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Biomedical subjects

H Higashida

Publications and source records attributed to H Higashida.

At least 127 records · Page 7Linked to original sources
PubMed1984

Bradykinin-induced rapid breakdown of phosphatidylinositol 4,5-bisphosphate in neuroblastoma X glioma hybrid NG108-15 cells.

External application of bradykinin to neuroblastoma X glioma hybrid NG108-15 cells produced a sustained depolarization preceded by a transient hyperpolarization. Bradykinin also increased the frequency of miniature end-plate potentials recorded from cultured striated muscle cells which had been innervated by NG108-15 cells. Parallelism between facilitative phases of miniature end-plate potentials and depolarization indicates that bradykinin caused an enhanced synaptic transmission from NG108-15 cells due to depolarization. Effects of bradykinin on phospholipid metabolism in the hybrid cells were then examined to shed light upon the mechanism by which bradykinin-receptor interaction leads to facilitation of synaptic transmission. Bradykinin induced specific incorporation of 32Pi into phosphatidic acid and phosphatidylinositol without affecting [3H]glycerol incorporation into these phospholipids by 10 min after its addition. The addition of bradykinin to hybrid cells prelabeled with 32Pi caused a transient decrease (maximal effect seen at 10-30 s) in the radioactivity from phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) which was followed by the accumulation of radioactivity in phosphatidic acid and phosphatidylinositol. A Ca2+ ionophore, A23187, failed to induce the initial degradation of PI-4,5-P2. The data show that the magnitudes of bradykinin-induced PI-4,5-P2 degradation and membrane potential changes in NG108-15 cells are both dependent on the concentration of bradykinin and that the degradation of PI-4,5-P2 precedes the electrophysiological responses. Taken together with the finding that bradykinin induced a transient increase in Ca2+ influx (at 10-20 s), it appears that a rapid and transient degradation of PI-4,5-P2 might be related to the initiation of the NG108-15 cell activities through mobilization of extracellular Ca2+ into the cells.

Animals↗
PubMed1984

Sensitive and insensitive states of cultured glioma cells to glutamate damage.

Cytotoxic effects of L-glutamate and related compounds were investigated on rat glioma C6 cells in vitro. Within 12-24 h, addition of glutamate to the culture medium, resulted in degeneration of the C6 cells. The ED50 for glutamate-induced damage was about 4 mM. Seventeen structural analogues of glutamate, including agonists and antagonists for glutamate receptors as well as glutamate-uptake inhibitor, were examined concerning their toxicity on C6 cells. Among them, L-aminoadipic acid, DL-aminopimelic acid, DL-homocysteic acid, L-cysteic acid, quisqualic acid, L-glutamic acid diethyl ester and 2-amino-4-phosphonobutyric acid elicited similar degeneration at comparable concentrations. The D-isomer of glutamate was not cytotoxic. Following differentiation of C6 cells with 1 mM dibutyryl cyclic AMP or 3 mM sodium butyrate, they were no longer susceptible to L-glutamate and L-aminoadipate. C6 cells treated with 10 microM hydrocortisone, which is known to induce glutamine synthetase activity, were also resistant to L-glutamate, but not to L-aminoadipate. The decomposition of cellular DNA in glutamate-treated cultures was confirmed by flow cytometer analysis. The results demonstrate that the sensitivity of C6 cells to glutamate-induced cytotoxicity was modified by cellular metabolic conditions. This indicates that cultured glioma C6 cells are a useful model system to investigate the molecular mechanism of glutamate gliotoxicity in vitro.

2-Aminoadipic Acid↗
PubMed1984

Cytotoxic action of retinoidal butenolides on mouse neuroblastoma and rat glioma cells.

Proliferation and death were measured in cultures of mouse neuroblastoma N18TG -2 and rat glioma C6BU -1 cells when treated with up to 100 micron retinoidal butenolides (RB 1-6). The number of viable cells in each case was measured with various concentrations of the compounds, of which RB-3 (5-hydroxy-4-[2-(2,6,6,-trimethyl-l- cyclohex en-l-yl) ethenyl ]-2(5H)- furanone ) was the most potent in destroying the cells after 2 days' incubation. ED50 of RB-3 was about 5 X 10(-7) M for both types of cell. RB-3 was 80 times more potent than retinoic acid. Ten analogues of RB-3 had a similar inhibitory effect on DNA synthesis in N18TG -2 cells. The degenerative changes caused by RB-3 in C6BU -l cells were irreversible even when the cells were exposed to it for 2 h. Tumor weights of N18TG -2 cells that had been inoculated subcutaneously onto the backs of A/J mice were 30-40% lower than those of untreated controls after 14 days of single daily i.p. injections of RB-3 doses of 100 mg/kg of body weight. The results indicate that RB-3 is cytotoxic in murine tumor cells originating from the nervous system and has an inhibitory effect on neuroblastoma-tumor growth in mice.

Animals↗
PubMed1984

Facilitation of synaptic transmission by prostaglandin D2 at synapses between NG108-15 hybrid and muscle cells.

The effect of prostaglandin (PG) D2 on neuronal functions was investigated in neuroblastoma X glioma NG108-15 hybrid cells. PGD2 caused a sustained increase in miniature end-plate potentials (MEPPs) recorded from cultured striated muscle cells which had formed junctions with NG108-15 cells. PGD2 initially hyperpolarized and then depolarized NG108-15 cells. The time course of depolarization fitted well to the facilitative phase of MEPPs. The same action on synaptic transmission and membrane potentials was detected with PGF2 alpha but not with PGE1. PGD2 (10(-4)M) produced a 3-fold increase of adenylate cyclase activity in NG108-15 cell homogenates through its receptors that are distinct from those of PGE1 and PGI2. These results show that PGD2 facilitates MEPP frequency from NG108-15 cells due to depolarization, and suggest that PGD2 may act as a physiological neuromodulator for synaptic transmission in vivo.

Adenylyl Cyclases↗
PubMed1984

Isolation and characterization of intraspecific cybrids. Effect of mitochondrial DNA on their cellular properties.

Cybrid clones were obtained by fusing whole cells of rat glioma C6BU-1, resistant to 5-bromodeoxyuridine (BrdU), with cytoplasts of embryonic rat 3Y1CAP cells, resistant to chloramphenicol (CAP), in selective medium with BrdU and CAP. The clones resistant to BrdU and CAP were confirmed to be cybrids by chromosome and mtDNA analyses. More than half the mtDNA of all the cybrid clones was from the 3Y1CAP cells. After cultivation of a cybrid clone Y22 for 3 months in the absence of CAP, subclones were isolated. One subclone Y22-22 contained predominantly mitochondrial DNA (mtDNA) from the 3Y1CAP cells. Using this subclone, the effects of the mitochondrial genome on cellular properties were examined. The growth patterns, expression of glioma-specific beta-adrenergic receptor, and composition of the major proteins of C6BU-1 cells were not affected by transmitted mtDNA from the 3Y1CAP cells. This procedure for isolating cells containing predominantly foreign mtDNA will be useful in studies on the interaction between genomes of the mitochondria and nucleus.

Animals↗
PubMed1984

Antiserum against neurite outgrowth factor in chick gizzard extract and its inhibitory effect on neuritic response in cultured ciliary neurons.

Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.

Animals↗
PubMed1984

A single class of neurotensin receptors with high affinity in neuroblastoma X glioma NG108-15 hybrid cells that mediate facilitation of synaptic transmission.

Receptor binding of [3H]neurotensin was examined on membrane preparations derived from neuroblastoma X glioma NG108-15 hybrid cells. The specific binding was saturable and reversible, and a dissociation constant (Kd) was calculated to be about 0.24 nM from the rate constants. Scatchard analysis of neurotensin binding at equilibrium revealed a single class of binding sites with a Kd of 0.86 nM and a maximal binding capacity (Bmax) of 250 fmol/mg of protein (7700 receptor sites/cell). [D-Arg9]-Neurotensin had a high affinity (IC50 = 0.5 nM) for the neurotensin receptors, but [D-Phe11]-neurotensin had a lower affinity (IC50 = 280 nM), while angiotensin II and bradykinin had almost no affinity for [3H]neurotensin-binding sites. Under similar conditions [3H]neurotensin binding to mouse and rat brain synaptosomal fractions showed two binding sites with high (0.86 and 0.44 nM) and low (13 and 19 nM) affinities. We have examined several possible physiological consequences of neurotensin receptor binding. Neurotensin (10 microM) exhibited no influence on adenylate cyclase activity, 45Ca uptake, or 32Pi incorporation into phosphatidylinositol fractions of NG108-15 cells. Electrophysiological study of isolated NG108-15 cells revealed neurotensin-induced transient hyperpolarization followed by sustained depolarization with enhanced membrane excitability. Application of neurotensin to NG108-15 cells that had formed synapses with cultured striated muscle cells caused a considerable increase in frequency of miniature endplate potentials from the muscle cells. These data show that NG108-15 cells possess a single class of neurotensin receptors similar to a high affinity site of synaptosomal membranes from the murine brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Tetanus toxin: a rapid and selective blockade of the calcium, but not sodium, component of action potentials in cultured neuroblastoma N1E-115 cells.

The slow action potential of mouse neuroblastoma N1E-115 cells, which was elicited in Na-free medium and whose amplitude was directly dependent on extracellular Ca concentrations, was blocked by tetanus toxin (TNTX) without change in resting membrane potential and resistance. The average value of action potential overshoot (peak potential level of spikes) in the absence and presence of TNTX was -1 and -11 mV, respectively. The concentration of TNTX required for 50% inhibition of Ca-dependent spikes (IC50) was about 65 ng/ml. The amplitude of spikes was decreased within 10-20 min after adding TNTX. The inhibition of Ca-dependent spikes was mimicked by 10 mM CoCl2 and MnCl2, but not by the boiled or neutralized toxin. The results indicate that TNTX inhibited electrogenesis of Ca, but not Na, components of action potentials of the neuroblastoma cells.

Animals↗
PubMed1983

Modulation of synapse formation by cyclic adenosine monophosphate.

Synapses between neuroblastoma-hybrid cells and myotubes exhibit a high degree of plasticity. Increase of cyclic adenosine monophosphate (AMP) levels of the hybrid cells for several days results in the appearance of functional voltage-sensitive Ca2+ channels, which are required for evoked secretion of acetylcholine. The results show that cyclic AMP regulates synaptogenesis by regulating the expression of voltage-sensitive Ca2+ channels, and suggest that cyclic AMP affects posttranslational modifications of some glycoproteins and cellular levels of certain proteins.

Animals↗
PubMed1983

Tetanus toxin blocks Ca spikes in neuroblastoma clone N1E-115 cells.

The effect of tetanus toxin on the Ca spike of the action potential evoked in neuroblastoma clone N1E-115 cells by constant current stimulation in Na+-free medium containing various concentrations of Ca2+(1.8-80 mM) was examined by intracellular recordings. Tetanus toxin (more than 140 ng/ml) blocked the Ca spikes. The resting membrane potentials and membrane input resistances of tetanus toxin-treated cells were similar to those of untreated cells. The blocking effect of tetanus toxin was evident in medium containing Ca2+ at concentrations of more than 30 mM. The difference between the peak levels of the action potentials of toxin-treated and untreated cells was statistically significant at Ca2+ concentrations of 7.2 mM and more.

Action Potentials↗
PubMed1983

Cytotoxic action of prostaglandin D2 on mouse neuroblastoma cells.

Addition to the culture medium of prostaglandin (PG) D2 resulted in the degeneration in a dose- and time-dependent manner of N18TG-2 cells cloned from mouse neuroblastoma. The ED50 for PGD2-induced cytotoxicity was about 10 microM. The degenerative changes were irreversible when the cells were exposed for more than 10 h. Scanning and transmission electron microscopic examination revealed that treatment with PGD2 resulted in appearance of numerous blebs of various sizes along the cell surface and also in destruction of surface membrane and of cytoplasmic organelles. Tumor weight of N18TG-2 neuroblastoma inoculated subcutaneously on the backs of A/J mice was about 35-70% less than that of controls after 14 days of single daily i.p. or s.c. injections of 0.5-1 mg/kg of PGD2. The results indicate that PGD2 has growth-inhibitory effects on mouse neuroblastoma cells in vitro and in vivo.

Animals↗
PubMed1983

Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes.

We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.

Action Potentials↗
PubMed1982

Receptor-associated changes of the catecholamine-sensitive adenylate cyclase in glioma cells doubly transformed with Moloney sarcoma virus.

A doubly transformed rat glioma cell line, designated C6V-1, was obtained from rat glioma C6 cells by infection with a rat-adapted variant of Moloney sarcoma virus (MSV-M-os). The C6V-1 cells show karyotypic changes in chromosome number (43) and structure, while C6 cells possess a normal male karyotype. C6V-1 and C6 cells were employed for characterization of a receptor-adenylate cyclase system of the surface membrane. C6V-1 cells showed lower adenylate cyclase activity than that of C6 cells, though the apparent Km for ATP in both types of cells was the same. The maximal stimulation of adenylate cyclase by isoproterenol was significantly reduced, and Kact for isoproterenol was approximately 18-fold lower in C6V-1 cells. When the concentration of beta-adrenergic receptors was measured by various concentrations of [3H] dihydroalprenolol (DHA), the maximal binding sites of C6 and C6V-1 cells were 760 and 230 fmol/mg protein, respectively, without any changes in the association constant for DHA. The concentration of isoproterenol required for 50% displacement of the [3H] DHA binding (Kd) was the same (around 1.5 X 10(-6)M) in both cells, measured in the presence of GTP. Thus the 19-fold drop in the Kd/Kact ratio in C6V-1 cells suggests an incomplete coupling of beta-receptors to adenylate cyclase. Cyclic AMP phosphodiesterase activity and cAMP content in C6V-1 were lower than in C6 cells. Mitochondrial monoamine oxidase and cytosomal enolase activities, however, were somewhat higher in C6V-1 cells. The results indicate that a set of changes in the receptors and in the cyclic AMP system of C6V-1 is one of the specific alterations by transformation, even though those may not be the cause of cell transformation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Coexpression of species-specific histone H1 and H2B genes in mouse-rat hybrid cells.

Five different clones of somatic hybrid cell lines between mouse and rat cells were examined for the expression of species-specific histone H1 and H2B subtypes. It was found that one hybrid (140-3) contained only mouse specific histones and the other four clones had both mouse and rat specific histones in various ratios. In three cases (141-B, NBr10A, NBr20A), the relative amount of mouse and rat specific histones was correlated with the karyotype of the hybrids. However, this was not true in one case (NG108-15). The results indicate that a single mammalian hybrid clone can express both parental types of histones, and the histone expression in these hybrids is not regulated by an allelic exclusion but may be expressed codominantly by gene dosage.

Animals↗
PubMed1982

Double minutes in mouse neuroblastoma cells and their hybrids.

Cytogenetic studies were carried out on three clones of mouse neuroblastoma cells and six interspecific hybrid cells derived from the mouse neuroblastoma cells with either rat glioma cells and liver cells or Chinese hamster brain cells. The hybrid cells possessed characteristic karyotypes with marker chromosomes originating from the neuroblastoma cells. The parental chromosome constitution in the hybrid cells was clone-specific, even in the clones derived from the same parental cells. Double minutes (DMs) were demonstrated in the neuroblastoma cells and in all the hybrid cells studied. In addition other chromosome aberrations, such as microchromosomes and chromosome pulverization, were also observed in these cells. DMs varied in number and morphology among the cells. The number of DMs per cell correlated positively with the level of ploidy and with the karyological constitution contributed by the parental neuroblastoma cells. The results indicate that DMs have a chromosome nature and that the DMs of neuroblastoma chromosomes were transferred into the hybrid cells.

Animals↗