Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator‐induced G‐protein coupled receptor activation

Baker, Susan M.; Kim, Nam-Ho; Gumpert, Anna M.; Segretain, Dominique; Falk, Matthias M.

doi:10.1016/j.febslet.2008.10.043

Cited by 50 publications

(72 citation statements)

References 33 publications

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“…Fate of Cell Surface Panx1-Internalization of K ϩ (34), Ca 2ϩ (35), Cl Ϫ (36), and gap junction channels (23,37,38) is mediated by clathrin and/or caveolin-driven pathways; however, this does not appear to be the case for Panx1 channels. Our data indicate that although Panx1 can co-exist and co-distribute in the same cell surface microdomain as clathrin, AP2, caveolins, and dynamin, its physical interaction with protein networks that contain any of these members of the endocytic machinery could not be detected.…”

Section: Discussionmentioning

confidence: 90%

Pathways Regulating the Trafficking and Turnover of Pannexin1 Protein and the Role of the C-terminal Domain

Gehi

Shao

Laird

2011

Journal of Biological Chemistry

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Pannexin1 (Panx1) is an integral membrane protein comprised of three species as follows: an unglycosylated core-Gly0, a high mannose-Gly1, and a complex glycosylated Gly2 species. Although Panx1 channels mediate several cellular responses, the domain regulating its oligomerization and cell surface trafficking and the mechanisms governing its internalization and degradation have not been identified. This study characterizes the role of the Panx1 C-tail domain by truncating the polypeptide at residue 307 and expressing the mutant in BICR-M1R k and HEK-293T cells. Enzymatic digestion and immunolabeling assays revealed that the Panx1 T307 -RFP was glycosylated primarily to the high mannose species consistent with its retention in the endoplasmic reticulum. Co-expression of Panx1 T307 -RFP with Panx1 followed by co-immunoprecipitation assays revealed that the mutant and Panx1 could interact, whereas biotinylation assays showed that this interaction inhibited Panx1 from maturing into the Gly2 species and reaching the cell surface. Additional inhibitor studies indicated that the degradation of the mutant was via proteasomes, whereas Panx1 was degraded by lysosomes. Analysis of the pathways important in Panx1 internalization revealed partial co-distribution of Panx1 with many molecular constituents of the endocytic machinery that include clathrin, AP2, dynamin II, caveolin-1, and caveolin-2. However, co-immunoprecipitation assays together with the disruption of lipid rafts by methyl-␤-cyclodextrin suggest that Panx1 does not engage this endocytic machinery. Furthermore, dominant-negative and pharmacological studies revealed that Panx1 internalization was dynamin II-independent. Collectively, these results indicate that the oligomerization and trafficking of Panx1 are regulated by the C-terminal domain, whereas internalization of long lived Panx1 channels occurs in a manner that is distinct from classical endocytic pathways.Pannexins were originally discovered due to their shared sequence homology with the invertebrate gap junction proteins, innexins (1). The pannexin family is comprised of three members as follows: Panx1, Panx2, and Panx3 (1). Although the scope of physiological significance of pannexin family members is only beginning to emerge, it has been reported that Panx1 holds importance in forming conduits for ATP release (2-4), propagation of Ca 2ϩ waves (5), and neuronal and immunological inflammasome (6 -8). Additionally, activation of Panx1 channels has been implicated in ischemic cell death (9) and seizure-like activities (10). Although Panx1 single channel conductances of 550 picosiemens (2) have been correlated with ionic dysregulation during ischemic conditions, subsequently leading to neuronal necrosis (9), the N-methyl-D-aspartate receptor-induced opening of Panx1 channels has been implicated in hippocampal epileptiform seizure-like activity (10). Other studies have associated Panx1 channel activation with apoptosis of Xenopus oocytes by forming a pore unit with the death complex of the P2X7 receptor (...

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Section: Discussionmentioning

confidence: 90%

Pathways Regulating the Trafficking and Turnover of Pannexin1 Protein and the Role of the C-terminal Domain

Gehi

Shao

Laird

2011

Journal of Biological Chemistry

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“…ET-1 is pro-inflammatory [25], and activation of ET-1 receptors on endothelial cells results in increased permeability [26] and enhanced neutrophil adhesion and migration [27]. In pigs and hamsters, the ETA receptor is associated with activation of inflammation [28,29].…”

Section: Discussionmentioning

confidence: 99%

Endothelin receptor expression in idiopathic pulmonary arterial hypertension: effect of bosentan and epoprostenol treatment

Hall¹,

Davie²,

Klein³

et al. 2011

Eur Respir J

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Endothelin receptor antagonists are used to treat idiopathic pulmonary arterial hypertension (IPAH), but human pulmonary arterial endothelin receptor expression is not well defined. We hypothesised that disease and treatment would modify normal receptor distribution in pulmonary resistance arteries of children.Using immunohistochemistry and semiquantitative analysis, we investigated endothelin receptor subtypes A and B (ETA and ETB, respectively), and endothelial nitric oxide synthase (eNOS) expression in peripheral pulmonary arteries of tissue from untreated children with IPAH (n57), following extended combined bosentan and epoprostenol therapy (n55) and from normal subjects (n55).Clinical, haemodynamic and pathological abnormalities were severe and advanced in all IPAH cases. ETA was detected in pulmonary arterial endothelial cells of all normal and diseased tissue and cultured cells. Endothelial ETA, ETB and eNOS expression was reduced in patent, plexiform and dilatation lesions of untreated cases, but in treated cases, ETA and ETB were normal and eNOS increased. In smooth muscle, ETA expression was reduced in treated cases but ETB expression increased in all arteries of both treated and untreated cases.In summary, ETA is expressed on human pulmonary arterial endothelium. In IPAH, combination treatment with bosentan and epoprostenol had a more marked influence on endothelin receptor expression of endothelial than smooth muscle cells.KEYWORDS: Endothelin receptor antagonists, endothelin receptor A and B, endothelium, paediatric idiopathic pulmonary arterial hypertension, peripheral pulmonary arteries I diopathic pulmonary arterial hypertension (IPAH) is a rare, incurable disorder occurring in both children and adults with a normally formed heart, characterised by irreversible occlusion of small intra-acinar pulmonary resistance arteries with development of plexiform lesions resulting in a sustained increase in pulmonary arterial pressure [1]. Death results from right heart failure. Treatment with prostacyclin, endothelin receptor antagonists and phosphodiesterase inhibitors aims to promote dilatation and reparative remodelling of the pulmonary arteries, and has improved survival and quality of life but is not curative [1]. The only therapeutic option for endstage disease is lung transplantation.The endothelin ET-1 is a potent vasoconstrictor and smooth muscle cell mitogen [2] that is important in the pathobiology of pulmonary arterial hypertension [3]. In humans with IPAH, plasma endothelin levels are elevated [4], endothelin-converting enzyme activity is enhanced [5] and lung expression of endothelin is increased [6]. Its action is mediated principally by two receptors, ETA and ETB [7]. Both mediate vasoconstriction of human smooth muscle cells whilst endothelial ETB receptors mediate vasorelaxation via endothelial nitric oxide synthase (eNOS) [8]. In experimentally induced pulmonary hypertension, the endothelin receptor antagonists diminish or abrogate endothelin-induced smooth muscle cell contract...

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“…G α q agonists reduce coupling in rat-1 fi broblasts as well, where uncoupling was caused entirely by reduced PIP 2 levels (van Zeijl et al, 2007). Uncoupling after G α q stimulation is also reported in several other studies (Giaume et al, 1991;Giaume et al, 1992;Hii et al, 1994 In endothelial cells, stimulation with endothelin-1 or thrombin reduced coupling by internalization of Cx43 (Baker et al, 2008). Both the proteasomal and lysosomal pathways are involved in degradation of Cx43 (reviewed by (Berthoud et al, 2004)) and ubiquitination is reported to precede internalization of Cx43 (Leithe & Rivedal, 2004a;Leithe & Rivedal, 2004b;Kimura & Nishida, 2010).…”

Section: Introductionmentioning

confidence: 65%

“…In primary cultures of pulmonary artery endothelial cells, G α q stimulation leads to internalization of Cx43 (Baker et al, 2008). In the present study, we investigated the acute effects of norepinephrine in cardiomyocytes further, with special emphasis on internalization of Cx43.…”

Section: Discussionmentioning

confidence: 99%

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Mollerup

Hofgaard

Braunstein

et al. 2011

Cell Communication & Adhesion

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G α q -stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α -adrenergic G α q -coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions : Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se .

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Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator‐induced G‐protein coupled receptor activation

Cited by 50 publications

References 33 publications

Pathways Regulating the Trafficking and Turnover of Pannexin1 Protein and the Role of the C-terminal Domain

Pathways Regulating the Trafficking and Turnover of Pannexin1 Protein and the Role of the C-terminal Domain

Endothelin receptor expression in idiopathic pulmonary arterial hypertension: effect of bosentan and epoprostenol treatment

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

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