Desensitization of beta-adrenergic receptors in lung injury induced by 2-chloroethyl ethyl sulfide, a mustard analog

Abstract

2-Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta-adrenergic receptors (beta-ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of beta-ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated-bovine serum albumin ([(125)I]-BSA) into lung tissue. Receptor-binding characteristics were determined by measuring the binding of [(3)H] dihydroalprenolol ([(3)H] DHA) (0.5-24 nM) to membrane fraction in the presence and absence of DLDL-propranolol (10 micro M). Both high- and low-affinity beta-ARs were identified in the lung. Binding capacity was significantly higher in low-affinity site in both control and experimental groups. Although CEES exposure did not change K(D) and B(max) at the high-affinity site, it significantly decreased both K(D) and B(max) at low affinity sites. A 20% decrease in beta(2)-AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES-induced lung injury is associated with desensitization of beta(2)-AR.

FIGURE 1

CEES-induced lung injury. Lung injury was expressed as permeability index, which was obtained by dividing total radioactive counts of [¹²⁵I]-BSA in lung by counts in 1 mL of blood from the same animal (n = 3). Asterisks indicate statistically different compared to control (p < 0.05).

FIGURE 2

Representative Scatchard analysis of [³H] DHA binding to β-ARs in guinea pig lung membranes: (A) high-affinity (control), (B) high-affinity (CEES-exposed), (C) low-affinity (control), (D) low-affinity (CEES-exposed). The binding of [³H] DHA to the membrane was measured as described in the “Materials and Methods” section (n = 3). B = Specific binding of ligand and F = Free ligand concentration.

FIGURE 3

Inhibition of [³H] DHA binding to the lung membrane by β-antagonists. (■– ■, Atenolol; ▲— ▲, ICI-118, 551). The binding of [³H] DHA to the membrane and IC₅₀ for the two antagonists were measured as described in the “Materials and Methods” section (n = 3).

FIGURE 4

RT-PCR products of β-ARs gene of lung; 1 = kb⁺ DNA ladder; 2 = Control; 3 = CEES-exposed.

FIGURE 5

Effect of mustard gas on guinea pig lung β₂-AR receptor gene expression. (A) A representative autoradiogram of Northern blot analysis, (B) bar graph of normalized densitometric values (n = 3).

FIGURE 6

Effect of mustard gas on guinea pig lung β₂-AR receptor levels. (A) Homogenate (n = 3), (B) membrane (n = 3).

FIGURE 7

Effects of mustard gas exposure on cAMP production in guinea pig lung. (A) Dose-response curve in the presence and absence of isoproterenol (n = 3), (B) time-response curve in the presence and absence of isoproterenol (n = 3), (C) response in the presence of isoproterol and IBMX singly or in combination (n = 3).