In the present study, we revealed that P 2 Y 1 and P 2 Y 11 receptors were in- volved in X. laevis head formation. The knockdown approach revealed loss of neural development, suggesting that these receptors would be required for the induction and/or maintenance of neural tissues. As P2Y receptors in X. laevis have already been classified into six subtypes, the cellular and molecular func- tions of these receptors, including P 2 Y 1 or P 2 Y 11, should be studied further to fully understand the roles they play. The fact that P2Y receptors exist in only vertebrates and are highly conserved, and that P 2 Y 1 or P 2 Y 11 is involved in head formation has led us to expect that P2Y receptors participate in the acquisi- tion of vertebrate head during evolution. As only the physiological and patho- physiological functions of P2Y receptors in the nervous system have been inves- tigated, it is important to study further the roles of these receptors and their re- lated molecules in early development.
18 Read more
that structural information resulting from these disclosures can assist in the design of new modulators for P2Y receptor subtypes, as many of the reported modulators still lack the potency and selectivity for viable chemical drug-like leads. Oral drug design has been hampered by the importance of retaining the negatively charged phosphates of nucleotides for their interaction with the P2Y receptors. This presents huge issues for drug development because of their metabolic instability, low bioavailability, nonspecific binding to biological membranes, and chemically due to their demanding purification. To date, the only clinically proven role for P2Y receptor antagonists lays in the antithrombotic area, where extracellular nucleotide signalling has been shown to be involved in platelet aggregation. Extracellular ADP has a crucial role in platelet activation through activation of cell surface P2Y 1 and P2Y 12
77 Read more
This study shows that recording ion currents in single cells can be useful in characterising the receptors expressed in tissues where multiple subtypes are present. A particular problem with P2Y receptors is ecto-nucleotidases, which are inhibited by PPADS in smooth muscle  and other tissues [38,39]. Recording from rapidly perfused, single cells minimises the problems created by extracellular metabolism in whole tissues, which may explain why PPADS potentiated contractions to UTP and UDP in the intact artery , but had no effect on activation of I Cl,Ca in single cells. Such studies also allow the regional variation in ion channel expression to be studied. Interestingly, we recorded I Cl,Ca in a similar proportion of rat SPA and LPA smooth muscle cells, whereas in rabbits it is more predominant in smaller pulmonary arteries . Limita- tions of the patch clamp technique encountered here were short recording times, wide variation in current amplitude between cells and a decline in the amplitude of I Cl,Ca over the recording period, all of which hampered quantitative analysis of antagonist action. It is not clear why rundown occurred, as loss of diffusible cytosolic factors into the recording pipette should have been minimised with the perforated-patch technique. Similar rundown was seen in previous patch clamp studies in these cells [4,5] and with ATP- and UTP-induced oscillations in cytosolic [Ca 2+ ]
11 Read more
Our primary aim was to discover new antagonists for P2Y receptors activated by UTP, so a protocol for detection of intracellular calcium mobilization was first established. We made a search in the literature to know concentrations of calcium indicator, Fluo-4, which were used in calcium assays and that could be applied in our protocol. We found in the literature a range of Fluo-4 used in calcium assays between 2 and 4 μM [34–37]. In order to use low doses of dye, the first step was to optimize the concentration of Fluo-4 to our conditions of the assay, making it more economical to high throughput screening. In this context, we investigated concentrations ranging from 1 to 8 μM. We used ATP [100 μM] to stimulate the cells due to its activity as a main physiological P2 receptor agonist, which is widely expressed in J774.G8 cells. As observed in Figure 1A, there is no significant difference in the quantification of calcium response between concentrations between 1 to 4 μM. Concentrations of 6 and 8 μM have significative difference only compared to 1 μM. Furthermore, our results are in a concentration range of Fluo-4 used in literature (2-4 μM) [34–37]. A similar result was obtained with stimulation of cells with ionomycin, a calcium ionophore that promotes a massive transport of this ion from the extracellular to intracellular medium. All concentrations evaluated have the similar profile of quantification of calcium response (Figure 1B). Thus, the selected concentration of Fluo-4 to use in our experiments was of 2 μM because calcium responses in this concentration did not differ from higher concentrations as well as being in literature range. As we could observe in Figure 1C, cells loaded with Fluo-4 at a concentration of 2 μM show a homogenous dye scattering in their cytosol and after a stimulus with ionomycin, they become more fluorescence.
31 Read more
Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future.
12 Read more
Conclusions: These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease.
Nucleotide receptors, also known as P2 recep- tors, together with P1 receptor subfamily, form the purinergic receptor family . P2 receptors consist of two classes of purinergic receptors. P2X receptors belong to cationic channels and P2Y receptors are G protein-coupled receptors (GPCR) . In mammals, seven different P2X subunits (P2X 1 -P2X 7 ) and eight P2Y subtypes have been identified . Eight P2Y receptors are subdivided into two distinct clusters based on different coupled G proteins: G q -coupled receptors (P2Y 1, 2, 4, 6, 11 ) and G i -coupled recep- tors (P2Y 12, 13, 14 ) . Among them, P2Y 6 is widely expressed in diverse tissues including placen- ta, spleen, thymus, intestine, leukocytes, heart, liver, blood vessels, ovary, and microglial and rat aorta, spleen, stomach, intestine, lung, dor- sal root ganglia, spinal cord, ovary, etc [5-9]. Extensive expression of P2Y indicates it may
11 Read more
ings alert us to the possibility that there may be more than one purinoceptor participate in the expression and release of these pro-inflammatory cytokines under physiological and pathological conditions. In our study, we noticed that ADP b s-induced increased expression of IL-1 b , IL-6 and TNF- a mRNA is only partly impaired after MRS2395 or MRS2211 pretreatment, which indicates that both P2Y 12 and P2Y 13 receptors are involved in this process. Ishimaru et al also showed that, in mice liver macrophages/Kupffer cells, P2Y 13 knockdown reduced lipopolysaccharide (LPS)- induced IL-6 production, but by < 50%, which also implies that P2Y receptors including P2Y 13 and others may be involved in LPS-induced IL-6 production. 65
13 Read more
Adrian et al.  showed alteration of expression of P2 receptors during granulocytic and monocytic differenti- ation of HL-60 cells. Heterologous desensitization with ADP and UTP was also observed on cobblestone areas in long-term bone marrow cultures (Fig. 5), suggesting that hetero-association may be present in primitive cells. Recently, the participation of P2 receptors in differentiation into myelocytes has been confirmed in hematopoietic stem cells [17, 22, 24]. Hetero-association among P2Y receptors would be a characteristic of the myelocytic lineage in mice.
11 Read more
Extracellular ATP and other nucleotides induce autocrine and/or paracrine purinergic signalling via activation of the P2 receptors on the cell surface, which represents one of the most common signalling mechanisms. Mesenchymal stem cells (MSC) are a type of multipotent adult stem cells that have many promising applications in regenerative medicine. There is increasing evidence to show that extracellular nucleotides regulate MSC functions and P2 receptor-mediated purinergic signalling plays an important role in such functional regulation. P2 receptors comprise ligand-gated ion channel P2X receptors and G-protein-coupled P2Y receptors. In this review, we provide an overview of the current understanding with respect to expression of the P2X and P2Y receptors in MSC and their roles in mediating extracellular nucleotide regulation of MSC proliferation, migration and differentiation.
37 Read more
in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y 12 receptors was verified by P2Y 12 receptor binding and cAMP assays. These novel data
14 Read more
Thienopyridines such as clopidogrel inhibit P2Y 12 , one of two platelet adenosine diphosphate (ADP) receptors (P2Y 1 , P2Y 12 ) and have been shown to confer clinical ben- efit in a variety of conditions characterized by the risk of arterial thrombosis [1-3]. Nonetheless, in the setting of coronary artery disease, about 1–1.9% of patients may experience acute or sub-acute stent thrombosis (ST) after implantation of a coronary stent [4,5] despite treatment with clopidogrel in combination with aspirin. Several mechanisms could explain a low platelet response to clopidogrel, including poor compliance to treatment,  variable absorption of the drug and/or variable generation of the active metabolite, and potential drug-drug interac- tions . Some studies contend that resistance to clopi- dogrel may be present in as many as 20% of subjects [8- 10]. Patients with a low response to clopidogrel are known to have an increased risk of cardiovascular events [11,12]. Thus, there is a clinical need for a reliable test of platelet response to clopidogrel therapy as a guide to indi- vidualizing dosing regimens. However, the ideal method for quantifying inhibition of platelet function by clopi- dogrel has yet to be agreed upon by the European Society of Cardiology and American College of Cardiology. Cur- rent assays which might be considered to be the gold standard, such as light transmission aggregometry (LTA), flow cytometric evaluation of platelet activation markers and flow cytometric measurement of the vasodilator-stim- ulated phosphoprotein (VASP) phosphorylation status, are technically complex and restricted to specialized labo- ratories and therefore none stands out as the clear investi- gation of choice.
G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, and G protein-linked receptors (GPLR), comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. GPCRs are involved in a wide variety of important physiological processes like visual sense, sense of smell, behavioral and mood regulation, regulation of immune system activity and inflammation, autonomic nervous system transmission and cell density sensing. There are two principal signal transduction pathways involving the G protein-linked receptors, cAMP signal pathway and Phosphatidylinositol signal pathway. Diabetes mellitus, often simply referred to as diabetes, is a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. One of the reasons for the growing public health concern over the rapidly increasing prevalence of obesity in society is its association with the growing incidence of type II diabetes, a combination of conditions often accompanied by increased cardiovascular risk factors. Much effort among health-care providers and pharmaceutical companies is now focused on the discovery of new treatments that alleviate this medical problem. As part of this effort, the G-protein-coupled receptors have recently attracted a lot of attention. GPR119 particularly is of importance because of evidence from in vitro systems and animal models that its modulation may produce favorable effects on glucose homoeostasis, food intake/body weight gain and possibly also β-cell preservation. Many modulators of GPCRs like GPR119, GPR40, GPR41, GPR43 and GPR120 can be used with a great added advantage of improvement in glucose handling and homeostasis in treating diabetes. Relatively high ‘druggability’ of G-protein coupled receptors as compared with many other molecular target classes may provide an insight into the treatment of diabetes. Hence, G protein coupled receptors show a great potential in drug targeting in the process of drug discovery and development. Provided that being clinically well tolerated, these GPCR effectors will prove a boon to the patients suffering from apathy of metabolic syndrome of diabetes.
11 Read more
to receive AZD6140 90 mg twice a day, AZD6140 180 mg twice a day, or a clopidogrel 300 mg loading dose plus 75 mg a day for up to 12 weeks. Patients in the AZD6140 group were further randomized to receive or not receive the 270 mg load- ing dose of the drug. The primary endpoint, ie, Kaplan–Meier rate of protocol-defined major or minor bleeding over four weeks, did not differ between the three groups (9.8%, 8.0%, and 8.1%, respectively). Rates of major bleeding were also close (7.1%, 5.1%, and 6.9%, respectively). Notably, the bleeding rates were not different regardless of previous treatment with clopidogrel, or administration of a loading dose of AZD6140 or platelet glycoprotein IIb/IIIa inhibitors. Asymptomatic ventricular pauses longer than 2.5 sec were more common with AZD6140, particularly at 180 mg twice a day (5.5%, 9.9%, and 4.3%, respectively; P = 0.58 and P = 0.01, respectively, versus clopidogrel). Remarkably also, the study highlighted for the first time that among patients undergoing CABG 1–5 days after stopping the drug, treatment with AZD6140 as opposed to clopidogrel was associated with a numerically lower incidence of major bleeding, a finding consistent with the reversible inhibition of the P2Y 12 receptor provided with AZD6140.
15 Read more
development, especially in the endochondral ossification process, the neonatal pups were skinned, eviscerated, and stained with alizarin red/alcian blue. After staining, the cartilage and ossified bone was clearly stained as blue colour by alcian blue and red colour by alizarin red respectively. All of the ossification centres in the vertebral bodies of the trunk in both KO and WT were present, as is expected at birth. In the coccyx, only coccygeal 1 to 5 had ossified in the majority of both KO and WT mice (Figure 1E). The whole length, mid shaft diameter, and ossification bone length of right femurs were measured as shown in Figure 1F using a calibrated vernier calliper, to address whether P2Y 13 receptor deficiency leads to
29 Read more
We have recently developed a quantitative HIV virion binding assay that documents rapid and extensive binding of HIV virions in seminal plasma to intact mucosal sur- faces [10,54]. In the course of these studies, we realized that both micro and macro structural heterogeneity were commonplace at the surface of randomly selected palatine tonsil samples and that surface structural aberrations could have a profound impact on susceptibility to all microbial infections, including HIV. The current study was designed to investigate the molecular basis for HIV virion binding to intact mucosal surfaces by characteriz- ing the expression patterns in palatine tonsil for HIV receptors, HIV co-receptors and other cell surface markers that have been implicated in HIV infection. Based on a comprehensive interpretation of the expression patterns revealed in this study, we conclude that multiple distinct interactions may be supporting HIV virion binding to mucosal surfaces and that the specific molecules involved with binding at any particular site will be directly deter- mined by the precise anatomical location of that site. For example, we have now recognized there are more poten- tial interactions that could support virion binding to retic- ulated epithelium in the tonsillar crypts than would be immediately available to support virion binding to the luminal surface of stratified squamous epithelium (Figure 5).
13 Read more
Host-seeking, ovipositional behavior and mating of insects are controlled mainly by odor per- ception through sensory organs such as antennae. Antennal chemoreception is extremely im- portant for insect survival. Several antennal chemosensory receptors are involved in mediating the odor detection in insects, especially the odorant receptors (ORs) and ionotropic receptors (IRs), to ensure the specificity of the olfactory sensory neuron responses. In the present study, we identified the chemosensory receptor gene repertoire of the parasitoid wasp Microplitis mediator, a generalist endoparasitoid that infests more than 40 types of Lepidopterous larvae and is widely distributed in the Palaearctic region. By transcriptome sequencing of male and female antennae we identified 60 candidate odorant receptors, six candidate ionotropic receptors and two gustatory receptors in M. mediator. The full-length sequences of these putative chemosensory receptor genes were obtained by using the rapid amplification of cDNA ends PCR (RACE-PCR) method. We also conducted reverse transcription PCR (RT-PCR) combined with real-time quantitative PCR (qPCR) for investigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues. The tissue- and sex-biased expression patterns may provide insights into the roles of the chemosensory receptor in M. mediator. Our findings support possible future study of the chemosensory behavior of M. mediator at the molecular level.
15 Read more
Abstract: Recent advances in the understanding of dry eye disease (DED) have revealed previously unexplored targets for drug therapy. One of these drugs is diquafosol, a uridine nucleotide analog that is an agonist of the P2Y 2 receptor. Several randomized controlled trials have demonstrated that the application of topical diquafosol significantly improves objective markers of DED such as corneal and conjunctival fluorescein staining and, in some studies, tear film break-up time and Schirmer test scores. However, this has been accompanied by only partial improvement in patient symptoms. Although evidence from the literature is still relatively limited, early studies have suggested that diquafosol has a role in the management of DED. Additional studies would be helpful to delineate how different subgroups of DED respond to diquafosol. The therapeutic combination of diquafosol with other topical agents also warrants further investigation.
the osteoblastic lineage, we tested the mRNA expression of the Runx2 transcription factor, which is one of the earliest determinants of osteoblast differentiation driving the differentiation of MSCs into pre-osteoblasts . Its pro-osteogenic activity implies not only the up-regulation of its expression but also post-translational modifications like phosphorylation, which depend on the Rho and MAP kinase signaling pathways [16, 20]. ADP does not modulate Runx2 expression in these cultures, but increases the mRNA level of OSX, another important transcription factor involved in osteoblast differentiation that is under the control of Runx2. ATP, the precursor of ADP, has been shown to stimulate Runx2 DNA-binding activity in osteoblast-like HOBIT cell line in an Erk1, 2-dependent way . Moreover, the RhoA/ROCK1 signaling pathway is linked to P2Y 13 R activation in
22 Read more
patients requiring long-term oral anticoagulant therapy Approximately 6–8% of patients undergoing PCI have a concomitant indication for long-term OAC with a vitamin K antagonist (VKA) or a non-VKA oral antico- agulant (NOAC) for conditions such as atrial fibrillation (AF), left ventricular thrombus, mechanical valve pros- thesis or venous thromboembolism. Current evidence to guide the management of patients undergoing PCI and requiring long-term OAC practice remains limited. Compared with VKA alone, dual therapy (clopidogrel and VKA) and triple therapy (VKA and DAPT with a P2Y 12 receptor inhibitor) in patients with AF following MI or PCI have been shown to increase the risk of major fatal and non-fatal bleeding. 62 Omission of aspirin while main-
16 Read more