Glucocorticoids reduce tachykinin NK2 receptor expression in bovine tracheal smooth muscle

Abstract

Neurokinin A is not only a potent bronchoconstrictor, but also has immuno-modulatory effects in animals and man, mediated via tachykinin NK2 receptors. We have examined the effect of the glucocorticoid, dexamethasone, on tachykinin NK2 receptor mRNA and the number of tachykinin NK2 receptors in bovine tracheal smooth muscle in vitro by Northern blot analysis using a human tachykinin NK2 receptor cDNA probe and receptor binding assay using [3H]SR48968 {(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2-(3,4-dichlorophenyl) butyl]benzamide}. Tachykinin NK2 receptor mRNA showed a time-dependent suppression (62% reduction after 6 h at 10-7 M of dexamethasone), as well as a concentration-dependent suppression after the incubation with dexamethasone (IC50 = 1.3 x 10-8 M). This suppression was abolished by the glucocorticoid receptor antagonist, mifepristone (RU38486), indicating that dexamethasone acts via the glucocorticoid receptor. It was also abolished by the protein synthesis inhibitor, cycloheximide (10 μg/ml), indicating that new protein synthesis is required on this suppression. Using the RNA polymerase inhibitor actinomycin D (5 μg/ml), we showed that the stability of tachykinin NK2 receptor mRNA was not affected by dexamethasone (t( 1/4 ) = 5 h). Nuclear run-on assays revealed a 51% reduction in the rate of tachykinin NK2 receptor gene transcription after treatment with dexamethasone for 6 h. Radioligand binding assay using an selective tachykinin NK2 receptor antagonist, [3H]SR48968 showed a significant decrease in the number of receptor binding sites after 16 h (B(max) = 262 ± 23 versus 213 ± 13 fmol/mg protein for vehicle and dexamethasone treatment respectively, P < 0.05), with no significant change at the earlier time points. These results suggest that glucocorticoids act on glucocorticoid receptors to decrease tachykinin NK2 receptor expression by decreasing the rate of tachykinin NK2 receptor gene transcription.