Activation of mitogen-activated protein kinase in porcine carotid arteries.
The thin-filament protein h-caldesmon (the high molecular weight isoform of caldesmon) is phosphorylated in resting and contracted porcine carotid arteries. Phosphorylation of h-caldesmon in intact tissue occurs at sites that are covalently modified by mitogen-activated protein kinase (MAPK) in vitro. In this study, we have evaluated MAPK activation in arteries in response to mechanical load and pharmacological stimulation. MAPK was extracted from resting and stimulated porcine carotid arteries and then partially purified by anion-exchange fast-performance liquid chromatography. MAPK activity was separated into two peaks corresponding to the tyrosine-phosphorylated 42- and 44-kD isoforms of MAPK (p42MAPK and p44MAPK, respectively). Of the total MAPK activity, 42% was associated with p42MAPK, and 58% was associated with p44MAPK, this percentage was not altered by stimulation of the muscles with either KCl (110 mmol/L) or phorbol 12,13-dibutyrate (PDBu, 1 mumol/L). Both p42MAPK and p44MAPK, purified from porcine carotid arteries, phosphorylated h-caldesmon at the same sites and to levels approaching or > 1 mol phosphate per mole protein. In unloaded muscle strips, MAPK activity was 39 pmol.min-1.mg protein-1 when assayed with the peptide substrate APRTPG-GRR. MAPK activity increased in response to incremental mechanical loading to a maximum of 99 pmol.min-1.mg protein-1 at 16 x 10(3) N/m2. MAPK activity could be further increased in loaded muscles by pharmacological stimulation. With KCl stimulation, MAPK activities rose to a peak of 205 pmol.min-1.mg protein-1 at 10 minutes and then declined to basal values at 30 and 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)
Adam
L P
LP
Department of Medicine, Indiana University School of Medicine, Indianapolis.
Franklin
M T
MT
Raff
G J
GJ
Hathaway
D R
DR
D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002339:Carotid Arteries / Q000201:enzymology* / Q000502:physiology; D004789:Enzyme Activation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases; D009119:Muscle Contraction; D010812:Physical Stimulation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013268:Stimulation, Chemical; D013552:Swine
D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases