Stable activation and desensitization of beta 2-adrenergic receptor stimulation of adenylyl cyclase by salmeterol: evidence for quasi-irreversible binding to an exosite.

The relaxation of tracheal smooth muscle by the beta 2-adrenergic receptor (beta AR) agonist salmeterol displays several unusual properties: (i) slow onset of action (t1/2 = 5-15 min), (ii) prolonged activation (t1/2 = 8-14 hr), and (iii) the ability to recover from beta AR blockade. These properties led to the hypothesis that salmeterol binds with very high affinity to an exosite in addition to the beta AR activating site. Despite extensive characterization of salmeterol-induced bronchodilation, little is known about the molecular actions of salmeterol. We report the unique properties of salmeterol binding to the beta AR, activation of adenylyl cyclase, and desensitization of the hamster beta AR expressed in L cells. First, we found that salmeterol activation of adenylyl cyclase, although rapid and potent (low EC50 relative to epinephrine), was nevertheless remarkably inefficient relative to the full agonist epinephrine. Reduced coupling efficiency of salmeterol was demonstrated using formulations recently introduced by our group. Second, we found that pretreatment of L cells with salmeterol led to a stable activation of adenylyl cyclase that survives extensive wash procedures and sucrose step gradient purification of plasma membrane fractions. This activation of basal adenylyl cyclase did not require salmeterol binding to the classic active site during pretreatment, as it occurred in the presence of an excess of a beta AR antagonist. Third, we found that the rapid phase of salmeterol-induced desensitization was much reduced relative to epinephrine, consistent with its poor coupling efficiency and with its prolonged activation of adenylyl cyclase. These unique properties of salmeterol support the proposal that it binds reversibly to the activating or active site and as well to an extremely high affinity exosite from which it has access to the active site.