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Cysteine-containing leukotrienes (cysteinyl-LTs) are potent bronchoconstrictors and play a key role in asthma. We found that histamine and LTD4 markedly constrict strips of human bronchi (HB) with similar efficacy. However, in human airway smooth-muscle (HASM) cells, LTD4, at variance with histamine, elicited only a small, transient change in intracellular calcium ion concentration. HASM cells express both Ca2+-dependent and -independent isoforms of protein kinase C (PKC) (i.e., PKC-alpha and PKC-alpha ). Western blot analysis showed that PKC-alpha is activated by histamine and, to a lesser extent, by LTD4, whereas only LTD4 translocates PKC-alpha. This translocation was specifically inhibited by the LTD4 antagonist pobilukast. Phorbol-dibutyrate ester (PDBu) (a PKC activator) contracted HB strips to the same extent in the presence as in the absence of extra- and intracellular Ca2+. In the absence of Ca2+, LTD4 contracted HB strips to the same extent as did PDBu, suggesting the involvement of a Ca2+-independent PKC in LTD4-mediated signal transduction. PDBu-induced desensitization and the PKC inhibitor H7 abolished the slow and sustained LTD4-triggered contraction of HB strips in the absence of Ca2+, although H7 did not greatly affect the response in the presence of the ion. Thus, in human airways, we identified a novel LTD4 transduction mechanism linked to bronchial smooth-muscle contraction, which is partly independent of Ca2+ and involves the activation of PKC-alpha.

We studied the effect of endogenous and exogenous prostaglandin E(2) (PGE(2)), a metabolite of arachidonic acid through the cyclooxygenase (COX) pathway, on interleukin (IL)-1 beta-induced COX-2 expression, using primary cultures of human bronchial smooth-muscle cells (HBSMC). Treatment with exogenous PGE(2) resulted in enhanced expression of IL-1 beta-induced COX-2 protein and messenger RNA (mRNA) as compared with the effect of the cytokine per se. Inhibition of PGE(2) production with a nonselective COX inhibitor (flurbiprofen, 10 microM) resulted in a significant reduction in IL-1 beta- induced COX-2 expression, supporting a role of endogenous COX metabolites in the modulation of COX-2 expression. None of the experimental conditions used in the study affected the expression of constitutive cyclooxygenase (COX-1). Treatment with cycloheximide to inhibit translation, and with dexamethasone or actinomycin D to inhibit transcription, linked the effect of PGE(2) to the transcriptional level of COX-2 mRNA rather than to a potential effect on protein and/or mRNA stabilization. PGE(2) increased adenylate cyclase activity in a concentration dependent manner, and forskolin, a direct activator of adenylate cyclase, caused a marked increase in IL-1 beta-dependent COX-2, suggesting the existence of a causal relationship between the two events. The same results were observed with salbutamol, a bronchodilator that acts by increasing cyclic adenosine monophosphate. The effect of PGE(2) on COX-2 expression may contribute to the hypothesized antiinflammatory role of PGE(2) in human airways, providing a self-amplifying loop leading to increased biosynthesis of PGE(2) during an inflammatory event.

The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in cultured human bronchial smooth-muscle cells challenged with interleukin-1 beta (IL-1 beta). Cells were incubated with IL-1 beta (10 to 50 U/ml) for 0 to 24 h. Prostaglandin E2 (PGE2) production was evaluated through the conversion of exogenous (14C)-arachidonic acid and specific enzyme immunoassay of endogenous products. IL-1 beta enhanced PGE2 formation in a concentration- and time-dependent manner, reaching its peak at 6 to 8 h and fading at 18 to 24 h. Immunoblot analysis showed that the inducible cyclooxygenase enzyme (COX-2) was expressed only in IL-1 beta treated cells, whereas the constitutive isoform of cyclooxygenase (COX-1) remained unaltered. COX-2 expression and PGE2 formation were inhibited by dexamethasone (2 microM), cycloheximide (10 microM), and IL-1-receptor antagonist (IL-1 ra) (250 ng/ml), independently. PGE2 synthesis was significantly reduced by compound SC-58125, a specific COX-2 inhibitor. The close parallelism between the kinetics of COX-2 protein expression and PGE2 accumulation, as well as the constitutive nature of COX-1 isoform, indicate that IL-1 beta-driven PGE2 formation in human bronchial smooth-muscle cells is mediated by de novo expression of COX-2 enzyme.

SCH 37224, 1-(1,2-dihydro-4-hydroxy-2-oxo-1-phenyl-1,8-naphthyridin-3-yl) pyrrolidinium, is a structurally novel compound that had been shown to inhibit leukotriene D4 formation in guinea pig lung in vitro. We tested whether SCH 37224 is able to inhibit both the formation of eicosanoids from human lung parenchyma in vitro and the binding of sulfidopeptide leukotrienes to membranes of lung parenchyma and bronchi. SCH 37224, at a concentration of 30 and 100 microM, was able to inhibit antigen-induced formation of sulfidopeptide leukotrienes, measured as leukotriene E4, while it did not significantly affect the formation of prostaglandin D2. At concentrations up to 100 microM, it did not affect either the binding of [3H]leukotriene C4 to membranes of human lung parenchyma or human bronchi, or the binding of [3H]leukotriene D4 to the parenchyma. In conclusion, SCH 37224 is a selective inhibitor of leukotriene formation in human lung in vitro, which might be of potential therapeutic interest in the treatment of asthma.

In vitro passive sensitization of human lung parenchyma with hyper-immune serum did not affect the release of prostaglandin D2 (PGD2) or leukotriene (LT)-like activity upon challenge with anti-IgE antibody with respect to control lung, despite a marked difference in IgE levels between control (C) and sensitized (S) tissue. Binding studies with [3H]LTC4, [3H]LTD4 and [3H]mepyramine (a histamine H1 antagonist) showed a statistically significant increase in the amount bound in sensitized vs control lung for [3H]mepyramine only. Contractile response to 5 x 10(-5) M histamine (H) in C and S lung parenchymal strips did not correlate with binding data. It is concluded that in vitro elevated IgE levels do not affect the interaction of sulfidopeptide leukotrienes with their putative receptors. As for the observed increase in [3H]mepyramine binding, this might not represent a true increase in histamine receptors on lung smooth muscle cells.

Esaprazole, N-cyclohexyl-1-piperazineacetamide monohydrochloride, was studied for its activity to prevent gastric mucosal damage induced by several necrotizing agents in the rat. Its effects on acid gastric secretion and the role of gastric mucosal prostaglandin generation were also investigated. Esaprazole, given orally, dose dependently prevented the formation of mucosal damage induced by absolute ethanol, 0.2 N NaOH or 0.6 N HCl. This activity occurred at doses lower than the antisecretory doses. Esaprazole was also found to increase the gastric mucosal prostaglandin content but at doses that exceeded the cytoprotective doses. The failure of indomethacin to impair the gastric mucosal protection provided by esaprazole suggests that mechanisms other than mobilization of endogenous prostaglandins may be involved.

We studied the ex vivo production of prostaglandin D2, prostaglandin E2, 6-ketoprostaglandin F1 alpha, and leukotriene C4 in the brain tissue of rats subjected to experimental subarachnoid hemorrhage. The ex vivo method allows the study of arachidonic acid metabolites released from brain slices at different times after subarachnoid hemorrhage induction and reflects the residual capacity for arachidonic acid metabolism after the pathologic event. The rats were sacrificed 30 minutes, 1 and 6 hours, and 2 days after subarachnoid hemorrhage was induced by the injection of 0.30 ml autologous arterial blood into the cisterna magna. Concentration of prostaglandin D2 and 6-ketoprostaglandin F1 alpha was increased significantly relative to control 2 days after induction. The concentration of prostaglandin E2 was increased significantly 6 hours after induction, while ex vivo production of leukotriene C4 was increased significantly at 1 and 6 hours and 2 days. The correlation between these results and the occurrence of vasospasm after subarachnoid hemorrhage is discussed. The results obtained from the ex vivo incubation of brain tissue slices after experimental subarachnoid hemorrhage suggest that after the hemorrhage there is a significant modification of brain eicosanoid metabolism, which could be of great importance in interpreting the pathogenesis of subarachnoid hemorrhage-related neuronal impairment.

1 Histamine (0.5 to 5 micrograms) and slow reacting substance of anaphylaxis (SRS-A, 0.05 to 0.3 u), injected in the isolated, perfused lungs of normal and ovalbumin-sensitized guinea-pigs, promote formation and release of thromboxane A2(TXA2) and other arachidonate metabolites, the effect being more pronounced in sensitized lungs. 2 Carbachol injected (1 to 10 micrograms) or perfused (1 micrograms ml-1 min-1) through normal or sensitized lungs does not elicit formation of TXA2 and prostaglandins. Furthermore the increased generation of arachidonate metabolites due to histamine is not altered by carbachol. 3 Atropine and ipratropium bromide (1 microgram ml-1 min-1) reduce significantly the increased rate of production of TXA2 caused by histamine and SRS-A both in normal and sensitized lungs, whereas hexamethonium (10 to 25 micrograms ml-1 min-1) is ineffective. 4 The mechanism of action of atropine in inhibiting the increased generation of TXA2 is clearly not related to its antimuscarinic or antihistaminic properties. The drug might act at the early events, involved in the activation of arachidonic acid metabolism. The results suggest new sites of action for atropine which, besides the control of the vagal bronchomotor tone, interferes directly with the primary mediators of anaphylaxis.

Experimental and clinical studies indicate that cerebral vasospasm following subarachnoid hemorrhage (SAH) may be caused by changed biochemical properties of the endothelium and vascular smooth muscle cell exposed to vasoactive substances synthetized by cerebral arteries and released in clotted blood. Many compounds have been identified in CSF from SAH patients: Thromboxanes A2 and B2, Prostaglandins F2 alpha, E2 and D2 are the major prostanoids incriminated in the causation of cerebral arterial spasm. We have monitored the CSF PGD2 concentrations with serial lumbar punctures at different intervals from the hemorrhage in 16 patients admitted for SAH: PGD2 was measured with radioimmunoassay as its 9-methoxy derivative. The lumbar CSF PGD2 concentration ranges from 0.11 to 1.53 ng/ml. In 7 cases vasospasm was angiographically demonstrated. 9 patients presented no clinical or radiological evidence of vasospasm. In 5 cases cisternal CSF samples were available at the operation by cisternal punctures. There was no correlation between CSF PGD2 concentration and clinical course. In the 7 cases with evidence of vasospasm a significant increase of CSF PGD2 corresponded to neurological deterioration. In all 9 cases without evidence of vasospasm CSF PGD2 concentration trend was in a steady-state. The cisternal CSF PGD2 concentration was higher than lumbar CSF concentration in cases with arterial spasm. This suggests the importance of the clotting phenomenon in vasospasm onset after SAH. PGD2 is one of the most important spasmogens in clotted blood. Although its role in the genesis of vasospasm onset remains to be defined, its vasospastic action, in addition to that of other analogous compounds, seems to be relevant.

Angiotensin II (AII) induces generation of prostacyclin (PGI2) in rabbit lung in vitro at different ages, i.e., fetus, newborn and adult. Particularly, neonatal rabbit lungs display a pattern of sensitivity to AII in producing PGI2, measured as 6-oxo-PGF1 alpha, which seems to be higher than that observed in fetal lungs. The PGI2 release appears to be specific for AII stimulation since the vasoconstriction induced by noradrenaline and ergotamine was not associated with generation of this lipidic material. The inability of PGI2 to relax the extralobar pulmonary artery in the newborn suggests that the lung microcirculation is the most likely site where the vasodilating and antiaggregatory functions of PGI2 may have a physiological role.

Various doses of histamine, bradykinin and leukotriene C4 caused bronchoconstriction in anaesthetized guinea-pigs which were breathing spontaneously or artificially ventilated; there was a simultaneous, dose-related increase in circulating TXA2. The animals were prepared for continuous recording of extracorporeal circulation in order to detect the appearance in the blood of bioassayable levels of TXA2 -like substance. Furthermore, a TXA2 derivative, the mono-O-Me-TXB2, was radioimmunoassayed. Atropine, oxytropium bromide and ipratropium bromide given in mumol doses prevented both the increased airway resistance and the release of TXA2-like substance in the blood Pirenzepine dihydrochloride was the least active of the drugs tested. The protecting activity of anticholinergics and the relationship with their ability to affect TXA2-like substance generation suggest a new site of action for these drugs besides the blockade of muscarinic receptors.

The stimulation of human lung parenchymal fragments with A-23187 induces formation of leukotrienes as well as of PGD2: LTE4 is the compound found in larger amount and independently of the intensity of the stimulus the % of metabolized precursor which is converted to leukotrienes or PGD2 is remarkably similar. However, under conditions of IgE-Anti IgE challenge the predominant conversion of arachidonic acid occurs to PGD2, LTB4 is almost negligible and LTD4 and E4 together represent less than 30% of the oxidative products of arachidonic acid. Whether PGD2 and leukotrienes derive from the same or different subset of cells is unresolved.

S-(+)-2-(4-Fluorophenyl)-alpha-methyl-5-benzoxazolacetic acid (flunoxaprofen, Priaxim is a new antiinflammatory compound, which in various biological systems interferes with the generation and release of arachidonic acid metabolites of the cyclo-oxygenase pathways without affecting the formation of 5- and 12-lipoxygenase products. Flunoxaprofen reduces the concentration of thromboxane (TX)B2 (ED50 = 35.4 mg/kg p.o.) and prostaglandin (PG)E2-like activity (ED50 = 39.9 mg/kg p.o.) in the inflammatory exudate of rats 8 h after implantation of sponges soaked with carrageenan, whereas the concentration of leukotriene (LT)B4 remains in the range of that of the untreated animals (3.1 ng/ml). Flunoxaprofen inhibits cell infiltration in the exudate and this suggests that LTB4 does not initiate cell recruitment but may represent a mechanism for amplifying the inflammatory response. Using human platelets challenged with collagen, flunoxaprofen only at higher concentration (10(-4) mol/l) reduces TXB2 formation without altering generation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid. This suggests a low capacity of flunoxaprofen of affecting platelet aggregation regulated by arachidonic acid metabolites. Flunoxaprofen prevents pulmonary changes due to secondary release of TXA2 in the guinea-pig. In fact this drug prevents changes due to immunological reaction and antagonizes bronchoconstriction due to exogenous administration of histamine and LTC4.(ABSTRACT TRUNCATED AT 250 WORDS)

Tiaramide hydrochloride (THC) is a benzothiazoline derivative with a remarkable antianaphylactic activity: in anaesthetized guinea-pig it shows protecting effects against histamine (H)- and bradykinin (Bk)-induced bronchoconstriction, preventing increase of lung resistance and decrease of dynamic compliance. At the same time THC inhibits formation of circulating thromboxane A2 brought about by H and Bk. THC is also able to antagonize the contractions induced by CaCl2 on isolated guinea-pig tracheal spirals and its mode of action seems to be of the competitive type. Furthermore THC abolishes the "tonic" phase of K+-induced contractions of guinea-pig taenia coli which are dependent on inward movement of calcium, whereas the "phasic" component of the contractions is left unaltered. THC is a very weak inhibitor of 3'-5' cyclic adenosine monophosphate phosphodiesterase which is affected only at very high dosage (10(-2) M). The calcium antagonistic activity of THC may explain both its bronchodilating and antianaphylactic properties.

Several naturally occurring compounds have been identified in human cerebrospinal fluid (CSF) after subarachnoid haemorrhage (SAH) as possible vasoactive agents involved in the biochemical mechanism of vasospasm. The authors have measured, in 30 patients admitted for SAH, CSF concentrations of two arachidonic acid metabolites. Prostacyclin and Prostaglandin D2, as representative of vasodilator and vasoconstrictor compounds. CSF samples were made available by lumbar punctures and intraoperative cisternal punctures. Nine patients presented with symptomatic vasospasm: lumbar CSF Prostaglandin D2 levels are significantly higher than in patients without vasospasm. The Cisternal Prostaglandin D2 level is significantly higher than the lumbar CSF concentration; CSF Prostacyclin levels do not significantly differ in the two groups of patients. These data suggest the presence of an imbalanced biochemical situation responsible for promoting vasospasm. The evaluation of cisternal levels of arachidonate metabolites support the hypothesis of the clotting phenomenon around the ruptured aneurysm wall as an important predictive pattern of vasospasm onset after SAH, as shown in computed tomography.

The selectivity of procaterol for beta 2-adrenoceptors vs beta 1-adrenoceptors in animals has been already described. In these studies the ability of procaterol to protect guinea-pig against bronchoconstriction induced by various spasmogens such as histamine, LTC4 and acetylcholine is demonstrated. The results obtained both in anaesthetized animals and in perfused lungs clearly show that procaterol antagonizes not only the direct effect of the bronchoconstrictors quoted above but also their ability to activate arachidonic acid metabolism and to augment the generation of TXA2. Procaterol (0, 3-3 micrograms/Kg i.v.) is more potent than salbutamol in protecting passively sensitized guinea-pig from massive bronchoconstriction following antigen challenge: a phenomenon which is paralleled by an increment of the circulating TXA2. The possibility that the adenylate-cyclase-stimulating properties of procaterol may explain its antiasthmatic activity is discussed.

Ca++-ionophore and antigen challenge of human lung fragments caused a massive release of leukotrienes, whose relative proportions varied considerably and were dependent on the experimental conditions. A broncho-selective beta 2-adrenoceptor agonist fenoterol was able to prevent the antigen-induced leukotriene release completely, whereas the Ca++-ionophore induced leukotriene release was only moderately inhibited. The HPLC data were confirmed by SRS-A bioassay.

When chopped human lung is stimulated with the Ca++ ionophore A23187 (0.25-10 microM) leukotriene (LT) B4, LTD4 and LTE4 are found in the incubation medium according to different patterns. LTD4 is released promptly and its levels increase up to 45 min after the stimulus (A23187, 10 microM) and decline later (120 min); LTE4 formation follows a sigmoidal shape and continues to accumulate even 2 hr after the challenge; LTB4 levels reach a plateau at 45 min. LTC4 was undetectable in most experiments but it was found to accumulate when reduced glutathione (10 mM) was added. Addition of exogenous LTC4 to unstimulated fragments of human lung shows that an effective interconversion to LTD4 and LTE4 takes place: A23187 stimulates formation of LT and cyclo-oxygenase products dose dependently; a statistically significant formation of LT occurs at A23187 concentration of 1 microM whereas thromboxane B2 (TXB2) and 6-K-prostaglandin F1 alpha levels increased significantly only at 2.5 microM A23187. Pretreatment with U-60257 (100 microM) prevented formation of LT without a concomitant increase of TXB2 levels. Indomethacin (1.5 microM) blocked the release of 6-K-prostaglandin F1 alpha and TXB2 without a shift of arachidonic acid towards LT-like activity. Addition of exogenous LTC4 did not trigger synthesis of TXB2 or 6-K-prostaglandin F1 alpha. Our results indicate that reduced glutathione levels and the activity of the enzymes involved in LT biosynthesis and/or metabolism play an important role in controlling the pattern of LT release from human lung.