Cold-shock-stimulated gamma-aminobutyric acid synthesis is mediated by an increase in cytosolic Ca2+, not by an increase in cytosolic H+

Synthesis of the nonprotein amino acid gamma-aminobutyric acid is stimulated within minutes by diverse environmental factors. Synthesis (L-Glu + H+ --> gamma-aminobutyric acid + CO2) is catalysed by L-Glu decarboxylase, a cytosolic enzyme having an acidic pH optimum. This study uses isolated Asparagus sprengeri (Regel) mesophyll cells to investigate the possible role of Ca2+ in stimulated gamma-aminobutyric acid synthesis. Abrupt cold shock (20 degrees C to 1 degrees C) stimulated gamma-aminobutyric acid levels from 2.7 to 5.6 nmol/10(6) cells within 15 min. This 100% increase was reduced to 28% in the presence of the Ca2+ channel blocker lanthanum, and was significantly reduced by incubation with 1 mM of the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide. Incubation at 20 degrees C with 25 mu M calcimycin, a Ca2+ ionophore, increased levels by 61% within 15 min. A fluorescent Ca2+ indicator demonstrated that cytosolic Ca2+ increased within 2 s of cold shock, followed by a return to initial levels within 25 s. In contrast, comparable experiments indicate a rapid and prolonged decrease in cytosolic H+. L-Glu decarboxylase isolated from asparagus cladophylls was stimulated 100% by addition of 500 mu M Ca2+ and 200 nM calmodulin. This activity was reduced to control values by the calmodulin antagonist. Collectively, the data indicate that cold shock initiates a signal transduction pathway in which increased cytosolic Ca2+ stimulates calmodulin-dependent L-Glu decarboxylase activity and gamma-aminobutyric acid synthesis. This mechanism appears independent of increased H+.