Chronic elevated endothelin-1 concentrations regulate mitogen-activated protein kinases ERK 1 and ERK 2 in vascular smooth muscle cells

Increased endothelin-1 (ET-1) levels are found in patients with atherosclerosis. ET-1 is known to increase the mitotic response of different growth factors already at threshold concentrations. The aim of this study was to investigate the influence of ET-1 on the mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase (ERK) 1 and ERK 2. Smooth muscle cells were incubated with ET-1 at a concentration of 10^-7M for 1–120h. ERK 1 and ERK 2 were determined in cell homogenates by electrophoresis. Specific antibodies were used to investigate the amount of ERK 1 or ERK 2 in the homogenate. The functional activity of ERK 1 and ERK 2 was determined. Immunofluorescence microscopy was performed to analyse the translocation of the MAP kinases into the nucleus. ET-1 incubation for 12h decreased ERK 1 concentration by -51%. After 36h of ET-1 application the concentration of ERK 1 increased to control levels again. When the cells were incubated for 120h ERK 1 rose by +65% above control. The incubation with ET-1 in the presence of an ET_A receptor antagonist inhibited the increase of ERK 1. ERK 2 showed a comparable time course with an initial decrease in the protein concentration followed by an increase after 120h. Incubation with an ET_A receptor antagonist inhibited the increase in protein concentration after 120h. However, the functional activity of both MAP kinases remained unchanged between 1 and 120h. Especially, after 120h of ET-1 incubation no translocation into the nucleus was observed. However, an additional stimulus with angiotensin II resulted in translocation of ERK into the nucleus. These data show that ET-1 increases the protein concentration of MAP kinases ERK 1 and ERK 2 but not their basal activity. Only an additional stimulation with angiotensin II leads to the translocation of ERK into the cell nucleus.