NUCLEOTIDES MODULATE THE LOW AFFINITY BINDING-SITES FOR [H-3] GLIBENCLAMIDE IN THE RAT-BRAIN

Receptors for hypoglycemic sulfonylureas. such as glibenclamide, are commonly linked to the activity of ATP-sensitive K+ channels (K-ATP). High and low affinity binding sites for glibenclamide were described previously in numerous tissues. High affinity binding sites have been thought to be responsible of the modulation of K-ATP, but new evidences suggest that low affinity ones could also regulate these channels. In order to clarify the properties of the two binding sites, with respect to their interaction with K-ATP, we characterized biochemically and pharmacologically [H-3]glibenclamide binding in the rat brain cortex. Competitive inhibition plots with [H-3]glibenclamide performed on membranes of adult and neonatal rat brain cortex exhibited a biphasic pattern with similar binding parameters, indicating the presence of two similar binding sites in adult as well as in neonatal animals. Membranes of adult rat cortex treated with thiol groups modifying agents, N-ethylmaleimide or 1,4-dithiothreitol, increased the inhibition constant of glibenclamide for the low affinity binding sites (K(i)L) by about 4-fold. The divalent cations Mg++ and Ca++ also increased K(i)L by 3- to 6-fold and enhanced the low affinity binding capacity (B(max)L) by 55 and 103%, respectively, both cations increasing B(max)L by 144%. Among the numerous nucleotides studied, adenine and guanidine triphosphate nucleotides were the most potent to affect the low affinity binding sites. ATP, ADP, GTP and respective nonhydrolysable nucleotides increased K(i)L by 7- to 12-fold and decreased B(max)L by 10 to 30%. The effects of nucleotides were not Mg++ dependent. Our extensive pharmacological study showed that only few compounds were able to inhibit the binding of [H-3]glibenclamide to its high or low affinity binding sites. Conversely, glibenclamide failed to inhibit a large number of selective radioligands for different receptors. It is concluded that the low affinity binding sites for glibenclamide are most sensitive to divalent cations and nucleotides than the high affinity ones and could interfere with ATP-sensitive or other nonrelated potassium channels.