BACKGROUND High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44). METHODS Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-alpha, PKC-delta, and PKC-epsilon translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation. RESULTS In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC-delta and PKC-epsilon but not PKC-alpha. In HG, the pattern of ET-1-stimulated PKC-delta and PKC-epsilon changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-alpha, PKC-delta, or PKC-epsilon in either NG or HG. In HG, both basal and ET-1-stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1. CONCLUSIONS In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC-delta and PKC-epsilon. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.