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Biomedical subjects

H Higashida

Publications and source records attributed to H Higashida.

At least 55 records · Page 3Linked to original sources
PubMed1995

Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma x fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes.

Intracellular free Ca2+ concentrations ([Ca2+]i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 microM acetylcholine (ACh) increased [Ca2+]i in all four subclones. The increased [Ca2+]i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca2+]i showed sinusoidal oscillations. ACh-induced increases in [Ca2+]i were not observed in cells treated with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ with ethylene-glycol-bis-(beta- aminoethyl)-N,N,N',N'-tetraacetate (EGTA) did not affect the initial [Ca2+]i increases, but reduced the late phases of delta [Ca2+]i in ml- and m3-transformed cells by 20-30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced delta [Ca2+]i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50-100 ng ml-1 Pertussis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1- and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic adenosine diphosphate ribose (cAD-PR) produced a rise in [Ca2+]i. ACh-induced [Ca2+]i oscillations were not observed in ryanodine-treated m2-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1995

Discrete acetylcholine release from neuroblastoma or hybrid cells overexpressing choline acetyltransferase into the neuromuscular synaptic cleft.

Neuroblastoma (clones NS-20Y, N1E-115, and Neuro2A) and neuroblastoma x glioma hybrid (NG108-15) cells were transfected with mouse choline acetyltransferase (ChAT) complementary DNA (cDNA) or vector DNA alone and stably transformed cell lines were established to examine their ability to secrete acetylcholine (ACh). Membrane potentials were recorded from either presynaptic neuroblastoma and hybrid cells or postsynaptic myotubes in co-culture. After transformation with ChAT, synapses were formed and miniature end-plate potentials (MEPPs) were recorded in myotubes co-cultured with Neuro2A and N1E-115 cells, while parental and mock-transfected control cells totally lacked this ability. The rate of synapse formation and/or MEPP frequency was higher in transformed NG108-15 hybrid and NS-20Y cells than that in the control cells. Action potentials of NS-20Y, Neuro2A or NG108-15 cells overexpressing ChAT were able to evoke end-plate potentials in myotubes, though the average quantum content of these cells was 0.04-0.14, which is as low as the control value. The results show that increased concentrations of ACh by ChAT cDNA transfection reveal a masked property in vesicular ACh release from Neuro2A and N1E-115 cells with no endogenous ChAT activity, or modify their secretory capacity upwardly from NG108-15 and NS-20Y cells with endogenous activity.

Acetylcholine↗
PubMed1995

Overexpression of choline acetyltransferase reconstitutes discrete acetylcholine release in some but not all synapse formation-defective neuroblastoma cells.

Secretion of acetylcholine (ACh) in neuroblastoma cells overexpressing choline acetyltransferase (ChAT) was examined. With transient transfection of ChAT cDNA, neuroblastoma cells, which have no endogenous ChAT and either adhere to myotubes or not, failed to form functional synapses, and thus no evidence for release of ACh was detected. Stable neuroblastoma cell lines overexpressing ChAT accumulated ACh inside the cell, and slowly released ACh to the outside of the cell in a calcium-independent fashion. However, after co-culturing them with rat muscle cells, these transformed cells adhered to myotubes and ACh was secreted in a discrete fashion into the synaptic cleft efficiently in some neuroblastoma cell lines but rather inefficiently in another cell line. The results show that the latent secretion machinery of ChAT overexpressing neuroblastoma cells either is competent or possess defect(s) in ACh release.

Acetylcholine↗
PubMed1995

Coupling of the cloned mu-opioid receptor with the omega-conotoxin-sensitive Ca2+ current in NG108-15 cells.

Voltage-dependent Ca2+ currents were measured in NG108-15 neuroblastoma x glioma hybrid cells transformed to express the rat mu-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba2+ as charge carrier. A mu-opioid receptor-selective agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin caused significant inhibition of voltage-dependent Ca2+ currents in mu-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a delta-opioid receptor-selective agonist, [D-penicillamine2,D-penicillamine5]enkephalin, induced inhibition of voltage-dependent Ca2+ currents in both control and mu-receptor-transformed cells, which is mediated by the delta-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca2+ currents induced by [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin [D-penicillamine2,D-penicillamine5]enkephalin was reduced by pretreatment of the cells with pertussis toxin or omega-contoxin GVIA. These results indicate that the mu-opioid receptor expressed from cDNA functionally couples with omega-contoxin-sensitive N-type Ca2+ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.

Animals↗
PubMed1995

Functional expression of transporter for beta-lactam antibiotics and dipeptides in Xenopus laevis oocytes injected with messenger RNA from human, rat and rabbit small intestines.

A heterologous gene expression system, Xenopus laevis oocytes, was used to prove the intestinal absorption of various beta-lactam antibiotics mediated by an H(+)-dipeptide cotransport system in rat, rabbit and human small intestines. The microinjection of mRNA (messenger RNA) from rat intestine into Xenopus laevis oocytes led to significantly higher uptakes of p.o. active cephalosporins including zwitter-ionic derivatives (cephalexin, cephradine and cefadroxil) and dianionic derivatives (cefixime and ceftibuten) in comparison with oocytes injected with water, whereas the uptake of cefazolin, a parenterally administered derivative, was negligible in both mRNA- and water-injected oocytes. The uptake of cefadroxil was reduced significantly in the presence of dipeptide and various beta-lactam antibiotics, but not in the presence of an amino acid. After sucrose density gradient centrifugation of mRNA, the highest expression of transport activities of both cefadroxil and ceftibuten was observed in the same mRNA fraction with a size of 2.20 to 3.75 kilobases. mRNA-injected oocytes showed a marked pH-dependency in the uptakes of cefadroxil and ceftibuten, whereas water-injected oocytes exhibited only modes uptakes. The most stimulated uptakes of cefadroxil and ceftibuten were observed at an external pH of 5.5 and 5.0, respectively. Furthermore, injection of mRNA isolated from either rat rabbit or human small intestine into oocytes produced significantly higher uptake of cefadroxil and ceftibuten compared with those by oocytes injected with water. Thus, intestinal absorption of p.o. active beta-lactam antibiotics was confirmed to be mediated by an H+ gradient-dependent transport system across the brush-border membrane of rats, rabbits and humans. The carrier-protein for this process is likely a dipeptide transport system.

Animals↗
PubMed1994

Functional expression of intestinal dipeptide/beta-lactam antibiotic transporter in Xenopus laevis oocytes.

An intestinal active transport system specific to small peptides and peptide-like drugs such as beta-lactam antibiotics was functionally expressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal mucosal cells. The transport activity was evaluated by measuring the uptake of a tripeptide-like cephalosporin antibiotic, ceftibuten, which has high affinity for the intestinal peptide/H+ co-transporter and is resistant to peptidases. Ceftibuten transport in mRNA-injected oocytes was pH dependent (a proton gradient is the driving force), stereo selective (uptake of the cis-isomer of ceftibuten was about 4-fold higher than that of the trans-isomer), saturable and temperature dependent. Furthermore, various dipeptides showed cis-inhibitory and trans-stimulatory effects on the uptake of ceftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipeptides are transported by a common carrier protein. These results are in accordance with the functional properties of native proton-coupled peptide transporter previously clarified by studies with isolated intestinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the membrane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive beta-lactam antibiotic, [3H]benzylpenicillin, followed by SDS-PAGE analysis of the radiolabeled protein. Furthermore, an experiment with mRNA size-fractionated by sucrose density gradient centrifugation indicated that the peptide transporter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtained using a heterologous gene expression technique, confirm that intestinal absorption of beta-lactam antibiotics occurs through a carrier-mediated mechanism and show that biologically stable beta-lactam antibiotics can be useful probes for molecular analysis of intestinal peptide transporter.

Affinity Labels↗
PubMed1994

Distinct induction of c-fos mRNA in NG108-15 cells transfected with muscarinic m1 and m3 receptors.

The differences of intracellular signalling mechanisms between muscarinic acetylcholine m1 and m3 receptors, which are coupled with polyphosphoinositide turnover, were examined by using m1- and m3-transfected NG108-15 cells. The c-fos mRNA was induced by 1 mM acetylcholine peak at 60 min in both m1 and m3 cells. The c-fos induction in m1 cells was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), but was not inhibited by prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), suggesting that intracellular Ca2+ and calmodulin are involved in the induction. The c-fos induction in m3 cells was inhibited by BAPTA-AM and prolonged treatment with TPA, but was not influenced by W-7, suggesting that protein kinase C is mainly involved in m3-induced c-fos expression. Acetylcholine induced an increase in inositol phosphates and a transient increase in the intracellular concentration of Ca2+ in both m1 and m3 cells. Sustained stimulation of acetylcholine strongly increased the inositol monophosphate content in m3 cells, but that of inositol trisphosphate and inositol diphosphate in m1 cells. These results suggest that the difference between m1- and m3-induced c-fos mRNA induction mechanisms is due to the difference in respective properties in polyphosphoinositide turnover.

Acetylcholine↗
PubMed1994

Activation of inward current associated with M-potassium current inhibition in m1-muscarinic receptor-transformed NG108-15 cells by KST-5452, a novel cognition enhancer.

The electrophysiological effects of KST-5452 [3-(m-phenoxybenzylidene)-quinuclidine], an M1 muscarinic acetylcholine receptor (muscarinic AChR) binding compound, were studied in NG108-15 neuroblastoma x glioma hybrid cells transfected with m1 muscarinic AChR cDNA. Application of KST-5452 to m1-transformed NGPM1-27 cells elicited a sustained inward current associated with decreased conductance and reduced M-current relaxations at a holding potential of -20 mV. The KST-5452-induced responses were blocked by pirenzepine, suggesting that KST-5452 acts as a potent excitant via M1 muscarinic AChRs in brain neurons.

Acetylcholine↗
PubMed1994

Ca2+ influx gated by inositol-3,4,5,6-tetrakisphosphate in NIH/3T3 fibroblasts.

The functional role of three inositol tetrakisphosphate isomers on Ca2+ influx was examined by intracellular application in NIH/3T3 mouse fibroblasts. Infusion of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol-1,3,4,6- tetrakisphosphate, and inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) from the patch pipette into the cytoplasm produced a transient increase in cytoplasmic free Ca2+ concentration by fura-2 recording in extracellular Ca(2+)-dependent fashion. Stimulation of NIH/3T3 cells with bradykinin increased the levels of Ins(1,3,4,5)P4 and Ins(3,4,5,6)P4. These results suggest that mouse fibroblast cells possess a Ca2+ influx pathway gated with naturally occurring inositol tetrakisphosphates in response to stimulation with bradykinin.

3T3 Cells↗
PubMed1994

B2 bradykinin receptors in NG108-15 cells: cDNA cloning and functional expression.

Two distinct cDNAs encoding bradykinin receptors (BKRs) were cloned from NG108-15 neuroblastoma-glioma hybrid cells. One was identical with rat uterus B2 BKR, whereas the other one (mBKR) had 91% amino acid homology to the rat B2 BKR and 82% homology to human B2 BKR. Southern blot analysis and genomic DNA cloning revealed that mBKR is derived from the mouse genome. The mBKR, expressed in Xenopus oocytes and COS-7 cells, produced functional BKRs that exhibited the properties of smooth muscle type B2 BKR. These results suggest that both the rat and mouse B2 BKRs of the smooth muscle type are expressed in NG108-15 cells.

Amino Acid Sequence↗
PubMed1994

Ca2+ influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts.

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca2+ concentration ([Ca2+]i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P4-induced increase in DT cells depended upon extracellular Ca2+, and was enhanced by membrane hyperpolarization. Identical [Ca2+]i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P4 and Ins(1,3,4,5)P4. These results suggest that Ins(3,4,5,6)P4 may serve as a second messenger for continuous Ca2+ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.

3T3 Cells↗
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↗