Metabolic constraints of swelling‐activated glutamate release in astrocytes and their implication for ischemic tissue damage

Abstract

Volume‐regulated anion channel (VRAC) is a glutamate‐permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes – the cell type which shows prominent swelling in stroke – to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post‐ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin–luciferase ATP assays and a Seahorse analyzer. Swelling‐activated glutamate release was quantified with the radiotracer D‐[3H]aspartate. The specific contribution of VRAC to swelling‐activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine‐rich repeat‐containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT‐PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2‐deoxy‐D‐glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP‐depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty‐four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

“…Astrocytic swelling has been implicated in volume regulated glutamate release relevant for ischemic stroke, through glutamate-permeable VRACs (Jayakumar and Norenberg, 2010; Wilson et al, 2019;Yang et al, 2019). At the same time, the significant glutamate uptake through astrocytic Na + -and ATP-dependent EAAT2 is impaired under metabolic stress, which sustains high extracellular glutamate concentration (Szatkowski et al, 1990;Rossi et al, 2000;Mahmoud et al, 2019).…”

Dysregulation of Astrocyte Ion Homeostasis and Its Relevance for Stroke-Induced Brain Damage

“…These channels permit Cl − efflux and allow regulatory volume decreases together with K + efflux through channels active under resting conditions. Volume-activated anion channels exhibit large pore diameters, and channel activation results in glutamate efflux and excitotoxic tissue damage [ 224 ]. Steric and electrostatic interactions with Cl − might result in changes of neurotransmitter efflux upon altered astrocytic [Cl − ] i .…”

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity

“…Inclusion of LRRC8D enhances the transport of all tested organic substrates (Lutter et al, 2017;Planells-Cases et al, 2015), whereas inclusion of LRRC8E rather specifically stimulates the transport of the negatively charged glutamate and aspartate (Lutter et al, 2017). VRACs are thought to be important for diverse processes including insulin secretion (Best et al, 2010;Kang et al, 2018;Stuhlmann et al, 2018), neuron-glia interaction (Yang et al, 2019), stroke (Wilson et al, 2019;Yang et al, 2019), apoptosis and cancer drug resistance (Planells-Cases et al, 2015), and sperm cell development (Luck et al, 2018). The importance of VRAC is further illustrated by the multiple tissue abnormalities and lethality of Lrrc8adeficient mice (Kumar et al, 2014;Platt et al, 2017).…”