Gilberto De Nucci

Superoxide anions enhance platelet adhesion and aggregation

1 Superoxide dismutase (SOD, 60 u ml−1) or ferricytochrome c (70 μm) significantly inhibited thrombin‐stimulated platelet adhesion to gelatin‐coated plastic, whereas catalase (1000 u ml−1) or mannitol (1 mm) had no effect. 2 The platelet aggregation induced by low concentrations of thrombin (causing less than 45% maximal change in light transmission) was inhibited by SOD. Catalase or mannitol had no effect on platelet aggregation. 3 Pyrogallol (an O2− generator) enhanced both platelet adhesion to gelatin‐coated plastic and platelet aggregation induced by thrombin; this enhancement was neutralized by SOD. 4 These results indicate that O2− increase both platelet adhesion and aggregation, whereas other free radicals such as hydrogen peroxide or hydroxyl radicals are not involved.

Kinins act on B1 or B2 receptors to release conjointly endothelium‐derived relaxing factor and prostacyclin from bovine aortic endothelial cells

1 Bradykinin (Bk) induced the coupled release of endothelium‐derived relaxing factor (EDRF) and prostacyclin (PGI2) from bovine aortic endothelial cells grown in culture. The B2 kinin receptor antagonist, [d‐ArgO,Hyp3,Thi5,8,d‐Phe7]‐Bk, abolished this release by Bk. 2 Des‐Arg9‐Bk, a B1 kinin receptor agonist, also induced the release of EDRF and PGI2, but much higher concentrations were required to obtain a similar release to that induced by Bk. 3 [Leu8],des‐Arg9‐Bk, a B1 receptor antagonist, significantly reduced the response to des‐Arg9‐Bk without affecting the release induced by Bk. 4 The release of EDRF and PGI2 induced by arachidonic acid or ADP was not significantly affected by the B2 or the B1 antagonist. 5 We conclude, therefore, that Bk and des‐Arg9‐Bk were acting respectively on B2 and B1 bradykinin receptors. 6 The possible role of kinin receptors in the release of EDRF and PGI2 from endothelial cells is discussed.

Prostaglandin E2 regulates inducible nitric oxide synthase in the murine macrophage cell line J774

We have evaluated the role of prostaglandin E2 (PGE2) in the synthesis of nitric oxide (NO) by the activation of the inducible form of nitric oxide synthase (NOS) in the murine macrophage cell line, J774, stimulated with different doses of lipopolysaccharide (LPS). The stimulation of the J774 line with suboptimal doses of LPS (0.1 microgram/mL) caused a production of endogenous PGE2 that was capable of stimulating NOS activity inducing an increase in the NO synthesis, as attested by the fact that cyclooxygenase enzyme inhibitor, indomethacin, significantly reduced NO secretion. On the contrary, a higher dose of LPS (1 microgram/mL) produced high levels of PGE2 that reduced the levels of NOS and, subsequently, NO production. Experiments carried out with exogenous PGE2 indicated that concentrations between 1 and 10 ng/mL are able to stimulate the expression of NOS and the release of NO, while higher concentrations (> 50 ng/mL) are inhibitory. Furthermore, our data indicate that there is a network of interaction which involves NO, PGE2, and tumor necrosis factor. High levels of PGE2 inhibited TNF alpha secretion, which in turn could exert inhibitory effects on NO synthesis.

Acute inhibition of nitric oxide synthesis induces anxiolysis in the plus maze test

The involvement of nitric oxide (NO) in anxiety was investigated in rats, using the elevated plus maze test. Acute, but not chronic, systemic treatment with N omega-nitro-L-arginine methyl ester (L-NAME, 10 and 60 mg.kg-1), an inhibitor of NO synthase, increased the time spent by the rats in the open arms. Both the acute and chronic treatments with L-NAME inhibited NO synthase in endothelial cells and in the central nervous system, as shown by the increase in mean arterial pressure and decreased NO synthase activity in brain tissue. Chronic treatment with L-NAME also decreased the serum nitrate levels. The anxiolysis induced by acute L-NAME treatment is unlikely to be due to hypertension, since two-kidney one-clip hypertension in non-L-NAME-treated rats failed to significantly change exploratory behaviour in the elevated plus maze. These results indicate that acute inhibition of NO synthesis decreases anxiety in rats.

Mast cell degranulation induced by two phospholipase A2 homologues : Dissociation between enzymatic and biological activities

Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 microg/paw) and bothropstoxin-II (12.5-50 microg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 microg/site) and bothropstoxin-II (0.125-5 microg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 microg/ml) and bothropstoxin-II (3 and 10 microg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.

Ticlopidine quantification in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study

A rapid, sensitive and specific analytical method was developed and validated to quantify gabapentin in human plasma using acetaminophen as an internal standard. The method employs a single plasma protein precipitation. The analytes are chromatographed on a C4 reversed-phase chromatographic column and analyzed by mass spectrometry in the multiple reaction monitoring (MRM) mode. The method has a chromatographic run time of 4 min and a linear calibration curve over the range 50-10 000 ng x ml(-1) (r > 0.999). The between-run precision, based on the relative standard deviation for replicate quality controls, was < or = 4.8 % (200 ng x ml(-1)), 6.0% (1000 ng x ml(-1)) and 4.4% (5000 ng x ml(-1)). The between-run accuracy was +/-2.6, 4.4 and 0.5% for the above-mentioned concentrations, respectively. This method was employed in a bioequivalence study of two gabentin capsule formulations (Progresse from Biosintética, Brazil, as a test formulation, and Neurotin from Parke-Davis, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 300 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval. The 90% confidence interval (CI) of the individual ratio geometric mean for Progresse/Neurotin was 87.9-115.6% for AUC(0-36 h) and 88.6-111.7% for Cmax. Since both 90% CI for AUC(0-36 h) and Cmax were included in the 80-125% interval proposed by the US Food and Drug Administration, Progresse was considered bioequivalent to Neurotin according to both the rate and extent of absorption.

Effect of Salt Intake and Inhibitor Dose on Arterial Hypertension and Renal Injury Induced by Chronic Nitric Oxide Blockade

Long-term nitric oxide blockade by N omega -nitro-L-arginine methyl ester (L-NAME) leads to severe and progressive hypertension. The role of salt intake in this model is unclear. To verify whether salt dependence in this model is related to the extent of nitric oxide inhibition, we gave adult male Munich-Wistar rats a low salt, standard salt, or high salt diet and oral L-NAME treatment at either 3 or 25 mg/kg per day. At 10 to 15 days of treatment, the slope of the pressure-natriuresis line was decreased in rats receiving low-dose L-NAME compared with untreated controls. In rats treated with the higher dose, the line was shifted to the right but remained parallel to that obtained in untreated controls. Renal vascular resistance was moderately increased in rats receiving low-dose L-NAME, whereas high-dose L-NAME induced a marked vasoconstriction that was aggravated by salt overload. Low-dose L-NAME treatment induced hypertension only when associated with sodium overload. In rats receiving high-dose L-NAME, hypertension was aggravated by sodium excess but was not ameliorated by sodium restriction. Long-term (6 weeks) L-NAME treatment was associated with progressive hypertension, which was aggravated by salt overload, and with the development of albuminuria, focal glomerular collapse, glomerulosclerosis, and renal interstitial expansion. These abnormalities were worsened by salt overload and largely prevented by salt restriction. In the model of chronic nitric oxide blockade, salt dependence is a function of the inhibitor dose, and renal injury varies directly with the level of salt intake.

Inhibition of carrageenin‐induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2

1 The effect of purified crotapotin, a non‐toxic non‐enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 μg/paw) and 5‐hydroxytryptamine (5‐HT) (3 μg/paw) in the rat hind‐paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea‐pig isolated lung were also investigated. 2 Subplantar co‐injection of crotapotin (1 and 10 μg/paw) with carrageenin or injection of crotapotin (10 μg/paw) into the contralateral paw significantly inhibited the carrageenin‐induced oedema. This inhibition was also observed when crotapotin (10–30 μg/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60°C) failed to inhibit carrageenin‐induced oedema. Subplantar injection of crotapotin (10 μg/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5‐HT‐induced oedema. 3 In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5‐HT stores. 4 Crotapotin (30 μg/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 mm) and platelet activating factor (1 μm) in human platelet‐rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml−1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 μg/paw) did not affect the release of 6‐oxo‐prostaglandin F1α and TXB2 induced by ovalbumin in sensitized guinea‐pig isolated lungs. 5 Our results indicate that the anti‐inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo‐oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.

The release of nitric oxide and superoxide anion by neutrophils and mononuclear cells from patients with sickle cell anaemia

The aim of this work was to investigate the release of nitric oxide and superoxide by neutrophils and mononuclear cells from patients with sickle cell anaemia. Nitric oxide release was assayed by the ability of leucocytes to inhibit thrombin‐induced washed platelet aggregation. Superoxide release was assessed by a cytochrome c reduction assay. Neutrophils from sickle cell anaemia patients released nitric oxide in a similar manner to those from healthy controls, because inhibition of platelet aggregation by neutrophils from sickle cell anaemia or from healthy controls was blocked by the inhibitor of nitric oxide synthesis N ω‐nitro‐l‐arginine methyl ester (300 μm), but not by N ω‐nitro‐d‐arginine methyl ester (300 μm) and was reversed by l‐arginine (1 mm). Additionally, a similar number of neutrophils from sickle cell anaemia patients and from healthy controls was required to inhibit platelet aggregation. Mononuclear cells from sickle cell anaemia patients inhibited platelet aggregation only in the presence of superoxide dismutase (60 U ml−1). Phorbol 12‐myristate 13‐acetate (PMA, 30 nm)‐ or zymosan (100 particles/cell)‐induced release of superoxide by mononuclear cells from sickle cell anaemia patients was significantly higher than that observed in mononuclear cells from healthy controls (P < 0.001 and P < 0.01 respectively, Mann‐Whitney test). The levels of superoxide released by neutrophils from sickle cell anaemia patients were similar to those from healthy controls. We conclude that mononuclear cells from sickle cell anaemia patients release more superoxide than those from healthy controls, when stimulated with PMA or zymosan in vitro. Considering that superoxide inactivates nitric oxide, that nitric oxide is an important endogenous vasodilator, and that superoxide produces oxidant damage, this greater production of superoxide by mononuclear cells from sickle cell anaemia patients may represent an additional risk factor for the obstruction of the microcirculation and tissue damage in these patients.

Simultaneous determination of nitrite and nitrate anions in plasma, urine and cell culture supernatants by high-performance liquid chromatography with post-column reactions.

A high-performance liquid chromatographic method for the determination of nitrite and nitrate anions derived from nitric oxide in biological fluids is presented. After separation on a strong anion-exchange column (Spherisorb SAX, 250 x 4.6 mm I.D., 5 microns), two on-line post-column reactions occur. The first involves nitrate reduction to nitrite on a copper-plated cadmium-filled column. In the second, the diazotization-coupling reaction between nitrite and the Griess reagent (0.05% naphtylethylendiamine dihydrochloride plus 0.5% sulphanilamide in 5% phosphoric acid) takes place, and the absorbance of the chromophore is read at 540 nm. This methodology was applied to biological fluids. Before injection into the chromatographic system, the samples were diluted and submitted to suitable clean-up procedures (urine and cell culture supernatant samples are passed through C18 cartridges, and serum samples were deproteinized by ultrafiltration through membranes with a molecular mass cut-off of 3000). The method has a sensitivity of 30 pmol for both anions, as little as 0.05-0.1 ml sample volume is required and linearity is observed up to 60 nmol for each anion.