Expression Of P2Y Receptors Research Articles

P2 receptor‐mediated stimulation of the PI3‐K/Akt‐pathway in vivo

ATP acts as a growth factor as well as a toxic agent by stimulating P2 receptors. The P2 receptor-activated signaling cascades mediating cellular growth and cell survival after injury are only incompletely understood. Therefore, the aim of the present study was to identify the role of the phosphoinositide 3 kinase (PI3-K/Akt) and the mitogen-activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathways in P2Y receptor-mediated astrogliosis after traumatic injury and after microinfusion of ADP beta S (P2Y(1,12,13) receptor agonist) into the rat nucleus accumbens (NAc). Mechanical damage and even more the concomitant treatment with ADP beta S, enhanced P2Y(1) receptor-expression in the NAc, which could be reduced by pretreatment with the P2X/Y receptor antagonist PPADS. Quantitative Western blot analysis indicated a significant increase in phosphorylated (p)Akt and pERK1/2 2 h after ADP beta S-microinjection. Pretreatment with PPADS or wortmannin abolished the up-regulation of pAkt by injury alone or ADP beta S-treatment. The ADP beta S-enhanced expression of the early apoptosis marker active caspase 3 was reduced by PPADS and PD98059, but not by wortmannin. Multiple immunofluorescence labeling indicated a time-dependent expression of pAkt and pMAPK on astrocytes and neurons and additionally the colocalization of pAkt, pMAPK, and active caspase 3 with the P2Y(1) receptor especially at astrocytes. In conclusion, the data show for the first time the involvement of PI3-K/Akt-pathway in processes of injury-induced astroglial proliferation and anti-apoptosis via activation of P2Y(1) receptors in vivo, suggesting specific roles of P2 receptors in glial cell pathophysiology in neurodegenerative diseases.

P2Y receptors mediate Ca2+signaling in duodenocytes and contribute to duodenal mucosal bicarbonate secretion

Since little is known about the role of P2Y receptors (purinoceptors) in duodenal mucosal bicarbonate secretion (DMBS), we sought to investigate the expression and function of these receptors in duodenal epithelium. Expression of P2Y(2) receptors was detected by RT-PCR in mouse duodenal epithelium and SCBN cells, a duodenal epithelial cell line. UTP, a P2Y(2)-receptor agonist, but not ADP (10 microM), significantly induced murine duodenal short-circuit current and DMBS in vitro; these responses were abolished by suramin (300 microM), a P2Y-receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB; 100 microM), a store-operated channel blocker. Mucosal or serosal addition of UTP induced a comparable DMBS in wild-type mice, but markedly impaired response occurred in P2Y(2) knockout mice. Acid-stimulated DMBS in vivo was significantly inhibited by suramin (1 mM) or PPADS (30 microM). Both ATP and UTP, but not ADP (1 microM), raised cytoplasmic-free Ca(2+) concentrations ([Ca(2+)](cyt)) with similar potencies in SCBN cells. ATP-induced [Ca(2+)](cyt) was attenuated by U-73122 (10 microM), La(3+) (30 microM), or 2-APB (10 microM), but was not significantly affected by nifedipine (10 microM). UTP (1 microM) induced a [Ca(2+)](cyt) transient in Ca(2+)-free solutions, and restoration of external Ca(2+) (2 mM) raised [Ca(2+)](cyt) due to capacitative Ca(2+) entry. La(3+) (30 microM), SK&F96365 (30 microM), and 2-APB (10 microM) inhibited UTP-induced Ca(2+) entry by 92, 87, and 94%, respectively. Taken together, our results imply that activation of P2Y(2) receptors enhances DMBS via elevation of [Ca(2+)](cyt) that likely results from an initial increase in intracellular Ca(2+) release followed by extracellular Ca(2+) entry via store-operated channel.

Apical versus Basolateral P2Y6 Receptor–Mediated Cl− Secretion in Immortalized Bronchial Epithelia

Apical and/or basolateral membranes of polarized epithelia express P2Y receptors, which regulate the transport of fluid and electrolytes. In the airway, P2Y receptors modulate Cl(-) secretion through the phospholipase C and calcium signaling pathways. Recent evidence suggests that P2Y(6) receptors are expressed in bronchial epithelium and coupled to the cAMP/protein kinase A (PKA) pathways. We examined P2Y receptor subtype expression, including P2Y(6,) and the effect of extracellular nucleotides on basal short-circuit current (I(SC)) and intracellular calcium concentration ([Ca(2+)](i)) in a human bronchial epithelial cell line (16HBE14o-). Real-time PCR demonstrated P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor expression and confirmed that transcript levels were not altered when cells were grown under varied conditions. It was determined that P2Y agonists (ATP, UTP, UDP) stimulated a concomitant increase in I(SC) and [Ca(2+)](i). Apical nucleotides stimulated an increase in [Ca(2+)](i) more efficiently than basolateral nucleotides; however, P2Y agonistic effects on I(SC) were greater when applied basolaterally. Since the P2Y(6) receptors differentially regulate apical and basolateral UDP-induced I(SC) and [Ca(2+)](i), we investigated membrane-resident P2Y(6) receptor functions using Cl(-) or K(+) channels blockers. Apical and basolateral UDP activation of I(SC) was inhibited by applying DIDS apically or TRAM-34 and clotrimazole basolaterally. Although both apical and basolateral UDP increased PKA activity, only apical UDP-induced I(SC) was sensitive to a CFTR inhibitor. These data demonstrate that P2Y agonists stimulate Ca(2+)-dependent Cl(-) secretion across human bronchial epithelia and that the cAMP/PKA pathway regulates apical but not basolateral P2Y(6) receptor-coupled ion transport in human bronchial epithelia.

Pharmacological properties of P2 receptors on rat otic parasympathetic ganglion neurons

To elucidate the pharmacological profile of P2X receptors and the probable expression of P2Y receptors in otic ganglion neurons from 17-day-old rats, single neurons were enzymatically isolated and maintained in tissue culture for up to 30 h. Whole-cell voltage-clamp recording was carried out at a holding potential of − 60 mV. Most otic ganglion neurons responded to adenosine 5′-triphosphate (ATP), 2-methylthio ATP (2-MeSATP) and α,β-methylene ATP (αβ-meATP) with sustained currents and EC 50 values of 19 µM, 47 µM and 94 µM, respectively. 2′,3′- O-trinitrophenyl-ATP (TNP-ATP) inhibited the response to αβ-meATP and ATP with an IC 50 values of 3.9 nM and 18.3 nM, respectively, which was closed to that observed in nodose neurons. The response to ATP was antagonized by suramin and cibacron blue. The dose–response curve of suramin against ATP response at a pH of 6.5 was shifted to the left compared to that at a pH of 7.4. Diinosine pentaphosphate (Ip 5I), which blocks P2X 3, but not P2X 2/3-mediated responses, had no effect on the currents evoked by ATP or αβ-meATP. In some neurons, uridine 5′-triphosphate (UTP) induced a tiny, but long-lasting current with a mean amplitude of 0.034 ± 0.011 nA. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the expression of mRNAs for P2X 2, P2X 3, P2X 4, P2X 6 and P2X 7, but not for P2X 1 and P2X 5 receptors in otic ganglion. In conclusion, in rat otic ganglion neurons, P2X 2/3 heteromultimer receptors dominate, but P2X 7 and P2Y 2 or P2Y 4 receptors also play roles.

Molecular and functional mapping of regional differences in P2Y receptor expression in the rat lens

Extracellular ATP has been shown to mobilize intracellular Ca 2+ in cultured ovine lens epithelial cells and in human lens epithelium, suggesting a role for purines in the modulation of lens transparency. In this study, we characterized the expression profiles of P2Y receptor isoforms throughout the rat lens at both the molecular and the functional levels. RT-PCR indicated that P2Y 1, P2Y 2, P2Y 4 and P2Y 6 are expressed in the lens, while P2Y 12, P2Y 13 and P2Y 14 are not. Immunohistochemistry, using isoform specific antibodies, indicated that the epithelium does not express P2Y 1 and P2Y 2, but that the underlying fiber cells, which differentiate from the epithelial cells, exhibit strong membranous labeling. Although co-expressed in fiber cells, differences in P2Y 1 and P2Y 2 expression were apparent. P2Y 1 expression extended deeper into the lens than P2Y 2, and its expression co-localized with Cx50 gap junction plaques, while P2Y 2 did not. Labeling for P2Y 4 and P2Y 6 receptors were observed in both epithelial cells and fiber cells, but the labeling was predominantly cytoplasmic in nature. While purine agonist (ATP, ADP, UTP and UDP) application to the lens induced mobilization of intracellular Ca 2+ in cortical fiber cells, little to no effect was observed in the anterior and equatorial epithelium. Thus the inability of UTP and UDP to mobilize intracellular Ca 2+ in the epithelium and the predominately cytoplasmic location of P2Y 4 and P2Y 6 suggests that these receptors may represent an inactive pool of receptors that may be activated under non-physiological conditions. In contrast, our results indicated that P2Y 1 and P2Y 2 are functionally active in fiber cells and their differential subcellular expression patterns suggest they may regulate distinct processes in the lens under steady state conditions.

Altered calcium handling following the recombinant overexpression of protein kinase C isoforms in HaCaT cells

Both changes in intracellular calcium concentration ([Ca(2+)](i)) and activation of certain protein kinase C (PKC) isoforms play a crucial role in keratinocyte functions. To better understand the interaction between these two signalling pathways we investigated the resting [Ca(2+)](i) and the extracellular ATP-induced changes in [Ca(2+)](i) on HaCaT cell clones overexpressing either the classical alpha or the beta PKC isoform. These PKC isoenzymes were previously shown to decrease (alpha) or increase (beta) cell proliferation and augment (alpha) or suppress (beta) cell differentiation. Keratinocyte clones with decreased proliferation rate were found to have unaltered resting [Ca(2+)](i), but responded with greater calcium transients to the application of 180 mum of ATP. In contrast, clones with increased proliferation rate had elevated resting [Ca(2+)](i) and suppressed calcium responses to ATP. Calcium transients on PKCbeta clones displayed a faster falling phase. Each clone had a distinct purinergic receptor expression pattern, some of which paralleled the altered proliferation rate and calcium handling. Keratinocytes overexpressing PKCbeta revealed decreased P2X1 and increased P2Y1 receptor expression as compared with the control or PKCalpha clones. The expression level of P2X7 was significantly increased in keratinocytes overexpressing PKCalpha. On the other hand neither the P2X2 nor the P2Y2 expression was altered significantly in the cell types investigated. These data indicate that a modified proliferation and differentiation pattern is associated with altered calcium handling in keratinocytes. The observations also suggest that different PKC isoenzymes have different effects on the phosphatidyl-inositol signalling pathway.

Molecular identification and localization of P2X receptors in the rat lens

There is growing evidence suggesting that purine-mediated signaling pathways play important roles in the ocular lens. We have previously reported the expression of P2Y receptors in the lens; and in this present study, we show that P2X receptors are also expressed. Transcripts for P2X1–7 receptors were detected in lens fiber cells using the reverse-transcription polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of all P2X receptor subtypes at the protein level. Immunohistochemistry revealed P2X1 and P2X7 expression in the cytoplasm of cortical fiber cells. P2X2 expression was confined to the apical–apical interface between epithelial and fiber cells with little expression in cortical fiber cells. In contrast, P2X3, P2X4, P2X5 and P2X6 were expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, immunolocalisation of these P2X receptors was predominantly cytoplasmic. However, deeper into the lens, P2X receptor immunolabeling became more membranous, indicating the recruitment of P2X receptors from cytoplasmic vesicles into the membranes of mature fiber cells in the lens core. Western blotting confirmed regional differences in P2X receptor expression. The differential expression of P2X receptors in the ocular lens indicates that the purinergic signaling pathways may be involved in the maintenance of lens homeostasis.

Transient Increase in P2Y2 Receptor Expression after Spinal Cord Injury in Rat Model

Spinal cord injury (SCI) increases inhibitory molecules release and reactive astrocytes proliferation at injury site producing nonpermissive environment for axonal regeneration. Reactive astrogliosis mechanism is unknown, but the release of nucleotides has been linked to this hypertrophic state. Our goal is to investigate the spatio‐temporal profile of P2Y2 after SCI. Molecular biology and immunohistochemical (IHC) studies were used to evaluate the expression and role of these receptors in rats injured at the T‐10 level using the NYU impactor device. P2Y2 gene temporal profile using standardized RT‐PCR showed a two‐fold increase after 4–7 days post‐injury (DPI) and returned to basal levels by 14 DPI. Double labeling IMH localized P2Y2 in cells bodies, axons, macrophages, oligodendrocytes and astrocytes. Also, levels of P2Y2 immunoreactivity were increased after injury in astrocytes. Therefore, the gene profile of P2Y2 after SCI showed that the levels of this mRNA and protein increased after trauma, suggesting a role in the establishment of the restrictive environment for axonal regeneration after SCI.Support by NIH‐MRISP 2R2MH48190‐14, NIH‐SNRP NS39405, MBRS‐SCORE S06‐GM008224, MBRS‐RISE GM‐68138, PR‐EPSCOR EPS‐9874782

Age‐dependent changes in P2Y receptors in the hearts of P2Y2 knockout mice

Genetic ablation in mice of the nucleotide receptor P2Y2 leads to salt‐resistant hypertension and changes in several organ systems. In the current study we employed this animal model to investigate age‐dependent changes in cardiac P2Y receptor expression. Using quantitative PCR, we determined the expression level of seven P2Y receptor subtypes, P2Y1, 2, 4, 6, 12, 13 and 14 in hearts of wild‐type (WT) and P2Y2 knockout (KO) mice at age of 4, 12 and 18 months. Hearts of WT mice have both P2Y1 and P2Y2 as the major receptor P2Y subtypes, with lower levels of RNA expression for P2Y6 and P2Y14. All four P2Y subtypes exhibit striking increases in expression in 18 month‐old mice whereas the other three P2Y subtypes have low expression levels and display no significant age‐dependent changes. In contrast, KO mice have elevated expression of all six of the other P2Y subtypes and demonstrate similar age‐dependent patterns of change as do WT mice, with increases in P2Y1, P2Y6 and P2Y14 in the 18‐month old group. These results demonstrate that P2Y2 receptors are an important subtype in the heart, such that their absence results in compensatory expression of other P2Y subtypes. Since P2Y2 KO mice are hypertensive, the observed changes in other cardiac P2Y receptors may represent adaptations to increase in blood pressure as well as to the absence of P2Y2 receptors. (Grant support from NIH, UCSD SIRA and the Ellison Medical Foundation)

Involvement of P2Y Receptors in TGFβ‐Induced EMT of MDCK Cells

Epithelial‐to‐mesenchymal transition (EMT) contributes to renal tubulointerstitial fibrosis, a common pathway leading to end‐stage renal disease. EMT can be induced in certain renal cells by transforming growth factor beta (TGFβ) and results in the phenotypic conversion of tubular epithelia to fibroblasts, reduction of E‐Cadherin expression and induction of α‐smooth muscle actin (αSMA). Since nucleotides can be released during renal injury, we hypothesized that P2Y (nucleotide) receptors contribute to EMT of renal epithelial cells. To test this hypothesis, we used a well‐characterized renal epithelial cell line, Madin Darby Kidney Cells (MDCK), and examined the role of P2Y receptors in TGFβ‐induced EMT. Quantative PCR experiments (used to identify P2Y receptor expression) revealed P2Y2 > P2Y11 > P2Y1 = P2Y14 > P2Y6 = P2Y13 > P2Y12 = P2Y4 as the relative order of abundance. TGFβ treatment reduced the expression of P2Y13 mRNA, elevated P2Y11 and P2Y14 mRNA levels without substantial changes in mRNA expression of P2Y1 and P2Y2. The P2Y agonist ATPγS inhibited protein expression of the EMT marker αSMA in response to TGFβ without influencing the E‐Cadherin decrease or morphological changes. These results indicate that TGFβ‐promoted EMT differentially regulates expression of P2Y receptor subtypes of MDCK cells and that P2Y receptor activation potentially blunts features of the EMT phenotype. Supported by grants from NIH.

Intestinal Inflammation Increases the Expression of the P2Y6 Receptor on Epithelial Cells and the Release of CXC Chemokine Ligand 8 by UDP

Epithelial cells participate in the immune response of the intestinal mucosa. Extracellular nucleotides have been recognized as inflammatory molecules. We investigated the role of extracellular nucleotides and their associated P2Y receptors in the secretion of cytokines by epithelial cells. The effect of intestinal inflammation on P2Y(6) receptor expression was determined by PCR in the mouse, rat, and human. Localization of the P2Y(6) receptor was determined by immunofluorescence microscopy in the colon of normal and dextran sulfate sodium-treated mice. The effect of P2Y(6) activation by UDP on cytokine expression and release by epithelial cells was determined using a combination of Western blots, luciferase assays, RT-PCR, cytokine Ab arrays, and ELISA. Inflammation up-regulates P2Y(2) as well as P2Y(6) receptor expression in the mucosa of the colon of colitic mice. In vitro, we demonstrated that UDP could be released by Caco-2/15 cells. We have confirmed the increased expression of P2Y(6) by challenging intestinal epithelial cell-6 and Caco-2/15 cells with TNF-alpha and IFN-gamma and showing that stimulation of epithelial cells by UDP results in an increased expression and release of CXCL8 by an ERK1/2-dependent mechanism. The increase in CXCL8 expression was associated with a transcriptional activation by the P2Y(6) receptor. This study is the first report demonstrating the implication of P2Y receptors in the inflammatory response of intestinal epithelial cells. We show for the first time that P2Y(6), as well as P2Y(2), expression is increased by the stress associated with intestinal inflammation. These results demonstrate the emergence of extracellular nucleotide signaling in the orchestration of intestinal inflammation.

Expression and function of rat urothelial P2Y receptors

The control and regulation of the lower urinary tract are partly mediated by purinergic signaling. This study investigated the distribution and function of P2Y receptors in the rat urinary bladder. Application of P2Y agonists to rat urothelial cells evoked increases in intracellular calcium; the rank order of agonist potency (pEC(50) +/- SE) was ATP (5.10 +/- 0.07) > UTP (4.91 +/- 0.14) > UTPgammaS (4.61 +/- 0.16) = ATPgammaS (4.70 +/- 0.05) > 2-methylthio adenosine 5'-diphosphate = 5'-(N-ethylcarboxamido)adenosine = ADP (<3.5). The rank order potency for these agonists indicates that urothelial cells functionally express P2Y(2)/P2Y(4) receptors, with a relative lack of contribution from other P2Y or adenosine receptors. Real-time PCR, Western blotting, and immunocytochemistry confirmed the expression of P2Y(2) and to a lesser extent P2Y(4) in the urothelium. Immunocytochemical studies revealed expression of P2Y(2) staining in all layers of the urothelium, with relative absence of P2Y(4). P2Y(2) staining was also present in suburothelial nerve bundles and underlying detrusor smooth muscle. Addition of UTP and UTPgammaS was found to evoke ATP release from cultured rat urothelial cells. These findings indicate that cultured rat urothelial cells functionally express P2Y(2)/P2Y(4) receptors. Activation of these receptors could have a role in autocrine and paracrine signaling throughout the urothelium. This could lead to the release of bioactive mediators such as additional ATP, nitric oxide, and acetylcholine, which can modulate the micturition reflex by acting on suburothelial myofibroblasts and/or pelvic afferent fibers.

Involvement of Purinergic P2 Receptors in Experimental Retinal Neovascularization

Purpose: The present study was aimed to investigate the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization. Methods: Immunohistochemistry was used to study the expression of purinergic P2Y2 and P2X2 receptors in the neonatal mouse retina during normal vascular development and after oxygen-induced retinopathy (OIR). The effect of the P2 antagonists, suramin and PPADS, on the extent of oxygen-induced retinal neovascularization was analyzed. Results: In normal mice, the expression of P2Y2 receptors was weak throughout the retina, whereas P2X2 receptor expression was detected in the outer plexiform layer. In mice treated with oxygen, P2Y2 expression was detected in the ganglion and in the nerve fiber layers, whereas P2X2 expression was found in the inner and outer plexiform layers. Oxygen-induced preretinal neovascularization was strongly inhibited by the P2 antagonists, suramin (p < 0.05) and PPADS (p < 0.05), and this was accompanied by a down-regulation of P2X2 receptor expression in the inner plexiform layer in suramin-treated mice. Conclusions: The data suggest that purinergic P2 receptors are involved in neovascularization associated with OIR.

Distinct P2Y Receptor Subtypes Regulate Calcium Signaling in Human Retinal Pigment Epithelial Cells

Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells. These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.

Regulation of Inositol 1,4,5-Trisphosphate Receptor-mediated Calcium Release by the Na/K-ATPase in Cultured Renal Epithelial Cells

It is known that the Na/K-ATPase alpha1 subunit interacts directly with inositol 1,4,5-triphosphate (IP(3)) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca(2+) release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase alpha1 subunit resulted in a parallel attenuation of ATP-induced ER Ca(2+) release. When the knockdown cells were rescued by knocking in a rat alpha1, the expression of rat alpha1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca(2+) release. Mechanistically, this defect in ATP-induced ER Ca(2+) release was neither due to the changes in the amount or the function of cellular IP(3) and P2Y receptors nor the ER Ca(2+) content. However, the alpha1 knockdown did redistribute cellular IP(3) receptors. The pool of IP(3) receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cepsilon activation and ER Ca(2+) release. The data showed that the alpha1 knockdown de-sensitized the ATP-induced ER Ca(2+) release but not PKCepsilon activation. Moreover, expression of the N terminus of Na/K-ATPase alpha1 subunit not only disrupted the formation of the Na/K-ATPase-IP(3) receptor complex but also abolished the ATP-induced Ca(2+) release. Finally, we observed that the alpha1 knockdown was also effective in attenuating ER Ca(2+) release provoked by angiotensin II and epidermal growth factor.

Deficiency of purinergic P2Y receptors in aganglionic intestine in Hirschsprung’s disease

Hirschsprung's disease is characterized by the absence of ganglion cells in the distal bowel and extends proximally for varying distances. In recent years, the purinergic P2Y receptors have begun to receive much attention as they have been recognised as major ATP receptors in many regions of the body, including the intestine. ATP has long been established as an inhibitory neurotransmitter in the enteric nervous system. The aim of our study was to analyse the expression of P2Y1 and P2Y2 receptors in the intestine of patients with Hirschsprung's disease. Frozen sections were cut from rectal tissue segments taken from both the aganglionic and ganglionic regions of patients with Hirschsprung's disease, as well as tissue from normal rectal biopsies, which were used as controls. Sections were incubated overnight with P2Y1 and P2Y2 receptor antibodies and results were analysed by light microscopy. Both P2Y1 and P2Y2 immunoreactivity was absent from the submucosal and myenteric plexuses of aganglionic tissue compared to ganglionic tissue and normal controls, in which large number of immunoreactive neurons were evident, arranged in ganglia in both plexuses and positive nerve fibres, in both the smooth and circular muscles. Our results show a lack of expression of P2Y1 and P2Y2 receptors in the aganglionic gut in Hirschsprung's disease. The absence of these receptors suggests the absence of the inhibitory neurotransmitter ATP, which may help to explain the contracted state of the aganglionic gut in Hirschsprung's disease.

Effects of UDP on ARPE cells: possible involvement of P2Y6 receptors

Abstract Purpose: We have studied the effects of nucleotides (ATPγS, UTP and UDP) on human retinal pigmental cells, in relation to their expression of functional P2Y receptors. Methods: ARPE cells were grown in culture and stimulated with ATPγS, UTP and UDP. Cell proliferation was analysed by BrdU incorporation. Calcium flux and cAMP were measured by fluorometry and radio‐immunoassay. IL‐8 production was investigated by semi‐quantitative RT‐PCR and ELISA. P2Y2, P2Y4 and P2Y6 expression was analysed by RT‐PCR. Results: UDP inhibited ARPE cell proliferation while ATPγS slightly stimulated it and UTP had no effect. This differential effect suggests the expression of other P2Y receptors than the already described P2Y2. Accordingly we detected transcripts for the P2Y6 receptor, which is a UDP receptor. UDP induced a calcium flux, like ATPγS and UTP, but no cAMP production. All nucleotides stimulated IL‐8 secretion. Conclusions: Our results suggest that ARPE cells express functional P2Y6 receptors.

Intestinal Ischemia-Reperfusion Injury Alters Purinergic Receptor Expression in Clinically Relevant Extraintestinal Organs

Intestinal ischemia-reperfusion (IIR) injury is known to initiate the systemic inflammatory response syndrome, which often progresses to multiple organ failure. We investigated changes in purinoceptor expression in clinically relevant extra-intestinal organs following IIR injury. Anesthetized adult male BalbC mice were randomized to sham laparotomy (control, n = 5), or 15 min of superior mesenteric artery occlusion. Experimental ischemia was followed by a period of reperfusion [1 min (n = 6) or 1 h (n = 6)]. Mice were then sacrificed and lung, kidney, and intestinal tissues were harvested. Following RNA extraction, purinoceptor mRNA expression for P2Y2, A3, P2X7, A2b, P2Y4, and P2Y6 was analyzed using real-time RT-PCR. Significant differences in purinoceptor expression were observed in the lungs and kidneys of mice exposed to IIR injury when compared to controls. Pulmonary P2Y2 receptor expression was increased in the 1 h IIR group when compared to control, while pulmonary A3 receptor expression was incrementally elevated following IIR injury. In the kidney, P2Y2 receptor expression was increased in the 1 h IIR group compared to both 1 min IIR and control, and A3 receptor expression was decreased in the 1 h IIR group compared to the 1 min IIR group. No significant changes were observed in the intestinal purinoceptor profiles. Purinoceptor expression is altered in the murine lung and kidney, but not intestine following experimental IIR injury. These findings may implicate extracellular nucleotides and purinoceptors as possible mediators of the extra-intestinal organ dysfunction associated with IIR injury.

Mice lacking P2Y2receptors have salt‐resistant hypertension and facilitated renal Na+and water reabsorption

Extracellular nucleotides (e.g., ATP) regulate many physiological and pathophysiological processes through activation of nucleotide (P2) receptors in the plasma membrane. Here we report that gene-targeted (knockout) mice that lack P2Y2 receptors have salt-resistant arterial hypertension in association with an inverse relationship between salt intake and heart rate, indicating intact baroreceptor function. Knockout mice have multiple alterations in their handling of salt and water: these include suppressed plasma renin and aldosterone concentrations, lower renal expression of the aldosterone-induced epithelial sodium channel alpha-ENaC, greater medullary expression of the Na-K-2Cl-cotransporter NKCC2, and greater furosemide-sensitive Na+ reabsorption in association with greater renal medullary expression of aquaporin-2 and vasopressin-dependent renal cAMP formation and water reabsorption despite similar vasopressin levels compared with wild type. Of note, smaller increases in plasma aldosterone were required to adapt renal Na+ excretion to restricted intake in knockout mice, suggesting a facilitation in renal Na+ retention. The results thus identify a previously unrecognized role for P2Y2 receptors in blood pressure regulation that is linked to an inhibitory influence on renal Na+ and water reabsorption. Based on these findings in knockout mice, we propose that a blunting in P2Y2 receptor expression or activity is a new mechanism for salt-resistant arterial hypertension.

Purinergic receptor activation inhibits mitogen-stimulated proliferation in primary neurospheres from the adult mouse subventricular zone

The expression pattern of purinergic receptors was examined in subventricular zone-derived primary neurospheres. Primary neurospheres expressed mRNA for P2X4 and P2X7 receptors, all P2Y receptors, with the exception of P2Y4, and the A1, A2a and A2b adenosine receptors. ATPγS, ADPβS and UTP evoked transient increases in cytoplasmic Ca2+ concentration in dissociated primary neurospheres, demonstrating the functional expression of P2Y1 and P2Y2 receptors. Ca2+ transients were not attenuated by the removal of extracellular Ca2+ and were reversibly inhibited by the P2Y1 selective antagonist, MRS 2179. P2Y and adenosine receptor agonists reduced the size and frequency of primary neurospheres. The effects of ADPβS and adenosine were reversed by subtype-selective receptor antagonists, demonstrating that P2Y1 and A2a receptors mediate inhibitory effects on primary neurosphere proliferation. The modulation of neural precursor cell proliferation by P2Y and adenosine receptors therefore represents a potential regulatory mechanism within the neurogenic microenvironment.