Interactions of glucagon with isolated rat-liver cells fate and subcellular localization of cell-associated hormone

Abstract

Listen

Translate article

The interaction of glucagon with isolated rat-liver cells was studied, and some results were compared with those obtained in parallel experiments with insulin. Attempts were made, in particular, to examine the functional relationships between receptor-binding and degradation, to assess the subcellular localization of cell-associated glucagon, and to compare the glucagonbinding properties of intact liver cells and whole-liver tissue. Binding determinations at the steady state are compatible with the existence of about 1.3 × 10 −5 sites per cell with a dissociation constant of 1.5 nM. Unlike that of liver membranes, the dissociation of bound glucagon is little affected by guanyl nucleotides. Glucagon in the incubation medium undergoes rapid inactivation and physical changes that suggest cleavage into iodotyrosyl peptides and free iodotyrosines. Most of the degrading activity is unrelated to the receptors as indicated by its low affinity (apparent K m 4 μM), its inhibition by polypeptides chemically unrelated to glucagon, and its partial association (about 50% of the total) with degradative components released extracellularly. In the absence of inhibitors, cell-associated glucagon also undergoes a progressive degradation, with a concomitant release of degraded products in the medium; this process may be, at least in part, receptor-mediated. However, in contrast with insulin, it represents only a minor fraction of the total degrading activity. About 80% of the glucagon, and 90% of the insulin associated to liver cells is recovered in the particulate fraction. On differential centrifugation, this material is almost exclusively recovered in the microsomal fraction; on sucrose density-gradient centrifugation, it is present to the extent of 70% in unidentified membranes of density 1.11–1.17. This pattern of distribution is unaffected by the time of incubation, indicating that if labeled hormone undergoes translocation, the latter is not detectable under the conditions used here. The subcellular distribution of the glucagon and insulinbinding activities is nearly the same whether the binding reaction is performed on intact cells or on isolated fractions, and, at saturation, intact cells bind about the same amount of hormone as does the particulate fraction derived from the cells. Thus, cell disruption does not affect the apparent accessibility of the binding sites. Crude particles and plasma membranes prepared from liver cells show a decrease in their ability to bind insulin and glucagon as compared with the same subcellular fractions obtained from whole liver; this is most pronounced with plasma membranes. Evidence that the collagenase preparation may be involved in these changes is presented.