Role of glial cells in the regulation of the brain ion microenvironment.
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Animals↗
Biomedical subjects
W Walz
Publications and source records attributed to W Walz.
pH shifts evoked by neuronal stimulation in slices of rat hippocampus.
Extracellular pH was measured with ion-selective microelectrodes in 500-microns thick slices of the CA1 region of the rat hippocampus. The center of the slice was 0.24 pH units more acidic than the surface, thus creating a decreasing pH gradient from the surface to the center, most likely owing to increased anaerobic metabolism. Stimulation at various frequencies created a transient alkaline shift of 0.03 pH unit, followed by a sustained acidification (0.05 pH unit above baseline). The same pattern was seen in both cell body and dendritic layers. The presence of the alkaline shift in slices in vitro is especially significant, implying that it is not due to alterations in blood flow. The HCO3- -Cl- transport inhibitor SITS and the carbonic anhydrase inhibitor acetazolamide increased the alkaline shift to 0.06 and 0.23, respectively. The acid shift was influenced by the Na+-H+ transport inhibitor amiloride, with a reduction of about 50%. Possible mechanisms for these stimulus-evoked changes as discussed. The most likely cause for the alkaline shift is bicarbonate accumulation in the extracellular space. Hydrogen ion and lactic acid release are seen as the major factors contributing to the sustained acid shift.
Acetazolamide↗
Is a rise in [CA]1 responsible for rapid changes in neuronal excitability, CA currents, extracellular ions and pH0 induced by brief anoxia?
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Calcium↗
Ionic alterations during anoxia in mammalian white matter.
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Action Potentials↗
Lactate production and release in cultured astrocytes.
Intracellular lactate content and release of lactate into the surrounding medium of mouse astrocytes in primary culture was measured using the lactate dehydrogenase method. During culturing the cellular content of astrocytes decreased from 400 to 200 nmol/mg protein. The total lactate released into the extracellular space, however, amounted to 75,000 nmol/mg within 98 h, corresponding to a lactate concentration of 10 mM in the cell culture dish. In another set of experiments, cytotoxic swelling was evoked by exposure of the cells to 60 mM K+, this situation caused a 40% increase in cellular volume and an increase in the KCl content of astrocytes. Within 3 h of a change to 60 mM K+ the intracellular lactate content was increased by 100 nmol/mg (one third) and the lactate release in the extracellular space by about 2000 nmol/mg (twice as high as during exposure to 3 mM K+). However, due to the increased intracellular water content, the lactate concentration inside the cells remained unchanged. It is concluded that astrocytes produce substantial amounts of additional lactate during cytotoxic swelling. This lactate, however, is not increasing the intracellular osmolarity and most of the lactate is released into the extracellular space. Depending on the transmembrane transport mechanism it could have the capability to decrease the strong ion difference and contribute to acid shifts in the extracellular space.
Animals↗
Electrophysiological properties of glial cells: comparison of brain slices with primary cultures.
Intracellular recordings were obtained from glial cells in the CA1 region of rat hippocampal slices to compare their electrophysiological properties with the previously reported properties of glial cells in primary tissue culture. The average resting potential was -77 mV and the average input resistance was 3.2 M omega. Barium (10 mM) depolarized glial cells in brain slices and increased input resistance, but barium action potentials which have been observed in primary cultures, were not observed in brain slices. gamma-aminobutyric acid (GABA) and glutamate depolarized glial cells. Spontaneous oscillations of membrane potential were observed occasionally. The mechanism underlying these responses are unknown as yet.
Action Potentials↗
Lactate release from cultured astrocytes and neurons: a comparison.
Lactate released into the surrounding salt solution as well as the cellular lactate content were measured in cerebral primary cultures of mouse astrocytes and of mouse neurons. Any newly produced lactate was immediately released as lactic acid into the extracellular compartment via a lactate/proton cotransport. The astrocytic release was about 2,000 nmol x mg-1 x hr-1; the neuronal release was about 300 nmol x mg-1 x hr-1. However, if election transport was blocked with dinitrophenol, the neuronal lactate release was as high as the astrocytic one under normal conditions. High glucose (30 mM) and K+ (60 mM) increased lactate release of astrocytes but not of neurons. In contrast it was found that insulin (1 microM) exposure mainly stimulated neuronal lactate release rather than glial release. Adenosine stimulated both neuronal and glial release. Neither intracellular lactate content nor concentration changed significantly in either cell type under any conditions tested. The pathophysiological implications of these measurements are discussed.
Adenosine↗
KCl movements during potassium-induced cytotoxic swelling of cultured astrocytes.
Astrocytes in primary cultures from mouse cortex were used to investigate the ionic mechanism of cytotoxic swelling which develops after exposure of the cells to 60 mM K+ under isosmotic conditions. Radiotracer analysis combined with a washing efflux method were used to analyze ion changes. We found that both the K+ and the Cl- content increased by about 100% within 1 min after increasing the external K+, and remained constant for 30 min thereafter. The mechanisms, i.e., Na+,K+-ATPase-mediated transport and NaCl/KCl contransport, that are involved in glial ion homeostasis in physiologic situations (extracellular K+ increase to 12 mM) are disturbed in this system and do not contribute to the cytotoxic swelling induced by pathologic K+ concentrations. The Na+,K+-ATPase-mediated K+ uptake is not additionally stimulated by the pathologic K+ increase. The NaCl/KCl cotransport was minimal, presumably due to a severe reduction of the Na+ driving force by the depolarization. The mechanisms of this pathological swelling were found to consist mainly of a passive KCl influx. This influx is mediated by a disturbance of the Donnan equilibrium. It probably involves an increase of the Cl- permeability by activation of a previously described voltage-dependent Cl- channel, which is closed under physiological conditions. However, it was found that the intracellular Cl- accumulation did not completely match the corresponding K+ accumulation. Possible reasons for this anion deficit are discussed.
Animals↗
Absence of decarboxylation of some aromatic-L-amino acids by cultured astrocytes.
The addition of L-3,4-dihydroxyphenylalanine, L-5-hydroxytryptophan or m-tyrosine to cultured isolated astrocytes does not elicit the synthesis of either dopamine, 5-hydroxytryptamine or m-tyramine, respectively. In contrast, the astrocytes show ability to take up and store dopamine and 5-hydroxytryptamine that are in turn respectively metabolized to 3,4-dihydroxyphenylacetic acid or 5-hydroxyindoleacetic acid. These findings suggest that astroglial cell cultures lack aromatic-L-amino acid decarboxylase and give further evidence that these cells contain monoamine oxidase.
5-Hydroxytryptophan↗
Culture environment and channel-mediated potassium fluxes in astrocytes.
Barium-sensitive channel-mediated unidirectional K+ influx into mouse and rat astrocytes and its dependency upon cell density and serum was investigated. The huge difference in flux velocity between mouse (about 1000-2000 nmol X mg-1 X min-1, depending on culture conditions) and rat astrocytes (100-20 nmol X mg-1 X min-1) could not be primarily explained by an effect of culture conditions. Some small effects of both cell density and the use of fetal calf serum were, however, seen on the fluxes in mouse as well as rat astrocytes. It seems that rat astrocytes in culture lose irreversibly a glial K+ channel. An alternative is that the results may reflect a lower level of K+ channels in rat astrocytes in situ.
Animals↗
Evaluation of the osmoregulatory function of taurine in brain cells.
Homogenous primary cultures of mouse astrocytes and cortical neurons were used to clarify the role of taurine in ion and osmoregulation in the CNS. This study indicates that both neurons and glial cells have uptake systems for taurine. The cell water content does not change during loading of cells with taurine. Chemical analysis indicates that part of the accumulated taurine is metabolized and that the product(s) are stored in the cells. Extracellular taurine (1 mM) has no effect on K+, Na+, Cl-, or Ca2+ movements in astrocytes. However, astrocytes loaded to a taurine content which corresponds a concentration of 60 mM (corresponds to normal mouse cortex levels) show a 50% reduction in their K+ accumulation by carriers and a 100% increase in Ca2+ turnover rates. Movements of Ca2+ and K+ are involved in neurotransmission. It appears that taurine stored in glial cells, has an important effect on ion homeostasis in the CNS and may act indirectly on neuronal excitability.
Animals↗
Extracellular hydrogen ions influence channel-mediated and carrier-mediated K+ fluxes in cultured mouse astrocytes.
Physiological (pH 7.2 and 7.0) and pathological (pH 6.8 and 6.6) changes of external pH, as they are measured in vivo, were imposed on mouse astrocytes in primary cultures and the effect on components of K+ transport pathways across the cell membrane was measured. Physiological pH changes had no effect at all. Pathological pH changes in inhibited K+ fluxes through channels by 40%. This effect occurred 3-6 min after a pH change and was not additive to a similar change induced by amiloride. This could be seen as an indication that the observed effects are mediated by subsequent changes in intracellular pH; however, direct intracellular pH measurements were not undertaken. A low pH of 6.6 inhibited the activity of the Na+, K+-ATPase by 65% and reduced the carrier-mediated K+ net accumulation into the cells by 60%. This suggests that measured extracellular pH changes in the brain may be functionally important, as they interfere with the K+ homeostasis of the nervous tissue.
Amiloride↗
Swelling and potassium uptake in cultured astrocytes.
The intracellular water content of astrocytes in primary cultures shows a biphasic swelling pattern on exposure to various increased external K+ concentrations over the range of 1.5-100 mM. The two phases (physiological, 1.5-12 mM K+; pathological, 25-100 mM K+) are based on two different mechanisms. Both can be blocked by low Cl- solutions and involve intensive net uptake of K+. However, the physiological phase consists of the activation of a KCl + NaCl carrier, while the Na+ in turn is pumped out by Na+-K+ ATPase, with a resultant net accumulation of KCl. At pathological K+ concentrations the KCl + NaCl carrier is less active because the Na+ driving force, its energy source, is reduced (owing to depolarization by K+). However, the Donnan equilibrium across the cell membrane is heavily disturbed, which leads to passive KCl accumulation. The results suggest that volume changes in cultured glial cells during exposure to high K+ should be taken into consideration since they disguise K+ accumulation when only ion activity is measured.
Animals↗
Calcium entry into cultured mouse astrocytes.
The barium-sensitive component of transmembrane influx of 45Ca into cultured mouse astrocytes was investigated in order to find clues for an involvement of a glial Ca2+ uptake system in Ca2+ movements of the CNS. The Ca2+ turnover was very high amounting to a rate constant of 1.4 min-1. The magnitude of the Ca2+ flux was, however, only about 2% of the K+ flux. It had the same magnitude as the Cl- flux. The Ca2+ influx was not sensitive to the organic Ca2+ channel blockers verapamil and nifedipine; however, cadmium, cobalt and barium blocked the flux in millimolar concentrations. The Ca2+ influx was a linear function of the external Ca2+ concentration up to 1.8 mM. A further increase in external Ca2+ did not increase the magnitude of the influx. Increases in external K+ to the physiological ceiling level of 12 mM (which corresponds to a 15 mV depolarization) and to 54 mM (48 mV depolarization) had no effect on the system. We conclude that the Ca2+ entry system of glial cells is not involved in the decrease of the external Ca2+ concentration during neuronal activity. The barium-evoked spontaneous depolarizations of astrocytes reported in the literature are probably due to the blockade of K+ channels by barium, since 50 microM barium did not affect the Ca2+ flux and increased the total Ca2+:K+ flux ratio from 1:50 to 1:2.5.
Animals↗
A transmembrane sodium cycle in astrocytes.
This study demonstrates that the Na+-K+ pump of mouse astrocytes in primary cultures is stimulated by increases of intracellular Na+. The data presented show that the coupling ratio of the pump varies as a function of intracellular Na+ but not of extracellular K+. A furosemide-sensitive K+ net uptake activated by increased external K+ was partly ouabain-sensitive and found to be dependent upon the Na+ driving force. These findings and those of other authors indicating that an increase of intracellular K+ of glial cells did not involve a concomitant decrease in intracellular Na+ are explained by a transmembrane Na+ cycle. Na+ would enter the cells by providing the driving force for the KCl carrier and would be pumped out by the Na+-K+ pump. The proposed Na+ cycle would function as a coupler and synchronizer of the Na+-K+ and KCl pump under conditions of physiologically elevated external K+.
Animals↗
AFA-I, a cloned afimbrial X-type adhesin from a human pyelonephritic Escherichia coli strain. Purification and chemical, functional and serologic characterization.
AFA-I, a mannose-resistant, P-independent, X-binding afimbrial Escherichia coli adhesin was purified from a recombinant strain and chemically, functionally and serologically characterized. AFA-I exists on the bacterial surface and free as a macromolecular aggregate in the supernatant of spent culture medium. It is composed of a single, repeating 16-kDa polypeptide subunit. The AFA-I protein amino acid composition is remarkable for the presence of 22% non-polar hydrophobic residues and 2.5-3.0 cysteines per subunit. Since AFA-I travels as a monomer in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under non-reducing conditions, no disulfide bonds exist between subunits and at least one free sulfhydryl per subunit is available. The AFA-I N-terminal amino acid sequence residues 1-24 was unrelated to E. coli fimbrial sequences; however, the N-terminus of AFA-I and GV-12, another E. coli afimbrial protein, was asparagine. HB101 (pIL 14), the AFA-I recombinant strain, agglutinated only human and gorilla erythrocytes, indicating a preference for receptor molecules on the red cells of man and the anthropoid apes. AFA-I did not bind glycophorin A or sialyl glycosides and is therefore distinct from the E. coli X-binding adhesins with M and S specificity. The AFA-I receptor was found to be abundant and diffusely distributed on HeLa tissue culture monolayer cell surfaces by indirect fluorescent microscopy. Anti-AFA-I sera bound AFA-I in Western blots of 4 out of 16 X-binding E. coli urine isolates. They did not bind MS or P pili. AFA-I may be exemplary of an adhesin class significant for the pathogenesis of human urinary tract infections.
Adhesins, Escherichia coli↗
Carrier-mediated KCl accumulation accompanied by water movements is involved in the control of physiological K+ levels by astrocytes.
Potassium accumulation and water transport into mouse astrocytes in primary cultures were investigated when external potassium was increased from 3 to 12 mM. The intracellular potassium content increased by 63% within 50 s of such a change. The increase consisted of a ouabain- and furosemide-sensitive component, both contributing in about the same amounts. Experiments with altered ion composition revealed that the furosemide-sensitive component consisted of a KCl accumulation. Water moved into the astrocytes without delay after such an external K+ increase and increased the cell water by 27%. This water increase was abolished in solutions with reduced Cl- and during application of furosemide. Thus, these results on a KCl uptake accompanied by water movements into astrocytes suggest a potential mechanism by which glial cells in situ can regulate external K+ levels.
Animals↗
Differences in cation transport properties of primary astrocyte cultures from mouse and rat brain.
42K and 22Na contents and unidirectional fluxes, as well as net accumulation of 42K in response to elevated extracellular K+, were investigated in primary cultures of astrocytes prepared from neonatal rat and mouse brain. The major difference between both species affected the unidirectional K+ influx which was up to 75 times higher in mouse as compared to rat cultures. The flux rates in mouse astrocytes were doubled by measuring uptake in salt solution instead of growth medium, while 42K influx in rat astrocytes was unaffected by such treatment. 22Na transport was very similar in astrocytes from both species. The length of culture period and treatment with DBcAMP (2',3'-dibutyryl cyclic adenyl monophosphate) modified K+ transport but not Na+ transport. Both types of cultures showed the same accumulation of 42K in response to raised medium K+. Amiloride inhibited 42K influx by 41% and 13% in mouse and rat cultures, respectively. In contrast, furosemide inhibited 42K uptake in rat astrocytes cultures by 50% but had no effect on mouse astrocyte cultures. 50 microM barium chloride markedly inhibited 42K uptake in mouse cultures by 96% (or 1491 nmol X mg-1 X min-1), but inhibited 42K uptake in rat cultures by only 23% (or 9 nmol X mg-1 X min-1). Ouabain was similarly effective in both types of astrocyte cultures. We conclude that Na+ transport as well as net K+ accumulation and Cl- transport (based on previous studies) properties are reasonably stable and reproduced in primary cultures from both mouse and rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Amiloride↗