Binding of Nicotinic Ligands to and Nicotine-Induced Calcium Signaling in Trypanosoma cruzi Mariana Bollo,* Graciela Venera,† Mirtha Biscoglio de Jime ´nez Bonino,† and Estela Machado-Domenech* ,1 *Quı´micaBiolo ´gica, Facultad de Ciencias Exactas, Fisico-Quı ´micas y Naturales, Universidad Nacional de Rı ´o Cuarto, 5800 Rı´o Cuarto, Co ´rdoba, Argentina; and †Departamento de Quı ´mica Biolo ´gica, Instituto de Quı´mica y Fisicoquı´mica Biolo ´gicas (UBA-CONICET), Junı ´n 956, 1113 Buenos Aires, Argentina Received January 12, 2001 This work provides evidence that nicotine (1  10 5 M) can cause changes in the intracellular cal- cium concentration of Trypanosoma cruzi epimasti- gotes, which can be blocked by -bungarotoxin but not by atropine. Moreover, parasite membranes also bind such nicotinic acetylcholine receptor antago- nist as well as agonists such as carbamylcholine (IC 50 : 7.6  10 7 M) and nicotine (IC 50 :1  10 7 M). Results suggest that there is a molecular species in the surface of the parasite able to bind nicotinic ligands; therefore, nicotine interaction could lead to the activation of the mechanisms involved in intra- cellular calcium concentration increase in the parasite. © 2001 Academic Press Key Words: Trypanosoma cruzi; nicotinic ligands; nicotine; -bungarotoxin; calcium; carbamylcholine. The characteristics of several signal transduction pathways in T. cruzi have been described (1–7). Meta- bolic changes occurring in the parasite after cholin- ergic stimulation have been reported (3–5). Carbamyl- choline induces changes in polyphosphoinositides and phosphatidic acid turnover as well as variations of inositol phosphates through phospholipase C activa- tion. Nonneuronal acetylcholine appears to be involved in the regulation of elementary cell functions such as cell mitosis and motion, ciliary activity; immune cells (macrophages, mast cells, lymphocytes) also seem to be regulated by the cholinergic system. Furthermore, ace- tylcholine has also been detected in very primitive plants and in unicellular organisms (including ciliated protozoa) indicating that the synthesis of acetylcholine occurred at a very early step in the evolutionary pro- cess. The existence of nonneuronal acetylcholine ex- plains the widespread expression of muscarinic and nicotinic receptors in cells not inervated by cholinergic neurons (8). Moreover, their ligand-recognition and channel domains may have originally been two differ- ent proteins that gradually fused over the lengthy course of evolution (9). T. cruzi is the agent causing Chagas disease, an extraordinarily complex process in which peripheral nervous system damage is frequently observed, with alterations of the neuromuscular junction in human chronic Chagas disease (10, 11). Goin et al. (12), showed the ability of human chagasic serum to bind nicotinic acetylcholine receptors, pentamers with high affinity for agonists as nicotine, acetylcholine, its ana- logue carbamylcholine and antagonists as -BgTx. In this paper we investigate the ability of nicotine to induce changes in the [Ca 2+ ] i and the binding of car- bamylcholine and nicotine to the T. cruzi epimastigote membranes. In addition, we describe herein the speci- ficity and saturability of such binding. MATERIALS AND METHODS Organisms and growth conditions. The T. cruzi Tulahuen strain was used in this study. Epimastigote forms were grown during 6 days at 28°C in a modified Warren medium (13), as described by Racagni et al. (2). Preparation of epimastigote membranes. The washed cells were suspended in 5 volumes of 50 mM Hepes (pH 7.4) containing 0.25 M sucrose, 5 mM KCl, 1mM EDTA and 4 g/ml leupeptin (homogenate buffer). This suspension was frozen at -180°C and thawed three times, homogenized and centrifuged at 100g for 15 min to remove unbroken cells and cell debris. The supernatant was then centri- fuged at 1000g, for 15 min to obtain the membrane fraction (14, 15) used throughout the work. The membranes were washed and resus- pended in the minimum possible volume of the homogenate buffer and stored at -80°C until used. Extraction and purification of the receptor. The receptor was affinity purified from frozen electric organ tissue of Torpedo califor- nica on an Affi-Gel 10 (bromoacetylcholine as the ligand) column according to Venera et al. (16). The receptor was stored at -70°C in Abbreviations used: -BgTx, -Bungarotoxin; [Ca 2+ ] i , intracellular calcium concentration. 1 To whom correspondence should be addressed. Fax: 54 358 467 6232. E-mail: emachado@exa.unrc.edu.ar. Biochemical and Biophysical Research Communications 281, 300 –304 (2001) doi:10.1006/bbrc.2001.4344, available online at http://www.idealibrary.com on 300 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.