Inhibition of ATP-sensitive K+ channel by a non-sulfonylurea compound KAD-1229 in a pancreatic beta-cell line, MIN 6 cell.
We studied the mechanism of action of KAD-1229, a non-sulfonylurea compound shown to stimulate insulin secretion, in a glucose responsive insulinoma cell line, MIN 6 cells. In microsomal fraction of MIN 6 cells, KAD-1229 displaced binding of [3H]glibenclamide in a concentration-dependent manner. The dissociation constant and the maximum binding capacity were 0.61 nM and 8.70 pmol/mg.protein, respectively. In inside out configuration of patch-clamp technique, KAD-1229 attenuated the opening of ATP-sensitive K+ channels. The effect of KAD-1229 was detected at 10(-8) M, and 10(-5) M KAD-1229 almost completely blocked the activity of ATP-sensitive K+ channel. When membrane potential was monitored by a perforated mode of patch clamp, KAD-1229 induced depolarization of plasma membrane, which was followed by a burst of action potentials. These action potentials were blocked by cobalt. In a fura-2-loaded single MIN 6 cell, KAD evoked an elevation of intracellular free Ca2+ concentration, [Ca2+]i. The KAD-1229-mediated elevation of [Ca2+]i was attenuated by either removal of extracellular Ca2+ or an addition of nifedipine. Finally, KAD-1229 augmented insulin secretion in MIN 6 cells in a concentration-dependent manner. KAD-1229 also enhanced the effect of glucose and nifedipine inhibited the action of KAD-1229 on insulin secretion. These results indicate that KAD-1229 stimulates insulin secretion by stimulating Ca2+ influx and that, despite the lack of sulfonylurea structure, KAD-1229 binds to sulfonylurea receptors and inhibits the activity of ATP-sensitive K+ channel in MIN 6 cells.
Mogami
H
H
Cell Biology Research Unit, Institute of Endocrinology, Gunma University, Maebashi, Japan.
Shibata
H
H
Nobusawa
R
R
Ohnota
H
H
Satou
F
F
Miyazaki
J
J
Kojima
I
I
D000200:Action Potentials / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003035:Cobalt / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D016257:Fura-2 / Q000737:chemistry; D005947:Glucose / Q000494:pharmacology; D005905:Glyburide / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology*; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects*; D054833:Isoindoles; D008564:Membrane Potentials / Q000187:drug effects; D009543:Nifedipine / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D014407:Tumor Cells, Cultured
D007004:Hypoglycemic Agents; D007211:Indoles; D007328:Insulin; D054833:Isoindoles; D026902:Potassium Channel Blockers; D003035:Cobalt; D000255:Adenosine Triphosphate; C087255:mitiglinide; D009543:Nifedipine; D005947:Glucose; D005905:Glyburide; D002118:Calcium; D016257:Fura-2