Changes in cytoplasmic ATP concentration were monitored in intact insulin-producing cells and correlated to changes in the activity of ATP-sensitive K+-channels (K(ATP) channels). Luciferase was introduced into HIT M2.2 cells and whole pancreatic islets by transient expression of firefly (Photinus pyralis) luciferase cDNA. In transfected cells, extracellular addition of luciferin increased the luminescence signal to a maximum within 50-120 s. Addition of 1 μM of the mitochondrial uncoupler FCCP decreased the luminescence, an effect partly reversed upon withdrawal of the compound. High concentrations of glucose increased cytoplasmic free ATP concentration. Changes in the luminescence signal were accompanied by changes in activity of the ATP-sensitive K+-channel. Transfection per se did not deteroriate cell function, as verified by experiments showing similar changes in cytoplasmic free Ca2+-concentration, [Ca2+](i), in both transfected and non-transfected cells. By measuring the cytoplasmic ATP concentration and K(ATP) channel activity under similar experimental conditions, it was possible to establish, for the first time, a direct relationship between these two parameters. This indeed suggests that the cytoplasmic ATP concentration has a crucial role in the regulation of K(ATP) channel activity under physiological conditions. Copyright (C) 1998 Federation of European Biochemical Societies.
ASJC Scopus subject areas
- Molecular Biology