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

Abstract

Synthesis of the nonprotein amino acid γ-aminobutyric acid is stimulated within minutes by diverse environmental factors. Synthesis (L-Glu + H+– γ-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 γ-aminobutyric acid synthesis. Abrupt cold shock (20 °C to 1 °C) stimulated γ-aminobutyric acid levels from 2.7 to 5.6 nmol/106cells 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 °C with 25 μM 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 μM 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 γ-aminobutyric acid synthesis. This mechanism appears independent of increased H+. Key words: cold shock, GABA, Ca2+, H+.