Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

2003 ◽  
Vol 284 (1) ◽  
pp. L187-L196 ◽  
Author(s):  
Theresa A. John ◽  
Stephen M. Vogel ◽  
Chinnaswamy Tiruppathi ◽  
Asrar B. Malik ◽  
Richard D. Minshall
Keyword(s):  
Specific Binding ◽  
Fluid Phase ◽  
Scatchard Analysis ◽  
Albumin Binding ◽  
Endothelial Monolayer ◽  
Site Model ◽  
Specific Binding Sites ◽  
High K ◽  
Secondary Antibodies ◽  
Uptake And Transport

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of 125I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol · min−1 · 106cells−1. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-β-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased 125I-albumin uptake 2.3-fold. At 37°C, 125I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC50= 1 μM). Using a two-site model, we estimated by Scatchard analysis the affinity ( K D) and maximal capacity (Bmax) of albumin uptake to be 0.87 μM ( K D1) and 0.47 pmol/106 cells (Bmax1) and 93.3 μM ( K D2) and 20.2 pmol/106 cells (Bmax2). At 4°C, we also observed two populations of specific binding sites, with high ( K D1 = 13.5 nM, 1% of the total) and low ( K D2 = 1.6 μM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.

scholarly journals Hepoxilin A3-specific binding in human neutrophils

1996 ◽  
Vol 313 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Denis REYNAUD ◽  
Peter DEMIN ◽  
Cecil R. PACE-ASCIAK
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Leukotriene B4 ◽  
Proteinase K ◽  
Human Neutrophils ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Specific Binding Sites ◽  
Binding Data ◽  
Hepoxilin A3

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

Specific albumin binding to microvascular endothelium in culture

1988 ◽  
Vol 254 (3) ◽  
pp. H425-H437 ◽  
Author(s):  
J. E. Schnitzer ◽  
W. W. Carley ◽  
G. E. Palade
Keyword(s):  
Specific Binding ◽  
Molecular Transport ◽  
Cell Number ◽  
Dissociation Rate ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Albumin Binding ◽  
Microvascular Endothelium ◽  
Rat Serum ◽  
Rate Analysis

The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degrees C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at approximately 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the endothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

Endothelin ETA and ETB receptors in postnatal intestine

2001 ◽  
Vol 280 (4) ◽  
pp. G555-G562 ◽  
Author(s):  
Craig A. Nankervis ◽  
David J. Dunaway ◽  
Charles E. Miller
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ischemia Reperfusion ◽  
Age Groups ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Site Model ◽  
Number Of Binding Sites ◽  
Competitive Binding Assays

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E− vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

Increased beta-adrenergic receptors in brown fat of winter-acclimatized Alaskan voles

1983 ◽  
Vol 245 (3) ◽  
pp. R357-R363 ◽  
Author(s):  
D. D. Feist
Keyword(s):  
Cold Acclimation ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Specific Binding ◽  
Brown Fat ◽  
Scatchard Analysis ◽  
Beta Adrenergic Receptors ◽  
Hormonal Stimulation ◽  
Beta Adrenergic ◽  
Specific Binding Sites

To assess a possible mechanism for the enhanced thermogenesis of cold-acclimated and winter-acclimatized red-backed voles (Clethrionomys rutilus), beta-adrenergic receptors of brown fat were characterized by specific binding of (-)-[3H]-dihydroalprenolol [( 3H]DHA) to isolated brown fat membranes from 23 degrees C-acclimated controls, cold-acclimated (5 wk or 5 mo at 5 degrees C), wild summer, and winter-acclimatized voles. Scatchard analysis to determine the equilibrium dissociation constant (Kd) and the maximum number of binding sites (Bmax) for control brown fat membranes gave a Kd of 4.45 nM [3H]DHA and Bmax of 249 fmol [3H]DHA bound per milligram of protein. beta-Adrenergic agonists competed for specific binding sites with an order of potency typical of the beta 1 subtype of adrenergic receptors: (-)-isoproterenol greater than (-)-norepinephrine greater than or equal to (-)-epinephrine. After cold acclimation for 5 wk or 5 mo, the Kd and Bmax for adrenergic binding sites were similar to those of controls. Brown fat mass was 1.5 times greater than that of controls after 5 wk cold acclimation but similar to controls after 5 mo cold acclimation. Winter voles had 1.7 times higher Bmax and 1.6 times more brown fat than summer voles. Thus seasonal acclimatization to winter in red-backed voles appears to involve an increase in beta-adrenergic receptors in brown fat, but cold acclimation does not. The results suggest quantitative and possibly qualitative differences in neural and hormonal stimulation of brown fat between cold acclimation and winter acclimatization in voles.

scholarly journals Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland

1998 ◽  
Vol 157 (1) ◽  
pp. 99-106 ◽  
Author(s):  
G Muccioli ◽  
C Ghe ◽  
MC Ghigo ◽  
M Papotti ◽  
E Arvat ◽  
...  
Keyword(s):  
Substance P ◽  
Human Brain ◽  
Pituitary Gland ◽  
Binding Sites ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Gh Secretagogues ◽  
Specific Binding Sites ◽  
Specific Receptors

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.

scholarly journals Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

1984 ◽  
Vol 219 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
Y A Lefebvre ◽  
J T Venkatraman
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Intact Animal ◽  
Binding Capacity ◽  
Specific Binding ◽  
Male Rat ◽  
Scatchard Analysis ◽  
Specific Binding Sites ◽  
Number Of Binding Sites

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3′,5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3′,5′-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

scholarly journals Studies on the fibronectin receptors of human peripheral blood leukocytes. Morphologic and functional characterization.

1984 ◽  
Vol 159 (1) ◽  
pp. 137-151 ◽  
Author(s):  
C G Pommier ◽  
J O'Shea ◽  
T Chused ◽  
K Yancey ◽  
M M Frank ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Specific Binding ◽  
Functional Characterization ◽  
Fluid Phase ◽  
Cell Types ◽  
Cell Type ◽  
Phagocytic Cells ◽  
Blood Leukocytes ◽  
Specific Binding Sites

We have investigated the interactions between plasma fibronectin (Fn) and human peripheral blood phagocytic cells. As shown by studies of the binding of Fn-coated fluorescent microspheres (Fn-ms), both polymorphonuclear leukocytes (PMN) and monocytes had specific binding sites for Fn at the plasma membrane. However, as purified from blood, only monocytes were stimulated by Fn to become more actively phagocytic. This increase in phagocytosis was reflected by an Fn-induced increase in the ingestion of IgG-coated erythrocytes and, more dramatically by an Fn-dependent initiation of phagocytosis of C3b-coated erythrocytes. Despite this difference between PMN and monocytes in the functional consequences of Fn binding, the cell surface molecules responsible for Fn binding on the two cell types shared many characteristics. On both cells, binding of Fn-ms was inhibited by sufficient concentrations of fluid-phase Fn; both PMN and monocytes bound fewer Fn-ms at 4 degrees C than at 37 degrees C; both achieved maximal binding at similar Fn-ms/cell ratios; and phenylmethylsulfonyl fluoride did not inhibit Fn-ms binding to either cell type. Most dramatically, monoclonal anti-Fn antibodies that inhibited binding of Fn-ms to one cell type inhibited binding to both; conversely, monoclonal anti-Fn antibodies that did not inhibit Fn-ms binding to either cell type did not inhibit binding to the other. Fn will stimulate PMN to a more actively phagocytic state, like that induced in monocytes, if the PMN are first exposed to C5a or N-formyl-methionyl-leucylphenylalanine. This effect occurs without apparent change in the number of Fn receptors. We conclude that the PMN and monocyte receptors for Fn are very similar, but that their milieu is very different in the two cells as purified from peripheral blood. Whereas Fn induces increased phagocytosis in monocytes, PMN must be activated before the Fn can be effective.

INTERACTION OF PLATELETS WITH DIFFERENT ISOTYPES OF HUMAN COLLAGEN: THE COLLAGEN-RECEPTOR AND SIGNAL-RES-P0NSE COUPLING:

1987 ◽  
Author(s):  
F Misselwitz ◽  
C Norden ◽  
S P Domogatsky ◽  
V S Repin ◽  
H Heine
Keyword(s):  
Specific Binding ◽  
Thrombus Formation ◽  
Scatchard Analysis ◽  
Primary Hemostasis ◽  
Specific Binding Sites ◽  
Extracellular Signal ◽  
Human Collagen ◽  
Coated Surfaces ◽  
Platelet Spreading ◽  
Collagen Receptor

Platelet-collagen interaction is important in primary hemostasis and thrombosis. However, little is known about the interaction of platelets with genetically distinct collagen types I, III, IV, and V (Cl, ClII, CIV, CV) in their triple helical form. Here we report on the interaction of gel- filtered platelets with surfaces coated with monomeric collagen isotypes and the binding of 125-iodine-labeled Cl to platelets. It was shown that the binding of monomeric Cl is dose and time-dependent, saturable and specific, since it is competitively inhibited by unlabeled Cl, but not by unlabeled CV. Scatchard analysis reveals a receptor-class with a Kd of 2.5 x 10−8M. The result suggests that the different collagen isotypes may bind to platelets through various specific binding sites. Confirming that, we demonstrated, that interaction of platelets with collagen-coated surfaces is associated with the activation of different intra- and extracellular signal transduction systems: Initial attachment of discoid platelets to a CV-substrate does not induce neither synthesis of prostanoids nor changes of cyclic AMP-level. Adhesion and massive spreading of platelets on a CIV-substrate result in a decrease of stimulated cAMP-level without any synthesis of prostanoids. Only thrombus-formation during interaction of platelets with Cl- or CHI-coated surfaces is accompanied by a parallel decrease of cAMP and massive synthesis of platelet prostanoids. Additionally, it was shown that the stable prostacyclin analogue, carbacyc-lin, inhibited thrombus-formation and platelet spreading, whereas the PGH2/TXA2-receptor antagonists, 13-Azaprostanoic acid ana BM 13.177, completely inhibited thrombus-formation without any effect on platelet spreading.

Sodium-dependent [3H]imipramine binding in rat hippocampus and its relationship to serotonin uptake

1987 ◽  
Vol 65 (12) ◽  
pp. 2422-2427 ◽  
Author(s):  
Pavel D. Hrdina
Keyword(s):  
Specific Binding ◽  
Serotonin Uptake ◽  
Neuronal Uptake ◽  
Scatchard Analysis ◽  
Serotonergic Neurons ◽  
Single Class ◽  
Site Model ◽  
High Affinity ◽  
Imipramine Binding ◽  
Sodium Dependent

The relationship of [3H]imipramine recognition sites and serotonergic function was investigated by simultaneously determining the desipramine-defined and sodium-dependent components of [3H]imipramine binding and the serotonin levels and uptake in hippocampus of rats without and with selective lesion of serotonergic neurons with 5,7-dihydroxytryptamine. In control rats, the desipramine-defined [3H]imipramine binding to hippocampal membranes showed a high affinity (Kd = 2 nM) and low affinity (Kd = 31 nM) component. In contrast, the Scatchard analysis of sodium-dependent binding revealed a single class of sites of high affinity (Kd = 1.5 nM). Displacement of sodium-dependent [3H]imipramine binding by cold imipramine resulted in a steep curve best fitted to a one-site model. Sodium-dependent binding of [3H]imipramine at 4 nM concentration represented only about 38% of desipramine-defined binding. 5,7-Dihydroxytryptamine treatment resulted in marked reduction of hippocampal serotonin concentration and uptake without any changes in norepinephrine levels. Virtually only the low affinity component of desipramine-defined [3H]imipramine binding was detected by Scatchard analysis in 5,7-dihydroxytryptamine lesioned rats. The desipramine-defined "specific" [3H]imipramine binding in hippocampi of lesioned rats was decreased by 46%, whereas the sodium-dependent binding was only 18% of that seen in controls. Desipramine-defined specific binding in absence of sodium was not altered by lesion to serotonergic neurons. The results suggest that desipramine-defined specific [3H]imipramine binding may not be appropriate for studying the role of imipramine sites in relation to serotonin neuronal uptake and that determination of sodium-dependent binding components of both [3H]imipramine binding and serotonin uptake should be used in future studies.

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