Junnan Xu

Astrocytic and neuronal accumulation of elevated extracellular K+ with a 2/3 K+/Na+ flux ratio—consequences for energy metabolism, osmolarity and higher brain function

Brain excitation increases neuronal Na+ concentration by 2 major mechanisms: (i) Na+ influx caused by glutamatergic synaptic activity; and (ii) action-potential-mediated depolarization by Na+ influx followed by repolarizating K+ efflux, increasing extracellular K+ concentration. This review deals mainly with the latter and it concludes that clearance of extracellular K+ is initially mainly effectuated by Na+,K+-ATPase-mediated K+ uptake into astrocytes, at K+ concentrations above ~10 mM aided by uptake of Na+,K+ and 2 Cl− by the cotransporter NKCC1. Since operation of the astrocytic Na+,K+-ATPase requires K+-dependent glycogenolysis for stimulation of the intracellular ATPase site, it ceases after normalization of extracellular K+ concentration. This allows K+ release via the inward rectifying K+ channel Kir4.1, perhaps after trans-astrocytic connexin- and/or pannexin-mediated K+ transfer, which would be a key candidate for determination by synchronization-based computational analysis and may have signaling effects. Spatially dispersed K+ release would have little effect on extracellular K+ concentration and allow K+ accumulation by the less powerful neuronal Na+,K+-ATPase, which is not stimulated by increases in extracellular K+. Since the Na+,K+-ATPase exchanges 3 Na+ with 2 K+, it creates extracellular hypertonicity and cell shrinkage. Hypertonicity stimulates NKCC1, which, aided by β-adrenergic stimulation of the Na+,K+-ATPase, causes regulatory volume increase, furosemide-inhibited undershoot in [K+]e and perhaps facilitation of the termination of slow neuronal hyperpolarization (sAHP), with behavioral consequences. The ion transport processes involved minimize ionic disequilibria caused by the asymmetric Na+,K+-ATPase fluxes.

Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation, MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling

Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α2 isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK1/2 and PI3K/AKT; interaction between EGF receptor, α1, and α2 isoforms of Na,K-ATPase, Src, ERK1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR(845) and EGFR(1068); (ii) ammonia-induced ERK1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK1/2 phosphorylation; (iv) ammonia stimulates association between the α1 Na,K-ATPase isoform, Src, EGF receptor, ERK1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK1/2, but in response to 3mM ammonia it does not appear until after 12h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.

Brain glycogenolysis, adrenoceptors, pyruvate carboxylase, Na(+),K(+)-ATPase and Marie E. Gibbs' pioneering learning studies.

The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., 2013). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present “perspective” suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either (1) activation of astrocytic receptors, perhaps especially β-adrenergic or (2) even small increases in extracellular K+ concentration above its normal resting level. It discusses (1) the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; (2) the established role of K+-stimulated glycogenolysis for K+ uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K+ homeostasis in the brain in vivo; and (3) the plausible role of transmitter-induced glycogenolysis, stimulating Na+,K+-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K+ concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP).

Role of Glycogenolysis in Stimulation of ATP Release from Cultured Mouse Astrocytes by Transmitters and High K+ Concentrations

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.

Role of the Astrocytic Na+, K+-ATPase in K+ Homeostasis in Brain: K+ Uptake, Signaling Pathways and Substrate Utilization

This paper describes the roles of the astrocytic Na+, K+-ATPase for K+ homeostasis in brain. After neuronal excitation it alone mediates initial cellular re-accumulation of moderately increased extracellular K+. At higher K+ concentrations it is assisted by the Na+, K+, 2Cl− transporter NKCC1, which is Na+, K+-ATPase-dependent, since it is driven by Na+, K+-ATPase-created ion gradients. Besides stimulation by high K+, NKCC1 is activated by extracellular hypertonicity. Intense excitation is followed by extracellular K+ undershoot which is decreased by furosemide, an NKCC1 inhibitor. The powerful astrocytic Na+, K+-ATPase accumulates excess extracellular K+, since it is stimulated by above-normal extracellular K+ concentrations. Subsequently K+ is released via Kir4.1 channels (with no concomitant Na+ transport) for re-uptake by the neuronal Na+, K+-ATPase which is in-sensitive to increased extracellular K+, but stimulated by intracellular Na+ increase. Operation of the astrocytic Na+, K+-ATPase depends upon Na+, K+-ATPase/ouabain-mediated signaling and K+-stimulated glycogenolysis, needed in these non-excitable cells for passive uptake of extracellular Na+, co-stimulating the intracellular Na+-sensitive site. A gradual, spatially dispersed release of astrocytically accumulated K+ will therefore not re-activate the astrocytic Na+, K+-ATPase. The extracellular K+ undershoot is probably due to extracellular hypertonicity, created by a 3:2 ratio between Na+, K+-ATPase-mediated Na+ efflux and K+ influx and subsequent NKCC1-mediated volume regulation. The astrocytic Na+, K+-ATPase is also stimulated by β1-adrenergic signaling, which further stimulates hypertonicity-activation of NKCC1. Brain ischemia leads to massive extracellular K+ increase and Ca2+ decrease. A requirement of Na+, K+-ATPase signaling for extracellular Ca2+ makes K+ uptake (and brain edema) selectively dependent upon β1-adrenergic signaling and inhibitable by its antagonists.

Roles of astrocytic Na+,K+-ATPase and glycogenolysis for K+ homeostasis in mammalian brain

Neuronal excitation increases extracellular K+ concentration ([K+]o) in vivo and in incubated brain tissue by stimulation of postsynaptic glutamatergic receptors and by channel‐mediated K+ release during action potentials. Convincing evidence exists that subsequent cellular K+ reuptake occurs by active transport, normally mediated by Na+,K+‐ATPase. This enzyme is expressed both in neurons and in astrocytes but is stimulated by elevated [K+]o only in astrocytes. This might lead to an initial K+ uptake in astrocytes, followed by Kir4.1‐mediated release and neuronal reuptake. In cell culture experiments, K+‐stimulated glycogenolysis is essential for operation of the astrocytic Na+,K+‐ATPase resulting from the requirement for glycogenolysis in a pathway leading to uptake of Na+ for costimulation of its intracellular sodium‐binding site. The astrocytic but not the neuronal Na+,K+‐ATPase is additionally stimulated by isoproterenol, a β‐adrenergic agonist, but only at nonelevated [K+]o. This effect is also glycogenolysis dependent and might play a role during poststimulatory undershoots. Attempts to replicate dependence on glycogenolysis for K+ reuptake in glutamate‐stimulated brain slices showed similar [K+]o recovery half‐lives in the absence and presence of the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol. The undershoot was decreased, but to the same extent as an unexpected reduction of peak [K+]o increase. A potential explanation for this difference from the cell culture experiments is that astrocytic glutamate uptake might supply the cells with sufficient Na+. Inhibition of action potential generation by tetrodotoxin caused only a marginal, nonsignificant decrease in stimulated [K+]o in brain slices, hindering the evaluation if K+ reaccumulation after action potential propagation requires glycogenolysis in this preparation.

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1 -adrenergic Na(+) /K(+) -ATPase stimulation and glycogenolysis.

The cotransporter of Na+, K+, 2Cl–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K+ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na+/K+‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na+, K+, and Cl– × Cl–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na+/K+‐ATPase activity is stimulated by elevated K+ or the β‐adrenergic agonist isoproterenol. Extracellular K+ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β1‐adrenergic antagonist betaxolol, but not by ICI118551, a β2‐adrenergic antagonist. A large part of the β1‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na+/K+‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot.

Signal Transduction in Astrocytes during Chronic or Acute Treatment with Drugs (SSRIs, Antibipolar Drugs, GABA-ergic Drugs, and Benzodiazepines) Ameliorating Mood Disorders

Chronic treatment with fluoxetine or other so-called serotonin-specific reuptake inhibitor antidepressants (SSRIs) or with a lithium salt “lithium”, carbamazepine, or valproic acid, the three classical antibipolar drugs, exerts a multitude of effects on astrocytes, which in turn modulate astrocyte-neuronal interactions and brain function. In the case of the SSRIs, they are to a large extent due to 5-HT2B-mediated upregulation and editing of genes. These alterations induce alteration in effects of cPLA2, GluK2, and the 5-HT2B receptor, probably including increases in both glucose metabolism and glycogen turnover, which in combination have therapeutic effect on major depression. The ability of increased levels of extracellular K+ to increase [Ca2+]i is increased as a sign of increased K+-induced excitability in astrocytes. Acute anxiolytic drug treatment with benzodiazepines or GABAA receptor stimulation has similar glycogenolysis-enhancing effects. The antibipolar drugs induce intracellular alkalinization in astrocytes with lithium acting on one acid extruder and carbamazepine and valproic acid on a different acid extruder. They inhibit K+-induced and transmitter-induced increase of astrocytic [Ca2+]i and thereby probably excitability. In several cases, they exert different changes in gene expression than SSRIs, determined both in cultured astrocytes and in freshly isolated astrocytes from drug-treated animals.

Glycogenolysis and Purinergic Signaling

Both ATP and glutamate are on one hand essential metabolites in brain and on the other serve a signaling function as transmitters. However, there is the major difference that the flux in the pathway producing transmitter glutamate is comparable to the rate of glucose metabolism in brain, whereas that producing transmitter ATP is orders of magnitude smaller than the metabolic turnover between ATP and ADP. Moreover, de novo glutamate production occurs exclusively in astrocytes, whereas transmitter ATP is produced both in neurons and astrocytes. This chapter deals only with ATP and exclusively with its formation and release in astrocytes, and it focuses on potential associations with glycogenolysis, which is known to be indispensable for the synthesis of glutamate. Glycogenolysis is dependent upon an increase in free intracellular Ca(2+) concentration (Ca(2+)]i). It can be further stimulated by cAMP, but in contrast to widespread beliefs, cAMP can on its own not induce glycogenolysis. Astrocytes generate ATP from accumulated adenosine, and this process does not seem to require glycogenolysis. A minor amount of the generated ATP is utilized as a transmitter, and its synthesis requires the presence of the mainly intracellular nucleoside transporter ENT3. Many transmitters as well as extracellular K(+) concentrations high enough to open the voltage-sensitive L-channels for Ca(2+) cause a release of transmitter ATP from astrocytes. Adenosine and ATP induce release of ATP by action at several different purinergic receptors. The release evoked by transmitters or elevated K(+) concentrations is abolished by DAB, an inhibitor of glycogenolysis.

Inhibition of brain swelling after ischemia-reperfusion by β-adrenergic antagonists: correlation with increased K+ and decreased Ca2+ concentrations in extracellular fluid.

Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl−, and water cotransporter NKCC1 and by β 1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β 1-adrenergic pathways, indicating failure of K+-mediated, but not β 1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β 1-adrenergic activation the only stimulus and its inhibition effective against edema.