Evidence that diazoxide promotes calcium influx in mouse keratinocyte cultures by membrane hyperpolarization

We have used fura-2/AM to investigate the effect of diazoxide on the cytosolic calcium concentration (Cai) in primary mouse keratinocyte cultures. Treatment of keratinocytes with 100 microM diazoxide induced a transient peak in Cai, followed by a sustained elevation. Depletion of medium calcium by addition of EGTA abolished the diazoxide-induced Cai response, indicating that the agent promoted calcium influx without release of calcium from intracellular stores. The diazoxide-induced rise in Cai was inhibited both by addition of 60 mM KCl to the assay buffer and by preincubation with glibenclamide, a specific K+ channel blocker. In addition, studies with the membrane potential-sensitive fluorescent probe bis-oxonol demonstrated that diazoxide hyperpolarized keratinocyte membranes. These findings suggest that keratinocytes possess K+ channels, and that the previously reported proliferation effects of K+ channel openers such as diazoxide on keratinocytes may result from hyperpolarization-induced elevation of Cai.