Effect of endogenous prostaglandins on acetylcholine release from dog trachealis muscle

We used a radioenzymatic technique to measure effects of the prostaglandin synthesis inhibitor indomethacin and of exogenous prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) on acetylcholine (ACh) efflux from canine tracheal smooth muscle (TSM) during sustained electrical field stimulation (EFS; 2 Hz, 2 ms pulse duration, 50 V for 15 min). ACh efflux from indomethacin (INDO, 10(-6) M)-pretreated and control TSM increased with consecutive stimulations. However, efflux of ACh was greater in INDO-treated than control muscles. INDO increased the tension produced by TSM in response to EFS. Neither PGE2 (10(-8) M) nor PGI2 (10(-6) M) had any effect on ACh efflux from INDO-pretreated TSM during the first of three periods of EFS. However, PGI2 and PGE2 prevented the progressive increase in ACh efflux observed on subsequent stimulations. PGE2 but not PGI2 decreased contractions of TSM caused by EFS. Our results demonstrate that endogenous prostaglandins, probably PGE2, do inhibit EFS-evoked ACh release from canine TSM in vitro, but suggest that these prostaglandins modulate EFS-evoked contractions predominantly by postsynaptic mechanisms.     

Transepithelial prostacyclin gradient in isolated canine trachea

Cyclooxygenase products of arachidonic acid, potential modulators of airway smooth muscle, have recently been described in bronchoalveolar lavage from canine lungs. To evaluate the possibility that airway epithelium represents a barrier to movement of prostacyclin (PGI2), an important bronchodilator synthesized by isolated airway, we measured the concentrations of 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable degradation product of PGI2, on the mucosal and serosal sides of isolated canine tracheal segments (CTS) mounted in Ussing chambers. 6-oxo-PGF1 alpha was measured by radioimmunoassay after purification by high-performance liquid chromatography. The concentration of 6-oxo-PGF1 alpha was significantly higher on the serosal than the mucosal side of CTS (1,262 +/- 252 vs. 390 +/- 168 pg.min-1.g-1, n = 8, P less than 0.05). A significant correlation was present between 6-oxo-PGF1 alpha measured on both sides of each CTS (r = 0.778, n = 26, P less than 0.01). 6-oxo-PGF1 alpha production from CTS stripped of mucosa was significantly greater than from isolated mucosa. Radiochromatograms obtained after incubation with [3H]arachidonic acid and calcium ionophore A23187 confirmed PGI2 as the predominant cyclooxygenase product of the submucosa, whereas the mucosa produced only small amounts of PGI2 in proportion to other cyclooxygenase products. PGI2 (10(-8) to 10(-6) M) applied to the mucosal surface of closed tracheal segments precontracted with histamine resulted in no significant relaxation, whereas serosal application showed a concentration-dependent effect. Radiolabeled 6-oxo-PGF1 alpha did not cross the isolated epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S).

The effects of prostacyclin (PGI2) and its stable metabolite 6-oxo-PGF1alpha on various bioassay tissues are compared with those of PGE2 and PGF2alpha, using the cascade superfusion method. On vascular smooth muscle, PGI2 caused relaxation of all tissues tested except the rabbit aorta. PGE2 relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF1alpha was ineffective at the concentrations tested. On gastro-intestinal smooth muscle, PGI2 contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE2 or PGF2alpha. None of these substances contracted the cat terminal ileum. 6-oxo-PGF1alpha was inactive on these tissues at the doses tested. PGI2 was less active than PGE2 or PGF2alpha in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF1alpha was active only on the rat uterus. Thus, PGI2 can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

Measurement of 6-oxo-PGF1 alpha in human plasma using gas chromatography-mass spectrometry.

The plasma concentration of the prostacyclin (PGI2) hydration product 6-oxo-PGF1 alpha has been assayed by stable isotope dilution GC-MS in six normal volunteers infused with increasing doses of PGI2 intravenously. The predosing levels of 6-oxo-PGF1 alpha ranged between 114 and 266 pg/ml. Infusion of PGI2 increased 6-oxo-PGF1 alpha concentration in plasma but the increments were lower than expected suggesting less conversion of the PGI2 to 6-oxo-PGF1 alpha at high infusion rates.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides.

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI2) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 micrograms ml(-1)). Both 15-HPAA (1-20 micrograms ml(-1) min (-1)) and 13-hydroperoxy linoleic acid (13-HPLA, 20 micrograms ml(-1) min(-1)) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI2 (5 micrograms ml(-1) min (-1)) or 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha, 5 micrograms ml(-1) min(-1)). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI2 (10 ng - 10 microgram ml(-1) min(-1)) but was inhibited by PGE2 (5 and 10 micrograms ml(-1) min (-1)). Antiserum raised to 5,6-dihydro prostacyclin (PGI1) in rabbits, which also binds PGI2, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Actions of prostacyclin (PGI2) and its product, 6-oxo-PGF1alpha on the rat gastric mucosa in vivo and in vitro.

The effects of prostacyclin (PGI2) and its breakdown product 6-oxo-PGF1alpha on various aspects of gastric function were investigated in the rat. PGI2 increased mucosal blood flow when infused intravenously. PGI2 was a more potent inhibitor of gastric acid secretion in vivo than PGE2. Like PGE2, PGI2 inhibited acid secretion from the rat stomach in vitro. PGI2 had comparable activity to PGE2 in inhibiting indomethacin-induced gastric erosions. Thus prostacyclin shares several of the activities of PGE2, and may be involved in the regulation of gastric mucosal function.     

Rat hypothalamic corticotropin-releasing hormone secretion in vitro is stimulated by interleukin-1 in an eicosanoid-dependent manner

We studied the effect of interleukin-1 alpha (IL-1) on corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami in vitro. We also assessed possible mediation of arachidonic acid metabolites on IL-1-stimulated CRH secretion, by preincubating hypothalami with the cyclooxygenase inhibitor indomethacin (INDO, 1 microM), the lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid (ETYA, 10 microM), or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, up to 30 microM). In additional experiments, prostaglandins (PG) E2 and F2 alpha were added to the cultures treated with INDO or ETYA. Finally, we investigated the effect of dexamethasone (DEX) on IL-1-stimulated CRH secretion. IL-1 stimulated immunoreactive CRH (iCRH) secretion by explanted hypothalami in a concentration-dependent fashion. Both INDO and ETYA inhibited IL-1-(10nM)-stimulated iCRH secretion, whereas NDGA did not have any effect. The addition of PGF2 alpha (10 nM) restored the secretion of iCRH inhibited by INDO. DEX treatment significantly inhibited IL-1-stimulated iCRH release. Our results suggest that the stimulatory effect of IL-1 on the hypothalamic CRH neuron is mediated by the cyclooxygenase metabolites of arachidonic acid, and, among others, by PGF2 alpha.     

Species dependent relaxation of intrapulmonary arteries (IPA) of rabbits, dogs and humans by prostacyclin

Helically cut strips of successive IPA segments of rabbits, dogs and human patients were set up for isometric recording in vitro. High tone was produced by norepinephrine (NE, 3 microM). This tone was markedly reduced by prostacyclin (PGI2) in the secondary, tertiary and quaternary branches of human and canine pulmonary trunk. The IC50 values for PGI2 ranged from 22 to 503 nM, the human vessels being more sensitive to prostacyclin than canine IPA. Under these conditions, the primary and secondary branches of the rabbit pulmonary trunk were not relaxed by PGI2. The contractile potency of NE was determined in each pulmonary vessel studied. The secondary segments of rabbit IPA were about ten times as sensitive to NE (EC50 for NE: 38 +/- 7 nM) as compared to the secondary IPA from dogs and humans (EC50 values: 370 +/- 84 and 440 +/- 50, respectively). When high tone was induced by equieffective contractile concentrations of NE (3 microM for canine and human IPA and 0.3 microM for rabbit vessels), PGI2 was still less effective (P less than 0.01) in relaxing secondary IPA of rabbits (IC25: 220 +/- 55) than the corresponding segments of dogs and humans (IC25: 51 +/- 12 and 17 +/- 4, respectively). The difference between canine and human vessels was also significant (P less than 0.02). These results indicate that there is an interspecies difference in the sensitivity of IPA to NE and PGI2.     

Endogenous AA metabolites and their possible role in tracheal smooth muscle tone in guinea pigs

The effects of endogenous arachidonic acid (AA) metabolites on inherent tone and histamine-induced constriction were studied in guinea pig tracheal smooth muscle. Inhibitors of either cyclooxygenase (indomethacin) or lipoxygenase (AA 861) significantly diminished the inherent tone of the muscle. Antagonists of prostaglandins (SC 19220) or leukotrienes (FPL 55712) also diminished the inherent tone, whereas an inhibitor of thromboxane synthase (OKY 046) had no significant effect. These results show that the metabolites of the lipoxygenase pathway as well as prostaglandins also participate in the maintenance of inherent tone. To reexamine the previously reported augmentation of histamine constriction induced by the inhibitors and the antagonists, we compared the active tension of the muscle measured from the maximum relaxed level as the base line to eliminate the fluctuation of inherent tone. Such comparison revealed that the inhibitors and the antagonists have no augmentative effect on either the maximum response to histamine or the concentration required to produce 50% of maximum active tension and that there is functional synergism between the exogenously added histamine and the endogenously produced AA metabolites. Therefore the zero active tension is useful as a base line to compare the contractile response of a drug-treated preparation with that of a nontreated preparation.     

Modulation of vascular reactivity to serotonin in the dog lung

Experiments were conducted to compare the effects of cyclooxygenase inhibition (COI) on vascular reactivity to serotonin (5-HT) in the isolated blood-perfused canine left lower lung lobe (LLL) and in isolated canine intrapulmonary lobar artery rings with and without a functional endothelium. LLLs (n = 6), perfused at constant blood flow, were challenged with bolus doses of 50, 100, and 250 micrograms 5-HT before COI, after COI with 45 microM meclofenamate, and after infusion of prostacyclin (PGI2) during COI. Lobar vascular resistance was segmentally partitioned by venous occlusion. Pulmonary arterial pressure increased from 13.5 +/- 1.0 to 16.3 +/- 0.8 cmH2O (P less than 0.01) after COI but declined to 13.1 +/- 1.1 cmH2O (P less than 0.01) subsequent to PGI2 infusion (91.3 +/- 14.5 ng.min-1.g LLL-1). The pulmonary arterial pressure changes were related to changes in postcapillary resistance. The dose-dependent pressor response to 5-HT was potentiated by COI (P less than 0.01) but reversibly attenuated (P less than 0.05) by PGI2 infusion. Isolated intrapulmonary artery rings (2-4 mm diam) exhibited a dose-related increase in contractile tension to 5-HT. The response to 5-HT was enhanced (P less than 0.05) in rings devoid of a functional endothelium. However, COI (10 microM indomethacin) did not alter (P greater than 0.05) the dose-related increase in contractile tension to 5-HT in rings with an intact endothelium. Our results suggest that both PGI2 and endothelium-derived relaxing factors modulate pulmonary vascular reactivity to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of cyclooxygenase inhibitors on the central histaminergic stimulations of hypothalamic - pituitary - adrenal axis.

Brain histamine participates in central regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Endogenous prostaglandins modulate signal transduction of different neurotransmitters involved in activation of HPA axis. In the present experiment we investigated whether endogenous prostaglandins are involved in the stimulation of ACTH and corticosterone secretion by histaminergic systems in the rat brain. Histamine (50 microg), histamine-trifluoromethyl-toluidine derivative (HTMT, 75microg) a selective and potent H(1)-receptor agonist, and amthamine (50 microg) a H(2)-receptor agonist given intracerebroventricularly (i.c.v.) to non-anesthetized rats considerably increased ACTH and corticosterone secretion 1h after administration. A non-selective cyclooxygenase inhibitor indomethacin (2 mg/kg i.p. or 10 microg i.c.v.), piroxicam (0.02 and 0.2 microg i.c.v.) a more potent antagonist of constitutive cyclooxygenase (COX-1) and compound NS-398 (0.1 and 1.0 microg i.c.v.), a selective inhibitor of inducible cyclooxygenase (COX-2) were given 15 min before histamine and histamine receptor agonists. One hour after the last injection trunk blood from decapitated rats was collected for hormones determination. The histamine-induced ACTH and corticosterone secretion was significantly diminished by piroxicam and was not markedly altered by indomethacin and compound NS-398. The HTMT-elicited increase in ACTH and corticosterone secretion was significantly prevented by indomethacin and was not affected by piroxicam or compound NS-398. The amthamine-evoked increase in ACTH and corticosterone secretion was not markedly influenced by any cyclooxygenase blocker applied in the present experiment. These results indicate that the histamine H(1)-receptor transmitted central stimulation of the HPA axis is considerably mediated by prostaglandins generated by consititutive cyclooxygenase, whereas stimulation transmitted via H(2)-receptor does not significantly depend on endogenous prostaglandins mediation.     

Effects of prostacyclin on coronary circulation, heart rate and myocardial contractile force in isolated hearts of guinea pig and rabbit - comparison with prostaglandin E2.

Infusions of prostacyclin (PGI2) (3 x 10(-10) - 3 x 10(-7)M) into the coronary circulation of isolated hearts from ginea pigs or rabbits resulted in a concentration-dependent decrease in the coronary perfusion pressure (CPP). There was a slight decrease in left ventricular systolic pressure in the heart of the rabbit, whereas the heart rate remained unchanged. PGE2 was without effect on the heart of the rabbit but was as potent as PGI2 in decreasing the CPP in the guinea pig heart. 6-oxo-PGF1 alpha (up to 3 x 10(-6) M) did not affect any of the parameters measured.     

The renal haemodynamic and excretory actions of prostacyclin and 6-oxo-PGF1 alpha in anaesthetized dogs.

Intrarenal arterial (i.a.) infusions of prostacyclin (PGI2) at 30-300 ng/min to anaesthetized dogs reduced renal vascular resistance (RVR) and filtration fraction (FF), increased mean renal blood flow (MRBF) but did not alter mean arterial pressure (MAP)or glomerular filtration rate (GFR). The urinary excretion of sodium (UNaV), potassium (UKV) and chloride ions (UC1V) were increased through inhibition of net tubular ion reabsorption. PGI2 (3000 ng/min, i.a.) reduced MAP and increased heart rate. Intravenous (i.v.) infusions of PGI2 (3000 gn/min) reduced MAP, GFR, FF, urine volume and ion excretion, with elevation of heart rate. The measured variables were unaltered by 6-oxo-PGF1 alpha (10,000 ng/min i.a.). Treatment of the dogs with the PG synthetase inhibitor meclofenamic acid (2.5 mg/kg i.v.) did not antagonise the elevation of MRBF to PGI2 (300 ng/min i.a.). Thus the renal effects of PGI2 were due to a direct action rather than through conversion to 6-oxo-PGF1 alpha or through stimulation of endogenous renal PG biosynthesis and release.     

Prostacyclin and thromboxane A2 in thrombotic thrombocytopenic purpura.

A study was conducted to find whether a deficiency in prostacyclin (prostaglandin I2; PGI2) is implicated in the pathogenesis of thrombotic thrombocytopenic purpura. Plasma samples from two patients with the disease before treatment and from 22 healthy controls were therefore assayed for concentrations of 6-oxo-PGF1 alpha and thromboxane B2, the stable metabolites of PGI2 and thromboxane A2, respectively. Neither of the patients responded to treatment, which in one case included an infusion of PGI2. Both patients had normal concentrations of 6-oxo-PGF1 alpha and thromboxane B2, thus implying that circulating amounts of PGI2 and thromboxane A2 were also normal. These findings suggest that 6-oxo-PGF1 alpha may be detectable in normal amounts in thrombotic thrombocytopenic purpura and that the condition need not be associated with a high concentration of thromboxane A2.     

Cyclooxygenase-2-derived endogenous prostacyclin reduces apoptosis and enhances embryo viability in mouse

The role of prostaglandins (PGs) in apoptosis in preimplantation mice embryo development is reported in this study. It is known that apoptosis plays a very important role in normal mice embryo development. Very few reports are available on this subject. Embryos (6-8 cells) were cultured in the presence of a selective cyclooxygenase (COX)1 inhibitor (SC560), a selective COX2 inhibitor (NS398) and a selective prostacyclin synthase (PGIS) inhibitor (U51605) in a 48-h culture. In another experiment, culture media were supplemented with prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2 or prostacyclin) analogues. The apoptosis was evaluated by detection of active caspase-3. It was strongly detected in the presence of selective COX-2 and PGIS inhibitors, which can be decreased by a PGI2 analogue. In our embryo transfer experiment, the implantation rate decreased with exposure to either the COX2 or the PGIS inhibitor which is increased further after PGI2 supplementation. The level of PGI2 is also higher at the 8-16-cell stage, compaction and blastocyst stage than PGE2. All these results indicate that COX2-derived PGI2 plays an important role in preimplantation embryo development and acts as an antiapopetic factor in in vitro culture.     

Dual actions of prostacyclin (PGI2) on the rat pregnant uterus.

Using strips of rat pregnant uterus, treated with indomethacin to suppress spontaneous contractility, the oxytocic activity of prostacyclin was compared with other prostaglandins. A prostacyclin concentration of 32 ng/ml elicited uterine contractions in all experiments. In this respect prostacyclin was 80 times more active than 6-oxo-PGF1 alpha but less active than PGE2 or PGF2 alpha. Apart from a direct stimulant effect, prostacyclin also exhibited an indirect potentiating action. In threshold concentrations prostacyclin caused a 3-fold potentiation of threshold doses of oxytocin. A lesser 1.5-fold potentiation of PGE2 alpha was also observed. The implications of these findings in relation to prostacyclin playing a role in parturition are discussed.     

Restoration of prostacyclin synthase in vascular smooth muscle cells after aspirin treatment: regulation by epidermal growth factor

Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important role in maintenance of vascular homeostasis. Aspirin irreversibly inactivates prostacyclin synthetase by acetylating the enzyme. Recovery of the enzyme following inactivation by aspirin was studied in rat aorta smooth muscle cells in tissue culture. Confluent cultures superfused with [14C]arachidonic acid, synthesized prostacyclin (PGI2) together with prostaglandins E2, D2, and F2 alpha. Brief treatment with physiological levels of aspirin (0.2 mM) completely inactivated prostacyclin synthesis. Following aspirin removal and addition of fresh growth medium, PGI2 synthesis recovered rapidly with a T 1/2 of only 30-40 min, compared to a doubling time of 24-30 hr for the cells. Recovery of PGE2, PGD2, and PGF2a synthesis paralleled that of PGI2, confirming that cyclooxygenase rather than endoperoxide-prostacyclin isomerase was the labile component. Recovery of PGE2 synthesis after aspirin was blocked by cycloheximide but not by actinomycin D. Recovery of aspirin-inactivated cells required a non-dialyzable component present in serum. All samples tested, including fetal bovine, new-born calf, human, and guinea pig, showed the activity. Fresh serum also induced a cycloheximide-sensitive 2- to 3-fold increase in cyclooxygenase levels in resting confluent cells within 1 to 2 hr. Serum factor was also required to restore PG synthesis after aspirin-inactivation in other cells, including 3T3 mouse fibroblasts, SV40-3T3 and K-Balb 3T3 transformed mouse fibroblasts, NRK rat kidney cells, and REF-9 rat embryonic fibroblasts. The activity was thermolabile, and was completely removed from the medium by growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of 6-oxo-prostaglandin F1 alpha and prostaglandin E2 by isolated glomeruli from normal and diabetic rats.

Production of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and prostaglandin E2 (PGE2) was measured by radioimmunoassay in supernatants of isolated glomeruli from rats with streptozocin-induced diabetes and non-diabetic rats. Production of 6-oxo-PGF1 alpha by discs of aortas from these rats was measured at the same time. As shown before, aortic discs from diabetic rats produced significantly less 6-oxo-PGF1 alpha than aortic discs from non-diabetic rats (diabetic 1.99 +/- SEM 0.27 ng v non-diabetic 2.92 +/- 0.46 ng/mg net weight aorta; p less than 0.05). In contrast production of 6-oxo-PGF1 alpha by isolated glomeruli was not reduced in the diabetic rats (diabetic 77 +/- 7 pg v non-diabetic 70 +/- 8 pg/micrograms glomerular DNA). Similarly production of PGE2 was not diminished in the diabetic glomeruli (diabetic 1.20 +/- 0.15 ng v non-diabetic 0.91 +/- 0.12 ng/microgram glomerular DNA). It is concluded that regional differences in production of prostacyclin and 6-oxo-PGF1 alpha occur in experimental diabetes. Diminished prostacyclin production may contribute to the increased susceptibility of diabetic patients to atherosclerosis but is less likely to have a role in the pathogenesis of microangiopathy.     

Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein.

Fluid flow and several other agonists induce prostacyclin (PGI2) production in endothelial cells. G proteins mediate the response of a large number of hormones such as histamine, but the transduction pathway of the flow signal is unclear. We found that GDP beta S and pertussis toxin inhibited flow-induced prostacyclin production in human umbilical vein endothelial cells. In addition, flow potentiated the histamine-induced production of PGI2. This suggests that flow stimulates prostacyclin production via a pertussis toxin-sensitive G protein and modulates the stimulus-response coupling of other agonists.     

Species dependent relaxation of intrapulmonary arteries (IPA) of rabbits, dogs and humans by prostacyclin

Helically cut strips of successive IPA segments of rabbits, dogs and human patients were set up for isometric recording . High tone was produced by norepinephrine (NE, 3 μM). This tone was markedly reduced by prostacyclin (PGI2) in the secondary, tertiary and quaternary branches of human and canine pulmonary trunk. The IC50 values for PGI2 ranged from 22 to 503 nM, the human vessels being more sensitive to prostacyclin than canine IPA. Under these conditions, the primary and secondary branches of the rabbit pulmonary trunk were not relaxed by PGI2. The contractile potency of NE was determined in each pulmonary vessel studied. The secondary segments of rabbit IPA were about ten times as sensitive to NE (EC50 for NE: 38±7 nM) as compared to the secondary IPA from dogs and humans (EC50 values: 370±84 and 440±50, respectively). When high tone was induced by equieffective contractile concentrations of NE (3 μM for canine and human IPA and 0.3 μM for rabbit vessels), PGI2 was still less effective (P<0.01) in relaxing secondary IPA of rabbits (IC25: 220±55) than the corresponding segments of dogs and humans (IC25: 51±12 and 17±4, respectively). The difference between canine and human vessels was also significant (P<0.02). These results indicate that there is an interspecies difference in the sensitivity of IPA to NE and PGI2.