Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at −40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor α (TNF-α) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.

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L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System

Blood ◽

10.1182/blood.v89.9.3228 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3228-3235 ◽

Cited By ~ 45

Author(s):

A. Zakrzewicz ◽

M. Gräfe ◽

D. Terbeek ◽

M. Bongrazio ◽

W. Auch-Schwelk ◽

...

Keyword(s):

Endothelial Cells ◽

Time Course ◽

Leukocyte Adhesion ◽

Flow Chamber ◽

Shear Stresses ◽

Selectin Ligands ◽

Tnf Α ◽

Factor Α

Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

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Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00173.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. L844-L855 ◽

Cited By ~ 47

Author(s):

Ming-Yuan Jian ◽

Mikhail F. Alexeyev ◽

Paul E. Wolkowicz ◽

Jaroslaw W. Zmijewski ◽

Judy R. Creighton

Keyword(s):

Endothelial Cells ◽

Acute Lung Injury ◽

Lung Injury ◽

Ex Vivo ◽

Endothelial Permeability ◽

Beneficial Effects ◽

Isolated Lung

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient ( Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

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P1238EVALUATION OF AN IN VITRO CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PERITONEAL DIALYSIS FLUIDS ON CARDIOVASCULAR OUTCOME

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1238 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Juan Manuel Sacnun ◽

Rebecca Herzog ◽

Maria Bartosova ◽

Claus Schmitt ◽

Klaus Kratochwill

Keyword(s):

Endothelial Cells ◽

Cell Death ◽

Peritoneal Dialysis ◽

Cardiovascular Risk ◽

Cell Damage ◽

Peritoneal Membrane ◽

Cytoskeleton Reorganization ◽

Harmful Effects

Abstract Background and Aims The composition of all currently available peritoneal dialysis (PD) fluids triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels, eventually leading to failure of the technique. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of these cytoprotective additives in PDF in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial and endothelial cells were co-cultured in transwell plates. Mesothelial cells were grown in the upper compartment and primary human umbilical vein endothelial cells (HUVEc) or primary microvascular cells were grown in the lower compartment. PDF with or without cytoprotective compounds, was added to the upper compartment to only expose mesothelial cells directly to different dilutions of the fluid. Effects on cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release and live-dead staining of cells. Proteome profiles were analysed for both cell-types separately and in combination using two-dimensional difference gel electrophoresis (2D-DiGE) and liquid chromatography coupled to mass spectrometry (LC-MS). In vitro findings were related to PD-induced arteriolar changes based on abundance profiles of micro-dissected omental arterioles of children treated with conventional PD-fluids and age-matched controls with normal renal function. Results Marked cellular injury of HUVEc after PD-fluid exposure was associated with a molecular landscape of the enriched biological process clusters ‘glucose catabolic process’, ‘cell redox homeostasis’, ‘RNA metabolic process’, ‘protein folding’, ‘regulation of cell death’, and ‘actin cytoskeleton reorganization’ that characterize PD-fluid cytotoxicity and counteracting cellular repair process respectively. PDF-induced cell damage was reduced by AlaGln and LiCl both in mesothelial and endothelial cells. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton reorganization. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. Comparison to human arterioles confirmed overlapping protein regulation between endothelial cells in vitro and in vivo, proving harmful effects of PD-fluids on endothelial cells leading to drastic changes of the cellular process landscape. Conclusion In summary, this study shows harmful effects of PD-fluids also effecting endothelial cells and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the peritoneal membrane for testing direct and indirect effects of cytoprotective additives in PDF. When cultured and stressed in close proximity cells may respond differently. Characterisation of PD-induced perturbations will allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

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The Indispensable Value of Clinical Trials in the Modernization of Traditional Chinese Medicine: 12 Years' Experience at CUHK and Future Perspectives

The American Journal of Chinese Medicine ◽

10.1142/s0192415x14500384 ◽

2014 ◽

Vol 42 (03) ◽

pp. 587-604 ◽

Cited By ~ 13

Author(s):

Willmann Liang ◽

David T. Yew ◽

Kam Lun Hon ◽

Chun Kwok Wong ◽

Timothy C. Y. Kwok ◽

...

Keyword(s):

Clinical Trials ◽

Chinese Medicine ◽

Herbal Medicines ◽

Chinese Herbs ◽

Protective Effects ◽

Beneficial Effects ◽

Artery Disease ◽

The Many

The last decade has seen a wealth of information reporting the beneficial effects of Chinese herbal medicines. While a lot more studies were done using in vitro and in vivo research platforms, much fewer investigations were conducted according to evidence-based requirements in clinical settings. The Institute of Chinese Medicine at the Chinese University of Hong Kong (CUHK) has had the opportunity to collaborate with clinicians over the years to initiate and conduct dozens of clinical trials investigating and verifying the therapeutic values of Chinese herbs in selected disease conditions. Of the many disorders, we chose to focus on those that are known for their difficulties achieving perfect results with conventional treatment methods. Examples include non-healing ulcers, allergic conditions, degenerative diseases and cancer. Protective effects of the herbs in such chronic diseases as coronary artery disease and osteoporosis were also part of our focus. Even in healthy individuals and those recovering from chemotherapy, Chinese herbs could help with the immune system and were studied in our clinical trials as well. This paper aims to highlight the important findings from these clinical studies while at the same time, stressing the indispensable value of clinical trials in modernizing the use of Chinese herbs in present-day medicine.

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Nicotinamide phosphoribosyltransferase inhibition reduces intraplaque CXCL1 production and associated neutrophil infiltration in atherosclerotic mice

Thrombosis and Haemostasis ◽

10.1160/th13-07-0531 ◽

2014 ◽

Vol 111 (02) ◽

pp. 308-322 ◽

Cited By ~ 27

Author(s):

Rafaela da Silva ◽

Rodrigo Fraga-Silva ◽

Sabine Steffens ◽

Mathias Fabre ◽

Inga Bauer ◽

...

Keyword(s):

Endothelial Cells ◽

Neutrophil Infiltration ◽

Nuclear Factor Κb ◽

In Vivo Model ◽

Atherosclerotic Plaques ◽

Nicotinamide Phosphoribosyltransferase ◽

Beneficial Effects ◽

Nuclear Levels

SummaryPharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.

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CD99 and CD99L2 act at the same site as, but independently of, PECAM-1 during leukocyte diapedesis

Blood ◽

10.1182/blood-2009-12-256388 ◽

2010 ◽

Vol 116 (7) ◽

pp. 1172-1184 ◽

Cited By ~ 55

Author(s):

M. Gabriele Bixel ◽

Hang Li ◽

Bjoern Petri ◽

Alexander G. Khandoga ◽

Andrej Khandoga ◽

...

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

Gene Disruption ◽

3 Dimensional ◽

Leukocyte Extravasation ◽

Or Gene ◽

Endothelial Basement Membrane ◽

Factor Α

Abstract Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1β-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-α, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.

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Inhibitory Effect of Piceatannol on Streptococcus suis Infection Both in vitro and in vivo

Frontiers in Microbiology ◽

10.3389/fmicb.2020.593588 ◽

2020 ◽

Vol 11 ◽

Author(s):

Guizhen Wang ◽

Yawen Gao ◽

Xiuhua Wu ◽

Xiue Gao ◽

Min Zhang ◽

...

Keyword(s):

Cell Damage ◽

Critical Role ◽

Streptococcus Suis ◽

Inhibitory Effect ◽

Drug Candidate ◽

Bacterial Colony ◽

Binding Effect ◽

Factor Α

Suilysin (SLY) plays a critical role in Streptococcus suis infections making it an ideal target to the combat infection caused by this pathogen. In the present study, we found that piceatannol (PN), a natural compound, inhibits pore-formation by blocking the oligomerization of SLY without affecting the growth of S. suis and the expression of SLY. Furthermore, PN alleviated the J774 cell damage and the expression of the inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1β) induced by S. suis in vitro. The computational biology and biochemistry results indicated that PN binds to the joint region of D2 and D4 in SLY, and Asn57, Pro58, Pro59, Glu76, Ile379, Glu380, and Glu418 were critical residues involved in the binding. The binding effect between PN and SLY hindered the SLY monomers from forming the oligomers, thereby weakening the hemolytic activity of SLY. This mechanism was also verified by hemolysis analysis and analysis of KA formation after site-specific mutagenesis. Furthermore, PN protected mice from S. suis infections by reducing bacterial colony formation and the inflammatory response in target organs in vivo. These results indicate that PN is a feasible drug candidate to combat S. suis infections.

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Effects of synthetic polycations on leucine incorporation, lactate dehydrogenase release, and morphology of human umbilical vein endothelial cells

Journal of Cell Science ◽

10.1242/jcs.91.2.231 ◽

1988 ◽

Vol 91 (2) ◽

pp. 231-238

Author(s):

D.M. Morgan ◽

J. Clover ◽

J.D. Pearson

Keyword(s):

Endothelial Cells ◽

Cell Damage ◽

Umbilical Vein ◽

Leucine Incorporation ◽

Human Umbilical Vein ◽

Lactate Dehydrogenase Release ◽

Dependent Inhibition ◽

Umbilical Vein Endothelial Cells

Naturally occurring cationic proteins secreted by human granulocytes have pro-inflammatory effects including induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. Synthetic cationic polyamino acids have been shown to exert similar inflammatory effects in vivo. We have therefore used a range of synthetic polycationic amino acids to investigate the characteristics required to cause endothelial cell damage, assessed by in vitro inhibition of leucine incorporation into macromolecules by human umbilical vein endothelial cells (HUVEC) in culture. Exposure of HUVEC to 20 nM-2 microM cationic polypeptides of similar Mr(av) (approximately 40,000) in the presence of 20% serum produced a dose-dependent inhibition of [3H]leucine incorporation by polymers of ornithine, arginine or lysine. Similar results were obtained using [3H]thymidine. Neutral or anionic polypeptides of similar Mr were without effect. The molar potency of polylysines increased over the range Mr 40,000-120,000, while polylysines of Mr(av) less than 25,000 had no effect. In the absence of serum, inhibition occurred more rapidly and at lower doses. Inhibition of leucine and thymidine incorporation was time-dependent, e.g. exposure to 800 nM-polylysine, Mr(av) 90,000, led to progressively increasing inhibition that was complete after 24 h exposure, and was irreversible. The effects of polycations could not be blocked by pretreatment of the cells with polyanions. Precoating of the culture surface with polylysines had no effect on leucine incorporation by HUVEC or their subsequent response to polylysines in solution. Exposure to the peptide Arg-Gly-Asp-Ser inhibited incorporation by 30% but did not increase susceptibility to polylysine.(ABSTRACT TRUNCATED AT 250 WORDS)

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656135 ◽

1997 ◽

Vol 77 (06) ◽

pp. 1182-1188 ◽

Cited By ~ 74

Author(s):

Ulrich M Vischer ◽

Claes B Wollheinn

Keyword(s):

Endothelial Cells ◽

Adenylate Cyclase ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Free Calcium ◽

Camp Content ◽

Von Willebrand ◽

Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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