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Rat thyroid hemilobes were incubated in presence of exogeneous, heterologous and homologous thyroglobulins. The median density of the thyroglobulin molecules originally added to the medium was compared with that of the molecules remaining at the end of a three-hour incubation period (37 degrees C). A certain degree of specificity in the reabsorption process of thyroglobulin was found: the uptake of homologous molecules (rat) was higher than heterologous (hog) molecules. The median density of the iodoproteins remaining after the incubation did not change for the heterologous whereas it shifted towards lower density for the homologous thyroglobulins (equilibrium labelled, 35 iodine atoms/molecule). In addition, rat follicular cells display selectivity in the endocytosis of homologous thyroglobulin. Among the rat molecules, the normally iodinated are taken up more actively than the lowly iodinated or newly synthesized ones. Dissimilarity in the median density of the thyroglobulin molecules before and after endocytosis was only evident for the equilibrium-labelled, normally iodinated rat preparation: the disappearance from the medium of 25-30% of exogenous iodoproteins was sufficient to lower significantly the median density of the remaining molecules. This means that the thyroglobulin molecules having higher density are taken up preferentially by the tissue. A mechanism involving specific interactions between the iodoproteins and the surface of thyroid cell is suggested.
In mice fed on an atherogenic diet for 4 to 8 months, the aortas were examined by transmission and scanning electron microscopy. After 8 months of being fed a high cholesterol diet, the animals developed aortic intimal lesions composed mainly of proliferated modified smooth muscle cells with an increase of connective tissues. Scanning electron microscopy showed changes in aortic luminal surface consisting primarily of altered distribution of microvilli. Quantitative analysis of these changes showed a statistically significant (P less than 0.001) increase in cholesterol-fed animals compared with controls and a significant (P less than 0.001) difference between at 4 months and at 8 months in cholesterol-fed mice. Ultrastructural study on the uptake of protein tracer, horseradish peroxidase (HRP), by aortic endothelial cells in vitro was performed. The uptake of HRP was essentially the same for controls and cholesterol-fed mice at 4 months, but a statistically significant (P less than 0.005) increased uptake of HRP was observed at 8 months. Additional mice were subjected to nephrectomy with DOCA administration for 4 months along with a cholesterol-feeding. These animals showed a 2-fold increase in HRP-uptake compared with nephrectomized mice without a cholesterol-feeding. These results suggest that the enhanced pinocytotic activity of aortic endothelial cells in vitro, especially in hypertensive condition, and altered distribution of microvilli might be correlated to the arteriosclerotic process.
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Human synovial tissue cells in monolayer can be shown to take up and digest a soluble protein, horseradish peroxidase (HRP). Uptake of HRP was linear with increasing concentrations of substrate and cell protein and with time for up to 4 h. Low temperature (4 degrees C), and sodium fluoride, an inhibitor of glycolysis were the most effective metabolic inhibitors of endocytosis. In addition, colchicine, an inhibitor of microtubule assembly, and yeast mannan, an inhibitor of mannose-specific receptors, reduced HRP uptake. Synovial cells from patients with rheumatoid arthritis (RSC) demonstrated a statistically significantly higher rate of endocytosis (247 +/- 107 ng HRP/100 micrograms cell protein per 2 h.) than cells from control, nonrheumatoid patients (NSC) (100 +/- 80 ng HRP/100 micrograms cell protein per 2 h). Thus, it is possible to discriminate RSC from NSC by their quantitatively different rates of endocytosis. Digestion of HRP by synovial cells is statistically significant by 6 h after uptake. A faster initial rate of digestion was seen in RSC. Over the first 6--8 h of incubation 42% of the endocytosed HRP was still cell-associated in RSC and 67% remained in NSC cultures. However, by 24 h 20--30% of endocytosed HRP was found in both types of cultures. These results indicate that endocytosed molecules may accumulate more rapidly in RSC and persist within their lysosomes for a longer time than in NSC. The quantitative determination of enhanced endocytosis by RSC compared with NSC suggests that this increased activity may have a role in the pathological function of synovial tissue in rheumatoid arthritis.
Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.
To understand how phosphatidylinositol 3-kinase (PI3K) modulates cell structure and function, we examined the molecular and cellular defects of a Dictyostelium mutant strain (pik1(Delta)2(Delta)) missing two (DdPIK1 and 2) of three PI3K genes, which are homologues of the mammalian p110 subunit. Levels of [32P]phosphatidylinositol 3, 4 bisphosphate (PI(3,4)P2) and [32P]phosphatidylinositol trisphosphate (PIP3) were reduced in pik1(Delta)2(Delta), which had major defects in morphological and functional correlates of macropinocytosis. This was accompanied by dramatic deficits in a subset of F-actin-enriched structures such as circular ruffles, actin crowns and pseudopodia. Although pik1(Delta)2(Delta) were mobile, they failed to aggregate into streams. Therefore we conclude that PIK1 and 2, possibly through modulation of the levels of PIP3 and PI(3,4)P2, regulate the organization of actin filaments necessary for circular ruffling during macropinocytosis, the extension of pseudopodia and the aggregation of cells into streams, but not the regulation of cell motility.
One aspect of human placental function which has not hitherto been studied is the ability of the placenta to digest proteins intracellularly and use the products of hydrolysis to supply its own and foetall complement of hydrolytic enzymes, including the acid proteases cathepsin C and D. We have used trophoblast cells in monolayer tissue culture as a model for the study of endocytosis and intracellular digestion of 125I-haemoglobin. The normal use of serum in tissue culture medium has shown up differences from the pattern observed with other phagocytic cells such as macrophages, in that serum allows endocytosis but prevents intracellular digestion of 125I-haemoglobin. Replacement of serum by lactalbumin hydrolysate enables both endocytosis and intracellular digestion of 125I-haemoglobin to occur as in other phagocytic cells. Digestion is followed by release into the medium of acid-soluble, lower-molecular-weight compounds. The reasons for this major difference between trophoblast and other cells are discussed in the light of our results and their possible relevance to placental function.
Flotation through a 5.6% Percoll gradient of pinosomes from Chinese hamster ovary (CHO) cells labelled during a 10 min internalization period with horseradish peroxidase (HRP), a solute, revealed two pinosomal populations, the expected low-buoyancy population and an unexpected buoyant population. The buoyant pinosomes that sedimented similarly to plasma membrane were not an artifact of HRP trapping during homogenization or of cell surface-adherent HRP. No trapping or cell surface adherence of HRP could be detected by biochemical or cytochemical assays, even after internalization periods as short as 15 s to 1 min. With short uptake times, the buoyant pinosome population was the major HRP positive vesicle population, suggesting a precursor-to-product relationship between the two populations. In pulse-chase experiments, the buoyant pinosome population was shown to be highly exocytic and the precursor to later pinosomes. By electron-microscope cytochemistry, rapidly labelled, HRP positive pinosomes (15 s to 1 min uptake) were typically smooth vesicles with a median diameter of approximately equal to 0.30 micron and a size range from approximately equal to 0.10 micron to greater than 1.0 micron in diameter. We suggest that these rapidly labelled structures are a very early stage in the intracellular processing of pinocytic vesicles.
In order to elucidate the physiological function of extrahepatic bile duct cells, we isolated epithelial cells from the rat extrahepatic bile duct by digesting resected segments of the extrahepatic bile duct with 0.15% trypsin in ice-cold Ca(2+)-free Hanks' balanced salt solution supplemented with 0.25 mM EDTA overnight. As a result, the epithelial cells were collected as aggregates and attached to culture dishes coated with type I collagen. Approximately 95% of the cells cultured for 24 hrs were found to be positive for gamma-glutamyl transpeptidase and cytokeratin-19, but negative for vimentin. These characteristics were identical to the features of rat extrahepatic biliary epithelial cells in situ. Ultrastructurally, the cells were long and columnar in configuration on the 2nd day in culture, and possessed numerous microvilli at the apical surface and well-developed junctional complexes at the lateral surface. These findings also indicate that the cells maintain an epithelial nature and are morphologically polarized. When the cells were exposed to a low dose of horseradish peroxidase (HRP) on the 2nd day in culture, which was followed by fixation and treatment with 3-3'-diaminobenzidine, HRP was found preferentially in the cytoplasmic vesicles near the apical surface. HRP was then observed in the intercellular spaces; however, the electron-dense tracer, ruthenium red, did not permeate into the intercellular spaces, and HRP was found in neither cytoplasms nor intercellular spaces when the cells were incubated in HRP-containing medium at 4 degrees C for 30 min. These results suggest that the extrahepatic bile duct epithelial cells are involved in the reabsorption of bile constituents.
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The endocytosis of tracer particles by sinusoidal endothelial cells was studied in the bone marrow and liver of the rabbit and in the bone marrow of the rat after intravenous injection. The tracer particles used included carbon particles and polystyrene particles of different sizes ranging from 0.11 to 5.7 micron in diameter. The sinusoidal endothelial cells of the rabbit liver and rat bone marrow took up carbon through the formation of bristle-coated vesicles at the luminal surface, but they eventually failed to ingest polystyrene particles of 0.11 micron or larger. On the other hand, the sinusoidal endothelial cells of the rabbit bone marrow took up carbon and polystyrene particles varying from 0.11 to 2.02 micron in diameter. These endothelial cells were found to sequester circulating tracer particles in the cytoplasm through the formation of two kinds of vesicles at the luminal surface: bristle-coated vesicles and "multiparticle-pinocytotic vesicles." This special type of pinocytotic vesicle was larger in diameter than the bristle-coated vesicle and variable in size according to the size or amount of the particles to be sequestered. Carbon and 0.11 micron polystyrene particles were taken up by means of both the bristle-coated vesicles and multiparticle-pinocytotic vesicles, whereas particles 0.30 micron in diameter or larger were taken up by the vesicles similar in nature to multiparticle-pinocytotic vesicles. A sequence for the morphological events occurring in the formation of multiparticle-pinocytotic vesicles in the sinusoidal endothelial cells of rabbit bone marrow shall be suggested and discussed.
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