Cell cycle regulation by ADP and IGF-1 in cultured late developing glia progenitors of the avian retina.

In the avian retina, ADP induces the proliferation of late developing glia progenitors. Here, we show that in serum-containing retinal cell cultures, ADP-induced increase in [3H]-thymidine incorporation can be prevented by the IGF-1 receptor antagonists AG1024 and I-OMe-Tyrphostin AG 538, suggesting the participation of IGF-1 in ADP-mediated progenitor proliferation. In contrast, no increase in [3H]-thymidine incorporation is observed in retinal cultures treated only with IGF-1. Under serum starvation, while no increase in cell proliferation is detected in cultures treated only with ADP or IGF-1, a significant increase in [3H]-thymidine incorporation and number of PCNA expressing cells is observed in cultures treated concomitantly with ADP plus IGF-1, suggesting that both molecules are required to induce proliferation of retinal progenitors. In serum-starved cultures, although an increase in cell viability is detected by MTT assays in IGF-1-treated cultures, no significant increase in viability of [3H]-thymidine labeled progenitors is observed, suggesting that IGF-1 may contribute to survival of postmitotic cells in culture. While only ADP increases intracellular calcium, only IGF-1 induces the phosphorylation of Akt in the retinal cultures. IGF-1 through the PI3K/Akt pathway induces a significant increase in the transcription and expression of CDK1 with a decrease in phospho-histone H3 expression that is concomitant with an increase in the expression of cyclins D1 and E and CDK2. These findings suggest that IGF-1 stimulates CDK-1 mRNA and protein expression that enable progenitors to progress through the cell cycle. However, signaling of ADP in the presence IGF-I seems to be required for DNA synthesis.

Potential Therapeutic Applications of P2 Receptor Antagonists: From Bench to Clinical Trials.

BACKGROUND: Extracellular purines and pyrimidines have important physiological functions in mammals. Purines and pyrimidines act on P1 and P2 purinergic receptors, which are widely expressed in the plasma membrane in various cell types. P2 receptors act as important therapeutic targets and are associated with several disorders, such as pain, neurodegeneration, cancer, inflammation, and thrombosis. However, the use of antagonists for P2 receptors in clinical therapy, with the exception of P2Y12, is a great challenge. Currently, many research groups and pharmaceutical companies are working on the development of specific antagonist molecules for each receptor subtype that could be used as new medicines to treat their respective disorders. OBJECTIVE: The present review compiles some interesting findings on the application of P2 receptor antagonists in different in vitro and in vivo experimental models as well as the progress of advanced clinical trials with these compounds. CONCLUSION: Despite all of the exciting results obtained on the bench, few antagonists of P2 receptors advanced to the clinical trials, and once they reach this stage, the effectiveness of the therapy is not guaranteed, as in the example of P2X7 antagonists. Despite this, P2Y12 receptor antagonists have a history of success and have been used in therapy for at least two decades to prevent thrombosis in patients at risk for myocardial infarctions. This breakthrough is the motivation for scientists to develop new drugs with antagonistic activity for the other P2 receptors; thus, in a matter of years, we will have an evolution in the field of purinergic therapy.

Intralesional uridine-5'-triphosphate (UTP) treatment induced resistance to Leishmania amazonensis infection by boosting Th1 immune responses and reactive oxygen species production.

Leishmania amazonensis is the etiologic agent of cutaneous leishmaniasis, an immune-driven disease causing a range of clinical symptoms. Infections caused by L. amazonensis suppress the activation and function of immune cells, including macrophages, dendritic cells, and CD4+ T cells. In this study, we analyzed the course of infection as well as the leishmanicidal effect of intralesional UTP treatment in L. amazonensis-infected BALB/c mice. We found that UTP treatment reduced the parasitic load in both footpad and lymph node sites of infection. UTP also boosted Th1 immune responses, increasing CD4+ T cell recruitment and production of IFN-γ, IL-1ß, IL-12, and TNF-α. In addition, the role of UTP during innate immune response against L. amazonensis was evaluated using the air pouch model. We observed that UTP augmented neutrophil chemoattraction and activated microbicidal mechanisms, including ROS production. In conclusion, our data suggested an important role for this physiological nucleotide in controlling L. amazonensis infection, and its possible use as a therapeutic agent for shifting immune responses to Th1 and increasing host resistance against L. amazonensis infection.

Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

BACKGROUND: All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. METHODS: Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. RESULTS: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. CONCLUSION: Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

Padronização da triagem de alto desempenho de antagonistas para receptores purinérgicos ativados por UTP a partir da mensuração de cálcio intracelular / Standardization of the screening of high performance antagonists For receivers purinergic activated by UTP from Measurement of calcium intracellular

Os receptores purinérgicos são proteínas expressas na membrana plasmática de diversos tiposcelulares e são ativadas por purinas e pirimidinas extracelulares. Eles são classificados emreceptores P1 e P2, de acordo com o tipo de ligante fisiológico, entretanto, os P2R são aindasubdivididos nas classes: P2XR, que abrange os subtipos ionotrópicos e P2YR, que agrupa ossubtipos acoplados à proteína G. Dentre os P2YR, os subtipos P2Y2 e P2Y4 vem sendoamplamente estudados em virtude de suas funções sobre diversos sistemas biológicos. Umavez ativados pelo UTP, estes receptores promovem a ativação e inibição de canais iônicos,controlam o fluxo de íons através da membrana, induzem um aumento de cálcio intracelular,além de estimular a proliferação celular. Em vista disso, muitos grupos de pesquisa já vemestudando o P2Y2 e o P2Y4 como alvos terapêuticos para o tratamento de diversas doenças,tais como fibrose cística, doença do olho seco, doença de Alzheimer, entre outras. Entretanto,a escassez de moléculas com propriedade antagonista seletiva sobre estes receptores dificultao avanço da caracterização de novos papéis funcionais associados a eles, e neste contexto, osprodutos naturais podem atuar como uma importante fonte para a descoberta de compostosque apresentem a característica almejada. Por isso, o objetivo deste trabalho foi padronizaruma metodologia de mensuração de cálcio intracelular induzido pelo UTP em células dalinhagem de macrófagos murinos J774.G8 e realizar a triagem de extratos oriundos deprodutos naturais a fim de identificar compostos com atividade antagonista sobre o P2Y2R eo P2Y4R. Para isso, foi padronizado um protocolo de carregamento celular com o indicadorde cálcio Fluo-4AM com posterior leitura dos sinais de cálcio no FlexStation III em células J774.G8...

The purinergic receptors are proteins expressed in plasma membrane of many cell types thatare activated by extracellular purines and pyrimidines. They are classified in P1 and P2receptors, according to physiological ligand. However, the P2R are further subdivided in twoclasses: P2X for ionotropic subtypes and P2Y for G-protein coupled ones. Among P2YR, theP2Y2 and P2Y4 subtypes has been widely studied due to their functions in many biologicalsystems. Once activated by UTP, these receptors promote the activation and inhibition of ionchannels, control the flow of ions through the membrane, induce an increase of intracellularcalcium and stimulate the cell proliferation. In view of this, many research groups have beenstudying the P2Y2 and P2Y4 as therapeutic targets for the treatment of several diseases likeCystic Fibrosis, Dry Eye Disease, Alzheimer, among others. However, the lack of moleculeswith selective antagonist property on these receptors difficults the advancement ofcharacterization of new functional roles associated with them, and in this context, naturalproducts may act as an important source for the discovery of compounds that present thisdesired feature. Therefore, the aim of this study was to standardize a methodology formeasurement of intracellular calcium mobilization induced by UTP in a murine macrophagecell line J774.G8 and perform the screening of extracts derived from natural products in orderto identify compounds with antagonist activity on the P2Y2R and P2Y4R. For this, it wasstandardized a protocol of cell labeling with calcium indicator Fluo-4AM in J774.G8 cellswith subsequent detection of calcium signals in FlexStation III...