Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells

Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.     

Expression of P2Y receptors in the rat anterior pituitary

In this study, the distribution patterns of P2Y₁, P2Y₂ P2Y₄, P2Y₆, P2Y₁₂, and P2Y₁₃ receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y₁ and P2Y₄ receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y₂ receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also stained with S100 protein immunoreactivity. P2Y₆ receptors were expressed in macrophages. P2Y₁₃ receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to be clarified. P2Y₁ and P2Y₄ receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs via P2Y₁ and P2Y₄ receptors, some lactotrophs via P2Y₂ receptors, and folliculo-stellate cells via P2Y₂ receptors in the rat anterior pituitary.     

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5′-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters’ cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y₄, P2Y₆ and P2Y₁₄ receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.     

Angiotensin II increases mRNA levels of all TGF‐β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF‐β (transforming growth factor‐β). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF‐β actions. Here, we studied the effect of AII on the mRNA levels of TGF‐β isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF‐β1, TGF‐β2 and TGF‐β3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF‐β isoforms, particularly TGF‐β2 and TGF‐β3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF‐β protein after AII treatment. The mRNA expression of TGF‐β isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF‐β expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.     

Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells

Depletion of peroxisome proliferator-activated receptor gamma (PPARgamma) accompanies myofibroblastic transdifferentiation of hepatic stellate cells (HSC), the primary cellular event underlying liver fibrogenesis. The treatment of activated HSC in vitro or in vivo with synthetic PPARgamma ligands suppresses the fibrogenic activity of HSC. However, it is uncertain whether PPARgamma is indeed a molecular target of this effect, because the ligands are also known to have receptor-independent actions. To test this question, the present study examined the effects of forced expression of PPARgamma via an adenoviral vector on morphologic and biochemical features of culture-activated HSC. The vector-mediated expression of PPARgamma itself is sufficient to reverse the morphology of activated HSC to the quiescent phenotype with retracted cytoplasm, prominent dendritic processes, reduced stress fibers, and accumulation of retinyl palmitate. These effects are abrogated by concomitant expression of a dominant negative mutant of PPARgamma that prevents transactivation of but not binding to the PPAR response element. PPARgamma expression also inhibits the activation markers such as the expression of alpha-smooth muscle actin, type I collagen, and transforming growth factor beta1; DNA synthesis; and JunD binding to the activator protein-1 (AP-1) site and AP-1 promoter activity. Inhibited JunD activity by PPARgamma is not due to reduced JunD expression or JNK activity or to a competition for p300. But it is due to a JunD-PPARgamma interaction as demonstrated by co-immunoprecipitation and glutathione S-transferase pull-down analysis. Further, the use of deletion constructs reveals that the DNA binding region of PPARgamma is the JunD interaction domain. In summary, our results demonstrate that the restoration of PPARgamma reverses the activated HSC to the quiescent phenotype and suppresses AP-1 activity via a physical interaction between PPARgamma and JunD.     

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue.Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL.Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels.In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.     

The P2Y nucleotide receptors in the human genome

Since the first identification of P2Y receptor sequences in 1993, it has quickly become apparent that this family of the G-protein coupled receptors is very diverse. Members of this receptor family are activated extra-cellularly by a wide variety of adenosine and uridine nucleotides including sugar-nucleotides. The recent decipherment of the Human Genome has enabled us to search for new, yet undiscovered P2Y receptor subtypes. In this article we examine the relationships of six orphan G-protein coupled receptor (GPCR) sequences which show considerable sequence homology to various P2Y receptors. The clustering at a few chromosomal loci of P2Y receptor genes and their related orphan genes further suggests that particular P2Y subsets were derived from the same ancestral gene during evolution.     

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.     

Apoptosis in activated rat pancreatic stellate cells

Proliferation and matrix synthesis by activated pancreatic stellate cells (PSC) participate in the development of chronic pancreatitis. Apoptosis of PSC may terminate this process but has not yet been studied in this particular cell type and was the aim of the present study. PSC were isolated from rat pancreas and characterized for expression of glial fibrillary acidic protein, alpha-smooth muscle actin, CD95, and tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) receptors. Apoptosis was determined by TdT-UTP nick end-labeling reaction, annexin V binding, and caspase-8 activation. Both CD95L and TRAIL induced apoptosis in PSC. The apoptotic response was minor in PSC cultured for 7 days but increased markedly thereafter. Sensitization of PSC with culture duration was accompanied by increased expression of CD95 and TRAIL receptor 2 and no alterations of Flip expression or protein kinase B phosphorylation but was paralleled by the appearance of a COOH-terminal cleavage product of receptor-interacting protein. PSC apoptosis was also induced by PK-11195, a ligand of the peripheral benzodiazepine receptor. PSC apoptosis may be important in terminating the wound-healing response after pancreas injury and exhibits features distinct from apoptosis induction in hepatic stellate cells.     

P2Y1, P2Y6, and P2Y12 receptors in rat splenic sinus endothelial cells: an immunohistochemical and ultrastructural study

Localization of three P2X and six P2Y receptors in sinus endothelial cells of the rat spleen was examined by immunofluorescent microscopy, and ultrastructural localization of the detected receptors was examined by immunogold electron microscopy. In immunofluorescent microscopy, labeling for anti-P2Y1, P2Y6, and P2Y12 receptors was detected in endothelial cells, but P2X1, P2X2, P2X4, P2Y2, P2Y4, and P2Y13 receptors was not detected. P2Y1 and P2Y12 receptors were prominently localized in the basal parts of endothelial cells. P2Y6 receptor was not only predominantly localized in the basal parts of endothelial cells, but also in the superficial layer. Triple immunofluorescent staining for a combination of two P2Y receptors and actin filaments showed that P2Y1, P2Y6, and P2Y12 receptors were individually localized in endothelial cells. Phospholipase C-β3, phospholipase C- γ2, and inositol-1,4,5-trisphosphate receptors, related to the release of the intracellular Ca²⁺ from the endoplasmic reticulum, were also predominantly localized in the basal parts of endothelial cells. In immunogold electron microscopy, labeling for P2Y1, P2Y6, and P2Y12 receptors were predominantly localized in the basal part of endothelial cells and, in addition, in the junctional membrane, basal plasma membrane, and caveolae in the basal part of endothelial cells. Labeling for phospholipase C-β3 and phospholipase C-γ2 was dominantly localized in the basal parts and in close proximity to the plasma membranes of endothelial cells. The possible functional roles of these P2Y receptors in splenic sinus endothelial cells are discussed.     

Expression and a role of functionally coupled P2Y receptors in human dendritic cells

We investigated the physiology and function of P2Y receptors expressed in human dendritic cells (DCs) differentiated in vitro from CD14+ cells (DC-14). These were obtained after a 10 day stimulation period in GM-CSF, IL-4 and monocyte conditioned medium. DC-14 were found to express high amounts of MHC class II, B7, CD40 as well as CD83. The functional analysis, using single cell Ca2+ imaging, demonstrated the expression of at least three subtypes of P2Y receptors. We further found using patch-clamp measurements that ATP evoked a pertussis toxin insensitive non-selective cation current with a peak current amplitude of -276+/-43 pA (holding potential -80 mV, n = 23). This current was not Ca(2+)-activated, since it was still observed under conditions of high intracellular Ca2+ buffering and could be blocked by Gd3+ (0.5 mM). In addition, intracellular application of GTP-gamma-S (0.3 mM) also activated the current. Interestingly, DC-14 redirected the orientation of their dendrites as well as cell shape towards a pipette containing ATP as observed with time lapse microscopy. These data suggest that in human DCs, ATP acts via P2Y receptors and induces chemokine effects.     

P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing

The amyloid precursor protein (APP) is proteolytically processed by beta- and gamma-secretases to release amyloid beta, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid beta domain by alpha-secretase releasing the non-amyloidogenic product sAPP alpha, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate alpha-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP alpha in a time- and dose-dependent manner. P2Y2 R-mediated sAPP alpha release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP alpha release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPalpha release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPP alpha release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPP alpha release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP alpha release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.     

Immunolocalisation of P2Y receptors in the rat eye

Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y₁ receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y₂ receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y₄ was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y₆ presented almost an identical distribution as the P2Y₄ receptor. The P2Y₁₁ was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function.     

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y₂ receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y₂ receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y₂ receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y₂ receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y₂ receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y₂ receptors. While ATP and UTP activated the murine and human P2Y₂ receptors with similar potencies (EC₅₀ values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC₅₀ = 9.1-21.2 M) than UTP (IC₅₀ = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.