Coupling efficiencies of human alpha 1-adrenergic receptor subtypes: titration of receptor density and responsiveness with inducible and repressible expression vectors.

We compared the efficiencies with which human alpha 1-adrenergic receptor (AR) subtypes activate inositol phosphate (InsP) formation and increase intracellular Ca2+ in transfected cell lines. Expression of human alpha 1a-, alpha 1b-, and alpha 1d-AR cDNAs under the repressible control of anhydrotetracycline in human embryonic kidney (HEK) 293 cells, which normally express no alpha 1-ARs, was used to compare responses to norepinephrine (NE) at different receptor densities. Maximal NE-stimulated InsP formation was found to increase with increasing density of each subtype, whereas basal levels and responses to sodium fluoride did not change. A comparison of multiple subclones over equivalent ranges of receptor expression showed that activation of each subtype resulted in different maximal responses (alpha 1a > alpha 1b > alpha 1d) in HEK 293 cells. Analogous studies were carried out in human SK-N-MC cells, which normally express low levels of all three alpha 1-AR subtypes, using an isopropyl-beta-D-thiogalactoside-inducible expression system. Induction with isopropyl-beta-D-thiogalactoside increased the density of individual alpha 1-AR subtypes by 4-6-fold over the level of endogenous expression. Increased expression of each of these subtypes in SK-N-MC cells did not alter the EC50 value for NE in stimulating InsP formation or releasing [Ca2+]i but did increase maximal responses to NE. Similar to our findings in HEK 293 cells, a comparison of responses at similar expression levels in SK-N-MC cells showed different maximal responses stimulated by each subtype, for both InsP (alpha 1a > alpha 1b > or = alpha 1d) and [Ca2+]i (alpha 1a > alpha 1b > alpha 1d) responses. These studies show that agonist-occupied human alpha 1-AR subtypes have different efficiencies in activating phospholipase C in human cell lines. In both HEK 293 and SK-N-MC cells, alpha 1a-ARs couple most efficiently, whereas alpha 1d-ARs couple very poorly.