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H Higashida

Publications and source records attributed to H Higashida.

At least 55 records · Page 3Linked to original sources
PubMed1990

Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells.

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.

Acetylcholine↗
PubMed1990

Bradykinin induces inositol 1,4,5-trisphosphate-dependent hyperpolarization in K+ M-current-deficient hybrid NL308 cells: comparison with NG108-15 neuroblastoma x glioma hybrid cells.

External application of bradykinin (BK) to mouse neuroblastoma X mouse fibroblast hybrid NL308 cells and mouse neuroblastoma X rat glioma hybrid NG108-15 cells produced a transient outward (hyperpolarizing) current. In NG108-15 cells, BK also induced an inward (depolarizing) current associated with a decrease in input membrane conductance, which results from the inhibition of a voltage-sensitive potassium current, the M-current. However, in NL308 cells, either no depolarization was elicited by BK or, even if the BK-induced depolarization was evoked, it was associated with an increased conductance. To explain the above difference, the intracellular second messenger system of NL308 cells was examined in detail. BK induced the rapid accumulation (three- to fivefold higher than the control level) of inositol 1,4,5-trisphosphate (InsP3) in NL308 cells. The cytosolic Ca2+ concentration was also elevated to 540 nM from 180 nM at a basal level. This seems to be enough to activate a voltage-independent and Ca2(+)-sensitive K+ current, resulting in the hyperpolarization. Intracellular injection of InsP3 replicated the hyperpolarization. NL308 cells possess protein kinase C (C-kinase), with specific activities of C-kinase in cytosolic and membrane fractions being 233 and 24 pmol/min/mg protein, respectively. The activity associated with particulates became higher after phorbol dibutyrate (PDBu) treatment. But NL308 cells did not show the characteristic inward relaxation by step hyperpolarizations and the outward rectification in the current-voltage relationship, indicating that the M current is deficient in NL308 cells. Therefore, application of PDBu failed to mimic the inward current. The results suggest the role of InsP3 and C-kinase in controlling two K+ currents.

Animals↗
PubMed1990

Cholinergic synapse formation between NG108-15 and muscle cells and modulation of transmission.

Mouse neuroblastoma x rat glioma hybrid NG108-15 cells form cholinergic synapses with rat or mouse muscle cells in culture. The rate of synapse formation is greatly dependent on intracellular cyclic AMP concentrations. The synapse formation is lower in the presence of glia maturation factor, a partially purified brain extract. Once the synapse between NG108-15 cells and myotubes has been formed, this synapse is stable for days. Extracellular application of serotonin, PGF2 alpha, PGD2, neurotensin and bradykinin on NG108-15 cells increases synaptic transmission. Since bradykinin increases the level of intracellular inositol 1,4,5-trisphosphate (InsP3), bradykinin-induced facilitation is due to InsP3-dependent elevation of intracellular Ca concentrations.

Animals↗
PubMed1989

Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. Primary structure and functional expression from cDNAs.

The complete amino acid sequences of two potassium channel proteins from NG108-15 neuroblastoma-glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.

Amino Acid Sequence↗
PubMed1989

Selective effector coupling of muscarinic acetylcholine receptor subtypes.

Attempts have been made by means of recombinant DNA technology to understand the molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor (mAChR). Molecularly defined mAChR subtypes have been produced from the cloned DNAs in Xenopus oocytes and NG108-15 neuroblastoma-glioma hybrid cells as transient and stable expression systems, respectively, and agonist-induced cellular responses have been examined. The results obtained provide evidence that mAChR subtypes are selectively coupled with different effector systems, albeit not exclusively.

Animals↗
PubMed1988

Ca2+-dependent K+ channels in neuroblastoma hybrid cells activated by intracellular inositol trisphosphate and extracellular bradykinin.

Bradykinin (BK) activation of phosphatidylinositide breakdown in NG108-15 neuroblastoma x glioma hybrid cells in the generation of an outward K+ current through the release of Ca2+ by the intermediary messenger inositol 1,4,5-trisphosphate (InsP3). Channels mediating this outward current were identified using cell-attached patch electrodes. Intracellular iontophoretic injection of InsP3 or Ca2+, or extracellular application of BK, evoked bursts of K+ channel activity coincident with cell hyperpolarization measured with an intracellular recording micropipette. The most frequent channels had a mean single-channel conductance of about 40 pS in symmetrical K+ solutions; additional openings of lower conductance (18 pS) channels were also detected. Bath application of phorbol dibutyrate (PDBu, 1 microM) increased the number and opening probability of the InsP3-induced channels.

Animals↗
PubMed1988

Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG108-15 cells.

The primary structures of two muscarinic acetylcholine receptor (mAChR) species, designated as mAChR I and mAChR II, have been elucidated by cloning and sequence analysis of DNAs complementary to the porcine cerebral and cardiac messenger RNAs, respectively. mAChR I and mAChR II expressed in Xenopus oocytes differ from each other both in acetylcholine-induced response and in antagonist binding properties. These results, together with the differential tissue location of the two mAChR mRNAs, have indicated that pharmacologically distinguishable subtypes of the mAChR represent distinct gene products. The primary structures of two additional mammalian mAChR species, designated as mAChR III and mAChR IV, have subsequently been deduced from the nucleotide sequences of the cloned cDNAs or genomic DNAs. We report here that mAChR I and mAChR III expressed in NG108-15 neuroblastoma-glioma hybrid cells, but not mAChR II and mAChR IV, efficiently mediate phosphoinositide hydrolysis, activation of a Ca2+-dependent K+ current and inhibition of the M-current, a voltage-dependent K+ current sensitive to muscarinic agonists.

Acetylcholine↗
PubMed1988

Voltage- and calcium-activated potassium currents in mouse neuroblastoma x rat glioma hybrid cells.

1. Membrane currents were recorded from voltage-clamped, microelectrode-impaled cells of the NG108-15 mouse neuroblastoma x rat glioma clonal cell line, differentiated with prostaglandin E1. 2. A slow outward tail current reversing at post-pulse potentials between -80 and -90 mV was evoked by depolarizing pre-pulses to near 0 mV. The tail current was inhibited by Cd2+ ions (0.2-1 mM) and hence attributed to activation of a Ca2+-dependent K+ current by a priming voltage-activated Ca2+ current. 3. Two components to this tail current could be distinguished pharmacologically: an early (less than or equal to 50 ms) component inhibited by 1-5 mM-tetraethylammonium (TEA), and a late component lasting several hundred milliseconds inhibited by apamin (0.1-0.4 microM) or d-tubocurarine (0.1-0.5 mM). 4. Ionophoretic injection of Ca2+ ions evoked a transient outward current with an apparent reversal potential (from ramped current-voltage curves) of -70 mV. This current was succeeded or sometimes replaced by an inward current with an apparent reversal potential between -20 and -10 mV. 5. The outward current induced by Ca2+ injections was unaffected or partly inhibited by TEA (1-5 mM), but was strongly inhibited by apamin or d-tubocurarine. 6. Hyperpolarizing voltage steps from between -30 and -40 mV induced inward current relaxations reversing at between -80 and -90 mV. These were considered to result from deactivation of the voltage-dependent sustained K+ current, IM. 7. Application of methacholine, muscarine or Ba2+ ions produced an inward current, reduced input conductance and reduced IM deactivation relaxations. 8. It is concluded that differentiated NG108-15 cells possess several of the K+ currents present in sympathetic neurones, including a delayed rectifier current, two species of Ca2+-activated K+ current and the M-current.

Action Potentials↗
PubMed1988

Membrane current responses of NG108-15 mouse neuroblastoma x rat glioma hybrid cells to bradykinin.

1. Membrane current responses to focal application of bradykinin (BK) were recorded in voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. 2. BK produced sequential outward and inward currents at clamp potentials between -60 and -30 mV, designated IBK(out) and IBK(in), respectively. 3. The outward current IBK(out) was accompanied by an increased membrane conductance. Ramp current-voltage (I-V) curves yielded a reversal potential (VBK) of -80 +/- 5.6 mV (mean +/- S.D., n = 9) in 5.4 mM [K+]o. VBK showed a positive shift on raising [K+]o, compatible with a primary increase in K+ conductance. Subtracted I-V curves indicated that the underlying conductance was not strongly voltage dependent between -120 and -40 mV. 4. IBK(out) was inhibited by d-tubocurarine (dTC, 0.1-0.5 mM) but was insensitive to tetraethylammonium (TEA) below 5 mM. 5. The inward current IBK(in) was accompanied by a fall in membrane conductance. This was associated with the inhibition of a time- and voltage-dependent K+ current, IM. In consequence, IBK(in) was strongly voltage dependent and dissipated, usually without reversal, on hyperpolarizing the cell beyond -70 mV in 5.4 mM [K+]o. Reversal to an outward current negative to -40 mV could be obtained on raising [K+]o to 54 mM. 5. Both IBK(in) and IBK(out) persisted when ICa was blocked with Co2+ or Cd2+. IBK(out) slowly diminished in Ca2+-free, Mg2+-substituted solution. 6. The Ca2+ spike current ICa and the Ca2+-activated K+ current IAHP were inhibited during IBK(out) or after Ca2+ injections. BK did not affect the voltage-activated K+ current IK(V) recorded in Co2+ solution. 7. It is concluded that the dual response to BK results from opposing effects on two different species of K+ current. IBK(out) results from activation of a Ca2+-dependent, voltage-insensitive K+ conductance, probably mediated by a transient rise in intracellular Ca2+. It is suggested that the Ca2+ is released from an intracellular store. IBK(in) results primarily from inhibition of the Ca2+-independent, voltage-gated K+ current, IM. This effect is not replicated by a rise of intracellular Ca2+ and must therefore be generated by another mechanism.

Action Potentials↗
PubMed1988

Inositol 1,4,5-trisphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastoma x rat glioma hybrid cells.

1. The role of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) as possible mediators of the membrane current responses of NG108-15 neuroblastoma x glioma hybrid cells to bradykinin (BK, Brown & Higashida, 1988b) has been tested using intracellular ionophoresis of InsP3 and external application of phorbol dibutyrate (PDBu) and 1-oleoyl-2-acetylglycerol (OAG). 2. Intracellular ionophoresis of InsP3 into cells clamped at -30 to -50 mV produced (i) a transient outward current, (ii) a transient outward current followed by an inward current, or (iii) an inward current. All currents were accompanied by an increased input conductance. 3. The transient outward current reversed at between -80 and -90 mV. The reversal potential was shifted to more positive potentials on raising extracellular [K+], suggesting that it resulted from an increased K+ conductance. 4. The outward current was inhibited by apamin (0.4 microM) or d-tubocurarine (0.2-0.5 mM); these drugs also inhibit the outward current produced by BK or by intracellular Ca2+ injections (Brown & Higashida, 1988 a, b). The outward current was also slowly reduced in 0 mM [Ca2+] or 0.5 mM [Cd2+] plus 2 mM [Co2+] solution. 5. Ionophoretic injection of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, guanosine trisphosphate or inorganic phosphate did not evoke an outward current but produced only an inward current with an increased conductance, reversing at between -10 and -20 mV. 6. Bath application of PDBu (10 nM-1 microM) or OAG (1-10 microM) produced an inward current with a fall in input conductance. The inward current was voltage dependent and was accompanied by an inhibition of the time-dependent current relaxations associated with activation or deactivation of the voltage-dependent K+ current, IM. 7. PDBu did not clearly reduce the Ca2+ current or the Ca2+-dependent K+ current recorded in these cells. During superfusion with PDBu, the outward current produced by intracellular ionophoresis of InsP3 was greatly enhanced. 8. The results support the view that the two membrane current responses to BK might both result from accelerated membrane phosphatidylinositide hydrolysis. One product, InsP3, releases Ca2+ and activates an apamin-curare-sensitive outward K+ current; this effect is imitated by intracellular InsP3 ionophoresis. The second product, DAG; activates protein kinase C to inhibit the voltage-dependent K+ current IM and generate an inward current; this effect is imitated by external application of PDBu or OAG.

Animals↗
PubMed1988

Acetylcholine release by bradykinin, inositol 1,4,5-trisphosphate and phorbol dibutyrate in rodent neuroblastoma cells.

1. The action of bradykinin (BK), inositol 1,4,5-trisphosphate (InsP3), and phorbol dibutyrate (PDBu) on the release of acetylcholine (ACh) was studied electrophysiologically on short-distance (less than 20 micron) synapses formed between cultured NG108-15 mouse neuroblastoma x rat glioma hybrid cells and rat muscle cells. Action potentials in NG108-15 cells did not usually evoke an excitatory junction potential (EJP) in the muscle cell in this system. 2. Ionophoretic application of BK onto the somatic surface of an NG108-15 cell produced an increase in frequency of miniature end-plate potentials (MEPPs) for 40-50s in the paired myotube. Some MEPPs were evoked during BK-induced hyperpolarization (10-20 s) of the hybrid cell soma. A few MEPPs were also elicited during BK-induced depolarization. 3. Ionophoretic injection of Ca2+ into an NG108-15 cell soma generated MEPPs for a very brief period (less than 3 s), coincident with somatic hyperpolarization. No increase was observed during a subsequent somatic depolarization induced by a larger current of Ca2+. 4. Ionophoretic injection of InsP3 into the cytoplasm of an NG108-15 cell soma transiently evoked MEPPs during the InsP3-induced hyperpolarizing phase. A large InsP3 injection caused sustained generation of MEPPs for 2-4 min, associated with InsP3-evoked depolarization. 5. Within 3-5 min after exposure of NG108-15-myotube pairs to 1 microM-PDBu, the MEPP frequency increased by 2-5 times and reached a plateau after 8 min. The increase continued after wash-out of the drug. The PDBu-induced increase of MEPPs was still observed when the membrane potential of the NG108-15 cell was clamped at -30 mV. 6. The data suggest that the BK-induced facilitation results from the action of two intracellular second messengers: an InsP3-dependent release of Ca2+ from the intracellular storage sites and protein phosphorylation by diacyclglycerol (DAG)-activated protein kinase C.

Acetylcholine↗
PubMed1987

Bradykinin inhibits potassium (M) currents in N1E-115 neuroblastoma cells. Responses resemble those in NG108-15 neuroblastoma x glioma hybrid cells.

Application of bradykinin to voltage-clamped N1E-115 mouse neuroblastoma cells evoked sequential outward and inward membrane currents, accompanied by an increase and decrease of membrane conductance, respectively. Methacholine produced an inward current with a decreased conductance. The outward current response to bradykinin was imitated by intracellular inositol 1,4,5-trisphosphate (IP3). Bath application of phorbol dibutyrate induced an inward current and potentiated the response to IP3. We conclude that the response of these cells to bradykinin is identical to that of NG108-15 hybrid cells, and therefore may be attributed to the dual effects of inositol trisphosphate and diacylglycerol formed by hydrolysis of phosphatidylinositide.

Animals↗