Isolated Guinea Pig Trachea Research Articles

Pharmacological activities of Lantana trifolia on isolated guinea pig trachea and rat phrenic nerve diaphragm

A methanol extract of L. trifolia produced bronchodilation of isolated guinea pig trachea comparable to that of salbutamol. The plant extract reduced bronchoconstriction induced by histamine, 5-HT and acetylcholine on isolated guinea pig trachea. Physostigmine failed to inhibit neuromuscular blocking activity of the extract on rat phrenic nerve diaphragm.

Involvement of cyclooxygenase 2 (COX-2) in intrinsic tone of isolated guinea pig trachea.

Indomethacin and related nonsteroidal anti-inflammatory drugs relax prostanoid-dependent intrinsic tone of isolated guinea pig trachea by inhibiting cyclooxygenase (COX). Recently, a second isoform of COX (COX-2) was discovered, which differed from COX-1 with respect to protein structure, transcriptional regulation, and susceptibility to inhibition by pharmacological agents. It is now known that indomethacin nonselectively inhibits COX-1 and COX-2, whereas NS-398 is a selective inhibitor of COX-2. In the present study we compared the activity of a selective (NS-398) and nonselective (indomethacin) COX-2 inhibitor on intrinsic tone of isolated guinea pig trachea. NS-398 > or = indomethacin produced a reversal of intrinsic tone with a similar concentration-dependent (10 nM to 1 microM) time course (Tmax approximately 20-45 min), potency (EC50 1.7 and 5.6 nM, respectively), and maximal response. Contractions to cholinergic nerve stimulation (45 V, 0.5 ms, 0.1-32 Hz) and histamine were similarly modulated in tissues relaxed with the selective or nonselective COX-2 inhibitors. Immunoblot analyses showed that COX-2 protein synthesis was induced in both the cartilage and smooth muscle portions of the trachea during changes in intrinsic tone. These findings are consistent with pharmacological results and provide the first demonstration that prostanoid tone in isolated guinea pig trachea is dependent on COX-2 activity. The results also suggest that the activity of indomethacin in this preparation is likely related to COX-2 inhibition.

Pharmacologic Characterization of Wool Dust Extract in Isolated Guinea Pig Trachea

Wool mill workers develop respiratory symptoms and lung function abnormalities associated with their work in the textile industry, As in other workplaces, which process organic materials, the dust generated in the manufacture of wool has been implicated as a cause of these respiratory problems. Pharmacologic studies of wool dust extract were performed in vitro on guinea pig tracheal (GPT) segments. A wool dust extract (WDE) was prepared from material collected from a mill previously surveyed. When the standardized WDE solution was added to an organ bath in increments of 10, 30, 100, 300, and 1000 μl it caused a consistent, dose-dependent constriction of GPT. Pretreatment of guinea pig tracheas, prior to WDE challenge, with atropine (10−6 M), pyrilamine (10−6 M), indomethacin (10−6 M), verapamil (10−6 M), TMB8 (10−6 M), BW755C (10−6 M), and LY171883 (10−6 M) was studied in order to evaluate receptor-dependent and -independent characteristics of WDE-induced constriction. WDE-induced bronchoconstriction was partially inhibited by the antihistamine pyrilamine. Atropine and leukotriene inhibitors (LY171883 and BW755C) were not found to have a significant protective effect on WDE-induced constriction. Both TMB8 and verapamil (intra- and extracellular calcium blocking agents) suppressed the effect of wool dust extract in the range tested. These findings suggest that in this model, WDE-induced airway constriction is only partly attributable to common mediators of bronchoconstriction (e.g., histamine). The airway effects of WDE may be modulated by calcium channel blocking agents.

Pharmacological Activities of Gardenia jovis-tonantis

A methanol extract derived from Gardenia jovis-tonantis showed bronchodilator activity on isolated guinea pig trachea. The extract also showed cardiorelaxant activity on isolated rabbit heart and caused a rapid fall in diastolic pressure in anaesthetised rats.

Endothelin-1 and Endothelin-3 Relax Isolated Guinea Pig Trachea Through Different Mechanisms

Endothelin-1 and Endothelin-3 Relax Isolated Guinea Pig Trachea Through Different Mechanisms

Endothelin-1 and Endothelin-3 Relax Isolated Guinea Pig Trachea Through Different Mechanisms

The effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) were investigated on carbachol-contracted guinea-pig trachea. ET-1 and ET-3 induced a dose-dependent relaxation of the precontracted preparations. ET-1-induced relaxation was attenuated by hemoglobin and methylene blue. By contrast, ET-3-induced relaxation was not affected by hemoglobin or methylene blue. The large conductance Ca(2+)-activated K(+)-channel blocker charybdotoxin antagonized ET-1- and ET-3-induced relaxations. These results show that charybdotoxin-sensitive K+ channels are involved in both ET-1 and ET-3 relaxant activities, whereas the nitric oxide pathway is involved only in ET-1 relaxant effects.

Effects of Sevoflurane on the Isolated Guinea Pig Trachea

Effects of Sevoflurane on the Isolated Guinea Pig Trachea

Evaporation-induced changes in airway surface liquid on an isolated guinea pig trachea.

An isolated preparation of the guinea pig trachea was developed. The trachea was exposed serosally to Krebs-Henseleit solution and mucosally to tidal airflow, designed to mimic conditions in vivo. The preparation establishes stable layers of airway surface liquid (ASL). Typical depth, transepithelial potential differences, and sodium activity are 200 microns, -3 mV, and 125 mM, respectively (approximate sodium concn 170 mM). When exposed to air with a vapor pressure deficit (VPD), evaporation of water occurs from ASL, ASL depth decreases, and the concentration of sodium ions in ASL increases. The water content of air passing over the trachea also increases. This measurement is compared with measurements of the change in volume of ASL, based on depth changes, to yield estimates of net water transport (NWT). Measurements of changes in the sodium content of ASL allow for the calculation of net sodium transport by the trachea. Evaporation rate, changes in the volume of ASL, NWT, and net sodium transport are all influenced by VPD. The results suggest that evaporation from ASL increases its sodium concentration (and osmotic pressure) and increases osmotically driven NWT to replace water lost. Evaporation-induced increases in the sodium concentration appear to be limited by enhanced sodium uptake at high VPD.

Modulation of rate at which serotonin-induced contraction decays in guinea pig trachea.

Stimulation of the type 2 serotonin (5-HT2) receptor in guinea pig trachea with 5-HT results in a contraction that decays in the continued presence of 5-HT. The decay of the 5-HT contraction has been proposed to be dependent on 5-HT2 receptor activation and to reflect desensitization of the receptor. The characteristics of the decay of the 5-HT contraction may also be dependent on other properties of the tissue. The effects of modulation of biochemical pathways implicated in airway smooth muscle contraction on the 5-HT contraction in isolated guinea pig trachea were determined with the use of a kinetic approach we developed previously. Decay of the 5-HT contraction was inhibited by cooling, increased by forskolin, 3-isobutylmethyl-1-xanthine, and 8-bromoadenosine 3',5'-cyclic monophosphate, and unaffected by staurosporine, H-7, H-8, phorbol 12,13-dibutyrate, and by inhibitors of the three major pathways of arachidonic metabolism. The results suggest that decay of the 5-HT contraction in guinea pig trachea is dependent on both the receptor and the biochemical state of the tissue.

P-479 - pharmacological study of ebastine,a novel histamine H1 receptor antagonist.

The anti-allergic activity of ebastine, a novel antihistamine, was assessed in comparison with several antihistamines. 1) Orally administered ebastine dose-dependently inhibited 7-day homologous passive cutaneous anaphylaxis (PCA), experimental allergic rhinitis and experimental asthma in guinea pigs or rats (ED50-values were 2.17, 0.29 and 0.35 mg/kg, respectively); and its anti-allergic activity was more potent than those of terfenadine and mequitazine. Moreover, its PCA-inhibitory activity was still observed 24 hr after the administration. 2) Orally administered ebastine also inhibited histamine-induced skin reaction in rats (ED50: 1.10 mg/kg). 3) In isolated guinea pig trachea, ebastine had no effect on histamine-induced contraction, but carebastine, a main metabolite of ebastine, inhibited this contraction (IC50: 0.12 microM). 4) Carebastine (30-100 microM) suppressed the histamine release from rat peritoneal mast cells and human basophils. 5) Ebastine at a high oral dose showed slight inhibition of the specific binding of 3H-mepyramine to the histamine H1-receptor in rat brain. This binding-inhibitory activity of ebastine was little more potent than that of terfenadine, but much less potent than those of mequitazine and ketotifen. These results indicated that ebastine has potent and long acting anti-allergic activity with few side effects based on the antihistaminic activity in the central nervous system. Furthermore, it was suggested that these effects of ebastine are due to the action of a main metabolite, carebastine.

The effects of IAPP and CGRP on guinea pig tracheal smooth muscle in vitro

Neither the novel peptide, islet amyloid polypeptide (IAPP), nor its homologue, calcitonin gene-related peptide (CGRP), contracted guinea pig isolated trachea (GPT), but on preparations contracted with KCl (40 m M), both caused concentration-related relaxation (1 n M–3 μ M). Relaxant curves to both were shallow and slow in development, with clear maxima not being obtained. The protease inhibitors, phosphoramidon (1 μ M), leupeptin (50 μ M), bestatin (100 μ M), soya bean trypsin inhibitor (1 μ M), and aprotinin (5 μ M), together had no effect on relaxations to CGRP, but depressed those to IAPP. Aprotinin appeared to be responsible for this depression. The specific CGRP antagonist, CGRP(8–37), 1–10 μ M, had no effect on relaxations to either peptide. These findings demonstrate that IAPP relaxes GPT in a similar manner to CGRP.

The effects of PACAP and VIP on guinea pig tracheal smooth muscle in vitro

Neither pituitary adenylate cyclase activating polypeptide-38 (PACAP) nor its homologue, vasoactive intestinal polypeptide (VIP), contracted guinea pig isolated trachea (GPT), but on preparations contracted with KCl (40 m M), both caused concentration-related relaxation (3 n M-3 μ M, VIP IC 50 = 72 n M , PACAP IC 50 = 224 n M ). Relaxant curves to PACAP were slower in reaching a maximum than those to VIP (∼ 150 and 50 min, respectively). The protease inhibitors, phosphoramidon (1 μ M), leupeptin (50 μ M), bestatin (100 μ M), soya bean trypsin inhibitor (1 μ M), and aprotinin (5 μ M), together caused a small enhancement of relaxations to VIP, but not to PACAP. The VIP antagonist, [4-Cl- d-Phe 6,Leu 17]VIP (1–10 μ M), did not inhibit the relaxation to either peptide, but did cause large contractions, which were enhanced by protease inhibition. These findings demonstrate that PACAP relaxes GPT in a similar manner to VIP.

Modulation of vasoactive intestinal peptide pulmonary relaxation by NO in tracheally superfused guinea pig lungs.

The mechanism of vasoactive intestinal peptide (VIP)-induced pulmonary relaxation in tracheally perfused guinea pig lungs was defined with the use of inhibitors of nitric oxide synthase (NOS) and by direct measurement of nitric oxide (NO) equivalents recovered from lung perfusion fluid. Lungs treated with 200 microM NG-nitro-L-arginine were resistant to the relaxant effects of VIP in these lungs; the 50% inhibitory dose (ID50) for VIP was 32 nmol/kg (95% confidence interval, 16-79), which was approximately 100-fold greater than the ID50 of control lungs which was 0.39 nmol/kg, (0.16-0.79, P < 0.0001). This inhibitory effect could be overcome with excess L- but not D-arginine. In contrast, VIP-induced relaxation of isolated guinea pig trachea was not modified by inhibitors of NOS. To confirm that VIP infusion resulted in NO generation in whole lungs, we measured NO equivalents in lung effluent by two distinct technologies. We found that VIP injection caused a significant increase in NO equivalents from 0.11 +/- 0.04 microM to 0.78 +/- 0.15 microM (P < 0.05) and that this increase preceded VIP-induced pulmonary relaxation. Lungs pretreated with the putative guanylyl cyclase inhibitor methylene blue were less responsive to VIP [ID50 4.0 nmol/kg (1.5-10), P < 0.005 compared with control lungs], consistent with a physiologically significant guanosine 3',5'-cyclic monophosphate-dependent mechanism. Our data demonstrate that VIP has the capacity to relax whole lungs in part by stimulating the generation of NO.

Pharmacological studies on Aristolochia papillaris Mast. (Aristolochiaceae)

The non-specific and reversible smooth muscle relaxant activities of the ethanolic extract (EE) of Aristolochia papillaris Mast., a fraction of EE containing tertiary alkaloids (TAF) and of 3 compounds isolated from TAF are reported. In the non-pregnant rat uterus, EE and TAF inhibited both the oxytocin-induced contractions and the amplitude of rhythmic spontaneous contractions. The IC 50 values for EE and TAF were 0.91 and 0.22 μg/ml, respectively in the first experiments while the corresponding values were 1.0 and 0.17 μg/ml in the second case. The rhythmic contractions of the uterus obtained from 21-day pregnant rats were also reduced by EE and TAF with IC 50 values of 25.5 and 11.2 μg/ml, respectively. The relaxation of isolated guinea pig trachea produced by EE and TAF were also observed with the compounds moupinamide, coclaurine and isoboldine isolated from TAF. The IC 50 values of these compounds were 1.58 × 10 −4 M, 3.98 × 10 −4M and 7.10 × 10 −4M, respectively. Propranolol significantly antagonized the effects of coclaurine and isoboldine but failed to inhibit the responses to moupinamide which suggests that the plant constituents produce muscle relaxation by β-adrenoceptor-dependent and -independent mechanisms.

Effects of an NK 1 receptor antagonist, FK888, on constriction and plasma extravasation induced in guinea pig airway by neurokinins and capsaicin

The effects of FK888, an NK 1 receptor antagonist, on airway constriction and airway plasma extravasation induced by neurokinins and capsaicin were investigated in guinea pigs. FK888 inhibited substance P (10 −8 M)- and neurokinin A (10 −9 M)-induced contraction of isolated guinea pig trachea, with IC 50 values of 3.2 × 10 −8 and 4.2 × 10 −6 M, respectively. FK888 given i.v. inhibited substance P (13.5 μg kg −1)-induced airway constriction with an ED 50 value of 0.40 mg kg −1 but did not inhibit neurokinin A (1.1 μg kg −1)- and capsaicin (3.1 μg kg −1)-induced airway constriction at a dose of 1 mg kg −1. On the other hand, FK888 given i.v. inhibited airway plasma extravasation induced by substance P (1.3 μg kg −1), neurokinin A (11 μg kg −1) and capsaicin (100 μg kg −1) with equal potency and ED 50 values of 0.011, 0.0063 and 0.019 mg kg −1, respectively. When FK888 was given locally (into the airway directly) inhibitory activities were more potent than following i.v. administration. In this case FK888 inhibited substance P-, neurokinin A- and capsaicin-induced airway constriction with ED 50 values of 3.2, 190 and 550 μg kg −1, respectively, suggesting that an about 100 times higher dose is reguired to inhibit neurokinin A- and capsaicin-induced airway constriction than substance P-induced constriction. FK888 given orally was also effective in substance P-, neurokinin A- and capsaicin-induced airway plasma extravasation with ED 50 values of 4.2, 5.9 and 9.5 mg kg −1. These results demonstrate that FK888 is an effective in vivo NK 1 receptor antagonist and the different inhibitory activity of FK888 on airway responses suggests that substance P-, neurokinin A- and capsaicin-induced airway plasma extravasation is solely mediated via NK 1 receptors whereas in airway constriction only substance P-induced reaction is mediated via NK 1 receptors.

Inhibition of β-adrenoceptor agonist relaxation of airway smooth muscle by Ca 2+-activated K + channel blockers

In isolated guinea pig trachea contracted by 0.5 mM acetylcholine, the cumulative relaxant concentration-response curves to the β 2-adrenoceptor agonist, salbutamol, were shifted to the right by depolarizing concentrations of KCl, as well as by charybdotoxin, iberiotoxin and tetraethylammonium ion, which are antagonists of the high-conductance Ca 2+-activated K + channel. The shifts produced by KCl (40 mM), charybdotoxin (100 nM), iberiotoxin (50 nM), and tetraethylammonium ion (2 mM) were approximately 230-fold, 10-fold, 78-fold, and 8-fold, respectively. The blockade of β 2-adrenoceptor agonist-induced relaxation by these agents was totally reversed by 0.3 μM nifedipine. Similar reversal was obtained with either 100 μM CdCl 2, or low Ca 2+ (50 μM) Krebs medium. These data suggest that charybdotoxin, iberiotoxin and tetraethylammonium ion, like KCl, cause membrane depolarization which in turn activates voltage-dependent Ca 2+ The influx of Ca 2+ via these channels provides an additional mode to that of release of intracellular Ca 2+ evoked by acetylcholine for maintaining cell Ca 2+ concentration at a high level. This is apparently sufficient to account functionally for the blockade of β 2-adrenoceptor agonist-induced relaxation. In view of this interpretation regarding the action of Ca 2+-activated K + channel antagonists, earlier proposals ascribing the relaxant effect of β 2-adrenoceptor agonists strictly to activation of these channels must be reevaluated.

The interaction of selective and non-selective antagonists with pre- and postjunctional muscarinic receptor subtypes in the guinea pig trachea

Muscarinic receptor antagonists were used to study prejunctional M 2 and postjunctional M 3 receptors in the isolated guinea pig trachea. The effects of four M 2-selective muscarinic receptor antagonists (gallamine, methoctramine, AQ-RA 741 and AF-DX 116) were studied on twitch contractions, elicited by electrical field stimulation, of tracheal ring preparations. M 1-selective (pirenzepine, (+)- and (−)-telenzepine), M 3-selective (4-DAMP-methobromide and UH-AH 371) and non-selective (atropine and ipratropium) muscarinic receptor antagonists were also used. The clear potentiation of the twitch contractions and the subsequent strong inhibition observed with M 2-selective antagonists demonstrate antagonism at prejunctional M 2 and postjunctional M 3 muscarinic receptors, respectively. The maximal potentiation correlated well with the M 2 M 3 - selectivity known from binding experiments: gallamine > methoctramine > AQ-RA 741 > AF-DX 116. Strong correlations were also found between the pEC 20 values for potentiation of the twitch response and the pK i values for bovine cardiac M 2 muscarinic receptors and between the pIC 50 values for inhibition of the twitch response and the pA 2 values for M 3 muscarinic receptors as determined on non-stimulated methacholine-contracted tracheal smooth muscle preparations. Thus, study of the effects of a wide concentration range of putative M 2-selective muscarinic receptor antagonists on the twitch contractions of single tracheal rings induced by low-intensity electrical field stimulation yields information about M 2 M 3 receptor selectivity and about prejunctional M 2 and postjunctional M 3 receptor affinity within the same experiment.

5-HT 3 receptors augment neuronal, cholinergic contractions in guinea pig trachea

Serotonin (5-HT) and the selective 5-HT 3 receptor agonist, 2-methyl-5-hydroxytryptamine enhanced electrical field stimulated contractions of the isolated guinea pig trachea. 5-HT (EC 50 = 3.5 μM) was twice as potent as 2-methyl-5-hydroxytryptamine (EC 50 = 7.4 μM). The effects of 5-HT and 2-methyl-5-hydroxytryptamine were antagonized by the selective 5-HT 3 receptor antagonist, zacopride (apparent pA 2 = 7.60 against 2-methyl-5-hydroxytryptamine). 2-Methyl-5-hydroxytryptamine (10 μM) had no effect on contractile responses to exogenous acetylcholine. Furthermore, the increase in electrical field stimulated contraction by 2-methyl-5-hydroxytryptamine was unchanged by hexamethonium (100 μM) but contractions were blocked by atropine (1 μM). These results suggest that excitatory 5-HT 3 receptors exist on postganglionic cholinergic nerves in the isolated guinea pig trachea.

Organ Culture with Proinflammatory Cytokines Reproduces Impairment of the β-Adrenoceptor-mediated Relaxation in Tracheas of a Guinea Pig Antigen Model

The challenge of previously sensitized guinea pigs with aerosolized ovalbumin resulted in impairment of the beta-adrenoceptor-mediated relaxation as measured by the in vitro isometric assay of tracheas preconstricted with endothelin-1 or carbamylcholine. Numbers and affinities of beta-adrenoceptors in lung membranes of these animals were not altered under these conditions, although the antigen challenge caused an inflammatory response, as evident from the accumulation of inflammatory cells in the bronchoalveolar lavage fluids. In order to investigate the pathophysiologic role of inflammation in hyperreactive airways, isolated guinea pig tracheas were cultured with proinflammatory cytokines such as human recombinant tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or interleukin-2 (IL-2). None of these cytokines affected the contractile response of tracheas to carbamylcholine. After preconstriction with carbamylcholine, the TNF-alpha- and IL-1 beta-pretreated tissues produced a significant reduction in the maximal relaxation induced by isoproterenol, whereas the IL-2 pretreatment had no effect. The reduction of the isoproterenol-mediated relaxation by the IL-1 beta treatment was time and dose dependent. Our present observations suggest that in vitro incubation of naive tracheas with proinflammatory cytokines is able to reproduce apparent beta-adrenoceptor impairment as seen in the airways of antigen-challenged guinea pigs of asthma model.

Modification by CO2 of endothelin-1-induced contraction of isolated guinea pig trachea

To investigate the effect of changes in CO2 tension on airway smooth muscle tone induced by various agonists, contractile responses to acetylcholine, histamine and endothelin-1 (ET-1) were studied in isolated guinea pig tracheae at very low PCO2 (16 +/- 0 mmHg, n = 38), moderately low PCO2 (28 +/- 0 mmHg, n = 23), normal PCO2 (38 +/- 1 mmHg, n = 70) or high PCO2 (94 +/- 1 mmHg, n = 32). The minimum concentration of ET-1 (10(-10) M) needed to induce contractions was lower than that of acetylcholine (10(-7) M) and histamine (10(-7) M) at normal PCO2. Changes in PCO2 did not significantly affect acetylcholine- or histamine-induced contractions. In contrast, very low and moderately low PCO2 attenuated the contractions induced by ET-1, but high PCO2 potentiated those induced by a high concentration of ET-1. Very low PCO2 with normal pH and with high pH attenuated the contractions caused by ET-1, whereas normal PCO2 with high pH did not. These results suggest that ET-1-induced airway smooth muscle contraction can be modified by PCO2 per se. Aspirin and indomethacin potentiated the responses to ET-1 at very low PCO2 more than at normal PCO2, but attenuated the responses to low concentration of ET-1 at high PCO2. These results also suggest that cyclooxygenase-related eicosanoids are involved in the effects of PCO2 on ET-1-induced contractions.