Distinct expression of protein kinase C (PKC) subspecies in the central nervous system suggested that each subspecies has a distinct neural function in the processing and modulation of a variety of physiological responses to external signals. In this study, the cellular and subcellular distributions of beta I-, beta II- and gamma-subspecies of PKC were demonstrated by using subspecies-specific antibodies in the rat spinal cord. By light microscopy both gamma- and beta II-subspecies immunoreactivities were found only in neurons of the substantia gelatinosa and axons of the dorsal corticospinal tract in the spinal cord. Use of a double staining method, however, revealed that beta II-subspecies immunoreactivity was localized in the outer part of the lamina II, whereas gamma-subspecies immunoreactivity was found in the inner part of lamina II. Immunoreactive neurons containing beta I-subspecies were scattered in the substantia gelatinosa. Beta I-subspecies immunoreactivity varied in neuronal types. Furthermore, electron microscopic analysis clearly showed the subcellular distribution of these subspecies to be different from one another. Dense gamma-subspecies immunoreactivity was found in the cytoplasm except within cell organelles of the perikarya and dendrites. Some nuclei were stained as strongly as the cytoplasm and others were stained less heavily. The nucleoli had faint or no immunoreactivity. Reaction products of beta II-subspecies were located against the inner plasma membrane but not seen in the nuclei or nucleoli. Beta I-subspecies immunoreactivity appeared to be associated with the Golgi complex. No immunoreactive products of any PKC subspecies were detected in the presynaptic terminals. The different patterns of expression described above imply that individual PKC subspecies may have a specific function in modulating the neuronal activity in the different neurons of the spinal cord.

A monoclonal antibody against protein kinase C (PKC) was used for immunocytochemical studies of the type I PKC encoded by gamma-cDNA sequence (gamma-subspecies) in rat Purkinje cells. Dense gamma-subspecies-like immunoreactivity was found on the cell membrane and in the cytoplasm except within cell organelles of the perikaryon, dendrites, axon, and axon terminals. The nucleus was also stained but less heavily, and the nucleoli remained unstained. Synaptic vesicles in the axon terminals were densely stained. The results suggest that gamma-subspecies might be functionally involved in modulation of nuclear function and of pre- and postsynaptic functions including transmitter release in the rat Purkinje cells.

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ABSTRACT

The distribution ofthe a subspecies ofprotein kinase C (PKC) in rat brain was demonstrated immunocytochemically by using polyclonal antibodies raised against a synthetic oligopeptide corresponding to the carboxyl-terminal sequence of a-PKC. The a-PKC-specific immunoreactivity was widely but discretely distributed in both gray and white matter. The immunoreactivity was associated predominantly with neurons, particularly with perikaryon, dendrite, or axon, but little was seen in the nucleus. (2), which correspond to y-, ,1-and 813,-, and a-PKC, respectively (3, 4). These PKC subspecies are subtly different from one another in their kinetic properties, mode of activation, and most likely substrate specificity (1,(5)(6)(7)(8). Early analysis of the mRNA levels has indicated that a-, 1I-, and 81,-PKC are expressed in a variety of tissues, whereas y-PKC is expressed only in the central nervous system (9-12). Enzymatic and immunochemical analysis has also shown that a-PKC (type III) is most commonly distributed in many tissues and cell types (13,14). On the basis of these studies, it has been suggested that a-PKC plays a role of crucial importance in the control of common processes in cell functions (1).Several laboratories have carried out immunocytochemical studies using antibodies specific to each type of . In earlier reports (20-24), with subspeciesspecific antibodies, it was shown that in the rat brain 1,-, 1I-, and y-PKC are differentially distributed in particular cell types, with limited intracellular localization. The present studies were undertaken to identify a-PKC in the rat brain by using immunocytochemistry, and the results show that this PKC subspecies is enriched in particular cell types. MATERIAL AND METHODSPreparation of Antibodies Against a-PKC. The carboxylterminal portion of a-PKC (residues 662-672; Gln-Phe-ValHis-Pro-Ile-Leu-Gln-Ser-Ala-Val) was selected as a sequence specific to a-PKC. The oligopeptide was coupled to keyhole limpet with m-maleimidobenzoic acid N-hydroxysuccinimide ester. Rabbits were immunized with the immunogen by the method described (23,24) and were bled 1 week after the third booster administration. The IgG fraction was obtained from the antisera by affinity chromatography on Sepharose CL-4B coupled to goat anti-rabbit IgG, and the fraction was used as a-PKC-specific antibodies.Immunoblotting Analysis. Specificity of the antibodies was examined by immunoblotting analysis using the three subtypes of rat brain PKC (types I, II, and III) and four subspecies of PKC (a-, 13-, 1I-, and -PKC). These subspecies were purified from COS-7 cells transfected with the respective cDNA-containing plasmids as described (3, 4). The enzyme samples were subjected to NaDodSO4/7.5% polyacrylamide slab gel electrophoresis as described by Laemmli (25) and transferred to nitrocellulose paper. The paper was incubated with the a-PKC-specific antibodies, and immunoreactive bands were visualized by the peroxidaseantiperoxidase method.Immunocytochemical Staining. Frontal sections of the rat brain...