Glutamine cycle enzymes in the crayfish giant nerve fiber: implications for axon-to-glia signaling

Abstract

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.