Characterization of the nipecotic binding to rat brain membranes

1. The specific binding of [3H]Nip and [3H]GABA to rat brain membranes has been reexamined. 2. Specific binding of [3H]Nip and [3H]GABA was exclusively dependent on sodium ions in a cooperative manner (Hill coefficient 2.2 +/- 0.2 and 2.4 +/- 0.3 for [3H]Nip and [3H]GABA binding, respectively). Potassium, lithium or choline chloride salts were ineffective for replacing NaCl. 3. Maximal binding was observed at 2 degrees C. The association constants were K+1 = 0.033 min-1 microM-1 for [3H]GABA. The dissociation constants obtained by the addition of 1 mM of non-labeled GABA were 0.087 +/- 0.01 min-1 and 0.139 +/- 0.025 min-1 for [3H]Nip and [3H]GABA, respectively. Scatchard curves were in agreement with these constants, KD = 1.6 microM for [3H]GABA and 2.6 microM for [3H]Nip with equal Bmax = 138 pmol per mg protein. 4. The dissociation constants for [3H]Nip bound increased from 0 to 0.035, 0.18 and 0.58 min-1 at 2, 16, 26 and 37 degrees C, respectively, contrary to the hydrophobic derivate of nipecotic acid, NO 328. 5. Pharmacological characterization and regional brain distribution of [3H]Nip and [3H]GABA binding and uptake, suggest that [3H]Nip specifically labels the neuronal GABA uptake system, absent in peripheral tissues.