Field Stimulation-induced Contractions Research Articles

The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic‐mediated contraction of mammalian urinary bladder smooth muscle

OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions. Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release). Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions. These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.

Propofol Attenuates Ovalbumin-Induced Smooth Muscle Contraction of the Sensitized Rat Trachea: Inhibition of Serotonergic and Cholinergic Signaling

Propofol is considered suitable for induction of anesthesia in patients with bronchial asthma. However, the mechanisms of its action on bronchi are not fully understood. We examined the effects of propofol on ovalbumin (OA)-induced contraction of OA-sensitized rat trachea. Male Wistar rats were sensitized by a single intraperitoneal injection of OA 10 microg mixed with aluminum hydroxide, 10 mg, as adjuvant. Fourteen days later, the experiment was performed using the tracheal rings. We observed the effects of ketanserin, a 5-HT2 receptor antagonist, and atropine on OA-induced contraction. Next, the effects of propofol on OA-, serotonin (5-HT)-, acetylcholine-, or electrical field stimulation-induced contractions were observed. OA-induced contraction was 90% attenuated by the combination of ketanserin and atropine. Propofol significantly attenuated OA-induced contraction in a dose-dependent manner. Propofol abolished 5-HT-induced contraction, attenuated acetylcholine-induced contraction, and also almost completely attenuated the enhancement by 5-HT of electrical field stimulation-induced contraction. These results suggest that the mechanism involved in the attenuation by propofol of OA-induced contraction is inhibition of the actions of 5-HT. Propofol should be a useful anesthetic in patients with immunoglobulin E-related asthma.

The Pharmacology of Two Novel Long-Acting Phosphodiesterase 3/4 Inhibitors, RPL554 [9,10-Dimethoxy-2(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one] and RPL565 [6,7-Dihydro-2-(2,6-diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido[6,1-a]isoquinolin-4-one

The pharmacology of two novel, trequinsin-like PDE3/4 inhibitors, RPL554 [9,10-dimethoxy-2(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido-[6,1-a]isoquinolin-4-one] and RPL565 [6,7-dihydro-2-(2,6-diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido[6,1-a]isoquinolin-4-one], has been investigated in a number of in vitro and in vivo assays. Electrical field stimulation-induced contraction of guinea pig superfused isolated tracheal preparations was significantly inhibited by RPL554 (10 microM) and RPL565 (10 microM) (percentage control; 93 +/- 1.2 and 84.4 +/- 2.7, respectively). Contractile responses were suppressed for up to 12 h after termination of superfusion with RPL554 demonstrating a long duration of action. RPL554 and RPL565 inhibited, in a concentration-dependent manner, lipopolysaccharide-induced tumor necrosis factor alpha release from human monocytes [IC50; 0.52 microM (0.38-0.69) and 0.25 microM (0.18-0.35), respectively] and proliferation of human mononuclear cells to phytohemagglutinin [IC50; 0.46 microM (0.24-0.9) and 2.90 microM (1.6-5.4), respectively]. The inhibitory effect of these drugs in vitro was translated into anti-inflammatory activity in vivo. RPL554 (10 mg/kg) and RPL565 (10 mg/kg) administered orally significantly inhibited eosinophil recruitment following antigen challenge in ovalbumin-sensitized guinea pigs. Likewise, inhalation of dry powder containing RPL554 by conscious guinea pigs (25% in micronized lactose) 1.5 h before antigen exposure significantly inhibited the recruitment of eosinophils to the airways. Exposure of conscious guinea pigs to inhalation of dry powder containing RPL554 (2.5%) and RPL565 (25%) in micronized lactose significantly inhibited histamine-induced plasma protein extravasation in the trachea and histamine-induced bronchoconstriction over a 5.5-h period. Thus, RPL554 and RPL565 are novel, long-acting PDE 3/4 inhibitors exhibiting a broad range of both bronchoprotective and anti-inflammatory activities.

Inhibitory effects of alendronate on cholinergic responses in rat lower esophageal sphincter

Alendronate is a potent inhibitor of osteoclast-mediated bone resorption, but its use results in serious esophageal damage. In order to clarify the latter, we examined the effects of alendronate on electrical field stimulation-induced responses in the rat lower esophageal sphincter. Electrical field stimulation induced atropine-sensitive contraction. Alendronate inhibited electrical field stimulation-induced contraction in a concentration-dependent manner. In the presence of N G-nitro- l-arginine ( l-nitroarginine), electrical field stimulation elicited a strong cholinergic contraction. This contraction was also inhibited by alendronate, to a similar extent as that seen in the absence of l-nitroarginine. In lower esophageal sphincter contracted by prostaglandin F 2α and treated with atropine, electrical field stimulation induced l-nitroarginine-sensitive relaxation. Alendronate did not affect relaxation. These results suggest that alendronate decreases the tone of lower esophageal sphincter by inhibiting cholinergic nervous activity.

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.

Relaxant Effects of the Essential Oil ofEucalyptus tereticornisand its Main Constituent 1,8-Cineole on Guinea-Pig Tracheal Smooth Muscle

The effects of the essential oil of Eucalyptus tereticornis Sm. (EOET) on guinea-pig tracheal smooth muscle were investigated. EOET (10 - 1000 microg/mL) relaxed the tracheal basal tonus with an EC (50) value of 125.3 [52.2 - 300.9] microg/mL. Its maximal relaxation (40 +/- 6 %) was significantly lower than that evoked by aminophylline (209 +/- 34 %). The K (+)-(60 mM)-induced contractions were significantly reduced by both EOET (200 - 1000 microg/mL) and its main constituent 1,8-cineole (600 - 1000 microg/mL). Acetylcholine (1 microgM)-induced contractions were significantly enhanced by 1,8-cineole (10 - 1000 microg/mL). However, they were significantly enhanced and reduced by lower (200 - 400 microg/mL) and higher (800 - 1000 microg/mL) concentrations of EOET, respectively. Electrical field stimulation-induced contractions were significantly increased by EOET (100 - 600 microg/mL). In conclusion, EOET produces myorelaxant effects on guinea-pig isolated trachea, an effect that seems to result from a complex interaction between its monoterpenoid constituents.

Effect of indomethacin on electrical field stimulation-induced contractions of isolated transverse and longitudinal rat gastric fundus strips

To study the effects of indomethacin on the isolated transverse and longitudinal rat gastric fundus strips. The strips were suspended in an organ bath containing oxygenated Krebs solution, and contractile responses to electrical field stimulation were recorded on a physiograph in an isotonic manner after administration of cumulative concentrations of indomethacin. The effects of indomethacin on the strips pretreated with K(ATP) channel modulators, diazoxide and glybenclamide were studied. Treatment of the transverse strips with indomethacin resulted in a concentration-dependent inhibitory response. In longitudinal strips, biphasic responses were seen, which included a stimulatory response at low concentrations of indomethacin, followed by an inhibitory response at higher concentrations. Diazoxide pre-treatment inhibited the stimulatory response of longitudinal strips. Glybenclamide pre-treatment not only blocked inhibitory effect of the low concentrations of indomethacin on transverse strips, but also increased the amplitude of contractions. Moreover, the drug decreased the amplitude of contractions in longitudinal strips. Responses of the isolated longitudinal and transverse rat gastric fundus strips to indomethacin are not similar, and are influenced by K(ATP) channel modulators.

Decreased sensory neuropeptide release in isolated bronchi of rats with cisplatin-induced neuropathy

We studied if attenuated neurogenic bronchoconstriction was associated with a change in sensory neuropeptide release in preparations from rats with cisplatin-induced neuropathy. Electrical field stimulation (100 stimuli, 20 V, 0.1 ms, 20 Hz) induced an increase in the release of somatostatin, calcitonin gene-related peptide (CGRP) and substance P determined by radioimmunoassay from baseline 0.18±0.01, 0.17±0.01 and 0.86±0.02, to 0.59±0.02, 1.77±0.04 and 5.96 fmol/mg wet tissue weight, respectively, in organ fluid of tracheal tubes from rats. This was significantly attenuated to post-stimulation values of 0.36±0.02, 0.45±0.02, 4.68±0.24 fmol/mg wet tissue weight for somatostatin, CGRP, and substance P, respectively, with a significant decrease in field stimulation-induced contraction of bronchial preparations from animals 11 days after a 5-day treatment period with cisplatin (1.5 mg/kg i.p. once a day). The cisplatin-treated animals developed sensory neuropathy characterized by a 40% decrease in femoral nerve conduction velocity. The results show that a decrease in tracheo-bronchial sensory neuropeptide release associates with feeble bronchomotor responses in rats with cisplatin-induced sensory neuropathy.

Cyclical elongation regulates contractile responses of isolated airways.

Bronchoconstrictor responses are quantitatively different when they are evoked under static conditions and during or after periods of deep inspiration. In vivo, deep inspirations produce bronchodilation and protect the lung from subsequent bronchoconstriction (termed bronchoprotection). These effects may be due in part to dynamic stretch on airways produced by cyclical expansion of airway diameter. However, airways also lengthen cyclically during breathing. The effects of cyclical airway elongation on evoked bronchoconstriction have not been examined. This study recorded evoked contractions of pig bronchial segments 1) at different airway lengths, 2) after a period of cyclical lengthening in relaxed airways, and 3) during cyclical lengthening in pretoned airways. Airway segments were mounted in organ baths and bathed in Krebs solution luminally and on the adventitia. Airways were cyclically lengthened by 5-30% of their deflated length at 0.5-2 Hz for 5 min. Contractions were evoked by electrical field stimulation or carbachol and were recorded under isovolumic conditions. Under static conditions, there was a blunt relationship between length and response to electrical field stimulation. After a period of airway length cycling, electrical field stimulation-induced contractions were increased. In airways pretoned with carbachol, cyclical lengthening produced a transient bronchodilation and a sustained increase in contraction. Contractile responses were not blocked by indomethacin. The results show that isolated airways respond actively to dynamic changes in length. Our results indicate that cyclical lengthening of airways could contribute to lung function in vivo but does not appear to account for the phenomenon of bronchoprotection.

Influence of maturation on constrictive response to stimulation of C-fiber afferents in isolated guinea pig airways.

We investigated whether the airway constrictive response to stimulation of bronchopulmonary C-fiber afferents is altered during the maturation process. Isometric tension was measured in airway rings isolated from three tracheobronchial locations (intrathoracic trachea and main and hilar bronchi) and compared in mature [M, 407 +/- 10 (SE) g body wt, n = 36] and immature (IM, 161 +/- 5 g body wt, n = 35) guinea pigs. Our results showed no difference in the ACh (10(-5) M)- or KCl (40 mM)-induced contraction between M and IM groups, regardless of the airway location. In sharp contrast, the concentration-response curves of 10(-8)-10(-6) M capsaicin were distinctly lower in IM hilar bronchi; for example, response to the same concentration of capsaicin (10(-6) M) was 89.2 +/- 15.3% of the response to 10(-5) M ACh in IM and 284.7 +/- 43.2% in M animals. Similar, but smaller, differences in the bronchoconstrictive response to capsaicin between IM and M groups were also observed in the trachea and main bronchus. Electrical field stimulation induced airway constriction in all three locations in M and IM groups. However, after administration of 10(-6) M atropine and 10(-6) M propranolol, electrical field stimulation-induced contraction was significantly smaller in the hilar bronchus of IM than M animals, and this difference was not prevented by pretreatment with 5 x 10(-5) M indomethacin. Although radioimmunoassay showed no difference in the tissue content of substance P between M and IM airways, the constrictive responses to exogenous substance P and neurokinin A were markedly greater in M airways at all three locations. In conclusion, the constriction of isolated airways evoked by C-fiber stimulation was significantly weaker in the IM guinea pigs, probably because of a less potent effect of tachykinins on the airway smooth muscle.

Ghrelin enhances gastric motility through direct stimulation of intrinsic neural pathways and capsaicin-sensitive afferent neurones in rats

Background: Ghrelin may stimulate gastric motility via the vagal nerve pathway. However, the mechanism of ghrelin-induced changes in gastrointestinal motility has not yet been clearly defined. The present study was designed to investigate whether ghrelin accelerates gastric emptying via capsaicin-sensitive afferent neurones and directly affects the enteric neuromuscular function. Methods: Gastric emptying of nutrient solids was assessed after intravenous administration of saline or ghrelin in conscious rats. The effects of ghrelin on gastric emptying were also examined in rats pretreated with capsaicin. Gastric emptying and intestinal transit of non-caloric liquids were evaluated using [Formula: See Text]Cr solution. The effects of ghrelin on spontaneous contractile activities of isolated strips from stomach and jejunum were also investigated and the influence of ghrelin on motor responses to carbachol and electrical field stimulation was examined. Results: Ghrelin significantly accelerated gastric emptying of both nutrient solids and non-caloric liquids in conscious rats. The intestinal transit of non-caloric liquids was also enhanced by ghrelin. Pretreatment with capsaicin prevented the ghrelin-induced acceleration of gastric emptying of nutrient solids. Ghrelin did not modulate spontaneous and carbachol-induced contractions of strips of gastric body, gastric antrum and jejunum. However, electrical field stimulation-induced contractions were significantly enhanced by ghrelin in the gastric body. Conclusions: The results suggest that the stimulatory effects of ghrelin on gastric motility are mediated by direct stimulation of the enteric neural pathway and capsaicin-sensitive afferent neurones.

P.5.012 The role of muscarinic receptors in the control of acute morphine withdrawal

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([3H]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (−logEC50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA2 values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (−logIC50) was 4-DAMP (8.35)>tropicamide (6.72)>himbacine (6.54)>pirenzepine (6.31)>AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M2 and/or M4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (Kd=0.92 nM; Bmax=126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pKi value and Hill coefficient in parentheses): atropine (8.22 and 0.93)>4-DAMP (8.18 and 0.94)>tropicamide (6.78 and 0.93)>pirenzepine (5.46 and 0.92)>AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M3 receptors present on smooth muscle cells.

Pharmacological Effects of KRP-197 on the Human Isolated Urinary Bladder

KRP-197, 4-(2-methylimidazol-l-yl)-2,2-diphenylbutyramide, is a newly synthesized antimuscarinic drug, developed for the treatment for overactive bladder. For evaluation of pharmacological characteristics of KRP-197, we investigated whether it influenced both prejunctional and postjunctional muscarinic receptors on the isolated human detrusor smooth muscles as compared with the effects of atropine, oxybutynin, and propiverine. Using the muscle bath technique, we investigated the effects of various antimuscarinic drugs on the contractions induced by carbachol, KCl, CaCl₂, and electrical field stimulation. Furthermore, using high-performance liquid chromatography with a microdialysis technique, we measured the acetylcholine release from the muscle strips during electrical field stimulation. The effects of various antimuscarinic drugs on acetylcholine releases were also evaluated. Pretreatment with various antimuscarinic drugs caused parallel shifts to the right in carbachol-induced concentration-response curves. The rank order of pA₂ values was KRP-197 ≧ atropine > oxybutynin > propiverine. Atropine and KRP-197 did not cause significant inhibition of KCl- and CaCl₂-induced contractions. All drugs caused concentration-dependent inhibitions in electrical field stimulation-induced contractions. Pretreatment with atropine and propiverine did not cause significant changes in electrical field stimulation-induced acetylcholine release. However, KRP-197, and oxybutynin caused significant decreases in acetylcholine release. The present study demonstrates that KRP-197 has an inhibitory effect on postjunctional muscarinic receptors as well as on prejunctional muscarinic receptors to modulate acetylcholine release in human detrusor smooth muscles. The findings suggest the usefulness of KRP-197 as a therapeutic drug for an overactive bladder with symptoms of frequency, urgency, and urge incontinence.

Nerve-mediated bladder contraction is impaired by cytokines: involvement of inducible nitric oxide synthase

We investigated the possible involvement of inducible nitric oxide synthase (iNOS) in the effect of cytokines on neuromuscular function in isolated rat bladder strips. Bladder strips were incubated in cell culture medium for 24 h with or without tumour necrosis factor-α (TNF-α)+interleukin-1β. Mechanical activity in response to electrical field stimulation and carbachol was recorded in organ baths. Both the electrical field stimulation- and carbachol-induced contractions were reduced by the incubation. The electrical field stimulation-induced contraction was significantly further impaired after prolonged exposure to TNF-α+interleukin-1β. This impairment was restored by dexamethasone, the iNOS inhibitor aminoguanidine and partially by brain-derived neurotrophic factor (BDNF). In contrast, carbachol-induced contractions were not affected by cytokines. iNOS protein expression was detected in cytokine-incubated bladder strips by immunohistochemistry and Western blot analysis. The results demonstrated that TNF-α+interleukin-1β impaired nerve-mediated bladder contractions. Aminoguanidine, and to some extent BDNF, exerted neuroprotective effects.

Investigation of the role of the serotonergic activity of certain subtype-selective alpha1A antagonists in the relaxant effect on the pregnant rat uterus in vitro.

Results from recent studies have shown that alpha(1A)-adrenergic receptor (alpha(1A)-AR) antagonists could offer a new alternative in the treatment of preterm delivery. However, members of this group [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil (5-MU)] are known to influence serotonin (5-hydroxy-tryptamine) (5-HT(1A)) receptors, too. Our objective was to clarify the role of their 5-HT(1A) activities in the uterus relaxant effect. RT-PCR was used to determine mRNA expression of the receptor subtypes in 22 day pregnant rat uteri. Isolated uteri were stimulated by 5-HT or electrical field to investigate the contraction-inhibiting effect and the 5-HT(1A) activity of the alpha(1A) antagonists. Both receptor subtypes are present in rat myometrium. 5-HT induced contractions were inhibited by the alpha(1A) antagonists. Besides shifting the dose-response curve of 5-HT to the right, 5-MU decreased its maximal effect. The alpha(1A) antagonists inhibited electrical field stimulation-induced contractions. 5-HT(1A) blockade increased the maximal effect of 5-MU but did not change that of WB4101. These results suggest that the contraction increase caused by 5-HT is mediated by alpha(1A) receptors. Serotonergic activity of alpha(1) antagonists and especially alpha(1A) antagonists should be investigated as it may alter their efficacy and could interfere with their side-effects. It is proposed that novel alpha(1A) antagonists should be designed with no 5-HT(1A) activity to achieve maximal relaxant effect.

Different effects of acute clenbuterol on vasomotor response in mesenteric arteries from young and old spontaneously hypertensive rats

We analysed the influence of aging on the acute effect of clenbuterol, a β 2-adrenoceptor agonist, on the vasoconstrictor response induced by electrical field stimulation in mesenteric arteries from young and old spontaneously hypertensive rats (SHRs). Clenbuterol increased the contraction elicited by electrical field stimulation in arteries from both groups, and this was prevented by propranolol. N G-nitro- l-arginine methyl ester ( l-NAME) also increased the electrical field stimulation-elicited contractions in arteries from both age groups. However, pretreatment with capsaicin increased the electrical field stimulation-induced contractions in young SHRs, but did not modify it in old SHRs. In segments from young SHRs, the treatment with the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP-(8–37), induced an increase in the electrical field stimulation-induced vasoconstrictor response that was not modified by the subsequent addition of capsaicin. Addition of clenbuterol to l-NAME-treated segments from both groups further increased the response to electrical field stimulation. In segments from young SHRs, clenbuterol failed to increase the electrical field stimulation-induced response in the capsaicin-treated segments, but the response was increased by the subsequent addition of l-NAME. The addition of l-NAME to the clenbuterol-treated segments from old SHRs did not modify the enhanced electrical field stimulation response. Electrical field stimulation induced a similar tritium release in arteries from young and old SHRs preincubated with [ 3H]noradrenaline. In arteries from young SHRs, isoproterenol increased this release and the increase was abolished by propranolol. Clenbuterol increased the stimulated tritium overflow and exogenous noradrenaline response only in segments from old SHRs, and both effects were abolished by propranolol. To summarize and conclude, clenbuterol increased the electrical field stimulation-induced contraction in segments from both age groups. In young SHRs, clenbuterol seems to inhibit CGRP release, while in old SHRs, it increases the release of and response to noradrenaline and decreases neuronal nitric oxide (NO) release.

Regional and muscle layer variations in cholinergic nerve control of the rat myometrium during the oestrous cycle

To determine regional and muscle layer differences in the cholinergic nerve control of uterine activity, functional and immunohistochemical experiments were carried out on the cervix, and circular and longitudinal muscle from the caudal and rostral uterine horn in cyclic rats. During oestrus, in vitro electrical field stimulation evoked contractions in the cervix and myometrium of the caudal horn, predominantly in circular muscle layer. All evoked responses were tetrodotoxin-sensitive and completely abolished by atropine, thus were cholinergic nerve-mediated. In contrast, no electrical field stimulation-induced contraction occurred in either the circular or longitudinal muscle from the rostral uterus. Concentration–response curves for carbachol showed that muscarinic receptor-mediated contractions occurred in all uterine regions and muscle layers during oestrus. Immunohistochemistry for the cholinergic nerve marker, vesicular acetylcholine transporter showed that the predominance of the acetylcholine-dependent contractions in circular muscle preparations were related to a layer-specific distribution of cholinergic nerve fibres, abundant in the circular muscle but scarce in the longitudinal muscle layer. In addition, the absence of electrical field stimulation-evoked acetylcholine-dependent contractions in the rostral uterus was correlated to a marked decrease in the density of cholinergic fibres along the caudo-rostral axis of the organ. In the uterus from diestrus rats, contractions were not elicited in response to electrical field stimulation in the cervix and circular or longitudinal muscle from the caudal as well as rostral uterine horn. Addition of cumulative doses of carbachol failed to increase in a concentration-dependent manner the frequency and amplitude of contractions in the cervix and myometrial layers from either the caudal and rostral uterine horn. The distribution and density of cholinergic nerve fibres along the uterus and between the muscle layers did not differ from the oestrus stage. We conclude that the cholinergic nerve control of uterine activity is layer-specific and predominant in the caudal uterine horn and the cervix. Impairment of this nerve control from oestrus to diestrus stages occurred in relation to a decrease in the myometrial sensitivity to muscarinic stimulation, not to a decrease in the density of cholinergic innervation.

Endogenous neurokinins facilitate synaptic transmission in guinea pig airway parasympathetic ganglia.

Neurokinin-containing nerve fibers were localized to guinea pig airway parasympathetic ganglia in control tissues but not in tissues pretreated with capsaicin. The purpose of the present study was to determine whether neurokinins, released during axonal reflexes or after antidromic afferent nerve stimulation, modulate ganglionic synaptic neurotransmission. The neurokinin type 3 (NK(3)) receptor antagonists SB-223412 and SR-142801 inhibited vagally mediated cholinergic contractions of bronchi in vitro at stimulation voltages threshold for preganglionic nerve activation but had no effect on vagally mediated contractions evoked at optimal voltage or field stimulation-induced contractions. Intracellular recordings from the ganglia neurons revealed that capsaicin-sensitive nerve stimulation potentiated subsequent preganglionic nerve-evoked fast excitatory postsynaptic potentials. This effect was mimicked by the NK(3) receptor agonist senktide analog and blocked by SB-223412. In situ, senktide analog markedly increased baseline tracheal cholinergic tone, an effect that was reversed by atropine and prevented by vagotomy or SB-223412. Comparable effects of intravenous senktide analog on pulmonary insufflation pressure were observed. These data highlight the important integrative role played by parasympathetic ganglia and indicate that activation of NK(3) receptors in airway ganglia by endogenous neurokinins facilitates synaptic neurotransmission.

β 2-Adrenoceptor-mediated inhibition of field stimulation induced contractile responses of the smooth muscle of the rat prostate gland

Isolated preparations of rat prostate responded to electrical field stimulation (2 strains every 60 s, 0.5 ms, 10 Hz, 80 V) with contractions. The adrenoceptor agonists adrenaline, isoprenaline and noradrenaline (0.1 nM–10 μM) elicited concentration-dependent inhibition of electrical field stimulation-induced contractions of the rat prostate. Phenylephrine had no effect on the amplitude of electrical field stimulation-induced contractions. The rank order of potency was isoprenaline≥adrenaline=noradrenaline>phenylephrine. Inhibition of electrical field stimulation-induced contractions by isoprenaline was attenuated by propranolol (1 μM). The selective β 1-adrenoceptor agonist (−)-1-(3,4-dimethoxy-phenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propanol)oxalate (RO363) and the selective β 2-adrenoceptor agonist salbutamol (1 nM–100 μM) were approximately equipotent in inhibiting electrical field stimulation-induced contractions but the selective β 3-adrenoceptor agonist sodium 4-(2-[2-hydroxy-{3-chlorophenyl}ethylamino]propyl)phenoxyacetate (BRL 37344, 1 nM–100 μM) did not inhibit electrical field stimulation-induced contractions. The selective β 2-adrenoceptor antagonist, (±)-1-[2,3-(dihydro-7-methyl-1 H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118 551, 0.1 μM) attenuated inhibitory responses to isoprenaline and salbutamol, while the selective β 1-adrenoceptor antagonist atenolol (3 μM) did not. Contractions induced by electrical field stimulation were also inhibited by forskolin (10 nM–3 mM) but unaffected by sodium nitroprusside (10 nM–1 mM) indicating the presence of an inhibitory cAMP mechanism. These data suggest that stimulation of β 2-adrenoceptors can inhibit contractions of the rat prostate induced by electrical field stimulation.

Age-related changes in cholinergic and purinergic neurotransmission in human isolated bladder smooth muscles.

We evaluated the correlation among age, cholinergic and purinergic neurotransmissions in the electrical field stimulation-induced contractions in human isolated urinary bladder smooth muscles, using the muscle bath technique. Human bladder specimens were divided into three groups (G1, under 50years; G2, 51-70years; G3, over 70years old), and each muscle strip was suspended in a thermostatically controlled organ bath filled with oxygenated Krebs-Henseleit solution, connected to an isometric force displacement transducer, and an isometric tension development was recorded. The contractile responses induced by KCl, carbachol, adenosine triphosphate (ATP) and electrical field stimulation, and the effects of atropine and alpha, beta methylene ATP on electrical field stimulation-induced contractions were observed. The contractile response to KCl and concentration-response curves for carbachol and ATP, and frequency-response curves for electrical field stimulation were not significantly different among the three groups. The atropine sensitive and resistant parts of contraction induced by electrical field stimulation were decreased and increased with age, respectively. There are significant positive and negative correlations between age and the purinergic, and age and the cholinergic neurotransmissions in human isolated bladder smooth muscles, respectively. The age-related changes in neurotransmissions may contribute to the changes in bladder function in the elderly.