Search PubMed⌕ Search

Biomedical subjects

H Higashida

Publications and source records attributed to H Higashida.

At least 73 records · Page 4Linked to original sources
PubMed1994

Sodium channel mRNAs I, II and III in the CNS: cell-specific expression.

The cellular localization of rat brain sodium channel alpha-subunit mRNAs I, II and III in the central nervous system (CNS) was examined by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes, one set recognizing sodium channel isoforms in the coding region and the other in the non-coding region of the sodium channel messages. The independent sets of probes demonstrated qualitatively similar patterns of sodium channel mRNA expression. In the hippocampus, sodium channel mRNA I was very weakly expressed in the pyramidal layer and in the granular layer of the dentate gyrus; in contrast, sodium channel mRNA II was strongly expressed by neurons in these regions. Sodium channel mRNA III exhibited low-to-moderate expression in some neurons of the pyramidal layer of the hippocampus and granular layer of the dentate gyrus, and was not detectable in others. In the cerebellum, sodium channel mRNA I was moderately expressed in some Purkinje cells, weakly expressed in scattered cells in the molecular layer and negligibly expressed in the granular layer. Sodium channel mRNA II was strongly expressed in Purkinje and granule cells, and was moderately expressed in some cells in the molecular layer. Sodium channel mRNA III was generally not detectable in the cerebellum. In the spinal cord, motor neurons and scattered neurons throughout the gray matter exhibited moderate-to-strong expression of both sodium channel mRNA I and II. A population of cells in the spinal zone of Lissauer showed heavy expression of mRNA II, but not mRNA I. Sodium channel mRNA III was not detectable in spinal cord neurons. These observations are consistent with a general regional distribution of sodium channel message isoforms, with mRNA II being preferentially expressed in rostral regions of the CNS and mRNA I in caudal regions. However, the results also indicate that different cell types, within a given region, display different patterns of sodium channel mRNA expression. Moreover, these data suggest that individual neurons may express multiple forms of sodium channel mRNA.

Animals↗
PubMed1994

Sodium channel mRNAs in cultured spinal cord astrocytes: in situ hybridization in identified cell types.

The expression of rat brain sodium channel alpha-subunit mRNAs I, II and III and a putative glial cell-specific sodium channel (NaG) mRNA was examined in cultured astrocytes from P-0 rat spinal cord by RNA blot hybridization and by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes. Sodium channel mRNA I was not detectable in the cultured astrocytes by RNA blot or in situ hybridization. Sodium channel mRNA II showed negligible-to-low levels of expression in flat, fibroblast-like and 'pancake' astrocytes at 4 days in vitro (div), while stellate, process-bearing astrocytes exhibited low-to-moderate levels of mRNA II expression. At 7 div, mRNA II expression ranged from low-to-moderate in flat astrocytes and was moderately high in most process-bearing astrocytes. In RNA blots, a weak band was observed at 9.5 kb. Sodium channel mRNA III expression was negligible in flat astrocytes and was detectable in low-to moderate levels in stellate astrocytes beginning at 4 div; by 7 div, mRNA III was detectable in low levels in flat astrocytes and low-to-moderate levels in stellate astrocytes. RNA blots showed two bands of nearly equal intensity, one at 9.0 kb and one at 7.2 kb. NaG mRNA showed increased expression with time in culture, being detectable in flat and stellate astrocytes at 4 div and becoming very prominent in flat astrocytes at extended times in culture. In RNA blots of cultured astrocytes at 7 div, a strong hybridizing signal with the NaG probe was observed. These observations demonstrate that flat and stellate astrocytes cultured from rat spinal cord express rat brain sodium channel mRNA II and III, and NaG, and suggest that astrocytes in vitro may co-express multiple forms of sodium channel mRNA.

Animals↗
PubMed1993

Bradykinin-induced cytosolic Ca2+ oscillations and inositol tetrakisphosphate-induced Ca2+ influx in voltage-clamped ras-transformed NIH/3T3 fibroblasts.

Microspectrofluorometry (fura-2) was combined with the whole-cell patch-clamp technique to study bradykinin-activated calcium (Ca2+) influx in single control or v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts. Application of bradykinin on DT cells, but not on control NIH/3T3 cells, evoked cytosolic Ca2+ oscillations in the presence of extracellular Ca2+, but not in the absence of external Ca2+. This effect of zero external Ca2+ concentration could be mimicked by holding at depolarized membrane potentials. Cytosolic Ca2+ oscillations observed at holding potentials of -20 to -80 mV were terminated by holding at -10 mV or more depolarized potentials. The frequency of Ca2+ oscillations increased with membrane hyperpolarization. Bradykinin significantly enhanced the hyperpolarization-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) upon membrane hyperpolarization only in DT cells but not in control cells. No [Ca2+]i increase upon hyperpolarization was observed in bradykinin-stimulated DT cells in the absence of external Ca2+, suggesting that bradykinin activates Ca2+ influx. [Ca2+]i increased upon application of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into control and DT cells in an extracellular Ca(2+)-dependent manner, indicating that NIH/3T3 fibroblasts have an Ins(1,3,4,5)P4-gated Ca2+ influx pathway. Ins-(1,3,4,5)P4, however, produced the sustained [Ca2+]i increase in DT cells, but not in control NIH/3T3 cells, suggesting that ras may lock the Ca2+ influx pathway at the activated state. Cytosolic Ca2+ oscillations, bradykinin-enhanced Ca2+ influx, and Ins(1,3,4,5)P4-induced Ca2+ influx were all similar in that activity was increased by membrane hyperpolarization. The results suggest that bradykinin-induced cytosolic Ca2+ oscillations in ras-transformed NIH/3T3 cells are maintained by bradykinin-activated continuous Ca2+ influx which may use Ins(1,3,4,5)P4 as an intracellular messenger.

3T3 Cells↗
PubMed1993

Coupling of m2 and m4 muscarinic acetylcholine receptor subtypes to Ca(2+)-dependent K+ channels in transformed NL308 neuroblastoma x fibroblast hybrid cells.

Muscarinic acetylcholine receptor (mAChR) subtype (m1-m4)-specific cDNAs were transfected into NL308 neuroblastoma-fibroblast hybrid cells and clones expressing each of the individual mAChR subtypes m1, m2, m3 and m4 obtained. Acetylcholine increased phosphoinositide (PI) turnover in m1- and m3-transformed cells, but did not produce detectable changes in m2- and m4-transformed cells. In cells expressing m1 and m3 subtypes, ACh produced an initial outward K+ current, followed by a cationic current. In cells expressing m2 and m4 receptors, only the initial K+ current was detected. The outward currents were associated with a rise in intracellular Ca2+ as measured with Fura-2 or Indo-1, and were inhibited by chelating intracellular Ca2+ with external BAPTA-AM, or by external charybdotoxin or Ba2+: hence they were attributed to the activation of a Ca(2+)-dependent K+ current. However, the outward current produced in m2- and m4-transformed cells was blocked by pretreatment with 5 ng ml-1 Pertussis toxin (PTX), whereas that in m1- and m3-transformed cells was not. These results suggest that m2- and m4-receptors in transformed NL308 cells coupled to PTX-sensitive G-protein which is capable of mobilizing intracellular Ca2+ and activate IK(Ca), whereas m1 and m3 receptors activate a similar process through a different, PTX-insensitive G-protein.

Acetylcholine↗
PubMed1993

Expression of the Na+ dependent uridine transport system of rabbit small intestine: studies with mRNA-injected Xenopus laevis oocytes.

Xenopus laevis oocytes were used as an expression system to prove and characterize the carrier-mediated transport of uridine in the small intestine. Significant Na+ dependency was observed for the uptake of [3H]uridine by Xenopus laevis oocytes injected with poly(A)+RNA prepared from rabbit small intestinal mucosa. By contrast, the uptake of [3H]uridine was negligible in water-injected oocytes. There was no significant difference in the Na+ dependent uptake rates of [3H]uridine among oocytes expressed by using mRNA prepared by three different methods. The uptake of [3H]uridine by mRNA-injected oocytes was enhanced by increasing the culturing time after mRNA injection. Concentration dependency for uridine transport was observed with the Michaelis constant of 8.27 microM, which was comparable to that reported in the study using the brush-border membrane vesicles from rabbit small intestine (6.4 microM). Furthermore, the uptake of [3H]uridine was significantly inhibited by adenosine and thymidine, but not by adenine and uracil. Consequently, the transport system of uridine expressed in mRNA-injected oocytes is clarified to be similar to that functioning in the brush-border membrane of the small intestine.

Animals↗
PubMed1993

[Cellular reactions after stimulation of receptors: research model for evaluation of effects and action mechanisms of drugs for discovery of innovative drugs].

When cellular stimulants such as neurotransmitters, hormones, autacoids, cytokines and growth factors stimulate their respective specific receptors in the plasma membranes of cells, a variety of responses are elicited. GTP-binding proteins are also involved in the reactions between receptors and cellular effectors. Stimulation of receptors are subsequently coupled to the activation of ion channels, turnover of inositol phospholipid metabolism, adenylate cyclase and guanylate cyclase, inhibition of adenylate cyclase and potentiation of all proliferation. Active substances such as the so-called second messengers are produced in the cells. In this article, two findings are described: 1) Ca2+, which increases by stimulation of receptors with neurotransmitters and hormones, stimulated Ca2+/calmodulin-dependent protein kinase II in cell systems such as NG108-15 neuroblastoma x glioma hybrid cells and primarily cultured neuronal cells of rat hippocampus. 2) Coupling preferences and possible transduction mechanisms from experiments on NG108-15 cells and NL308 neuroblastoma x fibroblast hybrid cells which have been stably transfected with DNA for m1, m2, m3 and m4 muscarinic acetylcholine receptors were examined. These results may provide a useful research model for examining and evaluating the effects and mechanisms of the drugs on a living system and may help develop useful methodology for the discovery of innovative drugs.

Animals↗
PubMed1993

Inositol trisphosphate/Ca2+ as a major signal transduction pathway from bradykinin receptors.

Enzymatic phosphatidylinositol-4,5-bisphosphate breakdown is not Ca(2+)-dependent in NG108-15 cells and the bradykinin-induced response consisted of at least two distinct components in the hybrid cells. The initial signal transduction from bradykinin receptors to acetylcholine secretion is composed of the inositol-1,4,5-trisphosphate-dependent Ca2+ pathway despite multiple pathways in the late phase.

Animals↗
PubMed1992

Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA.

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.

Acetylcholine↗
PubMed1992

Activation of Ca2+/calmodulin-dependent protein kinase II by stimulation with bradykinin in neuroblastoma x glioma hybrid NG108-15 cells.

To elucidate the mechanisms of the intracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca2+/calmodulin-dependent manner. The results suggest that the 50-kDa protein is the subunit of CaM kinase II in NG108-15 cells. The Ca2+/calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 microM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 microM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca2+ with 1 mM EGTA or by the pretreatment with 1 microM nifedipine. Stimulation of 32P-labeled NG108-15 cells with 1 microM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. These results suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.

Bradykinin↗
PubMed1992

Ion selectivity of Ba2+ inward current oscillations in ras-transformed fibroblasts that elicit cytoplasmic Ca2+ oscillations by bradykinin.

Ion selectivity of divalent cations on Ba2+ inward current oscillations was examined by voltage-clamp recording in v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts where repetitive transient increases in cytoplasmic Ca2+ concentration were evoked by bradykinin. Application of bradykinin onto DT cells in 50 mM Ba2+ solution initiated Ba2+ inward current oscillations. The inward currents were inhibited in equimolar Sr2+ or Ca2+ solutions. Ba2+ current oscillations were dependent upon extracellular Ba2+ concentration. The results suggest that inward current oscillations are highly selective to Ba2+.

3T3 Cells↗
PubMed1992

Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes.

Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.

Animals↗
PubMed1992

Ba2+ current oscillations modulated by cyclic AMP and phorbol esters in ras-transformed fibroblasts.

An oscillatory influx of divalent cations was measured as Ba2+ inward currents (Ba2+ current oscillations) by voltage-clamp recording in v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts after activation with bradykinin or serum. Application of forskolin or dibutyryl cyclic AMP onto DT cells initiated Ba2+ current oscillations. Increasing intracellular cyclic AMP reduced the amplitude but increased the frequency of the Ba2+ current oscillations. Activation of protein kinase C by phorbol esters terminated Ba2+ current oscillations. No inhibition of Ba2+ current oscillations by phorbol esters was observed in down-regulated cells that had been pretreated with phorbol esters for 24 hrs. The results suggest that Ba2+ current oscillations are regulated by intracellular second messengers.

3T3 Cells↗
PubMed1992

Dissection of bradykinin-evoked responses by buffering intracellular Ca2+ in neuroblastoma x glioma hybrid NG108-15 cells.

Signal transduction pathways from bradykinin (BK) receptors were investigated in NG108-15 neuroblastoma x glioma hybrid cells by buffering the intracellular calcium (Ca2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca2+ chelator. BK increased inositol-1,4,5-trisphosphate (Ins(1, 4,5)P3) formation at the same rate in the control and in BAPTA-acetoxy methyl ester (AM)-treated NG108-15 cells. However, a transient increase of intracellular Ca2+ concentrations in response to BK was significantly suppressed in Ca(2+)-buffered hybrid cells. Accordingly the BK-induced outward current was inhibited in BAPTA-AM-treated hybrid cells, while the subsequent inward current associated with a fall in membrane conductance was apparently increased. The initial phase of acetylcholine release from NG108-15 cells in response to BK was markedly inhibited in BAPTA-AM-treated coculture dishes when detected as miniature end-plate potentials of myotubes, though the late phase of acetylcholine secretion was observed. These results indicate that BK induces two distinct responses in NG108-15 cells: Ins(1,4,5)P3-dependent intracellular Ca2+ rise-sensitive and -insensitive components.

Animals↗
PubMed1992

Effects of toki-shakuyaku-san (Tsumura TJ-23) on electrical activity in neuroblastoma cells and frog neuromuscular junctions.

Toki-shakuyaku-san (Tsumura TJ-23) is a Chinese medicine which has been used for the treatment of gynecological symptoms in aged women. There are several reports on the usefulness of this drug in the treatment of cognitive disorders. We studied the effects of toki-shakuyaku-san on electrical activity in NG108-15 cells, a cell line of differentiated neuroblastoma x glioma hybrid cells, and on frog neuromuscular transmission. In the hybrid cells, an extract of toki-shakuyaku-san slightly depolarized the membrane potential, and strongly decreased the peak heights of the Na+ and Ca2+ current components of the action potential. The order of potency for NG108-15 cells of the 5 ingredients in toki-shakuyaku-san was soujyutsu >> shakuyaku, takusha, toki, senkyu. In voltage-clamped NG108-15 cells, toki-shakuyaku-san and soujyutsu decreased the Na+, K+, and Ca2+ current components. Toki-shakuyaku-san and soujyutsu also induced an increase in the intracellular calcium concentration. However, toki-shakuyaku-san did not affect neuromuscular transmission in the frog sartorius muscle. The results suggest that the effects of toki-shakuyaku-san on neurons are multiple, and tissue- and species-specific, and its effect derives mainly from soujyutsu.

Action Potentials↗