INHIBITION OF ATP-SENSITIVE K-SULFONYLUREA COMPOUND KAD-2229 IN A PANCREATIC BETA-CELL LINE, MIN-6 CELL( CHANNEL BY A NON)

We studied the mechanism of action of KAD-1229, a non-sulfonylurea com pound shown to stimulate insulin secretion, in a glucose responsive in sulinoma cell line, MIN 6 cells. In microsomal fraction of MIN 6 cells , KAD-1229 displaced binding of [H-3]glibenclamide in a concentration- dependent manner. The dissociation constant and the maximum binding ca pacity were 0.61 nM and 8.70 pmol/mg.protein, respectively. In inside out configuration of patch-clamp technique, KAD-1229 attenuated the op ening of ATP-sensitive K+ channels. The effect of KAD-1229 was detecte d at 10(-8) M, and 10(-5) M KAD-1229 almost completely blocked the act ivity of ATP-sensitive Kf channel. When membrane potential was monitor ed by a perforated mode of patch clamp, KAD-1229 induced depolarizatio n of plasma membrane, which was followed by a burst of action potentia ls. 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 additio n of nifedipine. Finally, KAD-1229 augmented insulin secretion in MIN 6 cells in a concentration-dependent manner. KAD-1229 also enhanced th e effect of glucose and nifedipine inhibited the action of KAD-1229 on insulin secretion. These results indicate that KAD-1229 stimulates in sulin secretion by stimulating Ca2+ influx and that, despite the lack of sulfonylurea structure, KAD-1229 binds to sulfonylurea receptors an d inhibits the activity of ATP-sensitive K+ channel in MIN 6 cells.