STAT3‐dependent regulation of the electrogenic Na^+/HCO₃⁻ cotransporter 1 (NBCe1) functional expression in cortical astrocytes

Abstract

The electrogenic Na + /HCO 3 − cotransporter (NBCe1) in astrocytes is crucial in regulation of acid-base homeostasis in the brain. Since many pathophysiological conditions in the brain have been associated with pH shifts we exposed primary mouse cortical and hippocampal astrocytes to prolonged low or high extracellular pH (pH o) at constant extracellular bicarbonate concentration and investigated activation of astrocytes and regulation of NBCe1 by immunoblotting, biotinylation of surface proteins, and intracellular H + recordings. High pH o at constant extracellular bicarbonate caused upregulation of NBCe1 protein, surface expression and activity via upregulation of the astrocytic activation markers signal transducer and activator of transcription 3 (STAT3) signaling and glial fibrillary acidic protein expression. High pH o-induced increased NBCe1 protein expression was prevented in astrocytes from Stat3 flox/flox ::Gfap Cre/+ mice. In vitro, basal and high pH o-induced increased NBCe1 functional expression was impaired following inhibition of STAT3 phosphorylation. These results provide a novel regulation mode of NBCe1 protein and activity, highlight the importance of astrocyte reactivity on regulation of NBCe1 and implicate roles for NBCe1 in altering/modulating extracellular pH during development as well as of the microenvironment at sites of brain injuries and other pathophysiological conditions.

Regulation of electrogenic Na+/HCO3− cotransporter 1 (NBCe1) function and its dependence on m‐TOR mediated phosphorylation of Ser245

“…The results also show that the observed change of NBCe1 activity following extracellular metabolic acidosis is not associated with changes of total NBCe1 or surface protein abundance or with activation of astrocytes (Figures 1 and 2). Thus, these results extend our previous observations following exposure of cortical astrocytes to metabolic alkalosis (Khakipoor et al, 2019) or to extracellular acidosis by decreasing extracellular pH at constant bicarbonate concentration (Giannaki et al, 2021). Moreover, the observation that in NBCe1 deficient astrocytes of the rate of alkalinization and acidification was almost abolished, as compared to WT cells (Figure 3) highlights that NBCe1 and not Na + /H + exchanger is the major regulator of intracellular pH in the presence of CO 2 /HCO 3 − in astrocytes.…”

“…To measure intracellular H + concentration ([H + ] i ) changes in cultured cortical astrocytes in the presence or absence of rapamycin, we used a Visitron imaging system (http://www.visitron.de/) and the acetoxymethyl ester of a proton‐sensitive dye, 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF‐AM), as described previously (Giannaki et al, 2021; Khakipoor et al, 2017, 2019). Cells were incubated with 3 µM BCECF‐AM in bicarbonate‐buffered saline solution for 15 min at room temperature.…”

“…Visitron imaging system (http://www.visitron.de/) and the acetoxymethyl ester of a proton-sensitive dye, 2′,7′-bis-(carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF-AM), as described previously (Giannaki et al, 2021;Khakipoor et al, 2017Khakipoor et al, , 2019…”

Cell-Type Dependent Regulation of the Electrogenic Na+/HCO3− Cotransporter 1 (NBCe1) by Hypoxia and Acidosis in Glioblastoma

“…First, NBCe1 protein abundance was determined by immunoblot analysis. Using an antibody raised against NBCe1, strong immunoreactive bands at ~110 kDa and ~130 kDa were detected in cell homogenates of all GBM cellular states, as previously described for mouse hippocampal and cortical astrocytes [ 14 , 15 , 16 ], ( Figure 1 A). These results were further confirmed by immunofluorescence, as shown in Figure 1 B,D.…”

Soluble tumor necrosis factor-alpha-induced hyperexcitability contributes to retinal ganglion cell apoptosis by enhancing Nav1.6 in experimental glaucoma

“…However, we found that blocking p38 MAPK and STAT3 signaling pathways completely eliminated the acute TNF-α application-induced enhancement of Na + currents, but did not influence the increased Nav1.6 protein expression due to longer TNF-α exposure (7 days), suggesting that these two signaling pathways may directly modulate Na + channels in a transcriptional-independent manner. Indeed, previous studies reported that p38 MAPK was involved in the upregulation of different subtypes of Na + currents [ 42 , 69 – 71 ], and activation of STAT3 directly increased the activity of electrogenic Na + /HCO 3 - cotransporter 1 [ 72 ]. However, it was also reported that activation of p38 MAPK reduced the amplitude of peak Nav1.6 currents in the DRG-derived cell line ND7/23 and hippocampal neurons [ 71 , 73 ].…”