Mitchell A. Hansen

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.

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.

Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X1 to P2X6 receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X1 to P2X6 receptor subtypes (about 0.4 µm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X1 to P2X6 receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X1–6 small receptor clusters so that they became colocalised, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalisation. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalised P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.

Distribution of purinergic P2X receptors in the rat heart.

The distribution of P2X purinergic receptor subtypes has been determined in relation to nerve varicosities in the rat heart with immunohistochemistry. Large clusters (about 1 microm diameter) of co-localised and sometimes co-extensive P2X1 and P2X3 receptors were found at sites of tyrosine hydroxylase (TH) positive axon varicosities in the atrium and the ventricle. Varicosities that were labelled with antibodies to the synaptic vesicle epitope SV2 were frequently labelled also with antibodies to P2X3, P2X5 and P2X6 but not always with antibodies to P2X1. Especially prominent were large numbers of small clusters (about 400 nm diameter) of co-localised P2X2 and P2X5 receptors on the sarcolemma unrelated to nerves at all. During development the 1 day-old heart possessed an abundance of co-localised P2X2 and P2X5 small receptor clusters on the sarcolemma. These observations are discussed in relation to the role of purinergic receptors in the mammalian heart.

Identification and localisation of ATP P2X receptors in rat midbrain.

P2X receptors that are gated by extracellular ATP are among the few known examples of ligand‐gated cation‐selective channels. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of tissues from the peripheral and central nervous systems and from many species. To determine the distribution of the P2X subtypes in the rat midbrain, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE)/Western blotting was combined with immunolocalisation using confocal microscopy. Subtypes P2X1‐6 were detected in the periaqueductal gray area and the ependymal layer bordering the ventricle with a widespread distribution.

Analysis of novel P2X subunit‐specific antibodies in rat cardiac and smooth muscle

P2X receptors are cation‐selective channels gated by extracellular adenosine triphosphate (ATP). There are relatively few known types of ligand‐gated receptors. In vertebrates they include acetylcholine (Ach), 5‐hydroxytryptamine (5‐HT), γ‐aminobutyric acid (GABA), glycine, and glutamate as well as ATP. Ach, 5‐HT, GABA and glycine ligand‐gated receptors are related in evolutionary terms, while glutamate and ATP receptors form separate groups. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of cells and species. We have carried out hydropathy investigations and sequence comparisons of each of the seven subunits in order to examine the putative transmembrane and cysteine‐rich extracellular domains. Probable locations of disulphide bridges are consistent with there being two separate extracellular folding domains. Assessment of the putative surface‐accessible regions was used to select small localised amino acid segments in nonglycosylated regions for raising antibodies against each of the P2X receptor subunits. To test the specificity of these novel P2X receptor antibodies and their presence in cardiac and smooth muscle, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE)/Western blotting was undertaken in homogenised rat heart, bladder, kidney, and vas deferens.

Autoradiography of [3H]α,β-methylene-ATP binding sites in medulla oblongata and spinal cord of the rat

Abstract Excitatory purinoceptors of P 2x -type have long been known to exist on smooth muscle cells, but recently it has been shown that they are also involved in synaptic transmission in the CNS. We have used a P 2x -specific agonist, α,β-methylene-ATP, as a 3 H-labelled radioligand, to study the distribution and characteristics of P 2x receptor-binding sites in the spinal cord and medulla oblongata. Using autoradiographic techniques, [ 3 H]α,β-methylene-ATP binding was found throughout the grey matter of the spinal cord with small areas of above-average density of binding sites in the marginal zone and substantia gelatinosa at all spinal levels and in the central grey matter of the thoracic spinal cord. In the medulla, [ 3 H]α,β-methylene-ATP binding was found to be strong in all cranial nerve nuclei, particularly those known to receive primary sensory fibres. We have found that the binding of [ 3 H]α,β-methylene-ATP in the spinal cord and medulla was inhibited by a broad-spectrum P 2 -receptor antagonist suramin (IC 50 ≈ 27 μM). This is in accordance with the data obtained previously in the forebrain and cerebellu. There was, however, no inhibition of [ 3 H]α,β-methylene-ATP binding by another close analogue of α,β-methylene-ATP and P 2x ligand β,γ-methylene-ATP (10 μM). The latter result is discussed in terms of possible involvement of Ca 2+ in the binding of [ 3 H]α,β-methylene-ATP to P 2x receptors in the CNS.

P2 purinoceptor blocker suramin antagonises NMDA receptors and protects against excitatory behaviour caused by NMDA receptor agonist (RS)‐(tetrazol‐5‐yl)‐glycine in rats

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P2 receptors, may antagonise N‐methyl‐D‐aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P2X subclass of P2 receptors are ligand‐gated Ca2+‐selective channels and, since the increased influx of Ca2+ into neurons has been linked to neurotoxicity, simultaneous inhibition of P2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [3H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch‐clamp studies. Suramin (1 mM) had no effect on [3H]kainate binding in vitro. In vivo, intracerebroventricular (ICV) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)‐(tetrazol‐5‐yl)‐glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by ICV administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression. J. Neurosci. Res. 49:627–638, 1997.