Mast cells and reactive oxygen species in citric acid-induced airway constriction.

The noncholinergic airway constriction is mediated by tachykinins, mainly neurokinin A and substance P, and this bronchoconstriction is usually enhanced during inflammatory episodes. We demonstrated previously that reactive oxygen species play an important role in capsaicin-, hyperventilation-, and citric acid (CA) inhalation-induced noncholinergic airway constriction. For understanding cellular involvement, we further investigated the relationship between mast cells, bradykinin (BK), reactive oxygen species, and noncholinergic airway constriction. Sixty-five guinea pigs were divided into seven groups: saline control; CA; BK + CA; cromolyn sodium (CS) + CA; BK + CS + CA; compound 48/80 + CA; and compound 48/80 + BK + CA. CS was used to stabilize mast cells, whereas a secretagogue, compound 48/80, was for the depletion of mast cells. Each animal was anesthetized, cannulated, paralyzed, and ventilated artificially. In control animals, CA aerosol inhalation caused decreases in dynamic compliance and forced expiratory parameters, indicating CA-induced noncholinergic airway constriction. Either CS or compound 48/80 significantly attenuated the CA-induced airway constriction. Also, we detected a significant increase in lucigenin-initiated chemiluminescence counts of the bronchoalveolar lavage sample in the BK + CA group. Furthermore, CA exposure caused an increase in bronchoalveolar lavage substance P level. Either CS or compound 48/80 prevented the above CA-induced increases in chemiluminescence and substance P. These results suggest that mast cells play an important role in CA aerosol inhalation-induced airway constriction via perhaps releasing constricting factors.     

Endothelin-1 in citric acid aerosol inhalation-induced airway constriction of guinea pigs

Endotheline-1 (ET-1) has been shown to enhance tachykinin-induced airway constriction. This study was designed to test whether ET-1 is involved in citric acid-induced bronchoconstriction. Forty-eight anesthetized-paralyzed guinea pigs were divided into six groups of 8 animals each: saline control; citric acid; ET-1; ET-1 + citric acid; BQ123 + ET-1 + citric acid; and BQ788 + ET-1 + citric acid. BQ123 and BQ788 are specific ETA and ETB receptor antagonists, respectively. Each animal in the saline control group received 50 breaths of 4 ml saline aerosol and in all citric acid-treated groups was given 50 breaths of 4 ml aerosol generated from 0.6 M citric acid. In all ET-1-treated groups, each animal was exposed to aerosol generated from 10(-8) M ET-1. The animal in the ET-1 + citric acid group was exposed to ET-1 5 min prior to the citric acid. For the last two groups, each animal was first exposed to aerosol generated from either 10(-5) M BQ123 or 10(-5) M BQ788. Five min later, the animal was exposed to ET-1; and then 5 min later was followed by citric acid. Dynamic respiratory compliance (Crs), forced expiratory volume in 0.1 sec (FEV(0.1)), and maximal expiratory flow at 30% total lung capacity (Vmax 30) were obtained before and 3-15 min after citric acid. Either citric acid or ET-1 inhalation caused significant decreases in Crs, FEV(0.1), and Vmax 30, indicating airway constriction. Citric acid-induced airway constriction, for most cases, was not significantly augmented by ET-1. However, either BQ123 or BQ 788 significantly attenuated the airway constriction induced by the combination of ET-1 and citric acid. Also, in an additional study, either BQ123 or BQ788 significantly attenuated citric acid-induced airway constriction. These data suggest that endogenous ET-1 plays an important role in citric acid aerosol-induced airway constriction in guinea pigs.     

Bronchial circulatory reversal of methacholine-induced airway constriction

Although a role for the bronchial circulation in clearance of bronchoactive agents has been frequently proposed, experimental evidence is limited. In this study, we determined the importance of bronchial blood flow (QBA) in the recovery from methacholine-(MCh) induced bronchoconstriction. In 10 pentobarbital-anesthetized ventilated sheep, the bronchial branch of the bronchoesophageal artery was cannulated and perfused (0.7 ml.min-1.kg-1) with blood pumped from the femoral artery. MCh was infused directly into the bronchial artery at increasing concentrations (10(-7) to 10(-5) M). MCh infusion caused a concentration-dependent increase in airway resistance at constant QBA. However, the time constant of recovery (TC) from airway constriction after cessation of the MCh infusion was not dependent on the MCh concentration or the magnitude of the increases in airway resistance. When QBA was at 50, 100, and 200% of control level, with constant MCh concentration, TC was 44 +/- 6, 25 +/- 2, and 24 +/- 2 (SE) s at each flow level, respectively. TC at 50% of control QBA was significantly greater than at control QBA (P less than 0.01). Thus the magnitude of QBA can alter the time course of recovery from MCh-induced increases in airway resistance. These results document the importance of QBA in reversing agonist-induced constriction and suggest that an impaired bronchial circulation may contribute to the mechanism of airway hyperreactivity.     

Amplification of airway constriction due to liquid filling of airway interstices

Luminal epithelial projections formed during bronchoconstriction define interstices in which liquid can collect. Liquid in these interstices could amplify the degree of luminal compromise due to muscular contraction in at least two distinct ways. First, the luminal cross-sectional area is reduced by simple filling of the interstices. Second, if the surface tension (gamma) of the air-liquid interface is positive, the pressure drop across the interface produces an additional inward force that can further constrict the airway. We present a theoretical treatment of these two mechanisms together with data which suggest that both may significantly amplify the luminal narrowing due to airway smooth muscle contraction, particularly in small airways when gamma is high. To qualitatively assess the effects of altered gamma, guinea pig lungs with normal and altered airway liquid lining layers were frozen and studied while fully hydrated by low-temperature scanning electron microscopy. Airway gamma was altered in these animals by intratracheal instillation of 0.5 mg lysoplatelet-activating factor (lyso-PAF). The interstices of normal airways were dry, whereas the interstices of airways with altered surface lining layers were liquid filled. In addition, the surfactant inhibitory properties of lyso-PAF, 2-arachidonyl-PAF, and dipalmitoyl phosphatidylcholine (DPPC) were measured with a pulsating bubble surfactometer, using surfactant TA as the model surfactant. Minimal gamma (gamma min) of surfactant TA alone was 4.0 +/- 0.2 dyn/cm; a 5% mixture of lyso-PAF with surfactant TA resulted in a significantly (P less than 0.02) greater gamma min of 8.8 +/- 1.8 dyn/cm. In contrast, 2-arachidonyl-PAF and DPPC had minimal effects on gamma min of surfactant TA.     

Phosphatidic acid-induced calcium mobilization in osteoblasts

Phosphatidic acid (PA) evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i) in osteoblasts isolated from neonatal mouse calvaria. This increase was observed in both low (below 150 microM) and high (1.26 mM) Ca2+-containing medium. In contrast, other phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, failed to increase [Ca2+]i in osteoblasts. In high Ca2+-containing medium, A23187 also increased [Ca2+]i in the cells, but the mode of the change was different from that in the case of PA. These results suggest that PA may induce Ca2+-mediated cellular responses through Ca2+ release from intracellular stores in osteoblasts.     

Noninvasive detection of airway constriction in awake guinea pigs

Tidal volume measured by the barometric method is very sensitive to increases in compression and expansion of alveolar gas, such as would be expected to occur during airway narrowing or closure. By comparing a barometric method tidal volume signal (VT') with a reference tidal volume (VT) obtained with a head-out pressure plethysmograph, a simple index related to gas compressibility effects was calculated (VT/VT'). Changes in this index were compared with decreases in dynamic compliance (Cdyn) during histamine aerosol challenge of 15 Charles River Hartley guinea pigs. Decreases in VT/VT' occurred during all aerosol challenges and were correlated with decreases in Cdyn (r = 0.84, P less than 0.001). Decreases in VT/VT' were most marked at Cdyn values of less than 50% of base line. At Cdyn of less than 15% of base line, VT' was 3.1-4.8 times the VT reference signal. No increase in total pulmonary resistance was noted, and Cdyn and VT/VT' returned to base line after histamine exposure was stopped. We conclude that gas compressibility effects become substantial during histamine-induced airway constriction in the guinea pig and that the VT/VT' ratio appears to provide a simple noninvasive method of detecting these changes.     

Detection of mediator-induced airway constriction by barometric plethysmography in mice

The barometric method has recently been employed to detect airway constriction in small animals. This study was designed to evaluate the barometric method to detect mediator-induced central and peripheral airway constriction in BALB/c mice. First, the central airway constrictor carbachol and the peripheral airway constrictor histamine were employed to induce airway constriction, which was detected by both the conventional body plethysmography and the barometric method in anesthetized mice. Second, bronchoconstriction induced by aerosolized carbachol or other mediators was detected with the barometric plethysmography in conscious, unrestrained mice. Carbachol inhalation caused about four-fold increase in pulmonary resistance (RL) and about two-fold increase in enhanced pause (Penh) in anesthetized mice. In contrast, in the same preparation, histamine aerosol induced a decrease in dynamic compliance (Cdyn), with no alteration in RL or Penh. In awake mice, carbachol and methacholine caused increases in Penh, frequency, and tidal volume (VT). On the other hand, histamine, histamine + bradykinin, and prostaglandin-D2 did not alter Penh but decreased VT in conscious mice. These data suggest that there was no sufficient evidence to indicate that Penh could be a good indicator of bronchoconstriction for the whole airways.     

Propionic acid-induced calcium mobilization in human neutrophils

The ability of propionic acid to elicit an increase in the level of cytoplasmic free calcium in human neutrophils was examined in detail. Propionic acid induced a rapid and dose-dependent mobilization of calcium that relied on both internal and external sources of calcium. The effects of propionic acid on the mobilization of calcium were inhibited by pertussis toxin, but not cholera toxin, implicating a guanine nucleotide binding protein. Furthermore, preincubation of the neutrophils with phorbol 12-myristate 13-acetate resulted in a decreased mobilization of calcium. This inhibitory activity of phorbol myristate acetate was antagonized by the protein kinase C inhibitor H-7. Preincubation of the cells with the synthetic chemotactic factor fMet-Leu-Phe caused a reduction in the magnitude of the calcium transient elicited by propionic acid. However, the calcium response to propionic acid was not affected by antagonists of fMet-Leu-Phe and platelet-activating factor binding or by an inhibitor of leukotriene synthesis. Propionic acid did not elicit a mobilization of calcium in monocytes, platelets, lymphocytes, or undifferentiated HL-60 cells. However, the treatment of the HL-60 cells with dimethylsulfoxide resulted in the appearance of a calcium response to propionic acid. The potential physiological significance of these findings are discussed.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

Partitioning of lung tissue response and inhomogeneous airway constriction at the airway opening

Suki, Béla, Huichin Yuan, Qin Zhang, and Kenneth R. Lutchen. Partitioning of lung tissue response and inhomogeneous airway constriction at the airway opening. J. Appl.Physiol. 82(4): 1349-1359, 1997.During abronchial challenge, much of the observed response of lung tissues isan artifactual consequence of inhomogeneous airway constriction.Inhomogeneities, in the sense of time constant inequalities, are aninherently linear phenomenon. Conversely, if lung tissues respond to abronchoagonist, they become more nonlinear. On the basis of thesedistinct responses, we present an approach to separate real tissuechanges from airway inhomogeneities. We developed a lung model thatincludes airway inhomogeneities in the form of a continuousdistribution of airway resistances and nonlinear viscoelastic tissues.Because time domain data are dominated by nonlinearities, whereasfrequency domain data are most sensitive to inhomogeneities, we apply acombined time-frequency domain identification scheme. This model wastested with simulated data from a morphometrically based airway modelmimicking gross peripheral airway inhomogeneities and shown capable ofrecovering all tissue parameters to within 15% error. Application toour previously measured data suggests that in dogs during histamine infusion 1) the distribution ofairway resistances increases widely and2) lung tissues do respond but lessso than previously reported. This approach, then, is unique in itsability to differentiate between airway and tissue responses to anagonist from a single broadband measurement made at the airway opening.

     

Assessment of heterogeneous airway constriction in dogs: a structure-function analysis

Obstructive lung diseases are often characterized by heterogeneous patterns of bronchoconstriction, although specific relationships between structural heterogeneity and lung function have yet to be established. We measured respiratory input impedance (Zrs) in eight anesthetized dogs using broadband forced oscillations at baseline and during intravenous methacholine (MCh) infusion. We also obtained high-resolution computed tomographic (HRCT) scans in 4 dogs and identified 20-30 individual airway segments in each animal. The Zrs spectra and HRCT images were obtained before and 5 min following a deep inspiration (DI) to 35 cmH(2)O. Each Zrs spectrum was fitted with two different models of the respiratory system: 1) a lumped airways model consisting of a single airway compartment, and 2) a distributed airways model incorporating a continuous distribution of airway resistances. For the latter, we found that the mean level and spread of airway resistances increased with MCh dose. Whereas a DI had no effect on average airway resistance during MCh infusion, it did increase the level of airway heterogeneity. At baseline and low-to-moderate doses of MCh, the lumped airways model was statistically more appropriate to describe Zrs in the majority of dogs. At the highest doses of MCh, the distributed airways model provided a superior fit in half of the dogs. There was a significant correlation between heterogeneity assessed with inverse modeling and the standard deviation of airway diameters obtained from HRCT. These data demonstrate that increases in airway heterogeneity as assessed with forced oscillations and inverse modeling can be linked to specific structural alterations in airway diameters.     

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (～250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ～70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.     

Functional antagonism of airway constriction in the canine lung periphery.

We studied the ability of a beta-adrenergic agonist (albuterol) to attenuate calcium chelator- and acetylcholine-induced airway constriction in the lung periphery of anesthetized mongrel and Basenji-Greyhound (BG) dogs. A wedged bronchoscope technique was used to measure collateral system resistance before and after challenges with aerosolized Na2EDTA and acetylcholine. Time course of the response to Na2EDTA differed significantly between mongrel and BG dogs. Peak response to challenge with 4% Na2EDTA occurred within 2 min for mongrel dogs and at 5 min for BG dogs. Albuterol (1 microgram/kg iv) significantly attenuated Na2EDTA-induced bronchoconstriction in both groups of animals (P less than 0.01, each group). Albuterol (1 microgram/kg iv) significantly attenuated acetylcholine-induced bronchoconstriction in mongrel (P less than 0.01) but not in BG dogs. We conclude that a qualitative difference exists in the mechanism of Na2EDTA-induced constriction in the lung periphery of BG compared with mongrel dogs. In addition, the lung periphery of BG dogs demonstrates reduced beta-adrenergic sensitivity with respect to a cholinergic challenge compared with mongrels, suggesting enhanced cholinergic inhibition of the beta-adrenergic system.     

Effects of tidal volume stretch on airway constriction in vivo.

Tidal stresses are thought to be involved in maintaining airway patency in vivo. The present study examined the effects of normal stresses exerted by the lung parenchyma during tidal ventilation on recovery from agonist-induced airway constriction. In seven anesthetized dogs, one lung was selectively ventilated with a Univent endotracheal tube (Vitaid, Lewiston, NY). Airway tone was increased either transiently (intravenous bolus) or continuously (intravenous infusion) with methacholine (MCh). During one-lung ventilation, changes in the airway size of both lungs were measured for up to 40 min during recovery from constriction by using high-resolution computed tomography. After recovery to baseline, the alternate lung was ventilated, and the protocol was repeated. The absence of tidal stresses led to an attenuated recovery from either transient or steady-state airway constriction. The effectiveness or lack thereof of normal tidal stress in stabilizing airway size may be one factor that contributes to the lack of reversal with tidal breathing and deep inspiration seen in asthmatic subjects.     

Arachidonic acid-induced release of calcium in permeabilized human neutrophils

The addition of arachidonic acid to a suspension of digitonin-permeabilized human neutrophils was found to induce, in a dose-dependent manner (ED50 about 15 microM), the release of calcium from internal stores. Arachidic acid was without effect, while linoleic acid and linolenic acid were (on a concentration basis) at least 5-times less active than arachidonic acid. The activity of arachidonic acid appears to be due to the fatty acid itself and not to one of its metabolites. The pool of calcium mobilized by arachidonic acid includes that sensitive to inositol 1,4,5-triphosphate. These results demonstrate a significant intracellular role for arachidonic acid at the level of the internal mobilization of calcium in human neutrophils.