Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.
The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.
Lazou
A
A
Department of Cardiac Medicine, National Heart and Lung Institute, London, UK.
Bogoyevitch
M A
MA
Clerk
A
A
Fuller
S J
SJ
Marshall C
J
J
Sugden
P H
PH
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002853:Chromatography, Liquid; D016232:Endothelins / Q000494:pharmacology; D004789:Enzyme Activation; D006321:Heart / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D009638:Norepinephrine / Q000494:pharmacology; D010477:Perfusion; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*
D016232:Endothelins; D007328:Insulin; D011994:Recombinant Proteins; D002217:Carbachol; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D007545:Isoproterenol; D009638:Norepinephrine