P2Y₁and Cysteinyl Leukotriene Receptors Mediate Purine and Cysteinyl Leukotriene Co-Release in Primary Cultures of Rat Microglia

Abstract

Inflammation is widely recognized as contributing to the pathology of acute and chronic neurodegenerative conditions. Microglial cells are pathologic sensors in the brain and activated microglia have been viewed as detrimental. Leukotriene, including cysteinylleukotrienes (CysLTs) are suggested to be involved in brain inftammation and neurological diseases andATP, by its receptors is a candidate for microglia activation.A23187 (l0f1M) stimulated microglia to co-release CysLTs and [3H]adenine based purines ([3H]ABPs), mainly ATP. The biosynthetic production of CysLTs was abolished by 10f1MMK-886, an inhibitor of 5-lipoxygenase-activating protein activity. RT-PCR analysis showed that microglia expressed both CysLT I / CysLT 2 receptors, P 2Y1 ATPreceptors and several members of the ATP binding cassette (ABC) transporters including MRP1, MRP4 and Pgpo The increase in [Ca 2+]i elicited by LTD4 (0.1 f1M) and 2MeSATP (l00J.1M), agonists for CysLT-and P 2Y1 -receptors, was abolished by the respective antagonists, BAYu9773 (0.5 f1M) and suramin (50 f1M). The stimulation of both receptor subtypes, induced a concomitant increase in the release of both [3H]ABPs and CysLTs that was blocked by the antagonists and significantly reduced by a cocktail ofABC transporter inhibitors, BAPTAIAM (intracellular Ca2+ chelator) and staurosporine (0.1 f1M, PKC blocker). P2Y antagonist was unable to antagonise the effects ofLTD 4 and BAYu9773 did not reduce the effects of2MeSATP. These data suggest that: i) the efffux of purines and cysteinyl-leukotrienes is specifically and independently controlled by the two receptor types, ii) calcium, PKC and the ABC transporter system can reasonably be considered common mechanisms underlying the release ofABPs and CysLTs from microglia. The blockade ofP 2Y1 orCysLT/CysLT 2 receptors by specific antagonists that abolished the raise in [Ca"]! and drastically reduced the concomitant efffux of both compounds, as well as the effects ofBAPTA and staurosporine support this hypothesis. In conclusion, the data of the present study suggest a cross talk between the purine and leukotriene systems in a possible autocrine/paracrine control ofthe microglia-mediated initiation and progression of an inftammatory response.Proinflammatory products of 5-lipoxygenase (5-LO) pathway, including cysteinylleukotrienes (LTC4, LTD4, LTE4) are suggested to be involved in brain inflammation and neurological diseases. In aging brain and in Alzheimer's disease an increase in the activity of 5-LO has been shown (41, 57). The inhibition of the enzyme or 5-LO activating protein (FLAP) reduced the microglia-mediated toxicity towards neuronal cells, whereas the toxicity was enhanced by the cysteinyl leukotriene LTD4 (35).

P2X7Receptor Activation in Rat Brain Cultured Astrocytes Increases the Biosynthetic Release of Cysteinyl Leukotrienes

“…We previously showed that either rat brain cultured microglia or astrocytes express functional subtypes ofABC proteins including MRP 1 (4). As expected the ABC-protein inhibitor cocktail significantly reduced the P2X 7-mediated efflux of CysLTs as it reduced the P2Y I-mediated CysLT release in microglia (3).…”

“…However, CysLTs levels have been reported to increase in brain injuries, including cerebral ischemia, hemorrhage and epileptic seizures (14,50). Recently we reported that astrocytes and microglia release CysLTs both under resting conditions and following stimulation with the calcium ionophore A23187 or with ATP through P2Y 1 metabotropic receptor subtypes (12,3).…”

“…In several cell types including macrophages, leukemia cells, eosinophils and microglia, the intracellular calcium rise play a key role in the biosynthetic production ofleukotrienes (2)(3)57).…”

Astrocytic P2Y1 receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia

“…In addition, in vitro studies have also confirmed P2Y 1 R expression in microglia (Ballerini et al, 2005;De Simone et al, 2010). However, in this report, we have shown that the P2Y 1 R is expressed on astrocytes before and after focal cerebral ischemia, using an in vivo model.…”

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

“…Crosstalk between purine and leukotriene systems has recently been reported, although the CysLT 1 receptor is not a P2Y nucleotide receptor [21]. P2Y 1 and CysLT receptors mediate co-release of purines and CysLTs in primary cultures of rat microglia [29].…”