- [Show abstract] [Hide abstract] ABSTRACT: The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR), promoted Ang II-induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II-induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein-dependent vascular hypertrophy but reduced b-arrestin-dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II-induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascularsmoothmuscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin-dependent proliferation to G protein- dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II.
- [Show abstract] [Hide abstract] ABSTRACT: Background/Objectives We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor and P2Y(12) receptor deficiency in vivo. Methods Deep vein thrombosis was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor or vehicle and in P2Y(12)-/- mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles. ResultsIncubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25min. Sham-operated mice, enoxaparin- and ticagrelor-pretreated wild-type and P2Y(12)-/- mice developed only small thrombi. After injection of the contrast agent, growing thrombi were delineated clearly as negative contrast on CEUS. Thrombus size on CEUS after 25min was significantly smaller in enoxaparin- (0.3 +/- 0.1mm(2)) and ticagrelor-treated (0.5 +/- 0.1mm(2)) wild-type and in P2Y(12)-/- mice (0.4 +/- 0.1mm(2)) as compared with vehicle-treated wild-type mice (2.0 +/- 0.3mm(2)) in the maximal sagittal plane (P<0.001, n=5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT. Conclusions Contrast-enhanced ultrasound allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y(12) receptor in early DVT formation.
- [Show abstract] [Hide abstract] ABSTRACT: Extracellular nucleotides promote vascular constriction through cell membrane P2 receptors. This effect involves neurogenic activation of vascular smooth muscle cell P2X1 (ATP) and some pyrimidine-sensitive (UDP, UTP) P2Y receptors. We used knockout mouse models to unravel the role of extracellular nucleotides in myogenic tone of resistance arteries. The contractile effects of exogenous UDP and UTP is abrogated in P2RY6-/- arteries suggesting that P2Y6 receptor fully underlies vascular contraction to uracyl nucleotides. Moreover, the deletion of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1 or CD39) the dominant ectoenzyme hydrolysing nucleotides at the smooth muscle surface (Entpd1-/- mice) unmasks potent constrictor effect of UDP and UTP in conductance (thoracic aorta) and resistance (mesenteric) arteries. Mirroring these observations, myogenic tone was diminished in P2RY6-/- while it was exaggerated in Entpd1-/- mesenteric arteries. This suggests some autocrine release of extracellular nucleotides participates in resistance arteries autoregulation trough P2Y6 receptor activation. Indeed, extracellular nucleotides release is well known to occur in response to cell strain (stretch). Involvement of Panx1 in this release is likely and was corroborated by pharmacological approach as well as dye uptake experiments. Finally, we propose that this signalling by extracellular nucleotides may contribute to the regulation of blood pressure in pathological conditions since P2RY6-/- mice are partially resistant to experimental hypertension.
- [Show abstract] [Hide abstract] ABSTRACT: The clearance of tissue debris by microglia is a crucial component of maintaining brain homeostasis. Microglia continuously survey the brain parenchyma and utilize extracellular nucleotides to trigger the initiation of their dynamic responses. Extracellular uridine diphosphate (UDP), which leaks or is released from damaged neurons, has been reported to stimulate the phagocytotic activity of microglia through P2Y(6) receptor activation. However, the intracellular mechanisms underlying microglial P2Y(6) receptor signals have not been identified. In this study, we demonstrated that UDP stimulation induced immediate and long-lasting dynamic movements in the cell membrane. After 60 min of UDP stimulation, there was an upregulation in the number of large vacuoles formed in the cell that incorporate extracellular fluorescent-labeled dextran, which indicates microglial macropinocytosis. In addition, UDP-induced vacuole formation and continuous membrane motility were suppressed by the protein kinase D (PKD) inhibitors, Gö6976 and CID755673, unlike Gö6983, which is far less sensitive to PKD. The inhibition of PKD also reduced UDP-induced incorporation of fluorescent-labeled dextran and soluble β-amyloid and phagocytosis of microspheres. UDP induced rapid phosphorylation and membrane translocation of PKD, which was abrogated by the inhibition of protein kinase C (PKC) with Gö6983. However, Gö6983 failed to suppress UDP-induced incorporation of microspheres. Finally, we found that inhibition of PKD by CID755673 significantly suppressed UDP-induced engulfment of IgG-opsonized microspheres. These data suggest that a PKC-independent function of PKD regulates UDP-induced membrane movement and contributes to the increased uptake of extracellular fluid and microspheres in microglia.
- [Show abstract] [Hide abstract] ABSTRACT: Extracellular nucleotides have widespread effects and various cell responses. Whereas the effect of a purine nucleotide (ATP) and a pyrimidine nucleotide (UTP) on myocardial infarction has been examined, the role of different purine and pyrimidine nucleotides and nucleosides in cardioprotection against hypoxic stress has not been reported. To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia. Rat cultured cardiomyocytes were treated with various extracellular nucleotides and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y₂ -/- , P2Y₄ -/-, and P2Y₂ -/-/P2Y₄ -/- receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. Extracellular tri/di-phosphates are apparently the molecule responsible for cardioprotection against hypoxic damage, probably by preventing free radicals formation.
- [Show abstract] [Hide abstract] ABSTRACT: Extracellular ATP contributes to the pathogenesis of asthma via signalling at purinergic receptors. However, the precise purinergic receptors subtypes mediating the pro-asthmatic effects of ATP have not been identified, yet. In vivo studies were performed using the OVA-alum model. Functional expression of the P2Y(2) purinergic receptor subtype on human monocyte-derived dendritic cells and eosinophils was investigated using real-time PCR, migration assays, and production of reactive oxygen species. Compared to wild-type animals P2Y(2) -/- mice showed reduced allergic airway inflammation which can be explained by defective migration of blood myeloid DCs towards ATP in vitro and in vivo, whereas the influence of ATP on maturation and cytokine production was not changed. Additionally, ATP failed to induce migration of bone marrow-derived eosinophils from P2Y(2) R-deficient animals. The relevance of our findings for humans was confirmed in functional studies with human monocyte-derived DCs and eosinophils. Interestingly, stimulation of human DCs derived from allergic individuals with house dust mite allergen induced functional up-regulation of the P2Y(2) R subtype. Furthermore, eosinophils isolated from asthmatic individuals expressed higher levels of P2Y(2) R compared to healthy controls. This was of functional relevance as these eosinophils were more sensitive to ATP-induced migration and production of reactive oxygen metabolites. In summary, P2Y(2) R appears to be involved in asthmatic airway inflammation by mediating ATP-triggered migration of mDCs and eosinophils, as well as reactive oxygen species production. Together our data suggest that targeting P2Y(2) R might be a therapeutic option for the treatment of asthma.
- [Show abstract] [Hide abstract] ABSTRACT: Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice. Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.
- [Show abstract] [Hide abstract] ABSTRACT: Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na+ channel (ENaC)-mediated Na+ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y2 and a luminal P2Y4 receptor, stimulate K+ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na+ absorption in distal colonic mucosa of mice treated on a low Na+ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R te): 24.1 ± 1.8 Ω cm2, equivalent short circuit current (I sc): −563.9 ± 63.8 μA/cm2 (n = 21). Amiloride completely inhibited I sc to −0.5 ± 8.5 μA/cm2. Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I sc by 160.7 ± 29.7 μA/cm2 (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC50 values of 10 μM and 3 μM respectively. In P2Y2 knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na+ absorption was absent. In contrast, in P2Y4 KO mice the inhibitory effect of luminal UTP on Na+ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na+ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y2 and P2Y4 receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y2 receptor mediates inhibition of electrogenic Na+ absorption.
- [Show abstract] [Hide abstract] ABSTRACT: Based on pharmacological criteria, we previously suggested that in the mouse aorta endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (ADP), P2Y2 (ATP) and P2Y6 (UDP) receptors. For UTP it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and ATPS-evoked relaxation, suggesting that in WT mice ATP and ATPS activate predominantly the P2Y2-subtype. The ATP/ATPS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, since they were sensitive to 2′-deoxy-N6-methyladenosine3′,5′-bisphosphate (MRS2179), a P2Y1 selective antagonist. In contrast to ATP, the UTP-evoked relaxation was not different among knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6 (like) receptor subtype. In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6 (like) receptor.
- [Show abstract] [Hide abstract] ABSTRACT: Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K+ secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP-stimulated K+ secretion was still present in P2Y2 receptor knock-out mice, but significantly reduced (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 2.08 +/- 0.52 mV, n = 9). In P2Y4 receptor knock-out mice the UTP-induced K+ secretion was similarly reduced. Luminal UTP-stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.
- [Show abstract] [Hide abstract] ABSTRACT: Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour.
- [Show abstract] [Hide abstract] ABSTRACT: Through the cAMP signaling pathway, TSH stimulates thyroid follicular cell proliferation, differentiation, and function. Although the autocrine production of IGF-I in the thyroid gland suggests an important physiological function for this factor in these processes, the exact role of the IGF-I/IGF-I receptor system in vivo remains unclear. Although the mitogenic action of TSH requires the presence of IGF-I or insulin in primary culture of dog and human thyroid cells, IGF-I has an effect equal to and independent of the effect of TSH on cell proliferation in rat thyroid cell lines and may even be the main growth regulator in this case. To investigate the in vivo function of the IGF-I/IGF-I receptor system, transgenic mice overexpressing human IGF-I, IGF-I receptor, or both in the thyroid were generated. Adult transgenic mice did not present external signs of thyroid dysfunction, but mice overexpressing both transgenes had significantly increased gland weight and follicular lumen area. A decreased TSH level together with a slightly increased serum T(4) concentration and increased thyroidal iodine uptake were also observed, suggesting that IGF-I and IGF-I receptor stimulate thyroid function to some extent in vivo.
- [Show abstract] [Hide abstract] ABSTRACT: Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531 s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.
- [Show abstract] [Hide abstract] ABSTRACT: Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.
Free University of Brussels
Bruxelles, Brussels Capital Region, Belgium
- Institute of Interdisciplinary Research (IRIBHM)