Biomedical subjects

Yasunobu Okada

Publications and source records attributed to Yasunobu Okada.

At least 19 recordsLinked to original sources

PubMed2007

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells.

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2006

Role of acid-sensitive outwardly rectifying anion channels in acidosis-induced cell death in human epithelial cells.

Recently, a novel type of anion channel activated by extracellular acidification has been found in a variety of mammalian cell types. However, the role of this acid-sensitive outwardly rectifying (ASOR) anion channel is not known. In human epithelial HeLa cells, reduction in extracellular pH below 5 rapidly activated anion-selective whole-cell currents. The currents exhibited strong outward rectification, activation kinetics at positive potentials, low-field anion selectivity, and sensitivity to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. When outside-out patches were exposed to acidic bathing solution, single-channel events of the anion channel could be observed. The unitary conductance was 4.8 pS in the voltage range between -80 and +80 mV. The single-channel activity prominently increased with depolarization and was suppressed by DIDS or phloretin. After 1-h incubation in acidic solution (pH 4.5), a significant population of HeLa cells suffered from necrotic cell injury characterized by stainability with propidium iodide and lack of caspase-3 activation. Upon exposure to acidic solution, HeLa cells exhibited immediate, persistent swelling. Both the necrotic volume increase and cell injury induced by extracellular acidification were inhibited by DIDS or phloretin. Therefore, it is concluded that the ASOR anion channel is involved in the genesis of necrotic cell injury induced by acidosis in human epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2006

Dysfunction of regulatory volume increase is a key component of apoptosis.

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.

Apoptosis↗

PubMed2006

Normotonic cell shrinkage induced by Na+ deprivation results in apoptotic cell death in human epithelial HeLa cells.

Apoptosis is a major form of cell death that occurs in response to a variety of signals in both physiological and pathological situations. A hallmark of apoptosis is normotonic cell shrinkage, called apoptotic volume decrease (AVD), the process of which involves fluxes of K(+), Cl(-), and Na(+). Na(+) influx was suggested to be required in Fas-induced apoptosis in human Jurkat T cells, whereas Na(+) efflux was found to be associated with AVD and apoptosis in human HL-60 cells. Here we examined the effects of extracellular Na(+) deprivation on cell volume and viability in human epithelial HeLa cells. The incubation of HeLa cells in normotonic Na(+)-free Ringer solution resulted in persistent cell shrinkage after > or = 30 min and reduction in cell viability after > or = 1 h. After exposure to Na(+)-free solution for 5 h, a marked reduction in cell viability was found to be associated with an activation of caspase-3 without showing significant LDH release, indicating that the cells underwent apoptosis but not necrosis. Na(+) deprivation-induced cell shrinkage and apoptotic cell death were significantly inhibited by a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC) or of the reverse-mode operation of Na(+)/Ca(2+) exchanger (NCX), but not by a blocker of Na(+)/H(+) exchanger (NHE). Therefore it is concluded that Na(+) deprivation causes persistent cell shrinkage resulting from Na(+) efflux mainly via NKCC and NCX and thereafter leads to apoptotic death of HeLa cells. It is also suggested that normotonic cell shrinkage per se, if persistent, provides a sufficient condition for apoptosis induction.

Apoptosis↗

PubMed2006

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells.

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg(2+)- and Gd(3+)-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg(2+) and Gd(3+). The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca(2+) was removed or Gd(3+) was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca(2+) influx pathway, it plays an important role in cell volume regulation.

Cell Line↗

PubMed2006

Single-channel properties of volume-sensitive Cl- channel in ClC-3-deficient cardiomyocytes.

It is controversial whether the ClC-3 protein, which is one of the voltage-dependent chloride channel ClC family members, is a candidate for the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel per se or its regulator. Here, for the first time, we examined the single-channel properties of the VSOR Cl(-) channel in ventricular myocytes isolated from ClC-3-deficient mice. The single-channel current induced by cell swelling exhibited Cl(-) selectivity, mild outward rectification, and an intermediate unitary conductance (around 38 pS). A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibited the outward current. These single-channel properties were identical with those in ClC-3 expressing wild-type ventricular myocytes. These results indicate that the single-channel activity of the VSOR Cl(-) channel is independent of the expression of ClC-3 proteins in mouse ventricular myocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2006

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress.

Astrocytes release glutamate upon hyperexcitation in the normal brain, and in response to pathologic insults such as ischemia and trauma. In our experiments, both hypotonic and ischemic stimuli caused the release of glutamate from cultured mouse astrocytes, which occurred with little or no contribution of gap junction hemichannels, vesicle-mediated exocytosis, or reversed operation of the Na-dependent glutamate transporter. Cell swelling and chemical ischemia activated, in cell-attached membrane patches, anionic channels with large unitary conductance (approximately 400 pS) and inactivation kinetics at potentials more positive than +20 mV or more negative than -20 mV. These properties are different from those of volume-sensitive outwardly rectifying (VSOR) Cl- channels, which were also expressed in these cells and exhibited intermediate unitary conductance (approximately 80 pS) and inactivation kinetics at large positive potentials of more than +40 mV. Both maxi-anion channels and VSOR Cl- channels were permeable to glutamate with permeability ratios of glutamate to chloride of 0.21 +/- 0.07 and 0.15 +/- 0.01, respectively. However, the release of glutamate was significantly more sensitive to Gd3+, a blocker of maxi-anion channels, than to phloretin, a blocker of VSOR Cl- channels. We conclude that these two channels jointly represent a major conductive pathway for the release of glutamate from swollen and ischemia-challenged astrocytes, with the contribution of maxi-anion channels being predominant.

Animals↗

PubMed2006

Cell volume-sensitive chloride channels: phenotypic properties and molecular identity.

Cell volume regulation is essential for the survival of cells. After osmotic swelling, animal cells show a regulatory volume decrease by releasing intracellular K(+), Cl (-)and water. In most cell types, volume-regulatory Cl(-) efflux is induced by activation of electroconductive anion pathways. Among these volume-activated Cl(-) channels, the most important and specific is a volume-sensitive outwardly rectifying (VSOR) Cl(-) channel. The phonotypical properties have been well described. Extracellular application of anionic forms of ATP and glibenclamide give rise to voltage-dependent open-channel block of this channel, the fact suggesting that its outer vestibule and pore are larger and smaller, respectively, than the sizes of ATP and glibenclamide. Consistent with this prediction, the pore radius of VSOR Cl(-) channel (0.63 nm) which has been recently determined is slightly smaller than the radii of ATP and glibenclamide. The activities of VSOR Cl(-) channels are implicated not only in regulatory volume decrease but also in many other physiological or pathophysiological cell events including cell death induction. Despite their ubiquitous expression and physiological/ pathophysiological significance, there is still a paucity of the molecular information of the VSOR Cl(-) channel.

Adenosine Triphosphate↗

PubMed2006

Inhibition of hypertonicity-induced cation channels sensitizes HeLa cells to shrinkage-induced apoptosis.

Hypertonicity-induced cation channels (HICCs) are an effective mechanism of regulatory volume increase (RVI), which is a restoration process of cell volume after osmotic cell shrinkage, in HeLa cells. Since a reduction of cell size is a hallmark of programmed cell death, we tested whether a blockage of HICCs sensitizes HeLa cells to shrinkage-induced apoptosis by using proliferation assays, apoptosis assays, and patch-clamp recordings. Under control conditions, increasing osmolality up to 600 mosmol/kg-H2O had no detectable effect on either cell proliferation or apoptosis. With HICCs blocked by flufenamate and Gd3+, however, a significant reduction of proliferation and a stimulation of apoptosis were observed. Both effects exhibited virtually identical sensitivity profiles to osmotic stress as well as to flufenamate and Gd3+. Moreover, the observed concentration dependency of flufenamate and Gd3+ on proliferation and apoptosis was in excellent accordance with that on HICC inhibition. These results suggest that persistent cell shrinkage may function as a specific signal in the induction of apoptosis. In addition, they provide further evidence for the interplay of proliferation vs. apoptosis and the actual role that mechanisms of cell volume regulation do play in these processes.

Apoptosis↗

PubMed2005

Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins.

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

Adenosine Triphosphate↗

PubMed2005

Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.

Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.

Animals↗

PubMed2005

HCO(3)(-)-independent rescue from apoptosis by stilbene derivatives in rat cardiomyocytes.

Apoptosis of rat cardiomyocytes induced by staurosporine is prevented by a stilbene derivative (DIDS), which is a known blocker of both Cl(-)/HCO(3)(-) exchangers and Cl(-) channels. To clarify its target, staurosporine-induced activation of caspase-3, DNA laddering and cell death were examined in cultured rat cardiomyocytes. Removal of ambient HCO(3)(-), which minimizes the function of Cl(-)/HCO(3)(-) exchangers, failed to affect the preventive effect of DIDS on apoptosis. A carboxylate analog Cl(-) channel blocker, which does not block Cl(-)/HCO(3)(-) exchangers, also inhibited apoptotic events. Thus, rescue by DIDS of cardiomyocytes from apoptosis is mediated by blockage of Cl(-) channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2005

Volume-sensitive chloride channels in mouse cortical neurons: characterization and role in volume regulation.

Because persistent swelling causes cell damage and often results in cell death, volume regulation is an important physiological function in both neuronal and non-neuronal cells. Brain cell swelling has been observed not only in various pathological conditions but also during physiological synaptic transmissions. Volume-sensitive anion channels have been reported to play an important role in the regulatory volume decrease occurring after osmotic swelling in many cell types. In this study, using a two-photon laser scanning microscope and patch-clamp techniques, we found that mouse cortical neurons in primary culture exhibit regulatory volume decrease after transient swelling and activation of Cl- currents during exposure to a hypotonic solution. The regulatory volume decrease was inhibited by Cl- channel blockers or K+ channel blockers. Swelling-activated Cl- currents exhibited outward rectification, time-dependent inactivation at large positive potentials, a low-field anion permeability sequence, an intermediate unitary conductance and sensitivity to known blockers of volume-sensitive Cl- channels. Thus, it is concluded that the activity of the volume-sensitive outwardly rectifying Cl- channel plays a role in the control of cell volume in cortical neurons.

Animals↗

PubMed2005

ClC-3-independent sensitivity of apoptosis to Cl- channel blockers in mouse cardiomyocytes.

It has been shown that Cl-/HCO3- exchangers and Cl- channels, both of which are sensitive to stilbene derivatives, have essential roles in the mechanism of apoptosis induction. Staurosporine-induced apoptosis in neonatal mouse cardiomyocytes was prevented by a stilbene derivative, DIDS. To clarify whether Cl-/HCO3- exchangers or Cl- channels are targets of DIDS and whether ClC-3 is involved in the apoptotic process, staurosporine-induced reduction of cell viability, DNA laddering and caspase-3 activation were examined in cultured mouse ventricular myocytes derived from wild-type and ClC-3-deficient mice. Staurosporine-induced apoptosis and its DIDS sensitivity in ambient HCO3(-)-free conditions in which operation of Cl-/HCO3- exchangers is minimized were indistinguishable from when HCO3- was present. Apoptosis was also prevented by application of a non-stilbene-derivative Cl- channel blocker, NPPB, which cannot block Cl-/HCO3- exchangers. Cardiomyocytes derived from ClC-3-deficient mice similarly underwent apoptosis after exposure to staurosporine; moreover, apoptosis was prevented by application of DIDS or NPPB. Thus, we conclude that in cardiomyocytes, apoptosis is critically dependent on operation not of Cl-/HCO3- exchangers but of Cl- channels which are distinct from ClC-3.

Animals↗

PubMed2005

Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia-reperfusion in mouse cardiomyocytes.

Apoptosis of cardiomyocytes following ischemia and reperfusion is of clinical importance. However, little is known about the mechanism by which it is induced. Recently, essential roles of a Cl- channel whose activity triggers the apoptotic volume decrease and of reactive oxygen species (ROS) in activation of this channel have been identified in mitochondrion-mediated apoptosis. Therefore, in this study, involvement of Cl- channels and ROS in apoptosis was studied in primary mouse cardiomyocyte cultures subjected to ischemia-reperfusion. Apoptotic cell death as measured by caspase-3 activation, chromatin condensation, DNA laddering, and cell viability reduction was observed tens of hours after reperfusion but never immediately after ischemia. A non-selective Cl-channel blocker (DIDS or NPPB) rescued cells from apoptotic death when applied during the reperfusion, but not ischemia, period. Another blocker relatively specific to the volume-sensitive outwardly rectifying (VSOR) Cl-channel (phloretin) was also effective in protecting ischemic cardiomyocytes from apoptosis induced by reperfusion. A profound increase in intracellular ROS was detected in cardiomyocytes during the reperfusion, but not ischemia, period. Scavengers for ROS, H2O2 and superoxide all inhibited apoptosis induced by ischemia-reperfusion. Thus, it is concluded that the mechanism by which cardiomyocyte apoptosis is induced by ischemia-reperfusion involves VSOR Cl- channel activity and intracellular ROS production.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2004

Detecting ATP release by a biosensor method.

Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

Adenosine Triphosphate↗

PubMed2004

Sizing the pore of the volume-sensitive anion channel by differential polymer partitioning.

Partitioning of ethylene glycol and its polymeric forms into the pore of the volume-sensitive outwardly rectifying (VSOR) anion channel was studied to assess the pore size. Polyethylene glycol (PEG) PEG 200-300 (Rh = 0.27-0.53 nm) effectively suppressed the single-channel currents, whereas PEG 400-4000 (Rh = 0.62-1.91 nm) had little or no effect. Since all the molecules tested effectively decreased electric conductivity of the bulk solution, the observed differential effects between PEG 200-300 and PEG 400-4000 on the VSOR single-channel current are due to their limited partitioning into the channel lumen. The cut-off radius of the VSOR channel pore was assessed to be 0.63 nm.

Anions↗

PubMed2004

Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions.

It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin-luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate- and stilbene-derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (approximately 390 pS). The channel was selective to anions and showed significant permeability to ATP4- (PATP/PCl approximately 0.1) and MgATP2- (PATP/PCl approximately 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels.

Adenosine Triphosphate↗