Domain-specific purinergic signaling in polarized rat cholangiocytes

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.     

Somatostatin stimulates ductal bile absorption and inhibits ductal bile secretion in mice via SSTR2 on cholangiocytes

With an in vitro model using enclosedintrahepatic bile duct units (IBDUs) isolated from wild-type andsomatostatin receptor (SSTR) subtype 2 knockout mice, we tested theeffects of somatostatin, secretin, and a selective SSTR2 agonist(L-779976) on fluid movement across the bile duct epithelial celllayer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1,SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected incholangiocytes in wild-type mice. In contrast, SSTR2A/2B werecompletely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 andSSTR4 were expressed in these cholangiocytes. Somatostatin induced adecrease of luminal area of IBDUs isolated from wild-type mice,reflecting net fluid absorption; L-779976 also induced a comparabledecrease of luminal area. No significant decrease of luminal area byeither somatostatin or L-779976 was observed in IBDUs from SSTR2knockout mice. Secretin, a choleretic hormone, induced a significantincrease of luminal area of IBDUs of wild-type mice, reflecting netfluid secretion; somatostatin and L-779976 inhibited (P < 0.01) secretin-induced fluid secretion. The inhibitory effect ofboth somatostatin and L-779976 on secretin-induced IBDUsecretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibitedsecretin-stimulated cAMP synthesis in cholangiocytes; depletion ofSSTR2 blocked these effects of somatostatin. These data suggest thatsomatostatin regulates ductal bile formation in mice not only byinhibition of ductal fluid secretion but also by stimulation of ductalfluid absorption via interacting with SSTR2 on cholangiocytes, aprocess involving the intracellular cAMP/cGMP second messengers.

     

P2Y(11), a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y(2) receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+](i)) and stimulate Ca2+-activated Cl- and K+ channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl- channels. ATP, 2-methylthio-ATP, and ATP-gamma-S stimulated a 4- to 10-fold cAMP increase with EC(50) of 10-100 microM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y(2) or P1 receptors. Although UTP stimulated an (125)I(-) efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl- conductances through P2Y(2)-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+](i). Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I(sc) increase, whereas serosal ATP stimulated a large cAMP-type I(sc) response. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl- conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y(11) receptor mRNA, the only P2Y receptor acting via cAMP.     

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.     

A primitive ATP receptor from the little skate Raja erinacea

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61-64% similarity to P2Y(1) receptors and 21-37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y(1) receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.     

Dual roles of P2 purinergic receptors in insulin-stimulated leptin production and lipolysis in differentiated rat white adipocytes

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP > UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.     

Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP > 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP > UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.     

P2Y receptors play a critical role in epithelial cell communication and migration

Cellular injury induces a complex series of events that involves Ca2+ signaling, cell communication, and migration. One of the first responses following mechanical injury is the propagation of a Ca2+ wave (Klepeis et al. [2001] J Cell Sci 114(Pt 23):4185-4195). The wave is generated by the extracellular release of ATP, which also induces phosphorylation of ERK (Yang et al. [2004] J Cell Biochem 91(5):938-950). ATP and other nucleotides, which bind to and activate specific purinergic receptors were used to mimic injury. Our goal was to determine which of the P2Y purinergic receptors are expressed and stimulated in corneal epithelial cells and which signaling pathways are activated leading to changes in cell migration, an event critical for wound closure. In this study, we demonstrated that the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors were present in corneal epithelial cells. A potency profile was determined by Ca2+ imaging for nucleotide agonists as follows: ATP > or = UTP > ADP > or = UDP. In contrast, negligible responses were seen for beta,gamma-meATP, a general P2X receptor agonist and adenosine, a P1 receptor agonist. Homologous desensitization of the Ca2+ response was observed for the four nucleotides. However, P2Y receptor internalization and degradation was not detected following stimulation with ATP, which is in contrast to EGFR internalization observed in response to EGF. ATP induced cell migration was comparable to that of EGF and was maximal at 1 microM. Cells exposed to ATP, UTP, ADP, and UDP demonstrated a rapid twofold increase in phosphorylation of paxillin at Y31 and Y118, however, there was no activation elicited by beta,gamma-meATP or adenosine. Additional studies demonstrated that wound closure was inhibited by reactive blue 2. These results indicate that P2Y receptors play a critical role in the injury repair process.     

Upregulation of secretin receptors on cholangiocytes after bile duct ligation

Secretin not only increases ductular bile secretion in vivo in rats after bile duct ligation (BDL) [1], but also increases cAMP levels and stimulates exocytosis in isolated cholangiocytes [2]. Although we have previously reported that secretin receptor mRNA was upregulated in cholangiocytes after BDL [3], the cholangiocyte secretin receptor has not been functionally characterized or quantified after BDL. In this work, we used a novel, photolabile and biologically active analogue of secretin to quantify and characterize secretin receptors on cholangiocytes isolated from normal and BDL rats. The cholangiocyte secretin receptor bound radioligand with high affinity and in a rapid, reversible, and temperature-dependent manner. While receptors on cholangiocytes from normal and BDL rats were functionally and biochemically identical, receptor density on cholangiocytes was increased 5-fold following BDL. The combination of increased cell number with increased functional secretin receptors per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion, explains the increased hormone-responsive choleresis observed after BDL and may reflect an adaptive response of the organism to cholestasis.     

ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the 'universal' agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.     

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.     

Molecular determinants of P2Y2 nucleotide receptor function

In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.     

P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors are coupled to Rho and Rho kinase activation in vascular myocytes

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.     

Extracellular uridine nucleotides initiate cytokine production by murine dendritic cells.

While it is recognized that activated dendritic cells perform their immune functions with greater efficacy, it is not altogether clear what factors are responsible for such activation. Recent evidence points to an important role for extracellular nucleotides in the modulation of leukocyte function. In the present study we investigated the ability of extracellular nucleotides to activate CD11c(+) murine dendritic cells. Mobilization of intracellular calcium was observed following treatment of these cells with UTP or UDP, but not ATP. Furthermore, this nucleotide receptor was pertussis toxin-sensitive, suggesting the presence of a P2Y nucleotide receptor. Such receptors were not present on murine peritoneal macrophages or on CD11c-negative leukocyte populations. Importantly, activation of these P2Y nucleotide receptors on dendritic cells provided a potent stimulus for cytokine mRNA expression and secretion. Thus, expression of a P2Y nucleotide receptor on CD11c(+) dendritic cells functions to mobilize intracellular calcium and to induce cytokine production.     

Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation

We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain: the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue non-specific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca(2+) transients in neurospheres that were exclusively mediated by the metabotropic P2Y(1) and P2Y(2) nucleotide receptors. In addition, agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y(1)-receptor antagonist MRS2179 and in neurospheres from P2Y(1)-receptor knockout mice. In situ hybridization identified P2Y(1)-receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.