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 P2X_1–6 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.     

Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder

Clusters of purinergic receptor subunits, about 1 μm diameter, are found on the smooth muscle cell membrane beneath junctional varicosities in the detrusor muscle of the rat urinary bladder. We have examined the extent of redistribution of the six different subunit clusters, P2X₁ to P2X₆, with respect to junctional varicosities during pregnancy, as it is known that the detrusor muscle undergoes changes in purinergic innervation during this period. Before pregnancy, clusters at junctional varicosities are principally composed of the subtypes P2X₁, P2X₂, P2X₃ and P2X₅. However this subtype distribution changes dramatically during pregnancy, such that by day 14 of pregnancy, the extent of P2X₁, P2X₂, P2X₃ and P2X₅ junctional clusters has decreased by more than 80% whereas the extent of P2X₄ and P2X₆ junctional clusters has increased by more than 80%. These changes were confirmed with Western blots for different subtypes. It is suggested that the changes in the purinergic innervation of the detrusor muscle during pregnancy reflect changes in the P2X subtypes found on the smooth muscle membrane beneath junctional varicosities.     

Electron-immunocytochemical localization of P2X1 receptors in the rat cerebellum

The distribution of the P2X₁ subtype of purinoceptors associated with the extracellular activities of ATP was studied in the rat cerebellum at the electron-microscope level. Receptors were labelled with peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex for immunocytochemistry. Immunoreactivity to P2X₁ receptors was localized in subpopulations of synapses between varicosities of parallel fibres of granule cells and dendritic spines of Purkinje cells. Unlabelled varicosities of parallel fibres formed asymmetric synapses with labelled dendritic spines, whereas labelled varicosities of parallel fibres formed asymmetric synapses with unlabelled dendritic spines. P2X₁ immunoreactivity was also localized in some astrocyte processes. The functional significance of these findings is discussed.     

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system

The distribution of the P2X₂ subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X₂ receptors was localised in: (i) paraventricular and supraoptic nuclei—in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis—in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X₂-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X₂-positive axons on P2X₂-positive endocrine cell bodies as well as on P2X₂-negative somata were also observed. The functional significance of these findings is discussed.     

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.     

18 μm replication units of chromosomal DNA fibers of differentiated cells of Pea (Pisum sativum)

A technique is presented for investigating possible early terminalisation of chiasmata, based on the analysis of labelling patterns in autoradiographs in X₂-labelled diplotene bivalents. Terminalisation is expected to produce unlabelled gaps in otherwise labelled bivalents which, given even a moderate amount of movement, is capable of resolution by this technique. The method has been evaluated using diplotene spermatocytes of Schistocerca gregaria as a test system. Very few unlabelled gaps were actually observed in X₂-labelled bivalents, in fact no more than in X₁-labelled bivalents, where chiasma terminalisation is not expected to produce gaps. Consequently it is concluded that the gaps observed are due to technical causes and that early terminalisation is an unimportant factor determining chiasma distribution in this system.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

Abstract. In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G₂ phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6–12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine ([³H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [³H]TdR labelling was simulated. The presence of two distinct sub-populations in G₂ phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G₁ phase is suggested. The sizes of the slowly cycling fractions in G₁ and G₂ showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3–5%) was arrested in G₂ phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G₂ cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Immunohistochemical evidence for different pathways immunoreactive to substance P and calcitonin gene-related peptide (CGRP) in the guinea-pig stellate ganglion

The colocalization of immunoreactivities to substance P and calcitonin gene-related peptide (CGRP) in nervous structures and their correlation with other peptidergic structures were studied in the stellate ganglion of the guinea pig by the application of double-labelling immunofluorescence. Three types of fibre were distinguished. (1) Substance P⁺/CGRP⁺ fibres, which sometimes displayed additional immunoreactivity for enkephalin, constituted a small fibre population of sensory origin, as deduced from retrograde labelling of substance P⁺/CGRP⁺ dorsal root ganglion cells. (2) Substance P⁺/CGRP^– fibres were more frequent; some formed baskets around non-catecholaminergic perikarya that were immunoreactive to vasoactive intestinal polypeptide (VIP). (3) CGRP⁺/substance P^– fibres were most frequent and were mainly distributed among tyrosine hydroxylase (TH)-immunoreactive cell bodies. The peptide content of fibre populations (2) and (3) did not correspond to that of sensory ganglion cells retrogradely labelled by tracer injection into the stellate ganglion. Therefore, these fibres are throught to arise from retrogradely labelled preganglionic sympathetic neurons of the spinal cord, in which transmitter levels may have been too low for immunohistochemical detection of substance P or CGRP. CGRP-immunoreactivity but no substance P-immunolabelling was observed in VIP-immunoreactive postganglionic neurons. Such cell bodies were TH-negative and were spared by substance P-immunolabelled fibre baskets. Retrograde tracing with Fast Blue indicated that the sweat glands in the glabrous skin of the forepaw were the targets of these neurons. The streptavidin-biotin-peroxidase method at the electron-microscope level demonstrated that immunoreactivity to substance P and CGRP was present in dense-cored vesicles of 50–130 nm diameter in varicosities of non-myelinated nerve fibres in the stellate ganglion. No statistically significant difference in size was observed between vesicles immunolabelled for substance P and CGRP. Immunoreactive varicosities formed axodendritic and axosomatic synaptic contacts, and unspecialized appositions to non-reactive neuronal dendrites, somata, and axon terminals. Many varicosities were partly exposed to the interstitial space. The findings provide evidence for different pathways utilizing substance P and/or CGRP in the guinea-pig stellate ganglion.     

Clustering of spin-labeled cholesterol analog diluted in bilayers of saturated and unsaturated phospholipids

Clustering of spin-labeled cholesterol analog, 3β-doxyl-5α-cholestane (DChl), diluted in bilayers comprised of either saturated dipalmitoyl-glycero-phosphocholine (DPPC) or unsaturated dioleoyl-glycero-phosphocholine (DOPC) phospholipids was studied. DChl molar fraction X varied between 0.005 and 0.04. EPR spectroscopy applied at low temperatures (200 K) enabled exploring magnetic dipole-dipole (d-d) interaction between spin labels. For DOPC bilayers, EPR spectra were found to broaden remarkably with X increase. The broadening was simulated for the models of 2-dimentional (2-D) clusters with enhanced local concentration, X_loc, which was several times larger than X, and for 1-dimensional (1-D) DChl clusters. The distance of closest approach in these simulations attained the intermolecular lateral distance in the membrane (~0.7 nm). For DPPC bilayers, EPR spectra showed only small broadening, which in these simulations could not be reproduced even if X_loc was taken as small as X. However strong concentration dependence was found for electron spin echo (ESE) decays. Both the EPR and ESE data for DPPC bilayers were explained within the model assuming encapsulation of DChl molecules in lipid shells so preventing them to approach each other closer than a certain distance, R_min. The R_min value was found to vary between ~2.5 nm and 5 nm, for X varying between 0.04 and 0.005; X_loc in these simulations was several times larger than X. So the DChl clustering in DOPC bilayers is driven by attractive lipid-mediated forces, while in DPPC bilayers long-range nanoscale lipid-mediated repulsive/attractive forces take place for distances smaller and larger R_min, correspondingly.     

Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency

Purinergic P2X receptors associated with the parasympathetic nerves supplying human bladder smooth muscle (detrusor) are implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalised with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder and in adults with the urodynamics findings of sensory urgency (SU) to determine how receptor distribution varied in patients with a small bladder capacity. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody (SV2) against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for haematuria (n=22, age 33–88 years) and adults with sensory urgency at cystoscopy/cystodistension (n=11, age 37–70 years). Normal adult specimens contained detrusor muscle innervated by parasympathetic nerves possessing large varicosities (1.2 m) distributed along their length. These mostly all showed colocalised patches of presynaptic P2X_1,2,3,5 subtypes while presynaptic subtypes P2X_4,6,7 were present in only 6–18% of varicosities. Detrusor nerve varicosities from SU patients revealed general loss of all presynaptic P2X subtypes with the proportion containing receptors reducing to only 0.5–5% depending on P2X subtype. The same loss was recorded from the sensory nerves in the surrounding lamina propria. This specific loss of P2X receptors may impair control of detrusor distension and contribute to the pathophysiology of sensory urgency.The study was funded by the National Health and Medical Research Council of Australia     

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding

The redistribution of purinergic P2X receptor subunits (P2X₁ to P2X₇) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X₁ receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X₄ clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X₅ clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X₃, P2X₆ and P2X₇ cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X₄ receptors may contribute to neointimal proliferation.     

Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: role of P2 and NGFRp75 receptors

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30–60 min inhibited P2Y₁ receptor protein, only weakly modulated P2X₁, P2X₂ and P2X₃, and up‐regulated by about two‐fold P2X₄, P2X₇ and P2Y₄. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X₇ and P2Y₄ proteins, but not P2X₄ or P2Y₁. Glucose starvation transiently increased (up to three‐fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10‐fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst‐positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia‐induced expression of P2X₇ and P2Y₄. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst‐positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia‐induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.     

Varicosities of single sympathetic nerve terminals possess syntaxin zones and different synaptotagmin N-terminus labelling following stimulation

A study has been made of the probability of exocytosis of synaptic vesicles at different varicosities in single sympathetic terminal axons in the mouse vas deferens. An antibody (SV2Ab) against SV2, a proteoglycan in synaptic vesicles, labelled an area of individual sympathetic varicosities that was slightly less than that occupied by dextran-rhodamine, previously orthogradely transported into the varicosities. In contrast plasma membrane bound protein syntaxin, found at active zones of motor nerve terminals, occupied an area of the varicosity that was approximately one-third that of SV2. This suggests that sympathetic varicosities possess specialized zones for exocytosis on their plasma membranes. Antibodies against the N-terminal sequence of synaptotagmin 1 (SNAb), a sequence exposed within synaptic vesicles, were used to determine the probability of exocytosis at different varicosities of single terminal branches. The area of SNAb labelling was not significantly different from that of the SV2 labelling, which implies vesicles that have undergone exocytosis may eventually return to the main pool of vesicles. Varicosities belonging to the same terminal axon, and identified with SV2Ab, showed different extents of labelling with SNAb when secretion was evoked with high potassium concentrations (80 mM) for 30 min in the presence of SNAb. There was up to an order of magnitude difference in the average intensity of SNAb labelling between different varicosities of the same terminal axon whereas there was little difference in the average intensity of SV2Ab labelling. These observations suggest that there is considerable variability in the probability of exocytosis at the specialized zones in different varicosities.     

RECYCLING OF RESTING CELLS IN THE JB-1 ASCITES TUMOUR AFTER TREATMENT WITH 1-β-D-ARABINOFURANOSYLCYTOSINE (Ara-C)

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of growth of the murine JB-1 ascites tumour (i.e. 10 days after 2–5 × 10⁶ cells i.p.) large fractions of non-cycling cells with G₁ and G₂ DNA content (Q₁ and Q₂ cells) are present, and the fate of these resting cells was investigated after treatment with l-β-d-arabinofuranosylcytosine (Ara-C). The experimental work consisted of growth curves, percentage of labelled mitoses curves after continuous labelling with ³H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with ³H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6–8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with ³H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with ³H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q_t cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. These results indicate recycling of resting cells first with G₂ and later with G_x DNA content, which contribute to the regrowth of the tumours.     

Nuclear labelling after prolonged H-uridine incorporation as visualized by high resolution autoradiography

Localization of radioactive labelling over the nuclei of BSC₁ cells is visualized after long periods of ³H-5-uridine incubation followed or not followed by periods of postincubation in nonradioactive medium for up to several days, using high resolution autoradiography combined with a preferential staining method for ribonucleoproteins.It is shown that when cells are labelled for 1 or 6 h with ³H-uridine and postincubated with a non-radioactive medium up to several days, there is always some radioactivity present in the nucleolus and nucleoplasm. When sections of cells fixed after 1 h of labelling followed by 24 h of postincubation are treated with RNase, part of the radioactivity found in the nucleus disappears almost completely only after a succeeding DNase digestion.The majority of interchromatin granules are weakly labelled after most incubation times, with the label localized rather at the periphery of clusters of granules, or are unlabelled.The results are discussed in the context of recent biochemical findings. It is proposed that interchromatin granules might represent a structure containing a limited quantity of slowly labelled nuclear RNA.     

ACTIVITY OF TUBULOSINE ON THE KINETICS OF ROOT MERISTEM CELL POPULATION

The action of tubulosine on the mitotic cycle was studied using continuous labelling with tritiated thymidine. This alkaloid provokes a lengthening of the G₁ and S phases and a blocking of G₂ is totally reversible when the treatment is followed by recovery in normal medium. At a dose of tubulosine which induces a reversible mitostasis in the shortest possible time the lengthening of the phases of the cell cycle was estimated by three different techniques: labelled mitoses for the determination of G₂; labelling intensity for the determination of S; binucleate cells for the determination of T, and an original technique using labelling index of binucleate cells for the determination of G₁. The limits of the technique of labelled mitosis together with the interest of the technique aiming at the direct determination of G₁ in the case of a perturbed cycle are then discussed.     

Altered expression of P2X3 in vagal and spinal afferents following esophagitis in rats

Purinergic P2X₃ receptors are predominantly expressed in small diameter primary afferent neurons and activation of these receptors by adenosine triphosphate is reported to play an important role in nociceptive signaling. The objective of this study was to investigate the expression of P2X₃ receptors in spinal and vagal sensory neurons and esophageal tissues following esophagitis in rats. Two groups of rats were used including 7 days fundus-ligated (7D-ligated) esophagitis and sham-operated controls. Esophagitis was produced by ligating the fundus and partial obstruction of pylorus that initiated reflux of gastric contents. The sham-operated rats underwent midline incision without surgical manipulation of the stomach. Expressions of P2X₃ receptors in thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophageal tissues were evaluated by RT–PCR, western blot and immunohistochemistry. Esophageal neurons were identified by retrograde transport of Fast Blue from the esophagus. There were no significant differences in P2X₃ mRNA expressions in DRGs (T1–T3) and NGs between 7D-ligated and sham-operated rats. However, there was an upregulation of P2X₃ mRNA in DRGs (T6–T12) and in the esophageal muscle. At protein level, P2X₃ exhibited significant upregulation both in DRGs and in NGs of rats having chronic esophagitis. Immunohistochemical analysis exhibited a significant increase in P2X₃ and TRPV1 co-expression in DRGs and NGs in 7D-ligated rats compared to sham-operated rats. The present findings suggest that chronic esophagitis results in upregulation of P2X₃ and its co-localization with TRPV1 receptor in vagal and spinal afferents. Changes in P2X₃ expression in vagal and spinal sensory neurons may contribute to esophageal hypersensitivity following acid reflux-induced esophagitis.     

Synthesis and structure–activity relationships of carboxylic acid derivatives of pyridoxal as P2X receptor antagonists

Carboxylic acid derivatives of pyridoxal were developed as potent P2X₁ and P2X₃ receptor antagonists with modifications of a lead compound, pyridoxal-5′-phosphate-6-azophenyl-2′,5′-disulfonate (5b, iso-PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic carboxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-5′-phosphate (1), 13 and 14, showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24–28) of compound 13 or 14 showed potent antagonistic activities similar to that of 5b at human P2X₃ receptors with 100 nM range of IC₅₀ values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic acid without affecting antagonistic potency at mouse P2X₁ and human P2X₃ receptors.