Changes in cytoplasmic ATP concentration parallels changes in ATP-regulated K+-channel activity in insulin-secreting cells.
Changes in cytoplasmic ATP concentration were monitored in intact insulin-producing cells and correlated to changes in the activity of ATP-sensitive K+-channels (KATP 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 microM 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 deteriorate cell function, as verified by experiments showing similar changes in cytoplasmic free Ca2+-concentration, [Ca2+li, in both transfected and non-transfected cells. By measuring the cytoplasmic ATP concentration and KATP 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 KATP channel activity under physiological conditions.
Köhler
M
M
Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden.
Norgren
S
S
Berggren
P O
PO
Fredholm
B B
BB
Larsson
O
O
Rhodes
C J
CJ
Herbert
T P
TP
Luthman
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D001517:Coleoptera; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary; D017930:Genes, Reporter; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D049449:Luminescence; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology*; D014162:Transfection
D018076:DNA, Complementary; D007328:Insulin; D015221:Potassium Channels; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D008156:Luciferases; D005947:Glucose; D002118:Calcium