scholarly journals Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis

2017 ◽  
Vol 8 (1) ◽  
pp. 204589321775315 ◽  
Author(s):  
Caleb L. Hamilton ◽  
Kevin A. Abney ◽  
Audrey A. Vasauskas ◽  
Mikhail Alexeyev ◽  
Li Ni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Heat Shock Protein 90 ◽  
Calcium Entry ◽  
Hek293 Cells ◽  
Wide Field ◽  
Cell Fractionation ◽  
Gap Formation ◽  
Tetratricopeptide Repeats ◽  
Store Operated Calcium Entry

Pulmonary endothelial cells express a store-operated calcium entry current ( Isoc), which contributes to inter-endothelial cell gap formation. Isoc is regulated by a heterocomplex of proteins that includes the immunophilin FKBP51. FKBP51 inhibits Isoc by mechanisms that are not fully understood. In pulmonary artery endothelial cells (PAECs) we have shown that FKBP51 increases microtubule polymerization, an event that is critical for Isoc inhibition by FKBP51. In neurons, FKBP51 promotes microtubule stability through facilitation of tau dephosphorylation. However, FKBP51 does not possess phosphatase activity. Protein phosphatase 5 (PP5C/PPP5C) can dephosphorylate tau, and similar to FKBP51, PP5C possesses tetratricopeptide repeats (TPR) that mediate interaction with heat shock protein-90 (HSP90) chaperone/scaffolding complexes. We therefore tested whether PP5C contributes to FKBP51-mediated inhibition of Isoc. Both siRNA-mediated suppression of PP5C expression in PAECs and genetic disruption of PP5C in HEK293 cells attenuate FKBP51-mediated inhibition of Isoc. Reintroduction of catalytically competent, but not catalytically inactive PP5C, restored FKBP51-mediated inhibition of Isoc. PAEC cell fractionation studies identified both PP5C and the ISOC heterocomplex in the same membrane fractions. Further, PP5C co-precipitates with TRPC4, an essential subunit of ISOC channel. Finally, to determine if PP5C is required for FKBP51-mediated inhibition of calcium entry-induced inter-endothelial cell gap formation, we measured gap area by wide-field microscopy and performed biotin gap quantification assay and electric cell-substrate impedance sensing (ECIS®). Collectively, the data presented indicate that suppression of PP5C expression negates the protective effect of FKBP51. These observations identify PP5C as a novel member of the ISOC heterocomplex that is required for FKBP51-mediated inhibition of Isoc.

Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP

1998 ◽  
Vol 275 (2) ◽  
pp. L203-L222 ◽  
Author(s):  
Timothy M. Moore ◽  
Paul M. Chetham ◽  
John J. Kelly ◽  
Troy Stevens
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Intracellular Signaling ◽  
Endothelial Permeability ◽  
Cell Permeability ◽  
Gap Formation ◽  
Pulmonary Endothelium ◽  
Intracellular Signals ◽  
Endothelial Cell Permeability ◽  
Cell Cell

Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+ concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]ipromotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]idecreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]iand cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]ipromotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]iand cAMP in mediating site-specific alterations in endothelial permeability.

scholarly journals Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gap Formation ◽  
Culture Dish ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

Abstract When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.

scholarly journals Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase

2002 ◽  
Vol 157 (7) ◽  
pp. 1267-1278 ◽  
Author(s):  
Donna L. Cioffi ◽  
Timothy M. Moore ◽  
Jerry Schaack ◽  
Judy R. Creighton ◽  
Dermot M.F. Cooper ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Physiological Role ◽  
Cell Types ◽  
Cytosolic Calcium ◽  
Calcium Entry ◽  
Gap Formation ◽  
Calcium Stimulation ◽  
Store Operated Calcium Entry ◽  
Stimulation Of

Acute transitions in cytosolic calcium ([Ca2+]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca2+]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC6; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca2+]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca2+]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC6 is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC8 to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca2+]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC8 resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC6 is essential for endothelial gap formation.

scholarly journals Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors

2015 ◽  
Vol 473 (1) ◽  
pp. 1-5 ◽  
Author(s):  
G. Cristina Brailoiu ◽  
Elena Deliu ◽  
Linda M. Console-Bram ◽  
Jonathan Soboloff ◽  
Mary E. Abood ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rat Brain ◽  
Critical Role ◽  
Calcium Entry ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Sigma 1 Receptor ◽  
Store Operated Calcium Entry ◽  
Sigma 1 ◽  
Microvascular Endothelial

We provide evidence that cocaine induces sigma-1 receptor-mediated inhibition of store-operated calcium entry (SOCE) in rat brain microvascular endothelial cells. Thus, we reveal sigma-1 receptors as SOCE blockers, adding novel insight regarding endothelial effects of cocaine and endogenous SOCE modulation.

Enhanced Adherence of Sickle Erythrocytes to Thrombin-Treated Endothelial Cells Involves Interendothelial Cell Gap Formation

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3445-3454 ◽  
Author(s):  
Annamaria B. Manodori ◽  
Neil M. Matsui ◽  
James Y. Chen ◽  
Stephen H. Embury
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Function ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gap Formation ◽  
Cell Contraction ◽  
Endothelial Cell Function ◽  
Sickle Erythrocyte ◽  
Sickle Erythrocytes

Abstract The adherence of sickle erythrocytes to vascular endothelium has the capacity to initiate vasoocclusion. The known effects of thrombin on endothelial cell function and the increased activity of thrombin in sickle cell disease led us to examine the effect of thrombin on the adhesivity of cultured endothelial cells for sickle erythrocytes. In particular, we studied whether the effect of thrombin on interendothelial cell gap formation (ICGF) was involved in endothelial cell adhesivity for sickle erythrocytes. Those endothelial cell monolayers stimulated by thrombin to maximal levels of static sickle erythrocyte adherence also underwent striking cell contraction and enlargement of interendothelial cell gaps. Adhesivity also increased when gaps were induced with antilaminin antibodies or EDTA. Maximally adhesogenic thrombin conditions failed to increase adhesivity when gap formation was prevented by pretreatment of the monolayers with 8-bromo-cyclic adenosine monophosphate (bromo-cAMP) or glutaraldehyde, agents that respectively inhibit actin-myosin–dependent cell contraction or cross-link adjacent cells in the monolayer. The influence of these two agents on EDTA-enhanced adhesivity was linked to their ability to prevent gap formation. Glutaraldehyde prevented both increased adherence and gap formation; bromo-cAMP prevented neither. Interendothelial cell gap formation may contribute to vasoocclusion by facilitating sickle erythrocyte adherence. © 1998 by The American Society of Hematology.

scholarly journals Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gap Formation ◽  
Culture Dish ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.

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