Y Oh

University of Alabama at Birmingham, Birmingham, AL, United States

Are you Y Oh?

Claim your profile

Publications (7)20.22 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.
    The American journal of physiology 10/1993; 265(3 Pt 1):C630-40. · 3.28 Impact Factor
  • Y Oh, D J Benos
    [Show abstract] [Hide abstract]
    ABSTRACT: We have used ion-exchange chromatography to isolate and partially purify an amiloride-sensitive Na+ channel protein from bovine renal papillae. The channel was purified by sequential chromatography on an anion-exchange and a cation-exchange cartridge. Five different approaches were applied to monitor the degree of purification of this Na+ channel. First, [3H]Br-benzamil binding activities to the purified proteins were determined. We found that no specific binding was measured in protein fractions eluted from a QUAT anion-exchange cartridge, whereas proteins eluted from an SP cation-exchange cartridge bound [3H]Br-benzamil specifically. Second, we examined the HPLC profiles of purified proteins through both ion-exchange chromatography steps and observed a major peak of very high molecular mass proteins (> 670 kDa) after final purification. Third, using anti-Na+ channel antibodies, Western and slot blot analyses were performed to assess qualitatively the enrichment of channel epitopes at each step of purification. Fourth, amiloride-sensitive 22Na+ influx could be demonstrated after the proteins eluted from the cation-exchange resin were reconstituted into liposomes. Fifth, the proteins eluted from the cation-exchange resin were reconstituted into planar lipid bilayers, and single amiloride-sensitive Na+ channel activity was measured. In summary, we have developed a new protocol applicable for speed- and scale-up purification of a functional amiloride-sensitive Na+ channel from bovine kidney papillae.
    Protein Expression and Purification 08/1993; 4(4):312-9. · 1.43 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.
    The American journal of physiology 08/1993; 265(1 Pt 1):C85-91. · 3.28 Impact Factor
  • Y Oh, D J Benos
    [Show abstract] [Hide abstract]
    ABSTRACT: We have purified an amiloride-inhibitable Na+ channel protein from bovine renal papillae using ion-exchange and immunoaffinity chromatography. In the present study, these purified Na+ channels were reconstituted into planar lipid bilayers, and their single-channel characteristics were studied. We observed both large- and small-conductance Na(+)-selective ion channels in planar lipid bilayers. Single-channel conductance for the large- and small-conductance channels saturated as a function of Na+ concentration. These relations could be fitted by a simple Langmuir isotherm with a Michaelis constant of 55 and 45 mM and a maximum open-state conductance of 56 or 8.4 pS, respectively. Both channels were perfectly cation selective, with a Na(+)-to-K+ permeability ratio of 6.7:1 for the large channel and 7.8:1 for the small channel, and their open single-channel current-voltage relations were linear when bathed with symmetrical Na+ solutions. The percent open time of the reconstituted large or small channels varied between 10 and 50% or 1 and 20%, respectively. After application of amiloride, both the large- and small-conductance Na+ channels were inhibited in a dose-dependent manner.
    The American journal of physiology 07/1993; 264(6 Pt 1):C1489-99. · 3.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.
    Journal of Biological Chemistry 10/1992; 267(26):18498-504. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to document the existence, assess the spatial localization, and characterize some of the transport properties of proteins antigenically related to epithelial Na+ channels in freshly isolated rabbit and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase digestion of lung tissue and purified by density-gradient centrifugation, were incubated with polyclonal antibodies raised against Na+ channel protein purified from beef kidney papilla (NaAb), followed by a secondary antibody (goat antirabbit immunoglobulin G conjugated to fluorescein isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at the level of the plasma membrane, which, in most cells, colocalized with that of the lectin Maclura pomiferra agglutinin, an apical surface marker. In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in rat ATII membrane vesicles. When patch clamped in the whole cell mode using symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited outwardly rectified Na+ currents that were diminished by amiloride (10-100 microM) instilled into the bath solution. Ion substitution studies showed that the conductive pathways were three times more permeable to Na+ than K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells (45% inhibition at 100 microM, with IC50 of approximately 1 microM for all inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM), well-known K+ channel blockers, had no effect on 22Na+ uptake. These results indicate that ATII cells express an amiloride-sensitive Na+ conductance, probably a channel, with a lower affinity for amiloride and its structural analogues than the well-established amiloride-sensitive Na+ channels found in bovine renal papila and cultured amphibian A6 kidney cells.
    The American journal of physiology 06/1992; 262(5 Pt 1):C1228-38. · 3.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Without Abstract
    Ergebnisse der Physiologie 02/1992; 120:31-113. · 1.00 Impact Factor

Publication Stats

90 Citations
20.22 Total Impact Points

Institutions

  • 1992–1993
    • University of Alabama at Birmingham
      • • Department of Cell, Developmental and Integrative Biology (CDIB)
      • • Department of Anesthesiology
      Birmingham, AL, United States