Characterization of ATP Release from Cultures Enriched in Cholinergic Amacrine-like Neurons Paulo F. Santos, Olga L. Caramelo, Arse ´ lio P. Carvalho, Carlos B. Duarte Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal Received 4 March 1999; accepted 27 April 1999 ABSTRACT: Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of -amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the lucife- rin/luciferase luminescent method. The ATP release was found to be largely Ca 2 dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and -Agatoxin IVA, but not by -Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca 2 influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca 2 channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adeno- sine deaminase, or in the presence of 1,3-dipropyl-8- cyclopentylxanthine (DPCPX), an adenosine A 1 receptor antagonist, showing that the release is tonically inhibited by the adenosine A 1 receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 340 –348, 1999 Keywords: amacrine-like cells; ATP release; adenosine; exocytosis; retina It is now recognized that adenosine 5'-triphosphate (ATP), the ubiquitous energy source of the cell, also plays a role as a neurotransmitter and as a neuromodu- lator in the nervous system (for reviews, see Burn- stock, 1996; Sperla ´gh and Vizi, 1996). The receptors for ATP have been found in a large number in nervous tissues (Barnard et al., 1997), and have been classified in the P2 purinoreceptors class, in contrast to the P1 purinoreceptors which are preferentially activated by adenosine (Burnstock, 1978). Based on their pharma- cological properties, Burnstock and Kennedy (1985) divided the P2 purinoreceptors in two subtypes, P2X and P2Y, which were later shown to also possess distinct molecular structures. The P2X receptors con- stitute a large family of at least seven subtypes (P2X 1 –P2X 7 ) of ionotropic receptors, whereas the receptors belonging to the P2Y family, with six sub- types (P2Y 1 –P2Y 7 ), are coupled to G proteins (for review, see Barnard et al., 1997). Molecular biology and functional studies have demonstrated the presence of P2X (Valera et al., 1994; Greenwood et al., 1997; Brandle et al., 1998) and P2Y (Keirstead and Miller, 1997; Newman and Zahs, 1997; Liu and Wakakura, 1998) receptors in the retina. However, not much attention has been given to the regulation of the release of endogenous ATP by retinal neurons, in contrast to considerable knowledge accumulated concerning the events leading to extra- cellular accumulation of adenosine in the retina (Blazinski et al., 1991; Paes de Carvalho et al., 1990; Santos et al., 1998a). The first evidence for the role of ATP as a retinal neurotransmitter emerged from the Correspondence to: C. B. Duarte Contract grant sponsor: Praxis XXI; contract grant numbers: 2/2.1/BIA/74/94, P/BIA/10181/1998 © 1999 John Wiley & Sons, Inc. CCC 0022-3034/99/030340-09 340