Glutamate Receptors Communicate with Na+/K+-ATPase in Rat Cerebellum Granule Cells: Demonstration of Differences in the Action of Several Metabotropic and Ionotropic Glutamate Agonists on Intracellular Reactive Oxygen Species and the Sodium Pump (original) (raw)
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Clinical and Experimental Pharmacology and Physiology, 2004
1. It has been suggested that Na + /K + -ATPase and Na +dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na + /K + -ATPase activity using Rb + uptake by 'minislices' (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb + was determined by estimating Rb + in aqueous extracts of the minislices, using atomic absorption spectroscopy.
Vulnerability of Medium Spiny Striatal Neurons to Glutamate: Role of Na + /K + ATPase
European Journal of Neuroscience, 1995
In Huntington's disease neuronal degeneration mainly involves medium-sized spiny neurons. It has been postulated that both excitotoxic mechanisms and energy metabolism failure are implicated in the neuronal degeneration observed in Huntington's disease. In central neurons, >40% of the energy released by respiration is used by Na+/K+ ATPase to maintain ionic gradients. Considering that impairment of Na+/K+ ATPase activity might alter postsynaptic responsivity to excitatory amino acids (EAAs), we investigated the effects of the Na+/K+ ATPase inhibitors, ouabain and strophanthidin, on the responses to different agonists of EAA receptors in identified medium-sized spiny neurons electrophysiologically recorded in the current-and voltage-clamp modes. In most of the cells both ouabain and strophanthidin (1-3 pM) did not cause significant change in the membrane properties of the recorded neurons. Higher doses of either ouabain (30 pM) or strophanthidin (30 pM) induced, per se, an irreversible inward current coupled to an increase in conductance, leading to cell deterioration. Moreover, both ouabain (1-1 0 yM) and strophanthidin (1-1 0 pM) dramatically increased the membrane depolarization and the inward current produced by subcritical concentrations of glutamate, AMPA and NMDA. These concentrations of Na+/Kt ATPase inhibitors also increased the membrane responses induced by repetitive cortical activation. In addition, since it had previously been proposed that dopamine mimics the effects of Na+/K+ ATPase inhibitors and that dopamine agonists differentially regulate the postsynaptic responses to EAAs, we tested the possible modulation of EAA-induced membrane depolarization and inward current by dopamine agonists. Neither dopamine nor selective dopamine agonists or antagonists affected the postsynaptic responses to EAAs. Our experiments show that impairment of the activity of Na+/K+ ATPase may render striatal neurons more sensitive to the action of glutamate, lowering the threshold for the excitotoxic events. Our data support neither the role of dopamine as an ouabain-like agent nor the differential modulatory action of dopamine receptors on the EAA-induced responses in the striatum.
Journal of Neurochemistry, 2002
In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca 2~'and Naĩ nto 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 mm (85% activation), and was maintained for at least 2 h. Glutamateinduced activation of Na,K~-ATPasewas dose dependent: It was appreciable (37%) at 0.1 ftM and peaked (85%) at 100
Glutamate transporter coupling to Na, K-ATPase
2009
Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The ␣1-␣3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using [ 3 H]D-aspartate, [ 3 H]L-glutamate, and rubidium-86 uptake into synaptosomes and cultured astrocytes. In synaptosomes, ouabain produced a dose-dependent inhibition of glutamate transporter and Na,K-ATPase activities, whereas in astrocytes, ouabain showed a bimodal effect whereby glutamate transporter activity was stimulated at 1 M ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [ 3 H]D-aspartate uptake indicated the selective involvement of Src kinases, which are probably a component of the Na,K-ATPase/glutamate transporter complex. These findings demonstrate that glutamate transporters and Na,K-ATPases are part of the same macromolecular complexes and operate as a functional unit to regulate glutamatergic neurotransmission.
The Journal of Biological Chemistry, 1994
Incubation of purified renal Na,K-ATPase with the fluorescent carboxyl-selective reagent, tl-(diazomethyl)-7-(diethylamino)-coum~n (DEAC), results in enzyme inactivation via disruption of the monovalent cation binding sites and loss of K+ and Na' binding capacity. ~odification of 1 or 2 carboxyl residues in the a-subunit in a K+ or Na+-preventable manner leaves the ATP binding unaltered, and the enzyme is still able to undergo the major conformational transitions (Argiiello, J. M., and
Cell Biochemistry and Function, 2004
Excessive excitatory action of glutamate and nitric oxide (NO) has been implicated in degeneration of striatal neurons. Evidence had been provided that Na þ K þ-ATPase might be involved in this process. Here we investigated whether glutamateregulated messengers, such as NO and cyclic GMP, could modulate the activity of membrane Na þ K þ-ATPase. Our results demonstrated that NO donors sodium nitroprusside (SNP at 30 and 300 mM) and S-nitroso-N-acetylpenicillamine (SNAP at 200 mM) increased 2,3 Na þ K þ-ATPase activity which was blocked by the NO chelator, haemoglobin and was independent of [Na þ ]. This regulation was associated with cGMP synthesis and mimicked by glutamate (300 mM) and 8-Br-cyclic GMP (4 mM). 8-Br-cGMP-induced stimulation of Na þ K þ-ATPase activity could be blocked by KT5823 (an inhibitor of cGMP-dependent protein kinase, PKG), but not by KT5720 (an inhibitor of cAMP-dependent protein kinase, PKA). N-Methyl-D-aspartate (NMDA) receptors appeared to be involved in the effect of glutamate, since MK-801 (NMDA receptor antagonist) produced a partial reduction in glutamate-induced activation of the enzyme. MK-801 was not synergistic to L-NAME (NOS inhibitor), suggesting that glutamate stimulates the NMDA-NOS pathway to activate 2,3 Na þ K þ-ATPase in rat striatum. This regulation was associated with cyclic GMP (but not cyclic AMP) synthesis. These data indicate the existence, in vitro, of a regulatory pathway by which glutamate, acting through NO and cGMP, can cause alterations in striatal 2,3 Na þ K þ-ATPase activity.
Cross-talk between glutamate and nucleotide receptors in cerebellar granule neurons in culture
ATP elicits Ca 2+ transients in cultured cerebellar granule neurons acting through specific ionotropic (P2X) and metabotropic (P2Y) purinergic receptors. In these neurons, application of L-Glutamate (L-Glu) immediately before ATP induced a prolonged reduction of ATP-mediated responses that remains at least 5 minutes after L-Glu wash out. alpha-amino-3-hydro-5-methyl-4-isoxazolpropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and 3,5-dihydroxyphenyl-glycine (DHPG), selective agonists of ionotropic non-NMDA, NMDA and Group I metabotropic glutamate receptors respectively, mimicked Glu-induced attenuating effects. The activity of calciumcalmodulin dependent protein kinase II (CaMKII) seems to be involved, at least at long term, because inhibitors of CaMKII, 1- , abolished the inhibitory effect of L-Glu on ATP-mediated responses. However, it is likely that other protein kinases could be involved in the cross-talk process between both groups of receptors at short term. Therefore, these results demonstrate that the activation of glutamate receptors is able to modulate nucleotide responses in cerebellar granule neurons.
FEBS Letters, 1999
Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10 33 M) for 25 min apparently swell, increase their [Ca 2+ ] i with obvious depolarization of the mitochondrial membrane. In 3 h after ouabain was omitted from the solution, 62 þ 3% of granule cells had pycnotic nuclei. The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosphonoheptanoate (10 34 M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of [Ca 2+ ] i. These data suggest that cellular Na + /K +-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca 2+ and Na + influxes into the cells through the channels activated by Glu. This process leads to cell swelling, mitochondrial deenergization and death of granule cells. Possibly, the decrease of Na + /K +-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders.
Effects of activation of glutamate receptors on neurons and blood vessels
International Congress Series, 2002
Glutamate dilates pial arterioles in piglets via activation of N-methyl-D-aspartate (NMDA) receptors and increases cortical glucose utilization. Administration of NMDA, the chemical compound which was used to characterize this glutamate receptor subtype, dilates cerebral arteries via activation of nitric oxide synthase (NOS) in neurons and subsequent actions of nitric oxide (NO) on vascular smooth muscle. Thus, administration of inhibitors of NOS attenuates NMDA-induced arteriolar dilation, while inhibitors of cyclooxygenase and cytochrome P-450 epoxygenase do not alter the vascular response. Additionally, inhibition of adenosine receptors and endothelial ''stunning'' do not alter arteriolar dilation to NMDA. Finally, NO metabolites accumulate on the cortical surface following NMDA application. We conclude that NMDA-induced arteriolar dilation is via direct actions on smooth muscle of NO synthesized and released by cortical neurons. NMDAinduced dilator responses are severely restricted after ischemia-reperfusion. Previous studies have shown that potassium channel activators given prior to ischemia preserve responses after ischemia. However, intracellular localization of this effect is unclear. We provide evidence to indicate that activation of K ATP on mitochondria by diazoxide is able to provide neuroprotection against ischemic stress, probably by restricting calcium entry into mitochondria. Use of selective activators of K ATP on mitochondria could be a new therapeutic approach to protect the brain against ischemic stress.