P2Y1 Nucleotide Receptors Research Articles

P2Y1 nucleotide receptor silencing and its effect on glioma C6 calcium signaling.

In our earlier studies of the signaling cross-talk between nucleotide receptors in an in vitro glioma model (C6 cell line) under prolonged serum deprivation conditions, a growth arrest of the cells and expression shift from P2Y(1) to P2Y(12) receptors was found. The aim of the present work was to test if siRNA silencing of P2Y(1) receptor changes P2Y(12) expression similarly as following the serum deprivation and which physiological downstream pathways it affects. Here we demonstrate for the first time the efficiency of siRNA technology in silencing P2Y nucleotide receptors in glioma C6 cell line. Moreover, P2Y(12) proved to be insensitive to the P2Y(1) receptor silencing. The effect of the P2Y(1) silencing on calcium signaling was less pronounced then the extent of the protein change itself, exactly as was the case for the serum starvation experiments. Phosphorylation of ERK and Akt kinases were studied as the downstream effect of P2Y(1)-evoked signaling and similar effects as in the case of serum deprivation were found for ERK, and even stronger ones for Akt phosphorylation.

Action of Nucleosides and Nucleotides at 7 Transmembrane-Spanning Receptors

Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y1 nucleotide receptor and the A3 adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.

Activation of P2Y1Nucleotide Receptors Induces Inhibition of the M-Type K+Current in Rat Hippocampal Pyramidal Neurons

We have shown previously that stimulation of heterologously expressed P2Y1 nucleotide receptors inhibits M-type K+ currents in sympathetic neurons. We now report that activation of endogenous P2Y1 receptors induces inhibition of the M-current in rat CA1/CA3 hippocampal pyramidal cells in primary neuron cultures. The P2Y1 agonist adenosine 5'-[beta-thio]diphosphate trilithium salt (ADPbetaS) inhibited M-current by up to 52% with an IC50 of 84 nM. The hydrolyzable agonist ADP (10 microM) produced 32% inhibition, whereas the metabotropic glutamate receptor 1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine] (10 microM) inhibited M-current by 44%. The M-channel blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] produced 73% inhibition at 3 microM; neither ADPbetaS nor ADP produced additional inhibition in the presence of XE991. The effect of ADPbetaS was prevented by a specific P2Y1 antagonist, MRS 2179 (2'-deoxy-N'-methyladenosine-3',5'-bisphosphate tetra-ammonium salt) (30 microM). Inhibition of the M-current by ADPbetaS was accompanied by increased neuronal firing in response to injected current pulses. The neurons responding to ADPbetaS were judged to be pyramidal cells on the basis of (1) morphology, (2) firing characteristics, and (3) their distinctive staining for the pyramidal cell marker neurogranin. Strong immunostaining for P2Y1 receptors was shown in most cells in these cultures: 74% of the cells were positive for both P2Y1 and neurogranin, whereas 16% were only P2Y1 positive. These results show the presence of functional M-current-inhibitory P2Y1 receptors on hippocampal pyramidal neurons, as predicted from their effects when expressed in sympathetic neurons. However, the mechanism of inhibition in the two cell types seems to differ because, unlike nucleotide-mediated M-current inhibition in sympathetic neurons, that in hippocampal neurons did not appear to result from raised intracellular calcium.

ATP potentiates the formation of AChR aggregate in the co-culture of NG108-15 cells with C2C12 myotubes

The role of adenosine 5′-triphosphate (ATP) and P2Y1 nucleotide receptor in potentiating agrin-induced acetylcholine receptor (AChR) aggregation is being demonstrated in a co-culture system of NG108-15 cell, a mouse neuroblastoma X rat glioma hybrid cell line that resembles spinal motor neuron, with C2C12 myotube. In the co-cultures, antagonized P2Y1 receptors showed a reduction in NG108-15 cell-induced AChR aggregation. Parallel to this observation, cultured NG108-15 cell secreted ATP into the conditioned medium in a time-dependent manner. Enhancement of ATP release from the cultured NG108-15 cells by overexpression of active mutants of small GTPases increased the aggregation of AChRs in co-culturing with C2C12 myotubes. In addition, ecto-nucleotidase was revealed in the co-culture, which rapidly degraded the applied ATP. These results support the notion that ATP has a role in directing the formation of post-synaptic apparatus in vertebrate neuromuscular junctions.

ATP Potentiates Agrin-induced AChR Aggregation in Cultured Myotubes: ACTIVATION OF RHOA IN P2Y1 NUCLEOTIDE RECEPTOR SIGNALING AT VERTEBRATE NEUROMUSCULAR JUNCTIONS

At vertebrate neuromuscular junctions, ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.

CDNA encodes Xenopus P2Y(1) nucleotide receptor: expression at the neuromuscular junctions.

A cDNA encoding P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of P2Y(1) receptor with 361 amino residues is 80-85% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding P2Y(1) receptor at approximately 3.2 kb was revealed in the brain, spinal cord and muscle of adult, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.

The P2Y(1) receptor closes the N-type Ca(2+) channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists.

The rat P2Y(1) nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro-injection of the receptor cRNA or cDNA. ADP inhibited the N-type Ca(2+) current by 64%, with EC(50) 8.2 nM, an action blocked competitively by the P2Y(1) receptor antagonist adenosine 3', 5'-bis-phosphate (K(i) 0.7 microM). 2-Methylthio-ADP inhibited the Ca(2+) current likewise, but with EC(50) 0.57 nM, giving the highest potency reported therewith for P2Y(1). Significantly, ATP and 2-methylthio-ATP were also agonists, the latter again at a very high potency (EC(50) 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N-type Ca(2+) currents and hence can reduce transmitter release or increase neuronal excitability.

Changes in P2Y1 nucleotide receptor activity during the development of rat salivary glands.

Experiments that used dispersed salivary gland cells from 1-day-old rats indicated the presence of the P2Y nucleotide receptor subtype, P2Y1, based on the agonist potency profile for mobilization of intracellular free Ca2+ [2-methylthio-ATP > ADP > adenosine 5'-O-(2-thiodiphosphate) > ATP, with UTP ineffective] and sequence analysis of reverse transcription-polymerase chain reaction (RT-PCR) products obtained with P2Y1 receptor-specific primers. P2Y1 receptor activity appears to be developmentally regulated, since Ca2+ mobilization in response to the P2Y1-selective agonist, 2-methylthio-ATP, decreased as animal age increased, with the maximal response of 129 +/- 23 nM obtained in 1-day-old animals, decreasing to 30 +/- 3 nM in 4-wk-old animals. However, the abundance of P2Y1 receptor mRNA, assessed by semiquantitative RT-PCR, did not change over this time period, suggesting that receptor activity is regulated by some mechanism other than changes in steady-state levels of P2Y1 receptor mRNA. These findings indicate that functional P2Y1 nucleotide receptors are expressed in immature salivary glands and that receptor activity decreases as the glands mature, suggesting that P2Y1 receptors may have an important role during salivary gland development.