RESUMO
The inhibition of guinea-pig heart (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) by calcium has been studied at pH 7.4, 6.8 and 6.4. 1. A decrease in pH reduced the threshold inhibitory concentration of calcium and the calcium concentration producing an inhibition of 50% of the enzyme activity. 2. Calcium reduced the apparent affinity of the enzyme of Na+, this effect occurred only at pH 7.4. 3. Calcium increased the apparent affinity of the enzyme for K+, this effect was enhanced at acidic pH. 4. Activation of the enzyme by Na+ for a constant Na+ : K+ ratio has been studied at pH 7.4 and at pH 6.8 in the absence and in the presence of 3.10(-4) M Ca 2+; the results of this experiment indicate that Ca2+ effect at pH 7.4 was not influenced by Na+ -- K+ competition and was probably due to a Na+ -- Ca2+ interaction. 5. At pH 7.4, the calcium inhibitory threshold concentration and the concentration producing 50% inhibition were reduced when Na+ was low; at pH 6.8, the calcium inhibition was not markedly modified by the change of Na+ concentration. 6. The Ca2+ -activated ATPase of myosin B which is related to the contractile behaviour of muscle and the Ca2+ -ATPase of the sarcoplasmic reticulum which is related to the ability of this structure to accumulate calcium were activated in a range of calcium concentration producing an inhibition of (Na2+ + K+) -ATPase. The present results indicate that the increase by acidity of the (Na2+ + K+) -ATPase sensitivity to calcium might be due to a suppression of a Na+ -Ca2+ interaction. On the basis of these observations, it is proposed that calcium might inhibit the Na+ -pump during the repolarization phase of the action potential and that, by this effect, it might control cell excitability.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Cálcio/farmacologia , Miocárdio/enzimologia , Sódio/farmacologia , Animais , Sítios de Ligação , Ativação Enzimática , Cobaias , Concentração de Íons de Hidrogênio , Miosinas , Potássio , Retículo Sarcoplasmático/enzimologiaRESUMO
OBJECTIVE: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. METHODS: For contractility studies, a total of 57 trabeculae were isolated from two non-failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three alpha isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. RESULTS: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p << 0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol.g-1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three alpha isoforms were expressed at the mRNA level in both normal and failing hearts. CONCLUSIONS: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition.
Assuntos
Cardiomiopatias/enzimologia , Isoenzimas/metabolismo , Contração Miocárdica/efeitos dos fármacos , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Northern Blotting , Humanos , Isoenzimas/análise , Miocárdio/enzimologia , RNA Mensageiro/análise , ATPase Trocadora de Sódio-Potássio/análise , ATPase Trocadora de Sódio-Potássio/genética , Estimulação QuímicaRESUMO
Two splice variants of the human uncoupling protein-3 (UCP3L and UCP3S) are highly expressed in skeletal muscle. The properties of UCP3L and S have been compared to those of UCP1 in a heterologous yeast expression system under the control of the galactose promoter. Both UCP3 isoforms were found to strongly impair the coupling efficiency of respiring cells thus resulting in increased thermogenesis. The uncoupling properties of both UCP3L and S could be clearly demonstrated also in isolated yeast mitochondria both in terms of coupled respiration and in the capacity to polarize the inner membrane in conditions of limited substrate availability. Contrary to what was observed with mitochondria containing UCP1, millimolar GDP and ATP had little if any effect on the uncoupling activity of UCP3. A very marked uncoupling of whole cells and isolated mitochondria was observed at very low expression levels of UCP3S indicating that the short isoform is more active than the long one.
Assuntos
Proteínas de Transporte/biossíntese , Calorimetria , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Galactose/metabolismo , Guanosina Difosfato/farmacologia , Humanos , Canais Iônicos , Malonatos/farmacologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Consumo de Oxigênio/efeitos dos fármacos , Regiões Promotoras Genéticas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/farmacologia , Proteína Desacopladora 1 , Proteína Desacopladora 3RESUMO
A reduction in fat intake may be achieved by making educated choices to reduce total calorie intake, to consume a lower quantity of total fats, or to modify the ratio of saturated-to-polyunsaturated lipids. Leptin agonists or NPY or CCK antagonists may prove to be useful to diminish appetite and thereby reduce the total intake of food. But eating has such cultural, social, and hedonistic attributes that such a single-pronged approach is unlikely to be successful. The use of fat substitutes may prove to be popular to provide a wide range of snack food options, but these are likely to be of minimal use in weight reduction programs because of their distribution of additives in only a limited number of foods. The inhibitors of lipid digestion will be modestly successful in the short term; their long-term success will be influenced by gastrointestinal adverse effects and the need to consume fat-soluble vitamin supplements to prevent the development of fat-soluble vitamin deficiencies. The inhibition of lipid absorption is an attractive targeted approach for the treatment of obesity, since this would reduce the uptake of visible as well as invisible fats, which would potentially offer convenient dosing, and could also be a means to inhibit secondarily the uptake of carbohydrate calories.
Assuntos
Absorção Intestinal , Metabolismo dos Lipídeos , Proteínas de Neoplasias , Obesidade/terapia , Proteínas Supressoras de Tumor , Animais , Ácidos e Sais Biliares/farmacologia , Proteínas de Transporte/metabolismo , Gorduras na Dieta/metabolismo , Digestão , Substitutos da Gordura/administração & dosagem , Substitutos da Gordura/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Absorção Intestinal/efeitos dos fármacos , Lipoproteínas/metabolismo , Proteína P2 de Mielina/metabolismo , Fenômenos Fisiológicos da Nutrição , Obesidade/tratamento farmacológico , Obesidade/metabolismo , SaciaçãoRESUMO
The inhibitory effect of formylated cardiac steroids (gitaloxin and its derivatives) on guinea-pig heart Na, K-ATPase was compared to that of other cardiac steroids with various hydroxy substituents. The decreasing order of potency of aglycones at equilibrium was as follows: gitaloxigenin greater than digitoxigenin greater than ouabagenin greater than digoxigenin greater than gitoxigenin greater than diginatigenin. This sequence was different to the sequence of drugs hydrophobic character. The compounds with hydroxy groups in the vicinity of the lactone ring (gitoxigenin, diginatigenin) were less potent than the hydrophilic compound ouabagenin. We propose that intramolecular bounding between 16 beta-OH and the lactone ring contributes to the relatively low potency of gitoxigenin and diginatigenin. The formylation of 16 beta-OH increased the potency of gitoxigenin by a factor of 41. The formylated compound (gitaloxigenin) was 5-fold more potent than digitoxigenin. The 3 beta-glycosylation of digoxigenin lead to pseudo-irreversible inhibitors of Na, K-ATPase. The half-time to achieve the equilibrium (for 5 mumol/l) was equal to 54 s, 90 s and 108 s respectively for digoxigenin monodigitoxoside, digoxin and desacetyllanatoside C. However, at equilibrium the three glycosides were equipotent, suggesting the existence of steric effects at the sugar site of the receptor. The sequence of potency observed for monodigitoxosides, monodigitalosides and tridigitoxosides after 60 min incubation was similar to that observed for the corresponding aglycones. These results suggest that the strongly negative inductive group 16 beta-OCHO is tightly bound to Na, K-ATPase, possibly to the same receptor site than that which is thought forming hydrogen and ionic bonds with the lactone ring. They show that the high toxicity of gitaloxin in guinea-pig heart is likely due to its high potency as Na, K-ATPase inhibitor.
Assuntos
Glicosídeos Digitálicos/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Digoxina/análogos & derivados , Digoxina/farmacologia , Cobaias , Técnicas In Vitro , Modelos Moleculares , Miocárdio/enzimologia , NAD/metabolismo , Fatores de TempoAssuntos
Miocárdio/metabolismo , Ouabaína/metabolismo , Potássio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Vanádio/farmacologia , Animais , Sítios de Ligação , Cobaias , Técnicas In Vitro , Cinética , Microssomos/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , VanadatosAssuntos
Cardiotônicos/isolamento & purificação , Miocárdio/análise , Acetona , Animais , Anticorpos/isolamento & purificação , Ligação Competitiva , Cardiotônicos/metabolismo , Cardiotônicos/farmacologia , Digoxina/imunologia , Cobaias , Técnicas In Vitro , Ouabaína/metabolismo , Cloreto de Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ÁguaRESUMO
The Na+, K+-pump controls a wide variety of cellular systems and its inhibition by cardiac glycosides modifies important physiological functions and evokes several pharmacological effects (refs 1, 2 and refs therein). However, not all the actions of cardiac glycosides can be attributed to Na+, K+-pump inhibition and several observations show that, at low doses, cardiac glycosides stimulate the pump. It has been proposed that their positive inotropic effect could be the sum of two processes: the inhibition of the pump and a still unknown additional inotropic mechanism. In guinea pig heart, low doses of ouabain interact with high-affinity binding sites, which differ from the lower-affinity sites responsible for Na+, K+-pump inhibition. It has been suggested that ouabain interaction with these high-affinity sites could be responsible for the additional inotropic mechanism. The existence of two classes of ouabain-binding sites has been documented not only in guinea pig heart, but also in dog, rat and human heart. Dihydroouabain, a derivative of ouabain in which the lactone ring is saturated, is about 50-fold less potent than ouabain as an inhibitor of Na+, K+-pump and does not stimulate the pump at low doses. Its inotropic effect can be entirely accounted for by the inhibition of the pump. We have examined the pharmacological action of ouabain in the presence of dihydroouabain and report here that dihydroouabain reduces ouabain inotropic action but not Na+, K+-pump inhibition.
Assuntos
Contração Miocárdica/efeitos dos fármacos , Ouabaína/análogos & derivados , Ouabaína/antagonistas & inibidores , Animais , Cobaias , Microssomos/metabolismo , Miocárdio/metabolismo , Ouabaína/farmacologia , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly documented. This study aimed to investigate EGF effects on fatty acid uptake and esterification in a human enterocyte cell line (Caco-2). EGF inhibited cell uptake of [14C]palmitate and markedly reduced its incorporation into triglycerides. In contrast, the incorporation in phospholipids was enhanced. To elucidate the mechanisms involved, key steps of lipid synthesis were investigated. The amount of intestinal fatty acid-binding protein (I-FABP), which is thought to be important for fatty acid absorption, and the activity of diacylglycerol acyltransferase (DGAT), an enzyme at the branch point of diacylglycerol utilization, were reduced. EGF effects on DGAT and on palmitate esterification occurred at 2-10 ng/ml, whereas effects on I-FABP and palmitate uptake occurred only at 10 ng/ml. This suggests that EGF inhibited palmitate uptake by reducing the I-FABP level and shifted its utilization from triglycerides to phospholipids by inhibiting DGAT. This increase in phospholipid synthesis might play a role in the restoration of enterocyte absorption function after intestinal mucosa injury.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Ácidos Graxos/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Neoplasias , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Proteínas Supressoras de Tumor , Aciltransferases/metabolismo , Células CACO-2 , Proteínas de Transporte/metabolismo , Coenzima A Ligases/metabolismo , Diacilglicerol O-Aciltransferase , Fator de Crescimento Epidérmico/farmacologia , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Intestinos/patologia , Proteína P2 de Mielina/metabolismo , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacocinéticaRESUMO
Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0. 72% of cytosolic proteins in fibroblasts (Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [(14)C]palmitate showed a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.
Assuntos
Proteínas de Transporte/genética , Mucosa Intestinal/metabolismo , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Proteínas Supressoras de Tumor , Células CACO-2 , Proteínas de Transporte/metabolismo , Diferenciação Celular , Divisão Celular , DNA Complementar , Esterificação , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Intestinos/citologia , Intestinos/ultraestrutura , Microscopia Eletrônica , Proteína P2 de Mielina/metabolismo , Ácido Palmítico/metabolismo , TransfecçãoRESUMO
Fatty acid-binding proteins (FABP) are small cytosolic proteins which are thought to play a key role in fatty acid metabolism. The intestine contains the intestinal (I-FABP) and the liver (L-FABP) isoforms, but their regulation is still poorly documented. In order to find suitable conditions for studying the regulation of the two FABP isoforms in Caco-2 cells, we investigated the effects of the presence of collagen during cell proliferation or differentiation. When collagen was present only during cell proliferation on culture dishes, I-FABP expression was enhanced, whereas sucrase-isomaltase was unaffected and L-FABP expression was merely accelerated. In contrast, when collagen was present during cell differentiation on filter inserts, both I-FABP and sucrase-isomaltase were strongly reduced, but L-FABP was not affected. Under the former conditions (the more suitable for studying FABP regulation), the peroxysome proliferator-activated receptor (PPAR) activators, clofibrate and alpha-bromopalmitate, enhanced the two isoforms. This study, which is the first one providing a quantitative protein analysis of I-FABP and L-FABP in Caco-2 cells, demonstrates different time courses of expression of these proteins during cell differentiation. It also shows that I-FABP is specifically regulated by collagen and that, under conditions optimal for their expression, both isoforms are modulated by metabolic factors.