ABSTRACT
Seasonality is an important aspect associated with population dynamic and structure of tropical insect assemblages. This study evaluated the effects of seasonality on abundance, richness, diversity and composition of an insect group, drosophilids, including species native to the Neotropical region and exotic ones. Three preserved fragments of the northern Atlantic Forest were surveyed, where temperatures are above 20 °C throughout the year and rainfall regimes define two seasons (dry and rainy). As opposed to other studies about arthropods in tropical regions, we observed that abundance of drosophilids was significantly higher in the dry season, possibly due to biological aspects and the colonization strategy adopted by the exotic species in these environments. Contrarily to abundance, we did not observe a seasonal pattern for richness. As for other parts of the Atlantic Forest, the most representative Neotropical species (Drosophila willistoni, D. sturtevanti, D. paulistorum and D. prosaltans) were significantly more abundant in the rainy season. Among the most abundant exotic species, D. malerkotliana, Zaprionus indianus and Scaptodrosophila latifasciaeformis were more importantly represented the dry season, while D. simulans was more abundant in the rainy period. The seasonality patterns exhibited by the most abundant species were compared to findings published in other studies. Our results indicate that exotic species were significantly more abundant in the dry season, while native ones exhibited an opposite pattern.
Subject(s)
Biodiversity , Drosophilidae , Seasons , Animals , Brazil , Female , Forests , MaleABSTRACT
Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1ß and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1ß secretion by means of its fimbriae in a purinergic P2X7 receptor-dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1ß processing and secretion by P. gingivalis-infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1ß secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1ß from P. gingivalis-infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1ß to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1ß secretion but also for intracellular pro-IL-1ß processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7(-/-) mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis.
Subject(s)
Bacteroidaceae Infections/metabolism , Porphyromonas gingivalis/pathogenicity , Receptors, Purinergic P2X7/physiology , Animals , Bacteroidaceae Infections/microbiology , Carrier Proteins/physiology , Caspase 1/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.
Subject(s)
Adenine Nucleotides/blood , Dermatitis, Contact/genetics , Dermatitis, Irritant/genetics , Receptors, Purinergic P2X7/genetics , Animals , Antigens, CD/blood , Apyrase/blood , Croton Oil/toxicity , Dermatitis, Contact/blood , Dermatitis, Contact/pathology , Dermatitis, Irritant/blood , Dermatitis, Irritant/pathology , Disease Models, Animal , Humans , Hydrolysis , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Nucleotide Deaminases/blood , Purinergic P2X Receptor Antagonists/administration & dosage , Receptors, Purinergic P2X7/bloodABSTRACT
Adenosine triphosphate (ATP) plays a role in cell signaling. It was soon proposed that ATP activates ionotropic P2X receptors, exerting an influence on neurons as well as on glial cells. In addition to the fact that the activation of P2X and P2Y receptors can stimulate or inhibit the release of glutamate from rat hippocampal neurons, the release of ATP has been implicated in hippocampal long-term potentiation (LTP). Through different behavioral paradigms, this study aimed to investigate the participation of P2X7R in genetically modified (knockout (KO)) mice with the suppressed expression of this receptor and in the pharmacological blockage of this receptor in rats, as well as to evaluate the effect of environmental enrichment on potential mnemonic deficits. The results suggest that P2X7R participates in aversive memory processes: pharmacological blockage with the selective P2X7R antagonist, A-740003, in different time frames elicited dose-dependent impairments in memory acquisition, consolidation and retrieval in rats that were submitted to the contextual fear-conditioning (FC) task, and the deletion of P2X7R hampered the aversive memory processes of mice that were subjected to the FC paradigm. Experiments using mice that were subjected to environmental enrichment suggest that this form of stimulation reverses mnemonic impairments that are ascribed to the absence of the P2X7R, suggesting that these receptors do not participate on such a reversal. Finally, no alterations were observed in the habituation memory of P2X7KO mice.
Subject(s)
Acetamides/pharmacology , Memory/drug effects , Memory/physiology , Purinergic P2X Receptor Antagonists/pharmacology , Quinolines/pharmacology , Receptors, Purinergic P2X7/physiology , Animals , CA1 Region, Hippocampal/drug effects , CA1 Region, Hippocampal/physiology , Conditioning, Psychological/drug effects , Conditioning, Psychological/physiology , Dose-Response Relationship, Drug , Environment , Fear/drug effects , Fear/physiology , Habituation, Psychophysiologic/drug effects , Habituation, Psychophysiologic/physiology , Housing, Animal , Male , Maze Learning/drug effects , Maze Learning/physiology , Mice, Inbred C57BL , Mice, Knockout , Pain Threshold/drug effects , Pain Threshold/physiology , Random Allocation , Rats, Wistar , Receptors, Purinergic P2X7/geneticsABSTRACT
Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7(-/-) mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Leishmaniasis/drug therapy , Leishmaniasis/immunology , Macrophages/immunology , Receptors, Purinergic P2X7/genetics , Adenosine Triphosphate/pharmacology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Bone Marrow Cells/parasitology , Histocompatibility Antigens Class II/biosynthesis , L-Lactate Dehydrogenase/metabolism , Leishmania/immunology , Leishmaniasis/parasitology , Macrophages/parasitology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/biosynthesis , Reactive Oxygen Species/metabolismABSTRACT
ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Cation Transport Proteins/metabolism , Protein Kinase C/metabolism , Receptors, Purinergic P2Y2/metabolism , Receptors, Purinergic P2/metabolism , Adenosine/metabolism , Animals , Cell Line , Enzyme Activation , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/metabolism , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , SwineABSTRACT
BACKGROUND AND PURPOSE: ATP is released in response to cellular damage, and P2X7 receptors have an essential role in the onset and maintenance of pathological changes. Haemorrhagic cystitis (HC) is a well-known adverse effect of therapy with cyclophosphamide used for the treatment of many solid tumours and autoimmune conditions. Here we have evaluated the role of P2X7 receptors in a model of HC induced by cyclophosphamide. EXPERIMENTAL APPROACH: Effects of pharmacological antagonism or genetic deletion of P2X7 receptor on cyclophosphamide-induced HC in mice was assessed by nociceptive and inflammatory measures. In addition, the presence of immunoreactive P2X7 receptors was assessed by immunohistochemistry. KEY RESULTS: Pretreatment with the selective P2X7 receptor antagonist A-438079 or genetic ablation of P2X7 receptors reduced nociceptive behaviour scores in the HC model. The same strategies decreased both oedema and haemorrhage indices, on macroscopic or histological evaluation. Treatment with A-438079 decreased the staining for c-Fos in the lumbar spinal cord and brain cortical areas. Treatment with A-438079 also prevented the increase of urinary bladder myeloperoxidase activity and macrophage migration induced by cyclophosphamide and reduced the tissue levels of IL-1ß and TNF-α. Finally, P2X7 receptors were markedly up-regulated in the bladders of mice with cyclophosphamide-induced HC. CONCLUSIONS AND IMPLICATIONS: P2X7 receptors were significantly involved in a model of HC induced by cyclophosphamide. Pharmacological inhibition of these receptors might represent a new therapeutic option for this pathological condition.
Subject(s)
Cyclophosphamide/toxicity , Cystitis/chemically induced , Hemorrhage/chemically induced , Inflammation/metabolism , Nociception/physiology , Receptors, Purinergic P2X7/metabolism , Animals , Cell Movement , Cystitis/metabolism , Cystitis/pathology , Cytokines/genetics , Cytokines/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation , Genes, fos , Hemorrhage/metabolism , Macrophages/physiology , Male , Mesna/pharmacology , Mice , Mice, Knockout , Purinergic P2X Receptor Antagonists/pharmacology , Pyridines/administration & dosage , Pyridines/pharmacology , Receptors, Purinergic P2X7/genetics , Tetrazoles/administration & dosage , Tetrazoles/pharmacology , Urinary Bladder/metabolismABSTRACT
BACKGROUND AND PURPOSE: The two longest C-termini of the purinergic P2X receptors occur in the P2X2 and P2X7 receptors and are thought to interact with multiple cytoplasmic proteins, among which are members of the cytoskeleton, including microtubules. In this work we asked whether disrupting the microtubule cytoskeleton might affect the functions of these receptors. EXPERIMENTAL APPROACH: Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTS: Disrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1ß release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1ß, interferon-γ and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONS: Colchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action - inhibition of pore formation induced by activation of P2X receptors - which could explain some of the anti-inflammatory effects of colchicine.
Subject(s)
Adenosine Triphosphate/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Colchicine/pharmacology , Ion Channel Gating/drug effects , Receptors, Purinergic P2X2/physiology , Receptors, Purinergic P2X7/physiology , Adenosine Triphosphate/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Cell Death/drug effects , Colchicine/therapeutic use , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Female , Fluorescent Dyes/pharmacokinetics , HEK293 Cells , Humans , Interferon-gamma/metabolism , Interleukin-1beta/metabolism , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/ultrastructure , Male , Mice , Microscopy, Fluorescence , Microtubules/drug effects , Microtubules/ultrastructure , Nitric Oxide/metabolism , Oocytes/drug effects , Oocytes/metabolism , Oocytes/ultrastructure , Rats , Reactive Oxygen Species/metabolism , Receptors, Purinergic P2X2/genetics , Receptors, Purinergic P2X7/genetics , Transfection , Xenopus laevisABSTRACT
P2X7 is a member of the purinergic receptors family, with extracellular adenosine triphosphate (ATP) as the main agonist, promoting cations influx and membrane permeabilization that can lead to cell death. We previously proposed that extracellular ATP is involved in thymus atrophy induced by Trypanosoma cruzi infection through the induction of CD4+/CD8+ double-positive cell death and that P2X7 could be involved in this process. To further elucidate this possibility raised by in vitro assays, in this study, we used P2X7-/- mice and observed no difference in thymus atrophy or parasitemia when compared to C57Bl/6. We then decided to investigate other aspects of purinergic receptor interplay that could be better evidenced by the infection and observed that (1) thymocytes from infected and noninfected C57Bl/6 mice express P2X4 and P2X7 receptors (Western blotting), but ATP-induced membrane permeabilization only occurs in thymocytes from infected mice; (2) peritoneal macrophages from noninfected C57Bl/6 mice (P2X4+ and P2X7+) are permeabilized by ATP. Although macrophages from infected C57Bl/6 mice are P2X7- but P2X4+, they are resistant to ATP, either through permeabilization or Ca++ influx (fluorimetry); (3) using noninfected P2X7-/- mice, C57Bl/6 infected mice, and different agonistic stimuli, we observed interesting cross-talks among P2X and P2Y receptors (flow cytometry).
Subject(s)
Receptors, Purinergic P2/metabolism , Thymus Gland/pathology , Thymus Gland/parasitology , Trypanosoma cruzi/physiology , Adenosine Triphosphate , Animals , Atrophy , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Membrane Permeability , Cells, Cultured , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Parasitemia , Receptors, Purinergic P2/deficiency , Receptors, Purinergic P2X7ABSTRACT
Receptors of the P2X7 type have been demonstrated in granulocytes, monocytes/macrophages, B and T lymphocytes, and have been involved in several cellular mechanisms including those related to inflammation and immunological response. This study attempted to investigate the role of these receptors on the inflammatory and fibrogenic response in the kidneys of unilateral ureteral obstruction (UUO), by using P2X7 knockout mice (-/-). C57Bl6 mice were submitted to left UUO and killed after 7 and 14 days. Histopathology using hematoxylin-eosin, periodic-acid Schiff and Sirius-red staining, immunohistochemistry for macrophages, myofibroblasts, transforming growth factor-beta (TGF-beta)1 and P2X7, and immunofluorescence for apoptotic cells (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling) were performed. Protocols were as follows: (1) control; (2) sham; (3) control P2X7 (-/-); (4) sham P2X7 (-/-); (5) UUO wild type (WT); (6) UUO P2X7 (-/-). Myofibroblasts and Sirius-red staining were significantly lower in UUO P2X7 (-/-) mice at days 7 and 14, compared to UUO WT. Kidneys from UUO P2X7 (-/-) mice showed reduced number of inflammatory cells at day 14 but not at day 7, compared to UUO WT. TGF-beta1 was less in UUO P2X7 (-/-) mice at days 7 and 14 when compared to UUO WT. Macrophage infiltration and tubular apoptosis were lower in UUO P2X7 (-/-) at day 14 but not at day 7, compared to UUO WT. P2X7 was expressed only in tubular epithelial cells at day 7 of UUO WT mice. These findings constitute the first evidence that P2X7 receptors are implicated in macrophage infiltration, collagen deposition and apoptosis in response to ureteral obstruction in mice.
Subject(s)
Inflammation/pathology , Inflammation/physiopathology , Receptors, Purinergic P2/physiology , Ureteral Obstruction/pathology , Ureteral Obstruction/physiopathology , Actins/genetics , Actins/metabolism , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Apoptosis/physiology , Atrophy/metabolism , Atrophy/pathology , Atrophy/physiopathology , Collagen/genetics , Collagen/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis/metabolism , Fibrosis/pathology , Fibrosis/physiopathology , Gene Expression Regulation/physiology , Inflammation/metabolism , Kidney Tubules/metabolism , Kidney Tubules/pathology , Kidney Tubules/physiopathology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7 , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Ureteral Obstruction/metabolismABSTRACT
In view of the evidence for a role for extracellular ATP in both pancreatic endocrine and exocrine functions, we have investigated the expression of P2X and P2Y receptors in this tissue in neonate and aged rat and mouse. Using immunohistochemistry it was shown that P2X(1), P2X(4), P2X(7), P2Y(1) and P2Y(2) receptors were present in different regions of the rat and mouse pancreas; P2X(3) and P2X(6) receptors were not found, and P2X(5) immunolabelling was only found in some nerves. The pancreatic vasculature of both rat and mouse expressed P2X(1), P2X(2), P2Y(1) and P2Y(2) receptors in the smooth muscle. P2X(1) and P2X(4) receptors were absent in the islets of the neonate pancreas, but were progressively upregulated with age after birth. In contrast, the greatest expression of P2Y(1) in cells from the duct system was in neonate pancreas, while there was no P2Y(1) expression in aged rat pancreas. P2X(7) receptors had a consistent pattern of distribution in all of the groups examined, being located in the outer periphery of the islet. Using antibodies raised against insulin, somatostatin and glucagon, double-labelling immunofluorescence was used to identify P2X(7)-positive cells in different islet of Langerhans cell populations. Our results demonstrated a clear immunoreaction to P2X(7) receptors in islet alpha cells, while no P2X(7) was expressed in beta and delta cells. The significance of the differential expression of P2 receptors in the pancreas during development and ageing, and a possible role for the proliferation and death of the islet cell population are discussed.
Subject(s)
Aging/metabolism , Islets of Langerhans/metabolism , Pancreas/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/metabolism , Animals , Animals, Newborn , Immunohistochemistry , Islets of Langerhans/chemistry , Islets of Langerhans/growth & development , Male , Mice , Mice, Inbred BALB C , Muscle, Smooth, Vascular/chemistry , Muscle, Smooth, Vascular/growth & development , Muscle, Smooth, Vascular/metabolism , Pancreas/chemistry , Pancreas/growth & development , Pancreatic Ducts/chemistry , Pancreatic Ducts/growth & development , Pancreatic Ducts/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/analysisABSTRACT
Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Apoptosis/drug effects , Chlamydia Infections/metabolism , Chlamydophila psittaci/metabolism , Macrophages/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/metabolism , Affinity Labels/pharmacology , Animals , Apoptosis/physiology , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Cell Survival/drug effects , Cell Survival/physiology , Chlamydia Infections/pathology , Chlamydia Infections/physiopathology , Chlamydophila psittaci/drug effects , DNA Fragmentation/drug effects , DNA Fragmentation/physiology , Extracellular Space/metabolism , HeLa Cells , Humans , Inflammation/metabolism , Inflammation/pathology , Inflammation/physiopathology , Macrophages/drug effects , Macrophages/microbiology , Magnesium/pharmacology , Mice , Nucleotides/pharmacology , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2X7ABSTRACT
Macrophages and thymocytes express P2Z/P2X7 nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7 receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.
Subject(s)
Apoptosis/physiology , Dendritic Cells/physiology , Receptors, Purinergic P2/physiology , Adenosine Diphosphate/pharmacology , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Apoptosis/drug effects , Caspase Inhibitors , Cell Membrane Permeability/drug effects , Cycloheximide/pharmacology , Enzyme Inhibitors/pharmacology , Gene Expression , Magnesium/pharmacology , Mice , Mice, Inbred BALB C , Purinergic P2 Receptor Antagonists , RNA, Messenger/metabolism , Receptors, Purinergic P2/genetics , Uridine Triphosphate/pharmacologyABSTRACT
It is well established that the process of thymocyte differentiation and maturation occurs in the thymus, where cell-to-cell communication is essential for providing the messages to T-cell precursors. At least two pathways are important for such communication: one via membrane surface molecules and the other via soluble mediators such as cytokines and some hormones. Recently, the presence of receptors for extracellular ATP has been demonstrated on thymocytes and microenvironment cells, and putative functions for this molecule have been proposed. Herein we focus on the recent evidence which supports the view of extracellular ATP and some related nucleotides as novel intrathymic signal molecules. In addition, we discuss the possible physiological implications of such purinergic receptors for the physiology of the thymus.
Subject(s)
Adenosine Triphosphate/physiology , Receptors, Purinergic P2/physiology , Signal Transduction , Thymus Gland/physiology , Animals , Apoptosis , Calcium/metabolism , Humans , Lymphocytes/metabolism , Models, BiologicalABSTRACT
The effects of extracellular nucleosides and nucleotides on many organs and systems have been recognized for almost 50 years. The effects of extracellular ATP (ATPo), UTPo, ADPo, and other agonists are mediated by P2 purinoceptors. One of the most dramatic effects of ATPo is the permeabilization of plasma membranes to low molecular mass solutes of up to 900 Da. This effect is evident in several cells of the lymphohematopoietic system and is supposed to be mediated by P2Z, an ATP(4-)-activated purinoceptor. Here, we review some basic information concerning P2 purinoceptors and focus our attention on P2Z-associated phenomena displayed by macrophages. Using fluorescent dye uptake, measurement of free intracellular Ca2+ concentration and electrophysiological recordings, we elucidate some of the events that follow the application of ATP to the extracellular surface of macrophages. We propose a regulatory mechanism for the P2Z-associated permeabilization pore. The presence of P2 purinoceptors in cells of the lymphohematopoietic system makes them potential candidates to mediate immunoregulatory events.
Subject(s)
Adenosine Triphosphate/physiology , Cell Membrane Permeability/physiology , Hematopoietic System/physiology , Receptors, Purinergic P2/physiology , T-LymphocytesABSTRACT
The effects of extracellular nucleosides and nucleotides on many organs and systems have been recognized for almost 50 years. The effects of extracellular ATP (ATP(o)), UTP (o), ADP(o), and other agonists are mediated by P2 purinoceptors. One of the most dramatic effects of ATP(o) is the permeeabilization of plasma membranes to low molecular mass solutes of up to 900 Da. This effect is evident in several cells of the lymphohematopoietic system and is supposed to be mediated by P2Z, and APT(4-) -activated purinoceptor. Here, we review some basic information concerning P2 purinoceptors and focus our attention on P2Z-associated phenomena displayed by macrophages. Using fluorescent dye uptake, measurement of free intracellular Ca2+ concentration and electrophysiological recordings, we elucidate some of the events that follow the application of ATP to the extracellular surface of macrophages. We propose a regulatory mechanism for the P2Z-associated permeabilization pore. The presence of P2 purinoceptors in cells of the lymphohematopoietic system makes them potential candidates to mediate immunoregulatory events.
Subject(s)
Adenosine Triphosphate/physiology , Cell Membrane Permeability/physiology , Hematopoietic System/physiology , In Vitro Techniques , Receptors, Purinergic P2/physiology , T-LymphocytesABSTRACT
Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 +/- 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-D-glucamine, and glutamate; their opening is favored at temperatures higher than 30 degrees C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3'-O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.
Subject(s)
Adenosine Triphosphate/pharmacology , Macrophages, Peritoneal/physiology , Receptors, Purinergic P2/physiology , Animals , Cell Line , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Cells, Cultured , Electric Conductivity , Glutamic Acid/metabolism , Macrophages, Peritoneal/drug effects , Meglumine/pharmacokinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Patch-Clamp Techniques , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2X7 , Tromethamine/pharmacokineticsABSTRACT
The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin-43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch-clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.
Subject(s)
Gap Junctions/physiology , Intercellular Junctions/physiology , Macrophages, Peritoneal/ultrastructure , 1-Octanol , Adenosine Triphosphate/metabolism , Animals , Biological Transport, Active , Bucladesine/pharmacology , Calcium/metabolism , Cell Communication , Coculture Techniques , Connexin 43/analysis , Connexin 43/genetics , Epithelial Cells , Extracellular Space/metabolism , Fluorescent Dyes/metabolism , Gap Junctions/chemistry , Hydrogen-Ion Concentration , Intercellular Junctions/chemistry , Interferon-gamma/pharmacology , Isoquinolines/metabolism , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Membrane Potentials , Mice , Mice, Knockout , Microinjections , Octanols/pharmacology , Patch-Clamp Techniques , Thymus Gland/cytology , Tumor Cells, CulturedABSTRACT
The thymic microenvironment is under intrinsic and extrinsic control circuits by several elements including hormones, neuropeptides, lymphokines, innervation and cell contact. P2 purinergic receptors have been described in a number of cells including macrophages, thymocytes, and other cells of the immune-inflammatory system. Here, we use the whole-cell patch-clamp technique and dye permeabilization assays to investigate the presence of ionic channels and purinergic receptors in one microenvironmental thymic component, namely the phagocytic cell of the thymic reticulum. At holding potentials ranging from -30 to -60 mV, applications of extracellular ATP in the vicinity of the cell membrane induce a transient and fast-activating inward current followed in most cells by an outward current. The whole event lasts 5-20 s. The inward current has a reversal potential close to 0 mV and the outward current can be ascribed to a Ca2+ -dependent K+ conductance. Both currents are inhibited by Mg2+, suggesting that the phenomenon is mediated by ATP4-. ATP-gamma-S can also induce both inward and outward currents. Exposure of phagocytic cells of the thymic reticulum to 5 mM ATP for 10 min induced permeabilization to lucifer yellow but not to the larger dyes trypan blue and rhodamine-dextran, suggesting a molecular weight cut-off smaller than 900. These observations lead us to conclude that phagocytic cells of the thymic reticulum express P2Z purinergic receptors that can mobilize Ca2+, induce the opening of ionic channels and permeabilize the cell membrane.