Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D(2)) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for `in-situ` hybridisation study using (35)S-labeled oligonucleotide probe complementary to D(2) receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D(2) receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D(2) receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Both human immunodeficiency virus-infected and human immunodeficiency virus-exposed, uninfected children living in Brazil, Argentina, and Mexico have similar rates of low concentrations of retinol, beta-carotene, and vitamin E

Our objective was to describe the prevalence of low concentrations of retinol, beta-carotene, and vitamin E in a group of human immunodeficiency virus (HIV)-infected Latin American children and a comparison group of HIV-exposed, uninfected children. Our hypothesis was that the rates of low concentrations of these micronutrients would be higher in the HIV-infected group than those in the HIV-exposed, uninfected group. This was a cross-sectional substudy of a larger cohort study at clinical pediatric HIV centers in Latin America. Serum levels of micronutrients were measured in the first stored sample obtained after each child`s first birthday by high-performance liquid chromatography. Low concentrations of retinol, beta-carotene, and vitamin E were defined as serum levels below 0.70, 0.35, and 18.0 mu mol/L, respectively. The Population for this analysis was 336 children (124 HIV-infected, 212 HIV-exposed, uninfected) aged I year or older to younger than 4 years. Rates of low concentrations were 74% for retinol, 27% for beta-carotene, and 89% for vitamin E. These rates were not affected by HIV status. Among the HIV-infected children, those treated with anti retrovirals were less likely to have retinol deficiency, but no other HIV-related factors correlated with micronutrient low serum levels. Low concentrations of retinol, beta-carotene, and vitamin E are very common in children exposed to HIV living in Brazil, Argentina, and Mexico, regardless of HIV-infection status. Published by Elsevier Inc.

The effect of ovariectomy on cardiac autonomic control in rats submitted to aerobic physical training

We have investigated the ovariectomy effects on the cardiovascular autonomic adaptations induced by aerobic physical training and the role played by nitric oxide (NO). Female Wistar rats (n =70) were divided into five groups: Sedentary Sham (SS): Trained Sham (TS); Trained Hypertensive Sham treated with N(C)-nitro-L-arginine methyl ester (L-NAME) (THS): Trained Ovariectomized (TO); and Trained Hypertensive Ovariectomized treated with L-NAME (THO). Trained groups were submitted to a physical training during 10 weeks. The cardiovascular autonomic control was investigated in all groups using different approaches: 1) pharmacological evaluation of autonomic tonus with methylatropine and propranolol; 2) analysis of heart rate (HR) and systolic arterial pressure (AP) variability; 3) spontaneous baroreflex sensitivity (BRS) evaluation. Hypertension was observed in THS and THO groups. Pharmacological analysis showed that TS group had increased predominance of autonomic vagal tonus compared to SS group. HR and intrinsic HR were found to be reduced in all trained animals. TS group, compared to other groups, showed a reduction in LF oscillations (LF=0.2-0.75 Hz) of pulse interval in both absolute and normalized units as well as an increase in HF oscillations (HF=0.75-2.50 Hz) in normalized unit. FIRS analysis showed that alpha-index was different between all groups. TS group presented the greatest value, followed by the TO, SS. THO and THS groups. Ovariectomy has negative effects on cardiac autonomic modulation in trained rats, which is characterized by an increase in the sympathetic autonomic modulation. These negative effects suggest NO deficiency. In contrast, the ovariectomy seems to have no effect on AP variability. (C) 2008 Elsevier B.V. All rights reserved.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1 beta, MIP-2, and LTB(4)

Objective. The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design. MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1 beta (IL-1 beta) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1 beta and MIP-2 exudates was measured by ELISA. Results. MTA induced dose-and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1 beta antibodies. In the exudates, IL-1 beta and MIP-2 were detected. Conclusions. This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1 beta, MIP-2, and LTB(4).

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.

Hemodynamic effects of inducible nitric oxide synthase inhibition combined with sildenafil during acute pulmonary embolism

While endogenous nitric oxide (NO) may be relevant to the beneficial hemodynamic effects produced by sildenafil during acute pulmonary embolism (APE), huge amounts of inducible NO synthase (iNOS)derived NO may contribute to lung injury. We hypothesized that iNOS inhibition with S-methylisothiourea could attenuate APE-induced increases in oxidative stress and pulmonary hypertension and, therefore, could improve the beneficial hemodynamic and antioxidant effects produced by sildenafil during APE. Hemodynamic evaluations were performed in non-embolized dogs treated with saline (n = 4), S-methylisothiourea (0.01 mg/kg followed by 0.5 mg/kg/h, n = 4), sildenafil (0.3 mg/kg, n = 4), or S-methylisothiourea followed by sildenafil (n = 4), and in dogs that received the same drugs and were embolized with silicon microspheres (n = 8 for each group). Plasma nitrite/nitrate (NOx) and thiobarbituric acid reactive substances (TBARS) concentrations were determined by Griess and a fluorometric assay, respectively. APE increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 25 +/- 1.7 mm Hg and by 941 +/- 34 dyn s cm(-5) m(-2), respectively. S-methylisothiourea neither attenuated APE-induced pulmonary hypertension, nor enhanced the beneficial hemodynamic effects produced by sildenafil after APE (>50% reduction in pulmonary vascular resistance). While sildenafil produced no change in plasma NOx concentrations, S-methylisothiourea alone or combined with sildenafil blunted APE-induced increases in NOx concentrations. Both drugs, either alone or combined, produced antioxidant effects. In conclusion, although iNOS-derived NO may play a key role in APE-induced oxidative stress, our results suggest that the iNOS inhibitor S-methylisothiourea neither attenuates APE-induced pulmonary hypertension, nor enhances the beneficial hemodynamic effects produced by sildenafil. (C) 2010 Elsevier Inc. All rights reserved.

Comparison of MB fraction of creatine kinase mass and troponin I serum levels with necropsy findings in acute myocardial infarction

Serum levels of troponin and heart-related fraction of creatine kinase (CK-MB) mass are used as diagnostic and prognostic criteria in myocardial infarction, but the relation between those levels and-the necropsy-determined size of necrosis has not been tested in human beings. In this retrospective study, 1-cm-thick transverse sections of the ventricles were cut from the base to the apex in the necropsy hearts of 27 patients aged 47 to 86 years (mean 66, median 69; 19 men). Total and necrotic areas were measured using a computer-linked image analysis system. The weights of the necrotic areas were also calculated. The correlations of the areas and weights of necrotic myocardium with the highest serum values of CK-MB mass and troponin 1, which had been quantified during life by chemiluminescence immunoassays, were verified by Pearson`s test; results were considered significant at p <= 50.05. Significant correlations were detected between CK-MB mass peak and infarct size (r = 0.63, p < 0.01) and weight (r = 0.69, p < 0.01) and between CK-MB mass and highest troponin level (r = 0.73, p < 0.01); however, the correlations between highest troponin level and myocardial infarct size (r = 0.31, p = 0.11) and weight (r = 0.35, p = 0.07) were small and nonsignificant. In conclusion, despite the well-established role of serum levels of troponin as a diagnostic tool for myocardial infarction, their highest values showed poor correlations with the extent of infarct. In contrast, the highest serum level of CK-MB mass was well correlated with myocardial infarct size. (c) 2008 Elsevier Inc. All rights reserved.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO2 in air) for I It in the following treatments either 500 mu L of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for I h either FM or FM with a L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) a L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) a L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 X 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha. L-PGDS: (1) 26.4 +/- 3.0; (2) 25.6 +/- 3.0; (3) 59.7 +/- 3.0; (4) 56.4 +/- 3.0; and (5) 57.1 +/- 3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 +/- 2.0%; (2) 87.5 +/- 2.0%; (3) 19.4 +/- 2.0%; (4) 27.2 +/- 3.1%; and (5) 14.1 +/- 3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization. (c) 2007 Elsevier B.V. All rights reserved.

Developing human minor salivary glands: morphological parallel relation between the expression of TGF-beta isoforms and cytoskeletal markers of glandular maturation

Morphogenesis of salivary glands involves complex coordinated events. Synchronisation between cell proliferation, polarisation and differentiation, which are dependent on epithelial-mesenchymal interactions and on the microenvironment, is a requirement. Growth factors mediate many of these orchestrated biological processes and transforming growth factor-beta (TGF-beta) appear to be relevant. Using immunohistochemistry and immunofluorescence, we have mapped the distribution of TGF-beta 1, 2 and 3 and compared it with the expression of maturation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. TGF-beta 1 first appeared during canalisation stage in the surrounding mesenchyme and, in the more differentiated stages, was expressed in the cytoplasm of acinar cells throughout the adult gland. TGF-beta 2 was detected since the bud stage of the salivary gland. Its expression was observed in ductal cells and increased along gland differentiation, TGF-beta 3 was detected from the canalisation stage of the salivary gland, being weakly expressed on ductal cells, and it was the only factor detected on myoepithelial cells. The data suggest that TGF-beta have a role to play in salivary gland development and differentiation.

Immunohistochemical approach reveals involvement of inducible nitric oxide synthase in rat late development

To better understand the role of nitric oxide (NO) in mammal development, specifically in the transition of the fetal stages at birth, we studied the timing of cell-specific expression of inducible NO synthase (iNOS) isoform during gestational periods of rats, mainly at the late stages of intra-uterine development. Before experimentation, the samples were collected (from 17th to 21st gestational days), fixed in 10% buffered formalin and embedded in paraffin for histological procedures. Hereafter, the sections (5 mu m thickness) obtained from different embryos were immunostained by avidin-biotin-immunoperoxidase technique, by using antibody against iNOS isoform. The most of cell immunopositive was suggestive of granulocyte-like cells and those cells were resident close to the blood vessels in different organs, such as: lung, liver or bone marrow environment. Sometimes we noted immunopositive cells in the blood flow, as reported in the thymus. In agreement, iNOS expression, obtained by western blotting analysis, showed the same profile. Together, our data shows that iNOS expression increased gradually during the late stages of rat development (from E17 to E21) and it was executed by cells close to blood vessels. Thus, we can clearly to predict that this expression was finely modulated and it contributes for time-line dependent NO production during rat late development.

Detection of cytokines and nitric oxide synthase in skin lesions of Jorge Lobo`s disease patients

Studies investigating the immunopathological aspects of Jorge Lobo`s disease have shown that the inflammatory infiltrate consists mainly of histiocytes and multinucleated giant cells involving numerous yeast-like cells of Lacazia loboi, with the T lymphocytes more common than B lymphocytes and plasma cells. The quantification of cytokines in peripheral blood mononuclear cells culture supernatant has revealed alterations in the cytokines profile, characterized by predominance of a Th2 profile. In view of these findings and of the role of cytokines in cell interactions, the objective of the present study was to investigate the presence of the cytokines IL-10, TGF-ss 1 and TNF-alpha, as well as iNOS enzyme in granulomas induced by L. loboi. Histological sections obtained from skin lesions of 16 patients were analyzed by immunohistochemistry for the presence of these cytokines and iNOS. The results showed that TGF-ss 1 was the cytokine most frequently expressed by cells present in the inflammatory infiltrate, followed by IL-10. There was a minimum to discrete positivity of cells expressing TNF-alpha and iNOS. The results suggest that the presence of immunosuppressive cytokines in skin lesions of patients with the mycosis might be responsible for the lack of containment of the pathogen as demonstrated by the presence of numerous fungi in the granuloma.

Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells

Background: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. Methods: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. Results: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. Conclusions: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

Nitric oxide synthase inhibitors improve prepulse inhibition responses of Wistar rats

Introduction: Cognitive and attentional deficits in schizophrenia include impairment of the sensorimotor filter as measured by prepulse inhibition (PPI). In this way, the study of animals that naturally present low PPI responses could be a useful approach for screening new antipsychotic drugs. Several pieces of evidence suggest that dopamine and nitric oxide (NO) can modulate PPI but their role in those animals is unknown. Objectives: The aim of this study was to investigate the role of dopamine and NO in Wistar rats with naturally low PPI response. Methods: Male Wistar rats with low PPI responses received an i.p. injection of the antipsychotics haloperidol (0.1, 0.3 or 1 mg/kg) or clozapine (0.5, 1.5 or 5 mg/kg), the anxiolytic diazepam (1 or 3 mg/kg) or the NO synthase (NOS) inhibitors, N(G)- nitro-L-arginine (L-NOARG; 40 mg/kg, acutely or sub-chronically) or 7-Nitroindazole (7-NI; 3, 10 or 30 mg/kg). All animals were submitted to the PPI test 1 h after injection. Striatal and cortical dopamine, DOPAC, and noradrenaline levels of rats with low PPI responses were compared to rats with normal PPI responses. Results: We found increased levels of catecholamines on the striatum and prefrontal cortex of Wistar rats with low PPI. In these animals, both antipsychotics, typical and atypical, and NOS inhibitors significantly increased PPI. Conclusion: Taken together, our findings suggest that the low PPI phenotype may be driven by an over-active catecholamine system. Additionally, our results corroborate the hypothesis of dopamine and NO interaction on PPI modulation and suggest that Wistar rats with low PPI may represent an interesting non-pharmacological model to evaluate new potential antipsychotics. (C) 2010 Elsevier B.V. All rights reserved.

Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (NG-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

Twisted sl(3, C)(k)((2)) current algebra: free field representation and screening currents

Motivated by application of twisted current algebra in description of the entropy of Ads(3) black hole, we investigate the simplest twisted current algebra sl(3, c)(k)((2)). Free field representation of the twisted algebra, and the corresponding twisted Sugawara energy-momentum tensor are obtained by using three (beta, gamma) pairs and two scalar fields. Primary fields and two screening currents of the first kind are presented. (C) 2001 Published by Elsevier Science B.V.