The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy.

Nongenomic bronchodilating action elicited by dehydroepiandrosterone (DHEA) in a guinea pig asthma model.

Primates secrete large amounts of the precursor steroid dehydroepiandrosterone (DHEA); in humans, its levels are low during childhood and start declining after the fourth decade. It has been postulated that the progressive decline in DHEA levels may be related with the severity of asthma associated with age. To determine whether DHEA may regulate the airway smooth muscle (ASM) activity, isolated tracheal rings with and without epithelium from male guinea pigs were isometrically recorded to characterize the response of ASM to DHEA at different concentrations on KCl- and carbachol (CCh)-induced contraction as well as on ovalbumin (OVA)-induced contraction in sensitized guinea pigs. Additionally, we used barometric plethysmography in sensitized guinea pigs in order to compare changes of the lung resistance increased by the antigen challenge to OVA in the absence and presence of different doses of DHEA. DHEA concentration-dependently abolished the contraction to KCl, CCh and OVA, and no differences were found in preparations with and without epithelium. DHEA-induced relaxation was not modified by the suppression of protein synthesis or transcription, pharmacological inhibition of nitric oxide (NO) synthase, nor by antagonist of ß2-adrenergic receptors or an inhibitor of the 3ß-HSD enzyme. Likewise, Ca(2+)-induced contraction in Ca(2+)-free depolarized tissues was antagonized by DHEA, and the contraction to the L-type voltage-dependent calcium channel activator (Bay K 8644) was inhibited by DHEA. Furthermore, DHEA prevented OVA-induced increases in lung resistance. These results indicate that DHEA-induced relaxation in ASM is a nongenomic (membrane) action and is not produced after its bioconversion. The data suggest that DHEA-induced relaxation is an epithelium- and NO-independent mechanism that involves a blockade of voltage-dependent calcium channels and possible non-selective cation channels.

Phytochemical study guided by the myorelaxant activity of the crude extract, fractions and constituent from stem bark of Hymenaea courbaril L.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenaea courbaril L. (Caesalpinoideae) is used in Brazilian folk medicine to treat anemia, kidney problems, sore throat and other dysfunctions of the respiratory system, such as bronchitis and asthma, although such properties are yet to be scientifically validated. AIM OF THE STUDY: In order to give a scientific basis to support the traditional use of Hymenaea courbaril, this study was designed to evaluate antioxidant, myorelaxant and anti-inflammatory properties of the ethanol extract from stem bark and its fractions. The myorelaxant effect of astilbin, a flavonoid isolated from the bioactive ethyl acetate fraction (EAF), has also been evaluated. MATERIAL AND METHODS: In the present study ethanol extract from stem bark (EEHC) and fractions were analyzed using bioassay-guided fractionation. The following activities were investigated: antioxidant by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, myorelaxant on rat tracheal smooth muscle, and anti-inflammatory using ovalbumin-induced leukocytosis and airway hyperresponsiveness in rats. RESULTS: The results of the present investigation show that the whole extract of Hymenaea courbaril and some of its fractions strongly scavenged DPPH radical. The extract showed myorelaxant activity on rat trachea, being EAF its highest efficient fraction. Bio-guided study allowed the isolation of astilbin, a well-known flavonoid. The activity induced by this compound indicates that it may be partly responsible for the myorelaxant effect of EAF. EAF reduced contractions that depended on divalent cation inflow through voltage-operated Ca(2+) channels (VOCCs) or receptor-operated Ca(2+) channels (ROCCs), but it was more potent to inhibit VOCC- than ROCC-dependent contraction induced by Ca(2+) addition in ACh-enriched Ca(2+)-free medium. Oral pretreatment of antigen-challenged animals with EAF prevented airway hyperresponsiveness on KCl-induced contraction and reduced the number of total white cells, particularly eosinophils and neutrophils in bronchoalveolar lavage. CONCLUSIONS: This study provided scientific basis that Hymenaea courbaril presents potential antioxidant, myorelaxant and anti-inflammatory actions, which support its use in folk medicine to treat inflammatory airway diseases.