Expression Of Purinergic Receptors Research Articles

Purinergic smooth muscle contractions in the human prostate: estimation of relevance and characterization of different agonists

Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)–induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84–124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12–24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,β-methylene-ATP (1.8–12.4%), 3.9% for β,γ-methylene-ATP (2.0–5.4%), 3.1% for 2-methylthio-ATP (− 0.1–6.3%), and 5.1% for ATPγS (1.0–9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib

Introduction: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition.Materials and Methods: Citrate-anticoagulated whole blood was stored with 5 nM and 1 μM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining.Results: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces.Conclusions: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.

Purinergic regulation: From a risky hypothesis to a triumphant theory

With the discovery of the ATP structure in 1929, significant progress was made in understanding the role of nucleosides and nucleotides in the body. One of the most important breakthroughs is associated with the determination of the function of an autacoid in ATP, a participant in purinergic signal transmission. For the first time, this function of ATP was pointed out by Professor Geoffrey Burnstock in 1972. Purinergic signaling activators are extracellular nucleotides including ATP, ADP, UTP, UDP, and adenosine nucleoside. The purinergic signaling pathway begins with the synthesis and intracellular accumulation of nucleotides, and then their release from the cell under various physiological and pathological conditions. In the extracellular spaces, nucleotides are hydrolyzed by various enzymes with the removal of phosphate groups, which leads to the appearance of various regulatory molecules that interact with P1 and P2 purinergic receptors. This ligand-receptor interaction changes the functional state of the target cell. In turn, the expression of purinergic receptors changes depending on the functional state of the cell. The participation of purinergic regulation in the development of many diseases indicates that by changing the concentration of signaling molecules, it is possible to change the course of pathological processes, in particular the activity of inflammation and the direction of immune responses. This article provides a brief review of the literature on the structure of nucleotide and nucleoside autacoids, enzymes involved in their metabolism, specific purinergic receptors.

Loganin Attenuates High Glucose-Induced Schwann Cells Pyroptosis by Inhibiting ROS Generation and NLRP3 Inflammasome Activation.

Diabetic peripheral neuropathy (DPN) is caused by hyperglycemia, which induces oxidative stress and inflammatory responses that damage nerve tissue. Excessive generation of reactive oxygen species (ROS) and NOD-like receptor protein 3 (NLRP3) inflammasome activation trigger the inflammation and pyroptosis in diabetes. Schwann cell dysfunction further promotes DPN progression. Loganin has been shown to have antioxidant and anti-inflammatory neuroprotective activities. This study evaluated the neuroprotective effect of loganin on high-glucose (25 mM)-induced rat Schwann cell line RSC96 injury, a recognized in vitro cell model of DPN. RSC96 cells were pretreated with loganin (0.1, 1, 10, 25, 50 μM) before exposure to high glucose. Loganin’s effects were examined by CCK-8 assay, ROS assay, cell death assay, immunofluorescence staining, quantitative RT–PCR and western blot. High-glucose-treated RSC96 cells sustained cell viability loss, ROS generation, NF-κB nuclear translocation, P2 × 7 purinergic receptor and TXNIP (thioredoxin-interacting protein) expression, NLRP3 inflammasome (NLRP3, ASC, caspase-1) activation, IL-1β and IL-18 maturation and gasdermin D cleavage. Those effects were reduced by loganin pretreatment. In conclusion, we found that loganin’s antioxidant effects prevent RSC96 Schwann cell pyroptosis by inhibiting ROS generation and suppressing NLRP3 inflammasome activation.

Functional expression of P2X1, P2X4 and P2X7 purinergic receptors in human monocyte-derived macrophages

This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA. In whole-cell patch clamp recordings of human MDMs, rapid application of ATP (0.01 mM) evoked fast current activation and two different desensitization kinetics: 1. a rapid desensitizing current antagonized by PPADS (1 μM), reminiscent of the P2X1 receptor's current; 2. a slow desensitizing current, insensitive to PPADS but potentiated by ivermectin (3 μM), similar to the P2X4 receptor's current. Application of 5 mM ATP induced three current modalities: 1. slow current activation with no desensitization, similar to the P2X7 receptor current, present in 69% of human macrophages and antagonized by A-804598 (0.1 μM); 2. fast current activation and fast desensitization, present in 15% of human MDMs; 3. fast activation current followed by biphasic desensitization, observed in 15% of human MDMs. Both rapid and biphasic desensitization kinetics resemble those observed for the recombinant human P2X1 receptor expressed in oocytes. These data demonstrate, for the first time, the co-expression of P2X1, P2X4, and P2X7 transcripts and confirm the presence of functional P2X1, P2X4, and P2X7 receptors in human macrophages.

Neurofibromatosis 1 - Mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/−) mice, purinergic control of phagocytosis was only affected in male Nf1+/− mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/−, microglia. These defects resulted from Nf1+/− male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/− microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain.

Neuroprotective role of resveratrol mediated by purinergic signalling in cerebral cortex of mice infected by Toxoplasma gondii.

The central nervous system of the intermediate host plays a central role in lifelong persistence of Toxoplasma gondii as well as the pathogenesis of congenital toxoplasmosis and reactivated infection in immunocompromised individuals. The purinergic system has been implicated in a wide range of immunological pathways for controlling intracellular responses to pathogens, including T. gondii. In the present study, we investigated the effect of resveratrol (RSV) on ectonucleotidases, adenosine deaminase (ADA), and purinergic receptors during chronic infection by T. gondii. For this study, Swiss mice were divided into control (CTL), resveratrol (RSV), infected (INF), and INF+RSV groups. The animals were orally infected with the VEG strain and treated with RSV (100mg/kg, orally). Ectonucleotidase activities, P2X7, P2Y1, A1, and A2A purinergic receptor density, ROS, and thiobarbituric acid reactive substances levels were measured in the cerebral cortex of mice. T. gondii infection increased NTPDase and reduced ADA activities. Treatment with RSV also affected enzymes hydrolysing extracellular nucleotides and nucleosides. Finally, RSV affected P1 and P2 purinergic receptor expression during T. gondii infection. Overall, RSV-mediated beneficial changes in purinergic signalling and oxidative stress, possibly improving cerebral cortex homeostasis in T. gondii infection.

Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1-/-, alias Thbs1-/-) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1-/- compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1-/- mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1-/- compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, andpurinergic receptors are decreased in TSP1-/- compared with WT mice.

The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness.

Introduction Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies.

Purinergic Signaling During Hyperglycemia in Vascular Smooth Muscle Cells

The activation of purinergic receptors by nucleotides and/or nucleosides plays an important role in the control of vascular function, including modulation of vascular smooth muscle excitability, and vascular reactivity. Accordingly, purinergic receptor actions, acting as either ion channels (P2X) or G protein-coupled receptors (GCPRs) (P1, P2Y), target diverse downstream effectors, and substrates to regulate vascular smooth muscle function and vascular reactivity. Both vasorelaxant and vasoconstrictive effects have been shown to be mediated by different purinergic receptors in a vascular bed- and species-specific manner. Purinergic signaling has been shown to play a key role in altering vascular smooth muscle excitability and vascular reactivity following acute and short-term elevations in extracellular glucose (e.g., hyperglycemia). Moreover, there is evidence that vascular smooth muscle excitability and vascular reactivity is severely impaired during diabetes and that this is mediated, at least in part, by activation of purinergic receptors. Thus, purinergic receptors present themselves as important candidates mediating vascular reactivity in hyperglycemia, with potentially important clinical and therapeutic potential. In this review, we provide a narrative summarizing our current understanding of the expression, function, and signaling of purinergic receptors specifically in vascular smooth muscle cells and discuss their role in vascular complications following hyperglycemia and diabetes.

Secretory Functions of Macrophages in the Human Pancreatic Islet Are Regulated by Endogenous Purinergic Signaling.

Endocrine cells of the pancreatic islet interact with their microenvironment to maintain tissue homeostasis. Communication with local macrophages is particularly important in this context, but the homeostatic functions of human islet macrophages are not known. In this study, we show that the human islet contains macrophages in perivascular regions that are the main local source of the anti-inflammatory cytokine interleukin-10 (IL-10) and the metalloproteinase MMP9. Macrophage production and secretion of these homeostatic factors are controlled by endogenous purinergic signals. In obese and diabetic states, macrophage expression of purinergic receptors MMP9 and IL-10 is reduced. We propose that in those states, exacerbated β-cell activity due to increased insulin demand and increased cell death produce high levels of ATP that downregulate purinergic receptor expression. Loss of ATP sensing in macrophages may reduce their secretory capacity.

Type 1 Diabetes Results in Significant Purinergic Receptor Remodeling in Podocytes

P2 (purinergic) receptors have been proposed to be potential viable targets for the treatment of various cardiovascular and renal disorders. Previous studies in our lab have demonstrated that the predominant purinergic signaling pathway in healthy podocytes is through P2Y1 metabotropic receptors. It was also reported that expression of purinergic receptors in different tissues, including kidney, is dynamic and might be changed under pathological conditions such as diabetic nephropathy. We hypothesized that prolonged type 1 diabetes (T1D) would result in substantial changes to podocyte purinergic calcium signaling and result in a transition from predominantly metabotropic signaling through P2Y1 to increased ionotropic signaling through P2X receptors. To do this we utilized the established T1D model of streptozocin‐injected Dahl Salt Sensitive (SS) rats, and measured intracellular calcium ([Ca2+]i) signaling in podocytes from their freshly isolated glomeruli. We determined that in comparison to non‐diabetic rats, which have a rapid elevation of [Ca2+]i levels in response to ATP (40 μM) that returns to baseline within 2 min, podocytes from T1D rats had reduced peak response amplitudes (0.53 ± 0.04 vs. 0.34±0.02, p<0.001). Furthermore, podocytes of glomeruli isolated from diabetic rats had prolonged sustained elevation of [Ca2+]i level, which was not observed in non‐diabetic rats. Additionally, we used P2 specific antagonists to determine which receptors resulted in the altered calcium response curve in T1D podocytes. We found that pharmacological inhibition of P2X7 and P2X4 (10 μM A438079 and 5‐BDBD, respectively) altered the calcium curve dynamics, but did not significantly reduce the total [Ca2+]i level over a 5 min time period, as determined by area under the curve (AUC) measurements. Similarly, inhibition of P2Y1 (1 μM MRS2500) changed the [Ca2+]i curve response, but did not reduce the AUC. Combining both P2X7 and P2X4 inhibitors reduced the AUC; however, did not completely eliminate podocyte purinergic signaling (51.42 ± 5.52 vs 30.39 ± 5.00). Combined inhibition of P2X7, P2X4, and P2Y1 resulted in the near complete elimination of [Ca2+]i release in response to ATP (AUC, 51.42 ± 5.52 vs 10.57 ± 2.56, p<0.001). These results demonstrate that T1D increases ionotropic purinergic signaling in podocytes; however, does not eliminate metabotropic signaling. These studies establish the potential for targeting both P2X4 and P2X7 for therapeutic intervention in T1D.Support or Funding InformationNational Institutes of Health grants T32 HL134643 and R35 HL135749, and Department of Veteran Affairs I01 BX004024

Maternal physical activity prevents the overexpression of hypoxia-inducible factor 1-\u03b1 and cardiorespiratory dysfunction in protein malnourished rats

Maternal physical activity attenuates cardiorespiratory dysfunctions and transcriptional alterations presented by the carotid body (CB) of rats. Rats performed physical activity and were classified as inactive/active. During gestation and lactation, mothers received either normoprotein (NP-17% protein) or low-protein diet (LP-8% protein). In offspring, biochemical serum levels, respiratory parameters, cardiovascular parameters and the mRNA expression of hypoxia-inducible factor 1-alpha (HIF-1α), tyrosine hydroxylase (TH) and purinergic receptors were evaluate. LP-inactive pups presented lower RF from 1st to 14th days old, and higher RF at 30 days than did NP-inactive and NP-active pups. LP-inactive pups presented with reduced serum protein, albumin, cholesterol and triglycerides levels and an increased fasting glucose level compared to those of NP-inactive and NP-active groups. LP and LP-inactive animals showed an increase in the cardiac variability at the Low-Frequency bands, suggesting a major influence of sympathetic nervous activity. In mRNA analyses, LP-inactive animals showed increased HIF-1α expression and similar expression of TH and purinergic receptors in the CB compared to those of NP groups. All these changes observed in LP-inactive pups were reversed in the pups of active mothers (LP-active). Maternal physical activity is able to attenuate the metabolic, cardiorespiratory and HIF-1α transcription changes induced by protein malnutrition.

P2X4 receptor as a modulator in the function of P2X receptor in CD4+ T cells from peripheral blood and adipose tissue

Obesity is characterized by immune cell infiltration and inflammation. Purinergic receptors such as P2X1, 4 and 7 are expressed on immune cells and their activation contributes with an inflammatory response. However, the simultaneous expression of P2X1, 4 and 7 during overweight or obesity have not been described. Therefore, the aim of this study was to determine single and simultaneously expression and function of the P2X1, 4 and 7 receptors in lymphocytes and CD4 + T cells from peripheral blood (PB) and adipose tissue (AT). Our results showed a higher expression of the P2X4 receptor on CD4 + T cells from PB regarding P2X7 and P2X1 receptor expression. In addition, P2X4 receptor expression on CD4 + T cells from PB and AT was increased in individuals with BMI ≥ 25 Kg/m2. Moreover, a higher simultaneous expression of the P2X4 and P2X7 receptors on CD4 + T cells from AT compared to CD4 + T cells expressing P2X1 and P2X7 receptors simultaneously. Besides, CD4 + T cells expressing P2X4 and P2X7 receptors from PB and AT were augmented in individuals with BMI ≥ 25 Kg/m2. In addition, the percentage of lymphocytes and also CD4 + T cells expressing P2X4 receptor were elevated both in PB and AT compared to cells expressing P2X7 or P2X1. However, CD4 + T cells expressing P2X4 and P2X7 were augmented in AT compared to PB. The function of the receptors showed a lower shedding of CD62 L in adipose tissue mononuclear cells (ATMC) compared with peripheral blood mononuclear cells (PBMC) and a greater participation of P2X4 in the mobilization of intracellular calcium. We concluded that it was possible to determine for the first time the simultaneous expression of purinergic receptors in ATMC, where the P2X4 receptor has a greater participation in the activation of CD4 + T cells possibly modulating the function of the other two receptors.

Fueling Melanocytes with ATP from Keratinocytes Accelerates Melanin Synthesis

Melanocyte homoeostasis and their response to ultraviolet radiation (UVR) are mediated to a large extent by keratinocyte-derived factors, many of which have been well-characterized. Lee et al. describe novel effects of adenosine 5'-triphosphate (ATP), which is secreted by keratinocytes and can stimulate melanogenesis by melanocytes following UVA exposure. The investigators attribute the melanogenic effect of ATP to binding purinergic receptors type 2 X7 (P2X7), which are expressed on human melanocytes, leading to activation of the protein kinase C pathway. This report is the first to identify expression of specific purinergic receptors on human melanocytes, and it suggests ATP as a signaling molecule that stimulates pigmentation. Follow up on these results should clarify the physiological role of ATP in mediating the tanning response to solar UVR.

Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors.

Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2 receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10–100 μM, but inhibiting at 300 μM ATP. On the other hand, 1–300 μM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

Enteric glial cells immunoreactive for P2X7 receptor are affected in the ileum following ischemia and reperfusion

The aim of this study was to analyze the effect of ischemia and reperfusion injury (IS) on enteric glial cells (EGCs) and neurons immunoreactive for the P2X7 receptor. Intestinal ischemia was induced by obstructing blood flow in the ileal vessels for 35 min. Afterwards, the vessels were reperfused for 14 days. Tissues were prepared for immunohistochemical labeling of P2X7 receptor, HuC/D (Hu) (pan-neuronal marker) and S100β (glial marker); HuC/D (Hu) and glial fibrillary acidic protein (GFAP, glial marker)/DAPI (nuclear marker); or S100β and GFAP/DAPI. Qualitative and quantitative analyses of colocalization, density, profile area and cell proliferation were performed via fluorescence and confocal laser scanning microscopy. The quantitative analyses revealed that a) neurons and EGCs were immunoreactive for P2X7 receptor; b) the P2X7 receptor immunoreactive cells and Hu immunoreactive neurons were reduced after 0 h and 14 days of reperfusion; c) the S100β and GFAP immunoreactive EGCs were increased; d) the profile area of S100β immunoreactive EGCs was increased by IS; e) few GFAP immunoreactive proliferated at 14 days of reperfusion; f) distinct populations of glial cells can be discerned: S100β+/GFAP+ cells, S100β+/GFAP– cells and S100β–/GFAP + cells; g) histological analysis revealed less alterations in the epithelium cells in the IS groups and h) myeloperoxidase reaction revealed increased of the neutrophils in the lamina propria in the IS groups. This study showed that IS is associated with significant neuronal loss, increase of glial cells and altered purinergic receptor expression and that these changes may contribute to intestinal disorders.

Phenotypic distinctions between the nodose and jugular TRPV1-positive vagal sensory neurons in the cynomolgus monkey.

Vagal capsaicin-sensitive afferent C-fibers play an important role in the maintenance of visceral homeostasis and contribute to symptoms in visceral diseases. Based on their developmental origin two functionally distinct types of vagal C-fibers are recognized: those with neurons in the vagal nodose ganglia (derived from epibranchial placodes) and in the vagal jugular ganglia (from neural crest). Studies in nonprimate species demonstrated that the vagal nodose and jugular C-fibers differ in activation profile, neurotrophic regulation, and expression of neurotransmitters. We hypothesized that the expression of selected markers related to key phenotypic properties of vagal C-fibers in the cynomolgus monkey is similar to that reported in nonprimate species. We performed single-cell RT-PCR on nodose and jugular putative C-fiber (TRPV1-positive) neurons isolated from the cynomolgus monkey. We found that the expression of purinergic P2X receptors that underlie selective responsiveness of nodose C-fiber terminals to ATP was conserved in that P2X2 and P2X3 subunits were expressed in nodose, but only P2X3 subunit was expressed in jugular TRPV1-positive neurons. Also conserved was the preferential expression of neurotrophic receptor TrkB in the nodose and preprotachykinin-A in the jugular TRPV1-positive neurons. Several key distinctions in gene expression between nodose and jugular TRPV1-positive (C-fiber) neurons that have been noted in mice, rats, and guinea pigs, are conserved in the cynomolgus monkey. Our results support the translatability of distinct vagal C-fiber phenotypes to primates.

‬The expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures during morphine withdrawal in rats

Morphine is one of the most potent analgesics used in medicine and it's long-term use is associated with the risk of the state of dependence. The cessation of chronic morphine administration leads to withdrawal signs which are associated with neurotransmitter dysregulations within mesolimbic system. Adenosine 5'-triphosphate (ATP) and purinergic system play an important role in the activity of central nervous system (CNS). Purinergic receptors are widely distributed in neurons and glial cells throughout the CNS taking part in integration of functional activity between neurons, glial and vascular cells. In the present study the mRNA and protein expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures (striatum, hippocampus and prefrontal cortex) during morphine withdrawal in rats was investigated by RT-PCR and Western Blot analysis. Two experimental models of morphine withdrawal were studied: single and repeated morphine withdrawal. We demonstrated that expression of P2X4 and P2X7 receptors was altered during morphine withdrawal period in rats. These alterations were varied in particular mesolimbic areas depending on the scheme of morphine administration. Our results extend the current knowledge on morphine withdrawal and for the first time high-light interactions between purinergic system and morphine withdrawal. It seems, the purinergic system may be a new, valuable tool in searching for a new strategy of management of opioid dependence.

Purinergic receptor expression and function in rat vagal sensory neurons innervating the stomach

The nodose ganglion (NG) is the main parasympathetic ganglion conveying sensory signals to the CNS from numerous visceral organs including digestive signals such as gastric distension or the release the gastrointestinal peptides. The response characteristics of NG neurons to ATP and ADP and pharmacological interrogation of purinergic receptor subtypes have been previously investigated but often in NG cells of undetermined visceral origin. In this study, we confirmed the presence of P2X3 and P2Y1 receptors and characterized P2X and P2Y responses in gastric-innervating NG neurons. Application of ATP-evoked large inward currents and cytosolic Ca2+ increases in gastric-innervating NG neurons. Despite the expression of P2Y1 receptors, ADP elicited only minor modulation of voltage-gated Ca2+ channels. Considering the sensitivity of NG neurons to comorbidities associated with disease or neural injury, purinergic modulation of gastric NG neurons in disease- or injury-states is worthy of further investigation.