valinomycin and Cell-Transformation--Viral

The purpose of the present study is to determine the role of K+ channel activity as an early event in UV-induced corneal epithelial cell apoptosis.. Both cell-attached and nystatin-perforated patch-clamping were performed to record K+ channel activity in rabbit corneal epithelial (RCE) and primary cultured rabbit corneal epithelial (PRCE) cells exposed to UV irradiation. On exposure of corneal epithelial cells or intact corneas to UV-C irradiation or treatment of corneal epithelial cells with etoposide, cell apoptosis was determined by DNA fragmentation, ethidium bromide-acridine orange nuclear stain and TdT-mediated dUTP nick-end labeling (TUNEL).. In the present study, UV-irradiation-induced corneal epithelial cell apoptosis through activation of a K+ channel in the cell membrane was an early event in response to UV irradiation. UV-C irradiation (42 microJ/cm(2)) activated robust K+ channel activity in RCE and PRCE cells at both the single-channel and whole-cell levels, when measured with the cell-attached and nystatin-perforated patch clamps, respectively. Suppression of UV-irradiation-induced K+ channel activity with the specific K+ channel blocker 4-aminopurydine (4-AP) prevented UV-irradiation-induced apoptosis in the RCE and PRCE cells, loss of the superficial layer of corneal epithelium, and apoptosis in the basal layer corneal epithelium. However, suppression of K+ channel activity did not protect RCE and PRCE cells from etoposide, a topoisomerase II inhibitor, which induced cell death by bypassing the membrane. Furthermore, application of valinomycin, a K+ ionophore, to mimic the effect of mass activation of the K+ channel in RCE and PRCE cells caused cell apoptosis.. The results indicate that UV irradiation induces superactivity of K+ channels in the membrane is an early event mediating signaling transduction and resulting in corneal epithelial cell death in response to UV irradiation.

Topics: 4-Aminopyridine; Animals; Apoptosis; Cell Line, Transformed; Cell Transformation, Viral; Epithelium, Corneal; Etoposide; Humans; In Situ Nick-End Labeling; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Rabbits; Simian virus 40; Topoisomerase II Inhibitors; Ultraviolet Rays; Valinomycin

The uptake of alpha-amino[(3)H]isobutyric acid (AIB) was studied in membrane vesicles from mouse fibroblasts transformed by simian virus 40 to examine the features of the Na(+)-stimulated and Na(+)-dependent AIB transport process. The simultaneous addition of NaCl and AIB to these vesicles produced a transient accumulation, or "overshoot," of amino acid 3-4 times the equilibrium value. Both the initial rate of uptake and the rate of fall of intravesicular AIB after maximal accumulation were sensitive to the temperature of incubation. The overshoot of AIB uptake was enhanced with Na(+) salts of highly permeant lipophilic anions, such as SCN(-) and NO(3) (-), and was decreased by the addition of SO(4) (2-), a relatively impermeant ion. Gramicidin D, which enhances the membrane conductance of Na(+) electrogenically, decreased the overshoot, while a potassium diffusion potential, induced by valinomycin (in K(+)-preloaded membrane vesicles), produced a Na(+)-dependent overshoot of AIB uptake. When vesicles were preincubated with both Na(+) and AIB, followed by the generation of an interior negative membrane potential (by the addition of SCN(-)), an overshoot of AIB uptake resulted. However, this did not occur in the absence of Na(+). It is concluded that, apart from its role in the generation of a transmembrane electrochemical potential, Na(+) is essential for the overshoot of AIB uptake.

Topics: Aminoisobutyric Acids; Anions; Biological Transport; Cell Membrane; Cell Transformation, Viral; Cell-Free System; Gramicidin; Simian virus 40; Sodium; Temperature; Thiocyanates; Valinomycin

Topics: Amino Acids; Biological Transport, Active; Cell Line; Cell Membrane; Cell Transformation, Viral; Fibroblasts; Kinetics; Membrane Potentials; Methylglucosides; Monensin; Nigericin; Onium Compounds; Potassium; Simian virus 40; Sodium; Trityl Compounds; Valinomycin