APICAL MEMBRANE AND INTRACELLULAR-DISTRIBUTION OF ENDOGENOUS ALPHA(2A)-ADRENERGIC RECEPTORS IN MDCK CELLS

The purpose of the current studies was to characterize the endogenous alpha(2)-adrenergic receptor (AR) subtypes present in Madin-Darby cani ne kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [H-3]MK-9 12, MDCK cells expressed high levels of alpha(2)-ARs with a maximum re ceptor density (B-max) of 798 +/- 55 fmol/mg protein and an equilibriu m dissociation constant (Ka) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine t o displace [H-3]MK-412 demonstrated inhibition constant (K-i) values o f 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expr essed transcripts encoding alpha(2A)-AR and not alpha(2B)-AR or alpha( 2C)-AR. Surface binding experiments suggested that similar to 60% of a lpha(2A)-ARs are distributed at the cell surface domain. Specific bind ing of [H-3]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha(2A)-ARs was limited to the apical domain of MDCK cells. No alpha (2A)-ARs were detected on the basolateral surface. We conclude that en dogenous alpha(2A)-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.