Cell Volume Regulation Mechanisms in Differentiated Astrocytes.

Abstract

BACKGROUND/AIMS: The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K+ channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4 (TRPV4), and Volume Regulated Anion Channel (VRAC). METHODS: The calcein quenching method was used to study water transport and cell volume regulation. Calcium imaging and electrophysiology (patch-clamp) were used for functional analyses of calcium dynamics and chloride currents. Western blot and immunofluorescence were used to analyse the expression and localization of the channel proteins of interest. RESULTS: We found that the increase in water permeability, previously observed in differentiated astrocytes, occurs simultaneously with more efficient regulatory volume increase and regulatory volume decrease. Accordingly, the magnitude of the hypotonic induced intracellular calcium response, typically mediated by TRPV4, as well as the hypotonic induced VRAC current, was almost twice as high in differentiated astrocytes. Interestingly, while we confirmed increased AQP4 expression in the membrane of differentiated astrocytes, the expression of the channels TRPV4 and Leucine-Rich Repeats-Containing 8-A (LRRC8-A) were comparable between differentiated and non-differentiated astrocytes. CONCLUSION: The reported results indicate that AQP4 up-regulation observed in differentiated astrocytes might promote higher sensitivity of the cell to osmotic changes, resulting in increased magnitude of calcium signaling and faster kinetics of the RVD and RVI processes. The implications for cell physiology and the mechanisms underlying astrocytic interaction with nanostructured interfaces are discussed.

MLC1: A New Calcium-regulated Protein Conferring Calcium Dependence to Volume-regulated Anion Channels (VRAC) in Astrocytes.

“…It is known that CaMKII is activated by CaM upon increase of cytosolic Ca 2+ . Increased cytosolic Ca 2+ due to cell swelling has also been shown to activate ICl,swell in astrocytes [63,64]. These results identify Ca 2+ as a key player in the activation pathways of both MLC1 and ICl,swell.…”

Decoding natural astrocyte rhythms: dynamic actin waves result from environmental sensing by primary rodent astrocytes

“…To observe actin dynamics in more in vivo -like cell culture conditions, we used two different approaches: i) astrocytes plated on a nanostructured surface, with starlike shapes and more realistic molecular and functional features devoted to homeostatic control of potassium concentrations and cell volume [79, 77], and ii) co-culture of astrocytes with primary cortical neurons.…”

“…Previous studies demonstrated that astrocytes grown on HTlc are more efficient in their homeostatic process. In particular, the expression and function of Kir4.1 and water channel aquaporin 4 (AQP4) as well as the functional response to anisotonic challenge are enhanced in differentiated [77,79] and mature [117][118][119][120][121][122] cells. A more efficient response to homeostatic challenges due to the higher levels of channel proteins may be related to the weaker actin dynamics observed in differentiated cells compared to polygonal cells (both with and without neurons).…”

“…Notably, the strength of actin dynamics changes depending on whether the exposure is to an increase in extracellular potassium or to hypotonic challenge, abbreviated as "high K + " and "hypotonic", respectively. We refer to the application of these solutions to cultures as "triggering" conditions because they have been shown to mimic pathophysiological changes in the extracellular milieu similar to epilepsy [69] or edema [70] and to cause responses in astrocytes that range from swelling to calcium waves [3,48,[71][72][73][74][75][76][77][78]. In addition, activity identified in the processes of differentiated astrocytes, induced by nanotopography [79], is weaker when compared to polygonal astrocytes, which have the shape characteristic of undifferentiated astrocytes grown in the absence of neurons [80][81][82][83][84].…”

dDAVP Downregulates the AQP3-Mediated Glycerol Transport via V1aR in Human Colon HCT8 Cells

“…Glycerol permeability in HCT8 cells was measured using a calcein-quenching method (Mola et al, 2009;Mola et al, 2021). Nearly 80% of confluent cells were grown on 40-mm glass coverslips.…”