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2; 171 Ci/mmol, New England Nuclear). After rinsing with Krebs buffer containing BSA, we subjected the cells to lipid extraction and counted radioactivity in the extracts. On average, neurons contained 245 ± 65 dpm per well and astrocytes 302 ± 20 dpm per well; nonspecific accumulation in astrocytes at 0° to 4°C was 355 ± 28 dpm per well (n = 6).
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max values were 29 pmol/min per milligram of protein without AM404 and 26 pmol/min per milligram of protein with AM404, respectively (n = 6).
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The amounts of cAMP in the presence of a concentration of WIN-55212-2 below threshold (1 nM, determined in preliminary experiments) were 96.7 ± 2.5% of forskolin alone and were not significantly affected by 10 μM AM404 (89.8 ± 2.6%), 10 μM AM403 (92.4 ± 2.3%), or 10 μM bromcresol green (92.9 ± 2.3%) (n = 3). In the presence of a concentration of glutamate below threshold (3 μM) (24), cAMP concentrations were 91.6 ± 2% of forskolin alone and were not significantly affected by AM404 (84.4 ± 4.9%), AM403 (89.5 ± 2.4%), or bromcresol green (84.4 ± 3%) (n = 3).
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The hot plate test (55.5°C) was carried out on male Swiss mice (25 to 30 g, Nossan, Italy) following standard procedures [F. Porreca, H. L. Mosberg, R. Hurst, V. J. Hruby, T. F. Burks, J. Pharmacol. Exp. Ther. 230, 341 (1994)]. Anandamide and AM404 were dissolved in 0.9% NaCl solution containing 20% dimethyl sulfoxide and injected intravenously at 20 mg/kg and 10 mg/kg, respectively. To determine whether cannabinoid receptors participate in the effect of anandamide, we administered anandamide (20 mg/kg intravenously) or anandamide plus SR141716-A (2 mg/kg, subcutaneously) to two groups of six mice each. In mice that received anandamide alone, latency to jump increased from 21.7 ± 1.5 s to 30.7 ± 0.8 s (P < 0.05, ANOVA) 20 min after injection. In contrast, in mice that received anandamide plus SR141716-A, the latency to jump was not affected (19.6 ± 3.1 s).
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1842307203
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note
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We thank E. di Tomaso and H. Cadas for help and E. Barker, L. Parsons, and P. Schweitzer for critical reading of the manuscript. Supported by the Neuroscience Research Foundation, which receives major support from Novartis.
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