Trehalose uptake through P2X7 purinergic channels provides dehydration protection

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.     

Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.     

Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages.

Extracellular ATP4- opens pores in the plasma membrane of mouse macrophages and the J774 macrophage-like cell line that allow molecules as large as fura-2 (831 daltons) to enter the cytoplasmic matrix of the cells. The functional similarity of the ATP-induced pores to gap junctions led us to examine whether these pores were related to members of the connexin family of gap junction proteins. Under conditions of high stringency, RNA isolated from J774 cells hybridized with cDNA for connexin-43 but not with cDNA for connexin-32, -26, or -46. RNA isolated from several variant J774 cell lines that do not permeabilize in response to extracellular ATP (ATPR cells) did not hybridize with connexin-43 cDNA. Immunoblots demonstrated that J774 cells, but not the variant ATPR B2 cell line, expressed connexin-43 protein. These studies demonstrate that mouse macrophages express the connexin-43 gap junction mRNA and protein and strongly suggest that in these cells connexin-43 forms "half-gap junctions" in response to extracellular ATP4-.     

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.     

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.     

Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.     

Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation

Cholesteryl ester accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that J774 macrophages accumulate large amounts of cholesteryl ester when incubated with unmodified low density lipoprotein (LDL) and that this is related to sluggish down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. To further explore intracellular cholesterol metabolism and regulatory events in J774 macrophages, we studied the effect of inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT) on the cells' ability to accumulate cholesterol and to down-regulate receptor and reductase. Treatment of J774 cells with LDL in the presence of ACAT inhibitor 58-035 (Sandoz) prevented both cholesteryl ester and total cholesterol accumulation. Furthermore, 58-035 markedly enhanced down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of LDL. In dose-response studies, down-regulation of the receptor by 58-035 paralleled its inhibition of ACAT activity. Compound 58-035 also increased the down-regulation of the J774 LDL receptor in the presence of 25-hydroxycholesterol and acetyl-LDL but not in the presence of cholesteryl hemisuccinate, which is not an ACAT substrate. The ability of 58-035 to enhance LDL receptor down-regulation was negated when cells were simultaneously incubated with recombinant high density lipoprotein3 discs, which promote cellular cholesterol efflux. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by 58-035. These data suggest that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, contributing to diminished receptor and reductase down-regulation, LDL-cholesterol accumulation, and foam cell formation.     

Involvement of the mannose receptor in infection of macrophages by influenza virus

Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774. The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family. Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus. In competitive binding studies, the binding of (125)I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains. The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages. Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan. A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of D-mannose, which down-modulated mannose receptor expression. Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives

Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb+ transport was measured directly from the uptake of 86Rb. The truncated gramicidins show enhanced selectivity for K+ and Rb+ when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered, i.e., Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+ for desfor and desval as compared to Cs+ greater than Rb+ greater than K+ = Na+ greater than Li+ for gramicidin. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and, consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that 86Rb exchange is stimulated by desfor and desval almost to the same extent as gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded beta 6.3-helical dimer ("channel") in its conductance characteristic and its structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cystic fibrosis transmembrane conductance regulator-independent phagosomal acidification in macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.     

Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X7 Receptor

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X₇. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X₇ receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X₇ receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X₇ blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X₇ receptor is involved in macrophage fusion.     

Induction of apoptosis in macrophage cell line,J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P. aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastase-positive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.     

Influence of extracellular monovalent cations on pore and gating properties of P2X7 receptor-operated single-channel currents

Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.     

Cytokine and nitric oxide release by J774A.1 macrophages is not regulated by estradiol.

Estradiol is able to regulate the release of inflammatory mediators by macrophages; however, the presence, extent, and direction of this modulation varies with species, tissue of origin, and cell culture conditions. This study examines the effects of 17-beta-estradiol (E2) on the release of inflammatory mediators by the J774A.1 mouse macrophage cell line. For experiments, cells were plated in phenol red-free DMEM containing 5% charcoal-dextran stripped calf serum. Western analysis showed that J774A.1 cells contain the estrogen receptor alpha (ER alpha) protein. We found that physiological and pharmacological levels of E2 (10(-12) M-10(-6) M) have no effect on the release of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), or monocyte chemoattractant protein-1 (MCP-1). This suggests that J774A.1 cells grown under these culture conditions would be useful for the investigation of non-estrogen-dependent mechanisms by which certain endocrine disruptors may affect their targets in macrophages.     

32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.     

Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages

Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-alpha or IFN-gamma, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not beta1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-kappaB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-kappaB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.     

Inhibitory effects of chloride on the activation of caspase-1, IL-1beta secretion, and cytolysis by the P2X7 receptor

The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1beta. Because previous studies indicated that extracellular Cl- exerts a negative allosteric effect on ATP-gating of P2X7R channels, we tested whether Cl- attenuates the P2X7R-->caspase-1-->IL-1beta signaling cascade in murine and human macrophages. In Bac1 murine macrophages, substitution of extracellular Cl- with gluconate produced a 10-fold increase in the rate and extent of ATP-induced IL-1beta processing and secretion, while reducing the EC50 for ATP by 5-fold. Replacement of Cl- with gluconate also increased the potency of ATP as an inducer of mature IL-1beta secretion in primary mouse bone marrow-derived macrophages and in THP-1 human monocytes/macrophages. Our observations were consistent with actions of Cl- at three levels: 1) a negative allosteric effect of Cl-, which limits the ability of ATP to gate the P2X7R-mediated cation fluxes that trigger caspase-1 activation; 2) an intracellular accumulation of Cl- via nonselective pores induced by P2X7R with consequential repression of caspase-1-mediated processing of IL-1beta; and 3) a facilitative effect of Cl- substitution on the cytolytic release of unprocessed pro-IL-1beta that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1beta from the lytic release of pro-IL-1beta. These results suggest that under physiological conditions P2X7R are maintained in a conformationally restrained state that limits channel gating and coupling of the receptor to signaling pathways that regulate caspase-1.     

Rapid turnover of low-density lipoprotein receptor by a non-lysosomal pathway in mouse macrophage J774 cells and inhibitory effect of brefeldin A

The low-density lipoprotein (LDL) receptor of molecular mass 155 kDa was expressed on the cell surface of cultured mouse macrophage J774 cells. The conversion rate of precursor to mature form of LDL receptor in J774 cells was comparable to that in mouse fibroblast L cells. The half-life of the LDL receptor of J774 cells was about 2 h, that of L cells was about 11 h. The rapid degradation of LDL receptor was not significantly inhibited by the lysosomotropic agents, chloroquine and NH4Cl, nor by the thiol-protease inhibitors leupeptin and E-64. By contrast, incubation at 18 degrees C retarded the degradation of LDL receptor. Treatment of J774 cells with brefeldin A, an inhibitor of membrane transport between the endoplasmic reticulum and the Golgi apparatus, inhibited the rapid turnover of the LDL receptor. Even after a 9-h chase in the presence of brefeldin A, LDL receptor 5-10 kDa smaller than the normal mature form was found to be stable. Rapid turnover of the LDL receptor in the macrophages appeared to occur after exit from the Golgi apparatus, possibly during transport of the LDL receptor to the plasma membrane.