Christopher J. Danpure

The uptake and subcellular distribution of gold in rat liver cells after in vivo administration of sodium aurothiomalate

Abstract Using the techniques of differential centrifugation, sucrose sedimentation-gradient and isopycnic-gradient centrifugation and gel chromatography through Sephadex G 75 and G 200, the uptake and subcellular distribution of gold in rat liver has been studied over a period of 1 hr to 36 days after intraperitoneal administration of 0.7 mg kg −1 to 80 mg kg −1 sodium aurothiomalate (“Myocrisin”). After 1 hr, gold concentrated in the lysosomes of liver cells and, under certain conditions, it was estimated that probably more than 90 per cent of the cytoplasmic gold was associated with these organelles. After longer time intervals partial redistribution of the gold took place, possibly due to sequestration into telolysosomes. The majority of the lysosomal gold appeared to be membrane-bound and increased with increasing aurothiomalate dose and time after injection. The distribution of gold among the proteins of the cytosol and the lysed granule-fraction supernatant has been investigated and compared with that in the plasma. Whereas in the latter more than 87 per cent of the gold was bound to albumin, the gold in the lysosomal supernatant was bound to molecules over a wide range of molecular weights, and that in the cytosol appeared to be bound to at least three macromolecules, one ~ 300,000, one ~ 40,000 (possibly ligandin) and one ~ 10,000. In addition 10–15 per cent of the cytosol gold appeared to be a much lower molecular weight species (less than 3,000), which was not found in the lysosomal supernatant or the plasma.

The interaction of aurothiomalate and cysteine.

Abstract Aurothiomalate and cysteine react under appropriate conditions to produce an insoluble complex of aurocysteine. Although alkylation of the SH group of cysteine completely blocks the reaction, the organomercurials have no effect. The results are compared with, and found to be compatible with, the interaction between aurothiomalate and the cysteinyl residue of human serum albumin.

The effect of cytochalasin B on the release of lysosomal enzymes and intra-lysosomally-stored polyvinylpyrrolidone in the isolated perfused rat liver

The uptake and subcellular distribution in vivo and release in vitro of 125I-polyvinylpyrrolidone (125I-PVP) has been studied in rat liver. Using the techniques of sucrose-density-gradient sedimentation and isopycnic centrifugation, it was shown that 4-15 days after injection of 125I-PVP the majority of the radioactivity was still in a high-molecular-weight form and associated with the lysosomes of the liver, the size and density properties of which were not significantly altered. Practically all of the 125I-PVP found in the lysosomes was free in the lumen and not associated with the lysosomal membrane, its intra-lysosomal distribution being much more similar to that of beta-galactosidase than arylsulphatase or beta-N-acetylglucosaminidase. In an isolated recirculating perfusion system the liver released all the enzymes studied (arylsulphatase, beta-galactosidase and lactate dehydrogenase) and, when previously loaded, 125I-PVP was also released into the perfusate. The magnitude of the release was always in the order 125I-PVP greater than beta-galactosidase greater than arylsulphatase. Preloading the lysosomes in vivo appeared to bring about an increase in the mean levels of release of all the enzymes, but the wide spread of data made this statistically significant only for lactate dehydrogenase. The microfilament poison cytochalasin B increased the release of arylsulphatase and beta-galactosidase, but not lactate dehydrogenase, in the perfused non-loaded livers, but failed to augment the release of any of the enzymes or 125I-PVP in the loaded livers. After perfusion, subcellular fractionation of the liver showed that the lysosomes had become enlarged and more fragile, especially so with those rich in 125I-PVP and beta-galactosidase rather than those rich in arylsulphatase and beta-N-acetylglucosaminidase.

The release of intralysosomally-stored 125I-Triton WR-1339 and lysosomal enzymes from the isolated perfused rat liver in the presence and absence of cytochalasin B.

The effect of in vivo loading of the lysosomotropic agent 125I-Triton WR-1339 on the release of lysosomal enzymes in isolated perfused rat liver has been studied in the presence and absence of the microfilament poison cytochalasin B, as has the release of the 125I-Triton WR-1339 itself. Perfused isolated rat livers released all the enzymes studied (arylsulphatase, beta-galactosidase and lactate dehydrogenase) and, when preloaded, 125I-Triton WR-1339 was also released into the perfusate. The magnitude of the net release (after 5 hr perfusion) was in the order beta-galactosidase = 125I-Triton WR-1339 greater than lactate dehydrogenase greater than arylsulphatase. Preloading of the lysosomes with the detergent appeared to bring about an increase in the release of all the enzymes studied (3.5 X for beta-galactosidase, 2.6 X for arylsulphatase and 1.7 X for lactate dehydrogenase). The addition of the microfilament poison cytochalasin B into the perfusate of non-loaded livers significantly increased the release of the lysosomal enzymes but not that of lactate dehydrogenase. However in the 125I-Triton WR-1339- loaded livers cytochalasin B had no effect on the release of lysosomal enzymes or detergent, but reduced the loss of lactate dehydrogenase by about 50%. This failure of cytochalasin B to potentiate the exocytosis of lysosomal contents in 125I-Triton WR-1339-loaded livers is similar to the effect found previously with 125I-PVP-loaded livers and may be related to the already enhanced loss of lysosomal enzymes apparently caused by the loading.