Pharmacological differences between human and mouse P2X4 receptor explored using old and new tools.

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.

Structural Basis of the Negative Allosteric Modulation of 5-BDBD at Human P2X4 Receptors.

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.

Transcriptional and Post-Translational Regulation of Junctional Adhesion Molecule-B (JAM-B) in Leukocytes under Inflammatory Stimuli.

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.

Constitutive P2Y2 receptor activity regulates basal lipolysis in human adipocytes.

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.

ATP Evokes Ca2+ Responses and CXCL5 Secretion via P2X4 Receptor Activation in Human Monocyte-Derived Macrophages.

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.

ATP-Evoked Intracellular Ca2+ Responses in M-CSF Differentiated Human Monocyte-Derived Macrophage are Mediated by P2X4 and P2Y11 Receptor Activation.

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).

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells.

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.

Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage.

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.

P2X4 Receptor-Dependent Ca2+ Influx in Model Human Monocytes and Macrophages.

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.

P2Y6 receptor inhibition perturbs CCL2-evoked signalling in human monocytic and peripheral blood mononuclear cells.

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 ∼80% of the intracellular Ca²âº 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 Ca²âº responses. Pharmacological inhibition of P2Y6 impaired CCL2-evoked Ca²âº 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 Ca²âº 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.

Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism.

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.

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes.

Elucidating mechanisms by which Ca(2+) signals are generated by monocytes is important for understanding monocyte function in health and disease. We have investigated mechanisms underlying Ca(2+) signals generated following disruption of lysosomes by exposure to the cathepsin C substrate glycyl-L-phenylalanine-ß-napthylamide (GPN). Exposure to 0.2 mM GPN resulted in robust increases in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence of extracellular Ca(2+). The response was antagonised by thapsigargin and evoked capacitative Ca(2+) entry. Dantrolene-sensitive Ca(2+) responses were observed at higher concentrations of GPN (0.4 mM) but not at 0.2 mM. Strikingly, GPN-evoked Ca(2+) responses and ß-hexosaminidase secretion were inhibited by the ATPase/ADPase apyrase. Simultaneous measurement of [Ca(2+)](i) and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca(2+) signalling. Furthermore, the ability of GPN to raise [Ca(2+)](i) was inhibited by P2Y receptor antagonists or by inhibiting vesicular exocytosis with N-ethylmaleimide (NEM). NEM treatment was associated with an inability of GPN to trigger ATP secretion, a drop in baseline [Ca(2+)](i) and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline [Ca(2+)](i). In summary, these data suggest that P2Y receptor activation contributes significantly to GPN-evoked Ca(2+) signalling, and that constitutive secretion of lysosomal ATP is a major determinant of Ca(2+) homeostasis in monocytes. Lysosomal Ca(2+) stores can communicate with ER Ca(2+) stores either directly through activation of ryanodine receptors, or indirectly through release of ATP and engagement of P2Y receptors.

A mechanism of intracellular P2X receptor activation.

P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen.

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum.

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.

Neuropeptide Y: Direct vasoconstrictor and facilitatory effects on P2X1 receptor-dependent vasoconstriction in human small abdominal arteries.

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.

STAT1- and NFAT-independent amplification of purinoceptor function integrates cellular senescence with interleukin-6 production in preadipocytes.

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.

The intestinal microbiota predicts COVID-19 severity and fatality regardless of hospital feeding method.

SARS-CoV-2-positive patients exhibit gut and oral microbiome dysbiosis, which is associated with various aspects of COVID-19 disease (1-4). Here, we aim to identify gut and oral microbiome markers that predict COVID-19 severity in hospitalized patients, specifically severely ill patients compared to moderately ill ones. Moreover, we investigate whether hospital feeding (solid versus enteral), an important cofounder, influences the microbial composition of hospitalized COVID-19 patients. We used random forest classification machine learning models with interpretable secondary analyses. The gut, but not the oral microbiota, was a robust predictor of both COVID-19-related fatality and severity of hospitalized patients, with a higher predictive value than most clinical variables. In addition, perturbations of the gut microbiota due to enteral feeding did not associate with species that were predictive of COVID-19 severity. IMPORTANCE SARS-CoV-2 infection leads to wide-ranging, systemic symptoms with sometimes unpredictable morbidity and mortality. It is increasingly clear that the human microbiome plays an important role in how individuals respond to viral infections. Our study adds to important literature about the associations of gut microbiota and severe COVID-19 illness during the early phase of the pandemic before the availability of vaccines. Increased understanding of the interplay between microbiota and SARS-CoV-2 may lead to innovations in diagnostics, therapies, and clinical predictions.

The Concise Guide to PHARMACOLOGY 2023/24: Ion channels.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Fluvastatin suppresses native and recombinant human P2X4 receptor function.

Statins have both cholesterol lowering and anti-inflammatory activities, whether mechanisms underlying their activities are independent remains unclear. The ATP-gated P2X(4) receptor is a pro-inflammatory mediator. Here, we investigate the action of fluvastatin and other cholesterol depleting agents on native and recombinant human P2X(4) receptor. Fluvastatin and mßCD suppressed P2X(4)-dependent calcium influx in THP-1 monocytes, without affecting P2Y receptor responses. mßCD or filipin III suppressed the current density of recombinant human P2X(4) receptors. Human P2X(2) was insensitive to cholesterol depletion. Cholesterol depletion had no effect on intrinsic P2X(4) receptor properties as judged by ATP concentration-response relationship, receptor rundown or current decay during agonist occupancy. These data suggest fluvastatin suppresses P2X(4) activity in monocytes through cholesterol depletion and not by modulating intrinsic channel properties.

Neuropeptide Y facilitates P2X1 receptor-dependent vasoconstriction via Y1 receptor activation in small mesenteric arteries during sympathetic neurogenic responses.

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.