Abstract:While many metabolic effects of insulin on various organs and tissues have been demonstrated, the underlying mechanism of the hormone action has remained obscure. In the present paper evidence will be presented to show that insulin has an immediate electrochemical action on unwashed red cells. In subsequent papers an attempt will be made to relate this action to the hormone's sustained metabolic effects.The distinction between 'immediate electrochemical' and 'metabolic' effects is based on experimental conditi… Show more
“…The concentration of extracellular superoxide can be increased by insulin using two different mechanisms: either eNOS secretes superoxide, or insulin activates the NADH/NADPHdependent transmembrane electron transport systems, which produce superoxide extracellularly. [22][23][24] In spite of the many similarities in behavior between endothelial and platelet eNOS functions, we do not have data indicating the existence of caveola-like parts of thrombocyte membranes. 25 …”
Section: Determination Of No • In Biological Systems Is Difficult Beccontrasting
Recent data support the possible role of nitric oxide (NO*) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO* production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO* by luminol is possible in the form of peroxynitrite produced in the reaction of NO* with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 micromol/l of desferrioxamine mesylate. Insulin, in the range of 0.084-840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO* synthase enzyme. N(omega)-nitro-L-arginine methyl ester (at concentrations of 2.0-4.0 mmol/l, P<0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72-144 IU/ml, P<0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO* and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.
“…The concentration of extracellular superoxide can be increased by insulin using two different mechanisms: either eNOS secretes superoxide, or insulin activates the NADH/NADPHdependent transmembrane electron transport systems, which produce superoxide extracellularly. [22][23][24] In spite of the many similarities in behavior between endothelial and platelet eNOS functions, we do not have data indicating the existence of caveola-like parts of thrombocyte membranes. 25 …”
Section: Determination Of No • In Biological Systems Is Difficult Beccontrasting
Recent data support the possible role of nitric oxide (NO*) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO* production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO* by luminol is possible in the form of peroxynitrite produced in the reaction of NO* with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 micromol/l of desferrioxamine mesylate. Insulin, in the range of 0.084-840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO* synthase enzyme. N(omega)-nitro-L-arginine methyl ester (at concentrations of 2.0-4.0 mmol/l, P<0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72-144 IU/ml, P<0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO* and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.
“…Ferricyanide reduction by intact erythrocytes is associated with increased intracellular ATP generation (15), no change in oxygen consumption (15), and proton movement out of the cells (19). The latter likely reflects an attempt of the cell to avoid ferricyanide-induced pH gradients across the cell membrane (20).…”
Ascorbic acid, or vitamin C, is a primary antioxidant in plasma and within cells, but it can also interact with the plasma membrane by donating electrons to the alpha-tocopheroxyl radical and a trans-plasma membrane oxidoreductase activity. Ascorbate-derived reducing capacity is thus transmitted both into and across the plasma membrane. Recycling of alpha-tocopherol by ascorbate helps to protect membrane lipids from peroxidation. However, neither the mechanism nor function of the ascorbate-dependent oxidoreductase activity is known. This activity has typically been studied using extracellular ferricyanide as an electron acceptor. Whereas an NADH:ferricyanide reductase activity is evident in open membranes, ascorbate is the preferred electron donor within cells. The oxidoreductase may be a single membrane-spanning protein or may only partially span the membrane as part of a trans-membrane electron transport chain composed of a cytochrome or even hydrophobic antioxidants such as alpha-tocopherol or ubiquinol-10. Further studies are needed to elucidate the structural components, mechanism, and physiological significance of this activity. Proposed functions for the oxidoreductase include stimulation of cell growth, reduction of the ascorbate free radical outside cells, recycling of alpha-tocopherol, reduction of lipid hydroperoxides, and reduction of ferric iron prior to iron uptake by a transferrin-independent pathway.
The findings reported in the preceding paper (Dormandy & Zairday, 1965) suggested that insulin has an immediate electrochemical effect on unwashed red cells suspended in a variety of simple inorganic saline solutions. The term 'electrochemical' was used to emphasize that experimental conditions and procedures were designed to show up immediate physical rather than sustained metabolic changes.
The findings reported in the preceding paper (Dormandy & Zairday, 1965) suggested that insulin has an immediate electrochemical effect on unwashed red cells suspended in a variety of simple inorganic saline solutions. The term 'electrochemical' was used to emphasize that experimental conditions and procedures were designed to show up immediate physical rather than sustained metabolic changes. The insulin effects on ionic partitions, Eh and pH, were consistent with the hypothesis that the hormone induces a shift in the trans-membrane redox-potential gradient; and this raised the question whether, under more physiological conditions, such a primary action might alter or reset the pattern of cell metabolism. The consensus of opinion among workers in the insulin field seems to be that red-cell metabolism is not sensitive to insulin; but it was noted that, while the immediate anion shifts and pH changes could no longer be observed in cell suspensions incubated at 370 in glucose-rich media, the addition of insulin to such preparations caused an immediate though transient rise in the extracellular inorganic phosphate concentration. It was therefore decided to reinvestigate the effects of the hormone on redcell metabolism centred on phosphorus (P) turnover rather than on such 'classical' insulin parameters as glucose uptake or C02 evolution. The findings are reported in the present paper.
METHODSAn account of the preparations of unwashed red-cell suspensions, of two ways of bicarbonate-CO2 buffering, and of a number of techniques of estimation have been given in the previous paper. Only additional material and procedures are here described. MaterialGluco8e-pho8phate-bicarbonate buffer (GPB). The composition in m-mole/l. was: NaCl, 140; KCI, 3; CaCl2, 0-2; MgCl1, 0-1; Na2HEPOj, 1 1; KH2PO4, 0 4; NaHCOS, 26 and glucose to 300 mg/100 ml.; equilibrated with nitrogen 95 %-O2 5 %.
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