Elisa Suzana Carneiro Pôças

Synthesis and preliminary pharmacological evaluation of coumestans with different patterns of oxygenation

Five coumestans with different patterns of oxygenation in rings A and D were synthesized from resorcinol and aromatic aldehydes, and screened for their antimyotoxic activity. The most potent compound (2b, IC50 = 1 microM) was selected for study of its pharmacological profile.

Natriuretic effect of bufalin in isolated rat kidneys involves activation of the Na+-K+-ATPase-Src kinase pathway.

Bufadienolides are structurally related to the clinically relevant cardenolides (e.g., digoxin) and are now considered as endogenous steroid hormones. Binding of ouabain to Na(+)-K(+)-ATPase has been associated, in kidney cells, to the activation of the Src kinase pathway and Na(+)-K(+)-ATPase internalization. Nevertheless, whether the activation of this cascade also occurs with other cardiotonic steroids and leads to diuresis and natriuresis in the isolated intact kidney is still unknown. In the present work, we perfused rat kidneys for 120 min with bufalin (1, 3, or 10 μM) and measured its vascular and tubular effects. Thereafter, we probed the effect of 10 μM 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4amine (PP2), a Src family kinase inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), a highly selective inhibitor of both MEK1 and MEK2, on bufalin-induced renal alterations. Bufalin at 3 and 10 μM profoundly increased several parameters of renal function in a time- and/or concentration-dependent fashion. At a concentration that produced similar inhibition of the rat kidney Na(+)-K(+)-ATPase, ouabain had a much smaller diuretic and natriuretic effect. Although bufalin fully inhibited the rat kidney Na(+)-K(+)-ATPase in vitro, its IC(50) (33 ± 1 μM) was threefold higher than the concentration used ex vivo and all its renal effects were blunted by PP2 and UO126. Furthermore, the phosphorylated (activated) ERK1/2 expression was increased after bufalin perfusion and this effect was totally prevented after PP2 pretreatment. The present study shows for the first time the direct diuretic, natriuretic, and kaliuretic effects of bufalin in isolated rat kidney and the relevance of Na(+)-K(+)-ATPase-mediated signal transduction.

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation

AIMS Cardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na(+)/K(+)-ATPase. MAIN METHODS Inhibition of Na(+)/K(+)-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released. KEY FINDINGS Analyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na(+)/K(+)-ATPase protomer. SIGNIFICANCE Considering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.

Insights into the mechanism of Na + ,K + -ATPase inhibition by 2-methoxy-3,8,9-trihydroxy coumestan

The molecular mechanisms involved in Na+,K+-ATPase inhibition by 2-methoxy-3,8,9-trihydroxy coumestan were investigated. We show that this compound decreases the free sulfydryl groups present in the enzyme and that its inhibitory effect is prevented by dithiothreitol and other two sulfydryl containing reagents. We propose a redox cycle culminating with the irreversible oxidation of sulfydryl groups essential for the catalytic activity of this enzyme and of two other related P-type ATPases.

Study of Propolis by High Temperature High Resolution Gas Chromatography-Mass Spectrometry

Abstract The underivatized hexane and acetone extracts of two propolis samples (from Brazil’s southwest) were analyzed by HT-HRGC (high temperature high resolution gas chromatogrphy) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds, including flavonoid aglycones, phenolic acids and high molecular weight compounds could be readily characterized in the crude extracts by HTHRGC-MS. HTHRGC and HTHRGC-MS were shown to be quick and informative tools for rapid analysis of crude extracts without need for prior derivatization and purification.

Inhibition of the alpha1beta1 isoform of the Na, K-ATPase by 8-methoxycoumestrol without positive inotropic effect in human myocardium--novel aspects of cardiac glycoside pharmacology.

Background: The Na,K-ATPase (NKA) is necessary for maintaining the resting membrane potential by transporting Na+ and K+ ions across the cell membrane. Although its 3 isoforms expressed in human heart (α1β1, α2β1, and α3β1) possess similar biochemical properties, their specific functions in human tissues remain unknown. In our search for an isoform-specific agent, which can serve to identify isoform-specific functions, we examined 8-methoxycoumestrol in its ability to inhibit the NKA and to produce inotropism in connection with the possibility to identify the NKA isoform-specific functions. Methods and Results: In radioligand binding experiments (membrane preparations of yeast expressing isoforms α1β1, α2β1, and α3β1; backdoor phosphorylation; and [3H]-ouabain, n = 3), 8-methoxycoumestrol (1-10 μM) produced no or only little inhibition of specific ouabain binding. However, when NKA activity of the α1β1 isoform was measured in membrane preparations from human kidney (reduced form of nicotinamide adenine dinucleotide-coupled assay, n = 3), a concentration-dependent full inhibition of the activity was induced by 8-methoxycoumestrol (IC50: 90 ± 97 nM), similar to that observed for classical cardiac glycosides digitoxin, digoxin, methyldigoxin, and β-acetyldigoxin (IC50 = 287 ± 190 nM, 409 ± 171 nM, 282 ± 482 nM, 587 ± 135 nM, P > 0.05). However, unlike the classical cardiac glycosides, 8-methoxycoumestrol did not increase cardiac contractility of electrically stimulated human right atrial trabeculae. Conclusions: These results indicate that 8-methoxycoumestrol inhibits the human α1β1 NKA by a mechanism different to that of cardiac glycosides. In addition, the inhibition of the α1β1 NKA activity seems not sufficient to evoke positive inotropy in human trabeculae, indicating that either the positive inotropic effect of cardiac glycosides is not mediated via the α1β1 isoform or the specific glycoside binding to α1β1 is needed for positive inotropy.

Study of endogenous profile of hCG in male Brazilian athletes

No mundo das drogas de otimizacao de desempenho, hormonio gonadotrofico humano (hCG) e crescentemente usado sozinho em tambem em combinacao com ciclos de Agentes Anabolicos Esteroides. Em homens o hCG mimetiza o hormonio Luteinizante (LH) e ajuda a aumentar a producao de testosterona nos testiculos. Assim sendo, hCG e comumente usado durante e depois de ciclos de esteroides para restaurar o tamanho testicular assim como a producao de testosterona endogena. Controle de dopagem para hCG requer atencao especial, visto que, deve ser feita diferenca entre a origem endogena e exogena de ambas substâncias. Na carencia de um padrao mais claro do que seria um nivel esperado de normalidade, os laboratorios de controle de dopagem tem aplicado valores de corte entre 5 e 25 mUI/mL. Por causa de grande diferenca em desenhos de ensaios e especificidade para diferentes formas de hCG, as concentracoes medidas sao altamente dependentes do ensaio usado e populacao analisada. O objetivo do estudo e o estabelecimento do valor de corte para a caracterizacao do abuso exogeno por atletas obtido com a aplicacao do Imunoensaio Enzimatico por Microparticulas.