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C Asher

Publications and source records attributed to C Asher.

36 records · Page 2Linked to original sources
PubMed1988

Sodium-dependent inhibition of the epithelial sodium channel by an arginyl-specific reagent.

Effects of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO) on the epithelial Na+ channel were evaluated by measuring the amiloride-blockable 22Na+ fluxes in membrane vesicles derived from the toad bladder epithelium. Incubating whole cells or isolated membranes with PGO readily and irreversibly blocked the channel-mediated tracer flux. Na+ ions present during the interaction of membranes with PGO could protect channels from inactivation by PGO. This effect required the presence of Na+ at the luminal side of the membrane and was characterized by an IC50 of 79 mM Na+. Amiloride, too, could desensitize channels to PGO, but its effect was significant only when whole cells were interacted with the protein-modifying reagent. The data are compatible with a model in which the conductive path of the channel contains a functional arginine, possibly forming a salt bridge with a carboxylic group, which is involved in Na+ translocation and amiloride binding. It was also shown that the augmentation of transport induced by incubating whole cells in Ca2+-free solution (Garty, H., and Asher, C. (1985) J. Biol. Chem. 260, 8330-8335) involves the activation or recruitment of channels that are not vulnerable to PGO prior to incubation.

Aldehydes↗
PubMed1988

Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.

The aldosterone-induced augmentation of Na+ transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive 22Na+ uptake in isolated membrane vesicles. Incubating bladders with 0.5 microM aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current (because of the activation or insertion of apical amiloride-blockable channels) but had no effect on the amiloride-sensitive Na+ transport in apical vesicles derived from the treated tissue. A longer incubation (e.g., 6 hr) produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone (present at 1000-fold excess) and the protein synthesis inhibitor cycloheximide (1 microM). In addition, triiodothyronine and butyrate, previously reported to partly inhibit the aldosterone-induced increase in short-circuit current, blocked the hormonal effect in vesicles. It is suggested that aldosterone elevates the apical Na+ permeability of target epithelia by two different mechanisms: a relatively fast effect (less than or equal to 3 hr), which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later (greater than 3 hr) response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.

Aldosterone↗
PubMed1988

Sodium channels in membrane vesicles from cultured toad bladder cells.

Electrical potential-driven 22Na+ fluxes were measured in membrane vesicles prepared from TBM-18(c123) cells (a clone of the established cell line TB-M). Fifty to seventy percent of the tracer uptake in vesicles derived from cells that were cultivated on a porous support were blocked by the diuretic amiloride. The amiloride inhibition constant was less than 0.1 microM, indicating that this flux is mediated by the apical Na+-specific channels. Vesicles prepared from cells that were not grown on a porous support exhibited much smaller amiloride-sensitive fluxes. Two Ca2+-dependent processes that down-regulate the channel conductance and were previously identified in native epithelia were found in the cultured cells as well. Vesicles isolated from cells that were preincubated with 5 X 10(-7) M aldosterone for 16-20 h exhibited higher amiloride-sensitive conductance than vesicles derived from control, steroid-depleted cells. Thus membrane derived from TBM-18(c123) cells can be used to characterize the epithelial Na+ channel and its hormonal regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Characterization of cAMP-induced activation of epithelial sodium channels.

Incubating toad bladder with 10 mU/ml vasopressin increases the amiloride-blockable Na+ flux in membrane vesicles derived from the epithelial cells by about twofold. This stimulation is further enhanced by 3-isobutyl-1-methylxanthine and can be mimicked by 8-bromoadenosine 3', 5'-cyclic monophosphate. Thus the natriferic action of cAMP involves a sustained change of the apical membrane preserved by the isolated vesicles. The possibility that transport is modulated by direct phosphorylation/dephosphorylation of the Na+ channel was tested. Trapping purified cAMP-dependent protein kinase, cAMP, and ATP in apical vesicles failed to alter Na+ transport even though the enzyme proved active and could phosphorylate intravesicular proteins. Trapping several phosphatases partially purified from toad bladder in vesicles was ineffective as well. These data suggest that the cAMP-induced increase in Na+ conductance involves processes other than phosphorylation of the channel protein or direct channel-cAMP interaction.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Effects of amiloride analogues on Na+ transport in toad bladder membrane vesicles. Evidence for two electrogenic transporters with different affinities toward pyrazinecarboxamides.

Most of the electrical potential-driven 22Na+ uptake in toad bladder membrane vesicles can be blocked by the diuretic amiloride. Analysis of the amiloride inhibition curve indicates the presence of two pathways with low and high affinities to the diuretic (Garty, H. (1984) J. Membr. Biol. 82, 269-279). The selectivity of these pathways to amiloride was explored by comparing the inhibition curve of this diuretic with those of 10 of its structural analogues. The relative potencies of various amiloride-like compounds as blockers of the flux component with high affinity to amiloride were in good agreement with the structure-activity relationships elucidated from transepithelial short-circuit current measurements. Thus, this pathway is most probably the apical Na+-specific channel. The other pathway with lower affinity to the diuretic was relatively insensitive to modifications of the amiloride molecule, and the structure-activity relationships measured for the inhibition of this pathway were different from those reported for any other amiloride-blockable process. Other experiments have established that the Na+ flux with low affinity to amiloride is electrogenic and is not mediated by a Na+/H+ or Na+/Ca2+ exchanger, Na+-hexose cotransporter, or the Na+/K+-ATPase. The data indicate that tracer flux measurements in toad bladder membrane vesicles monitor, in addition to the well-characterized apical Na+ channels, another amiloride-blockable electrogenic Na+ transporter. This pathway could be responsible for the basolateral amiloride-blockable Na+ conductance recently observed in nystatin-treated bladders (Garty, H., Warncke, J., and Lindemann, B. (1987) J. Membr. Biol. 95, 91-103).

Amiloride↗
PubMed1987

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions.

Direct inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable 22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati, J. Gen. Physiol. 81:643-666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive 22Na+ uptake could not be blocked by Ca2+ (up to 1 mM). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 microM at pH 7.8 to greater than 10 microM at pH 7.0. Strong pH effects were also demonstrated by measuring the pH dependence of 22Na+ uptake in vesicles that contained 0.5 microM Ca2+. This Ca2+ activity produced a maximal inhibition of 22Na+ uptake at pH greater than or equal to 7.4 but had no effect at pH less than or equal to 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+.

Amiloride↗
PubMed1986

Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium.

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Extracellular pH, transmembrane distribution and cytotoxicity of chlorambucil.

The effects of extracellular pH (6.2 to 7.3) on uptake and cytotoxicity of the weak acid anti-tumor drug chlorambucil were investigated. Decreasing extracellular pH from 7.3 to 6.5 had a negligible effect on the intracellular pH of Chinese hamster V79 fibroblasts, thus resulting in the formation of a transmembrane pH gradient (intracellular alkaline). Addition of high concentrations of acetate or bicarbonate partially collapsed the pH gradient. Chlorambucil (pKa = 5.8) behaved as a weak acid with enhanced accumulation and cytotoxicity at extracellular pH less than 7.0. As predicted for a weak acid, partial collapse of the transmembrane pH gradient decreased both uptake and cell killing. Since the interstitial pH of micrometastases and solid tumors of many cancers is low relative to normal tissues, these results have potential implications for both in vitro drug testing and in vivo therapy.

Animals↗
PubMed1985

Ca2+-dependent, temperature-sensitive regulation of Na+ channels in tight epithelia. A study using membrane vesicles.

Na+ fluxes were measured in toad bladder microsomes. Under favorable conditions, 60-90% of the tracer uptake was blocked by amiloride (Ki = 2.3 X 10(-8) M), i.e. mediated by the apical Na+-specific channels. Vesicles derived from cells maintained at 0 degrees C exhibited relatively small amiloride-sensitive fluxes. However, incubating the scraped cells at 25 degrees C prior to homogenization induced a nearly 5-fold increase of the amiloride-blockable flux in vesicles. This activation was fairly slow (t 1/2 = 5-10 min), irreversible, and strongly dependent on the incubation temperature. On the other hand, the Na+-specific apical conductance measured in mounted bladders was only slightly affected by the incubation temperature. The above activation process could be observed only in Ca2+-free EGTA-containing solutions. Adding Ca2+ (1 mM) to the cell suspension and subsequently removing it before homogenization blocked almost completely the amiloride-sensitive tracer uptake in the vesicles. The data are compatible with the model that the epithelial Na+ channels are down-regulated by a Ca2+-dependent reaction. The incubation of scraped, somewhat permeabilized cells in a Ca2+-free solution releases channels from this down-regulation and increases the Na+ conductance in a temperature-dependent process. The regulation of channels appears to involve a cytoplasmic factor which induces a stable modification of the apical membrane, preserved by the isolated vesicle.

Amiloride↗
PubMed1985

The removable quadhelix appliance.

The introduction of a preformed removable quadhelix appliance with curved rotation sheaths for insertion and removal has overcome many of the clinical problems associated with the fixed quadhelix appliance. The design, fitting and activation of the appliance are described. Various applications and modifications are discussed.

Equipment Design↗
PubMed1984

The effects of synthetic locust adipokinetic hormone on dispersed locust fat body cell preparations: cAMP induction, lipid mobilization, and inhibition of protein synthesis.

A procedure for the preparation of functional cells from adult locust fat bodies by collagenase treatment has been developed. The high variability of replicates encountered when whole fat bodies are incubated in vitro is greatly reduced in incubation of the dispersed cells. Synthetic locust adipokinetic hormone (AKH) (40 nM) stimulated release of lipids from the dispersed fat body cells at a rate comparable to that observed using whole fat bodies in vitro. Synthetic AKH elevated cAMP levels sixfold in dispersed cells. In addition, AKH inhibited protein synthesis to a maximum of 50-70% in a concentration-dependent manner. None of these actions of AKH required the presence of locust hemolymph components. These results demonstrate the utility of the isolated locust fat body cells for investigating hormonal action in vitro.

Adipose Tissue↗
PubMed1983

Determination of locust vitellogenin by radioimmunoassay with [3H]Propionyl-vitellogenin.

A simple procedure for radiolabeling of locust vitellogenin is described. This procedure involves coupling of [3H]propionyl succinimidate to purified vitellogenin with high yield and specific activity. Using this radiolabeled analog, a specific and sensitive radioimmunoassay was developed for determining locust vitellogenin content, with a lower detection limit of 1 ng. [3H]Propionyl-vitellogenin binds completely to rabbit anti-vitellogenin (locust) and can be completely competed out by locust vitellogenin. The structural similarity of locust vitellogenin with that of locust egg vitellin, male locust lipophorin (a diglyceride-carrying lipoprotein), Xenopus laevis vitellogenin, and chicken egg yolk lipovitellin was examined with this RIA procedure. Comparable binding competition was obtained with locust vitellin only. Male locust lipophorin, Xenopus vitellogenin, and chicken lipovitellin did not inhibit vitellogenin binding at concentrations 1000-fold greater than that of locust vitellogenin. The use of this RIA in determination of vitellogenin synthesis in vivo and in vitro, using isolated fat body preparations, is described.

Animals↗