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Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

ABSTRACT: Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered, indicating that bacitracin acted predominantly at the cell surface. In the presence of chloroquine, which dramatically increased the contribution of peak I to specific binding, 'intact' insulin was substantially overestimated when determined as the TCA-precipitable fraction. In addition, all peak I material and 50% of cell-associated label in peak II was trapped intracellularly, thereby pointing to the lysosomal or prelysosomal site of action of this drug.

SUBMITTER: Fleig WE

PROVIDER: S-EPMC1146952 | biostudies-other | 1986 Jul

REPOSITORIES: biostudies-other

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Project description:The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

Project description:Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [(73)As]arsenite (iAs(III); 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs(III) to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs(III) than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs(III) was associated with inhibition of DMAs production by moderate concentrations of iAs(III) and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to the interspecies differences in the hepatocyte capacity to methylate iAs.

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Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

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