP beta S), alpha,beta-MeATP, 2-methylthio adenosine 5'-triphosphate (2-MeSATP), ATP and uridine 5'-triphosphate (UTP). We also examined the activity of two putative P2 antagonists (p-chloromercuribenzensulfonic acid [PCMBS] and Reactive Blue 2 [RB-2]). The agonist rank order of potency was alpha,beta-MeATP = ADP beta S2-MeSATPATPUTP. Cumulative responses to alpha,beta-MeATP induced a very rapid desensitization, but responses to alpha,beta-MeATP and ADP beta S, both at 100 microM, were additive. PCMBS antagonized ADP beta S-induced contractions with a pKB of 6.49, but it was inactive against alpha,beta-MeATP. The putative P2Y antagonist RB-2 had no effect against ADP beta S-induced contraction. We conclude that human detrusor muscle contains two contractile purinoceptor subtypes. One is activated by alpha,beta-MeATP and is probably the P2X subtype; the other is activated by ADP beta S and appears to be different from those accepted by the current classification. The similarity between our results and those obtained by other investigators is discussed.