Urinary Bladder Strips Research Articles

Ovalbumin-induced neurogenic inflammation in the bladder of sensitized rats.

1. We have developed and characterized a model of immediate hypersensitivity/inflammation of the urinary bladder in vivo induced by local application of ovalbumin (OA) in OA- sensitive female rats. Two parameters of the inflammatory response were assessed following OA challenge: plasma protein extravasation (PPE) and changes in smooth muscle reactivity. The former was estimated by measurement of Evans blue extravasation at 0.5, 2, 4, 8 and 24 h time point following in vivo challenge. Changes in reactivity were determined by measurement of isotonic tension responses of urinary bladder strips following OA challenge in vitro. 2. Acute in vivo intravesical OA challenge (10 mg in 0.3 ml saline) in actively sensitized female Wistar rats caused a time-dependent PPE in the urinary bladder which was biphasic with peak responses at 2-4 and 24 h. 3. The PPE response to acute OA challenge, above base-line, at 2 h was abolished by systemic capsaicin pretreatment (50 mg kg(-1), s.c., 4 days before use) (P < 0.05) whilst the response at 24 h was unaffected. The 2 h time point was then used for further studies. 4. Degranulation of mast cells, achieved by pretreatment with compound 48/80 (5 mg kg(-1), s.c. for 3 consecutive days), completely abolished the PPE response to OA challenge at the 2 h time point. 5. The tachykinin NK1 receptor antagonist, SR 140333 (0.1 micromol kg(-1), i.v.), abolished the 2 h PPE response whilst the tachykinin NK2 receptor antagonist MEN 11420 (0.1 micromol kg(-1), i.v.) appeared to reduce the response by approximately 50% but this did not reach significance. The bradykinin B2 receptor antagonist, Hoe 140 (0.1 micromol kg(-1), i.v.), similarly to SR 140333, blocked the 2 h PPE response to OA, whereas the selective B1 receptor antagonist B 9858 (0.1 micromol kg(-1), i.v.) had no significant effect. Inhibition of cyclo-oxygenase (COX) achieved by pretreatment with the COX inhibitor dexketoprofen (5.3 micromol kg(-1), i.v.) also blocked the PPE response, whilst the leukotriene receptor antagonist ONO 1078 (1 micromol kg(-1), i.v.) significantly reduced PPE by about 80%. 6. In the rat isolated urinary bladder OA (1 mg ml(-1)) challenge produced a biphasic response with a rapidly achieved maximal contraction followed by a sustained contraction for approximately 25 min. In vitro capsaicin pretreatment (10 microM for 15 min) significantly attenuated the duration of the sustained contraction whilst having no effect on the maximum contractile response achieved. In vivo pretreatment of animals with compound 48/80 significantly attenuated (42%) the maximum contractile response. Combination of both treatments almost completely abolished the response. In vitro treatment with Hoe 140 (1 microM) had no significant effect on the response to OA and neither did ONO 1078 (1 microM). 7. These results show that both the early inflammatory response and alterations in smooth muscle reactivity to OA challenge in actively sensitized animals are dependent on mast cell degranulation and the activation of sensory C-fibres. Furthermore this model of allergic cystitis may be useful for investigating both the processes involved and potential novel therapies in the treatment of interstitial cystitis.

Effect of an atypical adrenergic beta3-agonist, GS-332: sodium (2R)-[3-[3-[2-(3-chlorophenyl)-2-hydroxyethylamino]cyclohexyl]phenoxy] acetate, on urinary bladder function in rats.

We have developed an atypical adrenergic beta3-agonist, GS 332: Sodium (2R)-[3-[3-[2-(3 Chlorophenyl)-2-hydroxyethylamino]cyclohexyl]phenoxy]acetate, which has an unique structure compared to other beta3 agonists. In vitro study, we compared effects of GS 332 on rat urinary bladder muscle strip contractility with those of clenbuterol hydrochloride (clenbuterol), an adrenergic beta2 agonist. GS-332 relaxed isolated rat urinary bladder strips in a concentration dependent manner with 50% effective concentration (EC50) of 15.7 nM, and the relaxant activity of GS 332 was as potent as that of clenbuterol(EC50; 30.8 nM). The concentration response curve of GS 332 on isolated rat urinary bladder was competed by a specific beta3-antagonist, SR59230A in a concentration-dependent manner, in which Schild slope was 1.1 and pA2 value of SR59230A was 7.1. In vivo study, cystometory investigated in anesthetized rats demonstrated that GS 332 was more potent in increasing the urinary storage volume than clenbuterol and less potent in inhibiting the contractile force of urinary bladder at micturition reflex than clenbuterol. These data demonstrate that GS 332, a new adrenergic beta3-agonist, may be more useful to maintain continence than clenbuterol, an adrenergic beta3 adrenergic agonist.

DMSO: EFFECT ON BLADDER AFFERENT NEURONS AND NITRIC OXIDE RELEASE

Purpose Interstitial cystitis (IC), a chronic disorder of the urinary bladder, is characterized by increased voiding frequency, urgency and pain. Patients with IC also exhibit reduced urinary nitric oxide synthase activity. Intravesical administration of dimethyl sulfoxide (DMSO) has been used to provide symptomatic relief in patients with IC. The present experiments were undertaken to determine if intravesical DMSO affects neural pathways involved in bladder function in the rat and if DMSO can influence the release of nitric oxide in the bladder or from afferent neurons. Materials and Methods The effects of intravesical DMSO (10% solution in saline) on reflex bladder activity, firing on bladder nerves and c-fos gene expression in spinal neurons was examined in urethane anesthetized female Wistar rats. The effect of DMSO (1-10%) on nitric oxide release from urinary bladder strips or acutely dissociated dorsal root ganglion cells was monitored in vitro with a porphyrinic microsensor. Results DMSO acutely increased reflex firing of pelvic nerve efferent axons, decreased bladder capacity and also increased neuronal c-fos expression in spinal cord regions that exhibit c-fos expression after chemical activation of capsaicin-sensitive bladder afferents. DMSO, like capsaicin, also directly released nitric oxide (NO) from both dissociated dorsal root ganglion neurons and from isolated strips of urinary bladder. Conclusions These results suggest that DMSO induced stimulation of bladder afferent pathways and NO release from afferent neurons may be a reflection of the initial event in the desensitization of nociceptive pathways in the lower urinary tract (LUT).

Antimuscarinic potency and bladder selectivity of PNU-200577, a major metabolite of tolterodine.

PNU-200577 (labcode DD 01 [(R)-N, N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphenyl)-3-phenylpropanamine ) is a major pharmacologically active metabolite of tolterodine, a new muscarinic receptor antagonist intended for the treatment of an overactive bladder. In vitro, PNU-200577 produced a competitive and concentration-dependent inhibition of carbachol-induced contraction of guinea-pig isolated urinary bladder strips (KB = 0.84 nM; pA2 = 9.14). In vivo, PNU-200577 was significantly more potent at inhibiting acetylcholine-induced urinary bladder contraction than electrically induced salivation in the anaesthetised cat (ID50 15 and 40 nmol.kg-1, respectively; P < 0.01). In radioligand binding studies carried out in homogenates of guinea-pig tissues and Chinese hamster ovary cell lines expressing human muscarinic m1-m5 receptors, PNU-200577 was not selective for any muscarinic receptor subtype. Thus, PNU-200577 is similar to tolterodine in terms of antimuscarinic potency, functional selectivity for the urinary bladder in vivo and absence of selectivity for muscarinic receptor subtypes in vitro. The results of this study clearly indicate that PNU-200577 contributes to the therapeutic action of tolterodine, in view of its high antimuscarinic potency, similar serum concentration and lower degree of protein binding.

The role of cyclic nucleotides in guinea-pig bladder contractility.

1. The effects of phosphodiesterase (PDE) inhibition and forskolin pretreatment on the contractile responses of guinea-pig urinary bladder strips to electrical field stimulation, carbachol, ATP and KCl were studied. 2. Inhibition of cyclic AMP-specific PDE4 isozymes by rolipram significantly reduced the contractile response of bladder strips to field stimulation. Rolipram also suppressed the contractile response to low concentrations of carbachol, but potentiated the response to high concentrations. The contractile response to ATP was significantly reduced by rolipram treatment, but that to KCl was unaltered. 3. Inhibition of cyclic GMP-specific PDE5 isozymes by zaprinast had no effects on the contractile response of bladder strips to field stimulation, ATP or KCl. Zaprinast suppressed the contractile responses to 1 microM carbachol and potentiated the response to high concentrations. 4. Contractile responses to field stimulation and to carbachol after pretreatment with the adenylyl cyclase activator, forskolin, were qualitatively similar to those caused by rolipram treatment. beta-Adrenoceptor blockade with propranolol partially reversed the inhibitory effects of rolipram on the response to field stimulation. 5. Rolipram significantly reduced the contractile response of bladder strips from sensitized guinea-pigs to ovalbumin challenge, but zaprinast was ineffective. PDE inhibition had similar effects on the responsiveness of control and of sensitized guinea-pig bladder strips to field stimulation, carbachol, ATP and KCl. 6. The data suggest that the contractile response of guinea-pig bladder strips can be modified by increases in cyclic AMP levels.

Neurokinin receptors (NK1,NK2) in the mouse: a pharmacological study

Experiments were performed on strips of mouse stomach and urinary bladder to characterize the receptors involved in the contractile responses of these tissues to neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide gamma (NP gamma). The neurokinin receptors were characterized by using assays with selective agonists as well as peptide and nonpeptide antagonists and by applying the two Schild criteria for receptor classification, namely, the order of potency of agonists and the apparent affinity of competitive antagonists. The mouse stomach contains primarily NK1 and NK2 functional sites and possibly some NK3 receptors, whereas the urinary bladder possesses only the NK2 receptor. The rank order of potency of agonists in the stomach is Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) > NKA > SP > [beta-Ala8]NKA-(4-10) > NKB > [MePhe7]NKB. Among the selective agonists, Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) is more active than SP and [Sar9,Met(O2)11]SP on the NK1 receptor, whereas the order of potency on the NK2 receptor is NKA > NP gamma > or = [beta-Ala8]NKA-(4-10) > [Nle10]NKA-(4-10). The order of potency of agonists in the bladder is NP gamma > NKA > [beta-Ala8]NKA-(4-10). The myotropic responses mediated by NK1 selective agonists are blocked by SR 140333 (pA2 8.57) and those mediated by the NK2 selective agonists are inhibited by SR 48968 (pA2 9.05). RP 67580 (pA2 8.41) is more active than CP 99994 (pA2 6.06) on the mouse NK1 receptor. The NK1 receptor of the mouse shows, therefore, a pharmacological profile similar to that of the NK1 receptor of the rat. Similarly, MEN 10627 (pA2 9.20) is more active than R 396 (pA2 6.21), suggesting that the mouse NK2 receptor is similar to that of the rabbit. The mouse NK2 receptor of the urinary bladder shows similarity with that of the stomach, but is less sensitive to [beta-Ala8]NKA-(4-10).

Kinin receptors in the diabetic mouse

It has been proposed that kinins are important inflammatory mediators involved in the pathogenesis of several diseases. In the present study, we attempted to determine the effects of kinins in a type I diabetic mouse model, using in vitro assays. Injection of streptozotocin (STZ) to the C57BL/Ks mdb mice causes an insulitis (inflammation of Langerhans islets) that leads to the diabetic condition. Ten days following the STZ treatment, the mice showed increased glycemia. We examined the effect of kinins and other agents (substance P, neurokinin A, acetylcholine) on the stomach fundus and urinary bladder of control and diabetic mice. Our results show that the sensitivity of the stomach fundus to bradykinin (BK) and desArg9BK (DBK), but not to other contractile agents, was substantially increased in the tissues of diabetic mice. The maximal contractions induced by BK and DBK were increased 1.5- to 2-fold in the stomachs from diabetic mice compared with those from normal mice. BK induced similar maximal contractions of urinary bladder strips from normal or STZ-treated mice, while DBK did not show any effect on this preparation. Interestingly, the apparent affinities of all agonists are similar in the two groups, normal and diabetic. These results suggest that B1 and B2 receptors are overexpressed in the stomach fundus but not in the urinary bladder of diabetic mice.

Tolterodine – a new bladder-selective antimuscarinic agent

Tolterodine is a new muscarinic receptor antagonist intended for the treatment of urinary urge incontinence and other symptoms related to an overactive bladder. The aim of the present study was to compare the antimuscarinic properties of tolterodine with those of oxybutynin, in vitro and in vivo. Tolterodine effectively inhibited carbachol-induced contractions of isolated strips of urinary bladder from guinea pigs ( K B 3.0 nM; pA 2 8.6; Schild slope 0.97) and humans ( K B 4.0 nM; pA 2 8.4; Schild slope 1.04) in a concentration-dependent, competitive manner. The affinity of tolterodine was similar to that derived for oxybutynin ( K B 4.4 nM; pA 2 8.5; Schild slope 0.89) in the guinea-pig bladder. Tolterodine (21–2103 nmol/kg (0.01–1 mg/kg); intravenous infusion) was significantly more potent in inhibiting acetylcholine-induced urinary bladder contraction than electrically-induced salivation in the anaesthetised cat. In contrast, oxybutynin displayed the opposite tissue selectivity. Radioligand binding data showed that tolterodine bound with high affinity to muscarinic receptors in urinary bladder ( K i 2.7 nM), heart ( K i 1.6 nM), cerebral cortex ( K i 0.75 nM) and parotid gland ( K i 4.8 nM) from guinea pigs and in urinary bladder from humans ( K i 3.3 nM). Tolterodine and oxybutynin were equipotent, except in the parotid gland, where oxybutynin bound with 8-times higher affinity ( K i 0.62 nM). Binding data on human muscarinic m1–m5 receptors expressed in Chinese hamster ovary cells showed that oxybutynin, in contrast to tolterodine, exhibits selectivity (10-fold) for muscarinic m3 over m2 receptors. The K B value determined for oxybutynin (4.4 nM) in functional studies on guinea-pig bladder correlated better with the binding affinity at muscarinic M 2/m2 receptors ( K i 2.8 and 6.7 nM) than at muscarinic M 3/m3 receptors ( K i 0.62 and 0.67 nM). The tissue selectivity demonstrated for tolterodine in vivo cannot be attributed to selectivity for a single muscarinic receptor subtype. However, the combined in vitro and in vivo data on tolterodine and oxybutynin may indicate either that muscarinic M 3/m3 receptors in glands are more sensitive to blockade than those in bladder smooth muscle, or that muscarinic M 2/m2 receptors contribute to bladder contraction.

Contractile responses and calcium mobilization induced by muscarinic agonists in the rat urinary bladder: Effects of age

In the current study, the contractile responses to muscarinic stimulation of urinary bladder strips from young rats and aged rats were compared. 1. 1. The EC 50 values of the two groups in response to bethanechol were similar. 2. 2. The magnitude and the velocity of tension generation was significantly lower in strips from the aged group. 3. 3. The magnitude as well as the velocity of tension generation in response to high K + solution of the two models were similar. 4. 4. The time to peak [Ca 2+]i in response to bethanechol was prolonged in strips from the aged group as compared with the young group. 5. 5. 45Ca 2+ influx in response to bethanechol was significantly reduced in the aged group as compared with the young group. It is concluded that the reduced contractile response to muscarinic stimulation of isolated urinary bladder strips from aged rats is mediated at least in part by a decreased rate of Ca 2+ entry.

Human Anti-IgE Monoclonal Antibody Blocks Passive Sensitization of Human and Rhesus Monkey Bladder

IgE antibodies may play an important role in noninfectious urinary inflammation, including interstitial cystitis (IC). In this study, urinary bladder strips were passively sensitized for 20 hours with serum from a ragweed-sensitive patient in the absence or presence of an anti-human IgE monoclonal antibody (MaE11) at 0, 1-, or 5-fold IgE concentration. The urinary bladder strips then were suspended in a superfusion apparatus for measurement of contraction and histamine release in response to antigen E (AgE) challenge. Non-sensitized tissues did not react to AgE challenge, whereas AgE challenge of passively-sensitized tissues resulted in time-dependent bladder contraction and histamine release. MaE11 abolished AgE-induced contraction and histamine release in a concentration-dependent manner. The safety of MaE11 was confirmed by its failure to contract or release histamine from passively-sensitized bladder tissues. The results of this study suggest that MaE11 may have immunotherapeutic benefit for amelioration of IgE-mediated diseases of the urinary system.

Evidence for the Presence of Regional Differences in the Subtype Specificity of Muscarinic Receptors in Rabbit Lower Urinary Tract

To elucidate the subtype specificity of muscarinic cholinergic receptors in mediating contractile responses in the lower urinary tract, we investigated contractile and biochemical properties of muscarinic receptors in bladder dome, bladder base and urethra of the rabbit. Isometric contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists. Bladder dome, bladder base, and urethra demonstrate different characteristics in terms of efficacy and potency with respect to carbachol-induced contractile responses. Emax values are significantly larger and ED50 values are significantly smaller in bladder dome and bladder base than in urethra. Calculation of the pA2 values, the negative logarithm of the antagonist affinity constant (KB), for a series of muscarinic antagonists, i.e., atropine (nonselective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective) indicate that the carbachol-induced contractile response in bladder dome and bladder base is mediated through the M3 receptor subtype whereas the carbachol-induced contractile response in urethra is probably mediated through the M1 and/or M3 and possibly M2 subtypes. Muscarinic cholinergic antagonists inhibit [3H]quinulidinyl benzilate binding to bladder dome, bladder base and urethra with the following rank order of affinities: atropine > 4-DAMP > methoctramine > pirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.

The relaxant effect of adrenomedullin on particular smooth muscles despite a general expression of its mRNA in smooth muscle, endothelial and epithelial cells.

1. By use of the reverse transcription polymerase chain reaction (RT-PCR), we determined the expression of adrenomedullin (AM) mRNA in the various tissues of the pig. To evaluate the significance of the expression of AM mRNA, we also determined the effects of AM on the cytosolic Ca2+ concentration ([Ca2+]i) and tension development of the porcine smooth muscle strips obtained from the coronary artery, pulmonary vein, trachea, ileum and urinary bladder. 2.AM mRNA was widely expressed in the porcine tissues examined, which included myocardium (left and right ventricle and right atrium), kidney, lung, endothelial cells (aorta and aortic valve), smooth muscles (aorta, main pulmonary artery, pulmonary vein, renal artery and vein, coronary artery, ileum, trachea and urinary bladder) and epithelial cells (trachea and urinary bladder). 3. AM induced a decrease in [Ca2+]i and tension of the coronary artery, but not the pulmonary vein. AM had no effects on either the [Ca2+]i or tension of the trachea and urinary bladder strips or on the tension development of strips of ileum. 4. These results indicated that AM has a role as an autocrine and/or paracrine regulator of the coronary arterial tone. AM probably does not have an important role in the regulation of the pulmonary venous, tracheal, ileac and urinary bladder smooth muscle tone, even though AM mRNA is expressed in these tissues; the functional significance of AM in these smooth muscles remains to be determined.

Kinin receptors in the diabetic mouse

It has been proposed that kinins are important inflammatory mediators involved in the pathogenesis of several diseases. In the present study, we attempted to determine the effects of kinins in a type I diabetic mouse model, using in vitro assays. Injection of streptozotocin (STZ) to the C57BL/Ks mdb mice causes an insulitis (inflammation of Langerhans islets) that leads to the diabetic condition. Ten days following the STZ treatment, the mice showed increased glycemia. We examined the effect of kinins and other agents (substance P, neurokinin A, acetylcholine) on the stomach fundus and urinary bladder of control and diabetic mice. Our results show that the sensitivity of the stomach fundus to bradykinin (BK) and desArg9BK (DBK), but not to other contractile agents, was substantially increased in the tissues of diabetic mice. The maximal contractions induced by BK and DBK were increased 1.5- to 2-fold in the stomachs from diabetic mice compared with those from normal mice. BK induced similar maximal contractions of urinary bladder strips from normal or STZ-treated mice, while DBK did not show any effect on this preparation. Interestingly, the apparent affinities of all agonists are similar in the two groups, normal and diabetic. These results suggest that B1 and B2 receptors are overexpressed in the stomach fundus but not in the urinary bladder of diabetic mice.

Potentiation by Bradykinin and Substance P of Purinergic Neurotransmission in Urinary Bladder

Purpose The higher than normal levels of substance P (SP) and the kinins in patients suffering from interstitial cystitis suggest that they may contribute to the complex symptoms of the condition. The purpose of our experiments was to determine whether SP and bradykinin (BK) influence the excitatory motor innervation of the urinary bladder. Materials and Methods Strips of guinea pig urinary bladder were placed in isolated tissue baths, and the influence of SP and BK on contractions induced by transmural electrical stimulation and cholinergic and purinergic agonists was evaluated. Results Substance P and BK potentiated responses to the purinergic component of the neurogenic stimulation (that part of the contractile response that remains after treatment with atropine) and potentiated responses to exogenously applied adenosine triphosphate (ATP). The peptides did not potentiate the response to the cholinergic component of the nerve-induced contraction (that part of the neurogenic response that remains after desensitization of purinoceptors with alpha, beta-methylene ATP) nor responses to carbachol. The potentiating actions of SP and BK were reduced but not abolished by treatment with meclofenamic acid. Conclusions Substance P and BK potentiate the neurogenic response of the bladder by influencing the purinergic component of the excitatory motor innervation, apparently at a postjunctional site. Prostaglandins may be involved in mediating some of the actions of these peptides.

Potentiation by bradykinin and substance P of purinergic neurotransmission in urinary bladder.

The higher than normal levels of substance P (SP) and the kinins in patients suffering from interstitial cystitis suggest that they may contribute to the complex symptoms of the condition. The purpose of our experiments was to determine whether SP and bradykinin (BK) influence the excitatory motor innervation of the urinary bladder. Strips of guinea pig urinary bladder were placed in isolated tissue baths, and the influence of SP and BK on contractions induced by transmural electrical stimulation and cholinergic and purinergic agonists was evaluated. Substance P and BK potentiated responses to the purinergic component of the neurogenic stimulation (that part of the contractile response that remains after treatment with atropine) and potentiated responses to exogenously applied adenosine triphosphate (ATP). The peptides did not potentiate the response to the cholinergic component of the nerve-induced contraction (that part of the neurogenic response that remains after desensitization of purinoceptors with alpha, beta-methylene ATP) nor responses to carbachol. The potentiating actions of SP and BK were reduced but not abolished by treatment with meclofenamic acid. Substance P and BK potentiate the neurogenic response of the bladder by influencing the purinergic component of the excitatory motor innervation, apparently at a postjunctional site. Prostaglandins may be involved in mediating some of the actions of these peptides.

3H][β-Ala 8]neurokinin A-(4–10): a novel, selective radioligand for the tachykinin NK 2 receptor

The binding characteristics of the novel radioligand [ 3H][β-Ala 8]neurokinin A-(4–10) were assessed in hamster urinary bladder membranes. This labelled compound bound in a reversible, highly specific and concentration-dependent manner to a single class of high affinity binding sites with a K d of 1.8 ± 0.2 nM and a B max of 139 ± 21 fmol/mg of protein. Specific binding of [ 3H][β-Ala 8]neurokinin A-(4–10) was displaced only by NK 2, but not by NK 1 or NK 3, tachykinin receptor agonists and antagonists. Neurokinin A, [β-Ala 8]neurokinin A-(4–10), L 659877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)], MEN 10376 (H-Asp-Tyr- d-Trp-Val- d-Trp- d-Trp-Lys-NH 2), MEN 10627 [cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β-5β)]and SR 48968 [( S)- N-methyl- N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]displaced the binding with K i values of 0.4 ± 0.1 nM, 1.9 ± 0.36 nM, 3.05 ± 0.1 nM, 7.9 ± 0.4 μM, 0.36 ± 0.02 nM and 2.5 ± 0.9 nM, respectively. Functional data, obtained in isolated hamster urinary bladder strips with the newly developed tachykinin NK 2 receptor antagonists (MEN 10627 and SR 48968), showed a good agreement with binding data. This novel radioligand could represent a new useful tool for the assessment of tachykinin NK 2 receptor antagonists.

Influence of strip size and location on contractile responses of rat urinary bladder body strips

1. 1. We investigated the influence of strip length and dorsal or ventral location of rat urinary bladder strips on contractile responsiveness. 2. 2. No differences occurred in the contractile responses of 0.5, 1.0 and 2.0 cm strips to field stimulation, carbachol, ATP, substance P or to KCl when the data were expressed as either absolute tension or as tension per cross-sectional area. However, correction for strip mass resulted in significant decreases in the contractile responses of the 2.0-cm strips compared with the 0.5-cm strips. 3. 3. No differences occurred in length-tension curves for ventral and dorsal bladder strips, even though the strips from the dorsal surface appeared thinner than those from the ventral surface. 4. 4. Strips from the ventral surface exhibited more variability in response to field stimulation and were less sensitive to atropine pre-treatment than were those from the dorsal surface. They were also less sensitive to the contractile effects of carbachol than dorsal strips. Dorsal and ventral strips were equally responsive to ATP, substance P and KCl. 5. 5. Our data indicate that the contractile responsiveness of rat urinary bladder strips is independent of strip length. Although there are some differences between the cholinergic responsiveness of strips from the ventral and dorsal surfaces of the bladder, the differences are so small that for most studies they will probably have no influence on data interpretation.

Chemical Constituents ofArtemisia arborescensand the Effect of the Aqueous Extract on Rat Isolated Smooth Muscle

Phytochemical analysis of the ground aerial parts of Artemisia arborescens resulted in the isolation from the ethanolic extract of the known compounds: artemitin, arborescin, sesamin, (+)-lirioresinol beta-dimethyl ether, chrysoeriol, apigenin, beta-sitosteryl glucoside, dihydroridentin, and chrysoeriol 4-glucoside. The last six compounds are isolated from this plant for the first time. The same fraction also yielded the new eudesmanolide jordanolide (1). The effect of an aqueous extract of the plant was studied on rat isolated ileum, uterus, and urinary bladder. The aqueous extract (AE) caused a concentration-dependent reduction in the amplitude of the phasic contractions and in the tone of the ileum. On the other hand, AE caused a significant increase in the frequency as well as the amplitude of the phasic contractions and increased the tone of the isolated uterus and the urinary bladder strips. On the uterus, quinacrine, an inhibitor of the release of arachidonic acid and its metabolites, and indomethacin, a cyclooxygenase inhibitor, potentiated rather than inhibited the effects of AE on this tissue. These observations are discussed in relation to the use of the plant extract in folk medicine.

K-channel opening activity of ZD6169 and its analogs: effect on 86Rb efflux and 3H-P1075 binding in bladder smooth muscle.

Zeneca ZD6169, (S)-N-(4-benzoylphenyl)-3,3,3-trifluoro- 2-hydroxy-2-methylpropionamide, is a novel compound which relaxes urinary bladder smooth muscle in vitro. The effect of ZD6169 and two of its analogs on 86Rb efflux and 3H-P1075 binding in guinea pig bladder strips was investigated to characterize the K-channel opening properties of this compound. ZD6169 concentration dependently increased the rate of 86Rb efflux from guinea pig bladder strips. 86Rb efflux evoked by ZD6169 and its analogs was blocked by glibenclamide (30 muM) but not by charybdotoxin, apamin or alpha-dendrotoxin, suggesting that this compound activates KATP channels in guinea pig bladder. In addition, interaction of ZD6169 with KATP channels was also confirmed in human bladder smooth muscle cells. Specific binding of 3H-P1075, a potent opener of KATP channels, to guinea pig urinary bladder strips was observed. 3H-P1075 binding was inhibited by known KATP openers. ZD6169 inhibited binding of 3H-P1075 to urinary bladder strips like other structurally different KATP openers, e.g. cromakalim and pinacidil. Potencies for inhibition of 3H-P1075 binding by ZD6169 and other potassium channel openers correlate well with potencies for increase in 86Rb efflux and bladder muscle relaxation studies. It is concluded that Zeneca ZD6169 is a potassium channel opener which activates ATP-sensitive K-channels in guinea pig urinary bladder strips as well as in human bladder cells. Furthermore, binding studies suggest that the effects of ZD6169 and its analogs are mediated by binding to the site labeled by 3H-P1075 in guinea pig bladder strips.

Functional Evidence for the Presence of &amp;beta;&lt;sub&gt;3&lt;/sub&gt;-Adrenoceptors in the Guinea Pig Common Bile Duct and Colon

To determine the existence of beta 3-adrenoceptors in functional assays in isolated preparations for which data are lacking, we compared the effects of SR 58611A, a selective beta 3-adrenoceptor agonist, and isoprenaline in the guinea pig common bile duct, distal colon and urinary bladder. SR 58611A and isoprenaline relaxed the common bile duct (EC50: 6.85 and 0.41 mumol/l, respectively). The effect of SR 58611A was resistant to CGP 20712A, ICI 118551, propranolol and tetrodotoxin, but was antagonized by alprenolol (pA2 = 6.86), while the effect of isoprenaline was antagonized by CGP 20712A, ICI 118551, propranolol and alprenolol (pA2 = 7.04, in the presence of propranolol to saturate beta 1- and beta 2-adrenoceptors). In colonic preparations, SR 58611A and isoprenaline relaxed circular muscle strips (EC50: 5.48 and 0.49 mumol/l, respectively). The effect of SR 58611A was resistant to CGP 20712A, ICI 118551, propranolol and tetrodotoxin, but was antagonized by alprenolol (pA2 = 7.01). The effect of isoprenaline was resistant to CGP 20712A, but was antagonized by ICI 118551, propranolol and alprenolol (pA2 = 6.88, in the presence of propranolol). In urinary bladder strips, SR 58611A had no effect, whereas isoprenaline reduced resting tone (EC50:0.87 mumol/l), an effect antagonized by alprenolol (pA2 = 8.14). These data provide functional evidence for the presence of beta 3-adrenoceptors in the guinea pig common bile duct and colon, but not in the urinary bladder. At the concentrations used, the effect of SR 58611A was probably mediated solely by activation of beta 3-adrenoceptors located on smooth muscle cells, whereas the effects of isoprenaline were due to beta 3- and also to beta 1-and/or beta 2-adrenoceptor activation.