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Glycyl-D-phenylalanine-2-naphthylamide (Gly-D-Phe-2-NNap) is a cytotoxic agent as exemplified by its effect on Vero cells in culture. This effect is inhibited to some extent by nigericin. On the other hand, Gly-D-Phe-2-NNap induces an increase of free activity of N-acetylglucosaminidase when incubated with a mitochondrial fraction of rat liver at pH 7.5. The phenomenon is inhibited by chloroquine, NH4Cl and nigericin, substances that are known to increase the intralysosomal pH. The latency of enzymes located in other subcellular structures - mitochondria, peroxisomes and endoplasmic reticulum - is not affected by Gly-D-Phe-2-NNap. Moreover, that compound does not cause a release of FITC-Dextran present in endosomes. Apparently Gly-D-Phe-2-NNap is a specific lytic agent for lysosomes. It is proposed that the molecule behaves like a lysosomotropic substance that is able to attack the lysosomal membrane from the interior of the organelle. Its cytotoxic properties could be explained by its effect on lysosomes.

When subjected to increasing pressure at 0 degree C, rat liver mitochondria become permeable to sucrose, causing them to swell and their outer membrane to rupture. Afterwards they are lysed and their matrix content is released into the medium. This permeation to sucrose may be prevented to some extent by increasing the temperature at which compression is carried out. 0.75 mM imipramine protects mitochondria against lysis caused by hydrostatic pressure, but does not oppose their permeation to sucrose nor the swelling resulting from the compression. At this concentration, the drug does not exhibit a significant effect on the lateral phase separations which take place in the inner mitochondrial membrane under pressure. The mitochondria of rat fetal liver (21 days), kidney and Morris hepatoma 16 become permeable to sucrose when they are subjected to compression; under these conditions, lateral phase separations occur in their inner membrane. Contrary to liver mitochondria, the former do not undergo lysis. Taking into account both present and previous results, events leading to mitochondrial membrane deterioration by hydrostatic pressure may be summarized in the following way. Pressure first leads to a phase transition of the membrane lipids, thus causing a permeation to sucrose; as a result the mitochondria swell because they have absorbed osmotic water. The membrane lipids freeze increasingly as the pressure increases; the inner membrane becomes fragile and finally, in the case of the adult liver organelles, can no longer resist the swelling. All these events can be avoided by increasing the temperature; imipramine only prevents inner membrane lysis.

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium. Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0 degrees C, when the pressure reaches 750 kg/cm2. Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal lead to gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes. These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.

When injected into rats, a certain amount of mannitol is taken up by the liver and is associated with sedimentable structures. Isopycnic centrifugation in a sucrose gradient shows that a large part of mannitol is present in mitochondria, what remains is located in the lysosomes. The hypotonic release of mannitol present in organelles shows that the polyol is shared between mitochondria and lysosomes. The trapping of mannitol in lysosomes could result from the heterophagic or autophagic function of the lysosomes; the mechanism of its accumulation in mitochondria is still unexplained.

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4',4'-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0 degree C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.

When rat-liver mitochondria are subjected to compression at 0 degrees C, they become permeable to sucrose and the latency of malate dehydrogenase disappears. Freeze fracture electron microscopy shows smooth areas devoid of particles in fracture faces of the membranes. The effects of pressure can be inhibited as the temperature increases. Such results are explained by supposing that compression increases the transition temperature of mitochondrial membrane lipids above 0 degrees C and causes their freezing.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.

The effects of pressure on late fetal and neonatal rat liver mitochondria have been investigated. High hydrostatic pressure, as produced by isopycnic centrifugation in sucrose and glycogen gradients, altered the mitochondrial membranes of 1- and 7-day-old rats. Most of the mitochondrial enzymes, chosen for their known submitochondrial location, had a trimodal distribution in the sucrose gradients. In the glycogen gradients, a shift of the mitochondria to a lower density was noticed. Fetal liver mitochondria were resistant to the hydrostatic pressure exerted during isopycnic centrifugation experiments under different conditions such as sucrose and glycogen density gradients. The submitochondrial compartment tracer enzymes exhibited an unimodal distribution. Experimental temperatures set at 15 degrees C had a protective effect from hydrostatic pressure alterations in the neonatal liver mitochondria, whereas no effects were noticeable in the fetal mitochondria. Experiments in a hydraulic compression chamber showed that outer membranes of fetal mitochondria were more fragile and the inner membranes more resistant to compression than in the early stages after birth.

We have investigated the effect of the centrifugation speed on the behavior of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium. The gradient was made with a macromolecular compound, glycogen dissolved in 0.25 M aqueous sucrose. The distribution curves of several mitochondrial enzymes change when the centrifugation reaches a certain speed: they are shifted toward regions of lower density. The results are plausibly explained by supposing that the inner mitochondrial membrane becomes permeable to sucrose at high centrifugation speeds, and that the granules swell. The main causal agent of the phenomenon is the hydrostatic pressure the mitochondria are subjected to during centrifugation. Morphological observations show that mitochondria are markedly deteriorated when centrifuged at high speed in the glycogen gradient; they are swollen and the outer membrane is broken; also frequently, a large electron-dense granule is seen in the matrix near the inner mambrane.