Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y(1) and P2Y(12) nucleotide receptors, 2-MeSADP, by blocking the beta-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y(1) and P2Y(12) receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human platelets, this derivative readily regenerates the parent agonist upon mild irradiation with long-wave UV light (360 nm). The functional effect of the regenerated agonist was demonstrated by a rise in intracellular calcium mediated by either P2Y(1) or P2Y(12) receptors in transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced to aggregate upon UV irradiation. At 1.0 microM MRS2703, full aggregation was achieved within 1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y receptor activation with light-directed spatial and temporal control.     

2,2'-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

The effect of 2,2'-pyridylisatogen tosylate (PIT) on the human P2Y(1) receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y(1) receptors in a non-competitive and concentration-dependent manner. The IC(50) for reduction of the maximal agonist effect was 0.14microM. In contrast, MRS2179, a competitive P2Y(1) receptor antagonist, parallel-shifted the agonist concentration-response curve to the right. PIT also concentration-dependently blocked the P2Y(1) receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y(1) receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y(1) receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y(1) receptor, [3H] MRS2279. It was found that PIT (0.01-10microM) did not inhibit [3H] MRS2279 binding to the human P2Y(1) receptor. PIT (10microM) had no effect on the competition for [3H] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y(1) receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y(2), P2Y(4), P2Y(6), P2Y(11) and P2Y(12) receptors. Thus, PIT selectively and non-competitively blocked P2Y(1) receptor signaling without affecting nucleotide binding.     

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

Novel effect of carvedilol on Ca2+ movement in renal tubular cells

The effect of carvedilol on intracellular free Ca(2+) levels ([Ca(2+)](i)) has not been explored previously. This study was aimed to examine the effect of carvedilol on Ca(2+) handling in renal tubular cells. Madin-Darby canine kidney cells were used as a model for renal tubular cells and fura-2 was used as a fluorescent Ca(2+) probe. Carvedilol increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 5 microM. Extracellular Ca(2+) removal partly inhibited the [Ca(2+)](i) signals. Carvedilol-induced Ca(2+) influx was verified by measuring Mn(2+)-induced quench of fura-2 fluorescence. Carvedilol-induced store Ca(2+) release was reduced by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) but not with 5 microM ryanodine or 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Carvedilol (30 microM)-induced Ca(2+) release was not affected by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-l)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; 2 microM), but was potentiated by increasing cAMP levels or inhibiting protein kinase C. The carvedilol-induced Ca(2+) mobilization was not significantly sequestered by the endoplasmic reticulum or mitochondria. This study shows that carvedilol increased [Ca(2+)](i) in renal tubular cells by causing Ca(2+) release from the endoplasmic reticulum and other unknown stores in an inositol-1,4,5-trisphosphate-independent manner, and by inducing Ca(2+) influx. The Ca(2+) release was modulated by cAMP and protein kinase C.     

Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5'-triphosphate analogues at the human P2Y2 and P2Y4 receptors

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 2'-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2'-amino-2'-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 2'-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3-30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPalphaS and 2'-deoxy-UTPalphaS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.     

Azaspiracid-4 inhibits Ca2+ entry by stored operated channels in human T lymphocytes

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6-11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca2+]i in freshly human lymphocytes. Cytosolic Ca2+ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca2+ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca2+ levels induced by thapsigargin (Tg). AZ-4 decreased Ca2+-influx induced by Tg but did not affect the Ca2+-release from internal stores induced by this drug. The effects of AZ-4 on Ca2+-influx induced by Tg were reversible and not regulated by adenosine 3',5'-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca2+ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca2+-influx by 5-10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca2+ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.     

Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.     

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.     

Inhibition of human and mouse plasma membrane bound NTPDases by P2 receptor antagonists

The plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase)-1, 2, 3 and 8 are major ectonucleotidases that modulate P2 receptor signaling by controlling nucleotides' concentrations at the cell surface. In this report, we systematically evaluated the effect of the commonly used P2 receptor antagonists reactive blue 2, suramin, NF279, NF449 and MRS2179, on recombinant human and mouse NTPDase1, 2, 3 and 8. Enzymatic reactions were performed in a Tris/calcium buffer, commonly used to evaluate NTPDase activity, and in a more physiological Ringer modified buffer. Although there were some minor variations, there were no major changes either in the enzymatic activity or in the profile of NTPDase inhibition between the two buffers. Except for MRS2179, all other antagonists significantly inhibited these ecto-ATPases; NTPDase3 being the most sensitive to inhibition and NTPDase8 the most resistant. Estimated IC(50) showed that human NTPDases were generally more sensitive to the P2 receptor antagonists tested than the corresponding mouse isoforms. NF279 and reactive blue 2 were the most potent inhibitors of NTPDases which almost completely abrogated their activity at the concentration of 100 microM. In conclusion, reactive blue 2, suramin, NF279 and NF449, at the concentrations commonly used to antagonize P2 receptors, inhibit the four major ecto-ATPases. This information may reveal useful for the interpretation of some pharmacological studies of P2 receptors. In addition, NF279 is a most potent non-selective NTPDase inhibitor. Although P2 receptor antagonists do not display a strict selectivity toward NTPDases, their IC(50) values may help to discriminate some of these enzymes.     

Functional calcium coupling with the human metabotropic glutamate receptor subtypes 2 and 4 by stable co-expression with a calcium pathway facilitating G-protein chimera in Chinese hamster ovary cells

The objective of the current study was to facilitate functional calcium assays, compatible with the fluorometric imaging plate reader platform, for the human metabotropic glutamate receptor (mGluR) subtypes 2 and 4, by co-expressing each receptor with a G-protein chimera comprising Galphaq with the C-terminal five amino acids replaced with those from Galphai3 (GqGi3). Transfection of GqGi3 into previously validated stable CHO cell lines expressing mGluR2 or mGluR4 allowed for the selection of new double transfectants in which application of L-glutamate and other mGluR agonists resulted in calcium coupling with a high signal:noise ratio (maximal changes in relative fluorescence units up to 20,000). The rank order of agonist potency for the stimulation of calcium mobilization in the mGluR2/GqGi3 stable cell line was LY354740>L-CCG-I=DCG-IV>L-glutamate>/=(2R,4R)-APDC>/=(1S,3R)-ACPD. In the mGluR4/GqGi3 stable cell line the rank order of agonist potency was L-AP4>L-SOP>/=ACPT-I=L-CCG-I>/=L-glutamate=(R,S)-PPG. By comparison, equivalent potency orders and a significant correlation in functional activities were observed when the same compounds were profiled in [35S]GTPgammaS binding assays for each mGluR subtype. These results validate the use of functional calcium assays, amenable to high-throughput applications on the fluorometric imaging plate reader, for the mGluR2 and mGluR4 subtypes when co-expressed in stable cell lines with the GqGi3 chimera.     

A novel adenosine analog, thio-Cl-IB-MECA, induces G0/G1 cell cycle arrest and apoptosis in human promyelocytic leukemia HL-60 cells

Human A3 adenosine receptor (A3AR) agonists have been shown to play important roles in several physiological and pathological processes, including growth inhibition of human cancer cells. On this line, we recently found that a novel adenosine analog, 2-chloro-N6-(3-iodobenzyl)-4'-thioadenosine-5'-N-methyluronamide (thio-Cl-IB-MECA) was a potent human A3AR agonist, and is superior to a known agonist Cl-IB-MECA [Jeong LS, Jin DZ, Kim HO, Shin DH, Moon HR, Gunaga P, et al. J Med Chem 2003;46:3775]. Here, we report that a novel A3AR agonist, thio-Cl-IB-MECA inhibited the growth of human promyelocytic leukemia HL-60 cells by arresting cell cycle and induction of apoptosis. Thio-Cl-IB-MECA induced the cell cycle arrest of G0/G1 in the early time and at lower concentration (up to 25 microM). At higher concentration (50 microM), the apoptotic cell deaths were manifested by observation of the increase of sub-G0 phase of cell cycle distribution, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, the down-regulation of checkpoint protein cyclin D1 and c-myc by thio-Cl-IB-MECA was well correlated with the arrest of cell cycle transition of G1 to S phase. Further study revealed that the growth inhibitory activity of thio-Cl-IB-MECA is also related with the modulation of Wnt signaling pathway. The levels of beta-catenin, phosphorylated forms of GSK-beta and Akt were down-regulated by the treatment of thio-Cl-IB-MECA (10 nM) in a time-dependent manner, providing one of plausible mechanistic evidence for the involvement of the Wnt signaling pathway in the HL-60 cell growth inhibitory effects by thio-Cl-IB-MECA. These results suggest that a novel A3AR agonist, thio-Cl-IB-MECA can down-regulate Wnt signaling, inhibit proliferation and induce apoptosis in HL-60 leukemia cells, and thus provide the possibility of this compound in the potential therapeutic value of the treatment of leukemia.     

Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0-30 °C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17 ≤ ΔH° ≤ 59 kJ/mol and 213 ≤ ΔS° ≤ 361 kJ/mol for agonists and −52 ≤ ΔH° ≤ −26 kJ/mol and −12 ≤ ΔS° ≤ 38 kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27 ≤ ΔH° ≤ 48 kJ/mol and 234 ≤ ΔS° ≤ 300 kJ/mol for agonists and −19 ≤ ΔH° ≤ −17 kJ/mol and 43 ≤ ΔS° ≤ 74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.     

Effect of hydrogen peroxide on Ca2+ mobilisation in human platelets through sulphydryl oxidation dependent and independent mechanisms

Using Fura-2-loaded human platelets we studied the nature of the mechanisms involved in Ca2+ signalling mediated by H2O2. In a Ca2+-free medium, H2O2 (10 microM-100 mM) induced a concentration-dependent increase in [Ca2+]i. Depletion of either agonist-sensitive or mitochondrial Ca2+ pools reduced this effect while depletion of both stores abolished it. Xestospongin C, an inositol 1,3,5-trisphosphate (IP3) receptor inhibitor, reduced Ca2+ release evoked by 1 mM H2O2 by 45%, indicating that H2O2-induced Ca2+ release involves interaction with IP3 receptors. Blockade of the IP3 turnover by lithium or treatment with U-73122 did not modify H2O2-induced Ca2+ release from the agonist-sensitive pool, suggesting the involvement of a mechanism independent of IP3 generation. H2O2 inhibited Ca2+ reuptake into the agonist-sensitive stores mediated by the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Thimerosal (5 microM), a sulphydryl reagent, induced Ca2+ release from the agonist-sensitive stores. This event was impaired by treatment with 2 mM DTT, which also inhibited H2O2-induced Ca2+ release from the agonist-sensitive pool but not from mitochondria. H2O2 reduced the ability of the plasma membrane Ca2+ ATPase (PMCA) to extrude Ca2+ by 75%, an effect that was unaffected by DTT. Consistent with this, thimerosal did not modify the PMCA activity. Finally, exposure to H2O2 triggered platelet aggregation, which was slower than that observed after agonist stimulation. We conclude that H2O2 induced Ca2+ release from agonist-sensitive stores by oxidation of sulphydryl groups in SERCA and the IP3 receptors independently of IP3 generation. In addition, H2O2 induced Ca2+ release from mitochondria and inhibited the PMCA activity by different mechanisms in human platelets.     

Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells

G protein-coupled receptors, the largest cell surface receptor family, have emerged as critical players in cell death and survival. High gene expression level of the G_q-coupled P2Y₁ nucleotide receptor in PC-3 prostate cancer cells was demonstrated using real-time quantitative PCR and confirmed by Western blotting and confocal laser scanning microscopy. A selective P2Y₁ receptor agonist, the ADP analogue MRS2365, concentration-dependently induced intracellular calcium mobilization (EC₅₀ 5.28 nM), which was diminished by P2Y₁ receptor-selective antagonist MRS2500. P2Y₁ receptor activation by MRS2365 induced apoptosis in assays of Caspase-3, LDH release, and annexin-V staining. The pro-apoptotic effect of MRS2365 was blocked by MRS2500, P2Y₁ siRNA, and an inhibitor of the MAP kinase pathway PD98059. MRS2365 significantly inhibited the proliferation of PC-3 cells, examined using a MTT assay. Thus, activation of the P2Y₁ receptor induced cell death and inhibited growth of human prostatic carcinoma PC-3 cells. Activation of the P2Y₁ receptor should be a novel and promising therapeutic strategy for prostate cancer.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Stimulation of intracellular Ca2+ elevation in neutrophils by thiol-oxidizing phenylarsine oxide

Phenylarsine oxide (PAO), a trivalent arsenical compound, stimulated [Ca2+]i elevation in rat neutrophils in a Ca2+-containing medium but caused no appreciable response in a Ca2+-free medium. PAO also induced external Mn2+ entry, which was inhibited by N-acetyl-L-cysteine (NAC), but failed to elicit any appreciable Ba2+ and Sr2+ entry. Pretreatment of neutrophils with thiol-reducing agents including dithiothreitol (DTT), NAC, 2,3-dimercapto-1-propanol (DMP), 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), all greatly inhibited PAO-induced [Ca2+]i elevation. Addition of Ni2+ or La3+ followed by PAO stimulation also attenuated the Ca2+ signals in a concentration-dependent manner. PAO had no significant effect on the production of reactive oxygen intermediates (ROI) and nitric oxide (NO) nor did it decrease cellular low molecular weight thiols levels. PAO-induced [Ca2+]i elevation was significantly inhibited by 1-[6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, genistein, a general tyrosine kinase inhibitor, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calyculin A, a cortical actin stabilizer, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase inhibitor, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), and cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), the blockers of receptor-gated and store-operated Ca2+ channels, whereas there was no appreciable effect exerted by aristolochic acid, a phospholipase A2 inhibitor, 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), the blockers of NO synthase, and by suspension in a Na+-deprived medium. In contrast, 2-aminoethoxydiphenyl borane (2-APB), the blocker of IP3 receptor and Ca2+ influx, enhanced the PAO-induced response. PAO had no effect on the plasma membrane Ca2+-ATPase (PMCA) activity in the pharmacological isolated neutrophil preparation and the neutrophil membrane fractions. These results indicate that PAO stimulates [Ca2+]i rise in rat neutrophils mainly through the oxidation of vicinal thiol groups on the cell surface membrane to activation of a non-store operated Ca2+ entry (non-SOCE) without affecting the activity of PMCA and the plasmalemmal Na+/Ca2+ exchanger.     

Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y13 receptor

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5'-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing G(alpha)16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5'-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5'-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5'-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP3) formation with pIC50 values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y1 receptor-mediated 1321N1 astrocytoma cells with IC50 values of >10 and 0.245 microM, respectively. Both analogues were inactive (IC50>10 microM) as antagonists of human P2Y12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y13 receptor in comparison to P2Y1 and P2Y12 receptors, while MRS2603 antagonized both P2Y1 and P2Y13 receptors.