Glutamate Induces a Calcineurin‐Mediated Dephosphorylation of Na⁺,K⁺‐ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract

In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+ and Na+ into the neuron. To maintain Na+ homeostasis, the excess Na+ entering through the ion channel should be removed by Na+,K+‐ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na+,K+‐ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate‐induced activation of Na+,K+‐ATPase was dose dependent: It was appreciable (37%) at 0.1 µM and peaked (85%) at 100 µM. The increase in Na+,K+‐ATPase activity by glutamate was prevented by MK‐801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12‐myristate 13‐acetate, an activator of protein kinase C, indicating that activation of Na+,K+‐ATPase is due to decreased phosphorylation by protein kinase C. W‐7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na+,K+‐ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K+‐ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na+,K+‐ATPase.

Searching for Mechanisms of N-Methyl-d-Aspartate-Induced Glutathione Efflux in Organotypic Hippocampal Cultures

“…Glutamate-mediated elevations of cAMP and Ca 2+ are attenuated by W7, presumably via inhibition of calmodulin-dependent activation of adenylate cyclase and CaMKII, respectively (71). Increased Na + ,K + -ATPase after glutamate application to cultured cerebellar neurones was reduced by W7 and by the calcineurin inhibitor cyclosporin, suggesting an involvement of calmodulin-calcineurin-dependent dephosphorylation (72). Interestingly, W7 prevents glutamate-induced depletion of ATP and neuronal death in cultured cerebellar neurones (73).…”

Glutamate modulates sodium‐potassium‐ATPase through cyclic GMP and cyclic GMP‐dependent protein kinase in rat striatum

“…In primary cultures of cerebellar neurons, glutamate induced activation of Na þ K þ -ATPase activity that was completely blocked by MK-801 (an antagonist of NMDA receptor). 37 This result could be linked to differences between slices and primary culture cells since the former do not only contain neurons. However, from both studies we can conclude that glutamate can stimulate Na þ K þ -ATPase by the NMDA pathway.…”

The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes

“…Data on the role of free-radical reactions in disturbing intracellular Ca 2+ concentrations are in some cases contradictory [29,32]. Deciphering of the mechanisms of glutamate neurotoxicity is also made difficult by the fact that they can differ according to the stage of development and local types of nerve cells [22,23,28,37]. A literature search yielded no systematic studies of the effects of glutamate and antioxidants on a variety of parameters characterizing calcium ion metabolism in nerve cells.…”