pH regulation after acid load in primary cultures of mouse astrocytes

Chow, S. Y.; Yen-Chow, Y. C.; White, H. Steve; Woodbury, Dixon M.

doi:10.1016/0165-3806(91)90156-d

Cited by 29 publications

(26 citation statements)

References 33 publications

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“…In our study, steady-state pHi (in C0,-free NaHEPES buffer) of C6 cells was 7.39, similar to the previously reported value of 7.35 in this cell line (Jean et al, 19861, while steady-state pHi of rat astrocytes was 7.15, somewhat higher than the average of previously reported values for mammalian astrocytes, between 6.89 and 7.02 (Boyarsky et al, 1993;Chow et al, 1991;Kaila et al, 1991;MellergArd et al, 1992). In mouse astrocytes, a very low value of 6.68 has been reported (Wuttke and Walz, 1990), but this value was measured with double-barrelled microelectrodes and might reflect cell damage.…”

Section: Discussionsupporting

confidence: 91%

“…Based on 36Cl-tracer studies, Kimelberg et al (1979) showed Cl-/HCO,-exchange in primary astrocytes. The existence of the Cl--/HCO,-exchanger has also been indicated in primary mouse (Chow et al, 1991) and rat (Mellergdrd et al, 1993) astrocytes. Thus, we expected that the Cl-/HCO,-exchanger would also be present in our cells.…”

Section: Naf -Independent Cl-/hc03-exchangementioning

confidence: 89%

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Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

Shrode

Putnam

1994

Glia

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We used the pH-sensitive fluorescent dye BCECF to study intracellular pH (pHi) 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 pHi was increased above the normal steady-state pHi, 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 pHi 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 pHi values above steady state; and 4) depolarization-induced alkalinization of astrocytes was seen only in the presence of CO2.

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Section: Discussionsupporting

confidence: 91%

Section: Naf -Independent Cl-/hc03-exchangementioning

confidence: 89%

Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

Shrode

Putnam

1994

Glia

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“…In this respect, it is worth mentioning that ouabain-like factor has been detected in ocular tissues (35). In apparent contrast to our findings, there is a report that 100 M ouabain failed to change the rate of pH recovery after ammonium chloride exposure in mouse astrocytes bathed in bicarbonate-containing buffer (16). Based on studies with low sodium solutions and amiloride, the authors suggested that in the presence of bicarbonate, NHE contributes minimally to pH recovery in mouse astrocytes.…”

Section: Discussioncontrasting

confidence: 77%

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Mandal¹,

Delamere²,

Shahidullah³

2008

American Journal of Physiology-Cell Physiology

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Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.

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“…To begin examining this, we used methazolamide, a carbonic anhydrase inhibitor, to acidify the cytoplasm of HCs. Inhibition of carbonic anhydrase should cause proton concentrations to rise in the cell body, an effect found previously in cultured astrocytes (Chow et al, 1991(Chow et al, , 1992. By acidifying the intracellular compartment, the proton driving force would be decreased, and, when hyperpolarized, HCs ought not be able to alkalinize the synaptic cleft via increased proton influx.…”

Section: Imaging Calcium Dynamics At the Photoreceptor Synapse Duringmentioning

confidence: 99%

Proton-Mediated Feedback Inhibition of Presynaptic Calcium Channels at the Cone Photoreceptor Synapse

Vessey

Stratis²,

Daniels³

et al. 2005

J. Neurosci.

115

136

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Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K ϩ increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K ϩ -induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

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pH regulation after acid load in primary cultures of mouse astrocytes

Cited by 29 publications

References 33 publications

Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Proton-Mediated Feedback Inhibition of Presynaptic Calcium Channels at the Cone Photoreceptor Synapse

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