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...

The distribution of a subspecies of protein kinase C (PKC) encoded by the P8n sequence in rat central nervous tissue was demonstrated immunocytochemically by using antibodies raised against an oligopeptide having a partial sequence specific for the p& PKC. The pn PKC immunoreactivity was widely but discretely distributed in the brain. The distribution of the Pu PKC immunoreactivity differed from that of the P1 and y PKC subspecies. The PHn PKC immunoreactivity was found in the perikarya, dendrites, and axons of neuronal cells. Few if any glial cells were stained. Immunoreactive neurons were present in the anterior olfactory nucleus, olfactory tubercle, amygdaloid complex, caudate-putamen, accumbens nucleus, claustrum, dorsal part of the lateral septal nucleus, CA1 region of the hippocampus, subiculum, medial habenular nucleus, cerebral cortex, nucleus of the spinal tract of the trigeminal nerve, nucleus of the solitary tract, and substantia gelatinosa of the spinal cord. In these neurons, the PI PKC immunoreactivity was seen mainly in the form of cytoplasmic dots and, in some cases, diffusely in the cytoplasm. Under electron microscopy, these immunoreactive large dots appeared to be associated with the Golgi complex, suggesting that the PBII PKC plays a specialized function at the Golgi complex in certain neuronal cell types.Protein kinase C (PKC), which is activated by 1,2-diacylglycerol in the presence of Ca2l and phospholipids, acts as a key enzyme for signal transduction in various physiological processes (1-3). Recent molecular cloning studies have revealed that PKC is a large family consisting of at least seven subspecies (a, 1 PI, y, 8, E, and O) with closely related but distinct structures (3). Enzymological studies have shown that PKC can be resolved into three distinct fractions, types I-III, by hydroxyapatite column chromatography (4). Comparison of these fractions with the enzymes expressed in COS-7 cells transfected by the respective cDNA-containing plasmids (5, 6) has indicated that PKC types I, II, and III are products of y, f, and a genes, respectively. Distribution of these three subspecies has been studied by biochemical and immunocytochemical procedures (7-13). The y (type I) PKC is found only in the neurons of the central nervous system, whereas the P8 and PII (type II) PKC and a (type III) PKC are detected in both central and peripheral tissues (7,(14)(15)(16) MATERIALS AND METHODS Production of Antibodies Against Pu Subspecies of PKC.The carboxyl-terminal portion of the PI, PKC (residues 660-673: Ser-Phe-Val-Asn-Ser-Glu-Phe-Leu-Lys-Pro-Glu-ValLys-Ser) was selected as an amino acid sequence specific to the PII PKC. The oligopeptide was synthesized and coupled to bovine serum albumin by glutaraldehyde (17). The product was mixed with complete Freund's adjuvant and injected intracutaneously into rabbits. The antigen, emulsified with incomplete Freund's adjuvant, was injected repeatedly at intervals of 2 weeks, and the rabbits were bled 4-6 days after each booster injection. The titer ...

No abstract

No abstract

Endothelin(ET) is a vasoconstrictor peptide derived from endothelial cells. We investigated the distribution of ET-like immunoreactivity in the cardiovascular system by use of immunohistochemical technique. The specimens were aorta, arteries, aortic valves, ventricles and pericardium of the heart obtained during operation, endomyocardial biopsy or autopsy. These were fixed in 10% buffered formalin and embedded in paraffin. The avidin-biotin peroxidase complex method was performed using the rabbit anti-human ET serum. ET-like immunoreactivity was observed in the cytoplasm of various endothelial cells, but not following immunoabsorption. The immunoreactivity was much higher in a variety of the endothelial cells under dynamically stressful conditions such as aortic and arterial aneurysm, Kawasaki disease, pericarditis, and hypertrophic cardiomyopathy. These results suggest that human cardiovascular endothelial. cells function as an endocrine and/or paracrine cells for ET secretion, especially in the pathological conditions.