Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

We used the pH-sensitive fluorescent dye BCECF to study intracellular pH (pH_i) regulation in primary cultures of rat astrocytes and C6 glioma cells. Both cell types contain three pH-regulating transporters: (1) alkalinizing Na⁺/H⁺ exchange; (2) alkalinizing Na⁺ + HCO₃ ?/Cl_? exchange; and (3) acidifying Cl^?/HCO₃ ^? exchange. Na⁺/H⁺ exchange was most evident in the absence of CO₂; recovery from acidification was Na⁺ dependent and amiloride sensitive. Exposure to CO₂ caused a cell alkalinization that was inhibited by DIDS, dependent on external Na⁺, and inhibited 75% in the absence of Cl^? (thus mediated by Na⁺ + HCO₃ ^?/Cl^? exchange). When pH_i was increased above the normal steady-state pH_i, a DIDS-inhibitable and Na⁺ -independent acidifying recovery was evident, indicating the presence of Cl^? /HCO₃ ^? exchange. Astrocytes, but not C6 cells, contain a fourth pH-regulating transporter, Na⁺ ?HCO₃ ^? cotransport; in the presence of CO₂, depolarization caused an alkalinization of 0.12 ⁺_? 0.01 (n = 8) and increased the rate of CO₂-induced alkalinization from 0.23 ± 0.02 to 0.42 ± 0.03 pH unit/min. Since C6 cells lack the Na⁺ -HCO₃ ⁺ cotransporter, they are an inferior model of pH_i regulation in glia. Our results differ from previous observations in glia in that: (1) Na⁺ /H⁺ exchange was entirely inhibited by amiloride; (2) Na⁺ + HCO₃ ^?/Cl^? exchange was present and largely responsible for CO₂?induced alkalinization; (3) Cl^? /HCO₃ ^? exchange was only active at pH_i values above steady state; and (4) depolarization-induced alkalinization of astrocytes was seen only in the presence of CO₂.     

Intracellular pH regulation in single cultured astrocytes from rat forebrain

We used the fluorescent pH-sensitive dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pH_i) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO₂/HCO₃^? -free medium buffered to pH 7.40 with HEPES at 37^°C, the cells had a mean pH_i of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO₂ and 25 mM HCO₃^? caused the steady-state pH_i to increase by an average of 0.35, suggesting the presence of a HCO₃^? -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO₃^?-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH₄⁺ caused pH_i to fall to a value substantially below the initial one. pH_i spontaneously recovered from this acid load, stabilizing at a value ～ 0.1 higher than the one prevailing before the application of NH₄⁺. In other experiments conducted on cells bathed in HEPES-buffered solutions, removing extracellular Na⁺ caused pH_i to decrease rapidly by 0.5. Returning the Na⁺ caused pH_i to increase rapidly, indicating the presence of an Na⁺-dependent/HCO₃^?-independent acid-extrusion mechanism; the final pH_i after returning Na⁺ was ～ 0.08 higher than the initial value. This pH_i recovery elicited by returning Na⁺ was not substantially affected by 50 μM ethylisopropylamiloride (EIPA), but was speeded up by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). Increasing [K⁺]_? from 5 to 25 mM caused pH_i to increase reversibly by ～ 0.2 in nominally CO₂/HCO₃^?-free solutions, and by ～ 0.1 in CO₂/HCO₃^?-containing solutions, although the initial pH_i was ～ 0.17 higher in the presence of CO₂/HCO₃^-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HCO₃^? dependent and -independent acid-base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pH_i is sensitive to changes in either membrane voltage or [K⁺]₀ per se. © 1993 Wiley-Liss, Inc.     

Intracellular pH regulation in cultured microglial cells from mouse brain

Intracellular pH (pH₁) and the mechanisms of pH₁ regulation have been investigated in cultured microglial cells from mouse brain using the pH-sensitive fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). Cells were acidified by a pulse of NH₄⁺ (4-5 min; 20 mM) and the subsequent pH₁ recovery from an acidification was studied. In HCO₃^--free saline, pH regulation was dependent on extracellular [Na⁺] and sensitive to amiloride, indicating the involvement of the Na⁺/H⁺ exchanger. In HCO₃^--containing solution 2 mM amiloride slowed but did not block pH₁ recovery; the recovery however was dependent on extracellular [Na⁺] and sensitive to 0.3 mM DIDS, suggesting the presence of Na⁺/HCO₃ cotransporter and/or Na⁺-dependent Cl^-/HCO₃^- exchanger. The involvement of a Na-dependent Cl^-/HCO₃^- exchanger was inferred from the observation that removal of Cl^- or application of 1 mM furosemide decreased but did not block the recovery rate. Increasing [K⁺]₀ resulted in an alkalinization by a process that was neither HCO₃^- nor Na⁺-dependent, nor DIDS- and amiloride-inhibitable. In conclusion, microglial cells express a distinct set of pH regulatory carriers which control for a defined level of pH₁. An increase in [K⁺]₀ can offset this level. © 1996 Wiley-Liss, Inc.     

Intracellular pH shifts capable of uncoupling cultured oligodendrocytes are seen only in low HCO3- solution

Electrical coupling between cultured mouse oligodendrocytes was transiently blocked when pHi was decreased below about 6.5 using the NH4+ prepulse method. This uncoupling could, however, only be achieved if the dominant pHi regulating mechanism in these cells, the Na+/HCO3- cotransporter, was blocked by lowering bath [HCO3-]. Under this condition, an NH4+ prepulse caused pHi to decrease toward the passive distribution for H+ (i.e., about pH 6.2). In the presence of normal bath [HCO3-] an NH4+ prepulse did not decrease pHi below 6.5 even when the second pHi regulating mechanism, the Na+/H+ exchanger, was blocked by amiloride, and consequently oligodendrocytes could not be uncoupled. Increasing CO2, which uncouples glial cells in situ (Connors et al: J. Neurosci. 4:1324-1330, 1984), did not uncouple cultured oligodendrocytes in the presence of normal bath [HCO3-], but did cause uncoupling in low [HCO3-] solution. These results indicate that electrical coupling between cultured oligodendrocytes is sensitive to pHi; in normal bath [HCO3-], however, the pHi regulation of these cells is so effective that standard techniques for intracellular acidification are unable to lower pHi to levels which cause the closure of oligodendrocyte gap junctions.     

Voltage-dependent Na+-HCO3− cotransporter and Na+/H+ exchanger are involved in intracellular pH regulation of cultured mature rat cerebellar oligodendrocytes

Intracellular pH (pH_i) was measured at 37°C in mature rat cerebellar oligodendrocytes dissociated in culture by using the pH-sensitive probe BCECF. Cells were identified by anti-galactocerebroside antibody. The mean steady-state pH_i was 7.02 in the absence of CO₂/bicarbonate (Hepes-buffered solution) at an external pH of 7.40 and 7.04 in 5% CO₂/25 mM bicarbonate-buffered solution at the same external pH; this value was modified neither by the removal of external chloride nor by the addition of the chloride-coupled transport blocker DIDS. In both external solutions steady-state pH_i values were strongly dependent on external pH. In Hepes-buffered solution pH_i recovery following an acid load required external Na⁺ and was completely inhibited by amiloride, indicating the presence of a Na⁺/H⁺ exchanger. In CO₂/bicarbonate-buffered solution amiloride partially reduced the pHi recovery rate, indicating the presence of a bicarbonate-dependent pH_i regulating mechanism. Membrane depolarization induced by increasing external K⁺ concentration elicited an alkalinization only in the presence of external Na⁺ and bicarbonate. Analysis of the calculated HCO₃ fluxes with respect to membrane potential indicated that these fluxes were mediated by a Na⁺-HCO₃ cotransport with a stoichiometry of 1:3. These results demonstrate that a Na⁺/H⁺ exchanger and a Na⁺ HCO₃ cotransporter are involved in pH_i regulation of mature oligodendrocytes. GLIA 19:74–84, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.     

Mechanisms of pH recovery from intracellular acid loads in the leech connective glial cell.

We used double-barrelled, neutral carrier, pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH (pHi) is regulated in the connective glial cells of the medicinal leech. In HEPES-buffered, nominally CO2/HCO3(-)-free solutions the recovery of pHi from intracellular acidosis is Na(+)-dependent and reduced by at least half in the presence of amiloride, suggesting the action of Na+:H+ exchange. The rate of pHi recovery by this mechanism can be increased by raising the extracellular buffering power or by increasing extracellular pH. The presence of CO2/HCO3(-)-greatly increases the rate of pHi recovery from intracellular acidosis. This CO2/HCO3(-)-stimulated recovery is also dependent on external Na+, largely Cl(-)-independent, inhibited by DIDS, and accompanied by membrane hyperpolarization. This is consistent with it being mediated by the electrogenic cotransport of Na+ and HCO3- into the cells. A Cl(-)-dependent component to Na(+)- and HCO3(-)-dependent regulation is most easily explained by the added presence of a Na(+)-dependent exchange of HCO3- and Cl-.     

腺病毒介导NT-3基因在雪旺细胞的表达

目的观察腺病毒介导的NT-3基因在培养雪旺细胞(Schwann cells, SCs)的表达.方法在293细胞中培养扩增NT-3重组腺病毒(adenovirus vector for NT-3,Ad-NT-3),用组织培养半数感染量法测定其滴度.然后用Ad-NT-3感染原代培养的SCs,逆转录酶-多聚酶链反应(RT-PCR)技术检测NT-3基因的表达.结果 Ad-NT-3扩增后获得了较高滴度的病毒.SCs经NT-3重组腺病毒感染24 h后有NT-3 mRNA的转录.结论腺病毒介导的NT-3基因可转入培养的SCs并高效表达.     

Intracellular pH regulation in isolated fast-twitch skeletal muscle from dystrophin-deficient mouse

In muscles from anaesthetized dystrophin-deficient mdx mice, exercise results in a stronger acidification and a slower intracellular pH recovery compared to control mice. We examined whether this observation could be attributed to defective H+-carriers in dystrophin-lacking muscles. Immunohistochemistry and Western blots revealed no defect in mdx muscles for the presence of the lactate-/H+co-transporter MCT4 and of the Na+/H+ antiporter NHE1, the main H+-carriers active in fast-twitch skeletal muscle after exercise. Functional tests of the H+-transporters, on isolated muscles submitted to identical flow of superfusion, were performed in conditions meant to lower intracellular pH: repetitive electrical stimulation or NH4Cl pre-pulse. These revealed no defect in intracellular pH recovery in mdx muscles. Therefore, we conclude that impaired intracellular pH regulation in anaesthetized mdx mice is not attributable to a reduced presence or activity of H+-extruders. We propose that CO2 washout might be slowed down in vivo in mdx muscles because of the defective vascular response in contracting muscles from these mice.     

Potassium channel regulation in Schwann cells during early developmental myelinogenesis

The presence of neuronal-like, voltage-gated ion channels on glia has raised questions concerning their physiological roles. Insights into glial channel function can be gained by examining regulation of channel expression during axoglial interactions. We examine the regulation of Schwann cell potassium channels in developing sciatic nerves of newborn rats when myelin is first laid down. During the initial postnatal week, cell-attached patch-clamp recordings at soma of Schwann cells with visible myelin revealed an inward rectifying potassium channel (KIR), to date described only in CNS glia but not Schwann cells, as well as an outward potassium channel (KO). Around the resting potential, the KO channel is virtually closed, while the KIR channel appears maximally open. Compared with the KO channel, the KIR channel is blocked by low concentrations of Cs+ and exhibits higher sensitivity to 4-aminopyridine (4AP). Further, the KIR channel appears similar to other mammalian inward rectifiers and rectification depends, in part, on cytoplasmic Mg2+. Channel regulation bears an interesting relation to early myelination: as the average number of myelin lamellae increases from 6 to 21 from day 2 to day 8, currents decrease by 80-90%. The reduction in KO current also parallels the known decrease in proliferation of Schwann cells as they are being committed to myelination, supporting the recently proposed notion of a functional link between potassium channels and proliferation. The KIR channels, by virtue of being open at the resting potential, may play a role in buffering activity-dependent K+ accumulation during early myelin formation. The subsequent reduction in somal channel density may parallel a diminished need for K+ buffering as electrogenesis is restricted to nodal regions.     

Proteasome dynamics during cell cycle in rat Schwann cells

The proteasome is responsible for most of the protein degradation that takes place in the cytoplasm and nucleus. Immunofluorescence and electron microscopy are used to study proteasome dynamics during the cell cycle in rat Schwann cells. During interphase, the proteasome is present in the nucleus and cytoplasm and shows no colocalization with cytoskeletal components. Some cytoplasmic proteasomes always localize in the centrosome both in interphase and in mitotic cells and only associate with microtubules during mitosis. The proteasome exits the nucleus during prophase. In anaphase, the proteasome becomes prominent in the region between the two sets of migrating chromosomes and in association with interzonal microtubules and stem bodies. In telophase, the proteasome begins to reenter the nucleus and is prominent in the midbody region until the end of cytokinesis. The proteasome does not colocalize with actin or vimentin during mitosis, except for colocalization with actin in the sheet-like lamellipodia, which serve as substrate attachments for the cell during mitosis. During S phase, nuclear proteasomes colocalize with foci of BrdU incorporation, but this association changes with time: maximal at early S phase and declining as S phase progresses to the end. These results are discussed in relation to the biochemical pathways involved in cell cycle progression.     

Proliferation of Schwann cells in demyelinated rat sciatic nerve

Summary Experimental demyelination was induced by intraneural injection of anti-galctocerebroside serum into the sciatic nerves of rats. Schwann cells undergoing mitotic division were observed between days 3 to 9 after the injection and demyelinated segments were still associated with macrophages. Dividing Schwann cells were often present in association with both unmyelinated and myelinated fibers. Whether or not, daughter Schwann cells migrate along the same fiber towards neighboring demyelinated segments remains unclear. When Schwann cells attached to axon membranes of demyelinated segments were studied at later time points, they were present in clusters randomly at various regions of the segments. There was no proximo-distal gradient for the wave of Schwann cell proliferation. Mean Schwann cell internuclear distances were around 40–50 m at the earliest time of remyelination. Schwann cell redistribution and remyelination progressed regardless of the length of demyelinated segments.Supported by grants for the study of disorders of peripheral nerve, subacute myelo-optic neuropathy, and immunological diseases of the nervous system from the Intractable Diseases Division, Public Health Bureau, Ministry of Health and Welfare, Japan     

GABA uptake by purified rat Schwann cells in culture

Purified rat Schwann cells maintained in culture for up to 6 months retained their ability to take up the neurotransmitter γ-aminobutyric acid (GABA) by a high-affinity mechanism. Although cultured fibroblasts also accumulated GABA, they did so by a low affinity mechanism. These results indicate that Schwann cells continue to express a high affinity GABA transport system in the absence of signals from neurons.     

Tumor necrosis factor α and interleukin-6 mRNA expression in neonatal Lewis rat Schwann cells and a neonatal rat Schwann cell line following interferon γ stimulation

There is increasing evidence that Schwann cells play an important role in the pathogenesis of autoimmune inflammatory peripheral nerve disease. Schwann cells have been reported to express major histocompatibility complex class I and II (MHC I and II) and intercellular adhesion molecule-1 (ICAM-1), and to produce interleukin-1 (IL-1), prostaglandin E2 and thromboxane A2. In this study we investigated freshly dissociated neonatal Lewis rat Schwann cells and a SV40 transfected neonatal rat Schwann cell line (Schwann cell line) for production of mRNA for the immunomodulatory cytokines IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFNγ), and tumor necrosis factor-alpha (TNFα) employing RT-PCR. Primary Schwann cells and Schwann cell line were examined following IFNγ stimulation and were found to express TNFα and IL-6 mRNA. These results further support a role for Schwann cell participation in inflammatory responses within the peripheral nervous system (PNS).     

Levetiracetam exhibits protective properties on rat Schwann cells in vitro

Oxidative stress and inflammation represent pathways causing substantial damage to the peripheral nervous system. Levetiracetam (LEV) is a commonly used antiepileptic drug targeting high-voltage activated N-type calcium channels. Recent evidence suggests that LEV may also act as a histone deacetylase inhibitor, suggesting that this drug exhibits both anti-inflammatory and anti-oxidative effects, and as such may represent an interesting candidate for treating inflammatory diseases affecting the peripheral nerve. Therefore, we analysed the influence of LEV ex vivo on purified Schwann cells from neonatal P3 rats as well as on dorsal root ganglia prepared from E15 rat embryos. LEV diminished a lipopolysaccharide (LPS)-induced increase of the pro-inflammatory signature molecules tumour necrosis factor alpha, matrix metalloproteinase 9 (MMP-9), and caspase 6. Furthermore, LEV decreased LPS-induced cell death and protected cells against oxidative stress in a glutamate-based oxidative stress model. MMP-2 activity, usually elevated during myelination and repair, was also found to be up-regulated following LEV, while LEV exhibited no negative effects on myelination. Intracellular sodium or calcium concentrations were unaltered by LEV. Thus, LEV may be a promising, well-tolerated drug that - besides its antiepileptic potential - mediates anti-inflammatory, anti-oxidative, and anti-apoptotic properties that may potentially be useful in treating diseases of the peripheral nerve.     

The influence of glucose on intracellular and extracellular pH in rat hippocampal slices during and after anoxia

In this study we investigated in rat hippocampal slices (1) how glucose availability affected tissue acidosis during and after anoxia, (2) whether the onset of anoxic depolarization was associated with a specific pH, (3) whether glycolysis was the major source of acidification before and during anoxic depolarization, and (4) whether improved recovery of synaptic function with elevated glucose levels was related to changes in tissue acidosis. Intracellular pH (pH_i) and extracellular pH (pH_o) were measured simultaneously before, during, and after anoxia in hippocampal slices bathed in 0, 5, 10, and 15 mM glucose. Slices exposed to 0 mM glucose were given 20 mM sodium lactate as a metabolic substrate. We found that the pH_i and pH_o at which anoxic depolarization occurred depended upon glucose concentration. We also found that elevated glucose availability increased acidification in both the intracellular and extracellular compartments during anoxia and delayed recovery of pH homeostasis after anoxia. Our results suggest that glycolysis is the primary source of acidosis before the onset of anoxic depolarization, but not during anoxic depolarization. Our results also suggest that moderate increases in acidosis resulting from increased glycolysis are potentially beneficial for anoxic survival.     

Proliferation of rat intraspinal Schwann cells following tellurium intoxication

Summary A stable population of intraspinal Schwann cells, which developed follwing early postnatal irradiation of the spinal cord, was challenged by the addition of tellurium (Te) to the diet beginning at 30 days of age. Schwann cells incorporating [³H]thymidine were identified by 1 m autoradiographs and by conventional electron microscopy of adjacent thin sections. Autoradiographs of areas with Schwann cell myclination showed extensive labelling of cells in the Te-fed animals. In contrast, control animals which were not fed Te showed little evidence of labelled Schwann cells. These data indicate that Schwann cells in the intraspinal environment show a proliferative response to the presence of Te in the rat's diet, as do Schwann cells in their normal extraspinal milieu.Supported by National Multiple Sclerosis Society grant 1791-A-1, and NIH grant No. NS-23124 to I.D.D. Supported in part by NIH grant 04761, a Jacob Javits Neuroscience Investigator Award to S.A.G.     

Ultrastructural and cytochemical characteristics of cultured rat Schwann cells

Summary Schwann cell cultures were established from sciatic nerve of 3 day-old rats. Described are the ultrastructural, histochemical and ultracytochemical properties of amyelic cultured rat Schwann cells. Ultrastructural characteristics of the cultured Schwann cells are compared to the Schwann cells of 3 day-old and adult rat sciatic nerve. These findings serve as a basis for comparison when studying experimentally induced alterations in the cultured Schwann cells as well as changes due to myelination in vitro.