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M Hagiwara

Publications and source records attributed to M Hagiwara.

At least 145 records · Page 8Linked to original sources
PubMed1991

Differential localization of protein kinase C isozymes in retinal neurons.

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, and III) in the rabbit retina using the monospecific monoclonal antibodies MC-1a, MC-2a, and MC-3a. Using immunoblot analysis of partially purified protein kinase C preparations of rabbit retina, types II and III isozymes alone were detected. The activity of type III was the stronger. By light microscopic immunohistochemical analysis, retinal neurons were negative for type I and positive for type II and type III isozymes. Type II was more diffusely distributed through the retinal layers, but was distinctive in ganglion cells, bipolar cells, and outer segments. The immunoreactivity was stronger for type III isozyme, and it was observed in mop (rod) bipolar cells and amacrine cells. By using immunoelectron microscopy, the cytoplasm of the cell body, the axon, and dendrites of the mop bipolar cells were strongly immunoreactive for type III. The so-called rod bipolar cells were for the first time seen to form synapses with rod photoreceptor cells. These differential localizations of respective isozymes in retinal neurons suggest that each isozyme has a different site of function in each neuron.

Animals↗
PubMed1991

Purification and characterization of calcium-calmodulin kinase II from human parathyroid glands.

Calmodulin has been identified in parathyroid cells and is thought to play an important role in the production or secretion of parathyroid hormone. However, a detailed investigation of calmodulin-binding proteins in parathyroid glands has not been conducted. In this study, we attempted to determine the presence of calmodulin-binding protein in human parathyroid adenoma by affinity chromatography. The eluted protein from a calmodulin-coupled Sepharose 4B column with EGTA was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis which revealed a major protein band of Mr 50,000. A Ca2+/calmodulin-dependent protein kinase activity was detected at the protein peak using dephosphorylated casein as a substrate. The 50 kDa band was identified as calcium/calmodulin-dependent protein kinase II (CaM-kinase II) by immunoblotting. The substrate specificity, pH dependency and affinity for calmodulin of this enzyme were identical to those of CaM-kinase II from rat brain. Also, the kinase activity was sensitive to KN-62, a specific inhibitor of CaM-kinase II. In total, 0.48 mg of this kinase was purified from 3 g human parathyroid adenoma.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1991

Immunohistochemical evidence for the overexpression of protein kinase C in proliferative diseases of human thyroid.

We report immunohistochemical evidence for the overexpression of protein kinase C in various proliferative diseases of human thyroid. Immunohistochemical characterization of various surgically removed thyroid tissues, viz., cancer tissues: papillary carcinoma and follicular carcinoma; adenoma tissues: tubular, trabecular and colloid adenomas; adenomatous goiter; and normal thyroid was done using the monospecific monoclonal antibodies MC-1a, MC-2a and MC-3a, each of which is specific for types I, II and III isozymes of protein kinase C, respectively. For protein kinase C type II, a remarkable difference in staining intensity was noted between the cancerous and normal tissues. The cytoplasm of papillary and follicular carcinoma cells stained more intensely than that of normal thyroid cells. In the benign tumor and adenomatous goiter tissues, stronger staining was noted in the papilliform-proliferating portion and cubic epithelial cells. In the normal thyroid tissues, epithelial cells of greater height were more strongly stained than simple squamous epithelial cells. These results indicated that protein kinase C type II isozyme is expressed in larger amounts in cancerous and proliferative tissues of the human thyroid.

Adenocarcinoma↗
PubMed1990

Assessment of protein kinase C isozymes by enzyme immunoassay and overexpression of type II in thyroid adenocarcinoma.

A two site enzyme immunoassay which quantitatively identifies types I, II, and III of protein kinase C isozymes has been designed. The soluble protein kinase C isozymes were selectively immobilized by type-specific monoclonal antibodies, MC-1a, -2a, and -3a (H. Hidaka et al., J Biol. Chem., 263: 4523-4526, 1988) which bind to the regulatory domain (NH2-terminal side) of protein kinase C. The amount of each isozyme was then determined using a horseradish peroxidase-conjugated polyclonal antibody raised against the COOH-terminal peptide of protein kinase C. By adding increasing concentrations of the antigen, the range of the assay proved to be 0.51-51, 0.081-8.1, and 0.31-31 nM for types I, II, and III, respectively. This sandwich method was used to determine the level of protein kinase C isozymes in rabbit tissues. Type I was mainly present in the cerebrum and cerebellum; the highest amount of type II isozyme was present in blood platelets [26.0 +/- 3.8 (SE) micrograms/g wet tissue]. We compared the protein kinase C isozyme levels in human normal thyroid gland and thyroid cancer tissues and found that type II protein kinase C specifically increased in thyroid cancer tissues. Immunocytochemical examination using MC-2a revealed that the cytoplasm of the cancer cells showed prominent immunoreactivity for type II isozyme.

Adenocarcinoma↗
PubMed1990

Immunoaffinity purification of type I protein kinase C.

We designed a simple procedure for the purification of type I protein kinase C, using immunoaffinity chromatography with a monoclonal antibody, MC-1b, obtained by rescreening hybridoma cells available for an affinity ligand. Western blotting demonstrated that MC-1b specifically reacted with type I protein kinase C, and the enzyme molecule dissociated from MC-1b-coupled Sepharose 4B with mild eluants such as thiocyanate retained the kinase activity. A 1148-fold purification was achieved and 210 micrograms of type I protein kinase C was obtained from three rabbit brains, by means of a two-step procedure, using DEAE-cellulose and immunoaffinity chromatography. The resultant preparation was homogeneous, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis hydroxylapatite chromatography, and immunological analysis using MC-1a, MC-2a, and MC-3a.

Animals↗
PubMed1990

d-Indobufen inhibits collagen-induced intracellular calcium mobilization and inositol phosphates formation in human platelets.

We obtained evidence that the cyclooxygenase inhibitor d-indobufen, (+)2[p-(1-oxo-2-isoindolinyl)phenyl] buthylic acid has a potent and specific inhibitory effect on collagen-induced aggregation and 40K-protein phosphorylation (Mamiya, S., Hagiwara, M., Ishikawa, T., and Hidaka, H. Thromb. Res. 54, 447, 1989). In Fura-2 loaded platelets, d-indobufen inhibited collagen-induced intracellular calcium mobilization in a dose dependent manner and this inhibitory effect on calcium mobilization paralleled that on aggregation, either in the presence or absence of extracellular free calcium ions. This compound inhibited inositol phosphates (IPs) formation in collagen-stimulated platelets. In arachidonic acid-stimulated platelets, d-indobufen caused a lag on calcium mobilization, as observed with arachidonic acid-induced aggregation. There was no significant effect on thrombin- or A23187-induced calcium mobilization or on aggregation. These observations suggest that the collagen receptor couples to a distinct intracellular calcium mobilization system possibly via inositol phospholipid metabolism and that d-indobufen blocks the collagen-induced aggregation by arresting mobilization of intracellular calcium.

Blood Platelets↗
PubMed1990

Circumvention of multidrug resistance in P388 murine leukemia cells by a novel inhibitor of cyclic AMP-dependent protein kinase, H-87.

A newly synthesized compound, H-87, N-[2-(p-bromo cinnamylmethylamino)ethyl]-5-isoquinolinesulfonamide was found to be a potent and selective inhibitor of cyclic AMP-dependent protein kinase. The effects of H-87 on in vitro sensitivities of various P388 murine leukemia cell lines resistant to several antitumor agents were examined. H-87 significantly potentiated the cytotoxic effects of Adriamycin (ADR), daunorubicin (DAU), vincristine (VCR) and vinblastine (VBL) on P388 cells resistant to these antitumor agents but hardly influenced the effects of mitomycin C (MMC), 5-fluorouracil (5-FU) and cisplatin (CDDP) on ADR-resistant P388 cells (P388/ADR) and P388 phenotypes resistant to the corresponding antitumor agents. H-87 promoted the accumulation of VBL much more in P388/ADR cells than in the sensitive cells by inhibiting the energy-dependent extrusion of the antitumor agent from the cells. These results suggest that this novel isoquinoline-sulfonamide derivative, H-87, overcomes the multidrug resistance by inhibiting the phosphorylation of an outward drug transport system through cyclic AMP-dependent protein kinase.

Animals↗
PubMed1990

zeta-Related protein kinase C in nuclei of nerve cells.

To determine whether or not PKC is present in the nuclei of nerve tissue we made use of biochemical and immunocytochemical techniques. A 219-fold purification of rabbit brain nuclear protein kinase C was achieved by sequential steps of Triton X-100 extraction of isolated nuclei, DEAE-cellulose, Butyl-toyopearl and hydroxylapatite chromatography. The major peak of protein kinase C activity was eluted from the hydroxylapatite column at the KPO4 concentration of 0.3 M. Both Ca2+ and Ptd Ser were required for stimulation of the enzyme. Immunoblot analysis revealed that the kinase fraction was immunoreactive with a polyclonal antibody, PC-zeta, that had been raised against a peptide synthesized according to the deduced sequence of rat zeta protein kinase C. Light-microscopy revealed strong immunoreactivity in the nuclei of Purkinje cells in cerebellum and pyramidal cells in the rat cerebral cortex. These observations suggest that a zeta-related protein kinase C is present in the nuclei of nerve cells.

Animals↗
PubMed1990

Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.

A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.

Adrenal Gland Neoplasms↗
PubMed1990

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II.

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Type-specific expression of protein kinase C isozymes in CNS tumor cells.

We examined specific expression of protein kinase C (PK-C) isozymes in cultured human glial and neuronal cell lines, using type-specific monoclonal antibodies MC-1a, -2a, and -3a (Hidaka H. et al., J. Biol. Chem., 263 (1988) 4523-4526). Immunoblotting experiments revealed that a 80 kDa band of three kinds of glioblastoma cells (A-172, SK-MG-1, SK-MG-4) was stained with MC-3a, whereas that of neuroblastoma cells (SK-N-MC) reacted with MC-2a. Immunoenzymetric assay showed that glioblastoma cells (A-172, SK-MG-1, SK-MG-4) contained 127.6 +/- 14.4, 248.8 +/- and 148.5 +/- 35.8 ng/mg protein of type III. respectively, while neuroblastoma cells (SK-N-MC) contained 389.5 +/- 20.7 ng/mg protein of type II. These results suggest that PK-C isozymes may be specifically expressed, depending on types of central nervous system (CNS) tumor cells.

Cell Line↗
PubMed1990

CSF beta-endorphin, HVA and 5-HIAA of dementia of the Alzheimer type and Binswanger's disease in the elderly.

Cerebrospinal fluid (CSF) concentration of beta-endorphin (beta-Ep), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) was measured in 15 patients with dementia of the Alzheimer type (DAT) and in 16 patients suspected of having Binswanger's disease (BD) by MRI, which sometimes resembles DAT clinically. These were classified into three stages according to severity of dementia, Stage 1 (mild dementia)-Stage 3 (severe dementia). CSF levels of HVA decreased significantly in severe dementia, but the level of 5-HIAA did not correlate with dementia severity in both dementia groups. beta-Ep levels did not differ significantly between any stages of DAT, and among controls. beta-Ep levels, however, in BD Stage 1 (27.5 +/- 5.9 pg/ml) were significantly higher (p less than 0.05), but level in Stage 3 (6.7 +/- 2.0) was significantly lower (p less than 0.001) than in the controls (19.2 +/- 4.5). These results suggest that CSF beta-Ep may depend on the cause of dementia rather than severity of dementia, and could possibly distinguish the closely resembling BD from true DAT.

Alzheimer Disease↗