Distribution of P2Y6 and P2Y12 receptor: their colocalization with calbindin, calretinin and nitric oxide synthase in the guinea pig enteric nervous system

The distribution of P2Y₆ and P2Y₁₂ receptor-immunoreactive (ir) neurons and fibers and their coexistence with calbindin, calretinin and nitric oxide synthase (NOS) has been investigated with single and double labeling immunostaining methods. The results showed that 30–36% of the ganglion cells in the myenteric plexus are strongly P2Y₆ receptor-ir neurons; they are distributed widely in the myenteric plexus of stomach, jejunum, ileum and colon, but not in the submucosal plexus, with a typical morphology of multipolar neurons with a long axon-like process. About 42–46% of ganglion cells in both the myenteric and submucosal plexuses show P2Y₁₂ receptor-ir. About 28–35% of P2Y₆ receptor-ir neurons were found to coexist with NOS and 41–47% of them coexist with calretinin, but there was no coexistence of P2Y₆ receptor-ir with calbindin. In contrast, all P2Y₁₂ receptor-ir neurons were immunopositive for calbindin, although occasionally P2Y₁₂ receptor-ir neurons without calbindin immunoreactivity were found, while none of the P2Y₁₂ receptor-ir neurons were found to coexist with calretinin or NOS in the gastrointestinal system of guinea pig. The P2Y₁₂ receptor-ir neurons coexpressing calbindin-ir in the small intestine are Dogiel type II/AH, intrinsic primary afferent neurons.     

Neuroprotective roles of the P2Y2 receptor

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation

Nucleotides released upon brain injury signal to astrocytes and microglia playing an important role in astrogliosis, but the participation of microglia in the purinergic modulation of astrogliosis is still unclear. Highly enriched astroglial cultures and co-cultures of astrocytes and microglia were used to investigate the influence of microglia in the modulation of astroglial proliferation mediated by nucleotides. In highly enriched astroglial cultures, adenosine-5’-triphosphate (ATP), adenosine 5’-O-(3-thio)-triphosphate (ATPγS), adenosine 5’-O-(3-thio)-diphosphate (ADPβS; 0.01–1 mM), and adenosine-5’-diphosphate (ADP; 0.1–1 mM) increased proliferation up to 382%, an effect abolished in co-cultures containing 8% of microglia. The loss of ATP proliferative effect in co-cultures is supported by its fast metabolism and reduced ADP accumulation, an agonist of P2Y_1,12 receptors that mediate astroglial proliferation. No differences in ADPβS and ATPγS metabolism or P2Y_1,12 receptors expression were found in co-cultures that could explain the loss of their proliferative effect. However, conditioned medium from microglia cultures or co-cultures treated with ADPβS, when tested in highly enriched astroglial cultures, also prevented ADPβS proliferative effect. None of the uracil nucleotides tested had any effect in proliferation of highly enriched astroglial cultures, but uridine-5′-triphosphate (UTP; 0.1–1 mM) inhibited proliferation up to 66% in co-cultures, an effect that was dependent on uridine-5’-diphosphate (UDP) accumulation, coincident with a co-localization of P2Y₆ receptors in microglia and due to cell apoptosis. The results indicate that microglia control astroglial proliferation by preventing the proliferative response to adenine nucleotides and favouring an inhibitory effect of UTP/UDP. Several microglial P2Y receptors may be involved by inducing the release of messengers that restrain astrogliosis, a beneficial effect for neuronal repair mechanisms following brain injury.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Association of P2Y2 receptor SNPs with bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients

The P2Y₂ receptor is a G-protein-coupled receptor with adenosine 5′-triphosphate (and UTP) as natural ligands. It is thought to be involved in bone physiology in an anti-osteogenic manner. As several non-synonymous single nucleotide polymorphisms (SNPs) have been identified within the P2Y₂ receptor gene in humans, we examined associations between genetic variations in the P2Y₂ receptor gene and bone mineral density (BMD) (i.e., osteoporosis risk), in a cohort of fracture patients. Six hundred and ninety women and 231 men aged ≥50 years, visiting an osteoporosis outpatient clinic at Maastricht University Medical Centre for standard medical follow-up after a recent fracture, were genotyped for three non-synonymous P2Y₂ receptor gene SNPs. BMD was measured at three locations (total hip, lumbar spine, and femoral neck) using dual-energy X-ray absorptiometry. Differences in BMD between different genotypes were tested using analysis of covariance. In women, BMD values at all sites were significantly different between the genotypes for the Leu46Pro polymorphism, with women homozygous for the variant allele showing the highest BMD values (0.05 > p > 0.01). The Arg312Ser and Arg334Cys polymorphisms showed no differences in BMD values between the different genotypes. This is the first report that describes the association between the Leu46Pro polymorphism of the human P2Y₂ receptor and the risk of osteoporosis.     

Quantification of extracellular UDP-galactose

The human P2Y₁₄ receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y₁₄ receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with subnanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1-4-β-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [γ-³²P]ATP and the formation of [γ-³²P]UTP is monitored by high-performance liquid chromatography. The overall conversion of UDP-Gal to [γ-³²P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. The occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule.     

Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways

Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y₆ receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091–1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G_12/13 signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia.     

P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX's MORFO domain

In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y(12) receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX's modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y(12) receptor is necessary for ATX's MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y(12) receptor can render cells responsive to the known effects of ATX's MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y(12) signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y(12) receptor provides the basis for an ATX-MORFO-P2Y(12) signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

膜周蛋白PICK1促进细胞膜上P2Y6受体表达及小胶质细胞的吞噬功能

膜周蛋白PICK1(protein interactingC-kinase-1)参与多种膜受体与膜上蛋白的运输并影响细胞的功能。本研究旨在探索小胶质细胞PICK1与P2Y6受体之间的相互作用是否可改变P2Y6受体在细胞膜上的表达,以及对小胶质细胞吞噬功能的影响。采用小鼠脑内皮层原代培养的小胶质细胞进行免疫共沉淀实验揭示,与PICK1敲除小鼠比较,野生小鼠皮层小胶质细胞内存在PICK1-P2Y6受体相互作用。生物素化、密度梯度离心结合蛋白质印迹实验证明,PICK1基因敲除小鼠的小胶质细胞膜表面P2Y6受体表达水平降低。荧光胶珠吞噬实验结合免疫组织化学染色显示,PICK1基因敲除小鼠的小胶质细胞对UDP（刺激）引起的荧光胶珠吞噬作用减弱。蛋白质印迹实验显示,与野生型小鼠比较,PICK1基因敲除小鼠小胶质细胞中的Akt 308T磷酸化水平明显降低|使用Akt抑制剂API-2能有效抑制Akt 在小胶质细胞内的（磷酸化）激活及UDP刺激引起的吞噬作用。上述结果表明,敲除PICK1能下调小胶质细胞膜上P2Y6受体的表达,并降低小胶质细胞的吞噬功能,且这一过程依赖Akt磷酸化修饰。总之,PICK1可促进P2Y6受体在细胞膜上的表达,是小胶质细胞吞噬功能的重要调节子|敲除PICK1可下调P2Y6膜受体表达,并降低小胶质细胞的吞噬功能。这一结果可加深对小胶质细胞的吞噬功能及机制的认识。     

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y₁₀ receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y₁₀ receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca²⁺ increases in the CHO cells stably expressing the P2Y₁₀ fused with a G_16α protein. These Ca²⁺ responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y₁₀ receptor into the P2Y₁₀-CHO cells effectively blocked both S1P- and LPA-induced Ca²⁺ increases. RT-PCR analysis showed that the mouse P2Y₁₀ was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y₁₀ receptor is the first receptor identified as a dual lysophospholipid receptor.