ABSTRACT
There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antagonists for P2X4, though agonist selectivity is less well studied. As there are some known pharmacological differences between P2X receptors from different species, it is important to understand these differences when designing a pharmacological strategy to probe P2X4 function in human tissue and mouse models. Here, we provide a systematic comparison of agonist and antagonist pharmacology in 1321N1 cells expressing either human or mouse P2X4 orthologues. We identify a rank order of agonist potency of ATP > 2-MeSATP > αßmeATP = BzATP > CTP = γ-[(propargyl)-imido]-ATP for human P2X4 and ATP > 2-MeSATP = CTP > ATPγS = γ-[(propargyl)-imido]-ATP = BzATP for mouse. Human P2X4 is not activated by ATPγS but can be activated by αßmeATP. We identify a rank order of antagonist potency of BAY-1797 = PSB-12062 = BX-430 > 5-BDBD > TNP-ATP = PPADS for human P2X4 and BAY-1797 > PSB-12062 = PPADS > TNP-ATP for mouse. Mouse P2X4 is not antagonised by 5-BDBD or BX-430. The study reveals key pharmacological differences between human and mouse P2X4, highlighting caution when selecting tools for comparative studies between human and mouse and ascribing cellular responses of some commonly used agonists to P2X4.
ABSTRACT
Neuropeptide Y (NPY) is co-released with norepinephrine and ATP by sympathetic nerves innervating arteries. Circulating NPY is elevated during exercise and cardiovascular disease, though information regarding the vasomotor function of NPY in human blood vessels is limited. Wire myography revealed NPY directly stimulated vasoconstriction (EC50 10.3 ± 0.4 nM; N = 5) in human small abdominal arteries. Maximum vasoconstriction was antagonised by both BIBO03304 (60.7 ± 6%; N = 6) and BIIE0246 (54.6 ± 5%; N = 6), suggesting contributions of both Y1 and Y2 receptor activation, respectively. Y1 and Y2 receptor expression in arterial smooth muscle cells was confirmed by immunocytochemistry, and western blotting of artery lysates. α,ß-meATP evoked vasoconstrictions (EC50 282 ± 32 nM; N = 6) were abolished by suramin (IC50 825 ± 45 nM; N = 5) and NF449 (IC50 24 ± 5 nM; N = 5), suggesting P2X1 mediates vasoconstriction in these arteries. P2X1, P2X4 and P2X7 were detectable by RT-PCR. Significant facilitation (1.6-fold) of α,ß-meATP-evoked vasoconstrictions was observed when submaximal NPY (10 nM) was applied between α,ß-meATP applications. Facilitation was antagonised by either BIBO03304 or BIIE0246. These data reveal NPY causes direct vasoconstriction in human arteries which is dependent upon both Y1 and Y2 receptor activation. NPY also acts as a modulator, facilitating P2X1-dependent vasoconstriction. Though in contrast to the direct vasoconstrictor effects of NPY, there is redundancy between Y1 and Y2 receptor activation to achieve the facilitatory effect.
Subject(s)
Neuropeptide Y , Receptors, Purinergic P2X1 , Humans , Neuropeptide Y/pharmacology , Vasoconstriction , Vasoconstrictor Agents/pharmacology , Receptors, Neuropeptide Y/metabolism , Arteries/metabolismABSTRACT
BACKGROUND AND PURPOSE: Senescent preadipocytes promote adipose tissue dysfunction by secreting pro-inflammatory factors, although little is known about the mechanisms regulating their production. We investigated if up-regulated purinoceptor function sensitizes senescent preadipocytes to cognate agonists and how such sensitization regulates inflammation. EXPERIMENTAL APPROACH: Etoposide was used to trigger senescence in 3T3-L1 preadipocytes. CRISPR/Cas9 technology or pharmacology allowed studies of transcription factor function. Fura-2 imaging was used for calcium measurements. Interleukin-6 levels were quantified using quantitative PCR and ELISA. Specific agonists and antagonists supported studies of purinoceptor coupling to interleukin-6 production. Experiments in MS1 VEGF angiosarcoma cells and adipose tissue samples from obese mice complemented preadipocyte experiments. KEY RESULTS: DNA damage-induced senescence up-regulated purinoceptor expression levels in preadipocytes and MS1 VEGF angiosarcoma cells. ATP-evoked Ca2+ release was potentiated in senescent preadipocytes. ATP enhanced interleukin-6 production, an effect mimicked by ADP but not UTP, in a calcium-independent manner. Senescence-associated up-regulation and activation of the adenosine A3 receptor also enhanced interleukin-6 production. However, nucleotide hydrolysis was not essential because exposure to ATPγS also enhanced interleukin-6 secretion. Pharmacological experiments suggested coupling of P2X ion channels and P2Y12 -P2Y13 receptors to downstream interleukin-6 production. Interleukin-6 signalling exacerbated inflammation during senescence and compromised adipogenesis. CONCLUSIONS AND IMPLICATIONS: We report a previously uncharacterized link between cellular senescence and purinergic signalling in preadipocytes and endothelial cancer cells, raising the possibility that up-regulated purinoceptors play key modulatory roles in senescence-associated conditions like obesity and cancer. There is potential for exploitation of specific purinoceptor antagonists as therapeutics in inflammatory disorders.
Subject(s)
Hemangiosarcoma , Receptors, Purinergic P2 , Mice , Animals , Interleukin-6 , Receptors, Purinergic P2/metabolism , Calcium/metabolism , Vascular Endothelial Growth Factor A/metabolism , Adenosine Triphosphate/metabolism , Receptors, Purinergic/metabolism , Cellular Senescence , Inflammation , STAT1 Transcription Factor/metabolismABSTRACT
Junctional adhesion molecules (JAMs; comprising JAM-A, -B and -C) act as receptors for viruses, mediate cell permeability, facilitate leukocyte migration during sterile and non-sterile inflammation and are important for the maintenance of epithelial barrier integrity. As such, they are implicated in the development of both communicable and non-communicable chronic diseases. Here, we investigated the expression and regulation of JAM-B in leukocytes under pathogen- and host-derived inflammatory stimuli using immunoassays, qPCR and pharmacological inhibitors of inflammatory signalling pathways. We show that JAM-B is expressed at both the mRNA and protein level in leukocytes. JAM-B protein is localised to the cytoplasm, Golgi apparatus and in the nucleus around ring-shaped structures. We also provide evidence that JAM-B nuclear localisation occurs via the classical importin-α/ß pathway, which is likely mediated through JAM-B protein nuclear localisation signals (NLS) and export signals (NES). In addition, we provide evidence that under both pathogen- and host-derived inflammatory stimuli, JAM-B transcription is regulated via the NF-κB-dependent pathways, whereas at the post-translational level JAM-B is regulated by ubiquitin-proteosome pathways. Anaphase-promoting ubiquitin ligase complex (APC/C) and herpes simplex virus-associated ubiquitin-specific protease (HAUSP/USP) were identified as candidates for JAM-B ubiquitination and de-ubiquitination, respectively. The expression and regulation of JAM-B in leukocytes reported here is a novel observation and contrasts with previous reports. The data reported here suggest that JAM-B expression in leukocytes is under the control of common inflammatory pathways.
Subject(s)
Junctional Adhesion Molecule B , Cell Movement , Humans , Inflammation/metabolism , Junctional Adhesion Molecule B/metabolism , Leukocytes/metabolism , Ubiquitins/metabolismABSTRACT
The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.
Subject(s)
Benzodiazepinones/chemistry , Benzodiazepinones/metabolism , Purinergic P2X Receptor Antagonists/chemistry , Purinergic P2X Receptor Antagonists/metabolism , Receptors, Purinergic P2X4/chemistry , Receptors, Purinergic P2X4/metabolism , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Benzodiazepinones/pharmacology , HEK293 Cells , Humans , Molecular Dynamics Simulation , Protein Structure, Secondary , Protein Structure, Tertiary , Purinergic P2X Receptor Antagonists/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: P2X4 is a ligand-gated cation channel activated by extracellular ATP involved in neuropathic pain, inflammation and arterial tone. EXPERIMENTAL APPROACH: Natural products were screened against human or mouse P2X4 activity using fura-2 loaded 1321N1 cells for measurement of intracellular Ca2+ responses. Whole-cell currents were measured by patch clamp. Human primary macrophage chemokine release was used to assess effect of taspine on inflammatory cell function. An enzymatic assay was performed to assess the effect of taspine on recombinant PI3-kinase. KEY RESULTS: A natural product screen identified taspine as an inhibitor of human P2X4 activity. Taspine inhibits human and mouse P2X4-mediated Ca2+ influx in 1321N1 cells expressing receptors but lacked activity at human P2X2, P2X3, P2X2/3 and P2X7 receptors. Taspine inhibited the maximal response at human and mouse P2X4 but effective on ATP potency. Taspine has a slow onset rate (~15 min for half-maximal inhibition), irreversible over 30 min of washout. Taspine inhibits P2X4-mediated Ca2+ signalling in mouse BV-2 microglia cells and human primary macrophage. Taspine inhibited P2X4-mediated CXCL5 secretion in human primary macrophage. Taspine reversed ivermectin-induced potentiation of P2X4 currents in 1321N1 stably expressing cells. The PI3-kinase inhibitor LY294002 mimicked the properties of taspine on P2X4-mediated Ca2+ influx and whole-cell currents. Taspine directly inhibited the enzymatic activity of recombinant PI3-kinase in a competitive manner. CONCLUSION AND IMPLICATIONS: Taspine is a novel natural product P2X4 receptor inhibitor, mediating its effect through PI3-kinase inhibition rather than receptor antagonism. Taspine can inhibit the pro-inflammatory signalling by P2X4 in human primary macrophage.
Subject(s)
Biological Products , Receptors, Purinergic P2X4 , Adenosine Triphosphate , Alkaloids , Animals , Mice , Phosphatidylinositol 3-Kinase , Phosphatidylinositol 3-Kinases , Receptors, Purinergic P2X7ABSTRACT
The known seven mammalian receptor subunits (P2X1-7) form cationic channels gated by ATP. Three subunits compose a receptor channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1 and TM2), intracellular N- and C-termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D structure and gating cycle of P2X receptors. The agonist-binding pocket is located at the intersection of two neighbouring subunits. In addition to the mammalian P2X receptors, their primitive ligand-gated counterparts with little structural similarity have also been cloned. Selective agonists for P2X receptor subtypes are not available, but medicinal chemistry supplied a range of subtype-selective antagonists, as well as positive and negative allosteric modulators. Knockout mice and selective antagonists helped to identify pathological functions due to defective P2X receptors, such as male infertility (P2X1), hearing loss (P2X2), pain/cough (P2X3), neuropathic pain (P2X4), inflammatory bone loss (P2X5), and faulty immune reactions (P2X7).
Subject(s)
Adenosine Triphosphate , Animals , Ligands , Male , Mice , Mice, Knockout , Receptors, Purinergic P2X2ABSTRACT
ATP, norepinephrine and NPY are co-released by sympathetic nerves innervating arteries. ATP elicits vasoconstriction via activation of smooth muscle P2X receptors. The functional interaction between neuropeptide Y (NPY) and P2X receptors in arteries is not known. In this study we investigate the effect of NPY on P2X1-dependent vasoconstriction in mouse mesenteric arteries. Suramin or P2X1 antagonist NF449 abolished α,ß-meATP evoked vasoconstrictions. NPY lacked any direct vasoconstrictor effect but facilitated the vasoconstrictive response to α,ß-meATP. Mesenteric arteries expressed Y1 and Y4 receptors, but not Y2 or Y5. Y1 receptor inhibition (BIBO3304) reversed NPY facilitation of the α,ß-meATP-evoked vasoconstriction. L-type Ca2+ channel antagonism (nifedipine) had no effect on α,ß-meATP-evoked vasoconstrictions, but completely reversed NPY facilitation. Electrical field stimulation evoked sympathetic neurogenic vasoconstriction. Neurogenic responses were dependent upon dual α1-adrenergic (prazosin) and P2X1 (NF449) receptor activation. Y1 receptor antagonism partially reduced neurogenic vasoconstriction. Isolation of the P2X1 component by α1-adrenergic blockade allowed faciliatory effects of Y1 receptor activation to be explored. Y1 receptor antagonism reduced the P2X1 receptor component during neurogenic vasoconstriction. α1-adrenergic and P2X1 receptors are post-junctional receptors during sympathetic neurogenic vasoconstriction in mesenteric arteries. In conclusion, we have identified that NPY lacks a direct vasoconstrictor effect in mesenteric arteries but can facilitate vasoconstriction by enhancing the activity of P2X1, following activation by exogenous agonists or during sympathetic nerve stimulation. The mechanism of P2X1 facilitation by NPY involved activation of the NPY Y1 receptor and the L-type Ca2+ channel.
Subject(s)
Mesenteric Arteries/innervation , Neuropeptide Y/pharmacology , Receptors, Neuropeptide Y/agonists , Receptors, Purinergic P2X1/metabolism , Sympathetic Nervous System/drug effects , Vasoconstriction/drug effects , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Adrenergic alpha-1 Receptor Antagonists/pharmacology , Animals , Benzenesulfonates/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/drug effects , Calcium Channels, L-Type/metabolism , Male , Mice, Inbred C57BL , Nifedipine/pharmacology , Prazosin/pharmacology , Receptors, Neuropeptide Y/metabolism , Suramin/pharmacology , Sympathetic Nervous System/metabolismABSTRACT
Tissues differentially secrete multiple colony stimulating factors under conditions of homeostasis and inflammation, orientating recruited circulating monocytes to differentiate to macrophage with differing functional phenotypes. Here, we investigated ATP-evoked intracellular Ca2+ responses in human macrophage differentiated with macrophage colony-stimulating factor (M-CSF). Extracellular ATP evoked (EC50 13.3 ± 1.4 µM) robust biphasic intracellular Ca2+ responses that showed a dependency on both metabotropic (P2Y) and ionotropic (P2X) receptors. qRT-PCR and immunocytochemistry revealed the expression of P2Y1, P2Y2, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, and P2X7. Pharmacological analysis revealed contribution of only P2X4 and P2Y11 to the Ca2+ response evoked by maximal ATP concentrations (100 µM). This study reveals the contribution of P2X4 and P2Y11 receptor activation to ATP-evoked intracellular Ca2+ responses, and makes comparison with macrophage differentiated using granulocyte colony-stimulating factor (GM-CSF).
Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Cell Differentiation , Macrophage Colony-Stimulating Factor/metabolism , Macrophages/cytology , Macrophages/metabolism , Receptors, Purinergic P2X4/metabolism , Receptors, Purinergic P2/metabolism , Calcium Signaling , Humans , Macrophages/immunologyABSTRACT
BACKGROUND AND PURPOSE: CCL2 is an inflammatory chemokine that stimulates the recruitment of monocytes into tissue via activation of the GPCR CCR2. EXPERIMENTAL APPROACH: Freshly isolated human monocytes and THP-1 cells were used. Fura-2 loaded cells were used to measure intracellular Ca2+ responses. Transwell migration to measure chemotaxis. siRNA-mediated gene knock-down was used to support pharmacological approaches. KEY RESULTS: CCL2 evoked intracellular Ca2+ signals and stimulated migration in THP-1 monocytic cells and human CD14+ monocytes in a CCR2-dependent fashion. Attenuation of DAG catabolism in monocytes by inhibiting DAG kinase (R59949) or DAG lipase (RHC80267) activity suppressed CCL2-evoked Ca2+ signalling and transwell migration in monocytes. These effects were not due to a reduction in the number of cell surface CCR2. The effect of inhibiting DAG kinase or DAG lipase could be mimicked by addition of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) but was not rescued by application of exogenous phosphatidylinositol 4,5-bisphosphate. Suppressive effects of R59949, RHC80267, and OAG were partially or fully reversed by Gö6983 (pan PKC isoenzyme inhibitor) but not by Gö6976 (PKCα and PKCß inhibitor). RNAi-mediated knock-down of DAG kinase α isoenzyme modulated CCL2-evoked Ca2+ responses in THP-1 cells. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that DAG production resulting from CCR2 activation is metabolised by both DAG kinase and DAG lipase pathways in monocytes and that pharmacological inhibition of DAG catabolism or application suppresses signalling on the CCL2-CCR2 axis via a mechanism dependent upon a PKC isoenzyme that is sensitive to Gö6983 but not Gö6976.
Subject(s)
Chemokine CCL2/metabolism , Diacylglycerol Kinase/metabolism , Lipoprotein Lipase/metabolism , Monocytes/metabolism , Receptors, CCR2/metabolism , Calcium/metabolism , Carbazoles/pharmacology , Cell Movement/drug effects , Diacylglycerol Kinase/antagonists & inhibitors , Humans , Indoles/pharmacology , Lipoprotein Lipase/antagonists & inhibitors , Maleimides/pharmacology , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , THP-1 CellsABSTRACT
BACKGROUND AND PURPOSE: The P2X3 receptor is an ATP-gated ion channel expressed by sensory afferent neurons and is used as a target to treat chronic sensitisation conditions. The first-in-class, selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK-7264 (gefapixant), has progressed to Phase III trials for refractory or unexplained chronic cough. We used patch clamp to elucidate the pharmacology and kinetics of MK-7264 and rat models of hypersensitivity and hyperalgesia to test its efficacy on these conditions. EXPERIMENTAL APPROACH: Whole-cell patch clamp of 1321N1 cells expressing human P2X3 and P2X2/3 receptors was used to determine mode of MK-7264 action, potency, and kinetics. The analgesic efficacy was assessed using paw withdrawal threshold and limb weight distribution in rat models of inflammatory, osteoarthritic, and neuropathic sensitisation. KEY RESULTS: MK-7264 is a reversible allosteric antagonist at human P2X3 and P2X2/3 receptors. Experiments with the slowly desensitising P2X2/3 heteromer revealed concentration- and state-dependency to wash-on, with faster rates and greater inhibition when applied before agonist compared to during agonist application. The wash-on rate (τ value) for MK-7264 at maximal concentrations was much lower when applied before compared to during agonist application. In vivo, MK-7264 displayed efficacy comparable to naproxen in inflammatory and osteoarthritic sensitisation models and gabapentin in neuropathic sensitisation models, increasing paw withdrawal threshold and decreasing weight-bearing discomfort. CONCLUSIONS AND IMPLICATIONS: MK-7264 is a reversible and selective P2X3 and P2X2/3 antagonist, exerting allosteric antagonism via preferential activity at closed channels. Its efficacy in rat models supports its clinical investigation for chronic sensitisation conditions.
Subject(s)
Carbolines , Hyperalgesia/drug therapy , Neuralgia/drug therapy , Osteoarthritis/drug therapy , Purinergic P2X Receptor Antagonists , Receptors, Purinergic P2X2/physiology , Receptors, Purinergic P2X3/physiology , Animals , Carbolines/blood , Carbolines/pharmacokinetics , Carbolines/pharmacology , Carbolines/therapeutic use , Cell Line, Tumor , Female , Freund's Adjuvant , Humans , Hyperalgesia/chemically induced , Iodoacetic Acid , Osteoarthritis/chemically induced , Physical Stimulation , Purinergic P2X Receptor Antagonists/blood , Purinergic P2X Receptor Antagonists/pharmacokinetics , Purinergic P2X Receptor Antagonists/pharmacology , Purinergic P2X Receptor Antagonists/therapeutic use , Rats, Sprague-Dawley , Sciatic Nerve/injuriesABSTRACT
White adipocytes are key regulators of metabolic homeostasis, which release stored energy as free fatty acids via lipolysis. Adipocytes possess both basal and stimulated lipolytic capacity, but limited information exists regarding the molecular mechanisms that regulate basal lipolysis. Here, we describe a mechanism whereby autocrine purinergic signalling and constitutive P2Y2 receptor activation suppresses basal lipolysis in primary human in vitro-differentiated adipocytes. We found that human adipocytes possess cytoplasmic Ca2+ tone due to ATP secretion and constitutive P2Y2 receptor activation. Pharmacological antagonism or knockdown of P2Y2 receptors increases intracellular cAMP levels and enhances basal lipolysis. P2Y2 receptor antagonism works synergistically with phosphodiesterase inhibitors in elevating basal lipolysis, but is dependent upon adenylate cyclase activity. Mechanistically, we suggest that the increased Ca2+ tone exerts an anti-lipolytic effect by suppression of Ca2+-sensitive adenylate cyclase isoforms. We also observed that acute enhancement of basal lipolysis following P2Y2 receptor antagonism alters the profile of secreted adipokines leading to longer-term adaptive decreases in basal lipolysis. Our findings demonstrate that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signalling in human adipocytes.
Subject(s)
Adipocytes/metabolism , Receptors, Purinergic P2Y2/metabolism , Adenosine Triphosphate/metabolism , Adenylyl Cyclases/metabolism , Adipocytes/cytology , Adipocytes/drug effects , Adult , Aged , Calcium/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Female , Humans , Lipolysis/drug effects , Middle Aged , Primary Cell Culture , Purinergic P2Y Receptor Antagonists/pharmacology , Signal TransductionABSTRACT
Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7-9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 µM) > ATP (EC50 2.2 ± 1.1 µM) = UTP (3.2 ± 2.8 µM). Cells were unresponsive to UDP (< 30 µM) and UDP-glucose (< 30 µM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 µM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.
Subject(s)
Mesenchymal Stem Cells/metabolism , Receptors, Purinergic P2Y2/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Diphosphate/metabolism , Calcium/metabolism , Humans , Uridine Diphosphate/metabolism , Uridine Triphosphate/metabolismABSTRACT
Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 µM, n = 3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whereas P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+ Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11, or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.
Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Chemokine CXCL5/metabolism , Macrophages/metabolism , Receptors, Purinergic P2X4/metabolism , Calcium Signaling , Cells, Cultured , Enzyme Activation/physiology , Gene Expression Regulation/immunology , Humans , Macrophages/immunology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2Y/metabolismABSTRACT
Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5'-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 µM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage.
Subject(s)
Calcium/metabolism , Macrophages/metabolism , Monocytes/metabolism , Receptors, Purinergic P2X4/metabolism , Action Potentials , Adenosine Triphosphate/metabolism , Biological Transport , Cell Line , Gene Knockdown Techniques , Gene Silencing , Humans , Immunohistochemistry , Macrophages/drug effects , Monocytes/drug effects , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X4/geneticsABSTRACT
Adenosine 5'-triphosphate is a well-known extracellular signaling molecule and neurotransmitter known to activate purinergic P2X receptors. Information has been elucidated about the structure and gating of P2X channels following the determination of the crystal structure of P2X4 (zebrafish), however, there is still much to discover regarding the role of this receptor in the central nervous system (CNS). In this review we provide an overview of what is known about P2X4 expression in the CNS and discuss evidence for pathophysiological roles in neuroinflammation and neuropathic pain. Recent advances in the development of pharmacological tools including selective antagonists (5-BDBD, PSB-12062, BX430) and positive modulators (ivermectin, avermectins, divalent cations) of P2X4 will be discussed.
ABSTRACT
Mechanisms controlling endoplasmic reticulum (ER) Ca2+ homeostasis are important regulators of resting cytoplasmic Ca2+ concentration ([Ca2+]cyto) and receptor-mediated Ca2+ signalling. Here we investigate channels responsible for ER Ca2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca2+ leak. Under these conditions ionomycin elevates [Ca2+]cyto revealing a Ca2+ leak response which is abolished by thapsigargin. IP3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca2+]cyto. In primary macrophage, translocon inhibition blocks Ca2+ leak but does not influence resting [Ca2+]cyto. We identify a role for translocon-mediated ER Ca2+ leak in receptor-mediated Ca2+ signalling in both model and primary human macrophage, whereby the Ca2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca2+ leak via the translocon in controlling resting cytoplasmic Ca2+ in model macrophage and receptor-mediated Ca2+ signalling in model macrophage and primary macrophage.
Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Macrophages/metabolism , Receptors, Purinergic/metabolism , Cells, Cultured , HumansABSTRACT
ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 µM ßγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd(3+) caused cell swelling while blocking any recovery by ßγ-imidoATP. ATP release was 4-fold higher in the presence of Gd(3+). Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd(3+), while NO donors rescued apyrase- and Gd(3+)-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd(3+)-sensitive receptor that is coupled with intracellular NO production.
Subject(s)
Adenosine Triphosphate/metabolism , Dictyostelium/metabolism , Stress, Physiological/physiology , Cell Size/drug effects , Cells, Cultured , Dictyostelium/drug effects , Exocytosis/drug effects , Exocytosis/physiology , Gadolinium/pharmacology , Nitric Oxide/metabolism , Stress, Physiological/drug effectsABSTRACT
The chemokine CCL2 serves to target circulating monocytes and other leukocytes to tissue during innate immune responses, and modulates the progression of chronic inflammatory disease through activation of the receptor CCR2. Here, we show that co-activation of the P2Y6 purinergic receptor (encoded by P2RY6) occurs when THP-1 cells and human peripheral blood mononuclear cells sense CCL2 through CCR2. Furthermore, P2Y6 receptor activation accounts for â¼80% of the intracellular Ca²âº signal evoked by CCL2. Scavenging extracellular nucleotides with apyrase caused a fourfold reduction in THP-1 sensitivity to CCL2, whereas inhibition of CD39-like ectonucleotidases potentiated CCL2-evoked Ca²âº responses. Pharmacological inhibition of P2Y6 impaired CCL2-evoked Ca²âº signalling and chemotaxis in peripheral blood mononuclear cells and THP-1 cells. Furthermore, stable P2Y6 receptor knockdown (of twofold) in THP-1 cells impaired CCL2-evoked Ca²âº signalling, chemotaxis and adhesion to TNFα-treated HUVECs. We demonstrate that THP-1 cells rapidly secrete ATP during signalling downstream of the CCL2-CCR2 axis and suggest this might act as a mechanism for P2Y6 receptor co-activation following CCL2 activation of the CCR2 receptor. The discovery that P2Y6 receptor mediates leukocyte responsiveness to CCL2 represents a new mechanism by which to modulate CCL2 signals.
Subject(s)
Chemokine CCL2/metabolism , Leukocytes, Mononuclear/metabolism , Monocytes/metabolism , Purinergic P2 Receptor Antagonists/pharmacology , Receptors, Purinergic P2/metabolism , Chemokine CCL2/blood , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Monocytes/cytology , Monocytes/drug effects , Receptors, Purinergic P2/blood , Receptors, Purinergic P2/genetics , Signal Transduction/drug effectsABSTRACT
Adenosine 5-triphosphate (ATP) is omnipresent in biology. It is therefore no surprise that organisms have evolved multifaceted roles for ATP, exploiting its abundance and restriction of passive diffusion across biological membranes. A striking role is the emergence of ATP as a bona fide transmitter molecule, whereby the movement of ATP across membranes serves as a chemical message through a direct ligand-receptor interaction. P2X receptors are ligand-gated ion channels that mediate fast responses to the transmitter ATP in mammalian cells including central and sensory neurons, vascular smooth muscle, endothelium, and leukocytes. Molecular cloning of P2X receptors and our understanding of structure-function relationships has provided sequence information with which to query an exponentially expanding wealth of genome sequence information including protist, early animal and human pathogen genomes. P2X receptors have now been cloned and characterized from a number of simple organisms. Such work has led to surprising new cellular roles for the P2X receptors family and an unusual phylogeny, with organisms such as Drosophila and C. elegans notably lacking P2X receptors despite retaining ionotropic receptors for other common transmitters that are present in mammals. This review will summarize current work on the evolutionary biology of P2X receptors and ATP as a signaling molecule, discuss what can be drawn from such studies when considering the action of ATP in higher animals and plants, and outline how simple organisms may be exploited experimentally to inform P2X receptor function in a wider context.