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H Hidaka

Publications and source records attributed to H Hidaka.

At least 361 records · Page 20Linked to original sources
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↗
PubMed1991

Protein kinase C activation and protooncogene expression in differentiation/retrodifferentiation of human U-937 leukemia cells.

Human U-937 leukemia cells differentiate along the monocytic lineage following 3-day exposures to 12-O-tetradecanoylphorbol-13-acetate (TPA). This induction of differentiation is accompanied by adherence and loss of proliferation, as well as expression/repression of differentiation-associated genes. Long term culture of TPA-differentiated U-937 cells in the absence of phorbol ester for 32-36 days resulted in a process of retrodifferentiation. The retrodifferentiated cells detached from the substrate and reinitiated proliferation. Other cellular parameters, such as glycosidase activities, cytokine release, and filament expression, returned to levels similar to that observed in uninduced cells. Treatment of U-937 cells with TPA resulted in a rapid translocation of protein kinase C (PKC) from the cytosol to cell membrane fractions within 2-8 min. Increased levels of membrane-associated PKC activity persisted until 17-29 days. However, longer periods of incubation were associated with a return to the distribution of PKC in control cells. Activation of PKC has been implicated in the regulation of certain immediate early response genes, and in the present studies, TPA rapidly induced c-fos and c-jun gene expression. Levels of c-fos and c-jun transcripts remained elevated during periods of PKC activation and also returned to levels observed in control cells by 30-36 days, when the cells entered retrodifferentiation. Staurosporine, a nonspecific inhibitor of PKC, partially blocked TPA-induced adherence and growth inhibition and concomitantly prevented TPA-induced c-fos and c-jun gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1990

Endothelin: a potential modulator of cerebral vasospasm.

1-(5-Isoquinolinesulfonyl)-homopiperazine, HA1077, is a calcium antagonist with anti-vasospastic properties. This compound blocks intracellular actions of calcium in a variety of experiments. In the present study, we examined the effects of HA1077 on the vascular actions of endothelin, an endothelium-derived vasoactive peptide, in dogs in vitro and in vivo. Intracisternal injections of endothelin (0.01 nmol) produced a significant vasospasm, as measured by angiography, similar to that seen in the canine hemorrhage model. Infusion of HA1077 led to a significant dilatation of the spastic basilar artery in endothelin-treated dogs. The rank order of in vitro contractile activity in canine cerebral arteries was a stable thromboxane A2 analog greater than endothelin greater than 5-hydroxytryptamine greater than prostaglandin F2 alpha greater than histamine greater than noradrenaline. HA1077 effectively antagonized the endothelin-induced contraction of canine basilar arterial strips in both calcium-containing and calcium-free medium. The present results indicate that HA1077 is an effective antagonist for endothelin in vitro and in vivo.

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

Apolipoprotein B-48 analysis by high-performance liquid chromatography in VLDL: a sensitive and rapid method.

Apolipoprotein (apo) B subspecies were separated by high-performance liquid chromatography (HPLC) to analyze Apo B-48 contents in very-low-density lipoproteins (VLDL) more easily. Apo B-100 and B-48 were eluted through two connected column of Shim-pack Diol-300 at a retention time of 24 min and 32.3 min, respectively. The molecular masses estimated by this method were approximately 600 kDa in apo B-100, 220 kDa in apo B-48. % apo B-48 in total apo B (% B-48) determined by measuring peak area of HPLC-separated apo B in a healthy subject was 76% in chylomicrons, 13% in fasting VLDL, and 20% in 2 h-postprandial VLDL. No peak of apo B-48 was detected in LDL. Recovery of Apo B determined by re-chromatography of HPLC-separated sample was 91 +/- 4.0% and 95 +/- 3.6% in Apo B-100 and apo B-48, respectively. 125I-labeled apo B in VLDL were also analyzed by both HPLC and SDS-PAGE. Percent radioactivity of apo B-48 fraction in the total apo B determined by the HPLC (17.3%) was similar to the value obtained through the measurement of radioactivity separated by the SDS-PAGE (17.6%). Coefficient of variation in % B-48 determined by the peak area was 2.5%. Percent B-48 determined by the HPLC method was significantly correlated with % B-48 by SDS-PAGE, but 6-8 times higher, which might be accounted for in part by the reported difference of chromogenicity between apo B-100 and apo B-48. % B-48 in VLDL separated from fasting plasma were 17.1 +/- 5.6% in 14 healthy subjects, and positively related to VLDL triglyceride and cholesterol concentrations. Apo B isoprotein analysis by the HPLC method is reliable and easy to perform for studying apo B metabolism in triglyceride-rich lipoproteins in physiological as well as pathological conditions.

Adult↗
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

Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin.

High-Mr caldesmon, which is involved in smooth muscle contraction, was phosphorylated by protein kinase C. By chymotryptic digestion, actin- and calmodulin-binding assays and immunoprecipitation with the antibody to the C-terminal 35-kDa fragment, we have identified that all phosphate groups are incorporated exclusively into this fragment, which is the functional domain for binding actin and calmodulin. Phosphorylation of high-Mr caldesmon and its C-terminal 35-kDa fragment reduced their binding abilities to both F-actin and calmodulin. Further, their inhibitory effects on the actin-activated ATPase activity of gizzard myosin were also reversed in proportion to the degree of phosphorylation. These results suggest that phosphorylation of high-Mr caldesmon by protein kinase C, which is restricted within the C-terminal 35-kDa domain, results in the modulation of its activity in the smooth muscle actin--myosin interaction.

Actins↗
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↗