P2X receptor immunoreactivity in the male genital organs of the rat

The distribution of ATP ionotropic P2X receptors in the genital organs of the male rat has been investigated with immunohistochemical techniques using specific antibodies to P2X1-7 receptors. In the excretory ducts of the testis (ductus epididymidis, vas deferens and its associated seminal vesicles), the major signals were seen with antibodies to P2X1 and P2X2 in the membranes of the smooth muscle layer, suggesting that these receptors are involved in the process of sperm transport and ejaculation. In the penis body, strong P2X1 and weaker P2X2 immunoreactivity was seen in the smooth muscle of blood vessels and the corpus cavernosum, suggesting a participation in the detumescence process. P2X5 immunoreactivity, a marker for differentiating cells in stratified squamous epithelia, was observed in the epithelia of the terminal urethra, the "horny spur" (spine-studded epithelium of the glans) and the inner surface of the prepuce. Antibodies to P2X3 reacted with nerve fibres in the adventitia of vas deferens, and the P2X6 receptor was localised in the basal lamina of the epithelium. In the prostate, there was immunostaining of the smooth muscle between the tubules with antibody for P2X1, but not with P2X2; P2X3 immunostaining of nerves and strong P2X7 immunostaining of the glandular epithelium of the prostate were also present.     

Distribution of P2X receptor subtypes in the rat female reproductive tract at late pro-oestrus/early oestrus

Using immunohistochemistry techniques, we have examined the female reproductive organs of the rat in late pro-oestrus/early oestrus for the presence of purine nucleotide P2X1-7 receptors. In contrast to the male genital organs and the urinary tract, P2X1 receptors were present weakly, if at all, on smooth muscle membranes, except in blood vessels, whereas P2X2 immunoreactivity in smooth muscle was present in ovary and uterus as well as in blood vessels. Neither P2X1 nor P2X2 receptors were present in fallopian tubes. P2X5 receptors were seen in the differentiating cell layers of the stratified squamous vaginal epithelium and also in the very early stages of ovarian follicular development; P2X6 receptors were present in secondary follicles. P2X7 receptors, markers for programmed cell death, were present in the keratinised vaginal epithelium and also in the exfoliating superficial endometrial cells. The possible biological significance of these signalling molecules in the female reproductive tract is discussed.     

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.     

Detection of P2X purinergic receptors on human B lymphocytes

B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family; however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.     

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.     

P2 receptors in the thymus: expression of P2X and P2Y receptors in adult rats, an immunohistochemical and in situ hybridisation study

The expression of the seven P2X receptor subtypes and of two P2Y receptors was examined immunohistochemically and by in situ hybridisation in thymi of adult male rats. P2X4, P2Y2 and 4 receptor mRNA colocalisation studies combining in situ hybridisation and immunohistochemistry were also carried out. P2X and P2Y receptors were found on thymocytes. P2X receptors were also abundant in cells of the thymic microenvironment, involved in control of T-cell maturation in vivo. We are the first to describe the expression of P2X4 receptors on thymocytes and confirm the finding of P2X1 and P2Y2 receptors on subpopulations of lymphocytes. P2X1,2,3,4 and 5 receptors were present in blood vessels of the thymus. P2X1,2 and 4 receptors were detected in vascular smooth muscle, while P2X3 receptors appeared to be associated with endothelial cells; some small arteries were positive for P2X5, possibly labelling vascular smooth muscle or fibroblasts in the adventitia. P2X2,3,6 and 7 receptors were found on thymic epithelial cells. P2X2 and 3 receptors were abundant on medullary epithelial cells, whilst P2X6 receptors were prominent in Hassall's corpuscles. P2X2 receptors were found on subcapsular and perivascular epithelial cells. P2X2,6 and 7 receptors were detected in epithelial cells along the thymic septa. Expression of P2X receptors was also investigated by Western blotting of crude thymic tissue extracts under reducing conditions. All seven P2X receptor subtypes were found to be dimers of approximately 70 kDa and 140 kDa molecular weight. ATP-mediated apoptosis and cell proliferation of thymocytes are discussed.     

P2X receptors in the rat duodenal villus

Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the rat using specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Of the antibodies to the seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus, which did not extend to the crypt region, suggesting that nucleotides may be involved in the uptake and transport of metabolites. Anti-P2X5 immunostained the membranes of the narrow "stem" of villus goblet cells, where the nucleus and cell organelles reside, possibly influencing synthesis and release of mucins. P2X7 receptor immunoreactivity was only seen in the membranes of enterocytes and goblet cells at the tip of the villus, where cells are exfoliated into the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.     

Distribution of immunoreactivity for P2X3, P2X5, and P2X6-purinoceptors in mouse retina

Previous findings have shown that P2X-purinoceptor-mediated signaling pathways regulate the release of ACh in the retina. We previously reported the existence of immunoreactivity for P2X1-, P2X2-, P2X4-, and P2X7-purinoceptors in mouse retina and speculated that P2X2 and P2X7-purinoceptors may modulate the activity of cholinergic amacrine cells. In the present study, we used an immunohistochemical technique to examine whether P2X3-, P2X5, and P2X6-purinoceptors are also important for the modulation of cholinergic amacrine cells in mouse retina. Immunoreactivity for P2X3-, P2X5-, and P2X6-purinoceptors was observed in mouse retina. Immunoreactivity for P2X3- purinoceptors was observed in the dendrites of cholinergic amacrine cells. Immunoreactivity for P2X5-purinoceptors existed in the soma of cholinergic amacrine cells. P2X6-purinoceptor immunoreactivity was not colocalized with the cholinergic amacrine cells. We concluded that, among the three P2X-purinoceptors that were examined, P2X3-purinoceptors seem to affect the function of cholinergic amacrine cells in the mouse retina.     

Co-localization of Pirt protein and P2X2 receptors in the mouse enteric nervous system

P2X2 receptors, with other P2X receptor subtypes, have an important role mediating synaptic transmission in regulating the functions of the gastrointestinal tract. Our recent work has found a new regulator of P2X receptor function, called phosphoinositide-interacting regulator of transient receptor potential channels (Pirt). In the present work, we have shown that Pirt immunoreactivity was localized in nerve cell bodies and nerve fibers in the myenteric plexus of the stomach, ileum, proximal, and distal colon and in the submucosal plexus of the jejunum, ileum, proximal, and distal colon. Almost all the Pirt-immunoreactive (ir) neurons were also P2X2-ir, and co-immunoprecipitation experiments have shown that Pirt co-precipitated with the anti-P2X2 antibody. This work provides detailed information about the expression of Pirt in the gut and its co-localization with P2X2, indicating its potential role in influencing P2X2 receptor function.     

Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Glial cells,but not interstitial cells,express P2X7, an ionotropic purinergic receptor,in rat gastrointestinal musculature

Purinergic (ATP) neurotransmission is a component of the inhibitory response of the musculature in various regions of the gastrointestinal tract. So far, seven ionotropic purinergic receptors (P2X1-7) have been cloned. As specific antibodies become available, their respective distribution in the gastrointestinal tract can be elucidated. Here, we used high-resolution tricolor confocal microscopy, to study the distribution of P2X7-immunoreactive (-ir) cells in the muscularis propria of the rat stomach, small intestine, and colon. Smooth muscle cells, KIT-ir interstitial cells of Cajal, and CD34/SK3-ir fibroblastlike cells were P2X7-negative, whereas P2X7 immunoreactivity was observed in nerves and S100-ir glial cells. In all regions studied, P2X7 immunoreactivity was also observed in myenteric and submucosal ganglia, where perineuronal nerve endings appeared brightly labeled. Our observations suggest that purinergic signaling could influence the enteric glia through P2X7 receptors.     

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.     

Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells

The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin-1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

Activation of P2X7R and downstream effects in bleomycin treated lung epithelial cells

Changes in intracellular calcium concentration [Ca(2+)](i) are believed to influence the proliferation and differentiation of airway epithelial cells both in vivo and in vitro. In the present study, using mouse alveolar epithelial E10 cells, we demonstrated that the treatment of lung epithelial cells with BLM resulted in elevated intracellular Ca(2+) levels. BLM further increased P2rx7 mRNA expression and P2X7R protein levels, paralleled by increased PKC-β1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-β1 from the cytoplasm to the membrane. The expression of PKC-β1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-β1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-β1 in Cav-1 containing lipid raft fractions. The comparison of lung tissues from wild-type and P2rx7(-/-) mice revealed decreased protein and mRNA levels of PKC-β1 and CaM as well as decreased immunoreactivity for PKC-β1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-β1. These data suggest that the effect of P2X7R on expression of PKC-β1 detected in alveolar epithelial cells is also functioning in the animal model. Immunohistochemical evaluation of fibrotic lungs derived from a BLM-induced mouse model revealed a strong increase in PKC-β1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-β1. We predict that increased Ca(2+) concentration stimulates PKC-β1, whereas the prerequisite for activating PKC-β1 after P2X7R increase remained to be determined. Our findings suggest that PKC-β1 is important in the pathogenesis of pulmonary fibrosis.     

A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.     

The P2X1 receptor and platelet function

Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca²⁺, leading to shape change, movement of secretory granules and low levels of α_IIbβ₃ integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques.     

Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X_1,2,4,7 and P2Y_1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X₇ receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X₇ receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X₇ receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X₇-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.