P2Y and P2X purinoceptor mediated Ca2+ signalling in glial cell pathology in the central nervous system

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Abstract

Activation of purinoceptors by extracellular ATP is an important component of the glial response to injury in the central nervous system (CNS). ATP has been shown to evoke raised cytosolic [Ca2+] in astrocytes, oligodendrocytes, and microglia, the three major glial cell types in the CNS. Glial cells express a heterogenous collection of metabotropic P2Y and ionotropic P2X purinoceptors, which respectively mobilise Ca2+ from intracellular stores and trigger Ca2+ influx across the plasmalemma. It is likely that different receptors have distinct roles in glial cell physiology and pathology. Our studies on optic nerve glia in situ indicate that P2Y1 and P2Y2/4 receptors are activated at low ATP concentrations, suggesting they are the predominant purinoceptors mediating physiological Ca2+ signalling. Glia also express P2X1 and P2X3 purinoceptors, which mediate fast, rapidly desensitising current and may also be important in signalling. At high concentrations, such as occur in CNS injury, ATP induces large and prolonged increases in glial [Ca2+]i with a primary role for P2Y purinoceptors and inositol trisphosphate (IP3)-dependent release of Ca2+ from intracellular stores. In addition, we found that high concentrations of ATP activated a significant P2X component that did not desensitise or saturate and was dependent on extracellular Ca2+. These are characteristic properties of the P2X7 subtype, and we provide in situ evidence that application of the P2X7 receptor agonist benzoyl-benzoyl ATP (BzATP) evokes raised [Ca2+]i in optic nerve glia, and that the dye YO-PRO-1, which passes through pore-forming P2X7 receptors, is taken up by astrocytes, oligodendrocytes and microglia. Glia also express P2X2 and P2X4 receptors that are also pore-forming in the presence of sustained high ATP concentrations and which may also be important in the glial injury response. There is evidence that activation of P2 purinoceptors is a key step in triggering reactive changes in glial cells, including expression of immediate early genes, induction of extracellular signal regulated kinase and cyclooxygenase-2, synthesis of phospholipase A2, release of arachidonic acid, production of prostaglandins and release of interleukins. We show that the ATP-mediated increase in glial [Ca2+]i is potentiated by arachidonic acid and reduced by the inhibition of phospholipase A2 inhibition. Together, the results implicate ATP as a primary signalling molecule in glial cells and indicate specific roles for P2Y and P2X purinoceptors in glial cell pathology.

Section snippets

Glial cells and CNS pathology

The mammalian central nervous system (CNS) consists of two main cell types, neurons and glia. Glia are subdivided into macroglia, namely astrocytes and oligodendrocytes, and microglia, together with Bergmann glia and Müller cells, which are specialised radial astrocytes of the cerebellum and retina, respectively. Astrocytes form multiple contacts with neurons at synapses and nodes of Ranvier, as well as with blood vessels and other glia, and have numerous functions, including structural

Ca2+ signalling in glial cells

Glia express plasmalemmal receptors that enable them to respond dynamically to extracellular factors (Verkhratsky and Kettenmann, 1996). The primary response of glial cells is an increase in intracellular calcium ([Ca2+]i) and glial Ca2+ signalling occurs in response to physiological and pathological stimuli. Astrocytes have been shown to signal to each other using Ca2+ waves, which propagate intercellularly through a network of gap junctions and also using ATP as a diffusible signal Dani et

Glial P2 purinoceptors

It may be no exaggeration to state that ATP is the primary extracellular signalling molecule for glial cells in the CNS, where it has both physiological and pathological functions. ATP mediates raised intracellular [Ca2+]i via P2 purinoceptors in all glial cell types in vitro and in situ to activate a number of signal transduction pathways (Table 1). Sources of extracellular ATP in the CNS include (1) neuronal release at synapses and along axons Edwards et al., 1992, Hamann and Attwell, 1996,

ATP-evoked glial Ca2+ signalling in situ is mediated via both P2Y and P2X purinoreceptors

In order to investigate P2 receptor signalling in glial cells in situ, we have used the optic nerve, because it is a simple white matter tract that contains axons and the glial cells that support them, but not neurons or synapses (James and Butt, 2001a). Accordingly, all cellular responses to agents superfused over the nerve surface are those of glial cells, which can be identified by their characteristic morphology and positions in the nerve (Butt and Ransom, 1993). We found that application

P2Y receptors and the propagation of glial Ca2+ signalling

We have found that the minimal concentrations that evoked a measurable increase in glial [Ca2+]i in the optic nerve were 100 nM for ATP and 2MeSATP, compared to 10 μM for α,β-metATP (Fig. 2C). The high sensitivity component of the response of optic nerve glia to ATP is mediated by both P2Y1 and P2Y2/4 receptors (James and Butt, 2001a), consistent with reports that glial cells in vitro and in slices express a heterogeneity of P2Y receptors Kirischuk et al., 1995a, Kirischuk et al., 1995b,

A role for P2X7 receptors in mediating the glial injury response

Our results indicated that P2X receptors only evoke large influxes of Ca2+ at high concentrations of ATP (Fig. 2C), suggesting they have a specific, but not exclusive, role in glial cell pathology. This possibility is supported by our observation that in reactive astrocytes, there is a relative down-regulation of P2Y receptors and up-regulation of P2X receptors (Fig. 2B). In addition, there are distinct differences in the response of glial cells depending on the concentration and duration of

Transduction mechanisms activated by P2 purinoceptors

As well as mobilisation of [Ca2+]i, activation of astroglial purinoceptors in vitro induces activation of protein kinase C, extracellular signal regulated kinase (ERK), cyclooxygenase-2, phospholipase A2 synthesis, arachidonic acid synthesis and release, eicosanoid release, stimulation of mitogen-activated protein (MAP) kinases, and induction of immediate early genes (Bruner and Murphy, 1993, Salter and Hicks, 1995, King et al., 1996, Bolego et al., 1997, Chen and Chen, 1998, Priller et al.,

Conclusions

One of the few undoubted functions of glial cells is their response to CNS damage, whereby astrocytes undergo reactive gliosis, oligodendrocytes and myelin degenerate, and microglia are activated. Activation of glial P2Y and P2X purinoceptors following CNS injury can initiate these events, and there may be at least three stages to the pathological process, depending on the level and duration of exposure to ATP and the purinoceptors activated, as summarised in Fig. 6. Stage 1: During

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