Julian A. Barden

Distribution of P2X receptors on astrocytes in juvenile rat hippocampus

Recent evidence suggested that ATP acting via ionotropic (P2X) and metabotropic (P2Y) purinergic receptors might be involved in signaling between glial cells and within glial–neuronal networks. In contrast to their neuronal counterpart, the identity of P2X receptors in CNS glial cells is largely unknown. In the present study, antibodies recognizing the subunits P2X1–P2X7 were applied together with the astroglial marker S100β and nuclear labeling with Hoechst 33342 to investigate semiquantitatively the distribution of the whole set of P2X receptors in astrocytes of the juvenile rat hippocampus. Expression of P2X1–P2X4, P2X6, and P2X7 subunits was observed in astrocytes of various hippocampal subregions, but the cells were completely devoid of P2X5 protein. S100β‐positive cells expressing subunits P2X3–P2X7 occurred evenly in the different subfields, while P2X1‐ and P2X2‐positive astrocytes were distributed more heterogeneously. The staining pattern of P2X subunits also differed at the subcellular level. Antibodies against P2X2 and P2X4 labeled both astroglial cell bodies and processes. Immunoreactivity for P2X1 and P2X6 was mainly confined to somatic areas of S100β‐positive cells, whereas the subunit P2X3 was primarily localized along astroglial processes. Knowledge of the distribution of P2X receptors might provide a basis for a better understanding of their specific role in cell–cell signaling. GLIA 36:11–21, 2001.

Comparative study on the distribution patterns of P2X1–P2X6 receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): Association with catecholamine cell groups

The present study investigated the topographical distribution of P2X1–P2X6 receptor subtypes in the rat and common marmoset hindbrain by immunohistochemistry. In addition, double‐labeling immunofluorescence was used to determine the extent of colocalization between catecholamine cell groups and the various P2X receptors. The data demonstrate a widespread distribution pattern for all six P2X receptors throughout both the rat hindbrain and the marmoset hindbrain, although distinctions between species, brain nuclei, and P2X receptor subtypes exist. In rat, dense staining for the P2X receptors was found in the nucleus of the solitary tract (NTS), medial vestibular nucleus, and medial and lateral parabrachial nuclei. Moderate staining was observed in the hypoglossal nucleus, cuneate nucleus, inferior olive, prepositus hypoglossi, rostral ventrolateral medulla (RVLM), and locus coeruleus. Staining was also observed in the gracile nucleus, the mesencephalic trigeminal nucleus, and the central pontine gray. In marmoset, prominent P2X receptor‐like immunoreactivity occurred in the NTS, medial cuneate nucleus, prepositus hypoglossi, and medial vestibular nucleus. Moderate staining was observed in the area postrema, dorsal motor nucleus of the vagus, lateral cuneate, lateral reticular, spinal trigeminal nucleus, RVLM, and inferior olive. Immunofluorescent double labeling of tyrosine hydroxylase (TH)‐containing cells revealed that all subtypes of P2X receptors show some degree of colocalization with TH. The highest proportion of TH and P2X receptor double labeling was in the A5 region (with the P2X2 subunit), whereas the lowest proportion of double‐labeled cells occurred in the C2 region of the NTS for the P2X5 subunit. These findings support a role for extracellular adenosine 5′‐triphosphate in fast synaptic neurotransmission within the brainstem. J. Comp. Neurol. 427:485–507, 2000.

P2X (Purinergic) receptor distributions in rat blood vessels

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised 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 mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.

Early prostate cancer detected using expression of non-functional cytolytic P2X7 receptors

Aims:  To detect early prostate cancer reliably by monitoring the expression of non‐functional P2X7 cytolytic purinergic receptors.

Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation.

SummaryThe contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated on this technique using actin, myosin and the actomyosin complex. These papers are reviewed with several aims in mind: (i) we assess the reliability and consistency of intra- and inter-molecular distances measured between the fluorescent probes attached to specific sites on these proteins; (ii) we determine whether the measurements can be assembled into an internally consistent model which can be fitted to the known dimensions of the actomyosin complex; (iii) several of the FRET distances are consistent with the available structural data from crystallographic and electron microscopic dimensions; (iv) the modelled FRET distances suggest that the assumed value of the orientation factor (K2 = 2/3) is reasonable; (v) we conclude that the model has a predictive value, i.e. it suggests that a small number of the published dimensions may be incorrect and predicts the magnitude of a larger number of measurements which have not yet been reported; and finally (vi) we discuss the contribution of FRET determinations to the current debate on the molecular mechanism of contraction.

Purinergic receptor distribution in endothelial cells in blood vessels: a basis for selection of coronary artery grafts.

Expression levels of the purinergic P2X receptor subunits (P2X(1) to P2X(7)) and P2Y(2) were examined in the endothelial cell layer of internal mammary artery (Ann. Thorac. Surg. 54 (1992) 652), radial artery (Ann. Thorac. Surg. 16 (1973) 111) and saphenous vein (Ann. Thorac. Surg. 20 (1975) 628) samples obtained at surgery for coronary artery bypass grafts using immunohistochemistry and confocal microscopy. Similar levels of P2X(1), P2X(2), P2X(3), P2X(7) and P2Y(2) were found in the endothelial cells in all vessels examined while the levels of P2X(5) and P2X(6) were uniformly lower. A clear difference was measured in P2X(4) expression between arteries and veins. Both radial and internal mammary arteries exhibited very low levels of P2X(4) whereas the level in the saphenous vein was 14.6 fold higher (P<0.0001), approaching that of the major receptor subtypes. These data showing strong expression of P2X(4) in veins have implications for the choice of vessels used in coronary artery bypass grafts given that P2X(4) is involved in calcium influx into endothelial cells, modulates blood vessel contractility and is up-regulated in situations involving intima proliferation suggesting vein grafts are more susceptible to developing atherosclerosis.

P2X1 receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras

The P2X(1) purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X(1) and a chimera of P2X(1) and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X(1)-GFP chimera was used to study the time course of P2X(1) receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X(1) and P2X(1)-GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 microm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X(1) antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED(50) for the binding of alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) to the P2X(1)-GFP chimera was similar to native P2X(1) receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X(1) or P2X(1)-GFP were similar. Exposure of HEK293 cells to alpha, beta-meATP for 10-20 min in the presence of 5 microM monensin led to the disappearance of P2X(1)-GFP fluorescence from the surface of the cells. These observations using the P2X(1)-GFP chimera demonstrate that P2X(1) receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.

Purinoceptors are involved in the induction of an osmolyte permeability in malaria-infected and oxidized human erythrocytes

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non‐infected or non‐oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch‐clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y1 protein. Moreover, P2Y1 deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection‐ or oxidation compared with wild‐type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y1‐deficient mice in vivo compared with wild‐type animals. In conclusion, induction of the osmolyte permeability in Plasmodium‐infected erythrocytes involves autocrine purinoceptor signaling.

Detection of P2X purinergic receptors on human B lymphocytes.

Abstract. 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.

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 μm and short axis 0.92 ± 0.17 μm and a smaller spherical cluster with a mean diameter of 0.40 ± 0.04 μm. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per μm2 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 μm 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.