Electrical Field Stimulation Responses Research Articles

Relaxant effect of quercetin on rabbit isolated bladder smooth muscles contractions

Introduction: Quercetin is a flavonoid compound found in many medicinal plants. Antispasmodic effect of quercetin has been reported in ileum and uterus smooth muscles but not in bladder. Therefore, the objective of this research was to investigate relaxant effect of quercetin in rabbit isolated bladder. Methods: Male rabbit was asphyxiated with carbon dioxide and then sacrificed. The whole bladder was dissected out and placed in oxygenated Tyrode’s solution. Isolated bladder was cut into longitudinal strips and placed in an organ bath for contraction studies. Contractions were induced with KCl (20mM), acetylcholine (5μM) and electrical field stimulation (EFS). Full inhibitory concentration–response curve was constructed for quercetin following addition of above spasmogens. Quercetin was added into the organ bath with 2 fold increments in concentration until maximum response was achieved. Nifedipine was used as positive control group and equivalent volume of quercetin vehicle (water + DMSO) was used as negative control group.Results: Quercetin (4 μg/mL to 640 μg/mL) in a concentration dependent manner inhibited isolated bladder strips contracted by KCl (IC50=159±25 μg/mL), acetylcholine (IC50=43±9.1 μg/mL) and EFS (IC50=38±9.3 μg/mL). In the highest used concentration, quercetin completely removed contractile responses to KCl, acetylcholine and electrical filed stimulation (EFS). Nifedipine totally inhibited KCl response (IC50=115±36 ng/mL) but only partially inhibited acetylcholine and EFS responses. Conclusion: These results confirm the relaxant effect of quercetin on rabbit bladder and if similar effects are seen in human studies, then quercetin would be a suitable drug candidate to be investigated for bladder incontinence.

Endothelium modulates electrical field stimulation-induced contractions of Chelonoidis carbonaria aortic rings

The role of endothelium in the electrical-field stimulation (EFS)-induced contractions of Chelonoidis carbonaria aorta was investigated. Contractions were evaluated in the presence and absence of L-NAME (100 μM), tetrodotoxin (1 μM), phentolamine (10 and 100 μM), phenoxybenzamine (1 and 10 μM), prazosin (100 μM), idazoxan (100 μM), atropine (10 μM), D-tubocurarine (10 μM) or indomethacin (10 μM). EFS-induced contraction was also carried out in endothelium-denuded rings. EFS-induced contraction was investigated by the sandwich assay. Concentration curves to endothelin-1 (0.1–100 nM) and U46619 (0.001–100 μM) were also constructed to calculate both Emax and EC50. EFS at 16 Hz contracted Chelonoidis aorta, which was almost abolished by the endothelium removal. The addition of L-NAME increased the EFS response (2.0 ± 0.4 and 8.3 ± 1.9 mN). In L-NAME treated aortic rings, tetrodotoxin did not change the EFS-response (5.1 ± 1.8 and 4.9 ± 1.7 mN). Indomethacin, atropine and d-tubucurarine also did not affect the EFS-response. Phentolamine at 10 μM did not change the EFS-induced contraction; however, at 100 μM, reduced it (3.9 ± 1 and 1.9 ± 0.3 mN). Prazosin and idazoxan did not change EFS-induced contractions. Phenoxybenzamine at 1 μM reduced by 76% (9.6 ± 3.4 and 2.3 ± 0.8 mN) and at 10 μM by 90% the EFS response. Immunohistochemistry identified tyrosine hydroxylase in the endothelium and brain, whereas S100 protein was found only in brain. In conclusion, endothelium modulates EFS-induced contractions in Chelonoidis aortic rings and this modulation may be due to endothelium-derived catecholamines, possibly dopamine.

Electrical field stimulation‐induced vasorelaxation responses decreased in anterior and middle cerebral arteries of endotoxemic rats

IntroductionEndotoxic shock is characterized by vascular dysfunction, hypotension and multiple organ dysfunction. Loss of vascular tone is a key factor responsible for death in endotoxic shock. Perivascular innervation has an important role in the regulation of vascular tone. Neural control of the arteries is mediated by adrenergic and nitrergic neurons. The nitrergic component is dominant in brain arteries while adrenergic component is dominant in mesenteric arteries. Lipopolysaccharide (LPS) alters the electrical field stimulation (EFS) responses in mesenteric arteries in vitro. Our aim is to study the alterations of EFS responses of cerebral and mesenteric arteries in endotoxemia in rats.Material and MethodsLPS (10 mg/kg) were given intraperitoneally to rats to make an animal model os sepsis. Rats were anesthetized with gaseous carbon dioxide and decapitated 20 hours after injection, Basilar, anterior cerebral, middle cerebral and 3rd branch of mesenteric arteries were isolated and mounted in a wire myograph covered with platinum electrodes. After U46619 precontraction, frequency‐dependent EFS responses were obtained using a constant current stimulator.ResultsHigh K+ responses did not change in brain and peripheral arteries. EFS‐dependent vasorelaxation responses decreased in anterior and middle cerebral arteries in endotoxemic rats. EFS‐induced vasorelaxation responses of basilar artery and vasoconstriction responses of mesenteric arteries did not change in endotoxemic rats.ConclusionsIn this study we demonstrate for the first time that EFS‐induced vasorelaxation responses of some cerebral arteries decreased in endotoxemic rats. This EFS‐dependent alterations could be explained by degeneration of perivascular innervations or an imbalance between adrenergic and nitrergic neurons.Support or Funding InformationThis study was funded by Hacettepe University Scientific Research Projects Coordination Unit (TSA‐2017‐12848).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A simple laboratory exercise with rat isolated esophagus and stomach fundus to reveal functional differences between striated and smooth muscle cells.

This study describes an undergraduate student laboratory activity using isolated preparations from rat gastrointestinal tissues that possess contractile profiles typically exhibited by striated and smooth muscle cells. While students are introduced to an ex vivo methodology, they can compare differences in trace experiments, twitch aspects, phasic and tonic properties, force-frequency relationships, and pharmacological responsiveness of esophageal (striated) and fundic (smooth muscle) segments. Muscle strips were subjected to electrical field stimulation (EFS) applied by platinum electrodes immersed in the physiological solution. The contractile profile of EFS responses varied between these two types of gut preparations. Atropine and tubocurarine revealed differential inhibitory influences in esophagus or fundus tissues; caffeine and procaine produced similar effects, i.e., potentiation and blockade of the EFS-induced contractile response in these tissues, respectively. Experimental results obtained during the activity helped the improvement of student learning about basic concepts previously discussed in theoretical lectures. To measure student learning with this laboratory exercise, a questionnaire was applied before and after the activity, and the number of expected correct answers, concerning the mechanisms of contraction in striated and smooth muscle, could be clearly evidenced.

Nerve-induced responses of mouse vaginal smooth muscle.

Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective α-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective α1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.

Signal Transduction Underlying the Inhibitory Mechanism of Fluoxetine on Electrical Field Stimulation Response in Rat Ileal Smooth Muscle

Fluoxetine (FLX), a well-known antidepressant drug under the class of selective serotonin reuptake inhibitor, exerts its action by inhibiting the reuptake of serotonin selectively. In some studies, it has been demonstrated that FLX relaxes the intestinal smooth muscle. In this study, we aimed at studying the signal transduction pathway underlying the muscle relaxation effect of FLX on electrically stimulated rat ileal muscle contraction. To investigate the possible mechanism involved, various antagonists were used. It was found that inhibition with L-N^G-nitroarginine methyl ester, ondansetron, GR113808 and bicuculline enhanced the relaxation effect of FLX. However, the effect of FLX was nullified under the presence of atropine, calcium channel modulator (calcium ionophore A23187), and potassium channel blockers (tetraethylammonium chloride, 4-aminopyridine and glybenclamide). Specific pathway-inhibiting antagonists, Y27632 (Rho-kinase inhibitor) and U73122 (phospholipase-C inhibitor) reversed the antagonistic effect of FLX, while ML-9 (myosin light chain kinase inhibitor) and chelerythrine (protein kinase C inhibitor) augmented the FLX-induced inhibition effect. Taken together, we concluded that FLX exerts the inhibitory effect on electric field stimulation response in rat ileal smooth muscle by the inhibition of muscarinic receptors, decrease of intracellular calcium level by inhibiting phospholipase C and opens the potassium channels.

Role of endogenous hydrogen sulfide in nerve-evoked relaxation of pig terminal bronchioles

Hydrogen sulfide (H2S) is a gasotransmitter employed for intra- and inter-cellular communication in almost all organ systems. This study investigates the role of endogenous H2S in nerve-evoked relaxation of pig terminal bronchioles with 260 μm medium internal lumen diameter. High expression of the H2S synthesis enzyme cystathionine γ-lyase (CSE) in the bronchiolar muscle layer and strong CSE-immunoreactivity within nerve fibers distributed along smooth muscle bundles were observed. Further, endogenous H2S generated in bronchiolar membranes was reduced by CSE inhibition. In contrast, cystathionine β-synthase expression, another H2S synthesis enzyme, however was not consistently detected in the bronchiolar smooth muscle layer. Electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked smooth muscle relaxation. Inhibition of CSE, nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and of ATP-dependent K+, transient receptor potential A1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) channels reduced the EFS relaxation but failed to modify the GYY4137 response. Raising extracellular K+ concentration inhibited the GYY4137 relaxation. Large conductance Ca2+-activated K+ channel blockade reduced both EFS and GYY4137 responses. GYY4137 inhibited the contractions induced by histamine and reduced to a lesser extent the histamine-induced increases in intracellular [Ca2+]. These results suggest that relaxation induced by EFS in the pig terminal bronchioles partly involves the H2S/CSE pathway. H2S response is produced via NO/sGC-independent mechanisms involving K+ channels and intracellular Ca2+ desensitization-dependent pathways. Thus, based on our current results H2S donors might be useful as bronchodilator agents for the treatment of lung diseases with persistent airflow limitation, such as asthma and chronic obstructive lung disease.

Impaired Excitatory Neurotransmission in the Urinary Bladder from the Obese Zucker Rat: Role of Cannabinoid Receptors.

Metabolic syndrome (MS) is a known risk factor for lower urinary tract symptoms. This study investigates whether functional and expression changes of cannabinoid CB1 and CB2 receptors are involved in the bladder dysfunction in an obese rat model with insulin resistance. Bladder samples from obese Zucker rat (OZR) and their respective controls lean Zucker rat (LZR) were processed for immunohistochemistry and western blot for studying the cannabinoid receptors expression. Detrusor smooth muscle (DSM) strips from LZR and OZR were also mounted in myographs for isometric force recordings. Neuronal and smooth muscle CB1 and CB2 receptor expression and the nerve fiber density was diminished in the OZR bladder. Electrical field stimulation (EFS) and acetylcholine (ACh) induced frequency- and concentration-dependent contractions of LZR and OZR DSM. ACh contractile responses were similar in LZR and OZR. EFS-elicited contractions, however, were reduced in OZR bladder. Cannabinoid receptor agonists and antagonists failed to modify the DSM basal tension in LZR and OZR In LZR bladder, EFS responses were inhibited by ACEA and SER-601, CB1 and CB2 receptor agonists, respectively, these effects being reversed by ACEA plus the CB1 antagonist, AM-251 or SER-601 plus the CB2 antagonist, AM-630. In OZR bladder, the inhibitory action of ACEA on nerve-evoked contractions was diminished, whereas that SER-601 did not change EFS responses. These results suggest that a diminished function and expression of neuronal cannabinoid CB1 and CB2 receptors, as well as a lower nerve fiber density is involved in the impaired excitatory neurotransmission of the urinary bladder from the OZR.

Biphasic Effect of Diabetes on Neuronal Nitric Oxide Release in Rat Mesenteric Arteries.

IntroductionWe analysed possible time-dependent changes in nitrergic perivascular innervation function from diabetic rats and mechanisms implicated.Materials and MethodsIn endothelium-denuded mesenteric arteries from control and four- (4W) and eight-week (8W) streptozotocin-induced diabetic rats the vasoconstriction to EFS (electrical field stimulation) was analysed before and after preincubation with L-NAME. Neuronal NO release was analysed in the absence and presence of L-arginine, tetrahydrobiopterine (BH4) and L-arginine plus BH4. Superoxide anion (O2-), peroxynitrite (ONOO-) and superoxide dismutase (SOD) activity were measured. Expressions of Cu-Zn SOD, nNOS, p-nNOS Ser1417, p-nNOS Ser847, and Arginase (Arg) I and II were analysed.ResultsEFS response was enhanced at 4W, and to a lesser extent at 8W. L-NAME increased EFS response in control rats and at 8W, but not at 4W. NO release was decreased at 4W and restored at 8W. L-arginine or BH4 increased NO release at 4W, but not 8W. SOD activity and O2- generation were increased at both 4W and 8W. ONOO- decreased at 4W while increased at 8W. Cu-Zn SOD, nNOS and p-NOS Ser1417 expressions remained unmodified at 4W and 8W, whereas p-nNOS Ser847 was increased at 4W. ArgI was overexpressed at 4W, remaining unmodified at 8W. ArgII expression was similar in all groups.ConclusionsOur results show a time-dependent effect of diabetes on neuronal NO release. At 4W, diabetes induced increased O2- generation, nNOS uncoupling and overexpression of ArgI and p-nNOS Ser847, resulting in decreased NO release. At 8W, NO release was restored, involving normalisation of ArgI and p-nNOS Ser847 expressions.

The signaling of amitriptyline-induced inhibitory effect on electrical field stimulation response in colon smooth muscle.

Amitriptyline, a well-known antidepressant, exerts inhibitory effect on electrically stimulated rat colon smooth muscle contraction. In this study, we investigated the signaling pathway of amitriptyline-induced inhibitory effect. Changes in isometric force of colon muscle were recorded on polygraph, and data were analyzed by measuring the inhibitory extent induced by amitriptyline. Firstly, muscles were contracted by stimulation with electric field stimulation (EFS), and then, amitriptyline was added cumulatively to determine its influence effect on EFS. Amitriptyline significantly inhibited EFS-induced contraction dose dependently. Then, the mechanism of inhibitory effect of amitriptyline was evaluated by pretreating with various antagonists such as L-NAME, methylene blue, atropine, 5-HT receptors blockers, guanethidine, prazosin, guanabenz, isoprenaline, Y27632 (Rho-kinase inhibitor), ML9 (myosin light chain kinase (MLCK) inhibitor), U73122 (PLC inhibitor), and chelerythrine (PKC inhibitor). Then, Ca(2+) channel blocker (nifedipine) and K(+)channel blockers, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and glybenclamide, were used to determine the involvement of ion channels. L-NAME, guanabenz, 5HT4 receptor blocker, ML9, and Y27632 enhanced the effect of amitriptyline. Meanwhile, methylene blue, atropine, guanethidine, prazosin, methylsergide, ondansetron, U73122, and chelerythrine blocked its effect. It was also shown that nifedipine enhanced but TEA and glybenclamide blocked amitriptyline-induced inhibitory effect on EFS. Our results indicated that amitriptyline may exert inhibitory effect in response to EFS by inhibiting muscarinic receptors and then PLC-mediated PKC pathway leading to opening of ATP-sensitive potassium channel.

Comparison of antispasmodic effect of hydroalcoholic extract of Dracocephalum kotschyi Boiss. in rat uterus and ileum.

Dracocephalum kotschyi Boiss. is a traditional medicine with antispasmodic activities. The objective of this research was to study antispasmodic activities of hydroalcoholic extract of D. kotschyi on rat isolated uterus contractions for comparison with isolated ileum. Hydroalcoholic extract was obtained from aerial part of D. kotschyi using percolation method. A portion of rat ileum or uterus was suspended in Tyrode's solution at 37°C and gassed with O2. Effect of D. kotschyi extract was assessed on ileum or uterus contractions induced by KCl (80 mM), acetylcholine (ACh, 500 nM), electrical field stimulation (EFS) or oxytocin (0.0005 IU/mL). The extract of D. kotschyi concentration-dependently inhibited ileum responses to KCl (IC50 = 65 ± 18 μg/mL), ACh (IC50 = 102 ± 18 μg/mL) and EFS (IC50 = 117 ± 29 μg/mL). The extract of D. kotschyi also concentration-dependently inhibited uterus responses to KCl (IC50 = 453 ± 64μg/mL), ACh (IC50 = 58 ± 9 μg/mL), EFS (IC50 = 22 ± 3 μg/mL) as well as oxytocin (IC50 = 70 ± 11 μg/mL). From this experiment it was concluded that D. kotschyi extract possesses antispasmodic activities on both smooth muscle of ileum and uterus. In comparison, the extract was more effective inhibitor of ACh and EFS responses in rat uterus than on the ileum. On the other hand, the extract was a more potent inhibitor of KCl response on rat ileum. However, the extract was found to be a potent inhibitor of oxytocin-induced contraction of rat uterus. These results indicate that D. kotschyi extract may contain components that might be useful lead compounds for prevention of uterus spasm.

SMN deficiency disrupts gastrointestinal and enteric nervous system function in mice

The 2007 Consensus Statement for Standard of Care in Spinal Muscular Atrophy (SMA) notes that patients suffer from gastroesophageal reflux, constipation and delayed gastric emptying. We used two mouse models of SMA to determine whether functional GI complications are a direct consequence of or are secondary to survival motor neuron (Smn) deficiency. Our results show that despite normal activity levels and food and water intake, Smn deficiency caused constipation, delayed gastric emptying, slow intestinal transit and reduced colonic motility without gross anatomical or histopathological abnormalities. These changes indicate alterations to the intrinsic neural control of gut functions mediated by the enteric nervous system (ENS). Indeed, Smn deficiency led to disrupted ENS signaling to the smooth muscle of the colon but did not cause enteric neuron loss. High-frequency electrical field stimulation (EFS) of distal colon segments produced up to a 10-fold greater contractile response in Smn deficient tissues. EFS responses were not corrected by the addition of a neuronal nitric oxide synthase inhibitor indicating that the increased contractility was due to hyperexcitability and not disinhibition of the circuitry. The GI symptoms observed in mice are similar to those reported in SMA patients. Together these data suggest that ENS cells are susceptible to Smn deficiency and may underlie the patient GI symptoms.

The Inhibitory Mechanism of Gentamicin on Electrical Field Stimulation Response in Rat Bladder Smooth Muscle.

To see the inhibitory mechanism of gentamicin in response to electrical field stimulation (EFS) using the rat bladder smooth muscle, atropine or guanethidine was treated but had no effect. Methylsergide, a non-selective 5-HT1, 5-HT2 receptor antagonist was also treated but had on effect. Kinase inhibitors, such as chelerythrine (PKC inhibitor), ML-9 (MLCK inhibitor), or Y27632 (rho kinase inhibitor) were pretreated before gentamicin treatment, but did not have effect. For U73122, a phospholipase C (PLC) inhibitor however, the inhibitory effect to gentamicin was significantly attenuated in all frequencies given by the EFS. Therefore gentamicin induced inhibitory effect on EFS response in rat bladder smooth muscle was not mediated by the activation of adrenergic, cholinergic, or serotonergic receptor. The inhibition of gentamicin might be mediated through the PLC dependent pathway, but not through the PKC, MLCK or rho kinase dependent pathway.

Fat deposition in the tunica muscularis and decrease of interstitial cells of Cajal and nNOS-positive neuronal cells in the aged rat colon

Little is known about the time course of aging on interstitial cells of Cajal (ICC) of colon. The aim of this study was to investigate the change of morphology, ICC, and neuronal nitric oxide synthase (nNOS)-immunoreactive cells in the aged rat. The proximal colon of 344 Fischer rats at four different ages (6, 31, 74 wk, and 2 yr) were studied. The immunoreactivity of c-Kit, nNOS, anti-protein gene product 9.5, and synaptophysin were counted after immunohistochemistry. The c-kit, stem cell factor (ligand of Kit), and nNOS mRNA were measured by real-time PCR. c-Kit and nNOS protein were assessed by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The area of intramuscular fat deposition significantly increased with age after 31 wk. c-Kit-immunoreactive ICC and nNOS-immunoreactive neurons and nerve fibers significantly declined with age. mRNA and protein expression of c-kit and nNOS decreased with aging. The functional study showed that the spontaneous contractility was decreased in aged rat, whereas EFS responses in the presence of atropine and L-NG-Nitroarginine methyl ester were increased in aged rat. In conclusion, the decrease of proportion of proper smooth muscle, the density of ICC and nNOS-immunoreactive neuronal fibers, and the number of nNOS-immunoreactive neurons during the aging process may explain the aging-associated colonic dysmotility.

In a non‐human primate model, aging disrupts the neural control of intestinal smooth muscle contractility in a region‐specific manner

BackgroundIncidences of gastrointestinal (GI) motility disorders increase with age. However, there is a paucity of knowledge about the aging mechanisms leading to GI dysmotility. Motility in the GI tract is a function of smooth muscle contractility, which is modulated in part by the enteric nervous system (ENS). Evidence suggests that aging impairs the ENS, thus we tested the hypothesis that senescence in the GI tract precipitates abnormalities in smooth muscle and neurally mediated contractility in a region-specific manner.MethodsJejunal and colonic circular muscle strips were isolated from young (4–10 years) and old (18+ years) baboons. Myogenic responses were investigated using potassium chloride (KCl) and carbachol (CCh). Neurally mediated contractile responses were evoked by electrical field stimulation (EFS) and were recorded in the absence and presence of atropine (1 μM) or NG-Nitro-l-arginine methyl ester (l-NAME; 100 μM).Key ResultsThe myogenic responses to KCl in the jejunum and colon were unaffected by age. In the colon, but not the jejunum, CCh-induced contractile responses were reduced in aged animals. Compared to young baboons, there was enhanced EFS-induced contractility of old baboon jejunal smooth muscle in contrast to the reduced contractility in the colon. The effect of atropine on the EFS response was lower in aged colonic tissue, suggesting reduced participation of acetylcholine. In aged jejunal tissue, higher contractile responses to EFS were found to be due to reduced nitregic inhibition.Conclusions & InferencesThese findings provide key evidence for the importance of intestinal smooth muscle and ENS senescence in age-associated GI motility disorders.

Functional Innervation of Guinea-Pig Bladder Interstitial Cells of Cajal Subtypes: Neurogenic Stimulation Evokes In Situ Calcium Transients

Several populations of interstitial cells of Cajal (ICC) exist in the bladder, associated with intramural nerves. Although ICC respond to exogenous agonists, there is currently no evidence of their functional innervation. The objective was to determine whether bladder ICC are functionally innervated. Guinea-pig bladder tissues, loaded with fluo-4AM were imaged with fluorescent microscopy and challenged with neurogenic electrical field stimulation (EFS). All subtypes of ICC and smooth muscle cells (SMC) displayed spontaneous Ca2+-oscillations. EFS (0.5 Hz, 2 Hz, 10 Hz) evoked tetrodotoxin (1 µM)-sensitive Ca2+-transients in lamina propria ICC (ICC-LP), detrusor ICC and perivascular ICC (PICC) associated with mucosal microvessels. EFS responses in ICC-LP were significantly reduced by atropine or suramin. SMC and vascular SMC (VSM) also responded to EFS. Spontaneous Ca2+-oscillations in individual ICC-LP within networks occurred asynchronously whereas EFS evoked coordinated Ca2+-transients in all ICC-LP within a field of view. Non-correlated Ca2+-oscillations in detrusor ICC and adjacent SMC pre-EFS, contrasted with simultaneous neurogenic Ca2+ transients evoked by EFS. Spontaneous Ca2+-oscillations in PICC were little affected by EFS, whereas large Ca2+-transients were evoked in pre-EFS quiescent PICC. EFS also increased the frequency of VSM Ca2+-oscillations. In conclusion, ICC-LP, detrusor ICC and PICC are functionally innervated. Interestingly, Ca2+-activity within ICC-LP networks and between detrusor ICC and their adjacent SMC were synchronous under neural control. VSM and PICC Ca2+-activity was regulated by bladder nerves. These novel findings demonstrate functional neural control of bladder ICC. Similar studies should now be carried out on neurogenic bladder to elucidate the contribution of impaired nerve-ICC communication to bladder pathophysiology.

Role of the enteric nervous system in the colon of patients with idiopathic megacolon.

To clarify the physiological significance of the enteric nervous system (ENS) in the colon of patients with idiopathic megacolon (IM), we investigated the enteric nerve responses on lesional and normal bowel segments derived from patients with IM and patients who underwent colon resection for colonic cancers. Eighteen preparations were taken from the lesional colon of 5 patients with IM (4 men and 1 woman, aged 46-70 yrs, mean age 55.8 yrs); 42 were taken from the normal colon of 14 patients with colonic cancer (10 men and 4 woman, aged 48-68 yrs, mean age 54.2 yrs). A mechanographic technique was used to evaluate in vitro muscle responses to electrical field stimulation (EFS) of the adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers. 1) Responses to EFS before blockade of adrenergic and cholinergic nerves. The muscle strips in both normal and IM colon demonstrated relaxation reactions rather than contraction reactions by EFS. The relaxation reaction to EFS in the IM was significantly stronger than that in the normal colon (p=0.0366). 2) Responses to EFS after blockade of the adrenergic and cholinergic nerves. The muscle strips in both normal and IM colon demonstrated relaxation reactions rather than contraction reactions by EFS. The relaxation reaction to EFS in the IM was stronger than that in the normal colon (p=0.0467). 3) Tetrodotoxin abolished the EFS responses in the muscle strips in both normal colon and abnormal colon with IM. An increase of NANC inhibitory nerves plays an important role in the impaired motility observed in the colon of patients with IM.

The Effect of Diabetes Mellitus on α1-Adrenergic Receptor Subtypes in the Bladder of Rats

To detect the possible alterations on density or sensitivity of α1-adrenergic subtypes in diabetic bladder by reverse transcriptase-polymerase chain reaction technology and in vitro studies. Experimental diabetes was induced by administration of streptozotocin with a single injection through the tail vein. Rats were divided into control and diabetic groups. Contractile responses of bladder strips from each group were obtained for postassium chloride, adenosine triphosphate, and electrical field stimulation (0.5-32 Hz) in organ bath. Electrical field stimulation responses of strips were evaluated in the presence of PPADS (nonselective P2 antagonist), atropine (cholinergic antagonist), 5 MU (α-1a-adrenergic antagonist), BMY-7378 (α-1d-adrenergic antagonist), and finally CED (α-1b-adrenergic antagonist). mRNA expression of α1-adrenergic subtypes was determined for each group. The difference between contractile responses related to electrical field stimulation with incubation with PPADS, atropine, 5 MU, BMY-7378, and CED, respectively, was not significant in the control and diabetic groups (P > .05). The electrical field stimulation responses of strips at 0.5-2 Hz without incubation were significantly different between the control and diabetic groups (P < .05). The contractile responses of strips with PPADS + atropine + 5 MU and BMY-7378 incubations in the diabetic group were significantly lower than in the control group in all doses (P < .05), The mRNA expression of α-1a-adrenergic in the diabetic group was significantly lower than in the control group (P < .05). No change was found in the expression of mRNA of α-1b-adrenergic. These results support the probability of changes in presynaptic and autonomic receptor sensitivity. We believe that α-1a-adrenergic and α-1d-adrenergic subtypes should be kept in mind in the treatment of diabetic cystopathy.

Allergen and Ozone Exacerbate Serotonin-Induced Increases in Airway Smooth Muscle Contraction in a Model of Childhood Asthma

Background: Serotonin (5-HT) modulates cholinergic neurotransmission and exacerbates airway smooth muscle (ASM) contraction in normal animal and nonasthmatic human tissue. Exposure to house dust mite allergen (HDMA) and ozone (O₃) leads to airway hyperreactivity and 5-HT-positive cells in the airway epithelium of infant rhesus monkeys. Research shows that concomitant exposure in allergic animals has an additive effect on airway hyperreactivity. Objectives: In this study, the hypothesis is that the exposure of allergic infant rhesus monkeys to HDMA, O₃ and in combination, acting through 5-HT receptors, enhances 5-HT modulation of postganglionic cholinergic ASM contraction. Methods: Twenty-four HDMA-sensitized infant monkeys were split into 4 groups at the age of 1 month, and were exposed to filtered air (FA), HDMA, O₃ or in combination (HDMA+O₃). At the age of 6 months, airway rings were harvested and postganglionic, and parasympathetic-mediated ASM contraction was evaluated using electrical-field stimulation (EFS). Results: 5-HT exacerbated the EFS response within all exposure groups, but had no effect in the FA group. 5-HT₂, 5-HT₃ and 5-HT₄ receptor agonists exacerbated the response. 5-HT concentration-response curves performed after incubation with specific receptor antagonists confirmed the involvement of 5-HT₂, 5-HT₃ and 5-HT₄ receptors. Conversely, a 5-HT₁ receptor agonist attenuated the tension across all groups during EFS, and in ASM contracted via exogenous acetylcholine. Conclusions: HDMA, O₃ and HDMA+O₃ exposure in a model of childhood allergic asthma enhances 5-HT exacerbation of EFS-induced ASM contraction through 5-HT₂, 5-HT₃ and 5-HT₄ receptors. A nonneurogenic inhibitory pathway exists, unaffected by exposure, mediated by 5-HT₁ receptors located on ASM.

Purinergic neuromuscular transmission is absent in the colon of P2Y1 knocked out mice

Purinergic and nitrergic co-transmission is the dominant mechanism responsible for neural-mediated smooth muscle relaxation in the gastrointestinal tract. The aim of the present paper was to test whether or not P2Y(1) receptors are involved in purinergic neurotransmission using P2Y(1)(−/−) knock-out mice. Tension and microelectrode recordings were performed on colonic strips. In wild type (WT) animals, electrical field stimulation (EFS) caused an inhibitory junction potential (IJP) that consisted of a fast IJP (MRS2500 sensitive, 1 μm) followed by a sustained IJP (N(ω)-nitro-L-arginine (L-NNA) sensitive, 1 mm). The fast component of the IJP was absent in P2Y(1)(−/−) mice whereas the sustained IJP (L-NNA sensitive) was recorded. In WT animals, EFS-induced inhibition of spontaneous motility was blocked by the consecutive addition of L-NNA and MRS2500. In P2Y(1)(−/−) mice, EFS responses were completely blocked by L-NNA. In WT and P2Y(1)(−/−) animals, L-NNA induced a smooth muscle depolarization but ‘spontaneous' IJP (MRS2500 sensitive) could be recorded in WT but not in P2Y(1)(−/−) animals. Finally, in WT animals, 1 μm MRS2365 caused a smooth muscle hyperpolarization that was blocked by 1 μm MRS2500. In contrast, 1 μm MRS2365 did not modify smooth muscle resting membrane potential in P2Y(1)(−/−) mice. β-Nicotinamide adenine dinucleotide (β-NAD, 1 mm) partially mimicked the effect of MRS2365. We conclude that P2Y(1) receptors mediate purinergic neurotransmission in the gastrointestinal tract and β-NAD partially fulfils the criteria to participate in rodent purinergic neurotransmission. The P2Y(1)(−/−) mouse is a useful animal model to study the selective loss of purinergic neurotransmission.