scholarly journals Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

1990 ◽  
Vol 97 (1) ◽  
pp. 127-138
Author(s):  
T.J. Raub ◽  
K.L. Audus
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Basolateral Membrane ◽  
Surface Membrane ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Bovine Brain ◽  
Membrane Recycling ◽  
Brain Microvessel

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t1/2 values for RCAI binding, internalization and efflux were 5, 18 and 13–14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 degrees C became localized within more caveolae within 2.5 min of warming to 37 degrees C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.

scholarly journals Demonstration of Acid Hydrolase Activity in Primary Cultures of Bovine Brain Microvessel Endothelium

1989 ◽  
Vol 9 (3) ◽  
pp. 280-289 ◽  
Author(s):  
Anna Baranczyk-Kuzma ◽  
Thomas J. Raub ◽  
Kenneth L. Audus
Keyword(s):  
Primary Cultures ◽  
Bovine Brain ◽  
Endocytic Pathway ◽  
Hydrolase Activity ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Cytochemical Localization ◽  
Brain Microvessel

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood–brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and β-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69–77%). The majority of β-galactosidase (≈48%) and total sulfatase (≈58%) activity was associated with the lysosome fraction of the BMECs. In contrast, ≈52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

scholarly journals Expression of a widely distributed, novel sulfated membrane glycoprotein by bovine endothelia and certain transporting epithelia.

1994 ◽  
Vol 42 (9) ◽  
pp. 1237-1250 ◽  
Author(s):  
T J Raub ◽  
G A Sawada ◽  
S L Kuentzel
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Choroid Plexus ◽  
Basolateral Membrane ◽  
Integral Membrane Protein ◽  
Bovine Brain ◽  
Cell Surface Antigens ◽  
Molecular Weights ◽  
Brain Microvessel

We generated monoclonal antibodies (MAbs) against cultured bovine brain microvessel endothelial cells (BMEC) for use as probes to study membrane protein traffic and polarity. One MAb recognized a heterogeneous family of acidic sulfoglycoproteins called gp4A4 with molecular weights of 50-65 KD and 85 KD. Gp4A4 is a long-lived integral membrane protein which resides mostly at the plasma membrane, and a portion appears to be in equilibrium with an intracellular pool via endocytosis. Gp4A4 is expressed by many endothelial cells, except for fenestrated capillaries in choroid plexus, and specific epithelial cells in bile duct, kidney, and choroid plexus. A comparison of indirect immunoperoxidase and immunofluorescence detection using semi-thin cryosections gave contrasting results on the apparent distribution of gp4A4 on the apical and basolateral membranes of cerebral endothelia and choroid plexus epithelia. Immunogold labeling of ultra-thin cryosections showed that gp4A4 was expressed by the apical and basolateral membrane domains of BMEC and choroid plexus epithelia. This was consistent with the results using indirect immunofluorescence microscopy. On an average, gp4A4 expression by cerebral endothelia was not asymmetric and was considerably variable between capillaries. These results emphasize the need to compare several different techniques in assessing polarized expression of cell surface antigens in vivo.

Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: studies with Tetrahymena thermophila

1992 ◽  
Vol 103 (2) ◽  
pp. 565-570
Author(s):  
V. Leick
Keyword(s):  
Bovine Serum Albumin ◽  
Cell Surface ◽  
Serum Albumin ◽  
Binding Sites ◽  
Bovine Serum ◽  
Tetrahymena Thermophila ◽  
Chemotactic Peptide ◽  
Dansyl Group ◽  
Peptide Derivatives

Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila. In vivo labelling of the cells with N-formylMet-Leu-[3H]Phe ([3H]fMLP) shows that the cells bind the ligand with high affinity (KD = 4 × 10(−9) M to 1 × 10(−8) M). Moreover, Scatchard transformations of the binding data show that there are about 5 × 10(5) binding sites per cell on the cell surface. Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdanLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface. Moreover, both derivatives have retained significant chemoattracting potentials. Fluorescence from dansMLP, but not from fMLPdansLys and dansyl-beta-endorphin, is internalized preferentially into small vesicles. The differences may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives. In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein. Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles).

scholarly journals Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R.

1992 ◽  
Vol 176 (2) ◽  
pp. 531-541 ◽  
Author(s):  
S D Voss ◽  
P M Sondel ◽  
R J Robb
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Binding Sites ◽  
Interleukin 2 ◽  
Affinity Binding ◽  
Beta Chain ◽  
Gamma Chain ◽  
Receptor Sites ◽  
Affinity Receptor

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.

scholarly journals IFNγ regulates retinal pigment epithelial fluid transport

2009 ◽  
Vol 297 (6) ◽  
pp. C1452-C1465 ◽  
Author(s):  
Rong Li ◽  
Arvydas Maminishkis ◽  
Tina Banzon ◽  
Qin Wan ◽  
Stephen Jalickee ◽  
...  
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Mapk Signaling ◽  
Therapeutic Interventions ◽  
Subretinal Space ◽  
Fluid Absorption ◽  
Blood Retinal Barrier

The present experiments show that IFNγ receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNγ applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNγ in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

scholarly journals Rab11a-positive compartments in proximal tubule cells sort fluid-phase and membrane cargo

2014 ◽  
Vol 306 (5) ◽  
pp. C441-C449 ◽  
Author(s):  
Polly E. Mattila ◽  
Venkatesan Raghavan ◽  
Youssef Rbaibi ◽  
Catherine J. Baty ◽  
Ora A. Weisz
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Chloride Ions ◽  
Endocytic Pathway ◽  
Kidney Cortex ◽  
Glomerular Filtrate ◽  
Electron Microscopy Analysis ◽  
Pt Cell

The proximal tubule (PT) reabsorbs the majority of sodium, bicarbonate, and chloride ions, phosphate, glucose, water, and plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell (PTC) function, the organization of the endocytic pathway in these cells remains poorly understood. We have used immunofluorescence and live-cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PTCs isolated from mouse kidney cortex. Cells from the S1 segment could be distinguished from those from more distal PT segments by their robust uptake of albumin and comparatively low expression of γ-glutamyltranspeptidase. Rab11a in these cells is localized to variously sized spherical compartments that resemble the apical vacuoles observed by electron microscopy analysis of PTCs in vivo. These Rab11a-positive structures are highly dynamic and receive membrane and fluid-phase cargo. In contrast, fluid-phase cargoes are largely excluded from Rab11a-positive compartments in immortalized kidney cell lines. The unusual morphology and sorting capacity of Rab11a compartments in primary PTCs may reflect a unique specialization of these cells to accommodate the functional demands of handling a high endocytic load.

Hexose uptake in primary cultures of bovine brain microvessel endothetial cells. I. Basic characteristics and effects of d-glucose and insulin

1991 ◽  
Vol 1070 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Yoshinobu Takakura ◽  
Sandra L. Kuentzel ◽  
Thomas J. Raub ◽  
Anthony Davies ◽  
Stephen A. Baldwin ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Bovine Brain ◽  
Basic Characteristics ◽  
Hexose Uptake ◽  
Brain Microvessel

scholarly journals Immunohistochemical localization and expression of the hyaluronan receptor CD44 in the epithelium of the pig oviduct during oestrus

Reproduction ◽  
2003 ◽  
pp. 119-132 ◽  
Author(s):  
P Tienthai ◽  
M Yokoo ◽  
N Kimura ◽  
P Heldin ◽  
E Sato ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Cumulus Cell ◽  
Secretory Cells ◽  
Specific Cell ◽  
Protein Determination ◽  
Sperm Reservoir ◽  
Hyaluronan Receptor

Hyaluronan is related to essential reproductive processes in pigs. Hyaluronan produced by cumulus cells builds, via specific cell surface receptors, an extracellular matrix responsible for cumulus cell cloud expansion during final oocyte maturation, a preparatory event for ovulation and fertilization. In addition, hyaluronan that has been localized in the pig oviduct both in the intraluminal fluid and on the surface of the lining epithelium of the preovulatory sperm reservoir, has proven beneficial during in vitro fertilization and embryo culture, thus indicating that it has a role in vivo. This study monitored the immunolocalization, protein determination and gene expression of the major cell surface hyaluronan receptor CD44 in the epithelial lining of the pig oviduct during selected stages of standing oestrus, in relation to spontaneous ovulation. The CD44 immunostaining in the lining epithelium was localized to the surface membrane and the supranuclear domain of mainly the secretory cells, particularly in the sperm reservoir of both treatment (inseminated) and control (non-inseminated) specimens. Up to four hyaluronan-binding protein (HABP) bands (60, 90, 100 and 200 kDa) were detected in the tubal epithelium, and the 200 kDa band was determined as CD44 by immunoblotting. The expression of CD44 mRNA was higher before than after ovulation (P < 0.05), most conspicuously in the uterotubal junction (UTJ). In addition, CD44 expression in the preovulatory UTJ and the ampullary-isthmic junction (AIJ) of control animals was higher than in those that were inseminated (P < 0.05 and P < 0.01 for UTJ and AIJ, respectively). The results demonstrate for the first time that the specific hyaluronan receptor CD44 is expressed by the oviduct epithelial cells during spontaneous oestrus, and is particularly abundant in the sperm reservoir before ovulation. Presence of spermatozoa in this segment seemed to downregulate the receptor. The variation in the expression of CD44 in relation to spontaneous ovulation and the presence of spermatozoa indicate that the hyaluronan CD44-signalling pathway may play a role in oviduct function during sperm storage and fertilization in pigs.

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