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

Abstract

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(-7) M for 1-120 h. 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 12 h decreased ERK 1 concentration by -51%. After 36 h of ET-1 application the concentration of ERK 1 increased to control levels again. When the cells were incubated for 120 h 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 120 h. Incubation with an ET(A) receptor antagonist inhibited the increase in protein concentration after 120 h. However, the functional activity of both MAP kinases remained unchanged between 1 and 120 h. Especially, after 120 h 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.