Astrocytes inhibit nitric oxide-dependent Ca²⁺dynamics in activated microglia: Involvement of ATP released via pannexin 1 channels

Abstract

Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca(2+) and produce high amounts of nitric oxide (NO). However, whether NO, Ca(2+) dynamics, and gliotransmitter release are reciprocally modulated is not fully understood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aim was to address if astrocytes could regulate NO-dependent Ca(2+) dynamics and ATP release in LPS-stimulated microglia. Griess assays and Fura-2AM time-lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca(2+) ]i that depended on the sequential activation of iNOS, COXs, and EP1 receptor. TGFβ1 released by astrocytes inhibited the abovementioned responses and also abolished LPS-induced ATP release by microglia. Luciferin/luciferase assays and dye uptake experiments showed that release of ATP from LPS-stimulated microglia occurred via pannexin 1 (Panx1) channels, but not connexin 43 hemichannels. Moreover, in LPS-stimulated microglia, exogenous ATP triggered activation of purinergic P2Y1 receptors resulting in Ca(2+) release from intracellular stores. Interestingly, TGFβ1 released by astrocytes inhibited ATP-induced Ca(2+) response in LPS-stimulated microglia to that observed in control microglia. Finally, COX/EP1 receptor signaling and activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS-stimulated microglia. Thus, Ca(2+) dynamics depended on the inflammatory profile of microglia and could be modulated by astrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the development of new treatments to reduce inflammatory cytotoxicity in several brain pathologies.

Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology?

“…2) [63,64]. Panx1 protein is expressed in neurons and is found to be present in microglia and astrocytes [65][66][67]. Important to note is that the latter has mostly been demonstrated in in vitro culture conditions [58,[68][69][70], albeit a more recent report also provides evidence for its astroglial expression in mouse hippocampal sections [71].…”

“…Both HCs, Panx1 channels and innexins, the invertebrate analog of Cxs and Panxs, have been proposed to function as potent ATP/glutamate release pathways [305,306,309]. Activated microglia express Cx32, Cx36, Cx43, and Panx1 [67,[310][311][312][313], and HCs, composed of Cx32 or Cx43, and Panx1 channels, have been shown to contribute to glutamate or ATP release upon microglial activation [67,314,315]. It should be noted that, although GJCs have been described in cultured microglial cells [312], GJIC between microglial cells as well as with other surrounding cells has been reported to be absent in vivo [316].…”

“…Proinflammatory cytokines TNF-a and IL1-b can furthermore reduce astroglial GJIC, while astroglial and microglial HCs and Panx1 channels are reported to open [45,52,336,[343][344][345][346]. It has been demonstrated in several scenarios, such as inflammatory conditions, ischemia and exposure to amyloid b, that a combined action of astrocytic Cx43 HCs, and microglial Cx43 HCs and Panx1 channels contributes to the reciprocal astrocyte-microglial communication and eventually to neuronal dysfunction [57,67,347,348]. Of note, channel-independent functions of Cx43 equally influence the proliferative and migratory response of astrocytes toward injury [349].…”

“…Secondly, these opposite functions could also relate to differences in the model system used [339], including the strategy that is applied to interfere with Cx43 channel function. By using Cx43 targeting peptides, it has been demonstrated that blocking HCs can counteract several pathological processes, such as glutamate and ATP release, excitotoxicity, and apoptosis, and it can influence the neuro-inflammatory process [47,52,67,90,336,367]. In addition, their application has been demonstrated to resort to beneficial effects in in vitro and in vivo models of CNS pathologies, including brain asphyxia [100], brain and retina ischemia [98,368,369], epilepsy [364,370], Alzheimer's Disease [57], optic neuropathy [371], spinal cord injury [88,337], and inflammation or ischemia-induced vascular leakage/inflammation-induced BBB permeability [89,98].…”

Regulation of gap junction channels by infectious agents and inflammation in the CNS

“…However, unresolved inflammation in the CNS can have devastating consequences, including impaired myelination (Akassoglou et al, 1998; Markoullis et al, 2012) sensorimotor deficits (Han et al, 2014), and loss of signaling that contributes to learning and memory (Koster-Patzlaff et al, 2009; Wang and Belousov, 2011). Activated microglia release pro-inflammatory factors, such as ATP (Orellana et al, 2013) and glutamate (Takeuchi et al, 2006), through uHCs that serve to amplify inflammation with devastating effects if left unchecked. Neuronal beading, a sign of neurotoxicity, was induced by activated microglial release of glutamate from Cx32 uHCs (Takeuchi et al, 2005, 2006).…”