Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle.
The effect of phorbol ester-induced down-regulation of protein kinase C (PKC) on diacylglycerol (sn-1,2-dioctanoylglycerol, diC8)- and G-protein-coupled Ca2+ sensitization and on the relationship between phosphorylation of the regulatory myosin light chains (MLC20) and force during Ca2+ sensitization were investigated in rabbit portal vein (PV), femoral artery (FA) and ileum smooth muscle. The effects of phorbol dibutyrate (PDBu), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and agonists on the membrane versus cytosolic distribution of PKC isoenzymes were also determined. Down-regulation of PKC abolished Ca2+ sensitization of force and the accompanying increases in MLC20 phosphorylation induced by PDBu, as well as Ca2+ sensitization of force by diC8, but not that by GTP[S], aluminum fluoride (AIF4-) or agonists (phenylephrine, endothelin or carbachol). Down-regulation also inhibited the PDBu-, but not the GTP[S]-induced increase in force under Ca(2+)-free conditions. In ileum, PDBu translocated PKCs alpha, beta 1, beta 2, epsilon and theta to the membrane fraction, and GTP[S] caused a small translocation of PKC-epsilon. Carbachol- and GTP[S]-induced Ca2+ sensitization remained unaffected in down-regulated ileum in which no cytosolic PKC-epsilon was detectable. We conclude that, although both phorbol ester-induced and G-protein-coupled Ca2+ sensitization of force are mediated by increased MLC20 phosphorylation, it is likely that PKCs alpha, beta 1, beta 2, epsilon and theta do not play an essential role in, although they may contribute to, the G-protein-coupled mechanism.
Jensen
P E
PE
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, USA.
Gong
M C
MC
Somlyo
A V
AV
Somlyo
A P
AP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D005263:Femoral Artery / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D007082:Ileum; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D009130:Muscle, Smooth / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010766:Phosphorylation; D011169:Portal Vein / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D013211:Staphylococcus aureus; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000494:pharmacology
D007527:Isoenzymes; D018994:Myosin Light Chains; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium