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-ß3, phospholipase C- γ2, and inositol-1,4,5-trisphosphate receptors, related to the release of the intracellular Ca(2+) 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-ß3 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.

Localization and possible function of P2Y(4) receptors in the rodent retina.

Extracellular ATP acts as a neurotransmitter in the retina, via the activation of ionotropic P2X receptors and metabotropic P2Y receptors. The expression of various P2X and P2Y receptor subtypes has been demonstrated in the retina, but the localization of P2Y receptors and their role in retinal signaling remains ill defined. In this study, we were interested in determining the localization of the P2Y(4) receptor subtype in the rat retina, and using the electroretinogram (ERG) to assess whether activation of these receptors modulated visual transmission. Using light and electron microscopy, we demonstrated that P2Y(4) receptors were expressed pre-synaptically in rod bipolar cells and in processes postsynaptic to cone bipolar cells. Furthermore, we show that the expression of P2Y(4) receptors on rod bipolar cell axon terminals is reduced following dark adaptation, suggesting receptor expression may be dependent on retinal activity. Finally, using the electroretinogram, we show that intravitreal injection of uridine triphosphate, a P2Y receptor agonist, decreases the amplitude of the rod PII, supporting a role for P2Y receptors in altering inner retinal function. Taken together, these results suggest a role for P2Y(4) receptors in the modulation of inner retinal signaling.

Automated analysis of the architecture of receptors, imaged by atomic force microscopy.

Fast neurotransmission involves the operation of ionotropic receptors, which are multi-subunit proteins that respond to activation by opening an integral ion channel. Examples of such channels include the GABA(A) receptor, the 5-HT(3) receptor and the P2X receptor for ATP. These receptors contain more than one type of subunit, although the exact subunit stoichiometry and arrangement around the receptor rosette is often unknown. We are using atomic force microscopy (AFM) of purified receptors to address these issues. Measurement of the molecular volume of the receptor permits the determination of the number of subunits that it contains. Furthermore, analysis of the geometry of complexes between receptors and subunit-specific antibodies reveals the subunit arrangement. Our AFM-based approach has so far been dependent on manual data processing, which is both time-consuming and prone to operator bias. In this study, we set out to develop a novel method capable of automatic segmentation and quantitative analysis of both single receptor particles and receptor-antibody complexes. The method was validated using images of wild type and mutant forms of the P2X(6) receptor. We suggest that the automated method will greatly facilitate further progress in the use of AFM for the determination of receptor and multi-protein architecture.

Electron microscopy and in situ hybridization: Expression of P2Y2 receptor mRNA in the cerebellum.

This chapter describes a pre-embedding in situ hybridization method utilizing an immunogold-silver intensification step to identify P2Y2 receptor mRNA transcripts in the adult rat cerebellum. The method was applied for ultrastructural (electron microscopic) examination. Transcripts for P2Y2 receptors, marked by the gold-silver grains, were revealed in Purkinje cells. Transcripts were essentially localized in the cytoplasm although they also appeared to be specifically associated with granular endoplasmic reticulum. This suggests that Purkinje cells may produce functional P2Y2 receptors involved in the ATP-related regulatory role in the cerebellum. The in situ hybridization method that was applied enabled simultaneous preservation of tissue ultrastructure and localization of labeled mRNA transcripts. This method may be adapted and used to study various tissues.

P2X2 receptors on ganglion and amacrine cells in cone pathways of the rat retina.

Extracellular ATP is known to mediate fast, excitatory neurotransmission through activation of ionotropic P2X receptors. In this study, the localization of the P2X(2) receptor (P2X(2)R) subunit was studied in rat retina by using immunofluorescence immunohistochemistry and preembedding immunoelectron microscopy. The P2X(2)R was observed in large ganglion cells as well as in a subset of amacrine cells. Double labeling revealed that 96% of all P2X(2)R-immunoreactive amacrine cells showed gamma-aminobutyric acid (GABA) immunoreactivity. Subsets of P2X(2)R-immunoreactive amacrine cells expressed nitric oxide synthase and substance P; however, no colocalization was observed with choline acetyltransferase, vasoactive intestinal peptide, or tyrosine hydroxylase. Nearest-neighbor analysis confirmed that P2X(2)Rs were expressed by a heterogeneous population of amacrine cells. The synaptic connectivity of P2X(2)R amacrine cells was also investigated. It was interesting that P2X(2)R-immunoreactive amacrine cell dendrites stratified in the sublaminae of the inner plexiform layer occupied by cone, but not rod bipolar cell axon terminals. Immunoelectron microscopy revealed that P2X(2)-immunoreactive amacrine cell processes were associated with cone bipolar cell axon terminals as well as other conventional synapses in the inner plexiform layer. Taken together, these data provide further evidence for the involvement of extracellular ATP in neuronal signaling in the retina, particularly within cone pathways.

Neuroprotective effects of inhibiting N-methyl-D-aspartate receptors, P2X receptors and the mitogen-activated protein kinase cascade: a quantitative analysis in organotypical hippocampal slice cultures subjected to oxygen and glucose deprivation.

Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the downstream pathways activated by intracellular calcium overload.

Visualization of the trimeric P2X2 receptor with a crown-capped extracellular domain.

The P2X2 purinergic receptor permeates cationic ions in response to stimulation by ATP and mediates fast synaptic transmission. Here, we purified the P2X2 receptor using baculovirus-Sf9 cell expression system and observed its structure using electron microscopy. The FLAG-tagged P2X2 receptor, which has intact ion channel function, was purified to be a single peak by affinity purification and gel filtration chromatography. It was confirmed to be a trimer by introducing cross-linking. Negatively stained P2X2 protein images were homogeneous and picked up by automated pick-up programs, aligned, and classified using the modified growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. These images demonstrate an inverted three-sided pyramid with the dimensions of 215 A in height and 200 A in side length. It is composed of a high-density trunk and a stain-permeable swollen extracellular domain of a crown-shaped structure. The internal cavities and constituent segments were clearly demonstrated in both the raw images and the averaged images. The threefold symmetrical top view demonstrates the first visual evidence of the trimeric composition of the P2X receptor family.

Co-localization of P2Y1 receptor and NTPDase1/CD39 within caveolae in human placenta.

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) is the dominant ecto-nucleotidase of vascular and placental trophoblastic tissues and appears to modulate the functional expression of type-2 purinergic (P2) G-protein coupled receptors (GPCRs). Hence, this ectoenzyme could regulate nucleotide-mediated signalling events in placental tissue. This immunohistochemical and immuno-electron microscopic study demonstrates the expression of NTPDase1/CD39, P2Y1 and P2Y2 receptors in different cell types of human placenta. Specifically P2Y1 has an exclusive vascular distribution whereas P2Y2 is localized on trophoblastic villi. Co-localization of P2Y1 and NTPDase1/CD39 are observed in caveolae, membrane microdomains of endothelial cells. The differential localization of these P2 receptors might indicate their unique roles in the regulation of extracellular nucleotide concentrations in human placental tissues and consequent effects on vascular tone and blood fluidity.

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor.

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.

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 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.

Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons.

1. The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage-clamp, in situ hybridization and immunohistochemistry. 2. Rapid application of ATP (100 microM) to isolated rat MPG neurons induced moderately large inward currents (0.33-5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly. 3. The concentration-response curve for ATP yielded an EC50 of 58.9 microM. The agonist profile was ATP> or =2MeSATP=ATPgammaS>BzATP, while alpha,beta-MeATP, beta,gamma-MeATP, UTP and ADP were all inactive at concentrations up to 100 microM. 4. The response to ATP was antagonized by suramin (pA2=5.6), reactive blue-2 (IC50=0.7 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 microM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 microM at pH 6.8, 7.4 and 8.0, respectively. 6. Co-application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 microM. In the presence of Zn2+ (30 microM), the EC50 for ATP was decreased to 4.57 microM. 7. In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity. 8. In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers. with P2X2 being the dominant component.

Ultrastructural localization of P2X3 receptors in rat sensory neurons.

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.

The distribution of single P2x1-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.

The distribution of purinergic (P2x1) receptors on smooth muscle cells in relation to autonomic nerve varicosities in the rat urinary bladder has been determined using immunofluorescence and confocal microscopy. P2x1 receptors were visualized using rabbit polyclonal antibodies against the extracellular domain of the P2x1 receptor, and varicosities were visualized using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2x1 receptor clusters were observed on the smooth muscle cells of the detrusor, namely, a large ellipse of mean long axis 1.23+/-0.21 microm and short axis 0.92+/-0.17 microm and a smaller spherical cluster with a mean diameter of 0.40+/-0.04 microm. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per microm2 or two orders of magnitude more than the larger-sized clusters. The large clusters are in general located beneath varicosities, with only 4.5% of P2x1 clusters not possessing an overlying varicosity. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2x1 and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2x1 receptor clusters. On occasions, two or more small SV2-labelled varicosities about 0.7 microm in diameter each with a receptor patch were found juxtaposed to each other; these might represent the splitting up of a single large varicosity. These observations are discussed in relation to the identity of the autonomic neuromuscular junction.