Modulation of nerve-evoked contractions by ß3-adrenoceptor agonism in human and rat isolated urinary bladder.

Activation of ß3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of ß3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of ß3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of ß3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αß-methylene adenosine triphosphate (αß-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the ß3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5-1µM) were inhibited 25% by isoproterenol (3µM) while contractions to 10Hz in the same strip were inhibited 67%. The selective ß3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αß-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of ß3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by ß3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional ß3-adrenoceptors may inhibit neurotransmitter release.

Differential effects of p38 mitogen-activated protein kinase and cyclooxygenase 2 inhibitors in a model of cardiovascular disease.

The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1beta. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.

Molecular mechanisms of antidiuretic effect of oxytocin.

Oxytocin is known to have an antidiuretic effect, but the mechanisms underlying this effect are not completely understood. We infused oxytocin by osmotic minipump into vasopressin-deficient Brattleboro rats for five days and observed marked antidiuresis, increased urine osmolality, and increased solute-free water reabsorption. Administration of oxytocin also significantly increased the protein levels of aquaporin-2 (AQP2), phosphorylated AQP2 (p-AQP2), and AQP3 in the inner medulla and in the outer medulla plus cortex. Immunohistochemistry demonstrated increased AQP2 and p-AQP2 expression and trafficking to the apical plasma membrane of principal cells in the collecting duct, and increased AQP3 expression in the basolateral membrane. These oxytocin-induced effects were blocked by treatment with the vasopressin V2 receptor antagonist SR121463B, but not by treatment with the oxytocin receptor antagonist GW796679X. We conclude that vasopressin V2 receptors mediate the antidiuretic effects of oxytocin, including increased expression and apical trafficking of AQP2, p-AQP2, and increased AQP3 protein expression.

Identification of atropine- and P2X1 receptor antagonist-resistant, neurogenic contractions of the urinary bladder.

Acetylcholine and ATP are excitatory cotransmitters in parasympathetic nerves. We used P2X1 receptor antagonists to further characterize the purinergic component of neurotransmission in isolated detrusor muscle of guinea pig urinary bladder. In the presence of atropine (1 microM) and prazosin (100 nM), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.1-100 microM) and suramin (1-300 microM) inhibited contractions evoked by 4 Hz nerve stimulation in a concentration-dependent manner (IC50 of 6.9 and 13.4 microM, respectively). Maximum inhibition was 50-60%, which was unaffected by coadministration of the ectonucleotidase inhibitor ARL67156 (6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP) (100 microM). The remaining responses were abolished by tetrodotoxin (1 microM). PPADS and suramin also reduced contractions to exogenous ATP (300 microM) by 40-50%, but abolished those to the P2X1 agonist alpha,beta-methyleneATP (alpha,beta-meATP) (1 microM). The P2X1 antagonists reactive blue 2, NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis-1,3,5-naphthalenetrisulfonic acid), MRS2159 (pyridoxal-alpha5-phosphate-6-phenylazo-4'-carboxylic acid) (100 microM), and NF449 [4,4',4,4-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid] (3 microM) abolished contractions to alpha,beta-meATP (1 microM; n = 4-5), but only reduced contractions evoked by 4 Hz nerve stimulation by approximately 40-60% (n = 4-6) and ATP by 30-60% (n = 4-7). However, prolonged exposure to alpha,beta-meATP (50 microM) abolished contractions evoked by all three stimuli (n = 5-12). PPADS (100 microM) and suramin (300 microM) reduced the peak neurogenic contraction of the mouse urinary bladder to 30-40% of control. At the same concentrations, the P2X1 antagonists abolished the nonadrenergic, purinergic component of neurogenic contractions in the guinea pig vas deferens (n = 4-5). Thus, P2X1 receptor antagonists inhibit, but do not abolish, the noncholinergic component of neurogenic contractions of guinea pig and mouse urinary bladder, indicating a second mode of action of neuronally released ATP. This has important implications for treatment of dysfunctional urinary bladder, for which this atropine- and P2X1 antagonist-resistant site represents a novel therapeutic target.

N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.

The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4-/- bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4alpha-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

A new method for producing urinary bladder hyperactivity using a non-invasive transient intravesical infusion of acetic acid in conscious rats.

INTRODUCTION: Animal models that closely resemble the pathophysiology of human overactive bladder are important for evaluating novel therapeutics to treat the disorder. We established a non-invasive hyperactive bladder model that is sensitive to anti-muscarinic drugs and without bladder inflammation. METHODS: Acetic acid solution was infused into the bladder for 5 min via the urethral orifice without any surgical procedures under isoflurane anaesthesia. After washing the bladder with saline, voiding frequency (VF) and total urine volume were determined for 9 h under conscious conditions. RESULTS: Infusion of a 0.5% acetic acid solution caused a significant increase in VF, without influencing total urine volume or inducing significant histopathological inflammatory alterations in the bladder urothelium. Oral administration of oxybutynin (3 and 10 mg/kg) significantly ameliorated increases in VF induced by 0.5% acetic acid. Infusion of 0.75% acetic acid induced intensive urinary inflammation and a decrease in total urine volume as well as an increase in VF. Oral treatment with oxybutynin (10 mg/kg) did not significantly improve the increased VF due to 0.75% acetic acid. Acetic acid (0.5%) infusion evoked bladder hyper-responsiveness whether applied at night or during the day. However, VF was increased more by the nighttime application of acetic acid, while there were no significant differences in basal levels of VF between daytime and nighttime. DISCUSSION: In this study, the non-invasive rat urinary hyperactive bladder model indicated minimizes the secondary effects of experimental procedures such as surgical operations and anesthesia on bladder function and is sensitive to oxybutynin. Thus, the model may be useful for investigating novel therapeutics for OAB treatment.

The discovery of GSK221149A: a potent and selective oxytocin antagonist.

Optimisation of a series of oxazole diketopiperazines has led to the discovery of a very potent and selective oxytocin antagonist GSK221149A. GSK221149A has been shown to inhibit oxytocin-induced uterine contractions in the anaesthetised rat.

The effects and selectivity of beta-adrenoceptor agonists in rat myometrium and urinary bladder.

Recent evidence supports a role for beta(3)-adrenoceptors in human non-pregnant and pregnant myometrium. The present study was designed to characterize the pharmacology of beta-adrenoceptors involved in the function of non-pregnant rat myometrium by comparison of the activity of several beta-adrenoceptor agonists and antagonists in isolated rat uterus and urinary bladder. Contractions of myometrial and detrusor strips were induced by adding 1 nM oxytocin and 15 mM KCl respectively. Cumulative concentration-response curves to the selective beta(3)-adrenoceptor agonists, CL 316243 and BRL 37344 and the selective beta(2)-adrenoceptor agonist ritodrine were obtained in the presence and absence of the selective beta(2)-adrenoceptor antagonist ICI 118551 and the non-selective beta-adrenoceptor antagonist bupranolol. Both BRL 37344 (pD(2)=6.79+/-0.09) and ritodrine (pD(2)=6.89+/-0.19) produced potent inhibition of oxytocin-induced contractions in myometrial strips; CL 316243 was inactive at concentrations up to 10 microM. Concentration effect curves to both BRL 37344 and ritodrine were shifted (10 to 30-fold) to the right in the presence of ICI 118551 (10 nM). BRL 37344 (pD(2)=8.51+/-0.21) and CL 316243 (pD(2)=8.61+/-0.24) produced potent inhibition of detrusor strips, while ritodrine was almost 100-fold less potent (pD(2)=5.83+/-0.17). The response to all agonists was significantly attenuated by pretreatment with bupranolol (10 microM), but only ritodrine was affected by ICI 118551 (1 microM). These results demonstrate that relaxation of rat myometrium is mediated by beta(2)-adrenoceptors while, consistent with previous reports, the beta(3)-subtype is primarily responsible for relaxation of rat detrusor.

Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle.

(1) The involvement of Rho-kinase (ROCK) in the contractile mechanisms mediating smooth muscle contraction of the rat urinary bladder was investigated using expression studies and the ROCK inhibitor Y-27632. (2) Both isoforms of ROCK (ROCK I and ROCK II) were detected in high levels in rat urinary bladder. (3) Y-27632 (10 micro M) significantly attenuated contractions of rat urinary bladder strips evoked by the G-protein coupled receptor agonists carbachol (58.1+/-10.5% at 0.3 micro M) and neurokinin A (68.6+/-12.7% at 1 micro M) without affecting contractions to potassium chloride (10-100 mM). In addition, basal tone was reduced by 47.8+/-2.0% by 10 micro M Y-27632 in the absence of stimulation. (4) Contractions of urinary bladder strips evoked by the P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-mATP; 10 micro M) were also attenuated by Y-27632 (30.0+/-7.2% at 10 micro M). (5) Y-27632 (10 micro M) significantly attenuated contractions evoked by electrical field stimulation (2-16 Hz). The effect of Y-27632 on the tonic portion of the neurogenic response (4-16 Hz) was not significantly different from the effect of atropine (1 micro M) alone. (6) While the mechanism underlying the ability of Y-27632 to inhibit alpha,beta-mATP-evoked contractions remains undetermined, the results of the present study clearly demonstrate a role for ROCK in the regulation of rat urinary bladder smooth muscle contraction and tone.

Voltage-gated Na+ channel blockers reduce functional bladder capacity in the conscious spontaneously hypertensive rat.

OBJECTIVE: To evaluate the consequence of pharmacologic inhibition of voltage-gated Na(+) channels (Nav) in the conscious rat, based on Nav having been implicated as modulators of rodent urodynamics using knockout as well as antisense oligodeoxynucleotide approaches. METHODS: The urodynamic response to standard Nav blockers, lamotrigine, amitriptyline, mexiletine, and carbamazepine were evaluated using conscious, continuous-filling cystometry in spontaneously hypertensive rats (SHRs). As a selectivity evaluation, the activity of the Nav blockers at muscarinic receptors was assessed via effect on carbachol-evoked bladder contractions. RESULTS: Lamotrigine, amitriptyline, mexiletine, and carbamazepine decreased peak micturition pressure, micturition interval, and void volume. These effects were markedly similar to observations with muscarinic antagonists. Therefore, we evaluated the selectivity of these agents against bladder muscarinic receptors. Lamotrigine, mexiletine, and carbamazepine had no effect on muscarinic bladder contractions, whereas amitriptyline displayed a robust antagonism of carbachol-induced contractility. CONCLUSION: Three Nav blockers--lamotrigine, mexiletine, and carbamazepine--demonstrated a reduction in micturition pressure and functional bladder capacity, similar to previous observations with muscarinic antagonists. These 3 Nav blockers are free of muscarinic antagonism, consistent with their cystometric effects being mediated via their Nav blocking activities. The negative findings reported here with Nav blockers suggest that Nav channel blockade is unlikely to reflect an improved treatment strategy for bladder disorders over currently prescribed muscarinic antagonists.

Fenoterol functionally activates the ß3-adrenoceptor in human urinary bladder, comparison with rat and mouse: implications for drug discovery.

Fenoterol has been reported to be a potent and selective ß(2)-adrenoceptor agonist and is currently used clinically to treat asthma. Electrical field stimulation (EFS) of isolated urinary bladder mimics the voiding contraction by stimulating parasympathetic nerves, resulting in neurogenic contractions. To determine if stimulation of ß(2)-adrenoceptors can inhibit this response, fenoterol was tested against EFS-induced contractions in human isolated urinary bladder and compared with mouse and rat. Bladder strips were mounted in organ baths and reproducible contractions induced by EFS. Fenoterol was added cumulatively in the presence of the ß(2)-adrenoceptor antagonist ICI118551 or the ß(3)-adrenoceptor antagonist L-748337. Fenoterol inhibited neurogenic contractions in all three species in a concentration-dependent manner with pEC(50) values of 6.66 ± 0.11, 6.86 ± 0.06 and 5.71 ± 0.1 in human, mouse and rat respectively. In human bladder strips ICI118551 (100 nM) did not affect responses to fenoterol, while L-748337 (0.3-3 µM) produced rightward shifts of the concentration-response curves with a pA(2) value of 8.10. In mouse bladder strips ICI118551 (30 nM) blocked the inhibitory effect of fenoterol (pA(2)=8.80), while L-748337 (10 µM) inhibited the response with a pA(2) of 5.79. In rat bladder ICI118551 (30 nM) was without effect, while L-748,337 (10 µM) inhibited the response to fenoterol with a pA(2) of 5.40. From these results it is clear that fenoterol potently activates ß(3)-adrenoceptors in human isolated urinary bladder to inhibit EFS-induced contractions. Fenoterol also activates ß(3)-adrenoceptors in rat, but ß(2)-adrenoceptors in mouse bladder to inhibit EFS-induced contractions.

Pyridyl-2,5-diketopiperazines as potent, selective, and orally bioavailable oxytocin antagonists: synthesis, pharmacokinetics, and in vivo potency.

A six-stage stereoselective synthesis of indanyl-7-(3'-pyridyl)-(3R,6R,7R)-2,5-diketopiperazines oxytocin antagonists from indene is described. SAR studies involving mono- and disubstitution in the 3'-pyridyl ring and variation of the 3-isobutyl group gave potent compounds (pK(i) > 9.0) with good aqueous solubility. Evaluation of the pharmacokinetic profile in the rat, dog, and cynomolgus monkey of those derivatives with low cynomolgus monkey and human intrinsic clearance gave 2',6'-dimethyl-3'-pyridyl R-sec-butyl morpholine amide Epelsiban (69), a highly potent oxytocin antagonist (pK(i) = 9.9) with >31000-fold selectivity over all three human vasopressin receptors hV1aR, hV2R, and hV1bR, with no significant P450 inhibition. Epelsiban has low levels of intrinsic clearance against the microsomes of four species, good bioavailability (55%) and comparable potency to atosiban in the rat, but is 100-fold more potent than the latter in vitro and was negative in the genotoxicity screens with a satisfactory oral safety profile in female rats.

Involvement of beta(3)-adrenoceptors in mouse urinary bladder function: role in detrusor muscle relaxation and micturition reflex.

beta(3)-adrenoceptor activation produces relaxation of human urinary bladder smooth muscle (detrusor). Therefore, beta(3)-adrenoceptor agonism is being investigated as a new therapeutic strategy for the treatment of overactive bladder. The aim of the current study was to identify the functional presence of beta(3)-adrenoceptors in mouse isolated urinary bladder using the selective beta(3)-adrenoceptor agonist CL316,243 and antagonists SR59230A and L748,337. The effects of CL316,243 on basal tone, spontaneous activity and electrical field stimulation (EFS)-induced contractions were investigated using in vitro techniques, while the in vivo effects of intravenously administered CL316,243 on the micturition reflex were investigated using cystometry. CL316,243 decreased basal tone (pEC(50)=6.4+/-0.4) as well as spontaneous activity (53+/-7% at 3 microM) and inhibited EFS-induced contractions (pEC(50)=7.0+/-0.2) of the detrusor muscle. The beta(3)-adrenoceptor antagonist SR59230A (1 microM) significantly inhibited the relaxing effects of CL316,243 on basal tone and neurogenic contractions (pA(2)=7.0 and 7.2, respectively). Another beta(3)-adrenoceptor antagonist L748,337 (1-10 microM) significantly blocked the CL316,243-evoked inhibition of neurogenic contractions in a concentration-dependent manner (pK(B)=6.8), while the selective beta(2)-adrenoceptor antagonist ICI118,551(30 nM) had no effect. In anesthetized mice, CL316,243 (0.03 and 0.1 mg/kg, i.v.) significantly increased bladder capacity and threshold pressure without a modification of bladder compliance. Moreover, it induced a significant decrease in the amplitude of both micturition and non-voiding contractions. Based on the current results obtained using the beta(3)-adrenoceptor agonist CL316,243 (as well as various beta-adrenoceptor antagonists), functional beta(3)-adrenoceptors appear to be present in mouse urinary bladder.

GW427353 (solabegron), a novel, selective beta3-adrenergic receptor agonist, evokes bladder relaxation and increases micturition reflex threshold in the dog.

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by beta3-adrenergic receptors (ARs) in human bladder. Thus, the use of selective beta3-AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective beta3-AR agonist, (R)-3'-[[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]ethyl]amino]-[1,1'-biphenyl]-3-carboxylic acid (GW427353; solabegron) on bladder function in the dog using in vitro and in vivo techniques. GW427353 stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human beta3-AR, with an EC50 value of 22 +/- 6 nM and an intrinsic activity 90% of isoproterenol. At concentrations of 10,000 nM, GW427353 produced a minimal response in cells expressing either beta1-ARs or beta2-ARs (maximum response <10% of that to isoproterenol). In dog isolated bladder strips, GW427353 evoked relaxation that was attenuated by the nonselective beta-AR antagonist bupranolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A) (reported to have beta3-AR antagonist activity). The relaxation was unaffected by atenolol, a selective beta1-AR antagonist, or (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118551), a selective beta2-AR antagonist. GW427353 increased the volume required to evoke micturition in the anesthetized dog following acetic acid-evoked bladder irritation, without affecting the ability of the bladder to void. GW427353-evoked effects on bladder parameters in vivo were inhibited by bupranolol. The present study demonstrates that selective activation of beta3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.

Use of a novel and highly selective oxytocin receptor antagonist to characterize uterine contractions in the rat.

Spontaneous and induced uterine contractions in the rat were found to be inhibited by a novel and selective oxytocin receptor antagonist GSK221149A (3R,6R)-3-Indan-2-yl-1-[(1R)-1-(2-methyl-1,3-oxazol-4-yl)-2-morpholin-4-yl-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione. GSK221149A displayed nanomolar affinity (K(i) = 0.65 nM) for human recombinant oxytocin receptors with >1,400-fold selectivity over human V1a, V1b, and V2 receptors. GSK221149A had similar affinity (K(i) = 4.1 nM) and selectivity for native oxytocin receptors from rat and produced a functional, competitive block of oxytocin-induced contractions in isolated rat myometrial strips with a pA(2) value of 8.18. Intravenous administration of GSK221149A produced a dose-dependent decrease in oxytocin-induced uterine contractions in anesthetized rats with an ID(50) = 0.27 +/- 0.60 mg/kg (corresponding plasma concentrations were 88 ng/ml). Oral administration of GSK221149A (5 mg/kg) was effective in inhibiting oxytocin-induced uterine contractions after single and multiple (4-day) dosing. Spontaneous uterine contractions in late-term pregnant rats (19-21 days gestation) were significantly reduced by intravenous administration of 0.3 mg/kg of GSK221149A. These results provide further evidence that selective oxytocin receptor antagonism may offer an effective treatment for preterm labor.

Effect of endothelin on bladder contraction: potential role in bladder hyperactivity.

In the present study, we demonstrate that the intravenous infusion of endothelin-1 (3 and 10 ng/kg/min) causes a decrease in the mean micturition volume of rats in addition to an increase in mean arterial pressure. These effects are blocked by both the ET(A)/ET(B)-non-selective and the ET(A)-selective endothelin antagonists SB 217242 and SB 247083 respectively (both 30 mg/kg). However, it was also observed that the ET(B)-selective agonist sarafotoxin 6c (3 and 10 ng/kg/min) had similar effects on both mean arterial pressure and micturition volume. Initial experiments indicated that spontaneously hypertensive rats have a much lower mean micturition volume than normal rats. Binding studies comparing the total number and ratio of ET(A)/ET(B) receptors in spontaneously hypertensive, Wister-Kyoto and Sprague-Dawley rats revealed no significant differences in receptor expression. However, the magnitude of the response to endothelin-1 was greater in spontaneously hypertensive versus normal rats.

Characterization of neuromedin U effects in canine smooth muscle.

Two endogenous receptors for the potent smooth muscle-stimulating peptide neuromedin U (NmU) have recently been identified and cloned. Pharmacological, binding, and expression studies were conducted in an attempt to determine the receptor(s) involved in the smooth muscle-stimulating effects of NmU. The NmU peptides caused a concentration-dependent contraction of canine isolated urinary bladder. NmU did not have this same effect in the urinary bladder from rat, guinea pig, rabbit, mouse, or ferret. Although NmU had no effect on canine uterus it did cause contraction of canine stomach, ileum, and colon. As well as causing contraction of canine bladder in vitro, NmU administered systemically resulted in a significant increase in urinary bladder pressure in vivo. High-affinity binding sites for NmU were identified in canine bladder. The four NmU peptides porcine NmU-8, rat NmU-23, human NmU-25, and porcine NmU-25 displaced (125)I-NmU-25 binding with similar K(i) values (0.08-0.24 nM). A different binding profile was revealed in human embryonic kidney-293 cells transiently expressed with the canine NmU-2 receptor where porcine NmU-8 (K(i) = 147.06 nM) was much less potent than the other NmU peptides. Using TaqMan, expression of NmU-1 was detected in human urinary bladder, small intestine, colon, and uterus. Expression of NmU-2 was much lower or absent in these human tissues and undetectable in canine bladder and stomach. The results of this study reveal significant species differences in the activity of NmU. The contractile activity in human and canine smooth muscle seems to be mediated by the recently cloned NmU-1 receptor.