Biomedical subjects

H Tohda

Publications and source records attributed to H Tohda.

At least 37 records · Page 2Linked to original sources

PubMed1994

Cl- regulation of a Ca(2+)-activated nonselective cation channel in beta-agonist-treated fetal distal lung epithelium.

Nonselective cation (NSC) channels have been identified in the apical membrane of fetal distal lung epithelium (FDLE). However, their physiological role in Na+ transport is uncertain. Because terbutaline, a beta 2-agonist, increases Na+ transport by FDLE, we studied its effect and selected signal transduction mechanisms on NSC channel activity. Using patch-clamp and single-cell imaging techniques, we found that terbutaline activated the NSC channel by 1) increasing its sensitivity to cytosolic Ca2+ concentration ([Ca2+]c) by 100- to 1,000-fold, 2) increasing [Ca2+]c from 35 nM to 3.3 microM, 3) producing a dependency of the NSC channel activity on the cytosolic Cl- concentration ([Cl-]c) at a physiological [Ca2+]c, and 4) inducing a reduction in the [Cl-]c from 45 to 25 mM, which directly activates the beta 2-treated NSC channel. These observations indicate that a beta 2-agonist physiologically activates an amiloride-blockable NSC channel in FDLE through an increase in its sensitivity to [Ca2+]c, resulting in the development of a [Cl-]c dependency at a physiological [Ca2+]c associated with both an increase in [Ca2+]c and a reduction in [Cl-]c. A development of the [Cl-]c dependency and a reduction in [Cl-]c act as a second messenger of the beta-agonist signal transduction pathway in this Na(+)-transporting epithelium.

Adrenergic beta-Agonists↗

PubMed1994

Endotoxin-stimulated alveolar macrophages impair lung epithelial Na+ transport by an L-Arg-dependent mechanism.

The Na+ transport function of alveolar epithelium represents an important mechanism for air space fluid clearance after acute lung injury. We studied the effect of endotoxin-stimulated rat alveolar macrophages on lung epithelial ion transport and permeability in vitro. Cultured rat distal lung (alveolar) epithelial monolayers incubated with both endotoxin and macrophages demonstrated a 75% decline in transepithelial resistance and a selective 60% reduction in amiloride-sensitive short-circuit current (Isc). Single-channel patch-clamp analysis demonstrated a 60% decrease in the density of 25-pS nonselective cation (NSC) channels on the apical membrane of epithelium exposed to both endotoxin and macrophages. A concurrent reduction in epithelial F-actin content suggested a role for actin depolymerization in mediating this effect. Incubation of cocultures with the methylated L-arginine (Arg) derivative NG-monomethyl-L-arginine prevented the reduction in epithelial Isc, as did substitution of L-Arg with D-Arg or incubation in L-Arg-free medium. Furthermore, the stable and products of Arg metabolism were found to have no effect on epithelial ion transport. These studies show that endotoxin-stimulated alveolar macrophages impair distal lung epithelial ion transport by an L-Arg-dependent mechanism by inactivating amiloride-sensitive 25-pS NSC channels. This may represent a novel mechanism whereby local inflammatory cells regulate lung epithelial ion transport. This could affect the ability of the lung to clear fluid from the air space.

Adenine↗

PubMed1994

Antidiuretic hormone-responding nonselective cation channel in distal nephron epithelium (A6).

Arginine vasotocin (AVT, 70 mU/ml) added from the basolateral side transiently activated a nonselective cation (NSC) channel with a single-channel conductance of 28.5 pS and almost identical selectivity for Na+ and K+ in the apical membrane of distal nephron cells (A6) cultured on permeable supports for 10-12 days in media containing 10% fetal bovine serum without supplemental aldosterone. The open probability (Po) of the NSC channel at the apical resting membrane potential in cell-attached patches was approximately 0.09 and increased when the apical membrane depolarized. The Po of the NSC channel was decreased by a rise in cytosolic Ca2+ concentration within a range of 30 nM-1 microM but not affected by cytosolic pH within a range of 6-8. The channel was activated by the application of negative pressure (10-60 cmH2O) into the patch pipette. Gadolinium (2 microM), an inhibitor of stretch-activated channels, decreased the Po by 40%. This blocking action of gadolinium was more effective after the channel was activated by stretch, i.e., 2 microM gadolinium decreased the Po by 70% when a negative pressure (60 cmH2O) was applied into the patch pipette. Amiloride (10 and 100 microM) showed a blocking action on the channel only when the NSC channel was activated by stretch.

Amiloride↗

PubMed1994

The stimulatory action of vasopressin on single Cl- channels in the apical membrane of distal nephron epithelium (A6).

A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl- channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without arginine vasopressin (AVP) pretreatment, the 3 pS Cl- channel, which was Ca(2+)-activated and had an outward rectification, was more frequently observed than the 8 pS Cl- channel, which had a linear current-voltage relationship. AVP increased the 3 pS channel's single channel conductance through an increase in the Ca(2+)-sensitivity about 100-fold and the open probability (Po) without significantly changing the Po or conductance of the 8 pS Cl- channel. On the other hand, AVP did not affect the number of the 3 pS Cl- channel, but increased the number of 8 pS Cl- channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., 1) increases the Po and single channel conductance of 3 pS Cl- channels and 2) increases the number of 8 pS Cl- channels.

Animals↗

PubMed1994

Cytosolic [Cl-] regulates Na+ absorption in fetal alveolar epithelium?: roles of cAMP and Cl- channels.

We studied the regulatory mechanism of Na+ absorption in fetal distal lung epithelium (FDLE), using patch clamp and single cell imaging techniques. A beta 2-agonist activated a 28 pS non-selective cation channel (NSCC) by: 1) producing a dependency of the NSCC activity on the cytosolic [Cl-] ([Cl-]c); 2) inducing a reduction in the [Cl-]c from 45 to 25 mM which directly activated the beta 2-treated NSCC. The beta 2-agonist-induced decrease in [Cl-]c was caused by activation of Ca(2+)-activated K+ channel and cAMP-activated Cl- channel. A development of the [Cl-]c-dependency and a reduction in [Cl-]c act as a second messenger of the beta 2 agonist signal transduction pathway in this Na+ transporting epithelium.

Action Potentials↗

PubMed1994

The crystalline structure of pancreatic calculi.

The present study was carried out to investigate the ultrastructure and composition of crystals of pancreatic calculi. Microradiography showed that they were irregularly mineralized with radial or stratified zones of low mineralization. The crystals were identified as calcite composed of CaCO3 in X-ray and electron diffraction studies. Scanning electron microscopy (SEM) revealed that the calculi consist of aggregations of rhombohedral, fang-shaped, botryoidal, foliaceous, and plate-shaped crystals. Some of them showed signs of partial dissolution on their surface. Transmission electron microscopy (TEM) showed crystals of various sizes and shapes either solitary or in clumps. The direct measurement of crystalline lattice intervals obtained from each crystal was 0.386 nm in accordance with the diffraction data. Exocrine pancreatic cell debris are present in pancreatic calculus matrices.

Calculi↗

PubMed1994

High resolution electron microscopic observation of hydroxyapatite in tooth crystals.

Structure images taken with a high resolution electron microscope with a resolution of 0.18 nm revealed that the atomic arrangement in tooth-enamel hydroxyapatite (HA) corresponds with the calculated image using crystallographic data from mineral HA (Ca10(PO4)6(OH)2). Direct information on the two types of Ca and the OH- groups in the HA crystals was obtained from the observed image through [001] projection. The established correspondence between the observed image contrast and the arrangements of the columnar Ca, the screw-axis Ca, and the OH- groups suggests that HREM observations are useful to identify defects in HA crystals.

Crystallography, X-Ray↗

PubMed1993

Effects of vasopressin on single Cl- channels in the apical membrane of distal nephron cells (A6).

We have investigated how two types of Cl- channels found in sodium transporting epithelium are regulated by arginine vasopressin (AVP). A6 cells cultured on permeable supports for 10 to 14 days have two types of Cl- channels in the apical membrane that have single channel conductances of 3 and 8 pS. In cells without AVP pretreatment, the 3 pS Cl- channel was more frequently observed than the 8 pS Cl- channel. AVP increased the open probability (Po) and single channel conductance of the 3 pS Cl- channel without significantly changing the Po or conductance of the 8 pS Cl- channels. On the other hand, AVP did not affect the number of the 3 pS Cl- channel, but increased the number of 8 pS Cl- channels. These observations suggest that AVP has two different pathways to increase apical membrane chloride conductance in distal nephron A6 cells; i.e., (1) increases the Po and single channel conductance of 3 pS Cl- channels and (2) increases the number of 8 pS Cl- channels.

Animals↗

PubMed1993

Okadaic acid, a protein phosphatase inhibitor, induces sister-chromatid exchanges depending on the presence of bromodeoxyuridine.

Okadaic acid (OA), a potent tumor promoter and an inhibitor of protein phosphatase 1 and 2A, induced sister-chromatid exchanges (SCEs) in human lymphoblastoid cells and Chinese hamster ovary cells at low concentrations of 2-10 nM, when the cells were grown for two cell cycles in the presence of OA and bromodeoxyuridine (BrdUrd). Prolonged treatment with OA prior to addition of BrdUrd did not induce SCEs, indicating an essential role of BrdUrd. A similar important role of BrdUrd in SCE induction has been reported in the cases of benzamide (BA) (Natarajan et al., 1981) and camptothecin (CPT) (Zhao et al., 1992), which are inhibitors of poly(ADP-ribose)polymerase and DNA topoisomerase I (topo I), respectively. Unlike many DNA-damaging agents, they are required to be present during S phase along with BrdUrd in the medium and/or in the parental DNA as BrdUMP. Thus OA, like BA and CPT, is a new type of SCE inducer. Exposing cells to a combined treatment with OA, BA and CPT, a significantly higher level of SCEs was induced than that expected if the numbers of SCE caused by these three inhibitors were additive, while no such synergistic increase was seen in every combination of two agents. Since both phosphorylation and poly(ADP-ribosyl)ation have been known to modify topo I activity, the results suggest a common involvement of topo I for SCE formation by OA, BA and CPT. In addition to SCE induction, OA resulted in an increase of mitotic cells which were characterized by a marked chromosome condensation. OA also induced chromosome fragmentation/pulverization in human lymphoblastoid cells and fragmented nuclei in Chinese hamster cells.

Animals↗

PubMed1993

Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis.

The adherence of Porphyromonas gingivalis to cell surfaces of periodontal tissues may play an important role in its pathogenicity. In this study, 12 strains of P. gingivalis, including both invasive and noninvasive strains, were investigated for adherence to gingival ligament components. The test strains of P. gingivalis adhered to collagen, fibronectin, and laminin to significantly different degrees. An overall positive correlation was noted between hydrophobicity and the number of cells that attached to collagen coated on hydroxyapatite beads. Invasive strains had low hydrophobicity and bound less to collagen than did noninvasive strains that possessed strong hydrophobic properties. 3H-fimbriae extracted from P. gingivalis were found to attach to collagen-coated hydroxyapatite. The fimbriae extracted from noninvasive strains bound strongly to collagen, whereas invasive strains' fimbriae with low hydrophobicity bound weakly to collagen or saliva-coated hydroxyapatite. These data suggest that 1) fimbriae play an important role in colonization through their hydrophobic activity; 2) fimbriae of noninvasive strains are associated with the major adhesin for attachment to gingival tissue, whereas fimbriae of invasive strains are weakly involved in adherence; and 3) there is no correlation between colonization and the invasiveness of P. gingivalis.

Bacterial Adhesion↗

PubMed1993

5-(N-Ethyl-N-isopropyl)amiloride sensitive Na+ currents in intact fetal distal lung epithelial cells.

To determine whether primary cultures of rat fetal distal lung epithelium (FDLE) possessed L-type Na+ channels on their plasma membrane we performed experiments with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and other amiloride analogs. Short-circuit current (Isc) was decreased by the apical application of amiloride and benzamil, but was unaffected by 10 microM dimethylamiloride (DMA). EIPA decreased Isc when added to either the apical or basal sides. Greatest effects were seen with bilateral EIPA, where half-maximal effects occurred in the micromolar range. Measurements of intracellular pH with the fluorescent dye BCECF demonstrated that DMA impaired (IC50 = 71 nM) the ability of FDLE to recover from intracellular acidification. Nystatin perforated patch clamp techniques showed that FDLE had nonrectifying Na+ currents but no detectable Cl- currents. The whole-cell currents were reversibly decreased by 20 microM concentrations of EIPA, benzamil, and amiloride but were unaffected by 20 microM DMA. These studies indicate that there are EIPA-sensitive Na+ conductances in intact FDLE and suggest the presence of L-type Na+ conductances on their apical membrane and EIPA-sensitive K+ channels on the basolateral membrane.

Adenine↗

PubMed1992

Cytosolic Ca(2+)-induced modulation of ion selectivity and amiloride sensitivity of a cation channel and beta agonist action in fetal lung epithelium.

The cytosolic Ca2+ concentration ([Ca2+]i) affects many cell functions, including the modulation of ion channel activity. In this study patch clamp experiments using primary cultures of fetal distal lung epithelium (FDLE) demonstrated that the elevation of [Ca2+]i modulated a 25pS amiloride-blockable non selective cation (NSC) channel's ion selectivity and sensitivity to amiloride. An elevation of [Ca2+]i from 0.1 microM to 1mM both increased open probability (Po) and decreased the ratio of the permeability to Na to the permeability to K (PNa/PK) from 1.96 +/- 0.11 (SEM, n = 6) to 0.88 +/- 0.04 (n = 6). Within the range of [Ca2+]i from 0.1 microM to 100 microM amiloride (0.5 microM) decreased Po, however amiloride (0.5 microM) no longer affected Po of the NSC channel when [Ca2+]i was increased to 1mM under physiologic membrane potentials. A beta adrenergic agonist (terbutaline, 10 microM) increased Po in cell-attached patches from almost 0 (Po less than 0.01; n = 9) to 0.39 +/- 0.09 (n = 9) and [Ca2+]i from 40 +/- 6nM (n = 9) to more than 1 microM. This suggested that amiloride-blockable NSC channel activity and ion permeability are modulated by changes in [Ca2+]i near physiologic membrane potentials and a beta adrenergic agonist increases [Ca2+]i to more than 1 microM (unlike other epithelial including adult alveolar cells) which is associated with activation the NSC channel.

Amiloride↗

PubMed1992

Effects of insulin and tyrosine kinase inhibitor on ion transport in the alveolar cell of the fetal lung.

We studied the effect of insulin and lavendustin-A (a tyrosine kinase inhibitor) on the short-circuit current (ISC) of primary cultures of fetal distal rat lung epithelium (FDLE). Insulin (2 microM) on the basolateral side of the monolayer increased ISC from 5.76 +/- 0.83 microA/cm2 (SEM, n = 7) to 7.23 +/- 1.00 microA/cm2 (p less than 0.01) under control conditions, and from 1.00 +/- 0.31 microA/cm1 to 1.53 +/- 0.34 microA/cm2 (p less than 0.05, n = 4) when amiloride (10 microM) was present on the apical side of the monolayer. Thus insulin increased both the amiloride-sensitive and insensitive ISC with the insulin-induced increase in ISC in the absence of amiloride (1.47 +/- 0.22 microA/cm2, n = 7) being significantly larger than that in the presence of 10 microM amiloride (0.53 +/- 0.14 microA/cm2, n = 4; p less than 0.025). Insulin's effect reached steady state in 1 hr. Lavendustin-A (10 microM), a tyrosine kinase inhibitor, applied to the apical side of the monolayer attenuated but did not completely block insulin's ability to increase in ISC; i.e., insulin increased ISC in lavendustin-A treated monolayers (0.63 +/- 0.09 microA/cm2, n = 5; p less than 0.0025) but the increase was significantly smaller than that without the pretreatment of lavendustin-A (p less than 0.05). In the presence of amiloride (10 microM) and lavendustin-A (10 microM) insulin was no longer able to increase ISC (change in ISC = 0.04 +/- 0.03 microA/cm2, n = 6), suggesting that lavendustin-A had blocked the insulin's effect on the amiloride-insensitive ISC. Lavendustin-A (10 microM) had no significant effect on the basal ISC in control and amiloride treated monolayers. Our studies demonstrate that insulin increases amiloride-insensitive ISC in FDLE via lavendustin-A sensitive tyrosine kinase and that insulin's action on the amiloride-sensitive ISC of FDLE is mediated through a lavendustin-A insensitive (and presumably tyrosine kinase-independent) pathway.

Amiloride↗

PubMed1992

Effects of F- on apatite-octacalcium phosphate intergrowth and crystal morphology in a model system of tooth enamel formation.

In order to study the effect of F- on tooth enamel-like apatite formation, crystal growth experiments were carried out in the presence of 0.1 approximately 2 ppm F- at 37 degrees C and at pH 6.5 in a model system of enamel formation where octacalcium phosphate (OCP) was stable. Morphology changed from long and thin ribbons to small needle-like plates, and the product changed from OCP to apatite with an increase in F- concentration. In the presence of 0.1-1 ppm F-, apatite-OCP intergrowth took place, and crystals composed of apatite and OCP lamellas were formed. These crystals showed long and thin plate-like morphology and embeded an OCP lamella in the center of the crystal. The OCP lamella and its (100) planes were parallel to the (100) planes of apatite. The thickness of OCP decreased and that of apatite increased with an increase in F- concentration. Some apatite crystals obtained at 1 ppm F- embedded a central plane instead of the distinct OCP lamella. The result indicates that initially formed, thin, plate-like OCP acted as a template for the subsequent epitaxial overgrowth of apatite and, moreover, F- played an important role in regulating the apatite-OCP intergrowth.

Apatites↗

PubMed1992

Camptothecin-induced sister-chromatid exchange dependent on the presence of bromodeoxyuridine and the phase of the cell cycle.

Camptothecin (CPT), a DNA topoisomerase I inhibitor, dose-dependently induced sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 when added with bromodeoxyuridine (BrdUrd) for 2 cell cycles. CPT given prior to the addition of BrdUrd scarcely induced SCEs. When cells with BrdUrd present for 2 cell cycles were treated with CPT in the first cell cycle, the SCE induction was evident, though its frequency was considerably lower than in cells treated in the second cell cycle, indicating that BrdUrd is essential for SCE induction by CPT. The replacement of BrdUrd with thymidine in the second cell cycle gave the same results in SCE induction and its removal without replacement resulted in a reduced but still clear induction by CPT treatment in the second cell cycle. These results indicate that BrdUrd, not only incorporated into DNA but also in the culture medium, plays an essential role in SCE induction by CPT. CPT treatment in the G1 phase of the second cell cycle also induced SCEs, as did treatment in the S phase. In phytohemagglutinin-stimulated peripheral lymphocytes, CPT given in the G1 phase of the second cell cycle, but not of the first one, also induced SCEs though to a lesser degree. These findings suggest that CPT is capable of inducing DNA lesions during the G1 phase when chromosomes contain BrdUrd-substituted DNA. The lesions are presumably formed in connection with transcription, which requires topoisomerase I activity, and are believed to be long-lived enough to induce SCEs in the following S phase.

Bromodeoxyuridine↗

PubMed1992

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6).

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Amiloride↗

PubMed1992

DNA repair pathways in mammalian cells analyzed by isolation of ACNU-sensitive Chinese hamster ovary cells.

1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) causes chloroethylation of DNA strand followed by cross linking through an ethylene bridge. We recently isolated two ACNU sensitive mutants from mutagenized Chinese hamster ovary cells, and found them to be new drug sensitive recessive mutants (Hata et al. 1991). The O6-methyl guanine DNA methyl transferase (MT) activities of these cells were undetectable as the parental cell line, indicating that the sensitivity of the mutant cell lines to ACNU was not due to the decreased cellular level of this enzyme. By complementation analysis with the 7 established UV-sensitive CHO cell lines, one of the mutants, UVS1, turned out to complement their UV-sensitivity and, therefore, build a new complementation group among all the CHO cell lines ever reported. The other mutant, CNU1 showed hypersensitivity only to chlorethylating agents (ACNU, CCNU) and exhibited a slightly reduced unscheduled DNA synthesis (UDS) induced by UV. It is, therefore, suggestive that this mutant is defective in a specific step of DNA repair systems, which is important for the processing of DNA damages produced by ACNU. Only cell lines from the complementation group 1 and 4 out of 7 established complementation groups of UV-sensitive CHO mutants were more sensitive to ACNU than UVS1 and CNU1, indicating some steps of excision repair pathways as well as specific repair system play important roles in repairing ACNU-induced DNA damages.

Animals↗

PubMed1992

A possible involvement of DNA topoisomerase I in "caffeine effect" after ultraviolet irradiation.

Caffeine has been known to enhance lethal and chromosome damaging effects of chemical and physical mutagens. In spite of numerous investigations, the mechanism is not fully elucidated. In this paper, we describe that 1) post-treatment with camptothecin (CPT), a specific inhibitor of DNA topoisomerase (topo) I, enhances SCE-induction by ultraviolet light (UV), as post-UV caffeine treatment does, 2) the lethal effect of UV is also enhanced but to a lesser degree than by post-UV caffeine treatment, and 3) caffeine, like CPT, inhibits calf thymus topo I activity, as determined by relaxation of pBR322 supercoiled DNA. These results suggest that the mechanism(s) of lethal and SCE enhancement involves the ability of caffeine to inhibit topo I.

Caffeine↗