The effect of a novel CCK-antagonist (lorglumide) on human and guinea pig gallbladder strips: a tensiometric study

The effect of a novel CCK-antagonist (lorglumide, CR 1409) was evaluated by "in vitro" tensiometric studies on 16 human (gallstone patients) and 12 guinea pig gallbladder smooth muscle strips. In the animal experiments, increasing doses of lorglumide (0.2-6.5 uM) caused a rightward shift of the dose-response curves of CCK-OP, with an increase of the ED50 from 8.2 nM +/- 1.62 SEM, n = 12; to 100 nM +/- 12, n = 4) without affecting the maximal effect (Emax). Schild plot gave an affinity constant of 7.19. In human gallbladders, the effect of lorglumide was also present (ED50 increased from 47 nM +/- 8 SEM, n = 16; to 300 nM +/- 10 SEM, n = 4) coexisting with a large inter-sample variation for CCK-OP ED50s and maximal contractions, most likely due to the histological changes of the wall in chronic cholecystitis. The affinity constant was similar to that found in animal experiments. We confirm the studies previously reported in animals on the existence of a competitive antagonism of lorglumide on CCK gallbladder receptors. Moreover, our results on gallbladders from gallstone patients show that lorglumide is a highly effective antagonist of CCK-induced contractions despite the presence of chronic cholecystitis. Our study might help for a better comprehension of the role of these new anti-CCK drugs in the treatment of biliary pain.     

Contraction of guinea pig trachea by epidermal growth factor--urogastrone

To evaluate further the action of epidermal growth factor - urogastrone (EGF-URO) in smooth muscle systems, we examined the effect of the peptide on guinea pig tracheal strips. The cumulative addition of EGF-URO to the organ bath resulted in a concentration-dependent tonic contraction without tachyphylaxis. The half-maximal contraction was obtained at 13 +/- 3 ng/mL EGF-URO (2 nM). The maximum contraction at 100 ng/mL approached 60% of that induced by 1 microM histamine. No significant difference in the EGF-URO-induced contraction was observed in the presence or absence of a functional epithelium. Preincubation with 1 microM indomethacin for 20 min abolished the action of EGF-URO. The contractile effect of EGF-URO was not affected by yohimbine, propranolol, atropine, tetrodotoxin, and esculetin. However, mepacrine caused inhibition by 37 +/- 7% (mean +/- SEM for n = 3). Verapamil (10 microM) inhibited the EGF-induced response by 62 +/- 5% (mean +/- SEM for n = 4); the response was also absent in Ca-free (1 mM EGTA) buffer. However, the response was restored after the readdition of calcium. Our results suggest that EGF-URO can modulate tracheal smooth muscle contractility via a cyclooxygenase product and raise the possibility that EGF-URO might play a role in controlling pulmonary smooth muscle tone in vivo.     

In vitro effect of calcitonin on guinea pig gallbladder

Calcitonin (CT) is a 32 amino acidic polypeptide hormone which has been found in almost all species and whose effects are mainly concerned with calcium and phosphorous homeostasis. Three preparations are employed for therapeutic uses: salmon (sCT), porcine (pCT) and human CT (hCT). The sCT is the most powerful one and in human volunteers a strong relaxing effect has been shown on gallbladder (GB) basal volume and emptying in response to a meal, intraduodenal instillation of a liquid meal and i.v. cholecystokinin (CCK) infusion. Our study was aimed at investigating if a direct sCT effect could be demonstrated on smooth muscle strips from guinea pig GBs "in vitro" (organ bath). Isometric contractions were measured in response to maximal doses of acetylcholine (ACh: 10(-4) M), KCl (80 mM) and cholecystokinin octapeptide (CCK-OP: 10(-6) M), in absence and in presence of four doses of sCT (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) and 1 x 10(-6) M). sCT did not affect the initial strip basal tone. ACh, CCK-OP and KCl caused, as expected, a powerful contraction of the strips, but no effect was shown when each of the sCT doses was administered before ACh (1.28+ 0.69 SEM without sCT vs 1.28g+ 0.69 with sCT; n = 6) and CCK-OP (1.46g+ 0.19 without sCT vs 1.46g+ 0.19 with sCT; n = 8) or 5 min after the induced KCl contraction. On the basis of these preliminary results, we conclude that no evidence of a direct sCT effect was found on guinea pig GBs when considering either basal smooth muscle tone or isometric contraction in response to ACh, KCl and CCK-OP. Further studies are therefore required to clarify the influence of CT on GB dynamics in vivo and to elucidate its the physiological significance.     

Tyrosine kinase inhibitors and the contractile action of epidermal growth factor-urogastrone and other agonists in gastric smooth muscle.

We examined the effects of the tyrosine kinase (TK) inhibitors, genistein, and tyrphostin (RG-50864) on the contractile action of epidermal growth factor - urogastrone (EGF-URO), transforming growth factor-alpha (TGF-alpha), and other agonists in two smooth muscle bioassay systems (guinea pig gastric longitudinal muscle, LM, and circular muscle, CM). We also studied the inhibition by tyrphostin of EGF-URO stimulated protein phosphorylation in identical smooth muscle strips. The selective inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, less than 7.4 microM (2 micrograms/mL); tyrphostin, less than 20 microM (4 micrograms/mL)) than those used in previously published studies with these TK inhibitors. In LM tissue, the IC50 values were for genistein 1.1 +/- 0.1 microM (0.30 micrograms/mL; mean +/- SEM) and 3.6 +/- 0.5 microM (0.74 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS: TP) of 1:3 in the longitudinal preparation. In CM tissue, the IC50 values were 3.0 +/- 0.3 microM (0.81 micrograms/mL) for genistein and 2.4 +/- 0.2 microM (0.49 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha mediated contraction was rapid (beginning within minutes) and was reversible upon washing the preparations free from the enzyme inhibitors. In intact tissue strips studied under bioassay conditions, tyrphostin (40 microM) also blocked EGF-URO triggered phosphorylation of substrates detected on Western blots using monoclonal antiphosphotyrosine antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Cholinergic Regulation of Intracellular Calcium in the Human Neuroblastoma, SH-SY5Y

The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 +/- 14 nM (mean +/- SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 microM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 +/- 52 (mean +/- SEM, n = 12) and 422 +/- 48 nM (mean +/- SEM, n = 7), respectively, in less than 4 s. This initial calcium transient declined to a plateau of 268 +/- 36 and 240 +/- 27 nM for carbachol and acetylcholine, respectively, in approximately 40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 +/- 0.05, 8.3 +/- 1.6, 411 +/- 36, and 240 +/- 46 nM (mean +/- SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 +/- 0.18, 13.5 +/- 8.5, 192 +/- 32, and 414 +/- 25 nM (mean +/- SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electromechanical effects of endothelin on ferret bronchial and tracheal smooth muscle

The effects of endothelin (ET) on transmembrane potential and isometric force were studied in ferret bronchial and tracheal smooth muscles. At rest, the muscle cells were electrically and mechanically quiescent. The mean resting potential for the bronchial cells was -70 +/- 1 mV (n = 25 cells/8 ferrets), and that of the tracheal cells was -60 +/- 1 mV (n = 7 cells/2 ferrets). ET depolarized and contracted both types of muscle cells in a concentration-dependent manner. At 1 nM ET, the bronchial muscle cells were significantly depolarized with concomitant force generation. In contrast, greater than 30 nM ET was required for the tracheal muscle cells to respond. The bronchial cells were further depolarized by 10 and 100 nM ET with electrical slow-wave activity present. The calcium channel antagonist verapamil substantially inhibited the contractions produced by 100 nM ET and abolished the slow-wave activity without affecting the base-line depolarization. Pretreatment of the bronchial muscle with 30 microM indomethacin did not affect the ET-induced contraction. These results suggest that ET modulates airway smooth muscle tone by direct activation and/or depolarization-induced activation of sarcolemmal calcium channels.     

Gastrin-releasing peptides from Xenopus laevis: purification, characterization, and myotropic activity

Two molecular forms of gastrin-releasing peptide (GRP) were isolated from an extract of the intestine of the tetraploid frog Xenopus laevis. The primary structure of GRP-1 (APTSQQHTEQ(10)LSRSNINTRG(20) SHWAVGHLM.NH(2)) differs from that of GRP-2 by a single amino acid substitution (Asn(15)--> Thr(15)). GRP-(20-29) peptide (neuromedin C) was also isolated from the extract. Synthetic GRP-1 produced concentration-dependent contractions of longitudinal smooth muscle strips from Xenopus cardiac stomach (pD(2) = 8.93 +/- 0.32; n = 6). The responses were unaffected by tetrodotoxin, atropine, and methysergide, indicating a direct action of the peptide on smooth muscle cells. GRP-1 elicited concentration-dependent relaxations of precontracted (5 microM carbachol) circular smooth muscle strips from the same region (pD(2) = 8.96 +/- 0.21; n = 8). The responses were significantly (P < 0.05) attenuated (71 +/- 24% decrease in maximum response; n = 6) by indomethacin, indicating mediation, at least in part, by prostanoids. Despite the fact that Xenopus GRP-1 differs from pig GRP at 15 amino acid sites, both peptides are equipotent and equally effective for both contractile and relaxant responses, demonstrating that selective evolutionary pressure has acted to conserve the functional COOH-terminal domain in the peptide. The data suggest a physiologically important role for GRP in the regulation of gastric motility in X. laevis.     

Pituitary adenylate cyclase activating polypeptide stimulates gallbladder motility in conscious dogs.

We investigated whether pituitary adenylate cyclase activating polypeptide (PA-CAP27 and PACAP38) had any effect on gallbladder motility in conscious dogs, in which force transducers were chronically implanted in the gastric antrum, duodenum and gallbladder. PACAP27 and PACAP38 were administered intravenously during the digestive and interdigestive states at doses of 30, 100 and 300 pmol/kg. By way of comparison, cholecystokinin octapeptide (CCK-OP) was administrated at doses of 3, 9 and 27 pmol/kg. As a result, each peptide evoked transient and tonic contractions both in the digestive and interdigestive states, and the effect on the motor index was dose dependent. PACAP27 and PACAP38 were 0.11 +/- 0.03 and 0.04 +/- 0.01 as potent as CCK-OP in the digestive state, and 0.18 +/- 0.04 and 0.02 +/- 0.01 in the interdigestive state, respectively, on a molar basis. Although PACAP27 and PACAP38 belong to the vasoactive intestinal polypeptide (VIP) family, intravenous administration of 300 pmol/kg of VIP had no effect on interdigestive gallbladder motility, but on the other hand inhibited gallbladder motility in the digestive state. The contractile effects of PACAP27 and PACAP38 were almost completely abolished by pretreatment with atropine or hexamethonium, but not with L364718. An in vitro study using canine gallbladder strips showed that PACAP27 and PACAP38 had no effect on spontaneous gallbladder motor activity evoked by electric field stimulation, CCK-OP or acetylcholine. It was concluded that PACAP27 and PACAP38 stimulate gallbladder motility in conscious dogs through a preganglionic cholinergic mechanism.     

AT1 Angiotensin Receptors Mobilize Intracellular Calcium in a Subclone of NG108–15 Neuroblastoma Cells

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.     

Microvascular and macrovascular endothelial cells produce different constrictor substances.

The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25-55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sprint training normalizes Ca(2+) transients and SR function in postinfarction rat myocytes.

Previous studies have shown that myocytes isolated from sedentary (Sed) rat hearts 3 wk after myocardial infarction (MI) undergo hypertrophy, exhibit altered intracellular Ca(2+) concentration ([Ca(2+)](i)) dynamics and abnormal contraction, and impaired sarcoplasmic reticulum (SR) function manifested as prolonged half-time of [Ca(2+)](i) decline. Because exercise training elicits positive adaptations in cardiac contractile function and myocardial Ca(2+) regulation, the present study examined whether 6-8 wk of high-intensity sprint training (HIST) would restore [Ca(2+)](i) dynamics and SR function in MI myocytes toward normal. In MI rats, HIST ameliorated myocyte hypertrophy as indicated by significant (P 相似文献   

Small-conductance, Ca(2+) -activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

Small-conductance Ca(2+)-activated K(+) (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing beta-galactosidase (beta-gal) under the direction of the SK2 promoter (SK2 beta-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2(-/-) mice) to assess SK2 function. In SK2 beta-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2(+/+)) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC(50) = 0.16 nM), whereas phasic contractions of SK2(-/-) strips were unaffected. Nerve-mediated contractions of SK2(+/+) UBSM strips were also increased by apamin, an effect absent in SK2(-/-) strips. Apamin increased the sensitivity of SK2(+/+) UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 +/- 4 Hz, n = 6; apamin, 12 +/- 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2(-/-) UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.     

The source of calcium for CCK-induced contraction of the guinea-pig gall bladder.

The sources of calcium for cholecystokinin octapeptide (CCK-OP)-induced gallbladder smooth muscle contraction are considered both extracellular and intracellular, but the relative need for intracellular calcium especially at low, physiological concentrations is not clear. To better define the calcium sources responsible for guinea-pig gallbladder contractions in vitro, we inhibited calcium influx using the calcium channel blocker, methoxyverapamil, and a calcium-free Krebs' solution. Availability and release of intracellular calcium stores were depleted by strontium substitution and ryanodine. CCK-OP was compared to bethanechol and potassium chloride (KCl). Preventing calcium influx with 10(-5) M methoxyverapamil depressed the responses to CCK-OP, bethanechol and KCl. Methoxyverapamil, however, had little effect on the time-dependent generation of tension to CCK-OP, but significantly reduced the response to bethanechol and KCl, each at ED50. The duration of the contractile response in the calcium-free Krebs' solution to CCK-OP was longer than that for bethanechol. Strontium (2.5 mM) significantly attenuated the response to CCK-OP and bethanechol, but not to KCl. Ryanodine significantly reduced contractions induced by CCK-OP but not for bethanechol, both at low dose ED25. These results indicate that contraction of the guinea-pig gallbladder induced by CCK-OP, bethanechol and KCl requires extracellular calcium influx. Further, the initiation and maintenance of contraction by CCK-OP and bethanechol necessitates calcium mobilisation from intracellular stores. CCK-OP may have a greater penchant for these calcium stores, particularly at physiological doses.     

Muscarinic receptor reserve of airway smooth muscle and the response to isoproterenol

It has been hypothesized that the muscarinic receptor reserve for contraction of airway smooth muscle is an important determinant of the potency with which isoproterenol relaxes submaximal muscarinic contractions. The goals of this study were to inactivate, with phenoxybenzamine, a fraction of the muscarinic receptors present in canine tracheal smooth muscle, and then to determine whether this decrease in muscarinic receptor reserve altered the potency with which isoproterenol relaxed submaximal muscarinic contractions. Strips of smooth muscle were suspended from force transducers in vitro and preincubated with either vehicle (untreated) or phenoxybenzamine (10(-5) M) for 30 min. For muscarinic contractions induced by carbachol that were approximately 70-80% of maximum, the half-maximally effective concentration of isoproterenol was 2.4 +/- 0.8 x 10(-7) M for untreated strips but 5.8 +/- 1.3 x 10(-9) M for strips treated with phenoxybenzamine (n = 6, P less than 0.05). We concluded that treatment with phenoxybenzamine increased the sensitivity of a submaximal muscarinic contraction to isoproterenol. The results support the hypothesis that the muscarinic receptor reserve for contraction is an important determinant of the potency with which isoproterenol relaxes submaximal muscarinic contractions.     

Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle

Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 microM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 +/- 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 +/- 7% and decreased ACh-induced contraction by 79 +/- 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.     

A role for Na+/H+ exchange in contraction of guinea pig airways by endothelin-1 in vitro.

Endothelin-1-induced contractions of guinea pig tracheal and bronchial strips were dose-dependently attenuated by the amiloride analogues 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 1-10 microM) and 5-(N,N-hexamethylene)amiloride (HMA, 1-10 microM). The calculated Ki values for EIPA and HMA were 0.11 +/- 0.02 microM and 0.06 +/- 0.02 microM in the trachea, and 0.28 +/- 0.11 microM and 0.70 +/- 0.25 microM in the bronchus, respectively. These values are in the same order of magnitude as those reported for inhibition of the Na+/H+ exchange in cells. Amiloride (1-10 microM) was ineffective. These data suggest that activation of the Na+/H+ exchange by ET-1 may be involved in mediating its myotropic action in guinea pig airway smooth muscle.     

M2 receptors in genito-urinary smooth muscle pathology

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats which had their major pelvic ganglion bilaterally removed (denervation, DEN) or from rats in which the spinal cord was injured (SCI) via compression. DEN induced both hypertrophy (505+/-51 mg bladder weight) and a supersensitivity of the bladders to carbachol (EC50=0.7+/-0.1 uM). Some of the SCI rats regained the ability to void spontaneously (SPV). The bladders of these animals weighed 184+/-17 mg, significantly less than the bladders of non voiding rats (NV, 644+/-92 mg). The potency of carbachol was greater in bladder strips from NV SCI animals (EC50=0.54+/-0.1 uM) than either bladder strips from SPV SCI (EC50=0.93+/-0.3 microM), DEN or control (EC50=1.2+/-0.1 microM) animals. Antagonist affinities in control bladders for antagonism of carbachol induced contractions were consistent with M3 mediated contractions. Antagonist affinities in DEN bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.5) and para fluoro hexahydrosilodifenidol (p-F-HHSiD, 6.6); were consistent with M2 mediated contractions, although the methoctramine affinity (6.5) was consistent with M3 mediated contractions. p-F-HHSiD inhibited carbachol induced contraction with an affinity consistent with M2 receptors in bladders from NV SCI (pKb=6.4) animals and M3 receptors in bladders from SPV SCI animals (pKb=7.9). Subtype selective immunoprecipitation of muscarinic receptors revealed an increase in total and an increase in M2 receptor density with no change in M3 receptor density in bladders from DEN and NV SCI animals compared to normal or sham operated controls. M3 receptor density was lower in bladders from SPV SCI animals while the M2 receptor density was not different from control. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediate smooth muscle contraction in bladders from DEN and NV SCI rats.     

Low levels of K(ATP) channel activation decrease excitability and contractility of urinary bladder

Activation of ATP-sensitive potassium (K(ATP)) channels can regulate smooth muscle function through membrane potential hyperpolarization. A critical issue in understanding the role of K(ATP) channels is the relationship between channel activation and the effect on tissue function. Here, we explored this relationship in urinary bladder smooth muscle (UBSM) from the detrusor by activating K(ATP) channels with the synthetic compounds N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (ZD-6169) and levcromakalim. The effects of ZD-6169 and levcromakalim on K(ATP) channel currents in isolated UBSM cells, on action potentials, and on related phasic contractions of isolated UBSM strips were examined. ZD-6169 and levcromakalim at 1.02 and 2.63 microM, respectively, caused half-maximal activation (K1/2) of K(ATP) currents in single UBSM cells (see Heppner TJ, Bonev A, Li JH, Kau ST, and Nelson MT. Pharmacology 53: 170-179, 1996). In contrast, much lower concentrations (K(1/2) = 47 nM for ZD-6169 and K1/2 = 38 nM for levcromakalim) caused inhibition of action potentials and phasic contractions of UBSM. The results suggest that activation of <1% of K(ATP) channels is sufficient to inhibit significantly action potentials and the related phasic contractions.