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...

In cardiac muscle, moderate degrees of hyperosmolality of the type encountered physiologically or clinically (i.e., less than 200 mosM above control) characteristically exert a positive inotropic effect, which presumably is mediated by increased Ca2+ availability for binding to troponin. In contrast, skeletal muscle displays significant contractile depression on exposure to hyperosmotic solutions, even at mild degrees of hypertonicity. To determine whether a similar potential for hyperosmolarity-induced depression also exists in cardiac muscle, right ventricular papillary muscles from cats were exposed to hypertonic solutions of mannitol or sucrose under circumstances in which positive inotropic effects were precluded by prior exposure to a bathing solution of 4.0 mM Ca2+ and paired electrical stimulation to maximize intracellular Ca2+ before addition of the hyperosmotic substances. In contrast to their usual positive inotropic effects, hypertonic solutions under these conditions caused cardiac depression at all osmolarities tested. Developed tension and its maximal rate of development (dT/dt) decreased by 18% at 50 mosM above control, by 30% at 100 mosM, by 36% at 150 mosM, and by 42% at 200 mosM (P less than 0.01 for all). Time to peak tension and resting tension were not changed significantly. When the muscles were returned to control solutions, tension development also returned toward normal. The data are compatible with the hypothesis that, within the range tested, all degrees of hyperosmolarity exert a significant negative inotropic influence on cardiac muscle, as is true in skeletal muscle; manifestation of this effect of increased tonicity normally would be obscured at low degrees of hyperosmolality, however, by an overriding positive influence that is absent in skeletal muscle.

A B S T R Apacing persisted in causing marked increases in developed tension and dT/dt even in the presence of D600, suggesting that their inotropic effects are not dependent on increased intracellular transfer of calcium during systole through cell membrane channels in which D600 acts as a competitive inhibitor.The results of these studies suggest that apparent functional saturation of intracellular calcium receptor sites eliminates any additional inotropic effect of hyperosmolality or paired pacing. The data are compatible with the hypothesis that the inotropic effects of hyperosmolality and of paired pacing result from an increase in calcium concentration at the myofilaments during contraction. The increase induced by hyperosmolality might occur because of an increase in the total amount of calcium released into the cytosol with each action potential and/or as a passive consequence of cellular dehydration. Norepinephrine has the capacity to increase contractility even when intracellular calcium receptor sites appear to be functionally saturated, suggesting that it may act at least in part by a mechanism that is independent of changes in net intracellular calcium concentration.

The direct effects of L-and D-triiodothyronine (T3) on cardiac protein metabolism were investigated using fetal mouse hearts in organ culture. This model allowed the production of "thyrotoxicosis" in isolated hearts in vitro in the absence of the usual systemic metabolic and hemodynamic effects of thyroid hormones. Hearts were studied during the first 24 h of T3 exposure in culture, before changes in beating rate due to T3 occurred. Phenylalanine release was decreased by 26 +/- 2.3% (P less than 0.001) by the optimal concentrations of T3 (10(-7) to 10(-6) M). Changes were similar in the presence or absence of insulin. D-T3 was also anabolic, decreasing phenylalanine release by 24 +/- 2.5% (P less than 0.001) at concentrations of 10(-6) to 10(-5) M. The L-isomer increased protein synthesis by 23 +/- 6.8% (P less than 0.05) and decreased protein degradation, as measured by phenylalanine release in the presence of cycloheximide, by 5 +/- 1.6% (P less than 0.01). The D-isomer also increased protein synthesis but had no measurable effect on protein degradation. We conclude that thyroid hormones can exert direct anabolic effects on heart in the absence of systemic hemodynamic and metabolic changes. These effects are mediated primarily through an acceleration of the rate of protein synthesis; in the case of L-T3, a small inhibition of proteolysis may also occur.

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