Inverse Agonist Activity of 3-Adrenergic Antagonists

Agonist-independent properties of the human f32-adrenergic receptor (fl2AR) were studied using the baculovirus expression system in Sf9 cells. In the absence of agonist but in the presence of GTP, membranes from cells expressing the fl2AR exhibited higher levels of cAMP production than did membranes from uninfected cells or from cells infected with wild-type baculovirus. The increase in cAMP production was proportional to the number of /32AR expressed, up to 40 pmol/mg of membrane protein, and it could be inhibited in a dose-dependent manner by I3AR antagonists. The increase and its reversal both were independent of the possible presence of contaminating catecholamines in the culture medium and thus appear to reflect spontaneous fl2AR activity and direct antagonist-receptor interactions, respectively. The maximal level of inhibition varied among the flAR ligands tested, to yield the following rank order of “inverse efficacy”: timolol propranolol > alprenolol pindolol > labetalol > dichloroisoproterenol. The same rank order was observed using membranes prepared from Chinese hamster fibroblasts expressing /32AR. The effect of timolol was partly blocked by labetalol and dichloroisoproterenol, in an apparently competitive manner. The intracellular cAMP content of Sf9 cells cultured in serumfree medium was also increased by the expression of fl2AR, and that increase was reversed by timolol and propranolol, consistent with observations in membrane preparations. The properties revealed by the expression of the fl2AR in Sf9 cells suggest two agonist-independent traits of G protein-linked receptors, i.e., 1) that unliganded receptors are able to activate G proteins both in membrane preparations and in whole cells and 2) that antagonists may mediate their effects not only by preventing the binding of agonists but also by decreasing the propensity of the receptor to assume an active state.