Activation of protein kinase C isozymes in primary mouse myotubes by carbachol

Abstract

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-θ to the membrane. This effect was dependent on both carbachol concentration and incubation time. The treatment also resulted in a drastic increase of PKC-α in the cytosol followed by an increase of PKC-α in the membrane. The regulation of PKC-α in response to carbachol was quite distinct from that produced by the PKC activator, PMA, which rapidly translocated PKC-α from the cytosol to the membrane without any increases in PKC-α in the cytosol. Confocal microscopy demonstrated an enhanced membrane localization of PKC-θ and overall increased intensity of PKC-α staining in the cytosol accompanied by a characteristic membrane staining of PKC-α 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.