Effects of angiotensin (5-8) microinfusions into the ventrolateral periaqueductal gray on defensive behaviors in rats.

Peptides of the renin-angiotensin system modulate blood pressure and hydro-electrolyte composition. Angiotensin (Ang) receptors are localized in brain areas related to the regulation of autonomic and endocrine control and involved in sensory perception, memory process and behavioral responses. Among these areas, the ventrolateral periaqueductal gray (vlPAG) is one of the most important structures of the neuronal circuitry controlling the autonomic and behavioral components of emotional states. Although Ang II metabolism in the vlPAG forms several Ang-peptides including Ang (5-8), the role of this tetrapeptide in the organization of defensive responses has not yet been described. To address this issue, the purpose of the present study was to determine the effects of intra-vlPAG injections of Ang (5-8) (0.2, 0.4 and 0.8 nmol/0.25 µL) in rats submitted to the elevated plus-maze (EPM) test. Additionally, it was evaluated the effects of intra-vlPAG Ang (5-8) on the expression of conditioned fear, assessed by the fear-potentiated startle and contextual conditioned freezing tests. The results showed that Ang (5-8) produced an intense, dose-related reduction in the entries into and time spent in the open arms of the EPM, decreased direct exploration and increased risk assessment behaviors. Moreover, intra-vlPAG injections of Ang (5-8) before the test session promoted pro-aversive effects in the FPS and enhanced contextual freezing. Taken together, these results point out to an important anxiogenic-like action for Ang (5-8) in the mediation of defensive behaviors organized in the vlPAG.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells.

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900 W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.

Immunolocalization of nitric oxide synthase isoforms in human archivaland rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50mM Tris–HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10mMsodium citrate buffer,pH6.0, for 15 min at 900 W.Inflammatory cells in ahumanlung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli.Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.

Ultrasonic tissue characterization of vulnerable carotid plaque: correlation between videodensitometric method and histological examination.

BACKGROUND: To establish the correlation between quantitative analysis based on B-mode ultrasound images of vulnerable carotid plaque and histological examination of the surgically removed plaque, on the basis of a videodensitometric digital texture characterization. METHODS: Twenty-five patients (18 males, mean age 67 +/- 6.9 years) admitted for carotid endarterectomy for extracranial high-grade internal carotid artery stenosis (> or = 70% luminal narrowing) underwent to quantitative ultrasonic tissue characterization of carotid plaque before surgery. A computer software (Carotid Plaque Analysis Software) was developed to perform the videodensitometric analysis. The patients were divided into 2 groups according to symptomatology (group I, 15 symptomatic patients; and group II, 10 patients asymptomatic). Tissue specimens were analysed for lipid, fibromuscular tissue and calcium. RESULTS: The first order statistic parameter mean gray level was able to distinguish the groups I and II (p = 0.04). The second order parameter energy also was able to distinguish the groups (p = 0,02). A histological correlation showed a tendency of mean gray level to have progressively greater values from specimens with < 50% to > 75% of fibrosis. CONCLUSION: Videodensitometric computer analysis of scan images may be used to identify vulnerable and potentially unstable lipid-rich carotid plaques, which are less echogenic in density than stable or asymptomatic, more densely fibrotic plaques.

Lectin KM+-induced neutrophil haptotaxis involves binding to laminin.

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.

The novel lectin KM+ detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum.

KM+ is a D(+)mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+)mannose, but not D(+)galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-alpha1,3-[man-alpha1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosyl-glycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates.

Microinjection of renin-angiotensin system peptides in discrete sites within the rat periaqueductal gray matter elicits antinociception.

The intracerebroventricular administration of renin substrate or angiotensin II evokes antinociception in rodents, but the brain sites where most of the renin-angiotensin system peptides act are not yet known. This study describes the antinociceptive effects of microinjecting porcine renin substrate tetradecapeptide (RS) or angiotensins I (AI), II (AII) or III (AIII) into different regions of the periaqueductal gray matter (PAG), using the rat tail flick test. All the above peptides were effective following administration into several PAG regions. Their antinociceptive effects were strongly evoked from the caudal ventrolateral and ventral PAG, including the dorsal raphe nucleus. A dose-dependent antinociception following administration into the ventrolateral PAG was demonstrated for all peptides studied. The effect of AII from the ventrolateral PAG was inhibited by the previous local administration of saralasin, a non-selective angiotensin receptor antagonist. Moreover, the peak effects of RS and AI occurred later than those of AII and AIII. The time-course of antinociception suggests that longer-chain peptides are locally processed to biologically active smaller-chain peptides. This study shows for the first time the antinociceptive effect of RS, AI, AII and III in well-defined PAG regions, an effect that is receptor mediated for AII.

High-performance liquid chromatographic separation of renin-angiotensin system peptides and most of their metabolic fragments.

We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.

Screening for prolactin isoforms in the follicular fluid of patients undergoing in vitro fertilization.

The purpose of this prospective and non-randomized study was to identify prolactin (PRL) isoforms in the follicular fluid (FF) of patients undergoing in vitro fertilization (IVF). The FF from 19 patients was obtained during oocyte retrieval at the University Hospital, Ribeirão Preto Medical School. The molecular weight of FF PRL was determined by column chromatography on Sephacryl S-200 HR combined with an immunometric assay. The only PRL isoform detected in FF exhibited a molecular weight of 20.5 kD. The molecular weights of FF PRL and small PRL are essentially identical. The 20.5-kD PRL accounted for 93.1 +/- 9.1% (mean +/- SD) of the recovered PRL after gel chromatography. Total PRL recovery was 74.2 +/- 19%. Thus, the only PRL detected in the FF was the small PRL, the most potent one.

Expression of nitric oxide synthases and in vitro migration of eosinophils from allergic rhinitis subjects.

The expression of nitric oxide (NO) synthases and the role of the NO cyclic GMP pathway on the migration of eosinophils from untreated patients with allergic rhinitis were investigated. Inducible NO synthase was strongly expressed in eosinophils from healthy individuals, but not in eosinophils from allergic rhinitis patients. The neuronal isoform was observed in eosinophils from each group studied, whereas no staining for the endothelial isoform was detected in either group. The chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-7) M) and eotaxin (100 ng/ml) was significantly potentiated in allergic rhinitis eosinophils. In both groups, N(omega)-nitro-L-arginine methyl ester (L-NAME, 1.0 mM) or 1H(1,2,4)-oxadiazolo(4,3,-a)quinoxalin-1-one (ODQ, 0.2 mM) markedly reduced the chemotaxis. The selective iNOS inhibitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W, 0.1-1.0 mM) significantly reduced the chemotaxis of eosinophils from healthy but not from allergic rhinitis subjects. The inhibition by L-NAME was restored by 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine, whereas the inhibition by ODQ was restored by dibutyryl cyclic GMP. In conclusion, both endothelial and inducible NO synthase isoforms are absent in allergic rhinitis eosinophils, suggesting that the NO cyclic GMP pathway in this cell type is maintained through the activity of a neuronal isoform.