Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues.
Homogeneous preparations of type I and type II regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinase (cAMP kinase) were utilized as antigens to obtain isozyme specific antisera. Injections of pure catalytic subunit (C) from the type I isozyme resulted in antisera that reacted with C subunit obtained from either isozyme type. Cross-reactivity of the antisera raised against isolated subunits of the kinase was assessed by immunodiffusion analysis and by measuring the cAMP binding and phosphotransferase activities of the subunits after immunoprecipitation. These antisera were used to localize subunits of type I and type II cAMP kinases in rat skeletal muscle, liver, and adrenal by using indirect immunofluorescence and immunoperoxidase techniques. Specificity of the immunofluorescence was shown by absorption of the antisera with pure homologous antigens. In skeletal muscle, both R and C subunits of the type I and type II cAMP kinases were localized in the area of the sarcoplasmic reticulum and in periodic crossbands. Specific fluorescence for these components was observed in both isotropic and anisotropic band regions of the sarcomere. Densitometric determinations of immunoperoxidase staining revealed a larger amount of RI, RII, and C subunits in the isotropic band than in the anisotropic band regions. In liver, C, RI, and RII subunits were distributed both in cytoplasmic and nuclear areas and along plasma membranes of hepatocytes; however, there were qualitative differences observed among these various subcellular sites. With each antiserum, fluorescence was blocked by prior absorption with homologous antigen. After treatment of rats with glucagon, dramatic changes in the relative distribution patterns of C and RII were noted in the nucleus. In the adrenal gland, RI, RII, and C subunits were localized in both cytoplasmic and nuclear areas, and an apparent redistribution of these subunits occurred after treatment of (dexamethasone-suppressed) rats with ACTH. The application of this immunocytochemical approach provides a tool for examining and monitoring the subcellular distribution of these components of cAMP kinase in biological systems.