Receptor binding characterization in kidney membrane of [3H]U-37883, a novel ATP-sensitive K+ channel blocker with diuretic/natriuretic properties

K Meisheri, M Fosset, S Humphrey and M Lazdunski

Upjohn Laboratories, Kalamazoo, Michigan 49001.

U-37883 (4-morpholinecarboximidine-N-1-adamantyl-N-cyclohexyl), a known blocker of ATP-sensitive K+ (KATP) channels, produces natriuresis/diuresis in vivo by a direct effect on the kidney. In the present study, the binding characteristics of the U-37883 receptor were investigated using pig kidney cortex microsomes. [3H]U-37883 (0.5-5 nM, 50 Ci/mmol) exhibited specific binding, which was reversible, increased linearly with protein concentration (50-500 micrograms/ml), and was destroyed after treatment with proteases. Scatchard plots derived from the competition experiments suggested the presence of a single class of low affinity binding sites, with a Kd of 225 nM and a Bmax of 7.8 pmol/mg of protein. A similar Kd value was derived from complementary studies dealing with association and dissociation kinetics. The binding of [3H]U-37883 was tissue specific, because very little specific binding could be detected in microsomes from rat insulinoma cells (RINm5F) and brain. In contrast, these membranes displayed high affinity specific binding of [3H]glyburide, another KATP channel blocker. Finally, analogs of U-37883 that were found to be active KATP channel blockers in isolated rabbit mesenteric artery and active in vivo as diuretics/natriuretics were also found to be active in displacing specific binding of [3H]U-37883, whereas the inactive analogs (no vascular KATP channel-blocking activity and no in vivo diuresis/natriuresis) were inactive in this binding assay. We suggest that the U-37883 binding site represents a functional receptor that mediates the KATP channel antagonism and natriuresis observed with this class of compounds.

Volume 47, Issue 1, pp. 155-163, 01/01/1995
Copyright © 1995 by American Society for Pharmacology and Experimental Therapeutics

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