Biochemical characteristics of the gamma-aminobutyric acid system in the insulinoma cell lines HIT-T15, RIN-m5F, beta TC3, and comparison with rat brain

Background. gamma -Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the mammalian brain. Both GABA and its synthesizing en zyme, L-glutamate decarboxylase (GAD), are also present in the insulin-secr eting pancreatic beta cells, in which its physiologic role is unclear. We h ave studied several aspects of the GABA system in the insulinoma cell lines HIT-TI5, RIN-m5F, and beta TC3 in comparison with rat brain tissue. Methods. Insulinoma cell lines and embryonic rat brain cortex neurons were cultured. GAD activity was determined by a radioenzymatic method and the pr esence of GAD(67) protein was assessed by immunocytochemistry. Amino acid c ontent and the effect of different conditions on the release of endogenous GABA were measured by HPLC and fluorometric detection after o-phthaldialdeh yde derivatization. [H-3]GABA was used for measuring the uptake of the amin o acid in the insulinoma cultures and in rat forebrain synaptosomes. Results. The three insulinoma lines possess GABA and GAD activity at levels of approximately 20% compared with adult rat brain cortex. Dissimilar from the latter, in insulinoma cultures enzyme activity was not enhanced by add ition of an excess of the coenzyme pyridoxal-5 ' -phosphate. Immunocytochem ical visualization of GAD showed that the cells in both neuronal cultures a nd insulinoma lines were GAD(67)-positive, Similar to Purkinje cell somata of adult rat cerebellar cortex. [H-3]GABA uptake in the cell lines was appr oximately 10% of that in rat forebrain synaptosomes and showed less ionic a nd temperature dependence, In both cultured cerebral neurons and RINm5F cel ls, the addition of arginine induced the release of GABA, whereas neither h igh K+ concentration nor glucose had any effect. Conclusions. The insulinoma cell lines studied possess the same GAD67 form of the enzyme present in brain. RIN line cells are capable of transporting glutamate. In these cells as well as in cultured cortical neurons, arginine stimulates the release of GABA and glutamate probably as the result of its electrogenic transport. Insulinoma cell lines may therefore be useful to s tudy GABA metabolism and function in pancreatic beta cells. (C) 2001 IMSS, Published by Elsevier Science Inc.