OXIDIZED GLUTATHIONE MEDIATES CATION CHANNEL ACTIVATION IN CALF VASCULAR ENDOTHELIAL-CELLS DURING OXIDANT STRESS

1. The oxidant, tert-butylhydroperoxide (tBuOOH) depolarizes calf pulm onary artery endothelial cells by activating a non-selective cation ch annel. To identify the molecular mediator of channel activation during oxidant stress, the patch-clamp technique was used to compare tBuOOH- induced changes in membrane potential and channel activity with those induced. by oxidized glutathione (GSSG), a cytosolic product of oxidan t metabolism. 2. When recording pipettes contained GSSG (2 mM), whole- cell zero-current potential measured immediately following pipette bre ak-in mras not different from control values (-57 mV). However, within 20 min of break-in, zero-current potential was depolarized to -7 mV. The time course of depolarization was dependent on the concentration o f GSSG and was accelerated by inhibition of GSSG metabolism. 3. In exc ised membrane patches, channels were activated by internal GSSG, but n ot by internal tBuOOH, reduced glutathione (GSH), or external GSSG. Ch annels were equal in size (28 pS) and in ionic selectivity to those ac tivated by incubation of intact cells with tBuOOH. As little as 20 mu M GSSG was sufficient to maximally activate channels. However, the tim e course of channel activation was concentration dependent between 20 mu M and 2 mM GSSG. 4. Channel activation by GSSG was reversed by GSH and by increasing the [GSH]:[GSSG] ratio. Likewise, channel activation by pre-incubation of intact cells with tBuOOH was reversed by GSH app lied after patch excision. 5. These results strongly suggest that GSSG is an endogenous intracellular mediator of channel activation and dep olarization during oxidant stress.