1Department of Medicine, University of Florida College of Medicine, and 2Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608-1197
Submitted 3 November 2003 ; accepted in final form 7 January 2004
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ABSTRACT |
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nitric oxide synthase; guanosine 3',5'-cyclic monophosphate; electron transfer element; lung perfusion
The NOS isoforms, including eNOS, are members of a novel family of enzymes containing heme oxygenase and cytochrome P-450 reductase domains that catalyze the oxidative metabolism of the cationic amino acid L-arginine to generate NO (9, 22). The NH2-terminal oxygenase domain of eNOS protein contains binding sites for heme, L-arginine, tetrahydrobiopterin, and zinc, and the COOH-terminal reductase domain consists of sites for flavin mononucleotide, flavin adenine dinucleotide, NADPH, and a 40-(604-RPEQHKSYKIRFNSISCSDPLVSSWRRKRKESSNTDSAGA-643) amino acid electron transfer control element or autoinhibitory domain (AID) (6, 16, 28, 38). The oxygenase and reductase domains are linked by a calcium/calmodulin binding region. The catalytic activity of eNOS is dependent on the rate of electron transfer from the reductase to the oxygenase domain, a required process for oxidative metabolism of L-arginine to generate NO (6, 28). A number of recent studies using molecular biology approaches and purified NOS preparations have characterized the role of the 40-amino acid segment or AID in the regulation of the catalytic activity of NOS isoforms (6, 16, 28, 38). The precise mechanism involved in the AID-mediated regulation of electron transfer within the eNOS protein remains to be determined. Because electron transfer within eNOS is regulated by AID and intrinsic eNOS activity can be increased or decreased by AID-mediated mechanisms, the present study was designed to determine the potential effects of the exogenous synthetic 40-amino acid (604-RPEQHKSYKIRFNSISCSDPLVSSWRRKRKESSNTDSAGA-643) peptide representing AID or its 11-amino acid (626-SSWRRKRKESS-636) internal fragment (AAF) in the modulation of eNOS activity, NO release, cGMP production, and NO-cGMP-mediated vasorelaxation using multiple models, including pulmonary artery (PA) endothelial cells (PAEC) in culture, PAEC/PA smooth muscle cell (PASM) cocultures, isolated PA segments, and isolated perfused lungs.
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MATERIALS AND METHODS |
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The potential effects of synthetic (90% purity) AID, AAF, and/or scrambled AAF (AAF-SR; Sigma-Genosys, Woodlands, TX) on the catalytic activity of eNOS were examined after treatment of isolated total membrane fractions of PAEC or intact PAEC. Total membrane fraction was isolated by differential centrifugation of cell homogenates in Tris·HCl buffer (50 mM Tris·HCl, 0.1 mM each of EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/l leupeptine, pH 7.4) (32, 36). Total membrane fractions (1 mg of protein) were treated with or without (control) the presence of varying concentrations (10100 µM) of AID or AAF for 30 min at 37°C. After being incubated, membranes were washed by centrifugation, resuspended in fresh Tris·HCl buffer, and used to monitor eNOS activity. To determine the potential effect of AID, AAF, or AAF-SR on the catalytic activity of eNOS and NO production, PAEC monolayers were incubated with varying concentrations (5100 µM) of AID, AAF, or AAF-SR (Sigma-Genosys) in RPMI 1640 for 2 h at 37°C. Controls were incubated in RPMI 1640 only under identical conditions. In some experiments, cells were treated with AAF or AAF-SR (50 µM each) for 1, 2, and 4 h at 37°C. After treatments, cells were used to measure the catalytic activity of eNOS and NO release. We also monitored NO release from PA segments treated with AAF or AAF-SR (25 µM each) with or without pretreatment with 100 µM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of eNOS.
Measurement of eNOS activity. eNOS activity was measured by monitoring the formation of L-[3H]citrulline from L-[3H]arginine in the total membrane fraction treated with AID or AAF and in total membrane fractions isolated from PAEC treated with AID, AAF, or AAF-SR. Total membranes (100200 µg of protein) were incubated (total vol 0.4 ml) in Tris·HCl buffer containing 1 mM NADPH, 100 nM calmodulin, 10 µM tetrahydrobiopterin, and 5 µM combined L-arginine and purified L-[3H]arginine for 30 min at 37°C. Purification of L-[3H]arginine and measurement of L-[3H]citrulline formation were carried out as previously described (32).
Western analysis of eNOS and phosphorylated eNOS. Control and AAF (50 µM for 30 min and 1 h)-stimulated PAEC were washed twice with PBS and then lysed in SDS-PAGE sample buffer (0.06 M Tris·HCl, 2% SDS, and 5% glycerol, pH 6.8). The cell lysate protein (20 µg) was fractionated on a 7.5% SDS-PAGE gel and blotted onto a polyvinylidene difluoride membrane (Bio-Rad). The blots were probed overnight (4°C) with polyclonal rabbit anti-eNOS antibody (Transduction Laboratories) at 1:1,000 dilution or with polyclonal rabbit anti-phospho-Ser1177-specific or anti-phospho-Thr495-specific antibodies (Cell Signaling Technology, Beverly, MA) at 1:500 dilution each and horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling) at 1:2,000 dilution for 1 h. The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham) with Kodak X-OMAT film exposed for 30 and 60 s for total eNOS and phospho-eNOS, respectively. The blots were scanned by laser densitometry to quantify eNOS and phosphorylated eNOS protein contents (34, 35).
Measurement of NO production. NO production was measured in real time using a membrane-permeable 4,5-diaminofluorescein diacetate (DAF-2 DA) probe that hydrolyzes inside the cells, releasing DAF-2 (17). In the presence of NO, DAF-2 converts into a detectable fluorescent product, DAF-2 triazole (27). For NO detection, PAEC in 96-well plates were incubated for 45 min in the presence of 10 µM DAF-2 DA in PBS or in PBS alone (control) in the dark at 37°C. After being incubated, cells were washed with PBS to remove excessive DAF-2 DA. Cells loaded with DAF-2 DA were then stimulated with AAF or AAF-SR (25 µM each) in PBS. In some experiments, cells were pretreated with L-NAME (100 µM) for 30 min before AAF or AAF-SR stimulation. The change in fluorescence was recorded for 15 min at room temperature using a microplate reader (FL 600, Bio-Tek) with excitation wavelength set at 485 and emission wavelength at 530 nm. All measurements of specific DAF-2 DA fluorescence were corrected by subtracting the nonspecific fluorescence in wells without addition of DAF-2 DA and in wells without cells.
We also measured release of stable products of NO [nitrite/nitrate (NOx)] in medium and in lung perfusate as an index of NO production. For measurement of NOx release in isolated porcine PA segments, PA segments (2-mm diameter x 4-mm length) were incubated in PBS with or without (control) 25 µM each of AAF or AAF-SR for 1 h at 37°C. In some experiments, PA segments were pretreated with 100 µM L-NAME for 30 min before AAF or AAF-SR stimulation. After a 1-h incubation, medium was collected to measure NOx release. Measurement of NOx release in the perfusates from isolated mouse lungs that were perfused with or without (control or basal) 5 µM AAF for 2050 min at 37°C was determined by conversion of accumulated nitrates to NO using electrochemistry techniques and an "in NOII" NO measurement system (Innovation Instruments) (39). Concentrations of NOx released were expressed as a percent ratio of AAF- or AAF-SR-stimulated NO release to control or basal NOx release.
Phospho-screen analysis of kinases and PKC- activity. PAEC monolayers were treated with or without (control) the presence of 25 µM each of AAF or AAF-SR for 1 h at 37°C. After being incubated, cell lysates were prepared as previously described (14). One set of lysates was sent to Kinexus Bioinformatics (Vancouver, BC, Canada) for identification of phosphorylation state of 70 kinases, including protein serine-threonine kinases using phosopho-screen analysis. The relative quantity of each protein detected on Kinexus scan screen was identified by molecular mass. The trace quantity of a band was measured as counts per minute (cpm) corrected to a scan time of 60 s. The data provided by Kinexus represent the normalized cpm following correction for differences in protein amount. Identification of the increased phosphorylation of PKC-
by phospho-screen analysis was further confirmed by monitoring the catalytic activity of PKC-
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For measurement of PKC- activity, cell lysates (2 mg of protein) were incubated with 5 µl of polyclonal anti-PKC-
(Calbiochem) antibody for 1 h at 4°C followed by addition of EZView red protein A affinity gel (Sigma Chemical) and overnight incubation on a rotator in a cold room. Blanks were incubated under identical conditions with a non-immuno IgG in the absence of cell samples. Immunoprecipitates were washed with Tris·HCl buffer and used to measure the catalytic activity as previously described (14). The catalytic activity is expressed as units, where one unit of enzyme transfers 1 nmol of phosphate to substrate/minute.
AAF-mediated cGMP production in PASM. To determine whether AAF stimulation of eNOS activity and NO production is associated with increased production of cGMP in PASM, a Transwell coculture system (Costar, Cambridge, MA) was used as previously described (7). In brief, PAEC were seeded on the microporous surface of the removable upper chamber (Transwell), and PASM cells were cultured independently in the lower chamber of a separate Transwell unit. Transwell units containing confluent PAEC and PASM in RPMI 1640 without serum were preincubated for 30 min at 37°C. After the 30-min incubation, AAF (50 µM) or RPMI 1640 medium only (control) was added to the upper chamber. The Transwell units containing both cell lines were incubated for 60 min at 37°C. After incubation control, AAF- and AAF-SR-stimulated cGMP levels in PASM and in medium from the PASM were measured. To confirm that AAF-induced production of cGMP is mediated through eNOS activation and NO production, in some experiments PAEC were preincubated with 50 µM L-NAME or with 10 µM indomethacin (Indo) before AAF or AAF-SR stimulation. The direct effect of AAF or AAF-SR on cGMP production in PASM was independently monitored. A cGMP enzyme immunoassay system kit (Amersham) was used to quantitate cGMP content as previously described (7). cGMP was not detectable in the medium in any experiment.
Vasorelaxation of porcine PA segments and intact mouse lungs. PA segments (1.5- to 2-mm diameter x 3- to 4-mm length) were isolated from the lungs of 6- to 7-mo-old pigs. PA segments were suspended in individual organ chambers (Rodnoti Four-Unit Tissue Bath System) with 5 ml of Krebs buffer and oxygenated with 95% O2 and 5% CO2 at 37°C. After equilibration of resting force of 1.5 g, smooth muscle and endothelium integrity were confirmed by monitoring 0.5 µM U-46619 (a thromboxane A2 mimetic)-mediated PA contraction and acetylcholine (5 µM)-mediated vasodilation before further experimentation. Smooth muscle and endothelium-responsive segments were washed and contracted with U-46619. After stable contraction, the segments were incubated with or without (control) AAF or AAF-SR (25 µM each) for 120 min. We also used endothelium-denuded PA segments to monitor the AAF-mediated response. In some experiments, the effects of L-NAME (100 µM), methylene blue (MB, 10 µM), and Indo (10 µM), inhibitors of eNOS, soluble guanylate cyclase, and prostacyclin (PGI2) production, respectively, on AAF-mediated vasodilation were assessed. The vascular tensions were continuously monitored with an isometric force transducer (Harvard Apparatus, Holliston, MA) and PO-NE-MAH PTP-Tissue Bath System software (Gould Instrument System, Vally View, OH). To standardize the data, the U-46619-induced stable increase in vascular tone was set as 100%.
To confirm the AAF-mediated vasodilation response in the intact pulmonary circulation, a mouse model of isolated lung perfusion was used. Specific pathogen-free female C57BL/6J mice weighing 3035 g were anesthetized with pentobarbital (60 mg/kg, ip), and the lungs were excised from the thorax, ventilated, and used for perfusion (Rodnoti Constant Flow Non-Recirculating Isolated Lung System) essentially as described previously (1, 15). In brief, the isolated lungs were placed in water-jacketed incubation chambers maintained at 37°C. The lungs were ventilated with air at 60 breaths/min with a SAR-830 series small animal ventilator (CWE, Ardmore, PA) at a tidal volume of 0.60.8 ml/100 g body wt. The lungs were perfused with RPMI 1640 containing 4% Ficoll with a constant flow at 0.81.0 ml/min. After a 30-min equilibration, 0.5 µM U-46619 was added to develop a stable PA contraction. The vasodilator response was monitored by addition of 5 µM each of AAF or AAF-SR or 500 µM L-NAME plus 5 µM AAF. The vasodilatory response was continuously monitored as described above using PO-NE-MAH P3P Lung Perfusion System software (Gould Instrument System).
Incorporation of fluorescein-labeled AAF in PAEC, PA, and intact lung. PAEC monolayers were incubated in HBSS containing 5 µM AAF-fluorescein (AAF-FTC; 90% purity, Sigma-Genosys, Woodlands, TX) for 515 min at 37°C. After being incubated, cells were washed three times with HBSS, fixed in 4% paraformaldehyde, and stained with 4',6'-diamidino-2-phenylindole (DAPI) dihydrochloride. For measurement of incorporation of AAF-FTC into isolated PA, 2- to 2.5-mm-diameter x 8- to 10-mm-length PA segments were isolated from porcine lungs. One end of the isolated PA was clamp sealed and filled with 50 µl of Krebs buffer solution containing 5 µM AAF-FTC. The open end was then clamp sealed, and the segment was incubated for 515 min at 37°C. After being incubated, PA segments were washed and fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4). For measurement of incorporation of AAF-FTC into an intact lung, mouse lungs were isolated, ventilated, and perfused with RPMI 1640 containing 4% Ficoll and 5 µM AAF-FTC with a constant flow of 0.8 ml/min for 3060 min. After repeated rinsing of PA segments and lungs with 0.2 M phosphate buffer and with PBS, the PA segments and lungs were infused with 15 and 30% saccharose, respectively, and then frozen in liquid nitrogen and sectioned using a cryostat. The sections were placed on gelatin-coated glass slides and were preincubated with a solution containing DAPI in antifade buffer for 20 min to avoid background staining.
Confocal microscopy. Fixed PAEC and sections of the PA and lung were observed and photographed using a Zeiss LSM 510 inverted confocal microscope equipped with an argon laser (1,488 nm, 100 mW) and two He-Ne lasers (1,543 and 633 nm, 5 mW). Collected images were digitized at a resolution of 12 bits into an array of 1,012 x 1,012 pixels using software provided by the manufacturer. Optical sections of the fluorescence specimens were obtained using a He-Ne laser (488 nm and a FITC set of filters) at a 6.5 µs/pixel scanning speed with an average of up to 16 images. The stacks of images obtained were processed using different software functions including correction for nonspecific fluorescence. Nonspecific background fluorescence was observed only in mouse lung samples.
Statistical analysis. Significance for the effect of AID, AAF, or AAF-SR and inhibitors of selective enzymes on eNOS activity, NO release, cGMP production in PAEC, PA segments, and isolated lungs or on vasorelaxation in PA and lung was determined with ANOVA and a Student's paired t-test (41). A value of P < 0.05 was considered statistically significant.
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RESULTS |
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AID and AAF, but not AAF-SR, activate eNOS. Concentration-dependent effects of AID, AAF, and AAF-SR on the catalytic activity of eNOS were determined. As shown in Fig. 2A, eNOS activity significantly increased after incubation of cells for 2 h at 37°C with 25 µM AID or AAF (P < 0.05 vs. control for both). This AID- and AAF-mediated activation of eNOS remained unchanged with increasing concentrations (up to 100 µM) of both peptides. In contrast, eNOS activity in PAEC treated with AAF-SR remained comparable to control at all concentrations. Because AID and AAF concentration-dependent eNOS activities were comparable, the remaining studies were conducted using AAF. Figure 2B shows the time-dependent effect of AAF and AAF-SR on eNOS activation. AAF-mediated activation was observed as early as 1 h and remained significantly elevated up to 4 h, whereas eNOS activity in AAF-SR-treated cells remained comparable to controls at all time points. Figure 2C shows that AAF, but not AAF-SR, stimulation increased NO release into the medium that was attenuated by pretreatment of cells with the eNOS inhibitor L-NAME.
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AAF does not alter expression or phosphorylation of eNOS. To examine whether AAF-stimulated activation of eNOS is linked with increased expression and/or phosphorylation of eNOS, porcine PAEC were stimulated with 50 µM AAF for 30 min and 1 h, after which eNOS expression and eNOS Ser1177 and Thr495 phosphorylation were analyzed by Western blot. AAF stimulation of PAEC for 30 min (Fig. 3A) or 1 h (Fig. 3B) did not alter expression or phosphorylation of eNOS.
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AAF stimulation increases PKC- phosphorylation and activity. To determine whether AAF stimulation alters the phosphorylation state of multiple kinases, phospho-screen analysis of 70 kinases was performed by Kinexus Bioinformatics. The results showed selective phosphorylation of PKC-
(Ser657). The phospho-screen quantitative analysis of Ser657 PKC-
of duplicate samples showed 849 cpm/50 µg of protein in control, 2,109 cpm/50 µg of protein in AAF-stimulated PAEC, and 998 cpm/50 µg of protein in AAF-SR-stimulated PAEC. AAF-mediated increased phosphorylation of PKC-
was confirmed by monitoring the catalytic activity of PKC-
. As shown in Fig. 4, AAF, but not AAF-SR, significantly increased the catalytic activity of PKC-
in PAEC (P < 0.05 vs. control or AAF-SR).
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AAF-stimulated NO release resulted in cGMP production in PASM. Basal (control) and 1-h AAF-stimulated levels of cGMP in PASM were 3.6 ± 0.7 and 3.9 ± 0.5 pmol/mg of protein, respectively (n = 6 in each set). As shown in Fig. 5, after 1-h preincubation of PASM and PAEC in Transwell coculture units, the basal level of cGMP in PASM was increased to 11.8 ± 0.9 pmol/mg of protein (n = 6) and remained at comparable levels for 2 h (13.1 ± 1.1 pmol/mg of protein, n = 6). However, after stimulation of PAEC with AAF, but not AAF-SR, (25 µM each) for 1 h, cGMP levels in PASM were significantly increased to 35.6 ± 4.0 (P < 0.01 vs. control at 1 h). Pretreatment of PAEC with 100 µM L-NAME significantly reduced the PAEC-AAF/AAF-SR-mediated cGMP production in PASM (P < 0.05 for all). Pretreatment of PAEC with Indo (10 µM), however, had no effect on the PAEC-AAF/AAF-SR-mediated cGMP production in PASM.
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AAF-mediated relaxation of PA segments is time and endothelium dependent. As shown in Fig. 6A, AAF caused vasorelaxation of U-46619-contracted PA segments in a time-dependent manner. AAF-mediated vasorelaxation was maximal at 120 min. AAF-SR-mediated relaxation at 120 min was slightly, but not significantly, increased compared with the respective control. To confirm the role of endothelium in the AAF-mediated relaxation of PA, endothelium-intact and endothelium-denuded PA segments were used in situ. Figure 6B shows that the AAF-mediated vasorelaxation response was significantly attenuated in endothelium-denuded PA (P < 0.05 vs. endothelium intact). AAF, but not AAF-SR, stimulation caused increased NO release (P < 0.05) in the medium, which was attenuated by pretreatment of PA with L-NAME (Fig. 6C).
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AAF-induced vasorelaxation is mediated via the NO-cGMP pathway. To determine whether AAF-induced vasorelaxation of PA is mediated via the NO-cGMP pathway, the effects of L-NAME and the soluble guanylate cyclase inhibitor MB were examined. We also determined the specificity of NO-mediated cGMP production using Indo, an inhibitor of PGI2 production. As shown in Fig. 7, AAF-induced vasorelaxation of U-46619-precontracted PA was abolished by pretreatment with L-NAME or MB but not by Indo.
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AAF causes vasorelaxation in intact lung. To confirm the AAF-mediated vasorelaxation response in the intact pulmonary circulation, the isolated perfused mouse lung model was used. As shown in Fig. 8A, AAF, but not AAF-SR, caused vasorelaxation of precontracted lungs. In the presence of L-NAME, however, the vasodilation of the pulmonary circulation by AAF was attenuated. To determine whether AAF-mediated vasorelaxation is associated with increased NO release, lung perfusates were analyzed. As shown in Fig. 8B, AAF-stimulated increase in NO release was detected as early as 20 min after initiation of perfusion, and this significantly increased with prolonged perfusion time (from 20 to 30 min) and remained elevated for up to 50 min of perfusion (P < 0.05).
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Exogenous AAF-FTC is taken up by PAEC, PA, and lung. Figure 9 shows confocal images of internalized AAF-FTC in PAEC in culture, in an isolated PA segment, and in an intact mouse lung. Cellular and tissue uptake and internalization of AAF-FTC appeared to be time dependent since fluorescence labeling of PAEC and lungs were enhanced with increasing time.
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DISCUSSION |
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Although the precise molecular mechanisms by which AID regulates electron transfer and the catalytic activity of eNOS remain to be determined, a number of intrinsic and extrinsic factors can distinctly influence the regulatory function of AID, leading to diminished or enhanced catalytic activity of eNOS in vitro or in vivo. For example, a previous study has shown that incubation of purified recombinant bovine eNOS with various synthetic fragments of AID, including a fragment representing AAF, diminished the catalytic activity of eNOS (38). We have also observed (Fig. 1) that incubation of isolated total membrane fractions from PAEC or incubation of PAEC homogenates with AID or AAF resulted in >50% loss of eNOS activity. In contrast, our present results demonstrate AID- or AAF-mediated activation of eNOS in intact PAEC, PA segments, and whole lung. The observation that AID or AAF activates eNOS in an intact cell or tissue but inhibits eNOS activity in isolated/disrupted preparations suggests the importance of structural integrity and implicates the involvement of signaling pathways in the regulatory processes, including phosphorylation events and the conformational change in eNOS protein. A number of external stimuli have been shown to increase eNOS phosphorylation and activation through activation of multiple kinases, including PKC, protein kinase B (PKB/Akt), protein kinase A (PKA), AMP-activated protein kinase, and calmodulin-dependent protein kinase in diverse systems (25, 11, 21). For example, increased phosphorylation of Ser1177 in human eNOS or Ser1179 in bovine eNOS is regulated by PKB/Akt- and PKA-dependent mechanisms without affecting the phosphorylation state of eNOS Ser116 and Thr495 (human)/Thr497 (bovine) (25). In addition to Ser1177/Ser1179, PKA also phosphorylates Ser635 in bovine eNOS (2, 3). However, PKA-mediated phosphorylation of Ser1177/Ser1179 and Ser635 in eNOS is regulated by a calcium-independent manner in response to shear stress (2, 3). Our studies involving identification of AAF- and AAF-SR-stimulated phosphorylation of multiple kinases, including PKB/Akt and various isoforms of PKC, revealed that AAF, but not AAF-SR, stimulation causes increased phosphorylation of the PKC- isoform as well as a threefold increase in the catalytic activity of PKC-
in PAEC. Recent studies suggest that PKC signaling results in enhanced phosphorylation of Thr497 and promotes dephosphorylation of Ser1177 in the eNOS protein (21, 26). However, these studies did not identify whether a specific isoform of PKC was associated with increased phosphorylation of Thr497 in eNOS protein. Although the absence of increased or decreased phosphorylation of Ser1177 or Thr495 in the eNOS protein following AAF stimulation of PAEC suggests that at least signaling associated with activation of PKC, PKA, and/or PKB/Akt is most likely not responsible for the AAF-mediated responses, the potential role of AAF-mediated increased phosphorylation of PKC-
is unclear in the present study. However, PKC-
-mediated enhanced phosphorylation of other proteins, including caveolin, calmodulin, and components of cytoskeletal proteins known to influence the catalytic activity of eNOS, cannot be ruled out (25, 30, 43). For example, a recent study demonstrates that PKC-
increases phosphorylation of the cationic amino acid transporter-1, which is known to form a functional unit with eNOS in caveolae of lung endothelial cells and is believed to play a critical role in the regulation of NO production (16, 23). The precise mechanisms involved in AAF-mediated eNOS activation and vasorelaxation in the pulmonary circulation remain to be determined.
Our results also demonstrate rapid internalization of AAF into PAEC, PA segments, and intact lung. Exogenous peptides of varying length are known to be internalized by multiple processes in biological systems. For example, the uptake processes of transport peptides in biological membranes can be regulated via receptor- or transporter-mediated internalization, by vesicular transport, by absorptive endocytosis, or via electrostatic interaction with membrane lipids/proteins (20). This latter possibility is of particular interest considering the nature of the amino acid residues present in AAF, namely Ser-Ser-Trp-Arg-Arg-Lys-Arg-Lys-Glu-Ser-Ser. The positive charges in the side chain of Arg/Lys and the presence of Trp or Phe residues in the peptide may be crucial factors determining cellular uptake through electrostatic interaction (20, 37). As such, AAF may be internalized through electrostatic interaction with membrane lipids and/or proteins in PAEC. It is tempting to speculate that the interaction of AAF with the eNOS protein in general or with the calmodulin-binding domain of the eNOS protein in particular can enhance the catalytic activity of eNOS through a conformational change in calcium/calmodulin binding, increased electron transfer from the reductase to the oxygenase domain, and/or modulation of phosphorylation/dephosphorylation of critical amino acid residues in eNOS. The studies to address these possibilities are currently in progress.
In summary, our results demonstrate that an exogenous synthetic peptide representing an internal fragment of native eNOS protein enhances the catalytic activity of eNOS, leading to NO-cGMP pathway-responsive vasorelaxation in isolated porcine PA segments and in the mouse pulmonary circulation. The physiological importance of these results is significant for peptide-based therapy in the regulation of pulmonary vascular function.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by the Medical Research Service of the Department of Veterans Affairs (to J. M. Patel) and by National Heart, Lung, and Blood Institute Grant HL-68666 (to J. M. Patel).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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