Autoinhibitory domain fragment of endothelial NOS enhances pulmonary artery vasorelaxation by the NO-cGMP pathway

Hanbo Hu,1 Meiguo Xin,1 Leonid L. Belayev,1 Jianliang Zhang,1 Edward R. Block,1,2 and Jawaharlal M. Patel1,2

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Catalytic activity of eNOS is regulated by multiple posttranscriptional mechanisms, including a 40-amino acid (604-643) autoinhibitory domain (AID) located in the reductase domain of the eNOS protein. We examined whether an exogenous synthetic AID, an 11-amino acid (626-636) fragment of AID (AAF), or scrambled AAF (AAF-SR), enhanced eNOS activity and NO-cGMP-mediated vasorelaxation using pulmonary artery (PA) endothelial/smooth muscle cell (PAEC/PASM) coculture, isolated PA segment, and isolated lung perfusion models. Incubation of isolated total membrane fraction of PAEC with AID or AAF resulted in concentration-dependent loss of eNOS activity. In contrast, incubation of intact PAEC with AID or AAF but not AAF-SR caused concentration- and time-dependent activation of eNOS. Because AID and AAF had similar effects on activation of eNOS, AAF and AAF-SR were used for further evaluation. Although AAF stimulation increased catalytic activity of PKC-{alpha} in PAEC, AAF-mediated activation of eNOS was independent of phosphorylation of Ser1177 or Thr495 and/or expression of eNOS protein. AAF stimulation of PAEC increased NO and cGMP production, which were attenuated by pretreatment with the eNOS inhibitor L-NAME. AAF caused time-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PA segments, and this response was attenuated by L-NAME. AAF, but not AAF-SR, also caused vasorelaxation in an ex vivo isolated mouse lung perfusion model precontracted with U-46619. Incubation with fluorescence-labeled AAF demonstrated translocation of AAF in PAEC in culture, isolated PA, and isolated intact lungs. These results demonstrate that AAF-stimulated vasodilation is mediated via activation of eNOS and enhanced NO-cGMP production in PA and intact lung.

nitric oxide synthase; guanosine 3',5'-cyclic monophosphate; electron transfer element; lung perfusion


VASCULAR ENDOTHELIUM is a metabolically active tissue that plays a critical role in the regulation of pulmonary vascular tone through multiple mechanisms, including metabolism, generation, and release of vasoactive mediators such as nitric oxide (NO) (8, 13, 35). Vascular endothelial cells generate NO from the metabolism of L-arginine via an oxidative catabolic reaction mediated by an endothelial cell constitutively expressed isoform of NO synthase (eNOS) (13, 31, 35). A number of external stimuli, including agonists, fluid shear stress, and growth factors, are known to modulate the catalytic activity of eNOS, NO release, and cGMP production, leading to NO-cGMP-mediated vasorelaxation (7, 19, 24, 40). The catalytic activity of eNOS is regulated by multiple posttranscriptional modifications, including phosphorylation-dephosphorylation state, redox modulation of active site cysteines, and protein:protein interactions, as well as by control of inter- and intradomain electron transfer mechanisms within the eNOS protein (12, 26, 34, 42).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and treatment. All animal protocols and procedures were approved by the Institutional Animal Care and Use Committee, VA Medical Center, Gainesville. All animals were euthanatized using appropriate methods as described in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. PAEC and PASM were obtained from the main PA of 6- to 7-mo-old pigs. Endothelial cells were propagated in monolayers as previously described (33). Third- to fifth-passage cells in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) were used in all experiments. PASM were isolated from middle media explants after removal of the endothelial, subendothelial, and adventitial layers of the arterial segments as previously described (10). Tissue explants were cultured for 2 wk in MEM-{alpha} containing 15% fetal bovine serum. After 2 wk, individual cell colonies grown from the explants were isolated and subcultured (7). All studies involving PAEC and PASM were carried out with cells at passages 3–5. In each experiment, PAEC and/or PASM were studied 1 or 2 days after confluence and were matched for cell line, passage number, and days after confluence.

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 (10–100 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 (5–100 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 (100–200 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 20–50 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-{alpha} 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-{alpha} by phospho-screen analysis was further confirmed by monitoring the catalytic activity of PKC-{alpha}.

For measurement of PKC-{alpha} activity, cell lysates (2 mg of protein) were incubated with 5 µl of polyclonal anti-PKC-{alpha} (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 30–35 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 0.6–0.8 ml/100 g body wt. The lungs were perfused with RPMI 1640 containing 4% Ficoll with a constant flow at 0.8–1.0 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 5–15 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 5–15 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 30–60 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AID and AAF inhibit eNOS activity in total membrane fractions. The catalytic activity of eNOS was determined after treatment of isolated total membrane fractions of PAEC with AID or AAF. As shown in Fig. 1, eNOS activity was decreased in a concentration-dependent fashion after incubation of isolated total membrane fractions for 30 min with AID or AAF. The significant loss of eNOS activity was observed at concentrations of AID or AAF as low as 25 µM. The loss of activity was enhanced with increasing concentrations of AID or AAF (P < 0.05 for 25–100 µM AID or AAF vs. control).



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Fig. 1. Concentration-dependent effect of autoinhibitory domain (AID) or fragment of AID (AAF) treatment on total membrane fraction endothelial nitric oxide synthase (eNOS) activity. Total membrane fractions (1 mg of protein) isolated from pulmonary artery endothelial cells (PAEC) were incubated with or without (control) the presence of 10–100 µM AID or AAF for 30 min at 37°C. After being incubated, membrane fractions were washed by centrifugation, resuspended in fresh Tris·HCl buffer, and eNOS activity was measured as described in MATERIALS AND METHODS. Data are means ± SE; n = 4 for each treatment group. *P < 0.05 vs. control for both AID and AAF.

 

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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Fig. 2. Concentration- and time-dependent effects of AID, AAF, and scrambled AAF (AAF-SR) on the catalytic activity of eNOS in PAEC. Cell monolayers were treated with RPMI 1640 containing 5–100 µM each of AID, AAF, AAF-SR, or RPMI 1640 only (0 or control) for 1 h at 37°C (A) or with RPMI 1640 with or without (control) AAF and AAF-SR (25 µM each) for 1, 2, or 4 h at 37°C (B). For measurement of NO release, cell monolayers were loaded with 10 µM 4,5-diaminofluorescein diacetate with or without the presence of 100 µM NG-nitro-L-arginine methyl ester (L-NAME) for 30 min before stimulation with 25 µM each of AAF or AAF-SR for 1 h at 37°C (C). Data are means ± SE; n = 4 for each treatment group in A–C. *P < 0.05 vs. control and AAF-SR in A–C; **P < 0.05 vs. all in C.

 

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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Fig. 3. Effect of AAF stimulation on expression and phosphorylation of eNOS in PAEC. Cell monolayers were treated with RPMI 1640 containing 50 µM AAF or RPMI 1640 only (control) for 30 min and 1 h at 37°C. After they were treated, cells were washed and lysed, and lysates were fractionated and immunoblotted with anti-eNOS antibody or with anti-phospho Thr495 (p-T495) eNOS antibody or phospho-Ser1177 (p-S1177) eNOS antibody as described in MATERIALS AND METHODS. Representative Western blots from 2 separate experiments with similar results are for total eNOS, p-T495 eNOS, and p-S1177 eNOS in A and B. Densitometric analysis represents an average from 2 blots for each experiment in A and B. C, control.

 

AAF stimulation increases PKC-{alpha} 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-{alpha} (Ser657). The phospho-screen quantitative analysis of Ser657 PKC-{alpha} 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-{alpha} was confirmed by monitoring the catalytic activity of PKC-{alpha}. As shown in Fig. 4, AAF, but not AAF-SR, significantly increased the catalytic activity of PKC-{alpha} in PAEC (P < 0.05 vs. control or AAF-SR).



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Fig. 4. Effect of AAF or AAF-SR treatment on the catalytic activity of PKC-{alpha} in PAEC. Cell monolayers were treated in RPMI 1640 containing 25 µM each of AAF or AAF-SR or RPMI 1640 only (control) for 1 h, and after treatment cell lysates were immunoprecipitated with anti-PKC-{alpha} antibody. Equal amounts of immunoprecipitated proteins were used to measure catalytic activity using PKC-{alpha} peptide substrate and [32P]ATP as described in MATERIALS AND METHODS. Data represent means ± SE; n = 4 for each treatment. *P < 0.05 vs. control or AAF-SR.

 

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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Fig. 5. cGMP production in porcine pulmonary artery (PA) smooth muscle cells (PASM) after AAF or AAF-SR stimulation of PAEC. Transwell units containing PAEC (top chamber) and PASM (bottom chamber) in RPMI 1640 medium were preincubated for 30 min at 37°C. After a 30-min incubation, AAF, AAF-SR (25 µM each), or RPMI 1640 medium (control) was added to PAEC and incubated for 60 min at 37°C (group 1). To determine the effect of NOS inhibition or inhibition of prostacyclin production, PAEC were preincubated (30 min) with L-NAME (100 µM; group 2) or indomethacin (Indo; 10 µM; group 3) before AAF or AAF SR stimulation. Control and AAF- or AAF-SR-stimulated cGMP levels were measured in PASM (as shown in group 1). Values are means ± SE; n = 8 in each group. **P < 0.05 vs. control and AAF-SR in the same group; *P < 0.05 vs. the respective treatment in group 1 and group 3.

 

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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Fig. 6. AAF-mediated vasodilation is time- and endothelium-dependent in porcine PA. A: vasorelaxation of U-46619 (500 nM)-contracted PA segments was measured in the presence of AAF, AAF-SR (25 µM each), or U-46619 only (control) for 10–120 min. B: vasorelaxation of U-46619-contracted endothelium-intact (EC-intact) and endothelium-denuded (EC-denuded) PA segments was measured in the presence of 25 µM AAF for 120 min. C: AAF- or AAF-SR (25 µM each)-mediated NO release from PA segments into the medium with or without the presence of L-NAME (100 µM) was measured at 60 min as described in MATERIALS AND METHODS. NO released is expressed as a percent ratio of AAF- or AAF-SR-stimulated release to control NO release. NOx, stable products of NO (nitrite/nitrate). Values are means ± SE; n = 4 experiments in each time point or in each treatment group. A: *P < 0.05 vs. control and AAF-SR at the same time. B: *P < 0.05 vs. EC-intact. C: *P < 0.05 vs. all other groups.

 

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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Fig. 7. AAF-stimulated vasodilation of porcine PA is mediated via the NO-cGMP pathway. Vasorelaxation of U-46619 (500 nM)-contracted endothelium-intact PA segments was measured in the presence of 25 µM AAF with or without the presence of 100 µM L-NAME (A), or with 10 µM methylene blue (MB; B), or with 10 µM Indo for 120 min (C). Values are means ± SE; n = 6 in each set of experiments. *P < 0.05 vs. AAF studied at the same time (A) or AAF treatment (B).

 

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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Fig. 8. AAF causes vasorelaxation and increases NO release in mouse lung. Mouse lungs were isolated, ventilated, and, after stable contraction with U-46619 (500 nM), perfused with RPMI 1640 containing 4% Ficoll and AAF or AAF-SR (5 µM) or L-NAME (500 nM) + AAF (5 µM) for 60 min, respectively (A). The vasodilatory response was continuously monitored as described in MATERIALS AND METHODS. Perfusates were analyzed for NO determination at 20, 30, and 50 min (B). NO release was expressed as a percent ratio of AAF-stimulated release to control NO release. Values are means ± SE; n = 4 for each time point. *P < 0.05 vs. AAF-SR or **P < 0.05 vs. AAF in A, and *P < 0.05 vs. control in B.

 

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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Fig. 9. Confocal images of AAF-fluorescein (FTC) internalization in porcine PAEC, PA, and mouse lung. PAEC monolayers were incubated in HBSS containing 5 µM AAF-FTC for 2 and 15 min at 37°C. PA segments were filled with Krebs-Henseleit solution containing 5 µM AAF-FTC for 60 min at 37°C. Ventilated mouse lungs were perfused with RPMI 1640 containing 5 µM AAF-FTC for 15 and 60 min. After being incubated, PAEC, PA segments, and lungs were prepared for confocal microscopy, and images were corrected for nonspecific fluorescence as described in MATERIALS AND METHODS. Blue color represents 4',6'-diamidino-2-phenylindole nuclear labeling in A–D. A and B: 2- and 15-min PAEC; C and D: 60 min, x50 and x1,000 magnification of PA segments; and E and F: 15- and 60-min perfused mouse lungs. Data are representative of 2 separate samples in each panel with similar observations.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We demonstrate here for the first time that an exogenous synthetic peptide representing an internal fragment of eNOS protein enhances the catalytic activity of eNOS and NO-cGMP-mediated vasorelaxation in the pulmonary circulation. Our results provide experimental evidence that PAEC stimulation with AID, a 40-amino acid segment in the reductase domain of eNOS, or AAF, an 11-amino acid internal fragment of AID, causes activation of eNOS and increased NO production. The results of the present studies using porcine PAEC/PASM cocultures, isolated porcine PA segments, and intact perfused mouse lung models demonstrate that 1) AAF-induced activation of eNOS and endothelium-derived release of NO are critical for enhanced production of cGMP in PASM in a coculture system, 2) AAF-mediated vasodilation response in PA segments is endothelium dependent, 3) AAF stimulation of eNOS activity and NO production was accompanied by NO-cGMP-mediated vasorelaxation in isolated porcine PA segments and in isolated perfused mouse lungs, and 4) exogenous AAF is taken up by PAEC, PA segments, and intact lung.

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-{alpha} isoform as well as a threefold increase in the catalytic activity of PKC-{alpha} 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-{alpha} is unclear in the present study. However, PKC-{alpha}-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-{alpha} 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.


    ACKNOWLEDGMENTS
 
We thank Bert Herrera for tissue culture assistance and Weihhong Han and Dihua He for technical assistance.

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).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Patel, Research Service (151), VA Medical Center, 1601 SW Archer Road, Gainesville, FL 32608-1197 (E-mail: Pateljm{at}medicine.ufl.edu).

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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