Modulation of monocyte adherence to endothelial cells by endothelin-1 involvement of Src (p60(src)) and JAK1-like kinases

Purpose: The purpose of this study was to determine the transmembrane signaling pathway by which endothelin-1 (ET-1) enhances monocyte adherence to human umbilical vein endothelial cells (HUVECs) and to investigate the role of tyrosine kinases in this mechanism. Methods: Adherence of purified human blood monocytes to HUVEC monolayers was assessed with radiolabeled monocytes. Tyrosine kinase activation was examined by immunoprecipitation and Western blotting. Results: ET-1 potentiated monocyte adherence to HUVECs in a biphasic manner with peaks at 10(-10) mol/L and 10(-7) mol/L. A potent antagonist to ET(B) receptors, when used alone, had no effect. However, the antagonist, when combined with ET-1, significantly enhanced monocyte adherence to HUVECs. Incubation of ET-1 (10(-12) mol/L to 10(-7) mol/L) with HUVECs activated tyrosine kinases in a biphasic manner as identified by immunoblotting with PY20 antibody to tyrosine phosphorylated proteins. Phosphorylated proteins with Mr 60, 110, and 130 kDa were observed after ET-1 stimulation of HUVECs. Of interest, ET(A) or ET(B) receptor antagonists failed to antagonize the effect of ET-1. Rather, these receptor antagonists significantly augmented ET-1 induced tyrosine phosphorylation in HUVECs. Immunoprecipitation with antibodies to p60(src) and JAK1 kinases followed by immunoblotting with PY20 antibody suggested that ET-1 receptor-response coupling in HUVECs involves the activation of p60(src) and JAK1-like kinases. Conclusions: These data suggest an association between activation of p60(src) and JAK1-like kinases and monocyte adherence in response to ET-1. ET-1-induced monocyte adherence is upregulated by ET(B) receptor antagonist, suggesting a negative feedback on cell adhesion through this receptor.