Gamma-aminobutyric acid uptake and localization in bovine chromaffin cells in primary culture

Abstract

γ-Aminobutyric acid (GABA) uptake was studied in bovine chromaffin cells maintained in primary culture. Uptake was found to be dependent on Na + but not on K + and Ca 2+ ions; it was found that 2 Na + ions were necessary for each molecule of GABA transported. 2,4-Dinitrophenol, ouabain and vanadate inhibited GABA uptake showing the energy dependency of the system. Two affinity sites were demonstrated, a high affinity site and a low affinity site with K m values of 10 μM and 170 μM, respectively. While the low affinity site did not show large variations with culture age, the K m of thehigh affinity site increased from 1 μM in freshly isolated cells to 10 μM in 3–9 day-old cells. GABA uptake was unaffected by glutamic acid, aspartic acid, glycine and catecholamines, while taurine, betaalanine, nipecotic acid and l-2,4 diaminobutyric acid inhibited GABA uptake. Nipecotie acid and l-2,4 diaminobutyric acid acted as competitive inhibitors modifying K m values of the high affinity site. Subcellular studies performed on [ 3H]GABA-loaded chromaffin cells showed that GABA was not in secretory granules but was recovered in the 100,000 g soluble fraction. The GABA uptake process associated with chromaffin cells may be an important mechanism for regulating the modulation of catecholamine secretion. In addition, the presence of GABA in the cytosol indicates that this molecule may be an effector of chromaffin cell activity in addition to modulating catecholamine secretion.