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P2 Receptor Antagonists (p2 + receptor_antagonist)
Selected AbstractsSpontaneous oscillation and mechanically induced calcium waves in chondrocytesCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2006Taisuke Kono Abstract The characteristics of spontaneous calcium (Ca2+) oscillation and mechanically induced Ca2+ waves in articular chondrocytes were studied. In some, but not all, chondrocytes in sliced cartilage and primary cultures, we observed spontaneous oscillation of intracellular Ca2+ that never spread to adjacent cells. In contrast, a mechanical stimulus to a single cell by touching with a glass rod induced an increase of intracellular Ca2+ that spread to neighboring cells in a wave-like manner, even though there was no physical contact between the cells. This indicated the release of some paracrine factor from the mechanically stimulated cells. Application of ultrasonic vibration also induced an oscillation of intracellular Ca2+. The application of a uridine 5,-triphosphate (UTP), UTP, induced a transient increase in intracellular Ca2+ and the release of adenosine 5,-triphosphate (ATP) in cultured chondrocytes. A P2 receptor antagonist (suramin) and blockers of Cl, channels, niflumic acid and 4,4,-diisothiocyanostilbene-2,2,-disulfonic acid (DIDS), reduced the UTP-induced ATP release. The results indicated that Cl, channels were involved in the extracellular release of ATP following mechanical or P2Y receptor stimulation. Thus, ATP stimulation of P2Y receptors elicits an increase in intracellular Ca2+, triggering further release of ATP from adjacent cells, thereby expanding the Ca2+ wave in chondrocytes. Copyright © 2005 John Wiley & Sons, Ltd. [source] Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cellsACTA PHYSIOLOGICA, Issue 3 2009H. A. Praetorius Abstract Aim:, Changes in perfusate flow produce increases in [Ca2+]i in renal epithelial cells. Cultured renal epithelia require primary cilia to sense subtle changes in flow. In perfused kidney tubules this flow response is caused by nucleotide signalling via P2Y2 receptors. It is, however, not known whether nucleotides are released by mechanical stress applied to renal primary cilia. Here we investigate whether nucleotides are released during the cilium-dependent flow response and contribute to the flow-induced, cilium-dependent [Ca2+]i signal. Methods:, MDCK cells loaded with Fluo-4-AM were observed at 37 °C in semi-open single or closed-double perfusion chambers. Results:, Our data suggest a purinergic component of the cilium-dependent flow-response: (1) ATP scavengers and P2 receptor antagonists reduced (55%) the cilium-dependent flow-response; (2) ATP added at subthreshold concentration sensitized the renal epithelia to flow changes; (3) increases in fluid flow transiently enhanced the ATP concentration in the superfusate (measured by biosensor-cells). To test if nucleotides were released in sufficient quantities to stimulate renal epithelia we used non-confluent MDCK cells without cilia as reporter cells. We confirmed that non-confluent cells do not respond to changes in fluid flow. Placing confluent, ciliated cells upstream in the in-flow path of the non-confluent cells made them responsive to fluid flow changes. This phenomenon was not observed if either non-confluent or de-ciliated confluent cells were placed upstream. The [Ca2+]i -response in the non-confluent cells with ciliated cells upstream was abolished by apyrase and suramin. Conclusion:, This suggests that subtle flow changes sensed by the primary cilium induces nucleotide release, which amplifies the epithelial [Ca2+]i -response. [source] Neurone-to-astrocyte communication by endogenous ATP in mixed culture of rat hippocampal neurones and astrocytesDRUG DEVELOPMENT RESEARCH, Issue 1 2003Schuichi Koizumi ATP is recognized as an important intercellular signaling molecule in the peripheral and CNS. Glutamate is reported to be an important neurone-to-glia mediator being released from neurones and astrocytes that activates astrocytic and neuronal Ca2+ responses, respectively. We demonstrate here that endogenous ATP could be an extracellular molecule for neurone-to-astrocyte communication in cocultured rat hippocampal neurones and astrocytes. Hippocampal neurones reveal synchronized Ca2+ oscillation, which was due to glutamatergic synaptic transmission. When analyzed in a fura-2 method, a slight and very slow increase in intracellular Ca2+ concentration ([Ca2+]i) elevation was observed in some population of astrocytes. Such astrocytic [Ca2+]i elevation was dramatically inhibited by apyrase, though apyrase itself had no effect on neuronal Ca2+ oscillation. For a detail analysis, we investigated changes in [Ca2+]i in cells using a confocal microscopy. When cocultured hippocampal neurones and astrocytes were depolarized electronically in the presence of glutamate-receptor antagonists, a transient elevation in [Ca2+]i was observed in neurones, which was followed by a slowly initiated and small rise in [Ca2+]i in astrocytes. Apyrase or P2 receptor antagonists almost abolished the [Ca2+]i rises in astrocytes, suggesting that depolarization-evoked ATP release from neurones should produce astrocytic [Ca2+]i elevation via P2 receptors. Using a luciferin,luciferase bioluminescence assay, we found that neurones could release ATP in an activity-dependent manner. These findings suggest that endogenous ATP should be an important intercellular mediator between neurones and astrocytes and that functions of these cells should be fine-tuned by endogenously released ATP in situ. Drug Dev. Res. 59:88,94, 2003. © 2003 Wiley-Liss, Inc. [source] Multiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp-Stimulated Rat Brown AdipocytesEXPERIMENTAL PHYSIOLOGY, Issue 6 2002Mariko Omatsu-Kanbe Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca2+ concentration ([Ca2+]i), which arises primarily from a release of Ca2+ from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca2+]i elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2,,4,-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 ,M) almost completely depressed the thapsigargin (100 nM)-evoked [Ca2+]i elevation mediated through store-operated Ca2+ entry. The inhibitory effect of ATP was antagonized by PPADS with IC50 of 0.7 ,M. In the presence of PPADS at concentrations of more than 5 ,M, the ATP-induced [Ca2+]i elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca2+]i elevation was reduced in a concentration-dependent manner by PPADS with an IC50 of 200 ,M. In contrast, the ATP-induced [Ca2+]i elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca2+]i responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca2+ stores, which is probably followed by the activation of store-operated Ca2+ entry. Extracellular ATP, however, would inhibit this Ca2+ entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca2+]i elevation in response to extracellular ATP. [source] Characterization of calcium-independent purinergic receptor-mediated apoptosis in hormone-refractory prostate cancerBJU INTERNATIONAL, Issue 3 2008Majid Shabbir OBJECTIVE To investigate the nature of purinergic signalling in hormone-refractory prostate cancer (HRPC) cells in vitro, as extracellular ATP inhibits the growth of HRPC in vitro via the activation of P2 purinergic receptors, and to characterize which P2 receptors subtypes and secondary mechanisms are involved. MATERIALS AND METHODS The effect of extracellular ATP on HRPC cell lines PC-3 and DU-145, and the normal prostate cell line PNT-2, were investigated. Reverse-transcription polymerase chain reaction was used to assess P2 purinergic receptors, which were pharmacologically characterized using various receptor agonists and antagonists. The effect of ATP on intracellular Ca2+ concentration ([Ca2+]i) was examined to asses its role in growth inhibition. The effect of combining ATP with the chemotherapeutic drug mitoxantrone was also assessed. RESULTS PC-3 cells expressed mRNA for P2X4,5,7, P2Y1,2,4,6; DU-145 cells expressed mRNA for P2X4,5, P2Y1,2,4,6,11; PNT-2 cells expressed mRNA for P2X4,5,7 and P2Y1,2,4,6,11. ATP (10,4m) inhibited HRPC PC-3 cell growth by ,,90%, an effect partially inhibited by the nonselective P2 receptor antagonists pyridoxal-5,-phosphate-6-azophenyl-2,,4, disulphonic acid (PPADS) and suramin. The order of potency of agonists was: adenosine 5,-O-(3 thiotriphosphate) > ATP > benzoyl benzoyl ATP >> 2-methylthio ATP. DU-145 cells responded similarly. Pharmacological profiling implicated P2X5 and/or P2Y11 receptors in the antineoplastic response in HRPC. ATP induced apoptosis in a [Ca2+]i -independent mechanism. ATP was significantly less effective on PNT-2 cells, which also had a different order of agonist potency. ATP combined with mitoxantrone in an additive manner in HRPC. CONCLUSIONS ATP effectively reduces growth of HRPC cells via calcium-independent apoptosis. Pharmacological profiling indicates P2X5 and/or P2Y11 receptors in this process, with a different functional purinergic receptor profile and sensitivity in normal vs cancer cells. [source] | |||||||||||||