Hyperosmolar solution effects in guinea pig airways. II. Epithelial bioelectric responses to relative changes in osmolarity

Osmotic challenge of airways alters the bioelectric properties of the airway epithelium and induces the release of factors that modulate smooth muscle tone. Recent studies in our laboratory suggested that methacholine-contracted airways relax in response to incremental increases in osmolarity, rather than from cell shrinkage or absolute solute concentration. In the present study, guinea pig tracheae were mounted in Ussing chambers to elucidate the bioelectric effects of challenge of the epithelium with hyperosmolar and isosmolar solutions. Transepithelial short-circuit current (Isc) across tracheae stimulated with basolateral methacholine was inhibited by apical amiloride, apical 5-nitro-2-(3-phenylpropylamino)benzoic acid, basolateral bumetanide, basolateral ouabain, and Cl(-)-free solution, but not by basolateral iberiotoxin. Apical hyperosmolar challenge with NaCl variably decreased or increased Isc, but D-mannitol (D-M) always inhibited Isc; bumetanide attenuated decreases in Isc. The effects of the transport blockers depended upon whether Isc was initially decreased or increased. Unique concentration-dependent changes in Isc and transepithelial resistance (Rt) were observed when ionic (NaCl and KCl), nonionic impermeant (D-M and sucrose), and nonionic permeant (urea) osmolytes were added to the apical and basolateral baths. At concentrations that doubled the osmolarity of the apical bath, D-M, urea, and N-methyl-D-glucamine-gluconate (NMDG-Glu) decreased Isc. Apical isosmolar NMDG-Glu solution decreased Isc, and additional NMDG-Glu caused a further decrease in Isc. Inclusion of one permeant ion, either Na+,K+, or Cl-, reversed the response to apical isosmolar and hyperosmolar solutions. Thus, bioelectric responses of the airway epithelium to hyperosmolar solution are induced by incremental increases in osmolarity.     

Hyperosmolar solution effects in guinea pig airways. I. Mechanical responses to relative changes in osmolarity

In the guinea pig isolated perfused trachea contracted with serosal methacholine (MCh), increasing the osmolarity of the mucosal bathing solution elicits relaxation of smooth muscle mediated by epithelium-derived relaxing factor (EpDRF). The present study was undertaken to determine whether a specific modality of the hyperosmolar stimulus induced the relaxation response. Mucosal hyperosmolar challenge with D-mannitol, N-methyl-D-glucamine (NMDG)-chloride, NMDG-gluconate (NMDG-Glu), or urea elicited relaxation with equal potency. In contrast, hyperosmolar solutions at the serosal surface induced diverse, osmolyte-specific responses. In tracheae contracted with MCh, abrupt replacement of the mucosal modified Krebs-Henseleit solution (MKHS) with isosmolar osmolyte solutions to stimulate cell shrinkage elicited five discrete response patterns related to the membrane permeance of the solute, but increasing the osmolarity of the isosmolar solution via the further addition of the same solute always induced relaxation. Similarly, perfusion of the lumen with water induced a transient contraction, but subsequent addition of MKHS, or isosmolar D-mannitol, urea, NMDG-Glu, NaCl, or KCl induced relaxation. Subsequent hyperosmolar addition of the same osmolyte-evoked relaxation. Compatible osmolytes had no effect on smooth muscle tone and did not affect responses to hyperosmolar challenge. The results suggest that the airway epithelium acts as an osmolarity sensor, which communicates with airway smooth muscle through EpDRF. The mechanical responses of the smooth muscle resulting from changes in the osmotic environment are associated with discrete modalities of the osmolar stimulus, including membrane reflection of the particles, incremental change in osmolarity and directionality, but not cell shrinkage.     

Hyperosmolar solution effects in guinea pig airways. III. Studies on the identity of epithelium-derived relaxing factor in isolated perfused trachea using pharmacological agents

Hyperosmolar challenge of airway epithelium stimulates the release of epithelium-derived relaxing factor (EpDRF), but the identity of EpDRF is not known. We examined the effects of pharmacological agents on relaxant responses of methacholine (3 x 10(-7) M)-contracted guinea pig perfused trachea to mucosal hyperosmolar challenge using D-mannitol. Responses were inhibited by gossypol (5 x 10(-6) M), an agent with diverse actions, by the carbon monoxide (CO) scavenger hemoglobin (10(-6) M), and by the heme oxygenase (HO) inhibitor zinc (II) protoporphyrin IX (10(-4) M). The HO inhibitor chromium (III) mesoporphyrin IX (10(-4) M) was not inhibitory, and the HO activator heme-L-lysinate (3 x 10(-4) M) did not evoke relaxant responses. The CO donor tricarbonyldichlororuthenium (II) dimer (2.2 x 10(-4) M) elicited small relaxation responses. Other agents without an effect on responses included: apyrase, adenosine, 6-anilino-5,8-quinolinequinone (LY83583), proadifen, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), diphenhydramine, glibenclamide, HgCl2, tetrodotoxin, nystatin, alpha-hemolysin, 8-bromoguanosine 3',5'-cyclic monophosphothioate, Rp-isomer, 12-O-tetradecanoylphorbol-13-acetate, cholera toxin, pertussis toxin, thapsigargin, nifedipine, Ca(2+)-free mucosal solution, hydrocortisone, and epidermal growth factor. Cytoskeleton inhibitors, includingerythro-9-(2-hydroxyl-3-nonyl)adenine, colchicine, nocodazole, latrunculin B, and cytochalasins B and D, had no effect on relaxation responses. The results suggest provisionally that a portion of EpDRF activity may be due to CO and that the release of EpDRF does not involve cytoskeletal reorganization.     

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.     

Changes in smooth muscle tone during osmotic challenge in relation to epithelial bioelectric events in guinea pig isolated trachea

The relationship between epithelial bioelectric events and epithelium-dependent relaxant and contractile responses of airway smooth muscle in response to hyperosmolar and hypo-osmolar solutions was investigated in guinea pig isolated trachea. Tracheae were perfused with normal or nonisosmotic modified Krebs-Henseleit solution while simultaneously monitoring transepithelial potential difference (VT) and contractile and relaxant responses of the muscle. Baseline VT was -10.1 to -13.3 mV (distal and proximal ends, respectively). Intraluminal amiloride (10(-4) M) induced a 3.7-mV depolarization, verifying that the VT was of epithelial origin. Extraluminal methacholine (3 x 10(-7) M; EC50) caused hyperpolarization and smooth muscle contraction; intraluminal methacholine had very little effect. Increasing intraluminal bath osmolarity via addition of 240 mOsM NaCl or KCl caused an immediate and prolonged depolarization and epithelium-dependent relaxation. Increasing intraluminal bath osmolarity with sucrose evoked similar responses, except that an immediate, transient hyperpolarization and contraction preceded the depolarization and relaxation. Increasing extraluminal bath osmolarity with 240 mOsM NaCl induced depolarization and a longer lasting epithelium-dependent relaxation, whereas extraluminally added 240 mOsM KCl induced a complex smooth muscle response (i.e., transient relaxation followed by contraction), which was accompanied by prolonged depolarization. Intraluminal hypo-osmolarity produced a transient hyperpolarization followed by depolarization along with contraction of the smooth muscle. Bioelectric responses always preceded smooth muscle responses. These results suggest that bioelectric events in the epithelium triggered by nonisosmotic solutions are associated with epithelium-dependent responses in tracheal smooth muscle.     

Influence of epithelium on guinea pig airway responses to tachykinins: role of endopeptidase and cyclooxygenase

We have studied the effect of epithelium removal on the contractile responses to exogenous tachykinins and to endogenous tachykinins released by capsaicin in guinea pig trachea. We also studied the effects of inhibition of endopeptidase (by phosphoramidon, 10 microM, and thiorphan, 100 microM), and of inhibition of cyclooxygenase (by indomethacin, 5 microM) on these responses. The order of potency of exogenous tachykinins was neurokinin A (NKA) greater than neurokinin B (NKB) greater than substance P (SP). Epithelium removal enhanced the sensitivity and magnitude of the contractile response to SP, and to a lesser extent NKA and NKB. Capsaicin induced only a weak contractile response in guinea pig trachea. Phosphoramidon and thiorphan increased the sensitivity to SP, but had no effect on acetylcholine responses. The leftwardshift due to epithelium removal was reduced, but not abolished, by phosphoramidon and thiorphan. NKA- and NKB-induced contractions were also enhanced significantly by phosphoramidon. The effect of epithelium removal was abolished for NKA, but not for NKB. Phosphoramidon also increased significantly the contraction to capsaicin in the presence of epithelium, without altering the response obtained in the absence of epithelium. Indomethacin potentiated the sensitivity and maximal contractile response to all the tachykinins with the greatest effect on SP responses, and to capsaicin. The combination of indomethacin with phosphoramidon or thiorphan abolished the effect of epithelium removal for all the tachykinins. We conclude that the effects of exogenous and endogenous tachykinins are enhanced by removal of epithelium and by inhibition of metalloendopeptidase and cyclooxygenase, suggesting that tachykinins may be degraded by epithelial enzymes, and may release relaxant prostanoids in airways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-response of inhaled diltiazem on airway reactivity to methacholine and exercise in subjects with mild asthma

Methacholine challenges were performed by 10 asthmatic subjects, 2 hours before and 15 minutes after placebo (diluent alone) and 5, 10, 15, 30, and 60 mg inhaled diltiazem given in a single-blind crossover manner. There was no significant change from placebo in the dose of methacholine required to produce a 20% decrease in forced expiratory volume in the first second (FEV1) (PD20); the fold increase in PD20 from baseline was 1.1 +/- 0.1 after placebo, 1.4 +/- 0.2 after 5 mg, 1.8 +/- 0.3 after 10 mg, 1.4 +/- 0.2 after 15 mg, 1.6 +/- 0.2 after 30 mg, and 1.2 +/- 0.1 after 60 mg. There was a 1% chance that we missed a twofold difference between placebo and the 10 mg dose because of inadequate sample size. Fifteen minutes before a standardized exercise challenge, 10 subjects received placebo, 10 mg, and the highest dose tolerated during the methacholine study (20 to 45 mg) in a randomized double-blind crossover design. The mean +/- SE maximum postexercise decrease in FEV1 was 28.8% +/- 5.7% after placebo, 23.4% +/- 4.6% after 10 mg, and 20.8% +/- 3.0% after high-dose diltiazem (P greater than 0.05). There was a 12% chance that we missed a 15% difference between placebo and the high-dose regimen because of inadequate sample size. We conclude that diltiazem does not attenuate airway reactivity to methacholine or exercise even when high concentrations are delivered to the lungs.     

Influence of cartilage on reactivity and on the effectiveness of verapamil in guinea pig isolated airway smooth muscle

The authors have examined the effects of cartilage removal on smooth muscle reactivity and the action of verapamil in guinea pig trachealis. In preparations devoid of cartilage, smooth muscle reactivity to both histamine and KCl was reduced. Reactivity to methacholine was unaffected by cartilage removal. In the absence of cartilage, verapamil had a greater depressant effect on the maximum responses to histamine and methacholine than in intact tissues. Similarly, verapamil was more potent against histamine- and methacholine-induced responses in the absence of cartilage where a greater shift to the right was seen in the concentration-response curves when compared with cartilage-containing controls. The spasmolytic action of verapamil on methacholine-induced responses was greater in the absence of cartilage and was greater than its antispasmogenic activity against methacholine (whether or not cartilage was present). Thus, cartilage removal reduces muscle reactivity and increases the potency of verapamil in guinea pig trachealis.     

Regional and time-dependent effects of inflammatory mediators on airway microvascular permeability in the guinea pig

1. Airway oedema resulting from increased microvascular permeability is a characteristic pathological finding of asthma. The regional effects of putative mediators involved in asthma on airway microvascular permeability have been studied. 2. The effects of histamine, leukotriene (LT) D4 and platelet-activating factor (PAF) on microvascular permeability in the nasal mucosa, larynx, trachea, main bronchi and intrapulmonary airways of the guinea pig were assessed by measuring the extravasation of intravenously administered Evans Blue dye. 3. PAF and LTD4 caused increased microvascular leakage throughout the respiratory tract, although their effects were maximal in different regions. Histamine had no significant effect on intrapulmonary airways. PAF was more potent than LTD4 and histamine at all airway levels. For example, in the trachea the doses required to cause leakage of 50% of maximal (ED50) were 10.4 nmol/kg, 138 nmol/kg and 11.2 mumol/kg, respectively, for PAF, LTD4 and histamine. 4. The effect of the three mediators was maximal 5 min after intravenous administration. Histamine, but neither LTD4 nor PAF, still caused significant leakage 30 min after administration. 5. The increased microvascular leakage induced by the mediators was inhibited by their respective specific receptor antagonists, suggesting that the effect was mediated via specific receptors. 6. Histamine, LTD4 and PAF have varying potencies in increasing microvascular permeability in the guinea-pig respiratory tract, exert their maximal effect in different regions and have varying durations of action.     

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.     

Antibody to VLA-4, but not to L-selectin, protects neuronal M2 muscarinic receptors in antigen-challenged guinea pig airways.

Antigen challenge of sensitized guinea pigs decreases the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung, potentiating vagally induced bronchoconstriction. Loss of M2 receptor function is associated with the accumulation of eosinophils around airway nerves. To determine whether recruitment of eosinophils via expression of VLA-4 and L-selectin is critical for loss of M2 receptor function, guinea pigs were pretreated with monoclonal antibodies to VLA-4 (HP1/2) or L-selectin (LAM1-116). Guinea pigs were sensitized and challenged with ovalbumin, and M2 receptor function was tested. In controls, blockade of neuronal M2 muscarinic receptors by gallamine potentiated vagally induced bronchoconstriction, while in challenged animals this effect was markedly reduced, confirming M2 receptor dysfunction. Pretreatment with HP1/2, but not with LAM1-116, protected M2 receptor function in the antigen-challenged animals. HP1/2 also inhibited the development of hyperresponsiveness, and selectively inhibited accumulation of eosinophils in the lungs as measured by lavage and histology. Thus, inhibition of eosinophil influx into the lungs protects the function of M2 muscarinic receptors, and in so doing, prevents hyperresponsiveness in antigen-challenged guinea pigs.     

An examination of the influence of the epithelium on contractile responses to peptidoleukotrienes and blockade by ICI 204,219 in isolated guinea pig trachea and human intralobar airways

The influence of the epithelium on antagonism by ICI 204,219 of contractile responses to peptide leukotriene (LT) agonists was examined in guinea pig tracheal and human bronchial rings. The -log molar KB values for ICI 204,219 were found to be independent of the epithelium in both tissues. Even though uninfluenced by the epithelium, the -log molar KB values for ICI 204,219 were about 10-fold smaller in human airways than in guinea pig trachea. Removal of the epithelium from guinea pig trachea resulted in small leftward shifts of the concentration-response curves to LTC4 and LTD4 and rightward shifts of the concentration-response curves to LTE4 when examined in the presence of indomethacin. The potentiation of LTC4 and LTD4 by epithelium removal was not seen in the presence of inhibitors of the transformation of LTC4 to LTD4 and LTD4 to LTE4. The influence of the epithelium on responses to LTE4 remained in the presence of these metabolic inhibitors. The lipoxygenase inhibitors nordihydroguaiaretic acid, B755C, Rev 5901 and AA861 antagonized responses to LTE4 in the presence, but not in the absence of epithelium. In human airways, epithelium removal resulted in a small leftward shift of the concentration-response curve to LTD4 whereas responses to LTC4 and LTE4 were unaltered. This effect was not observed in the presence of indomethacin, relating it to reduced release of cyclooxygenase products. These data suggest that contractile responses of guinea pig trachea to LTE4 are modulated by LTE4-induced release of 5-lipoxygenase product(s) only when the epithelium is present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phasic responses to carbachol in isolated guinea pig trachea are augmented by cooling and inhibited by nifedipine.

Steady-state and kinetic approaches were used to study the effects of cooling to 25 degrees C and nifedipine (1 microM) on the phasic and tonic responses to 0.3 and 10 microM carbachol (Carb) in isolated segments of epithelium-free guinea pig trachea. Cooling and nifedipine had no effect on the steady-state tensions of the contractile responses to Carb in Krebs-bicarbonate buffer, but did inhibit the response to 40 mM KCl. Cooling increased the tensions of the phasic responses to both concentrations of Carb in Ca(++)-depleted, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-containing buffer, whereas the rate constant of decay of the phasic response (kdecay) was decreased only at 0.3 microM Carb. Nifedipine inhibited the tension and increased the kdecay of the phasic response to 10 microM Carb at both 37 and 25 degrees C. The phasic response to 0.3 microM Carb was essentially completely inhibited by nifedipine. The tension of the tonic response to Carb was unaffected by cooling and was only inhibited by nifedipine at 0.3 microM Carb at 37 degrees C. Both cooling and nifedipine, at 37 degrees C, decreased the rate constant of onset of the tonic response (kon) to Carb. The predominant effect of cooling to augment the phasic component of the contractile response to Carb, whereas it does not provide the mechanism to explain the enhanced bronchoconstrictor response of airways to cold, does suggest that Ca++ mobilization is altered by cold and that this may contribute to the enhanced bronchoconstrictor response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody to very late activation antigen 4 prevents antigen-induced bronchial hyperreactivity and cellular infiltration in the guinea pig airways

This report examines the effect of an anti-VLA-4 monoclonal antibody (mAb) HP1/2 on antigen-induced bronchial hyperreactivity to methacholine, and on eosinophil and T lymphocyte infiltration in the airways of guinea pigs sensitized and challenged by aerosolized ovalbumin and used 24 h thereafter. The intravenous administration of 2.5 mg/kg of HP1/2, but not of its isotype-matched mAb 1E6, 1 h before and 4 h after antigen inhalation, markedly inhibited the increased bronchopulmonary responses to intravenous methacholine, as well as airway eosinophilia in bronchoalveolar lavage (BAL) fluid and in bronchial tissue. HP1/2 also suppressed the antigen-induced infiltration of the bronchial wall by CD4+ and CD8+ T lymphocytes, identified by immunohistochemical technique using specific mAbs that recognize antigenic epitopes of guinea pig T cells. Treatment with HP1/2 also resulted in a significant increase in the number of blood eosinophils, suggesting that inhibition by anti-VLA-4 mAb of eosinophil recruitment to the alveolar compartment may partially account for their accumulation in the circulation. These findings indicate that eosinophil and lymphocyte adhesion and subsequent infiltration into the guinea pig airways that follow antigen challenge are mediated by VLA-4. Furthermore, concomitant inhibition of antigen-induced bronchial hyperreactivity and of cellular infiltration by anti-VLA-4 mAb suggests a relationship between airway inflammation and modifications in the bronchopulmonary function.     

Negative and positive inotropic responses to muscarinic agonists in guinea pig and rat atria in vitro

In the guinea pig atria, carbachol, acetylcholine and bethanechol elicited negative inotropic and positive inotropic effects. In the rat atria, a negative inotropic response occurred, but the positive inotropic response was small. The positive and negative inotropic responses to carbachol and bethanechol (but not acetylcholine) were unaffected by pretreating the animals with reserpine and were antagonised by pirenzepine with pKB values of 6.7. Pretreatment with pertussis toxin abolished the negative inotropic responses, but was without effect on the positive inotropic responses in the guinea pig. Pretreatment of rats with pertussis toxin abolished the negative inotropic response and enhanced the positive inotropic response. The positive inotropic response was attenuated by pretreatment with dietary lithium for 2 weeks, whereas no effect was observed on the negative inotropic response. Negative and positive inotropic responses to muscarinic agonists in these species are mediated directly through an M2 muscarinic receptor. The ability of dietary lithium to selectively inhibit the positive inotropic response may provide evidence for the involvement of inositol phospholipid hydrolysis in this effect.     

The peroxisome proliferator-activated receptor alpha agonist fenofibrate decreases airway reactivity to methacholine and increases endothelial nitric oxide synthase phosphorylation in mouse lung

In the present study, we have investigated the effect of the peroxisome proliferator-activated receptor α (PPARα) agonist fenofibrate on airway reactivity and the role of the endothelial nitric oxide synthase (eNOS)/NO pathway in this effect. Airway reactivity to methacholine was assessed in C57BL/6 mice treated or not with fenofibrate by whole-body plethysmography. In some experiments, animals were administered with the NOS inhibitor L-NAME, one hour before airway reactivity measurement. Expression and phosphorylation of eNOS were evaluated in lung homogenates from fenofibrate and control animals using Western blotting. Fenofibrate dose and time dependently decreased airway reactivity to methacholine in mice. A statistically significant (P < 0.05) reduction was observed after a treatment of 10 days with a dose of 3 or 15 mg/day fenofibrate. Mice treated with fenofibrate and administered with l-NAME exhibited similar reactivity to methacholine than vehicle-treated mice administered with the NOS inhibitor, suggesting that NO mediates fenofibrate-induced decrease in airway reactivity. eNOS levels remained unchanged in the lung from mice treated with fenofibrate, but phosphorylation of the enzyme at Ser-1177 was increased by 118% (P < 0.05). Taken together, our data demonstrate that fenofibrate downregulates airway reactivity to methacholine in the mouse and suggest that this effect could involve an increase in NO generation through an enhanced eNOS phosphorylation.     

The inhibitory effects of cromakalim and its active enantiomer BRL 38227 against various agonists in guinea pig and human airways: comparison with pinacidil and verapamil.

The effects of the potassium channel activators, cromakalim, BRL 38227 and pinacidil, and the calcium antagonist, verapamil, have been compared against various spasmogens on airway responses in vitro and in vivo in the guinea pig and also in human isolated bronchi. In guinea pig tracheal spirals, potassium channel activators generally had a greater inhibitory effect than verapamil against tone induced by a wide range of spasmogens (spontaneous, 5-hydroxytryptamine, leukotriene D4, prostaglandin E2). The potassium channel activators had very little effect against potassium chloride- and carbachol-induced tone in guinea pig tracheal spirals [e.g., cromakalim (20 microM) induced relaxations of 0.21 +/- 0.03 (relative to an isoprenaline maximum = 1.0, mean +/- S.E.M.) against carbachol, compared to 0.77 +/- 0.03 against histamine]. In vivo, the potassium channel activators prevented histamine and 5-hydroxytryptamine-induced bronchoconstrictions, but had little inhibitory effect against acetylcholine. In contrast, in human bronchi, cromakalim was capable of inducing powerful concentration-dependent relaxations against carbachol-induced tone [cromakalim (20 microM) induced relaxations of 0.77 +/- 0.09 (relative to isoprenaline = 1.0, mean +/- S.E.M.) against carbachol, compared to 0.95 +/- 0.04 against histamine]. In human bronchi, all the inhibitory agents were more potent and more effective, except that verapamil did not have an increased maximum response. We conclude that potassium channel activators should be effective at relaxing contractions induced by a wide range of spasmogens in man.     

Cardiorespiratory, sympathetic and biochemical responses to T-2 toxin in the guinea pig and rat

The cardiorespiratory, sympathetic and biochemical effects of T-2 toxin were examined in conscious rats and guinea pigs. The pithed rat preparation was also used to evaluate possible direct effects of T-2 on the heart and vasculature. Injection of T-2 (0.5-2.0 mg/kg i.v.) into conscious rats produced prolonged (6-8 hr) hypertension and tachycardia, followed by hypotension. Total peripheral resistance was increased and cardiac output decreased. In guinea pigs, a steady decrease in pressure and rate occurred. Intravenous administration of T-2 to pithed rats did not alter blood pressure or heart rate at a time when, in conscious rats, both blood pressure and heart rate were increased. Significant elevations of arterial plasma norepinephrine, epinephrine and dopamine occurred after T-2, with metabolic acidosis, hypocarbia and hyperoxemia in both conscious rats and guinea pigs. In the rat, increase in plasma vasopressin and prostacyclin were elevated, but thromboxane and leukotriene C4-immunoreactivity were not changed. In pithed rats, T-2 did not increase basal or stimulated plasma catecholamines but produced the same changes in blood gases, pH and lactate. The LD50 values for i.v. T-2 in the rat and guinea pig were 0.74 and 1.30 mg/kg, respectively. The data are consistent with the hypothesis that T-2 toxin disrupts cellular aerobic metabolism, resulting in lactic acidosis, sympathoadrenomedullary activation, variable initial circulatory responses and eventual cardiovascular collapse.     

Antigen-induced enhancement of noncholinergic contractile responses to vagus nerve and electrical field stimulation in guinea pig isolated trachea.

Nonadrenergic, noncholinergic contractions were elicited by electrical field stimulation (EFS) (2 Hz, 1 msec, 12 V for 15 sec) of the distal aspect of guinea pig trachea pretreated with atropine (1 microM), propranolol (1 microM) and indomethacin (3 microM). The contractions were abolished by pretreatment with the sensory C-fiber toxin capsaicin or by a combination of the neurokinin (NK)1 receptor antagonist, CP 96,345 (0.1 microM), and the NK2 receptor antagonist, MEN 10376 (3 microM), and were markedly attenuated by tetrodotoxin. In animals actively sensitized to ovalbumin, the addition of threshold concentrations of antigen markedly increased the noncholinergic contractile responses to EFS (approximately 3- to 6-fold). This potentiation was long lasting, persisting virtually unchanged for 60 min, whereas the antigen-induced contractions were shorter lived, usually lasting less than 30 min. The ovalbumin-induced potentiation of the neuronal response was not observed in tissues pretreated with capsaicin or treated with tetrodotoxin. This antigen-induced potentiation of capsaicin-sensitive, EFS-induced contractions was not mimicked by serotonin or prostaglandin D2. However, it was mimicked by histamine. Moreover, the histamine H1 receptor antagonist pyrilamine (0.3 microM) reversed the potentiation elicited by ovalbumin. The effect of ovalbumin challenge was also examined on the distal trachea with the right vagus nerve intact. Noncholinergic contractions to EFS and vagus nerve stimulation were enhanced equally by threshold concentrations of antigen. The results support the hypothesis that antigen challenge releases histamine which acts via H1 receptors to enhance noncholinergic contractions due to the release of tachykinins from capsaicin-sensitive fibers in the guinea pig trachea.