The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro

Abstract

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.

Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro

“…The endocytic index obtained for [14C]sucrose in the present study is similar to that reported for sucrose uptake in the rat yolk sac and other systems (Pratten et al, 1980). Weisbecker et al (1983) reported values of between 9.1 and 1 3.4u1/h per mg of protein for the endocytic index of 125I-labelled IgG incubated with rat yolk sac in the presence of calf serum, which are higher than the values obtained here for the guinea pig yolk sac. The difference may be attributable to the fact that a lower ligand concentration (1 jug/ml) was used in the rat yolk sac experiments.…”

“…However, pepstatin, a specific inhibitor of cathepsin D, had no detectable effect on IgG degradation. In order to give the tissue an opportunity to attain greater intracellular concentrations of the cathepsin inhibitors, experiments were carried out in which tissue was preincubated with leupeptin and pepstatin for 30min prior to addition of ligand, as described by Weisbecker et al (1983). Under these conditions the inhibition of degradation seen with leupeptin was slightly greater (47%) but, once again, no effect of pepstatin was found (results not shown).…”

Interaction of a Cationic N-(2-hydroxypropyl)methacrylamide Copolymer with Rat Visceral Yolk Sacs Cultured in vitro and Rat Liver in vivo

“…The value measured for copolymer 2 is typical of many substrates in this tissue [20]. Faster rates of exocytosis have been observed previously when using 121 I-labelled immunoglobulin G (IgG) [29] and 121 I-labelled formaldehyde denatured bovine serum albumin (dBSA) [29] as pinocytic substrates. The apparently faster release of dBSA was shown to be due to release of 125 I-labelled low molecular weight degradation products, not macromolecular dBSA.…”

“…The apparently faster release of dBSA was shown to be due to release of 125 I-labelled low molecular weight degradation products, not macromolecular dBSA. However, 115 I-labelled IgG was rapidly released from the yolk sac in both its intact form and as low molecular weight degradation products [29]. This IgG transport mechanism is an important physiological adaptation of the tissue to permit transport of passive immunity from mother to the developing embryo.…”

Quantitative cumulative biodistribution of antibodies in mice

“…Accordingly, our experiments using indium-111-labeled IgGs provide a more direct approach for identifying the sites of cumulative distribution and potential catabolism of IgG under physiological conditions. It is difficult to argue from the 125 I-labeled IgG data alone that any one tissue has a dominant role in the metabolism of IgG, since it is simultaneously proposed that 125 I-labeled degradation products are being rapidly exocytosed or diffused from this tissue 25 . As such, alternative residualizing labels for studying IgG distribution, which are more efficiently retained in tissues, should permit a more definitive assessment of the role of various tissues in IgG tissue catabolism 26 .…”