Biomedical subjects

Yasunobu Okada

Publications and source records attributed to Yasunobu Okada.

12 recordsLinked to original sources

PubMed2003

Hypertonic activation of a non-selective cation conductance in HeLa cells and its contribution to cell volume regulation.

In whole-cell recordings on single HeLa cells, the hypertonic activation of a cation conductance with a selectivity ratio P(Na):P(Li):P(K):P(Cs):P(NMDG):P(Ca):P(Cl) of 1.00:0.86:0.84:0.56:0.10:0.07:0.15 was observed. This (non-selective) cation conductance was reduced to 59 and 30% of maximal stimulation by Gd(3+) and flufenamate, respectively, but it was insensitive to amiloride (with each compound applied at 100 microm/l). As was determined by the Coulter counter technique, the cation conductance was the main mechanism of regulatory volume increase (RVI) in HeLa cells. Whereas a significant contribution of Na(+)/H(+) antiport was also detectable, Na(+)-K(+)-2Cl(-) symport most likely did not contribute to RVI.

Cations↗

PubMed2003

Calcium-activated nonselective cationic channel in macula densa cells.

Patch-clamp experiments in cell-attached (c/a) and inside-out (i/o) configurations were performed to directly observe ionic channels in lateral membranes of macula densa (MD) cells from rabbit kidney. In the presence of 140 mM KCl in the pipette and normal Ringer solution in the bath, we repeatedly observed in c/a and in i/o configurations a 20- to 23-pS channel with a linear current-voltage (I-V) relationship reversing near 0 mV. Ionic replacement in the bath solution clearly indicated a cationic selectivity but with equal permeability for Na+ and K+. Single-channel kinetics was characterized by higher open probability at positive membrane potentials. In i/o experiments, elimination of bath Ca2+ (<or=1 microM) abolished channel activity in a reversible manner. This MD nonselective cationic channel was found to display a certain Ca2+ permeability because single-channel events could be detected when the pipette potential was very negative (-60, -80, and -100 mV) in the presence of 73 mM CaCl2 in the bath solution. The similarities between this channel and some channels of the transient receptor potential family suggest a possible role for this MD basolateral channel in controlling membrane potential and regulating Ca2+ entry during MD cell signaling.

Animals↗

PubMed2003

Upregulation of swelling-activated Cl- channel sensitivity to cell volume by activation of EGF receptors in murine mammary cells.

Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl- currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl- currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl- currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl- current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl- currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 micro M) was lower than that in control cells (31 micro M). However, the VSOR Cl- currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was markedly diminished by tyrphostin B46 but not significantly affected by tyrphostin AG1296. We thus conclude that the EGF receptor tyrosine kinase upregulates the activity of the VSOR Cl- channel, mainly by enhancing the volume sensitivity.

Algorithms↗

PubMed2003

Macula densa cell signaling involves ATP release through a maxi anion channel.

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl](L)) and transmit signals to the mesangial cellafferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch-clamp studies of macula densa cells, we identified an [NaCl](L)-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 microM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl](L), by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca(2+) concentration to extracellular application of ATP (EC(50) 0.8 microM). Importantly, we also found increases in cytosolic Ca(2+) concentration with elevations in [NaCl](L), when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y(2) receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

Adenosine Triphosphate↗

PubMed2003

Recovery from lactacidosis-induced glial cell swelling with the aid of exogenous anion channels.

Hypotonic challenge induces transient swelling in glial cells, which is typically followed by a regulatory volume decrease (RVD). In contrast, lactic acidosis (lactacidosis) induces persistent cell swelling in astrocytes without an accompanying RVD. In the present study, we studied the mechanisms by which lactacidosis interferes with normal volume regulation in rat astrocytic glioma C6 cells. Following exposure of C6 cells to a hypotonic challenge, a current was detected that exhibited properties consistent with those of volume-sensitive outwardly rectifying (VSOR) anion channels. When exposed to in vitro conditions designed to simulate lactacidosis, C6 cells failed to respond to hypotonic stress with an RVD, and VSOR anion currents were not activated. When added to C6 cells, an anion channel-forming protein purified from Helicobacter pylori, VacA, was found to form anion-selective channels in the plasma membrane, and the activity of the VacA channel was not affected by lactacidosis (pH 6.2). Cells preincubated with VacA and then exposed to lactacidotic conditions underwent transient swelling followed by RVD. In contrast, application of a cation ionophore, gramicidin, failed to inhibit lactacidosis-induced persistent cell swelling. From these results, we conclude that inhibition of a volume-sensitive anion channel contributes to persistent swelling induced by lactacidosis in glial cells. Introduction of anion channel activity into glial cells might provide a novel approach for treating cerebral edema, which is associated with lactacidosis in cerebral ischemia or head injury.

Acidosis, Lactic↗

PubMed2003

[Ionic mechanisms underlying the regulation of cell proliferation, differentiation and death].

Ion channels and transporters act as major components that regulate membrane excitability in neurons, muscles, and some secretory glands, but may also contribute to the regulation of proliferation, differentiation, and death in a greater variety of cells including non-excitable ones. The molecular basis of ionic mechanisms underlying the later regulation has been partly identified in the last several years and is a hot issue now. In this short review, some of the molecular mechanisms underlying these regulations and novel compounds acting on the mechanisms were introduced as exciting topics in this area. Several types of transient receptor potential (TRP), identified as Ca(2+)-permeable, non-selective cation channels, may play obligatory roles in functional complexes, which regulate multiple signal transduction pathways triggering proliferation, differentiation, or death of many cell types. In addition, the relation between Cl(-) pump activity and the induction of beta-amyloid protein toxicity for neuronal cell death in Alzheimer disease was described. Unique functions of H(+) channel and pump in osteoclasts in bone mineral homeostasis and remodeling were also discussed. Finally, topics about activation of specific types of Cl(-) channels and K(+) channels, which are responsible for the induction of apoptosis or proliferation in several types of cells, were introduced.

Alzheimer Disease↗

PubMed2002

Impaired activity of volume-sensitive anion channel during lactacidosis-induced swelling in neuronally differentiated NG108-15 cells.

Acidosis coupled to lactate accumulation, called lactacidosis, occurs in cerebral ischemia or trauma and is known to cause persistent swelling in neuronal and glial cells. It is therefore possible that mechanisms of cell volume regulation are impaired during lactacidosis. Here we tested this possibility using neuronally differentiated NG108-15 cells. These cells responded to a hypotonic challenge with osmotic swelling followed by a regulatory volume decrease (RVD) under physiological pH conditions in the absence of lactate. Under normotonic conditions, sustained cell swelling without subsequent RVD was induced by exposure to lactate-containing solution with acidic pH (6.4 or 6.2), but not with physiological pH (7.4). Under whole-cell patch-clamp, osmotic swelling was found to activate outwardly rectifying Cl(-) currents in cells exposed to control hypotonic solution. A Cl(-) channel blocker, NPPB, inhibited both RVD and the swelling-activated Cl(-) current. RVD and the volume-sensitive Cl(-) current were also markedly inhibited by lactacidosis (pH 6.4 or 6.2), but neither by application of lactate with physiological pH (7.4) nor by acidification without lactate (pH 6.2). RT-PCR analysis showed mRNA expression of two isoforms of proton-coupled monocarboxylate transporters, MCT1 and MCT8, in differentiated NG108-15 cells. Thus, we conclude that persistence of neuronal cell swelling under lactacidosis is coupled to an impairment of the activity of the volume-sensitive Cl(-) channel and to dysfunction of RVD. It is also suggested that the volume-sensitive Cl(-) channel is inhibited by intracellular acidification induced by MCT-mediated proton influx under lactacidosis.

Acidosis, Lactic↗

PubMed2002

IK channels are involved in the regulatory volume decrease in human epithelial cells.

Parallel activation of Ca(2+)-dependent K(+) channels and volume-sensitive Cl(-) channels is known to be responsible for KCl efflux during regulatory volume decrease (RVD) in human epithelial Intestine 407 cells. The present study was performed to identify the K(+) channel type. RT-PCR demonstrated mRNA expression of Ca(2+)-activated, intermediate conductance K(+) (IK), but not small conductance K(+) (SK1) or large conductance K(+) (BK) channels in this cell line. Whole cell recordings showed that ionomycin or hypotonic stress activated inwardly rectifying K(+) currents that were reversibly blocked by IK channel blockers [clotrimazole (CLT) and charybdotoxin] but not by SK and BK channel blockers (apamin and iberiotoxin). Inside-out recordings revealed the existence of CLT-sensitive single K(+)-channel activity, which exhibited an intermediate unitary conductance (30 pS at -100 mV). The channel was activated by cytosolic Ca(2+) in inside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin or iberiotoxin. Thus we conclude that the IK channel is involved in the RVD process in these human epithelial cells.

Action Potentials↗

PubMed2002

Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

Adenosine Triphosphate↗

PubMed2002

Regulation of an ATP-conductive large-conductance anion channel and swelling-induced ATP release by arachidonic acid.

Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume-dependent ATP-conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling-induced ATP release in the physiological concentration range with K(d) of 4- 6 microM. The former effect observed in the whole-cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate-metabolizing oxygenases, and by the observation that they were mimicked by cis-unsaturated fatty acids, which are not substrates for oxygenases. A membrane-impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non-charged arachidonate analogues as well as trans-unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonate's negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown (K(d) of 4- 5 microM) and reduced unitary conductance (K(d) of 13-14 microM) without affecting voltage dependence of open probability. ATP(4-)-conducting inward currents measured in the presence of 100 mM ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling-induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells.

Adenosine Triphosphate↗

PubMed2002

LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death.

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.

Animals↗

PubMed2002

Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.

Recently, multiphoton excitation fluorescence microscopy has been developed that offers important advantages over confocal imaging, particularly for in vivo visualization of thick tissue samples. We used this state-of-the-art technique to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney. This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal hemodynamics. We report, for the first time, on high-resolution three-dimensional morphology and Z-sectioning through isolated, perfused kidney glomeruli, tubules, and JGA. Time-series images show how alterations in tubular fluid composition cause striking changes in single-cell volume of the unique macula densa tubular epithelium in situ and how they also affect glomerular filtration through alterations in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. This imaging approach to the study of isolated, perfused live tissue with multiphoton microscopy may be applied to other biological systems in which multiple cell types form a functionally integrated syncytium.

Angiotensin II↗