PGI2 Formation Research Articles

Volatile and intravenous anesthetics selectively attenuate the release of endothelium-derived hyperpolarizing factor elicited by bradykinin in the coronary microcirculation.

In addition to nitric oxide (NO) and prostacyclin (PGI2) another endothelium-derived factor, which hyperpolarizes vascular smooth muscle cell via activation of K+ channels, contributes to the vasorelaxant effect of bradykinin in different vascular beds. Preliminary findings suggest that this endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation is attenuated by both volatile and intravenous anesthetics. Since EDHF may play an important role in the coronary microcirculation, we investigated the effects of isoflurane (2 vol.% equivalent to approximately 250 microM), etomidate (30 and 100 microM), phenobarbital (100 microM) and thiopental (30 and 100 microM) on the EDHF-mediated dilator response to bradykinin and on the endothelium-independent dilatation evoked by sodium nitroprusside (SNP) in the isolated saline-perfused rat heart (Langendorff preparation). None of the anesthetics tested affected the dilator response to bradykinin or SNP under basal conditions. However, following inhibition of NO and PGI2 formation with NG-nitro-L-arginine (100 microM) and diclofenac (1 microM) respectively, isoflurane, etomidate and thiopental, but not phenobarbital, significantly attenuated the NO/PGI2-independent, i.e. EDHF-mediated dilator response to bradykinin, while the vasorelaxant effect of SNP remained unaffected. Isoflurane, etomidate and thiopental, but not phenobarbital, display cytochrome P450-inhibiting properties, suggesting that these anesthetics impair the cytochrome P450-dependent synthesis of EDHF in the coronary microcirculation.

Role of cyclooxygenase metabolites in mediating platelet-induced baroreceptor dysfunction.

The goal of the study was to determine the role of cyclooxygenase metabolites in mediating platelet-induced suppression of baroreceptor activity. Exposure of the isolated carotid sinus of rabbits to thrombin-activated rabbit platelets (3 x 10(8) cells/ml Krebs buffer) decreased baroreceptor activity (P < 0.05) without significantly altering the slope of the pressure-activity relation (gain). The platelet-induced suppression of activity was not blocked but instead was even more pronounced after inhibition of cyclooxygenase with indomethacin; both maximum baroreceptor activity and gain were decreased markedly. The exacerbation of platelet-induced baroreceptor dysfunction contrasted with equivalent carotid vasoconstrictor responses to platelets before and after indomethacin. Furthermore, the stable thromboxane (TxA2) mimetic U-46619 caused similar vasoconstriction as platelets but did not influence baroreceptor gain or maximum activity. In contrast to indomethacin, the selective TxA2 synthesis inhibitor and receptor blocker CGS-22652 failed to influence platelet-induced suppression of activity. In summary, 1) rabbit platelet aggregating in carotid sinus suppress baroreceptor activity, which cannot be explained by the vasoconstriction, and 2) the suppression of activity is not mediated by TxA2 from platelets and is opposed by prostacyclin (PGI2) or other prostanoids produced in carotid sinus. The combination of impaired formation of PGI2 and platelet activation in atherosclerotic and thrombotic states may lead to profound baroreceptor dysfunction.

Prevention of the excitatory actions of bradykinin by inhibition of PGI2 formation in nodose neurones of the guinea‐pig.

1. Intracellular recordings were made from neurones in intact guinea-pig nodose ganglia in vitro and from acutely isolated adult guinea-pig and rabbit nodose neurons to study a bradykinin (BK)-mediated block of a slow spike after-hyperpolarization (AHPslow) that is prominent in 30-40% of these neurones. 2. BK (100 nM) reversibly blocked the AHPslow, resulting in an ablation of the spike accommodative properties of these neurones. The B1 BK receptor agonist [des-Arg9]-BK did not mimic or prevent the actions of BK. In contrast, the B2 BK receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) prevented BK-induced block of the AHPslow and the effect of BK on spike frequency adaptation. 3. The BK block of the AHPslow in acutely dissociated neurones was prevented by indomethacin, indicating that this BK effect was dependent upon a cyclo-oxygenase metabolite intrinsic to these neurones. 4. One to twenty femtomoles of the prostanoids PGE2, PGD2, 9 alpha, 11 beta-PGF2 (a metabolite of PGD2), PGF2 alpha, TxB2 and PGI2 were released spontaneously from a nodose ganglion in 15 min. BK (100 nM) selectively increased PGI2 release 2.8-fold without affecting the release of the other prostanoids. Treatment with 10 microM tranylcypromine (TCP), a putative PGI2 synthase inhibitor, completely prevented the BK-induced release of PGI2. 5. In the presence of 10 microM TCP, BK no longer produced significant effects on the AHPslow. In contrast, 10 microM TCP did not prevent PGI2 from blocking the AHPslow. 6. These results suggest that vagal afferents that exhibit AHPslow also possess the B2 type of BK receptor. Activation of these BK receptors results in the production of PGI2, which in turn controls spike frequency adaptation by affecting the amplitude of the AHPslow.

Signs of central nervous activation during mental practice in seniors

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinymethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro, as well as in rhesus monkey platelets ex vivo. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 at 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation,. Similar effects were obtained by oral administration of 1–5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxgenase inhibitors and equal to PGI2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cylooxygenase inhibitors suggesting that its efficacy dependened in part of endogenous PGI2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of patho[hysiological states.

Role of prostaglandin-mediated cyclic AMP formation in protein kinase C-dependent secretion of atrial natriuretic peptide in rat cardiomyocytes

The role of endogenous prostaglandin production in phorbol diester-induced myocardial atrial natriuretic peptide (ANP) secretion was investigated in cultured spontaneously beating ventricular rat cardiomyocytes. Incubation of cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 0.1 microM) led to a rapid response in ANP release, a response accompanied by increases in cellular prostacyclin (PGI2) production, cyclic AMP (cAMP) formation and spontaneous contraction frequency. Although PMA-induced ANP secretion exhibited the pharmacological profile of a protein kinase C (PKC)-mediated event, the response was abolished in the presence of the cyclo-oxygenase inhibitors indomethacin (10 microM) and diclofenac (1 microM), indicating that endogenous prostaglandin production is responsible for PMA-induced ANP secretion in this system. Confirming this, PMA-induced ANP secretion was strongly correlated with endogenous formation of 6-oxo-prostaglandin F1 alpha (r = 0.93, P < 0.0005, n = 11), and exogenously applied PGI2, prostaglandin E2 (PGE2) or prostaglandin F2 alpha (PGF2 alpha) elicited simultaneous increases in cAMP formation, contraction frequency and ANP secretion in these cells. Furthermore, PMA-induced cAMP formation was abolished in the presence of either diclofenac or indomethacin, whereas the cAMP-elevating agent forskolin (0.1 microM) mimicked the secretory and chronotropic effect of PMA in these cells. A role for cAMP in PMA-induced ANP secretion was also apparent insofar as PMA-induced ANP release was substantially decreased in the presence of the Rp-diastereomer of 3',5'-cyclic adenosine monophosphorothioate (Rp-cAMPS; 10 microM), whereas the cAMP-mimetic agent dibutyryl cAMP (10 microM) provoked a rapid increase in ANP secretion in this system. Finally, the Ca(2+)-channel antagonist nifedipine (0.1 microM) severely decreased PGI2-, PGE2- and PMA-induced ANP secretion without affecting PGF2 alpha-induced peptide release, suggesting that PGI2 and/or PGE2, but not PGF2 alpha, are the prostanoids involved in PMA-induced ANP release. Taken together, these results suggest that PKC activation induces ANP secretion in spontaneously beating rat ventricular cardiomyocytes via an autocrine pathway involving increased PGI2 and/or PGE2 formation, a response leading to the activation of a myocardial adenylate cyclase and, subsequently, to that of a nifedipine-sensitive Ca2+ channel.

Evidence that prostaglandins I2, E2, and D2 may activate ATP sensitive potassium channels in the isolated rat heart.

The aim was to study the contribution of ATP sensitive potassium channels (KATP channels) in the coronary vasodilatation produced by prostaglandins I2, E2, and D2 in rats. Isolated Langendorff rat hearts, perfused under constant flow conditions, and rat aortic rings were used. Dose-response curves to PGE2, PGD2, and iloprost, a PGI2 analogue, were performed before and during KATP channel blockade with glibenclamide. Arachidonic acid was used to increase the formation of endogenous PGI2. Infusions of PGE2, PGD2, and iloprost in isolated hearts induced marked vasodilatation, as reflected by the reduction in coronary perfusion pressure of 27(SEM 7), 30(6), and 43(6)%, for 0.1 microM PGE2, PGD2, and iloprost, respectively. Infusion of glibenclamide (0.3 microM) was accompanied by a 23(3)% increase in coronary perfusion pressure. The vasodilatation induced by levcromakalim (0.1 microM) was completely inhibited in the presence of glibenclamide, whereas that of papaverine (30 microM) was unaffected. Glibenclamide significantly reduced the vasodilatation induced by iloprost (at 3 to 100 nM), PGE2 (30 and 100 nM), and PGD2 (30 and 100 nM), at all concentrations studied. In contrast, glibenclamide (1 microM) had no effect on iloprost induced relaxation of aortic rings. Arachidonic acid infusion (from 0.1 to 3 microM) in isolated hearts induced a pronounced vasodilatation and a significant release of 6-keto-PGF1 alpha into the coronary effluent in a dose dependent fashion. Both responses to arachidonic acid were significantly reduced in the presence of the cyclo-oxygenase inhibitor diclofenac (1 microM). In an additional experimental series, the vasodilatation induced by arachidonic acid infusions was found to be significantly reduced in the presence of glibenclamide. Glibenclamide is a potent inhibitor of the coronary dilator action of prostaglandins I2, E2, and D2. This observation suggests that these prostaglandins may cause vasodilatation by opening KATP channels.

Prostaglandin E2 synthesis after oxidant stress is dependent on cell glutathione content

The role of glutathione in protecting prostaglandin (PG) generation after exposure of fibroblasts to oxidant stress was investigated. Exposure of 3T3 fibroblasts to H2O2, followed by washing and then 20 microM arachidonic acid, caused a dose-dependent decrease in PG synthesis as assessed by radioimmunoassay. PGE2 production decreased from 3.7 +/- 1.1 to 0.15 +/- 0.04 pmol/microgram protein, and prostacyclin (PGI2) formation decreased from 0.56 +/- 0.03 to 0.06 +/- 0.03 pmol/microgram protein after exposure to 200 microM H2O2. Decreasing intracellular glutathione with 50 micrograms/ml 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) enhanced the H2O2-induced decrease in PGE2 synthesis. Another glutathione-depleting agent, 1-chloro-2,4-dinitrobenzene (CDNB), also potentiated the H2O2-induced decrease in PGE2 formation. However, although PGI2 production was decreased by H2O2, neither BCNU nor CDNB potentiated this decrease. Without oxidant stress, extreme glutathione depletion decreased PGE2 synthesis and caused PGI2 synthesis to exceed PGE2. In summary, oxidant stress decreases both PGE2 and PGI2 formation. However, the primary effect of decreasing cell glutathione during oxidant stress is a reduction in PGE2 formation, not PGI2. This implies that the predominant effect of glutathione depletion during oxidant stress is on the PGE2 isomerase(s) and not PGH synthase or PGI2 synthase.

Reduced endothelium-dependent relaxation at enhanced NO release in hearts of hypercholesterolaemic rabbits.

1. Langendorff hearts, perfused at constant volume, were prepared from rabbits fed a cholesterol-enriched diet for 4 months. Coronary perfusion pressure and nitric oxide (NO) release (oxyhaemoglobin technique) into the coronary effluent were measured continuously. Prostacyclin (PGI2) in the effluents was determined by radioimmunoassay (6-oxo-PGF1 alpha). 2. Basal NO release was not different between control and hypercholesterolaemic rabbits. However, the coronary vasculature of hypercholesterolaemic rabbits showed a considerably (> 50%) reduced endothelium-dependent relaxation in response to short-term (3 min) infusion of bradykinin (50 nM) and substance P (50 nM) (P < 0.05, n = 8-9). Under these conditions, NO release into the vessel lumen was increased, by 26%, in hypercholesterolaemic hearts (P < 0.05, n = 8-9). NG-nitro-L-arginine (L-NOARG, 30 microM) significantly attenuated both bradykinin-induced NO formation and vessel relaxation in control hearts but only NO release in hypercholesterolaemia. L-Arginine (200 microM) restored the response to that before L-NOARG but did not improve the reduced endothelium-dependent relaxation in cholesterol-fed rabbits. 3. Superoxide dismutase (10 u ml-1) significantly improved vessel relaxation without changing the hypercholesterolaemia-related coronary dysfunction. Vasodilatation in response to exogenous NO donors (linsidomine) was diminished in hypercholesterolaemia as compared to controls. 4. Basal PGI2 release was unchanged in hypercholesterolaemic hearts. There was a tendency in these hearts for greater PGI2 formation after stimulation by substance P and bradykinin (P > or = 0.05). The coronary relaxation to iloprost was unchanged. 5. The data demonstrate impaired endothelium-dependent relaxation of coronary arterial resistance vessels in hypercholesterolaemia. This diminished vascular response was not due to reduced NO generation but probably a reduced action of released NO, either by accelerated degradation and/or disturbed signal transduction pathways to vascular smooth muscle cells. There was no significant change in PGI2 related pathways of vasomotor control in hypercholesterolaemia.

Cyclo-oxygenase products released by low pH have capsaicin-like actions on sensory nerves in the isolated guinea pig heart.

Previous work has shown that ischaemia releases calcitonin gene related peptide (CGRP) from capsaicin sensitive nerve terminals in the perfused heart. Prostacyclin (PGI2) is also released during ischaemia. The aim of this study was to investigate whether the release of CGRP by low pH and lactic acid was associated with PGI2 formation and if PGI2 mediated its effect through capsaicin receptors which could be inhibited by capsazepine. The isolated Langendorff perfused guinea pig heart was used with a constant perfusion pressure of 70 cm H2O. Low pH was accomplished by changing the Tyrode solution to buffers with pH 7, 6, and 5, or lactic acid (5 mM with pH 6.9). The outflow of CGRP and the stable PGI2 metabolite 6-keto-PGF1 alpha was measured by radioimmunoassay. Low pH (pH 7, 6, 5) and lactic acid evoked release of CGRP. At moderate acidosis (pH 7 and 6) the CGRP release was dependent on extracellular Ca2+, while at pH 5 approximately half of the peptide release persisted in the absence of extracellular Ca2+. This release was attenuated by diclofenac or indomethacin, two inhibitors of prostaglandin formation, as well as by the capsaicin receptor antagonist capsazepine. Both arachidonic acid and PGI2, the predominant cyclo-oxygenase product formed during myocardial ischaemia, evoked a capsazepine sensitive release of CGRP, while capsazepine did not influence the formation of PGI2 evoked by low pH or arachidonic acid. In the isolated guinea pig heart, moderate acidosis is associated with CGRP release dependent on influx of extracellular Ca2+ and formation of PGI2, with subsequent stimulation of capsazepine sensitive receptors. With more severe acidosis there is an additional non-PGI2-linked CGRP release. Capsazepine represents a novel pharmacological principle for inhibiting the effects of prostanoids on sensory nerves without influencing their formation.

Ca2+]i and protein kinase C in vasopressin-induced prostacyclin and ANP release in rat cardiomyocytes.

Exposure of cultured, spontaneously beating rat cardiomyocytes to arginine vasopressin (AVP) led to marked increases in the release of prostacyclin (PGI2) and atrial natriuretic peptide (ANP). These responses were accompanied by a rapid, transient rise of cytosolic free Ca2+ concentration ([Ca2+]i) and of membranous protein kinase C (PKC) activity. Ca2+ influx and PKC activity appeared to play important but distinct roles in AVP-induced cellular responses, insofar as only AVP-induced ANP secretion was abolished by the Ca2+ channel antagonist nifedipine, whereas both AVP-induced PGI2 production and ANP release were abolished by the PKC inhibitors staurosporine and CGP-41251. The AVP-induced increase in [Ca2+]i could also be mimicked with the vasopressin (V1-subtype) agonist Octapressin, but not with the V2-agonist 1-desamino-8-D-arginine vasopressin, and was fully abolished by the V1-antagonist [d(CH2)5Tyr(Me)]AVP, but not by d(CH2)5-D-Leu-VAVP (V1-/V2-antagonist). These results indicate that V1-vasopressinergic receptors mediate AVP-induced PGI2 production and ANP secretion in rat cardiomyocytes and that, whereas both Ca2+ influx and PKC activation are required for AVP-induced ANP secretion, AVP-induced PGI2 formation is mainly regulated by PKC.

Effects of ATP and bradykinin on endothelial cell Ca2+ homeostasis and formation of cGMP and prostacyclin.

ATP and bradykinin are known to activate Ca2+ release from intracellular Ca2+ pools as well as induce the influx of Ca2+ in many cell types. In adrenal medulla endothelial cells, we found that ATP and bradykinin could activate Ca2+ influx, although Ca2+ influx did not appear to be due to depletion of intracellular Ca2+ pools per se, since depletion of intracellular Ca2+ pools with thapsigargin reduced rather than enhanced both unidirectional and steady-state 45Ca2+ uptake. In addition, Ca2+ influx, activated by ATP but not bradykinin, was mostly abolished after agonist removal in cells in which intracellular Ca2+ pools had not been allowed to refill, suggesting that continued receptor occupancy was necessary for ATP to activate Ca2+ influx. The role of Ca2+ in activating guanosine 3',5'-cyclic monophosphate (cGMP) formation [a marker for nitric oxide (NO) secretion] and prostacyclin (PGI2) secretion was also studied. Bradykinin-induced cGMP and PGI2 formation and ATP-induced PGI2 formation each required Ca2+ release from intracellular Ca2+ pools, since depletion of these pools with thapsigargin inhibited their formation. In contrast, ATP-induced cGMP formation, particularly at early time points, did not appear to require either Ca2+ release or Ca2+ influx. This suggests that ATP, but not bradykinin, either induces Ca(2+)-independent NO formation or that ATP stimulates the generation of cGMP independently of NO. The latter supposition is supported by our observation that NO synthase inhibitors inhibited ATP-induced cGMP formation by at most 50%.

Angiopeptin enhances acetylcholine-induced relaxation and inhibits intimal hyperplasia after vascular injury.

The effects of the somatostatin analogue, angiopeptin (BIM-23014), on neoendothelial function, as evidenced by formation of prostaglandin (PG) I2 and by acetylcholine-induced relaxation (formation of endothelial-derived relaxing factor), were investigated in the rabbit aorta. A balloon catheter injury of the thoracic and abdominal aorta was induced in New Zealand White rabbits. Animals treated with angiopeptin for 2 or 4 wk were compared with untreated rabbits at 2 or 4 wk after the induction of injury, as well as to sham-operated controls. When the rabbits were killed, vascular rings were assessed for arachidonic acid-stimulated PGI2 formation, acetylcholine-induced relaxation, and the degree of intimal hyperplasia. Vascular rings from animals treated with angiopeptin exhibited enhanced acetylcholine-induced relaxation; however, angiopeptin treatment had no effect on arachidonic acid-stimulated PGI2 formation. Intimal hyperplasia in treated animals was reduced by 36%. Treatment with another somatostatin analogue, BIM-23030, did not enhance relaxation or inhibit intimal hyperplasia. These data suggest that treatment with angiopeptin may inhibit intimal hyperplasia in part by its beneficial effect on neoendothelial function.

Platelet Activating Factor Enhances Receptor-Operated Ca 2+-Influx and Subsequent Prostacyclin Synthesis in Human Endothelial Cells

Platelet activating factor (PAF) primes vascular actions of mediators such as histamine and stimulates human umbilical vein endothelial cells (HUVECs) to produce prostacyclin (PGI 2), which is important for the regulation of vascular tone, perfusion and hemostasis. We demonstrate that pretreatment of HUVECs with PAP enhances thrombin- or histamine-induced rises of cytosolic free Ca ++-concentration and subsequent Ca ++-dependent PGI 2-synthesis. Inhibition of Ca ++-influx and PGI 2-formation by SKF96365 (blocker of receptor-operated Ca ++-channels) in PAP-treated cells indicates that the enhancement of PGI 2-synthesis by PAP is due to sensitization of Ca ++-entry. This suggests cooperative effects of PAP on activation processes induced by thrombin or histamine in HUVECs.

Nitric oxide and prostacyclin influence coronary vasomotor tone in perfused rabbit heart and modulate endothelin-1 activity.

Using isolated perfused rabbit heart electrically paced, we assessed the relevance of both nitric oxide (NO) and prostacyclin (PGI2) in regulation of resting coronary perfusion pressure (CPP). In preparations in which NO-synthase was inhibited by NG-monomethyl-L-arginine (L-NMMA, 10 microM), resting CPP increased significantly; this phenomenon was potentiated by indomethacin infusion (3 microM), prevented by L-arginine (100 microM) and significantly reduced by iloprost (55 nM) and defibrotide (200 micrograms/ml). Furthermore, the increase in resting CPP induced by graded doses of endothelin-1 (ET-1 0.6-160 pmol), was further augmented by blocking of prostaglandin biosynthesis with indomethacin (3 microM) and was substantially reduced when the rate of formation of PGI2 was enhanced by defibrotide (200 micrograms/ml). Moreover, the coronary vasoconstriction induced by ET-1 (2, 4, and 8 pmol) was increased in hearts in which NO-synthase was blocked by L-NMMA (10 microM) and this event was abolished in preparations in which PGI2 synthesis was stimulated by defibrotide (200 micrograms/ml). These results further emphasize that rabbit coronary vessels are continuously dilated by NO released from endothelial cells. They also indicate that PGI2 takes part in NO generation in the endothelial-derived relaxing mechanism. Inactivation of this mechanism, owing to decreased formation of NO and PGI2 in rabbit heart, induces hyperreactivity of coronary smooth muscles to ET-1. Finally, an increase in PGI2 production (such as that caused by defibrotide) may counterbalance impaired NO generation and attenuate hyperreactivity of the coronary vasculature.

The effect of endothelins on nitric oxide and prostacyclin production from human umbilical vein, porcine aorta and bovine carotid artery endothelial cells in culture.

1. This study has investigated the effects of the endothelin isopeptides, endothelin-1 (ET-1), ET-2 and ET-3 on the production of the endothelium-derived relaxing factors, nitric oxide (NO) and prostacyclin (PGI2) from primary cultures of endothelial cells obtained from human umbilical vein (HUVECS), porcine aorta (PAECS) and bovine carotid artery (BCAECS). 2. NO generation was assessed indirectly by measuring production of cyclic GMP and PGI2 formation was measured by radioimmunoassay of 6-keto PGF1 alpha. 3. In HUVECS, histamine (1 microM) increased cyclic GMP and 6-keto PGF1 alpha production by 12.6 +/- 2.0 and 4.9 +/- 0.7 fold respectively over the corresponding basal values. Haemoglobin (10 microM) and the NO synthase inhibitor NG-monomethyl-L-arginine (10 microM) significantly inhibited the increase in cyclic GMP formation in response to histamine but had no effect on 6-keto PGF1 alpha production. In contrast to histamine, the endothelin isopeptides (ET-1, ET-2 and ET-3; 0.01-1000 nM) produced no significant change in either cyclic GMP or 6-keto PGF1 alpha production in HUVECS. 4. In a separate series of experiments, ET-3 (0.01-1000 nM) also failed to produce any significant change in cyclic GMP or 6-keto PGF1 alpha production from primary cultures of PAECS and BCAECS. In contrast, bradykinin (0.1 microM) and sodium nitroprusside (1 mM) were used as positive control agents and increased cyclic GMP production in these cells. 5. In conclusion, the endothelin isopeptides do not release NO and PGI2 from primary cultures of HUVECS, PAECS and BCAECS. This suggests that endothelin receptors are either absent from these cells or are not coupled to NO or PGI2 production.

Transdermal modification of platelet function. A dermal aspirin preparation selectively inhibits platelet cyclooxygenase and preserves prostacyclin biosynthesis.

Even low doses of oral aspirin inhibit prostacyclin (prostaglandin [PG] I2) formation and cause gastrointestinal toxicity. We examined the skin as a novel route for continuous low-dose aspirin administration and selective inhibition of platelet cyclooxygenase in humans. Aspirin 250 or 750 mg/d for 10 days induced a dose-dependent inhibition of serum thromboxane (TX) B2. At the highest dose, five of six subjects responded, with a mean reduction in serum TXB2 of 95 +/- 3% (P = .003). Urinary 2,3-dinor TXB2, an index of in vivo TXA2 formation, decreased by 68 +/- 7% and recovered slowly, consistent with inhibition of platelet cyclooxygenase in vivo. In contrast, PGI2 biosynthesis, determined as excretion of 2,3-dinor-6-keto PGF1 alpha, was 81 +/- 5% of baseline at 10 days. Intravenous bradykinin increased PGI2 biosynthesis 5.1 +/- 1.6-fold (n = 4) before aspirin treatment. Oral aspirin 75 mg/d for 14 days abolished bradykinin-induced PGI2 formation, whereas dermal aspirin 750 mg/d had no effect despite similar inhibition of TXA2 biosynthesis. In five subjects, plasma aspirin and salicylate were determined after a single application of 750 mg. Aspirin was absorbed slowly, with peak levels of 0.24 +/- 0.11 micrograms/mL at 3 hours. Salicylate levels peaked at 6 to 12 hours, with plasma levels of 0.79 +/- 0.14 micrograms/mL. Thus, it is possible to achieve selective inhibition of platelet cyclooxygenase by aspirin applied to the skin. This approach may be applicable to other antiplatelet agents and be useful in patients at risk for gastrointestinal bleeding or toxicity.

Endothelium-Derived Bradykinin

The effects of angiotensin-converting enzyme (ACE) inhibitors on endothelial autacoid formation were determined in human cultured endothelial cells and in endothelium-intact bovine coronary arteries under resting conditions and after stimulation with bradykinin. Incubation of cultured human endothelial cells with moexiprilat or ramiprilat (0.3 microM) caused a maintained increase in resting intracellular calcium [Ca2+]i, which was prevented by the selective B2-receptor antagonist Hoe 140 (0.1 microM). Both ACE inhibitors also significantly enhanced the increase in [Ca2+]i elicited by bradykinin (3 nM). In parallel with their effect on resting [Ca2+]i, moexiprilat and ramiprilat both induced an increase in intracellular cyclic GMP (cGMP). This increase was prevented by Hoe 140 (0.1 microM) and was abolished by NG-nitro-L-arginine (30 microM), indicating a kinin-induced nitric oxide (NO) formation in this response. The elevation in [Ca2+]i also led to an enhanced production of prostacyclin (PGI2), as indicated by an increase in the concentration of 6-keto prostaglandin F1 alpha (PGF1 alpha) in the cell supernatant. Similar effects of the ACE inhibitors on endothelial autacoid production were observed in endothelium-intact bovine coronary arteries. Like bradykinin (30 nM), moexiprilat (0.3 microM) elicited a nearly twofold increase in the cGMP content of these arteries, which was abolished by both NG-nitro-L-arginine and removal of the endothelium. The functional consequences of this ACE inhibitor-induced increase in vascular cGMP were reflected by a distinct relaxation of arteries preconstricted with PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin-dependent cyclic AMP formation in endothelial cells.

The effect of the stable prostacyclin (PGI2) mimetic iloprost on cyclic AMP levels was investigated in cultured porcine aortic endothelial cells. Iloprost (10(-10)-10(-5) mol/l) did not change cyclic AMP levels at passage 1 when endothelial cells were untreated but did so after inhibition of endogenous PGI2 formation by 48 or 72 h treatment with indomethacin or diclofenac (10(-5) mol/l). Iloprost increased cyclic AMP in a concentration-dependent manner and up to 6-fold above control when cells from passage 6 were used. In these cells, basal PGI2 generation was reduced to 20% of that at passage 1. Cyclic AMP stimulation by iloprost (10(-5) mol/l) in passage 6 cells was enhanced, reaching up to 11-fold the control level, when cells were cultured for 48 h in the presence of indomethacin or diclofenac (10(-5) mol/l). Cyclic AMP formation in LLC-PK1 cells, a kidney epithelial cell line without endogenous PGI2 biosynthesis, was markedly (25-fold above basal) stimulated by iloprost and unchanged by pretreatment with indomethacin and diclofenac. The data demonstrate that a continuous basal PGI2 generation occurs in porcine aortic endothelial cells that may be sufficient to completely desensitize PGI2-dependent adenylate cyclase activation, presumably at the receptor or GTP-binding protein level.

Production of platelet thromboxane A 2 and arterial prostacyclin I 2 from hypercholesterolemic rats

The plasma cholesterol, plasma malonaldehyde (MDA), platelet thromboxane A 2 (TXA 2) and vascular prostacyclin (PGI 2) were measured in male Sprague-Dawley rats fed diets supplemented with cholesterol (1%) and cholic acid (0.5%). For comparisons, measurements were made in rats fed normal diets. The concentration of cholesterol in the plasma of rats had reached a maximum in 1 week of feeding experimental diets. TXA 2 production from collagen and thrombin stimulated platelets was significantly decreased in animals fed experimental diets for 1 week. The production of MDA in the plasma of animals fed experimental diets for 8 weeks was significantly lower compared to the animals fed normal diets. There was a small but significant reduction in the formation of PGI 2 in rats fed experimental diets for 8 weeks. These data suggest that feeding cholesterol rich diets to rats alters the platelet membrane properties differently from human and rabbit. Furthermore, cholesterol feeding to rats had some damaging effect on the arterial PGI 2 synthesis.

Delivery of passivating proteins to sutures during passage through the vessel wall reduces subsequent platelet deposition by blocking fibrinogen adsorption.

Intraluminal vascular suture material, which attracts fewer than the expected number of platelets compared with the same biomaterial exposed to blood in vitro, differs from the untreated biomaterial in that it has been passed once through the vessel wall. The mechanism by which this apparently trivial maneuver reduces platelet deposition was investigated. Polypropylene suture (7-0 Prolene) was passed through human arteries (fetal and adult), and platelet deposition to the suture was measured in a standardized perfusion chamber. Single vessel passage of the sutures reduced platelet deposition by 68 +/- 23%, which contrasts sharply with the power of prostaglandin E1 (1 microM PGE1 is sufficient to abolish platelet shape change and aggregation), which inhibited only 11% of platelet deposition to the sutures. Aspirin treatment of the vessel (to prevent PGI2 formation) or endothelial stripping (to remove the ability to produce nitric oxide) had no effect on the degree of inhibition. Passage of the suture through a vessel analogue (expanded polytetrafluoroethylene) did not inhibit platelet deposition. 125I-fibrinogen adsorption to the suture after vessel passage was reduced to a degree similar to that of platelet deposition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins eluted from vessel-passed sutures revealed bands at 66, 47, and 16 kd. Western blotting indicated the presence of large amounts of albumin and hemoglobin, a moderate amount of haptoglobin, and only trace amounts of fibrinogen. When sutures were exposed to each of these proteins in vitro before perfusion, albumin and hemoglobin were found to reproduce the effect of vessel passage alone on platelet deposition. We conclude that albumin and hemoglobin adsorb to sutures during their passage through the vessel subendothelium.(ABSTRACT TRUNCATED AT 250 WORDS)