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 (Ca-i) in primary mouse keratinocyte cultures. Treatment of keratinocytes with 100 mu M diazoxide induced a transient peak in Ca-i, followed by a sustained elevation. Depletion of medium calcium by addition of EGTA abolished the diazoxide-induced Ca-i response, indicating that the agent promoted calcium influx withou release of calcium from intracellular stores. The diazoxide-induced rise in Ca-i 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 Ca-i.