Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates

Lum, Hazel; Aschner, Judy L.; Phillips, Patricia G.; Fletcher, Paul W.; Malik, Asrar B.

doi:10.1152/ajplung.1992.263.2.l219

Cited by 45 publications

(40 citation statements)

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“…The initial peak rise in thrombin-induced in [Ca 2ϩ ] i is attributed to release from intracellular Ca 2ϩ stores as the result of activation of G␣ q (30). Knockdown of PAR3 in endothelial cells using siRNA had no effect on the peak Ca 2ϩ rise.…”

Section: Discussionmentioning

confidence: 98%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

Proc. Natl. Acad. Sci. U.S.A.

160

164

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Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased G␣13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.T hrombin generation during thrombosis contributes to the pathophysiology of multiple thrombosis-related diseases such as acute lung injury resulting increased endothelial permeability (1), vascular inflammation (1, 2), and edema formation (for recent reviews, see refs. 3 and 4). These complications are the result of thrombin activation of protease-activated receptor-1 (PAR1) (5). Cleavage of the extracellular N terminus of PAR1 initiates signaling (6) by the concomitant activation of heterotrimeric GTP-binding proteins G␣ q and G␣ 12/13 (7-9). The mechanisms by which PAR1 selectively activates G protein signaling remain unknown. Because the nonsignaling receptor PAR3 is also expressed in endothelial cells (10), we surmised that PAR1 and PAR3 are capable of interacting in such manner as to regulate PAR1 signaling. We found that PAR3 directly dimerizes with PAR1 to induce a specific PAR1/G␣ 13 -binding conformation that favors G␣ 13 activation. We propose a model of PAR1 activation involving the interaction with PAR3, which alters the selectivity of PAR1 for G␣ 13 coupling and promotes endothelial barrier dysfunction. PAR3 functions as an essential allosteric modulator of PAR1 signaling through PAR1/3 dimerization and favors a distinct G␣ 13 -activated downstream pathway. Results PAR3 Knockdown in Endothelial Cells Shifts the Potency of ThrombinActivation of PAR1. Endothelial cells express PAR1, -2, and -3, but not PAR4 (10), and of these only PAR1 and PAR3 are thrombinsensitive (11). To address whether PAR3 alters thrombin signaling in endothelial cells, siRNA knockdown was used to reduce thrombin receptor expression in human pulmonary artery endothelial cells (HPAECs). PAR1 and PAR3 suppression was determined by semiquantitative RT-PCR. siRNA against PAR3 did not affect PAR1 expression, whereas it reduced PAR3 expression 8-fold. Conversely, siRNA against PAR1 only modestly affected PAR3 expression, whereas it suppres...

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Section: Discussionmentioning

confidence: 98%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

Proc. Natl. Acad. Sci. U.S.A.

160

164

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“…This process is reversible, and focal adhesion kinase plays a role in the reversal of the increased vascular permeability [64] . Thrombin-induced increase in vascular endothelial permeability contributes to the edema seen in inflammatory disorders such as acute lung injury [65,66] . Both the receptor and its downstream signaling pathways are targets for therapeutic intervention [67,68] .…”

Section: Gpcrs and Regulation Of Vascular Endothelial Permeabilitymentioning

confidence: 99%

Role of G protein-coupled receptors in inflammation

2012

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“…We have also shown that activation of endothelial cell surface protease-activated receptor-1 (PAR-1) by thrombin caused a rapid and transient increase in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) because of the release of stored Ca 2ϩ and a subsequent Ca 2ϩ entry triggered by store depletion (7,20,30). In endothelial cells, the plasma membrane cation channels known as store-operated cation channels (SOCs) mediate the entry of Ca 2ϩ into the cell (2,27,32).…”

Section: ϩmentioning

confidence: 99%

Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca²⁺influx and endothelial permeability

Paria

Vogel

Ahmmed

et al. 2004

American Journal of Physiology-Lung Cellular and Molecular Phys

142

152

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influx in response to thrombin exposure. We observed that thrombininduced Ca 2ϩ influx in TNF-␣-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated Ca 2ϩ influx and TRPC1 expression, we overexpressed TRPC1 by three-to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombininduced store Ca 2ϩ depletion in these cells caused approximately twofold greater increase in Ca 2ϩ influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPC1-transfected cells compared with control. To address the role of Ca 2ϩ influx via TRPC1 in signaling endothelial permeability, we measured actinstress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF-␣-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPC1-overexpressing HMEC compared with control cells. TNF-␣-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of Ca 2ϩ influx and signaling of the increase in endothelial permeability.tumor necrosis factor-␣; store-operated calcium ion influx; transient receptor potential channel 1; endothelial barrier dysfunction MICROVASCULAR ENDOTHELIAL cells regulate the vessel wall permeability of solutes, liquid, and macromolecules and produce numerous autocrine and paracrine factors, such as NO, that modulate the contractility of the underlying vascular smooth muscle (4,5,21

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Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates

Cited by 45 publications

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Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Role of G protein-coupled receptors in inflammation

Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca²⁺influx and endothelial permeability

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Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates

Cited by 45 publications

References 0 publications

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Role of G protein-coupled receptors in inflammation

Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca2+influx and endothelial permeability

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Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca²⁺influx and endothelial permeability