Protection of thrombin receptor expression under hypoxia

Thrombin receptor (ThR) plays a significant role in myocyte contractility a nd hypertrophy, Heart myocyte ischemic damage, caused by insufficient blood supply, is the leading cause of heart infarction, Here we demonstrate that when primary myocyte cultures are subjected to hypoxic stress, ThR mRNA le vels are reduced markedly. This takes place also in vivo in a model of isch emic pig heart, exhibiting reduced levels of ThR compared with normal heart sections. Prior activation of ThR however, by either thrombin receptor-act ivating peptide (TRAP) or by alpha-thrombin resulted in full protection of ThR mRNA levels under hypoxia, The effect appeared specific to ThR because the addition of TRAP did not affect the hypoxic damage as shown by the leve ls of lactic dehydrogenase release and up-regulated GLUT-1, a glucose trans porter gene. This protection effect took place not only in primary myocytes but also in NIH3T3 fibroblasts, ThR protection occurs via specific cell si gnaling events because activation of the receptor by TRAP, following interr uption of the signaling cascade by calphostin C, a protein kinase C inhibit or, resulted in loss of ThR mRNA protection. Because Ras and Src are part o f the ThR signaling cascade, the introduction of either dominant ras or src oncogenes to NIH3T3 murine fibroblasts gave rise to similar protection of ThR mRNA levels under hypoxic conditions without the exogenous addition of TRAP. Likewise, ThR mRNA protection was obtained after transfection with pr oto-oncogene vav. The 95-kDa protein Vav undergoes tyrosine phosphorylation after ThR activation, serving thus as part of the receptor machinery casca de. We therefore conclude that the initiation of the signaling cascades eit her exogenously by TRAP or within the cell via src or ras, as well as via a av oncogene interconnecting G-binding protein to the tyrosine kinase pathwa y, ultimately results in ThR protection under hypoxia, We present hereby, a novel concept of activated receptors, which under minimal oxygen tension p rotect their otherwise decaying mRNA Maintaining the level of ThR that play s an active role in normal myocyte function may provide a significant repai r mechanism in ischemic tissue, assisting in the regaining of normal myocyt e functions.