Cocaine’s Direct Effect on Either Sickle or Normal Erythrocytes Does Not Increase Phosphatidylserine Exposure, Calcium Pumping, or Scramblase Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3692-3692
Author(s):  
Kitty DeJong ◽  
Robert Hagar
Keyword(s):  
Flow Cytometry ◽  
Pulmonary Hypertension ◽  
Calcium Influx ◽  
Plasma Concentrations ◽  
Calcium Ionophore ◽  
Annexin V ◽  
Cell Types ◽  
Red Cell ◽  
Sickle Cells ◽  
Red Cell Membranes

Hypothesis: Cocaine directly affects the exposure of phosphatidylserine, calcium pumping, and scramblase activity in sickle cells. Background: Cocaine is highly associated with pulmonary hypertension in both normal and sickle cell disease patients. Given the growing interest in pulmonary hypertension as a major morbidity factor, the causes and determinants of pulmonary hypertension need to be elucidated to allow rational study designs. Phosphatidylserine (PS) exposure is known to occur in sickle cells and appears to be relevant for vascular damage. Although the mechanism underlying PS exposure is poorly understood, signal transduction processes appear to play a role. We wondered if cocaine could activate these processes and cause PS exposure, or interfere with the proteins involved in the transbilayer movement of PS. Methods: Sickle and normal erythrocytes were exposed to cocaine HCl (from a 10% topical solution, Roxane Labs) at concentrations between 100 and 1000 ng/ml, which is the range of determined plasma concentrations after cocaine use. PS exposure after cocaine treatment was measured by labeling with fluorescently conjugated annexin V (AV) and analysis by flow cytometry. Alterations in scramblase activity were determined by assessing the percentage of PS-exposing cells following loading of the cells with 0.1 mM calcium using calcium ionophore. Calcium influx and Ca-ATPase-mediated calcium efflux were monitored using the fluorescent probe Fluo4, and flippase activity was assessed using NBD-PS followed by analysis with flow cytometry. Results: Sickle cells differ from normal cells with respect to most of the measured parameters, such as having more PS-exposing cells, lower flippase activity, and alterations in calcium kinetics. However, cocaine did not have any effect on PS exposure, calcium-induced scrambling, calcium influx, calcium pump activity, or flippase activity. The cell scatter patterns did not show any gross changes in red cell shape or density after cocaine treatment. Discussion: The lack of effect implies that cocaine is working through other mechanisms than effects on red cell membranes. Therefore, future studies should focus on other vascular cell types and membrane pathways. Alternatively, cocaine metabolites should be evaluated for their activities on red cells.

Effect of LY171883 on endotoxin-induced lung injury in pigs

1990 ◽  
Vol 69 (4) ◽  
pp. 1315-1322 ◽  
Author(s):  
N. C. Olson ◽  
K. T. Kruse-Elliott ◽  
L. Johnson
Keyword(s):  
Pulmonary Hypertension ◽  
Lung Injury ◽  
Pulmonary Vascular Resistance ◽  
Vascular Resistance ◽  
Ex Vivo ◽  
Plasma Concentrations ◽  
Calcium Ionophore ◽  
Leukotriene D4 ◽  
Prostaglandin F2 Alpha ◽  
Capillary Membrane

We evaluated the role of sulfidopeptide leukotrienes as mediators of endotoxin-induced respiratory failure in pigs. Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h followed by 2 micrograms.kg-1.h-1 for 3 h in the presence and absence of LY171883, a specific leukotriene D4 (LTD4)/LTE4 receptor antagonist. Endotoxin caused hemoconcentration, granulocytopenia, decreased cardiac index, systemic hypotension, pulmonary hypertension, increased pulmonary vascular resistance, bronchoconstriction, hypoxemia, increased permeability of the alveolar-capillary membrane, pulmonary edema, and increased plasma concentrations of thromboxane B2 (TxB2), prostaglandin F2 alpha (PGF2 alpha), and 6-keto-PGF1 alpha. LY171883 did not modify endotoxin-induced cardiopulmonary and hematologic abnormalities, except for a modest attenuation of pulmonary hypertension (at 1 h) and increased pulmonary vascular resistance (at 1-2 h). Ex vivo stimulation of whole blood with calcium ionophore caused large increases in plasma concentrations of TxB2, PGF2 alpha, and LTB4. These increases were not significantly modified in blood derived from pigs treated with LY171883, indicating no inhibition of cyclooxygenase or 5-lipoxygenase. We conclude that LTD4 and LTE4 are not important mediators of endotoxin-induced lung injury in anesthetized pigs, although they may contribute modestly to pulmonary vasoconstriction.

scholarly journals Mechanism of spontaneous inside-out vesiculation of red cell membranes.

1988 ◽  
Vol 106 (6) ◽  
pp. 1893-1901 ◽  
Author(s):  
V L Lew ◽  
A Hockaday ◽  
C J Freeman ◽  
R M Bookchin
Keyword(s):  
Membrane Fusion ◽  
Cell Membranes ◽  
Membrane Surface ◽  
Cell Types ◽  
Red Cell ◽  
Membrane Lysis ◽  
Red Cell Membranes ◽  
Free Membrane ◽  
Mechanical Shearing ◽  
Inside Out

In certain conditions, human red cell membranes spontaneously form inside out vesicles within 20 min after hypotonic lysis. Study of the geometry of this process now reveals that, contrary to earlier views of vesiculation by endocytosis or by the mechanical shearing of cytoskeleton-depleted membrane, lysis generates a persistent membrane edge which spontaneously curls, cuts, and splices the membrane surface to form single or concentric vesicles. Analysis of the processes by which proteins may stabilize a free membrane edge led us to formulate a novel zip-type mechanism for membrane cutting-splicing and fusion even in the absence of free edges. Such protein-led membrane fusion represents an alternative to mechanisms of membrane fusion based on phospholipid interactions, and may prove relevant to processes of secretion, endocytosis, phagocytosis, and membrane recycling in many cell types.

TMEM16F Plays a Vital Role In Platelet Microparticle Generation and Thrombosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2294-2294 ◽  
Author(s):  
Ann-Desdemonia N Fowajuh ◽  
Debora K Mukaz ◽  
Dongjun Li ◽  
Pani A. Apostolidis ◽  
Aasma Khan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Thrombus Formation ◽  
Calcium Ionophore ◽  
Annexin V ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Microparticle Formation ◽  
Dual Agonist ◽  
High Resolution Microscopy

Abstract Transmembrane (TMEM)16f is member of the TMEM16 family of ion channels, recently shown to be essential for optimal Ca2+-dependent phospholipid scrambling in platelets, platelet-dependent pro-coagulant activity and thrombosis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were obtained from Andrea Vortkamp and the platelets of these mice were isolated, stimulated with either dual agonist (thrombin + convulxin) or calcium ionophore (A23187), and platelet surface exposure of phosphatidylserine (PS) and microparticle generation were assessed by both flow cytometry and high resolution immunofluorescent confocal microscopy. To measure PS exposure, annexin V binding to platelet surfaces was quantified: In response to thrombin and convulxin, only 10.65% ± 1.35% of TMEM16f-/- platelets exposed PS after dual agonist exposure, compared to 16.1% ± 2.3%PS+ WT platelets when analyzed by high resolution microscopy. When analyzed by flow cytometry, dual agonist exposure of WT platelets yielded 52.4% ± 7.0% of maximal annexin V binding achieved by 10μM A23187; in contrast, there was no significant increase in annexin V binding detected in TMEM16f-/- platelets treated with dual agonist. Responses to calcium ionophore were also reduced in TMEM16f-/- platelets relative to WT (17.3% ± 8.3% PS+ TMEM16f-/- platelets in response to 1μM A23187 compared to 40% ± 1.4% PS+ WT platelets, by high resolution microscopy). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by high resolution immunofluorescent microscopy and flow cytometry. On average, approximately 5.7 ± 0.33 microparticles were generated by untreated WT platelets however thrombin and convulxin treated platelets yielded 10.66 ± 0.92 microparticles per platelet whereas no significant increase in microparticle generation was observed in TMEM16f-/- platelets after dual agonist treatment. These data suggest that TMEM16f is required not only for PS exposure on platelet membranes, but also for the shedding of PS+ microparticles. TMEM16f-/- mice also have a significant defect in stable occlusive thrombus formation following 10% ferric chloride injury of the carotid artery (0/6 TMEM16f-/- mice form stable thrombi compared to 5/6 WT mice forming stable thrombi under the same conditions). TMEM16f-/- platelets had a slight (15%) reduction in total fibrinogen binding stimulated by PAR4 peptide agonist compared to WT, but showed no significant differences in aggregation to PAR4 agonist peptide or ADP compared to WT control platelets, suggesting that the defect in thrombus formation is likely due to PS- or microparticle-dependent procoagulant activity. This hypothesis is supported by preliminary results demonstrating that when microparticles isolated from ionophore-stimulated WT platelets were injected into TMEM16f knockout mice, 3/3 TMEM16f-/- mice formed stable thrombi, compared to 5/6 WT mice injected with vehicle control. In conclusion, TMEM16f-/- mice are deficient in platelet PS exposure, platelet-derived microparticle formation, and injury-induced thrombus formation, and platelet-derived microparticles appear to contribute to the defect in thrombus formation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Apoptotic Effects on HL60 Human Leukaemia Cells Induced by Lavandin Essential Oil Treatment

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 538 ◽  
Author(s):  
Valentina Laghezza Masci ◽  
Elisa Ovidi ◽  
Anna Rita Taddei ◽  
Giovanni Turchetti ◽  
Antonio Tiezzi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Essential Oil ◽  
Annexin V ◽  
Cell Types ◽  
Dependent Manner ◽  
Linalyl Acetate ◽  
Hl60 Cells ◽  
Morphological Alterations ◽  
Apoptotic Process ◽  
Human Leukaemia

Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding L. angustifolia × L. latifolia, was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.

Abstract 203: Cardiomyocyte T-tubule Membrane Turns Over, Releasing BIN1 Containing Microparticles into Blood

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Bing Xu ◽  
Ying Fu ◽  
TingTing Hong
Keyword(s):  
Flow Cytometry ◽  
Adult Mouse ◽  
Cardiac Tissue ◽  
Muscle Protein ◽  
Three Dimensional ◽  
Super Resolution ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Fluorescent Imaging

Bridging integrator 1 (BIN1) is a cardiac muscle protein that folds cardiomyocyte T-tubule membrane. BIN1 is intrinsic to cardiac health, and is reduced in acquired heart failure. Interestingly, we have found that BIN1 is also blood available, and that plasma BIN1 correlates with cardiac function, suggesting cardiac origin of plasma BIN1. We found that low plasma BIN1 correlates with failing muscle and predicts ventricular arrhythmia. However, the paradigm does not exist for an intracellular membrane associate cardiomyocyte protein to be homeostatically turned over into blood. In this study, using a mouse model with cardiac specific deletion of Bin1 gene, we identified with biochemical techniques that plasma BIN1 levels directly correlate with cardiac tissue BIN1 levels, indicating cardiac origin. Furthermore, investigations using both super-resolution fluorescent imaging and flow cytometry analysis revealed that adult ventricular cardiomyocytes constantly release BIN1 into blood via membrane microparticle production. Microparticles are small membrane vesicles shed from plasma membrane of a variety of cell types including platelets, leukocytes, and endothelial cells. Using super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we found similar to the blood cells, isolated adult mouse cardiomyocytes release Annexin V positive microparticles with diameters ranging between 0.1 to 1.0 μm. These microparticles also carry BIN1 protein. Flow cytometry was also used to detect and quantify microparticles <1.0 μm in size from medium bathing a pure population of adult mouse cardiomyocytes. BIN1 microparticle release is proportional to actin stability and amount of T-tubule membrane folds. Compared to wild type cardiomyocytes, microparticle release is significantly reduced from myocytes with heterozygous deletion of Bin1 gene. These data indicate that cardiomyocyte membrane undergoes dynamic turnover, releasing T-tubule folds into blood as microparticles. Furthermore, plasma BIN1 can be used as a direct measure of cardiomyocyte health and reserve.

scholarly journals Plasma membrane integrity in health and disease: significance and therapeutic potential

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Catarina Dias ◽  
Jesper Nylandsted
Keyword(s):  
Plasma Membrane ◽  
Therapeutic Potential ◽  
Calcium Influx ◽  
Membrane Integrity ◽  
Cell Types ◽  
Physical Parameters ◽  
Membrane Repair ◽  
Plasma Membrane Integrity ◽  
Repair Mechanisms ◽  
And Function

AbstractMaintenance of plasma membrane integrity is essential for normal cell viability and function. Thus, robust membrane repair mechanisms have evolved to counteract the eminent threat of a torn plasma membrane. Different repair mechanisms and the bio-physical parameters required for efficient repair are now emerging from different research groups. However, less is known about when these mechanisms come into play. This review focuses on the existence of membrane disruptions and repair mechanisms in both physiological and pathological conditions, and across multiple cell types, albeit to different degrees. Fundamentally, irrespective of the source of membrane disruption, aberrant calcium influx is the common stimulus that activates the membrane repair response. Inadequate repair responses can tip the balance between physiology and pathology, highlighting the significance of plasma membrane integrity. For example, an over-activated repair response can promote cancer invasion, while the inability to efficiently repair membrane can drive neurodegeneration and muscular dystrophies. The interdisciplinary view explored here emphasises the widespread potential of targeting plasma membrane repair mechanisms for therapeutic purposes.

BRIEF REPORT: Freeze-Cleaving of Red Cell Membranes in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
1967 ◽  
Vol 30 (6) ◽  
pp. 785-791 ◽  
Author(s):  
RONALD S. WEINSTEIN ◽  
ROGER A. WILLIAMS
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Cell Membranes ◽  
Red Cells ◽  
Red Cell ◽  
Electron Microscopic ◽  
Structural Differences ◽  
Microscopic Studies ◽  
Red Cell Membranes

Abstract Electron microscopic studies on dried isolated red cell ghosts have been reported to show lesions associated with cell membranes in paroxysmal nocturnal hemoglobinuria (PNH). In this study, carbon-platinum replicas of membranes of freeze-cleaved, partially hydrated PNH red cells and isolated PNH cell ghosts failed to confirm the existence of these abnormalities. This suggests that the previously described lesions are the products of drying artifacts, although they may reflect hidden structural differences between PNH and normal red cell membranes.

scholarly journals Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells.

1985 ◽  
Vol 85 (1) ◽  
pp. 123-136 ◽  
Author(s):  
J H Kaplan ◽  
L J Kenney
Keyword(s):  
Atpase Activity ◽  
Cell Membranes ◽  
Conformational Transition ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Red Cell ◽  
Na Pump ◽  
Na Ions ◽  
Red Cell Membranes ◽  
Increasing Temperature

Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chem., 243:1957), the phosphoenzyme formed at 0 degrees C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested that the conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1 approximately P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0 degrees C. Since the ATP:ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1 approximately P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0 degrees C. Simple saturation was observed instead with a K0.5 for Na of approximately 1 mM. The effect of increasing temperature on the ATP:ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0 degrees C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0 degrees C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 degrees C, there was an approximately 300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the E1 approximately P----E2P transition of the Na pump in human red cell membranes is blocked at 0 degrees C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP:ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0 degrees C, where E1 approximately P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme.

scholarly journals Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras.

1997 ◽  
Vol 17 (3) ◽  
pp. 1396-1406 ◽  
Author(s):  
N P Fam ◽  
W T Fan ◽  
Z Wang ◽  
L J Zhang ◽  
H Chen ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Calcium Influx ◽  
Calcium Ionophore ◽  
Mitogen Activated Protein Kinase ◽  
Northern Analysis ◽  
Free Form ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Iq Motif ◽  
Guanine Nucleotide Exchange

Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli. Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region. Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association with calmodulin in epithelial cells ectopically expressing Ras-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore-induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.

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