Distribution of active protein kinase C in smooth muscle

localize activated protein kinase C (PKC) in smooth muscle cells, an antibo dy directed to the catalytic site of the enzyme was used to assess PKC dist ribution by immunofluorescence techniques in gastric smooth muscle cells is olated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluoresce nce were obtained from a series of images collected through focus with a di gital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 mu M carbachol for 1 min, 1 mu M phorbol le-myris tate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 mu m 4-0 phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, act ivated PKC and vinculin were located and organized at the cell surface. Cel l cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated P KC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of ac tivated PKC observed after PMA but not 4-alpha PMA were similar to those ob served with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, suppo rting a possible role in modulation of contraction.