Amanda B. MacKenzie

Rapid Secretion of Interleukin-1β by Microvesicle Shedding

The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.

Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor

1 Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole‐cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO‐PRO‐1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2 The ATP analogue 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (30 μm) induced membrane blebbing within 30‐40 s of sustained application; this was 5‐10 times slower when extracellular sodium was replaced by larger cations. 3 Fluorescence of YO‐PRO‐1 was detectable within 3 s, and the uptake reached a steady rate within 10‐20 s; YO‐PRO‐1 uptake was greatly enhanced by removing extracellular sodium. 4 Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively dilated during 10‐20 s to a diameter greater than 1 nm (10 Å). With short agonist applications (3‐5 s) the pore dilatation and YO‐PRO‐1 uptake were reversible and repeatable. 5 Polyethylene glycols having molecular weights ≥ 5000 blocked the increase in cation permeability, YO‐PRO‐1 uptake and membrane blebbing. 6 We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3‐5 nm from an initial minimum pore diameter of 0.8 nm.

ACTIVATION OF RECEPTOR-OPERATED CATION CHANNELS VIA P2X1 NOT P2T PURINOCEPTORS IN HUMAN PLATELETS

We have investigated the purinoceptor subtypes responsible for calcium signaling in human platelets, which previous studies have shown to involve both Ca influx via receptor-operated cation channels and release of Ca from intracellular stores. Fura-2 measurements of [Ca] in stirred platelet suspensions showed that both ADP (40 μM) and the non-hydrolyzable ATP analogue αβ-meATP (α,β-methyleneadenosine 5′-triphosphate, 10 μM) activated a rapid Ca influx whereas only ADP mobilized Ca from internal stores. In “nystatin” whole-cell patch clamp recordings, ATP, ADP, and the non-hydrolyzable ATP analogues, α,β-meATP and ATPS (adenosine 5′-O-(3-thiotriphosphate), all activated a cation channel permeable to both monovalent and divalent cations with a single-channel conductance of 11 picosiemens in NaCl saline. The current response to ATP (40 μM) was activated within 20 ms and desensitized with a time constant of 47-107 ms in the continued presence of agonist, which are characteristics of P receptors in other tissues. We conclude that human platelets possess a P purinoceptor, which mediates a rapid phase of ADP- or ATP-evoked Ca entry via a cation channel, whereas one or more separate ADP-selective P purinoceptors evoke release of calcium from intracellular stores.

Pseudoapoptosis Induced by Brief Activation of ATP-gated P2X7 Receptors

P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors.

Functional and Molecular Diversity of Purinergic Ion Channel Receptors

ABSTRACT: P2X receptors are membrane ion channels gated by extracellular adenosine 5′‐triphosphate (ATP); nucleotides also activate a family of seven transmembrane G protein‐coupled receptors (P2Y). P2X receptors are widely expressed on mammalian cells, where they can be broadly differentiated into three groups. The first group is almost equally well activated by ATP and its analog αβmethyleneATP (αβmeATP), whereas a second group is not activated by αβmeATP. A third‐group type of receptor (termed P2Z) is distinguished by the fact that the channel opening is followed by cell permeabilization and lysis if the agonist application is continued for more than a few seconds. Seven cDNAs have been cloned that encode P2X receptor subunits. When expressed individually in heterologous systems, P2X1 and P2X3 subunits form channels activated by ATP or αβmeATP; whereas P2X2, P2X4, and P2X5 form channels activated by ATP but not αβmeATP. P2X6 receptors do not express readily, and P2X7 receptors correspond closely in their properties to P2Z. Further phenotypes can be produced when two subunits are coexpressed, indicating heteromultimerization. This chapter compares the properties of the native P2X receptors with those of the cloned and expressed subunits.

A Key Role for Redox Signaling in Rapid P2X7 Receptor-Induced IL-1β Processing in Human Monocytes

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that trigger caspase-1 activation in the presence of TLR ligands. Inflammatory caspase-1 is responsible for the proteolytic activation of IL-1β. However, the signaling events that couple P2X7Rs to caspase-1 activation remain undefined. In this study we demonstrate that ATP-induced cellular oxidation is critical for caspase-1 activation and subsequent IL-1β processing. Purinergic receptor stimulation, including P2X7Rs, of endotoxin-primed human monocytes augments NADPH oxidase activity whereas concurrent purinergic receptor stimulation triggers protein denitroyslation, leading to the formation of peroxynitrite. IL-1β cleavage is blocked under conditions where superoxide anion formation is blocked or monocytes are treated with antioxidants or a peroxynitrite scavenger. Nigericin, a K+/H+ antiporter, also increases NADPH oxidase activity, leading to IL-1β and caspase-1 processing that is blocked by a peroxynitrite scavenger or inhibition of NADPH oxidase. These data demonstrate that signaling via NADPH oxidase activity is fundamental for the processing of mature IL-1β induced by P2X7R stimulation.

NADPH Oxidase NOX2 Mediates Rapid Cellular Oxidation following ATP Stimulation of Endotoxin-Primed Macrophages

The phagocytic NADPH oxidase (NOX2) plays a fundamental role in host defense and innate immunity. Here we demonstrate that external ATP triggers rapid cellular oxidation inhibited by diphenyleneiodonium in endotoxin-primed J774 macrophages and primary murine bone marrow-derived macrophages. To identify the source of reactive oxygen species (ROS), we compared responses between wild-type and NOX2-deficient macrophages. ATP-mediated ROS production was strongly attenuated in NOX2-deficient macrophages where responses were comparable to inhibition with diphenyleneiodonium. Notably, spatial differences in superoxide anion formation were observed where ROS formation was partially antagonized by extracellular superoxide dismutase in primary bone marrow-derived macrophages but unaffected in J774 macrophages. Loss of NOX2 was not observed to affect ATP-induced cell death. However, ATP-evoked cell death was found to be partially dependent on caspase-1 and cathepsin B activation. In conclusion, NOX2 plays a fundamental role in conferring macrophages with the ability to respond to extracellular ATP stimulation with robust changes in cellular oxidation.

A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells.

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.

Species and agonist dependent zinc modulation of endogenous and recombinant ATP-gated P2X7 receptors

Zinc (Zn2+) and copper (Cu2+) are key signalling molecules in the immune system and regulate the activity of many ion channels. Both Zn2+ and Cu2+ potently inhibit rat P2X7 receptors via a binding site identified by mutagenesis. Here we show that extracellular Cu2+ also potently inhibits mouse P2X7 receptors. By contrast, the receptor expression system and agonist strongly influence the action of extracellular Zn2+ at mouse P2X7 receptors. Consistent with previous reports, Zn2+ inhibits recombinant rat P2X7 receptors. However, recombinant mouse P2X7 receptors are potentiated by Zn2+ when activated by ATP4- but inhibited when stimulated with the ATP analogue BzATP4-. Endogenous murine macrophage P2X7 receptors are not modulated by Zn2+ when stimulated by ATP4- however Zn2+ inhibits BzATP4- mediated responses. In summary, these findings provide a fundamental insight into the differential actions of Zn2+ and Cu2+ between different P2X7 receptor species.

Mitochondrial superoxide generation enhances P2X7R-mediated loss of cell surface CD62L on naïve human CD4+ T lymphocytes

Migration of naive CD4+ T lymphocytes into lymphoid tissue is essential for their activation and subsequent roles in adaptive immunity. The adhesion molecule L-selectin (CD62L), critical for this process, is highly expressed on naive CD4+ T lymphocytes and is downregulated upon T lymphocyte activation. We demonstrate protein expression of P2X7R on naive CD4+ T lymphocytes and show functional channel activity in whole-cell patch clamp recordings. CD62L downregulation occurs rapidly in response to extracellular ATP, a process that is blocked by selective antagonists of P2X7R. This loss of surface CD62L expression was not associated with externalization of phosphatidylserine. While investigating the mechanisms for this process, we revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R-dependent CD62L downregulation by increasing ATP potency. Enhanced superoxide generation in the mitochondria of rotenone- and antimycin A–treated cells was observed and may contribute to the enhanced sensitivity of P2X7R to ATP. P2X7R-dependent exposure of phosphatidylserine was also revealed by preincubation with mitochondrial uncouplers prior to ATP treatment. This may present a novel mechanism whereby P2X7R-dependent phosphatidylserine exposure occurs only when cells have enhanced mitochondrial reactive oxygen species generation. The clearance of apoptotic cells may therefore be enhanced by this mechanism which requires functional P2X7R expression.