iloprost and octimibate

1. Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [(3)H]-cyclic AMP and [(3)H]-inositol phosphate production. 2. The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E(1), stimulated adenylyl cyclase activity with EC(50) values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 - 40 fold lower potency than stimulation of adenylyl cyclase. 3. The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC(50) values of 219, 166 and 398 nM, respectively, but failed to stimulate [(3)H]-inositol phosphate production. 4. Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [(3)H]-inositol phosphate production in a non-competitive manner. 5. Activation of the endogenously-expressed P(2) purinergic receptor by ATP led to an increase in [(3)H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [(3)H]-inositol phosphate production. 6. A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP(3) receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors. 7. The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant Galpha(q)RC, Galpha(14)RC and Galpha(16)QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant Galpha(s)QL. 8. These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [(3)H]-inositol phosphate production by targeting G(q/11) and/or phospholipase C in CHO cells, and that this effect is independent of IP receptors.

Topics: Acetates; Adenylyl Cyclases; Alprostadil; Animals; Cell Survival; CHO Cells; Cricetinae; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epoprostenol; Iloprost; Imidazoles; Inositol Phosphates; Oxazoles; Phenoxyacetates; Pyridines; Receptors, Epoprostenol; Receptors, Prostaglandin; Transfection; Tritium; Type C Phospholipases

[3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid (BMY 45778) inhibits human (IC50 = 35 nM), rabbit (136 nM) and rat (1.3 microM) platelet aggregation. This compound activates adenylyl cyclase (ED50 = 6-10 nM) and stimulates GTPase in human platelet membrane preparations. The potency (EC50) of BMY 45778 stimulating adenylyl cyclase is comparable to iloprost. However, maximal stimulation of GTPase by BMY 45778 is approximately half the iloprost-stimulated activity, and BMY 45778 limits the GTPase stimulation by iloprost suggesting that BMY 45778 is a partial agonist at the IP receptor. BMY 45778 completely prevents [3H]]Iloprost binding to platelet membranes (IC50 = 7 nM). In whole platelets, BMY 45778 causes elevation of platelet cAMP levels (cAMP content doubles at 13 nM) and activation of the cAMP-dependent protein kinase (cAMP-protein kinase ratio is twice basal at 2 nM). BMY 45778 treatment of whole platelets also desensitizes the adenylyl cyclase activation by iloprost. These results indicate that BMY 45778, which is structurally different from prostacyclin and most prostacyclin agonists, acts by stimulating prostacyclin (IP) receptors.

Topics: Acetates; Adenylyl Cyclases; Animals; Binding, Competitive; Cell Membrane; Cyclic AMP; Epoprostenol; GTP Phosphohydrolases; Humans; Iloprost; Imidazoles; Oxazoles; Phenoxyacetates; Platelet Aggregation Inhibitors; Protein Kinases; Rabbits; Rats

The specific prostacyclin (IP) receptor agonist cicaprost relaxed human pulmonary artery preparations precontracted with phenylephrine [50% inhibitory concentration (IC50) approximately 0.6 nM], U-46619 (IC50 approximately 0.9 nM), and K+ (approximately 40% maximal relaxation); endothelium removal had little effect on relaxant activity. Ranking of relaxant potencies for prostacyclin and five of its analogs was 17 alpha, 20-dimethyl-delta 6,6a-6a-carba PGI1 (TEI-9063) > or = cicaprost > iloprost > prostacyclin > taprostene > benzodioxane prostacyclin > 15-deoxy-16 alpha-hydroxy-16 beta,20-dimethyl-delta 6,6a-6a-carba PGI1 (TEI-3356). The potency of the isocarbacyclin TEI-3356 may have been under-estimated because of its contractile (EP3 receptor agonist) activity. The potency ranking of four nonprostanoid prostacyclin mimetics was 3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy] acetic acid (BMY 45778; IC50 approximately 2.5 nM) > > 2-[3-[2-(4, 5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid (BMY 42393) > octimibate > CU 23 (a novel diphenylindole). From IP receptor binding affinities obtained on human platelet membranes, it is suggested that the slightly shallower log concentration-response curves for BMY 45778, BMY 42393, and CU 23 may reflect the near-maximal receptor occupancy required for complete relaxation. A fifth nonprostanoid, CU 602, had much shallower log concentration-response curves than cicaprost against phenylephrine tone but not against U-46619 tone; this may indicate IP receptor partial agonism coupled with TP receptor antagonism. The relaxant actions of the nonprostanoid mimetics were more persistent than those of the prostacyclin analogs on washout of the organ bath; by the inhalation route, this type of compound may be retained within pulmonary tissue and thus afford greater pulmonary/systemic selectivity than currently used pulmonary vasodilators.

Topics: Acetates; Aged; Cardiovascular Agents; Child; Child, Preschool; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epoprostenol; Fatty Acids; Humans; Iloprost; Imidazoles; Indoles; Middle Aged; Muscle, Smooth, Vascular; Oxazoles; Phenoxyacetates; Platelet Aggregation Inhibitors; Prostaglandins, Synthetic; Pulmonary Artery; Receptors, Epoprostenol; Receptors, Prostaglandin; Structure-Activity Relationship; Vasoconstrictor Agents; Vasodilator Agents

Topics: Acetates; Animals; Cell Aggregation; Cyclic AMP; Epoprostenol; Iloprost; Imidazoles; Isoquinolines; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxazoles; Peritoneal Cavity; Phenoxyacetates; Pyrrolidinones; Rats; Rolipram; Second Messenger Systems; Sulfonamides

Topics: Acetates; Cell Line; Cyclic AMP; Epoprostenol; Humans; Iloprost; Imidazoles; Kinetics; Neuroblastoma; Neurons; Oxazoles; Phenoxyacetates

The effects of three non-prostanoid prostacyclin mimetics on rat peritoneal neutrophil activity have been investigated and compared with the effects of the prostacyclin analogues cicaprost and iloprost. Cicaprost, iloprost, BMY 22389 (octimibate), BMY 42393 and BMY 45778 inhibited N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophil aggregation with IC50 values of 2.1, 4.5, 286, 462 and 20 nM, respectively. Cicaprost and iloprost produced clear concentration-related increases in [3H]cyclic AMP accumulation; EC50 values were 20 and 44 nM, respectively. In contrast, the three BMY compounds showed low efficacy as activators of adenylyl cyclase. The inhibitory effect of prostacyclin mimetics does not appear to depend on effects on intracellular calcium concentration, or on KATP channels. Extensive studies using cyclic AMP mimetics and antagonists suggest that the anti-aggregatory activity of the non-prostanoid prostacyclin mimetics on rat neutrophils may involve highly localized increases in cyclic AMP.

Topics: Acetates; Animals; Calcium; Cell Aggregation; Cyclic AMP; Drug Design; Epoprostenol; Glyburide; Iloprost; Imidazoles; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxazoles; Phenoxyacetates; Phosphodiesterase Inhibitors; Prostaglandins, Synthetic; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin

A series of phenylated pyrazoloalkanoic acid derivatives were synthesized and evaluated as inhibitors of ADP-induced human platelet aggregation. 3,4,5-Triphenyl-1H-pyrazole-1-nonanoic acid (8d), with an IC50 of 0.4 microM, was the most potent inhibitor identified in this study. Biochemical studies determined that 8d increased intraplatelet cAMP accumulation and stimulated platelet membrane-bound adenylate cyclase in a concentration-dependent fashion. Displacement of [3H]iloprost by 8d from platelet membranes indicated that the platelet prostacyclin (PGI2) receptor is the locus of biological action. Structure-activity studies demonstrated that the minimum structural requirements for binding to the platelet PGI2 receptor and inhibition of ADP-induced platelet aggregation within this series are a vicinally diphenylated pyrazole substituted with an omega-alkanoic acid side chain eight or nine atoms long. Potency depended upon both side-chain length and its topological relationship with the two phenyl rings.

Topics: Adenylyl Cyclases; Animals; Blood Platelets; Epoprostenol; Humans; Iloprost; Imidazoles; In Vitro Techniques; Platelet Aggregation Inhibitors; Pyrazoles; Rabbits; Rats; Receptors, Epoprostenol; Receptors, Prostaglandin; Structure-Activity Relationship

1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist-stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP-inhibited phosphodiesterase (phosphodiesterase-ITI), which is the major cyclic AMP-degrading enzyme in human platelets.5. Octimibate inhibits, apparently competitively, the binding of [3H]-iloprost (a stable PGl2 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6. The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fol

Topics: Adenylyl Cyclases; Animals; Blood Platelets; Calcium; Cats; Cattle; Cell Membrane; Cyclic AMP; Dogs; Guinea Pigs; Iloprost; Imidazoles; In Vitro Techniques; Macaca fascicularis; Platelet Aggregation; Platelet Aggregation Inhibitors; Protein Kinases; Rats; Receptors, Epoprostenol; Receptors, Prostaglandin; Species Specificity; Sterol O-Acyltransferase