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 rece ptor (AR) subtypes activate inositol phosphate (InsP) formation and in crease intracellular Ca2+ in transfected cell lines. Expression of hum an alpha(1a)-, alpha(1b)-, and alpha(1d)-AR cDNAs under the repressibl e control of anhydrotetracycline in human embryonic kidney (HEK) 293 c ells, which normally express no alpha(1)-ARs, was used to compare resp onses to norepinephrine (NE) at different receptor densities. Maximal NE-stimulated InsP formation was found to increase with increasing den sity of each subtype, whereas basal levels and responses to sodium flu oride did not change. A comparison of multiple subclones over equivale nt ranges of receptor expression showed that activation of each subtyp e resulted in different maximal responses (alpha(1a) > alpha(1b) > alp ha(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 expr ession system. Induction with isopropyl-beta-D-thiogalactoside increas ed 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 EC(50) value for NE in st imulating InsP formation or releasing [Ca2+](i) but did increase maxim al responses to NE. Similar to our findings in HEK 293 cells, a compar ison 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) greater than or equal to alpha(1d)) and [Ca2+] (i) (alpha(1a) > alpha(1b) > alpha(1b)) responses, These studies show that agonist-occupied human alpha(1)-AR subtypes have different effici encies 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.