Glutamate homeostasis during glutamatergic neurotransmission is predominantly maintained via functioning of the glutamate-glutamine cycle. However, the glutamate-glutamine cycle explains only the fate of the carbon atoms but not that of the accompanying transfer of nitrogen from neurons to astrocytes. In this respect, a putative branched-chain amino acid (BCAA) shuttle has been suggested for transfer of amino nitrogen. Metabolism of BCAAs was investigated in cultured cerebellar astrocytes in a superfusion paradigm employing (15)N-labeled leucine, isoleucine, or valine. Some cultures were exposed to pulses of glutamate (50 microM; 10 sec every 2 min; 75 min in total) to mimic conditions during glutamatergic synaptic activity. (15)N labeling of glutamate, aspartate, glutamine, alanine, and the three BCAAs was determined by using mass spectrometry. Incorporation of (15)N into intracellular glutamate from [(15)N]leucine, [(15)N]isoleucine, or [(15)N]valine amounted to about 40-50% and differed only slightly among the individual BCAAs. Interestingly, label (%) in glutamate from [(15)N]valine was not decreased upon exposure to exogenous glutamate, which was in contrast to a marked decrease in labeling (%) from [(15)N]leucine or [(15)N]isoleucine. This suggests an up-regulation of transamination involving only valine during repetitive exposure to glutamate. It is suggested that valine in particular might have an important function as an amino acid translocated between neuronal and astrocytic compartments, a function that might be up-regulated during synaptic activity.