Activation of protein kinase C isozymes in primary mouse myotubes by carbachol.
The activation of muscle PKC isozymes following treatment with carbachol, an acetylcholine receptor agonist, has been investigated. Primary mouse myotubes were treated with carbachol, and protein extracts from the cytosol and membrane fractions of the myotubes were subjected to Western blot analyses. Carbachol treatment resulted in a rapid translocation of PKC-theta; to the membrane. This effect was dependent on both carbachol concentration and incubation time. The treatment also resulted in a drastic increase of PKC-alpha in the cytosol followed by an increase of PKC-alpha in the membrane. The regulation of PKC-alpha in response to carbachol was quite distinct from that produced by the PKC activator, PMA, which rapidly translocated PKC-alpha from the cytosol to the membrane without any increases in PKC-alpha in the cytosol. Confocal microscopy demonstrated an enhanced membrane localization of PKC-theta; and overall increased intensity of PKC-alpha staining in the cytosol accompanied by a characteristic membrane staining of PKC-alpha in the myotubes treated with carbachol. Taken together, the results suggested that the activation of PKC isozymes in response to the receptor agonist is quite distinct, which indicates their diverse role in the muscle upon the release of neurotransmitter at the neuromuscular junction.
Kim
Sunghee
S
Laboratory of Developmental Neurobiology, NICHD, NIH, Bethesda, MD 20892, USA. sunghee_kim@hgsi.com
Bondeva
Tzvetanka
T
Nelson
Phillip G
PG
D000818:Animals; D000831:Animals, Newborn; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta; D011978:Receptors, Nicotinic / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D018679:Cholinergic Agonists; D007527:Isoenzymes; D011978:Receptors, Nicotinic; D012694:Serine; D002217:Carbachol; C496630:Prkca protein, mouse; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta