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Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.

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We have previously reported that the occupancy of P2z purinergic receptors in BAC1.2F5 macrophages stimulates both the formation of nonselective membrane pores/channels and a novel phospholipase D (PLD) activity. In the present study, the relationship between these two P2z receptor-mediated responses was investigated. In [3H]oleic acid-labeled macrophages, isoosmotic replacement of extracellular Na+ with choline completely abolished 3'-O-(4-benzoyl)benzoyl-ATP (BzATP)-stimulated [3H]phosphatidylethanol ([3H]PEt) accumulation. This inhibitory effect of choline was mimicked when Na+ was replaced with other amine-containing cations (N-methylglucamine, ethanolamine, Tris+), but not with inorganic cations (K+, Li+). In contrast, substitution of Na+ with choline (or other cationic amines) did not prevent BzATP-induced Ca2+ influx and membrane depolarization. Thus, choline, which enters the cells through the BzATP-induced pores, can act to inhibit P2z receptor-stimulated PLD activity but not pore formation. The two responses also exhibit differential sensitivities to the pH of the incubation medium, to depletion of intracellular ATP, and to chronic phorbol ester treatment. These observations indicate that P2z receptor-mediated pore formation can be functionally uncoupled from the activation of PLD. However, both pore formation and PLD activity are similarly inhibited when the cells are: 1) partially desensitized by previous treatment with P2z agonists and 2) preincubated with 2',3'-dialdehyde-ATP or 4',4'-diisothiocyanostilbene-2,2'-disulfonate. This suggests that these two distinct responses are mediated by the same, or similar, P2z receptors.

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Extracellular ATP activates two distinct types of P2 purinoreceptor-mediated signaling pathways in macrophages, 1) the rapid formation of nonselective pores/channels in the plasma membrane and 2) a guanine nucleotide-binding protein-dependent stimulation of phosphotidylinositol-specific phospholipase C, with subsequent mobilization of intracellular Ca2+. Several studies have suggested that the pore-forming, or P2z, purinoreceptor may be involved in the cytolytic effects of ATP on macrophages and other cell types. We have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) and UTP as selective agonists for the P2z purinoreceptor and Ca(2+)-mobilizing nucleotide receptor, respectively, in BAC1.2F5 macrophages. In this paper we demonstrate that BzATP and ATP (but not UTP) activate membrane depolarization in BAC1.2F5 cells and also stimulate appropriate electrophysiological responses, consistent with the expression of the P2z purinoreceptor, in Xenopus oocytes injected with poly(A)+ RNA derived from BAC1.2F5 cells. Micromolar concentrations of BzATP or millimolar concentrations of ATP induced a sustained increase in the membrane holding current in these voltage-clamped oocytes. This response was significantly potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P2z purinoreceptor for tetrabasic nucleotides. The sustained currents induced by BzATP or ATP were distinct from the transient and/or oscillating increases in Ca(2+)-dependent Cl- currents that were stimulated by UTP but not BzATP. UTP-stimulated transient currents and nucleotide-dependent increases in aequorin luminescence in poly(A)+ RNA-injected oocytes were independent of extracellular Ca2+ and were correlated with the mobilization of intracellular Ca2+ stores. Sucrose fractionation of the poly(A)+ RNA from BAC1.2F5 cells resulted in the enrichment of mRNA species encoding the components of the P2z purinoreceptor, as well as the Ca(2+)-mobilizing nucleotide receptor, in fractions containing 2.5-4.0-kilobase species.

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Macrophages express two distinct types of nucleotide (P2 purinergic) receptors for extracellular ATP: one type induces a Ca(2+)-mobilizing response via the activation of phosphatidylinositol-phospholipase C (PI-PLC) while the second type induces the rapid formation of nonselective pores which are permeated by ions and small (< 1 kDa) organic molecules. We have confirmed the presence of these two ATP receptor types in the BAC1.2F5 murine macrophage cell line and have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) as a selective and potent agonist for the so-called P2z or pore-forming ATP receptor type. Several lines of evidence indicated that occupation of these P2z receptors is also accompanied by a rapid and large increase in the activity of a phosphatidylcholine-selective phospholipase D (PLD) effector enzyme. In cells metabolically labeled with [3H]oleic acid or [3H]glycerol and stimulated in the presence of ethanol, ATP and BzATP induced a severalfold increase in the rate and extent of [3H]phosphatidylethanol (PEt) accumulation. These responses were stimulated only by ATP, BzATP, and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate) with the rank order of potency: BzATP >> ATP > ATP gamma A; there was no response to other adenine nucleotides or to non-adenine nucleotides. Significantly, the ability of P2z receptor agonists to stimulate this PLD activity was not dependent on the presence of extracellular [Ca2+] or elevation of cytosolic [Ca2+]. The inability of ionomycin, gramicidin, digitonin, UTP, platelet-activating factor, or phorbol ester to quantitatively mimic these nucleotide effects suggested that activation of this PLD by P2z receptor agonists was not a secondary response due to: 1) enhanced Ca2+ influx; 2) membrane depolarization; 3) nonselective permeabilization of the plasma membrane; 4) stimulation of Ca(2+)-mobilizing ATP receptors; 5) stimulation of a primary PI-PLC pathway; or 6) activation of protein kinase C. These findings suggest that activation of a novel PLD-based signaling pathway may play an important role in the modulation of macrophage function by pore-forming P2z receptors for extracellular ATP.

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Extracellular ATP increases the free cytosolic calcium concentration ([Ca2+]i) of thymocytes by changing the membrane permeability to divalent cations: (1) no [Ca2+]i elevation is observed in the absence of extracellular calcium and (2) ATP also promotes Mn2+ entry into thymocytes, as shown by a stimulated quench of the fluorescence of Quin2-loaded cells. Using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5- hexatriene, a dye which usually does not pass through membranes, we showed that ATP permeabilizes thymocyte membranes not only to cations but also to low-molecular-weight solutes. The ATP effect is dose-dependent, specific and reversible; ATP4- appears to be the active species and probably acts through specific receptor(s). Both medullary and cortical thymocytes respond to ATP whereas splenocytes do not; this shows that the ATP effect is restricted to certain populations of lymphocytes. The thymoma-derived cell line, EL-4, the permeability of which is also sensitive to ATP, could provide a useful model for studying the putative ATP4- receptor(s) of thymocytes.

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We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5'-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4 is probably the active species and that a phosphotransferase activity is not involved in its effects. There ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP greater than ADP much greater than AMP greater than adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.

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We have previously demonstrated that exogenous ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis. In the present study, using the highly sensitive calcium indicator Indo-1, we have measured the effect of exogenous nucleotides on the cytosolic-free calcium concentration [Ca2+]i of thymocytes, and determined inositol phosphate (IP) formation in the same cells, in parallel assays. The results were compared to those obtained with the mitogenic lectin concanavalin A (ConA) in similar experiments. They show that ATP does not mobilize calcium from its internal stores but stimulates its influx from the extracellular medium. Nevertheless, these data do not rule out the possibility that the nucleotide acts through specific P2 purinergic sites.

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1. The apparent Ki values of (-)-noradrenaline (NA), (+)- and (-)-adrenaline (Ad), phenylephrine and the mono-fluorinated NAs (in position 2, 5 or 6) for alpha 1-adrenoceptors of intact BC3H1 cells labelled with [3H]-prazosin were greatly dependent on the incubation temperature. 2. The EC50 values of these compounds for stimulation of the inositol phosphate (IP) accumulation at 37 degrees C were intermediate between their apparent dissociation constants at 2 degrees C (Ki2 degrees) and at 37 degrees C (Ki37 degrees). 3. The fact that an irreversible blockade of 46% +/- 6% (n = 3) of the [3H]-prazosin binding sites by phenoxybenzamine reduced the maximal IP-formation induced by NA by 57% +/- 5% (n = 3) shows that there is a direct coupling between alpha 1-adrenoceptors and phospholipase C in BC3H1 cells. 4. The Ki37 degrees s of all agonists tested were in the same range (0.1 to 1 mM) and showed no simple correlation with their EC50 values. 5. The Ki2 degrees values for all the agonist correlated linearly with their EC50 values but were about 20-100 times lower than the respective EC50 values (except for the partial agonist methoxamine). In order to explain this difference, we propose that the apparent high affinity in the cold could be due to an [3H]-prazosin-induced alteration of the active site of the alpha 1-adrenoceptor, increasing its apparent affinity for catecholamines.

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Increased rates of monovalent cation fluxes are implicated in the activation of lymphocytes by mitogens. Our report shows that amiloride and dimethylamiloride, two inhibitors of the Na+/H+ antiport, dose dependently prevent the proliferation of mitogen-activated human peripheral blood lymphocytes. The action of these drugs follows several mechanisms, since their inhibitory effect can be reversed by extensive washing when they are used at low concentrations (150 microM for amiloride, 40 microM for dimethylamiloride), while at higher non cytotoxic concentrations this reversibility is no longer observed. We have studied the mechanism whereby amiloride inhibits the blastogenesis by measuring their effect on: 1) IL 2 production, 2) acquisition of IL 2 responsiveness and induction of IL 2 receptors, 3) IL 2-induced proliferation. Unlike the expression of IL 2 receptors, IL 2 production and IL 2-dependent proliferation were not inhibited by the low drug concentrations which indeed prevent blastogenesis. Moreover under these conditions, an enhanced accumulation of IL 2 was observed in the supernatants of stimulated cells. These results show that the drugs interact differently with the different cell populations involved in T cell proliferation: increase of an amiloride-dependent sodium influx is an obligatory step required to induce the early increase of the ouabain-dependent potassium influx which is needed for the expression of IL 2 receptors. On the contrary, the influx of potassium necessary for the IL 2-dependent proliferation does not seem to be controlled by the amiloride-dependent sodium flux.

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Exogeneous nucleotides or nucleosides may influence lymphocyte functions such as proliferation and cytotoxicity. We report that ATP, and to a lesser extent ADP, at concentrations as low as 0.3 mM, are highly mitogenic for medullary mature thymocytes, when added in combination with phorbol myristate acetate (PMA), which is only weakly mitogenic by itself. Under the same conditions, the other nucleotides (AMP; GTP, ITP, 2'd-deoxyATP), the non-hydrolysable ATP analogs (p[NH]ppA, pp[CH2]pA) and adenosine are unable to trigger thymocyte blastogenesis. p[NH]ppA, a potent inhibitor of ATP hydrolysis, potentiates the ATP mitogenic effect. In contrast, T-cell-enriched splenocytes do not proliferate in response to ATP + PMA. These data and measurements of interleukin 2 synthesis suggest that ATP may efficiently deliver in thymocytes the calcium signal necessary for the initiation of blastogenesis (in medullary cells). Indeed, among all nucleotides tested, only ATP or ADP were able to increase the intracellular free calcium level in thymocytes, but not in splenocytes. Our results led us to suggest that thymocytes express on their surface receptors specific for ATP, which might be P2 type nucleotide receptors and could be involved in the lymphocyte response through the regulation of intracellular free calcium levels.

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