The Synthesis of [gamma]-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH

Abstract

[gamma]-Aminobutyric acid (GABA) synthesis (L-glutamic acid + H+ -> GABA + CO2) is rapidly stimulated by a variety of stress conditions including hypoxia. Recent literature suggests that GABA production and concomitant H+ consumption ameliorates the cytosolic acidification associated with hypoxia or other stresses. This proposal was investigated using isolated asparagus (Asparagus sprengeri Regel) mesophyll cells. Cell acidification was promoted using hypoxia, H+/L-glutamic acid symport, and addition of butyrate or other permeant weak acids. Sixty minutes of all three treatments stimulated the levels of both intracellular and extracellular GABA by values ranging from 100 to 1800%. At an external pH of 5.0, addition of 5 mM butyrate stimulated an increase in overall GABA level from 3.86 (0.56 [plus or minus] SE) to 20.4 (2.16 [plus or minus] SE) nmol of GABA/106 cell. Butyrate stimulated GABA levels by 200 to 300% within 15 s, and extracellular GABA was observed after 10 min. The acid load due to butyrate addition was assayed by measuring [14C]butyrate uptake. After 45 s of butyrate treatment, H+-consuming GABA production accounted for 45% of the imposed acid load. The cytosolic location of a fluorescent pH probe was confirmed using fluorescent microscopy. Spectrofluorimetry indicated that butyrate addition reduced cytosolic pH by 0.60 units with a half-time of approximately 2 s. The proposal that GABA synthesis ameliorates cytosolic acidification is supported by the data. The possible roles of H+ and Ca2+ in stimulating GABA synthesis are discussed.