L. A. Ginsel

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Horseradish peroxidase, lactoperoxidase, and ferritin as markers

SummaryThe occurrence of endocytotic mechanisms in human small intestinal absorptive cells was investigated by culturing biopsy specimens in the presence of horseradish peroxidase (HRP), lactoperoxidase (LPO), and ferritin. The results indicate that both HRP and LPO entered the cells by apical endocytosis, after which they were transported via apical vesicles and tubules to the lysosome-like bodies. Ferritin, which showed a distinct affinity for the cell-coat glycoproteins, was not interiorized by the absorptive cells.These findings suggest that although human absorptive cells have an endocytotic mechanism, possibly fluid-phase endocytosis, cell-coat glycoproteins are not taken up by the cells, as indicated by the absence of ferritin in the apical vesicles and tubules, as well as the lysosome-like bodies. These findings provide indirect support for our hypothesis that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport via a crinophagic mechanism (fusion of apical vesicles and tubules with lysosome-like bodies) rather than via an exocytotic-endocytotic mechanism.

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages.

SummaryThe binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.

A cytochemical method for the demonstration of 5′-nucleotidase in mouse peritoneal macrophages, with cerium ions used as trapping agent

SummaryA method was developed for the demonstration of 5′-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5′-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5′-nucleotidase.

The effect of colchicine on the intracellular transport of 3H-fucose-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue

SummaryThe effect of colchicine on the intracellular transport of 3H-fucose-labelled glycoproteins in the absorptive cells of cultured biopsy specimens of the human intestine was investigated by light- and electron-microscopical autoradiography and by biochemical methods. The results showed a decrease in the radioactivity of the cell coat on the microvilli and an increase in the Golgi apparatus and in the apical vesicles and tubules. This divergence is attributed to a colchicine-induced impairment of the normal transport of cell-coat glycoproteins from the Golgi apparatus, via the apical vesicles and tubules, to the apex of the cell.The radioactivity of the lysosome-like bodies in the absorptive cells cultured with colchicine also increased. This finding supports a crinophagic function of these organelles in the degradation of excess cell-coat material.The investigations were supported in part by the Foundation for Medical Research (FUNGO), which is subsidized by the Netherlands Organisation for the Advancement of Pure Research

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Effect of cytochalasin B and D

SummaryThe effect of cytochalasin B (CB) and cytochalasin D (CD) on the endocytotic uptake of horseradish peroxidase (HRP) by intestinal absorptive cells was investigated by morphometric methods. The results showed that CD inhibited endocytosis considerably, and without any detrimental side-effects. CB had hardly any effect on the endocytosis of HRP, but caused a significant decrease in the number of apical vesicles and tubules involved in the transport of cell-coat glycoproteins from the Golgi apparatus to the brush border.Electron-microscopic autoradiographic analysis of the effect of CD showed that although endocytosis is inhibited significantly by the drug, the amount of radiolabelled cell-coat material entering the lysosome-like bodies was unaltered compared with control cultures. These observations support our hypothesis that the cell-coat glycoproteins of the absorptive cells enter the lysosome-like bodies by a crinophagic rather than by an exocytotic-endocytotic mechanism.

The effect of chloroquine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue

SummaryThe effect of chloroquine, an inhibitor of intralysosomal catabolism, on the synthesis, transport, and degradation of cell-coat glycoproteins in absorptive cells of cultured human small-intestinal tissue was investigated by morphometrical, autoradiographical, and biochemical methods. Neither synthesis nor transport of cell-coat material was affected by the drug, but culturing of the absorptive cells in the presence of chloroquine led to a dose- and time-dependent enlargement of the dense bodies; other cell structures showed no alterations. 3H-fucose-labelled material accumulated in the dense bodies of the absorptive cells of these cultures. Since no increase of β-glucuronidase and acid phosphatase activity (both lysosomal enzymes of glycoprotein nature) was found, this accumulation of radiolabelled material can be explained as a chloroquine-mediated inhibition of the degradation of cell-coat glycoproteins. These macromolecules probably enter the lysosome-like bodies by a crinophagic mechanism, i.e., fusion of these organelles with the apical vesicles and tubules involved in intracellular transport. These findings suggest that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport in human intestinal absorptive cells, i.e., the degradation of excess cell-coat material.

Qualitative and quantitative preservation of the fine structure of absorptive cells in cultured biopsies of human small-intestine

SummaryThe fine structure of the absorptive cells in human small-intestinal biopsies cultured for 6, 24, and 48 h was analyzed qualitatively and quantitatively. The findings show generally good preservation of the cultured absorptive cells and a normal distribution, size, and relative volume of their cell organelles, but there was a systematic decrease in the apical cell surface and an increase in the number of apical vesicles and tubules after culturing. Since the apical vesicles and tubules are thought to have a function in the transport of cell-coat material from the Golgi apparatus to the cell surface, these findings raise the question of whether a delayed transport or extrusion of cell surface material occurs.The diminished relative volume of the mitochondria and the increased signs of autophagy in some poorly preserved absorptive cells, are assumed to be an adaption to less favourable culture conditions.

Fine structure and silver-staining patterns of lysosome-like bodies in absorptive cells of the small intestine in normal children and children with a lysosomal storage disease.

SummaryThe absorptive cells of the small intestine in normal children and children with a lysosomal storage disease were investigated with special attention to the fine structure and silver-staining pattern of lysosome-like bodies and related cell organelles.In children with a lysosomal storage disease the absorptive cells contain enlarged lysosomelike bodies. The average diameter of these dense bodies is significantly larger than that of the dense bodies in normal children, whereas the size of the multivesicular bodies is unchanged. The silver-staining pattern showed great similarity between the lysosome-like bodies, Golgi apparatus, apical vesicles and tubules, and cell coat.A possible crinophagic role of the lysosome-like bodies in the transport or secretion of cell coat material is discussed.

Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in cultured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease

SummaryThe transport of3H-fucose-and3H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light-and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter’s type of lysosomal storage disease, a similar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies.

Resolution of a gold latensification-elon ascorbic acid developer for IIford L4 emulsion

SummaryThe electron-microscopical autoradiographical resolution of a gold latensification-elon ascorbic acid (GEA) developer for IIford L4 emulsion was determined experimentally, using radioactive line sources of tritiated albumin (Heijnen and Geuze, 1977). For sections with a thickness of 62 nm (SD±11), which were covered with a carbon layer about 5 nm thick and a slightly overlapping monolayer of L4 silver bromide crystals, the measured half-distance (HD) of resolution was 115 nm. This improvement in resolution, the high efficiency of the GEA developer for L4 emulsion (Wisse and Tates, 1968), and the excellent visibility of the cellular structures under the small silver grains, mean that the L4-GEA combination deserves preverence as a method for quantitative electron-microscopical autoradiography.