Biomedical subjects

D E Golan

Publications and source records attributed to D E Golan.

At least 37 records · Page 2Linked to original sources

PubMed1996

A multiparameter analysis of sickle erythrocytes in patients undergoing hydroxyurea therapy.

During 24 weeks of hydroxyurea treatment, we monitored red blood cell (RBC) parameters in three patients with sickle cell disease, including F-cell and F-reticulocyte profiles, distributions of delay times for intracellular polymerization, sickle erythrocyte adherence to human umbilical vein endothelial cells in a laminar flow chamber, RBC phthalate density profiles, mean corpuscular hemoglobin concentration and cation content, reticulocyte mean corpuscular hemoglobin concentration, 1H-nuclear magnetic resonance transverse relaxation rates of packed RBCs, and plasma membrane lateral and rotational mobilities of band 3 and glycophorins. Hydroxyurea increases the fraction of cells with sufficiently long delay times to escape the microcirculation before polymerization begins. Furthermore, high pretreatment adherence to human umbilical vein endothelial cells of sickle RBCs decreased to normal after only 2 weeks of hydroxyurea treatment, preceding the increase in fetal hemoglobin levels. The lower adhesion of sickle RBCs to endothelium would facilitate escape from the microcirculation before polymerization begins. Hydroxyurea shifted several biochemical and biophysical parameters of sickle erythrocytes toward values observed with hemoglobin SC disease, suggesting that hydroxyurea moderates sickle cell disease toward the milder, but still clinically significant, hemoglobin SC disease. The 50% reduction in sickle crises documented in the Multicenter Study of Hydroxyurea in Sickle Cell Disease is consistent with this degree of erythrocyte improvement.

Adult↗

PubMed1996

Adhesion of sickle neutrophils and erythrocytes to fibronectin.

The pathophysiology of vaso-occlusive crisis in sickle cell disease involves interactions among blood cells, plasma proteins, and vessel wall components. The initial goal of this work was to quantify the adhesion of sickle red blood cells (RBCs) to fibronectin immobilized on glass under both static and dynamic shear stress conditions. High-power microscopic inspection of static assay plates showed striking numbers of adherent neutrophils as well as RBCs. Sickle neutrophils and RBCs were significantly more adherent to fibronectin than the corresponding normal cells in static adhesion assays. Adhesion of both sickle neutrophils and sickle RBCs in dynamic adhesion assays was promoted by a period of static incubation preceding initiation of shear stress conditions. Adherent neutrophils remained attached at shear stresses up to 51 dyne/cm2; most adherent RBCs were attached at shear stresses up to 13 dyne/cm2, but detached at a shear stress of 20 dyne/cm2. Sickle neutrophil adhesion was enhanced significantly by autologous plasma. Elevated levels of plasma interleukin-6 (IL-6; but not IL-1 or IL-8) were found in 6 of 9 sickle cell disease samples examined, and elevated levels of tumor necrosis factor were found in 2 of 9 samples. Plasma IL-6 levels correlated positively with both the number of sickle neutrophils adherent to fibronectin and the ability of sickle plasma to enhance adhesion of normal neutrophils to fibronectin. These data suggest possible roles for neutrophil activation and for fibronectin in mediating sickle neutrophil and RBC adhesion.

Adult↗

PubMed1996

Red cell membrane remodeling in sickle cell anemia. Sequestration of membrane lipids and proteins in Heinz bodies.

In red cells from patients with sickle cell anemia, hemoglobin S denatures and forms Heinz bodies. Binding of Heinz bodies to the inner surface of the sickle cell membrane promotes clustering and colocalization of the membrane protein band 3, outer surface-bound autologous IgG and, to some extent, the membrane proteins glycophorin and ankyrin. Loss of transbilayer lipid asymmetry is also found in certain populations of sickle red cells. The lateral distribution of sickle cell membrane lipids has not been examined, however. In this report, we examine by fluorescence microscopy the incorporation and distribution of the fluorescent phospholipid analogues 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phosphatidylserine and NBD-phosphatidylcholine in sickle red cells. Both phospholipid analogues are observed to accumulate prominently at sites of Heinz bodies. Accumulation at sites of Heinz bodies is also shown by 1,'1-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent lipid analogue that readily crosses membranes, but not by fluorescein-phosphatidylethanolamine, an analogue that is localized to the outer leaflet of the membrane. Double labeling and confocal microscopy techniques show that NBD-lipids, band 3 protein, protein 4.1, ankyrin, and spectrin are all sequestered within sickle red cells and colocalized at sites of Heinz bodies. We propose that Heinz bodies provide a hydrophobic surface on which sickle red cell membrane lipids and proteins are sequestered.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

ATP depletion causes translational immobilization of cell surface transferrin receptors in K562 cells.

We have used quantitative fluorescence microscopy and fluorescence photobleaching recovery to examine the role of metabolic energy in the translational movement of transferrin receptors in the plasma membrane of K562 erythroleukemia cells. Cellular ATP depletion caused a significant decrease in the translational mobility of cell surface transferrin receptors and a significant increase in the number of receptors on the cell surface. ATP repletion restored receptor translational mobility and cell surface expression to control values. Inhibition of ATP hydrolases by orthovanadate also immobilized cell surface transferrin receptors and altered cell surface receptor expression, in a concentration-dependent manner. Vanadate-induced changes in receptor mobility and cell surface expression were reversible upon washing out the drug. Cellular ATP depletion did not affect the translational mobility of plasma membrane glycophorins or a fluorescent phospholipid analogue. We conclude that the translational movement of cell surface transferrin receptors specifically requires metabolic energy and ATP hydrolysis.

Adenosine Triphosphate↗

PubMed1996

Immune interferon gamma inhibits translational mobility of a plasma membrane protein in preimplantation stage mouse embryos: a T-helper 1 mechanism for immunologic reproductive failure.

OBJECTIVE: Immune interferon gamma adversely affects mouse embryo development and has been proposed as a mediator of reproductive failure involving T-helper 1 immunity. We hypothesized that one mechanism by which interferon gamma could exert an adverse effect on embryos was by altering plasma membrane organization and transmembrane protein mobility. STUDY DESIGN: The fluorescence photobleaching recovery technique was used to measure the effect of the T-helper 1 cytokine interferon gamma on the translational mobility of a specific embryonic surface glycoprotein recognized by the monoclonal antibody S75. RESULTS: Compared with controls interferon gamma significantly decreased the fractional mobility of fluorescein isothiocyanate S75 in one- and two-cell mouse embryos. CONCLUSION: Interferon gamma may alter plasma membrane domains or cytoskeletal organization in early-stage embryos. By restricting plasma membrane protein mobility interferon gamma could effect T-helper 1-mediated reproductive failure.

Abortion, Spontaneous↗

PubMed1996

Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems.

An image-based technique of fluorescence recovery after photobleaching (video-FRAP) was used to measure the lateral diffusion coefficients of a series of nine fluorescent probes in two model lipid bilayer systems, dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (40 mol%), as well as in human stratum corneum-extracted lipids. The probes were all lipophilic, varied in molecular weight from 223 to 854 Da, and were chosen to characterize the lateral diffusion of small compounds in these bilayer systems. A clear molecular weight dependence of the lateral diffusion coefficients in DMPC bilayers was observed. Values ranged from 6.72 x 10(-8) to 16.2 x 10(-8) cm2/s, with the smaller probes diffusing faster than the larger ones. Measurements in DMPC/cholesterol bilayers, which represent the most thorough characterization of small-solute diffusion in this system, exhibited a similar molecular weight dependence, although the diffusion coefficients were lower, ranging from 1.62 x 10(-8) to 5.60 x 10(-8) cm2/s. Lateral diffusion measurements in stratum corneum-extracted lipids, which represent a novel examination of diffusion in this unique lipid system, also exhibited a molecular weight dependence, with values ranging from 0.306 x 10(-8) to 2.34 x 10(-8) cm2/s. Literature data showed that these strong molecular weight dependencies extend to even smaller compounds than those examined in this study. A two-parameter empirical expression is presented that describes the lateral diffusion coefficient in terms of the solute's molecular weight and captures the size dependence over the range examined. This study illustrates the degree to which small-molecule lateral diffusion in stratum corneum-extracted lipids can be represented by diffusion in DMPC and DMPC/cholesterol bilayer systems, and may lead to a better understanding of small-solute transport across human stratum corneum.

Cholesterol↗

PubMed1996

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1996

Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area.

Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) KdS suggest that cell-cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion.

Antibodies, Monoclonal↗

PubMed1996

Reorganization of microfilament structure induced by ac electric fields.

AC electric fields induce redistribution of integral membrane proteins. Cell-surface receptor redistribution does not consistently follow electric field lines and depends critically on the frequency of the applied ac electric fields, suggesting that mechanisms other than electroosmosis are involved. We hypothesized that cytoskeletal reorganization is responsible for electric field-induced cell-surface receptor redistribution, and used fluorescence video microscopy to study the reorganization of microfilaments in human hepatoma (Hep3B) cells exposed to low-frequency electric fields ranging in strength from 25 mV/cm to 20 V/cm (peak to peak). The frequency of the applied electric field was varied from 1 to 120 Hz and the field exposure duration from 1 to 60 min. In control cells, cytoplasmic microfilaments were aligned in the form of continuous parallel cables along the longitudinal axis of the cell. Exposure of cells to ac electric fields induced alterations in microfilament structure in a manner that depended on the frequency of the applied field. A 1 or 10 Hz ac field caused microfilament reorganization from continuous, aligned cable structures to discontinuous globular patches. In contrast, the structure of microfilaments in cells exposed to 20-120 Hz electric fields did not differ from that in control cells. The extent of microfilament reorganization increased nonlinearly with the electric field strength. The characteristic time for microfilament reorganization in cells exposed to a 1 Hz, 20 V/cm electric field was approximately 5 min. Applied ac electric fields could initiate signal transduction cascades, which in turn cause reorganization of cytoskeletal structures.

Actin Cytoskeleton↗

PubMed1995

Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human umbilical vein endothelial cells.

BACKGROUND: The peptido-leukotrienes (LTs) and lipoxins (LX) are produced by platelets through the transcellular conversion of leukocyte-derived LTA4 at sites of vascular inflammation and injury, such as during coronary artery balloon angioplasty. We studied the actions of these eicosanoids on vascular endothelium. METHODS AND RESULTS: We found that stimulation of cultured human umbilical vein endothelial cells (EC) with LTC4 and LTD4 resulted in the release of high-molecular-weight multimers of von Willebrand factor (vWF) in a concentration- and time-dependent fashion, as measured by ELISA. Neither LXA4 nor LXB4 stimulated vWF release. LTC4 and LTD4 also stimulated a rapid increase in the surface expression of P-selectin indicated by increased binding of anti-P-selectin monoclonal antibody-coated beads. Fluorescence cytometry detected prolonged peaks of [Ca2+]i in EC in response to concentrations of thrombin and LTD4 that induce near-maximal vWF secretion. In contrast, concentrations of LTC4 that induce similar levels of vWF secretion produced only asynchronous oscillations of [Ca2+]i in most EC and rarely induced prolonged peaks of [Ca2+]i. Depletion of external Ca2+ had no apparent impact on LT-stimulated [Ca2+]i transients and vWF secretion, implicating an intracellular pool as the source of this response. Staurosporine, sphingosine, and H-7 each had only modest effects on peptido-LT-induced vWF secretion, suggesting that protein kinase C is not a primary mediator of peptido-LT-induced exocytosis. Inhibitors of cyclooxygenase and platelet-activating factor had no effect on peptido-LT-mediated vWF secretion. CONCLUSIONS: Through the induction of vWF secretion and P-selectin surface expression, peptido-LTs are likely to play an important role in the interrelated processes of hemostasis and inflammation.

Calcium↗

PubMed1995

Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton.

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1995

Alterations in the frequency and shape of Ca2+ fluctuations in GH4C1 cells induced by thyrotropin-releasing hormone and Bay K 8644.

We have examined statistically the actions of thyrotropin-releasing hormone (TRH) and Bay K 8644, an L-type Ca(2+)-channel agonist, on the frequency and shape of cytosolic Ca2+ spikes in individual GH4C1 rat pituitary cells. TRH induced a brief (0-40 s) suppression of Ca2+ spikes followed by a period (40-200 s) of increased spike frequency. TRH treatment reduced the rate of rise and amplitude of Ca2+ spikes, and increased the rate of fall, relative to spontaneous spikes before treatment. TRH had no significant effect on the correlation between spike amplitude and the spike decay time constant tau, suggesting that the increased rate of fall was due to enhanced Ca2+ extrusion and not to decreased Ca(2+)-induced Ca2+ release. Bay K rapidly (t1/2 = 9-13 s) induced a 2-fold increase in the rate of rise of spikes with no change in the total rise time, leading to an increase in spike amplitude. It increased by 2-fold the fall time of spikes, as predicted solely by the previously observed relationship between spike amplitude and fall time. Bay K therefore appeared to increase the number of Ca2+ channels participating in each spike event without altering the kinetics of channel activation or deactivation, and without influencing Ca2+ extrusion. After addition of Bay K, the interval between spikes gradually (t1/2 approximately 100 s) increased, whereas the rate of rise remained constant and maximal. To explain these actions of TRH and Bay K, we postulate that a fraction of L-type Ca2+ channels are inactivated during each spike and must be re-activated in order to participate in a subsequent spike. We conclude further that the changes in spike frequency and profiles induced by these secretagogues are most consistent with a model in which TRH induces increases in both Ca2+ influx and efflux while Bay K induces a large increase in Ca2+ influx but has little effect on efflux.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Direct presentation of nonpeptide prenyl pyrophosphate antigens to human gamma delta T cells.

Human V gamma 2V delta 2+ T cells recognize mycobacterial nonpeptide antigens, such as isopentenyl pyrophosphate, and their synthetic analogs, such as monoethyl phosphate, through a TCR-dependent process. Here, we examine the presentation of these antigens. V gamma 2V delta 2+ T cells recognized secreted prenyl pyrophosphate antigens in the absence of other accessory cells but, under such conditions, required T cell-T cell contact. Recognition required neither the expression of classical MHC class I, MHC class II, or CD1a, CD1b, and CD1c molecules, nor MHC class I or class II peptide loading pathways. Fixed accessory cells also presented the prenyl pyrophosphate antigens to gamma delta T cells. Thus, in contrast with the presentation of conventional peptide antigens, protein antigens, and superantigens to alpha beta T cells, prenyl pyrophosphate antigens are presented to gamma delta T cells through a novel extracellular pathway that does not require antigen uptake, antigen processing, or MHC class I or class II expression. This pathway allows for the rapid recognition of bacteria by gamma delta T cells and suggests that gamma delta T cells play a role in the early response to bacterial infection.

Antigen Presentation↗

PubMed1995

Intracellular mediators regulate CD2 lateral diffusion and cytoplasmic Ca2+ mobilization upon CD2-mediated T cell activation.

CD2 is a T cell surface glycoprotein that participates in T cell adhesion and activation. These processes are dynamically interrelated, in that T cell activation regulates the strength of CD2-mediated T cell adhesion. The lateral redistribution of CD2 and its ligand CD58 (LFA-3) in T cell and target membranes, respectively, has also been shown to affect cellular adhesion strength. We have used the fluorescence photobleaching recovery technique to measure the lateral mobility of CD2 in plasma membranes of resting and activated Jurkat T leukemia cells. CD2-mediated T cell activation caused lateral immobilization of 90% of cell surface CD2 molecules. Depleting cells of cytoplasmic Ca2+, loading cells with dibutyric cAMP, and disrupting cellular microfilaments each partially reversed the effect of CD2-mediated activation on the lateral mobility of CD2. These intracellular mediators apparently influence the same signal transduction pathways, because the effects of the mediators on CD2 lateral mobility were not additive. In separate experiments, activation-associated cytoplasmic Ca2+ mobilization was found to require microfilament integrity and to be negatively regulated by cAMP. By directly or indirectly controlling CD2 lateral diffusion and cell surface distribution, cytoplasmic Ca2+ mobilization may have an important regulatory role in CD2 mediated T cell adhesion.

Actin Cytoskeleton↗

PubMed1995

Millisecond measurement of transport during and after an electroporation pulse.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.

Biological Transport↗

PubMed1994

Microtubule inhibitors differentially affect translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 cells.

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleaching parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37 degrees C and 21 degrees C were calculated to be 0.10-0.15 min-1 and 0.02-0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by beta-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtubule inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells.

Biopolymers↗

PubMed1994

Prolonged peak elevations in cytoplasmic free calcium ions, derived from intracellular stores, correlate with the extent of thrombin-stimulated exocytosis in single human umbilical vein endothelial cells.

We have used indo-1-loaded human endothelial cells (EC) in monolayer culture and quantitative laser scanning fluorescence microscopy techniques to investigate the magnitude and duration of the change in cytoplasmic free calcium ([Ca2+]i) required for thrombin-stimulated von Willebrand factor (vWF) secretion in individual EC. Both alpha-thrombin and a 14 amino acid thrombin receptor activating peptide stimulate an increase in EC [Ca2+]i that is agonist dose dependent. Low-dose agonist treatment generates asynchronous oscillations (i.e., repetitive spikes < 80 sec duration) in [Ca2+]i. Stimulation with higher agonist concentrations generates a prolonged single peak elevation in [Ca2+]i. Both the number of cells displaying prolonged [Ca2+]i peaks and the mean amplitude of the peaks increase as a function of agonist concentration. Higher doses of agonist also cause sustained elevations in [Ca2+]i that depend upon extracellular Ca2+. Oscillations in [Ca2+]i are not sufficient to stimulate significant vWF secretion, and sustained elevations in [Ca2+]i are not required for maximal secretion. Both the number of cells displaying prolonged peaks and the mean peak amplitude correlate with increasing levels of vWF secretion from the culture. We have used the expression of P-selectin, a secretory granule membrane protein, as a marker for measuring thrombin-induced exocytosis in individual EC. Both the number of secreting cells and the amount of secretion per cell increase as a function of thrombin concentration. The graded responses in [Ca2+]i amplitudes and the graded exocytotic response may be causally related.

Antibodies, Monoclonal↗

PubMed1994

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage. Removal of oxygen may allow excited state fluorophores in close proximity to the plasma membrane to react with neighboring proteins or lipids during photobleaching. This effect has important implications for the ability of the fluorescence photobleaching recovery technique to report accurate lateral mobilities of cell membrane molecules under hypoxic conditions.

Anion Exchange Protein 1, Erythrocyte↗