RESUMO
We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K(+)-congener Rb(+) in the Na(+)/K(+)-ATPase. Eosin decreases the equilibrium levels of occluded Rb(+), this effect being fully attributable to a decrease in the apparent affinity of the enzyme for Rb(+) since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na(+)/K(+)-ATPase, both to the Rb(+)-free and to the Rb(+)-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb(+) occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that, like ATP, it increases the velocity of deocclusion of Rb(+). Interactions between eosin and ATP on Rb(+)-release experiments seem to indicate that eosin binds to the low-affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.
Assuntos
Amarelo de Eosina-(YS)/química , Amarelo de Eosina-(YS)/farmacocinética , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Radioisótopos de Rubídio/farmacocinética , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/farmacocinética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacocinética , Animais , Sítios de Ligação , Modelos Químicos , Dinâmica não Linear , Ligação Proteica , Conformação Proteica , Suínos , Termodinâmica , Fatores de TempoRESUMO
The K+ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K+-mediated mitochondrial swelling observed in the absence of phosphate (PO4) and in the presence of a respiratory substrate has led to propose that large K+ movements occur in yeast mitochondria. Thus, the uptake of K+ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K+ transport; these were mitochondrial swelling and the uptake of the radioactive K+ analog 86Rb+. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO4 concentrations. The high protein concentrations used to measure 86Rb+ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO4 but not at 0 or 4 mM PO4. At 4 mM PO4 swelling was inhibited while, in contrast, 86Rb+ uptake was still observed. The results suggest that an energy-dependent K+ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K+ uptake system, the Mg2+ and quinine sensitivity of both swelling and 86Rb+ uptake were evaluated. Under the conditions where the unspecific pore was closed, K+ transport sensitivity to Mg2+ and quinine increased. In addition, when Zn2+ was added as an antiport inhibitor, uptake of 86Rb+ increased. It is suggested that in yeast mitochondria, the K+ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.
Assuntos
Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Canais de Potássio/fisiologia , Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte de Íons , Magnésio/farmacologia , Potenciais da Membrana , Mitocôndrias/ultraestrutura , Dilatação Mitocondrial , Permeabilidade , Fosfatos/farmacologia , Quinina/farmacologia , Radioisótopos de Rubídio/farmacocinética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiaeRESUMO
We examined the rat proximal tubule (PT) response to endothelin-1 (ET-1) in terms of 20-hydroxyeicosatetraenoic acid (HETE) dependency. Arachidonic acid (AA) (1 microM) decreased ouabain-sensitive (86)Rb uptake from 2.1 +/- 0.1 to 0.3 +/- 0.08 ng Rb. 10 microg protein(-1). 2 min(-1) (P < 0.05); 20-HETE (1 microM) had similar effects. Dibromododecenoic acid (DBDD) (2 microM), an inhibitor of omega-hydroxylase, abolished the inhibitory action of AA on (86)Rb uptake whereas the PT response to 20-HETE was unaffected. ET-1 at 0.1, 1, 10, and 100 nM reduced (86)Rb uptake from 2.8 +/- 0.3 in control PTs to 2.4 +/- 0.2, 1.7 +/- 0.1, 0.67 +/- 0.08, and 0.1 +/- 0.03 ng Rb. 10 microg protein(-1). 2 min(-1), respectively. DBDD (2 microM) abolished the inhibitory effect of ET-1 on (86)Rb uptake as did BMS182874 (1 microM), an ET(A)-selective receptor antagonist. ET-1 (100 nM) significantly increased PT 20-HETE release by approximately 50%, an effect prevented by DBDD. N(omega)-nitro-L-arginine-methyl ester (L-NAME), given for 4 days to inhibit nitric oxide synthase (NOS), increased arterial pressure from 92 +/- 12 to 140 +/- 8 mmHg and increased endogenous release of 20-HETE from isolated PTs (measured by gas chromatography/mass spectrometry). In L-NAME-treated PTs, but not in control PTs, 0.1 microM AA inhibited ouabain-sensitive (86)Rb uptake by > 40%; the response to AA was attenuated by DBDD. We conclude that, in the PTs, 1) 20-HETE is a second messenger for ET-1 and 2) conversion of AA to 20-HETE is augmented when NOS is inhibited.
Assuntos
Ácido Araquidônico/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Endotelina-1/metabolismo , Túbulos Renais Proximais/metabolismo , Animais , Transporte Biológico/fisiologia , Antagonistas dos Receptores de Endotelina , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A , Receptores de Endotelina/metabolismo , Radioisótopos de Rubídio/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Radioiodine is used to treat thyroid cancer and hyperthyroidism. In order to reduce radiation hazard to the patient and to people in contact with the patient it would be desirable to obtain the same therapeutic effect with lower activities of the radioisotope. This could be achieved by the simultaneous administration of a compound that increases tissue radiosensitivity. In this study we analyzed the use of nicotinamide (NA) as a radiosensitizer to radioiodine, to increase 131I efficacy. NA administered during 30 days to Wistar rats failed to alter thyroid weight. The influence of NA on radiothyroidectomy induced by increasing doses of 131I was examined in otherwise nontreated rats. NA produced a significant increase in the ablation caused by radioiodine. Goiter was then induced by the administration of methylmercaptoimidazol (MMI) to rats, followed by the treatment with radioiodine with and without simultaneous administration of NA. Thyroid weight per 100 g of body weight ratio was not changed by NA alone; 131I administration caused a 25% decrease in goiter size, while 131I plus NA produced a reduction of the ratio of 46% (p < 0.01 vs. NA). No changes were observed in adenosine diphosphate (ADP)-ribosilation of thyroid nuclear protein in NA-treated rats. Thyroid blood flow (determined by 86Rb uptake) was increased by 84% by NA. In conclusion, nicotinamide has a significant radiosensitizing effect to 131I both in normal and goitrous rats. This action is because of an increase in thyroid blood flow, which probably enhances tissue oxgenation.