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.

P2X receptors are ligand-gated ion channels activated by the binding of extracellular adenosine 5'-triphosphate (ATP). Brief (< 1 s) applications of ATP to nodose ganglion neurons or to cells transfected with P2X2 or P2X4 receptor cDNAs induce the opening of a channel selectively permeable to small cations within milliseconds. We now show that, during longer ATP application (10-60 s), the channel also becomes permeable to much larger cations such as N-methyl-D-glucamine and the propidium analog YO-PRO-1. This effect is enhanced in P2X2 receptors carrying point mutations in the second transmembrane segment. Progressive dilation of the ion-conducting pathway during prolonged activation reveals a mechanism by which ionotropic receptors may alter neuronal function.

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. 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. 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. 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. Polyethylene glycols having molecular weights ≥ 5000 blocked the increase in cation permeability, YO‐PRO‐1 uptake and membrane blebbing. 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.

There are few antagonists selective for subtypes of the several P2X receptors, but these are needed to identify the receptors expressed on native cells and tissues. In particular, P2X(4) and P2X(7) receptor subunits are colocalized on immune, epithelial, and exocrine gland cells, but both are relatively insensitive to suramin and pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid derivative. In this article, we show that Coomassie Brilliant Blue G selectively inhibits P2X(7) receptors with nanomolar affinity. We measured currents in response to P2X receptor activation in HEK293 cells heterologously expressing human or rat P2X(1), P2X(2), P2X(3), P2X(2/3), P2X(4), P2X(1/5), and P2X(7) receptors. Brilliant Blue G produced a noncompetitive inhibition of rat and human P2X(7) receptors with IC(50) values of 10 and 200 nM, respectively. IC(50) values for inhibition of the other receptors ranged from 2 to >30 microM; the rat and human P2X(4) receptors showed IC(50) values of >10 and 3.2 microM. Coomassie Blue G also blocked YO-PRO1 uptake and membrane blebbing, which are uniquely associated with activation of P2X(7) receptors. Thus, Brilliant Blue G is at least 1000-fold more potent at rat P2X(7) receptors than at rat P2X(4) receptors.

We have investigated the purinoceptor subtypes responsible for calcium signaling in human platelets, which previous studies have shown to involve both Ca 2؉ influx via receptor-operated cation channels and release of Ca 2؉ from intracellular stores. Fura-2 measurements of [Ca 2؉ ] i in stirred platelet suspensions showed that both ADP (40 M) and the non-hydrolyzable ATP analogue ␣␤-meATP (␣,␤-methyleneadenosine 5-triphosphate, 10 M) activated a rapid Ca 2؉ influx whereas only ADP mobilized Ca 2؉ from internal stores. In "nystatin" whole-cell patch clamp recordings, ATP, ADP, and the non-hydrolyzable ATP analogues, ␣,␤-meATP and ATP␥S (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 M) 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 2X1 receptors in other tissues. We conclude that human platelets possess a P 2X1 purinoceptor, which mediates a rapid phase of ADP-or ATP-evoked Ca 2؉ entry via a cation channel, whereas one or more separate ADP-selective P 2 purinoceptors evoke release of calcium from intracellular stores.In many cell types, extracellular ATP and ADP interact with a family of P 2 purinoceptors (1). Two subgroups have been recently cloned: ionotrophic (P 2X1 , P 2X2 , P 2X3 , P 2X4 ) and Gprotein-coupled (P 2Y , P 2U ) purinoceptors (2-8). Data suggest putative P 2D and P 2Z purinoceptors (9, 10) also exist. Human platelets are reported to possess a unique ADP-selective purinoceptor, termed P 2T , which mediates shape change and aggregation (11). Major actions of ADP in platelets include mobilization of intracellular calcium stores (13) and activation of a non-selective cation channel (12). In cell-attached patch clamp recordings (14), ADP evoked single channel activity if included in the pipette but not when added to the bath saline, demonstrating that this channel is activated by a direct receptoroperated or G-protein-linked mechanism rather than via a diffusible second messenger. Whether the actions of ADP in Ca 2ϩ signal generation in human platelets are mediated by one or multiple purinoceptors is, however, uncertain. In the present study we have used both whole-cell patch clamp recordings and fura-2 intracellular calcium measurements to investigate the purinoceptor subtypes involved in human platelet calcium signaling. MATERIALS AND METHODSSolutions and Reagents-Unless otherwise stated, standard platelet saline contained (mM) 150 NaCl, 10 Hepes, 1 MgCl 2 , 1 EGTA at pH 7.35 (with NaOH). BaCl 2 saline contained (mM) 110 BaCl 2 , 10 Hepes, 1 MgCl 2 at pH 7.35 (with N-methyl D-glucamine base). The pipettes were filled with a solution containing (mM) 50 KCl, 70 K 2 SO 4 , 10 Hepes, 5 MgCl 2 , 0.1 EGTA, pH 7.2 (KOH). 50 -100 M nystatin was added to the internal pipette saline from a 50 mM stock, made in dimethyl sulfoxide, immediately ...