Neurons respond directly to mechanical deformation with pannexin‐mediated ATP release and autostimulation of P2X₇ receptors

Abstract

Key points• Neurons can be damaged when tissues are stretched or swollen; while astrocytes can contribute to this process, the mechanosensitive response from neurons is unclear.• We show here that isolated retinal ganglion cell neurons respond to mechanical strain with a rapid, sustained release of the neurotransmitter ATP.• The conduit for ATP release was through pannexin hemichannels, with probenicid, carbenoxelone and 10 panx inhibiting release.• Once released, this ATP acts back on the neurons to autostimulate lethal P2X 7 receptors, as A438079, AZ 10606120 and zinc reduced currents in whole cell patch clamp recordings.• Blocking release of ATP through pannexin channels, or activation of P2X 7 receptors, might be neuroprotective for stretched or swollen neurons.• Stretch-dependent release of ATP through neuronal pannexins, combined with the autostimulation of the P2X 7 receptors, provides a new pathway by which neuronal activity and health can be altered by mechanical strain independently of glial activity.Abstract Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide 10 panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X 7 receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X 7 receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X 7 receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of J. Xia and J. C. Lim contributed equally to this work. purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X 7 receptor, is neuroprotective for stretched neurons.

Hypoxia-induced changes in Ca2+ mobilization and protein phosphorylation implicated in impaired wound healing

“…Another contributing factor could be changes in the regulation of extracellular ATP levels, which may be mediated by membrane pores such as those formed by pannexins. Previous studies found a direct relationship between pannexin channels and the regulation of released ATP-mediated signal transmission (17,27,28,56). Because we observed a decrease in released ATP levels after injury, it is possible that hypoxic stress may induce changes in the modulation of pannexin pore formation along with other downstream targets, ultimately contributing to impaired Ca 2ϩ mobilization.…”

Rat, Mouse, and Primate Models of Chronic Glaucoma Show Sustained Elevation of Extracellular ATP and Altered Purinergic Signaling in the Posterior Eye

“…22 Both RGCs and astrocytes release ATP upon mechanical strain, with pannexin hemichannels a likely pathway for release in both cell types. [23][24][25] While these reports indicate that acute increases in IOP and/or mechanical strain lead to release of ATP in the retina, most patients with glaucoma suffer from an elevation in IOP for extended periods. Evidence for a sustained release of ATP associated with a prolonged elevation of IOP would thus have relevance for the most common forms of the disease.…”

Purinergic dysregulation causes hypertensive glaucoma–like optic neuropathy

“…ATP and nucleotides are present in the aqueous humor (5). Nucleotides are released from various tissues and cells, including lens (6), trabecular meshwork (7), whole retina (8), corneal endothelial cells (9), retinal ganglion cells (RGCs) (10), retinal astrocytes (11), and ciliary body (CB) (12). ATP levels in the aqueous humor are highly elevated in glaucoma patients (13,14), and although IOP and ATP levels in the aqueous humor are positively correlated in patients (14), their interaction is undefined.…”