Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis

Qureshi, Omar; Paramasivam, Anbalakan; Yu, Jowie C.H.; Murrell‐Lagnado, Ruth D.

doi:10.1242/jcs.010348

Cited by 197 publications

(256 citation statements)

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“…In contrast, the lysomotropic compound, chloroquine [24], rapidly increased P2X 4 R currents. Our results are in accord with a recent study showing that other lysomotropic agents increased surface expression of P2X 4 R in macrophages [17] and suggest that alterations in endocytic and lysosomal trafficking pathways are primarily responsible for the regulation of functional P2X 4 R in macrophages. Phagocytosis is one of the most important innate effector functions of macrophages, serving to clear particulate matter, apoptotic cells and bacteria [32].…”

Section: Discussionsupporting

confidence: 93%

“…These results allow us to conclude that the ATP-evoked currents we record under these conditions result solely from activation of P2X 4 R. P2X 4 R in resting macrophages was predominantly localized to lysosomal compartments as indicated by co-localization of P2X 4 R and the lysosomal marker, CD68 (Fig 2A). In agreement with the previous study on mouse peritoneal macrophages [17], we found that agents that induce lysosomal secretion increase P2X 4 Rmediated membrane currents (Fig. 2C).…”

Section: P2x 4 R Protein and Functional Expression In Unstimulated Masupporting

confidence: 93%

“…Qureshi et al [17] have recently found that phagocytosis of zymosan or latex beads by mouse peritoneal macrophages lead to accumulation of P2X 4 R at the phagosome membrane; when lysosomal exocytosis was induced by the calcium ionophore, ionomycin, a significant increase in the plasma membrane surface expression of P2X 4 R occurred with concomitant increase in the ATP-evoked currents. P2X 4 R are highly permeable to calcium [18]; thus, dynamic changes in P2X 4 R expression density at the cell surface may be expected to result in significant modulation or regulation of numerous calcium-dependent signalling processes.…”

Section: Introductionmentioning

confidence: 99%

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Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation

Stokes

Surprenant

2009

Eur J Immunol

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ATP-gated P2X 4 receptors (P2X 4 R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X 4 R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X 4 R protein expression by Western blot and biotinylation assays and functional expression by whole-cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X 4 R expression by 2-to 7-fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN-c and TNF-a or IFN-c and LPS reduced surface and functional P2X 4 R expression by 3-fold without altering total P2X 4 R protein levels. Alternative activation with IL-4 or IL-13 did not alter total, surface or functional expression of P2X 4 R. This is the first study of the regulation of P2X 4 R in macrophages by physiological stimuli and presents a picture whereby P2X 4 R become functional in response to initial phagocytic stimuli but return to a non-functional state during sustained activation by classical macrophage activation.Key words: Inflammation . Ion channels . Patch-clamp . Purine receptors Introduction Extracellular ATP acts as a ''danger signal'' when it is released from cells during tissue damage and inflammation; it acts on metabotropic (G-protein coupled) P2Y and ionotropic (cation channel) P2X purinergic receptors to affect several inflammatory and immune responses [1,2]. There are seven members of the P2X receptor family (P2X 1-7 R) that show widespread distribution in both neuronal and non-neuronal tissues [3]. Two of these seven members, P2X 4 R and P2X 7 R, have generated increasing interest as potential anti-inflammatory drug targets [2,4,5]. The case for P2X 7 R is now well established: Stimulation of P2X 7 R in activated monocytes, macrophages and microglia leads to rapid release of pro-inflammatory IL-1b and IL-18 cytokines, to phospholipase D activation and, if stimulation is sustained, to apopotosis [1,[3][4][5]. Classical activation of macrophages, generally induced by IFN-g and/or LPS, up-regulates P2X 7 R mRNA and protein levels and functional expression [6,7] and this correlates well with a role for P2X 7 R in bacterial killing [8,9]. Recently, highly selective and potent P2X 7 R antagonists have been identified [10] with one of these showing initial positive results in Phase II clinical trials for rheumatoid arthritis [11]. The case for P2X 4 R remains tentative, largely due to the absence of selective P2X 4 R antagonists and the complicating presence of P2X 7 R, which is generally co-expressed with P2X 4 R in ...

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Section: Discussionsupporting

confidence: 93%

Section: P2x 4 R Protein and Functional Expression In Unstimulated Masupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

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Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation

Stokes

Surprenant

2009

Eur J Immunol

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“…Surprisingly, a large amount of P2X4R protein within microglia (and macrophages) localizes predominantly to intracellular lysosomal compartments71, and P2X4R protein remains stable within the proteolytic environment of lysosomes. How P2X4R protein is recruited to the cell surface of microglia remains elusive, but recent studies have shown that trafficking of P2X4R protein to the cell surface occurs when microglia are stimulated by a toll‐like receptor 4 agonist, lipopolysaccharide72, 73, or the Ca 2+ ionophore, ionomycin71. Furthermore, the chemokine CCL2 increased P2X4R protein levels on the cell surface (without changing total cellular expression) through chemotactic cytokine receptor 274.…”

Section: Spinal Microglia Are Crucial For Neuropathic Painmentioning

confidence: 99%

Microglia in the spinal cord and neuropathic pain

Tsuda

2015

J of Diabetes Invest

217

147

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In contrast to physiological pain, pathological pain is not dependent on the presence of tissue‐damaging stimuli. One type of pathological pain – neuropathic pain – is often a consequence of nerve injury or of diseases such as diabetes. Neuropathic pain can be agonizing, can persist over long periods and is often resistant to known painkillers. A growing body of evidence shows that many pathological processes within the central nervous system are mediated by complex interactions between neurons and glial cells. In the case of painful peripheral neuropathy, spinal microglia react and undergo a series of changes that directly influence the establishment of neuropathic pain states. After nerve damage, purinergic P2X4 receptors (non‐selective cation channels activated by extracellular adenosine triphosphate) are upregulated in spinal microglia in a manner that depends on the transcription factors interferon regulatory factor 8 and 5, both of which are expressed in microglia after peripheral nerve injury. P2X4 receptor expression on the cell surface of microglia is also regulated at the post‐translational level by signaling from CC chemokine receptor chemotactic cytokine receptor 2. Furthermore, spinal microglia in response to extracellular stimuli results in signal transduction through intracellular signaling cascades, such as mitogen‐activated protein kinases, p38 and extracellular signal‐regulated protein kinase. Importantly, inhibiting the function or expression of these microglial molecules suppresses the aberrant excitability of dorsal horn neurons and neuropathic pain. These findings show that spinal microglia are a central player in mechanisms for neuropathic pain, and might be a potential target for treating the chronic pain state.

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“…The early hints and recent evidence for localization of purinoceptors on intracellular sites, including lysosomes [2][3][4], mitochondria [5,6] and in nuclei where they open ion channels and appear to influence mRNA activity [7,8], offers up a whole new aspect of purinergic signalling.Purinergic signalling, ATP acting as an extracellular signalling molecule, was proposed in 1972 [9]. Separate families of purinergic receptors were recognised, named P1 receptors for adenosine and P2 receptors for ATP and ADP [10].…”

mentioning

confidence: 99%

Intracellular expression of purinoceptors

Burnstock

2015

Purinergic Signalling

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Purinoceptors, receptors for nucleosides and nucleotides, have been identified on the plasma membranes of many cell types [1]. The early hints and recent evidence for localization of purinoceptors on intracellular sites, including lysosomes [2][3][4], mitochondria [5,6] and in nuclei where they open ion channels and appear to influence mRNA activity [7,8], offers up a whole new aspect of purinergic signalling.Purinergic signalling, ATP acting as an extracellular signalling molecule, was proposed in 1972 [9]. Separate families of purinergic receptors were recognised, named P1 receptors for adenosine and P2 receptors for ATP and ADP [10]. Two subtypes of P2 receptors were shown in 1985, based on pharmacology [11] and in the early 1990s P1, P2X and P2Y receptor subtypes were cloned and characterised: four subtypes of P1 receptors (A 1 , A 2A , A 2B and A 3 ), seven subtypes of P2X ion channel receptors (P2X1-7) and eight subtypes of P2Y G protein-coupled receptors (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 and P2Y 14 ) [1,12]. Cloning of receptors made it possible to generate polyclonal antisera for immunohistochemical studies of their expression and distribution [13]. Many papers using this technique were published [14][15][16][17]. As expected, the receptors were located on the plasma membranes of cells, but sometimes there was also intracellular immunostaining. This was often dismissed by referees as artefacts. However, later studies have revealed that intracellular localization of purinoceptors is genuine. For example, it was suggested that uptake of P2X1 receptors into smooth muscle cells of the rat vas deferens was responsible for desensitization ([18] and see [19]). P2Y 2 receptor internalization via the clathrin-mediated pathway was observed in HEK293 cells using receptors tagged with green fluorescent protein (GFP), to be colocalized with endosomes and lysosomes [2]. GFP was also used to show internalization of P2Y 1 receptors in HEK-293 cells [20,21]. Ser352 and Ser354 in the carboxyl terminus of human P2Y 1 receptors were shown to be needed for internalization in MDCK cells [22]. P2X3 receptors transfected into HEK-293 cells and expressed endogenously in dorsal root ganglion sensory neurons undergo rapid constitutive endocytosis, targeting the late endosomal/lysosomal system [23]. The role of internalised P2X7 receptors on lysosomes in macrophages in the killing of mycobacteria is discussed in a review [24]. A Rab5-dependent pathway was described for internalisation of P2X4 receptors in HEK-293 cells [25]. The internalised P2X4 receptors are located on lamellar bodies, lysosomes, vesicles and vacuoles in HEK-293 cells, hippocampal neurons and alveolar type II cells [3,4]. P2X4 receptor channel activity was directly measured in intact lysosomes in HEK-239 cells [26]. Both ATP and P2X4 receptors were present in lysosomes and the lysosomal P2X4 receptors were activated by ATP at the luminal side in a pH-dependent manner. The lysosomal P2X4-mediated responses were potentiated by ivermectin, b...

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Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis

Cited by 197 publications

References 65 publications

Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation

Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation

Microglia in the spinal cord and neuropathic pain

Intracellular expression of purinoceptors

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Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis

Cited by 197 publications

References 65 publications

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation

Microglia in the spinal cord and neuropathic pain

Intracellular expression of purinoceptors

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Resources

About

Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation

Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation