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PubMed1979

Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.

Animals↗
PubMed1983

Receptors, acceptors, and the action of polypeptide hormones: illustrative studies with epidermal growth factor (urogastrone).

The action of polypeptide hormones is discussed in the context of observations made both with receptors, like the ones for epidermal growth factor (urogastrone) (EGF-URO), insulin, and gonadotropin-releasing hormone (GnRH), and with acceptors, like the ones that participate in the adsorptive pinocytosis of substances like cholesterol (low density lipoprotein acceptor), cobalamin (transcobalamin-II acceptor), and asialoglycoproteins (galactose acceptor). The overall processes that both pharmacologic receptors and acceptors undergo subsequent to ligand binding comprise (a) microclustering; (b) aggregation at both coated ("coated pits") and uncoated plasma membrane regions; (c) internalization, with the formation of endocytic vesicles (endosomes or receptosomes); and (d) either recycling of endosomes (receptosomes) to the plasma membrane or fusion of endosomes (receptosomes) with lysosomes, accompanied by proteolytic degradation of the internalized ligand-receptor complex. The relevance of these processes to hormone action is discussed. Further, illustrative studies with EGF-URO are used to highlight general mechanisms that are thought to participate in the action of a variety of peptide hormones. The microclustering event and the concomitant receptor-regulated membrane phosphorylation reactions are emphasized as key plasma-membrane-localized processes that are very likely involved in the rapid (minutes to tens of minutes) of many peptide hormones. For the delayed actions of hormones (hours to tens of hours; e.g., DNA synthesis), the potential importance of nonlysosomal receptor processing, mediated by SH-requiring proteases that generate intracellular receptor fragments, is pointed out. A scheme for the action of EGF-URO is outlined and a model for peptide hormone action is presented that illustrates how multiple chemical mediators of hormone action may result from the combination of a single hormone with its unique receptor.

Animals↗
PubMed1998

Estrogens modulate bovine vascular endothelial cell permeability and HSP 25 expression concomitantly.

The atheroprotective properties of estrogens are supported by clinical data from postmenopausal women who use estrogen replacement therapy. However, the mechanisms mediating activity remain unknown, and it has been suggested that estrogens may help to modulate endothelial permeability to atherogenic lipoproteins. In these studies we used bovine vascular endothelial cells as an in vitro model to show that estrogens were able to regulate low-density lipoprotein transport and permeability of the endothelial monolayer. Macromolecular transport was observed to be a second-order polynomial function of estrogen concentration. Moreover, this regulation was correlated with expression of heat shock protein (HSP) 25, which is known to influence fluid phase pinocytosis and cytoskeleton remodeling, thus suggesting a role for HSP 25 in the estrogenic control of transcellular permeability of the endothelium monolayer.

Animals↗
PubMed1978

Possible role of the proximal convoluted tubules of human kidney in chronic glomerulonephritis. A quantitative electron-microscopic study.

The amount of forming pinocytic (coated) microvesicles on the apical plasma membrane of kidney proximal tubule cells was assessed in kidney biopsies of 10 patients suffering from chronic glomerulonephritis. A significant correlation was found between the amount of these vesicles and diurnal proteinuria levels (r = 0.889; p less than 0.01). The possible mechanisms of protein reabsorption via pinocytosis in both normal and pathological conditions are considered.

Adolescent↗
PubMed1991

Effects of geraniin on morphology and function of macrophages.

The effects of geraniin, a tannin, isolated from Geranium thunbergii Sieb. et Zucc. on the morphology and function of macrophages were studied. Geraniin caused a marked retardation of the recovery from fully spread surface membrane and a highly reorganized cytoskeleton of macrophages, whereas endocytotic activity, phagocytosis and pinocytosis in the cells were significantly inhibited.

Actin Cytoskeleton↗
PubMed1978

Relationship of incorporation of radioprecursors into protein and phospholipids of the plasmalemma of Guinea pig (neonate) intestinal epithelium and the cessation of uptake of macromolecules (closure).

The incorporation of radioprecursors into intestinal epithelial cell proteins and phospholipids was studied to assess the role of protein and phospholipid synthesis and/or turnover in the cessation of the absorption of macromolecules (closure). Radiophosphorus incorporation into cellular phospholipids was enhanced when pinocytosis was stimulated. The specific activity of cellular and brush border phospholipids and specific phosphatides increased during the period of active endocytosis. No alteration in specific phosphatide percent composition was observed. Radioamino acid incorporation into cellular protein was not influenced by age. These data are consistent with the idea that the period of active macromolecule absorption by the guinea pig intestinal epithelium is defined by the exhaustion of membrane available for endocytosis.

Age Factors↗
PubMed1971

Synthesis and transport of lipoprotein particles by intestinal absorptive cells in man.

The site of synthesis and some new details of lipoprotein particle transport have been demonstrated within the jejunal mucosa of man. In normal fasting volunteers, lipoprotein particles (88%, 150-650 A diameter) were visualized within the smooth endoplasmic reticulum and Golgi cisternae of absorptive cells covering the tips of jejunal villi. Electron microscopic observations suggested that these particles exited through the sides and bases of absorptive cells by reverse pinocytosis and then passed through the extracellular matrix of the lamina propria to enter lacteal lumina. When these lipid particles were isolated from fasting intestinal biopsies by preparative ultracentrifugation, their size distribution was similar to that of very low density (S(f) 20-400) lipoprotein (VLDL) particles in plasma. After a fatty meal, jejunal absorptive cells and extracts of their homogenates contained lipid particles of VLDL-size as well as chylomicrons of various sizes. The percentage of triglyceride in isolated intestinal lipid particles increased during fat absorption. Our interpretation of these data is that chylomicrons are probably derived from intestinal lipoprotein particles by addition of triglyceride.

Biological Transport↗
PubMedDate not supplied

Activation of tumor-associated macrophages by thymosin alpha 1.

It was shown earlier that the progressive growth of a transplantable T-cell lymphoma of spontaneous origin, designated as Dalton's lymphoma (DL), in a murine host is associated with an inhibition of macrophages (TAM) along with an involution of thymus. However, it remained unclear if a decline in the level of thymic peptides in DL-bearing host, due to thymic regression, has any implications in the inhibited responses of TAM. Therefore, the present investigation was under taken to study whether the TAM of DL-bearing host can be activated to tumoricidal state by peptides of thymic origin. It was observed that intraperitoneal administration of thymosin alpha 1 to DL-bearing mice resulted in activation of TAM. Such TAM were found to produce enhanced amount of interleukin-1 (IL-1), tumor necrosis factor (TNF), reactive oxygen intermediates (ROI), nitric oxide (NO) and showed an increased abilities of pinocytosis, phagocytosis, antigen presentation and tumor cytotoxicity. The TAM were found to be directly responsive to thymosin alpha1 as in vitro treatment with thymosin alpha 1 could activate TAM to tumoricidal state. Treatment of TAM with thymosin alpha 1 also enhanced their LPS responsiveness for an augmented state of activation. The findings of this study demonstrate for the first time that the TAM of a T cell lymphoma can be activated to tumoricidal state by thymosin alpha 1.

Animals↗
PubMed2006

Dendritic cell function in cytomegalovirus-infected patients with mononucleosis.

Dendritic cells (DCs) are important target cells for human cytomegalovirus (HCMV) infection, and the virus has been shown to hamper the differentiation and maturation pathways of these cells in vitro. In the present study, we examined the function of monocyte-derived DCs obtained from immunocompetent individuals undergoing symptomatic HCMV infection in terms of immunophenotypic characteristics, pinocytosis, lymphocyte stimulation capacity, and cyto-chemokine secretion in comparison with DCs obtained from healthy controls. Immature and lipopolysaccharide (LPS)-stimulated DCs obtained from patients actively infected with HCMV expressed significantly lower levels of major histocompatibility complex (MHC) class II molecules. The inhibition of expression of MHC class II molecules by HCMV appeared to be functionally relevant, as mature DCs obtained from patients with HCMV mononucleosis were inefficient in stimulating proliferation of allogenic lymphocytes. Finally, the pattern of cyto-chemokines secreted by DCs obtained from patients with HCMV mononucleosis was characterized by a proinflammatory profile with an increased production of interleukin (IL)-1beta, tumor necrosis factor alpha, CC chemokine ligand 2 (CCL2) and CCL3, and reduced secretion of IL-10 upon LPS stimulation. During symptomatic HCMV infection in the immunocompetent host, DCs exhibit an impaired immunophenotype and function. These effects may contribute to the viral-induced immunomodulation, which is often observed in HCMV-infected patients.

Adult↗
PubMed2004

Mercury intake by inflammatory phagocytes: in vivo cytology of mouse macrophages and neutrophils by X-ray elemental microanalysis coupled with scanning electron microscopy.

Phagocytes remove and store mercury (Hg) that enters the body. Macrophages and granulocytes respond in opposite ways to Hg: macrophages loose cell viability, and neutrophils become protected from apoptosis. We have investigated the cytology of early intake of Hg by macrophages and neutrophils after a short period (2-4 min) of in vivo exposure to HgCl2. The two types of phagocytes were attracted either to a subcutaneous air pouch or to the peritoneal cavity of BALB/c mice by in situ BSA injection. BSA caused, 72 hours later, inflammatory exudates where neutrophils (air-pouch cavity) or macrophages (peritoneal cavity) were the predominant cell type. A lethal dose of HgCl2 (25 mg) was then injected in the two inflammatory cavities. The mice died 2-4 min later and the cell exudates were harvested and studied by scanning electron microscopy coupled with X-ray elemental microanalysis (SEM-XRM). More than half of the phagocytes showed ingested Hg; a higher percentage of macrophages (around 70%) than neutrophils (around 50%) were positive for the metal. Intracellular particles of Hg were spheroid and presented a small diameter (less than 20 nm). They could be seen in large numbers inside phagocytes (up to 20-30 Hg dots per cell); they were scattered throughout the cytoplasm of the cells. The ability of phagocytes to ingest Hg increased as the BSA-induced inflammation progressed. We conclude that (i) Hg is quickly ingested as small particles by phagocytes; (ii) endocytosis of Hg increases with the degree of activation of phagocytes; and (iii) phagocytes internalize Hg by pinocytosis.

Animals↗
PubMed1980

Modulation of the intracellular cystine content of cystinotic fibroblasts by extracellular albumin.

Cystinotic fibroblasts contain highly elevated amounts of intracellular non-protein cystine reaccumulation by cystine-depleted cystinotic fibroblasts and the steady-state cystine content of nondepleted cystinotic fibroblasts can be modulated by the addition of bovine serum albumin to the culture medium. This effect is not seen in cultures of normal and cystinotic heterozygote fibroblasts. The cystinotic homozygote cells accumulate cystine under there conditions from proteolysis of the albumin. An increased rate of pinocytosis or proteolysis of albumin does not account for the observed cystine accumulation by the cystinotic fibroblasts. Comparison of the amount of cystine accumulated to the amount of albumin degraded shows that less than one percent of the cystine moieties released by proteolysis is retained within these cells.

Cells, Cultured↗
PubMed1984

Microsurgical carotid endarterectomy in rats.

The purpose of this study was to develop an inexpensive experimental model of carotid endarterectomy. Unilateral common carotid endarterectomies 3 mm in length were performed on 25 rats using microsurgical techniques. The pattern of endothelial regrowth after endarterectomy, the pinocytosis of regenerated endothelial cells, and the effects of heparin and thromboxane A2 synthetase inhibitor on thrombus formation were examined 7 days after the resumption of flow. Pinocytotic vesicular transport in endothelial cells was evaluated using horseradish peroxidase. All endarterectomized vessels in both control and drug-treated groups were patent 7 days after operation without apparent mural thrombus formation. Regenerated endothelial cells had spindle-shaped nuclei and cell bodies with long axes oriented parallel to the direction of blood flow. There were several instances where the leading edge of the endothelium at the distal end was reflected back in the distal direction. The extent of the endothelial ingrowth was often greater from the proximal end of the endarterectomy than from the distal end. These observations suggest that hemodynamic force affects endothelial regeneration. Pinocytotic activity was less in the regenerated endothelium than in either the endothelium remaining at the periphery of the endarterectomy or the endothelium of the control animals.

Animals↗
PubMed1995

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Animals↗
PubMed2001

The WASp-like protein scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium.

Scar, a member of the WASp protein family, was discovered in Dictyostelium discoideum during a genetic screen for second-site mutations that suppressed a developmental defect. Disruption of the scar gene reduced the levels of cellular F-actin by 50%. To investigate the role of Scar in endocytosis, phagocytosis and endocytic membrane trafficking, processes that depend on actin polymerization, we have analyzed a Dictyostelium cell line that is genetically null for Scar. Rates of fluid phase macropinocytosis and phagocytosis are significantly reduced in the scar- cell-line. In addition, exocytosis of fluid phase is delayed in these cells and movement of fluid phase from lysosomes to post-lysosomes is also delayed. Inhibition of actin polymerization with cytochalasin A resulted in similar phenotypes, suggesting that Scar-mediated polymerization of the actin cytoskeleton was important in the regulation of these processes. Supporting this conclusion, fluorescence microscopy revealed that some endo-lysosomes were ringed with F-actin in control cells but no F-actin was detected associated with endo-lysosomes in Scar null cells. Disruption of the two genes encoding the actin monomer sequestering protein profilin in wild-type cells causes defects in the rate of pinocytosis and fluid phase efflux. Consistent with a predicted physical interaction between Scar and profilin, disrupting the scar gene in the profilin null background results in greater decreases in the rate of fluid phase internalization and fluid phase release compared to either mutant alone. Taken together, these data support a model in which Scar and profilin functionally interact to regulate internalization of fluid and particles and later steps in the endosomal pathway, probably through regulation of actin cytoskeleton polymerization.

Actins↗
PubMed1981

Structural heterogeneity of endocytic membranes in macrophages as revealed by the cholesterol probe, filipin.

The polyene antibiotic, filipin, by specifically interacting with cholesterol, produces approximately 25-nm protuberances (filipin-sterol complexes) in freeze-fractured membranes, and the addition of filipin to aldehyde fixatives has been recently introduced as a cytochemical technique for the localization of cholesterol in cell membranes. In a previous study we showed that, in fibroblasts filipin-sterol complexes are absent from endocytic coated pits. To establish whether the absence of filipin-sterol complexes is a phenomenon restricted to coated pits or is correlated with endocytosis in general, we applied the filipin probe to cultured mouse peritoneal macrophages, in which different forms of endocytosis take place. The macrophages were incubated with bovine albumin or concanavalin A (Con A) to induce pinocytosis, and with heat-killed straphylococci or opsonized erythrocytes to induce phagocytosis, then fixed in glutaraldehyde/filipin and freeze-fractured. Filipin-sterol complexes were plentiful on the plasma membrane, on the smooth-membrane invaginations and vesicles induced by albumin, on the large endocytic vacuoles induced by Con A, and on the membrane of phagosomes but, in contrast, they were absent from coated pits and vesicles, as well as from coated segments of invagination or vesicles. These results indicate that the membranes involved in different types of endocytosis do not react in the same way with filipin and may, therefore, have a different cholesterol content. This could reflect different mechanisms of formation for the various types of endocytic vesicles.

Animals↗
PubMedDate not supplied

Evidence for a vacuolar-type proton ATPase in Entamoeba histolytica.

Entamoeba histolytica is a primitive eukaryote that lacks mitochondria. Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin A1, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concomitantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

Animals↗
PubMed2004

Superparamagnetic iron oxide-labeled Schwann cells and olfactory ensheathing cells can be traced in vivo by magnetic resonance imaging and retain functional properties after transplantation into the CNS.

Schwann cell (SC) and olfactory ensheathing cell (OEC) transplantation has been shown experimentally to promote CNS axonal regeneration and remyelination. To advance this technique into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Previous studies, using complex modification processes to enable uptake of contrast agents, have shown that cells labeled in vitro with paramagnetic contrast agents transplanted into rodent CNS can be visualized using magnetic resonance imaging (MRI). Here we show that SCs and OECs efficiently internalize dextran-coated superparamagnetic iron oxide (SPIO) from the culture medium by fluid phase pinocytosis. After transplantation into focal areas of demyelination in adult rat spinal cord both transplanted SPIO-labeled SCs and OECs produce a signal reduction using T(2)-weighted MRI in anesthetized rats that persists for up to 4 weeks. Although signal reduction was discernable after transplantation of unlabelled cells, this is nevertheless distinguishable from that produced by transplanted labeled cells. The region of signal reduction in SPIO-labeled cell recipients correlates closely with areas of remyelination. Because the retention of functional integrity by labeled cells is paramount, we also show that SPIO-labeled SCs and OECs are able to myelinate normally after transplantation into focal areas of demyelination. These studies demonstrate the feasibility of noninvasive imaging of transplanted SCs and OECs and represent a significant step toward the clinical application of promising experimental approaches.

Animals↗
PubMed1985

Fine structural and cytochemical aspects of granuloma formation derived from Kupffer cells in mice injected with latex particles.

The fate of Kupffer cells in mice for 3 days following injection with polystyrene latex particles (0.2 and 2.0 micron in diameter) was studied by electron microscopy. Kupffer cells took up the latex particles by pinocytosis as well as by phagocytosis. The particles ingested were in contact or fused with lysosomes in the cell. Two days after the final injection, most Kupffer cells were already stuffed with the particles. Within one month, cell clumps or cell aggregates, which could be called granuloma, were formed in the liver connective tissue space, i.e., Disse spaces, interlobular connective tissue spaces and subperitoneal connective tissue spaces. They were mostly composed of cells laden with numerous latex particles. The large granulomas were 80-120 micron in diameter. In each granuloma, endogenous peroxidase was localized in the cisternae of the nuclear envelope and of the rough endoplasmic reticulum of most cells, and in the cytoplasmic granules of some other cells. The former is known to be a Kupffer cell, and the latter, a monocyte. The granuloma further contained a few intermediate cells, showing peroxidase activity in the cisternae of the nuclear envelope and of the rough endoplasmic reticulum, and also in the cytoplasmic granules, phagocytic cells without peroxidase activity, granulocytes, and plasma cells. Hepatic sinusoidal endothelial cells, attenuated in shape, also took up latex particles of a 0.2 micron diameter and rarely with a 2.0 micron diameter in their cytoplasm. Some Kupffer cells in the granuloma filled with numerous latex particles were labeled with 3H-thymidine (2.5 mCi in total dose) after subcutaneous injection ten times for 80 hr. In the animals 8 months after the injection of the latex particles, numerous large granulomas were distributed throughout the liver in the interlobular or subperitoneal connective tissue space. The formation of the granuloma of Kupffer cells is considered to play a great role in the disposing of foreign materials from the functional liver parenchymatous tissue.

Animals↗