G-protein linked melatonin binding in isolated mouse hepatocytes and melatonin injection effects on blood glucose

Abstract

Melatonin receptors in the mouse liver were investigated using 2-[125I]iodomelatonin ([125I]MEL). [125I]MEL binding on hepatocytes isolated from the mouse using collagenase was found to be stable, saturable, reversible and of high affinity. The equilibrium dissociation constant (Kd) obtained from saturation studies was 10.0 B 0.4 pM (n = 18), and the maximum number of binding sites (Bmax) was 2.7 B 0.3 fmol/mg protein (n = 18). The Hill coefficient obtained from Hill analysis was 1.1 B 0.02 (n = 18), suggesting that a single class of [125I]MEL binding sites was present in the mouse hepatocytes. The Kd value of 6.87 B 1.24 pM (n = 3) obtained from the kinetics studies was comparable to the Kd obtained from saturation studies. The binding sites are specific as demonstrated by competition studies with several structural analogs and neurotransmitters. Adenosine nucleotides have no effects on [125I]MEL binding while guanine nucleotides inhibited the binding. GTPÁS and Gpp(NH)p inhibited the binding with IC50 of 2.67 ! 10–6 M and 1.71 ! 10–5 M, respectively. Results showed that the binding sites are G-protein linked. At concentrations of 10 ÌM and 50 ÌM, GTPÁS increased the Kd (28.9 B 2.0 pM and 79.7 B 8.3 pM) without changing the Bmax (1.2 B 0.2 fmol/mg protein and 1.4 B 0.3 fmol/mg protein) when compared to the control (Kd = 10.4 B 0.1, Bmax = 1.5 B 0.2). This finding proved that the mouse hepatocyte melatonin receptor belongs to ML-1· subtype [1]. The functional significance of melatonin receptors in the liver is unknown. The fact that liver is involved in glucose metabolism and the conflicting reports of hyper-[2, 3] and hypoglycemic [4, 5] effects of melatonin have prompted us to hypothesize that melatonin may act directly on the hepatocytes to regulate the plasma glucose. This is supported in part by our findings that intraperitoneal injection of 4 mg/kg and 40 mg/kg melatonin at mid-light resulted in a respective 20% and 30% increase in plasma glucose between 45 and 60 min. Further study has to be carried out to investigate the effects of melatonin injection on hepatocyte melatonin receptors. References: 1) Pang SF, et al: Biol Signals 1993;2:27–36. 2) Ortega-Corona BG, et al: Proc West Pharmacol Soc 1991; 34:75–77. 3) John TM, et al: Gen Comp Endocrinol 1990;79:226–232. 4) Shima T, et al: Neurosci Lett 1997;226:119–122. 5 Rodriguez V, et al: Pineal Res 1989;6:77–88.