Desensitization of the biologic response to thromboxane A2 (TXA2) mimetics has been observed ex vivo in human platelets due to TXA2 receptor uncoupling and downregulation. To define more clearly the mechanisms of homologous TXA2 receptor downregulation, the effects of the TXA2 mimetics U44069 [(15S)-hydroxy-9,11-(epoxymethano) prostadienoic acid] and I-BOP ([1 S-(1α2β(5Z),3α(1E,3S),4α]))-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1- butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid) on receptor-mediated calcium fluxes and on ligand binding to TXA2 receptors were studied in the K562 cultured human leukemic cell line which possesses many platelet characteristics. Incubation with U44069 resulted in a time-dependent decrease in the amplitude of TXA2 receptor-mediated intracellular free calcium transients. Under the same conditions, binding of [125I]BOP demonstrated a concurrent loss of K562 plasma membrane binding sites to approximately one-third the original number. The loss of [125I]BOP binding was prevented by coincubation with the TXA2 antagonist SQ29548 ([1S-1α,2β (5Z), 3β,4α))-7-(3-((2- ((phenyl-amino)-carbonyl) hydrazino) methyl)-7-oxabicyclo-(2.2.1)- heptan-2-yl)-5-heptenoic acid]) and was reversed upon removal of U44069 from the culture medium. SQ29548 alone had no affect on receptor density or affinity. Loss of surface receptors was demonstrated to be mediated by agonist-occupied receptor internalization which was inhibited by incubation at 4°C and did not occur with antagonist occupation. The results indicate that homologous downregulation of TXA2 receptors in K562 cells occurs by agonist-mediated active internalization of plasma membrane TXA2 receptors.
|Number of pages||7|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Jan 1 1991|