Influence of interleukin 1alpha on superoxide anion, platelet activating factor release and phospholipase A2 activity of naive and sensitized guinea-pig alveolar macrophages.

1. We studied the effect exerted by hr-interleukin-1alpha (IL-1alpha) on responsiveness of alveolar macrophages (AM) from naive and sensitized guinea-pigs, through O2.- production (by ferricytochrome C reduction), platelet-activating factor (PAF) release (by platelet aggregation), prostaglandin E2 (PGE2) release (by a radioimmunoassay), and cytosolic phospholipase A2 (cPLA2) activity (by hydrolysis of radioactive substrate). 2. In naive guinea-pig AM, 0.06 nM hr-IL-1alpha pretreatment decreased by 65% O2.- release stimulated with 10 nM fMLP. In contrast, O2.- production was not affected in sensitized guinea-pig AM. 3. O2.- release elicited by fMLP stimulation in both cell groups was affected by PLA2 inhibitors (10 microM bromophenacyl bromide, BPB or 10 microM methylprednisolone, MP). In contrast, 10 microM arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA2 inhibitor, was ineffective. 4. In naive AM, PAF release was elicited by hr-IL-1alpha pretreatment and by separate fMLP-stimulation, but when the stimulus was added to hr-IL-1alpha-pretreated cells inhibition of PAF release was observed. In sensitized AM, PAF release was lower than that found in naive guinea-pig AM in both hr-IL-1alpha-pretreated and fMLP-stimulated cells. 5. PGE2 release was unaffected by hr-IL-1alpha pretreatment and it was decreased by fMLP in both naive and sensitized AMs. The latter released less PGE2 than naive cells in basal conditions and after fMLP treatment. 6. Sensitized AM showed a greater cPLA2 activity in all experimental conditions in comparison to naive cells. cPLA2 activity assayed in the cytosolic fraction was found to be enhanced by hr-IL-1alpha pretreatment and by fMLP stimulation in naive but not in sensitized AM. However, when the stimulus was added to hr-IL-1alpha-pretreated cells we observed a decrease in cPLA2 activity in the cytosol and an increase in the membranes, thus suggesting a translocation of enzymatic activity. 7. In conclusion, hr-IL-1alpha can modulate the responsiveness of AM from naive and sensitized guinea-pigs, as suggested by changes found in the release of PAF and O2.- and in cPLA2 activity; therefore, sensitization itself may affect cellular responsiveness.

Mechanical stretch reveals different components of endothelial-mediated vascular tone in rat aortic strips.

1. Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone. 2. Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium-preserved strips were precontracted with either phenylephrine or prostaglandin F(2 alpha) (PGF(2 alpha)). 3. In control conditions, acetylcholine (ACh) dose-dependently relaxed phenylephrine-precontracted strips independently of resting tension. 4. At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh-induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose-response curve to the right. 5. After preincubation with indomethacin (5 microM) or ibuprofen (10 and 100 microM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium-dependent, dose-dependent contraction. ACh (10(-6) M) increased the contraction up to two times greater the phenylephrine-induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF(2 alpha). 6. Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX-derived metabolites, can relax precontracted strips. COX inhibition up-regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase-dependent, ACh-induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh-induced relaxation marginally.

Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.

1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

Effect of 21-aminosteroid U74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bis(diethylamino)-2-pyridinyl)-1-piperazinyl)-16-methyl-, HCl (16 alpha)) on rat brain cortex lipid peroxidation induced "in vivo" by iron-carbohydrate.

Compounds derived from glucocorticoids, 21-aminosteroids, were reported to inhibit in vitro lipid peroxidation in CNS tissue. In order to evaluate the possible scavenging and/or iron chelating activities in vivo of the 21-aminosteroid U74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bisdiethylamino)-2-pyridinyl)-1-piperazinyl)-16-methyl- , HCl (16 alpha)), the drug was administered for seven days to rats. These rats had been induced by iron-saccharate complex injection a slow process of lipid peroxidation into their right brain hemicortex. The drug was injected also to intact rats (normal rats). Seven days after the operation the extent of iron-induced lipid peroxidation in both the hemicortices and the effect of the drug, were assessed by the evaluation of lipid-soluble fluorescence and of conjugated diene formation. The assessment was performed both in vehicle (control) and in U74500A-treated rats. In the iron-injected rat groups the drug induced a significant dose-related reduction of fluorescence values. Formation of conjugated dienes showed a significant decrease when U74500A (48 mg/kg every 48 hr) was administered to cortico-cerebrally iron-injected animals. The lipid peroxidation of cortices in normal rats was evaluated as thiobarbituric acid reactant substances in both the drug-treated and the control animals. In normal rats, U74500A (48 mg/kg every 48 hr) caused a significant decrease of TBARS values, as compared to those observed in the control group. The iron content in the iron-injected hemicortices, which was evaluated by the ferrozine method, was not modified by drug treatment. U74500A appears to have in vivo antioxidant properties and not to affect the iron content in the neural tissue. An interaction of this drug with the metal, however, cannot be excluded.

Hemoglobin affects lipid peroxidation and prostaglandin E2 formation in rat corticocerebral tissues in vitro.

Variations of lipid peroxidation and arachidonic acid (AA) metabolism products were found when experimental subarachnoid hemorrhage or ischemia and reperfusion were performed in an animal brain model. In a previous study, we showed that hemoglobin (Hb) produces prostaglandins when incubated in AA. To elucidate how Hb affects lipid peroxidation and AA metabolism in the CNS, we measured lipid hydroperoxides (LOOH), PGE2 and thiobarbituric acid reactant substances (TBARS) in corticocerebral homogenates and slices of rats (normal rats) after incubation with different concentrations (10(-9) to 10(-5) M) of Hb. In addition, brain cortices of indomethacin-treated (40 mg/Kg) rats (IN-treated rat) were incubated in the presence of 10(-5) M indomethacin (IN) to exclude the interference of prostaglandin enzyme synthetase. Hb was able to affect LOOH, PGE2, and TBARS production in both normal and IN-treated rat brain cortex homogenates and slices. In all cases, we found an increase in prostaglandin when 10(-8) M Hb was used, whereas no effect was noticed with 10(-9) M. On the other hand, with higher Hb concentrations (10(-6)-10(-5) M), the LOOH and PGE2 values did not reach statistical significance, and TBARS significantly increased. In all cases, when 10(-4) M scavenger or metal-chelating compounds were added to an incubation mixture with 10(-8) M Hb, PGE2 formation was inhibited, whereas no variation occurred when 10(-4) M IN was further added to IN-treated rat corticocerebral homogenate or slices. We hypothesize that in in vivo experimental neuropathologies, Hb must attain the 10(-8) M concentration in the reaction cellular microenvironment to stimulate PGE2 production, and that an evaluable part of this PGE2 production may be directly ascribable to the iron-heme oxy-redoxy activity of Hb.

Peroxidase catalysed formation of prostaglandins from arachidonic acid.

Horseradish peroxidase and bovine lactoperoxidase (EC 1.11.1.7), when incubated aerobically with arachidonate, gave rise to the formation of substances identified by bioassay as prostaglandin F2 alpha (PGF2 alpha)- and prostaglandin E2 (PGE2)-like compounds. Boiling of enzymes, which suppressed their capacity to peroxidize guaiacol, also destroyed their capacity to convert arachidonate into PG-like compounds. The rates of formation of PG-like compounds rapidly declined with time, approaching zero after 10 and 20 min for PGE2 alpha- and PGE2-like compounds, respectively. Addition of more enzyme further promoted the reaction. Horseradish and lacto-peroxidases showed optimum pH values of 9.0 and 10.0, respectively. Both enzymes exhibited apparent Km values of about 5 x 10(-5) M for arachidonate. Some reducing agents such as ascorbic acid, NADH and adrenaline dose-dependently inhibited this reaction. The haem poison, phenylhydrazine, also inhibited, with an IC50 of 1 x 10(-7) M. Indomethacin inhibited only the formation of PGE2-like compounds with an IC50 of about 3 x 10(-6) M. As compared to a standard commercial preparation of horseradish peroxidase, the purified horseradish basic and acidic isoenzymes exhibited a higher activity, towards arachidonate whereas other haemoproteins, possessing peroxidase activity, were less active. TLC and GC-MS analyses performed on the reaction products led to the identification of PGF2 alpha, PGE2 and PG6K1 alpha and other unidentified arachidonate derivatives. At 25 degrees, pH 9.5, horseradish peroxidase, acting on saturating concentration of arachidonate, catalysed the formation of 60 mumol/min/mmole enzyme of PGE2 + PGF2 alpha. This appears to be the first report of the synthesis of prostaglandins catalysed by peroxidases.

Histamine release from rat mast cells induced by metabolic activation of polyunsaturated fatty acids into free radicals.

Polyunsaturated fatty acids (PUFA: arachidonic and linoleic acid) release histamine from isolated purified rat serosal mast cells only in the presence of oxidizing systems such as phenobarbital-induced rat liver microsomes, prostaglandin-H-synthetase (PHS) or soybean lipoxygenase. The release of mast cell histamine by activated PUFA has a long time-course and the electron microscopical features are consistent with an exocytotic secretion in the case of arachidonic acid and cell lysis in the case of linoleic acid. The phenomenon is associated with a significant increase in malonyldialdehyde (MDA) and conjugated diene generation, suggesting a relationship between histamine release and membrane lipid peroxidation. The secretion of histamine was inhibited by anti-free radical interventions such as D-mannitol, reduced glutathione and alpha-tocopherol. Some cyclooxygenase and lipoxygenase inhibitors, cimetidine and carnitine derivatives, are differentially active in the inhibition of mast cell histamine release by activated arachidonic acid. These results suggest that free radical derivatives of PUFA, generated by metabolic activation, trigger mast cell histamine release.

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.

Ovalbumin sensitization of guinea-pigs reduces fMLP-induced calcium signal in alveolar macrophages.

In this study we analyzed the N-formyl-Met-Leu-Phe (fMLP)-induced calcium signal in alveolar macrophages (AM) isolated from ovalbumin-sensitized (OA-sensitized AM) and naive (naive AM) guinea-pigs. Guinea-pigs were sensitized by subcutaneous injection of OA and AM were isolated by bronchoalveolar lavage 6 weeks thereafter. On the following day, we measured in resting and fMLP-stimulated cells: intracellular calcium concentration by fura-2 imaging analysis, forskolin-induced cyclic AMP production and superoxide dismutase inhibitable superoxide anion release of adherent AM. Resting calcium was 82+/-5.0 nM (n=217) and 144+/-9.3 nM (n=213, P<0.001) in naive and OA-sensitized AM respectively. fMLP (10(-11)-10(-7)M) induced a dose-dependent calcium increase, 10(-8)M being the maximal effective dose in both naive and OA-sensitized AM. However, at all doses tested, this fMLP effect was lower in OA-sensitized than in naive AM. While in resting condition 10(-5)M forskolin increased cyclic AMP both in naive and OA-sensitized AM, in fMPL-stimulated AM forskolin was effective only in OA-sensitized AM. Superoxide anion release measured 10 min after fMLP stimulus was higher in naive than in sensitized AM. These data suggest that the fMLP-induced intracellular signal is different in OA-sensitized AM compared to naive cells.

The polysaccharide from Tamarindus indica (TS-polysaccharide) protects cultured corneal-derived cells (SIRC cells) from ultraviolet rays.

The aim of this work was to investigate the possible protective effect of a new viscosising agent, TS-polysaccharide, on corneal-derived cells (SIRC) exposed to ultraviolet-B rays. To verify this, SIRC cells were first exposed, in the absence or in the presence of TS-polysaccharide (1% w/v), for 9 s at the UV-B source and then post-incubated for 45 min at 37 degrees C. After this period the hydrogen peroxide (H(2)O(2)) accumulated in the medium and the concentration of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) in cell DNA was measured. In addition, the amount of (3)H-methyl-thymidine incorporated in cellular DNA was evaluated after 18 h from irradiation. Our results show that cells exposed to UV-B rays accumulate H(2)O(2), and have higher levels of 8OHdG and a lower amount of (3)H-methyl-thymidine incorporated in DNA than control cells. In the presence of TS-polysaccharide, the H(2)O(2) and 8-OHdG accumulation, and the (3)H-methyl-thymidine incorporation were significantly reduced with respect to the values measured in cells exposed in the absence of the polysaccharide. We propose a protective role of the polysaccharide in reducing UV-B derived DNA damage to eye cells. This finding could be of some clinical importance when the polysaccharide is used as a delivery system for ophthalmic preparations.

Pharmacokinetics of oxatomide in preterm infants.

The pharmacokinetics and tolerability of oxatomide oral suspension were investigated in preterm infants to evaluate the feasibility of planning a further study to assess its antiinflammatory effects and its effectiveness in preventing chronic lung disease (CLD). Following the administration of oxatomide 1 mg/kg, the peak plasma concentration (Cmax), the elimination half-life (t1/2), the volume of distribution (Vd), and the area under the curve (AUC) 0-36 h were measured and the following results were obtained: 42.2 +/- 15 ng/ml at 2 h after oxatomide administration, 41.4 +/- 2.0 h, 37.4 +/- 4.2 l/kg, and 468 +/- 52 ng/ml/h, respectively. Our study, therefore, demonstrated that a dose of 1 mg/kg/day oxatomide was effective in reaching therapeutic plasma levels in preterm infants without inducing adverse effects.

Estradiol and progestin receptors, 17-beta-hydroxysteroid-dehydrogenase and histopathologic grade in endometrial carcinoma.

The possible correlation between steroid receptor systems, 17-beta-HSD and histopathologic examinations were investigated. The well-differentiated tumors showed higher steroid receptor and 17-beta-HSD values than undifferentiated carcinomas. The steroid receptors did not present a statistically significant correlation with 17-beta-HSD. Nevertheless, some neoplastic endometria (29%) show higher values of progestin receptors and 17-beta-HSD, with a progestin:estrogen receptor ratio greater than one.

[Endoscopic insertion of a prosthesis in the palliative treatment of tumors of the head of the pancreas]. / L'apposizione di protesi per via endoscopica nel trattamento palliativo dei tumori della testa del pancreas.

The Authors report their experience of biliary endoscopic endoprosthesis placement in 42 patients for palliative treatment of carcinoma of the pancreatic head. The morbidity, the mortality and the duration of hospitalization are considerably less than with surgical biliodigestive anastomosis. The mean survival is almost the same in both cases.

[Low-malignancy duodenal leiomyosarcoma]. / Il leiomiosarcoma duodenale a bassa malignità.

The authors report a case of duodenal leiomyosarcoma presenting a low degree of histological malignancy. The main anatomoclinical features of the neoplasm are summarized. The authors conclude indicating not aggressive surgical therapy as adequate in such cases. However, a prolonged post-operative follow-up is always necessary, in order to detect recurrences as early as possible.