Characterization of P2 receptors in thymic epithelial cells.

The presence of P2 receptors was investigated in three distinct preparations of murine thymic epithelial cells (TEC): 2BH4 murine cell line, IT45-R1 rat cell line, and a primary murine cell derived from the Nurse cell lympho-epithelial complex. In all preparations, application of ATP to the extracellular milieu triggered intracellular calcium signals indicating the presence of P2 receptor(s) in these cells. After an initial peak of calcium concentration, a plateau phase that could last more than 10 min was frequently observed. Ion replacement and channel blockage experiments indicated that the initial peak was associated with the release of calcium from intracellular stores, while the plateau phase was associated with an influx from the extracellular medium. ATP and UTP induced similar calcium signals, suggesting the presence of P2Y2 receptors in all three cell types. The murine 2BH4 cells also expressed P2X7/P2Z receptor, since under exposure to millimolar concentrations of ATP, a continuous rise in intracellular calcium concentration was observed and their plasma membranes became permeabilized to the fluorescent dyes Lucifer yellow and ethidium bromide. In addition, this permeabilization phenomenon was blocked by the P2Z-specific antagonist, oxidized ATP. RT-PCR assays confirmed the presence of mRNAs for the P2Y2 molecule in all TEC, while mRNA for the P2X7 molecule was detected only in 2BH4 cells. Our data indicate that P2Y2 purinergic receptors are widely expressed by thymic epithelial cells, whereas the expression of the P2X7 receptor appears to be more restricted, raising the possibility that its expression is related only to a particular epithelial microenvironment within/the thymus.     

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.     

P2X1 receptors and the endothelium

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, alpha, beta methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.     

Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5'-triphosphate, UTP, and UDP each increased Ca. Basolateral addition of adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), but not ATP, to the perifusing bath increased Ca. IBDU perfusion with ATP-gamma-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.     

Distribution of P2X receptors in the rat adrenal gland

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.     

P2Y receptor subtypes evoke different Ca2+ signals in cultured aortic smooth muscle cells

Adenine and uridine nucleotides evoke Ca(2+) signals via four subtypes of P2Y receptor in cultured aortic smooth muscle cells, but the mechanisms underlying the different patterns of these Ca(2+) signals are unresolved. Cytosolic Ca(2+) signals were recorded from single cells and populations of cultured rat aortic smooth muscle cells, loaded with a fluorescent Ca(2+) indicator and stimulated with agonists that allow subtype-selective activation of P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Activation of P2Y1, P2Y2, and P2Y6 receptors caused homologous desensitisation, while activation of P2Y2 receptors also caused heterologous desensitisation of the other subtypes. The Ca(2+) signals evoked by each P2Y receptor subtype required activation of phospholipase C and release of Ca(2+) from intracellular stores via inositol 1,4,5-trisphosphate (IP(3)) receptors, but they were unaffected by inhibition of ryanodine or nicotinic acid adenine dinucleotide phosphate (NAADP) receptors. Sustained Ca(2+) signals were independent of the Na(+)/Ca(2+) exchanger and were probably mediated by store-operated Ca(2+) entry. Analyses of single cells established that most cells express P2Y2 receptors and at least two other P2Y receptor subtypes. We conclude that four P2Y receptor subtypes evoke Ca(2+) signals in cultured aortic smooth muscle cells using the same intracellular (IP(3) receptors) and Ca(2+) entry pathways (store-operated Ca(2+) entry). Different rates of homologous desensitisation and different levels of receptor expression account for the different patterns of Ca(2+) signal evoked by each P2Y receptor subtype.     

P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation

Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X1, P2X3, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors. Protein expression for P2Y1 and P2Y2 receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X4 > P2X7 > P2X1 > P2X3 > P2X6 > P2X5 > P2X2. The functional presence of P2X7, P2Y1, P2Y2, and P2Y4 receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X7 and P2Y1 receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y2 receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.     

Localization of P2X and P2Y receptors in dorsal root ganglia of the cat.

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X(5) receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y(2) = P2Y(4)>P2X(3)>P2X(2) = P2X(7)>P2X(6)>P2X(1) = P2X(4)>P2Y(1). P2X(3), P2Y(2), and P2Y(4) receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y(2), P2X(1), P2X(3), P2X(4), and P2X(6) receptor staining was detected in small- and medium-diameter neurons. However, P2Y(4), P2X(2), and P2X(7) staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X(3) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y(4) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y(2) receptor-positive neurons also stained for IB(4), CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X(3)-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y(1) receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.     

P2 nucleotide receptors on C2C12 satellite cells

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12) family members while among P2X receptors P2X(4), P2X(5) and P2X(7) were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca(2+) ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X(5) play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y(2)-dependent Ca(2+) signaling, while the mRNA expression of P2Y(2) receptor reminds still on the high level. Moreover, P2X(2) receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.     

P2 purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production

Extracellular nucleotides elicit multiple responses in eosinophils but no information on expression of purinergic receptors in these cells is available so far. In the present study we show that human eosinophils express the following P2Y and P2X subtypes: P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), and P2X(1), P2X(4), P2X(7), whose stimulation results in intracellular Ca(2+) increase and production of large amounts of reactive oxygen intermediates. These events are stimulated or inhibited, respectively, by P2 receptor agonists or antagonists.     

Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing

In view of the evidence for a role for extracellular ATP in both pancreatic endocrine and exocrine functions, we have investigated the expression of P2X and P2Y receptors in this tissue in neonate and aged rat and mouse. Using immunohistochemistry it was shown that P2X(1), P2X(4), P2X(7), P2Y(1) and P2Y(2) receptors were present in different regions of the rat and mouse pancreas; P2X(3) and P2X(6) receptors were not found, and P2X(5) immunolabelling was only found in some nerves. The pancreatic vasculature of both rat and mouse expressed P2X(1), P2X(2), P2Y(1) and P2Y(2) receptors in the smooth muscle. P2X(1) and P2X(4) receptors were absent in the islets of the neonate pancreas, but were progressively upregulated with age after birth. In contrast, the greatest expression of P2Y(1) in cells from the duct system was in neonate pancreas, while there was no P2Y(1) expression in aged rat pancreas. P2X(7) receptors had a consistent pattern of distribution in all of the groups examined, being located in the outer periphery of the islet. Using antibodies raised against insulin, somatostatin and glucagon, double-labelling immunofluorescence was used to identify P2X(7)-positive cells in different islet of Langerhans cell populations. Our results demonstrated a clear immunoreaction to P2X(7) receptors in islet alpha cells, while no P2X(7) was expressed in beta and delta cells. The significance of the differential expression of P2 receptors in the pancreas during development and ageing, and a possible role for the proliferation and death of the islet cell population are discussed.     

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.     

P2Y1, P2Y6, and P2Y12 receptors in rat splenic sinus endothelial cells: an immunohistochemical and ultrastructural study

Localization of three P2X and six P2Y receptors in sinus endothelial cells of the rat spleen was examined by immunofluorescent microscopy, and ultrastructural localization of the detected receptors was examined by immunogold electron microscopy. In immunofluorescent microscopy, labeling for anti-P2Y1, P2Y6, and P2Y12 receptors was detected in endothelial cells, but P2X1, P2X2, P2X4, P2Y2, P2Y4, and P2Y13 receptors was not detected. P2Y1 and P2Y12 receptors were prominently localized in the basal parts of endothelial cells. P2Y6 receptor was not only predominantly localized in the basal parts of endothelial cells, but also in the superficial layer. Triple immunofluorescent staining for a combination of two P2Y receptors and actin filaments showed that P2Y1, P2Y6, and P2Y12 receptors were individually localized in endothelial cells. Phospholipase C-β3, phospholipase C- γ2, and inositol-1,4,5-trisphosphate receptors, related to the release of the intracellular Ca²⁺ from the endoplasmic reticulum, were also predominantly localized in the basal parts of endothelial cells. In immunogold electron microscopy, labeling for P2Y1, P2Y6, and P2Y12 receptors were predominantly localized in the basal part of endothelial cells and, in addition, in the junctional membrane, basal plasma membrane, and caveolae in the basal part of endothelial cells. Labeling for phospholipase C-β3 and phospholipase C-γ2 was dominantly localized in the basal parts and in close proximity to the plasma membranes of endothelial cells. The possible functional roles of these P2Y receptors in splenic sinus endothelial cells are discussed.     

克隆的P2受体亚型的药理学研究进展

细胞外嘌呤（腺苷,ADP,ATP）及嘧啶（UDP,UTP）为重要的信使分子,通过细胞表面P2受体介导产生不同的生物效应,P2嘌吟受体的概念于1978年被提出,随后根据药理学特征又被分为P2X及P2X嘌呤受体,90年代,采用分子生物学手段,一系列配体门控的P2X受体及G蛋白耦联的P2Y受体被克隆及功能表达,迄今为止,已有七型P2X受体亚型（P2X1－7）及六型P2Y受体亚型被克隆（P2Y1,2,4,6,11,12）,各型具有不同的分子结构,药理学特征及组织分布,本文还讨论了目前可用于区分各亚型激动剂及拮抗剂。     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.     

Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells

In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.