In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (51 A), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained it5 chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and as5ociated with directed cell migration. Furthermore, itseffect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristdte acelate (PMA) on the translocation of PKC activity in endothelial cells. We ob5erved that both SLA and PMA induced the translocation of PKC activity trom the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (M, 23.4 and 36.5 kDa) in intact "P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.
Recently, we demonstrated that the protease inhibitor, E-64, stimulated the centrosomal orientation and locomotor responses of cultured bovine arterial endothelial cell monolayers subjected t o a wounding stimulus (Mascardo and Eilon, 1988). To clarify whether the stimulatory action of E-64 was dependent on the molecule's protease inhibitory effect, we synthesized a n analogue of E-64, SLA, which lacked the epoxy group needed for its protease inhibitory activity. We demonstrate in this paper that SLA is an effective and rapidly acting chemokinetic agent in wounded endothelial cells, and that its action depends on PKC activation.
METHODS
Synthesis of SLA
SLA (Fig. ) was prepared from N-carbobenz0xy-N'-L-leucyl-l,4-diaminobutane in five steps , by acylation with ethyl succinyl chloride (CH,