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Margaret Colden-Stanfield William P. Schilling Lourival D. Possani Diana L. Kunze 《The Journal of membrane biology》1990,116(3):227-238
Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]
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, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2+-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100nm [Ca2+]
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. The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of86Rb– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2+-activated membrane currents. The results confirm that bradykinin-stimulated86Rb+ efflux occurs via Ca2+-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2+-activated K+ current in the response of BAECs to bradykinin. 相似文献
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Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium 总被引:2,自引:0,他引:2
Colden-Stanfield Margaret; Gallin Elaine K. 《American journal of physiology. Cell physiology》1998,275(1):C267
Resting membrane potential (RMP) and whole cell currents wererecorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs),lipopolysaccharide (LPS)-treated HUVECs, immobilizedE-selectin, or vascular cell adhesion molecule 1 (VCAM-1)using the patch-clamp technique. RMP after 5 h on polystyrene was24.3 ± 1.7 mV (n = 42) with delayed rectifier K+(Idr) andCl currents(ICl) presentin >75% of the cells. Inwardly rectifying K+ currents(Iir) werepresent in only 14% of THP-1 cells. Adherence to unstimulated HUVECsor E-selectin for 5 h had no effect on Iir orICl but decreasedIdr. Five hoursafter adherence to LPS-treated HUVECs, outward currents were unchanged,but Iir waspresent in 81% of THP-1 cells. A twofold increase inIir and ahyperpolarization (41.3 ± 3.7 mV,n = 16) were abolished by pretreatmentof THP-1 cells with cycloheximide, a protein synthesis inhibitor, orherbimycin A, a tyrosine kinase inhibitor, or by pretreatment of theLPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interactionbetween THP-1 cells and LPS-treated HUVECs was required toinduce Iir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similarconductances to cells adherent to LPS-treated HUVECs. Thus engagementof specific integrins results in selective modulation of differentK+ conductances. 相似文献
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VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes 总被引:1,自引:0,他引:1
Hyperpolarization in human leukemia THP-1 monocytes adherent tovascular cell adhesion molecule (VCAM)-1 is due to an induction ofinwardly rectifying K+ currents(Iir) (Colden-Stanfield M and Gallin EK,Am J Physiol Cell Physiol 275: C267-C277, 1998).We determined whether the VCAM-1-induced hyperpolarization issufficient to augment the increase in intracellular free calcium([Ca2+]i) produced by Ca2+ storedepletion with thapsigargin (TG) and readdition of external CaCl2 in fura 2-loaded THP-1 monocytes. Whereas there was a2.1-fold increase in [Ca2+]i in monocytesbound to glass for 5 h in response to TG and CaCl2 addition, adherence to VCAM-1 produced a 5-fold increase in[Ca2+]i. Depolarization of monocytes adherentto VCAM-1 by Iir blockade or exposure to high[K+] abolished the enhancement of the peak[Ca2+]i response. In monocytes bound toglass, hyperpolarization of the membrane potential with valinomycin, aK+ ionophore, to the level of hyperpolarization seen incells adherent to VCAM-1 produced similar changes in peak[Ca2+]i. Adherence of monocytes to E-selectinproduced a similar peak [Ca2+]i to cellsbound to glass. Thus monocyte adherence to the physiological substrateVCAM-1 produces a hyperpolarization that is sufficient to enhanceCa2+ entry and may impact Ca2+-dependentmonocyte function. 相似文献
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Clustering of very late antigen-4 integrins modulates K(+) currents to alter Ca(2+)-mediated monocyte function 总被引:1,自引:0,他引:1
Colden-Stanfield M 《American journal of physiology. Cell physiology》2002,283(3):C990-C1000
Endothelialcell vascular cell adhesion molecule-1 (VCAM-1) activates adherentmonocytes by clustering their very late antigen-4 (VLA-4) receptors,resulting in the modulation of the inwardly rectifying(Iir) and delayed rectifying(Idr) K+ currents, hyperpolarizationof the cells, and enhanced Ca2+ influx (Colden-Stanfield Mand Gallin EK. Am J Physiol Cell Physiol 275:C267-C277, 1998; Colden-Stanfield M and Scanlon M. Am JPhysiol Cell Physiol 279: C488-C494, 2000). The present studywas undertaken to test the hypothesis that monoclonal antibodies(MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4integrins, which play a key role in signaling an increase in thesecretion of the proinflammatory cytokine interleukin-8 (IL-8). Wholecell ionic currents and IL-8 secretion from THP-1 monocytes that wereincubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or anisotype-matched MAb against CD45 (MAbCD45) were measured. Clustering ofVLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalentMAbVLA-4, modulated the K+ currents in an identical mannerto incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 orVCAM-1 augmented Ca2+-mediated IL-8 secretion from THP-1monocytes and was completely abolished by exposure to CsCl, anIir blocker. Thus VLA-4 integrin clustering bycross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently tobe associated with events leading to monocyte differentiation, enhancedCa2+-mediated macrophage function, and possiblyatherosclerotic plaque formation. 相似文献
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GDKV-induced antiphospholipid antibodies enhance thrombosis and activate endothelial cells in vivo and in vitro. 总被引:6,自引:0,他引:6
A E Gharavi S S Pierangeli M Colden-Stanfield X W Liu R G Espinola E N Harris 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(5):2922-2927
Antiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). beta2-Glycoprotein I (beta2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with beta2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of beta2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274-Cys288 in the fifth domain of human beta2GPI, which represents the phospholipid-binding site of beta2GPI. The Abs generated had aPL and anti-beta2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of beta2GPI are thrombogenic and activate endothelial cells. 相似文献
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