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 rec eptor (beta AR) agonist salmeterol displays several unusual properties : (i) slow onset of action (t(1/2) = 5-15 min), (ii) prolonged activat ion (t(1/2) = 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 acti vating site. Despite extensive characterization of salmeterol-induced bronchodilation, little is known about the molecular actions of salmet erol. We report the unique properties of salmeterol binding to the bet a AR, activation of adenylyl cyclase, and desensitization of the hamst er beta AR expressed in L cells. First, we found that salmeterol activ ation of adenylyl cyclase, although rapid and potent (low EC(50) relat ive to epinephrine), was nevertheless remarkably inefficient relative to the full agonist epinephrine. Reduced coupling efficiency of salmet erol was demonstrated using formulations recently introduced by our gr oup. Second, we found that pretreatment of L cells with salmeterol led to a stable activation of adenylyl cyclase that survives extensive wa sh procedures and sucrose step gradient purification of plasma membran e 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. Thi rd, we found that the rapid phase of salmeterol-induced desensitizatio n was much reduced relative to epinephrine, consistent with its poor c oupling efficiency and with its prolonged activation of adenylyl cycla se. These unique properties of salmeterol support the proposal that it binds reversibly to the activating or active site and as well to an e xtremely high affinity exosite from which it has access to the active site.