Pharmacologic properties of brewery dust extracts in vitro

STUDY OBJECTIVES: To study the effects of extracts of brewery dust on isolated guinea pig tracheal smooth muscle in vitro. DESIGN: Parallel pharmacologic intervention on guinea pig tracheal rings that were obtained from the same animal. SETTING: Mount Sinai School of Medicine, Department of Pulmonary Medicine. MATERIAL: The isolated guinea pig tracheal tissue of 18 guinea pigs. INTERVENTIONS: Pretreatment of guinea pig rings by mediator-modifying agents before challenge with the brewery dust extracts. MEASUREMENTS AND RESULTS: The effect of brewery dust extracts on isolated guinea pig tracheal smooth muscle was studied using water-soluble extracts of dust obtained from brewery materials, including hops, barley, and brewery yeast. Dust extracts were prepared as a 1:10 (wt/vol) aqueous solution. Dose-related contractions of nonsensitized guinea pig tracheas were demonstrated using these extracts. The dust extracts contained significant quantities of bacterial components (eg, endotoxin and n-formyl-methionyl-leucyl-phenylalanine), but these agents were not thought to contribute directly to the constrictor effect of the dusts. Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with the following drugs known to modulate smooth muscle contraction: atropine; indomethacin; pyrilamine; LY171883; nordihydroguaiaretic acid; captopril; thiorphan; verapamil; and TMB8. The constrictor effects of the dust extracts were inhibited by a wide variety of agents, the patterns of which depended on the dust extract. Atropine consistently and strikingly reduced the contractile effects of these extracts. These observations may suggest an interaction of the extracts with parasympathetic nerves or, more directly, with muscarinic receptors. The inhibition of contraction by the blocking of other mediators was less effective and varied with the dust extract. CONCLUSIONS: We suggest that brewery dust extracts cause a dose-related airway smooth muscle constriction by nonimmunologic mechanisms involving a variety of airway mediators and, possibly, cholinergic receptors. This effect is not dependent on presensitization of the guinea pigs.     

Pharmacologic Effects of Grain Weevil Extract on Isolated Guinea Pig Tracheal Smooth Muscle

The grain weevil, an insect (pest) that infects grain, is a frequent contaminant of processed wheat, and its presence may contribute to respiratory abnormalities in grain workers. We studied the in vitro effects of an extract of grain weevil (GWE) on airway smooth muscle. Pharmacologic studies included in vitro challenge of guinea pig trachea with GWE, in parallel organ baths, pretreated with mediator-modifying agents or a control solution. Dose-related contractions of nonsensitized guinea pig trachea (GPT) were demonstrated using this extract. Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with drugs known to modulate smooth muscle contraction: atropine, indomethacin, pyrilamine, acivicin, NDGA, BPB, TMB8, captopril, and capsaicin. Atropine, pyrilamine, BPB, and capsaicin significantly reduced the contractile effects of the extract at most of the challenge doses (p < 0.01 or p < 0.05). Inhibition of GWE-induced contraction by blocking of other mediators was less complete. We suggest that GWE causes dose-related airway smooth muscle constriction of the GPT by nonimmunologic mechanisms involving a variety of airway mediators and possibly cholinergic receptors.     

Pharmacological studies of the effect of wheat grain extract

BACKGROUND: Agricultural farm workers exposed to wheat grain dust are at risk of developing respiratory abnormalities. The pathogenesis of this injury is only partially understood. OBJECTIVES: To determine the effect of wheat grainextract on isolated guinea pig tracheal smooth muscle. METHODS: In the current study, pharmacologic properties of wheat grain extract (WGE) were tested using guinea pig tracheas studied in vitro. Dose-related contractions of nonsensitized guinea pig trachea were demonstrated using these extracts. Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with drugs known to modulate smooth muscle contraction: atropine 10(-6)M, indomethacin 10(-6)M, pyrilamine 10(-6)M, acivicin 10(-5)M, nordihydroguaretic acid (NDGA) 10(-5)M, bromophenacyl bromide (BPB) 10(-5 )M, 3,4,5-trimethoxybenzoic acid-8-(diethylamino)- octyl ester TMB8 10(-5)M, captopril 10(-5)M and capsaicin 5 x 10(-6)M. RESULTS: WGE causes a dose-dependent constriction of guinea pig tracheal smooth muscle. Atropine, pyrilamine, TMB8 and acivicin significantly reduced the contractile effects of the WGE. Inhibition of contraction by blocking of other mediators was significant but less complete. CONCLUSION: We conclude that WGE causes a dose-related constriction of airway smooth muscle by nonimmunological mechanisms involving a variety of airway mediators and possibly cholinergic receptors.     

Pharmacologic effects of tobacco dust extract on isolated Guinea pig trachea

STUDY OBJECTIVE: To determine the effects of tobacco dust extract (TDE) on isolated guinea pig tracheal smooth muscle. DESIGN: A controlled, in vitro smooth-muscle study of the effect of pharmacologic agents on TDE. METHODS: The effect of TDE on isolated guinea pig tracheal smooth muscle was tested using water-soluble extracts of dust obtained from machines in a cigarette manufacturing plant. Dose-related contractions of nonsensitized guinea pig trachea were demonstrated using these extracts. The dust extracts contained significant quantities of bacterial components (eg, endotoxin). Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with drugs known to modulate smooth-muscle contraction: atropine, indomethacin, pyrilamine, nordihydroguaretic acid, acivicin, bromophenacyl bromide, 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester, captopril, and capsaicin. RESULTS: Atropine strikingly reduced the contractile effects of these extracts. Inhibition of contraction by blocking other mediators was less complete. There was no inhibition of contraction by hexamethonium (10(-4) mol/L, 10(-5) mol/L, 10(-6) mol/L), suggesting that nicotine was not the major contractile mediator of TDE. A separate analysis using different molecular weight fractions of TDE indicated that the constrictor activity appears to be primarily in the fraction with a molecular weight < 10 kd. Additionally, the constrictor effect resided entirely in the nonlipid fraction of the extract. We suggest that TDE causes dose-related airway smooth-muscle constriction by nonimmunologic mechanisms involving a variety of airway mediators and possibly cholinergic receptors. CONCLUSIONS: The bronchoconstrictor activity of TDE resides primarily in its low molecular weight, nonlipid fraction, and hexamethonium studies suggest that this agent is not nicotine.     

Response to Oyster Mushroom (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>) Extract by Sensitized and Nonsensitized Guinea Pig Trachea

Occupational exposure to oyster mushroom (Pleurotus ostreatus) has been associated with obstructive lung disease. Previously, we studied an extract of oyster mushroom (OME) and determined that it causes dose-dependent contractions of nonsensitized guinea pig trachea (GPT). We extend these studies to the investigation of sensitized tissue. In the present study 24 animals were sensitized using ovalbumin (OA) and subsequently challenged with an aerosol of 2.5% OA. A control group of 12 nonsensitized GPs was also studied. Tracheas were removed and were divided into rings in which the epithelium was retained (EP+) or removed (EP−). Dose-related contractions of sensitized and nonsensitized GPTs were elicited with OME. In nonsensitized animals the EP+ GPTs demonstrated a significantly greater response to OME (100–1000 μl) than did the EP− GPTs (p < 0.01). By contrast, in sensitized GPTs with and without epithelium there was no difference to challenge with OME. Finally, sensitized GPTs with and without epithelium and nonsensitized GPTs with epithelium responded similarly to challenge with OME. These findings suggest that in nonsensitized animals there is an enhancement of contractile response to OME which is in part mediated by the GPT epithelium. In sensitized animals with or without epithelium, the level of bronchoconstrictor response is similar to that of the nonsensitized animals with epithelium, suggesting an enhanced constrictor response independent of epithelium in the sensitized animals.     

Effects of Recycled Paper Dust Extracts on Isolated Guinea Pig Trachea

The effect of paper dust collected at two different locations in a paper recycling plant (PD1 and PD2) on isolated nonsensitized guinea pig tracheal smooth muscle was studied in vitro. Dust extracts were prepared as a 1:10 w/v aqueous solution. Dose-related contractions of guinea pig tracheal rings were elicited with both PD1 and PD2. Pharmacologic studies were performed with atropine (10⁻⁶ M), indometacin (10⁻⁶ M), pyrilamine (10⁻⁶ M), LY171883 (10⁻⁵ M), nordihydroguaiaretic acid (10⁻⁵ M), and TMB8 (10⁻⁵ M). The possible role of endogenous neuropeptides in this constrictor process was studied by depleting neural mediators with capsaicin (5 × 10⁻⁶ M) before challenge with dust extracts. Constrictor effects were partially inhibited by a wide variety of the mediator blocking agents. The effects of both extracts were almost totally inhibited by the anticholinergic agent atropine, suggesting that a principal pathway mediating this response may involve the parasympathetic nervous system. The intracellular calcium-blocking agent TMB8 also induced a reduction of the contractile responses to PD1 and PD2 concsistent with the well established role of intracellular calcium in smooth muscle constriction. Pretreatment with capsaicin significantly increased the contractile activity of paper dust extracts but only at the higher doses of these extracts. This suggests that the effect of paper dust is not initiated by the release of mediators stored in sensory nerves but that the prerelease of these mediators may enhance the constrictor effects of these dusts. We suggest that paper dust extracts cause dose-related airway smooth muscle constriction possibly associated with the release of cholinergic as well as other mediators. The constrictor effect does not require tissue presensitization or the release of neuropeptides from sensory nerves. Accepted for publication: 21 March 1997     

Ozone exposure may enhance airway smooth muscle contraction by increasing Ca(2+) refilling of sarcoplasmic reticulum in guinea pig

Ozone induces airway hyperresponsiveness, but there is controversy about effects of ozone on smooth muscle per se. We therefore investigated effects of in vivo ozone exposure on intracellular calcium mobilization in relation to tracheal smooth muscle contractility in the guinea pig in vitro. Guinea pigs underwent ozone exposure or sham exposure (3 ppm, 2 h). Then, a tracheal smooth muscle strip was mounted in an organ bath to record isometric tension. Effects of ozone exposure on acetylcholine-induced contraction of smooth muscle were as follows. Contraction was not altered in normal Krebs solution, but was increased in Ca(2+)-free solution in ozone-exposed animals. Decline of tension on repetitive application of acetylcholine in Ca(2+)-free solution was reduced, while the tension decline rate while acetylcholine was washed out with Ca(2+)-free solution was facilitated in ozone-exposed animals. Tension decline during the continuous administration of acetylcholine in Ca(2+)-free solution was slowed. Contraction occurred more quickly in Ca(2+)-free solution in ozone-exposed animals. Results suggest that ozone has a direct action on airway smooth muscle by changing Ca(2+) mobilization; Ca(2+) refilling via a Ca(2+) pump and Ca(2+) release via Ca(2+) channels in the sarcoplasmic reticulum were increased, while Ca(2+) extrusion via the plasma membrane Ca(2+) pump was unchanged.     

New evidence for transmitter role of VIP in the airways: impaired relaxation by a catalytic antibody

The identity of the transmitter(s) of nonadrenergic, noncholinergic airway smooth muscle relaxation has long been investigated. Recently, nitric oxide (NO) has been proposed as the main, if not the only transmitter. We earlier suggested vasoactive intestinal peptide (VIP) as a candidate transmitter and target for pathogenic catalytic autoantibodies (VIPases) found in certain humans. To re-examine the role of VIP, we studied the airway transport and effects of a model monoclonal antibody (Ab) capable of binding and cleaving VIP. In vitro receptor binding assays indicated the catalytic light chain subunit of the VIPase Ab to inhibit the saturable binding of (Tyr(10-125)I) VIP by guinea pig lung membranes, whereas a catalytically deficient mutant of the Ab light chain was without significant inhibitory activity. Systemically administered IgG preparations of the VIPase Ab accumulated in the airway lavage fluid of guinea pigs at levels close to those in blood, suggesting that the Ab reaches the airways freely. Electrical field stimulation (EFS)-induced relaxations of tracheal strips were weaker and shorter in VIPase-treated animals than in control nonimmune IgG-treated animals. The inhibitory effect of the VIPase was dose-dependent. VIPase-mediated inhibition of EFS-induced relaxation was evident both in the absence and presence of blockade of beta-adrenergic and cholinergic receptors. Thus, circulating VIP binding and cleaving antibodies can reach the airways and attenuate the neurogenic relaxation of guinea pig tracheal smooth muscle, probably by neutralizing endogenously released VIP. The findings support a role for VIP as a major mediator of neurogenic relaxation of guinea pig tracheal smooth muscle. Lack of complete abrogation of relaxation is consistent with a co-transmitter role for NO.     

Nonadrenergic noncholinergic inhibitory nervous system in guinea pig airway]

It has been reported that tracheal smooth muscle of guinea pig is innervated by both the adrenergic and nonadrenergic noncholinergic (NANC) inhibitory nervous system. However, NANC inhibitory nerve supply to the lower airway of guinea pig has not yet been demonstrated in in vivo experiments. We performed the present study to evaluate the physiological role of NANC inhibitory nerves in tracheal smooth muscle of guinea pig in vitro, and in anesthetized guinea pigs in vivo. Innervation of NANC inhibitory nerves to tracheal smooth muscle was much greater than that of adrenergic nerves (77.8 +/- 3.8%, 22.2 +/- 3.8%, respectively, p less than 0.01). Neither decrease in RL nor increase in CL, however, was observed with vagal stimulation during serotonin infusion after the administration of propranolol. The role of NANC inhibitory nerves in histamine-induced bronchoconstriction (HIB) was investigated. HIB was enhanced by vagotomy in guinea pig pretreated with propranolol (p less than 0.01). These results suggest the role of the NANC inhibitory nervous system in the attenuation of HIB in vivo in the guinea pig.     

吸烟对沙丁胺醇和布地奈德抑制气道平滑肌细胞收缩作用的影响

目的：研究长期吸烟对于哮喘治疗常用药物沙丁胺醇和布地奈德药效的影响。方法原代培养吸烟与非吸烟豚鼠气道平滑肌细胞,分别检测其在静息态、组胺激发态及以药物(沙丁胺醇和布地奈德)抑制平滑肌收缩后再以组胺激发细胞内游离钙离子的水平,取峰值并比较两组间抑制率的差异。结果吸烟组气道平滑肌细胞静息态、激动态及药物(沙丁胺醇和布地奈德)作用后胞内游离钙离子的水平均显著高于不吸烟组(P <0.05或 P <0.01);吸烟豚鼠气道平滑肌细胞内2种药物抑制胞内钙水平升高的作用均削弱(P <0.05)。结论吸烟可使豚鼠呼吸道平滑肌内游离钙离子升高,加强其收缩强度,同时吸烟会影响沙丁胺醇和布地奈德对胞内钙升高的抑制作用,降低药物疗效。     

Subchronic exposure to diisocyanates increases guinea pig tracheal smooth muscle responses to acetylcholine

OBJECTIVE: In order to study the threshold concentrations of isocyanates (IC) for induction of lung disorders, constrictive responses of tracheal smooth muscles to acetylcholine (ACH) in guinea pigs with and without diisocyanate [toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and diphenylmethane diisocyanate (MDI)] exposure were investigated. METHODS: An IC-induced increase in smooth muscle responsiveness was studied by measuring cumulative ACH dose responses (10(-10) to 10(-4) M ACH). Basal ACH dose-response curves, measured twice in intervals of 1 h using tracheal preparations of 11 guinea pigs previously not exposed to IC, were reproducible. RESULTS: Subchronic in vivo exposures to TDI, HDI, and MDI atmospheres of 10 and 20 parts per billion (ppb) on 5 consecutive days led to significantly (p < 0.05) increased ACH responsiveness of tracheal smooth muscle, whereas concentrations of 2.5 and 5 ppb were not effective. Exposure to HDI atmospheres of 10 ppb for 1, 2, 4, or 8 weeks resulted in a time-dependent increase in ACH responses (p < 0.05) of guinea pig tracheal smooth muscle. Increased tracheal muscle responses to ACH were transient since tracheal preparations from animals exposed to 10 and 20 ppb MDI for 4 weeks and with an exposure-free interval of 8 weeks before preparation did not show enlarged ACH responses, which were present in preparations at the end of the exposure period (p < 0.05). Exposure to low IC concentrations as present in workplaces cause increased ACH responsiveness of guinea pig tracheal smooth muscle. The increased responsiveness of the airways seems to be largely reversible, since normal responses were found after 8 weeks of IC avoidance. CONCLUSION: Reversibility of IC-induced airway hyperresponsiveness is of great occupational and preventive medical importance. Workers with acquired airway hyperresponsiveness might escape lung damage if the changes are detected in an early stage before alterations in lung function are in a chronic stage.     

Relaxant effect of brain natriuretic peptide in nonsensitized and passively sensitized isolated human bronchi

Brain natriuretic peptide (BNP) relaxes guinea pig tracheal smooth muscle in vitro and is effective in preventing ovalbumin-induced bronchoconstriction and microvascular leakage in guinea pigs in vivo. Nonetheless, published studies on BNP in human airways in vitro are still lacking in the literature. The aim of this study was to investigate the effect of BNP in isolated human bronchi. The relaxant effect of BNP (1 nM to 10 μM) was assessed in nonsensitized and in passively sensitized human bronchial airways pre-contracted with submaximal concentration (EC₇₀) of carbachol or histamine. At the end of the experiment, papaverine (500 μM) was then added. BNP induced a weak relaxant activity on carbachol-contracted bronchi in nonsensitized (relaxation: 4.23 ± 0.51%) and passively sensitized bronchi (relaxation: 11.31 ± 2.22%). On the other hand, BNP induced a relaxant activity on His-contracted bronchi in nonsensitized (relaxation: 42.52 ± 9.03%) and in passively sensitized (relaxation: 60.57 ± 9.58%). All these findings are a clear documentation of the modest relaxant role of BNP in asthma and, likely, COPD.     

Relaxant effects of forskolin on guinea pig tracheal smooth muscle

We investigated the relaxant effects of forskolin, a diterpene derivative isolated from the roots of Coleus forskohlii, on guinea pig airway smooth muscle by measuring the isometric tension of tracheal smooth muscle in vitro and transcutaneous Po2 during the histamine inhalation test (HIT) in vivo. Forskolin (10(-9)-10(-5) M) caused dose-dependent relaxant effects on resting tone and on leukotriene C4 (10(-7) M)-, leukotriene D4 (10(-7) M)-, and carbachol (3 X 10(-6) M)-induced contraction of tracheal smooth muscle. Moreover, with propranolol pretreatment the relaxant effect of forskolin on tracheal smooth muscle did not change, whereas with the same pretreatment the relaxant effect of isoproterenol diminished. Forskolin (10(-8)-10(-6) M) raised tissue cyclic AMP levels dose-dependently in tracheal smooth muscle (6.7-359.9 pmol/mg protein). Forskolin (1 mg/kg) administered subcutaneously raised the respiratory threshold of (RT-histamine in the HIT. The determination of the RT-histamine by measuring tcPo2 was possible without anesthesia. These results suggest that forskolin relaxes airway smooth muscle in guinea pigs in vitro and in vivo by raising tissue cyclic AMP levels and that its actions are independent of beta-adrenoceptors.     

Antigen-induced release of airway epithelium-derived inhibitory factor

The ability of guinea pig trachea to produce a vasoactive epithelium-derived inhibitory factor (EpDIF) in response to mast cell-derived mediators was assessed in a coaxial bioassay system. The mast cell degranulating agent compound 48/80 (10 micrograms/ml) and histamine caused reductions in phenylephrine-induced tone in endothelium-denuded rat aorta preparations mounted coaxially within epithelium-intact guinea pig tracheal tube tissue. Relaxation responses to histamine and to compound 48/80 (10 micrograms/ml) were markedly reduced in the presence of mepyramine (50 microM) or when the epithelium was removed from coaxially mounted guinea pig trachea, indicating that they were mediated via the release of EpDIF. Coaxial bioassay assemblies were also prepared using EpDIF donor tracheal tissue obtained from guinea pigs actively sensitized to ovalbumin. Subsequent challenge with ovalbumin (10(-7) to 10(-1) mg/ml) produced concentration-dependent relaxation mediated by EpDIF. Ovalbumin-induced relaxation responses were not inhibited in the presence of either mepyramine (20 and 100 microM) or SKF 104353-Z2 (10 microM) alone but were significantly reduced when both mepyramine (20 microM) and SKF 104353-Z2 (10 microM) were present. Antigen-induced relaxation was apparently mediated by EpDIF in response to mast cell-derived histamine and leukotrienes. Rat tracheal airway smooth muscle did not relax in response to EpDIF, suggesting selectivity of action on vascular smooth muscle. Vasoactive EpDIF may play a role in protecting against antigen-induced bronchoconstriction by regulating bronchial circulation flow.     

Relaxant effects of forskolin on guinea pig tracheal smooth muscle

We investigated the relaxant effects of forskolin, a diterpene derivative isolated from the roots ofColeus forskohlii, on guinea pig airway smooth muscle by measuring the isometric tension of tracheal smooth muscle in vitro and transcutaneous Po₂ during the histamine inhalation test (HIT) in vivo. Forskolin (10⁻⁹–10⁻⁵ M) caused dose-dependent relaxant effects on resting tone and on leukotriene C₄ (10⁻⁷ M)-, leukotriene D₄ (10⁻⁷ M)-, and carbachol (3 × 10⁻⁶ M)-induced contraction of tracheal smooth muscle. Moreover, with propranolol pretreatment the relaxant effect of forskolin on tracheal smooth muscle did not change, whereas with the same pretreatment the relaxant effect of isoproterenol diminished. Forskolin (10⁻⁸–10⁻⁶ M) raised tissue cyclic AMP levels dose-dependently in tracheal smooth muscle (6.7–359.9 pmol/mg protein). Forskolin (1 mg/kg) administered subcutaneously raised the respiratory threshold of (RT-histamine in the HIT. The determination of the RT-histamine by measuring tcPo₂ was possible without anesthesia. These results suggest that forskolin relaxes airway smooth muscle in guinea pigs in vitro and in vivo by raising tissue cyclic AMP levels and that its actions are independent ofβ-adrenoceptors.     

The effect of platelet-activating factor on histamine and muscarinic receptor function in guinea pig airways

Platelet-activating factor (PAF) administered i.v. or by aerosol to guinea pigs elicited an increase in airway responsiveness to both acetylcholine and histamine when compared with guinea pigs exposed to the vehicle bovine serum albumin (BSA). After i.v. PAF, the ED100 for acetylcholine and histamine was 5.4 and 3.4 micrograms/kg, respectively, in comparison with 17 and 6 micrograms/kg, respectively, before PAF, representing approximately a 3- and 2-fold increase in responsiveness, respectively. After aerosol PAF (500 micrograms), the ED100 for acetylcholine and histamine was 13 and 7 micrograms/kg, respectively, whereas after aerosolized BSA, the ED100 for acetylcholine and histamine was 23 and 12 micrograms/kg, respectively, representing approximately a 2-fold increase in responsiveness. However, airway smooth muscle obtained from these PAF- or BSA-treated animals did not exhibit any differences in contractile response to histamine or acetylcholine in vitro. Likewise, there were not significant differences in the binding affinity or receptor density between PAF- and BSA-treated tissues with [3H]quinuclidinylbenzilate or [3H]pyrilamine binding, which were used to identify muscarinic and H1-histamine receptors, respectively. Furthermore, histamine and carbachol-induced phosphoinositide hydrolysis was similar in PAF- and BSA-treated tracheal smooth muscle preparations. Thus, PAF induces airway hyperresponsiveness in the guinea pig that is not related to changes in airway smooth muscle or to changes in muscarinic and histamine (H1) receptor density or function.     

Ontogenesis of myosin light chain phosphorylation in guinea pig tracheal smooth muscle

Increased airway responsiveness occurs in normal young individuals compared to adults. A maturation of airway smooth muscle (ASM) contractility is likely a mechanism of this juvenile airway hyperresponsiveness. Indeed, we showed in guinea pig tracheal smooth muscle (TSM) that maximum shortening velocity decreases dramatically after the first 3 weeks of life. Because the phosphorylation of the 20-kDa myosin light chain (MLC(20)) was shown to be a key event in ASM contractility, in the present work we sought to investigate it during ontogenesis. In three age groups (1-week-old, 3-week-old, and adult guinea pigs), we assessed the amount of MLC(20) phosphorylation achieved either in TSM crude protein homogenates exposed to Mg(2+) . ATP . CaCl(2) or in tracheal strips during electrical field stimulation (EFS). Phosphorylated and unphosphorylated MLC(20) were separated on nondenaturing 10% polyacrylamide gels, and the ratio of phosphorylation was obtained by densitometric analysis of chemiluminescent Western immunoblots. Maximum MLC(20) phosphorylation (% of total MLC(20)) in TSM tissue homogenate was, respectively, 32.6 +/- 5.7, 32.2 +/- 5.7, and 46.8 +/- 5.8 in 1-week, 3-week, and adult guinea pigs. Interestingly, in nonstimulated intact tracheal strips, we found a substantial degree of MLC(20) phosphorylation: respectively, 42.2 +/- 5.8, 36.5 +/- 7.8, and 46.4 +/- 4.7 in 1-week, 3-week, and adult guinea pigs. Maximal EFS-induced MLC(20) phosphorylation (% increase over baseline) in the 3-week age group was attained after 3 sec of EFS, and was 161.2 +/- 17.6, while in 1-week and adult guinea pigs, it was attained at 1.5 sec of EFS and was, respectively, 133.3 +/- 9.3 and 110.2 +/- 3.9 (P < 0.05). We conclude that MLC(20) phosphorylation in guinea pig intact tracheal strips correlates with ontogenetic changes in shortening velocity and changes in myosin light chain kinase content. These results further suggest that the maturation of ASM contractile properties plays a role in the greater airway responsiveness reported in children and young animals.     

Development of a prolonged eosinophil-rich inflammatory leukocyte infiltration in the guinea-pig asthmatic response to ovalbumin inhalation

Considerable attention has recently focused on the role of inflammation in the pathophysiology of asthma, with special emphasis on "late-phase" bronchoconstriction and increased airway hyperreactivity after antigen challenge in sensitized subjects. The present report describes the histopathologic changes in guinea-pig lung and trachea at various time intervals after ovalbumin inhalation in nonsensitized (control) and sensitized animals. Bronchoalveolar lavage (BAL) was also used to assess the accompanying accumulation of intraluminal leukocytes. A distinct leukocyte margination, consisting of neutrophils and eosinophils, was observed in the peribronchial vasculature as early as 8 min postchallenge in sensitized guinea pigs. At 6 h, the eosinophils predominated and migrated to the peribronchiolar smooth muscle layer. Between 6 h and 18 h, eosinophils were seen in tracts between the smooth muscle cell layers, accumulating in large numbers in the bronchial mucosal epithelium. This pattern persisted for at least 7 days postchallenge during which eosinophils remained the dominant cell type present. Peribronchiolar accumulation of neutrophils and mononuclear cells was minimal at all time points studied. Intraluminal mucus eosinophilia developed between 18 h and 7 days. A similar pattern of eosinophil infiltration was observed in the tracheal epithelium. Control, nonsensitized, guinea-pig lungs showed minor changes with little or no eosinophil infiltration at any time after antigen challenge. These findings correlated well with the BAL study in which sensitized guinea pigs exhibited a marked delayed increase in eosinophil counts between 18 h and 7 days compared with that in nonsensitized animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Airway hypocapnia increases microvascular leakage in the guinea pig trachea.

We have previously shown that airway hypocapnia induced bronchoconstriction in the guinea pig lung by releasing tachykinins. To examine whether airway hypocapnia could also cause an increase in airway microvascular leakage, a tracheal segment was isolated in vivo in anesthetized guinea pigs and unidirectionally ventilated (200 ml/min) for 1 h with fully conditioned air (0% CO2) or isocapnic gas (5% CO2). The lungs were ventilated through a distally placed tracheal cannula. Microvascular leakage was quantitated by the injection of Evans blue (EB) and its extraction from the tracheal segment. EB extravasation was increased in tracheae exposed to 0% CO2 (52.3 +/- 2.0 micrograms/g wet tissue) compared with tracheae exposed to 5% CO2 (26.4 +/- 2.9 micrograms/g; p less than 0.05) and to tracheae from spontaneously breathing guinea pigs (25.2 +/- 2.3 micrograms/g; p less than 0.05). Groups of animals in which trachea were unidirectionally ventilated with 0% CO2 were then pretreated with a range of drugs in an attempt to determine the mediators responsible for the microvascular leakage with 0% CO2. Capsaicin and morphine pretreatment did not significantly alter 0% CO2-induced EB extravasation, and phosphoramidon prevented rather than increased extravasation, suggesting that tachykinins did not play a role. The hypocapnia-induced increase in microvascular leakage was, however, prevented by indomethacin pretreatment and significantly attenuated by dazmegrel, a thromboxane synthetase inhibitor. We conclude that airway hypocapnia causes microvascular leakage in the guinea pig trachea and that this effect is mediated by prostaglandins and/or thromboxane.