Contraction Of Rat Urinary Bladder Research Articles

The effects of nitrofurantoin on rat urinary bladder contraction

Nitrofurantoin is a drug used in the antibacterial treatment of urinary tract infections (UTIs) for over 60 years. The aim of our study was to investigate the possible effects of nitrofurantoin, which is commonly used as a urinary tract antiseptic, on bladder contractions in male rats. Bladder tissues obtained from male Sprague-Dawley rats were used in the study (n=24). After decapitation, bladder tissues were suspended in an isolated organ bath of 5 ml containing a Krebs-Ringer bicarbonate solution by applying a tension of 1.5 grams. Nitrofurantoin was administered to three groups at doses of 50, 500 and 1000 μM, respectively. The area, peak-to-peak (p-p) and frequency values of bladder contractions were analyzed before and after administration of nitrofurantoin. The data obtained from the analysis were evaluated using the Paired T-Test in the IBM SPSS Statistics Software. Nitrofurantoin was observed to have an inhibitory effect on bladder contractions at all doses. The decrease in the area and peak-to-peak values was statistically significant at all doses (P

Preparation, characterization and pharmacological evaluation of tolterodine hydrogels for the treatment of overactive bladder

In this study, transdermal gel formulations for tolterodine were developed to investigate the effects of gel matrix and chemical enhancers on drug skin permeation from tolterodine hydrogels. In vitro permeation studies of tolterodine through excised mouse skin were carried out using Franz-type diffusion cells. In the optimum gel formulation, Carbopol 940 was selected as the gel matrix. Compared to gels without enhancer, tolterodine hydrogels with N-methyl pyrrolidone (NMP) showed significant enhancing effect on transdermal permeation of tolterodine (p<0.05). The results of in vitro percutaneous delivery experiment showed that the relationship of the steady accumulative percutaneous amount (Q, μg cm(-2)) of tolterodine hydrogels and time was Q4-12h=770.19t(1/2)-966.99. Tolterodine permeated at the steady-state speed of 770.19 μg cm(-2)h(-1) and its release coincided with Higuchi Equation. The pharmacokinetic properties of the optimized tolterodine formulation were studied in rabbits. The absolute bioavailability of tolterodine was 11.47%. Since the absence of hepatic first-pass metabolism, only a single active compound-tolterodine was detected in the plasma. A skin irritation study was also carried out on rabbits, and the results showed tolterodine hydrogels had no skin irritation. In the pharmacodynamic study, the significant effects of tolterodine hydrogels on the inhibition of pilocarpine-induced rat urinary bladder contraction were last to 12h, indicating that tolterodine hydrogels could produce prolonged pharmacological responses. In conclusion, tolterodine hydrogels were formulated successfully using Carbopol 940 and NMP and these results helped in finding the optimum formulation for percutaneous drug release. It is quite evident that tolterodine hydrogels may offer a possibility to avoid the first-pass effect, resulting in a single active compound of tolterodine in plasma, which may profit on the patient under the dose control and the reduction of potential adverse effect from two active compounds in the body.

Long-acting formulation of a new muscarinic receptor antagonist for the treatment of overactive bladder

A new muscarinic receptor antagonist, 5-hydroxymethyl tolterodine (5-HMT), was successfully encapsulated into PLGA microspheres. With an increase of PLGA concentration from 15% to 40%, encapsulation efficiency of 5-HMT increased from 55.39% to 76.32%, and the particle size of microsphere increased from 34.33 to 70.48 µm. Increasing the homogenisation speed from 850 to 2300 rpm, the particle size was reduced about 65%.The in vitro and in vivo studies in beagle dogs show that the release profile of 5-HMT-loaded microspheres (5-HMT MS) prepared with 503H is characterised by a low initial burst followed by slow release that lasted for 2 weeks. A Cmax of 1.617 ± 0.392 ng/mL was found on the sixth day. When evaluated for inhibition of the carbachol-induced contraction of rat urinary bladder, 5-HMT MS showed a much longer and more potent effect than tolterodine tablets. The mean urination time of the rats in the 5-HMT MS group was significantly decreased (p < 0.05 or p < 0.01) to less than 2 weeks.

1,4-Dioxane, a Suitable Scaffold for the Development of Novel M3 Muscarinic Receptor Antagonists

In this study the modulation of the pharmacological profile from agonist to antagonist was successfully obtained by replacing the methyl group in position 6 of the 1,4-dioxane scaffold of the potent M(2)/M(3) muscarinic agonist 1 with bulkier groups. In particular, the 6,6-diphenyl substitution provided the potent M(3) preferring antagonist (±)-17, which in in vivo study proved to be effective in reducing the volume-induced contractions of rat urinary bladder and was devoid of cardiovascular effects.

A Novel Pathway Underlying the Inhibitory Effects of Melatonin on Isolated Rat Urinary Bladder Contraction

The aim of the present study was to elucidate the direct effects of melatonin on bladder activity and to determine the mechanisms responsible for the detrusor activity of melatonin in the isolated rat bladder. We evaluated the effects of melatonin on the contractions induced by phenylephrine (PE), acetylcholine (ACh), bethanechol (BCh), KCl, and electrical field stimulation (EFS) in 20 detrusor smooth muscle samples from Sprague-Dawley rats. To determine the mechanisms underlying the inhibitory responses to melatonin, melatonin-pretreated muscle strips were exposed to a calcium channel antagonist (verapamil), three potassium channel blockers [tetraethyl ammonium (TEA), 4-aminopyridine (4-AP), and glibenclamide], a direct voltage-dependent calcium channel opener (Bay K 8644), and a specific calcium/calmodulin-dependent kinase II (CaMKII) inhibitor (KN-93). Melatonin pretreatment (10-8~10-6 M) decreased the contractile responses induced by PE (10-9~10-4 M) and Ach (10-9~10-4 M) in a dose-dependent manner. Melatonin (10-7 M) also blocked contraction induced by high KCl ([KCl]ECF; 35 mM, 70 mM, 105 mM, and 140 mM) and EFS. Melatonin (10-7 M) potentiated the relaxation response of the strips by verapamil, but other potassium channel blockers did not change melatonin activity. Melatonin pretreatment significantly decreased contractile responses induced by Bay K 8644 (10-11~10-7 M). KN-93 enhanced melatonin-induced relaxation. The present results suggest that melatonin can inhibit bladder smooth muscle contraction through a voltage-dependent, calcium-antagonistic mechanism and through the inhibition of the calmodulin/CaMKII system.

Studies on the preparation, characterization and pharmacological evaluation of tolterodine PLGA microspheres

In this study, poly( d, l-lactide-co-glycolide) (PLGA) microspheres of tolterodine depot formulation were prepared using oil in water (o/w) method to investigate their potential pharmacokinetic and pharmacodynamic advantages over tolterodine l-tartrate tablets. Morphological studies of the microspheres showed a spherical shape and smooth surface with mean size of 50.69–83.01 μm, and the encapsulation efficiency was improved from 62.55 to 79.10% when the polymer concentration increased from 180 to 230 mg/ml. The addition of stearic or palmitic acids could significantly raise the drug entrapment efficiency but only slightly affected the in vitro release. A low initial burst followed by a proximately constant release of tolterodine was noticed in the in vitro release profiles. The in vivo study was carried out by intramuscular (i.m.) administration of tolterodine-loaded microspheres on beagle dogs, and a sustained release of drug from the PLGA microspheres was achieved until the 18th day with a low initial burst. Since the absence of hepatic first pass metabolism, only a single active compound—tolterodine was detected in the plasma. This avoided the coexistence of two active compounds in plasma in the case of oral administration of tolterodine, which may lead to a difficulty in dose control due to the different metabolic capacity of patients. In the pharmacodynamic study, the influence of tolterodine PLGA microspheres on the inhibition of carbachol-induced rat urinary bladder contraction was more significant than that of tolterodine l-tartrate tablets. There were invisible changes in rat bladder slices between tolterodine-loaded PLGA microspheres group and tolterodine l-tartrate tablets group. These results indicate that the continuous inhibition of muscarinic receptor may offer an alternative therapy of urge incontinence.

Does Phospholipase C Mediate Muscarinic Receptor-Induced Rat Urinary Bladder Contraction?

Muscarinic acetylcholine receptors, particularly M(3) receptors, are physiologically the most important mechanism to induced urinary bladder smooth muscle contraction. Their prototypical signaling response is a stimulation of phospholipase C (PLC), and this also has been shown in the urinary bladder. Nevertheless, it has remained controversial whether PLC signaling mediates bladder contraction induced by muscarinic receptor agonists. Studies in favor and against a role for PLC differed in their experimental protocol (single versus repeated concentration-response curves within a single preparation) and in the PLC inhibitors that have been used. We have now tested whether previous differential conclusions regarding a role for PLC are related to inhibitors and/or experimental protocols. In a single curve protocol, U-73,122 [1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione] did not attenuate carbachol responses. In a repeated curve protocol, ET-18-OCH(3) (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine) lacked significant inhibition relative to vehicle time controls. In contrast, D609 (O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt) depressed maximal carbachol effects but also nonspecifically inhibited contraction induced by KCl. Neomycin did not affect the carbachol-induced rat urinary bladder contraction. We conclude that previously reported differences relate to the use of inhibitors rather than experimental protocols and that the overall data do not support a role for PLC in M(3) muscarinic receptor-mediated rat bladder contraction.

Role of capsaicin-sensitive sensory nerves in protease-activated receptor-2-mediated contraction of rat urinary bladder

The capsaicin receptor, transient receptor potential vanilloid receptor 1 (TRPV1), colocalizes with protease-activated receptors (PARs) in the urinary bladder, but functional interaction between TRPV1 and PARs remains to be clarified. In the present study, the role of capsaicin-sensitive sensory neurons in contraction induced by activation of PAR-2 was investigated in the rat urinary bladders. The contractile responses to trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH 2 were significantly attenuated by either desensitization of sensory neurons achieved by capsaicin (10 μM) or by the TRPV1 antagonist capsazepine (30 μM). These results suggest that stimulation of capsaicin-sensitive sensory fibers contributes, at least in part, to PAR-2-mediated bladder contractions in rats.

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10–100 μM) inhibits capsaicin (0.03 μM), allyl isothiocyanate (100 μM) and cinnamaldehyde (300 μM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 μM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10–1000 μM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [ 3H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca 2+ influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 μM) or indomethacin (1 μM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 μM) or suramin (300 μM). The exposure of urinary bladders to allyl isothiocyanate (100 μM) causes an increase in the prostaglandin E 2 and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca 2+ influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E 2. Thus, TRPA1 appears to exert an important role in urinary bladder function.

M2 and M3 Muscarinic Receptor Activation of Urinary Bladder Contractile Signal Transduction. I. Normal Rat Bladder

The muscarinic receptor subtype-activated signal transduction mechanisms mediating rat urinary bladder contraction are incompletely understood. M(3) mediates normal rat bladder contractions; however, the M(2) receptor subtype has a more dominant role in contractions of the hypertrophied bladder. Normal bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a single cumulative concentration-response curve to the muscarinic receptor agonist carbachol. The outcome measures were the maximal contraction, the potency of carbachol, and the affinity of the M(3) -selective antimuscarinic agent darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH(3)) reduces carbachol potency and reduces darifenacin affinity, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximal contraction. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol potency and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine.HCl (HA-1077) reduces the carbachol maximal contraction, carbachol potency, and darifenacin affinity. Inhibition of protein kinase C (PKC) with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine.2HCl (H7) reduces the carbachol maximum and carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H89) reduces carbachol maximum and carbachol potency. Both the M(2) and the M(3) receptor subtype are involved in normal rat bladder contractions. The M(3)subtype seems to mediate contraction by activation of PI-PLC, PC-PLC, and PKA, whereas the M(2) signal transduction cascade may include activation of rho kinase, PKC, and an additional contractile signal transduction mechanism independent of rho kinase or PKC.

A ROLE FOR MUSCARINIC RECEPTORS OR RHO-KINASE IN HYPERTENSION ASSOCIATED RAT BLADDER DYSFUNCTION?

Essential arterial hypertension is a frequent condition. Spontaneously hypertensive rats (SHRs) show bladder dysfunction similar to that seen in patients with overactive bladder. Since muscarinic receptors and rho-kinase have a key role in the regulation of bladder contractility, we determined whether alterations of either one might contribute to hypertension associated bladder dysfunction. The bladders of SHRs and normotensive Wistar Kyoto rats (WKYs) were compared in in vitro radioligand binding and contractility studies. The mean total number of muscarinic receptors +/- SEM (181 +/- 14 vs 191 +/- 22 fmol/mg protein) and the relative roles of their subtypes were similar in SHRs and WKYs. Contractile responses to the muscarinic agonist carbachol (maximum effect 2.04 +/- 0.24 vs 2.05 +/- 0.14 mN/mm strip length and -log EC50 5.61 +/- 0.07 vs 5.64 +/- 0.04) and to KCl in a receptor independent manner were similar in the 2 strains. The M3 selective antagonist darifenacin inhibited carbachol responses much more potently than the M2 selective antagonist methoctramine but the potency of the 2 drugs was similar in each strain. The rho-kinase inhibitor Y27,632 attenuated carbachol induced contraction in a quantitatively similar manner in SHRs and WKYs. An altered function of muscarinic receptor subtypes or rho-kinase does not appear to contribute to bladder dysfunction in SHRs.

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. II. Protein kinases.

We have investigated the role of several protein kinases in carbachol-stimulated, M(3) muscarinic receptor-mediated contraction of rat urinary bladder. Concentration-response curves for the muscarinic receptor agonist carbachol were generated in the presence of multiple concentrations of inhibitors of various protein kinases, their inactive controls, or their vehicles. Bladder contraction was not significantly inhibited by three protein kinase C inhibitors (chelerythrine, 1-10 microM; calphostin C, 0.1-1 microM; and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Gö 6850), 1-10 microM), by the tyrosine kinase inhibitor genistein or its inactive control daidzein (3-30 microM each), or by two inhibitors of activation of mitogen-activated protein kinase [10-100 microM 2'-amino-3'-methoxyflavone (PD 98,059) and 3-30 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 124)] or their negative control 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 126) (3-30 microM). Although high concentrations of wortmannin (3-30 microM) inhibited bladder contraction, this was not mimicked by another inhibitor of phosphatidylinositol-3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294,002) (3-30 microM) and, hence, was more likely due to direct inhibition of myosin light chain kinase by wortmannin than to an involvement of phosphatidylinositol-3-kinase. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration-dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of rat urinary bladder does not involve protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinases, or extracellular signal-regulated kinases; in contrast, rho-associated kinases appear to play an important role in the regulation of bladder contraction.

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. I. Phospholipases and Ca2+ sources.

We have reexamined the muscarinic receptor subtype mediating carbachol-induced contraction of rat urinary bladder and investigated the role of phospholipase (PL)C, D, and A2 and of intra- and extracellular Ca2+ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M2 receptor-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]-piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M3 receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M3 receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122; 10 microM). Nevertheless, U 73,122 (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. Carbachol had only little effect on PLD activity in bladder slices, but the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol (0.3% each), caused detectable inhibition of carbachol-induced bladder contraction. The cytosolic PLA2 inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbachol-induced contraction at a concentration of 300 microM, but the cyclooxygenase inhibitor indomethacin (1-10 microM) remained without effect. The Ca2+ entry blocker nifedipine (10-100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 microM), an inhibitor of store-operated Ca2+ channels, caused little inhibition. We conclude that carbachol-induced contraction of rat bladder largely depends on Ca2+ entry through nifedipine-sensitive channels and, perhaps, PLD, PLA2, and store-operated Ca2+ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.

Possible involvement of Ca2+-independent phospholipase A2 in protease-activated receptor-2-mediated contraction of rat urinary bladder.

Possible involvement of Ca(2+)-independent phospholipase A2 (iPLA2) was examined in protease-activated receptor-2 (PAR-2)-mediated contraction of the rat urinary bladder. Both PAR-2 activating peptide (PAR-2 AP; SLIGRL-NH2) and trypsin produced a concentration-dependent contractile response in the urinary bladder preparations. These contractions were significantly (p<0.01) attenuated by indomethacin (10 microM), an inhibitor of cyclooxygenase, or bromoenol lactone (BEL; 10 micro M), an inhibitor of iPLA2. On the other hand, the contractile responses to bradykinin were not significantly affected by BEL, although they were reduced by indomethacin. Arachidonyltrifluoromethyl ketone (AACOCF3; 30 microM), an inhibitor of cytosolic Ca(2+)-dependent phospholipase A2, did not affect the trypsin- and bradykinin-induced contractions. Both indomethacin and BEL had no inhibitory effect on the prostaglandin E2-induced contractions. These results suggest that PAR-2 activators and bradykinin stimulate the release of prostaglandins and thereby contract the rat urinary bladder smooth muscles. The release of prostaglandins by PAR-2 activators seems to be partly mediated by the iPLA2.

The role of cholinesterases in rat urinary bladder contractility.

This study examines the effects of inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) on acetylcholine (ACh)-induced contraction in rat urinary bladder smooth muscle. Neostigmine, a non-selective ChE inhibitor, caused concentration-dependent contractions in rat urinary bladder strips, whereas tetraisopropylpyrophosphoramide (iso-OMPA; a BuChE inhibitor) failed to affect the resting tone of the preparations. Neostigmine (1 microM) markedly augmented the contractile responses to ACh. Although iso-OMPA (10 microM) also potentiated ACh-induced contraction, the effect was less than that evoked by neostigmine. The activities of AChE in rat urinary bladder strips were significantly (P<0.05) higher than those of BuChE. These results indicated that AChE, rather than BuChE, plays an important role in controlling ACh-induced contractions of rat urinary bladder.

EFFECTS OF THE NUCLEAR FACTOR-κB INHIBITORS 2-HYDROXY-4-TRIFLUOROMETHYLBENZOIC ACID AND ASPIRIN ON MICTURITION IN RATS WITH NORMAL AND INFLAMED BLADDER

We examined the effects of intravenous administration of the 2 nuclear factor-kappaB inhibitors aspirin and 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) on bladder filling and voiding in anesthetized and conscious rats. Disappearance of isovolumic bladder contractions after intravenous administration of different doses of aspirin and HTB in anesthetized, transurethrally catheterized rats was evaluated. Cystometry was performed in conscious rats during bladder infusion with saline or diluted acetic acid as well as in those with cyclophosphamide induced cystitis. Changes in bladder capacity and voiding pressure were evaluated after intravenous administration of test compounds. Aspirin induced a dose dependent disappearance of isovolumic bladder contractions in anesthetized rats with an extrapolated dose of 2.1 mg./kg. inducing 10 minutes of bladder quiescence. HTB was practically inactive, inducing a dose independent block of 3 to 4 minutes after intravenous administration of 1 to 10 mg./kg. In conscious rats with a bladder infused with saline aspirin was poorly active on bladder capacity, inducing a 20% increase 60 minutes after intravenous administration of 30 and 100 mg./kg. In rats with a bladder infused with acetic acid aspirin was much more active when injected at the initiation of inflammation and after 1 hour of irritant infusion. In this latter situation aspirin increased bladder capacity up to 60% after intravenous administration of 30 and 100 mg./kg. Similar results were obtained in rats with cyclophosphamide induced cystitis in which the bladder was infused with saline. In these cystometrography models 30 mg./kg. HTB intravenously was completely inactive. The results show that HTB is devoid of significant effects on the micturition reflex in the absence or presence of bladder inflammation, suggesting that acute inhibition of nuclear factor-kappaB does not influence bladder urodynamics in rats. In contrast, aspirin, which is a cyclooxygenase and nuclear factor-kappaB inhibitor, was always effective, indicating the important role of cyclooxygenase enzymes.

Contractility of urinary bladder and vas deferens after sensory denervation by capsaicin treatment of newborn rats

1. Capsaicin, a selective sensory neurotoxin, was given to newborn rats and at the age of 3 months the contractile activity of the urinary bladder detrusor muscle and vas deferens evoked by either electrical field stimulation (EFS) or exogenous adenosine 5'-triphosphate (ATP) and carbachol (urinary bladder), or ATP and noradrenaline (vas deferens) were tested. 2. EFS of the urinary bladder evoked contractions which consisted of cholinergic and purinergic components, since they could be partially blocked by either the muscarinic cholinoceptor antagonist, atropine (0.3 microM) or by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP (10 microM). In capsaicin-treated rats, contractions of the urinary bladder evoked by EFS were significantly larger than those of control (vehicle-treated) animals, and this difference remained after the purinergic component of the contractions was blocked by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP. However, when the cholinergic component of the contractions was blocked with atropine, the difference between the groups at 8 Hz and 16 Hz was abolished; EFS caused significantly larger contractions of the capsaicin-treated rat bladder only at frequencies of 2 Hz and 4 Hz. 3. EFS evoked contractions of the vas deferens consisted of adrenergic and purinergic components since they could be partially blocked by either the alpha-adrenoceptor antagonist, phentolamine (3 microM) or by alpha,beta-methylene ATP (10 microM). The contractions of the vas deferens were significantly larger than in the capsaicin-treated rats only at a frequency of 16 Hz. There were no differences between vas deferens contractions of the two groups either after desensitization of P2X-purinoceptors by alpha,beta-methylene ATP or in the presence of phentolamine.4. Contractions of the capsaicin-treated rat urinary bladder evoked by exogenous carbachol (0.1-100 microM) were not significantly different from those of controls, the pD2 values being 1.78 +/- 0.23 micro M and 1.90 +/- 0.20 micro M respectively. There was also no significant difference between the groups in contractions of the bladder evoked by ATP (10 micro M-3 mM).5. Contractions of the vas deferens evoked by either ATP (10 micro M-3 mM) or noradrenaline (1-1000 micro M) in the capsaicin-treated group showed no significant difference between control and capsaicin treated rats.6. In conclusion, the present results indicate that chronic capsaicin treatment increases the amplitude of contractions of the rat urinary bladder, an effect which preferentially involves the cholinergic component of the response; since the response to carbachol is unaffected, the change involves prejunctional mechanisms. In contrast, both the purinergic and adrenergic components of contraction in the vas deferens are unaffected by capsaicin. It is suggested that sensory nerves have a trophic influence on the development of parasympathetic nerves in the rat bladder; removal of sensory nerves shortly after birth results in an increase mainly in the cholinergic, and to a lesser extent purinergic component.

Effects of Ca 2+ channel antagonists and benzodiazepine receptor ligands in normal and skinned rat urinary bladder

The effects of Ca 2+ channel antagonists and benzodiazepine receptor ligands against concentration-dependent contractions of rat urinary bladder induced by CaCl 2 (0.1–50 mM, in K +-depolarized tissues), KCl (1–100 mM) and acetylcholine (0.1 μM to 1 mM) were studied. Nifedipine (0.001–0.1 μM), verapamil (0.01–1 μM), diltiazem (0.01–1 μM), cinnarizine (1–100 μM), and trifluoperazine (1–100 μM) each produced a concentration-related inhibition of the log concentration-effect curve for CaCl 2. The rank order of potencies of these antagonists, measured as the IC 50 against Ca 2+ (25 mM)-induced contraction of depolarized bladder, was nifedipine (0.01 μM) > diltiazem (0.36 μM) ≈ verapamil (0.41 μM) ≥ cinnarizine (2.57 μM) > trifluoperazine (17.4 μM). These antagonists depressed KCl-induced contractions with an effectiveness and potency similar to that displayed against CaCl 2-induced contractions. Nifedipine, verapamil, and diltiazem but not cinnarizine and trifluoperazine had a preferential inhibitory effect on the contractions elicited by KCl when compared to those elicited by acetylcholine. Ro 5-4864, diazepam, midazolam and the non-benzodiazepine PK 11195, each at 1–100 μM, depressed CaCl 2- and KCl-induced contractions (IC 50 values in the micromolar range). Benzodiazepines and PK 11195, all at 100 μM, markedly depressed acetylcholine-induced contractions. Flumazenil was scarcely effective. Cinnarizine (100 μM) and trifluoperazine (100 μM), but not the other Ca 2+ channel antagonists and benzodiazepine receptor ligands tested, depressed Ca 2+ (20 μM)-evoked contractions of skinned bladder. It is concluded that the action of nifedipine, verapamil, and diltiazem is restricted to the plasmalemma whereas cinnarizine and trifluoperazine also act on the intracellular contractile apparatus. The inhibitory effects of micromolar concentrations of benzodiazepines appear to be related to an interaction with low-affinity sites functionally linked to L-type Ca 2+ channels but intracellular actions are present at high concentrations.

Antagonism of substance P and related peptides by RP 67580 and CP-96,345, at tachykinin NK 1 receptor sites, in the rat urinary bladder

Tonic contraction of rat urinary bladder was elicited in vitro and in vivo by substance P, two selective NK 1 receptor agonists, septide ([pGlu 6, Pro 9]substance P-(6–11)) and [Sar 9, Met(O 2) 11]substance P, and an NK 2 agonist, [Lys 5, MeLeu 9, N Nle 10]neurokinin A-(4–10), but not by senktide (succinyl[Asp 6, MePhe 8]substance P-(6–11)), an NK 3 agonist. Substance P only stimulated the NK 1 receptors of smooth muscle. The non-peptide selective NK 1 receptor antagonists, RP 67580 and CP-96,345, both inhibited substance P-induced contraction (pK B values 6.7 and 5.7; ED 50=1.4 and 5.0 mg/kg i.v., respectively) and septide-induced contraction (pK B values 7.5 and 6.5; ED 50=0.076 and 0.250 mg/kg i.v., respectively). Both antagonists, at lower doses, also inhibited substance P- and septide-induced plasma extravasation. That both antagonists blocked the effects of septide much more than the effects of substance P suggests the existence of an NK 1 receptor subtype or isoform. Selective NK 1 receptor antagonists, by blocking both spasm and plasma extravasation in the urinary bladder, would be useful for treating substance P-related motor disorders and cystitis.

In vivo and in vitro pharmacology of SR 48,968, a non-peptide tachykinin NK 2 receptor antagonist

The activity of SR 48,968, a novel non-peptide antagonist of tachykinin NK 2 receptors was evaluated in vitro in several bioassays for the NK 2 receptor (rabbit pulmonary artery, rabbit bronchus, hamster trachea, rat vas deferens) and compared to that of the peptide antagonists, MEN 10,376, L 659,877 and MDL 29,913. SR 48,968 behaved as a potent and competitive antagonist in the four isolated preparations (pA 2 values between 8.3 and 9.6 in different preparations), being more potent (about 10 times) in the rabbit pulmonary artery and rabbit bronchus than in the hamster trachea or rat vas deferens. The antagonistic profile of SR 48,968 resembled that of MEN 10,376, and contrasted with those of L 659,877 and MDL 29,913 which were distinctly more potent on the hamster trachea and rat vas deferens. In vivo, SR 48,968 (0.1 μmol/kg i.v.) blocked the contraction of rat urinary bladder stimulated by [βAla 8]neurokinin A-(4–10) (NK 2 receptor agonist) without affecting that produced by [Sar 9]substance P sulfone (NK 1 receptor agonist). The hypotension and salivary secretion produced by the latter agonist were not modified by SR 48,968. In contrast, (±)-CP 96,345 (10 μmol/kg i.v.) blocked bladder contraction, salivary secretion and hypotensive responses elicited by the NK 1 receptor agonist while leaving unaffected the bladder contraction produced by the NK 2 receptor agonist. SR 48,968 is a potent and competitive antagonist of the tachykinin NK 2 receptor with a limited but distinct ability to discriminate between putative subtypes/species variants of the NK 2 receptor. The high potency and selectivity of SR 48,968 make this novel compound an important tool for studying the distribution and function of tachykinin NK 2 receptors.