Responses Of Guinea-pig Trachea Research Articles

Hyperosmolarity-Induced Dilation and Epithelial Bioelectric Responses of Guinea Pig Trachea in Vitro: Role of Kinase Signaling

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (V(t)), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, D-mannitol applied intraluminally (< or = 80 mosM) increased V(t) and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased V(t). In tracheas exposed to 30 mosM D-mannitol (approximately EC(50)), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH(2)-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO(2) (mitogen-activated protein kinase stress inducer) and Na(3)VO(4) (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na(3)VO(4) did not affect the V(t) responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.

Effects of Phosphodiesterase V Inhibition on Nitric Oxide-Mediated Relaxation Responses in Guinea Pig Trachea

In the present study, we aimed to evaluate the effects of PDE V inhibition on NO-mediated relaxation responses in isolated guinea pig trachea. Under the NANC conditions, tracheal preparations were contracted with histamine (100 µm/l). When contraction had reached a plateau, relaxation responses to electrical field stimulation (EFS, 60 V, 0.5 ms, 5–10 Hz) were determined before and after incubation of the tracheal ring with L-NAME (1 mmol/l), a NO synthase inhibitor. L-NAME significantly inhibited the relaxation responses and this inhibitory effect was reversed by L-arginine (1 mmol/l), a precursor of NO, but was not affected by D-arginine. In addition, cumulative application of the NO donors, 3-morpholino-sydnonimine (SIN-1) and sodium nitroprusside (SNP), caused concentration-dependent relaxation of tissues precontracted with histamine. The selective PDE type V inhibitor zaprinast at EC₅₀ concentration (30 µmol/l) significantly potentiated EFS-induced NANC relaxations and relaxant responses to SIN-1 and SNP. In conclusion, these data support the hypothesis that NO is a mediator of NANC relaxations of guinea pig tracheal rings and PDE V inhibition potentiates NO-mediated relaxation.

Effect of PAR-activation on smooth muscle contractile response of guinea-pig trachea and ileum.

Effect of PAR-activation on smooth muscle contractile response of guinea-pig trachea and ileum.

Involvement of L-arginine-nitric oxide system in the response of isolated trachea to reactive oxygen species.

The aim of this study was to investigate the effect of reactive oxygen species (ROS), generated by electrolysis of Krebs-Henseleit solution (GE-KHS), on isolated guinea pig trachea and to assess the possible involvement of nitric oxide (NO) in the observed effects. The isolated trachea was superfused in GE-KHS, generating H2O2 and hypochlorous acid (HOCl), both of which slowly increased in the organ bath and reached final stable concentrations of 42 and 63 microM, respectively, at the rate of 20 ml/min(-1), and 261 and 245 microM, respectively, at the rate of 5 ml/min(-1). ROS GE-KHS-induced relaxation of tracheal rings was preceded by a small transient contraction in 40% and 65% of experiments when tracheal rings were superfused at the rate of 20 ml/min(-1) and 5 ml/min(-1), respectively. Removal of tracheal epithelium abolished the relaxation of the trachea induced by ROS GE-KHS and unmasked or potentiated trachealis contraction. The ROS GE-KHS-induced changes in trachealis tension were accompanied by an increase in thiobarbituric acid reactive substances (TBARS) and a decrease in nonprotein (NP) thiols in the trachea. These changes were inhibited by treatment with the antioxidant N-acetylcysteine (100 microM). Pretreatment of tracheal rings with the inhibitor of NO synthase (NOS) N(omega)-nitro-L-arginine (L-NOARG; 100 microM) for 20 min prior to exposure to ROS GE-KHS decreased the ROS GE-KHS-induced relaxation. When L-NOARG (100 microM) was present in the superfusing solution, not only 20 min before but also during superfusion with ROS GE-KHS, the evoked trachealis relaxation was reduced in the first 15 min but was enhanced in the 30th min. This late enhancement of relaxation was accompanied by a 12-fold increase in nitric oxide metabolites (NO(x)). ROS GE-KHS-induced elevation of TBARS levels in the trachea was decreased to 63% by pretreatment with L-NOARG (100 microM). Elevation of TBARS levels induced by incubation of brain liposomes with a hydroxyl radical generating system was decreased to 90% by L-NOARG (10, 100 microM), while the antioxidant stobadine (100 microM) nearly completely inhibited the evoked lipid peroxidation. In comparison with Trolox, L-NOARG exerted a slight scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl radical. The presence of L-arginine and D-arginine in the superfusion fluid for 15-20 min before and during exposure of the trachea to ROS GE-KHS inhibited trachealis relaxation. Results indicate that epithelium derived NO may participate in the response of guinea pig trachea to ROS GE-KHS. The presence of L-NOARG in the bathing fluid during superfusion with ROS GE-KHS gave rise to NO(x), with relaxing activity. L- and D-arginine induced an inhibition of the relaxatory response to ROS GE-KHS and partially prevented a ROS-induced decrease in NP thiols. The involvement of the small antioxidant effects of L-NOARG and L- and D-arginine in the above mentioned actions of L-NOARG and L-arginine requires additional investigation.

Modulation of acetylcholine release from parasympathetic nerves innervating guinea-pig and human trachea by endomorphin-1 and -2

Endomorphin-1 and -2 (μ-opioid receptor agonists) produced a concentration-dependent and naloxone-sensitive inhibition of cholinergic contractile responses in guinea-pig trachea (at 10 μM, 46.1±8.0% and 33.8±8.6%, respectively). Endomorphin-1 and -2 also inhibited electrically-evoked acetylcholine release from cholinergic nerves innervating guinea-pig (at 0.1 μM, 41.8±10.9%; at 1 μM 60.1±6.3%, respectively) and human trachea (at 10 μM, 76.2±18.1%, and 77.7±14.3%, respectively). Naloxone prevented the inhibition by endomorphin-1 and -2 in both guinea-pig and human trachea, suggesting that these peptides can inhibit cholinergic, parasympathetic neurotransmission to the airways via the activation of classical opioid receptors.

P-538 - Possible involvement of COX and LOX metabolites on arachidonic acid-induced responses in guinea pig trachea

P-538 - Possible involvement of COX and LOX metabolites on arachidonic acid-induced responses in guinea pig trachea

Phosphodiesterase inhibitors and endothelin as modulators of respiratory neurotransmission.

1. Airway smooth muscle receives cholinergic, adrenergic and non-adrenergic, non-cholinergic (NANC) neural input. In guinea-pig airways cholinergic and NANC nerves provide contractile innervation, while adrenergic and NANC nerves provide relaxant pathways. In contrast, the major relaxant innervation in human airways is NANC in nature. 2. The present review describes the effects of selective phosphodiesterase (PDE) inhibitors on NANC relaxant and contractile responses in guinea-pig trachea as well as on NANC relaxations in human bronchus. 3. The effects of endothelin-1 on cholinergic contractile responses obtained in a variety of species are also assessed.

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.

H-7, A protein kinase C inhibitor, inhibits spontaneous tone and spasmogenic responses in normal and sensitized guinea pig trachea

1. 1. H-7, a protein kinase C inhibitor, fully inhibited the spontaneous and stimulated (KC120 mM or histamine 0.5 mM) tone of trachea from normal and sensitized guinea pig. 2. 2. H-7 depressed the concentration-contraction curves to KCI, histamine or 5-hydroxytryptamine in epithelium-denuded, indomethacin-treated, trachea from normal and sensitized guinea pigs while responses to CaCI 2 (in Ca 2+-free, K +-depolarized tissues) and acetylcholine were not affected. 3. 3. H-7 (100 gM) did not depress Ca 2+ (20 μM)-induced contraction of Triton X-100 skinned trachea. 4. 4. These results suggest the involvement of PKC in the maintenance of spontaneous tone and spasmogenic responses of guinea pig trachea.

Nitric Oxide: Role as a Relaxant Agonist and Transmitter of Nonadrenergic Noncholinergic Inhibitory Nerves in Guinea Pig Trachea

Nitric oxide (NO) has been proposed as an inhibitory neurotransmitter in guinea pig trachea. Studies were undertaken to examine the relaxant actions of NO and its possible role as a transmitter of nonadrenergic noncholinergic inhibitory (NANCI) nerves and as a modulator of contractile responses in guinea pig trachea.

Age-related alterations in sensitivity of muscarinic and serotonergic responses of guinea pig trachea to removal of extracellular Ca 2+

Contractile responses to carbamylcholine and 5-hydroxytryptamine (5-HT) in the presence and absence of extracellular Ca 2+ were compared in tracheal rings from 3–4-week-old and 7–8-week-old guinea pigs. Contraction to carbamylcholine and 5-HT did not differ in potency or maximal response between age groups in the presence of extracellular Ca 2+. However, removal of extracellular Ca 2+ inhibited maximal contraction mediated by both agonists to a greater extent in younger versus older guinea pigs. These data suggest that carbamylcholine- and 5-HT-induced contraction of guinea pig trachea from younger animals is more dependent on extracellular Ca 2+ as compared to mature animals, and may be related to an age-dependent maturation of sarcoplasmic reticulum function in smooth muscle.

Furosemide and bumetanide, but not nedocromil sodium, modulate nonadrenergic relaxation in guinea pig trachea in vitro.

Furosemide has recently been shown to be effective in inhibiting various indirect challenges in asthmatic patients, but its mode of action is not yet clear. There is some evidence that furosemide has an inhibitory effect on sensory and cholinergic nerves in the airways. We have investigated the effects of furosemide, bumetanide, and nedocromil sodium on inhibitory nonadrenergic, noncholinergic (iNANC) responses in guinea pig trachea in vitro using electrical field stimulation (50 V, 0.5 ms, 2 to 32 Hz for 30 s) and exogenously applied vasoactive intestinal peptide (VIP) or nitroprusside. In the presence of atropine (1 microM), indomethacin (10 microM), and propranolol (1 microM), both furosemide and bumetanide but not nedocromil sodium produced a concentration-dependent inhibition of the iNANC response (maximum inhibition, 31.2 +/- 5.6% with 100 microM furosemide at 16 Hz and 44.2 +/- 4.1% with 10 microM bumetanide at 4 Hz). Furthermore, after pretreatment of the tissues with L-NG-monomethyl arginine (90 microM), alpha-chymotrypsin (2 U/ml), or both, furosemide and bumetanide produced a further inhibition of the iNANC relaxation. Neither loop diuretic had any effect on the concentration-response curves to exogenous VIP (10(-9) to 10(-7) M) or nitroprusside (10(-8) to 10(-6) M). These results indicate that loop diuretics may inhibit nonadrenergic relaxation in guinea pig trachea in vitro by a prejunctional mechanism, probably through inhibition of nerve activation, the exact mechanism of which is still undefined.

The effect of EDTA on contractile responses of guinea-pig trachea in calcium-free medium and on the recovery of the contractile responses in calcium-containing solution

1. 1. Acetylcholine (0.1 mmol/l) and KCl (80 mmol/l) induce contractile responses in guinea-pig trachea. 2. 2. In the absence of extracellular Ca, contractile responses to ACh or KCl decrease as the preincubation time or the EDTA concentration increases. 3. 3. Exposure of the tissue to Ca-free medium decreases the basal tone. 4. 4. Prolonged incubation in the presence of EDTA (1 mmol/l) promotes a delay in the recovery of the tonic component of the contraction elicited subsequently by acetylcholine and KCl in physiological salt solution, and abolishes the phasic one.

Contractile Responses of Guinea Pig Trachea to Oxybarbiturates and Thiobarbiturates

To determine what mechanisms are involved in barbiturate-induced tracheal constriction and whether a relationship exists between barbiturate structure and the ability of the barbiturate to induce constriction, we compared the effects of thiamylal, thiopental, methohexital, pentobarbital, and phenobarbital at increasing airway tone in an intact guinea pig tracheal preparation in the presence and absence of cyclooxygenase and thromboxane synthetase inhibition. Whole tracheas were suspended between two cannulas in 50-ml tissue baths and perfused at a constant flow rate with Krebs-Henseleit solution. The contractile responses were assessed by measuring the pressure differential between the tracheal inlet and outlet ports. Barbiturates were added to the bath of each trachea, which was washed between each drug. Each drug was added to produce final bath concentrations of 10(-6), 10(-5), 10(-4), 10(-3), and 3 x 10(-3) M. Tracheas were also pretreated with meclofenamate (10(-6) M) (a cyclooxygenase inhibitor) and UK 37,248 (10(-8) to 10(-5) M) and OKY 046 (10(-9) to 10(-6) M) (thromboxane synthetase inhibitors), and the thiamylal protocol was repeated. All data were normalized to a concentration of carbachol (2 x 10(-6) M) that has been shown to produce maximum constriction in this preparation. Thiamylal and thiopental produced constriction beginning at 10(-4) M and reached a maximum at 10(-3) M (P less than 0.0001). Methohexital, pentobarbital, and phenobarbital did not produce any significant change in airway tone. Pretreatment with meclofenamate (10(-6) M), UK 37,248 (5 x 10(-5) M), and OKY 046 (10(-6) M) prevented thiamylal-induced tracheal constriction.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial modulation of thromboxane A2 and PAF involvement in IgE- and IgG-mediated guinea pig anaphylaxis

The role of prostanoids and platelet-activating factor (PAF) was studied inthe in vitro response of guinea pig trachea to immunochallenge according to the presence or the absence of the epithelial layer and to the sensitization procedure leading to the preferential synthesis of immunoglobulin E (IgE) or immunoglobulin G (IgG) antibodies. Indomethacin, a cyclooxygenase inhibitor, potentiated the antigen-induced contractions both in IgE and IgG models, suggesting the involvement of relaxant prostaglandins (PGs), independently of the presence of the airway epithelium. UK-38485, a thromboxane synthetase inhibitor, did not modify the tracheal response to antigen in the IgE model. However, this compound enhanced the maximum contractile response to antigen of the intact tracheal strips of IgG-sensitized guinea pig, but reduced the contractile response of the epithelium-free tracheal strips. Two potent non-structurally related PAF antagonists, Ro 19-3704 and BN 52021, reduced antigen-induced contraction of the epithelium-free tracheal strips in the IgE model. In contrast, these compounds did not affect the contractile responses of the preparations in the IgG model. These results suggest the selective implication of thromboxane A2 and PAF, in IgG- and IgE-mediated guinea pig anaphylaxis respectively. Finally, these results indicate that thromboxane A2 (TXA2) and PAF are potent inducers of epithelium-derived mediators.

Modulation of bradykinin responses in airway smooth muscle by epithelial enzymes

We have studied the effect of epithelium removal on responses of guinea pig trachea to bradykinin (BK). BK (1 nM - 10 microM) gave a concentration-dependent relaxation when epithelium was present (E+: EC50 = 10 +/- 3 nM). Epithelium removal resulted in a biphasic response to BK with relaxation at low concentrations (E-: EC50 = 3.0 +/- 1.0 nM) and a recontraction to baseline at higher concentrations (EC50 = 2.0 +/- 1 microM). Phosphoramidon (10 microM), an inhibitor of neutral endopeptidase (NEP), which cleaves BK into inactive peptides, potentiated relaxation (EC50 = 1.0 +/- 0.9 nM in E+ and E respectively) and contraction in trachea with intact epithelium (EC50 = 0.08 +/- 0.03 microM). Inhibition of cyclooxygenase by indomethacin (5 microM), inhibited relaxation to BK in E+ tracheal segments, resulting in a slight contraction (EC50 = 1.0 microM), whereas a potent contractile response was observed in E- segments (EC50 1.6 microM, maximal contraction greater than 1 g). In the presence of both indomethacin and phosphormidon BK caused contraction, even in the presence of epithelium (EC50 = 0.2 +/- 0.11 microM), and the response in the absence of epithelium was similar to the response observed in trachea with intact epithelium (EC50 = 0.25 +/- 0.1 microM). The contractile effect of BK on airway smooth muscle may be inhibited by a protective role of epithelium, due to release of relaxant prostanoids and by degradation by epithelial NEP. In asthma, bronchoconstrictor responses to BK may be partly explained by loss of airway epithelium.

Effect of aging on response of guinea pig trachea to isoprenaline

Effect of aging on response of guinea pig trachea to isoprenaline

Bradykinin B 1- and B 2-receptor mediated responses in guinea-pig trachea

Bradykinin B 1- and B 2-receptor mediated responses in guinea-pig trachea

Effect of Aging on the Response of Guinea Pig Trachea to Isoprenaiine

To study the effect of aging on the β-adrenergic receptor potency of isoprenaiine, tracheas from 3-, 6-, 10-, 40- and 100-week-old guinea pigs were used as test tissues. The pD2 values (potency) of isoprenaiine increased with age in tracheas treated with corticosterone, but decreased in untreated tracheas. The pA2 value of propranolol against isoprenaiine estimated in the treated tracheas did not change with age. Specific binding of [3H]-dihydroalprenolol to the microsomal fractions from the tracheal muscles from 6-, 10- and 40-week-old guinea pigs was analyzed with Scatchard plots. The capacity of maximum binding sites of [3H]-dihydroalprenolol increased with increasing age, while its dissociation constant did not change. Age-related increase in the potency of isoprenaline in the tracheas treated with corticosterone is considered to be due to the increase in the total amount (density) of β-adrenoceptors. However, the potency of the drug in untreated tracheas decreased with age. These results suggest that extraneuronal uptake plays an important role in the β-adrenergic action of isoprenaline in older (40 and 100 weeks) guinea pigs.

Effects of thromboxane A2 synthetase inhibitor, DP-1904 on the action of vasoactive substances in guinea pig trachea and lung tissue strips

Thromboxane A2 (TxA2) is a potent platelet aggregator and vascular or bronchial constrictor. DP-1904, a newly synthesized imidazol TxA2 synthetase inhibitor, is a potent and long-acting agent. The present investigation was conducted to explore the effect of DP-1904 on the contractile or relaxing responses in guinea pig trachea and lung tissue strips induced by various vasoactive substances. Fourteen guinea pigs, weighing 300-350 g, were sacrificed. Trachea and lungs were removed, cut spirally, set up in bioassay glass jackets and superfused with Krebs-Henseleit solution at 37 degrees C, saturated with oxygen and carbon dioxide. Contraction of tissues was detected by an isotonic transducer and displayed on a polyrecorder. Arachidonic acid-induced relaxing responses in guinea pig trachea strips were attenuated significantly by the continuous infusion of DP-1904 in a dose-dependent fashion. Arachidonic acid-induced contractile responses in guinea pig lung strips were attenuated significantly by the continuous infusion of DP-1904, dose-dependently. Acetylcholine-, histamine- and prostaglandin F2 alpha-induced contractile responses in guinea pig lung and trachea strips were attenuated significantly by the continuous infusion of DP-1904, dose-dependently. The above results suggest that DP-1904 might be a useful therapeutic agent for the treatment of chronic obstructive lung diseases.