Adenosine-induced bronchoconstriction of isolated lung and trachea from sensitized guinea-pigs.

1. The bronchoconstriction of airway-perfused lungs and contraction of superfused tracheal spirals from guinea-pigs in response to adenosine were examined. 2. In lungs from untreated animals, adenosine had little effect unless the perfusion pressure was raised with carbachol (1.1 microM), when it caused a fall in perfusion pressure. However, if removed from guinea-pigs sensitized with ovalbumin (5 mg and 10 mg i.p. 14 and 12 days before use), adenosine was bronchoconstrictor, exerting bronchodilator effects only at high (1 mg) doses. The constrictor response to adenosine (300 micrograms) was significantly greater than that in lungs from untreated or sham-injected animals. 3. In superfused trachea from untreated animals, adenosine exerted only relaxant responses. In tissues from ovalbumin-sensitized guinea-pigs adenosine produced contractile responses, with relaxation appearing only at high (1 mg) doses. 4. Thus sensitization by antigen challenge revealed a bronchoconstrictor response of isolated airway preparations to adenosine. This is related to the clinical situation where only asthmatic subjects respond to adenosine by bronchoconstriction and suggests that the sensitization may destabilize inflammatory cells for mediator release by adenosine. 5. The response to a second exposure to adenosine was consistently reduced (lungs) or converted to a relaxation (trachea) indicating tachyphylaxis and consistent with a mediator release mechanism. 6. The P1-purinoceptor antagonist, 8-phenyltheophylline (3.9 microM), antagonized the relaxant responses to higher doses of adenosine. However, it did not affect the contractile responses to lower doses of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-specific Airway Hyperreactivity in Isolated Respiratory Preparations from Guinea-pigs Sensitized and Challenged with Ovalbumin

Summary: Airway hyperreactivity to physical, chemical, immunological and pharmacological stimuli is well documented in vivo. The aim of this study was to investigate whether tissues taken from guinea-pigs that had been shown to display hyperreactivity in vivo after antigen challenge were also hyperreactive in vitro. Isolated airway-perfused lungs from ovalbumin-sensitized guinea-pigs challenged 24 h beforehand with an aerosol of ovalbumin showed a significant (P<0.05) increase in responsiveness to the bronchoconstrictor response to a bolus dose of carbachol (10 μg) when compared with saline challenged animals. The contractile responses to single doses of carbachol (10 μg) and histamine (30 μg) in immersed tracheal spiral preparations taken from sensitized animals exposed to the ovalbumin were also significantly enhanced (P<0.05). A non-significant leftward shift was observed in the concentration-response curve for histamine in challenged perfused lungs from ovalbumin-challenged animals compared with an NaCl challenge. Concentration-response curves to carbachol and histamine in immersed tracheal spirals were virtually superimposed. Therefore, this study has shown non-specific airway hyperreactivity of isolated airway perfused lungs at 24 h following a challenge of sensitized gainea-pigs with aerosolized ovalbumin, although this was not evident from concentration-response curves in immersed trachea. The isolated perfused lung therefore provides a simple method for further evaluation of the mechanisms of airway hyperreactivity.     

Beta-adrenoceptor reactivity after epithelium removal in guinea-pig trachea in vitro

In guinea-pig isolated tracheae precontracted with pilocarpine (2 x 10(-5)M) mechanical removal of the epithelium did not significantly modify the degree of relaxation induced by three different adrenergic agonists: epinephrine (adrenaline), isoproterenol (isoprenaline) and salbutamol. The failure of epithelium removal to modify isoproterenol relaxant activity was observed in both spontaneous tone and pilocarpine precontracted tracheae. In tracheae obtained from actively sensitized (ovalbumin) guinea-pigs, log-concentration response curves of epinephrine and salbutamol were unchanged by epithelium damage, whereas that of isoproterenol was slightly shifted to the left. In ovalbumin sensitized tracheae exposed to the antigen (ovalbumin, 50 micrograms/ml) epithelium removal enhanced the relaxant activity of the three adrenergic agonists used. Beta-adrenoceptor desensitization (isoproterenol, 10(-6)M for 20 min twice) carried out in ovalbumin actively sensitized tracheae with or without epithelium, shifted the log-concentration response curves of isoproterenol to a similar rightward position. The present data suggest that the airway epithelium plays a minor role in the regulation of guinea-pig tracheal sensitivity to beta-adrenoceptor agonists, which is evident only in a classical model of experimental asthma.     

Partial inhibition by epithelium of tracheal smooth muscle relaxation induced by the potassium channel activator, BRL 38227.

1. A method is described whereby either the serosal (Out) or epithelial (In) sides of rat isolated tracheae were selectively perfused. Perfusion with BRL 38227 (10(-8)-5 x 10(-6) M; In/Out) of preparations with intact epithelium (+ EP) precontracted with carbachol (10(-6) M; Out/In) produced complete relaxation. Perfusion with aminophylline (10(-5)-10(-3) M; In) of + EP preparations precontracted with carbachol (10(-6) M; Out) also produced complete relaxation. 2. In preparations precontracted with carbachol (10(-6) M) epithelium removal (- EP) increased the sensitivity to the relaxant effect of BRL 38227 (In), but not BRL 38227 (Out) [- log EC50, + EP/- EP; carbachol (In), BRL 38227 (Out): 6.76 +/- 0.11 vs 6.67 +/- 0.15; carbachol (Out), BRL 38227 (In): 5.93 +/- 0.06 vs 6.25 +/- 0.07]. Removal of the epithelium increased also the sensitivity to BRL 38227 (In) of preparations precontracted with a lower concentration (5 x 10(-7) M) of carbachol (Out). [- log EC50, + EP/- EP, carbachol (Out), BRL 38227 (In): 6.19 +/- 0.14 vs 6.58 +/- 0.17]. 3. Removal of the epithelium did not affect the sensitivity to BRL 38227 (In) of preparations precontracted with a higher concentration (5 x 10(-6) M) of carbachol (Out). 4. In both + EP and - EP preparations precontracted with carbachol (10(-6) M; Out), BRL 38227 (In) had a more potent relaxant effect than aminophylline (In) (EC50, BRL 38227 vs aminophylline, + EP/- EP: 5.93 +/- 0.06 vs 3.66 +/- 0.11/6.25 +/- 0.07 vs 3.77 +/- 0.11).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of lipoxygenase inhibitors and leukotriene antagonists on anaphylaxis

The experiments whose results are reported here were carried out with the aim of showing a possible role for lipoxygenase products in the modulation of the Schultz-Dale reaction. For this purpose, the actions of nordihydroguaieretic acid (NDGA) and of FPL 55712 were tested during anaphylaxis in guinea-pig ileum and trachea in vitro. Isolated preparations from guinea-pigs, which had been subcutaneously sensitized with ovalbumin and incomplete Freund adjuvant, were challenged with increasing concentrations of antigen; in preparations isolated from the same animal an antigen-concentration anaphylactic-reaction curve was performed in the presence of the drugs. NDGA 3.3 X 10(-6) M was capable of inhibiting anaphylaxis in the trachea to a maximum extent of 40% but it did not affect anaphylactic reaction in the intestinal smooth muscle. FPL 55712 2 X 10(-6) M did not exert any activity on anaphylaxis in either preparations. The difference between SRS-A and histamine as mediators of anaphylaxis in the tissue preparations used could explain the fact that NDGA acted on the trachea alone.     

Nicotine potentiates the nitrergic relaxation responses of rabbit corpus cavernosum tissue via nicotinic acetylcholine receptors

The presence of neuronal nicotinic acetylcholine receptors in rabbit corpus cavernosum tissue and possible mechanisms underlying the potentiation of electrical field stimulation induced relaxation by nicotine were analyzed. In corpus cavernosum tissue strips nicotine (3 x 10(-5) M) and acetylcholine (10(-3) M) produced potentiation on electrical field stimulation (amplitude 50 V; frequency 4 Hz; width 0.8 ms) induced relaxation responses. This nicotine-induced potentiation was not altered by atropine (10(-6) M), guanethidine (5 x 10(-6) M) and indomethacin (10(-5) M), but abolished by hexamethonium chloride (10(-5) M) and L-nitro arginine methyl ester (10(-5) M). Nicotine did not cause any alteration on a single dose of carbachol (3 x 10(-5) M) and sodium nitroprusside (10(-5) M) induced relaxation responses. The results suggest that, nicotine-induced potentiation is NO and nicotinic acetylcholine receptor dependent but independent from prostaglandin synthesis, activation of muscarinic receptors and does not require intact adrenergic neurons. Nicotine did not affect smooth muscle and endothelium directly. In conclusion, in this study we showed for the first time that, nicotine acts on the nicotinic acetylcholine receptors located on the nitrergic nerves, thereby evoking the release of NO from these nerve terminals inducing relaxation response in rabbit corpus cavernosum tissue.     

Influence of tracheal contraction on relaxant effects in vitro of theophylline and isoprenaline.

1 Relaxation by (-)-isoprenaline (Iso) and theophylline (Theo) was measured in guinea-pig isolated trachea, in the presence or absence of carbachol. 2 With basal tone or with carbachol at a concentration of 5.4 x 10(-7) M, causing 70% maximal contraction, Iso and Theo relaxed the trachea to the same extent. 3 With carbachol concentrations of 5.4 x 10(-6) M and 5.4 x 10(-5) M (96% and 100% maximal contractions) Iso caused no more than 63% and 34%, respectively, of the maximum relaxation to Theo. 4 When calculated at 25% of the maximum Theo relaxation, the Iso/Theo potency ratio was gradually reduced from 14,160 when evaluated at basal tone to 1,560 at the highest carbachol concentration. 5 In combination, at their maximally effective concentrations, Theo and Iso produced no larger a relaxation than did Theo alone. 6 At the two highest concentrations of carbachol, concentration-response curves to Theo were virtually superimposable whether determined in the absence or the presence of Iso at its maximally effective concentration. 7 It is concluded that Theo causes a greater relaxation of highly contracted tracheal muscle than Iso.     

The relaxation induced by nicotine in the rat isolated renal artery.

The mechanism of nicotine-induced relaxation was investigated in the rat isolated renal artery. Nicotine (10(-3) M) produced a relaxation when preparations were precontracted by phenylephrine (3 x 10(-6) M). Nicotine-induced relaxation was 27.3 +/- 2.5% of phenylephrine contraction and was not affected by atropine (10(-5) M), guanethidine (10(-5) M), hexamethonium (10(-4) M), indomethacin (10(-5) M), N(G)-nitro-L-arginine (10(-4) M), methylene blue (10(-5) M), glibenclamide (10(-5) M), quinacrine (3 x 10(-6) M), tetrodotoxin (10(-6) M), capsaicin (10(-6) M), tetraethylammonium (10(-3) M), 4-aminopyridine (10(-3) M), and ouabain (10(-6) M) (n = 6, Mann-Whitney U-test). A calcium antagonizing effect of nicotine was not observed. Therefore, it appears that nicotine relaxes rat isolated renal artery by a nonspecific action on the vascular smooth muscle.     

Effects of esculetin (6,7-dihydroxycoumarin) on guinea-pig tracheal chains in vitro

Esculetin (6,7-dihydroxycoumarin) was tested in isolated guinea-pig tracheal chains from normal and ovalbumin-sensitized animals. The drug showed a concentration-dependent relaxant effect, with an EC50 of 60 microM. Pretreatment with esculetin (30-60 microM) was not able to reduce the contractile response induced by either carbachol (0.1 microM), histamine (10 microM), BaCl2 (10 mM) or K+ (80 mM). Esculetin increased the contraction evoked by PGF2 alpha and by histamine, but reduced A23187-induced contraction. Mepacrine (10 microM), a phospholipase A2 inhibitor, reduced the relaxant effect of the lower esculetin concentrations (3-60 microM). Indomethacin (0.1 microM) increased the relaxation induced by esculetin (3-10 microM). In tracheal chains from ovalbumin-sensitized guinea-pigs, esculetin (60 microM) significantly reduced the duration of the recovery phase after antigen challenge contraction. In conclusion, in our in vitro conditions, this natural coumarin derivative seems to act by interfering with the arachidonic acid cascade.     

Bronchodilator-mediated relaxation of normal and ovalbumin-sensitized guinea-pig airways: lack of correlation with lung adenylate cyclase activation.

Isoprenaline, vasoactive intestinal peptide (VIP), prostaglandin E2 (PGE2) and forskolin caused a dose-dependent relaxation of normal and ovalbumin-sensitized guinea-pig tracheal spirals and lung parenchymal strips in vitro. There was no difference in magnitude of relaxation or sensitivity to these relaxants between normal and sensitized tissues. The rank order of potency (concentration of each drug at which 50% of the maximum is obtained) for these relaxants on both trachea and parenchyma was VIP greater than isoprenaline greater than PGE2 greater than forskolin, although the parenchyma was more sensitive than the trachea. The rank order of efficacy of the drugs used in relaxing both the trachea and lung parenchyma was isoprenaline (10 microM) greater than forskolin (30 microM) greater than VIP (0.1 microM) greater than PGE2 (10 microM). PGE2 at concentrations greater than 1 microM sometimes contracted the lung strip. Pretreatment with indomethacin (8.5 microM), a cyclo-oxygenase inhibitor, reduced the resting tone of tracheal spirals, but did not significantly affect the tone of lung strips. Indomethacin-pretreatment did not affect drug-induced relaxations of either normal or sensitized tracheal spirals. However, both normal and sensitized indomethacin-pretreated lung strips relaxed significantly less (P less than 0.05) to isoprenaline, PGE2 and forskolin. Indomethacin-pretreatment did not affect sensitivity of normal and sensitized trachea or parenchyma to the relaxant drugs. All the relaxant drugs used stimulated adenylate cyclase activity in normal or sensitized lung parenchyma membrane preparations. The rank order of efficacy (maximal activation) was forskolin greater than isoprenaline = VIP greater than PGE2. There was no difference in response between normal and sensitized lungs. Adenylate cyclase activity of normal lung was stimulated as follows: forskolin (100 microM), 500.0 +/- 50.0%; isoprenaline (100 microM), 186.0 +/- 29.0%; VIP (10 microM), 213.0 +/- 19.0% and PGE2 (100 microM), 155.0 +/- 23.0% of basal activity. Similar values were obtained for sensitized lung parenchyma. Indomethacin-pretreatment did not significantly affect normal or sensitized lung adenylate cyclase stimulation by isoprenaline, VIP, forskolin or PGE2. It was concluded that: Immunological sensitization to ovalbumin does not induce hypoactivity of relaxant drug receptors and/or the adenylate cyclase system of the airway tissues of the guinea-pig.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of vitamin E in ovalbumin-sensitized guinea pigs

It has been suggested that epithelium destruction, mucus secretion, edema and bronchoconstriction may play a role in asthma pathogenesis. Additionally, histamine, serotonine, leukotrienes and other mediators are released and free oxygen radicals are produced during bronchoconstriction. We studied the role of antioxidants on the treatment of asthma by testing alpha-tocopherol (vitamin E; 5, 25, 50 and 100 mg/kg/day i.p.), a scavenger of oxygen radicals, on male Guinea pigs. The animals were sensitized by injecting ovalbumin. After sensitization, isolated tracheal preparations were studied in vitro. The effects of carbachol 10(-4) M, ovalbumin 10(-3) M and vitamin E (10(-5) M, 10(-4) M, 10(-3) M concentrations) were evaluated in an isolated organ bath. It was observed that alpha-tocopherol reduced the contractile effects of ovalbumin and carbachol. The 5, 25 and 50 mg/kg/day doses of vitamin E were injected intraperitoneally to Guinea pigs that were sensitized with ovalbumin. We observed significant differences between the contractile responses to carbachol and vitamin E with carbachol. These treatments significantly reduced the contractility of tracheal preparations (p < 0.001). There was no significant difference with the 100 mg/kg/day i.p. dose of vitamin E in the contractile response to carbachol E (p > 0.05). The upper part of the tracheal preparations was the most sensitive region to the contractile effect of 10(-3) M ovalbumin in all groups (p < 0.001). The contractile response to 10(-3) M ovalbumin was reduced by 25 and 50 mg/kg/day doses of vitamin E (p < 0.001).     

The isolated lung strip and single open tracheal ring: a convenient combination for characterizing Schultz Dale anaphylactic contractions in the peripheral and central airways of the guinea-pig

1. The use of the isolated lung strip and single open tracheal ring for studying Schultz Dale anaphylactic contractions in both the peripheral and central airways is described. 2. This method is particularly relevant to studies of anaphylaxis because many preparations may be obtained from a single sensitized animal. 3. Isolated preparations from control guinea-pigs did not respond to ovalbumin, whereas the lung strip and trachea taken from guinea-pigs sensitized to ovalbumin, contracted markedly to that antigen. 4. The peripheral and central airways from sensitized animals responded in the same way to antigen challenge.     

Forskolin reverses tachyphylaxis to the bronchodilator effects of salbutamol: an in-vitro study on isolated guinea-pig trachea

The objective of this study was to assess the relaxant responses of salbutamol, a beta2 agonist, and forskolin, an activator of adenylate cyclase, and the possible role of forskolin in reversing tachyphylaxis to salbutamol. The in-vitro bronchodilator effects of salbutamol and forskolin (10(-9)-10(-5) M) were tested on isolated guinea-pig tracheal rings precontracted with carbachol (10(-7) M). Both salbutamol and forskolin elicited concentration-dependent relaxation. Potency (EC50; the dose resulting in 50% relaxation) was determined from cumulative concentration-response curves. Salbutamol was more potent than forskolin in relaxing the tracheal preparations (-log molar EC50 7.68+/-0.14 and 6.3+/-0.17, respectively). Reproducible relaxant responses to salbutamol could be elicited after 24 h incubation in Krebs solution. Tachyphylaxis to the relaxant effects of salbutamol was experimentally induced by incubation (24h) of the preparations in Krebs solution containing salbutamol (10(-6) 3x10(-6) or 10(-5) M). This pretreatment of the tissues resulted in a significant reduction in the potency of salbutamol. The potency of salbutamol was reduced to 6.85+/-0.2, 6.8+/-0.1 and 5.9+/-0.27 after 24h incubation with salbutamol 10(-6), 3x10(-6) or 10(-5) M, respectively. The potency of salbutamol was increased from 7.35+/-0.2 to 7.76+/-0.28 by addition of forskolin (3x10(-7) M) under control conditions. Moreover, forskolin (3x10(-7) M) reversed the development of tachyphylaxis to salbutamol-induced relaxation in tissues pretreated with salbutamol. The potency of salbutamol was increased to 7.29+/-0.41, 7.37+/-0.17 and 7.23+/-0.35 after the addition of forskolin (3x10(-7) M) to preparations pre-incubated (24h) with salbutamol 10(-6), 3x10(-6) or 10(-5) M respectively. These results show that in guinea-pig tracheal ring preparations, forskolin shares with salbutamol the ability to relax airway smooth muscle and produces an apparent reversal of tachyphylaxis to the bronchodilator effects of salbutamol, particularly in the low concentration range. This effect could provide an alternative therapy for long term use, particularly with high doses of beta2 agonists in bronchial asthma.     

Nitric oxide and relaxation of pig lower urinary tract.

1. We studied the non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation induced by electrical stimulation in pig isolated lower urinary tract smooth muscle, and the possible involvement of the L-arginine (L-ARG)/nitric oxide (NO) pathway in this response. 2. Trigonal strips, precontracted by noradrenaline (NA), carbachol or endothelin-1 (ET-1), relaxed frequency-dependently in response to electrical stimulation. Maximum relaxation was obtained at 6-8 Hz, and amounted to 56 +/- 2%, 77 +/- 3% and 62 +/- 6% of the agonist-induced tension in preparations contracted by NA, carbachol, or ET-1, respectively. Exposure to NG-nitro-L-arginine (L-NOARG; 10(-7)-10(-5) M) concentration-dependently reduced the relaxant response in preparations contracted by NA. L-NOARG (10(-6) M) reduced the maximal response to 51 +/- 8% of control. L-NOARG (10(-5) M) abolished all relaxation, and unmasked a contractile component; D-NOARG had no effect. Also in trigonal preparations, where the tension had been raised by carbachol or ET-1, L-NOARG (10(-5) M) markedly reduced relaxations evoked by electrical stimulation. 3. In trigonal preparations contracted by NA, maximal relaxation was increased after pretreatment with L-ARG (10(-3) M), and the inhibitory effect of L-NOARG (10(-6) M) was prevented. Incubation of the trigonal strips with methylene blue had no effect on relaxations elicited at frequencies less than 6 Hz, but a small inhibition was observed at higher frequencies. 4. Administration of NO (present in acidified solution of NaNO2) induced concentration-dependent relaxations in trigonal preparations contracted by NA, carbachol, or ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible mechanism of endothelium-dependent relaxation induced by pirarubicin and carbachol in rat isolated aorta.

The mechanism of endothelium-dependent relaxation induced by pirarubicin, (2'R)-4'-O-tetrahydropyranyladriamycin, THP, or carbachol was investigated in the rat isolated aorta. The relaxant effect of THP (1.5 x 10(-6)-4.5 x 10(-5) M) or carbachol (10(-8)-10(-4) M) on the aorta with endothelium was decreased by lowering Ca2+ in the medium. The relaxation induced by THP was not inhibited by pretreatment with verapamil (10(-6)-10(-5) M), and that induced by carbachol was only partially inhibited. However, on replacement of all but 20 mM Na+ with either Li+ or choline, the THP- or carbachol-induced relaxation was inhibited. Furthermore, the relaxing effect of THP or carbachol was inhibited by pretreatment with amiloride (10(-4)-3 x 10(-4) M), with ouabain (10(-4)-10(-3) M), or with K(+)-depletion. These results suggest that the THP- or carbachol-induced relaxation depending on endothelium was affected by modifying the calcium ion concentration, and that a Na(+)-Ca2+ exchange process is involved.     

The effect of nitric oxide synthase blockade on responses to morphine in rat aortic rings

1 It has been suggested that opioids may play an indirect role in the regulation of the peripheral circulation through the control of nitric oxide (NO) release in vascular tissue. The current study was undertaken to investigate the effect of nitric oxide synthase (NOS) blockade on responses to morphine in phenylephrine (PE)- or KCl-precontracted rat aortic rings. 2 Morphine (3 x 10(-8) - 3 x 10(-5) M) administration did not cause any significant effect on basal tonus of endothelium-intact or endothelium-denuded preparations. Morphine produced concentration-dependent relaxation responses in endothelium-intact as well as in endothelium-denuded rat aortic rings precontracted by PE or KCl. Removal of endothelium did not significantly alter the relaxation responses to morphine. 3 The relaxant responses to morphine were significantly and partially inhibited by pretreatment of tissues with naloxone (NAL, 3 x 10(-5) M) for 5 min. The inhibitory effect of NAL on relaxant responses to morphine in PE- or KCl-precontracted rings did not differ significantly between endothelium-intact and endothelium-denuded preparations. 4 Incubation of endothelium-intact or endothelium-denuded rat aortic rings with NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) for 20 min did not cause a significant inhibition on relaxation responses to morphine. 5 These findings confirmed the presence of opiate receptors in rat thoracic aorta, but suggested that mechanisms other than NO release play a role in the relaxant effect of morphine on rat aortic rings.     

A pharmacological and histochemical study of hamster urethra and the role of urothelium.

1. Electrical field stimulation (EFS) of circular strips of hamster proximal urethra caused frequency-dependent relaxations at raised tone. Phentolamine (10(-6) M), propranolol (10(-6) M) and atropine (10(-6) M) were present throughout the experiment. Neurogenic relaxation was attenuated by L-NG-nitroarginine methyl ester (L-NAME) (10(-4) M), was restored by L-arginine (3 x 10(-3) M) but not by D-arginine (3 x 10(-3) M) and completely blocked by tetrodotoxin (10(-6) M). Neurogenic relaxation was also reduced by suramin (10(-4) M) and totally blocked by suramin together with L-NAME. Strips of hamster urethra devoid of urothelium showed little, if any, relaxant response to EFS. 2. An immunohistochemical study showed nitric oxide synthase-immunoreactive nerves in the smooth muscle layers and in the lamina propria, just beneath the urothelium, but no nitric oxide synthase (NOS) staining in the urothelial layer. 3. Noradrenaline elicited a significantly greater contraction in strips without urothelium than in control strips. L-NAME (10(-4) M) did not affect noradrenaline-induced contraction in both control and urothelium-free strips. The contractile response to acetylcholine was not dependent on the presence or absence of urothelium. Nevertheless the response induced by exogenous acetylcholine (10(-3) M) was increased by L-NAME (10(-4) M), both in intact and in urothelium-free strips. 4. Prostaglandin E2 (10(-8)-5 x 10(-6) M) and 2-methyl-thio-ATP (10(-9)-10(-5) M) relaxed proximal urethra. Suramin (10(-4) M) significantly inhibited the relaxation induced by 2-methyl-thio-ATP. The amplitude of these responses was not significantly different between intact and urothelium-free strips and was not blocked by L-NAME (10(-4) M). 5. These results suggest that nitric oxide (NO) is the principal transmitter involved in the non-adrenergic, non-cholinergic (NANC) relaxation of hamster proximal urethra possibly together with another inhibitory transmitter released from nerves. NO can be released from nerves located in the circular smooth muscle layer and in the lamina propria rather than in the urothelium. The reduced neurogenic relaxation in urothelium-free preparations suggests that a NO-dependent inhibitory factor is released from the urothelium. In addition, ATP and prostaglandin E2 may be involved, together with NO, in the urethra during micturition.     

Mechanism involved in the spasmolytic effect of a mixture of two triterpenes, cycloartenol and cycloeucalenol, isolated from Herissanthia tiubae in the guinea-pig ileum

The smooth muscle relaxant properties of a mixture of the two triterpenoids cycloeuclalenol and cycloartenol (CC) isolated from Herissanthia tiubae (Malvaceae) were studied in several smooth muscle preparations. CC inhibited contractions induced by carbachol, histamine and KCl in the guinea-pig ileum, but no spasmolytic activity was found in guinea-pig trachea or rat aorta. In guinea-pig ileum, concentration-response curves to carbachol and CaCl (2) in high K(+) were shifted to the right by CC in a concentration-dependent manner with slopes of the Schild plot differing from the unity. IC(50) values were 3.4 +/- 0.8 x 10 (-5) M and 8.44 +/- 1.87 x 10 (-5) M for carbachol and CaCl(2), respectively. The phorbol ester TPA, which activates protein kinase C (PKC), potentiated contractions induced by submaximal concentrations of carbachol. This potentiation was inhibited by CC. Desensitization of PKC by TPA completely abolished the inhibition produced by CC on carbachol-induced contractions. Together, our results indicate that inhibition of PKC is involved in the spasmolytic effect of CC in the guinea-pig ileum.     

Deficiency of nitric oxide in allergen-induced airway hyperreactivity to contractile agonists after the early asthmatic reaction: an ex vivo study.

1. Using a guinea-pig model of allergic asthma, we investigated the role of nitric oxide (NO) in allergen-induced airway hyperreactivity after the early asthmatic reaction, by examining the effects of the NO-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) on the responsiveness to methacholine and histamine of isolated perfused tracheae from unchallenged (control) animals and from animals 6 h after ovalbumin challenge. 2. All animals developed airway hyperreactivity to inhaled histamine at 6 h after ovalbumin challenge, with a mean 3.11 +/- 0.45 fold increase in sensitivity to the agonist (P < 0.001). 3. In perfused tracheal preparations from the ovalbumin-challenged guinea-pigs, the maximal responses (Emax) to methacholine and histamine were significantly enhanced compared to controls, both after intraluminal (IL) and extraluminal (EL) administration of the contractile agonists. In addition, a small but significant increase in the pD2 (-log10 EC50) for IL and EL methacholine and for IL histamine was observed. As a consequence, the delta pD2 (EL-IL) for histamine was slightly decreased from 1.67 +/- 0.13 to 1.23 +/- 0.14 (P < 0.05). However, the delta pD2 for methacholine was unchanged (1.85 +/- 0.11 and 1.77 +/- 0.12, respectively; NS). 4. Incubation of control tracheae with 100 microM L-NAME (IL) significantly enhanced the Emax for both IL and EL methacholine and histamine to approximately the same degree as observed after ovalbumin challenge, with no effect on the pD2 and delta pD2 for both agonists. On the contrary, L-NAME had no effect on Emax and pD2 values of tracheal preparations from ovalbumin-challenged guinea-pigs. 5. L-NAME (10 microM-1 mM) had no effect on methacholine-induced contraction of isolated tracheal strip preparations obtained from control animals, indicating that L-NAME has no antimuscarinic effect on tracheal smooth muscle. 6. Histological examination of the intact tracheal preparations indicated epithelial and subepithelial infiltration of eosinophils after ovalbumin challenge. However, no apparent damage of the airway epithelium was observed in these preparations. 7. The results indicate that a deficiency of NO contributes to allergen-induced airway hyperreactivity after the early asthmatic reaction and that this deficiency appears not to be due to epithelial shedding.     

Contribution of RhoA kinase and protein kinase C to weak relaxant effect of pinacidil on carbachol-induced contractions in sensitized guinea-pig trachealis

The exact mechanisms underlying the weak bronchodilator effect of K_ATP channel openers on cholinergic stimulations is unknown. The present study was designed to examine the relaxant efect of pinacidil in guinea-pig trachea stimulated with carbachol by the presence of calcium sensitizer inhibitors; HA 1077, a rhoA kinase inhibitor, and chelerythrine, a protein kinase C inhibitor. Adenosine (10 μM) was used as other contractile agent for comparison. Tracheal tissues were isolated from ovalbumin sensitized guineapigs and changes in tension were recorded isometrically. Pinacidil (1–100 μM, cumulatively) and HA 1077 (0.01–30 μM, cumulatively) produced concentration-dependent relaxations in unstimulated tisues. The relaxant response to pinacidil decreased in carbachol contracted tissues, but increased in adenosine-stimulated tissues. Pretreatment of the tissues with HA 1077 (0.1 μM) and chelerythrine (10 μM) increased the pinacidil-induced relaxations by ∼%100 and %40, respectively. Glibenclamide, a KATP channel blocker, partially antagonized the pinacidil response in contracted tissues. Glibenclamide also inhibited the carbachol and adenosine induced contractions. These results suggest that diminish effect of pinacidil may have related to the enhanced calcium sensitization by cholinergic stimulation. Rho kinase inhibitors appear more effective than PKC inhibitors to achieve of this failure. Two authors made an equal contribution to this study.