Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor

Ozone causes persistent airway hyperreactivity in humans and animals. One day after ozone exposure, airway hyperreactivity is mediated by release of eosinophil major basic protein that inhibits neuronal M2 muscarinic receptors, resulting in increased acetylcholine release and increased smooth muscle contraction in guinea pigs. Three days after ozone, IL-1β, not eosinophils, mediates ozone-induced airway hyperreactivity, but the mechanism at this time point is largely unknown. IL-1β increases NGF and the tachykinin substance P, both of which are involved in neural plasticity. These experiments were designed to test whether there is a role for NGF and tachykinins in sustained airway hyperreactivity following a single ozone exposure. Guinea pigs were exposed to filtered air or ozone (2 parts per million, 4 h). In anesthetized and vagotomized animals, ozone potentiated vagally mediated airway hyperreactivity 24 h later, an effect that was sustained over 3 days. Pretreatment with antibody to NGF completely prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone and significantly reduced the number of substance P-positive airway nerve bundles. Three days after ozone, NK1 and NK2 receptor antagonists also blocked this sustained hyperreactivity. Although the effect of inhibiting NK2 receptors was independent of ozone, the NK1 receptor antagonist selectively blocked vagal hyperreactivity 3 days after ozone. These data confirm mechanisms of ozone-induced airway hyperreactivity change over time and demonstrate 3 days after ozone that there is an NGF-mediated role for substance P, or another NK1 receptor agonist, that enhances acetylcholine release and was not present 1 day after ozone.

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Localization of eosinophils to airway nerves and effect on neuronal M2 muscarinic receptor function

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l93 ◽

1997 ◽

Vol 273 (1) ◽

pp. L93-L103 ◽

Cited By ~ 34

Author(s):

R. W. Costello ◽

B. H. Schofield ◽

G. M. Kephart ◽

G. J. Gleich ◽

D. B. Jacoby ◽

...

Keyword(s):

Guinea Pigs ◽

Acetylcholine Release ◽

Basic Protein ◽

Receptor Function ◽

Major Basic Protein ◽

Parasympathetic Nerves ◽

M2 Muscarinic Receptor ◽

Fatal Asthma ◽

Eosinophil Major Basic Protein ◽

M2 Muscarinic Receptors

Neuronal M2 muscarinic receptors inhibit acetylcholine release from pulmonary parasympathetic nerves but are dysfunctional in antigen-challenged animals and asthmatics. Deletion of pulmonary eosinophils protects M2 receptor function in antigen-challenged guinea pigs. Therefore, the association of eosinophils with airway nerves was investigated. Nerve-associated eosinophils were significantly increased in challenged animals compared with controls (0.75 +/- 0.05 vs. 0.28 +/- 0.05 eosinophils/nerve). In antigen-challenged animals, eosinophil density was greatest around airway nerves, suggesting recruitment to the nerves. M2 receptor function was inversely correlated with the number of eosinophils per nerve, thus eosinophils are associated with airway nerves in antigen-challenged guinea pigs, where they impair M2 receptor function. In airways from three patients with fatal asthma, 196 of 637 eosinophils (30%) were associated with nerves, and release of eosinophil major basic protein was evident; conversely, in three control patients 1 of 11 (9%) eosinophils were in contact with nerves. Thus eosinophils and their granule proteins are also seen in association with airway nerves in patients with asthma.

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The changing role of eosinophils in long-term hyperreactivity following a single ozone exposure

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00377.2004 ◽

2005 ◽

Vol 289 (4) ◽

pp. L627-L635 ◽

Cited By ~ 26

Author(s):

Bethany L. Yost ◽

Gerald J. Gleich ◽

David B. Jacoby ◽

Allison D. Fryer

Keyword(s):

Smooth Muscle ◽

Vagal Stimulation ◽

Airway Hyperreactivity ◽

Ozone Exposure ◽

Single Exposure ◽

Eosinophil Major Basic Protein ◽

Changing Role ◽

Eosinophil Accumulation

Ozone hyperreactivity over 24 h is mediated by blockade of inhibitory M2 muscarinic autoreceptors by eosinophil major basic protein. Because eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to M2 dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, M2 function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone-induced hyperreactivity to vagal stimulation persisted over 3 days. Although hyperreactivity one day after ozone is mediated by eosinophils, AbVLA-4 did not inhibit either eosinophil accumulation in the lungs or around the nerves or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs, were decreased, and neuronal M2 receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from M2 receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL in lungs and around nerves, and M2 receptors were again dysfunctional. At this point, airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4, or cyclophosphamide protected M2 function 3 days after ozone and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significantly potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over 3 days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair.

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Substance P-induced airway hyperreactivity is mediated by neuronal M2 receptor dysfunction

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.3.l477 ◽

2000 ◽

Vol 279 (3) ◽

pp. L477-L486 ◽

Cited By ~ 25

Author(s):

Christopher M. Evans ◽

Kristen E. Belmonte ◽

Richard W. Costello ◽

David B. Jacoby ◽

Gerald J. Gleich ◽

...

Keyword(s):

Substance P ◽

Guinea Pigs ◽

Inflammatory Cells ◽

Airway Hyperreactivity ◽

Vagus Nerves ◽

Intravenous Heparin ◽

Release Of Acetylcholine ◽

Neurokinin 1 ◽

Eosinophil Major Basic Protein

Neuronal muscarinic (M2) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M2 autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M2 receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar9,Met(O2)11]- substance P, guinea pigs were hyperreactive to vagal stimulation and M2 receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK1) receptor antagonist (SR-140333) all prevented substance P-induced M2dysfunction and hyperreactivity. Intravenous heparin acutely reversed M2 receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK1 receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M2 receptors by MBP, resulting in increased release of acetylcholine.

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Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.276.5.l709 ◽

1999 ◽

Vol 276 (5) ◽

pp. L709-L714 ◽

Cited By ~ 21

Author(s):

Richard W. Costello ◽

Christopher M. Evans ◽

Bethany L. Yost ◽

Kristen E. Belmonte ◽

Gerald J. Gleich ◽

...

Keyword(s):

Guinea Pigs ◽

Acetylcholine Release ◽

Basic Protein ◽

Major Basic Protein ◽

Vagus Nerves ◽

Parasympathetic Nerves ◽

Atropine Treatment ◽

Vagal Reflex ◽

Eosinophil Major Basic Protein

M2muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2 receptors is due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2 receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.

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Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00253.2005 ◽

2007 ◽

Vol 292 (4) ◽

pp. L915-L923 ◽

Cited By ~ 10

Author(s):

Jaime Chávez ◽

Patricia Segura ◽

Mario H. Vargas ◽

José Luis Arreola ◽

Edgar Flores-Soto ◽

...

Keyword(s):

Substance P ◽

Guinea Pigs ◽

Smooth Muscle Contraction ◽

In Vivo Experiments ◽

Intracellular Ca ◽

Neurokinin 1 ◽

Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

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Acute and chronic respiratory effects of sulfur mustard intoxication in guinea pig

Journal of Applied Physiology ◽

10.1152/jappl.1994.76.2.681 ◽

1994 ◽

Vol 76 (2) ◽

pp. 681-688 ◽

Cited By ~ 71

Author(s):

J. H. Calvet ◽

P. H. Jarreau ◽

M. Levame ◽

M. P. D′Ortho ◽

H. Lorino ◽

...

Keyword(s):

Substance P ◽

Guinea Pigs ◽

Sulfur Mustard ◽

Neutral Endopeptidase ◽

Chemical Warfare Agent ◽

Airway Hyperreactivity ◽

Tracheal Epithelium ◽

Endopeptidase Activity ◽

Respiratory System Resistance ◽

Cell Shedding

Sulfur mustard (SM) has been used as a vesicant chemical warfare agent. To investigate the respiratory damages it causes, we studied the effects on guinea pigs of an intratracheal injection of 0.3 mg/kg of SM 5 h and 14 days after injection. Five hours after SM intoxication, respiratory system resistance and microvascular permeability were increased. These alterations were not prevented by pretreatment with 50 mg/kg sc of capsaicin 2 wk before SM intoxication. Histological studies showed columnar cell shedding all along the tracheal epithelium, bronchoconstriction, and peribronchial edema. Fourteen days after SM intoxication, guinea pigs demonstrated airway hyperreactivity to aerosolized substance P and histamine. Pretreatment with phosphoramidon caused a further increase in airway responsiveness to substance P. Neutral endopeptidase activity in the tracheal epithelium was decreased by twofold in SM-intoxicated guinea pigs. At this stage, the tracheal epithelium was disorganized and atrophic. These results demonstrate that in guinea pigs SM intoxication induces severe lesions to the tracheal epithelium, which might account for the airway hyperresponsiveness observed 14 days after intoxication.

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Mechanisms of smooth muscle contraction elicited by cationic proteins in guinea pig trachealis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.270.1.l133 ◽

1996 ◽

Vol 270 (1) ◽

pp. L133-L140 ◽

Cited By ~ 3

Author(s):

M. E. Strek ◽

F. S. Williams ◽

G. J. Gleich ◽

A. R. Leff ◽

S. R. White

Keyword(s):

Nervous System ◽

Smooth Muscle ◽

Guinea Pig ◽

Basic Protein ◽

Parasympathetic Nervous System ◽

Smooth Muscle Contraction ◽

Cationic Proteins ◽

Subsequent Response ◽

Eosinophil Major Basic Protein

Cationic proteins elicit contraction of airway smooth muscle, but the mechanisms by which this occurs are not completely understood. We studied potential mechanisms by which eosinophil major basic protein (MBP) and the synthetic cationic proteins poly-L-lysine (PL) and poly-L-arginine (PA) cause contraction of isolated guinea pig tracheal smooth muscle (TSM) in vivo. Topical application of 10(-8) mol/cm2 of each protein to an isolated tracheal segment elicited TSM contraction with potency PL > MBP > PA. Pretreatment with atropine blocked the subsequent response to MBP but did not block the response to either PL or PA. Pretreatment with indomethacin blocked the subsequent response to both MBP and PL but did not block the response to PA. We demonstrate that MBP causes contraction of guinea pig TSM both through stimulation of the parasympathetic nervous system and secretion of a cyclooxygenase mediator. Neither PL nor PA, while of similar molecular weight and charge as MBP, cause TSM contraction via the parasympathetic nervous system, though some cationic proteins may act via a prostanoid mediator. Thus the cationic charge of MBP is not solely responsible for its effects on TSM in the guinea pig.

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

Journal of Applied Physiology ◽

10.1152/jappl.1992.72.3.1133 ◽

1992 ◽

Vol 72 (3) ◽

pp. 1133-1141 ◽

Cited By ~ 13

Author(s):

C. G. Murlas ◽

Z. Lang ◽

G. J. Williams ◽

V. Chodimella

Keyword(s):

Substance P ◽

Airway Resistance ◽

Neutral Endopeptidase ◽

Exposed Group ◽

Airway Hyperreactivity ◽

Ozone Exposure ◽

Response Curves ◽

Air Exposure ◽

Mean Values ◽

Specific Airway Resistance

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)

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