iloprost and Leukemia--Erythroblastic--Acute

Like Ras, farnesylation of the IP (prostacyclin receptor) is required for its efficient intracellular signalling, and hence the IP represents a potential target for inhibition by FTIs [FTase (farnesyl protein transferase) inhibitors]. Herein, the effect of SCH66336 on the isoprenylation and function of the human and mouse IPs overexpressed in human embryonic kidney 293 cells, and by the IP endogenously expressed in human erythroleukaemia cells, was investigated. SCH66336 yielded concentration-dependent decreases in IP-mediated cAMP generation (IC50 0.27-0.62 nM), [Ca2+]i mobilization (IC50 26.6-48.3 nM) and IP internalization, but had no effect on signalling by the non-isoprenylated beta2 adrenergic receptor or b isoform of the TP (prostanoid thromboxane A2 receptor). Additionally, SCH66336 impaired IP-mediated crossdesensitization of TPa signalling (IC50 56.1 nM) and reduced farnesylation of the molecular chaperone protein HDJ-2 (IC50 3.1 nM). To establish whether farnesylation of the IP is inhibited and/or whether its 'CaaX motif' might undergo alternative geranylgeranylation in the presence of SCH66336, a series of chimaeric Ha (Harvey)-Ras fusions were generated by replacing its CaaX motif (-CVLS) with that of the IP (-CSLC) or, as controls, of Ki (Kirsten)-Ras 4B (-CVIM) or Rac 1 (-CVLL). Whereas SCH66336 had no effect on Ha-RasCVLL isoprenylation in vitro or in whole cells, it supported alternative geranylgeranylation of Ha-RasCVIM, but completely impaired isoprenylation of both Ha-RasCVLS and Ha-RasCSLC. These data confirm that the -CSLC motif of the IP is a direct target for inhibition by the FTI SCH66336, and in the presence of strong FTase inhibition, the IP does not undergo compensatory geranylgeranylation

Topics: Adrenergic beta-Agonists; Alkyl and Aryl Transferases; Amino Acid Motifs; Animals; Calcium Signaling; Carrier Proteins; Cell Line; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Endocytosis; Epoprostenol; Farnesyltranstransferase; Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Humans; Iloprost; Isoproterenol; Kidney; Leukemia, Erythroblastic, Acute; Mice; Mutagenesis, Site-Directed; Organophosphorus Compounds; Piperidines; Proline; Propanolamines; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); Pyridines; Receptors, Adrenergic, beta-2; Receptors, Epoprostenol; Receptors, Thromboxane A2, Prostaglandin H2; Recombinant Fusion Proteins; Signal Transduction; Transfection

Prostacyclin, one of the cyclooxygenase metabolites, causes various biological effects, including vasodilation and antithrombogenicity, and is also involved in several pathophysiological effects, such as inflammatory pain and bladder disorders. The prostacyclin receptor (IP receptor) agonists iloprost, cicaprost, and carbacyclin have been useful for clarifying the role of the IP receptor signaling, since the endogenous ligand, prostacyclin, is very unstable. On the other hand, only a few IP receptor antagonists have been reported to date. Here, we characterized the biological activities of 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]-3-phenyl-propionic acid (compound A) in various in vitro systems. Compound A inhibited the accumulation of the second messenger cyclic AMP in the UMR-108 rat osteosarcoma cell line and primary cultured rat dorsal root ganglion (DRG) neurons in a concentration-dependent manner up to 10 microM, without affecting other eicosanoid receptors. Functionally, the IP receptor plays an important role in DRG neuron sensitization, which is measured by release of the neurotransmitter substance P. Although the effects of iloprost or Lys-bradykinin, an inflammatory peptide, alone on substance P release were limited, stimulation of the neurons with both these ligands induced substantial amounts of substance P release. This synergistic effect was suppressed by compound A. Collectively, these results suggest that compound A is a highly selective IP receptor antagonist that inhibits iloprost-induced sensitization of sensory neurons. Furthermore, these findings suggest that IP receptor antagonist administration may be effective for abnormal neural activities of unmyelinated sensory afferents. Compound A should prove useful for further investigations of the IP receptor in various biological processes.

Topics: Animals; Antineoplastic Agents; Calcium; Cell Line, Tumor; Cells, Cultured; CHO Cells; Cricetinae; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Ganglia, Spinal; Humans; Iloprost; Inhibitory Concentration 50; K562 Cells; Kallidin; Leukemia, Erythroblastic, Acute; Molecular Structure; Neurons, Afferent; Osteosarcoma; Rats; Receptors, Epoprostenol; Substance P

The purpose of this study was to characterize the prostanoid receptors coupled to intracellular calcium in human erythroleukemia (HEL) cells, a cell line with platelet/megakaryocytic characteristics. Both prostaglandin E1 (PGE1) and iloprost increased cyclic AMP (cAMP) in HEL cells, but modulated [Ca2+]i by different mechanisms. Iloprost (10(-9) to 10(-6) M) had no effect on basal [Ca2+]i, but greatly potentiated the increase in [Ca2+]i produced by thrombin. This effect was mimicked by cholera toxin and other Gs-coupled receptors, and involved calcium influx since iloprost had no effect on [Ca2+]i in cells incubated in Ca2+-free buffer. Furthermore, iloprost did not increase the generation of baseline or thrombin-induced inositol phosphates at these concentrations. In contrast, PGE1 (10(-7) to 10(-5) M), but not iloprost, increased basal [Ca2+]i through a pertussis toxin-sensitive mechanism that involved stimulation of inositol phosphate generation and mobilization of intracellular calcium. The order of potencies of other prostaglandins that increased [Ca2+]i was not consistent with known IP, EP, DP, FP, or TP receptors. 11-Deoxy-16,16-dimethyl PGE2 was the most potent of the analogs tested (EC50 = 28 nM). In summary, at least two prostaglandin receptors are functionally coupled to intracellular calcium in HEL cells: a putative IP receptor coupled to Gs proteins that increases cAMP and enhances calcium influx, and a novel prostanoid receptor that evokes calcium mobilization through stimulation of phospholipase C by a pertussis toxin-sensitive pathway.

Topics: 16,16-Dimethylprostaglandin E2; Alprostadil; Calcium; Cyclic AMP; Humans; Iloprost; Inositol Phosphates; Leukemia, Erythroblastic, Acute; Receptors, Epoprostenol; Receptors, Prostaglandin; Signal Transduction; Thrombin; Tumor Cells, Cultured

Topics: Animals; Blood Platelets; CHO Cells; Cloning, Molecular; Cricetinae; Cyclic AMP; Gene Library; Humans; Iloprost; Kinetics; Leukemia, Erythroblastic, Acute; Receptors, Epoprostenol; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The group of prostaglandin (PG) E2- and prostacyclin receptors consists of different subtypes, which exhibit different affinities for prostaglandins and synthetic analogues. PGE2 activities the E-type PG receptor subtypes EP1, EP2 and EP3, whereas the PGE2 analogue, sulprostone, binds only to the EP1 and EP3 receptor subtypes. The stable PGI2 analogues, iloprost and cicaprost, both activate the PGI2 receptor (IP) and iloprost, additionally, bind to the EP1 subtype. Using these subtype-selective PG receptor agonists, we studied the interaction of PG receptor subtypes with Gs and Gi-type heterotrimeric guanine nucleotide-binding proteins (G proteins) in membranes from the human erythroleukaemia cell line, HEL. Sulprostone stimulated high-affinity GTPase in HEL membranes in a pertussis toxin (PTX)-sensitive manner. In contrast, the stimulations induced by PGE2, iloprost and cicaprost were only partially inhibited by PTX. PGE2, sulprostone, iloprost and cicaprost stimulated cholera toxin-catalysed ADP-ribosylation as well as labelling with GTP azidoanilide of membrane proteins comigrating with immunologically identified Gi protein alpha subunits. Furthermore, PGE2, iloprost and cicaprost enhanced GTP azidoanilide-labelling of Gs protein alpha subunits, whereas sulprostone did not. We suggest that in HEL cells (1) EP1 and EP3 receptor subtypes activate G1 proteins, that (2) the EP2 receptor subtype activates Gs proteins and that (3) the IP receptor activates both Gi and Gs proteins.

Topics: Cell Membrane; Dinoprostone; Epoprostenol; GTP-Binding Proteins; Humans; Iloprost; Leukemia, Erythroblastic, Acute; Prostaglandins E; Receptors, Epoprostenol; Receptors, Prostaglandin; Receptors, Prostaglandin E; Signal Transduction; Tumor Cells, Cultured

In the human erythroleukemia cell line, HEL, prostaglandin E2 (PGE2) and the stable prostacyclin analogue, iloprost, increase cytosolic Ca2+ concentration ([Ca2+]i) via pertussis toxin-sensitive and -insensitive pathways. Unlike iloprost, the stable prostacyclin analogue cicaprost (ZK 96480), is devoid of agonistic properties at prostaglandin E2 receptors. We compared the effects of cicaprost, iloprost and PGE2 on [Ca2+]i in HEL cells. Cicaprost, iloprost and PGE2 were similarly potent to increase [Ca2+]i in HEL cells. However, unlike the effects of PGE2, those of the prostacyclin analogues were not inhibited by pertussis toxin. The prostaglandins studied increased [Ca2+]i through both mobilization from internal stores and Ca2+ influx from the extracellular space. Prostacyclin analogue- and PGE2-induced rises in [Ca2+]i were desensitized in a homologous manner. Additionally, there was cross-desensitization between cicaprost and iloprost, but not between the prostacyclin analogues and PGE2. Our data suggest that in HEL cells (i) cicaprost and iloprost act through prostacyclin receptors and (ii) that these receptors couple to pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (iii) resulting in an increase in [Ca2+]i by Ca2+ mobilization from internal stores and sustained influx.

Topics: Calcium; Cytosol; Dinoprostone; Epoprostenol; GTP-Binding Proteins; Humans; Iloprost; Kinetics; Leukemia, Erythroblastic, Acute; Pertussis Toxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Prostaglandin-regulated cyclic AMP metabolism in human erythroleukaemia (HEL) cells was similar to that previously described in platelets [Ashby (1989) Mol. Pharmacol. 36, 866-873], displaying prostaglandin-concentration-dependent desensitization that could be explained by the presence of separate stimulatory and inhibitory prostaglandin receptors. Pertussis toxin abolished prostaglandin-concentration-dependent desensitization, indicating that the process is mediated through a pertussis toxin-sensitive GTP-binding protein. Treatment of HEL cells for 4 days with the inducer dimethyl sulphoxide enhanced prostaglandin-concentration-dependent desensitization, but did not alter the initial rate of cyclic AMP synthesis or the amount of Gi2 alpha measured by immunoblotting, suggesting that the inhibitory receptor was selectively induced by changing the cells to a more platelet-like form.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Alprostadil; Cyclic AMP; Dimethyl Sulfoxide; Epinephrine; Humans; Iloprost; Leukemia, Erythroblastic, Acute; Pertussis Toxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.

Topics: Cell Line; Cell Membrane; Cytosol; Epoprostenol; Humans; Iloprost; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Leukemia, Erythroblastic, Acute; Membrane Proteins; Phosphorylation; Protein Kinases; Thrombin; Tumor Cells, Cultured

Monoclonal antibody M90 recognizes a specific epitope of the ras-encoded p21 protein. This region comprises amino acids 107-130 containing the residues 116-119, which are related to GTP binding. This antibody strongly reacts on Western Blots with a 22kDa protein from human erythroleukemia (HEL) cells. Treatment of HEL cells with iloprost, an agonist that increases cellular cyclic AMP levels, produces the appearance of a protein with an apparent molecular mass of 24kDa. This protein is also recognized by antiserum M90 on Western Blots; its appearance parallels a decrease of the 22kDa protein, and it can be labeled with 32P. This effect is also observed with dibutyryl cyclic AMP, which indicates phosphorylation of the 22kDa protein by cyclic AMP-dependent protein kinase. This phosphorylation produces an electrophoretic mobility change of the 22kDa protein to a 24kDa region on gels. The change of mobility of the 22kDa protein induced by iloprost in HEL cells is also observed when the protein is labeled with [35S]methionine and immunoprecipitated with antiserum M90. This information indicates a coupling mechanism involving phosphorylation of an oncogene product in HEL cells.

Topics: Antibodies, Monoclonal; Bucladesine; Cell Line; Cell Membrane; Cytosol; Epitopes; Epoprostenol; Humans; Iloprost; Leukemia, Erythroblastic, Acute; Membrane Proteins; Molecular Weight; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Theophylline; Tumor Cells, Cultured

We have identified both high-affinity (KD = 36 +/- 3 nM) and low-affinity (KD = 2.1 +/- 0.8 microM) prostacyclin (PGI2)-receptor sites on human erythroleukemia (HEL) cells using the radiolabelled prostacyclin analogue. [3H]iloprost. The addition of the phorbol ester, TPA, to the culture medium caused a 5-10-fold increase in the number of both the low- and the high-affinity sites, without any change in their affinity constants. Iloprost stimulated HEL cell membrane adenylate cyclase activity 5-fold. This stimulation was potentiated in the presence of GTP, indicating a conventional PGI2 receptor-G2-adenylate cyclase system. HEL cells represent a source of prostacyclin receptor mRNA which may be of value in expression cloning of this receptor.

Topics: Adenylyl Cyclases; Affinity Labels; Binding Sites; Cell Membrane; Enzyme Induction; Epoprostenol; Guanosine Triphosphate; Humans; Iloprost; Leukemia, Erythroblastic, Acute; Phorbol Esters; Receptors, Epoprostenol; Receptors, Prostaglandin; RNA, Messenger; Tumor Cells, Cultured