Intracellular disruption of rat heart lysosomes by leucine methyl ester: effects on protein degradation.

Abstract

Perfusion of rat hearts with Krebs-Henseleit medium containing 10 mM L-leucine methyl ester leads to swelling of lysosomes and loss of lysosmal integrity within 30-60 min. No morphological changes can be detected in the nuclei, mitochondria, sarcoplasmic reticulum, or Golgi complex as a result of the treatment with leucine methyl ester, and the hearts continue to beat normally during the treatment period. Homogenates of rat hearts perfused with the methyl ester exhibit a decrease in the sedimentability of cathepsin D activity compared to controls, thus providing additional evidence for a loss of lysosomal integrity. Swelling and disruption of the lysosomes presumably occurs because of the extensive accumulation of leucine within the organelles resulting from the intralysosomal hydrolysis of the freely permeating methyl ester. The lysosomal dysfunction that occurs with exposure to leucine methyl ester produces a 30% decrease in cardiac protein degradation. These results provide an estimate ofthe contribution oflysosomes to total protein degradation in the rat heart, and they also suggest that the enzymes released as a result oflysosomal disruption are relatively inactive in hydrolyzing cellular constituents under the perfusion conditions used here. The use of amino acid methyl esters to produce rapid, specific loss of lysosomal integrity in situ provides an approach to the study of lysosomal function in intact cells.Goldman and her colleagues (1)(2)(3)(4) demonstrated that the methyl esters of certain amino acids at concentrations of 0.1-10 mM cause a loss of latency and sedimentability of the enzyme activities of isolated lysosomes. This was assumed to be due to the extensive accumulation of amino acids within the lysosome, resulting from the intralysosomal hydrolysis of the readily permeating methyl ester. At sufficiently high concentrations, the osmotic effects of the accumulated amino acid would lead to swelling and disruption of the lysosomes. Reeves (5) showed recently that amino acids do in fact accumulate against a high concentration gradient when lysosomes are incubated with sublytic concentrations of 3H-labeled amino acid methyl esters. Amino acid accumulation under these conditions was shown to be a specific property of lysosomes (5).The high permeability of biological membranes for amino acid methyl esters and the specificity of amino acid accumulation for lysosomes suggest that lysosomes in intact cells might become swollen and disrupted if the cells were exposed to millimolar concentrations ofan amino acid methyl ester. We chose heart tissue to test this hypothesis because of our long-standing interest in delineating the contributions oflysosomes to protein degradation and to the effects of ischemia in this tissue. In this report, we show that perfusion of rat hearts with 10 mM L-leucine methyl ester (Leu-OMe) causes loss of lysosomal integrity without damage to other cellular organelles. The lysosomal dysfunction is accompanied by a 30% decline in the rate ofprotein degradation, as measu...

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization

“…This suggests that SKI-1 localizes in the Golgi but may sort to other organelles, including lysosomal and͞or endosomal compartments. An indication of lysosomal͞ endosomal localization was provided by the analysis of SKI-1 immunofluorescence within cells preincubated for 4 h with 10 mM leucine methyl ester, a specific lysosomal͞endosomal protease inhibitor (25). The results showed a net increase in the proportion of cells exhibiting punctate staining as compared with control cells (Fig.…”

Regulation of cellular function by products of lysosomal enzyme activity: elimination of human natural killer cells by a dipeptide methyl ester generated from L-leucine methyl ester by monocytes or polymorphonuclear leukocytes.

“…Furthermore, these compounds cause lysosomal disruption in isolated subcellular organelles (7,8), intact cells (6), and whole organ preparations (9). Such an action explains the loss of lysosomal function within cells exposed to amino acid methyl esters (9). In particular, the lysis of tumor cell targets by NK is dependent on intact lysosomal function (13) and can be inhibited by a wide variety of amino acid methyl esters (1).…”

“…As is the case for the majority of weakly basic alkylamines, methyl esters of amino acids and dipeptides are lysosomotropic (6)(7)(8)(9)12). Furthermore, these compounds cause lysosomal disruption in isolated subcellular organelles (7,8), intact cells (6), and whole organ preparations (9).…”

“…PBM lymphocytes (1,6). Amino acid methyl esters are known to be lysosomotropic compounds (7)(8)(9), and in previous studies it was found that the lysosomal inhibitors chloroquine and NH4Cl prevented Leu-OMe-induced MO toxicity (6) le-OMe this system whereas the supernatants or sonicated-cell extracts of Leu-OMe-treated PMN or MO also depleted NK from MO-depleted lymphocytes. These results therefore suggested that interaction of Leu-OMe with the lysosomal compartment of MO or PMN produced a product that was directly toxic to NK through a mechanism that was no longer dependent on lysosomal processing within either the NK or an additional cell type.…”

Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment.

“…To test this, we conducted pulse-chase studies of 8 in 21.2.2 and 2114 cells in the absence and presence of several inhibitors of lysosomal function: 50 mM NI-hCI, 20 mM methionine methyl ester (MME), 100 IxM chloroquine, 100 ~tg/ml leupeptin, and 7 pg/ml monensin (Wibo and Poole, 1974;Livesey et al, 1980;Seglen, 1983;Rote and Rechsteiner, 1983;Ohkuma and Poole, 1981;Poole and Ohkuma, 1981;Reeves et al, 1981;Tartakoff, 1983;Libby and Goldberg, 1978). Fig.…”