Characterization of an Na+/K+/Cl− co-transport in primary cultures of rat astrocytes (original) (raw)

A linked active transport system for Na+ and K+ in a glial cell line

Brain Research, 1976

Ouabain (5 × l0-4 M) induced a 6-fold increase in intracellular Na + and a 65 % loss of cellular K + in C6 glial cells which was accompanied by a 12 mV decrease in the resting membrane potential. Following ouabain washout intracellular ion concentrations and the membrane potential returned to control levels suggesting that C6 is capable of active Na + transport which is linked to uptake of K +. A portion of K + uptake under steady-state conditions is also active since K + influx was reduced 32 % by ouabain. Five mM cyanide significantly increased cell Na + and significantly decreased cell K + and the membrane potential. The similarity in the ratio of Na + gained/K + lost (ouabain 1.24, cyanide 1.41) suggests that the two agents inhibit the same ion transport system. Decreased temperature had the paradoxical effect of increasing intracellular K + while significantly decreasing both membrane potential and K + influx. Part of this effect may be due to the marked reduction in K + efflux at low temperature. At 6 °C cell loss of K + was much less than loss of K + with ouabain at 37 °C. The observation of a linked Na+-K + transport system in C6 cells confirms the hypothesis that coupled active Na+-K + exchange occurs in glial cells and suggests that ionic transport may regulate certain aspects of glial metabolism.

The Na+/K+/Cl cotransport in C6 glioma cells. Properties and role in volume regulation

European Journal of Biochemistry, 1988

The role of the Naf/K+/CI-cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide > bumetanide > piretanide > furosemide.

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock

European Journal of Biochemistry, 1986

1. The properties of the N a t / H + exchange system in the glial cell lines c 6 and NN were studied from "Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethylcarboxyfluorescein. In both cell types, the N a f / H + exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride > amiloride > benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H'. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in c 6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines.

Evidence for chloride ions as intracellular messenger substances in astrocytes

Journal of neurophysiology, 1999

Cultured rat hippocampal astrocytes were used to investigate the mechanism underlying the suppression of Ba2+-sensitive K+ currents by GABAA receptor activation. Muscimol application had two effects on whole cell currents: opening of the well-known Cl- channel of the GABAA receptor and a secondary longer-lasting blockade of outward K+ currents displaying both peak and plateau phases. This blockade was independent of both Na+ (inside and outside) and ATP in the pipette. It also seemed to be independent of muscimol binding to the receptor because picrotoxin application showed no effect on the K+ conductance. The effect is blocked when anion efflux is prevented by replacing Cl- with gluconate (both inside and out) and is enhanced with more permeant anions such as Br- and I-. Moreover, the effect is reproduced in the absence of muscimol by promoting Cl- efflux via lowering of extracellular Cl- levels. These results, along with the requirement for Cl- efflux in muscimol experiments, show...

Na + -Ca 2+ cosignaling in the stimulation of the glucose transporter GLUT1 in cultured astrocytes

Glia, 2008

Glutamate triggers an acute stimulation of the glucose transporter GLUT1 in cultured astrocytes, a phenomenon thought to facilitate energy delivery to active areas in the brain. Here we have explored the cell signaling mechanisms involved in this response. Half-stimulation of GLUT1 occurred at low micromolar glutamate, thus within the physiological range estimated in brain interstitium. The effect was mimicked by D-aspartate and inhibited by Lthreo-beta-benzyloxyaspartate or Na 1 replacement with NMDG 1 , showing the participation of the Na 1-glutamate co-transporter. AMPA and the mGLURI agonist DHPG had no effect. The stimulation of GLUT1 was fully inhibited by ouabain, but independent activation of the Na 1 /K 1 ATPase pump with gramicidin did not affect glucose transport. Simultaneous with the Na 1 rise, glutamate and D-aspartate triggered a Ca 21 signal, whose inhibition with BAPTA prevented the stimulation of GLUT1. However, an isolated Ca 21 signal, triggered with endothelin 1, ATP or DHPG, did not affect glucose transport. The stimulation of GLUT1 could finally be mimicked by simultaneous induction of Na 1 and Ca 21 signals. The requirement for both cations in the stimulation of the astrocytic glucose transporter, may help to explain how glucose metabolism in the brain is strongly activated by glutamate, but not by GABA or by inter-astrocytic signaling. V

Water permeability of Na+-K+-2Cl- cotransporters in mammalian epithelial cells

The Journal of Physiology, 2005

Water transport properties of the Na +-K +-2Cl − cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide-sensitive component of 86 Rb + uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (L p) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide-sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l −1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi-osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide-sensitive component of the L p was 21 kcal mol −1 , which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na + , K + and Cl −. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive 86 Rb + , the NKCCs of this tissue did not contribute any bumetanide-sensitive L p. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.

Characterization of 14C-acetate uptake in cultured rat astrocytes

Brain Research, 2009

To investigate the regulation mechanism of 14 C-acetate uptake in astrocytes, uptake experiments with cultured rat astrocytes were performed. In this study, we used a relatively low concentration (0.0185 mM) of 14 C-acetate. The uptake was dependent on pH and acetate concentration, and saturated by 10 mM acetate. Furthermore, the selective inhibition by p-(chloromercuri) benzenesulfonic acid (pCMBS) but not by α-cyano-3-hydroxycinnamate (CHC) showed that the monocarboxylate transporter-1 (MCT-1)-mediated transport system is essentially important in cultured rat astrocytes. A significant reduction (more than 30%) in 14 C-acetate uptake was observed with 0.5 mM fluorocitrate treatment, which indicated 14 C-acetate uptake in this study might reflect not only the transport process, but also the metabolic process. Glutamate significantly increased 14 C-acetate uptake. An increase in extracellular potassium concentration had no effect on 14 C-acetate uptake. The Ca 2+ ionophore A23187 caused a 40% reduction, and ouabain (inhibitor of Na +-K +-ATPase) and monensin (Na + ionophore) significantly decreased 14 Cacetate uptake by astrocytes. The combined use of ouabain and monensin caused significantly greater decreases than the addition of either chemical alone. These results suggest that glutamate stimulation and changes in Ca 2+ and Na + concentrations might have important roles in regulation of 14 C-acetate uptake in cultured rat astrocytes.

Glial Na(+) -dependent ion transporters in pathophysiological conditions

Glia, 2016

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na(+) signaling influences and regulates important glial activities, and plays a role in neuron-glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na(+) pumps and Na(+) -dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na(+) homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na(+) -dependent ion transporters, including Na(+) /K(+) ATPase, Na(+) /Ca(2+) exchangers, Na(+) /H(+) exchangers, Na(+) -...

The effects of metabolism on Na(+)-K(+)-Cl- co-transport in ferret red cells

The Journal of physiology, 1991

1. The effects of altering metabolism on Na(+)-K(+)-Cl- co-transport were studied in ferret red cells. Na(+)-K(+)-Cl- co-transport was measured as the bumetanide-sensitive uptake of 86Rb. 2. Glucose, but not inosine or adenosine, sustained metabolism and maintained cell ATP content ([ATP]i) at the physiological level. [ATP]i could be reduced by prolonged incubation of cells in a substrate-free medium or more quickly by incubating cells with 2-deoxyglucose or with a mixture of iodoacetamide and glucose. 3. Na(+)-K(+)-Cl- co-transport activity was inhibited when [ATP]i was reduced to below 100 mumol (1 cell)-1 by starvation or by treatment with 2-deoxyglucose. However, a unique relationship between [ATP]i and activity could not be found. [ATP]i and the method and time course of ATP depletion all influenced activity. The inhibition of Na(+)-K(+)-Cl- co-transport, caused by reducing [ATP]i could be partially reversed by restoring [ATP]i to normal. 4. Increasing the concentration of intr...