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H Nakabayashi

Publications and source records attributed to H Nakabayashi.

At least 91 records · Page 5Linked to original sources
PubMed1989

Disopyramide induces insulin secretion and plasma glucose diminution: studies using the in situ canine pancreas.

The mechanism of disopyramide-induced hypoglycemia, a life-threatening complication in the antiarrhythmic drug treatment, is still controversial. To elucidate this, we have evaluated plasma insulin (IRI) and glucagon (IRG) responses in the pancreatic vein (PV) of the in situ pancreas as well as responses of plasma IRI, IRG, and glucose in the femoral artery (FA) to disopyramide phosphate administration in anesthetized dogs. First, infusion of disopyramide at a dose of 50 mg for ten minutes directly into the pancreatic artery, but not the vehicle, increased significantly plasma IRI concentration in the PV (P less than .05 or less), where the IRI response started within three minutes and reached a peak of 2.8-fold preinfusion value at 30 minutes after starting the infusion (n = 7). Plasma IRI concentration in the FA also increased slightly but significantly (P less than .05). Plasma IRG concentration in the PV initially decreased significantly (P less than .05 or less) and in the FA at one point (P less than .05) during the infusion, and then increased significantly after cessation of the infusion, showing a peak of 1.9-fold preinfusion value at 60 minutes in the PV and the FA (P less than .05). Plasma glucose concentration in the FA decreased slowly and significantly after the infusion (P less than .05 or less) and fell by 16% of the baseline value at 60 minutes (P less than .05). Second, serum disopyramide concentration of 13.7 +/- 2.8 micrograms/mL at ten minutes, which corresponds to a twofold to threefold concentration of the human therapeutic level (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A major myristylated substrate of protein kinase C and protein kinase C itself are differentially regulated during murine B- and T-lymphocyte development and activation.

The regulation and expression of protein kinase C (PKC) and phosphomyristin C (PMC) (a principal substrate of PKC which is the major myristylated protein in lymphocyte and glioma lines that express it) in murine B and T lymphocytes were investigated. Both PMC and PKC are differentially regulated during T-cell development. The level of PMC expression is highest in CD4-8-, intermediate in CD4+8+, and lowest in J11d-, CD4, or CD8 single-positive thymocytes. PKC is equally expressed by all three thymic populations. In striking contrast to thymocytes, resting peripheral lymph node T cells and T-cell clones express little if any PMC and reduced levels of PKC. Neither PKC nor PMC is significantly induced upon the activation of lymph node T cells: treatment with anti-CD3 antibodies or anti-CD3 and interleukin-2 fails to induce PKC, whereas PMC is not induced by anti-CD3 alone and is only slightly induced by anti-CD3 and interleukin-2. In contrast to the situation with T cells, PMC and PKC are constitutively expressed at moderate levels in mature B cells. PMC is greatly increased in B-cell blasts generated by cross-linking the antigen receptor with anti-immunoglobulin. These results demonstrate that PMC and PKC are differentially regulated during the development and activation of B and T cells, suggesting that cellular events that rely upon PKC and PMC may differ during ontogeny and activation of different lymphocyte subsets.

Animals↗
PubMed1989

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2.

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.

Aminoquinolines↗
PubMed1989

Interleukin 2 promotes expression of an interleukin 2 receptor and proliferation of T cells stimulated with non-mitogenic dose of concanavalin A.

Interleukin 2 (IL-2) initiated proliferation of the cells of T-enriched population stimulated with non-mitogenic dose of concanavalin A (Con A), whereas it could not induce proliferation of the cells treated with non-mitogenic lectin wheat germ agglutinin (WGA). Use of monoclonal antibody (MAb) directed against interleukin 2 receptor (IL-2R) showed that IL-2 significantly increased expression of IL-2R on the cells of T-enriched population stimulated with Con A, whereas it could not cause any significant enhancement of expression of IL-2R on the cells treated with WGA. We concluded that IL-2 activated T cells in combination with non-mitogenic does of Con A, and induced both expression of IL-2R and proliferation.

Concanavalin A↗
PubMed1989

Expression and function of protein kinase C isozymes.

Three protein kinase C (PKC) isozymes, type I, II, and III, have been identified as the major Ca2+/phospholipid-stimulated protein kinases in the various animal tissues. Based on the immunochemical analysis it was demonstrated that PKC I was encoded by gamma cDNA, PKC II by the alternatively spliced beta I and beta II cDNAs, and PKC III by alpha cDNA. The expression of these enzymes appears to be tissue-specific and developmentally regulated. The central nervous system expresses high level of all three isozymes and the peripheral tissues mainly PKC II and III. During brain development, the expression of PKC I appears to follow the progress of synaptogenesis, whereas PKC II and III increase progressively from fetus up to 2-3 weeks of age. The level of PKC I in adult brain is highest in the cerebellum, hippocampus, amygdala, and cerebral cortex especially in those cortical regions being important for visual information processing and storage. The role of PKC II and III in cellular regulation was investigated by treatment of rat basophilic leukemia cells with the phorbol ester, phorbol 12-myristate 13-acetate. This phorbol ester caused a faster degradation of PKC II than III, indicating a differential down-regulation of these two enzymes by this compound. The results presented in this study support the contention that each species of PKC has a distinct function in the regulation of a variety of cellular processes.

Animals↗
PubMed1989

Sheehan's syndrome with hypomagnesemia and polymorphous ventricular tachycardia.

A 40 year old woman with Sheehan's syndrome demonstrated polymorphous ventricular tachycardia and hypomagnesemia. The diagnosis of Sheehan's syndrome was based on her history, physical examination and endocrinological data. Serum magnesium level was 1.5 mg/dl. After the treatment of polymorphous ventricular tachycardia by lidocaine, an electrocardiogram showed low voltage, T-wave inversion or flattening and QT-prolongation, and these electrocardiographic abnormalities gradually improved after the initiation of cortisone therapy. A chest roentgenogram, an echocardiogram, thallium-201 scan of the myocardium and hemodynamic parameters indicated no abnormal findings. It is suspected that hypomagnesemia caused polymorphous ventricular tachycardia. However, the cause of hypomagnesemia and its relation to Sheehan's syndrome in the present case were not determined, although, hypomagnesemia was improved after the administration of cortisone.

Adult↗
PubMed1988

Biochemical characterization of rat brain protein kinase C isozymes.

Biochemical characteristics of three rat brain protein kinase C isozymes, types I, II, and III, were compared with respect to their protein kinase and phorbol ester-binding activities. All three isozymes appeared to be alike in their phorbol ester-binding activities as evidenced by their similar Kd for phorbol 12,13-dibutyrate and requirements for Ca2+ and phospholipids. However, differences with respect to the effector-mediated stimulation of protein kinase activity were detectable among these isozymes. The type I enzyme could be stimulated by cardiolipin to a greater extent than those of the type II and III enzymes. In the presence of cardiolipin, the concentrations of dioleoylglycerol or phorbol 12,13-dibutyrate required for half-maximal activation (A1/2) of the type I enzyme were nearly an order of magnitude lower than those for the type II and III enzymes. In the presence of phosphatidylserine, differences in the A1/2 of dioleoylglycerol and phorbol 12,13-dibutyrate for the three isozymes of protein kinase C were less significant than those measured in the presence of cardiolipin. Nevertheless, the A1/2 of these two activators for the type I enzyme were lower than those for the type II and III enzymes. At high levels of phosphatidylserine (greater than 15 mol %), binding of phorbol 12,13-dibutyrate to the type I enzyme evoked a corresponding stimulation of the kinase activity, whereas binding of this phorbol ester to the type II and III enzymes produced a lesser degree of kinase stimulation. For all three isozymes, the concentrations of phosphatidylserine required for half-maximum [3H]phorbol 12,13-dibutyrate binding were almost an order of magnitude less than those for kinase stimulation. Consequently, neither isozyme exhibited a significant kinase activity at lower levels of phosphatidylserine (less than 5 mol %) and phorbol 12,13-dibutyrate (50 nM), a condition sufficient to promote near maximal phorbol ester binding. In addition to their different responses to the various activators, the three protein kinase C isozymes also have different Km values for protein substrates. The type I enzyme appeared to have lower Km values for histone IIIS, myelin basic protein, poly(lysine, serine) (3:1) polymer, and protamine than those for the type II and III enzymes. These results documented that the three protein kinase C isozymes were distinguishable in their biochemical properties. In particular, the type I enzyme, which is a brain-specific isozyme, is distinct from the type II and III enzymes, both have a widespread distribution among different tissues.

Animals↗
PubMed1988

Increased degradation of protein kinase C without diminution of mRNA level after treatment of WEHI-231 B lymphoma cells with phorbol esters.

Immunoblot analysis of WEHI-231 B lymphoma cell homogenates revealed that both type II, a major component, and type III, a minor component, protein kinase C (PKC) were present. Northern blot analysis of PKC mRNA showed a higher level of beta II and beta I mRNA (encoding type II PKC) than of alpha mRNA (encoding type III PKC). Short term (3 min) treatment with phorbol 12-myristate 13-acetate (PMA) caused a rapid loss of PKC in cytosol and a concomitant increase in the particulate fraction. After prolonged (24 hr) exposure, the level of both PKC isozymes were decreased. However, the corresponding mRNA levels remained intact. PMA did not inhibit the anti-IgM-mediated increase in [Ca2+]i in PKC-depleted cells.

Animals↗
PubMed1988

Tissue distribution and developmental expression of protein kinase C isozymes.

Protein kinase C is a ubiquitous enzyme found in a variety of mammalian tissues and is especially highly enriched in brain and lymphoid organs. Based on biochemical and immunological analyses, we have identified three types of protein kinase C isozyme (designated types I-III) from rat brain. Monospecific antibodies against each of the protein kinase C isozymes were prepared for the determination of tissue distribution, subcellular localization, and developmental changes of these enzymes. The various protein kinase C isozymes were found to be distinctively distributed in different tissues: the type I enzyme in brain; the type II enzyme in brain, pituitary and pineal glands, spleen, thymus, retina, lung, and intestine; and the type III enzyme in brain, pineal gland, retina, and spleen. The rat brain enzymes were differentially distributed in different subcellular fractions. The type I enzyme appeared to be most lipophilic and was recovered mostly in the particulate fractions (80-90%) regardless of the EGTA- or Ca2+-containing buffer used in the homogenization. Significant amounts (30-40%) of the type II and III enzymes were recovered in the cytosolic fraction with EGTA-containing buffer. The expressions of different protein kinase C isozymes appear to be differently controlled during development. In rat brain, both type II and III enzymes were found to increase progressively from 3 days before birth up to 2-3 weeks of age and remained constant thereafter. However, the expression of the type I enzyme displayed a different developmental pattern; it was very low within 1 week, and an abrupt increase was observed between 2 and 3 weeks of age. In thymus, the type II enzyme was found to be maximal shortly after birth; whereas the same kinase in spleen was very low within 2 weeks of age, and a significant increase was observed between 2 and 3 weeks. These results demonstrate that protein kinase C isozymes are distinctively distributed in different tissues and subcellular locales and that their expressions are controlled differently during development.

Animals↗
PubMed1988

Monoclonal antibodies against type II rat brain protein kinase C.

Monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C were used for the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II protein kinase C in a dose-dependent manner but did neither to the type I nor III isozyme. Immunoblot analysis of the tryptic fragments from protein kinase C revealed that all three antibodies recognized the 27-38-kDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-kDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained 70-80% of the kinase activity which was dependent on Ca2+ and phosphatidylserine and further activated by diacylglycerol or tumor-promoting phorbol ester. With antibody 8/1, the kinetic parameters with respect to Km for ATP and histone and K alpha for phosphatidylserine and phorbol 12,13-dibutyrate were not significantly influenced. However, the antibody causes variable effects on the K alpha for Ca2+ under different assay conditions. When determined in the presence of phosphatidylserine, the K alpha for Ca2+ was reduced by an order of magnitude (37 +/- 8 to 2.0 +/- 1.8 microM); in the presence of phosphatidylserine and phorbol 12,13-dibutyrate, the K alpha for Ca2+ was not significantly altered; and in the presence of phosphatidylserine and dioleoylglycerol, the kinase became an apparently Ca2+-independent enzyme. The effects of antibody 8/1 on the kinetic parameters of the enzyme for phorbol ester binding were different from those for kinase activity. This antibody causes a 20-30% reduction in phorbol ester binding and a 2-fold increase (1.9 +/- 0.2 to 3.9 +/- 0.3 micrograms/ml) in the concentration of phosphatidylserine required for half-maximal binding, but is without significant influence on those parameters for Ca2+ and phorbol 12,13-dibutyrate. The differential effects of antibody 8/1 on kinase activity and phorbol ester binding with respect to the kinetic parameter of phosphatidylserine suggest that the roles of this phospholipid in supporting phorbol ester binding and kinase activation are different. In the presence of the antibody, the autophosphorylations of the phospholipid/phorbol ester-binding domain and the kinase domain were reduced; the reduction was more pronounced for the former than for the latter. These results suggest that the epitope for antibody 8/1 is localized within the phospholipid/phorbol ester-binding domain at the region adjacent to the kinase domain so that the autophosphorylations of both domains are affected.

Animals↗
PubMed1988

Immunocytochemical localization of protein kinase C isozymes in rat brain.

Recently, we isolated 3 protein kinase C (PKC) isozymes from rat brain (Huang et al., 1986a). Using isozyme-specific antibodies for immunoblot, we have determined the relative levels of each isozyme in various regions of the rat brain (Huang et al., 1987b). The present paper describes the cellular distributions of PKC isozymes in rat brain as determined by light microscopic immunocytochemistry. Staining with PKC antibodies revealed strong immunoreactivities in neuronal somata and their dendrites and weak to no reaction in axon and the astroglial structures. In the cerebellum, the type I PKC antibodies stained the Purkinje cell bodies and dendrites; the type II PKC antibodies stained the granule cells; and the type III PKC antibody stained both Purkinje and granule cells. In the cerebral cortex, all antibodies stained neurons resembling pyramidal cells and their apical dendrites in layers II to VI, while layer I was nearly devoid of staining. However, the various isozyme-specific antibodies revealed distinct laminar distribution patterns of the positively stained neurons, and the type III PKC-positive neurons exhibited a higher density than those of type I or II PKC-positive ones, especially in layer II of cingulate (retrosplenial) and piriform cortices. In the hippocampal formation, both pyramidal cells of the hippocampus and granule cells of the dentate gyrus were stained by all PKC antibodies. Subcellularly, type III PKC appeared mostly in the cytoplasm of these neurons, whereas type I and II PKC seemed to associate with the nucleus as well. In the olfactory bulb, both type II and III PKC antibodies stained the periglomerular and granular cells, and the latter also stained the mitral cells. The distinct cellular and subcellular distribution of PKC isozymes suggests that each isozyme plays a unique role in the various neural functions.

Animals↗
PubMed1987

Immunochemical identification of protein kinase C isozymes as products of discrete genes.

Immunocytochemical studies of rat cerebellum using specific antibodies against type I, II, and III PKC revealed the presence of the type I PKC in the Purkinje cells where transcripts of tau cDNA were localized, the type II PKC in the granule cells where transcripts of beta cDNA were detected, and the type III PKC in both the Purkinje and granule cells. Immunoblot analysis revealed that the expressed PKC in COS cells transfected with either alpha, beta, or tau cDNA of PKC were recognized by specific antibodies against the type III, II, and I PKC isozymes, respectively. These results prove that the type I, II, and III PKC are products of PKC genes, tau, beta, and alpha, respectively. With these specific antibodies we have identified the presence of multiple species of PKC in a variety of cell types.

Animals↗
PubMed1987

Differential distribution of protein kinase C isozymes in the various regions of brain.

We have previously identified three types of protein kinase C (a Ca2+-activated phospholipid-dependent kinase) isozymes, designated types I, II, and III, from rat brain (Huang, K.-P., Nakabayashi, H., and Huang, F. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8535-8539). These enzymes are different in their elution profile from hydroxylapatite column, sites of autophosphorylation, and immunoreactivity toward two types of monoclonal antibodies. Now we describe the purification of similar protein kinase C isozymes from monkey brain and their regional distribution in the brain. These primate enzymes all have the same molecular weight of 82,000, and each type of isozyme cross-reacts with the purified monospecific antibodies against its corresponding rat brain counterpart isozyme. These purified antibodies were used to quantify the relative contents of three types of protein kinase C isozymes in various regions of rat and monkey brains. In rat brain, cerebellum contained a high level of the type I isozyme; cerebral cortex, thalamus, and corpus callosum were high in the type II enzyme; and olfactory bulb was highest in the type III enzyme. In monkey brain, the type I isozyme was found to be enriched in cerebellum, hippocampus, and amygdala; the type II enzyme was at very high level in caudate, frontal and motor cerebral cortices, substantia nigra, and thalamus; and the type III enzyme was at the highest level in olfactory bulb. These results indicate that protein kinase C isozymes are differentially distributed in various regions of rat and monkey brains and suggest a unique role for each isozyme in controlling the different neuronal functions in the brain.

Animals↗
PubMed1987

Role of protein kinase C in the regulation of rat liver glycogen synthase.

Rat liver glycogen synthase was phosphorylated by purified protein kinase C in a Ca2+- and phospholipid-dependent fashion to 1-1.4 mol PO4/subunit. Analysis of the 32P-labeled tryptic peptides derived from the phosphorylated synthase by isoelectric focusing and two-dimensional peptide mapping revealed the presence of a major radioactive peptide. The sites in liver synthase phosphorylated by protein kinase C appears to be different from those phosphorylated by other kinases. Prior phosphorylation of the synthase by protein kinase C has no significant effect on the subsequent phosphorylation by glycogen synthase (casein) kinase-1 or kinase Fa, but prevents the synthase from further phosphorylation by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, phosphorylase kinase, or casein kinase-2. Additive phosphorylation of liver glycogen synthase can be observed by the combination of protein kinase C with the former set of kinases but not with the latter. Phosphorylation of liver synthase by protein kinase C alone did not cause an inactivation nor did the combination of this kinase with glycogen synthase (casein) kinase-1 or kinase Fa produce a synergistic effect on the inactivation of the synthase. Based on these findings we conclude that the phorbol ester-induced inactivation of glycogen synthase previously observed in hepatocytes cannot be accounted for entirely by the activation of protein kinase C.

Animals↗
PubMed1987

Pancreatic vagal nerve is receptive to somatostatin in rats.

To investigate whether somatostatin, one of the pancreatic islet hormones, influences the afferent firing activity of the vagus innervating the pancreas and whether there is any relevant morphological basis for the electrophysiological event, cyclic somatostatin-14 was injected into the rat pancreatic artery. The somatostatin injection at a physiological (0.61 pmol) or a pharmacological (3.05 pmol) dose significantly increased the afferent firing discharge rate of the pancreatic vagus in rats anesthetized with urethan and chloralose. The present histological examination disclosed a glomuslike neurovascular body in the rat peripancreatic sinusoidal vein. The body included many small corpuscles, and the corpuscle contained a structure like the afferent nerve ending. The immunohistochemical study revealed that the structure preferentially bound exogenous somatostatin. These electrophysiological and histological findings suggest that the vagal pancreatic nerve is receptive to somatostatin and that there exists a local neural monitoring system for somatostatin secreted from the islets.

Afferent Pathways↗