Thrombin-Induced Refractoriness Of PGI2 And Adenine Nucleotide Release From The Vascular Endothelium

Abstract

Primary monolayer cultures of human umbilical vein endothelium release both PGI2 (measured by radioimmunoassay of 6-keto-PGF1α) and adenine nucleotides (AN) (measured as release of 3H from monolayers prelabeled with [3H]adenosine) when incubated with thrombin (0.5 U/ml) or A23187 (10 μM) .The release of PGI2 and AN in response to A23187 was greater than that observed in response to thrombin by an average of 2- and 3.5-fold, respectively. Preincubation of the endothelium with aspirin (100 μM) for 30 min resulted in a total inhibition of thrombin or A23187 induced release of PGI2, but had no significant effect on the release of AN. Thus, AN release does not appear to be dependent on PGI2 biosynthesis. After an initial exposure to thrombin, the endothelium became refractory to both PGI2 and AN release. Prior exposure of the endothelium to thrombin also decreased A23187 induced release of PGI2 by an average of 50%, but had no effect on AN release. Preincubation of the endothelium for 10 min with the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (4 mM) increased the intracellular concentration of cyclic AMP (measured by radioimmunoassay) by an average of 3.5-fold, and caused a total inhibition of thrombin and A23187 induced release of PGI2 and AN. Although PGI2 (400 nM) and ADP (10 μM) increased cyclic AMP levels by several-fold in IBMX-treated endothelium, no significant increase was observed in the absence of IBMX. These findings suggest that the phenomenon of thrombin-induced refractoriness is not the result of a simple negative feedback mechanism involving the activation of adenylate cyclase by PGI2 and AN released from the endothelium. Complex mechanisms involving the thrombin binding mechanism, depletion of Ca2+ gradients or inactivation of Ca2+-dependent reactions are more likely to be responsible.