Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction.
The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein kinase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphorylation are not activated and determine if the PKC pathway is activated under these conditions. The phosphorylation of caldesmon, myosin light chain (MLC20), and the specific PKC substrate, MARCKS (myristoylated, alanine-rich C-kinase substrate) was measured in bovine carotid arterial smoothmuscle (BCASM) stimulated with phorbol 12,13-dibutyrate (PDBu) under Ca2+-containing and Ca2+-free conditions. PDBu stimulation led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca2+. PDBu stimulation but did not lead to increases in MLC20 phosphorylation over basal levels in Ca2+-free conditions. Immunoblot analysis of BCASM using PKC isoform-specific antibodies demonstrated the presence of one "Ca2+- dependent" PKC isoform: alpha, and two of the "Ca2+-independent" isoforms: epsilon and zeta. These data suggest that Ca2+-independent isoforms of PKC may play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation.
Throckmorton
D C
DC
Department of Surgery, Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Packer
C S
CS
Brophy
C M
CM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D002339:Carotid Arteries / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D009119:Muscle Contraction / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014661:Vasoconstriction / Q000502:physiology*; D000099221:Protein Kinase C zeta
D002118:Calcium; D002148:Calmodulin-Binding Proteins; D002273:Carcinogens; D002614:Chelating Agents; D004533:Egtazic Acid; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D018994:Myosin Light Chains; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate; D011189:Potassium Chloride; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011506:Proteins; D000099221:Protein Kinase C zeta; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester