Cell physiology of the rat visceral yolk sac: a study of pinocytosis and lysosome function

The rat visceral yolk sac is active in pinocytosis. Macromolecules accumulated by the tissue are, in general, routed to the lysosomes, where they either accumulate (if non-digestible by the lysosomal enzymes) or are degraded to their monomeric components. The yolk sac cells engage in adsorptive pinocytosis, which leads to the preferential uptake of macromolecules bearing certain surface features, such as a hydrophobic or a cationic domain. Substrates that enter the yolk sac by adsorptive pinocytosis can in some cases act as bivalent ligands, carrying in a second substance by "piggy-back" pinocytosis. Pinocytosis and intralysosomal digestion of plasma proteins by the organogenesis-stage rat embryo play an important nutritional role, supplying a high proportion of the embryo's amino acid requirement. Teratogenic effects can be induced by substances that inhibit either pinocytosis or intralysosomal proteolysis at this sensitive stage of gestation.     

A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages.

A method for the culture of rat peritoneal macrophages in vitro is described, in which pinocytic uptake of colloidal [198 Au]gold, 125I--labelled poly(vinylpyrrolidone) and [14C]sucrose proceeds at contant and fairly reproducible rates for several hours. The rat of uptake of colloidal [198 Au]gold, which wxhibited some inter-batch variation, was approx. 100 times that of the other two substrates. Colloidal gold did not affect the rate of uptake of 125I-labelled poly(vinylpyrrolidone) and therefore its own high rate of uptake could not be attributed to a stimulation of the formation of pinocytic vesicles. It conclude that uptake of collodial gold is highly dependent on adsorption on binding sites on the plasma membrane. Uptake of formaldehyde-treated 125I-labelled bovine serum albumin was followed by the release of [125I]iodo-L-tyrosine into the culture medium and took place at a rate intermediate between those of collodial [198Au]gold and the other two non-digestible substrates, 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose.     

Mass spectrometry in the study of lysosomal storage disorders.

Lysosomal storage disorders represent a group of over 45 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from non-lysosomal proteins that are involved in lysosomal biogenesis. A common biochemical feature of this group of disorders is the accumulation within lysosomes of undegraded or partially degraded substrates that are normally degraded within, and transported out of the lysosome. The particular substrates stored and the site(s) of storage vary with disease type and enzyme/protein deficiency. The nature of the substrate can be used to group the disorders into broad categories including the mucopolysaccharidoses, lipidoses, glycogenoses and oligosaccharidoses. These categories show many clinical similarities within groups as well as significant similarities between groups. For most lysosomal storage disorders the relationship between the stored substrates (type, amount and location) and the disease pathology is not well understood. The use of mass spectrometry and in particular tandem mass spectrometry provides a powerful tool for the investigation of stored substrates in this group of disorders. In this review we will describe the use of mass spectrometry for the analysis of stored substrates. We will discuss progress in the field, limitations of current methods, and summarise issues relating to the diagnosis and treatment of some of the more prevalent lysosomal storage disorders.     

Ito cells in lysosomal storage disorders. An ultrastructural study

An ultrastructural study was performed in a series of liver biopsies from patients with various lysosomal storage diseases to evaluate the extent of lysosomal hypertrophy and hyperplasia in Ito cells (ICs). In previous studies this has been considered to be absent or only rudimentary. Lysosomal storage was recognized by the presence of storage cytosomes surrounded by limiting membranes and by the appearance of their content which was identical to that in other hepatic storage lysosomes. Storage was found in sphingomyelinase deficiency (Niemann-Pick disease types A, B), in Wolman's disease, GM1 gangliosidosis, mucopolysaccharidosis and in multiple sulphatase deficiency. In type C Niemann-Pick disease it was virtually absent with the exception of cases with prominent hepatic symptomatology. Storage was of variable degree and was accompanied by a decrease in the physiological fat content (cytoplasmic lipid droplets). The degree to which ICs were affected correlated only with the extent to which nonspecific fibroblasts were involved in the specimens studied and thus seems to reflect storage in the fibroblastic population.     

Glucocerebrosidase deficiency and lysosomal storage of glucocerebroside induced in cultured macrophages

A cell culture model stimulating the genetic deficiency of glucocerebrosidase has been developed, utilizing macrophages and conduritol B epoxide (CBE), the specific irreversible inhibitor of the enzyme. Rat peritoneal macrophage glucocerebrosidase was completely inhibited when cells were treated with 10 microM CBE for 16 h or 100 microM CBE for 2 h. The t1/2 of inactivation was 30 min at 10 microM concentration. When cells were washed free of CBE, the enzyme activity reappeared linearly with time, reaching 50% of control activity 48 h after removal of the inhibitor. CBE-treated macrophages have normal phagocytic activity toward [3H]glycine-coupled latex beads and a normal number of mannose receptors. CBE was found to have no effect on other lysosomal enzymes. When [14C]glucocerebroside, encapsulated in multilamellar liposomes with alpha-D-mannopyranoside covalently coupled to the surface, was fed to glucocerebrosidase-depleted macrophages, the radiolabelled glycolipid accumulated and was undegraded. Subcellular fractionation on a Percoll density gradient demonstrated that the stored glucocerebroside in the CBE-treated macrophages was localized in lysosomes.     

Assessment of lysosomal function by quantitative histochemical and cytochemical methods

Summary Quantitative histochemistry and cytochemistry enables a direct link to be made between metabolic functions such as the activity of lysosomal enzymes and the morphology of a tissue or a type of cell. Several approaches exist such as microchemistry based on (bio)chemical analysis of a single cell or a small piece of tissue dissected from a freeze-dried section. This technique has been routinely used for prenatal diagnosis of inherited enzyme defects and especially of lysosomal storage diseases. Other approaches are cytofluorometry or cytophotometry, which are based on the principle that a fluorescent or coloured final reaction product is precipitated at the site of the enzyme. The amount of final reaction product is analysed per cell or per unit volume of tissue using either a microscope cytofluorometer or flow cytometer for fluorescence measurements or an image analysing system or scanning and integrating cytophotometer for absorbance measurements.In principle, fluorescence methods are to be preferred over chromogenic methods because they are more sensitive and enable multiparameter analysis. However, only a limited number of fluorogenic methods are at hand that give a final reaction product which is sufficiently water-insoluble to guarantee good localisation. The best results have been obtained with methods based on naphthol AS-TR derivatives and with methods for the demonstration of protease activity using methoxynaphthylamine derivatives as substrates and 5-nitrosalicylaldehyde as coupling reagent. Chromogenic methods are far better with respect to localisation properties and, therefore, most commonly used for quantitative histochemical analysis of lysosomal enzyme activities. Besides the measurement of enzyme reactions in tissues and cells, chromogenic methods have been applied for the analysis of kinetic parameters of lysosomal enzymesin situ which could be a better reflection of enzyme kineticsin vivo than those obtainedin vitro with biochemical means in diluted solutions. Chromogenic methods have also been used in the lysosomal fragility test which is based on the lag phase occurring when a lysosomal enzyme reaction is analysed against time. The duration of the lag phase is a parameter for the stability of the lysosomal membrane and is affected by toxic compounds or under pathological conditions. This paper reviews briefly fundamental aspects and applications of quantitative histochemical and cytochemical methods in the study of lysosomes.     

A quantitative study of some lysosomal enzymes in the bovine endometrium during early pregnancy.

Endometrium was obtained from cattle slaughtered at various stages of early pregnancy and of the oestrous cycle. Analyses for protein, RNA, DNA, glucose and some lysosomal enzymes were carried out on this tissue. The results are considered with respect to the general influence of the hormonal status of pregnancy and the specific influence of the hormonal status of pregnancy and the specific influence of the blastocyst in one uterine horn.     

Normal and experimentally induced lysosomal activity in the larval fat body of Calliphora erythrocephala Meigen

Summary Acid phosphatase activity was demonstrated by EM-cytochemistry in 4 day old third instar larvae of the fly Calliphora erythrocephala Meigen, but not in younger stages. During larval development, the activity increased, reaching a maximum at the onset of pupariation. The reaction product was localized in Golgi vesicles and sacculi, in vacuoles and in protein granules of varying size and composition, confirming the autophagic character of the protein granules. Throughout larval development, the reaction product was restricted to membrane-bound structures and no indications of free cytoplasmic activity that might be related to cytolysis were found.Enzyme activity could be evoked by transplanting inactive fat body lobes into host larvae of a later developmental stage. High enzyme activity was induced in these transplants within 18h. The sites of activity were roughly the same, but a portion of the activity in the transplants was found in the vacuoles. The induction could be inhibited by cycloheximide.     

Kinetics of lysosomal storage of indigestible matter.

In lysosomal storage diseases and in accumulation of lipofusion in the lysosomes there is a gradual eroding of the lysosomal system due to overloading the lysosomes by molecules which cannot be digested or expelled. The kinetics of this accumulation is examined for tissue cultures in terms of the cell growth rate, lysosomal production rate, and of generation of the indigestible element.     

Effect of L-arginine and L-lysine on lysosomal hydrolases and membrane bound phosphatases in experimentally induced myocardial infarction in rats

The protective effect of L-arginine and L-lysine on lysosomal enzymes and membrane bound ATPases was examined on isoproterenol induced myocardial infarction in rats. Lysosomal enzymes play an important role in the inflammatory process. The rats given isoproterenol (150 mg kg^–1 daily) intraperitoneally for 2 days showed significant changes in the marker enzymes, lysosomal enzymes and membrane bound phosphatases. Histopathological studies also confirmed the induction of myocardial infarction in isoproterenol administered rats. Prior oral treatment with L-arginine (250 mg kg^–1 daily) and L-lysine (5 mg kg^–1 daily) for 5 days significantly prevented these alterations and restored the enzyme activities to near normal. These findings demonstrate the protective effect of L-arginine and L-lysine in combination against isoproterenol induced cardiac damage.     

Pinocytosis and locomotion of amoebae: XII. Dynamics and motive force generation during induced pinocytosis in A. proteus

Summary The mechanism of induced pinocytosis was investigated in Amoeba proteus by light and electron microscopy. The application of nine different inducing substances revealed that pinocytotic channel formation, elongation, vesiculation, shortening and disappearance are the result of the successive or simultaneous action of both traction and pressure forces, which are produced by the contractile activity of a plasma membrane-associated layer of filaments ranging from a few hundred nm to several in thickness. The initial phase of channel formation is caused by traction forces according to the membrane flow concept, whereas channel elongation and vesiculation mainly result from pressure forces in conjunction with the extrusion of small hyaline pseudopodia. Shortening and disappearance of the pinocytotic channels are brought about by local contractions of the cortical filament layer in the basal region of the hyaline pseudopodia. Experiments using latex beads as marker particles together with inducing substances show that a rapid membrane turnover during pinocytosis can be excluded, and that the plasma membrane slides as an entire structure over the underlying cytoplasm.The authors are most grateful to Mrs. J. Ruch for technical assistance     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.