30 research outputs found
Two distinct P2Y receptors are involved in purine- and pyrimidine-evoked Ca2+ elevation in mammalian brain astrocytic cultures
No full textATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca2+](i)) in rat striatal astrocytes (Centemeri et al. (1997) Br J Pharmacol 121:1700-1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca2+](i) increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca2+](i) elevation (EC(50)s = 0.58 muM +/- 0.4 and 31 muM +/- 6, respectively). Pyrimidine-evoked [Ca2+](i) elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca2+](i) increments. Guanosine-5'-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca2+](i) rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y(1) receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca2+](i) elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y(1) and P2Y(6), subtypes
Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes
No full text1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry. 2. ATP triggered a fast and transient elevation of [Ca2+]i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), as well as uridine-5'-triphosphate (UTP) resembled that of ATP, while alpha, beta-methylene-ATP (alpha, beta-meATP) and beta, gamma-methylene-ATP (beta, gamma-meATP) were totally ineffective. 3. Suramin (50 microM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 microM) significantly depressed the maximum response. 4. Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses. 5. Furthermore, after preincubation with 10 microM thapsigargin, the nucleotide-evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved. 6. Both application of the G-protein blocker guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml-1) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 microM), significantly reduced the ATP-evoked [Ca2+]i rise. 7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC
Two distinct P2Y receptors are involved in purine- and pyrimidine-evoked Ca2+ elevation in mammalian brain astrocytic cultures
No full textATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca2+](i)) in rat striatal astrocytes (Centemeri et al. (1997) Br J Pharmacol 121:1700-1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca2+](i) increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca2+](i) elevation (EC(50)s = 0.58 muM +/- 0.4 and 31 muM +/- 6, respectively). Pyrimidine-evoked [Ca2+](i) elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca2+](i) increments. Guanosine-5'-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca2+](i) rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y(1) receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca2+](i) elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y(1) and P2Y(6), subtypes. Drug Dev. Res. 52:122-132, 2001
Heterogeneous platelet-activating factor (PAF) receptors and calcium increase in platelets and macrophages
No full textWe used the increase in cytosolic Ca2+ levels, [Ca2+]i, as a way to characterize PAF (platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptors in human platelets and rat and human macrophages. [Ca2+] was measured by means of the fluorescent probe fura-2/acetoxymethylester. PAF recognized heterogeneous receptors in human macrophages only (curve slope <1). The PAF antagonist SCH 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5.6]cyclohepta[1,2-b]pyridine -11-ylidine)piperidine) abolished [Ca2+]i elevation in human platelets, while in rat and human macrophages the maximal inhibition was 76% and 85%, respectively. On the contrary, the antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6Hthieno[3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]-diazepin-2-yl]-1-(4-morpholiny)-1-propanon, apafant) totally inhibited the effect of PAF in both platelets and macrophages. The WEB 2086 concentration-response curves had a slope <1 in the three cell types, indicating interaction with heterogeneous receptors. Accordingly, 3H-WEB 2086 bound to two different classes of sites. Both phases of [Ca2+]i elevation (influx or release) were equally affected by the antagonists. These data support the notions that: 1) PAF receptors are heterogeneous; 2) the two antagonists have a different selectivity toward the receptor subtypes: WEB 2086 recognizes two different receptors both in platelets and in macrophages, while SCH 37370 does not discriminate between receptor subtypes in platelets, and only interacts with one subtype in macrophages; and 3) both SCH 37370 and WEB 2086 display different potencies in rat and human macrophages
Purinoceptors involved in ATP- and UTP-induced calcium elevation in rat striatal astrocytes
No full textATP-induced intracellular calcium elevation in rat striatal astrocytes is mediated exclusively by G-protein coupled purinoceptors
No full textCharacterization of an ATP receptor coupled to calcium release in rat striatal astrocytes
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