Search

Pretreatment of NG108-15 cells for 1 h with 1 microM phorbol 12-myristate,13-acetate produced no significant effect on the subsequent stimulation of adenylate cyclase activity by the IP receptor agonist, iloprost, the adenosine A2 receptor agonist, N-ethylcarboxamidoadenosine (NECA), or sodium fluoride, suggesting that protein kinase C activation does not produce desensitization in this system. Pretreatment of cells with 10 microM iloprost or forskolin for 17 h produced a decrease in the specific binding of [3H]iloprost, consistent with a decrease in IP receptor number. Iloprost pretreatment produced a decrease in responses to iloprost, NECA and sodium fluoride, whereas forskolin pretreatment produced a decrease in subsequent responsiveness to iloprost and NECA, but the response to sodium fluoride remained unaffected. The desensitization produced by forskolin could be completely inhibited by the inhibitor of protein kinase A and protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), but H7 had no effect on the desensitization produced by iloprost.

Pretreatment of NG108-15 cells with 0.03-25 microM prostaglandin E1 (PGE1) produced decreases in the maximal stimulation of adenylate cyclase activity produced by iloprost, N-ethylcarboxamidoadenosine and sodium fluoride. The rate of desensitization to all three agents was dependent on the concentration of PGE1 used, but at each concentration of PGE1 the rate of loss of responsiveness to each agent was the same, suggesting that the decreases in responsiveness may be mediated by a single process. Functional desensitization was accompanied by a decrease in the specific binding of [3H]iloprost, consistent with a 75-80% decrease in IP receptor number, with no change in the coupling of the remaining IP receptors to G protein. At each concentration of PGE1 used, the times taken for half maximal decreases in receptor number and functional responsiveness were similar, suggesting that IP receptor down-regulation is a relatively early event in desensitization. IP receptor down-regulation could be inhibited partially by 100 microM chloroquine, suggesting that lysosomal breakdown of receptors may be occurring.

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F 2 alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [Is-[1 less than a, 2 less than b(5z), 3 less than b, 4 less than a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo- [2.2.1]hept-2-yl]-5- heptenoic acid (SQ29548) greater than 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) greater than 4-[2-(phenylsulphonamido)-ethyl]phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [3H]SQ29548 and 9,11-dimethylmethano-16(3-[125I]iodo-4-hydroxyphenyl)-13,14-dih ydr o-13-aza- 15-omega-o-tetranor-thromboxane ([125I]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [3H]SQ29548 and [125I]PTA-OH were approximately 10 nM and approximately 0.3 nM, respectively, and the density of sites labelled by either ligand was approximately 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [3H] SQ29548 or [125I]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.

A component of the displaceable binding of the stable prostacyclin analogue, [3H]iloprost, to membranes from human platelets and the somatic hybrid cell lines NG108-15 and NCB20, was inhibited by guanine nucleotides. The order of potency of a range of nucleotides for this effect was GTP gamma S greater than GppNHp greater than GTP greater than GDP = GMP; ATP, UTP and CTP were ineffective at concentrations up to 1 mM. In the presence of 100 microM GppNHp, iloprost binding curves were displaced to the right of curves obtained in the absence of guanine nucleotide, and their Hill slopes were greater. This was consistent with a conversion of a minor population of high affinity agonist binding sites to lower affinity sites in the presence of guanine nucleotides. These effects of guanine nucleotides on the binding of the agonist ligand [3H]iloprost were consistent with an interaction with a G protein coupled receptor.

1. Octimibate is a potent inhibitor of human platelet aggregation, and appears to act (at least in part) through the prostacyclin receptor, as described in the preceding paper. Here, the vascular effects, both in vitro and in vivo, of octimibate have been compared to those of the stable prostacyclin (PGI2) mimetic, iloprost. Since octimibate shows extensive species variation and is potent at inhibiting platelet aggregation in primates, all of the experiments reported here have been carried out with primate tissue or in vivo in cynomolgus monkeys. 2. Activation of adenylyl cyclase in human lung membranes appears to involve stimulation of the vascular PGI2 receptor. Octimibate, as well as iloprost, stimulates adenylyl cyclase in this preparation. The EC50 values for iloprost and octimibate are 50 nM and 340 nM respectively. These values are similar to those seen with human platelet membranes. As with platelets, the maximal activation achievable with octimibate is 60% of that seen with iloprost. This result suggests that octimibate is a partial agonist for stimulation of adenylyl cyclase. 3. Iloprost (10-100 nM) relaxes human coronary and mesenteric artery precontracted with KCl, and also relaxes cynomolgus monkey aorta precontracted with phenylephrine. Octimibate appears to be a partial agonist for relaxation of human coronary artery precontracted with KCl; the intrinsic activity of octimibate (10 microM) is 0.15 compared to iloprost, and octimibate surmountably antagonizes the relaxant effects of iloprost with a Kp of 200 nM. Octimibate (up to 10 microM) evokes only weak relaxation of human mesenteric artery (precontracted with KCl) and cynomolgus monkey aorta (precontracted with phenylephrine). 4. The effects of iloprost and octimibate were compared in vivo in cynomolgus monkeys. In addition to inhibiting ex vivo platelet aggregation, both compounds cause hypotension with little effect on heart rate. The dose-response curves for inhibition of ex vivo platelet aggregation and a fall in mean arterial blood pressure were compared. The dose-separation (i.e., the relative differences in effective concentrations) for the two responses is similar with both iloprost and octimibate. 5. Since the pern; beral resistance vessels are intimately involved in regulation of systemic arterial blood pressure, the effects of both agents were tested on human peripheral resistance vessels (150-400pm diameter) in vitro. These vessels are relaxed by both iloprost and octimibate following precontraction with KCI. The IC50 value for iloprost is 44nM, and 1.7 microM octimibate evokes 50% of the maximal relaxation obtained with iloprost. Thus, the relative potencies of the two compounds in relaxing human subcutaneous resistance vessels are similar to their relative potencies in inhibiting platelet responses. This result correlates with the lack of platelet versus vascular selectivity seen with the in vivo monkey studies. 6. These results suggest that octimibate, a partial agonist at the prostacyclin receptor, is unable to discriminate between platelet and vascular prostacyclin receptors in primates.

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 fold with no obvious difference between primates and non-primates. 7. Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non-prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes.

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.