P2X₃ Knock-Out Mice Reveal a Major Sensory Role for Urothelially Released ATP

The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X₃ receptor knock-out (P2X₃ ⁻⁄⁻) and wild-type control (P2X₃ ⁺⁄⁺) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X₃. P2X₃ -positive staining was completely absent in the subepithelial plexus of the P2X₃ ⁻⁄⁻ mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder–pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X₃ ⁻⁄⁻ mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder–pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X₃ ⁺⁄⁺ and P2X₃ ⁻⁄⁻ mice, although P2X₃ ⁻⁄⁻ bladder had an increased capacity compared with that of the P2X₃ ⁺⁄⁺ bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X₃ ⁻⁄⁻ mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X₃ ⁺⁄⁺, but not P2X₃ ⁻⁄⁻, were rapidly activated by intravesical injections of P2X agonists (ATP or α,β-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2′,3′-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2′,4′-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X₃ receptors on a subpopulation of pelvic afferent fibers

TRPV1 enhances the afferent response to P2X receptor activation in the mouse urinary bladder

Both TRPV1 and P2X receptors present on bladder sensory nerve fibres have been implicated in mechanosensation during bladder filling. The aim of this study was to determine possible interactions between these receptors in modulating afferent nerve activity. In wildtype (TRPV1+/+) and TRPV1 knockout (TRPV1−/−) mice, bladder afferent nerve activity, intravesical pressure, and luminal ATP and acetylcholine levels were determined and also intracellular calcium responses of dissociated pelvic DRG neurones and primary mouse urothelial cells (PMUCs). Bladder afferent nerve responses to the purinergic agonist αβMethylene-ATP were depressed in TRPV1−/− mice (p ≤ 0.001) and also in TRPV1+/+ mice treated with the TRPV1-antagonist capsazepine (10 µM; p ≤ 0.001). These effects were independent of changes in bladder compliance or contractility. Responses of DRG neuron to αβMethylene-ATP (30 µM) were unchanged in the TRPV1−/− mice, but the proportion of responsive neurones was reduced (p ≤ 0.01). Although the TRPV1 agonist capsaicin (1 µM) did not evoke intracellular responses in PMUCs from TRPV1+/+ mice, luminal ATP levels were reduced in the TRPV1−/− mice (p ≤ 0.001) compared to wildtype. TRPV1 modulates P2X mediated afferent responses and provides a mechanistic basis for the decrease in sensory symptoms observed following resiniferatoxin and capsaicin treatment for lower urinary tract symptoms

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT3 receptors, respectively. We have investigated the changes in P2X and 5-HT3-mediated signalling in pelvic afferent neurons in mice deficient in P2X2 and/or P2X3 subunits by whole-cell recording of L6–S2 dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X3−/− neurons, whereas sustained currents were absent in P2X2−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X2/P2X3Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT3 receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X 2−/−, P2X3−/− and P2X2/P2X3Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X2/P2X3Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X2/P2X3Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X2/P2X3Dbl−/− preparations. These data suggest that P2X receptors containing the P2X2 and/or P2X3 subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes. This effect is more pronounced when both subunits are absent

ATP release from the human ureter on distension and P2X3 receptor expression on suburothelial sensory nerves

It is not clear how the increase in intraluminal pressure behind an obstructing ureteric calculus causes an increase in action potential frequency in ureteric sensory nerves so the pain messages are transmitted to the brain. It has been proposed that ureteric distension causes urothelial release of ATP, which activates purinoceptors on suburothelial nociceptive sensory nerves. The purpose of this study was to determine whether distension of the human ureter results in the release of ATP and whether the nociceptive P2 receptor, P2X3, is expressed on suburothelial sensory nerves in the human ureter. Human ureter segments were perfused with Krebs solution and intermittently distended to a range of pressures. Samples of perfusate were collected throughout and the ATP concentration ([ATP]) was determined using a luciferin-luciferase assay. Sections of ureter were stained using antibodies against P2X3 and capsaicin receptors (TRPV1). [ATP] rose to more than 10 times baseline levels after distension beyond a threshold of 25–30 cmH2O. Immunofluorescence studies on consecutive frozen sections showed that suburothelial nerves stained positively for P2X3 and capsaicin receptors, with no staining in controls. These findings are consistent with the hypothesis that purinergic signalling is involved in human ureteric mechanosensory transduction, leading to nociception

Blood neutrophil counts are associated with exacerbation frequency and mortality in COPD

BACKGROUND: Identifying patients with COPD at increased risk of poor outcomes is challenging due to disease heterogeneity. Potential biomarkers need to be readily available in real-life clinical practice. Blood eosinophil counts are widely studied but few studies have examined the prognostic value of blood neutrophil counts (BNC). METHODS: In a large population-based COPD registry in the East of Scotland (TARDIS: Tayside Allergic and Respiratory Disease Information System), BNC were compared to measures of disease severity and mortality for up to 15 years follow-up. Potential mechanisms of disease modification by BNC were explored in a nested microbiome substudy. RESULTS: 178,120 neutrophil counts were obtained from 7220 people (mean follow up 9 years) during stable disease periods. Median BNC was 5200cells/μL (IQR 4000-7000cells/μL). Mortality rates among the 34% of patients with elevated BNCs (defined as 6000-15000cells/μL) at the study start were 80% higher (14.0/100 person years v 7.8/100py, P < 0.001) than those with BNC in the normal range (2000-6000cells/μL). People with elevated BNC were more likely to be classified as GOLD D (46% v 33% P < 0.001), have more exacerbations (mean 2.3 v 1.3/year, P < 0.001), and were more likely to have severe exacerbations (13% vs. 5%, P < 0.001) in the following year. Eosinophil counts were much less predictive of these outcomes. In a sub-cohort (N = 276), patients with elevated BNC had increased relative abundance of Proteobacteria and reduced microbiome diversity. CONCLUSIONS: High BNC may provide a useful indicator of risk of exacerbations and mortality in COPD patients

Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201

CD38 Exacerbates Focal Cytokine Production, Postischemic Inflammation and Brain Injury after Focal Cerebral Ischemia

BACKGROUND: Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion. METHODOLOGY/PRINCIPAL FINDINGS: We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8(+) cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model. CONCLUSION/SIGNIFICANCE: CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant

Writing in Britain and Ireland, c. 400 to c. 800

No abstract available

Bladder sensory desensitization decreases urinary urgency

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