Type II sodium channels in spinal cord astrocytes in situ: Immunocytochemical observations

The expression of sodium channel α-subunit isoforms in astrocytes in adult rat spinal cord and optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and subtype-specific sequences of the sodium channel. In adult rat spinal cord, astrocytes within the dorsal and ventral funiculi were immunolabelled with antibody SP20, which recognizes a conserved sequence within sodium channel types I, II, and III. In addition, astrocytes within these spinal cord white matter tracts were immunostained with antibody SP11-II, which recognizes sodium channel type II. Antibodies SP11-I and SP32-III, which are directed against subtype-specific sequences in sodium channel types I and III, respectively, did not label astrocytes in the dorsal and ventral funiculi of the spinal cord. In optic nerves, astrocytes were immunostained with antibody SP20. However, no detectable labelling of cells within the optic nerve was observed with antibodies SP11-I, SP11-II, and SP32-III. These observations demonstrate that sodium channel II is expressed by astrocytes in spinal cord white matter. Moreover, these data suggest that regional factors regulate the level of sodium channel isoform expression in astrocytes.     

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.     

Properties of single potassium channels in cultured primary astrocytes

The patch-clamp technique was used to characterize the single channel ion currents in primary cultures of rat astrocytes. The most dominant channel type, which was found in over half of the inside-out membrane patches, was a potassium channel. The measured reversal potential was -67 mV, which is close to the calculated Nernst potential for potassium ions (-80 mV). These potassium channels activated with bursts of very brief openings. Once activated the channels did not inactivate. The measured probabilities of the channels to be closed showed at least 3 different modes of channel behaviour: one voltage-independent and two voltage-dependent modes. During each activity-mode a 'main' conductance level plus two other conductance levels were observed. In some recordings a pronounced outward rectification could also be seen.     

Mechanotransducing ion channels in C6 glioma cells

Mechanotransducing (MS) ion channels and images of the patch membrane were studied in cell-attached patches in C6 glioma cells. MS channel density was ∼0.08 to 0.5 channels/μm², channel conductance was ∼40 pS (at -40 mV), and the reversal potential was +15 mV. Replacement of NaCl with KCl, CsCl, or Na gluconate in the pipette solution was without substantial effect on the current-voltage relationship. Replacement of NaCl with NMDG (N-Methyl-D-Glucamine) Cl or reducing NaCl decreased the amplitude of inward currents at negative membrane potentials and caused the reversal potential to shift in the negative direction. Rapid application of suction to the back of the pipette usually elicited a fast (<0.1 s) appearance of channel activity. The peak (phasic) in channel activity was followed by a decrease to a constant (tonic) level of activity. The reduction in channel activity—called adaptation—was reduced at depolarizing membrane potentials and disappeared if too much pressure was applied. Positive pressure caused the patch membrane to curve toward the pipette tip, move in the direction of the tip, and evoke MS channel activity. Removal of the positive pressure caused the patch to move back to the original position. Conversely, negative pressure caused the patch membrane to curve away from the pipette tip, move away from the tip, and elicit MS channel activity. Gigohm seal resistances were always maintained during translational movement of the patch membrane. Tonic MS channel activity was not associated with translational movements of the patch membrane. Phasic and tonic channel activity were independent of the sign of curvature of the patch membrane. C6 glioma cells have rapidly adapting voltage-dependent MS ion channels, which are non-selective for monovalent cations, and belong to the stretch-activating class of mechanosensory ion channels. Adaptation in MS channels may allow the cell to limit the influx of cations in response to mechanical input. The selective loss of adaptation suggests that the MS channel's gate receives input from two sources. A minimal viscoelastic mechanical model of adaptation and two alternative models for translational movement of the patch are presented. © 1996 Wiley-Liss, Inc.     

Isoform-specific expression of sodium channels in astrocytes in vitro: Immunocytochemical observations

The expression of sodium channel α-subunit isoforms in astrocytes cultured from P-0 rat spinal cord and P-7 rat optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and isoform-specific amino acid sequences of the rat brain sodium channel. In spinal cord cultures at 5 days in vitro (DIV), both stellate and flat astrocytes were immunostained with antibody SP20, which recognizes a conserved sequence common to sodium channel types I, II/IIA, and III. Antibody SP11-I, which is directed against a subtype-specific sequence in sodium channel I, did not yield detectable staining in spinal cord astrocytes. Antibody SP11-II, which is directed against a subtype-specific sequence in sodium channel II, immunostained both stellate and flat spinal cord astrocytes, although with less intensity than SP20. Antibody SP32-III, which is directed against a subtype-sequence in sodium channel III, immunostained stellate but not flat spinal cord astrocytes. SP20, SP11-II, and SP32-III staining persisted in stellate spinal cord astrocytes through 14–21 DIV, while SP20 and SP11-II immunostaining in flat spinal cord astrocytes was attenuated with time in culture. In optic nerve cultures at 5 DIV, SP20 staining was present in both stellate and flat astrocytes, but at reduced levels compared to spinal cord astrocytes. With increased time in culture, SP20 staining was maintained in stellate optic nerve astrocytes but was gradually lost in flat optic nerve astrocytes. Stellate optic nerve astrocytes exhibited low levels of staining with SP11-I, SP11-II, and SP32-III. Flat optic nerve astrocytes lacked or displayed very low SP11-II staining, and SP11-I and SP32-III staining was not detectable. These observations demonstrate that cultured astrocytes are immunoreactive to antibodies generated against conserved and isotype-specific peptide sequences of rat brain sodium channels, and further suggest that there are different patterns of sodium channel expression between flat vs. stellate astrocytes and in astrocytes derived from different regions of the CNS. © 1995 Wiley-Liss, Inc.     

Voltage-dependent ion channels in glial cells

Glial cells, although non-excitable, express a wealth of voltage-activated ion channels that are typically characteristic of excitable cells. Since these channels are also observed in acutely isolated cells and in brain slices, they have to be considered functional in the intact brain. Numerous studies over the past 10 years have yielded detailed characterizations of glial channels permitting comparison of their properties to those of their neuronal counterparts. While for the most part such comparisons have demonstrated a high degree of similarity, they also provide evidence for the expression of some uniquely glial ion channels. An increasing number of studies indicate that the expression of “glial” channels is influenced by the cells' microenvironment. For example, the presence of neurons can induce or inhibit (depending on the preparation and type of channel studied) the expression of glial ion channels. Like ion channels in excitable cells, glial channels can be functionally regulated by activation of second-messenger pathways, allowing for short-term modulation of their membrane properties. Although the extent to which most of the characterized ion channels are involved in glial function is presently unclear, a growing body of data suggests that certain channels play an active role in glial function. Thus inwardly rectifying K⁺ channels in concert with delayed rectifying K⁺ channels are thought to be involved in the removal and redistribution of excess K⁺ in the brain, a process referred to as “spatial buffering.” Glial K⁺ channels may also be crucial in modulating glial proliferation. Cl^? channels and stretch-activated cation channels are believed to be involved in volume regulation. Na⁺ channels appear to be important in fueling the glial Na⁺/K⁺ -pump, and Ca²⁺ channels are likely involved in numerous cellular events in which intracellular Ca²⁺ is a critical second messenger. © 1994 Wiley-Liss, Inc.     

Glutamate opens Na+/K+ channels in cultured astrocytes

Glial cells from different brain regions and species are depolarized by the neurotransmitter glutamate. The depolarization or, if voltage-clamped at the resting membrane potential, the inward current induced by glutamate could be due either to activation of receptor-coupled ion channels or electrogenic uptake of the transmitter. In the present study we applied the patch-clamp technique in the whole-cell recording mode to analyze glutamate-induced currents in cultured astrocytes from rat cerebral hemispheres. At the resting membrane potential, glutamate induced an inward current ranging from 40 to 300 pA. This current decreased in size with depolarization and reversed at about 0 mV. The resulting current-to-voltage curve was linear and depended strongly on the transmembrane Na+ but not on the Ca++ or Cl- gradient. In the presence of glutamate, current noise increased at potentials positive or negative from the reversal potential indicating that ionic channels are activated by glutamate. Both kainate and quisqualate mimicked the effect of glutamate. We conclude that glutamate opens a Na+/K+ channel in cultured astrocytes because of activation of a receptor which shares many properties with the neuronal kainate/quisqualate receptor.     

A primary encephalocele culture yields a pure population of human astrocytes

In order to develop a reliable source of human astrocytes for in vitro studies, we established a primary explant culture of a human encephalocele. This culture yielded a population of cells which were karyotypically normal, morphologically resembled astrocytes, expressed glial fibrillary acid protein, and responded mitogenically to exogenous growth factors. We conclude that white matter derived from human encephaloceles can be used to generate pure populations of normal astrocytes.     

Voltage-dependent ion channels in T-lymphocytes

The gigaohm seal 'patch-clamp' technique has recently enabled exploration of the electrical properties of cells of the immune system. In this paper we review progress made to date in cataloguing the ion channels present in the cell membranes of T-lymphocytes and present new data on the types of ion channels present in a number of human and murine T-cell-derived cell lines. The ion channels thus far described in these cells are strikingly similar to those found in nerve and muscle cells. Voltage-gated potassium channels resembling delayed rectifier potassium channels in excitable cells are present in most T-lymphocytes, T-lymphocyte-derived cell lines and macrophages. Sodium channels indistinguishable from those in excitable cells are present in a small fraction of T-cells and T-cell lines, and in some natural killer cells. Calcium channels have been reported in B-lymphocyte-derived cell lines, but have not been found in T-lymphocytes or in any T-cell-derived cell line. Potassium channels are required for activation of T-lymphocytes by mitogen, allogeneic cells, or by antigen, for lysis of target cells by natural killer cells, and may be involved in the triggering mechanism for activation of T-cells. The prevailing conception of early events in T-lymphocyte activation, the 'calcium hypothesis', involves an elevation of cytoplasmic free calcium levels as the proposed 'second messenger' in activation, giving rise to a cascade of subsequent events resulting eventually in cell division. A major focus of this paper is to evaluate specific mechanisms which have been proposed to account for experimental evidence, both in the literature and also presented here, pertaining to the calcium hypothesis. One such mechanism involves calcium channels, which have been postulated to account for the early calcium influx in activated T-lymphocytes. Since calcium channels have not been detected in T-cells, we explore the possibility that existing data can be accounted for without calcium channels. In particular, we show that many of the effects of 'calcium channel antagonists' such as verapamil, nifedipine, diltiazem and some polyvalent cations, can be accounted for by their blocking of voltage-gated potassium channels.     

Norepinephrine-mediated protein phosphorylation in astrocytes

Studies were conducted to determine if norepinephrine activates both protein kinase C and the cyclic AMP-dependent protein kinase in cultured rat astrocytes using phosphoproteins as markers. Norepinephrine was found to decrease 32P incorporation into an acidic 80,000 M(R) protein. A similar response was observed with isoproterenol and cyclic AMP analogs. In contrast, phorbol myristate acetate (PMA) increased 32P incorporation into this protein. Further studies looked at phosphorylation sites on glial fibrillary acidic protein and vimentin using two-dimensional tryptic phosphopeptide maps. The pattern of phosphorylation of these two proteins by norepinephrine resembles that of 8-bromo cyclic AMP and isoproterenol, and not that of PMA. Additionally, the effect of norepinephrine on the phosphorylation of GFAP and vimentin was blocked by alprenolol. One difference noted between norepinephrine and isoproterenol was the phosphorylation of an 18,000 M(R) protein. Norepinephrine increased, and isoproterenol decreased, 32P incorporation into this protein; however, the mechanism which mediates the norepinephrine effect remains to be determined. Overall, these studies indicate that the most prominent phosphorylation events mediated by norepinephrine are the consequence of the activation of cyclic AMP-dependent protein kinase.     

Effects of volatile anaesthetics on the membrane potential and ion channels of cultured neocortical astrocytes

Volatile anaesthetics cause changes in the membrane resting potential of central neurons. This effect probably arises from actions on neuronal ion channels, but may also involve alterations in the ion composition of the extracellular space. Since glial cells play a key role in regulating the extracellular ion composition in the brains of mammals, we analyzed the effects of halothane, isoflurane and enflurane on the membrane conductances and ion channels of cultured cortical astrocytes. Astrocytes were dissociated from the neocortex of 0–2-day old rats and grown in culture for 3–4 weeks. Anaesthetic-induced changes in the membrane potential were recorded in the whole cell current-clamp configuration of the patch-clamp technique. We further studied the effects of halothane and enflurane on single ion channels in excised membrane patches. At concentrations corresponding to 1–2 MAC (1 MAC induces general anaesthesia in 50% of the patients and rats), membrane potentials recorded in the presence of enflurane, isoflurane and halothane did not differ significantly from the control values. At higher concentrations, effects of enflurane and halothane, but not of isoflurane, were statistically significant. Single-channel recordings revealed that halothane and enflurane activated a high conductance anion channel, which possibly mediated the effects observed during whole cell recordings. In less than 10% of the membrane patches, volatile anaesthetics either increased or decreased the mean open time of K⁺-selective ion channels without altering single-channel conductances. In summary, it seems unlikely that the actions of volatile anaesthetics described here are involved in the state of general anaesthesia. Statistically significant effects occurred at concentrations ten times higher than those required to cause half-maximal depression of action potential firing of neocortical neurons in cultured brain slices. However, it cannot be excluded that the changes observed in the membrane conductance of cortical astrocytes disturb the physiological function of these cells, thereby influencing the membrane resting potential of neurons.     

Microglia, but not astrocytes, react to sciatic nerve injury in aging rats

Peripheral nerve axotomy activates microglia and astrocytes within regions of brainstem or spinal cord from which the nerve arises. The present study demonstrates that unilateral sciatic axotomy in rats 2 to 18 months of age results in differing responses with age between these two glial populations. By 4 days postaxotomy, both astrocytes and microglia become activated in 2-month-old rats, whereas only the microglial population shows evidence of activation in rats 8 to 18 months of age.     

Potassium and sodium channels in human malignant glioma cells

Human malignant glioma cells from 5 different cell lines were voltage clamped and examined for the presence of depolarization-activated ion channels. Outward K-currents were elicited at membrane potentials > 40 mV, which had two main components, one which was delayed and blocked by externally applied tetraethylammonium (TEA, 10 mM), and another which was instantaneous and insensitive to TEA in the outside solution. The proportion of the two K-current components varied between cell lines. An increase in [Ca²⁺] in the range 0–4 mM, decreased the leak conductance and shifted the activation of the instantaneous outward K-current towards more positive potenttials. Mg²⁺, Zn²⁺ and Co²⁺ had qualitatively similar effects. Patch recordings with 150–160 mM K⁺-solution on both sides of the membrane revealed that the delayed outward K-current was carried through large conductance (250–300 pS) channels. Changes in free [Ca²⁺]_i from 0 to 2 × 10⁻⁸ M increased the activation of the large conductance K-channel. Small Na-currents were identified in cells from one cell line (Tp-378MG). The Na-conductance rangedfrom 0.5 to 7.5 nS in 25% of the cells, and was less than 0.5 nS in 75%. The Na-channels were activated and inactivated at 30–40 mV more positive potentials than in the mammalian peripheral nerve. Tetrodotoxin (100 mM) blocked g_Na almost completely.     

Gliosis in human brain: relationship to size but not other properties of astrocytes

Gliosis is the most frequent and therefore important neurocellular reaction to brain insult occurring in diseases ranging from AIDS to infarction. Neuropathological diagnosis is bases on morphological changes of brain glial cells. Changes commonly agreed to reflect gliosis are qualitative increases in size, number and glial fibrillary acidic protein (GFAP) immunoreactivity of astrocytes. These parameters were morphometrically quantified in brain tissues of 22 individuals who died with 7 diseases and statistically compared to the extent of gliosis independently determined by 3 qualified observers. The data indicate that the extent of gliosis correlated with the increase in size of astrocytes in white matter (π = 0.67) and this relationship was statistically significant (P = 0.0006). In contrast, the extent of gliosis was not correlated with the density of astrocytes nor the intensity of GFAP staining.     

Monoclonal antibody, KK1, recognizes human retinal astrocytes and distinguishes a subtype of astrocytes in mouse brain

Astrocytes exhibit a diverse morphology and numerous functions in the central nervous system as well as in the retina. In order to obtain markers for the analysis of astrocytes, we prepared monoclonal antibodies that recognized antigens specific to astrocytes. Monoclonal antibody (mAb), designated KK1, reacted with the processes of astrocytes in the nerve fiber layer and the ganglion cell layer in the human retina as detected by indirect immunofluorescence. Normal Müller cells, whose processes are localized vertically in retina, were not labeled by KK1 mAb. In mouse brain, KK1 mAb reacted specifically with astrocytes in the white matter, but not with those in the gray matter. Studies employing a high-resolution confocal laser scanning microscope and double-labeling with KK1 mAb and commercially available anti-glial fibrillary acidic protein (GFAP) mAb (GA5) revealed that KK1 mAb visualized the processes that were not recognized by anti-GFAP rnAb (GA5) in both human retina and mouse brain. In cultured mouse astrocytes. KKI mAb reacted only with anti-GFAP mAb (GA5)-positive cells, but a small percentage of anti-GFAP mAb (GA5)-positive cells were labeled with KK1 mAb. In addition, the subcellular distribution of the KK1 antigen in cultured astrocytes apparently differed from that of GFAP labeled by anti-GFAP mAb (GA5). The antigen that was purified from the normal mouse brain by KK1 mAb-conjugated beads reacted with anti-GFAP mAb(GA5) in immunoblotting. No reactivity of KK1 mAb was observed in immunohistochemical analysis in GFAP − / − mutant mouse brain. These results demonstrate that KK1 mAb specifically recognized an epitope of GFAP that did not react with other anti-GFAP mAb (GA5). Retinal astrocytes and a subtype of astrocytes in the white matter of mouse brain shared the epitope that was recognized by KKI mAb. KKI mAb might be a powerful tool to investigate a subtype of astrocytes.     

NPPB对星形胶质细胞增殖的抑制作用

目的观察体积调节性阴离子通道阻滞剂NPPB对体外培养星形胶质细胞增殖的影响。方法采用原代培养的大鼠大脑皮层星形胶质细胞,实验分为对照组(正常培养基培养)与NPPB干预组;在不同时间点(0、6、12、24、48h),应用流式细胞技术以及免疫细胞荧光双标法(Brdu/DAPI)检测各实验组星形胶质细胞增殖及细胞周期进展的情况。结果与对照组相比,NPPB干预组在12、24h时星形胶质细胞增殖率较正常对照组降低(P<0.05),同时处于S期细胞的百分率亦相对正常对照组明显下降(P<0.01)。结论体积调节性氯离子通道阻滞剂NPPB可以显著抑制体外培养星形胶质细胞增殖及细胞周期进展,提示VRAC通道参与了体外培养星形胶质细胞的增殖。     

Thorn-shaped astrocytes: possibly secondarily induced tau-positive glial fibrillary tangles

Argyrophilic and tau-positive abnormal structures occurring in glial cells are called glial fibrillary tangles. In the astrocyte, a conspicuous tau-positive structure is known to appear in progressive supranuclear palsy (PSP). In this report, another type of argyrophilic and tau-positive astrocytes is reported. The morphology of this new type is quite different from that of the previously reported tau-positive astrocyte in PSP and they are designated here as thorn-shaped astrocytes (TSA). TSA have an apparently argyrophilic cytoplasm with a few short processes and often have a small eccentric nucleus, whose appearance resembles that of a reactive astrocyte. Immunohistochemically, TSA are positive to anti-tau antibodies but are negative for ubiquitin. Simultaneous immunostaining revealed the coexistence of tau and glial fibrillary acidic protein epitopes in the same cytoplasm. Electron microscopically, bundles of 15-nm straight tubules were included in the cytoplasm together with abundant glial filaments. In the vicinity of a cluster of TSA, related structures of perivascular or subpial tau-positive linings, which correspond to astrocytic end-feet, are sometimes observed. In almost all cases, a few TSA are generally located in a confined area of subpial and subependymal regions. Although TSA appear to be intimately associated with some diseases, they are also found in a wide range of cytoskeletal disorders including the aged brain with neurofibrillary tangles. TSA are presumed to be a secondarily induced product in relation to astrocytic reaction.     

Characterization of stretch-activated ion channels in cultured astrocytes

The presence of a stretch-activated channel in rat cerebellar astrocytes in culture is described. This stretch-sensitive channel is K⁺ -selective and its open probability increases with suction following a Boltzmann-like distribution with half activation at 45 mm Hg. Kinetic analysis of the single-channel data indicated that there are two open and two closed states and that the shortest time constants of both open and closed states are the most sensitive to suction. A symmetrical two-barrier-one-site permeation model can quantitatively describe the inward rectification of the single-channel current to voltage relations. It is suggested that this stretch-activated channel plays a role in the regulatory volume response of astrocytes to hyposmotic conditions. © 1993 Wiley-Liss, Inc.     

Tamoxifen对星形胶质细胞增殖的作用

目的观察体积调节性阴离子通道阻滞剂Tamoxifen对体外培养星形胶质细胞增殖的的影响。方法采用原代培养的大鼠大脑皮层星形胶质细胞,实验分为对照组(正常培养基培养)与Tamoxifen干预组;在不同时间点(0h、12h、24h、48h),应用流式细胞技术以及免疫细胞荧光双标法(Brdu/DAPI)检测各实验组星形胶质细胞增殖及细胞周期进展的情况。结果与对照组相比,Tamoxifen干预组在12h、24h时星形胶质细胞增殖率较正常对照组降低(P0.05);处于S期细胞的百分率相对正常对照组明显下降,处于G0-G1期细胞的百分率较正常组增高(P0.01)。结论体积调节性阴离子通道阻滞剂Tamoxifen可以显著抑制体外培养星形胶质细胞增殖及细胞周期进展,提示VRAC参与了体外培养星形胶质细胞的增殖。