Regulation of endothelial cell cyclic nucleotide metabolism by prostacyclin.

An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.

Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis in human polymorphonuclear leukocytes.

Acetyl glyceryl ether phosphorylcholine (AGEPC) and leukotriene B4 (LTB4) induce concentration-dependent neutrophil aggregation. On a molar basis, LTB4 is approximately 10 to 100 times more potent than AGEPC. AGEPC-induced aggregation is attenuated by two inhibitors of arachidonate lipoxygenation, eicosatetraynoic acid and nordihydroguaiaretic acid, and to a lesser extent by the cyclooxygenase inhibitor, indomethacin. LTB4-induced aggregation is not readily reduced by the above inhibitors of arachidonic acid metabolism. Reverse phase high performance liquid chromatography, coupled with selective ion gas chromatography/mass spectrometry, shows that AGEPC stimulates neutrophils to synthesize sufficient LTB4 to account for the AGEPC response. In addition, the rate of LTB4 biosynthesis in response to AGEPC correlates well with the rate of AGEPC- and/or LTB4-induced neutrophils aggregation, and desensitization experiments indicate that AGEPC and LTB4 cross-desensitize. These data suggest that AGEPC-induced neutrophil aggregation may be mediated by LTB4.

Stimulation of human eosinophil and neutrophil polymorphonuclear leukocyte chemotaxis and random migration by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid.

A human platelet lipoxygenase-generated product of arachidonic acid, identified by thin-layer chromatographic and mass spectrometric properties as 12-L-hydroxy-5,8,10,14-eicosatertraenoic acid (HETE), was selectively chemotactic in vitro for human polymorphonuclear leukocytes (PMN), as compared to mononuclear leukocytes, with a preference for eosinophils. Preincubation of PMN with partially-purified HETE at peak chemotactic concentrations of 8-24 mug/ml reduced their random and chemotactic migration and stimulated the activity of their hexose monophosphate shunt; minimally chemotactic concentrations of 0.03-1 mug/ml enhanced PMN random migration without influencing other functions. HETE may thus be capable of preferentially attracting eosinophils to foci of tissue reaction associated with platelet activation.

Specific inhibition of the polymorphonuclear leukocyte chemotactic response to hydroxy-fatty acid metabolites of arachidonic acid by methyl ester derivatives.

The human polymorphonuclear (PMN) leukocyte chemotactic activity of the hydroxy-fatty acid metabolites of arachidonic acid, 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), is eliminated by methylation. Both methyl esters are specific competitive inhibitors of the PMN leukotactic responses to the parent stimuli, and exert no effect on the responses to formyl-methionyl peptides or chemotactic fragments of the fifth component of complement. 50% inhibition of the in vitro chemotactic responses of PMN leukocytes to HETE and HHT was achieved by an equimolar concentration of the corresponding methyl esters, whereas reciprocal cross-inhibition was observed at molar ratios of HETE methyl ester to HHT and HHT methyl ester to HETE which reflected the three- to fivefold greater chemotactic potency of HETE relative to HHT. Methyl esters of structurally related, but nonchemotactic, fatty acids did not competitively inhibit the chemotaxis elicited by HETE or HHT. The intraperitoneal injection of HETE in guinea pigs evoked an eosinophil response at 30 min and a neutrophil response at 5 h, which were prevented by a one-to twofold molar ratio of HETE methyl ester. The competitive inhibition of the in vitro chemotactic activity and the in vivo leukotactic effect of the unsaturated hydroxy-fatty acids by homologous methyl ester derivatives suggests that the cellular component of natural inflammatory reactions may be susceptible to specific regulation by receptor-directed modulation of the activity of the predominant chemotactic principles.

Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2).

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.

Regulatory role of cyclic adenosine 3',5'-monophosphate on the platelet cyclooxygenase and platelet function.

Thromboxane A2 plays an important role in arachidonic acid- and prostaglandin H2-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I2, prostaglandin I1 and prostaglandin E1) and dibutyryl cyclic AMP inhibit both thromboxane A2 formation and arachidonate-induced aggregation in platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A2 is still formed. Prostaglandin H2-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A2 production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cyclooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate model for studying the relationship between the platelet cyclooxygenase and platelet function.

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor.

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid. Two selective thromboxane synthetase inhibitors.

Two selective thromboxane A2 synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B2 and prostaglandin E2 accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B2 formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H2. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A2 formation with an accompanying, parallel, suppression of the platelet aggregation. Imidazole selectively suppressed thromboxane A2 formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H2 on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A2 formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A2 in arachidonate mediated platelet aggregation.

Antagonism of the prostaglandin endoperoxide imhibition of hormone-stimulated adenylate cyclase by guanosine triphosphate and 5'-guanylyl-imidodiphosphate.

The prostaglandin endoperoxide prostaglandin H2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid) inhibits basal and hormone-stimulated adenylate cyclase in fat cell ghosts. This inhibition by prostaglandin H2 has been found to be antagonized by GTP and Gpp(NH)p. Dose response studies have shown GTP and Gpp(nh)p to be maximally effective at 3.3 muM, the lowest concentration tested. Although the system is exceedingly sensitive to modulation by GTP or Gpp(NH)p UTP, CTP, GMP, and cyclic GMP did not antagonize the antihormone activity of prostaglandin H2. Kinetic studies indicate that the GTP or Gpp(NH)p antagonism of prostaglandin H2 is observable on initial rates of cyclic AMP synthesis, and persists throughout the adenylate cyclase measurements. Preincubation of fat cell ghosts with GTP followed by washing and resuspension results in a prostaglandin H2-sensitive adenylate cyclase system. However, the same preincubation experiment with Gpp(NH)p produces an irreversible antagonism of the prostaglandin H2 inhibition of hormone-stimulated adenylate cyclase. It is suggested that prostaglandin H2 stabilizes the fat cell adenylate cyclase system in a state that is resistant to hormone stimulation, and GTP or Gpp(NH)p overcome this stabilization.

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3',5'-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4.

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I2, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B4 synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B4 itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B4 is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B4 synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B4 can limit their own activity in neutrophils.

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer.

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets. Evidence for thromboxane A2 dependence.

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.

Tröger's base. An alternate synthesis and a structural analog with thromboxane A2 synthetase inhibitory activity.

The synthesis of 2,8-dimethyl-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine (Tröger's base) from p-toluidine and of two Tröger's base analogs from other anilines by reaction with hexamethylenetetramine in trifluoroacetic acid is described. 2,3,6,7-Tetrahydro-9-methyl-2,6-di-p-tolyl-1H,5H-pyrimido[5,6,1-ij] quinazoline is formed as a secondary product in the reaction of p-toluidine and hexamethylenetetramine. One of the Tröger's base analogs, 2,8-bis(3'-pyridylmethyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]d iazocine (5), is an effective inhibitor of the enzyme, thromboxane A2 (TxA2) synthase, with an ED50 of 30 ng/mL in a specified in vitro assay. Three analogs having substituents on the bridging methylene group of the bicyclic nucleus of the Tröger's base structure were prepared, but all were considerably less active than the aforementioned compound in the inhibition assay. The structures of these inhibitors of TxA2 synthase fall outside the classical structure-activity relationship that has been established for this class of enzyme inhibitors.

Thromboxane A2 synthase inhibitors. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acids.

The synthesis and screening of a series of 5-(3-pyridylmethyl)benzofuran-2-carboxylic acids as selective thromboxane A2 (TxA2) synthase inhibitors is outlined. The ability of these compounds to inhibit TxA2 biosynthesis was assayed using microsomal enzyme from human platelets. Substitution of the benzofuran ring caused small changes in potency; modification of the carboxylic acid group caused modest reductions in potency, and substitution of the pyridine ring resulted in large reductions of potency. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acid sodium salt (9b, sodium furegrelate) was chosen for further evaluation as a TxA2 synthase inhibitor.

Novel molecules that antagonize leukotriene B4 binding to neutrophils.

A series of LTB4 analogues have been synthesized that replace carbons 7-9 of the cis-trans-trans triene unit of LTB4 with a stable ring structure. Meta-substituted pyridine analogues are more potent inhibitors than benzene or furan analogues. C-1 alcohols are often more potent inhibitors than free carboxylic acids, and 5,6-cis double bond compounds are more potent than 5,6-trans compounds. Compounds such as these may prove to be useful in the treatment of inflammatory diseases.

Thromboxane synthase activity and platelet function after furegrelate administration in man.

Furegrelate sodium (U-63,557A), a pyridine-derivative thromboxane synthase inhibitor, was administered orally in single doses of 200 to 1600 mg to normal male subjects. Furegrelate produced a dose-related inhibition of thromboxane synthesis for 8-12 hours when measured either ex vivo from platelet-rich plasma (PRP) or in vivo from urine. In general, the extent of thromboxane synthesis inhibition was greater in PRP than in urine. Furegrelate significantly inhibited platelet aggregation, but the effect was variable and measurements of thromboxane synthase did not predict the impact on platelet aggregation. Bleeding times and coagulation parameters were not altered significantly. Furegrelate was well absorbed orally with Tmax = 1 hr and t1/2 = 3.5 to 5 hrs. There was no marked metabolism; elimination was primarily by renal excretion of parent compound. Thus, furegrelate is an effective inhibitor of thromboxane synthase in man with a relatively long biologic and circulating half-life.