Activity‐dependent astrocyte swelling is mediated by pH‐regulating mechanisms

During neuronal activity in the mammalian brain, the K⁺ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na⁺/K⁺/2Cl^? cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity‐dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion‐sensitive microelectrodes to measure changes in ECS, [K⁺]_o and [H⁺]_o/pH_o during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K⁺ and glutamate released into the extracellular space (the K⁺/Cl^? cotransporter, KCC, glutamate transporters and G protein‐coupled receptors) did not modulate the extracellular space dynamics. The ‐transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na⁺/ cotransporter 1 (NBCe1), is activated by the K⁺‐mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by ～25% without affecting the K⁺ transients, pointing to NBCe1 as a key contributor to the stimulus‐induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like‐wise, reduced the ECS shrinkage by ～25% without compromising the K⁺ transients. Isosmotic reduction of extracellular Cl^? revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus‐evoked astrocytic cell swelling does not appear to occur as a direct effect of the K⁺ clearance, as earlier proposed, but partly via the pH‐regulating transport mechanisms activated by the K⁺‐induced astrocytic depolarization and the activity‐dependent metabolism.     

Contributions of the Na+/K+‐ATPase,NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

Network activity in the brain is associated with a transient increase in extracellular K⁺ concentration. The excess K⁺ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na⁺/K⁺/2Cl^? cotransporter 1 (NKCC1), and/or Na⁺/K⁺‐ATPase activity. Their individual contribution to [K⁺]_o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na⁺/K⁺‐ATPase and to resolve their involvement in clearance of extracellular K⁺ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K⁺]_o increases above basal levels. Increased [K⁺]_o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K⁺ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K⁺ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K⁺]_o increase. In contrast, inhibition of the different isoforms of Na⁺/K⁺‐ATPase reduced post‐stimulus clearance of K⁺ transients. The astrocyte‐characteristic α2β2 subunit composition of Na⁺/K⁺‐ATPase, when expressed in Xenopus oocytes, displayed a K⁺ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K⁺]_o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na⁺/K⁺‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K⁺ recovery in native hippocampal tissue while Kir4.1 and Na⁺/K⁺‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622     

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. © 2014 Wiley Periodicals, Inc.     

Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning

Initial clearance of extracellular K⁺ ([K⁺]_o) following neuronal excitation occurs by astrocytic uptake, because elevated [K⁺]_o activates astrocytic but not neuronal Na⁺,K⁺-ATPases. Subsequently, astrocytic K⁺ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K⁺]_o increase and allowing neuronal Na⁺,K⁺-ATPase-mediated re-uptake. Na⁺,K⁺-ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K⁺]_o increases to open L-channels for Ca²⁺, activating [K⁺]_o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K⁺]_o is decreased to levels mainly affecting astrocytic membrane potential or Na⁺,K⁺-ATPase or by abnormalities in its α2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K⁺]_o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocytic-oligodendrocytic gap junctions also promotes learning.     

Involvement of vH+‐ATPase in synaptic vesicle swelling

Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G_αi3‐PLA2‐mediated involvement of water channel AQP‐1 in the regulation of secretory vesicle swelling in exocrine pancreas and the G_αo‐mediated AQP‐6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH⁺‐ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer‐scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH⁺‐ATPase in GTP‐G_αo‐mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP‐G_αo‐mediated vesicle swelling is vH⁺‐ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin‐sensitive vesicle acidification, following the GTP‐G_αo‐induced swelling stimulus. Water channels are bidirectional and the vH⁺‐ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP‐mastoparan stimulated swelling, suggesting that vH⁺‐ATPase is upstream of AQP‐6, in the pathway leading from G_αo‐stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP‐6‐mediated gating of water into synaptic vesicles. © 2009 Wiley‐Liss, Inc.     

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

Salt‐loading (SL) impairs GABA_A inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down‐regulating the activity of K⁺/Cl^? co‐transporter2 (KCC2) and up‐regulating Na⁺/K⁺/Cl^? co‐transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl^?]_i in SON AVP neurones. The study combined virally‐mediated chloride imaging with ClopHensorN with a single‐cell western blot analysis. An adeno‐associated virus with ClopHensorN and a vasopressin promoter (AAV2‐0VP1‐ClopHensorN) was bilaterally injected in the SON of adult male Sprague‐Dawley rats that were either euhydrated (Eu) or salt‐loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl^?]_i in response to focal application of the GABA_A agonist muscimol (100 μmol L^‐1). SON AVP neurones from Eu rats showed muscimol‐induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β‐actin. Neurones that did not respond during chloride imaging did not show KCC2 and β‐actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L^‐1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L^‐1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol‐induced Cl^? influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA‐12) (50 μmol L^‐1) and protein kinase inhibitor (K252a) (100 nmol L^‐1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt‐loading increases [Cl^?]_i in SON AVP neurones via a TrKB‐KCC2‐NKCC1‐dependent mechanism in rats.     

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH₄⁺/NH₃). Here, we examined the effects of NH₄⁺/NH₃ on steady‐state intracellular pH (pH_i) and sodium concentration ([Na⁺]_i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH₄⁺/NH₃ on glutamate transporter activity by measuring D ‐aspartate‐induced pH_i and [Na⁺]_i transients. In 20–34 days in vitro (DIV) astrocytes, NH₄⁺/NH₃ decreased steady‐state pH_i by 0.19 pH units and increased [Na⁺]_i by 21 mM. D ‐Aspartate‐induced pH_i and [Na⁺]_i transients were reduced by 80–90% in the presence of NH₄⁺/NH₃, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pH_i and [Na⁺]_i were minimally affected by NH₄⁺/NH₃, and D ‐aspartate‐induced pH_i and [Na⁺]_i transients were reduced by only 30–40%. Next we determined the contribution of Na⁺, K⁺, Cl^?‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH₄⁺/NH₃‐evoked changes in steady‐state pH_i and [Na⁺]_i and attenuated the reduction of D ‐aspartate‐induced pH_i and [Na⁺]_i transients by NH₄⁺/NH₃ to 30% in 20–34 DIV astrocytes. Our results suggest that NH₄⁺/NH₃ decreases steady‐state pH_i and increases steady‐state [Na⁺]_i in astrocytes by an age‐dependent activation of NKCC. These NH₄⁺/NH₃‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE. © 2008 Wiley‐Liss, Inc.     

Astrocyte‐mediated infantile‐onset leukoencephalopathy mouse model

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile‐onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte‐specific Mlc1 overexpression causes infantile‐onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na⁺/K⁺ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na⁺/K⁺ pump. In contrast, no changes in Na⁺/K⁺ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na⁺/K⁺ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile‐onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile‐onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150–168     

Astrocytic poly(ADP‐ribose) polymerase‐1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP‐1‐induced depletion of cellular NAD⁺, glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP‐dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP‐1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP‐1 activation by the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC₅₀ = 78.2 ± 2.7 μM). HPLC revealed MNNG‐induced time‐dependent reductions in NAD⁺ (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [³H]glutamate uptake rate (V_max) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 ± 2.8 in control cells to 2.1 ± 0.8 nmol/min/mg protein 4 h post‐MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP‐1, directly indicating PARP‐1 involvement, and by adding exogenous NAD⁺ (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co‐culture with PARP‐1^−/− astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP‐1 can raise glutamate levels sufficiently to have receptor‐mediated effects at neighboring neurons. Taken together, these experiments showed that PARP‐1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity. © 2009 Wiley‐Liss, Inc.     

Voltage‐induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation‐secretion coupling is assumed to be dependent only on extracellular calcium ([Ca²⁺]_o). Ryanodine receptor (RyR)‐sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca²⁺]_i) that are voltage‐dependent. We hypothesised that voltage‐elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage‐gated calcium channels and/or from such voltage‐sensitive ryanodine‐mediated calcium stores. Increases in [Ca²⁺]_i upon depolarisation in the presence of voltage‐gated calcium channel blockers, or in the absence of [Ca²⁺]_o, still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca²⁺]_o, there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Furthermore, such depolarisations lead to increases in [Ca²⁺]_i. Pre‐incubation with BAPTA‐AM resulted in > 50% inhibition of peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Nifedipine but not nicardipine inhibited both the high K⁺ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage‐induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation‐induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine‐sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.     

Clearance of activity‐evoked K+ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform‐selective AQP4 inhibitor

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4‐deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4^?/? mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4‐expressing Xenopus laevis oocytes monitored by a high‐resolution volume recording system, we here demonstrate that the compound TGN‐020 is such a selective AQP4 inhibitor. TGN‐020 targets the tested species of AQP4 with an IC₅₀ of ~3.5 μM, but displays no inhibitory effect on the other AQPs (AQP1‐AQP9). With this tool, we employed rat hippocampal slices and ion‐sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN‐020‐mediated inhibition of AQP4 did not prevent stimulus‐induced extracellular space shrinkage, nor did it slow clearance of the activity‐evoked K⁺ transient. These data, obtained with a verified isoform‐selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K⁺ clearance or the associated extracellular space shrinkage.     

Dopamine D3 receptor activation promotes neural stem/progenitor cell proliferation through AKT and ERK1/2 pathways and expands type‐B and ‐C cells in adult subventricular zone

The neurotransmitter dopamine acts on the subventricular zone (SVZ) to regulate both prenatal and postnatal neurogenesis, in particular through D₃ receptor (D₃R) subtype. In this study, we explored the cellular mechanism(s) underlying D₃R‐mediated cell proliferation and tested if systemic delivery of a D₃R agonist would induce SVZ multipotent neural stem/precursor cell (NSC/NPC) proliferation in vivo. We found that treatment with the D₃R agonist, 7‐OH‐DPAT, enhances cell proliferation in a dose‐dependent manner in cultured SVZ neurospheres from wild‐type, but not D₃R knock‐out mice. Furthermore, D₃R activation also stimulates S‐phase and enhances mRNA and protein levels of cyclin D1 in wild‐type neurospheres, a process which requires cellular Akt and ERK1/2 signaling. Moreover, chronic treatment with low dose 7‐OH‐DAPT in vivo increases BrdU⁺ cell numbers in the adult SVZ, but this effect was not seen in D₃R KO mice. Additionally, we probed the cell type specificity of D₃R agonist‐mediated cell proliferation. We found that in adult SVZ, GFAP⁺ astrocytes, type‐B GFAP⁺/nestin⁺ and type‐C EGF receptor (EGFR⁺)/nestin⁺ cells express D₃R mRNA, but type‐A Doublecortin (Dcx)⁺ neuroblasts do not. Using flow cytometry and immunofluorescence, we demonstrated that D₃R activation increases GFAP⁺ type‐B and EGFR⁺ type‐C cell numbers, and the newly divided Dcx⁺ type‐A cells. However, BrdU⁺/Dcx⁺ cell numbers were decreased in D₃R KO mice compared to wildtype, suggesting that D₃R maintains constitutive NSC/NPCs population in the adult SVZ. Overall, we demonstrate that D₃R activation induces NSC/NPC proliferation through Akt and ERK1/2 signaling and increases the numbers of type‐B and ‐C NSC/NPCs in the adult SVZ. © 2013 Wiley Periodicals, Inc.     

Synaptic impairment induced by paroxysmal ionic conditions in neocortex

Purpose: Seizures are associated with a reduction in extracellular Ca²⁺ concentration ([Ca²⁺]_o) and an increase in extracellular K⁺ concentration ([K⁺]_o). The long‐range synchrony observed between distant electrodes during seizures is weak. We hypothesized that changes in extracellular ionic conditions during seizures are sufficient to alter synaptic neuronal responses and synchrony in the neocortex. Methods: We obtained in vivo and in vitro electrophysiologic recordings combined with microstimulation from cat/rat neocortical neurons during seizures and seizure‐like ionic conditions. In vitro the [K⁺]_o was 2.8, 6.25, 8.0, and 12 mm and the [Ca²⁺]_o was 1.2 and 0.6 mm . Key Findings: During seizures recorded in vivo, we observed abolition of evoked synaptic responses. In vitro, the membrane potential of both regular‐spiking and fast‐spiking neurons was depolarized in high [K⁺]_o conditions and hyperpolarized in high [Ca²⁺]_o conditions. During high [K⁺]_o conditions, changes in [Ca²⁺]_o did not affect membrane potential. The synaptic responsiveness of both regular‐spiking and fast‐spiking neurons was reduced during seizure‐like ionic conditions. A reduction in [Ca²⁺]_o to 0.6 mm increased failure rates but did not abolish responses. However, an increase in [K⁺]_o to 12 mm abolished postsynaptic responses, which depended on a blockade in axonal spike propagation. Significance: We conclude that concomitant changes in [K⁺]_o and [Ca²⁺]_o observed during seizures contribute largely to the alterations of synaptic neuronal responses and to the decrease in long‐range synchrony during neocortical seizures.     

The search for NKCC1-selective drugs for the treatment of epilepsy: Structure–function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A

The Na⁺–K⁺–Cl⁻ cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl⁻ concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~ 5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure–function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC₅₀s) of bumetanide were 0.68 (hNKCC1A) and 4.0 μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC₅₀s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.     

Molecular mechanisms of K+ clearance and extracellular space shrinkage—Glia cells as the stars

Neuronal signaling in the central nervous system (CNS) associates with release of K⁺ into the extracellular space resulting in transient increases in [K⁺]_o. This elevated K⁺ is swiftly removed, in part, via uptake by neighboring glia cells. This process occurs in parallel to the [K⁺]_o elevation and glia cells thus act as K⁺ sinks during the neuronal activity, while releasing it at the termination of the pulse. The molecular transport mechanisms governing this glial K⁺ absorption remain a point of debate. Passive distribution of K⁺ via Kir4.1-mediated spatial buffering of K⁺ has become a favorite within the glial field, although evidence for a quantitatively significant contribution from this ion channel to K⁺ clearance from the extracellular space is sparse. The Na⁺/K⁺-ATPase, but not the Na⁺/K⁺/Cl⁻ cotransporter, NKCC1, shapes the activity-evoked K⁺ transient. The different isoform combinations of the Na⁺/K⁺-ATPase expressed in glia cells and neurons display different kinetic characteristics and are thereby distinctly geared toward their temporal and quantitative contribution to K⁺ clearance. The glia cell swelling occurring with the K⁺ transient was long assumed to be directly associated with K⁺ uptake and/or AQP4, although accumulating evidence suggests that they are not. Rather, activation of bicarbonate- and lactate transporters appear to lead to glial cell swelling via the activity-evoked alkaline transient, K⁺-mediated glial depolarization, and metabolic demand. This review covers evidence, or lack thereof, accumulated over the last half century on the molecular mechanisms supporting activity-evoked K⁺ and extracellular space dynamics.     

Methylmercury-induced astrocytic swelling is associated with activation of the Na/H antiporter, and is fully reversed by amiloride

Astrocytes are a known ‘sink' for brain methylmercury (MeHg) deposition. Yet, the significance of the preferential accumulation of MeHg within these cells is imprecisely defined. To determine whether MeHg in isotonic buffer has the potential to interfere with homeostatic functions, we measured its effect on astrocytic volume using an electrical impedance method [E.R. O'Connor, H.K. Kimelberg, C.R. Keese, I. Giaever, Electrical impedance method for measuring volume changes in astrocytes, Am. J. Physiol. 264 (1993) C471–C478.]. In addition, we have characterized the alterations in astrocytic ion permeability associated with exposure to this organometal. The results show that MeHg rapidly induces astrocytic swelling, and that this effect is secondary to increased astrocytic Na⁺ uptake. Furthermore, the effect of MeHg on astrocytic swelling is completely inhibited by amiloride, but not by SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid), furosemide, or bumetanide. Accordingly, increased cellular permeability to Na⁺ via the Na⁺/H⁺ antiporter is invoked as the primary mechanism of MeHg-induced astrocytic swelling.     

Manganese‐induced downregulation of astroglial glutamine transporter SNAT3 involves ubiquitin‐mediated proteolytic system

SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn²⁺). The present studies were performed to identify the processes responsible for this effect. One of the well‐established mechanisms for protein‐level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4‐2 (neural precursor cells expressed developmentally downregulated 4‐2), and that Mn²⁺ increases both Nedd4‐2 mRNA and protein levels. Additionally, we have found that Mn²⁺ exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn²⁺ effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid‐inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4‐2. Additional findings establish that Mn²⁺ increases astrocytic caspase‐like proteolytic proteasome activity and that the Mn²⁺‐dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N‐acetyl‐leu‐leu‐norleucinal and lactacystin. Combined, these results demonstrate that Mn²⁺‐induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin‐mediated proteolytic system. © 2010 Wiley‐Liss, Inc.     

Role of mitogen‐activated protein kinases in the mechanism of oxidant‐induced cell swelling in cultured astrocytes

Cytotoxic brain edema, usually a consequence of astrocyte swelling, is an important complication of stroke, traumatic brain injury, hepatic encephalopathy, and other neurological disorders. Although mechanisms underlying astrocyte swelling are not fully understood, oxidative stress (OS) has generally been considered an important factor in its pathogenesis. To better understand the mechanism(s) by which OS causes cell swelling, we examined the potential involvement of mitogen‐activated protein kinases (MAPKs) in this process. Cultures exposed to theoxidant H₂O₂ (10, 25, 50 μM) for different time periods (1–24 hr) significantly increased cell swelling in a triphasic manner. Swelling was initially observed at 10 min (peaking at 30 min), which was followed by cell shrinkage at 1 hr. A subsequent increase in cell volume occurred at approximately 6 hr, and the rise lasted for at least 24 hr. Cultures exposed to H₂O₂ caused the activation of MAPKs (ERK1/2, JNK and p38‐MAPK), whereas inhibition of MAPKs diminished cell swelling induced by 10 and 25 μM H₂O₂. These findings suggest that activation of MAPKs is an important factor in the mediation of astrocyte swelling following oxidative stress. © 2010 Wiley‐Liss, Inc.