Interleukin-1 beta increases airway epithelial cell mitogenesis partly by stimulating endothelin-1 production.

To investigate the influence of interleukin-1 beta (IL-1 beta) on airway epithelial cell growth, we measured [3H]thymidine incorporation and cell numbers of cultured porcine tracheal epithelial cells in the presence or absence of human recombinant IL-1 beta with or without the following: goat antiporcine polyclonal antibody to platelet-derived growth factor (PDGF); IL-1 receptor antagonist; indomethacin; PD-145065, a combined endothelin-A and -B receptor antagonist; BQ-123, an antagonist selective for endothelin-A receptors; or phosphoramidon, an inhibitor, in part, of endothelin-converting enzymes, including neutral endopeptidase. We found that IL-1 beta stimulated the proliferation of airway epithelial cells, and this response was inhibited by the IL-1 receptor antagonist and by PD-145065 or BQ-123. However, neither indomethacin nor PDGF antibody was influential. The endothelin receptor antagonists also decreased basal thymidine incorporation by these cells as did phosphormidon, although to a lesser degree. Data from radioimmunoassays indicated that phosphormidon reduced the endogenous production of endothelin-1 from the cells, and IL-1 beta clearly increased it over time. We conclude that IL-1 beta is a stimulant of airway epithelial cell growth, and its mitogenic effects are mediated, in part, by endogenous endothelin-1 production.

Endothelin-1 stimulates proliferation of normal airway epithelial cells.

To investigate the influence of endothelins on airway epithelial cell growth, we measured [3H]thymidine incorporation and cell numbers of cultured porcine tracheal epithelial cells in the presence or absence of endothelin-1 or -3 with or without PD-145065 (a combined endothelin-A and -B receptor antagonist), BQ-123 (an antagonist specific for endothelin-A receptors) or phosphoramidon (an inhibitor, in part, of endothelin converting enzymes). We found that endothelin-1 stimulated the proliferation of airway epithelial cells and this response was progressively inhibited by increasing concentrations of either PD-145065 or BQ-123. In contrast to endothelin-1, airway epithelial cells were not responsive to endothelin-3. They also appeared to be producing endothelin-1 endogenously. Phosphoramidon significantly decreased basal growth of cells incubated in the absence of exogenous endothelin, and was associated with a significant diminution in their endothelin production. We conclude that endothelin-1 is mitogenic for porcine airway epithelial cells and may be involved in both autocrine and paracrine control of them.

Dexamethasone reduces tachykinin but not ACh airway hyperreactivity after O3.

We investigated whether dexamethasone pretreatment affected the acute increase in airway reactivity produced by high-level ozone exposure. Reactivity to intravenous IV substance P (SP), IV acetylcholine (ACh), or aerosolized capsaicin (CAP) before and 1 hr after ozone exposure (3 ppm for 2 hr) was determined by measuring specific airway resistance in anesthetized, spontaneously breathing guinea pigs, half of whom had been pretreated for 2 days pre-ozone with dexamethasone (2 mg/kg intramuscularly [IM] daily). The amount of IV SP, IV ACh, or inhaled capsaicin necessary to increase baseline specific airway resistance by 100% (ED200ACh or ED200SP) or 35% (ED135CAP) was determined by interpolation from dose-response curves. Compared to their pre-ozone status on the day of exposure, we found that dexamethasone-pretreated animals manifested significantly less of an increase in airway reactivity postozone to IV SP or inhaled CAP than did untreated animals. Changes in logEDs of the pretreated group were 0.18 +/- 0.03 (mean +/- SE) for SP and 2.20 +/- 0.11 for CAP compared to 0.27 +/- 0.04 and 3.38 +/- 0.34, respectively, for the untreated groups post-ozone (p < 0.05 and n = 4 for each). In contrast, dexamethasone pretreatment had no effect on IV ACh reactivity postozone: changes in logED200ACh were 0.27 +/- 0.08 and 0.28 +/- 0.04 for the pretreated and untreated groups, respectively (n = 4). In animals pretreated with captopril to block possible dexamethasone stimulation of angiotensin-converting enzyme synthesis that could influence tachykinin reactivity, we found that the corticosteroid effect on post-ozone SP reactivity was as marked as that seen in animals without captopril (n = 4). Because these reactivity studies were consistent with the possibility that dexamethasone may ameliorate ozone-induced, tachykinin hyperreactivity by stimulating airway neutral endopeptidase (NEP), we measured NEP activity by high-performance liquid chromatography (HPLC) of each tracheal homogenate made from other groups of animals. Homogenates from ozone-exposed, dexamethasone-pretreated animals demonstrated significantly greater NEP activity (81 +/- 24%) than that from ozone-exposed, untreated animals (p < 0.05, n = 5). We conclude that corticosteroid pretreatment reduces the acute increase in airway reactivity to exogenous and endogenous tachykinins caused by ozone. This reduction may be at least partly due to stimulation of airway NEP activity, perhaps most of which is nonmucosal in that ozone acutely inactivates mucosal NEP.

Dexamethasone increases airway epithelial cell neutral endopeptidase by enhancing transcription and new protein synthesis.

Neutral endopeptidase (NEP; also known as EC 3.4.24.11, CALLA) is a widely distributed membrane-bound enzyme that hydrolyzes many biologically important endogenous peptides. To evaluate the influence of glucocorticoids on airway epithelial cell NEP expression, we used the human airway epithelial cell line Calu-1. Cells, grown to confluency in Dulbecco's modified Eagle medium with 10% fetal bovine serum and penicillin-streptomycin, were incubated with different concentrations of dexamethasone or vehicle alone in the presence or absence of actinomycin D or cycloheximide for planned times. NEP activity was assayed at the end of treatment employing reverse-phase, high-pressure liquid chromatography. In some experiments, changes in NEP-specific mRNAs in the presence or absence of dexamethasone and/or the inhibitors were also evaluated by Northern blot analysis. We found that dexamethasone increased Calu-1 NEP activity in a dose- and time-dependent manner. Northern blot analysis indicated that NEP-specific mRNAs were also increased by dexamethasone. Furthermore, neither actinomycin D nor cycloheximide inhibited the increases in NEP activity and NEP-specific mRNAs caused by dexamethasone stimulation. We speculate, therefore, that dexamethasone increases NEP expression of these airway epithelial cells by enhancing transcription and new protein synthesis.

Neutral endopeptidase of a human airway epithelial cell line recovers after hypochlorous acid exposure: dexamethasone accelerates this by stimulating neutral endopeptidase mRNA synthesis.

Hypocholorous acid (HOCl) exposure of Calu-1 cells in situ leads to a relatively rapid and substantial decrease in whole cell neutral endopeptidase (NEP) activity that may result from the internalization of NEP from plasma membrane surfaces. To confirm this, and to assess the time course of changes in cell NEP after oxidant exposure and the potential influence of corticosteroid treatment on these, we evaluated Calu-1 NEP activity by high performance liquid chromatography and NEP-specific mRNA over the ensuing 48 h after HOCl in the presence or absence of 1 microM dexamethasone. Cells, grown to confluency in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, were exposed for 5 min to 100 microM HOCl and then maintained in culture for 48 h thereafter. Before exposure, some cell plates were cooled to 4 degrees C and/or incubated for 5 min in 1 mM sodium azide. In some experiments, post-HOCl changes in NEP-specific mRNA in the presence or absence of dexamethasone were also evaluated using Northern blot analysis. We found that sodium azide at 4 degrees C totally blocked the effect of HOCl on Calu-1 NEP (n = 6). In the absence of sodium azide, NEP activity spontaneously recovered to preexposure levels within 24 h. This recovery occurred 6 h earlier in the presence of 1 microM dexamethasone. Furthermore, dexamethasone increased NEP activity at 24 and 48 h after HOCl. Northern blot analysis indicated that NEP-specific mRNA did not change during spontaneous recovery, but was increased by dexamethasone 24 h after HOCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosolized neutral endopeptidase reverses ozone-induced airway hyperreactivity to substance P.

We investigated the effects of ozone exposure (3.0 ppm, 2 h) on airway neutral endopeptidase (NEP) activity and bronchial reactivity to substance P in guinea pigs. Reactivity after ozone or air exposure was determined by measuring specific airway resistance in intact unanesthetized spontaneously breathing animals in response to increasing doses of intravenous substance P boluses. The effective dose of substance P (in micrograms) that produced a doubling of baseline specific airway resistance (ED200SP) was determined by interpolation of cumulative substance P dose-response curves. NEP activity was measured in tracheal homogenates made from each animal of other groups exposed to either ozone or room air. By reverse-phase high-pressure liquid chromatography, this activity was characterized by the phosphoramidon-inhibitable cleavage of alanine-p-nitroaniline from succinyl-(Ala)3-p-nitroaniline in the presence of 100 microM amastatin. Mean values of the changes in log ED200SP were 0.27 +/- 0.07 (SE) for the ozone-exposed group and 0.08 +/- 0.04 for the air-exposed group. We found that phosphoramidon significantly increased substance P reactivity in the air-exposed animals (P less than 0.01), but it had no effect in the ozone-exposed group. This finding was associated with a significant reduction in tracheal homogenate NEP activity of ozone-exposed animals compared with controls: mean values were 18.1 +/- 1.9 nmol.min-1.mg protein-1 for the ozone-exposed group and 25.1 +/- 2.4 nmol.min-1.mg protein-1 for air-exposed animals (P less than 0.05). Inhalation of an aerosolized NEP preparation, partially purified from guinea pig kidney, reversed the substance P hyperreactivity produced by ozone exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung peptidases, including carboxypeptidase, modulate airway reactivity to intravenous bradykinin.

We investigated the effect of inhibition of carboxypeptidase, neutral endopeptidase, or angiotensin converting enzyme on airway reactivity to intravenous bradykinin in guinea pigs. Bradykinin reactivity in intact, unanesthetized, spontaneously breathing animals was determined by measuring specific airway resistance in response to increasing doses of intravenous bradykinin or acetylcholine. We found that phosphoramidon and/or captopril (specific antagonists of neutral endopeptidase and angiotensin converting enzyme, respectively) increased airway reactivity to bradykinin, but the combination had no effect on muscarinic reactivity. Although 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA, a carboxypeptidase inhibitor) alone did not alter bradykinin reactivity, MGTA in the presence of both phosphoramidon and captopril significantly potentiated bradykinin-induced airway reactivity. In comparison, this did not affect reactivity to acetylcholine. Having found that carboxypeptidase inhibition could augment kinin-induced airway reactivity, we subsequently assayed for and identified carboxypeptidase M activity in guinea pig lung. We found considerable carboxypeptidase M activity in guinea pig lung subcellular fractions, the 100,000 x g membrane pellet having the highest specific activity. Our data indicate that airway reactivity to intravenous bradykinin is modulated by the activity of endogenous neutral endopeptidase, angiotensin converting enzyme, and carboxypeptidase, all of which are present in lung cell membranes. This study also suggests that the influence of carboxypeptidase per se may be substantially enhanced if endogenous pulmonary neutral endopeptidase and angiotensin converting enzyme activities are reduced.

HOCl exposure of a human airway epithelial cell line decreases its plasma membrane neutral endopeptidase.

It has recently been demonstrated that luminal exposure of airway segments in vitro to HOCl produces airway muscle hyperresponsiveness to substance P and a decrease in neutral endopeptidase (NEP) activity of tissue segment homogenates, suggesting that HOCl may decrease airway epithelial cell NEP activity. To confirm that this effect occurs in humans and to investigate possible subcellular mechanisms for it, we assessed HOCl exposure of the human airway epithelial cell line Calu-1. These cells, grown to confluency in Dulbecco's modified Eagle medium with 10% fetal bovine serum and penicillin-streptomycin, were exposed in situ for 5 min to 100 microM HOCl in a phosphate-buffered saline solution (PBS; pH 7.0 at 37 degrees C) or to PBS alone. Thereafter, cells were rinsed and assayed for NEP activity employing reverse-phase high-pressure liquid chromatography. This activity was characterized by the generation of phosphoramidon-inhibitable product (ANA) cleaved from the synthetic substrate succinyl-(ala)3-p-nitroaniline during a 30 min incubation at 37 degrees C. Cell viability was assessed by changes in LDH release, trypan blue exclusion, and cell volume. In some experiments, crude plasma membrane and soluble components of exposed cells were isolated and differential NEP activity was assayed. We found that a 5 min exposure to HOCl decreased whole cell NEP activity from 74.1 +/- 4.4 (mean +/- SE) to 54.3 +/- 6.0 pmoles of ANA/min/10(6) cells (p less than 0.05), while no parameter of cell viability was affected. NEP activity in the crude membrane fraction decreased 36.3 +/- 3.1% after exposure (p less than 0.01), whereas NEP activity in the soluble fraction increased 4.0 +/- 0.6%. Isolated membrane NEP exposed by itself was not affected. Subsequent experiments with reducing agents demonstrated that NEP activity of cell cultures pretreated with 100 mM of either beta-mercaptoethanol or dithiothrietol before HOCl exposure was not significantly different from control values. We conclude that whole cell HOCl exposure decreases Calu-1 plasma membrane NEP. This loss appears to occur by internalization of cell membrane NEP.

O3-induced mucosa-linked airway muscle hyperresponsiveness in the guinea pig.

We investigated the effects of ozone exposure (3.0 ppm, 2 h) on the responsiveness of guinea pig airway muscle in vitro from animals developing bronchial hyperreactivity. Muscarinic reactivity in vivo was determined by measuring specific airway resistance (sRaw) in response to increasing concentrations of aerosolized acetylcholine (ACh) administered before and 30 min after exposure. Immediately after reactivity testing, multiple tracheal rings from ozone- and air-exposed animals were prepared and the contractile responses to increasing concentrations of substance P, ACh, or KCl were assessed in the presence of 10 microM indomethacin with or without 1 microM phosphoramidon, an inhibitor of neutral endopeptidase. Isometric force generation in vitro was measured on stimulation by cumulative concentrations of the agonists, and force generation (in g/cm2) was calculated after determination of muscle cross-sectional area. The smooth muscle of mucosa-intact airways from guinea pigs with ozone-induced bronchial hyper-reactivity proved to be hyperresponsive in vitro to substance P and ACh but not to KCl. Pretreatment with phosphoramidon abolished the increase in substance P responsiveness but had no effect on muscarinic hyperresponsiveness after ozone exposure. Furthermore, substance P responsiveness was not augmented in ozone-exposed airways in which the mucosa had been removed before testing in vitro. Likewise, muscarinic hyperresponsiveness was not present in ozone-exposed airways without mucosa. Our data indicate that airway smooth muscle responsiveness is increased in guinea pigs with ozone-induced bronchial hyperreactivity and suggest that this hyperresponsiveness may be linked to non-cyclooxygenase mucosa-derived factors.

HOCl causes airway substance P hyperresponsiveness and neutral endopeptidase hypoactivity.

We investigated whether exposure of guinea pig tracheal tissue to hypochlorous acid (HOCl) or hydrogen peroxide (H2O2) by perfusion through the airway lumen affected the responsiveness of airway muscle to ACh, KCl, or substance P in the presence or absence of 1 microM phosphoramidon, an inhibitor of neutral endopeptidase (NEP). Pairs of tracheal segments were immersed in a Krebs solution (pH 7.40 at 37 degrees C) and connected to perfusion circuits so that the lumen of one segment of each pair could be perfused with Krebs solution while the other was perfused for the same time (10 min) with either 0.1 microM HOCl or 10 mM H2O2. Segments after perfusion were cut into rings of similar size and placed in muscle chambers so that airway muscle force generation in vitro could be measured on stimulation by cumulative agonist doses. In addition, cell homogenates were made from other, similarly perfused tracheal segments to assess NEP activity using reverse-phase, high-pressure liquid chromatography (HPLC). We found that smooth muscle of mucosa-intact guinea pig airways perfused with HOCl, but not H2O2, was hyperresponsive to substance P but not to ACh or KCl. HOCl-perfused rings were not different from Krebs solution-exposed rings pretreated with phosphoramidon. There was no increase in substance P responsiveness of HOCl-exposed airways in which the mucosa had been removed before testing in vitro. The substance P hyperresponsiveness of HOCl-exposed, mucosa-intact airways was associated with decreased NEP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Guinea pig ozone-induced airway hyperreactivity is associated with increased N-acetyl-beta-D-glucosaminidase activity in bronchoalveolar lavage fluid.

High level ozone exposure is known to cause acute, neutrophil-independent airway hyperreactivity in the guinea pig. The precise biochemical mechanisms involved remain unclear. Because of its potential pathophysiologic importance, we examined whether a lysosomal hydrolase, N-acetyl-beta-D-glucosaminidase (NAGA) was released from the airways in vivo and from bronchoalveolar cells, specifically macrophages. Muscarinic reactivity was determined by measuring specific airway resistance (sRaw) in response to increasing doses of aerosolized acetylcholine in guinea pigs that were either exposed to air or to ozone (3.0 ppm, 2 h). The ozone-exposed animals showed substantial muscarinic hyperreactivity 30 min after exposure. In addition, both total and percent released NAGA in bronchoalveolar lavage fluid obtained immediately after reactivity testing were significantly greater in the ozone-exposed group. It was also found that substantially more NAGA was released from mixed bronchoalveolar lavage cells in response to 20 microM A23187. Moreover, bronchoalveolar macrophages of ozone-exposed animals secreted more NAGA upon stimulation in vitro by either 20 microM A23187 or 200 micrograms/ml opsonized zymosan. We conclude that ozone-induced airway hyperreactivity in guinea pigs is associated with the presence of increased NAGA activity in bronchoalveolar fluid. Our data suggest that bronchoalveolar macrophages may, at least in part, be responsible for release of this enzyme into the airways after ozone exposure.

Electromechanical coupling of ferret airway smooth muscle in response to leukotriene C4.

We investigated the possible electrophysiological basis for the slow, prolonged force generation by airway smooth muscle (ASM) produced by leukotriene C4 (LTC4). Preparations of ASM were made from ferret trachea and placed in tissue microchambers for study. Some of these preparations were arranged so that force transducers and intracellular microelectrodes (with tip resistances of 30-80 M omega) could be used to measure isometric force and cell membrane potential (Em) simultaneously from ASM cells stimulated by LTC4. We found that ferret tracheal muscle was relatively sensitive to LTC4 and that this sensitivity was not significantly affected by atropine (1 microM), phentolamine (1 microM), propranolol (3 microM), and pyrilamine (1 microM). In a 1 nM solution of LTC4, Em was -54.0 +/- 1.2 mV from 18 impalements (n) from 6 animals (N) compared with a base-line value of -61.6 +/- 0.8 mV (n/N = 29/8, P less than 0.0005). This change did not lead to force generation, however. Higher concentrations of LTC4 led to progressive decreases in Em to which force generation was closely coupled. Concentrations greater than or equal to 70 nM led to phasic oscillations in Em of 0.6-0.8 Hz and 1.7 mV in amplitude, which were abolished by 10 microM verapamil, although the base-line Em was unaffected by this concentration. Although 300 nM LTE4 by itself caused only a small depolarization of ferret trachealis, it substantially antagonized the electromechanical responsiveness of this smooth muscle to LTC4. We conclude that ferret ASM is relatively sensitive to LTC4 and that there is an electrical basis for the slow, prolonged force generation caused by this mediator.

Prednisone pretreatment leads to histaminic airway hyporeactivity soon after resolution of the immediate allergic response.

We assessed the effect of prednisone pretreatment (50 mg/day for three days) on the development of the early increase in histamine reactivity that occurs soon after resolution of the immediate response in allergic humans. Four allergic subjects who were known to develop only isolated immediate responses upon Kentucky bluegrass inhalation, as well as four mild allergic asthmatic subjects known to develop typical dual phase responses, were evaluated. All testing was done more than nine weeks after the grass pollen season had ended. Allergen inhalation produced an immediate response in all subjects. However, upon resolution of the immediate response to allergen in these pretreated subjects, the PC200His in all dual responding asthmatics and in three of the four isolated immediate responders had substantially increased above baseline values. We conclude that prednisone pretreatment leads to histaminic hyporeactivity soon after resolution of the immediate allergic response in both dual responding asthmatics and isolated immediate responders. It would seem that this prednisone effect is independent of its potential influence on the influx of inflammatory cells into diseased airways.

Electromechanical effects of leukotriene D4 on ferret tracheal muscle and its muscarinic responsiveness.

We investigated the possible electrophysiological processes by which leukotriene D4 (LTD4) affects airway smooth muscle and its responsiveness to acetylcholine (ACh). For study in vitro, preparations of ferret tracheal muscle (dissected free of overlying mucosal and submucosal layers) were used. These preparations were arranged so that force transducers and glass intracellular microelectrodes (having tip resistances of 35-60 megohm) could be used to measure isometric force generation and cell membrane potential (Em) simultaneously from muscle stimulated by LTD4. At rest, the muscle was electrically and mechanically quiescent and had an Em of -59 +/- 0.2 mV (mean +/- SEM). We found that ferret tracheal muscle cells were relatively sensitive to LTD4, and that both the resulting depolarization (beginning at 10(-10) M LTD4) and force generation (produced by higher concentrations) progressed in a concentration-dependent manner. Depolarization by 10(-9) M LTD4 elicited electrical oscillations. These oscillations were accompanied by phasic contractile activity at 5 x 10(-9) M LTD4. Verapamil abolished these oscillations and diminished force substantially. We also found that ACh depolarized and contracted the muscle in a concentration-dependent manner. It caused electrical oscillations at greater than or equal to 10(-6) M. Diltiazem abolished these oscillations and markedly diminished force generation without affecting Em. Preexposure of airway muscle preparations for 20 min to a concentration (10(-10) M) of LTD4 that, by itself, did not produce significant force, substantially augmented the voltage-tension relationship of the muscle upon ACh stimulation. We conclude that there is an electrical basis for the slow, prolonged force generation of airway muscle caused by LTD4, and that LTD4 potentiates the electromechanical responsiveness of the airway muscle to muscarinic stimulation.

Toluene diisocyanate-induced airway hyperreactivity in guinea pigs depleted of granulocytes.

The influence of cyclophosphamide-induced granulocyte depletion on toluene diisocyanate (TDI)-related changes in airway reactivity and pathology was assessed in guinea pigs. Twelve cyclophosphamide-treated and 12 control animals comprising each group were studied physiologically before and 2 h after a single 10-min exposure to 3 ppm of TDI. Reactivity was determined in intact unanesthetized animals by measuring specific airway conductance before and during intravenous acetylcholine infusion. After testing, tracheal tissue for light microscopic examination was obtained from three hyperreactive guinea pigs in each exposed group and compared with tissue from treated and control animals (n = 3 each) that had not been TDI exposed. Cyclophosphamide treatment caused substantial decreases in both circulating and airway granulocyte counts. However, the incidence and degree of bronchial hyperreactivity that occurred 2 h post-TDI was similar in the untreated and treated groups. Our results indicate that TDI-induced bronchial hyperreactivity 1) occurs shortly after a brief high concentration exposure and 2) appears independent of circulating or airway granulocyte counts.

Bronchial reactivity increases soon after the immediate response in dual-responding asthmatic subjects.

To determine whether bronchial reactivity is augmented soon after an allergen-induced immediate asthmatic response, we compared reactivity to histamine before and immediately upon resolution of the immediate asthmatic response in seven subjects with mild asthma who were known to develop dual asthmatic responses after inhalation of Kentucky bluegrass allergen. Using a body plethysmograph and quiet breathing technique, specific airway resistance (SRaw) and reactivity to aerosol histamine were assessed on two days prior to allergenic challenge. The dose of histamine that doubled SRaw (PC200His) was determined by interpolation from histamine dose-response curves. On the day of allergenic challenge, each subject inhaled a concentration that was sufficient to induce a dual asthmatic response. Upon resolution of the immediate asthmatic response (45 to 105 minutes) after allergen, the PC200His in all cases had decreased more than 50 percent of its original value (PC200His for the group was 0.29 +/- 1.42 mg/ml [mean +/- SE], compared to 0.84 +/- 1.23 mg/ml initially). Seven or more days after the allergen, each subject had a PC200His comparable to original values. Our data indicate that airway reactivity in dual-responding asthmatic subjects markedly increases soon after the immediate asthmatic response and much before the late asthmatic response manifests clinically. Whether this early increase in bronchial reactivity is a putative requirement for, or shares common characteristics with the late asthmatic response requires further study.

cAMP suppresses CA2+-dependent electrical activity of airway smooth muscle induced by TEA.

Using intracellular microelectrodes, we investigated whether exogenous dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) or forskolin influenced the electrical effects of tetraethylammonium (TEA) on canine tracheal smooth muscle. We found that 20 mM TEA depolarized airway smooth muscle cells from a resting membrane potential (Em) of -59 +/- 4 mV (mean +/- SD) to -45 +/- 2 mV and caused spontaneous action potentials (AP's) to develop, which were 33 +/- 2 mV in amplitude. These were totally abolished in 0 Ca2+ solution. DBcAMP (1 mM) suppressed the development of this TEA-induced electrical activity and the phasic contractions electrically coupled to it. DBcAMP had no significant effect on Em in the absence of TEA however. Forskolin (1 microM) produced similar effects. Our findings suggest that Ca2+ is the principal ion responsible for the inward current associated with the TEA-induced AP's in airway smooth muscle, and that adenosine 3',5'-cyclic monophosphate may suppress the electrogenesis of this current.

Effects of leukotriene D4 and antagonists of arachidonic acid metabolism and calcium entry on guinea pig tracheal muscle responsiveness.

We studied the effects of antagonists of arachidonic acid metabolism and calcium entry on the responsiveness of airway smooth muscle to acetylcholine (ACh), potassium and norepinephrine. Responsiveness in vitro was assessed by measuring isometric contraction of guinea pig trachealis muscle upon chemical stimulation. We found that indomethacin potentiated the response of airway muscle to ACh and KCl, but not to norepinephrine. The indomethacin-induced potentiation observed was inhibited by both BW 755C and nifedipine. As occurred with indomethacin pretreatment, we also found that a noncontractile concentration of leukotriene D4 (LTD4) potentiated the responsiveness of tracheal muscle to both ACh and KCl. Our data suggest that indomethacin- and LTD4-potentiation of guinea pig airway muscle responsiveness to certain bronchoconstrictors may be mediated, at least in part, by enhanced extracellular Ca++ entry.

Toluene diisocyanate-induced airway hyperreactivity and pathology in the guinea pig.

We assessed the nature and progression of airway mucosal disease and histaminic reactivity in English short-haired guinea pigs at 2, 24, 72, 168, and 504 hours after toluene diisocyanate (TDI) exposure (4 hours of 3 ppm of TDI for 5 consecutive days). To also determine whether TDI-specific, IgE-like antibodies developed in TDI-exposed animals, passive cutaneous anaphylaxis testing was done 28 days after TDI. Bronchial reactivity was determined serially by measuring specific airway conductance as a function of increasing doses of aerosolized histamine in six exposed and three control animals studied intact and unanesthetized. The remaining 10 exposed and 10 control guinea pigs were sacrificed in groups of two at each time point to obtain airway tissue for light microscopic examination. We found that airway hyperreactivity to histamine occurred after TDI in all animals tested. It was maximal 2 hours after the 5-day exposure and remitted by 72 hours. In addition, marked airway obstruction occurred after TDI that persisted for at least 168 hours. There were dramatic signs of airway mucosal damage associated with the bronchial hyperreactivity that included substantial decreases in epithelial cilia, mucin content, and mast cells, as well as squamous metaplasia, numerous mitotic figures, and a prominent polymorphonuclear leukocytic infiltrate. Passive cutaneous anaphylaxis tests in exposed animals were negative. Our results suggest that TDI-induced bronchial hyperreactivity may be related to airway mucosal injury and inflammation.