EFS-induced Contractions Research Articles

Effect of peripheral obestatin on food intake and gastric emptying in ghrelin‐knockout mice

The finding that obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's stimulatory effect on food intake and gastric emptying has been questioned. The effect of obestatin has been mostly investigated in fasted rodents, a condition associated with high blood levels of ghrelin which may mask the effect of obestatin. We therefore investigated the effect of obestatin on food intake, gastric emptying and gastric contractility in ghrelin knockout mice. The effect of obestatin on 6-h cumulative food intake was studied in fasted wildtype (ghrelin+/+) and ghrelin knockout (ghrelin-/-) mice. In both genotypes, the effect of obestatin and/or ghrelin was studied in vivo on gastric emptying measured with the (14)C-octanoic acid breath test and in vitro on neural responses elicited by electrical field stimulation (EFS) of fundic smooth muscle strips. Administration of obestatin did not influence fasting-induced hyperphagia or gastric emptying in both genotypes. Injection of ghrelin accelerated gastric emptying in ghrelin+/+ and ghrelin-/- mice but the effect was not reversed by co-injection with obestatin. In fundic strips from ghrelin+/+ and ghrelin-/- mice, ghrelin increased EFS-induced contractions, but obestatin was without effect. However, co-administration with obestatin tended to reduce the excitatory effect of ghrelin in both genotypes. In ghrelin-/- mice, obestatin failed to affect food intake and gastric motility. These results suggest that endogenous ghrelin does not mask the effect of obestatin and confirm that obestatin administered peripherally is not a major regulator of satiety signalling or gut motility.

Effects of Ischemia on Tachykinin-Containing Nerves and Neurokinin Receptors in the Rabbit Bladder

Our previous studies showed marked changes in efferent nerve structure and reactivity in the ischemic bladder. The goal of this study was to examine the effects of bladder ischemia on tachykinin (TK) containing sensory nerves and neurokinin receptors (NKR) in a rabbit model. We recorded bladder blood flow and spontaneous contractions in treated animals at week 8 after the induction of iliac arteries atherosclerosis and in age-matched controls. Bladder tissues were processed for studies of isometric smooth muscle tension in the organ bath, NK2R gene expression using quantitative real-time polymerase chain reaction (PCR), immunohistochemical staining of TK containing nerves and epithelial TK expression, and transmission electron microscopy. Atherosclerosis-induced ischemia significantly increased the frequency of spontaneous bladder contractions in vivo. Electrical field stimulation (EFS)-induced smooth muscle contractions were significantly greater in the ischemic tissues. Inhibition of NK1R diminished contractions to low-frequency EFS in control tissues while having no significant effect on the ischemic tissues. In contrast, NK2R inhibition significantly decreased contractions to both low- and high-frequency EFS in the ischemic tissues. Inhibition of NK3R had no significant effect on EFS-induced contractions. Real-time PCR showed a significant increase in NK2R gene expression in the ischemic bladder. The number of TK immunopositive nerves and epithelial TK immunoreactivity were significantly greater in the ischemic bladder. These alterations were associated with marked ultrastructural reactions to bladder ischemia. Alterations of NK2R reactivity and gene expression, increased number of TK immunopositive nerves, and greater epithelial TK immunoreactivity may imply activated bladder afferents to signal ischemic insult.

Total urinary bladder regeneration with restoration of native structure and pharmacological response

Neo‐bladder histology and pharmacological response were investigated in 7 dogs at 51 (n=1), 89 (n=3), and 179 (n=3) days (d) after radical cystectomy and implantation of a Neo‐Bladder Replacement Construct (Construct). Pharmacological responses of neo‐bladder tissue strips (51, 89, and 179 d; 3, 18, and 27 strips, respectively) were compared to age‐matched native bladder (n=13; 49 strips).By 51 d post‐implantation (p.i.) regenerated bladder was histologically consistent with native bladder including mucosal and serosal linings, detrusor muscle, vascular, and nerve composition. Logistic analysis revealed similar EC50 and slope factor values for contraction of bladder tissue strips derived from all groups induced by carbachol (Car) and electrical field stimulation (EFS). A statistically significant and progressive increase in the mean Emax values occurred in response to both Car and EFS over time. While contractile responses to Car and EFS increased over time, they were lower than native bladder responses at 51 and 89 d p.i. Car‐induced contractions, but not EFS‐induced contractions, were equivalent to native tissue at 179 d p.i.A Neo‐Bladder Replacement Construct is capable of regenerating urinary bladders in vivo with histological and pharmacological responses similar to native bladder by 179 d p.i. There was no evidence of abnormal cell growth, immune response, or adverse systemic effects.

Detrusor muscle contractility and compliance are impacted by diet in Ossabaw miniature pigs with metabolic syndrome (MetS)

The etiology of diabetic bladder dysfunction is not understood. Previous work suggests that MetS Ossabaw pigs have detrusor muscle that is stiffer than bladder muscle from lean pigs. The purpose of the current study was to determine if the loss of bladder muscle compliance is exacerbated in pigs manifesting robust MetS. Detrusor muscle strips from Ossabaw pigs maintained on either an excess kcal atherogenic diet or a control diet were mounted initially at slack length in muscle baths. Following equilibration, muscle strips were stimulated with supramaximal electrical field stimulation (EFS: 20 V, 60 Hz, AC, 10 sec). Following relaxation, strips were stretched in 0.5–1.5 mm increments. The cycles of incremental stretches followed by EFS‐induced contractions were repeated every 5 minutes until maximum total tension was achieved or the tissue yielded. Normalized passive and active length‐tension (L‐T) curves were generated. Due to small sample sizes (n=3/group), no statistical analyses are reported. However, both mean passive and active L‐T curves for bladder muscle from MetS pigs are elevated above the respective control curves indicating that MetS results in increased contractility in addition to decreased compliance of the bladder muscle. Stiffer bladders that are hypercontractile could explain both the incomplete voiding and increased urinary frequency experienced in diabetes which is characteristic of metabolic syndrome.(R Sharma is an APS Undergraduate Summer Research Fellow; B Harden was an Indy Project Seed Scholar; Support: NIH RR013223, HL062552)

Inhibitory effect of the herbal antidepressant St. John’s wort (Hypericum perforatum) on rat gastric motility

St. John's wort (Hypericum perforatum) is a highly popular and effective herbal antidepressant that clinically interacts with a number of conventional drugs. Because alterations in gastric emptying can cause pharmacokinetic interactions, in the present study we evaluated the effect of a standardized extract prepared from the flowering tops of Hypericum perforatum (SJW extract) on rat gastric motility. Orally administered SJW extract delayed gastric emptying in vivo. In vitro studies showed that SJW extract was significantly more active in inhibiting acetylcholine (or prostaglandin E2)-induced contractions than electrical field stimulation (EFS)-induced contractions. The effect of SJW extract on EFS-induced contractions was unaffected by drugs that inhibit intrinsic inhibitory nerves or by tachykinin antagonists, but it was reduced by the 5-hydroxytryptamine antagonist methysergide. The inhibitory effect of SJW extract on acetylcholine-induced contractions was reduced by the sarcoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid, but not by the L-type Ca2+ channel blocker nifedipine or by methysergide. Among the chemical constituents of SJW extract tested, hyperforin and, to a lesser extent, the flavonoids kaempferol and quercitrin, inhibited acetylcholine-induced contractions. It is concluded that SJW has a direct inhibitory effect on smooth muscle and could also possibly modulate gastric neurotransmission. If extended to humans, the inhibitory effect of SJW extract on gastric emptying in vivo could contribute, at least in part, to the clinical pharmacokinetic interactions between conventional medicines and this herbal antidepressant.

Change in acetylcholine release from rat bladder with partial outlet obstruction

To investigate changes in acetylcholine release from the bladder of rats with partial bladder outlet obstruction (BOO), as partial BOO leads to hypertrophy and an alteration in the contractions of the detrusor smooth muscle, and acetylcholine plays an important role in urinary bladder contractions but there is little available information on acetylcholine release after BOO. Partial BOO was induced in adult female rats by ligating the proximal urethra over a 1 mm angiocatheter; sham-operated rats served as controls. The rats were killed 2 weeks, 3 and 6 months after induction of BOO. We investigated the contractions induced by carbachol, KCl (80 mm), ATP and electrical-field stimulation (EFS, 2.5-40 Hz), and collected the dialysate obtained from a microdialysis probe inserted into the muscle strips during EFS, and measured the amount of acetylcholine in the dialysate fraction by high-performance liquid chromatography with electro-chemical detection. S-100 immunohistochemical staining of the bladder preparations was used for histological examination in BOO and control rats. The bladder weight gradually increased after BOO. There were no significant changes in KCl-induced contractions throughout the experimental period in either group. There were no significant changes in carbachol-induced contractions until 3 months after BOO but there was a significant reduction at 6 months. ATP-induced contractions were significantly increased 2 weeks and 3 months after BOO. EFS-induced contractions were gradually reduced after BOO. Acetylcholine release from the bladder strips was not significantly different between the groups until 2 weeks after BOO. However, acetylcholine release in BOO rats was significantly decreased 3-6 months after BOO, being significantly lower than that of the control rats. In the histological study, the number of nerve fibres in the BOO rats was significantly lower than in the control rats. We suggest that the prolonged BOO caused a decrease in EFS-induced acetylcholine release and the number of nerves in the rat urinary bladder, which might contribute to bladder underactivity in BOO.

Effects of ( R,R)- and ( R,R/S,S)-Formoterol on Airway Relaxation and Contraction in an Experimental Rat Model

Background: Racemic ( R,R/S,S)-formoterol is a long-acting β-agonist composed of a 50:50 mixture of ( R,R)- and ( S,S)-enantiomers. Objective: The aim of this study was to determine whether ( R,R)-formoterol and ( R,R/S,S)-formoterol have differing effects on airway contraction and relaxation in vitro. Methods: Cylindrical airway segments 3-mm long were isolated from the mid-trachea of healthy Sprague-Dawley rats and placed in a modified Krebs-Henseleit solution. Dose-response curves of bethanechol-induced contraction (measured as milligrams of tension) and the concentration of bethanechol that elicited 50% to 75% of maximal contraction (EC 50–75) were determined. The air-way cylinders were then precontracted with bethanechol at the EC 50–75 and exposed to different concentrations of ( R,R)-formoterol (0.0001–1.0 μM) or ( R,R/S,S)-formoterol (0.0002–2.0 μM). Each concentration of the 2 formoterol formulations contained the same amount of ( R,R)-enantiomer (eg, [ R,R]-formoterol 0.0001 μM and [ R,R/S,S]-formoterol 0.0002 1JM contained the same amount of [ R,R]-enantiomer). The relaxation percentage in response to formoterol was calculated as a reduction in tension (in milligrams) in relation to baseline tension in the precontracted state, with each tracheal cylinder serving as its own control. To determine the effect of ( R,R)-formoterol on airway contraction, tracheal cylinders were incubated with ( R,R)- or ( R,R/S,S)-formoterol before electrical field stimulation (EFS). Results: Tracheae from 56 three-week-old Sprague-Dawley rats were used in the study. The relaxation percentage of precontracted trachea was significantly greater after exposure to ( R,R)-formoterol than to ( R,R/S,S)-formoterol at a 2-fold higher concentration ( P = 0.03; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in a post hoc analysis, the mean (SE) relaxation percentage of precontracted trachea was significantly greater only after exposure to ( R,R)-formoterol 0.01 μM than to ( R,R/S,S)-formoterol 0.02 μM (15.6% [5.8%] vs 39.0% [5.6%]; P < 0.05, unpaired t test). EFS-induced airway contraction was significantly less in tracheal cylinders incubated in ( R,R)-formoterol compared with those incubated in ( R,R/S,S)-formoterol at a 2-fold higher concentration ( P = 0.05; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in the post hoc analysis, mean (SE) EFS-induced tracheal contraction was significantly less only in ( R,R)-formoterol 0.01 μM compared with ( R,R/S,S)-formoterol 0.02 μM at 10 V (1070 [55] mgvs 1225 [28] mg; P < 0.05, unpaired t test). Conclusion: We found that ( R,R)-formoterol may induce greater relaxation of precontracted airway smooth muscle cells than ( R,R/S,S)-formoterol and that ( R,R)-formoterol may have a greater inhibitory effect on the endogenous cholinergic and excitatory nonadrenergic, noncholinergic contractile airway responses than ( R,R/S,S)-formoterol. We speculate that the presence of the ( S,S)-enantiomer in ( R,R/S,S)-formoterol may impair airway relaxation of pre-contracted trachea in rats.

Succinylcholine potentiates acetylcholine-induced contractile and phosphatidylinositol responses of rat trachea

Although succinylcholine (SCh) is often used as a muscle relaxant in electroconvulsive therapy, its influence on airway reactivity has not been fully investigated. We examined the effects of SCh on acetylcholine (ACh)-, carbachol (CCh)-, and electrical field stimulation (EFS)-induced contractions, and on the ACh-induced phosphatidylinositol (PI) response of rat trachea. Thirty-two male Wistar rats weighing 250-350 g were used. The trachea was rapidly isolated and cut into 3-mm-wide rings. The resting tension was adjusted periodically to 1.0 g during the equilibration period. ACh, 1 microM; carbachol (CCh), 0.05 microM; or neither of them, was added, and SCh was then added at 1-300 microM final concentrations, and ring tension was examined. Contractions were elicited by EFS in the presence or absence of 100 microM SCh. Tracheal slices were incubated with [3H] myo-inositol, 1 microM ACh, and various concentrations of SCh. The accumulation of [3H] inositol monophosphate (IP1) was measured. SCh did not affect the tension by itself without ACh, or with CCh, but SCh potentiated the ACh-induced contraction of rat trachea at concentrations of 10 microM or more (50% effective concentration [EC50]; 43.6 microM). SCh produced a significant increase in the amplitude and duration of EFS-induced contractions. SCh, at concentrations of 10 microM and 100 microM, potentiated ACh-induced IP1 accumulation. SCh potentiated ACh-induced, but not CCh-induced, contractile and PI responses, and enhanced EFS-induced contraction of rat trachea, suggesting that competition for butyrylcholinesterase (BChE) in airway smooth muscle could be involved in the potentiation by SCh of ACh-induced airway smooth muscle contraction.

Effects of Varicocele on Electrical Field Stimulation-Induced Biphasic Twitch Responses in the Ipsilateral and Contralateral Rat Vasa Deferentia

Aim: Although little is known about the mechanisms, varicocele is considered as one of the factors leading to male infertility. Since reduced motility of the vas deferens was shown to contribute to male infertility, in this study we aimed to investigate the effect of varicocele on electrical field stimulation (EFS)-induced biphasic contractions of the vas deferens in order to evaluate the effect of varicocele on the motility of the vas deferens. Material and Methods: A total of 26 Sprague-Dawley rats (200–250 g) were assigned randomly into two groups: sham (n = 10) and varicocele (n = 16). Varicocele was produced by partial obstruction of the left renal vein. Four weeks after the surgical procedure, vasa deferentia were harvested and EFS-induced responses were recorded from the strips prepared from ipsilateral and contralateral sides via Grass isometric force displacement transducers. Exogenous α-β methyl ATP was applied at the concentration of 10^–5M to the vasa deferentia strips, and exogenous noradrenalin was applied cumulatively at the concentrations between 10^–7 and 10^–4M. At the end of each experiment, 80 mM KCl was applied to induce contractions. All contractions were expressed as the percentage of the 80 mM KCl-induced contractions. Results: Varicocele significantly inhibited both phases of EFS-induced biphasic contractions in the ipsilateral side, whereas in the contralateral site it did not produce any change. However, there was no change in exogenously applied α-β methyl ATP, noradrenalin and KCl-evoked contractions of the vasa deferentia obtained from both sides. Conclusions: These results suggest that varicocele affects the ipsilateral vas deferens motility by reducing neurotransmitter release.

Effect of indacaterol, a novel long-acting 2-agonist, on isolated human bronchi

Indacaterol is a novel beta2-adrenoceptor agonist in development for the once-daily treatment of asthma and chronic obstructive pulmonary disease. The present study evaluated the relaxant effect of indacaterol on isolated human bronchi obtained from lungs of patients undergoing surgery for lung carcinoma. Potency (-logEC50), maximal relaxant effect (Emax) and onset of action were determined at resting tone. Duration of action was determined against cholinergic neural contraction induced by electrical field stimulation (EFS). At resting tone, -logEC50 and Emax values were 8.82+/-0.41 and 77+/-5% for indacaterol, 9.84+/-0.22 and 94+/-1% for formoterol, 8.36+/-0.16 and 74+/-4% for salmeterol, and 8.43+/-0.22 and 84+/-4% for salbutamol, respectively. In contrast to salmeterol, indacaterol did not antagonise the isoprenaline response. Indacaterol's onset of action (7.8+/-0.7 min) was not significantly different from that of formoterol (5.8+/-0.7 min) or salbutamol (11.0+/-4.0 min), but it was significantly faster than that of salmeterol (19.4+/-4.3 min). EFS-induced contractions were inhibited with -logIC50 values of 6.96+/-0.13 (indacaterol), 8.96+/-0.18 (formoterol), 7.18+/-0.34 (salmeterol) and 6.39+/-0.26 (salbutamol). Duration of action was >12 h for indacaterol and salmeterol, and 35.3+/-8.8 and 14.6+/-3.7 min for formoterol and salbutamol, respectively. In isolated human bronchi, indacaterol behaved as a long-acting beta2-adrenoceptor agonist with high intrinsic efficacy and fast onset of action.

Functional roles of muscarinic M 2 and M 3 receptors in mouse stomach motility: Studies with muscarinic receptor knockout mice

Functional roles of muscarinic acetylcholine receptors in the regulation of mouse stomach motility were examined using mice genetically lacking muscarinic M 2 receptor and/or M 3 receptor and their corresponding wild-type (WT) mice. Single application of carbachol (1 nM–30 μM) produced concentration-dependent contraction in antral and fundus strips from muscarinic M 2 receptor knockout (M 2R-KO) and M 3 receptor knockout (M 3R-KO) mice but not in those from M 2 and M 3 receptors double knockout (M 2/M 3R-KO) mice. A comparison of the concentration–response curves with those for WT mice showed a significant decrease in the negative logarithm of EC 50 (pEC 50) value (M 2R-KO) or amplitude of maximum contraction (M 3R-KO) in the muscarinic receptor-deficient mice. The tonic phase of carbachol-induced contraction was decreased in gastric strips from M 3R-KO mice. Antagonistic affinity for 4-diphenylacetoxy- N-methyl-piperidine (4-DAMP) or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) indicated that the contractile responses in M 2R-KO and M 3R-KO mice were mediated by muscarinic M 3 and M 2 receptors, respectively. Electrical field stimulation (EFS, 0.5–32 Hz) elicited frequency-dependent contraction in physostigmine- and N ω-nitro- l-arginine methylester ( l-NAME)-treated fundic and antral strips from M 2R-KO and M 3R-KO mice, but the cholinergic contractile components decreased significantly compared with those in WT mice. In gastric strips from M 2/M 3R-KO mice, cholinergic contractions elicited by EFS were not observed but atropine-resistant contractions were more conspicuous than those in gastric strips from WT mice. Gastric emptying in WT mice and that in M 2/M 3R-KO mice were comparable, suggesting that motor function of the stomach in the KO mice did not differ from that in the WT mice. The results indicate that both muscarinic M 2 and M 3 receptors but not other subtypes mediate carbachol- or EFS-induced contraction in the mouse stomach but that the contribution of each receptor to concentration–response relationships is distinguishable. Although there was impairment of nerve-mediated cholinergic responses in the stomach of KO mice, gastric emptying in KO mice was the same as that in WT mice probably due to the compensatory enhancement of the non-cholinergic contraction pathway.

Effect of Cooling on Electrical Field Stimulation and Norepinephrine-Induced Contraction in Isolated Hypertrophic Human Nasal Mucosa

Exposure to cold causes cutaneous vasoconstriction to reduce body heat loss, while the nasal cavity warms up the inspired cold air. This suggests cooling might evoke a different response in nasal mucosa blood vessels than in cutaneous blood vessels. Thus, the aim of this study was to evaluate the effect of cooling (to 24 degrees C) on the vascular response of isolated nasal mucosa. An in vitro isometric contraction of nasal mucosa excised from patients suffered from chronic nasal congestion was continuously recorded. Either electrical field stimulation (EFS) or exogenous norepinephrine (NE) was applied to the turbinate mucosa strip at 37 and 24 degrees C, and the influence of cooling (to 24 degrees C) was evaluated. EFS (1, 2, 4, and 8 Hz) produced frequency-dependent contractions at 37 and 24 degrees C. Cooling did not alter the magnitude but significantly prolonged the duration of EFS-induced contraction. Exogenous NE (10(-8) to 10(-4) M) produced concentration-dependent contractions at 37 and 24 degrees C. Cooling significantly enhanced the contractile responses evoked by NE between 3 x 10(-6) and 1 x 10(-4) M. Cooling (to 24 degrees C) prolonged the EFS-induced and enhanced NE (3 x 10(-6) to 1 x 10(-4) M)-induced contraction of isolated human nasal mucosa.

Effects of Mexiletine on Electrical Field Stimulation-Induced Contractile Responses in the Ipsilateral and Contralateral Vasa Deferentia after Unilateral Testicular Torsion/Detorsion

Aim: To investigate testicular torsion-induced changes on the electrical field stimulation (EFS)-induced contractions in rabbit vasa deferentia and to evaluate the effect of mexiletine. Methods: 18 male New Zealand albino rabbits were used in this experiment. Rabbits were divided into three groups: (1) control group (n = 6); (2) torsion group (n = 6), and (3) mexiletine group (n = 6). In the control group, vasa deferentia on both sides were harvested. In the torsion and mexiletine groups, the left testes of the rabbits were subjected to 720° of clockwise torsion for 2 h and then detorsion was performed. In the mexiletine group, 50 mg/kg i.p. mexiletine was administered 1 h before detorsion. Following 24 h of the torsion, vasa deferentia on both sides were harvested and 2-cm strips including both the prostatic and epididymal portions were prepared to record EFS-induced contractions. Results: Testicular torsion caused a significant inhibition in both phases of EFS-induced biphasic contractions of the ipsi- and contralateral vasa deferentia. Mexiletine treatment did not affect these inhibitory responses. Torsion/detorsion of the spermatic cord did not alter exogenously applied noradrenaline-induced contractions in both vasa deferentia. However, KCl-induced contractions diminished significantly in ipsilateral vas deferens of the torsion group and mexiletine restored this inhibition. Conclusions: Unilateral testicular torsion/detorsion leads to inhibition in both phases of EFS-induced biphasic contractions of the ipsi- and contralateral vasa deferentia by causing a defect in presynaptic nerve transmission. However, mexiletine has no effect on this inhibition. Inhibition of the KCl-induced contractions in the ipsilateral vas deferens, which indicates postsynaptic tissue damage, is restored by administering mexiletine 1 h prior to detorsion.

Acid-Induced Release of Platelet-Activating Factor by Human Esophageal Mucosa Induces Inflammatory Mediators in Circular Smooth Muscle

In a human in vitro model of esophagitis, we investigated the genesis of esophagitis-associated dysmotility by examining HCl-induced production of inflammatory mediators in the mucosa and investigating their effect on esophageal circular muscle. Muscularis propria was removed from organ donors' esophagi, leaving the mucosal tube intact. The tube was tied at both ends, forming a sac, and filled with HCl at pH 4. After 3 h of incubation, the supernatant surrounding the sac was analyzed or applied to circular muscle strips. HCl alone did not affect circular muscle contraction in response to electrical field stimulation (EFS), but supernatant of HCl-treated mucosa abolished contraction. The inhibition was reversed by the platelet-activating factor (PAF) antagonist CV3988 [(+/-)-3-(N-octadecylcarbamoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate], whereas the PAF analog 2-O-methyl platelet-activating factor C-16 (PAF-16) inhibited EFS-induced contraction and acetylcholine (ACh) release in circular muscle strips. The hydrogen peroxide scavenger catalase reversed the inhibition in contraction, to the same extent as CV3988. We therefore measured PAF and hydrogen peroxide (H(2)O(2)) in mucosa, mucosa supernatant, and circular muscle. HCl increased PAF and interleukin (IL)-1beta (but not IL-6, prostaglandin E(2), or H(2)O(2)) in mucosa, and only PAF was released into the supernatant, presumably to affect circular muscle. In circular muscle, exogenous PAF induced sequential formation of IL-6, H(2)O(2), IL-1beta, and PAF. Release of PAF by the mucosa inhibits ACh release from circular muscle layer neurons and initiates sequential formation of inflammatory mediators in muscle, resulting in production of PAF by the muscle itself, possibly initiating in a self-sustaining cycle.

The prokinetic-like activity of ghrelin in rat isolated stomach is mediated via cholinergic and tachykininergic motor neurones

Ghrelin increases electrically evoked, neuronally mediated contractions of rat isolated forestomach, a prokinetic-like activity. Since the nerve type sensitive to ghrelin is unclear, we examined the activity of ghrelin in the presence of antagonists at receptors for the main gastric motor neurotransmitters. Electrical field stimulation (EFS; 5 Hz, 0.5 ms, ± 50 V, 30 s every 3 min) of circular muscle preparations evoked tetrodotoxin 1 μM-sensitive responses, consisting of a small initial contraction followed by a further contraction or more usually, by muscle relaxation. Termination of EFS evoked a large rapidly developing after-contraction. Atropine 1μM prevented contractions during EFS, increased any relaxations and prolonged the after-contractions. N ω-Nitro- l-arginine-methyl-ester-hydrochloride (L-NAME) 0.3mM prevented relaxations during EFS, changing the triphasic response into a monophasic contraction. The tachykinin NK 1 and tachykinin NK 2 receptor antagonists N-acetyl- l-tryptophan-3,5-bistrifluoromethyl-benzyl-ester (L-732,138 1 μM) and Cyclo[Gln–Trp–Phe–Gly–Leu–CH 2N(CH 3)–Leu] (MDL-29,913 1 μM) each reduced EFS-evoked relaxations; the latter also reduced the after-contractions. The tachykinin NK 3 receptor antagonist (−)-( S)- N-(α-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB-235375, 0.1 μM) had no effects. The combination of tachykinin NK 1,2,3 receptor antagonists reduced the after-contractions and abolished relaxations during EFS, replacing this with a contraction. In control tissues, ghrelin 1 μM increased EFS-induced contractions and tended to reduce any relaxations. In the presence of atropine 1 μM, L-NAME 0.3 mM or the tachykinin receptor antagonists (as above), ghrelin 1 μM increased any EFS-induced contraction but in the presence of atropine had no effects on EFS-evoked relaxations. We conclude that EFS evokes responses mediated by acetylcholine, nitric oxide and tachykinins. Ghrelin facilitates both cholinergic and tachykininergic excitatory pathways, consistent with activity within the enteric nervous system and possibly the vagus nerve.

Perivascular adipose tissue promotes vasoconstriction: the role of superoxide anion

Recent studies have demonstrated that perivascular adipose tissue (PVAT) releases vascular relaxation factor(s). In this study, we examined if PVAT releases other vasoactive factors in response to perivascular nerve activation by electrical field stimulation (EFS). In Wistar-Kyoto rats, rings of superior mesenteric artery (MA) with intact PVAT (PVAT (+)) showed a greater contractile response to EFS than rings with PVAT removed (PVAT (-)). Superoxide dismutase (SOD) reduced the contractile response to EFS more in PVAT (+) MA than in PVAT (-) MA. Inhibitors of NAD(P)H oxidase and cyclooxygenase exerted a greater inhibition on EFS-induced contraction in PVAT (+) MA than in PVAT (-) MA. Inhibitors of tyrosine kinase (tyrphostin A25) and MAPK/ERK (U 0126) attenuated EFS-induced contraction in PVAT (+) MA in a concentration-related manner, while inactive forms of these inhibitors (tyrphostin A1 and U 0124) did not inhibit the response. Exogenous superoxide augmented the contractile response to EFS and to phenylephrine in PVAT (-) MA, and this augmentation was blunted by inhibition of tyrosine kinase and MAPK/ERK. EFS increased superoxide generation in isolated PVAT and PVAT (+)/(-) MA, which was attenuated by NAD(P)H oxidase inhibition. RT-PCR showed the mRNA expression of p(67phox) subunit of NAD(P)H oxidase and immunohistochemical staining confirmed its localization in the adipocytes of PVAT. These results show that PVAT enhances the arterial contractile response to perivascular nerve stimulation through the production of superoxide mediated by NAD(P)H oxidase, and that this enhancement involves activation of tyrosine kinase and MAPK/ERK pathway.

Effect of hypothyroidism on the nitrergic relaxant responses of corpus cavernosal smooth muscle in rabbits

The incidence of hormonal dysfunction as a cause of impotence remains controversial. However, several recent studies have reported evidence of hormonal abnormalities in 25-35% of impotent men. Hypothyroidism has been reported to occur in 6% of impotent men. In the present study, we examined nitrergic responses in hypothyroidism in rabbit corpus cavernosum and compared them with controls. Carbachol-induced relaxation responses and electrical field stimulation (EFS)-induced frequency-dependent relaxations decreased significantly in hypothyroid rabbits. Papaverine and sodium nitroprusside (SNP)-induced relaxation responses did not change significantly in hypothyroid rabbits. The contraction responses of phenylephrine and EFS-induced frequency-dependent contractions were significantly decreased in the hypothyroid group. We can speculate that the reduction of relaxant responses to EFS and carbachol in hypothyroid rabbits can depend on a decreased release of nitric oxide (NO) from nitrergic nerves and endothelium or a reduction of muscarinic receptor density. Also, decreases in contraction responses may depend on diminished adrenoceptor density.

Analysis of Vasoreactivity of Isolated Human Radial Artery

Radial artery (RA) is increasingly used as graft for coronary artery bypass grafting due to its good long-term patency. However, the mechanism of peri- and post-operative spasm is still unclear. Because of that, the aim of our study is to analyze the contractility of RA and to determine whether the presence of functional endothelium alters its contractile properties. Contractions of isolated RA rings were provoked by exogenously applied vasoconstrictors or by electrical field stimulation (EFS, 20 Hz). The order of vasoconstrictors potency based on their EC50 values was as follows: angiotensin II > phenylephrine > 5-hydroxytriptamine. Presence of endothelium increased both EC50 and maximal contraction to phenylephrine and angiotensin II, but inhibited reactivity of RA to 5-hydroxytriptamine. Spontaneous rhythmic contractions (SRC, <4 mHz) and EFS-induced contractions of RA are endothelium-independent and weaker than contractions induced by exogenously applied vasoconstrictors. Our study concludes that RA shows marked sensitivity and reactivity to angiotensin II, phenylephrine, and 5-hydroxytriptamine. Further investigations are necessary to answer why angiotensin II and phenylepehrine induce stronger contractions in the presence of endothelium. In addition, SRC as well as contractions of neurogenic origin may take part in developing vascular spasm of RA.

The Role of Nitric Oxide in the Electrical Field Stimulation-Induced Contractions of Sphincter of Oddi and Gallbladder Strips in Guinea Pigs

The aim of this study was to investigate the modulatory role of nitric oxide (NO) in the electrical field stimulation (EFS)-induced contractions of isolated sphincter of Oddi (SO) and gallbladder strips from guinea pigs. EFS was used to activate the intrinsic nerves in SO and gallbladder strips. EFS produced frequency-dependent biphasic contractile responses in the SO strips. A smaller contraction, "on response", occurred during EFS, which was followed by a bigger contraction, "off response". Both responses were completely and irreversibly abolished by tetrodotoxin (TTX) (10(-6) M). Atropine (10(-6) M) inhibited the "on response", but not the "off response". EFS produced frequency-dependent monophasic contractile responses in gallbladder strips, which were completely and irreversibly abolished by TTX (10(-6) M) and atropine (10(-6) M). A nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (10(-4) M and 3 x 10(-4) M, in SO and gallbladder strips, respectively), significantly increased all EFS-induced contractions of SO and gallbladder strips. L-Arginine, but not D-arginine reversed the effect induced by the NOS inhibitor, at all frequencies, in both strips. These results suggested that NO released from nitrergic nerve endings might play a regulatory role in the cholinergic neurotransmission of guinea pig SO and gallbladder strips. The "off response" in he SO preparations might be a rebound increase that was modulated by the nonadrenergic, noncholinergic inhibitory mediator NO.

Rho kinase expression and its central role in ovine gallbladder contractions elicited by a variety of excitatory stimuli

Rho kinase has contractile activity, which induces Ca 2+ sensitization in various cells. Several receptors are linked to the Rho/Rho-kinase pathway. Therefore, in this study we aimed to demonstrate the central importance of this novel pathway for diverse excitatory stimuli in the smooth muscle of the sheep gallbladder. Accordingly, the effects of a Rho kinase inhibitor, (+)-( R)- trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10 − 8 –3 × 10 − 5 M), were investigated on cholecystokinin-8 (CCK-8, 10 − 8 M), endothelin-1 (10 − 8 M), carbachol (10 − 6 –10 − 5 M), 5-hydroxytryptamine (5-HT, 10 − 6 –10 − 5 M), histamine (10 − 6 –10 − 5 M), phenylephrine (10 − 5 –10 − 4 M), neurokinin A (10 − 7 –10 − 6 M), electrical field stimulation (40 V, 0.5 ms, 2, 4, 8, 16, 32 Hz, 15 s, 3 min intervals) and potassium chloride (KCl, 25–50 mM)-induced contractions as well as spontaneous contractile activity. Electrical field stimulation evoked tetrodotoxin (3 × 10 − 6 M)-sensitive reproducible contractions, which were inhibited by atropine (2 × 10 − 6 M) and potentiated by eserine (5 × 10 − 7 M). EFS-induced contraction was significantly inhibited by Y-27632 (10 − 5 M). In addition, spontaneous contractile activity was suppressed in the presence of the compound (10 − 6 –10 − 5 M). This Rho kinase inhibitor also dramatically decreased the contractions elicited by 5-HT, neurokinin A and carbachol. KCl-induced contraction, which was not atropine-sensitive, was also conspicuously attenuated by Y-27632. Moreover, Y-27632 (10 − 8 –3 × 10 − 5 M) relaxed gallbladder strips that were contracted by histamine, endothelin-1, CCK-8 and phenylephrine in a concentration-dependent manner. pEC 50 values for Y-27632 were 6.25 ± 0.10, 5.79 ± 0.12, 5.83 ± 0.09 and 5.70 ± 0.13 for the contraction elicited by histamine, CCK-8, endothelin-1 and phenylephrine, respectively. Furthermore, we also demonstrated Rho kinase protein expression (ROCK-1 and ROCK-2) by Western blot analysis. In conclusion, ROCK is expressed in the smooth muscle of the ovine gallbladder, and it has a central role in the contractile activity induced by diverse excitatory stimuli.