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 protein kinase C (PKC) subspecies and their colocalization with neurotransmitters were examined in the rat striatum and substantia nigra (SN), using immunocytochemistry. The alpha- and beta I-PKC immunoreactivies were seen predominantly in the perikarya of the neurons in the striatum and SN. In contrast, the beta II- and gamma- PKC immunoreactivities were abundant in both the perikarya and the neuropils in the striatum and only in the neuropils in the SN. From electron microscopic studies, the alpha- and beta I-PKC immunoreactivities were seen adjacent to the plasma membrane, while the beta II-PKC immunoreactivity was observed in the cytoplasm around the Golgi complex. The gamma-PKC immunoreaction was dense throughout the cytoplasm. The double-staining and lesion studies revealed that the alpha-PKC-immunopositive neurons in the striatum were intrinsic cholinergic neurons, and that most of the alpha-PKC-immunoreactive neurons in the SN were dopaminergic neurons. The beta I-PKC- immunoreactive neurons were intrinsic GABAergic neurons in the striatum. Moreover, most of the beta II- and gamma-PKC-immunoreactive neurons were medium-sized neurons projecting to the SN, and over 90% of GABAergic neurons in the caudate-putamen contained beta II-PKC. The beta II-PKC-immunoreactive neurons showed no gamma-PKC immunoreactivity, and the gamma-PKC-immunoreactive neurons were not beta II-PKC immunoreactive. These findings suggest that alpha-PKC is related to the function of the nigral dopaminergic and the striatal cholinergic neurons, and that the beta I-PKC is involved in the function of the striatal intrinsic GABAergic neurons. The beta II- and gamma-PKC may also modulate a specific neuronal function in the striatonigral system.
Restrictive cardiomyopathy (RCM) is a rare disease characterized by increased ventricular stiffness and preserved ventricular contraction. Various sarcomere gene variants are known to cause RCM; however, more than a half of patients do not harbor such pathogenic variants. We recently demonstrated that cardiac fibroblasts (CFs) play important roles in inhibiting the diastolic function of cardiomyocytes via humoral factors and direct cell–cell contact regardless of sarcomere gene mutations. However, the mechanical properties of CFs that are crucial for intercellular communication and the cardiomyocyte microenvironment remain less understood. In this study, we evaluated the rheological properties of CFs derived from pediatric patients with RCM and healthy control CFs via atomic force microscopy. Then, we estimated the cellular modulus scale factor related to the cell stiffness, fluidity, and Newtonian viscosity of single cells based on the single power-law rheology model and analyzed the comprehensive gene expression profiles via RNA-sequencing. RCM-derived CFs showed significantly higher stiffness and viscosity and lower fluidity compared to healthy control CFs. Furthermore, RNA-sequencing revealed that the signaling pathways associated with cytoskeleton elements were affected in RCM CFs; specifically, cytoskeletal actin-associated genes (ACTN1, ACTA2, and PALLD) were highly expressed in RCM CFs, whereas several tubulin genes (TUBB3, TUBB, TUBA1C, and TUBA1B) were down-regulated. These results implies that the signaling pathways associated with cytoskeletal elements alter the rheological properties of RCM CFs, particularly those related to CF–cardiomyocyte interactions, thereby leading to diastolic cardiac dysfunction in RCM.
Background Dilated cardiomyopathy (DCM) is a major cause of heart failure in children. Despite intensive genetic analyses, pathogenic gene variants have not been identified in most patients with DCM, which suggests that cardiomyocytes are not solely responsible for DCM. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. They have several roles in maintaining cardiac function; however, the pathological role of CFs in DCM remains unknown. Methods and Results Four primary cultured CF cell lines were established from pediatric patients with DCM and compared with 3 CF lines from healthy controls. There were no significant differences in cellular proliferation, adhesion, migration, apoptosis, or myofibroblast activation between DCM CFs compared with healthy CFs. Atomic force microscopy revealed that cellular stiffness, fluidity, and viscosity were not significantly changed in DCM CFs. However, when DCM CFs were cocultured with healthy cardiomyocytes, they deteriorated the contractile and diastolic functions of cardiomyocytes. RNA sequencing revealed markedly different comprehensive gene expression profiles in DCM CFs compared with healthy CFs. Several humoral factors and the extracellular matrix were significantly upregulated or downregulated in DCM CFs. The pathway analysis revealed that extracellular matrix receptor interactions, focal adhesion signaling, Hippo signaling, and transforming growth factor‐β signaling pathways were significantly affected in DCM CFs. In contrast, single‐cell RNA sequencing revealed that there was no specific subpopulation in the DCM CFs that contributed to the alterations in gene expression. Conclusions Although cellular physiological behavior was not altered in DCM CFs, they deteriorated the contractile and diastolic functions of healthy cardiomyocytes through humoral factors and direct cell–cell contact.
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