Biochemical characteristics of the gamma-aminobutyric acid system in the insulinoma cell lines HIT-T15, RIN-m5F, betaTC3, 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 enzyme, L-glutamate decarboxylase (GAD), are also present in the insulin-secreting pancreatic beta cells, in which its physiologic role is unclear. We have studied several aspects of the GABA system in the insulinoma cell lines HIT-T15, RIN-m5F, and betaTC3 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 presence of GAD(67) protein was assessed by immunocytochemistry. Amino acid content and the effect of different conditions on the release of endogenous GABA were measured by HPLC and fluorometric detection after o-phthaldialdehyde derivatization. [3H]GABA was used for measuring the uptake of the amino 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 addition of an excess of the coenzyme pyridoxal-5'-phosphate. Immunocytochemical visualization of GAD showed that the cells in both neuronal cultures and insulinoma lines were GAD(67)-positive, similar to Purkinje cell somata of adult rat cerebellar cortex. [3H]GABA uptake in the cell lines was approximately 10% of that in rat forebrain synaptosomes and showed less ionic and temperature dependence. In both cultured cerebral neurons and RINm5F cells, the addition of arginine induced the release of GABA, whereas neither high K(+) concentration nor glucose had any effect.


CONCLUSIONS
The insulinoma cell lines studied possess the same GAD(67) 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 study GABA metabolism and function in pancreatic beta cells.
Salazar
P
P
Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico.
del Carmen Sánchez-Soto
M
M
Hiriart
M
M
Tapia
R
R
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015415:Biomarkers; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000737:chemistry; D005968:Glutamate Decarboxylase / Q000032:analysis*; D006801:Humans; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology*; D009363:Neoplasm Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000378:metabolism; D009928:Organ Specificity; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D020033:Protein Isoforms / Q000032:analysis; D051381:Rats; D014407:Tumor Cells, Cultured / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
D000596:Amino Acids; D015415:Biomarkers; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase