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      PECAM-1 (CD31) regulates a hydrogen peroxide–activated nonselective cation channel in endothelial cells

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          Hydrogen peroxide (H 2O 2) released by neutrophils is an important mediator of endothelial cell (EC) injury and vascular inflammation via its effect on EC-free Ca 2+, [Ca 2+] i. Although the underlying mechanisms are not well understood, platelet endothelial cell adhesion molecule (PECAM)-1/CD-31 is a critical modulator of neutrophil–EC transmigration. PECAM-1 is also known to regulate EC calcium signals and to undergo selective tyrosine phosphorylation. Here, we report that PECAM-1 molecules transduce EC responses to hydrogen peroxide. In human umbilical vein EC and REN cells (a PECAM-1–negative EC-like cell line) stably transfected with PECAM-1 (RHP), noncytolytic H 2O 2 exposure (100–200 μM H 2O 2) activated a calcium-permeant, nonselective cation current, and a transient rise in [Ca 2+] i of similar time course. Neither response was observed in untransfected REN cells, and H 2O 2-evoked cation current was ablated in REN cells transfected with PECAM-1 constructs mutated in the cytoplasmic tyrosine–containing domain. The PECAM-dependent H 2O 2 current was inhibited by dialysis of anti–PECAM-1 cytoplasmic domain antibodies, required Src family tyrosine kinase activity, was independent of inositol trisphosphate receptor activation, and required only an intact PECAM-1 cytoplasmic domain. PECAM-1–dependent H 2O 2 currents and associated [Ca 2+] i transients may play a significant role in regulating neutrophil–endothelial interaction, as well as in oxidant-mediated endothelial response and injury.

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          Most cited references 62

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          Store depletion and calcium influx.

          Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.
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            hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel beta subunit family.

            We cloned two beta subunits of large-conductance calcium-activated potassium (BK) channels, hKCNMB3 (BKbeta1) and hKCNMB4 (BKbeta4). Profiling mRNA expression showed that hKCNMB3 expression is enriched in testis and hKCNMB4 expression is very prominent in brain. We coexpressed BK channel alpha (BKalpha) and BKbeta4 subunits in vitro in CHO cells. We compared BKalpha/beta4 mediated currents with those of smooth muscle BKalpha/beta1 channels. BKbeta4 slowed activation kinetics more significantly, led to a steeper apparent calcium sensitivity, and shifted the voltage range of BK current activation to more negative potentials than BKbeta1. BKalpha/beta4 channels were not blocked by 100 nM charybdotoxin or iberiotoxin, and were activated by 17beta-estradiol.
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              Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice.

              The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H(2)O(2)) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H(2)O(2) to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K(+) channel opener levcromakalim. Exogenous H(2)O(2) elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H(2)O(2) upon stimulation by ACh and that the H(2)O(2) production was markedly reduced in eNOS-KO mice. These results indicate that H(2)O(2) is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.

                Author and article information

                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                1 April 2002
                : 157
                : 1
                : 173-184
                [1 ]Division of Pulmonary, Allergy, and Critical Care Medicine
                [2 ]Institute for Environmental Medicine, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
                [3 ]Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853
                Author notes

                Address correspondence to Christopher D. O'Brien, Dept. of Pulmonary/Critical Care, University of Pennsylvania, 838 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Tel.: (215) 746-6709. Fax: (215) 573-4469. E-mail: christoo@

                Copyright © 2002, The Rockefeller University Press


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