Journal of Neurochemisrry zyxwvutsrqponm Raven Press, zyxwvutsrqpo Ltd., New York zyxwvutsrqpon 0 1990 International Society for Neurochemistry Effect of zyxw 5’-(N-Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chrornaffin Cells Esmerilda G. Delicado, *Alexandra Rodrigues, Raquel P. Sen, *Ana M. Sebastiao, *J. Alexandre Ribeiro, and M. Teresa Miras-Portugal zyx Departamento de Bioquhica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain; and *Laboratory of Pharmacology, Gulbenkian Institute of Science, Oeiras, Portugal Abstract: Extracellular adenosine is transported into chro- maffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5‘-(N-Ethylcarboxamido)adenosine (NECA), an ag- onist of AZ receptors, activated adenosine transport. K, values for adenosine were 4.6 zyxwvutsrq k 1.0 (n = 5) and 10.2 ? 3.0 & 4 ( n = 5) for controls and 100 M N E C A , respectively. The V,,, values were 66.7 k 23.5 and 170.2 ? 30 pmol/106 cells/min for controls and 100 M N E C A , respectively. The Al agonist N6-cyclohexyladenosine, the zyxwvuts A I antagonist 8-cyclopentyl- 1, 3-dipropylxanthine, and the A,-A2 antagonist I ,3-dipropyl- 8- { 4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl}- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treat- ment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]- nitrobenzylthioinosine, which quantifies the plasma mem- brane transporters, showed a B,, of 19,200 * 800 and 23,200 k 700 transporters/cell for controls and 100 nM NECA, re- spectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase ac- tivation, because its action was not imitated by forskolin. Key Words: Adenosine receptors-Adenosine transport- Chromaffin cells-5’-(N-Ethylcarboxamido)adenosine-Ni- trobenzylthioinosine-Nucleoside transport. Delicado E. G. et al. Effect of 5’-(N-ethylcarboxamido)adenosine on aden- osine transport in cultured chromaffin cells. J. Neurochem. 54, 1941-1946 (1990). The granular content of catecholamines and ATP is released from chromaffin cells during exocytosis (Bur- goyne, 1984; Winkler, 1988). The ATP so released can be degraded to extracellular adenosine by the action of ectonucleotidases (Gordon et al., 1986; Ribeiro and Sebastiao, 1987; Richardson et al., 1987). Both ATP and adenosine modulate the secretion of catechol- amines from chromaffin cells (Chern et al., 1987, 1988). It is now well documented that adenosine modulates neural functions via its interaction with adenosine A, and A2 receptors, which couple in an inhibitory and in a stimulatory manner, respectively, to adenylate cy- clase (Daly et al., I98 1; Williams, 1987). Extracellular adenosine is transported into the cell by a high-affinity transport system, which terminates the actions of adenosine on extracellular receptors. The kinetic and pharmacological characterization of adenosine or nucleoside transport in chromaffin cells was camed out (Miras-Portugal et al., 1986; Torres et al., 1986) and proved to be very similar to that in other neural tissues (Bender et al., 1980; Wu and Phillis, 1984). A wide variety of drugs can inhibit the nucleo- side transport; two of the most commonly used are dipyndamole and nitrobenzylthioinosine (NBTI). These compounds, when radioactively labeled, are useful tools in quantifying the adenosine transporter density in biological samples (Marangos et al., 1985; Davies and Hambley, 1986; Jarvis, 1986; Torres et al., 1986, 1988; Plagemann and Woffendin, 1988). Although adenosine transport terminates the effects of adenosine on the plasma membrane receptors, no work has been reported on the possible interactions between adenosine transport sites and A, and A2 sites. The aim of the present work was to study the effects of the adenosine A2 agonist 5’-(N-ethylcar- boxamido)adenosine (NECA) on the kinetics of aden- Received May 23, 1989; revised manuscript received September 8, 1989; accepted October 26, 1989. Address correspondence and reprint requests to Prof. M. T. Miras- Portugal at Departamento de Bioquimica, Facultad de Veterinana, Universidad Complutense, 28040 Madrid, Spain. Abbreviations used: CHA, p-cyclohexyladenosine; DPCPX, 8- cyclopentyl-1,3-dipropylxanthine; NBTI, nitrobenzylthioinosine; NECA, 5’-(N-ethylcarboxamido)adenosine; XAC, 1,3-dipropyl-8-{4- [(2-aminoethyl)amino]-carbonylmethyloxyphenyl~xanthine. 1941