Thiol-dependent Redox Mechanisms in the Modification of ATP-Sensitive Potassium Channels in Rabbit Ventricular Myocytes

Cellular redox state is known to be perturbed during ischemia and that Ca2+ and K+ channels have been shown to have functional thiol groups. In this study, the properties of thiol redox modulation of the ATP-sensitive K+ (KATP) channel were examined in rabbit ventricular myocytes. Rabbit ventricular myocytes were isolated using a Langendorff column for coronary perfusion and collagenase. Single-channel currents were measured in excised membrane patch configuration of patch-clamp technique. The thiol oxidizing agent 5, 5'-dithio-bis- (2-nitro-benzoic acid) (DTNB) inhibited the channel activity, and the inhibitory effect of DTNB was reversed by dithiothreitol (disulfide reducing agent; DTT). DTT itself did not have any effect on the channel activity. However, in the patches excised from the metabolically compromised cells, DTT increased the channel activity. DTT had no effect on the inhibitory action by ATP, showing that thiol oxidation was not involved in the blocking mechanism of ATP. There were no statistical difference in the single channel conductance for the oxidized and reduced states of the channel. Analysis of the open and closed time distributions showed that DTNB had no effect on open and closed time distributions shorter than 4 ms. On the other hand, DTNB decreased the life time of bursts and increased the interburst interval. N-ethylmaleimide (NEM), a substance that reacts with thiol groups of cystein residues in proteins, induced irreversible closure of the channel. The thiol oxidizing agents (DTNB, NEM) inhibited of the KATP channel only, when added to the cytoplasmic side. The results suggested that metabolism-induced changes in the thiol redox can also modulate KATP channel activity and that a modulatory site of thiol redox may be located on the cytoplasmic side of the KATP channel in rabbit ventricular myocytes.

The effects of melatonin on cisplatin-induced renal cortical cell injury in rabbits

Melatonin, a pineal gland hormone, is believed to act as an antioxidant via the stimulation of radical detoxifying enzymes and scavenging of free radicals. In this study, effects of in vitro and in vivo treatments of melatonin on the cisplatin-induced lipid peroxidation, LDH release and plasma creatinine were determined in rabbit renal cortical cells. The level of malondialdehyde (MDA) was assayed as an index of lipid peroxidation and the level of LDH release as an indicator of cellular damage. In in vitro studies, cisplatin increased the levels of MDA and LDH release in a concentration-and time-dependent manner. Melatonin inhibited the cisplatin-induced lipid peroxidation and LDH release in a concentration-dependent manner. The minimal effective concentration of melatonin that significantly reduced the 300 muM cisplatin-induced lipid peroxidation and LDH release was 1 mM. In in vivo studies, the levels of lipid peroxidation and LDH release in renal cortical cells increased significantly 24 or 48 hours after a single injection of cisplatin (6 mg/kg). When the cisplatin-injected rabbits were pretreated with 10 mg/kg of melatonin, a significant reduction in both lipid peroxidation and LDH release was observed. The plasma creatinine level increased from 0.87+/-0.07 mg/dl in control to 6.33+/-0.54 mg/dl in cisplatin-injected rabbits (P<0.05). Melatonin partially prevented the increase in serum creatinine level (1.98+/-0.11 mg/dl) by cisplatin (P<0.05). In the proximal tubules from cisplatin-treated group, tubular cells had microvilli of variable heights. Necrotic debris was seen in tubular lumens. In most of cells, the mitochondria and lysosomes were increased in frequency. The endocytic vacuoles were not prominent and distribution of the brush border was irregular and shortened. These cisplatin-induced morphological changes were moderate in the melatonin-pretreated group. These results suggest that the toxicity of cisplatin is associated with the generation of reactive oxygen free radicals and that melatonin is a powerful antioxidant, which prevents some of the adverse effects of cisplatin.