scholarly journals The GLUT5 hexose transporter is also localized to the basolateral membrane of the human jejunum

1995 ◽  
Vol 309 (1) ◽  
pp. 7-12 ◽  
Author(s):  
S J Blakemore ◽  
J C Aledo ◽  
J James ◽  
F C Campbell ◽  
J M Lucocq ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Unit Length ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Morphological Evidence ◽  
Hexose Transporter ◽  
Gold Particles ◽  
Quantitative Analyses ◽  
Gold Labelling ◽  
Quantitative Immunoblotting

The intestine is a major site of expression of the human GLUT5 hexose transporter, which is thought to be localized exclusively to the brush border membrane (BBM) where its major role is likely to be in the absorption of fructose. In this study we present novel biochemical and morphological evidence showing that the GLUT5 transporter is also expressed in the basolateral membrane (BLM) of the human intestine. BBM and BLM were isolated by fractionation of human jejunum. BBM were enriched with alkaline phosphatase activity by over 9-fold relative to a crude jejunal homogenate and contained immunoreactive sucrase-isomaltase and GLUT5 proteins. By contrast the BBM fraction was substantially depleted of immunoreactive a1 subunits of the Na,K-ATPase and GLUT2 glucose transporters which were abundantly present in the BLM fraction. This BLM fraction was enriched by over 11-fold in potassium-stimulated phosphatase activity relative to the crude homogenate; BLM also reacted to immunological probes for GLUT5 but showed no observable reactivity with antibodies directed against sucrase-isomaltase. Quantitative immunoblotting revealed that the BBM and BLM contained near equal amounts of GLUT5 per mg of membrane protein. Immunogold localization of GLUT5 on ultrathin sections of human jejunum showed that GLUT5 was present in both apical BBM and BLM. This gold labelling was absent when antiserum was pre-incubated with the antigenic peptide corresponding to a specific C-terminal sequence of human GLUT5. Quantitative analyses of the number of gold particles per unit length of BBM and BLM indicated that the mean density of gold labelling was marginally greater in the BBM (0.399 gold particles/micrometer) than in the BLM (0.293 gold particle/micrometer). The localization of GLUT5 in the BLM of the human jejunum may suggest that it specifically participates in the transfer of fructose across the basal membrane of the enterocyte.

scholarly journals Signal-mediated nuclear transport in proliferating and growth-arrested BALB/c 3T3 cells.

1991 ◽  
Vol 115 (4) ◽  
pp. 933-939 ◽  
Author(s):  
C M Feldherr ◽  
D Akin
Keyword(s):  
High Cell Density ◽  
Unit Length ◽  
3T3 Cells ◽  
Proliferating Cells ◽  
Gold Particles ◽  
Nuclear Uptake ◽  
Large Particles ◽  
The Mean ◽  
Transport Channels ◽  
Relative Uptake

Mediated transport across the nuclear envelope was investigated in proliferating and growth-arrested (confluent or serum starved) BALB/c 3T3 cells by analyzing the nuclear uptake of nucleoplasmin-coated colloidal gold after injection into the cytoplasm. Compared with proliferating cells the nuclear uptake of large gold particles (110-270 A in diameter, including the protein coat) decreased 5.5-, 33-, and 78-fold, respectively, in 10-, 14-17-, and 21-d-old confluent cultures; however, the relative uptake of small particles (total diameter 50-80 A) did not decrease with increasing age of the cells. This finding suggests that essentially all pores remain functional in confluent populations, but that most pores lose their capacity to transport large particles. By injecting intermediate-sized gold particles, the functional diameters of the transport channels in the downgraded pores were estimated to be approximately to 130 and 110 A, in 14-17- and 21-d-old cultures, respectively. In proliferating cells, the transport channels have a functional diameter of approximately 230 A. The mean diameters of the pores (membrane-to-membrane distance) in proliferating and confluent cells (728 and 712 A, respectively) were significantly different at the 10%, but not the 5%, level. No differences in pore density (pore per unit length of membrane) were detected. Serum-deprived cells (7-8 d in 1% serum or 4 d in 0.5% serum) also showed a significant decrease in the nuclear uptake of large, but not small, gold particles. Thus, the permeability effects are not simply a function of high cell density but appear to be growth related. The possible functional significance of these findings is discussed.

Identification of intracellular sites of superoxide production in stimulated neutrophils

1998 ◽  
Vol 111 (1) ◽  
pp. 81-91 ◽  
Author(s):  
T. Kobayashi ◽  
J.M. Robinson ◽  
H. Seguchi
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Time Course ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Morphological Evidence ◽  
Marker Enzyme ◽  
Intracellular Compartments

In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.

The Localization of Alkaline Phosphatase during the Post-embryonic Development of Drosophila melanogaster

1950 ◽  
Vol s3-91 (13) ◽  
pp. 89-105
Author(s):  
T. YAO
Keyword(s):  
Alkaline Phosphatase ◽  
Drosophila Melanogaster ◽  
Embryonic Development ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Larval Instar ◽  
Morphological Evidence ◽  
Simultaneous Increase ◽  
Endocrine Organs ◽  
Pericardial Cells

1. The localization of alkaline phosphatase during the post-embryonic development of Drosophila melanogaster has been described. 2. In the larvae, nuclear phosphatase is always demonstrable, but cytoplasmic phosphatase shows a more restricted distribution. Salivary glands, mid-gut, Malpighian tubes, and pericardial cells are richest in cytoplasmic phosphatase. 3. The larva prior to puparium formation is characterized by a decrease of alkaline phosphatase in the internal organs with a simultaneous increase in the hypodermis. 4. The phosphatase data support the view that the prepupa is actually an intrapuparial larval instar. 5. Pupation is accompanied, by a very noticeable increase of alkaline phosphatase which is mainly confined to the cytoplasm. The high enzyme activity is maintained for the first day and a half after head eversion: there is a subsequent decline until at the time of emergence most organs are inactive. However, certain organs retain their alkaline phosphatase activity. 6. As in embryogenesis, alkaline phosphatase seems to be more concerned with histo-differentiation than with chemo-differentiation. 7. Alkaline phosphatase (and also acid phosphatase) actively participates in the process of histolysis or cellular degeneration. 8. The alkaline phosphatase activity of the pericardial cells, together with other morphological evidence, indicates that these cells are endocrine organs which play important roles in Drosophila metamorphosis. 9. Cytochemical evidence suggests that alkaline phosphatase in Drosophila is probably playing a part in the carriage of organic substances across the membrane barrier.

Basolateral impalement of intestinal villus cells: electrophysiology of Cl- transport

1989 ◽  
Vol 256 (5) ◽  
pp. C1022-C1032 ◽  
Author(s):  
J. F. White ◽  
D. Ellingsen
Keyword(s):  
Membrane Potential ◽  
Cell Layer ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Direct Access ◽  
Intestinal Villus ◽  
Cl Transport ◽  
Conductive Pathway ◽  
Flat Sheet ◽  
Tip Cells

A method of dissecting the serosal muscle layers is described that transforms the villus of isolated Amphiuma small intestine into a flat sheet one cell layer thick, allowing rapid equilibration of the serosal medium with the basolateral membrane of the villus tip cells and direct access of the basal membrane to microelectrodes. The "villus sheet" preparation was used to examine the luminal and basolateral mechanisms of Cl- transport. The serosal membrane potential (Vs), measured with conventional microelectrodes, averaged -79.7 mV in tissues bathed in Cl- -free medium; the mucosal membrane potential (Vm) averaged -80.9 mV. Fractional resistance measured directly was 0.82 and 0.14 for the mucosal and serosal membranes, respectively. Elevation of bath [K] reduced Vm and Vs by 30.3 and 44.5 mV, respectively. Cl- (20 mM) added to the luminal medium reduced Vm by 23.9 mV and stimulated Cl- transport; luminal addition of furosemide then increased Vm by 5.6 mV and reduced Cl- transport. Addition of Cl- (20 mM) to the Cl- -free serosal fluid increased Vs 2.0 +/- 1.9 mV. On reducing the serosal [Cl] 10-fold Vs decreased 2.0 +/- 2.2 mV. These and other results indicate that basolateral Cl- exit is not over a conductive pathway. The villus sheet affords new opportunities for studying enterocyte function in the intact mucosa.

scholarly journals Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

1989 ◽  
Vol 37 (3) ◽  
pp. 353-363 ◽  
Author(s):  
T Iwano ◽  
A Yamamoto ◽  
K Omori ◽  
M Akayama ◽  
T Kumazawa ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Stria Vascularis ◽  
Basolateral Membrane ◽  
Lateral Wall ◽  
Alpha Subunit ◽  
Membrane Domain ◽  
Cochlear Duct ◽  
Gold Particles ◽  
Marginal Cells

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.

scholarly journals Antibodies to mouse lung capillary endothelium.

1988 ◽  
Vol 36 (7) ◽  
pp. 741-749 ◽  
Author(s):  
M C Rorvik ◽  
D P Allison ◽  
J A Hotchkiss ◽  
H P Witschi ◽  
S J Kennel
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Interstitial Cells ◽  
Mouse Lung ◽  
Capillary Endothelium ◽  
Specific Cell ◽  
Alveolar Wall ◽  
Gold Particles ◽  
Capillary Endothelial Cells ◽  
Quantitative Analyses

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.

scholarly journals Identification and functional assay of an extracellular calcium-sensing receptor in Necturusgastric mucosa

1997 ◽  
Vol 273 (5) ◽  
pp. G1051-G1060 ◽  
Author(s):  
Robert R. Cima ◽  
Ivan Cheng ◽  
Mary E. Klingensmith ◽  
Naibedya Chattopadhyay ◽  
Olga Kifor ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Membrane Resistance ◽  
Immunohistochemical Localization ◽  
Functional Assay ◽  
Antral Mucosa ◽  
Electrical Potentials ◽  
Necturus Maculosus

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca2+-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Basolateral membrane Cl- and K+ conductances of the dark-adapted chick retinal pigment epithelium

1993 ◽  
Vol 70 (4) ◽  
pp. 1656-1668 ◽  
Author(s):  
R. P. Gallemore ◽  
E. Hernandez ◽  
R. Tayyanipour ◽  
S. Fujii ◽  
R. H. Steinberg
Keyword(s):  
Retinal Pigment Epithelium ◽  
Time Course ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Diffusion Potential ◽  
Ion Concentration ◽  
Concentration Changes ◽  
Cl Conductance

1. We characterized the basolateral membrane Cl- and K+ conductances of the dark-adapted chick neural retina-retinal pigment epithelium (RPE)-choroid preparation. Conventional microelectrodes were used to measure apical (V(ap)) and basolateral (Vba) membrane voltage, and double-barreled Cl- and K+ selective microelectrodes were used to follow the time course and magnitude of ion concentration changes outside the basolateral (basal) membrane. 2. In response to a fivefold decrease in basal [Cl-]o, Vba rapidly depolarized by 6.4 +/- 0.7 (SE) mV, and the apparent resistance of the basolateral membrane (Rba) increased. The Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) suppressed the Vba depolarization by 40% and blocked the Rba increase. Estimates of the relative Cl- conductance (transference number, TCl) from the DIDS-sensitive component of the Cl- diffusion potential gave an average value for TCl of 0.22 +/- 0.03. 3. Further evidence for a Cl- conductance was obtained by measuring changes in intracellular Cl- activity (aCli) induced by transtissue current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. TCl estimated from these data averaged 0.23 +/- 0.02. 4. In response to a sixfold increase in basal [K+]o, Vba depolarized 6.1 +/- 0.8 mV. The amplitude of this K+ diffusion potential was inhibited 44 and 67% by 5 and 10 mM Ba2+, respectively. TK was estimated to be 0.61 +/- 0.05. 5. The rapid c-wave membrane hyperpolarizations in response to the light-evoked decrease in subretinal [K+]o were used to calculate the equivalent resistances of the apical membrane (R(ap)), basolateral membrane (Rba), and the paracellular shunt pathway (Rs). They were 152 +/- 10, 615 +/- 38, and 138 +/- 7 omega.cm2 (n = 11 tissues), respectively. From these data the equivalent electromotive force for the basal (Eba) and apical (Eap) membranes were estimated to be -45 +/- 2 and -77 +/- 1 mV, respectively. This estimate of Eba is in the range of that predicted from our estimates of TCl and TK, indicating that, in the dark-adapted chick retina, the resting conductance of the basal membrane can largely be accounted for by the Cl- and K+ conductances described here.

IMMUNOCYTOCHEMICAL LOCALIZATION OF FIBRINOGEN DURING THROMBIN-INDUCED AGGREGATION OF HUMAN PLATELETS

1987 ◽  
Author(s):  
H Suzuki ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
K Tanoue ◽  
H Yamazaki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Immunocytochemical Localization ◽  
Albumin Solution ◽  
Gold Particles ◽  
Gold Labelling ◽  
Lowicryl K4m ◽  
Induced Aggregation

The association of fibrinogen (Fbg) with washed platelets was studied during thrombin-induced aggregation in Tyrode-albumin solution with 2 mM Ca2+ or no added Ca2+. Platelets were fixed, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman Fbg, washed, reacted with gold-labelled rabbit anti-goat IgG and prepared for electron microscopy. To ensure that Fbg could be detected with this method, platelets were pretreated with chymotrypsin and aggregated with Fbg; gold particles were apparent on the surface and between adherent platelets and in the alpha granules. In a Ca2+-containing medium in the absence of external Fbg, washed platelets did not have Fbg on their surface although there was extensive gold labelling of the platelet alpha granules. Thrombin (0.05 U/ml) caused platelet aggregation, centralization and apparent fusion of alpha granules. By 60 sec large aggregates had formed, many platelets appeared degranulated but few gold particles were seen between adherent platelets. At 5 min, little Fbg remained in the aggregated platelets. In a few regions gold accumulated in fused granule material, and in occasional clusters between adherent platelets. In the presence of external Fbg (0.4 mg/ml) thrombin caused aggregation, centralization and fusion of granules, and discharge of granule contents. However, numerous gold particles were readily detectable between adherent platelets and on the platelet membrane. Fibrin formed and was abundantly labelled with immuno-gold. Similar findings were obtained in the medium without added Ca2+ (20 μM Ca2+). These results agree with observations obtained from measurements of 125I-Fbg binding and show that in the presence of external Fbg thrombin causes Fbg binding to platelets during aggregation. In the absence of added Fbg, thrombin aggregates platelets without extensive binding of released Fbg to the platelets.

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