Contraction In Rats Research Articles

1446: Participation of Endothelin Eta Receptors in Pre-micturition Contractions in Rats with Bladder Outlet Obstruction

1446: Participation of Endothelin Eta Receptors in Pre-micturition Contractions in Rats with Bladder Outlet Obstruction

Muscular mechanical hyperalgesia revealed by behavioural pain test and c‐Fos expression in the spinal dorsal horn after eccentric contraction in rats

Delayed onset muscle soreness (DOMS) is quite common, but the mechanism for this phenomenon is still not understood; even the existence of muscle tenderness (mechanical hyperalgesia) has not been demonstrated in experimental models. We developed an animal model of DOMS by inducing eccentric contraction (lengthening contraction, ECC) to the extensor digitorum longus muscle (EDL), and investigated the existence of mechanical hyperalgesia in the EDL by means of behavioural pain tests (Randall-Selitto test and von Frey hair test, applied to/through the skin on the EDL muscle) and c-Fos expression in the spinal dorsal horn. We found that the mechanical withdrawal threshold measured with the Randall-Selitto apparatus decreased significantly between 1 and 3 days after ECC, while that measured by von Frey hairs did not. The group that underwent stretching of the muscle only (SHAM group) showed no change in mechanical pain threshold in either test. These results demonstrated that the pain threshold of deep tissues (possibly of the muscle) decreased after ECC. c-Fos immunoreactivity in the dorsal horn (examined 2 days after ECC/SHAM exercise) was not changed by either ECC or compression (1568 mN) to the EDL muscle by itself, but it was significantly increased by applying compression to the EDL muscle 2 days after ECC. This increase was observed in the superficial dorsal horn of the L4 segment of the ipsilateral side, and was clearly suppressed by morphine treatment (10 mg kg(-1), i.p.). These results demonstrated the existence of mechanical hyperalgesia in the muscle subjected to ECC. This model could be used for future study of the neural mechanism of muscle soreness.

Effect of pregnancy on the roles of nitric oxide and prostaglandins in 5‐hydroxytryptamine‐induced contractions in rat isolated thoracic and abdominal aorta

1. Vascular resistance and sensitivity to circulating pressor and vasoconstrictor substances are blunted during pregnancy. This has been attributed mainly to an increased production of endothelium-derived mediators. The aim of the present study was to determine whether pregnancy changes the relative participation of nitric oxide (NO) and prostaglandins (PG) in the modulation of the contractile response to 5-hydroxytryptamine (5-HT) in two anatomically distint segments of the rat aorta. 2. Full concentration-response curves to 5-HT were obtained in isolated rings from the thoracic and abdominal portion of the aorta from pregnant and non-pregnant rats in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 10 micromol/L) or the PG synthesis inhibitor indomethacin (10 micromol/L). Cyclo-oxygenase (COX)-1, COX-2 and endothelial (e) NOS protein expression were determined in the same tissues by immunoblot. 3. The effects of pregnancy were accentuated in the abdominal compared with the thoracic aorta. In addition, the relative participation of the NO and PG pathways seems to be changed during pregnancy. Although NO seems to be the mediator mainly responsible for the effect of pregnancy in the thoracic aorta, our results suggest a complex interaction between NO and PG in the abdominal aorta. Indomethacin significantly reduced the contractile response of both segments of the aorta, whereas expression of COX-1, COX-2 and eNOS were increased only in the abdominal segment of pregnant animals. 4. These results show that the effect of pregnancy is not homogeneous along the aorta. There seems to be a mutual interaction between PG and NO in the abdominal, but not in the thoracic, aorta from pregnant rats: the role of NO becomes evident in the absence of vasodilatory PG, whereas the participation of the latter increases in the absence of NO working as a compensatory mechanism.

Effect of stimulation of anterior hypothalamic area on urinary bladder function of the anesthetized rat.

The hypothalamus is a key area for the integration of the autonomic features of affective behavior. Hypothalamic defence area (HDA) stimulation evokes major cardiorespiratory changes as well as modifications of general autonomic activity both in the anesthetized and conscious animal. Micturition is due to an increase in pelvic parasympathetic activity and, in the cat, the anterior hypothalamus has been implicated in urinary bladder control with the demonstration of a dorsolateral vesicoconstrictor pathway and a ventromedial inhibitory pathway. In this study we have investigated the effect of electrical and chemical stimulation of the HDA on bladder pressure and contractions in rat. Female rats (n = 15) were anesthetized, paralyzed and ventilated artificially. Arterial blood pressure, heart rate, urinary bladder pressure and pelvic nerve activity were recorded. HDA was electrically (1 ms, 100 Hz, 5-10 s train at intensities up to 150 micro A) and chemically (sodium glutamate, 50 nl, 2mM) stimulated. For statistical analysis the t-test was used, data were expressed as mean +/- SEM. Values of t were taken as significant when p < 0.05.HDA stimulation at 100-150 micro A evoked changes of both mean blood pressure (mBP) and bladder pressure (BlP). However, stimulation at < 30 micro A allowed a distinction within HDA of two different regions, at the same antero-posterior and lateral level, but separated 100-150 micro m in depth, which evoked differential effects on blood pressure and urinary bladder pressure. Results show that low intensity stimulation at ventral sites evoked a significant increase of mBP (from 102 +/- 5.9 to 127 +/- 8.6 mmHg, n = 10, p < 0.0001) with little changes of BlP (from 12 +/- 2.2 to 16 +/- 2.9 cmH(2)O, n = 10, p < 0.0005), whilst at more dorsal sites significant increases of BlP were elicited (from 12 +/- 8.3 to 38 +/- 4.6 cmH(2)O,n = 10, p < 0.0001) with only a small rise of mBP (from 102 +/- 6.2 to 111 +/- 9.8 mmHg, n = 10, p < 0.005). Glutamate injections at dorsal sites evoked a rise of BlP (from 11 +/- 2.2 to 30 +/- 3.0 cmH(2)O (n = 5; p < 0.0001) with small changes in BP, whilst at ventral sites (n = 4) glutamate microinjections evoked changes in BP but not of BlP. In conclusion stimulation at different sites within HDA can elicit separate changes in BP and BlP.

Bupivacaine inhibits thromboxane A2-induced vasoconstriction in rat thoracic aorta.

Plasma levels of thromboxane A2 (TXA2), an inflammatory mediator inducing platelet aggregation, bronchoconstriction, and vasoconstriction, are increased in the perioperative period. A major role in the pathogenesis of perioperative thromboembolic and ischemic syndromes is attributed to this prostanoid. Local anesthetics (LA) inhibit signaling of TXA2 receptors expressed in cell models. Therefore, we hypothesized that LA may inhibit vasoconstriction induced by the TXA2 analog U46619 in rat thoracic aorta. Rings (3-mm length) of the rat thoracic aorta were mounted in organ baths and isometric contractile force was measured. Rings, with or without endothelium, were incubated for 60 min in bupivacaine (10(-6) or 10(-5) M) or Krebs-Henseleit solution (control group) and subsequently exposed to cumulative concentrations of U46619 (10(-10) to 10(-6) M). The reversibility of the TXA2-induced vasoconstriction by bupivacaine was also studied. Pretreatment of rings with bupivacaine concentration-dependently diminished TXA2-induced contraction in rat aortic rings. We found no significant differences in relaxing effect of bupivacaine between rings with and without endothelium. Contraction in rings established with U46619 could not be reversed by cumulative concentrations of bupivacaine. Bupivacaine inhibited carbachol-induced vascular relaxation. This study provides experimental evidence that bupivacaine is an endothelium-independent inhibitor of TXA2-induced vasoconstriction of rat thoracic aorta.

Urethral Dysfunction in Diabetic Rats

We investigated the effects of diabetes mellitus (DM) on urethral relaxation mechanisms during reflex bladder contractions in rats. Five weeks after streptozotocin injection (65 mg/kg intraperitoneally) the effects of DM on urethral relaxation mechanisms were evaluated by simultaneous recordings of intravesical pressure under isovolumetric conditions and urethral perfusion pressure (UPP) using urethane anesthesia. In diabetic rats the UPP nadir during urethral relaxation and intravesical pressure thresholds for inducing urethral relaxation were significantly higher (199% and 92%, respectively) than in normal rats, while baseline UPPs were not significantly different. The mean rate and amplitude of high frequency oscillations of urethral striated muscle in diabetic rats were also significantly lower (17% and 64%, respectively) compared with normal rats. Following alpha-bungarotoxin treatment to eliminate striated muscle sphincter contractions intravenous administration of L-arginine (200 mg/kg) [corrected] , the substrate of nitric oxide (NO) synthase, decreased the UPP nadir (36% and 22%, in diabetic and normal rats) as well as intravesical pressure thresholds (49% and 22%, respectively). The effect was greater (61% to 126%) in diabetic rats than in normal rats. In each group of rats the effect of L-arginine was inhibited by Nomega-nitro-L-arginine (100 mg/kg intravenously) [corrected], a NO synthase inhibitor. During reflex bladder contractions streptozotocin induced diabetic rats exhibited smooth and striated muscle dysfunctions of the urethral outlet. L-arginine therapy, which could augment urethral smooth muscle relaxation by increasing NO production, may be useful for partially restoring the urethral relaxation mechanism in DM.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

To investigate the pathway (s) mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent, bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer. Isometric tension was recorded. Cumulative concentration-response curves were obtained for (+)-cis-dioxolane (cD), a nonspecific muscarinic agonist, at 10(-8)-10(-4) mol/L, in the presence of tetrodotoxin (TTX, 10(-7) mol/L). Results were normalized to cross sectional area. A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1 (pirenzepine), M2 (methoctramine) and M3 (darifenacin) muscarinic receptor subtypes. The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment. The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. A dose-dependent contractile response observed with bethanechol, was not affected by TTX. The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol. Lack of calcium as well as the presence of the L-type calcium channel blocker, nifedipine, also inhibited the cholinergic contraction, with a reduction in response from 2.5+/-0.4 g/mm2 to 1.2+/-0.4 g/mm(2) (P<0.05). The dose-response curves were shifted to the right by muscarinic antagonists in the following order of affinity: darifenacin (M(3))>methocramine (M(2)) >pirenzepine (M(1)). The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s) involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels. The presence of the residual contractile response after the treatment with nifedipine, suggests that an additional pathway could mediate the cholinergic contraction. The involvement of more than one muscarinic receptor (functionally predominant type 3 over type 2) also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

Effects of TAK-802, a novel acetylcholinesterase inhibitor, on distension-induced rhythmic bladder contractions in rats and guinea pigs

In the present study, we investigated the effects of 8-[3-[1-[(3-fluorophenyl)methyl]-4-piperidinyl]-1-oxopropyl]-1,2,5,6-tetrahydro-4 H-pyrrolo[3,2,1- ij]quinolin-4-one (TAK-802), a novel acetylcholinesterase inhibitor, on distension-induced rhythmic bladder contractions in urethane-anesthetized rats and guinea pigs. TAK-802 potently inhibited human-erythrocyte-derived acetylcholinesterase activity with an IC 50 value of 1.5 nM, which represented a potency 30 and 250 times greater than that of the two carbamate acetylcholinesterase inhibitors, neostigimine and distigmine, respectively. Unlike the carbamate acetylcholinesterase inhibitors, TAK-802 exhibits high selectivity for acetylcholinesterase inhibition over butyrylcholinesterase inhibition. In an assay conducted to measure the muscarinic and nicotinic actions, TAK-802 was found to exhibit higher selectivity for muscarinic actions over nicotinic actions in comparison to distigmine. Both TAK-802 and distigmine increased isovolumetric bladder contractions in rats and guinea pigs in a dose-dependent manner, with a minimum effective dose (MED) of 0.01 and 0.03 mg/kg i.v., respectively, in rats, and 0.01 and 0.1 mg/kg i.v., respectively, in guinea pigs. The effects of both the drugs were completely abolished by atropine. These results suggest that TAK-802 and other acetylcholinesterase inhibitors can effectively increase reflex bladder contractions by increasing the efficacy of acetylcholine released by nerve impulses. On the other hand, bethanechol, a muscarinic agonist, markedly changed the pattern of distension-induced bladder contractions when administered at the dose of 1 mg/kg i.v., and it did not necessarily augment well-coordinated bladder contractions. Thus, considering that it has some selectivity for muscarinic action, TAK-802 might be expected to be useful in the treatment of voiding dysfunction caused by impaired detrusor contractility.

Amitriptyline eliminates calculi through urinary tract smooth muscle relaxation

We investigated the effects of amitriptyline in the urinary tract smooth muscle and urolithiasis. Cats presenting with obstructive acute renal failure (ARF) received amitriptyline, and renal function and survival rates were analyzed. Isometric contractions and membrane potentials of rat, pig, or human isolated urinary tract smooth muscle were recorded in the presence or absence of amitriptyline. Twenty cats with obstructive ARF caused by urethral plugs received amitriptyline. In all cases, plugs were completely eliminated, and renal function returned to normal, with a 100% survival rate in the follow-up. Amitriptyline produced potent relaxations in rat urethral strips, accompanied by significant reductions in urethral ring membrane potential. This effect was prevented by pretreatment of urethral rings with 4-aminopyridine (4-AP), a voltage-dependent potassium channel blocker. Amitriptyline abolished in a reversible manner acetylcholine-, bradykinin-, and KCl-induced contractions in rat isolated bladder, and this effect was also prevented by 4-AP. Of interest, spontaneous and KCl-induced contractions of pig and human isolated ureter were also blocked by amitriptyline. Our results indicate that amitriptyline is an effective and potent relaxant of urinary tract smooth muscle and this effect is mediated by opening of voltage dependent-potassium channels. We suggest that amitriptyline administration may help to promote elimination of urinary calculi.

Cobalt injections into the pedunculopontine nuclei attenuate the reflex diaphragmatic responses to muscle contraction in rats.

Previous studies have suggested that neurons in the pedunculopontine nucleus (PPN) are activated during static muscle contraction. Furthermore, activation of the PPN, via electrical stimulation or chemical disinhibition, is associated with increases in respiratory activity observed via diaphragmatic electromyogram recordings. The present experiments address the potential for PPN involvement in the regulation of the reflex diaphragmatic responses to muscle contraction in chloralose-urethane anesthetized rats. Diaphragmatic responses to unilateral static hindlimb muscle contraction, evoked via electrical stimulation of the tibial nerve, were recorded before and subsequent to bilateral microinjections of a synaptic blockade agent (CoCl2) into the PPN. The peak reflex increases in respiratory frequency (9.0 +/- 1.0 breaths/min) and minute integrated diaphragmatic electromyogram activity (14.6 +/- 3.3 units/min) were attenuated after microinjection of CoCl2 into the PPN (2.6 +/- 0.9 breaths/min and 4.6 +/- 2.1 units/min, respectively). Consistent diaphragmatic responses were observed in the subset of animals that were barodenervated. Control experiments suggest no effects of PPN synaptic blockade on the cardiovascular responses to muscle contraction. The results are discussed in terms of a potential role for the PPN in modulation of the reflex respiratory adjustments that accompany muscular activity.

Effect of melatonin on estrogen and progesterone receptors in relation to uterine contraction in rats

The present study was designed to investigate the possible modulator effect of melatonin on uterine estrogen and progesterone receptors in rats as well as the uterine response to oxytocin. Non-pregnant rats were pretreated with melatonin in a dose of 0.8 mg kg −1 per day for 15 consecutive days. Control animals received the vehicle. The uterus was dissected out and uterine contraction in one horne was recorded in vitro for each animal as a response to oxytocin (0.5×10 −11 to 2×10 −11 M). The other uterine horne was subjected to estrogen and progesterone receptors detection by immunohistochemical and image analysis techniques. The results reveal a significant reduction (59%) in the number of uterine estrogen receptors with concomitant increase in the progesterone receptors (53%) in melatonin-pretreated rats as compared to the control ones. In addition, our data show an inhibitory effect of melatonin on the uterine contraction as a response to oxytocin (0.5×10 −11, 1×10 −11, and 2×10 −11 M) amounting to 48, 77, and 59.5% reduction, respectively, in the amplitude of contraction as well as 62, 19.9, and 47% reduction in the area under the curve (AUC) of uterine contractions, respectively. The data, so far obtained, may indicate a possible relationship of melatonin-induced modulation of the number of estrogen and progesterone receptors and its inhibitory effect on uterine contraction. These findings merit further investigations on the possible beneficial role of melatonin in a plethora of hormone-dependant uterine disorders.

In vivo and in vitro pharmacological studies of a new hypotensive compound (QF0301B) in rat: comparison with prazosin, a known α 1-adrenoceptor antagonist

In this work, we studied the in vivo and in vitro pharmacological effects of the novel compound QF0301B (2-[2-( N-4- o-methoxyphenyl- N-1-piperazinyl)ethyl]-1-tetralone) and compared with those of prazosin. In anaesthetized normotensive rats, both QF0301B and prazosin (0.1–0.2 mg/kg iv) caused a pronounced and prolonged fall in mean arterial blood pressure accompanied by bradycardia. Neither QF0301B nor prazosin (0.2 mg/kg iv) significantly modified the cardiovascular effects of either 5-hydroxytryptamine (serotonin, 5-HT, 75 μg/kg iv) or the selective α 2-adrenoceptor agonist B-HT 920 (0.2 mg/kg iv), but both markedly inhibited the hypertensive effect of noradrenaline (5 μg/kg iv), a nonselective α-adrenergic receptor agonist. In isolated rubbed rat aorta rings, QF0301B and prazosin showed marked α 1-adrenoceptor blocking activity, with pA 2 values of 9.00±0.12 and 9.75±0.14, respectively. In addition, QF0301B reversed and competitively antagonized the inhibitory action produced by clonidine in electrically stimulated rat vas deferens and inhibited the force and rate of contraction in rat isolated atria ( pA 2=5.91±0.43), competitively antagonized the contractile effect of 5-HT in rat aorta ( pA 2=6.75±0.06) and in rat stomach fundus ( pA 2=7.13±0.48) and the contractions induced by histamine in isolated guinea pig longitudinal ileal muscle ( pA 2=7.40±0.40). QF0301B showed noncompetitive low action in 5-HT 3, muscarinic and nicotinic receptors, or as Ca 2+ antagonist. These results indicate that a α 1-adrenoceptor blocking lead has been obtained with a new chemical structure and interesting pharmacological properties, which only α 1-adrenoceptor blocking activity seems to be responsible for its cardiovascular effects.

Elucidation with the protease alpha-chymotrypsin of the inhibitory modulating action of endogenous neuropeptide Y over sympathetic neurotransmission in rat vas deferens.

The physiological role of endogenous neuropeptide Y (NPY) in sympathetic neurotransmission was examined in rat and guinea pig vas deferens (VD), using alpha-chymotrypsin (alpha-CT). NPY-like immunoreactivity was detected in the longitudinal muscle layer of VD densely in rats but sparsely in guinea pigs, and it disappeared following surgical denervation. Under blockade of the prejunctional alpha(2)-adrenergic autoinhibition, alpha-CT potentiated the phasic contraction in rat, but not guinea pig, VD induced by trains of transmural nerve stimulation (TNS) in a frequency-dependent manner, which was reproducible during repeated applications and not affected by pretreatment with capsaicin. In contrast, alpha-CT did not potentiate the twitch response or contractions induced respectively by a single pulse TNS or by direct electrical stimulation to the smooth muscle. Exogenously applied NPY suppressed the twitch response, which was cancelled by alpha-CT, and excitatory junction potentials, although it affected neither spontaneous junction potentials nor the direct electrical stimulation-induced contraction. These observations provided further evidence to support that NPY is released endogenously by TNS at high frequency, acting prejunctionally to suppress sympathetic neurotransmission. Thus, the protease alpha-CT proved itself to be a useful tool to reveal a functional role of endogenously released peptides.

Investigation of the involvement of rho-kinase (ROK) in agonist-induced contractions of rat and human uterine smooth muscle

Investigation of the involvement of rho-kinase (ROK) in agonist-induced contractions of rat and human uterine smooth muscle

Antagonistic effects of CompB on orphanin FQ-induced colonic contractions in rats

Orphanin FQ has been shown to stimulate colonic contraction without affecting upper gastrointestinal motility in rats. We studied whether a new putative orphanin FQ receptor antagonist, 1-[(3 R,4 R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one (CompB), has antagonistic effects on orphanin FQ-induced colonic contractions in vivo and in vitro in rats. Orphanin FQ-(1–17) and orphanin FQ-(1–13) caused contractions of the circular muscle of the distal colon in a concentration dependent manner (10 −10–10 −6 M) in vitro. CompB (10 −7–10 −6 M) caused a significant inhibition on orphanin FQ-(1–17) and orphanin FQ-(1–13)-induced contractions without affecting the spontaneous contractions. Orphanin FQ-(1–17) also caused contractions of the distal colon in vivo. ED 50 dose of orphanin FQ-(1–17) (400 pmol/kg)-induced contractions were significantly antagonized by CompB (30–300 nmol/kg) in a dose-dependent manner. In contrast, CompB had no inhibitory effects on dynorphin A-induced contractions in vivo and in vitro. These indicate that CompB is a selective orphanin FQ receptor antagonist of the rat colon.

Pharmacological characterization of SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide), a novel peptidic urotensin-II receptor antagonist.

1. Human urotensin-II (hU-II), a cyclic undecapeptide, is amongst the most potent mammalian vasoconstrictors identified, suggesting that hU-II and its G-protein-coupled receptor (UT) may regulate cardiovascular homeostasis. Such a hypothesis would benefit greatly from the development of selective UT antagonists. 2. Although the somatostatin (SST) antagonist SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide) is purported to block U-II-induced contractions in rat isolated aorta, little is known about its specific pharmacological properties. 3. SB-710411 (10 micro M) inhibited hU-II-induced contraction in rat isolated aorta causing a significant, parallel shift in the agonist concentration-response curve (pK(b) 6.28+/-0.11; n=8) with no suppression of the E(max). In contrast, SB-710411 did not alter the contractile actions of angiotensin-II, phenylephrine, or KCl. Paradoxically, however, SB-710411 potentiated the contractile response to endothelin-1 (pEC(50) 8.02+/-0.16 and 8.54+/-0.11, P<0.01; n=8). Rather than being specific toSB-710411, this phenomenon appears to be related to somatostatin receptor affinity and not intrinsic activity since the SST agonist somatostatin-14 and antagonist cyclo-somatostatin also potentiated endothelin-1-induced contraction. 4. SB-710411 (10 micro M) did not inhibit carbachol, sodium nitroprusside, IBMX, isoprenaline, and levcromakalim-induced reversal of tone established with noradrenaline. In contrast, however, SB-710411 significantly inhibited the reversal of tone established with endothelin-1 using the same vasorelaxants. 5. In summary, although SB-710411 inhibits the vasoconstrictor actions of hU-II in a competitive, surmountable manner, it also possesses additional pharmacological actions. Thus, whilst the present study is amongst the first to detail the properties of a functional U-II receptor antagonist, the data suggest caution be used when assessing data generated utilizing this moiety and other SST analogues.

The subtypes of muscarinic receptors for neurogenic bladder contraction in rats

We evaluated in vivo functional selectivity profiles for muscarinic M 2 and M 3 subtypes of four muscarinic antagonists: Compound A (a novel muscarinic receptor antagonist with M 2-sparing antagonistic activity), darifenacin, (a muscarinic M 3 receptor antagonist); methoctramine (a muscarinic M 2 receptor antagonist) and tolterodine (a nonselective muscarinic receptor antagonist), and compared the inhibition potency on distention-induced bladder contraction in rats. In an in vivo functional study, Compound A (0.03–10 mg/kg, i.v.) showed antimuscarinic activity with high selectivity for M 3 (salivation) over M 2 (bradycardia) (>100-fold). Darifenacin (0.01–0.3 mg/kg, i.v.) showed only slight selectivity for M 3 over M 2 (3.7-fold). Methoctramine (0.003–1 mg/kg, i.v.) showed the reverse selectivity profile (0.077-fold). Tolterodine (0.003–0.3 mg/kg, i.v.) showed less selectivity (1.2-fold). Compound A at M 3 inhibitory doses (0.1 and 0.3 mg/kg, i.v.) showed inhibition in a distention-induced neurogenic bladder contraction model, and its maximal inhibitory effects were about 60% at an even higher dose (3 mg/kg). Methoctramine at M 2 inhibitory doses (0.03 and 0.1 mg/kg, i.v.) did not significantly affect distention-induced bladder contraction. When tolterodine and darifenacin caused inhibition of distention-induced bladder contraction, its maximal inhibitory effects were similar to that of Compound A. Therefore, these findings suggest that Compound A would be an excellent pharmacological tool to give a better understanding of which subtypes of muscarinic receptors act in bladder function so far, and muscarinic M 3, but not M 2, receptors mainly mediate the cholinergic component of distention-induced bladder contraction.

Control of the unstable urinary bladder by graded thermoelectric cooling of the spinal cord.

To evaluate local lumbosacral spinal-cord cooling (a novel technique for neuromodulating urinary bladder reflexes) for its feasibility in possible clinical use, by determining the efficacy and the optimum temperature for suppressing reflex urinary incontinence in two rat models of neurogenic urinary bladder instability. Overactivity of the detrusor muscle was induced by inflammation of the urinary bladder in a group of rats. A second group of rats was examined 6 weeks after complete midthoracic spinal cord transection, when all animals had developed neurogenic bladder hyper-reflexia. The intravesical pressure, urethral pressure and electromyographic (EMG) activity of the external urethral sphincter (EUS) were recorded simultaneously during repetitive local cooling and re-warming of the dorsal L6/S1 spinal cord segments, using a thermoelectric cooling device. Spinal cord cooling at L6/S1 had no influence on the recorded values at >26 degrees C, but markedly suppressed detrusor contraction frequency at 21- 25 degrees C. Cooling to <20 degrees C completely and reversibly eliminated inflammation-induced bladder contractions in rats with an intact neural axis and significantly reduced the contraction amplitudes (mean reduction 61%) and duration of contractions in spinally transected rats. Cooling simultaneously increased tonic EUS EMG activity and urethral perfusion pressure in both experimental groups, indicating closure of the urethral outlet. Cooling of adjacent spinal cord segments had no influence on bladder and urethral functions. Cooling the dorsal spinal cord at the origin of the parasympathetic innervation of the bladder can be used to reversibly suppress bladder instability with simultaneous closure of the urethral outlet. Therefore, local spinal cord cooling, e.g. as an implantable thermoelectric device, may offer a suitable method to treat detrusor overactivity and restore continence.

495 The intensity of uterine contractions in rats during pregnancy and parturition can be estimated from electromyography signals

495 The intensity of uterine contractions in rats during pregnancy and parturition can be estimated from electromyography signals

Increased expression of G12 and G13 proteins in bronchial smooth muscle of airway hyperresponsive rats

To obtain information on the activation pathway of the monomeric G protein, RhoA, in bronchial smooth muscle, the expression of G alpha12 and G alpha13 in bronchial smooth muscle of the rat was determined. The levels of these G proteins were also compared between antigen-induced airway hyperresponsive and normal control groups. Actively sensitized rats were repeatedly challenged by antigen inhalation. Twenty-four hours after the final antigen challenge, membrane preparations of bronchial smooth muscles were prepared. Immunoblottings were performed, and the density ratios of G alpha12/beta-actin and G alpha13/beta-actin were calculated to quantify the levels of these G-protein alpha subunits. Both G alpha12 and G alpha13 proteins were expressed in rat bronchial smooth muscle. The levels of bronchial G alpha12 and G alpha13 proteins in the repeatedly antigen challenged rats were significantly increased as compared with those in control animals; the magnitude of upregulation in the airway-hyperresponsive group was 89% and 68% in the control group, respectively. G alpha12 and G alpha13 proteins were expressed in rat bronchial smooth muscle. Considering the probable involvement of G12 and G13 proteins in Ca2+ sensitization through Rho protein, the augmented expression of such G proteins after repeated antigen challenge may be responsible for the hyperresponsiveness of bronchial smooth muscle contraction in rats.