Proteolysis and phosphorylation-mediated regulation of thrombin receptor activity in in situ endothelial cells

The regulatory mechanism of thrombin receptor responsiveness in in situ end othelial cells was investigated by evaluating elevations of cytosolic Ca2concentration ([Ca2+](i)) in fura-2-loaded porcine aortic valvular strips. Once stimulated with thrombin, endothelial cells did not respond to the sec ond thrombin stimulation within 90 min. However, applying thrombin receptor activating peptide (TRAP7) at 15 min after the thrombin stimulation caused [Ca2+](i) elevation, which was smaller than that seen without preceding st imulation. After 90 min, response to TRAP7 recovered to the control level. When stimulated with TRAP7, the subsequent responses to thrombin and TRAP7 were attenuated at 15 min, and fully recovered after 90 min. Staurosporine partially prevented the TRAP7-induced desensitization. The recovery of resp onsiveness was inhibited completely by calyculin-A and partially by okadaic acid. Proteolysis and phosphorylation thus play an important role in throm bin receptor desensitization in in situ endothelial cells. Both cleaved and uncleaved receptors were desensitized through phosphorylation in part by s taurosporine-sensitive kinase, and restored the responsiveness through deph osphorylation by type 1 phosphatase. The mechanism of regulation of thrombi n receptor activity in in situ endothelial cells differed from those report ed in cultured endothelial cells. We suggest that the cell-specific regulat ory mechanism may be altered by culture conditions. (C) 2000 Elsevier Scien ce B.V. All rights reserved.