FLUID SHEAR-STRESS STIMULATES MITOGEN-ACTIVATED PROTEIN-KINASE IN ENDOTHELIAL-CELLS

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinas es have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the sig nal events involved in the endothelial cell response to fluid shear st ress. Flow at physiological shear stress (3.5 to 117 dynes/cm(2)) acti vated 42-kD and 44-kD MAP kinases present in cultured bovine aortic en dothelial cells, with maximal effect at 12 dynes/cm(2). Activation of a G protein was necessary, as demonstrated by complete inhibition by t he nonhydrolyzable GDP analog GDP-beta S. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine o r downregulating PKC with phorbol 12,13-dibutyrate. Both Ca2+-dependen t and -independent PKC activity, measured by translocation and substra te phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2+ mobilization, since Ca2+ chelati on had no inhibitory effect. On the basis of these findings, it is pro posed that flow activates two signal-transduction pathways in endothel ial cells. One pathway is Ca2+ dependent and involves activation of ph ospholipase C and increases in intracellular Ca2+. A new pathway, desc ribed in the present study, is Ca2+ independent and involves a G prote in and increases in PKC and MAP kinase activity.