Extracellular ATP stimulates an amiloride-sensitive sodium influx in human lymphocytes

Extracellular ATP has been shown to increase the Na+ permeability of human lymphocytes by 3 to 12-fold. The kinetics of this ATP-induced response were studied by measuring 22Na+ influx into chronic lymphocytic leukemic lymphocytes incubated in low-sodium media without divalent cations. ATP-stimulated uptake of 22Na-ions was linear over 4 min incubation and this influx component showed a sigmoid dependence on ATP concentration. Hill analysis yielded a K1/2 of 160 microM and a n value of 2.5. The nucleotide ATP-gamma-S (1-2 mM) gave 30% of the permeability increase produced by ATP, but UTP (2 mM) and dTTP (2 mM) had no effect on 22Na influx. The amiloride analogs 5-(N-ethyl-N-isopropyl) amiloride and 5-(N,N-hexamethylene) amiloride, which are potent inhibitors of Na(+)-H+ countertransport, abolished 72-95% of the ATP-stimulated 22Na+ influx. However, the involvement of Na(+)-H+ countertransport in the ATP-stimulated Na+ influx was excluded by three lines of evidence. Sodium influx was stimulated 7-fold by extracellular ATP but only 2.4-fold by hypertonic conditions which are known to activate Na(+)-H+ countertransport. Addition of ATP to lymphocytes produced no change in intracellular pH when these cells were suspended in isotonic NaCl media. Finally ATP caused a membrane depolarization of lymphocytes which is inconsistent with stimulation of electroneutral Na(+)-H+ exchange. These data suggest that ATP acts cooperatively to induce the formation of membrane channels which allow increased Na+ influx by a pathway which is partially inhibited by amiloride and its analogs.     

Exogenous ATP enhances calcium influx in intact thymocytes

Recent observations have indicated that exogenous adenosine triphosphate (ATP) may influence lymphocyte functions such as proliferation and cytoxicity. Here we report a novel activity of extracellular ATP--it specifically increases Ca2+ uptake in murine lymphocytes. ATP added to thymocytes increases the rate of [45Ca2+] uptake by up to 20-fold. The increased rate is seen with ATP concentrations as low as 500 microM and is half-maximal at approximately 2 mM ATP. The magnitude of stimulation by ATP is dependent on Mg2+ concentration, and ATP-Mg2+ complex is probably the true activator. Of the high-energy phosphate-containing compounds tested, including deoxy-ATP, only GTP showed a modest stimulation of calcium uptake. ADP, AMP, cyclic AMP, and adenosine did not significantly increase calcium uptake. Cellular integrity as indicated by trypan blue exclusion and ethidium bromide/acridine orange staining was unaffected by ATP. Ca2+ influx is the major mode of action of ATP in raising intrathymocyte Ca2+ levels, because neither the Ca2+ efflux nor the [45Ca2+]-Ca2+ exchange was significantly altered in the presence of ATP. Verapamil, a Ca2+ channel blocking agent, could not prevent the ATP effect, suggesting that ATP may be acting by a mechanism other than the voltage-dependent Ca2+ channel. An analysis of intracellular and extracellular ATP levels by chemiluminescence assay indicated no significant ATP entry into intact lymphocytes. Also, ATP added to the medium containing thymocytes was destroyed (approximately 50% by 20 min). The nonhydrolyzable ATP analogs, AMPPCP and AMPPNP, were unable to stimulate a significant amount of Ca2+ uptake, suggesting the involvement of a cell surface phosphotransferase activity. This was supported by the demonstration of a threefold to fivefold increase in the labeling of protein and phospholipid fractions obtained from intact thymocytes exposed to [gamma 32P]ATP for 30 min. Ca2+ is believed to play an important role in a variety of lymphocyte functions, including mitogenesis and natural killer cell activity. The data herein thus provide a potential mechanism for the action of exogenous ATP on these lymphocyte functions.     

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands

The effect of ATP on mitochondrial membrane depolarization in rat submandibular glands was investigated. Exposure of the cell suspension to high concentrations of ATP induced a sustained depolarization of mitochondrial membrane. This effect was blocked in the presence of magnesium and reproduced by low concentrations of 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), suggesting the implication of the P2X(7) purinergic receptor. This point was confirmed by comparison of the response to ATP by wild-type and P2X(7) knock-out (P2X(7)R(-/-)) mice. Mitochondria took up calcium after ATP stimulation but the depolarization of the mitochondrial membrane by ATP was not affected by the removal of calcium from the extracellular medium. It was nearly fully suppressed in the absence of sodium and partially blocked by the mitochondrial Na/Ca exchanger inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Both ATP and monensin increased the uptake of extracellular sodium (as shown by the depolarization of the plasma membrane) but the sodium ionophore did not affect the mitochondrial membrane potential. It is concluded that the activation of P2X(7) receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.     

Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic [Ca2+], and inhibits phagocytosis in mouse macrophages

In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.     

Extracellular ATP induces a nonspecific permeability of thymocyte plasma membranes

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.     

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in “reverse” mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.     

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of K_ATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.     

Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca²⁺]_o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X₇ receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X₇ expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.     

Extracellular ATP regulates cell death of lymphocytes and monocytes induced by membrane-bound lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium

The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT-4, and a monocytic cell line, HL-60, was upregulated by ATP added extracellularly in a dose-dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate-oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase-3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate-oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.     

Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.     

Control of membrane permeability by external ATP in mammalian cells: isolation of an ATP-resistant variant from Chinese hamster ovary cells

External ATP causes a great increase in the passive permeability of the plasma membrane for phosphorylated metabolites and other small molecules in cultured mammalian cells. We previously demonstrated that in CHO-K1 cells an ATP-dependent permeability change was induced in the presence of a mitochondrial inhibitor (KCN or rotenone), a cytoskeleton-attacking agent (vinblastine) and a calmodulin antagonist (trifluoperazine). These permeability changes were reversible but long exposure, for 30-60 min, to ATP together with a mitochondrial inhibitor significantly reduced the cell viability of the treated cells. Since this cell lysis was shown to be due to the ATP-dependent permeability change, we could isolate several clones resistant to the action of the external ATP from CHO-K1 cells after repeated treatment with ATP and rotenone. In 9.1 cells, one of the isolated clones, little or no ATP-dependent permeability change was observed in the presence of either a mitochondrial inhibitor, vinblastine or trifluoperazine. This CHO variant could be specifically resistant as to the change in membrane permeability induced by external ATP, since the permeabilities for the 2-deoxyglucose and drugs used in the present studies were similar to those in the case of the parent cells. These results suggest that a specific defect or alteration in the plasma membrane is involved in the ATP-dependent permeability change. It is also reported that Mg2+-dependent ATPase activity was found on the cell surface of both CHO-K1 and 9.1 cells, and this activity was shown to be not involved in the permeability change controlled by external ATP.     

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous ATP induces electrical membrane responses in fibroblasts

Mouse fibroblastic L cells responded to exogenous ATP (0.2 mM) with a transient hyperpolarization due to increased membrane permeability to K⁺. By contrast, intracellular injection of ATP (up to about 3 mM) produced no noticeable effects on the membrane potential. The effects of a non-hydrolysable analogue of ATP (AMP-PNP) were similar to those of ATP. After successive applications of ATP, the cell membrane became virtually unresponsive (desensitized). Extracellular ADP was also effective, but AMP or adenosine was not. Antazoline suppressed the ATP response. Thus, exogenous ATP and ADP appear to stimulate P₂-purinoceptors. Similar responses to ATP (or ADP) were also observed in human normal diploid fibroblasts (Flow 1000 line).     

Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.     

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.     

Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation

Extracellular ATP (ATPo) caused dose-dependent lysis of YAC-1 and P-815 mouse tumor cells. This event, assessed by 51Cr release, was accompanied by sustained depolarization of the plasma membrane potential and Ca2+ influx. Plasma membrane depolarization and Ca2+ influx occurred within a few seconds of ATPo addition to both cell types, whereas 51Cr was released without apparent lag in YAC-1 cells and after 2 h in P-815 cells. Furthermore, a rise in [Ca2+]i was required for ATPo-dependent lysis of YAC-1 but not P-815 cells. In P-815 cells, ATPo caused an early and [Ca2+]i-independent DNA fragmentation that occurred at lower nucleotide concentrations than those required to trigger 51Cr release. Instead in YAC-1 cells very low concentrations of ATPo caused early lysis (ED50 for lysis about 200 microM) accompanied by only barely detectable DNA fragmentation. Previous studies disclosed that lymphokine-activated killer cells are fully resistant to the membrane-perturbing effects of ATPo. We show that lymphokine-activated killer cells also do not undergo DNA fragmentation even in the presence of high ATPo concentrations. This study complements previous observations on the lytic effects of ATPo and shows that this nucleotide can also cause DNA fragmentation, one of the earliest target cell alterations observed during CTL-mediated lysis.     

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose‐ and time‐dependent reduction in viability, and the agonist‐treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca²⁺ levels, resulting in Ca²⁺ uptake by the mitochondria and subsequent H₂O₂ accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP‐induced increase of intracellular ATP, essential for the activation of caspase‐like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca²⁺ concentration but plays a negligible role in eATP‐stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.     

ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes

Extracellular ATP induces cation fluxes in thioglycolate-elicited mouse peritoneal macrophages and the J774 macrophage cell line apparently due to ligation of a plasma membrane receptor for ATP4-. We report that ATP permeabilizes the plasma membrane of J774 cells to 6-carboxyfluorescein (376 Da), lucifer yellow (457 Da), and fura-2 (831 Da) but not to trypan blue (961 Da), Evans blue (961 Da), or larger dye conjugates. We employed fluorescence microscopy and quantitative fluorimetry to study entry of lucifer yellow into the cytoplasm of J774 cells. Permeabilization to lucifer yellow appears to be mediated by the same ATP4- receptor that induces cation fluxes because it was inhibited by divalent cations and low pH, was mediated by the nonhydrolyzable analog adenosine 5'-(beta, gamma-imido)triphosphate, and because a variant J774 cell line resistant to ATP-induced Rb+ efflux did not take up lucifer yellow when exposed to ATP. ATP permeabilization was reversed within 5 min by removal of ATP or by addition of divalent cations. ATP also caused a transient increase in lucifer yellow uptake by pinocytosis. These data suggest that ATP4- ligates a receptor on macrophages which induces the formation of a channel admitting molecules less than or equal to 831 daltons into the cytoplasmic matrix and that removal of ATP4- from the medium causes rapid channel closure.     

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.