A NOVEL D-PHENYLALANINE-DERIVATIVE HYPOGLYCEMIC AGENT A-4166 INCREASES CYTOSOLIC-FREE CA2+ IN RAT PANCREATIC BETA-CELLS BY STIMULATING CA2+ INFLUX

It has recently been shown that N-[(trans-4-isopropylcyclohexyl) carbonyl]D-phenylalanine (A-4166), a new nonsulfonylurea oral hypoglycemic agent, reduces blood glucose levels in nondiabetic and diabetic animals in a quicker and shorter lasting manner than sulfonylureas, and that the hypoglycemic effect of A-4166 is due to the stimulation of insulin release. However, the mechanism by which A-4166 stimulates insulin release is still. unknown. In the present study, we investigated the effect of A-4166 on the cytosolic free Ca2+ concentration ([Ca2+](i)) in pancreatic beta-cells from normal rats by dual wavelength fura-2 microfluorometry. In the presence of 2.8 mM glucose, A-4166 produced a rapid increase in [Ca2+](i) in a concentration-dependent manner over the range of 3-30 mu M. The increase in [Ca2+](i) was transient, oscillatory, or sustained. A-4166 did not evoke any decrease in [Ca2+](i), whereas a high concentration of glucose (16.7 mM), a metabolized secretagogue, produced an initial decrease and a subsequent increase in [Ca2+](i). In the presence of 16.7 mM glucose, low concentrations (0.03-1 mu M) of A-4166 produced an increase in [Ca2+](i) in some of the beta-cells tested. The [Ca2+](i) response to A-4166 was completely and reversibly inhibited under Ca2+-free conditions as well as by nitrendipine, a blocker of the L-type Ca2+ channel. Nitrendipine also inhibited insulin release from perfused rat pancreases stimulated by A-4166. Diazoxide, an opener of the ATP-sensitive K+ channel, blocked the [Ca2+](i) response to A-4166. Sulfonylureas such as tolbutamide and glibenclamide increased [Ca2+](i) in a manner similar to A-4166. These results indicate that at basal glucose concentrations, A-4166 increases [Ca2+](i) in rat pancreatic beta-cells by stimulating Ca2+ influx through L-type Ca2+ channels, and that this effect is markedly augmented at elevated glucose concentrations. It appears that the increase in [Ca2+](i) is related to the stimulation of insulin release by A-4166. Inhibition of ATP-sensitive potassium channels, but not stimulation of beta-cell metabolism, may be involved in the increase in [Ca2+](i) by A-4166.