[3H]gamma‐Aminobutyric acid uptake into neuroglial cells of rat superior cervical sympathetic ganglia.

Abstract

The influx of [3H]GABA into isolated rat superior cervical ganglia was measured by radioassay, supplemented by autoradiography. Ganglia were incubated in oxygenated Krebs solution at 25.degree. C, containing 10 .mu.M-amino-oxyacetic acid. Under these conditions more than 95% of accumulated 3H was unmetabolized [3H]GABA. Ganglionic radioactivity increased linearly with incubation time, to yield an intracellular fluid/extracellular fluid concentration ratio (Ci/Co) of .apprx. 200 after 6 h in 0.5 .mu.M-external [3H]GABA. Uptake showed saturation, with an apparent transport constant (KT) of 6.8 .mu.M and maximum influx velocity .**GRAPHIC**. of 7 .mu.mol/l cell fluid per min. The influx rate at Co = 0.5 .mu.M was unaltered by raising intracellular GABA from 0.2 to 1 mM. Influx velocity increased with temperature (5-35.degree. C) in a monotonic manner with an apparent activation energy of 14 kcal/mol. Concentrative uptake was depressed by reducing external [Na+) with ouabain, by raising [K+]o above 20 mM, or by removing external Cl-. Uptake was not particularly sensitive to Ca2+ or Mg2+. Uptake of [3H]GABA (0.5 .mu.M) was inhibited by .beta.-guanidinopropionic acid (apparent KI [inhibition constant] 28 .mu.M), .beta.-alanine (KI, 55 .mu.M), .gamma.-amino-.beta.-hydroxybutyric acid (KI , 220 .mu.M), .beta.-amino-n-butyric acid (KI, 708 .mu.M), 3-aminopropanesulfonic acid (KI, 832 .mu.M) and taurine (KI > 1 mM). Uptake was not depressed by 1 mM-glycine, .alpha.-alanine, leucine, serine, methionine or .alpha.-amino-isobutyric acid. Radioactively labeled methionine, leucine, glycine, serine, .beta.-alanine and taurine (concentrations .ltoreq. 5 .mu.M) were taken up by ganglia. Only uptake of .beta.-alanine and taurine were significantly depressed by 1 mM-GABA. Autoradiographs confirmed that [3H]GABA and [3H].beta.-alanine were taken up predominantly into extraneuronal sites (presumed to be neuroglial cells). Methionine, leucine, glycine and serine showed preferential accumulation in neurons. Neuronal uptake of leucine was not prevented by inhibiting protein synthesis. Calculations of net fluxes from unidirectional tracer fluxes suggest that the sympathetic glial cells are capable of promoting net uptake of GABA at external concentrations > 1 .mu.M.