Lithium Suppresses Signaling and Induces Rapid Sequestration of 2-Adrenergic Receptors Sergey Doronin,* Elena Shumay,* Hsien-yu Wang,† and Craig C. Malbon* ,1 *Department of Molecular Pharmacology, University Medical Center, SUNY/Stony Brook, Stony Brook, New York 11794-8651; and †Department of Physiology and Biophysics, Diabetes and Metabolic Diseases Research Program, University Medical Center, SUNY/Stony Brook, Stony Brook, New York 11794-8661 Received September 4, 2001 Lithium is a monovalent cation used therapeutically to treat a range of affective disorders (1), although the cellular mechanisms of lithium regulation that might contribute to its therapeutic effects at the level of neurotransmitter receptors are not known. Herein we report the ability of lithium to stimulate the internal- ization of 2-adrenergic receptors. Lithium treatment of A431 human epidermoid carcinoma cells resulted in a rapid, prominent desensitization and internaliza- tion of 2-adrenergic receptors. The ability of these receptors to generate a cyclic AMP response was strongly inhibited by lithium, at concentrations ther- apeutic in humans. Receptors for the serotonin (5HT1c) and for opiates (-opioid), in sharp contrast, resisted the effects of lithium on internalization. These data provide the first receptor-based mecha- nism to be described for lithium that could explain, in part, the therapeutic effects of lithium on affective disorders. © 2001 Academic Press Key Words: lithium; 2-adrenergic receptors; signal- ing; sequestration; Na-channels. Lithium has a long history as a useful drug in the treatment of various affective disorders, especially bi- polar disease. In the last decade, insights in possible mechanisms of lithium action have been revealed, in- cluding its effects on nerve conduction, neurotransmit- ter uptake, and neuronal excitability (2). Neurotrans- mitters, such as the catecholamine norepinephrine, have been implicated in affective disorders (3) and act via a well-known signaling paradigm, in which norepi- nephrine binds to receptors that couple via heterotri- meric G-proteins to effectors such as adenylylcyclase (4, 5). In spite of the level of knowledge that has accu- mulated, little is known to what extent, if any, are the therapeutic effects of lithium exerted directly at the level of these G-protein coupled receptors (GPCRs). MATERIALS AND METHODS Cell culture. Human epidermoid carcinoma cells (A431) were maintained in Dulbecco’s modified Eagle medium (DMEM) supple- mented with 10% fetal bovine serum (HyClone, Logan, UT), penicil- lin (60 g/ml), and streptomycin (100 g/ml) and grown in a humid- ified atmosphere of 5% CO 2 and 95% air at 37°C (6). Transfection of GFP-tagged 2-adrenergic, 5HT1c, and -opioid receptor. A431 cells were transfected with expression vectors har- boring the GFP-tagged  2 AR using Lipofectin (Life Technologies, Inc.), according to the manufacturer’s protocol and viable clones selected in 400 g/ml of the neomycin analogue G418. Resistant colonies were subcloned and screened for GFP-fusion protein expres- sion by epifluorescence microscopy. Transfections with the 5HT1c and opioid receptor expression vectors were transient. Epifluorescence imaging. Microscopy of live cells was performed on the Eclipse TE300 (Nikon) inverted microscope equipped with 40 oil objective. Images were acquired using MicroMAX Imaging System (Princeton Instruments Inc.) and WinView32 software (7). Inhibitor studies. A431 cells and stably transfected clones were routinely challenged without or with either lithium (5 min) and then with or without isoproterenol (10 M) for 30 min and the trafficking of the GFP-tagged  2 AR monitored by epifluorescence. Cells were serum-deprived for 8 h prior to remove growth factors and catechols from the cell media. For studies of the effects of inhibitors on the trafficking of the GFP-tagged receptor in response to either isopro- terenol or lithium (or both), the inhibitors were added 30 – 40 min in advance of the challenge with drugs. The inhibitors and the concen- trations at which they were employed are as follows: amiloride (100 M); genistein (5 M); and LY294002 (10 M) to inhibit 1-phosphatidylinositol 3-kinase (8). RESULTS AND DISCUSSION To address the effects of lithium on GPCRs, A431 human epidermoid carcinoma cells propagated in cul- ture were treated with a -adrenergic agonist and their cyclic AMP responses measured (Fig. 1A). Treatment with lithium at a final concentration of 1 mM resulted in a 50% decline in the ability of the cells to respond 1 To whom correspondence should be addressed. Fax: 516-444- 7696. E-mail: craig@pharm.sunysb.edu. Biochemical and Biophysical Research Communications 288, 151–155 (2001) doi:10.1006/bbrc.2001.5755, available online at http://www.idealibrary.com on 151 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.