Lysosomal degradation of cell organelles. I. Ultrastructural analysis of uptake and digestion of intravenously injected mitochondria by Kupffer cells.

An experimental model comparable to autophagocytosis is presented to evaluate lysosomal degradation of biologic membranes in vivo. This design takes advantage of the efficient phagocytic capacity of Kupffer cells. For this purpose, mitochondria were isolated from one rat liver (inbred strain) and subsequently injected intravenously into a series of rats, which were sacrificed at various time points. The uptake and intralysosomal digestion were examined by electron microscopy. Mitochondria were seen in the sinusoids and attached to the surface of Kupffer cells 1 to 3 minutes after injection. One or two mitochondrial profiles were trapped in cavelike structures often embraced by long hyaloplasmic pseudopods. By 5 and 10 minutes, the mitochondria were in single membrane-bound phagosomes within the Kupffer cells and early signs of mitochondrial alterations were present, e.g., loss of cristae and condensation. At 30 minutes and 1 hour the mitochondria were seen in large vacuoles which contained up to 20 profiles in a single section. The mitochondria showed evident signs of digestion such as membrane fragmentation and flocculent densities. By 4 hours and especially by 8 hours, most digestive vacuoles were laden with flocculent membrane debris. By 24 hours, the Kupffer cells showed multiple lysosomal structures which were irregular in shape and small. These often contained a number of lipid-like droplets of various electron densities and pentalaminar structures. By 2 and 5 days, the Kupffer cells had returned to their normal appearance although the lysosomal apparatus was still prominent. No uptake of mitochondria was seen in endothelial cells, fat-storing cells, or hepatocytes. It is concluded that lysosomes have an efficient capacity to digest mitochondria. Some lipid remnants are not completely or slowly degraded by Kupffer cell lysosomes but remain in "residual bodies" for up to 1 or 2 days.     

Lysosomal degradation of cell organelles. II. Ultrastructural analysis of uptake and digestion of intravenously injected microsomes and ribosomes by Kupffer cells.

Rough and smooth microsomes, "mixed" or total microsomes, and ribosomes were isolated from one single rat liver and subsequently injected intravenously into a series of inbred rats. The uptake and the degradation of the injected organelles by Kupffer cells were followed by means of electron microscopic analysis. By 1 minute after injection, microsomes were seen attached to the surface of Kupffer cells separated by a gap of 200 to 300 A. No attachment to hepatocytes, fat-storing cells, or endothelial cells was seen. By 5 and 10 minutes, most microsomes were phagocytosed and sequestered in large numbers within single membrane-enclosed vacuoles or phagosomes. The engulfment proceeded by two mechanisms: (1) most frequently, flaplike processes of cytoplasm embraced aggregates of microsomes, concomitant with the formation of indention of the cytoplasm; (2) occasionally, single microsomal profiles were taken up by bristle-coated endocytic vacuoles. Ribosomes were also seen penetrating into the wormlike structures (micropinocytosis vermiformis) at the cell surface. At 30 minutes after injection, clear signs of alteration were noted starting with vesicle aggregation, clumping, and elongation of the microsomal profiles. The ribosomes were quickly stripped from their microsomal membranes and marginated to the inside of the vacuoles but separated from the limiting membrane by a distance of 200 to 300 A. By 1 and 2 hours, disruption of the vesicles into membrane fragments and formation of dense material in and between the profiles occurred. By 8 hours it was difficult to recognize the degradation products as membrane derivatives. The digestive vacuoles retained their size at this time interval. Typical pentalaminar structures were observed. By 14 to 24 hours the digestive vacuoles became electron lucent and appeared to shrink, and in addition to containing various types of granular material, many were laden with lipid-like droplets presumed to be conglomerates of phospholipid remnants. Rough microsomes, when compared to smooth microsomes, gave rise to more granular material within the digestive vacuoles. Ribosomes were still identifiable 24 hours after injection, indicative of a somewhat slower rate of degradation. Accumulation of various types of lipid-like droplets in the "residual bodies" was typical after microsomal injections. It is concluded that although microsomes appear to be phagocytosed at a quicker rate than mitochondria, they are digested within the lysosomal apparatus of the Kupffer cells at a somewhat slower rate. This especially seems to be the case for ribosomes. Heterophagy of microsomes is one source of residual bodies.     

Modification of lysosomal proteolysis in mouse liver with taxol.

Perturbation of lysosomal degradation pathways by drugs such as chloroquine and vinblastine has given us important insights into the means for segregation and degradation of cytoplasm by the lysosomes. Vinblastine (VBL) is a microtubule depolymerizer which increases the number of autophagosomes and also impairs the maturation of the autophagosomes into secondary lysosomes. The authors have tested the effects of the microtubule stabilizer taxol on the proteolytic activity of the lysosomal system in VBL-treated mice. Liver proteins of CD-1 mice were radioisotopically labeled. Taxol and/or vinblastine were subsequently administered. Proteolysis in liver homogenates was determined during in vitro incubation by measuring the release of trichloroacetic acid-soluble radioactivity over time. Taxol enhanced proteolysis in homogenates from VBL-treated mice. The site of the enhanced proteolysis was lysosomal, because it was demonstrated in a crude lysosomal fraction, it manifested an acid pH optimum, and it could be suppressed with cycloheximide. Morphometric analysis indicated that taxol caused a shift in hepatocyte lysosomes of VBL-treated mice toward the later stages of the degradation cycle, coupled with a decrease in the number of small dense body profiles. The results indicate that taxol enhances the maturation of the autophagosomes into proteolytically active secondary lysosomes. It is concluded that the microtubule stabilizer taxol enhances fusion between VBL-induced autophagosomes and lysosomes (small dense bodies) and does not affect the segregation of cytoplasm into autophagosomes.     

Gentamicin nephrotoxicity in rats. I. Acute biochemical and ultrastructural effects.

This investigation characterizes the acute biochemical and ultrastructural alterations within the rat kidney following the single injection of 10, 40, 80, or 160 mg. of gentamicin per kilogram of rat weight. Gentamicin was given intraperitoneally and rats were killed 80 minutes later. To assess a possible inhibitory effect upon protein synthesis 3H-leucine was injected 20 minutes postantibiotic and renal cortex was assayed for whole tissue and the protein fraction amino acid uptake. To exclude the possibility of gentamicin-induced lysosomal membrane instability with subsequent release of acid hydrolases, the activity of acid phosphatase was assayed in the supernatant and the residue of cortical homogenates containing the lysosomal fraction. Ultrastructural changes were concomitantly studied. To determine the intracellular localization of gentamicin, levels of the antibiotic were measured in subcellular fractions of cortical homogenates obtained from rats 2 hours following a single subcutaneous injection of 20 mg. of gentamicin per kilogram of rat weight. No gentamicin-induced alterations either of protein synthesis or of acid phosphatase distribution were demonstrated. Ultrastructural changes were most marked at 160 mg. per kg. consisting mainly of dilation of the endoplasmic reticulum, altered mitochondria, and increased cytosegresomes. Significant quantities of gentamicin were distributed within the nuclear, mitochondrial, and microsomal fractions. These studies indicate that although cytoplasmic alterations are prominent within the proximal tubular epithelial cells, they are not likely the result of either inhibition of protein synthesis or release of acid phosphatase from lysosomes.     

Response of autophagic protein degradation to physiologic and pathologic stimuli in rat hepatocyte monolayer cultures

The lysosomes of hepatocytes increase in numbers and size during acute cell injury in vivo. To elucidate the mechanism of this change, we have studied in vitro the response of the autophagic lysosomal system to several physiologic mediators of autophagy, and to agents known to induce injury and/or the accumulation of lysosomes in vivo. To this end, monolayer cultures of rat hepatocytes were labeled with [14C]leucine to measure hepatocyte protein degradation; ultrastructural analyses were carried out to measure the volume fraction of lysosomes in the hepatocytes. Dibutyryl cyclic AMP increased protein degradation in the hepatocytes either in the presence or absence of serum and insulin. Deprivation of serum and insulin also increased hepatocyte protein degradation. Morphometric analysis indicated parallel increases in the volume fraction of lysosomes in the hepatocytes. The calcium ionophore ionomycin (5 microM), in the presence of 1.3 mM extracellular calcium, increased protein degradation (but not the volume fraction of lysosomes), and this increase was abolished by the addition of ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. On the other hand, vasopressin (5 nM) caused an increase in protein degradation coupled with an increase in volume fraction of lysosomes. The microtubule depolymerizer vinblastine decreased protein degradation. The microtubule stabilizer taxol did not prevent the inhibitory effects caused by vinblastine. Parallel decreases in the lysosomal compartment were found in the hepatocytes exposed to vinblastine or taxol. Dimethylnitrosamine inhibited protein degradation as well as decreased the volume fraction of lysosomes. Finally, carbon tetrachloride also decreased protein degradation. These data indicate that in physiologic conditions, increases in numbers of hepatocyte lysosomes reflect increased sequestration and degradation of cytoplasmic proteins in response to changes in the levels of hormones, serum factors and nutrients as well as cyclic AMP. The induction of acute cell injury in vitro by calcium ionophore, microtubule active agents, and hepatotoxins inhibits lysosomal proteolysis and causes a decrease in the volume fraction of lysosomes. We conclude that the increase in lysosomal size and numbers occurring in acutely injured hepatocytes in vivo is induced primarily by altered levels of nutritional and hormonal regulators of lysosomal protein degradation.     

Lysosomal alterations in hypoxic and reoxygenated hearts. I. Ultrastructural and cytochemical changes.

Rabbit hearts perfused under hypoxic conditions underwent progressive subcellular damage, which becomes irreversible by one hour. During the first 20 minutes of perfusion, minor dilation of mitochondria and condensation of nuclear chromatin were the only salient features of cell injury. By 40 minutes moderate mitochondrial swelling was evident in hypoxic myocytes. Moreover, an increase in degenerating mitochondria and autophagic vacuoles was apparent. Reperfusion after either 20 or 40 minutes of hypoxia restored contractility, and injured myocytes underwent a cellular repair process that involved a dramatic increase in lysosomal autoplagy. One hour of hypoxia yielded irreversibly injured myocytes. Upon reoxygenation, some of these cells displayed typical changes of necrosis, but others apparently underwent an abortive repair process involving the formation of large, probably nonfunctional lysosomes. These observations suggest that lysosomal autophagy is important in the efforts at repair that cardiac cells initiate during and after hypoxia.     

Cytochemical localization of lysosomal enzyme activity in normal and ischemic dog myocardium.

The effect of ischemia on the integrity of myocardial lysosomes was observed 3 1/2 and 24 hours after the production of infarcts in 20 anesthetized closed-chest dogs by electrically induced thrombosis of the left anterior descending coronary artery. Biopsies from normal, marginal and infarcted areas were fixed and incubated to localize the lysosomal enzymes acid phosphatase and aryl sulphatase. Reaction product in normal cells was localized in small circular or oblong profiles between bundles of myofilaments and adjacent to mitochondria. In addition, curvilinear, membrane-bound profiles containing reaction product were found in close apposition to transverse tubules and near the free margins of the myocardial cells. Thus the distribution of elements of the sarcoplasmic reticulum. Additional reaction product was also seen in residual bodies, on myelin figures, and in the few conventional appearing spherical lysosomes. Little or no acid phosphatase or aryl sulphatase reaction product was seen in the sarcoplasmic reticulum of infarcted myocardium. The degree of cellular degeneration correlated with disappearance of enzyme activity from the sarcoplasmic reticulum and included disruption of membranes and loss of mitochondrial matrix and erosion of I but not A bands. Marginal areas showed variable amounts of cellular degeneration. Separation of myofilament bundles and loss of glycogen correlated with the localized disappearance of acid phosphatase and aryl sulphatase activity in marginal tissue. Disruption of mitochondrial and erosion of I bands correlated with extensive loss of these enzymes. The data suggest that degeneration of myocardial cells following ischemic injury is associated with release of endogenous lysosomal enzymes from the sarcoplasmic reticulum.     

Uptake and degradation of glycogen by Kupffer cells.

Rat liver glycogen was isotopically labeled with [¹⁴C] glucose and isolated. The isolated glycogen was injected intravenously into a series of rats and its vascular clearance, uptake and degradation in liver was analyzed by means of labeling and ultrastructural techniques. Injected glycogen was quickly removed from serum with a $\text{t}\text{1}\text{2}$ of less than 15 min. Glycogen particles, identified in the electron microscope, were never seen to attach to the surface of Kupffer cells or hepatocytes. These particles appeared to be taken up by Kupffer cells by nonspecific pinocytosis “fluid endocytosis” e.g., as a solute with engulfed liquid. By 10 min the particles were present within single membrane bound vacuoles of Kupffer cells. At this early time point, the vacuoles did not seem to have fused with preexisting prelabeled secondary lysosomes containing ferritin. At later time points, glycogen particles were seen intermingled with ferritin. By 1, 2, and 4 hr, increasing numbers of vacuoles containing granular material believed to represent glycogen were observed. These vacuoles often showed extensive enlargement (“swelling”). By 24 hr, most glycogen particles had disappeared and granular material was prresent only in occasional lysosomes which no longer appeared swollen. The estimated half-life for glycogen in Kupffer cell lysosomes was in the range of 12 to 16 hr. This is considerably longer than for membrane proteins and lipids introduced into Kupffer cell lysosomes by means of heterophagy. Because of possible reutilization of isotope it was difficult to define the half-life of glycogen more exactly. It is concluded that glycogen is degraded in Kupffer cell lysosomes, although at a relative slow rate, in comparison with the capability of lysosomal hydrolases to digest proteins and lipids. This conclusion is in line with the general notion that glycogen degradation takes place in the cell sap and is not primarily associated with any particular organelle.     

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Subunit c is normally present as an inner mitochondrial membrane component of the F₀ sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid-lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. The rate of degradation of subunit c as measured by pulse–chase and immunoprecipitation showed a marked delay of degradation in patients' fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes through an autophagic process. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. © 1995 Wiley-Liss, Inc.     

Involvement of cathepsins B and H in lysosomal degradation of horseradish peroxidase endocytosed by the proximal tubule cells of the rat kidney: II. Immunocytochemical studies using protein A-gold technique applied to conventional and serial sections

Immunoelectron microscopic localizations of cathepsins B and H and injected horseradish peroxidase (HRP) in the lysosomal system of rat kidney proximal tubules were investigated by a protein A-gold technique. Kidneys were fixed by perfusion at fixed intervals after intravenous injection of HRP. At 5 to 15 minutes after the injection, the endocytic apparatus--including the apical vesicles, tubules, and vacuoles (endosomes)--were stained for HRP, but they were negative for cathepsins. Within 15 to 30 minutes after the HRP injection, HRP-containing endosomes were fusing with preexisting lysosomes. In the S1 segment, they accumulated in the apical cytoplasm and formed giant phagosomes, which increased markedly in number and size after 1 hour. These phagosomes were composed of a peripheral clear matrix and electron-dense inclusions. The clear matrix was stained heavily for cathepsins and HRP, whereas the electron-dense inclusions were consistently negative for cathepsins and HRP. The same results also were obtained after the double-labeling and serial sectioning techniques. The dense inclusions were fragmented gradually as the phagosomes decreased in size. After 3 hours, the size and number of phagosomes returned to their normal state (before the HRP injection). These results indicate that the endocytic apparatus of the proximal tubule cells does not contain cathepsins. Phagosomes are formed by the fusion of endosomes containing the internalized protein with the preexisting lysosomes. The degradation of HRP in giant phagosomes occurred rapidly. The coexistence of cathepsins B and H with the endocytosed HRP suggests that these cystein proteinases are involved in the degradation of protein in heterophagosomes of the proximal tubule cells.     

Morphometric and cytochemical analysis of lysosomes in rat Peyer's patch follicle epithelium: their reduction in volume fraction and acid phosphatase content in M cells compared to adjacent enterocytes

M cells are specialized epithelial cells over lymphoid follicles in Peyer's patches which take up viruses, bacteria, and antigenic macromolecules from the intestinal lumen. Unlike ordinary enterocytes which sequester pinocytosed material in lysosomes, M cells transport such material across the epithelium to antigen-processing areas in lymphoid follicle domes, suggesting a difference in lysosomal activity or a different route for movement of endocytic vesicles. Ileal Peyer's patches in rats were examined by electron microscopy to identify lysosomes by acid phosphatase activity. Acid phosphatase was found in dense bodies in enterocytes but not in M cells. Stereological analysis showed the volume fraction occupied by dense bodies in M cells to be 16 times less than in enterocytes (P less than .0005), even though the volume fractions of cytoplasm occupied by mitochondria in M cells and enterocytes were not significantly different. The small volume fraction of dense bodies and the absence of acid phosphatase activity in M cells thus correlate with absence of lysosomal degradation of luminal microorganisms during transport into lymphoid follicles by M cells and may provide not only a complete array of microbial antigens for initiation of immune responses, but also a route through the mucosal barrier for microorganisms which can evade local containment mechanisms.     

Defective Function of Renal Lysosomes in Mice with the Chediak-Higashi Syndrome

Morphologically abnormal lysosomes demonstrated in individuals with the Chediak-Higashi syndrome (CHS) suggested a defect in the function of these abnormal lysosomes. To gain direct experimental evidence of such a defect, horseradish peroxidase (HRP) was injected intravenously into CHS and control mice, the mice killed at varying intervals and the kidneys studied by ultrastructural cytochemistry. No morophologic difference was observed in the absorption and uptake of HRP by proximal convoluted tubules in the two groups of mice. In CHS mice, however, some of the HRP fused with enlarged lysosomes. By 48 hours after injection, the lysosomes of normal mice had digested all but trace amounts of HRP, whereas large amounts were still present in CHS mice at this time. In CHS mice, moderate amounts were still present at 72 hours and trace amounts 96 hours post injection. This slowed rate of digestion of HRP by lysosomes of the proximal convoluted tubule cells of CHS mice suggests a similar defect in all cells in CHS individuals in which there is a lysosomal degradation of protein or other matter obtained by endocytosis. Such a defect may explain some manifestations of impaired host defense observed in CHS.     

Acute hemorrhagic pancreatic necrosis in mice: the activity of lysosomal enzymes in the pancreas and the liver.

The activity of lysosomal enzymes of the pancreas and the liver has been studied during induction and onset of acute hemorrhagic pancreatic necrosis with fat necrosis (AHPN) in mice. We induced AHPN by feeding the animals a choline-deficient (CD) diet containing 0.5% DL-ethionine (CDE). Control animals were fed either laboratory chow or a plain CD DIET. Increased total activities of cathespin B1, beta-galactosidase, and acid phosphatase were found to occur in pancreas homogenates of mice fed the CDE diet for 2 and 3 days. Release of cathespin B1 into pancreas cytosol was observed after 1 day of feeding. beta-galactosidase and acid phosphatase were increased in pancreas cytosol after 2 and 3 days of feeding. Changes in total activity and location of the lysosomal enzymes did not occur in the liver. Feeding the CD and CDE diets resulted in an increase in the free activity of lysosomal enzymes of both the pancreas and the liver, suggesting the existence of alterations in the lysosomal membrane. Pancreas and liver homogenates were stored on ice up to 3 hours, and the free activity of acid phosphatase and beta-galactosidase were determined at various time intervals. The free activity of both enzymes increased progressively for 3 hours in the pancreas but not in the liver. It is concluded that: 1) induction of AHPN in mice is accompanied by an increase in the activity of lysosomal enzymes of the acinar cells of the pancreas; 2) cathepsin B1 may be responsible for triggering an intraparenchymal activation of zymogens, and 3) pancreatic lysosomes are labilized more easily than liver lysosomes.     

The effect of thyroid antigens on the in vitro migration of leucocytes from patients with Hashimoto thyroiditis

A total of fifty-two patients with Hashimoto thyroiditis were tested for delayed hypersensitivity to thyroid antigens using the leucocyte migration test. The percentage of patients showing abnormal migration in the presence of crude thyroid extract, thyroglobulin, thyroid mitochondria and thyroid microsomes was 75, 44, 54 and 34% respectively. Fifty-three control patients were studied concurrently with the same antigens and the percentage showing abnormal migration was 4, 6, 6 and 6% respectively. The antigenic activity of the mitochondrial fraction was not organ specific; both liver and kidney mitochondria interfered with the migration of leucocytes from patients with Hashimoto thyroiditis.     

Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermediate voltage electron microscopy was used to examine monocyte-derived macrophages (monocyte/macrophages) during early in vitro uptake of beta migrating very-low-density lipoproteins (beta VLDL). Lysosomes were identified using acid phosphatase cytochemistry, and in control cells these organelles constituted 3.5% of the total cytoplasmic volume. Both primary and secondary lysosomes were observed. Upon beta VLDL uptake, the total volume of acid-phosphatase-positive organelles increased threefold over 30 minutes, and the reaction product was found in three additional morphologically distinct structures: tubular lysosomes, membrane stacks, and endoplasmic reticulum with widened cisternae. The proportion of the cell occupied by each of the five acid-phosphatase-positive organelles was quantitated at 10 minutes, 30 minutes, 1 hour, and 4 hours of beta VLDL incubation, and their relative abundance was compared with controls that were processed either with no lipoprotein challenge or albumin incubation for 1 hour. Secondary lysosomes compartment volume peaked at 30 minutes; over the ensuing 3.5 hours, however, the reaction progressively shifted to three new membrane-limited locations. Our observations document the complex 3D organization and spacial relationships among the acid-phosphatase-positive structures induced by lipoprotein uptake. The 3D organization patterns for acid-phosphatase-positive lysosomes in lipoprotein-stimulated pigeon monocyte/macrophages were similar in several aspects to the complex lysosomes previously observed in the macrophages of pigeon arterial lesions.     

Intracellular localization of the copper-zinc and manganese superoxide dismutases in rat liver parenchymal cells

Both the copper-zinc (CuZn) and the manganese (Mn) containing superoxide dismutases (SOD) have been immunolocalized in rat liver sections using protein A-gold labeling on ultrathin cryosections. The CuZnSOD was found to distribute uniformly throughout the nuclear and cytoplasmic matrix. The CuZn enzyme was excluded from membrane-bound compartments such as the nuclear envelope, endoplasmic reticulum, Golgi elements, secretory vesicles, and mitochondria. The primary exception was that lysosomes frequently labeled heavily for CuZnSOD. The lysosomal nature of these membrane-bound bodies was confirmed by double immunolabeling with a lysosomal enzyme, cathepsin-D. The MnSOD was located in mitochondria, particularly the matrix between the cristae. Light but distinct labeling for the MnSOD occurred in the cytoplasmic matrix.     

Uptake--microautophagy--and degradation of exogenous proteins by isolated rat liver lysosomes. Effects of pH, ATP, and inhibitors of proteolysis

Isolated rat liver lysosomes were incubated with [14C]methemoglobin under various conditions. Optimal pH for the in vitro proteolysis was found to be 4-5. To evaluate whether or not degradation of added proteins could be due to enzyme leakage the integrity of the lysosomes was measured. Isolated lysosomes were found to be stable for up to 10 min of incubation at pH 5.5 and for 30 min at pH 7. The degradation of three different proteins (methemoglobin, ovalbumin, and lysozyme) was analyzed. No correlation was detected between rate of breakdown and physical properties of the proteins. Leupeptin, chloroquine, and propylamine inhibited proteolysis of added proteins by 45-65% in both neutral and acid milieu. Possible energy requirement was tested by the addition of Mg2+ and ATP to the incubation medium. A dose-dependent increase in proteolytic rate was found when ATP was added to the lysosomal suspension, a finding most likely due to acidification of the lysosomes and ensuing increased degradation. GTP and ITP were somewhat less effective. The noncleavable ATP analogue 5'-adenylylimidodiphosphate gave no stimulation. The ATP-driven proteolysis was inhibited by ethylmaleimide. Isolated lysosomes were also incubated with ferritin in order to visualize a possible uptake process of a protein in the electron microscope. Following incubation, ferritin particles were seen inside intralysosomal vesicles which appeared to be formed by invagination of the lysosomal membrane, a process designated microautophagy. The results thus support the notion that isolated lysosomes may micropinocytose and degrade exogenously added proteins and that this process is ATP dependent.     

Effect of gastric antigens on the in vitro migration of leucocytes from patients with atrophic gastritis and pernicious anaemia

A total of eighteen patients with pernicious anaemia (PA) ten patients with atrophic gastritis and achlorhydria but without PA, and fourteen control subjects were tested for delayed hypersensitivity to gastric antigens using the leucocyte migration test. The percentage of PAs showing inhibition of migration in the presence of a crude extract of gastric mucosa, liver mitochondria, stomach mitochondria and stomach microsomes was 50, 55, 50 and 50% respectively. The results in atrophic gastritis were 0, 20, 20 and 0% respectively and those in the fourteen control subjects were 0, 14, 7 and 14% respectively.

There was a significant difference between PA and controls with all four antigens. There was a significant difference using crude extract and stomach mitochondria and microsomes between PA and atrophic gastritis but not with liver mitochondria. There was no significant difference between atrophic gastritis and controls although the results tended to be midway between PA values and controls.

These results indicate that cell-mediated immunity is more strongly implicated in PA than it is in atrophic gastritis which has not progressed to PA.

     

Degradation of short- and long-lived proteins in perfused liver and in isolated autophagic vacuoles--lysosomes

The lysosomal contribution to breakdown of prelabeled short- and long-lived cell proteins in the perfused rat liver was monitored in two ways. In the first, either leupeptin or chloroquine was injected into rats as well as added to the perfusate. The extent of suppression of proteolysis compared to that of controls was equated with the lysosomal share of protein breakdown. Both compounds inhibited degradation of short-lived proteins by some 30% and long-lived proteins by some 60%. In the second approach, lysosomal-autophagic vacuolar (LAV) fractions were isolated from livers pretreated and perfused as above. The LAV fractions generated different amounts of degradation products during subsequent incubations. They were, in decreasing order of magnitude, that of the LAV fraction isolated from livers perfused with chloroquine, without inhibitor, and with leupeptin. The LAV fraction from control livers was considered to yield the most reliable estimate of the lysosomal share of proteolysis as recorded during liver perfusions. It was 54% for short-lived proteins and 75% for long-lived proteins. These figures are higher than those obtained in the perfusion experiments in the first approach. It is therefore suggested that quantitative assessment concerning the lysosomal share of overall proteolysis may be underestimated when based on inhibition experiments performed on intact cells. The ultrastructure of hepatocytes and LAV fractions was also investigated. Leupeptin-treated hepatocytes displayed more frequent and larger AVs than those of control livers. Cell vacuolation was an additional characteristic of chloroquine-exposed hepatocytes. The LAV fractions isolated from control and leupeptin-treated livers mirrored the observations made in liver tissue. The constituents of the LAV fraction isolated from chloroquine-treated tissue were, however, less swollen than in situ, probably due to the extraction of water during isolation.     

Effects of mercury on lysosomal protein digestion in the kidney proximal tubule.

The effect of mercury on renal lysosomal protein digestion was studied after administration of mercury in vitro and in vivo. Mercuric chloride or methylmercury chloride was added in vitro to lysosomal enzymes isolated from normal rats, and subsequently, digestion experiments were carried out using 125I-labeled lysozyme or cytochrome c as substrate proteins. Both mercury compounds produced a concentration-dependent inhibition of the degradation of the proteins, mercuric chloride being the strongest inhibitor. Mercuric chloride was also administered to rats in vivo for 5 to 8 months. Renal lysosomal enzymes from these animals also had a decreased ability to digest for the two substrate proteins. Furthermore, the digestion of lysozyme intravenously injected into mercury-intoxicated rats was decreased in renal cortical slices incubated in vitro. Electron microscope autoradiography showed that intravenously injected labeled lysozyme was located primarily over lysosomes in proximal tubule cells 1 hour after injection in both control animals and mercury-intoxicated rats. These results suggest a decreased catabolism of low molecular weight proteins in the kidney during chronic mercury intoxication.