Immunodetection of activated mitogen-activated protein kinase in vascular tissues.
Mitogen-activated protein kinase (MAPK) is a key modulator of cytoplasmic-nuclear signal transmission, and for this reason measurement of MAPK activity has become very popular. Monitoring of MAPK activity may be particularly relevant to the cardiovascular system where it has already been shown that the stimulation of cardiomyocytes and smooth muscle cells by stretch and by growth factors activates MAPK. Since both growth factors and mechanical stress are causal agents for certain pathologies, enhanced MAPK activity may be a good predictor of disease progression. A variety of methods have been designed to measure the activation of this enzyme including an in vitro assay coupled to either gel electrophoresis or binding to P81 paper, an activity gel assay to detect p42/44 isoforms and, more recently, monitoring MAPK phosphorylation using immunoblot detection. The validity of the latter method is based on the correlation between MAPK activity and the degree of phosphorylation. The antibodies have also been of use in the detection of MAPK translocation in cell monolayers. In this report, we discuss the advantages and disadvantages of all MAPK detection methods and demonstrate an additional application for the MAPK antibodies using an in vitro restenosis model. In addition, the utility of MAPK measurements to smooth muscle pathophysiology and vascular injury (as a predictor of injury) has been assessed.
Yau
L
L
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Winnipeg, Canada.
Zahradka
P
P
D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000276:immunology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000201:enzymology; D004789:Enzyme Activation; D007150:Immunohistochemistry; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D013552:Swine
D017871:Calcium-Calmodulin-Dependent Protein Kinases