Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.     

Regulatory mechanism of eosinophil peroxidase release from guinea pig eosinophils

The regulatory mechanism of degranulation of guinea pig peritoneal eosinophils was studied by determination of eosinophil peroxidase (EPO) release. Beta-agonists, such as isoproterenol, salbutamol and fenoterol, effectively inhibited A23187-induced EPO release from guinea pig eosinophils. The inhibitory effects of beta-agonists were attenuated by pretreatment with either propranolol, a non-selective beta-antagonist, or ICI 118,551, a selective beta2-antagonist. Both theophylline and dibutyryl-cAMP (db-cAMP) also significantly inhibited A23187-induced EPO release. The inhibition of EPO release induced by db-cAMP was attenuated by pretreatment with KT5720, a protein kinase A inhibitor. In addition, calphostin C as well as cytochalasin D effectively inhibited A23187-induced EPO release. From the results of the present study, it was concluded that an increase in intracellular Ca2+ concentration may lead to exocytosis of eosinophil granules through activation of protein kinase C and microfilaments. Beta-agonists and theophylline were effective in inhibiting degranulation of eosinophils by increasing intracellular cAMP level coupled with the activation of protein kinase A.     

P2Y2- and P2Y4 purinergic receptors are over-expressed in human colon cancer

1. A characteristic of cancer is altered signal transduction leading to uninhibited growth. Adenosine-5'-triphosphate (ATP), a natural ligand at P2X- and P2Y purinergic receptors may regulate cell growth in non-neoplastic, as well as neoplastic tissues. In the human colon cancer cell line, HT-29, we previously demonstrated the expression of purinergic receptors of the P2Y(2)- and P2Y(4) subclasses. 2. The aim of the current study was to investigate whether these two purinergic receptors are expressed also in human colon cancer, and, if so, how such expression is related to that in tumour-free colonic tissue. 3. The immunohistochemical findings of both P2Y(2)- and P2Y(4) receptors in the tumours from three patients, prompted us to conduct an investigation of a consecutive series of patients utilizing Western blotting for protein detection and densitometry for quantitation. 4. Both P2Y(2)- and P2Y(4) purinergic receptors could be identified in tumour-free tissue, and both were significantly over-expressed in each of the 10 colon cancers.     

Interaction of P2 purinergic receptors with cellular macromolecules

Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via G_s, G_q/11 or G_i/o protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Gα-dependent signalling with other G protein-coupled receptors and via Gβγ (or indirectly Gα)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects.     

Substance P induces tumor necrosis factor-alpha release from human skin via mitogen-activated protein kinase

Substance P plays an important role in neurogenic inflammation with granulocyte infiltration. To investigate cytokines involved in the substance P-induced inflammation and the mechanism of cell activation, we studied the release of TNF (tumor necrosis factor)-alpha and histamine from human skin slices in response to substance P and antigen. Substance P induced the release of histamine and TNF-alpha in a dose-dependent manner at concentrations from 0.8 to 100 microM. PD 098059 (2'-amino-3'-methoxyflavone) selectively inhibited the release of TNF-alpha, but not the release of histamine induced by either substance P or antigen. SB 203580 ([4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-++ +imida zole]) slightly inhibited TNF-alpha release induced by antigen, but not that induced by substance P, and slightly enhanced histamine release induced by either stimulation. The release of TNF-alpha in response to either stimulation was inhibited by 1 nM-1 microM dexamethasone, but histamine release was not affected. These results suggest that substance P, in addition to antigen, induced TNF-alpha release from human skin by a mitogen-activated protein (MAP) kinase, predominantly extracellular signaling-regulated protein kinase (ERK)-dependent, and dexamethasone-sensitive pathway, which is separate from that for histamine release from mast cells.     

Stimulation of rat erythrocyte P2X7 receptor induces the release of epoxyeicosatrienoic acids

BACKGROUND AND PURPOSE: Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation. EXPERIMENTAL APPROACH: Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry. KEY RESULTS: EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs. CONCLUSIONS AND IMPLICATIONS: EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR.     

The roles of P2 purinergic receptors in nociception and antinociception

Extracellular adenosine 5‘-triphosphate (ATP) has been established as a neurotransmitter or neuromodulator in both the periphe- ral and central nervous systems,in addition to diverse intracellular roles of it.P2 purinergic receptors,the receptors of ATP,are classified into two subfamilites,ionotropic P2X and metabotropic P2Y receptors.Recent studies suggest that ATP play a significant role in facilitating perpheral and spinal nociceptive transmission via P2X receptors.However,we demonstrated that at the supraspinal level P2X receptor agonists produce an antinociception.On the other hand,the activation of some subtypes of P2Y receptors in the spinal cord caused inhibitory effects on nociceptive transmission.Thus,P2X and P2Y receptors are suggested to be related to diverse roles in nociceptive functions at peripheral,spinal and supraspinal levels.We would like to take an overview about the significance of P2X and P2Y receptors in nociception and antinociception.     

P2 purinergic receptors for diadenosine polyphosphates in the nervous system

• 1.1. The actions of diadenosine polyphosphates, diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A) and diadenosine hexaphosphate (Ap₆A) in the nervous system have been reviewed.
• 2.2. In the peripheral nervous system, diadenosine polyphosphates bind to P_2Y-purinergic receptors such as the P_2Y in chromaffin cells and Torpedo synaptosomes, P_2X in vas deferens and urinary bladder and also Torpedo synaptosomes and P_2U in endothelial chromaffin cells.
• 3.3. In the central nervous system Ap_nA compounds can act through P_2X-purinoceptors opening cation channels in nodose ganglion neurones. Diadenosine polyphosphates bind to a P_2d-purinergic receptor in rat brain synaptic terminals and hippocampus, linked to protein kinase C (PKC) activation.
• 4.4. P₄-purinoceptors are specific receptors for diadenosine polyphosphates, coupled to the Ca²⁺ influx, in the central synapses. This purinoceptor is not activated by ATP and synthetic analogs. The P₄-purinoceptor could act as a positive modulator of the synaptic transmission, giving even more importance to diadenosine polyphosphates as neurotransmitters.

     

Demonstration of P2Y4 purinergic receptors in the HT-29 human colon cancer cell line

1 The aim of the current study was to investigate the existence of P 2 Y(4) purinergic receptors in the HT-29 human colon cancer cell line. 2 We utilized Western blots and immunocytochemistry for the analysis. 3 Western blotting demonstrated two bands that could not be found after the antibody had been preabsorbed with the control peptide, suggesting that both bands are related to the P 2 Y(4) purinergic receptor. 4 Immunocytochemistry showed immunoreactivity for the P 2 Y(4) purinergic receptor localized in the cytoplasm of the HT-29 cells. 5 This is the first demonstration of the protein expression of P 2 Y(4) purinergic receptors in a human colon cancer cell line.     

ATP release mechanisms, ATP receptors and purinergic signalling along the nephron

1. Autocrine and paracrine signalling along the nephron of the kidney has been a widely held hypothesis for several decades. The lumen of the nephron is an ideal autocrine and paracrine signalling microenvironment. Any agonist, filtered at the glomerulus or released in the proximal tubule or other proximal segments, is subsequently trapped in the lumen of the nephron and present to interact with luminal receptors. Similar signalling in the renal interstitium is also possible and likely. Indeed, receptors for many autocrine and paracrine agonists have been characterized on the luminal membrane and serosal membrane of multiple nephron segments. 2. An important family of autocrine and paracrine agonists in the kidney are the purinergic agonists. Extracellular ATP, as well as its metabolites (ADP, 5'-AMP and adenosine), is released by renal epithelial cells. These compounds are also freely filtered at the glomerulus and are found in the final urine. Receptors for ATP and adenosine are also expressed on the luminal and serosal side of many nephron segments. 3. The present review discusses purinergic signalling by nucleotide agonists in an integrated manner, from ATP release to ATP receptors to extracellular ATP-mediated effects on renal epithelial function. These themes are the focus of our laboratory in normal and polycystic kidneys as well as in normal and diseased epithelial cells from other tissues. The physiological roles of extracellular purinergic signalling in the kidney and other tissues are only beginning to emerge.     

嗜酸性粒细胞阳离子蛋白mRNA在哮喘中的表达及意义

目的探讨嗜酸性粒细胞阳离子蛋白（eosinophil cationic protein，EcP）mRNA在哮喘中的表达及意义。方法采用逆转录聚合酶链反应技术（RT—VCR），对40例哮喘患者及20例正常对照者的ECPmR—NA表达水平进行半定量分析。同时，研究了外周血ECPmRNA表达水平与患者嗜酸性粒细胞数量、肺功能的关系。结果（1）哮喘患者组的嗜酸性粒细胞计数（0．86±0．52）和ECPmRNA表达水平（0．37±0．11）均远高于正常对照组[（0．21±0．10），P〈0．001和（0．17±0．04），P〈0．001]；（2）在哮喘患者中，中度持续哮喘组ECPmRNA表达水平（0．42±0．05）和重度持续哮喘组（0．47±0．05），远高于轻度持续哮喘组ECPmRNA表达水平[（0．25±0．06），P〈0．001]。同时，在中度和重度持续哮喘组间，ECPmRNA含量也存在显著差异（P〈0．05）。结论哮喘患者中ECPmRNA表达水平的增高可能与哮喘病情的严重程度密切相关。ECP在哮喘疾病的发展过程中起着非常重要的作用。     

The cytotoxicity of jet fuel aromatic hydrocarbons and dose-related interleukin-8 release from human epidermal keratinocytes

Many jet fuel aromatic hydrocarbons are known carcinogens with the ability to both readily penetrate the skin with high absorptive flux and cause skin irritation. In order to evaluate the in vitro cutaneous toxicity of individual aromatic hydrocarbons in jet fuels and their potential for inducing skin irritation, we evaluated the LD(50), the highest non-cytotoxic (5% mortality) dose (HNTD), and interleukin-8 (IL-8) release activity of nine major jet fuel aromatic hydrocarbons in human epidermal keratinocytes (HEK). LD(50) ranged from 1.8 mM (0.03%) for cyclohexylbenzene to 82.9 mM (0.74%) for benzene, with a rank order potency of cyclohexylbenzene >trimethylbenzene >/=xylene >dimethylnaphthalene >ethylbenzene >toluene >benzene. The HNTD values ranged from 0.1 mM (0.001%) for cyclohexylbenzene to 48.2 mM (0.43%) for benzene. Naphthalene and methylnaphthalene could not be ranked in this comparison since their concentrations, presented as percentage saturation, were not comparable to the others presented as solutes in solution. There was a dose-related differential response in IL-8 release at 24 h. Toluene, xylene, trimethylbenzene, cyclohexylbenzene and dimethylnaphthalene significantly decreased IL-8 release at the respective HNTDs, while IL-8 release did not continue to decrease, or significantly increased (cyclohexylbenzene and dimethylnaphthalene), at the LD(50). IL-8 significantly increased with both doses of methylnaphthalene and naphthalene. The presence of hexadecane and mineral oil greatly attenuated the cytotoxicity elicited by individual aromatic hydrocarbons in HEK cells.     

Pharmacological modulation of interleukin-17-induced GCP-2-, GRO-alpha- and interleukin-8 release in human bronchial epithelial cells

The cytokine interleukin-17 may play a role in the recruitment of airway neutrophils, and interleukin-17 protein is increased in the airways of patients with asthma. In this study, we characterised the effect of interleukin-17 on the release of the neutrophil-recruiting cytokines granulocyte chemotactic protein (GCP)-2, growth-related oncogene (GRO)-alpha and interleukin-8 in human bronchial epithelial (HBE) cells. We also characterised the involvement of mitogen-activated protein (MAP) kinases as well as the effect of beta-adrenoceptor and glucocorticoid receptor stimulation and calcineurin and P-glycoprotein inhibition on these epithelial responses to interleukin-17. We found that interleukin-17 (1-1000 ng/ml) increased the release of GCP-2, GRO-alpha and interleukin-8 in a concentration-dependent manner. This interleukin-17-induced release of C-X-C chemokines was sensitive to inhibition of the p38 MAP kinase pathway and to stimulation of glucocorticoid receptors. In contrast, stimulation of beta-adrenoceptors increased the release of interleukin-8 and did not markedly alter the release of GCP-2 and GRO-alpha. Inhibition of calcineurin and of P-glycoproteins did not exert any substantial effect on the release of C-X-C chemokines. In conclusion, interleukin-17 bears the potential to increase neutrophil recruitment into the airways by releasing several, different C-X-C chemokines, including GCP-2, GRO-alpha and interleukin-8 in human bronchial epithelial cells. Inhibition of the p38 MAP kinase pathway and glucocorticoid receptor stimulation constitute two credible therapeutic strategies against this interleukin-17-induced release of neutrophil-recruiting cytokines.     

Adenosine A3 receptors on human eosinophils mediate inhibition of degranulation and superoxide anion release.

The role of adenosine A3 receptors on human eosinophil degranulation and superoxide anion (O2-) release was studied in vitro using the complement fragment C5a as the main stimulus and employing a number of selective agonists and antagonists. In the presence of cytochalasin B (CB), C5a induced a dose-dependent release of the granular eosinophil peroxidase (EPO), but not O2-, whereas in the absence of CB O2- , but not EPO, was released. C5a-induced EPO release was inhibited dose-dependently by the selective A3 agonist N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N6-2-(4-amino-3-iodophenyl) ethyladenosine (APNEA). The IC50 (95% CI) for IB-MECA was 6.8 microM (3.1-12.0 microM). At concentrations up to 100 microM, neither adenosine nor the selective A1 agonist N-cyclopentyladenosine (CPA) and the selective A2 agonist 2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamidoadenosine (CGS 21680) had any significant effect. The inhibitory effect of IB-MECA was almost completely abolished by pre-treatment with 1 microM of the selective A3 antagonist 9-chloro-2-(2-furyl)-5-phenylactylamino[1,2,4]triazolo[1,5-c]quina zoline (MRS 1220), but not the selective A1 antagonist 1,3-dipropyly-8-cyclopentylxanthine (DPCPX) or the selective A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). IB-MECA also significantly inhibited C5a-induced O2- release with IC50 (95% CI) of 9.5 microM (4.6-13.1 microM) whereas adenosine and the A1 agonist CPA potentiated this effect at low concentrations. The potentiation appeared to be a result of their direct O2- release from these cells, probably mediated via A1 receptors. The inhibition by IB-MECA was selectively reversed by MRS 1220. These results show that the A3 receptors on human eosinophils mediate inhibition of both degranulation and O2- release and suggest a therapeutic potential for A3 agonists in diseases such as asthma in which activated eosinophils are involved.     

Roles of ectodomain and transmembrane regions in ethanol and agonist action in purinergic P2X2 and P2X3 receptors

The present work investigated sites of ethanol action in ATP-gated P2X receptors (P2XRs) using chimeric strategies that exploited the differences in ethanol response between P2X2R (inhibition) and P2X3R (potentiation). We tested ethanol (10–200 mM) effects on ATP- and α,β-methylene-ATP (α,β-meATP)-induced currents in wildtype P2X2, P2X3 and chimeric P2X2/P2X3Rs expressed in Xenopus oocytes using two-electrode voltage-clamp (−70 mV). Exchanging ectodomain regions of P2X2 and P2X3Rs reversed wildtype ethanol responses. Substituting back portions of the P2X2R ectodomain at TM interfaces in chimeras that contained the P2X3R ectodomain restored wildtype P2X2R-like ethanol response. Point mutations that replaced non-conserved ectodomain residues at TM interfaces of P2X3Rs with homologous P2X2R residues identified positions that reversed the direction (304) or changed the magnitude (53, 55 and 313) of ethanol response. Homologous substitutions in P2X2Rs did not significantly alter wildtype P2X2R-like ethanol responses. These findings suggest that ectodomain segments at TM interfaces play key roles in determining qualitative and quantitative responses to ethanol of P2X2 and P2X3Rs. Studies that substituted TM regions of P2X3R with respective P2X2R TMs indicate that the TM1, but not the TM2, region plays a role in determining the magnitude of ethanol response. Studies with ATP and α,β-meATP support prior indications that TM regions are important in agonist desensitization and suggest that both ectodomain and TM regions play roles in determining agonist potency and selectivity. Overall, these findings are the first to identify potential targets for ethanol in P2X2 and P2X3Rs and should provide insight into the sites of ethanol action in other P2XRs.     

Altered expression of P2Y2 and P2X7 purinergic receptors in the isolated rat heart mediates ischemia–reperfusion injury

The aim of this study is to analyze the expression of purinergic receptors in the heart after ischemia–reperfusion, and their possible role in ischemia–reperfusion injury. Rat hearts were perfused according to the Langendorff technique and subjected to 30 min ischemia followed by 15 min reperfusion. Ischemia–reperfusion reduced the gene expression and protein content of purinergic receptors of the P2Y2 subtype, and increased the gene expression and protein content of the P2X7 subtype. Treatment with the agonist of the P2Y2 subtype 2-thio-UTP and with the antagonist of the P2X7 subtype Brilliant Blue improved myocardial function parameters, reduced cell death and increased the myocardial expression of antiapoptotic markers after ischemia–reperfusion. These results suggest that the myocardial expression of the protective P2Y2 subtype of purinergic receptors is reduced, whereas that of the harmful subtype P2X7 subtype is increased during coronary ischemia–reperfusion. This may contribute to myocardial injury in this condition.     

Efficacy of purine nucleotides on purinergic P2 platelet receptors by small angle light scattering

The effect of purines on the activation and aggregation of thrombocytes in rats and rabbits was studied by the method of small-angle light scattering. The EC50 values of ADP, inducing the activation and aggregation of thrombocytes, reflect the sequence of the agonist action on various receptors: P2X1, 20-40 nM; P2Y1, 90-110 nM; P2YADP, 120-240 nM. It was demonstrated that ADP behaves as partial agonist not only with respect to P2X1 receptors, but with respect to P2Y1 receptors as well. Thrombocytes activated by 20 nM ADP or 100-nM ATP pass into a refracter state in the absence of further stimulation. The reaction halftime is tau 1/2 = 6.0 +/- 0.2 min for the cells activated with ADP and tau 1/2 = 16.5 +/- 0.2 min for ADP.