Effects of glycosaminoglycan from Urechis unicinctus on the P2Y1 receptor pathway and expression of related factors in rat platelets.

The aim of this study was to examine the effects of glycosaminoglycan (GAG) from Urechis unicinctus on the P2Y1 receptor pathway and expression of related factors in rat platelets. The concentration of calcium ion (Ca2+) in rat platelets was determined by double wavelength Fura-2 fluorescence spectrophotometry, and the concentrations of inositol trisphosphate (IP3) and glycoprotein IIb/IIIa (GPIIb/IIIa) in rat platelets were measured using the enzymatic immunoassay method. The phosphorylation levels of phospholipase C (PLC), phospholipase A2 (PLA2), protein kinase C (PKC), and p38 mitogen-activated protein kinase (p38MAPK) were also detected by Western blot. It was found that the GAG from U. unicinctus significantly reduced the Ca2+ and IP3 levels in rat platelets (p<0.05, p<0.01). Moreover, medium and high concentrations of GAG significantly reduced the concentration of the platelet membrane GPIIb/IIIa in rats (p<0.05, p<0.01). The phosphorylation levels of PLC, PLA)2), PKC and p38MAPK in rat platelets were also inhibited by GAG and P)2)Y)1) receptor blocker MRS2179 (p<0.05, p<0.01). However, the degree of inhibition of GAG was lower than that of MRS2179. The results laid a foundation for further utilization of the glycosaminoglycan.

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors.

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

Identification of contractile P2Y1, P2Y6, and P2Y12 receptors in rat intrapulmonary artery using selective ligands.

ATP and UDP constrict rat intrapulmonary arteries, but which receptors mediate these actions is unclear. Here, we used selective agonists and antagonists, along with measurements of P2Y receptor expression, to characterize the receptor subtypes involved. Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Expression of P2Y receptor subtype expression was determined by using reverse transcription-polymerase chain reaction with receptor-specific oligonucleotide primers. The selective P2Y(1) agonist (N)-methanocarba-2-methylthioadenosine-5'-O-diphosphate (MRS2365) induced small, concentration-dependent contractions that were inhibited by the P2Y(1) antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179). Contractions evoked by ATP were unaffected by MRS2179, but inhibited by approximately one-third by the P2Y(12) antagonist N(6)-(2-methylthiomethyl)-2-(3,3,3-trifluoropropylthio)dichloro-methylene ATP (AR-C69931MX). Combined blockade of P2X1 and P2Y(12) receptors virtually abolished the response to ATP. ADP also evoked contractions that were abolished by AR-C69931MX. The selective P2Y(6) receptor agonist 3-(2-oxo-2-phenylethyl)-UDP (PSB 0474) evoked concentration-dependent contractions and was approximately three times more potent than UDP, but the P2Y(14) agonist UDP-glucose had no effect. Contractions evoked by UDP were inhibited by the P2Y(6) receptor antagonist N,N'-1,4-butanediylbis-N'-(3-isothiocyanatophenyl)thiourea (MRS2578), but not the cysteinyl leukotriene 1 (CysL(1)) antagonist 3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid (MK571). Higher concentrations of MRS2578 inhibited contractions to KCl, so they were not studied further. mRNA for P2Y(1), P2Y(6), and P2Y(12) receptors was identified. Our working model is that P2Y(12) and P2X1 receptors are present in rat intrapulmonary arteries and together mediate ATP-induced vasoconstriction. Contractile P2Y(6), but not P2Y(14) or CysLT(1), receptors are also present and are a major site through which UDP evokes constriction.

Purinergic receptors expressed in human skeletal muscle fibres.

Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death.

The P2Y-like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time-dependent increase in the number of highly branched GPR17-positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4-positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4-positive cells. Under both culture protocols, appearance of more mature CNPase- and MBP-positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide-induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double-edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.