Relaxation Of Guinea-pig Trachea Research Articles

The Flavone Luteolin, an Endocrine Disruptor, Relaxed Male Guinea Pig Gallbladder Strips.

BackgroundLuteolin (3',4',5,7-tetrahydroxyflavone) is a flavone with a yellow crystalline appearance present in numerous plants such as broccoli, green chili, and carrot. Luteolin is considered to be an endocrine disruptor with potent estrogen agonist activity and potent progesterone antagonist activity. Luteolin has effects on smooth muscle. Luteolin relaxed guinea pig trachea smooth muscle as it inhibited both phosphodiesterase and reduced intracellular Ca2+. Luteolin also caused vasorelaxation in rat thoracic aorta smooth muscle by inhibiting intracellular Ca2+ release, inhibition of sarcolemmal Ca2+ channels, and activation of K+ channels. Luteolin or its glycosides from artichoke extracts may have an ameliorating effect on irritable bowel syndrome. The purpose of this study was to determine if luteolin had an effect on gallbladder motility.MethodsAn in vitro pharmacologic technique was utilized. Either cholecystokinin octapeptide (CCK) or KCl were used to induce tension in male guinea pig gallbladder strips maintained in Sawyer-Bartlestone chambers. Luteolin relaxed either the CCK- or KCl-induced tension in a concentration dependent manner. Various blockers were added to the chambers to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant.ResultsTreatment of the gallbladder strips with luteolin prior to either KCl or CCK significantly (P < 0.001) decreased the amount of either KCl- or cholecystokinin-induced tension. The 2-aminoethoxydiphenylborane was used to ascertain if the release of intracellular Ca2+ mediated the luteolin-induced relaxation. It significantly (P < 0.001) decreased the amount of luteolin-induced relaxation. To ascertain if PKA mediated the luteolin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < 0.01) reduced the amount of luteolin-induced relaxation. Neither KT5823, NG-methyl-L-arginine acetate salt, genistein, tetraethylammonium, nor fulvestrant had a significant effect. To ascertain if PKC mediated the luteolin-induced relaxation, the PKC inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together. They had no significant effect.ConclusionsLuteolin relaxed cholecystokinin- or KCl-induced tension by blocking extracellular Ca2+ entry as well as intracellular Ca2+ release. In addition, the actions of PKA are also involved in mediating the luteolin effect.

Spasmolytic activity of trachylobanes ent-7α-acetoxytrachyloban-18-oic acid and ent-7α-hydroxytrachyloban-18-oic acid isolated from Xylopia langsdorfiana A. St-Hil. & Tul. (Annonaceae)

We aimed to investigate the possible spasmolytic activity of ent-7α-acetoxytrachyloban-18-oic acid (1) and ent-7α-hydroxytrachyloban-18-oic acid (2) on smooth muscle models. In male rat aorta and rat uterus, both diterpenes were unable to trigger spasmolytic action. However, 2 relaxed guinea-pig trachea: Compounds 1 and 2 antagonised, significantly and concentration-dependently, carbachol- and histamine-induced phasic contractions in guinea-pig ileum. Moreover, they induced a significant and concentration-dependent relaxation in pre-contracted (KCl, carbachol or histamine) guinea-pig ileum, with 2 being 15 times more potent than 1 in histamine-contracted ileum. These dissimilar results may be due to chemical differences between them. Thus, we demonstrated that 1 and 2 seem to be promising spasmolytic agents, although further studies are required to elucidate the spasmolytic action mechanism.

Gnaphaliin A and B relax smooth muscle of guinea-pig trachea and rat aorta via phosphodiesterase inhibition†

To explore the relaxant mechanism of action of gnaphaliin A and gnaphaliin B in guinea-pig trachea and rat aorta, and to investigate the theoretical and experimental phosphodiesterase (PDE) inhibitory activity of these flavones. The relaxant effect and the inhibition of calcium chloride induced contractions of both flavones were evaluated on guinea-pig trachea and rat aorta rings. The PDE inhibitory activity was evaluated using a cyclic nucleotide PDE colorimetric assay kit with cAMP and cGMP as substrates. The docking analysis was carried out with AutoDock4 software and X-ray structure of PDE type 5. The activity of both gnaphaliins was compared with the activity of sildenafil, rolipram, aminophylline, IBMX and enoximone. Gnaphaliin A and B were more actives as relaxants on rat aorta than guinea-pig trachea. They were less potent in the relaxation of guinea-pig trachea and rat aorta than sildenafil, but they were equal or more potent than the other PDE inhibitors tested. The relaxant effect of these flavones was potentiated by nitroprusside and forskolin, and blocked by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one but not by 2',5'-dideoxyadenosine in guinea-pig trachea. L-NAME did not modify the relaxant effect of gnaphaliins. Gnaphaliins were more potent as PDE inhibitors when cGMP was used as substrate. Docking analysis revealed that gnaphaiins bind to the same binding site of sildenafil at PDE type 5. The results suggest that the main relaxant mechanism of action of gnaphaliin A and B is inhibition of PDEs with a preference to inhibit the degradation of cGMP. The docking study suggested that these flavones bind with high specificity to the same binding site of sildenafil at PDE type 5.

Effects of pulmonary metabolites of prostaglandins E2 and F2α on guinea-pig respiratory tract

The effects of three pulmonary metabolites of prostaglandins E2 and F2alpha on the guinea-pig tracheal muscle in vitro and on lung resistance in vivo were investigated. Prostaglandin F2alpha, 13,14-dihydro PGF2alpha and 13,14-dihydro 15-oxo PGF2alpha produced contractions on the tracheal muscle in vitro. 15-oxo PGF2alpha relaxed some tracheal preparations but stimulated others, the stimulant action having a threshold dose range some 10-25 times lower than the relaxant doses. Prostaglandin F2alpha and 13,14-dihydro PGF2alpha increased lung resistance in anaesthetized guinea-pigs. Prostaglandin E2 and its three pulmonary metabolites produced relaxation of guinea pig trachea in vitro and decreased lung resistance in vivo. All three metabolites were less active than PGE2 in both the systems. The in vitro effects of PGF2alpha and its metabolites were selectively blocked by polyphloretin phosphate.

The relaxant effect of extract of Phyllanthus urinaria in the guinea-pig isolated trachea. Evidence for involvement of ATP-sensitive potassium channels.

This study analyses the relaxation induced by the hydroalcoholic extract of stems, leaves and roots from Phyllanthus urinaria (Euphorbiaceae) in the guinea-pig trachea (GPT) pre-contracted by carbachol. The hydroalcoholic extract of P. urinaria (0.1-10 mg mL-1) caused a graded relaxation in GPT with or without epithelium, with mean EC50 values of 1.94 (1.41-2.67) and 2.00 (1.47-2.78) mg mL-1 and Emax of 717 mg (+/- 16) and 627 mg (+/- 12), respectively. The relaxation in response to hydroalcoholic extract, like that to cromakalim (EC50 3.57 (2.75-4.64 microM) in GPT without epithelium, was fully abolished in the presence of high KCl concentrations (80 mM), and was significantly attenuated by tetraethylammonium (10 or 30 mM) or glibenclamide (0.1 or 3 microM). However, the relaxation caused by the hydroalcoholic extract was unaffected by apamin (0.1 or 1.0 microM), nitro-L-arginine (L-NOARG, 100 microM), methylene blue (10 microM) or by calcitonin gene-related peptide (CGRP) (8-37) (a CGRP antagonist, 0.1 microM). Both propranolol (1 or 3 microM) and [D-p-Cl-Phe6,Leu17]VIP (a vasoactive intestinal peptide (VIP) receptor antagonist, 0.1 microM) produced a significant displacement to the right (about 2-fold) of the relaxation response to hydroalcoholic extract of P. urinaria. Thus, the present results indicate that the ATP-activated potassium channels sensitive to glibenclamide, but not the small conductance calcium-activated potassium channels sensitive to apamin, largely contribute to the relaxation effect of the hydroalcoholic extract of P. urinaria in GPT. In addition, both beta 2 and VIP-mediated responses seem to account, at least in part, for the relaxation effect of the hydroalcoholic extract, as its relaxant response was partially attenuated by both propranolol and VIP receptor antagonist.

Spasmolytic effect of galetin 3,6-dimethyl ether, a flavonoid obtained from Piptadenia stipulacea (Benth) Ducke

Piptadenia stipulacea (Benth) Ducke is a tree of the Caatinga, in Northeast Brazil, popularly known as "Jurema-branca", "Jurema malícia-da-serra", "Carcará" and "Calumbi". In folk medicine, a decoction or tincture of its bark and leaves are used to treat wounds and as healing agents. Galetin 3,6-dimethyl ether (FGAL) is a flavonoid isolated from the aerial components of Piptadenia stipulacea (Benth) Ducke. We decided to investigate a possible FGAL spasmolytic effect on preparations of both the guinea pig ileum and trachea, the rat uterus and the male rat aorta. FGAL inhibited oxytocin (IC(50) = 2.2 ± 0.4 × 10(-5) M) and carbachol (CCh)-induced (IC(50) = 7.7 ± 1.3 × 10(-5) M) phasic contractions in the rat uterus, but was more effective in the inhibition of the oxytocin-induced contractions. In the guinea pig ileum, FGAL equipotently inhibited CCh (IC(50) = 2.8 ± 0.4 × 10(-5) M) and histamine-induced (IC(50) = 2.3 ± 0.5 × 10(-5) M) phasic contractions. FGAL equipotently and concentration-dependently relaxed guinea pig trachea preparations pre-contracted with CCh, both in the absence (EC(50) = 0.8 ± 0.1 × 10(-5) M) and presence (EC(50) = 1.0 ± 0.1 × 10(-5) M) of a functional epithelium. FGAL also relaxed preparations of the rat aorta pre-contracted with phenylephrine in both the absence (EC(50) = 5.0 ± 1.1 × 10(-6) M) and presence (EC(50) = 5.4 ± 1.2 × 10(-6) M) of a functional endothelium. FGAL shows a non-selective spasmolytic effect on each of the smooth muscle preparations we have tested, but with a greater effect on those from the rat aorta. The relaxant effect on preparations of both the guinea pig trachea and the rat aorta seems to not involve the epithelium or endothelium-derived relaxing factors.

Mechanisms involved in the relaxant action of the ethanolic extract of propolis in the guinea-pig trachea in-vitro.

This study examines the mechanisms by which the standardised ethanolic extract of propolis induces relaxation of the guinea-pig trachea in-vitro. In guinea-pig trachea with or without epithelium and contracted by histamine, the propolis extract caused reproducible and graded relaxation, with a mean EC50 value of 3.8 or 10.5 microg mL(-1) and Emax of 100%, respectively. The propolis extract-induced relaxation was markedly reduced (26+/-9 and 96+/-3%) when guinea-pig tracheas were exposed to Krebs solution containing elevated K+ in the medium (40 or 80 mM). Pre-incubation of guinea-pig tracheas with tetraethylamonium (100 mM) or with 4-aminopyridine (10mM) reduced the propolis extract-induced relaxation by 31+/-10% and 28+/-2%. Likewise, apamin (0.1 microM), charybdotoxin (0.1 microM) or iberiotoxin (0.1 microM) caused marked inhibition of propolis extract-mediated relaxation in guinea-pig trachea (percentage of inhibition: 65+/-3%, 60+/-5% and 65+/-9%, respectively). Also, glibenclamide (1 microM) inhibited the relaxant response caused by the propolis extract by 57+/-4%. Omega-conotoxin GIVA (0.1 microM) or capsaicin (1 microM) produced small but significant inhibition (30+/-5% or 47+/-7%, respectively) of the propolis extract-induced relaxation. The vasoactive intestinal peptide (VIP) antagonist D-p-Cl-Phe6,Leu17[VIP] porcine (0.1 microM) inhibited relaxation by 55+/-5%, while propranolol (1 microM) induced a parallel rightward displacement (about 20 fold) of the propolis extract concentration-response curve. Finally, the propolis extract-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-N(G)-nitroarginine (L-NOArg, 100 microM) (48+/-6%), and by the soluble guanylatecyclase inhibitormethylene blue (10 microM) (37+/-6%), whilethe moreselectivesoluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolol[4,3-alquinoxalin-1-one (ODQ, 1 microM) produced only a parallel (about 3 fold) rightward displacement of the propolis extract concentration-response curve. Collectively, these results support the notion that the propolis extract-mediated relaxation in the guinea-pig trachea involves the release of nitric oxide, probably from sensory neurons, besides the activation of soluble guanylate cyclase and activation of Ca2+- and ATP-sensitive K+ channels. Furthermore, the stimulation of beta2-adrenergic and VIP receptors also seems to account for its relaxant action.

Bronchodilator activity of Mikania glomerata Sprengel on human bronchi and guinea-pig trachea.

The effects of aqueous extracts and hydro-alcoholic extract (HAE), and of a dichloromethane fraction (MG1) obtained from the HAE of Mikania glomerata leaves on isolated respiratory and vascular smooth muscle have been investigated. Aqueousextracts and HAE induced a significant inhibition on the histamine contractions on the isolated guinea-pig trachea. HAE extract induced a concentration-dependent relaxation on guinea-pig trachea pre-contracted with histamine (IC50 0.34 (0.29-0.39) mg mL(-1)), acetylcholine (IC50 0.72 (0.67-0.77) mg mL(-1)) or K+ (IC50 1.41 (1.18-1.64) mg mL(-1)) and on isolated human bronchi precontracted with K+ (IC50 0.34 (0.26-0.42) mg mL(-1)). The dichloromethane fraction induced a concentration dependent relaxation in guinea-pig trachea precontracted with K+ (IC50 0.017 (0.012-0.022) mg mL(-1)). The dichloromethane fraction had also a small vasodilator effect on the isolated mesenteric vascular bed and on the isolated rat aorta, and a significant reduction of the oedema induced by subplantar injections of Bothropsjararaca venom in mice. When tested on plasmid DNA, MG1 did not damage the DNA. Chromatographic analysis showed the presence of 11.4% w/w coumarin in MG1. The results supported the indication of M. glomerata products for the treatment of respiratory diseases where bronchoconstriction is present.

Effects of hyperosmolarity and airway epithelial ion transport inhibitors on sodium nitroprusside-induced relaxation of guinea pig trachea

Increased airway surface osmolarity has been shown to abolish the airway relaxant effects of inhaled nitric oxide. We have investigated the effects of increased airway surface osmolarity on airway relaxation induced by nitric oxide. The physiological responses, obtained by a guinea pig tracheal perfusion method, were compared to the ion content of the tracheal tissues, and the effects of amiloride or furosemide were studied. Hyperosmolarity decreased the ability of sodium nitroprusside (SNP) to relax carbachol-constricted trachea. Light microscopy showed shrinkage of the epithelial cells and X-ray microanalysis showed increased epithelial ion content under conditions of intraluminal hyperosmolarity, suggesting dehydration of the epithelium. Amiloride treatment reduced the increase in epithelial ion content but had no effect on shrinkage or SNP-induced relaxation. Furosemide had no effect on the altered ion content, shrinkage, or on SNP-induced relaxation. In conclusion, neither amiloride nor furosemide can counteract the shrinkage of the airway wall induced by increased osmolarity in the lumen of guinea pig trachea in vitro, nor can they affect the reduction in relaxing response to the NO-donor SNP.

Effects of cannabinoids on non-adrenergic non-cholinergic-mediated relaxation in guinea-pig trachea

The effects of cannabinoid receptor agonists on the non-adrenergic non-cholinergic (NANC) inhibitory responses to electrical field stimulation in guinea-pig trachea were assessed. R-(+)-[2,3-dihydro-5-methyl-3-[(morpholilinyl) methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2; 10 −5 M) significantly enhanced the frequency-dependent response to electrical stimulation. The same concentration of R-( N)-(2-hydroxy-1-methylethyl)-5 Z,8 Z,11 Z,14 Z-eicosatetraenamide (R(+)methanandamide) and 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015) did not affect significantly the electrically induced inhibitory NANC responses. The effect of WIN 55,212-2 was not modified by the cannabinoid CB 1 and CB 2 receptor-selective antagonists, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10 −5 M) and N-(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528; 10 −5 M), respectively. Moreover, the nitric oxide synthase inhibitor, l- N G-nitro-arginine methyl ester ( l-NAME; 10 −4 M), but not the peptidase, α-chymotrypsin (2 U/ml), blocked the effect of WIN 55,212-2. Postsynaptically, WIN 55,212-2 did not produce any change of tracheal smooth muscle tone, either basal or histamine-induced, and did not interfere with the relaxant activity of the nitric oxide donor, sodium nitroprusside (10 −8–10 −4 M). In conclusion, our results suggest that (a) cannabinoid CB 1 and CB 2 receptor stimulation does not alter the inhibitory NANC transmission in guinea-pig trachea, and (b) WIN 55,212-2 potentiates the NO-mediated component of the NANC relaxant response to electrical stimulation through a cannabinoid receptor-independent mechanism.

Role of BK Ca channels and cyclic nucleotides in synergistic relaxation of trachea

β-Adrenoceptor agonists, nitric oxide (NO), and NO donors have been shown to mediate their effects through large conductance Ca 2+-activated K + (BK Ca) channels. The mechanism of the synergistic effect of the β 2-adrenoceptor agonist, salbutamol, and an NO donor, sodium nitroprusside, was studied in guinea pig tracheal preparations. Salbutamol (0.1 nM) and sodium nitroprusside (0.33 μM) alone relaxed the acetyl-β-methylcholine chloride (methacholine)-contracted preparations only by 0.5% and 28%, respectively, but their combination caused a maximum of 60% relaxation (at 3 min), which stabilized to 40% (at 10 min). Iberiotoxin, a selective inhibitor of the BK Ca channels, did not abolish the synergistic effect. 3-isobutyl-1-methylxanthine (IBMX) did not modify relaxation evoked by the drugs. Concentrations of cyclic nucleotides did not correlate with relaxations as a function of time. The mechanism of synergy remains to be clarified. The results show that NO is an important modulator in the relaxation of guinea pig trachea induced by β 2-adrenoceptor agonists in vitro.

Interactions between β 2-adrenoreceptor agonist and NO donors in the relaxation of guinea pig trachea in vitro

The effects of interaction between the β 2-adrenoreceptor agonist, salbutamol, and the nitric oxide donors, sodium nitroprusside and 3-morpholinosydnonimine (SIN-1), on guinea pig trachea contraction were studied. Cumulatively increased concentrations (0.1–10 nM) of salbutamol together with a single concentration of SNP (0.33 μM) or with SIN-1 (1 μM) showed significant ( p<0.001) synergy for the inhibition of 1 μM metacholine-induced contraction. Significant synergy ( p<0.05) was also found for the inhibition of this contraction by cumulatively increased concentrations (0.1–33 μM) of SNP and a single concentration (1 nM) of salbutamol. No synergistic effect was found on the 40 mM KCl-induced contraction. We suggest that the combination of NO donors with salbutamol has a synergistic effect on metacholine contraction.

Flufenamic and tolfenamic acids and lemakalim relax guinea-pig isolated trachea by different mechanisms

The effects of K + channel inhibitors on the relaxations induced by flufenamic and tolfenamic acids and lemakalim were examined in guinea-pig isolated trachea precontracted with prostaglandin F 2α (PGF 2α, 1 μM). Flufenamic and tolfenamic acids (0.1–33 μM) and lemakalim (0.01–33 μM) relaxed guinea-pig trachea in a concentration-dependent manner. Tetraethylammonium (0.5–2 mM), a nonspecific inhibitor of K + channels, inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation Charybdotoxin (ChTX, 33–100 nM), an inhibitor of the large Ca 2+-activated K + channels (BK Ca), also inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Glipizide (3.3–33 μM), an inhibitor of the ATP-sensitive K + channels (K ATP) inhibited lemakalim-induced relaxation without affecting those induced by flufenamic and tolfenamic acids. Our results indicate that the relaxations of guinea-pig isolated trachea by flufenamic and tolfenamic acids are due to activation of BK Ca. The relaxant mechanism of flufenamic and tolfenamic acids thus differs from that of lemakalim, an activator of K ATP

SCA 40: studies of the relaxant effects on cryopreserved human airway and vascular smooth muscle

1. 6-Bromo-8-methylaminoimidazol[1,2-a]pyrazine-2carbonitrile (SCA 40) has been claimed to induce relaxation in guinea-pig trachea by opening high conductance, calcium-activated potassium (BKCa) channels. The mechanism of action of SCA 40 has now been further investigated in ring preparations from cryopreserved human airway and vascular smooth muscle preparations in vitro. 2. Human bronchi with spontaneous tone relaxed in response to SCA 40 in a biphasic way. A high affinity component (pD2 8.61 +/- 0.21; mean +/- s.e.mean) accounted for 30% of the response and a low affinity component (pD2 6.53 +/- 0.14) for the remaining 70%. In contrast, in bronchi contracted with carbachol, 1 microM, the concentration-response curve to SCA 40 was monophasic and yielded a pD2 of 6.31 +/- 0.29. 3. SCA 40 relaxed pulmonary and mesenteric arteries and peripheral veins which had been precontracted by 10 nM U46619 nearly completely and in a monophasic way; the pD2 values were 6.37 +/- 0.08, 6.17 +/- 0.15 and 5.45 +/- 0.25, respectively. 4. Lemakalim, an opener of ATP-dependent potassium (KATP) channels, also relaxed human bronchi under spontaneous tone and the vascular tissues. NS 1619, a recognised opener of BKca channels, was inactive up to 10 microM on bronchial and vascular tissues. 5. The SCA 40-induced relaxation of human bronchi was reduced concentration-dependently in the presence of high potassium chloride (20 and 80 mM). However, in the presence of 80 mM KCl and nifedipine, 30 nM, SCA 40 fully relaxed the remaining contractile response with pD2 values of 8.08 +/- 0.13 and 5.27 +/- 0.13 for the high and low affinity component, respectively. 6. Relaxation responses to SCA 40 in human bronchi were resistant to blockade by glibenclamide at concentrations up to 10 microM (which blocked the relaxant response to lemakalim), quinine (30 microM), apamin (100 nM), tetraethylammonium (0.1-1 mM) and charybdotoxin (10-100 nM), thus excluding the involvement of a variety of K+ channels including KATP and KCa channels. 7. In bronchi contracted with carbachol, 1 microM, the nature of the interaction between SCA 40 and the beta 2-adrenoceptor agonist, salbutamol, was synergistic. 8. These experiments establish that SCA 40 is a potent relaxant of human bronchial smooth muscle manifesting spontaneous tone. A low affinity relaxant component has its counterpart in the relaxation seen in both human arterial and venous smooth muscle. The consensus of the evidence suggests that K+ channel opening is not the basis of the relaxant response to SCA 40. Furthermore, BKCa channels appear to be of minor importance in the regulation of human airway smooth muscle tone. Our data suggest that inhibition of an adenosine 3':5'-cyclic monophosphate phosphodiesterase may contribute, at least to the low affinity relaxant component of SCA 40. However, the exact mechanism mediating the SCA 40-induced relaxation of human airways remains to be defined.

Role of nitric oxide in nonadrenergic, noncholinergic relaxation of whole tracheal tube preparations isolated from guinea pigs

1. 1. Frequency-dependent nonadrenergic, noncholinergic (NANC) relaxant responses were induced by transmural stimulation of whole tracheal tube preparations. 2. 2. Responses at lower frequencies (⩽10 Hz) were abolished by L-nitroarginine methyl ester ( L-NAME). 3. 3. Responses at higher frequencies (⩾20 Hz) consisted of a rapid, short-lasting relaxation, followed by a slow, long-lasting relaxation. The former and the latter were reduced by L-NAME and α-chymotrypsin, respectively. 4. 4. α-Chymotrypsin had little effect on the magnitude of NANC responses, but reduced the duration of responses at higher frequencies (⩾20 Hz). 5. 5. The results suggest that NANC relaxation of guinea pig trachea may be mediated primarily by nitric oxide, with and without concomitant release of vasoactive intestinal peptide or related peptides, and nitric oxide may act as predominant mediator providing the magnitude of relaxant response.

Relaxation of guinea-pig trachea by sodium nitroprusside: cyclic GMP and nitric oxide not involved.

1. Sodium nitroprusside (SNP) completely relaxed the guinea-pig isolated, perfused trachea in a concentration-dependent manner. Although SNP was less potent by about 2 orders of magnitude, its maximal effect was 25% higher compared to isoprenaline. 2. SNP (3.2 microM) increased cyclic GMP levels by 300% and relaxed guinea-pig isolated, perfused trachea by 54%. The SNP-induced relaxations of the preparations were not affected by the guanylate cyclase inhibitor, methylene blue. Moreover, zaprinast, a cyclic GMP-specific phosphodiesterase inhibitor which was supposed to enhance SNP-induced relaxations, decreased the maximal relaxation by 22% (P < 0.001). 3. In contrast, 8Br-cyclic GMP (10 microM) increased the cyclic GMP levels by 1100% without inducing a marked relaxation. 4. SNP (10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; a direct donor of nitric oxide; 10 microM), relaxed the tissues by 75% and 25%, respectively, without any nitric oxide (NO) release by SNP (< 1 pmol 100 microliters-1), but a substantial NO release by SNAP (560 pmol 100 microliters-1). 5. It is concluded that the SNP-induced tracheal relaxations are probably not mediated by cyclic GMP and NO.

Effects of in vitro 5-HT 1 receptor activation in guinea pig trachea

5-hydroxytryptamine (5-HT) has been reported to show some effects in respiratory tissues by activation of different subtype receptors. It has been demonstrated that 5-HT 2 receptor activation causes in vivo and in vitro airways contraction and enhances effects of cholinergic nerve-mediated responses, whereas 5-HT 1, receptor activation seems to be related to a relaxant effect. Moreover, in isolated guinea pig ascendens colon preparations 5-HT 1, activation causes relaxation by involvement of nitric oxide (NO). The aim of this study was to investigate the effects of 5-HT 1 receptor activation in guinea pig trachea as well as NO probable role in this activation. In tissues pretreated with both ketanserin (10 μM), an antagonist of 5-HT 2 receptors, and ondansetron (10 μM), an antagonist of 5-HT 3 receptors, 5-HT (from 10 nM to 10 mM) relaxed guinea pig trachea precontracted with acetylcholine (ACh, 100 μM). Carboxamidotryptamine (5-CT, from 10 nM to 10 mM), an agonist of 5-HT 1 receptors, as well relaxed guinea pig trachea precontracted with ACh (100 μM). A pretreatment with NAN-190 (from 10 nM to 100 μM), a 5-HT 1A selective antagonist, reduced the 5-HT and 5-CT relaxant effects but only at very high concentrations. Finally, a pretreatment with L-nitro-arginine-methyl-ester (L-NAME, 1 mM), an inhibitor of NO-synthase, and L-arginine (L-ARG, 1 mM), a precursor of NO synthesis, did not modify 5-HT and 5-CT responses in guinea pig trachea. In conclusion, this study suggests a 5-HT relaxant activity in guinea pig trachea via a 5-HT 1 receptor activation without any NO pathway involvement. However, further investigations are needed to clarify which 5-HT 1 receptor subtype is involved in 5-HT relaxant effect.

Induction of guinea pig airway hyperresponsiveness by inactivation of guanylate cyclase

To examine the role of cyclic 3′,5′-guanosine monophosphate (cGMP) in airway responsiveness the effects of substances known to interfere with nitric oxide (NO) or cGMP were investigated on guinea pig airways. Using a perfused organ bath system, it was possible to apply the chemicals from either the serosal or the mucosal side independently. In addition, levels of intracellular cGMP were determined in tissues after various treatments. Sodium nitroprusside (a donor of NO), zaprinast (a specific inhibitor of cGMP phosphodiesterase) and 8-bromo-cGMP (8-Br-cGMP) caused a concentration-dependent relaxation of guinea pig trachea. These results indicate that cGMP is an important second messenger mediating tracheal relaxations. The above mentioned drugs caused a more profound relaxation when applied to the serosal side compared to the mucosal side, suggesting a barrier function of the epithelial layer. Incubation on the mucosal side of the tissues with 100 μM pyrogallol (a generator of superoxide that may inactivate NO) increased the contractile response to histamine at concentrations 0.3–3.2 μM ( P < 0.05). Treatment of the preparations with 1 mM cystamine (an inactivator of guanylate cyclase) caused a 5-fold increase in the sensitivity to histamine ( P < 0.05), indicating the involvement of the NO/cGMP pathway in the development of airway hyperresponsiveness. Incubation of the tissues with 100 μM histamine elevated the intracellular cGMP levels 10-fold; this effect was completely prevented by incubation of the tissues with methylene blue (a potent inactivator of guanylate cyclase). Mucosal incubation of the tracheal tubes with 10 μM methylene blue induced an 8-fold increase in sensitivity to histamine ( P < 0.01) and the E max was slightly increased. 25 min after instillation of 0.4 μmol methylene blue into the airways of anaesthetized guinea pigs, the lung resistance in response to histamine was elevated up to 395 ± 82% (P < 0.001). The present study revealed that inactivation of NO or guanylate cyclase enhances the histamine-induced contractions of guinea pig tracheas. Therefore, it is suggested that the NO/cGMP pathway may be implicated in the pathogenesis of airway hyperresponsiveness and that drugs which enhance cGMP levels in airway smooth muscle may be of significance in the treatment of airway obstruction and enhanced reactivity.

Different mechanisms involved in relaxation of guinea-pig trachea by endothelin-1 and -3

The effects of endothelin-1 and endothelin-3 were investigated on carbachol-contracted guinea-pig isolated trachea. Endothelin-1 and endothelin-3 (0.1–100 nM) induced partial dose-dependent relaxation of the precontracted preparations. The endothelin-1-induced relaxation was markedly attenuated by haemoglobin (10 μM) and methylene blue (10 μM) and by epithelium removal. In contrast, endothelin-3-induced relaxation was not affected by haemoglobin, methylene blue or epithelium removal. The large conductance Ca 2+-activated K +-channel blocker, charybdotoxin, antagonized the endothelin-1- and the endothelin-3-induced relaxation to the same extent. These results show that both endothelin-1 and endothelin-3 relaxant activities are modulated by charybdotoxin-sensitive K +-channels, while the nitric oxide pathway is only involved in endothelin-1 relaxant effects.

Mechanisms involved in the relaxant response of bradykinin in epithelium intact strips of the guinea-pig trachea

Kinins caused graded relaxations in guinea-pig trachea with epithelium under spontaneous or carbachol-induced tone. The order of potency was: [Tyr 8]bradykinin > lysyl-bradykinin > bradykinin > methionyl-lysyl-bradykinin. The bradykinin B 1 receptor agonist des-Arg 9-bradykinin (1 μM) was inactive. Relaxation in response to bradykinin (100 nM) was unaffected by tetrodotoxin (0.3 μM), nicardipine (1 μM), Ca 2+-free solution without or plus ryanodine (10 μM), propranolol (1 μM), glibenclamide (1 μM), staurosporine (0.3 μM), nickel chloride (100 μM) or [ d- p-Cl-Phe 6,Leu 17]VIP (a vasoactive intestinal peptide receptor antagonist, 0.03 μM), but was partially inhibited by apamin (0.3–1 μM). Both HOE 140 ( d-Arg-[Hyp 3,Thi 5, d-Tic 7,Oic 8]bradykinin) and NPC 17761 ( d-Arg 0[Hyp 3, d-Hype E( trans-thiophenyl) 7,Oic 8]bradykinin) (0.1–1000 nM) caused graded, reversible and selective inhibition of the bradykinin (100 nM) relaxation, with IC 50 values of 1.4 and 19.1 nM, respectively. HOE 140 and NPC 17761 (0.1–10 nM) produced a graded shift to the right of the bradykinin concentration-response curves associated with a reduction of the maximum relaxation. The kinin B 1 receptor antagonist, des-Arg 9-[Leu 8]bradykinin (1 μM), was inactive. Thus, bradykinin-induced relaxation in guinea-pig trachea results from activation of bradykinin B 2 receptors and can be antagonized with high affinity in a selective and reversible manner, through noncompetitive mechanism, by both HOE 140 and NPC 17761. In addition, the bradykinin response does not involve neural pathways, extracellular Ca 2+ influx or mobilization of intracellular Ca 2+ stores sensitive to ryanodine, but is modulated by small conductance Ca 2+-activated K + channels.