Selective blockade and recovery of cell surface alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells. Studies with the irreversible antagonist benextramine

RM McKernan, WR Strickland and PA Insel

Department of Pharmacology, University of California, La Jolla 92093.

alpha 2-Adrenergic receptors are present on human erythroleukemia (HEL) cells, both on the cell surface and in a sequestered compartment. In the current study we show that benextramine, a hydrophilic irreversible antagonist, can be used to investigate alpha 2-adrenergic receptor compartmentation in these cells. In membranes prepared from HEL cells, benextramine competed for all alpha 2-adrenergic receptors ( [3H]yohimbine sites). In intact cells, at 4 degrees, benextramine exhibited a biphasic competition curve for alpha 2-adrenergic receptors, with EC50 values of approximately 10 microM and greater than 1 mM for the high and low affinity components, respectively. We propose that the alpha 2-adrenergic receptors preferentially blocked by benextramine are those on the surface of the cell, whereas those with low affinity are sequestered receptors because: 1) only epinephrine- accessible sites [i.e., cell surface sites; McKernan et al., Mol. Pharmacol. 32:258-265 (1987)] are removed by prior treatment of cells with benextramine, 2) a preparation enriched with surface membranes is also enriched in receptors with a high affinity for benextramine; and 3) after blockade of cell surface receptors (54 +/- 6% of total sites, n = 7) by benextramine, the ability of the alpha 2-adrenergic agonists epinephrine and UK-14,304 to inhibit forskolin-stimulated cAMP accumulation is lost. The latter result implies that only cell surface and not sequestered receptors are functionally coupled to adenylate cyclase. The return of receptors from the sequestered compartment to the cell surface and the recovery of alpha 2-adrenergic receptor function were measured after HEL cells were treated with benextramine (50 microM for 1 hr at 4 degrees). The recovery of receptor binding (t1/2 = 25 min) was somewhat slower than the recovery of function (t1/2 approximately 8 min). This is consistent with the existence of "spare receptors" and also suggests that the sequestered compartment of alpha 2-adrenergic receptors can rapidly exchange with those on the surface. When all alpha 2-adrenergic receptors were blocked by incubation of HEL cells with benextramine for 1 hr at 37 degrees, repopulation of surface and sequestered receptors was much slower (t1/2 = 9 hr for recovery of total receptors). Surface receptors recovered even more slowly than did total cellular receptors consistent with the idea that alpha 2- adrenergic receptors must traverse through intracellular locations before insertion into the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 33, Issue 1, pp. 51-57, 01/01/1988
Copyright © 1988 by American Society for Pharmacology and Experimental Therapeutics

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