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M Sokabe

Publications and source records attributed to M Sokabe.

At least 19 recordsLinked to original sources
PubMed2003

In vitro reconstitution of signal transmission from a hair cell to the growth cone of a chick vestibular ganglion cell.

Signal transmission from a chick hair cell to the growth cone of a vestibular ganglion cell was examined by placing an acutely dissociated hair cell on the growth cone of a cultured vestibular ganglion cell. Electrical stimuli were applied to the hair cell while monitoring the intracellular Ca(2+) concentration ([Ca(2+)](i)) at the growth cone or recording whole-cell currents from the vestibular ganglion cell. Electrical stimulation of the hair cell induced [Ca(2+)](i) increases at the growth cone and inward currents in the vestibular ganglion cell. The [Ca(2+)](i) increase was blocked by 6-cyano-7-nitroquinoxaline (CNQX) (10 microM) but not by 2-amino-5-phosphonovaleric acid (APV; 50 microM). Glutamate (100 nM-300 microM) applied to the vestibular ganglion cell by the Y-tube method induced inward currents which were also antagonized by CNQX, but not by APV. These results indicate that the electrical stimulation of a hair cell induced glutamate or glutamate like agent release from the hair cell, which activated non-N-methyl-D-aspartate receptors at the growth cone of the vestibular ganglion cell, followed by action potentials and [Ca(2+)](i) elevation in the vestibular ganglion cell. This is the first demonstration of in vitro reconstitution of functional signal transmission from a hair cell to a vestibular ganglion cell.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2001

Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation.

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd(3+) or extracellular Ca(2+) removal which may inhibit intracellular Ca(2+) increase caused by the activation of cation selective (Ca(2+)-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation.

Amino Acid Substitution↗
PubMed2001

SA channel mediates superoxide production in HUVECs.

Superoxide production in response to cyclic stretch (1 Hz, 20% in length) was investigated in human umbilical vein endothelial cells (HUVECs). The basal production of superoxide without stretch increased gradually, while the production of superoxide with stretch increased significantly as compared to that without stretch and it became significant 80 min after the onset of cyclic stretch (P<0.05, n=8-14). The superoxide production increased in a stretch-dependent manner and became significant when stretch was more than 10% (p<0.05, n=11-16). To investigate the involvement of SA channel, we added Gd3+ or EGTA in the reaction solution and examined the stretch-induced superoxide production. In cells stretched in the presence of 20 microM Gd3+, the stretch-induced superoxide production was significantly inhibited (at 120 min, p<0.05, n=8-18). The cyclic stretch-induced superoxide production was also significantly inhibited by the removal of extracellular Ca2+ with 5 mM EGTA (at 120 min, p<0.05, n=8-18). Neither the application of Gd3+ nor the removal of extracellular Ca2+ significantly changed the basal production of superoxide. These data suggest that the stretch-induced superoxide production increases in time- and stretch-dependent manner and that the stretch-induced superoxide production in HUVECs is regulated by Ca2+ influx through SA channels.

Calcium↗
PubMed2001

Calcium-sensitive nonselective cation channel identified in the epithelial cells isolated from the endolymphatic sac of guinea pigs.

We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 +/- 1.3 pS (n = 8) in our standard solution. The relative ionic permeability of the channel was in the order K+ = Na+ > Ca2+ >> Cl-. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 microm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 microm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 microm to 10 microm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.

Adenosine Triphosphate↗
PubMed2001

Molecular design and synthesis of artificial ion channels based on cyclic peptides containing unnatural amino acids.

A series of novel cyclic peptides composed of 3 to 5 dipeptide units with alternating natural-unnatural amino acid units, have been designed and synthesized, employing 5-(N-alkanoylamino)-3-aminobenzoic acid with a long alkanoyl chain as the unnatural amino acid. All cyclic peptides with systematically varying pore size, shape, and lipophilicity are found to form ion channels with a conductance of ca. 9 pS in aqueous KCl (500 mM) upon examination by the voltage clamp method. These peptide channels are cation selective with the permeability ratio P(Cl(-))/P(K(+)) of around 0.17. The ion channels formed by the neutral, cationic, and anionic cyclic peptides containing L-alanine, L-lysine, and L-aspartate, respectively, show the monovalent cation selectivity with the permeability ratio P(Na(+))/P(K(+)) of ca. 0.39. On the basis of structural information provided by voltage-dependent blockade of the single channel current of all the tested peptides by Ca(2+), we inferred that each channel is formed from a dimer of the peptide with its peptide ring constructing the channel entrance and its alkanoyl chains lining across the membrane to build up the channel pore. The experimental results are consistent with an idea that the rate of ion conduction is determined by the nature of the hydrophobic alkanoyl chain region, which is common to all the channels.

Amino Acid Sequence↗
PubMed2001

Functional connections between the HVC and the shelf of the zebra finch revealed by real-time optical imaging technique.

Auditory-motor interaction is essential for the understanding of avian song learning and maintenance. The HVC is thought to be the most important station for auditory-motor integration in the avian song system. However, little is known about where and how auditory inputs are conveyed into the HVC. The shelf, a multi-auditory recipient region located along the ventral border of the HVC, is thought to be a possible source of auditory inputs to the HVC. Here, we investigated the functional neural connections between the HVC and surrounding area of the HVC including the shelf in zebra finch brain slices, using the real-time optical recording technique. Unexpectedly electrical stimulation of the shelf region did not induce signal propagation into the HVC, but stimulation within the HVC propagated strong signals into the shelf. These results suggest that the shelf is the region that receives output signals from the HVC, rather than a major source of auditory inputs to the HVC.

2-Amino-5-phosphonovalerate↗
PubMed2001

Protamine augments stretch induced calcium increase in vascular endothelium.

1. Human umbilical vein endothelial cells cultured on a transparent silicone chamber were subjected to a short stretch pulse (ca. 1 s, 5-25% stretch) of their substrate and following increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured by fluorescence intensity ratiometry using fura-2. 2. In response to mechanical stretch, the cells in HEPES buffered saline exhibited a Ca(2+) transient in a dose dependent way. The response was completely dependent on external Ca(2+) and inhibited by gadolinium (Gd(3+)), suggesting that it was mediated by the activation of a stretch activated cation channel (SACatC). 3. Interestingly, the stretch induced Ca(2+) transient was significantly augmented in the presence of basic polypeptide, protamine. This augmented Ca(2+) response was inhibited neither by Gd(3+) nor by the deprivation of external Ca(2+), indicating that the SACatC is not responsible for this phenomenon. 4. In contrast, this augmentation was inhibited by depletion of intracellular Ca(2+) stores with thapsigargin or by the pretreatment with phospholipase inhibitors such as U73122 and manoalide. 5. These results suggest the presence of a metabotropic mechanoreceptor distinct from the SACatC in vascular endothelium. This augmented [Ca(2+)](i) increase may contribute to the vasodilating response induced by protamine during heparin neutralization in cardiac surgery.

Calcium↗
PubMed2001

Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy.

Human umbilical vein endothelial cells were stained with FITC-labeled anti-beta(1) integrin antibody and plated on a glass cover slip to elucidate the mechanism of integrin clustering during focal contact formation. The process of integrin clustering was observed by time-lapse total-internal-reflection fluorescence microscopy, which can selectively visualize the labeled integrins at the basal surface of living cells. The clustering of integrins at focal contacts started at 1 hour after plating and individual clusters kept growing for approximately 6 hours. Most integrin clusters (approximately 80%) elongated towards the cell center or along the cell margin at a rate of 0.29+/-0.24 microm minute(-1). Photobleaching and recovery experiments with evanescent illumination revealed that the integrins at the extending tip of the clusters were supplied from the intracellular space. Simultaneous time-lapse imaging of exocytosis of integrin-containing vesicles and elongating focal contacts showed that most exocytosis occurred at or near the focal contacts followed by their elongation. Double staining of F-actins and integrins demonstrated that stress fibers were located near the integrin clusters and that intracellular punctate integrins were associated with these stress fibers. These results suggest that the clustering of integrins is mediated by actin-fiber-dependent translocation of integrins to the extending tip of focal contacts.

Actins↗
PubMed2000

Stretch-induced morphological changes of human endothelial cells depend on the intracellular level of Ca2+ rather than of cAMP.

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine 3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca2+]i. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs. Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca2+]i resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca2+]i and resulted in cell shape changes. On the other hand, 2'5'-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca2+]i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca2+]i rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca2+ rather than cAMP.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Extracellular ATP-dependent activation of plasma membrane Ca(2+) pump in HEK-293 cells.

1. It is well known that extracellular ATP (ATP(o)) elevates the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) influx or mobilizing Ca(2+) from internal stores via activation of purinoceptors in the plasma membrane. This study shows that ATP(o) also activates the plasma membrane Ca(2+) pumps (PMCPs) to bring the elevated [Ca(2+)](i) back to the resting level in human embryonic kidney-293 (HEK-293) cells. 2. The duration of ATP(o)-induced intracellular Ca(2+) transients was significantly increased by PMCP blockers, La(3+) or orthovanadate. In contrast, replacement of extracellular Na(+) with NMDG(+), a membrane-impermeable cation, had no significant effect on duration, thus suggesting that Na(+)/Ca(2+) exchangers do not participate in the ATP(o)-induced Ca(2+) transient. 3. A rapid and significant decrease in [Ca(2+)](i), which was not dependent on extracellular Na(+), was induced by ATP(o) in cells pretreated with thapsigargin (TG). This decrease was blocked by orthovanadate, indicating that it was caused by PMCPs rather than sarco/endoplasmic reticulum Ca(2+) pumps (SERCPs). 4. UTP and ATPgammaS also caused a decrease in [Ca(2+)](i) in cells pretreated with TG, although they were less effective than ATP. The effect of UTP implies the involvement of both P2Y(1) and P2Y(2) receptors, while the effect of ATPgammaS implies no significant role of ectophosphorylation and agonist hydrolysis in the agonist-induced [Ca(2+)](i) decreases. 5. These results point to a role of PMCPs in shaping the Ca(2+) signal and in restoring the resting [Ca(2+)](i) level to maintain intracellular Ca(2+) homeostasis after agonist stimulation.

Adenosine Triphosphate↗
PubMed2000

Molecular cloning and characterization of a putative cyclic nucleotide-gated channel from Drosophila melanogaster.

We have cloned a cDNA encoding a putative cyclic nucleotide-gated (CNG) channel from Drosophila melanogaster. The N-terminal half of the predicted protein, designated as CNGL, shows a high degree of sequence similarity with the known CNG channel proteins. CNGL has a long hydrophilic C-terminal stretch that is absent in other CNG channels. Northern blot analysis revealed that the messenger RNA (mRNA) corresponding to the size of the cloned cDNA is expressed in Drosophila heads. Immunolocalization studies showed that CNGL is expressed in the brain, including the medulla, lobulla and lobulla plate, the antennal lobe glomeruli, and mushroom bodies. These results suggest a possible role of the putative CNGL channel in the processing of visual and olfactory information in the nervous system of Drosophila.

Amino Acid Sequence↗
PubMed2000

P2X(4) receptors mediate ATP-induced calcium influx in human vascular endothelial cells.

ATP induces Ca(2+) influx across the cell membrane and activates release from intracellular Ca(2+) pools in vascular endothelial cells (ECs). Ca(2+) signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca(2+) influx in ECs have not been thoroughly clarified. Here we demonstrate evidence that a P2X(4) receptor for an ATP-gated cation channel is predominantly expressed in human ECs and is involved in the ATP-induced Ca(2+) influx. Northern blot analysis distinctly showed the expression of P2X(4) mRNA in human ECs cultured from the umbilical vein, aorta, pulmonary artery, and skin microvessels. Competitive PCR revealed that P2X(4) mRNA expression was much higher in ECs than was the expression of other subtypes, including P2X(1), P2X(3), P2X(5), and P2X(7). Treatment of ECs with antisense oligonucleotides designed to target the P2X(4) receptor decreased the P2X(4) mRNA and protein levels to approximately 25% of control levels and markedly prevented the ATP-induced Ca(2+) influx.

Adenosine Triphosphate↗
PubMed1999

Attachment of growth cones on substrate observed by multi-mode light microscopy.

Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rhodamine B). Dynamic changes of the cell substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed.

Animals↗
PubMed1999

Impairments of long-term potentiation in hippocampal slices of beta-amyloid-infused rats.

In this study, we investigated the neuronal activity of hippocampal slices from the beta-amyloid protein-infused (300 pmol/day for 10-11 days) rats using the extracellular recording technique. Perfusion of nicotine (50 microM) reduced the amplitude of electrically evoked population spikes in the CA1 pyramidal cells of the vehicle control rats, but not in those of the beta-amyloid protein-infused rats, suggesting the impairment of nicotinic signaling in the beta-amyloid protein-infused rats. Long-term potentiation induced by tetanic stimulations in CA1 pyramidal cells, which was readily observed in the vehicle control rats, was also impaired in the beta-amyloid protein-infused rats. Nicotinic blockade by adding hexamethonium into the perfused solution inhibited long-term potentiation induction. Taken together, our previous and present results suggest that beta-amyloid protein infusion impairs the signal transduction mechanisms via nicotinic acetylcholine receptors. This dysfunction may be responsible, at least in part, for the impairment of long-term potentiation induction and may lead to learning deficits.

Amyloid beta-Peptides↗
PubMed1999

Sex differences in the vocal motor pathway of the zebra finch revealed by real-time optical imaging technique.

Male zebra finches sing, whereas female zebra finches do not. To elucidate the neural mechanisms underlying sexual dimorphism in song behavior, the spatio-temporal properties of neural activity in the vocal motor pathway of the zebra finch were examined in sliced brain preparations using a real-time optical recording technique. Electrical stimulation to higher vocal center (HVC) fibers induced within 20 ms neural activities in the robust nucleus of the archistriatum (RA) of both male and female finches, although the amplitude was smaller and the latency was greater in females than in males. Bicuculline, a GABA(A) receptor antagonist, induced a robust activity in female RA, but had little effect in males. This suggests that neural connections from HVC to RA in female zebra finches are inhibited by GABAergic inputs. The results provide first evidence that an inhibitory neurotransmitter is involved in the sex difference in the motor vocal pathway of song birds.

Animals↗
PubMed1999

Molecular identification of a eukaryotic, stretch-activated nonselective cation channel.

Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Mid1 in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Mid1 acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Mid1 appears to be a eukaryotic, SA Cat channel.

Amino Acid Sequence↗
PubMed1999

Structure-function study on a de novo synthetic hydrophobic ion channel.

Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) >> Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore.

Binding Sites↗