ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

Abstract

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 microM) activated a small EAA release in 70-80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5-10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

Cytotoxic edema: mechanisms of pathological cell swelling

“…Genetic ablation of NKCC1, as well as its block by bumetanide,17 causes a decrease in intracellular Cl − in hypoxic neurons145 and blocks Na + -and Cl − -mediated cell swelling in astrocytes. 101,142,143 In vivo studies have shown that intracerebral bumetanide administered via a microdialysis probe, either prior to or during ischemia/hypoxia insult caused by temporary MCA occlusion, is neuroprotective, ameliorates brain damage, and reduces brain edema in rat focal ischemia,141,172,173 reinforcing the in vitro data. Focal cerebral ischemia in rats results in elevated NKCC1 protein levels in the ipsilateral cortex and striatum.…”

Synaptotagmin IV regulates glial glutamate release

“…Changes in osmolarity evoke the release of glutamate from astrocytes (Fig. 5d) by a mechanism that is thought to involve anion transporters (43,44). Because it is possible that ATP-stimulated Ca 2ϩ -dependent glutamate release may involve the exocytotic insertion into the plasma membrane of such transporters, we determined whether hypoosmolarity-induced loss of this transmitter from astrocytes was affected by manipulations of Syt IV expression.…”

Mechanisms of glutamate release from astrocytes

“…For example, NPPB can inhibit the vesicular chloride transporter and 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) has been implicated in blocking VGLUTs and V-ATPases (reviewed in Evanko et al, 2004)]. Although buffering cytoplasmic Ca 2+ can interfere with osmotically-induced amino acid release from astrocytes (Mongin et al, 1999), tetanus toxin had no effect on swelling-induced release, indicating that this release does not appear to be through a vesicle mediated pathway (Mongin and Kimelberg, 2002). This also emphasizes how difficult it can be to isolate the effects of one release mechanism from another.…”