A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes

doi:10.1016/S0165-1838(00)00152-1

Journal of the Autonomic Nervous System

Volume 81, Issues 1–3, 3 July 2000, Pages 3-9

This paper is dedicated to Professor Geoffrey Burnstock, without whom research in the purinergic neurotransmission field would not have been possible.

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Abstract

In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog α,βmethyleneATP (α,βmeATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. α,βmeATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X₇ receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both α,βmeATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

Introduction

Extracellular ATP exerts its effects through ligand-gated ion channels (P2X receptors) or G-protein-coupled receptors (P2Y receptors) (Abbracchio and Burnstock, 1994, Abbracchio and Burnstock, 1998, Ralevic and Burnstock, 1998). While P2X receptors have been implicated in fast neuro-neuronal communication (North, 1996), P2Y receptors have been mainly found on astroglial cells, where they seem to mediate induction of reactive astrogliosis following massive ATP release during trauma and ischemia (Neary et al., 1996, Abbracchio et al., 1999, Neary and Abbracchio, 2000). We have previously shown that a brief (2 h) challenge of rat striatal astrocytes with the ATP analogs α,βmethyleneATP (α,βmeATP) and β,γmethyleneATP (β,γmeATP) results, 3 days later, in a marked concentration-dependent elongation of astrocytic processes which intensively stain for the astroglial marker GFAP (glial fibrillary acidic protein) (Bolego et al., 1997). This effect, which reproduces in vitro the astrocytic hypertrophy known to occur in in vivo reactive astrogliosis (Hatten et al., 1991, Ridet et al., 1997) was abolished by pertussis toxin, suggesting the involvement of a novel P2Y receptor, whose pharmacological profile significantly differs from the ones of the already cloned P2Y receptors (Bolego et al., 1997). We have more recently shown that purine-induced reactive astrogliosis is accompanied by significantly increased expression of cyclooxygenase (COX)-2 (Brambilla et al., 1999). Excessive COX-2 activation has been implicated in a variety of neurological disorders, including chronic pain and inflammation (Dolan et al., 1998), acute (e.g., ischemia, Ohtsuki et al., 1996, Planas et al., 1995) as well as chronic (e.g., Alzheimer’s disease; Ho et al., 1999) neurodegenerative diseases characterized by a marked inflammatory component and activation of astroglial cells (Ohtsuki et al., 1996, Blom et al., 1997, Dolan et al., 1998, Ho et al., 1999). The selective COX-2 inhibitor NS-398 completely abolished α,βmeATP-induced astrocytic activation, suggesting a causal relationship between COX-2 induction and purine-induced astrogliosis (Brambilla et al., 1999). On the basis of these data, we suggested that a novel P2Y receptor mediates reactive astrogliosis via induction of COX-2, and that antagonists selective for this receptor may represent a new class of anti-neurodegenerative agents of potential interest in both acute and chronic neurological diseases. The present study has been undertaken to further characterize this novel gliotic receptor and also to assess its possible expression in non-striatal brain areas of potential interest for chronic neurodegenerative diseases (i.e., cortex), as well as in human cells of the astroglial lineage (ADF astrocytoma cells).

Section snippets

Rat astrocytic primary cultures

Primary astrocytic cultures were established from rat cortex and corpus striatum of 7 days old pups as previously described (Bolego et al., 1997). Cells were initially plated in serum-supplemented medium and after 24 h placed in chemically defined serum-free medium.

Human astrocytoma cells (ADF)

Cell were maintained in culture as previously described (Abbracchio et al., 1997). For elongation experiments, cells were plated at a density of 3000 cells/well in 24 well dishes, grown for 24 h in serum-supplemented medium and then

Results

As previously demonstrated (Bolego et al., 1997, Brambilla et al., 1999), challenge of primary striatal astrocytes with 10⁻⁵ M α,βmeATP resulted in the formation of reactive astrocytes, as suggested by elongation of GFAP-positive astrocytic processes (Fig. 1). Oxidized ATP, an antagonist of the P2X₇ receptor, did not revert α,βmeATP-induced reactive astrogliosis. In fact, in cultures treated with α,βmeATP in the presence of 300 μM oATP, elongation of GFAP-positive processes was 125.3±4.1 (% of

Discussion

Our findings confirm the existence of a novel P2 receptor, whose presence is functionally detected on both rat (striatal and cortical) and human astrocytes (ADF cells), and whose activation is responsible for the induction of reactive astrogliosis. The presence of this receptor in cortex also highlights its possible involvement in neurological disorders characterized by marked functional impairment of cortical areas, such as Alzheimer’s disease. Moreover, the expression of this gliotic receptor

Acknowledgements

This work was partially supported by the European Union BIOMED 2 programme BMH4 CT96-0676. Authors are grateful to Matteo Laurita, Institute of Pharmacological Sciences, University of Milan for skilful assistance, and to Prof. Francesco Di Virgilio, University of Ferrara, Italy, for providing oATP and for helpful advice.

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The receptor mechanisms regulating the ATP-induced free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) changes in cultured rat cortical type-1 astrocytes were analyzed using fura-2-based Ca²⁺ imaging microscopy. Upon prolonged ATP challenge (1–100 μM), astroglial cells displayed a biphasic [Ca²⁺]_i response consisting of an initial peak followed by a sustained elevation. Suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid blocked both components, albeit to a different extent. By contrast, the selective P2X7 antagonist oxidized ATP irreversibly abrogated the sustained [Ca²⁺]_i signal without affecting the transient phase. Finally, astrocyte challenge with the selective P2X7 agonist 3′-O-(4-benzoyl)benzoyl-ATP evoked a sustained [Ca²⁺]_i elevation, which occluded that induced by ATP. We can conclude that in cultured cortical astrocytes the ATP-mediated sustained [Ca²⁺]_i rise does not implicate capacitative Ca²⁺ entry but involves Ca²⁺ influx through P2X7-like receptors.
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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 P2Y_2/4 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.

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A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes

Abstract

Introduction

Section snippets

Rat astrocytic primary cultures

Human astrocytoma cells (ADF)

Results

Discussion

Acknowledgements

Pharmac. Ther.

Biochem. Biophys. Res. Commun.

Prog. Brain Res.

Brain Res.

Chem. Biol. Interact.

Trends Neurosci.

Brain Res.

Neurosci. Lett.

Trends Neurosci.

Purinergic signalling: pathophysiological roles

Jpn. J. Pharmacol.