Search

Simple Summary: Bladder cancer is one of the most common malignancies. The prognosis is particularly poor for advanced cancer. There is a need for new prognostic markers to guide treatment decisions and promote the development of novel therapeutic strategies to increase the success rate of current treatment regimens. The tumor microenvironment is characterized by an increase in extracellular ATP levels. ATP-recognizing P2X receptors have been implicated in the growth and metastasis of various malignant cancers. Here, we analyzed the potential of different P2X receptor subtypes as prognostic markers in muscle-invasive bladder cancer. In vitro experiments confirmed a growth-promoting effect of extracellular ATP and P2X receptors on bladder cancer cells. In agreement, our analyses of cancer tissue samples from 173 patients showed that expression of P2X1 and P2X7 receptors is an independent negative predictor of survival and a potential therapeutic target in muscle-invasive bladder cancer. Bladder cancer is amongst the most common causes of cancer death worldwide. Muscle-invasive bladder cancer (MIBC) bears a particularly poor prognosis. Overexpression of purinergic P2X receptors (P2XRs) has been associated with worse outcome in several malignant tumors. Here, we investigated the role of P2XRs in bladder cancer cell proliferation in vitro and the prognostic value of P2XR expression in MIBC patients. Cell culture experiments with T24, RT4, and non-transformed TRT-HU-1 cells revealed a link between high ATP concentrations in the cell culture supernatants of bladder cell lines and a higher grade of malignancy. Furthermore, proliferation of highly malignant T24 bladder cancer cells depended on autocrine signaling through P2X receptors. P2X1R, P2X4R, and P2X7R expression was immunohistochemically analyzed in tumor specimens from 173 patients with MIBC. High P2X1R expression was associated with pathological parameters of disease progression and reduced survival time. High combined expression of P2X1R and P2X7R increased the risk of distant metastasis and was an independent negative predictor of overall and tumor-specific survival in multivariate analyses. Our results suggest that P2X1R/P2X7R expression scores are powerful negative prognostic markers in MIBC patients and that P2XR-mediated pathways are potential targets for novel therapeutic strategies in bladder cancer. [ABSTRACT FROM AUTHOR]

Platelets express P2X1 receptors and our data also show the expression of P2X7 receptors. We studied the role of both receptors in platelet apoptosis by incubation of PRP with P2X agonists, then centrifuged to remove viable platelets, and analyzed the supernatant by flow cytometry to identify a sparse platelet-derived population that stained with MitoTracker dyes and CD41. BzATP, a potent agonist of P2X receptors, and ABT737, an activator of intrinsic apoptosis, produced altered platelets that stained moderately for annexin V and corresponded to an early stage apoptotic platelet (ESAP). Over a range of BzATP concentrations, we observed a dose-dependent formation of ESAPs between 5 and 500 uM BzATP, together with a variable formation of ESAPs at nanomolar ATP or BzATP (50–200 nM). Production of ESAPs occurred with αβ-meATP, while responses with either BzATP or αβ-meATP showed desensitization at a higher agonist concentration. Formation of ESAPs by either 100 nM or 0.5 mM BzATP was inhibited by preincubation of platelets with latrunculin A, an inhibitor of the actin cytoskeleton that prevents apoptosis. ESAP production was totally inhibited by preincubation of platelets with methyl-beta-cyclodextrin, which removes cholesterol from lipid rafts. Our data show that both P2X1 and P2X7 receptors are localized in platelet lipid rafts where P2X-agonists act to produce early stage apoptotic platelets.

Introduction. Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promoting the trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs. Similarly, defective expression of these receptors on transplanted HSPCs or in the bone marrow (BM) microenvironment of graft recipient mice led to defective homing, engraftment, and delayed hematopoietic reconstitution. This correlated with decreased activation of intracellular pattern recognition receptor Nlrp3 inflammasome. The P2X receptor family consists of seven purinergic receptors (P2X1-7) and we noticed that in addition to P2X4 and P2X7, HSPCs also highly express rapidly signaling the P2X1 receptor. Therefore, we asked if P2X1 receptor is also involved in HSPCs trafficking. Material and methods. We employed in vitro and in vivo murine models to study the role of P2X1 receptor blocked on HSPCs or bone marrow microenvironment cells by specific small molecular inhibitor NF499. First, we performed in vitro cell migration assays of bone marrow mononuclear cells (BMMNCs) isolated from normal mice that were exposed to NF499 and compared them to unexposed control cells. Next, in experiments in vivo we mobilized mice exposed to NF499 with G-CSF or AMD3100 and compared mobilization to control unexposed animals. Flow cytometry was employed to identify cell populations and clonogenic assays to enumerate the number of mobilized clonogenic progenitors. Similarly, in homing and engraftment experiments BMMNCs or recipient mice were exposed to NF499 and we evaluated homing and engraftment of transplanted cells by enumerating the number of cells labeled with fluorochromes in recipient mice BM and by evaluating the number of clonogenic progenitors in BM and spleen 24 hours and 12 days after transplantation. We also evaluated the potential involvement of Nlrp3 inflammasome in P2X1 receptor-mediated HSPCs trafficking. Results. We report that the functional P2X1 receptor is highly expressed on murine and human HSPCs. We could demonstrate that the P2X1 receptor promotes the trafficking of murine cells in Nlrp3 inflammasome-dependent manner. Mice after exposure to P2X1 receptor inhibitor poorly mobilized HSPCs from the bone marrow into the peripheral blood. Mice transplanted with BMNNCs exposed to NF499 or recipient mice pretreated with this inhibitor demonstrated defective homing and engraftment as compared to control animals transplanted with cells not exposed to P2X1 inhibitor. Similar effects were noticed for control recipient mice that were not exposed to NF499. Conclusions. This study demonstrates for the first time the novel role of the P2X1 receptor in HSPCs trafficking in the mouse. Furthermore, it supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in the mobilization, homing and engraftment of HSPCs. (Folia Histochemica et Cytobiologica 2022, Vol. 60, No. 3, 280-290). [ABSTRACT FROM AUTHOR]

Platelets express P2X1 receptors and our data also show the expression of P2X7 receptors. We studied the role of both receptors in platelet apoptosis by incubation of PRP with P2X agonists, then centrifuged to remove viable platelets, and analyzed the supernatant by flow cytometry to identify a sparse platelet-derived population that stained with MitoTracker dyes and CD41. BzATP, a potent agonist of P2X receptors, and ABT737, an activator of intrinsic apoptosis, produced altered platelets that stained moderately for annexin V and corresponded to an early stage apoptotic platelet (ESAP). Over a range of BzATP concentrations, we observed a dose-dependent formation of ESAPs between 5 and 500 uM BzATP, together with a variable formation of ESAPs at nanomolar ATP or BzATP (50–200 nM). Production of ESAPs occurred with αβ-meATP, while responses with either BzATP or αβ-meATP showed desensitization at a higher agonist concentration. Formation of ESAPs by either 100 nM or 0.5 mM BzATP was inhibited by preincubation of platelets with latrunculin A, an inhibitor of the actin cytoskeleton that prevents apoptosis. ESAP production was totally inhibited by preincubation of platelets with methyl-beta-cyclodextrin, which removes cholesterol from lipid rafts. Our data show that both P2X1 and P2X7 receptors are localized in platelet lipid rafts where P2X-agonists act to produce early stage apoptotic platelets. [ABSTRACT FROM AUTHOR]

• P2X4 receptor on CD4 + T cells from PB and AT was increased in individuals with BMI ≥ 25 Kg/m2. • CD4 + T cells expressing P2X4 and P2X7 from PB and AT were augmented in individuals with BMI ≥ 25 Kg/m2. • CD4 + T cells with P2X4 and P2X7 showed a higher percentage in cells isolated from AT compared to PB. • ATP-induced shedding of CD62 L was higher in PBMC than in ATMC. • Influx of Ca2+ dependent of ATP-induced P2X4 activation in PBMC was higher than in ATMC. 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. [ABSTRACT FROM AUTHOR]

With immunohistochemical and Western blot techniques, P2X1 receptors were detected in the whole mouse gastrointestinal tract and pancreatic islets of mouse and human. (1) δ Cells containing somatostatin (SOM) in the stomach corpus, small intestines, distal colon, pancreatic islets of both mouse and human express P2X1 receptors; (2) strong immunofluorescence of P2X1 receptors was detected in smooth muscle fibers and capillary networks of the villus core of mouse intestine; and (3) P2X1 receptor-immunoreactive neurons were also detected widely in both mouse myenteric and submucosal plexuses, all of which express SOM. The present data implies that ATP via P2X1 receptors is involved in SOM release from pancreatic δ cells, enteric neurons, and capillary networks in villi. [ABSTRACT FROM AUTHOR]

Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promote trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs as well as defective expression of these receptors on transplanted HSPCs or in bone marrow (BM) microenvironment of graft recipient mice lead to defective homing, engraftment and delayed hematopoietic reconstitution. We explained these defects as result of an impaired activation of downstream target from P2X4 and P2X7 receptors that is intracellular pattern recognition receptor Nlrp3 inflammasome. P2X receptor family consists of seven purinergic receptors (P2X1-7). We report that one of the members of this receptor family ― P2X1 receptor is highly expressed on HSPCs and promotes migration of leucocytes and lymphocytes. This prompted us to investigate its role in trafficking of more primitive hematopoietic cells. Our data indicates that P2X1 receptor is involved in eATP mediated trafficking of HSPCs, and this occurs in Nlrp3 inflammasome-dependent manner. This study sheds light on novel role of P2X1 receptor in normal hematopoiesis and further supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in mobilization, homing and engraftment of HSPCs. [ABSTRACT FROM AUTHOR]

P2X1 receptors and adrenoceptors are mainly responsible for vasoconstriction in a variety of blood vessels. However, previous studies have shown that α,β-methylene adenosine 5′-triphosphate (α,β-MeATP), a stable analogue of ATP, can induce both pressor and depressor responses in laboratory animals. In this study, the effects of increasing intravenous doses of α,β-MeATP and noradrenaline (NA) (0–30 nmol/kg) administered at 20 min intervals on systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure in groups of anesthetized mice (n=6) were compared. Both α,β-MeATP and NA caused transient, dose-dependent increases in SBP, DBP and MBP but the effect of α,β-MeATP was more rapid and significantly larger at doses of 10 and 30 nmol/kg (P

Purinergic signaling involves the activation of cell surface P1 and P2 receptors by extracellular nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP), respectively. P2 receptors comprise P2X and P2Y receptors, and have well-established roles in leukocyte and platelet biology. Emerging evidence indicates important roles for these receptors in red blood cells. P2 receptor activation stimulates a number of signaling pathways in progenitor red blood cells resulting in microparticle release, reactive oxygen species formation and apoptosis. Likewise, activation of P2 receptors in mature red blood cells stimulates signaling pathways mediating volume regulation, eicosanoid release, phosphatidylserine exposure, hemolysis, impaired ATP release, and susceptibility or resistance to infection. This review summarizes the distribution of P2 receptors in red blood cells, and outlines the functions of P2 receptor signaling in these cells and its implications in red blood cell biology.

The pathologic hallmark of Parkinson's disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation and/or aggregation. Using 2 neuronal cell models, we found that extracellular adenosine triphosphate (ATP) induced a significant increase in intracellular αsyn levels between 24 and 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells' response to extracellular ATP. Although Ca 2+ influx via P2X1 receptor is necessary for αsyn accumulation, Ca 2+ influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD. [ABSTRACT FROM AUTHOR]

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca] in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca] by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP. [ABSTRACT FROM AUTHOR]

Adenine nucleotides acting at P2X 1 receptors are potent vasoconstrictors. Recently, we demonstrated that activation of adenosine A 2B receptors on human coronary smooth muscle cells inhibits cell proliferation by the induction of the nuclear receptor subfamily 4, group A, member 1 (NR4A1; alternative notation Nur77). In the present study, we searched for long-term effects mediated by P2X 1 receptors by analyzing receptor-mediated changes in cell proliferation and in the expression of NR4A1. Cultured human coronary smooth muscle cells were treated with selective receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The P2X receptor agonist α,β-methylene-ATP and its analog β,γ-methylene-ATP inhibited cell proliferation by about 50 % after 5 days in culture with half-maximal concentrations of 0.3 and 0.08 μM, respectively. The effects were abolished or markedly attenuated by the P2X 1 receptor antagonist NF449 (carbonylbis-imino-benzene-triylbis-(carbonylimino)tetrakis-benzene-1,3-disulfonic acid; 100 nM and 1 μM). α,β-methylene-ATP and β,γ-methylene-ATP applied for 30 min to 4 h increased the expression of NR4A1; NF449 blocked or attenuated this effect. Small interfering RNA directed against NR4A1 diminished the antiproliferative effects of α,β-methylene-ATP and β,γ-methylene-ATP. α,β-methylene-ATP (0.1 to 30 μM) decreased migration of cultured human coronary smooth muscle cells in a chamber measuring changes in impedance; NF449 blocked the effect. In conclusion, our results demonstrate for the first time that adenine nucleotides acting at P2X 1 receptors inhibit the proliferation of human coronary smooth muscle cells via the induction of the early gene NR4A1. [ABSTRACT FROM AUTHOR]

Highlights: [•] Alexa-647-ATP is a potent fluorescent agonist of P2X receptors. [•] Three agonist molecules are required to desensitize the P2X1 receptor. [•] Agonist remains trapped in a high affinity desensitized state of P2X1 receptor. [•] Slow agonist unbinding appears to be the rate limiting step in resensitization. [•] Recovery from desensitization is a cooperative process. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: P2X1 receptors and adrenoceptors are mainly responsible for vasoconstriction in a variety of blood vessels. However, previous studies have shown that α,β-methylene adenosine 5′-triphosphate (α,β-MeATP), a stable analogue of ATP, can induce both pressor and depressor responses in laboratory animals. In this study, the effects of increasing intravenous doses of α,β-MeATP and noradrenaline (NA) (0–30nmol/kg) administered at 20min intervals on systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure in groups of anesthetized mice (n=6) were compared. Both α,β-MeATP and NA caused transient, dose-dependent increases in SBP, DBP and MBP but the effect of α,β-MeATP was more rapid and significantly larger at doses of 10 and 30nmol/kg (P<0.01). At the dose of 30nmol/kg, α,β-MeATP increased SBP, DBP and MBP by 65.8±7.0, 65.7±5.0 and 65.7±5.5mmHg, respectively, compared to increases of 36.8±4.6, 33.3±4.9 and 34.5±4.7mmHg, respectively, produced by NA. These results indicate P2X1 receptors play an important role in BP regulation although purinergic vasoconstriction alone may not explain the more potent pressor response to α,β-MeATP in the anesthetized mouse. [Copyright &y& Elsevier]

Background: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. Methods: We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. Results: The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Conclusions: Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression of P2X1 receptors and connexin 43 in gap junctions between smooth muscle cells. Contraction mediated by P2X receptors is known to occur in the bladder and male reproductive tract, and cell–cell coupling of smooth muscle via gap junctions is essential for synchronized rhythmic activity of these tissues. Design: We selected for this study rat myometrial smooth muscle during pregnancy and at postpartum day l. Setting: University medical school. Animal(s): Laboratory rats. Intervention(s): Rats were mated and became pregnant. Main Outcome Measure(s): Immunostaining and fluorescence and confocal microscopy. Result(s): The level of P2X1 receptor expression remained low throughout pregnancy (days 4 to 20) but was greatly up-regulated at day 22 (postpartum day 1). Connexin 43 expression showed a pattern of up-regulation, with progression through pregnancy and peaking near labor, but exhibited a rapid down-regulation after parturition. Conclusion(s): The functional significance of the changes in connexin 43 and P2X1 receptor expression that have been observed is discussed in relation to triggering and modulation of uterine contractility during and after pregnancy. [Copyright &y& Elsevier]

Acetylcholine and ATP are excitatory cotransmitters in parasympathetic nerves. We used P2X1 receptor antagonists to further characterize the purinergic component of neurotransmission in isolated detrusor muscle of guinea pig urinary bladder. In the presence of atropine (1 µM) and prazosin (100 nM), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (0.1-100 µM) and suramin (1-300 µM) inhibited contractions evoked by 4 Hz nerve stimulation in a concentration-dependent manner (IC50 of 6.9 and 13.4 µM, respectively). Maximum inhibition was 50-60%, which was unaffected by coadministration of the ectonucleotidase inhibitor ARL67156 (6-N,N-diethyl-D-β,γ-dibromomethyleneATP) (100 µM). The remaining responses were abolished by tetrodotoxin (1 µM). PPADS and suramin also reduced contractions to exogenous ATP (300 µM) by 40 -50%, but abolished those to the P2X1 agonist α,β-methyleneATP (α,β-meATP) (1 µM). The P2X1 antagonists reactive blue 2, NF279 (8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1- phenylenecarbonylimino)] bis-1,3,5-naphthalenetrisulfonic acid), MRS2159 (pyridoxal-α5-phosphate-6-phenylazo-4′-carboxylic acid) (100 µM), and NF449 [4,4′,4 ,4 -(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid] (3 µM) abolished contractions to α,β-meATP (1 µM; n = 4 -5), but only reduced contractions evoked by 4 Hz nerve stimulation by ∼40-60% (n = 4-6) and ATP by 30-60% (n = 4 -7). However, prolonged exposure to α,β-meATP (50 µM) abolished contractions evoked by all three stimuli (n=5-12). PPADS (100 µM) and suramin (300 µM) reduced the peak neurogenic contraction of the mouse urinary bladder to 30-40% of control. At the same concentrations, the P2X1 antagonists abolished the nonadrenergic, purinergic component of neurogenic contractions in the guinea pig vas deferens (n = 4 -5). Thus, P2X1 receptor antagonists inhibit, but do not abolish, the noncholinergic component of neurogenic contractions of guinea pig and mouse urinary bladder, indicating a second mode of action of neuronally released ATP. This has important implications for treatment of dysfunctional urinary bladder, for which this atropine- and P2X1 antagonist-resistant site represents a novel therapeutic target. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of histamine on field-stimulated contractions of the guinea-pig prostate using isolated preparations in organ bath experiments. The histamine receptor subtype involved in potentiating the twitch responses was characterised, and the presence of any post-synaptic effects was determined. In addition, the effects of histamine on nerve-stimulated contractions of the ventral and dorsal prostate as well as the coagulating gland were compared in preliminary experiments. Histamine (300 μM) approximately doubled the magnitude of the twitch contractions in the ventral and dorsal lobes of the prostate (nerve stimulation parameters were 10 Hz for 2 s every 50 s, 0.5 ms duration at supramaximal voltage). In the coagulating glands, histamine increased the contractions more than fourfold. Further experiments were performed only on the ventral glands. The H1 receptor antagonist, mepyramine, exhibited an apparent competitive antagonism against the histamine-induced potentiations of the twitch responses (apparent pKB value = 9.21±0.17 ( n=5). The H2 receptor antagonist, ranitidine, produced a small, significant shift to the right, as did the time control. The H3 receptor antagonist, thioperamide, had no significant effect on the concentration–response curve. The effects of histamine (10 μM) on exogenously applied acetylcholine (Ach), noradrenaline (NA) and adenosine 5′-triphosphate (ATP) were determined. In each case, the contractile responses were significantly potentiated: ATP (5.8-fold P<0.0001, n=8), NA (1.6-fold P<0.05, n=10) and Ach (2.1-fold P<0.0007, n=10). This is the first study that has shown the effects of histamine on field-stimulated contractions in the prostate of any species. It is concluded that histamine, acting on H1 receptors, elicits a considerable potentiation of the nerve-stimulated twitch contractions in the guinea-pig prostate. One mechanism whereby histamine exerts its potentiating effect is a post-synaptic enhancement of the response to ATP in particular, but also to NA and Ach. It is suggested that H1-receptor and purinoceptor antagonists may have a role to play in the treatment of benign prostatic hyperplasia (BPH). [ABSTRACT FROM AUTHOR]

Background: It has been suggested that gap-junctional conductance between cardiomyocytes is regulated through a specific ligand-receptor interaction between ATP and connexins. In this study we examined the localization of P2X1 ionotropic receptors and their relation to connexin43 in gap junctions in human left ventricles. Methods and results: Using immunohistochemistry, we detected P2X1 expression predominantly in the intercalated discs. Labelling of the P2X1 receptor and the gap junction protein connexin43 showed close association in some gap junctions, while in others the two proteins often appeared to be spatially discrete. Western blotting detected four major bands at 45, 60, 95 and 120 kDa in the protein extracts from human left ventricles corresponding to equivalent bands from rat vas deferens. The most prominent band in human left ventricles was at 95 kDa, possibly a dimer of the native P2X1 receptor, whereas in rat vas deferens it was at 60 kDa. After preincubation of the antibody with its epitope peptide, the 45 and 60 kDa bands almost disappeared and the 95 and 120 kDa bands were significantly attenuated. Conclusions: P2X1 receptors in human myocardium are densely localized in gap junctions at intercalated discs between muscle cells. Close association of P2X1 receptors and connexin 43 occurred in some regions of some gap junctions, but in others they were spatially separate. Little difference in the pattern of distribution of P2X1 receptors was found in failing left ventricles of patients with dilated cardiomyopathy, although Western blots showed an enhancement of P2X1 receptor protein. [Copyright &y& Elsevier]

1 ATP is an important vasoactive mediator, which acts via two receptor classes: P2X and P2Y. Activation of P2X receptors has traditionally been associated with the well-characterised vasoconstrictor properties of ATP. 2 In the current study, we have shown that the P2X(1 & 3) receptor ligand, alpha, beta methylene ATP, induces vasodilation of rat isolated mesenteric arteries and that P2X1 receptors are abundantly expressed in the endothelium of these vessels. 3 Second-order rat mesenteric arteries were mounted in myographs and vasomotor responses recorded. Both ATP and alpha, beta methylene ATP induced a constriction followed by a vasodilation. The dilator effects of either ATP or alpha, beta methylene ATP were slower in onset than those induced by acetylcholine. By contrast, the traditional vasodilator P2Y ligand, ADP, induced vasodilation without contraction. 4 Vasodilation induced by alpha, beta methylene ATP was endothelial dependent, but was not affected by treatment of the vessels with L-NAME plus indomethacin alone. Dilation was, however, partially inhibited by the combination of apamin plus charybdotoxin and blocked by treating vessels with all four drugs. 5 Using confocal microscopy, P2X1 receptor immunoreactivity was localised to the endothelial, smooth muscle and adventitial layers of mesenteric vessels. P2X1 protein migrated as a primary band at around 50-60 kDa in vascular tissue. 6 These results show for the first time that P2X1 receptors are expressed on the endothelium and that a selective ligand of this receptor results in vasoconstriction followed by vasodilation. These observations have important implications for our understanding of the role of purines in biological responses. [ABSTRACT FROM AUTHOR]

The four highly conserved intracellular tyrosine residues of the P2X1 ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X1 activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X1 immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X1 endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X1 domains. [Copyright &y& Elsevier]

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, a, b methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.