GABA PRODUCTION IN RAT ISLETS OF LANGERHANS

Homogenates of pancreatic islets catalyzed breakdown of L-glutamate to GABA with a rate of 0.24 +/- 0.04 nmol . min-1 . mg-1 protein at 37-degrees-C. The formation of GABA was stimulated by addition of pyridoxal phosphate in the range 0.05-1 muM (0.97 +/- 0.02 nmol . min-1 . mg protein-1 at a saturating cofactor concentration), which indicates that the process was catalyzed by glutamic acid decarboxylase. The half-maximal effect was obtained with 0.1 muM PLP. Kinetic analyses of the results showed that the V(max) and K(m) for the reaction were 1.12 nmol . min-1 . mg protein-1 and 0.66 mM, respectively. The pH optimum was 7.0. Subcellular fractionation revealed that 51% of GAD activity was present in the cytosol, 17% in microsomes, 9% in secretory granules, 5% in mitochondria, and 11 % in cell debris. Comparison of the kinetic properties of the cytosolic and microsomal forms of the enzyme showed that their K(m) for glutamate was the same, but that the cytosolic GAD had a lower K(m) for PLP. GABA synthesis in the nominal absence of PLP was enhanced by malate (twofold increase at 5 mM) and citrate (threefold increase at 5 mM), but was unaffected by ATP and chloride. However, if the islet homogenate was prepared and incubated in the presence of PLP, neither malate nor citrate influenced enzyme activity. Aspartate and AOA were powerful inhibitors of glutamate breakdown. Freshly isolated islets contained approximately 4 mM GABA, whereas the concentration was <0.1 mM in whole pancreas. Perifusion of islets in the absence of amino acids decreased GABA concentration by approximately 40% in 60 min; this decline could be prevented by addition of either GABA itself or gabaculine, an inhibitor of GABA-transaminase. inclusion of 5 mM GABA in the perifusion buffer raised the tissue ATP/ADP beyond the value observed in the absence of any external oxidizable substrate; the effect was eliminated by gabaculine. It is concluded that pancreatic islets contain active GAD, an enzyme responsible for GABA formation; malate and citrate may act as physiological stimulators of GAD, whereas aspartate may function as its inhibitor; and GABA serves as a source of fuel for generation of intracellular ATP in islets perifused in the absence of glucose.