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Biomedical subjects

S Okuya

Publications and source records attributed to S Okuya.

At least 19 recordsLinked to original sources

Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein kinase.

Leptin at 1-5 nM, the concentrations observed in obese subjects, caused an increase in the active form of mitogen-activated protein kinase (MAPK) that was accompanied by increased tyrosine phosphorylation of STAT-1 and STAT-3 in a mouse pancreatic beta cell line, MIN6. Leptin also increased DNA synthesis and cell viability in MIN6 cells based on the results of [3H]-thymidine incorporation and colorimetric MTT assay, respectively. The specific MAPK-inhibitor PD98059 blocked not only the MAPK activation but also the increment in DNA synthesis and cell viability caused by leptin. Thus, leptin stimulates both the MAPK and the Janus kinase (JAK)-STAT cascade as well as inducing proliferation through the MAPK cascade in MIN6 cells. This mechanism might account, at least in part, for obesity-induced pancreatic islet hypertrophy.

Animals

Direct stimulation of basal insulin secretion by physiological concentrations of leptin in pancreatic beta cells.

We examined a possible mechanism underlying the link between obesity and hyperinsulinemia, focusing on leptin, a peptide released from adipocytes which affects the satiety center in the brain. The leptin receptor isoforms, Ob-Ra and Ob-Rb, are present in the pancreatic beta cell line MIN6 and in rat pancreatic islets, based on RT-PCR. A 2 hr, but not a 30 min, incubation with 1 nM recombinant mouse leptin, the concentration observed in obese subjects, stimulated basal (at 5 mM glucose) insulin secretion by approximately 40% in both MIN6 and rat islets. Stimulatory effects were not observed without glucose or when the incubation medium containing 1 nM leptin had been preincubated with the immobilized leptin antibody. In contrast to the stimulatory effects on basal insulin secretion at 1 nM, the maximally stimulated insulin secretion at 25 mM glucose was not significantly changed by 1 nM leptin in isolated rat islets. In addition, 10 and 100 nM leptin exerted small but significant inhibitory effects on 16.7 mM glucose-stimulated insulin secretion. Thus, leptin acts directly on pancreatic beta cells, and stimulation of basal insulin secretion by physiological concentrations of leptin may account in part for the fasting hyperinsulinemia observed in obese subjects.

Animals

Altered Gs alpha N-terminus affects Gs activity and interaction with the G beta gamma subunit complex in cell membranes but not in solution.

The stimulatory G protein (Gs) mediates activation of adenylylcyclase by a ligand-receptor complex. Gs is heterotrimeric (alpha beta gamma) and activation can be accomplished by dissociation of the alpha-subunit (Gs alpha) from the beta gamma-subunit complex (G beta gamma). Gs alpha is also a substrate for choleragen catalyzed ADP-ribosylation when it is associated with G beta gamma but not as free Gs alpha. Using recombinant DNA techniques we modified the cDNA for the 52,000 M(r) form of Gs alpha (Gs alpha 52) to produce a protein with a 2,400 M(r) N-terminal extension (Gs alpha 54.4). This N-terminal extension could be removed with the protease Factor Xa. In vitro transcription and translation of the recombinant plasmid containing the cDNA's for Gs alpha 52 and Gs alpha 54.4 produced a 52,000 M(r) and a 54,000 M(r) protein, respectively. In solution the properties of Gs alpha 52 and Gs alpha 54.4 were indistinguishable. Both proteins: (a) formed a heterotrimer with G beta gamma and their affinities for the subunit complex were the same; (b) could be ADP-ribosylated by choleragen in the presence but not in the absence of G beta gamma; (c) bound the non-hydrolyzable GTP analogue, GTP gamma S, and were protected from chymotryptic proteolysis by the guanine nucleotide; and (d) could activate in vitro translated type IV adenylylcyclase. Gs alpha 54.4 and Gs alpha 52 were incorporated into S49 cyc-membranes, which lack Gs alpha. After incorporation, both Gs alpha 52 and Gs alpha 54.4 were protected from chymotryptic proteolysis when GTP gamma S was present, revealing that both proteins were able to bind the nucleotide and undergo a conformational change characteristic of Gs alpha activation. When Gs alpha 52 was incorporated into cyc-membranes it could mediate both hormone and GTP gamma S stimulation of adenylylcyclase and could be ADP-ribosylated by choleragen, but Gs alpha 54.4 could do neither of these things, indicating that the properties of Gs alpha 54.4 were altered by the membrane. Deletion of the N-terminal extension by treatment with Factor Xa in solution converted Gs alpha 54.4 to Gs alpha 52, and upon incorporation into cyc-membranes it behaved like Gs alpha 52 in every regard, showing that the effect of the N-terminal extension was reversible. A lack of other differences in the functional properties of Gs alpha 52 and Gs alpha 54.4 suggests a correlation between the interaction of Gs alpha with G beta gamma and its ability to activate adenylylcyclase.

Adenosine Diphosphate Ribose

GTP binding to Gs does not promote subunit dissociation.

The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.

Adenosine Triphosphate

Donor leukocyte transfusions and discontinuation of immunosuppressants to achieve an initial remission after allogeneic bone marrow transplantation in a patient with primary refractory acute leukemia.

We present a female patient who received an allogeneic bone marrow transplantation for primary refractory Philadelphia-positive acute biphenotypic leukemia. Since leukemic blasts were persistently present in peripheral blood and bone marrow, in spite of the evidence for engraftment of male donor hematopoiesis, we performed donor leukocyte transfusions and discontinued immunosuppression. An initial complete remission was obtained 15 weeks after allogeneic bone marrow transplantation, and lasted for 24 weeks. We concluded that the prominent mechanism for the eradication of the refractory leukemic clone in the patient was the graft-versus-leukemia effect.

Adult

Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer but not its free alpha-subunit.

The heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) mediates activation of adenylylcyclase. Gs is inactive when GDP is bound to the guanine nucleotide binding site of the alpha-subunit (Gs alpha). Gs can be activated by fluoroaluminate or by binding GTP or GTP analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is also required for the activation of Gs, and Gs alpha is a substrate for ADP-ribosylation catalyzed by choleragen (CT). Gs activation can also be accompanied by dissociation of Gs alpha from the G beta gamma-subunit complex. When dissociated Gs subunits were separated by chromatography, isolated Gs alpha could not be ADP-ribosylated by CT unless G beta was added back. RM/1 antiserum against Gs alpha was used to immunoprecipitate Gs, and the subunit composition of the immunoprecipitate was determined. When Gs was incubated with 2 mM MgCl2, the Gs heterotrimer was immunoprecipitated, and Gs alpha could be ADP-ribosylated by CT. Activation of Gs with GTP gamma S or fluoroaluminate in the presence of 2 mM MgCl2 did not cause Gs subunit dissociation nor did it affect the ability of Gs alpha to be ADP-ribosylated by CT. MgCl2 caused a dose-dependent decrease in the amount of G beta that coprecipitated with Gs alpha in the absence as well as the presence of GTP gamma S or fluoroaluminate. Gs subunit dissociation was accompanied by a corresponding decrease in CT-catalyzed ADP-ribosylation of Gs alpha regardless of whether or not GTP gamma S or fluoroaluminate was bound to Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose

Myelofibrosis and systemic lupus erythematosus: reversal of fibrosis with high-dose corticosteroid therapy.

A case of myelofibrosis in association with systemic lupus erythematosus (SLE) is reported. Acute thrombocytopenia and a bleeding tendency developed in a 24-year-old woman with SLE. Bone marrow aspiration was unsuccessful due to myelofibrosis. Pulse therapy with methylprednisolone reversed both thrombocytopenia and myelofibrosis. A review of the literature revealed that the coexistence of SLE and myelofibrosis is a rare occurrence. Only 7 cases, to our knowledge, have ever been reported in detail. The present case is the 3rd in which myelofibrosis was reversed by corticosteroids.

Adult

Effect of brain-gut peptides upon neurons in centrally regulating sites for drinking.

The effects of angiotensin II (A II) and ANP on spontaneously active neurons in the subfornical organ (SFO), anteroventral third ventricle (AV3V) and supraoptic (SON) and paraventricular nucleus (PVN) were investigated using slice preparations and extracellular recordings. Application of A II (10(-7)M) excited the neural activity of 66% of the SFO neurons, 28% of the AV3V neurons and 44% of the SON neurons. The threshold concentration to produce responses in SFO and AV3V neuron was less than 10(-10)M, while that in SON neurons was 10(-9)M. The excitatory effects of A II were reversibly antagonized by saralasin and persisted after synaptic blockade in a low Ca2+ and high Mg2+ medium. Application of ANP (10(-7)M) inhibited the neural activity of 41% of the AV3V neurons, 22% of the PVN neurons and only 14% of the SFO neurons but had no effect on SON neurons. The threshold concentration for ANP in the AV3V was 10(-11)M. Interestingly, ANP inhibited A II induced excitation in most of the SFO neurons (87%), while ANP had little effects on their spontaneous firing rates. These results show that both peptides of A II and ANP have direct central actions on hypothalamic neurons although ANP can not directly influence magnocellular neurons, suggesting that these blood borne peptides are detected in the SFO and AV3V and that they are acting as a neurotransmitter or a neuromodulator in the central nervous system to regulate water homeostasis.

Angiotensin II

Oxytocin predominantly excites putative oxytocin neurons in the rat supraoptic nucleus in vitro.

To determine the oxytocin (OXT) sensitivity of neurons in the supraoptic nucleus (SON), extracellular recordings were made from the rat hypothalamic slice preparation. OXT added to the bathing medium (3 X 10(-7) M) excited 13 (93%) of 14 cells which fired continuously (average 4.9 +/- 0.7 spikes/s) and 26 (81%) of 32 cells which fired slowly and irregularly (average 1.4 +/- 0.4 spikes/s). By contrast, only 2 (8%) of 26 phasically firing neurons were excited and none of the SON cells tested were inhibited. The excitation was reversibly antagonized by a synthetic OXT analogue, 1-deamino-[2-(O-methyltyrosine), 4-valine, 8-D-arginine]vasopressin. The results suggest that OXT exerts predominantly excitatory effects in the SON and that putative OXT cells are more likely to be affected than putative vasopressin cells.

Action Potentials

Angiotensin II sensitive neurons in the supraoptic nucleus, subfornical organ and anteroventral third ventricle of rats in vitro.

The angiotensin II (AII) sensitivity of neurons in the supraoptic nucleus (SON), subfornical organ (SFO) and the region near the anteroventral part of the third ventricle (AV3V) was investigated using extracellular recording in the rat brain slice preparation by adding AII (10(-10)-10(-6) M) to the perfusion medium. Forty seven (44%) of 106 SON neurons, 62 (66%) of 94 SFO neurons and 28 (33%) of 86 AV3V neurons were excited by AII. One cell was inhibited by AII in the SON and one in the SFO. The threshold concentration to evoke responses in the SON neurons was approximately 10(-9) M, but neurons in the SFO and AV3V showed clear excitatory responses to AII at 10(-10) M. In the SON, 18 (40%) of 45 phasic firing neurons (putative vasopressin neurons) and 29 (48%) of 61 nonphasic firing neurons (including putative oxytocin neurons) were excited by AII. The excitatory effect of AII was reversibly antagonized by a specific antagonist saralasin and persisted after synaptic blockade in medium with low [Ca2+] and high [Mg2+]. We conclude that AII can stimulate both vasopressin and oxytocin release, acting directly upon SON neurons and also that both the SFO and AV3V are important receptive sites for AII (although the SFO is relatively more sensitive) which contributes SON input and modulates release of these hormones.

Angiotensin II

Effects of atrial natriuretic polypeptide on rat hypothalamic neurones in vitro.

1. Extracellular recordings were made from 175 spontaneously active cells in the rat coronal hypothalamic slice preparation. Reconstruction of the recording sites showed that fifteen were in the supraoptic nucleus (s.o.n.), ten in the magnocellular portion of the paraventricular nucleus (p.v.n.) which could be antidromically activated by stimulation lateral to the nucleus, seventy-seven other cells in the p.v.n. and seventy-three in the anteroventral third ventricle (a.v.3.v.) region. 2. The mean firing rates (mean +/- S.E. of mean) of the spontaneously firing cells in the s.o.n., p.v.n. and a.v.3.v. were 2.8 +/- 0.4 spikes/s, 2.9 +/- 0.2 spikes/s and 5.0 +/- 0.4 spikes/s, respectively. Antidromically identified p.v.n. cells fired spontaneously with a mean firing rate of 1.5 +/- 0.5 spikes/s. 3. Bath application of atrial natriuretic polypeptide (a.n.p.; 10(-7) M) had no effect on fifteen s.o.n. cells tested but nineteen (22%) of eighty-seven p.v.n. cells (including two of the ten antidromically activated cells) and thirty (41%) of seventy-three a.v.3.v. cells showed inhibitory responses. Three (3%) cells in the p.v.n. were excited by a.n.p. 4. The dose dependence of the response to a.n.p. was tested in two p.v.n. and five a.v.3.v. cells. As a.n.p. concentration increased, the firing rates of all seven cells generally decreased. However, one a.v.3.v. neurone was excited at low concentrations (less than 10(-8) M) but inhibited at high concentrations (10(-7) and 10(-6) M) of a.n.p. The threshold concentration to evoke inhibitory responses in the p.v.n. was 10(-10) M and in the a.v.3.v. was 10(-11) M. 5. With the exception of the two antidromically activated p.v.n. cells, the inhibitory effect of a.n.p. still persisted after synaptic transmission had been suppressed with a low-Ca2+ and high-Mg2+ medium. 6. Thirty-six cells in the a.v.3.v. were tested with both a.n.p. and angiotensin II applied at 10(-7) M. Twelve showed inhibitory responses to a.n.p. and nine showed excitatory responses to angiotensin II. In other experiments, a.n.p., angiotensin II and arginine-vasopressin were each applied to neurones in the p.v.n. Of the forty cells tested with all three peptides at 10(-7) M, seven were inhibited by a.n.p., fourteen were excited by angiotensin II and twenty were excited by arginine-vasopressin. No neurones in either the p.v.n. or a.v.3.v. were inhibited by a.n.p. and excited by angiotensin II, but four neurones in the p.v.n. were inhibited by a.n.p. and excited by arginine-vasopressin.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials

Fructose-6-phosphate,2-kinase activity in human erythrocytes.

The hemolysate partially purified from human red cells was demonstrated to be capable of synthesizing fructose-2,6-bisphosphate (F-2,6-P2) from fructose-6-phosphate in the presence of adenosine triphosphate (ATP) indicating that human red cells contain fructose-6-phosphate,2-kinase. The effect of F-2,6-P2 on the rate-limiting enzymes of glycolysis, ie, hexokinase, phosphofructokinase (PFK), and pyruvate kinase, has also been examined. PFK was activated by this metabolite and the half-maximum activation was obtained at a concentration of 10(-7) mol/L. Neither hexokinase nor pyruvate kinase was affected by F-2,6-P2. These results suggest that human erythrocytes may contain this metabolite as one of the positive effectors for PFK.

Erythrocytes

Electrophysiological properties of neurons in the caudal ventrolateral medulla projecting to the paraventricular nucleus of the hypothalamus in rats.

Extracellular recordings were made from neurons in the caudal ventrolateral medulla in urethane-chloralose-anesthetized rats. Stimulation of the paraventricular nucleus (PVN) in the hypothalamus evoked antidromic action potentials in 71 neurons. On the basis of antidromic spike latencies, these neurons could be divided into fast- (24 neurons) and slow-conducting cell groups (47 neurons). Slow-conducting cells showed irregular and slow spontaneous discharges, while a majority of the fast-conducting cells did not show spontaneous discharges. The spontaneous activity of slow-conducting cells was suppressed by i.v. clonidine administration. The effects of clonidine could be consistently reversed by administration of the alpha 2-adrenergic antagonist, yohimbine. The responses by clonidine and yohimbine remained unimpaired in baroreceptor-denervated rats. Vagus nerve stimulation produced an excitation in 80% of slow-conducting cells tested. Baroreceptor activation induced by i.v. administration of phenylephrine inhibited about half of slow-conducting cells tested. Similar elevation of blood pressure in baroreceptor-denervated rats did not show any effect. These physiological and pharmacological properties of slow-conducting cells were similar to those previously reported for catecholaminergic cells in other parts of the brain. The results show the existence of two different populations among neurons in the caudal ventrolateral medulla which project directly to the PVN, and suggest that the presumed A1 catecholaminergic cells are involved in the afferent pathway from cardiovascular baroreceptors and the vagus nerve to the PVN.

Animals

Characterization of hypothalamic noradrenaline receptors in the supraoptic nucleus and periventricular region of the paraventricular nucleus of mice in vitro.

In an attempt to determine the basis for apparently conflicting reports of the effects of noradrenaline (NA) on the neurohypophyseal system and its effects on the parvocellular periventricular region of the paraventricular nucleus (PVN), recordings were made from the neurons in the supraoptic nucleus (SON) and the periventricular region in the mouse hypothalamic slice preparation. Of 47 SON neurons, 43 (91%) were excited and two (4%) were inhibited by NA. Seven SON neurons increased the firing rate with increase of NA concentration (10(-7)-10(-4) M). Both the alpha 1-agonists phenylephrine and methoxamine also increased the activity of all SON neurons tested whereas application of the alpha 2-agonist clonidine and the beta-agonist isoproterenol had weak and inconsistent effects. While the alpha 2-antagonist yohimbine had no consistent influence, the alpha 1-antagonist prazosin blocked or reversed the effects of NA. Another group of 37 neurons in the periventricular region of the PVN was also tested; 13 (35%) were excited and 22 (59%) inhibited by application of NA (10(-5) M). When tested with phenylephrine or methoxamine, 6 of the 7 neurons were excited and one inhibited but all the 4 neurons tested were excited by isoproterenol. Clonidine strongly depressed the activity of all 12 neurons tested. The NA-induced excitatory effects were suppressed or reversed by pre-application of prazosin and the beta-antagonist propranolol while the inhibitory ones were suppressed or reversed by yohimbine. Synaptic blockade did not affect the excitatory responses of SON cells to NA nor the inhibitory responses of periventricular neurons to NA or clonidine. We conclude that SON neurons receive adrenergic excitatory effects mainly through alpha 1-receptors. The periventricular neurons receive the excitatory effects through alpha 1- or beta-receptors and receive the inhibitory effects through alpha 2-receptors.

Animals

Circadian cycles in VIP content and VIP stimulation of cyclic AMP accumulation in the rat pineal gland.

VIP content in the rat pineal gland and cyclic AMP accumulation in response to VIP in the daily light and dark cycle were examined. VIP content in the pineal varied significantly during the day and night; the content decreased during exposure to light and was lowest at the onset of darkness, 6 p.m. (mean +/- SE, 23 +/- 5 pg/pineal), and increased during the night and was highest at the onset of light, 6 a.m. (72 +/- 12 pg/pineal). Response of cyclic AMP accumulation to VIP varied with a periodicity inversely related to the daily light and dark cycle of VIP content; cyclic AMP accumulation in response to 10(-7) M VIP increased in proportion to periods of exposure to light and peaked at 6 p.m., and decreased with the onset of darkness.

Animals