P2X1 Expression Research Articles

Characterizing purinergic receptors in the gastrointestinal epithelium and immune cells

Background: Extracellular release of nucleotides (e.g., ATP, ADP, UTP, UDP) are well established as potent intercellular signaling molecules during homeostasis but are also released from cells during pathogenic microbial infection and tissue injury to serve as danger signals. These important extracellular signaling molecules activate purinergic receptors, of which there are 19 different purinergic receptor subtypes that are classified into P1 and P2 receptors. P1 receptor subtypes are activated by adenosine, while P2 receptors are differentially activated by ATP, ADP, UTP, UDP, and UP-glucose. P2 receptors are further divided into P2Y receptors (P2YRs) and P2X receptors (P2XRs) based on their structures and distinct signal-transduction mechanisms. Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, yet the majority of work to date has focused on purinergic signaling in immune cells. Our group has previously identified a role for P2Y1 in mediating ADP-driven calcium waves during rotavirus infection. While purinergic signaling is a well-established mode of signaling in the enteric nervous system, the functional roles of purinergic signaling in the gastrointestinal tract remain incompletely understood. Hypothesis: Since epithelial cells serve as the first barrier against irritants and infection, we hypothesized that gut epithelium express multiple purinergic receptors that regulate cellular responses to extracellular nucleotide signals. Methods & Results: Using the Human Protein Atlas, we queried single cell RNA sequencing data for the P2 purinergic receptors. We examined these receptors along the length of the gastrointestinal tract, including esophagus, small intestine, colon, and rectum as well as in flow-sorted immune cells, peripheral blood mononuclear cells and and bone marrow derived immune cells. In silico analysis revealed high expression of P2Y1, P2Y2, P2Y11 and P2X4 among the purinergic receptors throughout the gastrointestinal tract. Interestingly, P2Y1 showed highest expression in esophageal epithelial cells, small intestinal enterocytes, and goblet cells. P2Y6 was also observed in promixal enterocytes of the small intestine, but was largely absent from the colon and rectum. In contrast, we found that immune cells from bone marrow, peripheral blood and flow sorted cells had relatively high expression of P2X1 and P2X4. We noted P2X5 expression in plasma cells and B -cells in all sets. Pathway analysis from the Reactome database revealed P2Y receptors are associated with signal transduction, metabolism and hemostasis pathways, while P2X receptors were primarily involved in immune response and infectious disease pathways. Conclusions: These findings confirm that the gastrointestinal tract possess robust P2Y1, P2Y2, P2Y11 and P2X4 receptor expression and can participate in purinergic signaling pathways. R01 DK115507 (Hyser); R01 AI158683 (Hyser); APS Postdoctoral Fellowship (Engevik); F32 DK130288 (Engevik) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dysfunction of low-density neutrophils in peripheral circulation in patients with sepsis

Low-density neutrophils (LDNs) have been described in tumors and various autoimmune diseases, where they exhibit immune dysfunction and alter disease progression. Nevertheless, LDNs have been rarely reported in sepsis. We studied sepsis patients admitted to the intensive care unit. Wright-Giemsa stain assay and Transmission electron microscopy were performed to detect the morphology of neutrophils. Flow cytometry was used to analyze the number and function of LDNs. Concentration of cytokines was measured using ELISA. Neutrophil chemotaxis was examined using an under-agarose chemotaxis model. We found that LDNs were significantly elevated in patients with sepsis. Phenotypes and morphological characteristics suggest that LDNs may be formed by mixtures of neutrophils at various maturation stages. In vitro experiments showed that LDN formation was closely associated with neutrophil degranulation. We preliminarily discussed changes in immune function in LDNs. Compared with high-density neutrophils, expression levels of CXC chemokine receptor 4 on LDN surfaces were increased, phagocytotic capacity was decreased, and life span was prolonged. The chemotactic ability of LDNs was significantly reduced, possibly related to the increased expression of P2X1. These data suggest that LDNs are essential components of neutrophils in sepsis. To clarify the source and dysfunction mechanism of LDN in sepsis may be helpful for the diagnosis and treatment of sepsis in the future.

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female.

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

Functional expression of P2X1, P2X4 and P2X7 purinergic receptors in human monocyte-derived macrophages

This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA. In whole-cell patch clamp recordings of human MDMs, rapid application of ATP (0.01 mM) evoked fast current activation and two different desensitization kinetics: 1. a rapid desensitizing current antagonized by PPADS (1 μM), reminiscent of the P2X1 receptor's current; 2. a slow desensitizing current, insensitive to PPADS but potentiated by ivermectin (3 μM), similar to the P2X4 receptor's current. Application of 5 mM ATP induced three current modalities: 1. slow current activation with no desensitization, similar to the P2X7 receptor current, present in 69% of human macrophages and antagonized by A-804598 (0.1 μM); 2. fast current activation and fast desensitization, present in 15% of human MDMs; 3. fast activation current followed by biphasic desensitization, observed in 15% of human MDMs. Both rapid and biphasic desensitization kinetics resemble those observed for the recombinant human P2X1 receptor expressed in oocytes. These data demonstrate, for the first time, the co-expression of P2X1, P2X4, and P2X7 transcripts and confirm the presence of functional P2X1, P2X4, and P2X7 receptors in human macrophages.

Low androgen status inhibits erectile function by up-regulating the expression of P2X receptors in rat corpus cavernosum.

The aim of our study was to investigate whether low androgen level inhibits the erectile function of rats by regulating the expression of P2X receptors. Thirty-six 8-week-old male SD rats were randomly divided into six groups: sham-operated groups (4w-sham, 8w-sham), castration groups (4w-cast, 8w-cast) and androgen replacement after castration groups (4w-cast+T, 8w-cast+T). The maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP), the levels of serum testosterone (T) and nitric oxide (NO), and the expression of P2X1, P2X2, P2X3, eNOS, p-eNOS, ROCK1 and ROCK2 in the cavernous tissue of rats were determined. The serum T, ICPmax/MAP and NO levels in penile corpus cavernosum in the castration groups were significantly lower than those in other groups (p<.01). The protein expression of P2X1, P2X2, P2X3, ROCK1 and ROCK2 in the castration groups was significantly higher than those in other groups (p<.01). P-eNOS/eNOS of the castration groups were significantly lower than those of other groups (p<.01). The serum T level was negatively correlated with the expression of P2X1, P2X2 and P2X3 in the corpus cavernosum. Low androgen level inhibits erectile function by up-regulating the expression of P2X1, P2X2, P2X3 and RhoA/Rho-kinase resulting in reducing the ratio of p-eNOS/eNOS and the level of NO in corpus cavernosum of rats.

Purinergic receptor mediated calcium signalling in urothelial cells

Non-neuronal ATP released from the urothelium in response to bladder stretch is a key modulator of bladder mechanosensation. Whilst non-neuronal ATP acts on the underlying bladder afferent nerves to facilitate sensation, there is also the potential for ATP to act in an autocrine manner, modulating urothelial cell function. The aim of this study was to systematically characterise the functional response of primary mouse urothelial cells (PMUCs) to ATP. PMUCs isolated from male mice (14–16 weeks) were used for live-cell fluorescent calcium imaging and qRT-PCR to determine the expression profile of P2X and P2Y receptors. The majority of PMUCs (74–92%) responded to ATP (1 μM–1 mM), as indicted by an increase in intracellular calcium (iCa2+). PMUCs exhibited dose-dependent responses to ATP (10 nM–1 mM) in both calcium containing (2 mM, EC50 = 3.49 ± 0.77 μM) or calcium free (0 mM, EC50 = 9.5 ± 1.5 μM) buffers. However, maximum iCa2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X1–6, and P2Y1–2, P2Y4, P2Y6, P2Y11–14, but not P2X7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.

P2X4 receptor as a modulator in the function of P2X receptor in CD4+ T cells from peripheral blood and adipose tissue

Obesity is characterized by immune cell infiltration and inflammation. Purinergic receptors such as P2X1, 4 and 7 are expressed on immune cells and their activation contributes with an inflammatory response. However, the simultaneous expression of P2X1, 4 and 7 during overweight or obesity have not been described. Therefore, the aim of this study was to determine single and simultaneously expression and function of the P2X1, 4 and 7 receptors in lymphocytes and CD4 + T cells from peripheral blood (PB) and adipose tissue (AT). Our results showed a higher expression of the P2X4 receptor on CD4 + T cells from PB regarding P2X7 and P2X1 receptor expression. In addition, P2X4 receptor expression on CD4 + T cells from PB and AT was increased in individuals with BMI ≥ 25 Kg/m2. Moreover, a higher simultaneous expression of the P2X4 and P2X7 receptors on CD4 + T cells from AT compared to CD4 + T cells expressing P2X1 and P2X7 receptors simultaneously. Besides, CD4 + T cells expressing P2X4 and P2X7 receptors from PB and AT were augmented in individuals with BMI ≥ 25 Kg/m2. In addition, the percentage of lymphocytes and also CD4 + T cells expressing P2X4 receptor were elevated both in PB and AT compared to cells expressing P2X7 or P2X1. However, CD4 + T cells expressing P2X4 and P2X7 were augmented in AT compared to PB. The function of the receptors showed a lower shedding of CD62 L in adipose tissue mononuclear cells (ATMC) compared with peripheral blood mononuclear cells (PBMC) and a greater participation of P2X4 in the mobilization of intracellular calcium. We concluded that it was possible to determine for the first time the simultaneous expression of purinergic receptors in ATMC, where the P2X4 receptor has a greater participation in the activation of CD4 + T cells possibly modulating the function of the other two receptors.

Toxoplasma gondii ROP18 inhibits human glioblastoma cell apoptosis through a mitochondrial pathway by targeting host cell P2X1

BackgroundApoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed.MethodsApoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting.ResultsThe apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation.ConclusionsToxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

Erythrocyte P2X1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

BackgroundPore-forming proteins released from bacteria or formed as result of complement activation are known to produce severe cell damage. Inhibition of purinergic P2X receptors markedly reduces damage inflicted by cytolytic bacterial toxin and after complement activation in both erythrocytes and monocytes. P2X expression generally shows variation throughout the population. Here, we investigate correlation between P2X receptor abundance in blood cell plasma membranes and haematocrit during sepsis, in patients admitted to the emergency department (ED) or intensive care unit (ICU).MethodPatients admitted to the ED and successively transferred to ICU with the diagnosis sepsis (< 2 systemic inflammatory response syndrome (SIRS) criteria and suspected infection), were grouped as either blood pathogen-positive (14 patients) or blood pathogen-negative (20 patients). Blood samples drawn at ICU admission were analysed for P2X1 and P2X7 receptor abundance using indirect flow cytometry.ResultsHere, we find inverse correlation between P2X1 receptor expression and change in haematocrit (rs − 0.80) and haemoglobin (rs − 0.78) levels from admission to ED to arrival at ICU in patients with pathogen-positive sepsis. This correlation was not found in patients without confirmed bacteraemia. Patients with high P2X1 expression had a significantly greater change in both haematocrit (− 0.59 ± 0.36) and haemoglobin levels (− 0.182 ± 0.038 mg/dl) per hour, during the first hours after hospital admission compared to patients with low P2X1 expression (0.007 ± 0.182 and − 0.020 ± 0.058 mg/dl, respectively).ConclusionHigh levels of P2X1 are correlated with more pronounced reduction in haematocrit and haemoglobin in patients with confirmed bacteraemia. This supports previous in vitro findings of P2X activation as a significant component in cell damage caused by pore-forming bacterial toxins and complement-dependent major attack complex. These data suggest a new potential target for future therapeutics in initial stages of sepsis.

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.

Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries

BackgroundThe internal pudendal arteries (IPAs) supply blood to the penis and are highly susceptible to vascular remodeling in rodent models of diabetes, hypertension, aging, and chronic kidney disease, thus contributing to erectile dysfunction. Interestingly, vascular remodeling primarily occurs in the distal and not in the proximal IPA, suggesting distinct local physiologic signaling differences within the IPA. AimTo examine the role of purinergic signaling and neurotransmitter release by electrical field stimulation (EFS) in the regulation of proximal and distal IPA vascular tone. MethodsProximal and distal IPAs were mounted in wire myographs and vascular responses to phenylephrine, acetylcholine, and 2-(N,N-diethylamino)-diazenolate-2-oxide, diethyl-ammonium salt (DEA NONOate) were measured. EFS-mediated contraction and non-adrenergic non-cholinergic (NANC) relaxation were evaluated in the absence and presence of a nitric oxide synthase antagonist. Purinergic agonist and NANC relaxation responses were assessed in the presence and absence of P2X1 and P2Y1 antagonists. Protein expression of P2X1 and P2Y1 receptors was measured by western blot. Main Outcome MeasuresProximal and distal IPA contraction and relaxation were measured during increasing agonist administration and EFS in the presence and absence of antagonists. ResultsProximal and distal IPA concentration response curves to phenylephrine, acetylcholine, and DEA NONOate did no differ. Interestingly, distal IPA exhibited greater EFS-mediated contraction and NANC relaxation compared with proximal IPA. Nitric oxide synthase inhibition completely inhibited distal IPA NANC relaxation but did not affect proximal IPA relaxation. P2X1 or P2Y1 receptor antagonism during NANC relaxation increased distal IPA relaxation but decreased proximal IPA relaxation. Combined P2X1 and P2Y1 receptor antagonism had no effect on proximal IPA relaxation but significantly increased distal IPA NANC relaxation. Clinical TranslationUnderstanding neurovascular regulation of IPA vascular tone through nitrergic and purinergic mechanisms could yield new therapeutic targets to improve IPA blood flow and treat vasculogenic erectile dysfunction. Strengths and LimitationsThis study is the first to illustrate the differences in mechanisms responsible for regulating vascular tone in the proximal and distal IPAs. All presented findings are currently limited to ex vivo vascular function. ConclusionThe regulation of vascular tone differs regionally in the IPA. The distal IPA is controlled through neurotransmitter-mediated NO-dependent mechanisms and increased sensitivity to purinergic P2X1 and P2Y1 receptor inhibition.Odom MR, Pak ES, Brown DA, Hannan JL. Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries. J Sex Med 2017;14:1285–1296.

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.

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits.

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10μmol/l and αβ-meATP of 10μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca(2+)]i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca(2+)]i by about 70%. ATP of 10μmol/l and αβ-meATP of 10μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Enhanced uridine adenosine tetraphosphate-induced contraction in renal artery from type 2 diabetic Goto-Kakizaki rats due to activated cyclooxygenase/thromboxane receptor axis

The dinucleotide uridine adenosine tetraphosphate (Up4A), which has both purine and pyrimidine moieties, was reported as a novel endothelium-derived contracting factor. Recently, growing evidence has suggested that Up4A plays an important role in regulation of the cardiovascular function. We previously demonstrated that Up4A-induced vasoconstrictions are altered in arteries from DOCA-salt hypertensive rats. We have assessed responses to Up4A shown by renal arteries from type 2 diabetic Goto-Kakizaki (GK) rats (42-46weeks old) and identified the molecular mechanisms involved. Concentration-dependent contractions to Up4A were greater in renal arterial rings from the GK than age-matched control Wistar group. In both groups, the inhibition of nitric oxide synthase (with N (G)-nitro-L-arginine) increased the response to Up4A, whereas the inhibition of cyclooxygenase (COX) (with indomethacin) decreased the response. Specific inhibitors of COX-1 (valeroyl salicylate) and COX-2 (NS398), a thromboxane (TX) receptor (TP) antagonist (SQ29548), and P2 receptor antagonist (suramin) also decreased the response to Up4A. Protein expressions of COXs in renal arteries were greater in the GK than Wistar group. The production of TXB2 (a metabolite of TXA2) by Up4A did not differ between these groups. Concentration-dependent contractions to U46619, an agonist of the TP receptor, were greater in renal arteries from the GK than Wistar group. The expression of P2X1 and P2Y2 receptors did not differ between these groups. These results suggest that enhancement of the Up4A-induced contraction in renal arteries from GK rats may be attributable to the increased activation of COXs/TP receptor signaling.

The purinergic component of human bladder smooth muscle cells’ proliferation and contraction under physiological stretch

ObjectiveTo investigate whether cyclic stretch induces proliferation and contraction of human smooth muscle cells (HBSMCs), mediated by P2X purinoceptor 1 and 2 and the signal transduction mechanisms of this process. MethodsHBSMCs were seeded on silicone membrane and stretched under varying parameters; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (Frequency: 0.05Hz, 0.1Hz, 0.2Hz, 0.5Hz, 1Hz). 5-Bromo-2-deoxyuridine assay was employed for proliferative studies. Contractility of the cells was determined using collagen gel contraction assay. After optimal physiological stretch was established; P2X1 and P2X2 were analyzed by real time polymerase chain reaction and Western Blot. Specificity of purinoceptors was maintained by employing specific inhibitors; (NF023 for P2X1, and A317491for P2X2), in some experiments. ResultsOptimum proliferation and contractility were observed at 5% and 10% equibiaxial stretching respectively, applied at a frequency of 0.1Hz; At 5% stretch, proliferation increased from 0.837±0.026 (control) to 1.462±0.023%, p<0.05. Mean contraction at 10% stretching increased from 31.7±2.3%, (control) to 78.28 ±1.45%, p< 0.05. Expression of P2X1 and P2X2 was upregulated after application of stretch. Inhibition had effects on proliferation (1.232±0.051, p<0.05 NF023) and (1.302±0.021, p<0.05 A314791) while contractility was markedly reduced (68.24±2.31, p<0.05 NF023) and (73.2±2.87, p<0.05 A314791). These findings shows that mechanical stretch can promote magnitude-dependent proliferative and contractile modulation of HBSMCs in vitro, and P2X1 and 2 are at least partially responsible in this process.

Expression of P2X1 and P2X4 receptors in rat trigeminal ganglion neurons

Extracellular ATP, an essential pain mediator, is received by cell-surface ionotropic P2X and/or metabotropic P2Y receptors. Although the contribution of P2X₃ and/or P2X(2/3) receptors toward the pain mechanism is well described in trigeminal ganglion neurons, the expression of other subtypes of P2X receptor remains to be clarified. We examined expression of P2X receptor mRNA and measured intracellular free Ca²⁺ concentration ([Ca²⁺]i) by the activation of these receptors by fura-2 fluorescence in primary cultured rat trigeminal ganglion neurons. Real-time reverse transcription-PCR analysis revealed mRNA expression of P2X receptor subtype P2X₁, P2X₃, and P2X₄ in trigeminal ganglion neurons. In the presence of extracellular Ca²⁺, the application of P2X receptors agonists, ATP, α,β-methylene ATP or β,γ-methylene ATP induced Ca²⁺ influx significantly. The ATP-induced increase in [Ca²⁺]i was inhibited by a series of selective antagonists for P2X₁, P2X₃, or P2X₄ receptors. These results indicate that trigeminal ganglion neurons functionally express P2X₁, P2X₃, and P2X₄ receptors and that these receptors are involved in the mediation of not only nociceptive but also neuropathic pain in the orofacial area.

Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells

P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 microM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by alphabetamethylene ATP (10 microM, 94% cells, n= 31) and was antagonized in LAD2 cells by NF 449 (1 microM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 microM). A P2X7-like non-desensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n= 24; 54% HLMCs, n= 24) which was antagonized by AZ11645373 (1 microM). P2X7-like responses were also evoked in LAD2 cells by 2'(3')-0-(4-benzoylbenzoyl)ATP (300 microM). A P2X4-like current was evoked by 100 microM ATP (80% LAD2, n= 10; 21% HLMCs, n= 29), the amplitude and duration of which was potentiated by ivermectin (3 microM). Our data confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in LAD2 cells and HLMCs.

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus

Expression of P2X1, P2X2, P2X3, P2X4, P2X5 and P2X6 receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X2, P2X4, P2X5 and P2X6 receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X2 and P2X6 on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.

Changes in the expression of P2X1 and P2X2 purinergic receptors in facial motoneurons after nerve lesions in rodents and correlation with motoneuron degeneration

Involvement of P2X1 and P2X2 purinergic receptors in motoneuron response to injury was investigated with Western blotting and immunohistochemistry and correlated with motoneuron loss, Bcl-2 expression, nitric oxide synthase induction and glial activation. P2X1 was highly induced in rat facial motoneurons after nerve resection, which causes slowly occurring neurodegeneration. P2X1 induction was lower and less persistent after nerve crush, permissive for fiber regeneration. P2X2 expression was found in nuclei of rat facial motoneurons, with nuclear export in the cytoplasm after nerve resection. P2X1 induction in axotomized facial motoneurons was impaired in superoxide dismutase (SOD)1-G93A-mutant mice, a model of motoneuron disease. The data in rats point to a correlation of P2X1 induction with motoneuron degeneration, which also involves P2X2 intracellular changes, rather than with axon regeneration effort. The data in mice show that the SOD1 mutation interferes with injury-elicited P2X1 induction, suggesting alterations of ATP release from mutant motoneurons after damage.

Sp1/3 and NF-1 mediate basal transcription of the human P2X1 gene in megakaryoblastic MEG-01 cells.

BackgroundP2X1 receptors play an important role in platelet function as they can induce shape change, granule centralization and are also involved in thrombus formation. As platelets have no nuclei, the level of P2X1 expression depends on transcriptional regulation in megakaryocytes, the platelet precursor cell. Since nothing is known about the molecular mechanisms regulating megakaryocytic P2X1 expression, this study aimed to identify and functionally characterize the P2X1 core promoter utilized in the human megakaryoblastic cell line MEG-01.ResultsIn order to identify cis-acting elements involved in the transcriptional regulation of P2X1 expression, the ability of 4.7 kb P2X1 upstream sequence to drive luciferase reporter gene expression was tested. Low promoter activity was detected in proliferating MEG-01 cells. This activity increased 20-fold after phorbol-12-myristate-13-acetate (PMA) induced differentiation. A transcription start site was detected 365 bp upstream of the start codon by primer extension. Deletion analysis of reporter constructs indicated a core promoter located within the region -68 to +149 bp that contained two Sp1 sites (named Sp1a and Sp1b) and an NF-1 site. Individual mutations of Sp1b or NF-1 binding sites severely reduced promoter activity whereas triple mutation of Sp1a, Sp1b and NF-1 sites completely abolished promoter activity in both untreated and PMA treated cells. Sp1/3 and NF-1 proteins were shown to bind their respective sites by EMSA and interaction of Sp1/3, NF-1 and TFIIB with the endogenous P2X1 core promoter in MEG-01 cells was demonstrated by chromatin immunoprecipitation. Alignment of P2X1 genes from human, chimp, rat, mouse and dog revealed consensus Sp1a, Sp1b and NF-1 binding sites in equivalent positions thereby demonstrating evolutionary conservation of these functionally important sites.ConclusionThis study has identified and characterized the P2X1 promoter utilized in MEG-01 cells and shown that binding of Sp1/3 and NF-1 to elements in the direct vicinity of the transcription start site is essential for basal transcription. Targeting the function of these transcription factors in megakaryocytes may therefore provide a basis for the future therapeutic manipulation of platelet P2X1 function.