Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1 beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE(2), and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1 beta (10 ng/ml), TNF-alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1 beta- and TNF-alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE(2) (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE, was ablated in the IL-1 beta-treated TSM that were pretreated with either the muscarinic M(2)-receptor antagonist, methoctramine (10(-6) M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) Gi protein expression was significantly enhanced in membrane fractions isolated from IL-1 beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the G(i) alpha(2) and G(i) alpha(3) subunits. Collectively, these observations provide new evidence demonstrating that IL-1 beta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M(2)-receptor/G(i) protein-coupled expression and function.