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The influence of Ca 2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD + -NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD + -NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca 2+ -free perfusion (no Ca 2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b) In the presence of Ca 2+ , the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca 2+ -free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c) In the presence of Ca 2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca 2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca 2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca 2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis; Key wordsBraz J Med Biol Res 30 (7) 1997 A.C. Marques-da-Silva et al.the only difference was that inhibition rather than stimulation of oxygen uptake was observed for a lactate to pyruvate ratio equal to 0.01; progressive stimulation was observed in the 0.1 to 100 range. f) In the absence of Ca 2+ the effects of glucagon on oxygen uptake were different; the dependence curve was sigmoidal at the onset, with a welldefined maximum at a lactate to pyruvate ratio equal to 1; this maximum was followed by a steady decline at higher ratios; at the ratios of 0.01 and 0.1 inhibition took place; oxygen uptake stimulation caused by glucagon was generally lower in the absence of Ca 2+ except when the lactate to pyruvate ratio was equal to 1. The results of the prese...
The influence of Ca 2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD + -NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD + -NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca 2+ -free perfusion (no Ca 2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b) In the presence of Ca 2+ , the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca 2+ -free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c) In the presence of Ca 2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca 2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca 2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca 2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis; Key wordsBraz J Med Biol Res 30 (7) 1997 A.C. Marques-da-Silva et al.the only difference was that inhibition rather than stimulation of oxygen uptake was observed for a lactate to pyruvate ratio equal to 0.01; progressive stimulation was observed in the 0.1 to 100 range. f) In the absence of Ca 2+ the effects of glucagon on oxygen uptake were different; the dependence curve was sigmoidal at the onset, with a welldefined maximum at a lactate to pyruvate ratio equal to 1; this maximum was followed by a steady decline at higher ratios; at the ratios of 0.01 and 0.1 inhibition took place; oxygen uptake stimulation caused by glucagon was generally lower in the absence of Ca 2+ except when the lactate to pyruvate ratio was equal to 1. The results of the prese...
NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by β-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10–4M), a β-adrenergic antagonist, and was not modified in the presence of prazosin, an α-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to β-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuriben-zoate (PCMB, 10–5M) and N-ethylmaleimide (NEM, 10–3M), indicating the involvement of –SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10–3M) and GTPγS (10–4M), suggesting that the specific alkylated –SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10–2 M) and fluoroaluminate AlF–4 (F–, 10–2M + Al3+, 10–5M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.
After summarizing the discrimination of the several transport systems for neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends t o run torward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali-ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate-aspartate shuttle. After depletion of the energy reserves in the Ehrlich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restored by pyruvate, but in poor correlation with the restoration of alkali-ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly sensitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali-ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na+-independent system, as well as to one entering by the principal Na+-dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali-ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali-ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.Key words: amino acid transport in animal cells, energization of transport systems, discrimination of transport systems, reverse operation of transport systems, Ehrlich cell, NADH dehydrogenase, alkali-ion gradients, phenazine methosulfate, ouabain TRANSPORT SYSTEMS AND THEIR INTERACTIONThe transport systems for neutral amino acids in the Ehrlich cell are taken t o be approximately representative of those of the cells of the higher animal in general, with more and more evidence supporting that interpretation. Most conspicuous are a broad-
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