RESUMO
Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania, which has very limited treatment options and affects poor and underdeveloped populations. The current treatment is plagued by many complications, such as high toxicity, high cost and resistance to parasites; therefore, novel therapeutic agents are urgently needed. Herein, the synthesis, characterization and in vitro leishmanicidal potential of new complexes with the general formula [RuCl3(TMP)(dppb)] (1), [PtCl(TMP)(PPh3)2]PF6 (2) and [Cu(CH3COO)2(TMP)2]·DMF (3) (dppb = 1,4-bis(diphenylphosphino)butane, PPH3 = triphenylphosphine and TMP = trimethoprim) were evaluated. The complexes were characterized by infrared, UV-vis, cyclic voltammetry, molar conductance measurements, elemental analysis and NMR experiments. Also, the geometry of (2) and (3) were determined by single crystal X-ray diffraction. Despite being less potent against promastigote L. amazonensis proliferation than amphotericin B reference drug (IC50 = 0.09 ± 0.02 µM), complex (2) (IC50 = 3.6 ± 1.5 µM) was several times less cytotoxic (CC50 = 17.8 µM, SI = 4.9) in comparison with amphotericin B (CC50 = 3.3 µM, SI = 36.6) and gentian violet control (CC50 = 0.8 µM). Additionally, complex (2) inhibited J774 macrophage infection and amastigote number by macrophages (IC50 = 6.6 and SI = 2.7). Outstandingly, complex (2) was shown to be a promising candidate for a new leishmanicidal therapeutic agent, considering its biological power combined with low toxicity.
Assuntos
Antiprotozoários , Complexos de Coordenação , Cobre , Leishmania/crescimento & desenvolvimento , Leishmaniose/tratamento farmacológico , Platina , Rubídio , Trimetoprima , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Cristalografia por Raios X , Leishmaniose/metabolismo , Leishmaniose/patologia , Camundongos , Estrutura Molecular , Platina/química , Platina/farmacologia , Rubídio/química , Rubídio/farmacologia , Trimetoprima/química , Trimetoprima/farmacologiaRESUMO
A comprehensive study of the interaction between Na(+) and K(+) with the Na(+)/K(+)-ATPase requires dissecting the incidence of alternative cycling modes on activity measurements in which one or both of these cations are absent. With this aim, we used membrane fragments containing pig-kidney Na(+)/K(+)-ATPase to perform measurements, at 25°C and pH=7.4, of ATPase activity and steady-state levels of (i) intermediates containing occluded Rb(+) at different [Rb(+)] in media lacking Na(+), and (ii) phosphorylated intermediates at different [Na(+)] in media lacking Rb(+). Most relevant results are: (1) Rb(+) can be occluded through an ATPasic cycling mode that takes place in the absence of Na(+) ions, (2) the kinetic behavior of the phosphoenzyme formed by ATP in the absence of Na(+) is different from the one that is formed with Na(+), and (3) binding of Na(+) to transport sites during catalysis is not at random unless rapid equilibrium holds.
Assuntos
Trifosfato de Adenosina/metabolismo , Medula Renal/enzimologia , Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cinética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Rubídio/farmacologia , Sódio/farmacologia , SuínosRESUMO
This work presents a detailed kinetic study that shows the coupling between the E2âE1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2âE1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2âE1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2âE1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2âE1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).
Assuntos
Rim/metabolismo , Rubídio/farmacologia , ATPase Trocadora de Sódio-Potássio/química , Vanadatos/farmacologia , Trifosfato de Adenosina/química , Animais , Biofísica/métodos , Amarelo de Eosina-(YS)/química , Cinética , Magnésio/química , Modelos Biológicos , Ligação Proteica , Conformação Proteica , Rubídio/química , Suínos , Fatores de Tempo , Vanadatos/químicaRESUMO
We used suspensions of partially purified Na(+)/K(+)-ATPase from pig kidney to compare the effects of Rb(+), as a K(+) congener, on the time course and on the equilibrium values of eosin fluorescence and of Rb(+) occlusion. Both sets of data were collected under identical conditions in the same enzyme preparations. The incubation media lacked ATP so that all changes led to an equilibrium distribution between enzyme conformers with and without bound eosin and with and without bound or occluded Rb(+). Results showed that as Rb(+) concentration was increased, the equilibrium value of fluorescence decreased and occlusion increased along rectangular hyperbolas with similar half-maximal values. The time courses of attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolution of our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functions of time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the time course than in the case of occlusion. Comparison between experimental and simulated results suggests that fluorescence changes express a process that is coupled to Rb(+) occlusion but that is completed before occlusion reaches equilibrium.
Assuntos
Amarelo de Eosina-(YS)/química , Rubídio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Algoritmos , Animais , Amarelo de Eosina-(YS)/metabolismo , Fluorescência , Transporte de Íons/efeitos dos fármacos , Cinética , Modelos Químicos , Ligação Proteica , Rubídio/metabolismo , Rubídio/farmacologia , ATPase Trocadora de Sódio-Potássio/química , SuínosRESUMO
In steady-state conditions and for concentrations of the K(+)-congener Rb(+) less than 2.5 mM, Rb(+)-dependent ATPase activity is significantly higher than the steady-state rate of breakdown of Rb(+)-occluded states, a discrepancy that disappears at sufficiently high [Rb(+)]. Direct experimental evidence is provided that supports the explanation that the binding of a single Rb(+) to the phosphoenzyme conformer E(2)P accelerates dephosphorylation without leading to the occlusion of the cation.
Assuntos
Rubídio/farmacocinética , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Rim/enzimologia , Cinética , Fosforilação , Rubídio/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , SuínosRESUMO
We used the direct route of occlusion to study the equilibrium between free and occluded Rb(+) in the Na(+)/K(+)-ATPase, in media with different concentrations of ATP, Mg(2+), or Na(+). An empirical equation, with the restrictions imposed by the stoichiometry of ligand binding was fitted to the data. This allowed us to identify which states of the enzyme were present in each condition and to work out the schemes and equations that describe the equilibria between the ATPase, Rb(+), and ATP, Mg(2+), or Na(+). These equations were fitted to the corresponding experimental data to find out the values of the equilibrium constants of the reactions connecting the different enzyme states. The three ligands decreased the apparent affinity for Rb(+) occlusion without affecting the occlusion capacity. With [ATP] tending to infinity, enzyme species with one or two occluded Rb(+) seem to be present and full occlusion seems to occur in enzymes saturated with the nucleotide. In contrast, when either [Mg(2+)] or [Na(+)] tended to infinity no occlusion was detectable. Both Mg(2+) and Na(+) are displaced by Rb(+) through a process that seems to need the binding and occlusion of two Rb(+), which suggests that in these conditions Rb(+) occlusion regains the stoichiometry of the physiological operation of the Na(+) pump.
Assuntos
Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Rim/enzimologia , Cinética , Magnésio/farmacologia , Modelos Teóricos , Rubídio/farmacologia , Sódio/farmacologia , SuínosRESUMO
The threshold for spreading depression (SD) in chicken retina elicited by rose bengal photoactivation was raised by Trolox (water soluble vitamin E) suggesting the participation of reactive oxygen species in SD. The typical increases in K+ concentration associated with SD were preceded by small K+ oscillations that were more sensitive to photoactivation than was SD. It is hypothesised that all these phenomena could be accounted for by a free-radical mediated transient increase in membrane permeability and that this may be relevant to ischemic brain damage.
Assuntos
Corantes Fluorescentes/farmacologia , Cloreto de Potássio/metabolismo , Retina/fisiologia , Rosa Bengala/farmacologia , Animais , Antioxidantes/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Galinhas , Cromanos/farmacologia , Radicais Livres/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatologia , Sulfato de Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Periodicidade , Fotoquímica , Retina/efeitos dos fármacos , Rubídio/farmacologiaRESUMO
The influence of rubidium-substituted physiological salt solution (Rb-PSS) on the relaxant effects of K+ channel openers was investigated in the human saphenous vein. In tissues precontracted with 20 mM KCl (in K-PSS) levcromakalim and P1060 produced complete, sustained relaxations. However, in Rb-PSS (containing 20 mM RbCl) these effects were inhibited and, although complete relaxations still occurred, were transient. When caffeine was applied at the beginning of this fade of levcromakalim-induced relaxation in Rb-PSS its contractile effect was potentiated. Similarly, the contraction to noradrenaline was potentiated when applied at the beginning of this fade of levcromakalim-induced relaxation, whereas this response was attenuated in control tissues bathed in 20 mM KCl (in K-PSS). Our results show that the relaxant effects of K+ channel openers in human saphenous vein are inhibited in Rb-PSS, in agreement with previous studies in animal tissue, and suggest that an increased Ca2+ uptake into intracellular stores may be contributory to vasorelaxation.
Assuntos
Benzopiranos/farmacologia , Cálcio/fisiologia , Guanidinas/farmacologia , Canais de Potássio/efeitos dos fármacos , Pirróis/farmacologia , Vasodilatadores/farmacologia , Cafeína/farmacologia , Cloretos/farmacologia , Cromakalim , Humanos , Técnicas In Vitro , Nifedipino/farmacologia , Norepinefrina/farmacologia , Cloreto de Potássio/farmacologia , Rubídio/farmacologia , Veia Safena/efeitos dos fármacos , Veia Safena/fisiologiaRESUMO
The mechanoinhibitory effects of two structurally dissimilar K+ channel openers, levcromakalim and P1060, and verapamil were compared in strips of human myometrium bathed in either K-PSS (normal Krebs solution) or Rb-PSS (K+ salts replaced by Rb+ equivalents). In Rb-PSS the effects of levcromakalim and P1060 on amplitude and frequency of spontaneous contractions were inhibited by more than 20- and 138-fold, respectively, whereas those of verapamil were unaltered. These results indicate that K+ channel openers possess Rb-sensitive and Rb-insensitive mechanoinhibitory actions on the human uterus, the former being more important in the effects of P1060 than levcromakalim.
Assuntos
Benzopiranos/farmacologia , Guanidinas/farmacologia , Canais de Potássio/efeitos dos fármacos , Pirróis/farmacologia , Rubídio/farmacologia , Contração Uterina/efeitos dos fármacos , Cromakalim , Feminino , Humanos , Técnicas In Vitro , Verapamil/farmacologiaRESUMO
The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.