The effect of modulators of large-conductance Ca2+-modulated K+ channels on rat AS-30D ascites hepatoma cells and isolated liver mitochondria treated with Cd2+

Abstract

ATP-producing cell organelles, mitochondria, are a primary target of heavy metals, major environmental pollutants causing a variety of diseases and pathologies. The mechanism of heavy metal toxic effect was established to include changes both in the intracellular production of reactive oxygen species (ROS) and mitochondrial dysfunction due to respiratory chain disorders and activation of the Ca2+-dependent non-selective pore in the mitochondrial inner membrane. Te role of other ion channels including such selective potassium channels as large-conductance Ca2+-activated K+ channels, BK(Ca), supposed to be cytoprotective, remains poorly studied. In the present work conducted on rat AS-30D ascites hepatoma cells and liver mitochondria, we studied the effect of different BK(Ca) effectors in the presence or absence of Cd2+ ions in the incubation medium, specifically of two activators, NS1619 and NS004, and one blocker, paxilline. After 24-h incubation of AS-30D cells with 10 μM of both NS1619 and NS004, the number of apoptized cells was found to increase significantly versus control; moreover, the presence of these BK(Ca) activators in the incubation medium exerted an additive effect on Cd2+-induced apoptosis of AS-30D cells. The same concentration of NS1619 and NS004 did not affect significantly AS-30D cellular respiration after 3, 24 and 48 h of incubation, although increasing after 3 h the intracellular production of ROS. In experiments on isolated rat liver mitochondria, NS1619 and NS004 added at the same concentration to the KCl-containing medium did not affect the rates of respiratory State 3 (after Chance) and maximally uncoupled respiration (both in the presence and absence of Cd2+); concurrently, they induced a weak uncoupling effect by increasing both basal and resting-state (State 3 after Chance) respirations, and also enhanced a high-amplitude mitochondrial swelling induced by Cd2+ in this medium. Paxilline (at 1μM) was shown to reduce the mortality of AS-30D cells after 3-, 24- and 48 h-incubation in the presence of Cd2+ and to increase the intracellular ROS production in control after 3 and 24 h of exposure. Paxilline added at a concentration exerting a long-term protective effect did not affect cellular respiration of rat AS-30D cells and isolated liver mitochondria (both in the presence and absence of Cd2+) and did not reduce mitochondrial swelling observed in the presence of Cd2+ and BK(Ca) activators. Possible molecular action mechanisms of BK(Ca) modulators are discussed.