Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Ceramide-activated protein kinases A and C zeta inhibit kidney proximal tubule cell Na-ATPase

The basolateral membranes of kidney proximal tubule cells have (Na⁺+K⁺)-ATPase and Na⁺-ATPase activities, involved in Na⁺ reabsorption. We showed that ceramide (Cer) modulates protein kinase A (PKA) and protein kinase C (PKC), which are involved in regulating ion transporters. Here we show that ceramide, promotes 60% inhibition of Na⁺-ATPase activity (I₅₀ ≈ 100 nM). This effect was completely reversed by inhibiting PKA but did not involve the classic PKC signaling pathway. In these membranes we found the Cer-activated atypical PKC zeta (PKCζ) isoform. When PKCζ is inhibited, Cer ceases to inhibit the Na⁺-ATPase, allowing the cAMP/PKA signaling pathway to recover its stimulatory effect on the pump. There were no effects on the (Na⁺+K⁺)-ATPase. These results reveal Cer as a potent physiological modulator of the Na⁺-ATPase, participating in a regulatory network in kidney cells and counteracting the stimulatory effect of PKA via PKCζ.     

Cardiac Glycosides Ouabain and Digoxin Interfere with the Regulation of Glutamate Transporter GLAST in Astrocytes Cultured from Neonatal Rat Brain

Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na⁺, K⁺)-dependent ATPase (Na⁺/K⁺-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na⁺/K⁺-ATPase. We now report that Na⁺/K⁺-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 μM). Therefore, while we may not have established a direct link between GLAST regulation and Na⁺/K⁺-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic.     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Tubular Fluid Secretion in the Seminiferous Epithelium: Ion Transporters and Aquaporins in Sertoli Cells

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K⁺ than the blood and express various membrane and water transporters (Na⁺/K⁺-ATPase; Ca²⁺-ATPase; V-type ATPase; Cl⁻ channels; CFTR Cl⁻ channels; K⁺ channels; L-, T- and N-type Ca²⁺ channels; Na⁺/H⁺ exchangers; Na⁺-driven HCO₃ ⁻/Cl⁻ exchangers (NDCBEs); Na⁺/HCO₃ ⁻ cotransporters (NBCes); Na⁺–K⁺–2Cl⁻ cotransporter; Na⁺/Ca²⁺ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.     

Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na⁺ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na⁺ + K⁺-ATPase and the ouabain-insensitive, K⁺-insensitive, and furosemide-sensitive Na⁺-ATPase, which participate in the active Na⁺ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na⁺-ATPase activity (C1P₅₀ ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na⁺-ATPase. Conversely, we did not observe any effect on the Na⁺ + K⁺-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na⁺-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

Activité phosphatase alcaline dans le labyrinthe du placenta de chatte,et problème de la localisation de l'ATPase de transport: étude ultrastructurale

Summary The ultrastructural localizations of alkaline phosphatase (ALPase) and of Na⁺–K⁺-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) were studied in the placental labyrinth of the cat during the last days of gestation. ALPase activity could be detected in the syncytiotrophoblast but was absent from maternal tissues. Enzyme activity was observed only along plasma membranes of microvilli and absorption tubules on the maternal surface of the syncytium and also on the podocytes-like cytoplasmic processes of the fetal face. The localization of the Na⁺–K⁺-ATPase activity as obtained with the method of Ernst was identical with that of ALPase. This activity was not very ouabaine sensitive or K⁺ dependent, but was almost completely inhibited by levamisole. The strong ALPase activity of the syncytiotrophoblast does not allow a specific detection of Na⁺–K⁺-ATPase. However, the localization of these enzymes activities on syncytiotrophoblast surfaces directly related to fetal and maternal capillaries could suggest that these surfaces are associated with transport mechanisms of the trophoblast.

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Activité phosphatase alcaline dans le labyrinthe du placenta de chatte, et problème de la localisation de l'ATPase de transport: étude ultrastructurale

Résumé Les localisations de la phosphatase alcaline (ALPase) et de l'adénosine triphosphatase Na⁺–K⁺-dependante (Na⁺–K⁺-ATPase) ont été recherchées dans le labyrinthe du placenta de chatte en fin de gestation. L'activité ALPase est localisée de manière discontinue le long de la membrane plasmique du syncytiotrophoblaste: microvillosités et tubules d'absorption de la face maternelle, digitations cytoplasmiques à allure podocytique de la face foetale. La localisation de l'activité Na⁺–K⁺-ATPase par la méthode de Ernst est identique à celle de l'ALPase. Cette activité, très peu sensible à la ouabaïne et à l'absence de K⁺, est presque totalement inhibée par le tétramisole. Cette présence importante d'activité ALPase empêche la mise en évidence spécifique de la Na⁺–K⁺-ATPase. La localisation de ces enzymes dans des zones en rapport avec les capillaires foetaux et maternels, permet de soutenir la thèse d'une spécialisation de ces zones dans les mécanismes de transport.

     

(Na---K)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells

The migration of intestinal epithelial cells from the crypt area to the villus tip is associated with progressive differentiation of these cells. The distribution of (Na⁺---K⁺) stimulated adenosinetriphosphatase ((Na⁺---K⁺)-ATPase; EC 3.6.1.3) along the intestinal villus may have functional as well as developmental implications. To define this distribution, rat jejunal and ileal segments were incubated in vitro with a citrate solution that dissociates epithelial cells sequentially from villus tip to crypt area. ATPase activity in cell collections from villus tips and crypt areas were compared. The specific activity of (Na⁺---K⁺)-ATPase was higher in the villus tip than in the crypt cells of both jejunum and ileum. Crypt cell (Na⁺---K⁺)-ATPase activity in the jejunum and ileum were similar. Thus, (Na⁺---K⁺)-ATPase activity of villus tip cells in the jejunum was greater than in the ileum. There was no difference in villus tip and crypt cell Mg²⁺-ATPase activity in either jejunum or ileum. The steep gradient for (Na⁺---K⁺)-ATPase along the intestinal villus may signify an improtant difference in Na⁺ transport between the villus tip and crypt area. The higher level of (Na⁺---K⁺)-ATPase activity in the jejunal villi is consistent with the more important role of the jejunum in Na⁺ and substrate-linked Na⁺ transport.     

Salt,Na+,K+-ATPase and hypertension

Chronic hypertension is characterized by a persistent increase in vascular tone. Sodium-rich diets promote hypertension; however, the underlying molecular mechanisms are not fully understood. Variations in the sodium content of the diet, through hormonal mediators such as dopamine and angiotensin II, modulate renal tubule Na⁺,K⁺-ATPase activity. Stimulation of Na⁺,K⁺-ATPase activity increases sodium transport across the renal proximal tubule epithelia, promoting Na⁺ retention, whereas inhibited Na⁺,K⁺-ATPase activity decreases sodium transport, and thereby natriuresis. Diets rich in sodium also enhance the release of adrenal endogenous ouabain-like compounds (OLC), which inhibit Na⁺,K⁺-ATPase activity, resulting in increased intracellular Na⁺ and Ca²⁺ concentrations in vascular smooth muscle cells, thus increasing the vascular tone, with a corresponding increase in blood pressure. The mechanisms by which these homeostatic processes are integrated in response to salt intake are complex and not completely elucidated. However, recent scientific findings provide new insights that may offer additional avenues to further explore molecular mechanisms related to normal physiology and pathophysiology of various forms of hypertension (i.e. salt-induced). Consequently, new strategies for the development of improved therapeutics and medical management of hypertension are anticipated.     

Angiotensin II decreases system A amino acid transporter activity in human placental villous fragments through AT1 receptor activation

Reduced transport of amino acids from mother to fetus can lead to fetal intrauterine growth restriction (IUGR). The activities of several amino acid transport systems, including system A, are decreased in placental syncytiotrophoblast of IUGR pregnancies. Na(+)-K(+)-ATPase activity provides an essential driving force for Na(+)-coupled system A transport, is decreased in the placenta of IUGR pregnancies, and is decreased by angiotensin II in several tissues. Several reports have shown activation of the fetoplacental renin-angiotensin system (RAS) in IUGR. We investigated the effect of angiotensin II on placental system A transport and Na(+)-K(+)-ATPase activity in placental villi. Placental system A activity in single primary villous fragments was measured as the Na(+)-dependent uptake of alpha-(methylamino)isobutyric acid, and Na(+)/K(+) ATPase activity was measured as ouabain-sensitive uptake of (86)rubidium. Angiotensin II decreased system A activity in a concentration-dependent fashion (10-500 nmol/l). Angiotensin II type 1 receptor (AT1-R) antagonists losartan and AT1-R anti-peptide blocked the angiotensin II effect, but the angiotensin II type 2 receptor antagonist PD-123319 was without effect. System A activity was not altered by preincubation with AT1-R-independent vasoconstrictors, and antioxidants did not prevent the decrease in activity mediated by angiotensin II. Angiotensin II decreased Na(+)-K(+)-ATPase activity by an AT1-R dependent mechanism, and inhibition of Na(+)-K(+)-ATPase activity decreased system A activity in a dose-response fashion. These data suggest that angiotensin II, via AT1-R signaling, decreases system A activity by suppressing Na(+)-K(+)-ATPase in human placental villi, consistent with possible adverse effects of enhanced placental RAS on fetal growth.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na⁺ + K⁺)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na⁺ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na⁺ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na⁺ transport sensitively. This technique relies on the presence of Na⁺-Ca²⁺ exchange and ATP-driven Na⁺ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na⁺ uptake is monitored by a subsequent Na_i⁺-dependent Ca²⁺ uptake reaction (Na⁺-Ca²⁺ exchange) using ⁴⁵Ca²⁺. We present evidence that the Na⁺-Ca²⁺ exchange will be linearly related to the prior active Na⁺ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na⁺ isotopes. Applying this method, we measure cardiac ATP-dependent Na⁺ transport and (Na⁺ + K⁺)-ATPase activities in identical ionic media. We find that the (Na⁺ + K⁺)-ATPase and the Na⁺ pump have identical dependencies on both Na⁺ and ATP. The dependence on [Na⁺] is sigmoidal, with a Hill coefficient of 2.8. Na⁺ pumping is half-maximal at [Na⁺] = 9 mM. The K_m for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na⁺ pumping. This approach should allow other new investigations on on ATP-dependent Na⁺ transport across cardiac sarcolemma.     

Digitalis-induced cell signaling by the sodium pump: On the relation of Src to Na/K-ATPase

In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na⁺/K⁺-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na⁺/K⁺-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na⁺/K⁺-ATPase, Mg²⁺, and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na⁺/K⁺-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na⁺/K⁺-ATPase under physiological conditions.     

Transport of amino acids through the placenta and their role

Amino acids are transported across the human placenta mediated by transporter proteins that differ in structure, mechanism and substrate specificity. Some of them are Na⁺-dependent systems, whereas others are Na⁺-independent. Among these there are transporters composed of a heavy chain, a glycoprotein, and a light chain. Moreover, they can be differently distributed in the two membranes forming the syncytiotrophoblast. The transport mechanisms involved and their regulation are only partially known. In the placenta itself, part of the amino acids is metabolized to form other compounds important for the fetus. This occurs for instance for arginine, which gives rise to polyamines and to NO. Interconversion occurs among few other amino acids Transport is altered in pregnancy complications, such as restricted fetal growth.     

Role of Vacuolar Membrane Transport Systems in Plant Salinity Tolerance

About 20% of all irrigated land is adversely affected by salinity hazards and therefore understanding plant defense mechanisms against salinity will have great impact on plant productivity. In the last decades, comprehension of salinity resistance at molecular level has been achieved through the identification of key genes encoding biomarker proteins underpinning salinity tolerance. Implication of the vacuolar transport systems in plant salinity tolerance is one example of these central mechanisms rendering tolerance to saline stress. One important organelle in plant cells is the central vacuole that plays pivotal multiple roles in cell functioning under normal and stress conditions. This review thus attempts to address different lines of evidence supporting the role of the vacuolar membrane transport systems in plant salinity tolerance. Vacuolar transport systems include Na⁺(K⁺)/H⁺ antiporters, V-ATPase, V-PPase, Ca²⁺/H⁺ exchangers, Ca²⁺-ATPase, ion channels, aquaporins, and ABC transporters. They contribute essentially in retaining a high cytosolic K⁺/Na⁺ ratio, K⁺ level, sequestrating Na⁺ and Cl^? into vacuoles, as well as regulation of other salinity responsive pathways. However, little is known about the regulation and functions of some of the vacuolar transporters under salinity stress and therefore need more exploration and focus. Numerous studies demonstrated that the activities of the vacuolar transporters are upregulated in response to salinity stress, confirming their central roles in salinity tolerance mechanism. The second line of evidence is that manipulation of one of the genes encoding the vacuolar transport proteins results in some successful improvement of plant salinity tolerance. Therefore, transgene pyramiding of more than one gene for developing genotypes with better and strong salinity tolerance and productivity should gain more attention in future research. In addition, we should move step further and verify the experimental data obtained from either a greenhouse or controlled environment into field trials in order to support our claims.

     

The Na<Superscript>+</Superscript> pump and intracellular signaling mechanisms

The main properties of Na⁺/K⁺-ATPase as a natural receptor for cardiotonic steroids are considered. Primary attention is focused on structural and functional differences between the α-subunit isoforms of Na⁺/K⁺-ATPase in different tissues. General information on the role of the Na⁺ pump in signaling cascades in kidney epithelial cells, cardiomyocytes and neurons is presented. The data obtained indicate that, in neurons, several α-isoforms of Na⁺/K⁺-ATPase possessing different sensitivity to ouabain may have different signaling functions.     

Glutamate Transporters and Mitochondria: Signaling,Co-compartmentalization,Functional Coupling,and Future Directions

In addition to being an amino acid that is incorporated into proteins, glutamate is the most abundant neurotransmitter in the mammalian CNS, the precursor for the inhibitory neurotransmitter γ-aminobutyric acid, and one metabolic step from the tricarboxylic acid cycle intermediate α-ketoglutarate. Extracellular glutamate is cleared by a family of Na⁺-dependent transporters. These transporters are variably expressed by all cell types in the nervous system, but the bulk of clearance is into astrocytes. GLT-1 and GLAST (also called EAAT2 and EAAT1) mediate this activity and are extremely abundant proteins with their expression enriched in fine astrocyte processes. In this review, we will focus on three topics related to these astrocytic glutamate transporters. First, these transporters co-transport three Na⁺ ions and a H⁺ with each molecule of glutamate and counter-transport one K⁺; they are also coupled to a Cl^? conductance. The movement of Na⁺ is sufficient to cause profound astrocytic depolarization, and the movement of H⁺ is linked to astrocytic acidification. In addition, the movement of Na⁺ can trigger the activation of Na⁺ co-transporters (e.g. Na⁺–Ca²⁺ exchangers). We will describe the ways in which these ionic movements have been linked as signals to brain function and/or metabolism. Second, these transporters co-compartmentalize with mitochondria, potentially providing a mechanism to supply glutamate to mitochondria as a source of fuel for the brain. We will provide an overview of the proteins involved, discuss the evidence that glutamate is oxidized, and then highlight some of the un-resolved issues related to glutamate oxidation. Finally, we will review evidence that ischemic insults (stroke or oxygen/glucose deprivation) cause changes in these astrocytic mitochondria and discuss the ways in which these changes have been linked to glutamate transport, glutamate transport-dependent signaling, and altered glutamate metabolism. We conclude with a broader summary of some of the unresolved issues.

     

Requirement of Glycogenolysis for Uptake of Increased Extracellular K+ in Astrocytes: Potential Implications for K+ Homeostasis and Glycogen Usage in Brain

The importance of astrocytic K⁺ uptake for extracellular K⁺ ([K⁺]_e) clearance during neuronal stimulation or pathophysiological conditions is increasingly acknowledged. It occurs by preferential stimulation of the astrocytic Na⁺,K⁺-ATPase, which has higher K_m and V_max values than its neuronal counterpart, at more highly increased [K⁺]_e with additional support of the cotransporter NKCC1. Triggered by a recent DiNuzzo et al. paper, we used administration of the glycogenolysis inhibitor DAB to primary cultures of mouse astrocytes to determine whether K⁺ uptake required K⁺-stimulated glycogenolysis. KCl was increased by either 5 mM (stimulating only the Na⁺,K⁺-ATPase) or 10 mM (stimulating both transporters) in glucose-containing saline media prepared to become iso-osmotic after the addition. DAB completely inhibited both uptakes, the Na⁺,K⁺-ATPase-mediated by preventing Na⁺ uptake for stimulation of its intracellular Na⁺-activated site, and the NKCC1-mediated uptake by inhibition of depolarization- and L-channel-mediated Ca²⁺ uptake. Drugs inhibiting the signaling pathways involved in either of these processes also abolished K⁺ uptake. Assuming similar in vivo characteristics, partly supported by literature data, K⁺-stimulated astrocytic K⁺ uptake must discontinue after normalization of extracellular K⁺. This will allow Kir1.4-mediated release and reuptake by the less powerful neuronal Na⁺,K⁺-ATPase.