Peptide-Stimulation Enhances Compartmentalization and the Catalytic Activity of Lung Endothelial NOS

Hutchinson, Tarun E.; Kuchibhotla, Sudeep; Block, Edward R.; Patel, Jawaharlal M.

doi:10.1159/000257487

Cited by 8 publications

(13 citation statements)

References 44 publications

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“…The caveolae enriched membrane fractions were isolated using detergent-free method as we previously described [10, 24]. The isolated membrane fractions were analyzed by western blots using 50 μg of protein to determine the level of the caveolae marker protein Cav-1.…”

Section: Methodsmentioning

confidence: 99%

“…The immunoprecipitation of caveolar proteins by anti Cav-1 antibody were carried out as previously described [24]. In brief, 10 μg of Cav-1 antibody was added to the tube containing cold pre-cleared cell lysate obtained from EC treated with or without P1 or PKC/PLC inhibitors with or without P1 and incubated at 4°C overnight.…”

Section: Methodsmentioning

confidence: 99%

“…The cell lysate (100–200 μg of protein) were incubated in 0.4 ml of Tris HCl buffer containing 1 mM NADPH, 100 nM calmodulin, 10 μM tetrahydrobiopterin, and 5 μM combined L-arginine and purified L-[ 3 H] arginine for 30 min at 37°C [24]. Purification of L-[ 3 H] arginine and measurement of L-[ 3 H] citrulline formation were carried out as previously described [26].…”

Section: Methodsmentioning

confidence: 99%

“…Our previous reports demonstrated that an 11-amino acid (626-SSWRRKRKESS-636) peptide (P1) derived from 40 amino acid autoinhibitory domain of eNOS protein modulated EC and lung function. For example, P1-stimulation of EC and/or PA resulted in : i) activation of eNOS independent of increased expression or phosphorylation of eNOS leading to NO-dependent vasorelaxation of contracted PA [23] and ii) enhanced compartmentalization and activation of eNOS in lung EC [24]. More recently we reported that P1-stimulation modulates caveolar PKC-α signaling that enhances eNOS activation, NO production, intracellular Ca 2+ release, and phosphorylation of PKC-α and regulates internalization of P1 in lung EC [10].…”

Section: Introductionmentioning

confidence: 99%

“…More recently we reported that P1-stimulation modulates caveolar PKC-α signaling that enhances eNOS activation, NO production, intracellular Ca 2+ release, and phosphorylation of PKC-α and regulates internalization of P1 in lung EC [10]. P1-mediated activation of eNOS is associated with increased phosphorylation of Cav-1 and Cav-1/eNOS dissociation in caveolae-enriched fractions of EC and in an intact EC [24]. In addition, P1-mediated time-dependent increased phosphorylation of PKC-α and Cav-1 was independent of the basal level of these proteins (24).…”

Section: Introductionmentioning

confidence: 99%

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Peptide-stimulated angiogenesis: Role of lung endothelial caveolar signaling and nitric oxide

Hutchinson

Patel

2015

Nitric Oxide

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Endothelial nitric oxide (NO) synthase (eNOS)-derived NO plays a critical role in the modulation of angiogenesis in the pulmonary vasculature. We recently reported that an eleven amino acid (SSWRRKRKESS) cell penetrating synthetic peptide (P1) activates caveolar signaling, caveloae/eNOS dissociation, and enhance NO production in lung endothelial cells (EC). This study examines whether P1 promote angiogenesis via modulation of caveolar signaling and the level of NO generation in EC and pulmonary artery (PA) segments. P1-enhanced tube formation and cell sprouting were abolished by caveolae disruptor Filipin (FIL) in EC and PA, respectively. P1 enhanced eNOS activity and angiogenesis were attenuated by inhibition of eNOS as well as PLCγ-1, PKC-α but not PI3K-mediated caveolar signaling in intact EC and/or PA. P1 failed to enhance the catalytic activity of eNOS and angiogenesis in caveolae disrupted EC by FIL. Lower (0.01 mM) concentration of NOC-18 enhanced angiogenesis without inhibition of eNOS activity whereas higher concentration of NOC-18 (1.0 mM) inhibited eNOS activity and angiogenesis in EC. Inhibition of eNOS by L-NAME in the presence of P1 resulted in near total loss of tube formation in EC. Although P1 enhanced angiogenesis mimicked only by lower concentrations of NO generated by NOC-18, this response is independent of caveolar signaling/integrity. These results suggest that P1-enhanced angiogenesis is regulated by dynamic process involving caveolar signaling-mediated increased eNOS/NO activity or by the direct exposure to NOC-18 generating only physiologic range of NO independent of caveolae in lung EC and PA segments.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Peptide-stimulated angiogenesis: Role of lung endothelial caveolar signaling and nitric oxide

Hutchinson

Patel

2015

Nitric Oxide

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Subcellular Redistribution of NOS

Röszer

2012

The Biology of Subcellular Nitric Oxide

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Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells

et al. 2011

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We previously reported that the vasoactive peptide 1 (P1, “SSWRRKRKESS”) modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 μM) for 0–120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-β-cyclodextrin (CD) and filipin (FIL) inhibited the internalization of P1 in ECs suggesting that P1 internalizes via caveolae. P1-stimulation also enhances phosphorylation of caveolar PKC-α and increases intracellular calcium (Ca2+) release in intact cells suggesting that P1 internalization is regulated by PKC-α in ECs. To confirm the roles of increased phosphorylation of PKC-α and Ca2+ release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca2+ scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-α is associated with significant reduction in P1 internalization. In contrast, PKC-α deficiency and reduced phosphorylation of PKC-α enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-α appears to be associated with increased intracellular calcium (Ca2+) release since the Ca2+ scavenger BAPTA-AM enhanced P1 internalization. These data indicate that caveolar integrity and P1-mediated increased phosphorylation of caveolar PKC-α play crucial roles in the regulation of P1 internalization in lung ECs.

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Peptide-Stimulation Enhances Compartmentalization and the Catalytic Activity of Lung Endothelial NOS

Cited by 8 publications

References 44 publications

Peptide-stimulated angiogenesis: Role of lung endothelial caveolar signaling and nitric oxide

Peptide-stimulated angiogenesis: Role of lung endothelial caveolar signaling and nitric oxide

Subcellular Redistribution of NOS

Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells

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