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Biomedical subjects

K Fukunaga

Publications and source records attributed to K Fukunaga.

At least 163 records · Page 9Linked to original sources
PubMed1996

Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-62 inhibits adrenal medullary chromaffin cell functions independent of its action on the kinase.

KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced 45Ca2+ influx, which is essential for functional responses. Carbachol-stimulated 22Na+ influx, veratridine-induced 22Na+ influx, and 56 mM K(+)-evoked 45Ca2+ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Cyclic AMP inhibits activation of mitogen-activated protein kinase and cell proliferation in response to growth factors in cultured rat cortical astrocytes.

The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 mM and isoproterenol at 10 microM inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with 32P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.

Adrenergic beta-Agonists↗
PubMed1996

Cyclosporin A stimulation of glucose-induced insulin secretion in MIN6 cells.

Effects of the immunosuppressant cyclosporin A (CsA), a specific inhibitor of Ca2+/calmodulin-dependent protein phosphatase (PP2B), were examined with regard to the induction of insulin secretion from MIN6 cells, a glucose-responsive cell line derived from mouse insulinoma. CsA had no effect on basal insulin secretion from MIN6 cells, but did increase glucose-, tolbutamide-, and KCl-induced insulin secretion. Treatment of the cells with CsA resulted in a dose-dependent increase in insulin secretion, which was maximal at 3 microM. CsA inhibited PP2B activity in a dose-dependent manner, and the increase in insulin secretion correlated with the decrease in PP2B activity. In 32P-labeled cells, treatment with CsA for 30 min increased phosphorylation of synapsin I-like protein by 50 +/- 5.7%. As revealed by one-dimensional phosphopeptide mapping of 32P-labeled synapsin I-like protein, treatment with CsA for 30 min increased phosphorylation of site II of synapsin I-like protein by 59 +/- 8%, which is phosphorylated by calmodulin kinase II. Messenger RNAs, which hybridize with complementary DNAs of calcineurin A and B subunits from rat brain, were detected in MIN6 cells. Western blot analysis showed a 61-kDa band, which interacts with rat brain calcineurin A antibody. Similar increases in secretagogue-induced insulin secretion with CsA were observed for HIT-T15 cells. These results suggest that CsA stimulates glucose-induced insulin secretion by inhibiting the activity of PP2B, an event that may be involved in mechanisms governing glucose-induced insulin secretion via dephosphorylation of synapsin I-like protein in MIN6 cells.

Animals↗
PubMed1996

[Comparative evaluation of 99mTc-MIBI (hexakis2-methoxy isobutyl isonitrile) and 201Tl-chloride in primary lung cancer].

We performed comparative study, using 99mTc-MIBI and 201Tl-chloride in patients with primary lung cancer. There were 42 patients with primary lung cancer. All patients underwent dual radionuclide imaging with 201Tl-chloride and 99mTc-MIBI. Regions of interest were placed over the tumor area (T) and contralateral normal lung area (N). A tumor to normal lung ratio (T/N ratio) and retention index were calculated by early and delayed ratios. In the SPECT images, the positive rate was 98% in both the early and delayed images for 201Tl-chloride and 95% in the early and 88% in the delayed images for 99mTc-MIBI. Both early and delayed T/N ratios of 201Tl-chloride were significantly higher than those of 99mTc-MIBI. The retention index of 201Tl-chloride was higher than that of 99mTc-MIBI. T/N ratio and retention index did not significantly correlated with reference to histological types. We conclude that 99mTc-MIBI SPECT can be helpful in localizing primary lung cancers. However, in our study the tumor accumulation of 99mTc-MIBI was significantly lower compared with 201Tl-chloride.

Adult↗
PubMed1996

[Evaluation of reserved hepatic function in patients with hepatobiliary tumor by 99mTc-GSA: effect of hyperbilirubinemia and usefulness of regional reserved hepatic functional imaging].

The evaluation of the reserved hepatic function was performed by 99mTc-galactosyl serum albumin (99mTc-GSA) in seventy patients with hepatobiliary tumor. The dynamic study was performed to evaluate global reserved hepatic function following the intravenous bolus injection of 99mTc-GSA, and the hepatic single photon emission computed tomography (SPECT) was obtained to assess the regional reserved hepatic function. The functional hepatic index (LHL15) was derived from liver time-activity data, and it was compared with serum total-bilirubin level, serum albumin level and plasma disappearance rate of indocyanine green (ICG15). In the patients with hepatocellular carcinoma, LHL15 value agreed well with ICG15 value, serum total-bilirubin level, and serum albumin level. Moderate or severe hepatic dysfunction was observed at 65.4% of these patients. In the patients with cholangiocellular carcinoma, a discrepancy of LHL15 value and ICG15 value was observed. Increment of the ICG15 value was correlated with that of the serum total-bilirubin level, whereas the correlations was not observed between the LHL15 value and the serum total-bilirubin level. These results indicate that 99mTc-GSA scintigraphy can evaluate the reserved hepatic function without the embellishment of jaundice. This method is useful for assessing the global and regional reserved hepatic function.

Aged↗
PubMed1996

[Second phase 99mTc-HMDP accumulation using three phase bone scintigraphy in a case of primary breast cancer].

Accumulation of 99mTc-HMDP was recognized by three-phase bone scintigraphy in a case of primary breast cancer. A 67-year-old female was found to have a 14 x 13 mm mass in the upper lateral area of the left breast. At operation the histopathological finding was a proven solid-tubular carcinoma. On the first phase images, there was no abnormal accumulation in the tumor. On the second phase images, there was abnormal accumulation in the tumor, however, abnormal accumulation was observed followed by a decrease in accumulation. The third phase image showed no abnormal extra-skeletal accumulation. Color Doppler ultrasound sonography showed an abundance of color signals within the breast tumor. Increased 99mTc-HMDP accumulation in the second phase image might reflect tumor hypervascularization. It is concluded that three-phase bone scintigraphy using 99mTc-HMDP has the ability of positive description in primary breast cancer.

Adenocarcinoma↗
PubMed1995

High-performance liquid chromatographic determination of plasma malondialdehyde level without a solvent extraction procedure.

A highly sensitive and simple HPLC method for measuring malondialdehyde (MA) in plasma was developed. For sample preparation, an aliquot of plasma (10 microliters) was mixed with 2.0% thiobarbituric acid (TBA) in 2 M sodium acetate buffer (containing 1 mM DTPA and 0.05% BHT), pH 3.5. After incubating at 95 degrees C for 45 min, the reaction solution was passed through a 0.2-micron membrane filter. An aliquot of 10 microliters filtrate was injected into HPLC system without any extraction procedures. The thiobarbituric acid-malondialdehyde (TBA-MA) complex was separated on reversed-phase HPLC and quantitated with a fluorescence detector. The mobile phase was mixture of acetonitrile:water (7:3, v/v) under isocratic conditions at ambient temperature, and a single analysis was completed in ca. 2.5 min. The minimum detection level for MA was 0.01 pmol. A very close correlation was observed between this method and the Yagi method, which is widely used for clinical diagnosis. The simple and inert mobile phase along with a very simple sample preparation makes this method applicable to numerous clinical samples without any difficulties.

Animals↗
PubMed1995

Increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in the induction of long-term potentiation.

Induction of long-term potentiation in the CA1 region of hippocampal slices is associated with increased activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D. (1993) J. Biol. Chem. 268, 7863-7867). Here we report that application of high but not low frequency stimulation to two groups of afferents in the CA1 region of 32P-labeled slices resulted in the phosphorylation of two major substrates of this enzyme, synapsin I and microtubule-associated protein 2, as well as in the autophosphorylation of CaM kinase II. Furthermore, immunoblotting analysis revealed that long term potentiation induction was associated with an increase in the amount of CaM kinase II in the same region. All these changes were prevented when high frequency stimulation was applied in the presence of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These results indicate that activation of CaM kinase II is involved in the induction of synaptic potentiation in both the postsynaptic and presynaptic regions.

Animals↗
PubMed1995

Stimulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase with brain gangliosides.

The holoenzyme of cAMP-dependent protein kinase (cAMP-kinase) partially purified from the particulate fraction of rat brain was stimulated by gangliosides. Among various gangliosides tested, GM1 was most potent, giving Ka value of 19.5 microM. The maximal activation of the kinase was obtained with 100 microM GM1 using kemptide as substrate. Gangliosides inhibited the kinase activity of the catalytic subunit of cAMP-kinase. Of various substrates tested, the ganglioside-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I and myelin basic protein, but not histone H1 and casein. The molecular mechanisms of the stimulatory effect of gangliosides were investigated. The kinase activated with GM1 was inhibited by the addition of PKItide, a specific inhibitor for cAMP-kinase. However, GM1 did not dissociate the holoenzyme into the catalytic and regulatory subunits and did not interfere with the binding ability of cAMP to the holoenzyme. These results suggest that the gangliosides can directly activate cAMP-kinase in a different manner from cAMP.

Animals↗
PubMed1995

Glutamate-induced loss of Ca2+/calmodulin-dependent protein kinase II activity in cultured rat hippocampal neurons.

The exposure of cultured rat hippocampal neurons to 500 microM glutamate for 20 min induced a 55% decrease in the total Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca(2+)-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N-methyl-D-aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca2+ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.

Animals↗
PubMed1995

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by proline-directed protein kinases and its dephosphorylation.

We identified two major substrates for the proline-directed protein kinases--cdc2 kinase and tau protein kinase II (TPKII)--in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80-kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine-rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80-kDa protein and purified MARCKS, we concluded that the 80-kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two-dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline-directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.

Animals↗
PubMed1995

Activation of mitogen-activated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors.

Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca2+ ionophore, remained intact. Lavendustin A, a tryrosine kinase inhibitor, was without effect. In experiments with 32P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c-raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca(2+)-dependent pathways and that the activation of Raf-1 is not involved.

Animals↗
PubMed1995

Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.

Animals↗
PubMed1994

Bradykinin-evoked non-specific cationic current in neuroblastoma-glioma hybrid (NG108-15) cells and its down-regulation through differentiation.

Effects of bradykinin (BK) on the membrane conductance and level of cytoplasmic free Ca2+ in undifferentiated and differentiated neuroblastoma-glioma hybrid (NG108-15) cells were studied using the nystatin-perforated patch-clamp technique and fura-2 fluorometry. Under voltage clamp at -20 mV, undifferentiated cells responded to BK at > 10(-9) M, producing a biphasic current composed of an apamin-sensitive Ca(2+)-activated K+ outward current and non-specific cationic inward current. Both current components corresponding to a biphasic elevation of [Ca2+]i were completely prevented by an intracellular perfusion with EGTA (1 mM) under conventional whole cell recording condition. Undifferentiated cells revealed almost no voltage sensitive Ca2+ current. In NG108-15 cells differentiated with 8-Br-cyclic AMP (1 mM) or rolipram (1 mM), an inhibitor of type IV phosphodiesterase, BK concentration required for the non-specific cationic current with amplitude of > 100 pA was much greater than that of undifferentiated cells. This suggests that the differentiated cells decreased BK-sensitivity in induction of the non-specific cationic current. The non-specific cationic channel is suggested to play roles as a source of Ca2+ entry in undifferentiated NG108-15 cells.

Bradykinin↗
PubMed1994

Neostriatal mosaic and type II Ca2+/calmodulin-dependent protein kinase: an immunohistochemical study on the adult rat striatum.

The present immunohistochemical study is concerned with the expression of type II Ca2+/calmodulin-dependent protein kinase (CaM-kinase II), which is supposed to play an essential role in the intracellular Ca2+ signal transduction, in the striatum of adult rats. CaM-kinase II immunoreactivity was differentially concentrated in irregularly shaped compartments within the nucleus in a mosaic-like fashion. The compartment of heightened CaM-kinase II-immunolabeling corresponded to the extrastriosomal matrix visualized by calbindin-D28k-immunostaining. Light microscopic observation showed neurons immunoreactive for CaM-kinase II to be less densely distributed in the striosomes than in the matrix compartment. The present data suggest that these two striatal compartments may differ in an intracellular Ca(2+)-signaling process associated with protein phosphorylation.

Animals↗