Receptors, Free Ca2+ Oscillations, and Activation of Glycogenolysis

Abstract

High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 1251-ET-1 with its site was specific, saturable, and time dependent (kob. = 0.019±0.001 min-'), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4±9.8 pM and a B.,. of 1084±118 fmol/mg protein. ET-3 and bigET-1 (1-38) (human) inhibited '25I-ET-1 binding with IC50 values of 1.85±1.03 nM and 43±6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained CaQ2 transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation ofglycogenolysis in rat hepatocytes.A 1.8-fold maximal increase in glycogen phosphorylase a was observed at 1 pM ET-1, with an EC5 of0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency ofrelated peptides was similar to that in binding experiments (ET-1 > ET-3 > big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2' and a potent glycogenolytic effect.We conclude that ET-1 behaves as