Glucocorticoids reverse IL-1beta-induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-protein-coupled receptor kinases - PubMed (original) (raw)

Glucocorticoids reverse IL-1beta-induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-protein-coupled receptor kinases

Judith C W Mak et al. Br J Pharmacol. 2002 Feb.

Abstract

1. The aim of the present study was to examine the effects of glucocorticoid dexamethasone on airway responsiveness to albuterol after intratracheal instillation of saline or IL-1beta in Brown-Norway rats in vivo and to elucidate the molecular mechanism of this effect. 2. IL-1beta caused a significant reduction in albuterol-mediated relaxation to protect against MCh-induced bronchoconstriction. Dexamethasone attenuated the IL-1beta-induced impaired relaxation while alone had no effect when compared to rats treated identically with saline. 3. The density of beta(2)-adrenoceptors was significantly reduced in lung membranes harvested from IL-1beta-treated rats, which was associated with impaired isoproterenol- and forskolin-stimulated cyclic AMP accumulation and adenylyl cyclase (AC) activity ex vivo. Dexamethasone did not prevent IL-1beta-induced down-regulation of beta(2)-adrenoceptors but completely blocked IL-1beta-induced impairment of cyclic AMP accumulation and AC activity stimulated by isoproterenol and forskolin. 4. The inhibitory G-protein subtypes, G(ialpha1), G(ialpha2) and G(ialpha3), were detected in lung membranes prepared from all groups of rats but the intensity of G(ialpha1) and G(ialpha2) was markedly increased in IL-1beta-treated rats, which were not prevented by dexamethasone. 5. The activity of cytosolic GRK and the expression of GRK2 and GRK5 were elevated in the lung of IL-1beta-treated rats, which were completely abolished by dexamethasone. 6. These results indicate that treatment of rats with IL-1beta results in desensitization of pulmonary beta(2)-adrenoceptors. In light of data obtained in this study, we propose that both the decrease in AC activity and the increase in GRK activity, which are reversed by dexamethasone, may underlie beta(2)-adrenoceptor desensitization.

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Figures

Figure 1

Figure 1

Time-course of per cent change in RL after inhalation of 4 mg ml−1 methacholine (MCh). The bronchoconstrictor response to the inhalation of MCh was rapid and was maximal by 5 min. The bronchoconstrictor response was stable for 45 min. All groups of animals showed a similar time-course and there were no significant differences between the groups. _n_=3 in each group.

Figure 2

Figure 2

Effect of IL-1β instillation (500 U), dexamethasone (DEX; 3 mg kg−1, i.p.) and/or combined treatments on the protective actions of albuterol against methacholine-induced bronchoconstriction. IL-1β attenuated the relaxant effect of albuterol. DEX alone did not have any effects while DEX reversed the IL-1β-induced attenuation of albuterol effect. *P<0.05, **P<0.01 in comparison to saline-treated group.

Figure 3

Figure 3

Effect of IL-1β and/or dexamethasone (DEX) on the density (as measured by the maximal binding, Bmax) and the affinity (as measured by the dissociation constant, K d) of β2-adrenoceptors (β2ARs). Total [125I]-iodocyanopindolol ([125I]-ICYP) was displaced by the specific β1-receptor antagonist, CGP-20712A (100 n

M

), or isoproterenol (200 μ

M

, to give nonspecific binding). The affinity of β2-adrenoceptors was not altered by either IL-1β or by dexamethasone. However, IL-1β caused a significant reduction in the density of receptors, an effect that was not prevented by dexamethasone. Data are means±s.e.mean of four animals in each group. **P<0.01.

Figure 4

Figure 4

Effect of IL-1β and/or dexamethasone (DEX) on cyclic adenosine monophosphate (cyclic AMP) accumulation stimulated by isoproterenol (10 μ

M

) and forskolin (10 μ

M

) (A), and on adenylyl cyclase activity stimulated by sodium fluoride (10 m

M

), isoproterenol and forskolin (B). Intratracheal instillation of IL-1β caused significant attenuation in isoproterenol- and forskolin-stimulated cyclic AMP accumulation and adenylyl cyclase activity, which was reversed by DEX. Data are means±s.e.means of 4 – 6 animals in each group. *P<0.05, **P<0.01.

Figure 5

Figure 5

Comparison of Western blots for the expression of Giα isoforms in lung membranes prepared from each group of animals. The left-hand panel shows a representative example of Western blot analysis from a rat from each experimental group. Note enhanced expression of Giα1 and Giα2 in IL-1β-treated (lane 2), and dexamethasone (DEX) plus IL-1β-treated (lane 4) compared with saline (lane 1), and DEX plus saline (lane 3) groups. Right-hand panels (A,B) Densitometric measurements of Giα1 and Giα2 immunoreactivity were normalized to Giα3. There was a significant increase in Giα1 and Giα2 induced by IL-1β, but dexamethasone had no significant effect. Data are means±s.e.mean of 4 – 6 animals in each group. *P<0.05.

Figure 6

Figure 6

Assessment of GRK activity in lung cytosolic fractions from different treatment groups. (A) Autoradiograph depicting light-dependent phosphorylation of rhodopsin (∼38 kD). Rat lungs from saline (lane 1), IL-1β-treated (lane 2), DEX plus saline (lane 3), and DEX plus IL-1β-treated (lane 4) were shown. (B) Quantification of GRK activity measurements in each group of animals. Data are means±s.e.mean of four animals in each group. *P<0.05.

Figure 7

Figure 7

(A) Representative autoradiograph of Western blot depicting immuno-detectable GRK2 (∼80 kD) in lung cytosolic fractions from different treatment groups. Rat lungs from saline (lane 1), IL-1β-treated (lane 2), DEX plus saline (lane 3), and DEX plus IL-1β-treated (lane 4) were shown. (B) Densitometric measurements of GRK2 immunoreactivity. Data are means±s.e.mean of 4 – 6 animals in each group. *P<0.05, **P<0.01.

Figure 8

Figure 8

Representative autoradiographs of Northern blot for steady-state level of GRK2, GRK5 and house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in lung from different treatment groups. Rat lungs from saline (lane 1), IL-1β-treated (lane 2), DEX plus saline (lane 3), and DEX plus IL-1β-treated (lane 4) were shown.

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