The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

Maria Grazia Mola,Angelo Sparaneo,David C Spray,Antonio Frigeri,Eliana Scemes,Grazia Paola Nicchia,Maria Svelto,Concetta Domenica Gargano

doi:10.1002/glia.22921

Abstract

Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.

Highlights

Under physiological conditions, cells are able to regulate their volume to balance the perturbations due to osmotic pressure (Hoffmann et al, 2009), which constantly challenge intracellular and extracellular osmolarity, causing cell swelling or shrinkage
In this study we have focused on the role played by two different Aquaporins (AQPs), AQP4 and AQP1, in triggering Regulatory Volume Decrease (RVD) and in mediating calcium signaling in astrocytes exposed to hypotonic stimulus
The time constant of astrocyte swelling measured by total internal reflection fluorescence (TIRF) analysis showed that, at 10°C, AQP4 KO astrocytes displayed a significantly reduced rate of water transport with a 4-fold increase in the time constant of cell swelling (6.57±1.36 s for WT vs 24.39±2.19 s for KO astrocytes, Fig. 1C), a result that is in agreement with previous reports (Nicchia et al, 2005; Solenov et al, 2004)

Summary

Introduction

Cells are able to regulate their volume to balance the perturbations due to osmotic pressure (Hoffmann et al, 2009), which constantly challenge intracellular and extracellular osmolarity, causing cell swelling or shrinkage. Abnormal astrocyte swelling is induced by intracellular acidification, states associated with energy depletion and hypothermia. Under these circumstances, astrocyte swelling itself may cause ischemia due to compression of small vessels (Kimelberg et al, 1995). While astrocytes exhibit a rise in intracellular calcium concentration in response to hypotonic stimulus (Morales-Mulia et al, 1998; O’Connor and Kimelberg, 1993), the role of calcium as a triggering signal of RVD is still a matter of debate (Benfenati et al, 2011; Morales-Mulia et al, 1998; O’Connor and Kimelberg, 1993; Sanchez-Olea et al, 1995)

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