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Biomedical subjects

H Katagiri

Publications and source records attributed to H Katagiri.

At least 73 records · Page 4Linked to original sources
PubMed1997

Calcium- and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density.

We have detected immunoreactivities of AMPA receptor subunits GluR1-4 in post-synaptic density (PSD) fraction and tested whether they can be phosphorylated by endogenous kinases. Incubation of PSD with Ca2+ and calmodulin increased phosphorylation of GluR1 and GluR2/3. The phosphorylation of GluR1 was largely blocked by a Ca2+/calmodulin-dependent protein kinase type II inhibitor. Thus Ca2+/calmodulin-dependent phosphorylation of glutamate receptor may be a mechanism underlying enhanced post-synaptic receptor responsiveness in LTP.

Animals↗
PubMed1997

Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3-L1 adipocytes.

The dominant negative p85alpha regulatory subunit (delta p85alpha) of phosphatidylinositol (PI) 3-kinase or dominant negative Ras (N17Ras) was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Functional expression of delta p85alpha and N17Ras was confirmed by marked inhibition of insulin-stimulated PI 3-kinase activity and mitogen-activated protein kinase activity, respectively. N17Ras expression did not affect glucose transport activity, whereas delta p85alpha expression inhibited insulin-stimulated glucose transport with impairment of GLUT-4 translocation, although inhibition of glucose transport activity was less remarkable than that of PI 3-kinase activity in delta p85alpha-expressing cells. Thus the Ras signaling pathway does not play a major part in either translocation or intrinsic activity of glucose transporters, but PI 3-kinase activation, via phosphotyrosyl proteins and heterodimeric PI 3-kinase, plays a pivotal role in insulin-stimulated glucose transport. However, a discrepancy was observed between PI 3-kinase activity and glucose transport activity, suggesting a possibility that a different pathway(s) is involved in insulin-stimulated intrinsic activity of glucose transporters.

3T3 Cells↗
PubMed1997

Targeting of GLUT1-GLUT5 chimeric proteins in the polarized cell line Caco-2.

Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus approximately sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL) approximately C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus approximately IL)-GLUT5 (TM7 approximately C terminus) and GLUT1 (N terminus approximately TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus approximately TM6)-GLUT1(IL)-GLUT5(TM7 approximately C-terminus) chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.

Animals↗
PubMed1997

The pulmonary hemodynamic effects of 1,1,1-trichloroethane inhalation.

We examined the hemodynamic effects of 1,1,1-trichloroethane (TCE) inhalation in anesthetized dogs in acute inhalation experiments. Six adult mongrel dogs with spontaneous respiration were intubated, connected to a one-way valve. TCE was delivered by the tubular system connected to a 30-liter tedlar bag reservoir filled with 1 v/v% of TCE. After TCE inhalation, the animals revealed a significant decrease in systemic arterial pressure following a decrease in systemic vascular resistance. Pulmonary arterial pressure, pulmonary vascular resistance and right cardiac work increased significantly. These changes were compatible with clinical manifestations of TCE intoxication in human beings. In conclusion, inhalation of TCE may not only decrease peripheral vascular resistance, but also induce transient disturbance of pulmonary blood flow with subsequent pressure overloading in the right ventricle.

Analysis of Variance↗
PubMed1997

Concentration in blood and organs of dogs after high dose 1,1,1-trichloroethane inhalation.

Dogs were exposed to 1% (v/v) (10,000 ppm) vapor of 1,1,1-Trichloroethane (1,1,1-TCE) by inhalation for 3 min repeated four times at 4 hr intervals under continuous anesthesia. Changes in the 1,1,1-TCE concentration in blood with time, as well as distribution of 1,1,1-TCE in the organs and tissues (lungs, liver, kidneys, heart, brain, and fat around the kidneys and on the abdominal wall) upon completion of the four exposures were studied. Concentrations of 1,1,1-TCE in blood showed the highest level immediately after exposure, and fairly decreased in about 30 min after exposure. The half life of 1,1,1-TCE in blood was 4-12 min after exposure. Upon completion of the exposures (3 min inhalations repeated four times), 1,1,1-TCE concentrations per gram wet weight of each organ ranged from 0.1 to 0.5 microgram/g in the lungs, liver, kidneys, heart and brain. On the other hand, the 1,1,1-TCE concentration in fat ranged from 16.9 to 54.6 micrograms/g, greatly exceeding those in other organs.

Adipose Tissue↗
PubMed1997

A polysomnographic study on periodic limb movements in patients with restless legs syndrome and neuroleptic-induced akathisia.

Eighteen patients with restless legs syndrome (RLS) and 4 patients with neuroleptic-induced akathisia (NIA) underwent all-night polysomnographic recordings before and during clonazepam treatment. Ten normal control subjects and 4 non-akathitic psychiatric patients treated with neuroleptics underwent polysomnographic recordings, which were compared with those of the RLS and NIA patients, respectively. Daily treatment with 0.5 to 3 mg clonazepam improved subjective complaints of 17 out of 18 patients with RLS and all the 4 patients with NIA. All the 18 patients with RLS exhibited periodic limb movements (PLM) on the polysomnograms before treatment, but only 2 of 10 control subjects exhibited PLM. Three of the 4 patients with NIA exhibited PLM, but none of the 4 controls on neuroleptics showed PLM. Clonazepam decreased the total number of PLM per hour in patients of both RLS and NIA.

Aged↗
PubMed1996

Interaction between the two signal transduction systems of the histamine H2 receptor: desensitizing and sensitizing effects of histamine stimulation on histamine-dependent cAMP production in Chinese hamster ovary cells.

The histamine H2 receptor is a member of the family of G-protein-coupled receptors and is linked to the activation of adenylate cyclase phospholipase C (PLC). In this study we examined the effects of protein kinase C (PKC) activation in Chinese hamster ovary (CHO) cells stably expressing canine histamine H2 receptors. Pretreatment with 100 nM phorbol 12-myristate 13-acetate at 37 degrees C for 15 min led to significant potentiation of histamine-dependent and forskolin-dependent cAMP production, whereas the biologically inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate, was without effect. These potentiating effects were abolished by preincubation with 0.5 microM bisindolylmaleimide, a PKC inhibitor. Thus the activation of PKCs seems to be involved in the potentiation of cAMP production by acting on a post-receptor mechanism. Preincubation of a CHO cell line, CHO-H2R, with 10 microM histamine for 30 min had two effects. Maximal histamine-dependent cAMP production and forskolin-dependent cAMP production were potentiated by 36% and 105.2% respectively. The other effect was a desensitization of the histamine-dependent adenylate cyclase response as demonstrated by a three-fold increase in EC50. Administration of 0.5 microM bisindolylmaleimide before preincubation of CHO-H2R with 10 microM histamine did not alter the desensitizing effect on cAMP production, but did abolish the sensitizing effect. Preincubation of CHO-H2R cells with 10 nM histamine resulted in moderate potentiation, which was also abolished by bisindolylmaleimide, but not in desensitization of the histamine-dependent cAMP production. Thus these results suggest that preincubation with histamine had a sensitizing effect on cAMP production mediated by PLC and PKC activation, as well as a desensitizing effect on the H2 receptor. The former effect is dependent on the intensity of PLC and PKC signals delivered by H2 receptors. The latter effect requires a higher concentration of histamine.

Animals↗
PubMed1996

Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase.

Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic beta-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic beta-cell line MIN6. Two novel cDNAs, designated PI4P5K-Ialpha and PI4P5K-Ibeta, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.

Amino Acid Sequence↗
PubMed1996

Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.

To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.

3T3 Cells↗
PubMed1996

A novel 55-kDa regulatory subunit for phosphatidylinositol 3-kinase structurally similar to p55PIK Is generated by alternative splicing of the p85alpha gene.

Phosphatidylinositol 3-kinase, which is composed of a 110-kDa catalytic subunit and a regulatory subunit, plays important roles in various cellular signaling mechanisms. We screened a rat brain cDNA expression library with 32P-labeled human IRS-1 protein and cloned cDNAs that were very likely to be generated by alternative splicing of p85alpha gene products. These cDNAs were demonstrated to encode a 55-kDa protein (p55alpha) containing two SH2 domains and an inter-SH2 domain of p85alpha but neither a bcr domain nor a SH3 homology domain. Interestingly, p55 alpha contains a unique 34-amino acid sequence at its NH2 terminus, which is not included in the p85alpha amino acid sequence. This 34-amino acid portion was revealed to be comparable with p55PIK (p55gamma) in length, with a high homology between the two, suggesting that these NH2-terminal domains of p55alpha and p5 gamma may have a specific role that p85 does not. The expression of p55alpha mRNA is most abundant in the brain, but expression is ubiquitous in most rat tissues. Furthermore, it should be noted that the expression of p85alpha mRNA in muscle is almost undetectably low by Northern blotting with a cDNA probe coding for the p85alpha SH3 domain, while the expression of p55alpha can be readily detected. These results suggest that p55 alpha may play an unique regulatory role for phosphatidylinositol 3-kinase in brain and muscle.

Alternative Splicing↗
PubMed1996

A human pancreatic islet inwardly rectifying potassium channel: cDNA cloning, determination of the genomic structure and genetic variations in Japanese NIDDM patients.

Ligand gated potassium channels, such as the ATP-regulated potassium channel, play crucial roles in coupling of stimuli to insulin secretion in pancreatic beta cells. Mutations in the genes might lead to the insulin secretory defects observed in patients with non-insulin-dependent diabetes mellitus (NIDDM). We isolated a cDNA encoding a putative subunit of a ligand gated potassium channel from a human islet cDNA library. The channel, which we designated hiGIRK2, appeared to be an alternative spliced variant and a human homologue of recently reported mbGIRK2, KATP-2/BIR1. Transcripts were detected in human brain and pancreas, but not in other tissues including cardiac muscle. The sizes of transcripts in the pancreas differed from those in the brain, suggesting tissue-specific alternative splicing and possible isoforms. We then isolated human genomic clones, determined the complete genomic structure and localized the gene to chromosome 21 (21q22). The gene was comprised of four exons and the protein was encoded by three exons. The entire coding region of the hiGIRK2 gene was scanned by polymerase chain reaction-single strand conformation polymorphism analysis in 80 Japanese NIDDM patients. We found five nucleotide substitutions; three were silent mutations of the third base of codons, one in the first intron, 9 bases upstream of exon 2, and one in the 3'-untranslated region. We conclude that mutations in the gene encoding hiGIRK2, a (subunit of) ligand gated potassium channel, is not a major determinant of the susceptibility to NIDDM in Japanese.

Alternative Splicing↗
PubMed1996

Association between HLA and islet cell antibodies in diabetic patients with a mitochondrial DNA mutation at base pair 3243.

Islet cell antibodies (ICA), autoantibodies to glutamic acid decarboxylase (GAD) and HLA genotypes were examined in 31 patients with diabetes and a mitochondrial gene mutation located at base pair 3243 (mtDNA 3243 mutation). ICA was detected in 42% (13/31) of these patients compared to 0 of 90 among healthy control subjects. The ICA showed a "non-restricted" pattern of staining in all 13 ICA-positive patients. In a sensitive radioligand assay only 2 of 31 (6%) diabetic patients with the mutation were positive for both GAD65 autoantibodies and ICA, while the remaining 29 patients were GAD65 antibody negative. The ICA-positive patients had an increased frequency of the HLA-DQA1*0301 allele compared to control subjects (p < 0.05). Of the diabetic patients with the mutation 45% (14/31) had progressive clinical course of beta-cell failure. These results indicate that patients with an mtDNA 3243 mutation may develop islet autoimmunity associated with ICA and GAD autoantibodies. We hypothesize that the presence of HLA-DQA1*0301 in individuals with the mtDNA 3243 mutation increases the risk for diabetes and associated autoantibodies against islet cell antigens.

Adult↗
PubMed1996

Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells.

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.

Adenoviridae↗
PubMed1996

Beta-cell loss and glucose induced signalling defects in diabetes mellitus caused by mitochondrial tRNALeu(UUR) gene mutation.

Japanese diabetic patients with diabetic mothers were screened, using peripheral leukocytes, for an A to G transition at nucleotide pair 3243, a tRNALeu(UUR) mutation of the mitochondrial gene. This mutation was identified in four pedigrees from among 300 unrelated patients. Diabetes mellitus cosegregated with the mutation, except in one young subject, and was maternally inherited. Long-term follow-up revealed that the underlying disorder, in affected individuals, is a progressive impairment of insulin secretion. In accordance with this finding, the mutation was found to be highly prevalent in a diabetes mellitus subset termed slowly progressive IDDM; the mutation was identified in 3 out of 27 subjects enrolled in the prospective study of islet cell antibody (ICA)-positive, initially non-insulin-dependent diabetic Japanese patients, who are at high risk of progressing to insulin dependence over several years. The histologic characteristics of slowly progressive insulin-dependent diabetes mellitus include substantial loss, though incomplete, of pancreatic beta-cells. Mitochondrial gene defects in beta-cells could therefore cause glucose-induced signalling defects as well as beta-cell loss. This would explain the wide range of diabetic phenotypes, from NIDDM to IDDM, in patients with this mitochondrial gene mutation.

Adult↗
PubMed1995

Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance.

We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice.

Animals↗
PubMed1995

Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action.

G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N-glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s).

1-Methyl-3-isobutylxanthine↗
PubMed1995

A novel isoform of syntaxin-binding protein homologous to yeast Sec1 expressed ubiquitously in mammalian cells.

munc-18/n-Sec1/rbSec1, a brain homologue of the yeast Sec1p protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. We have screened the mouse cDNA library of an MIN6 cell line, derived from pancreatic beta cells, for its novel isoform and have identified a cDNA encoding a 593-amino acid protein having 63, 53, and 30% identity with munc-18/n-Sec1/rbSec1, Caenorhabditis elegans unc18, and Saccharomyces cerevisiae Sec1p, respectively. While munc-18/n-Sec1/rbSec1 expression has been reported to be neural-specific, RNA blot analysis has revealed that the novel isoform, which we refer to as muSec1 (mammalian ubiquitous Sec1), is expressed ubiquitously. We have also identified mouse munc-18/n-Sec1/rbSec1 from the MIN6 cDNA library, indicating that different isoforms of a protein participating in vesicular transport exist in a single cell. muSec1 bound to glutathione S-transferase-syntaxin 1A and, although with lower affinity, to glutathione S-transferase-syntaxin 4 fusion protein. These findings suggest that muSec1 is, via its binding to the syntaxin family, involved in the protein trafficking from the Golgi apparatus to the plasma membrane and that the fundamental mechanisms of protein trafficking have been conserved from yeast through virtually all mammalian cells.

Amino Acid Sequence↗