1Department of Pediatrics, Northwestern University, Chicago, Illinois 60611; and 2Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59802
Submitted 10 July 2003 ; accepted in final form 15 December 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
guanosine 3',5'-cyclic monophosphate
Endogenous NO is an endothelially derived regulator of vascular tone that is formed by nitric oxide synthase (NOS; see Ref. 13). NO can then freely diffuse to the smooth muscle cell and activate soluble guanylate cyclase (sGC), increasing the conversion of GTP to the intracellular signaling molecule cGMP (39). cGMP works, at least in part, by activating cGMP-dependent protein kinase (PKG), another important intracellular signaling enzyme (1). It is presumed that, when NO is given by inhalation, it freely diffuses through the pulmonary epithelium to the vascular smooth muscle where it exerts its vascular dilating effect in a similar fashion (13).
Approximately 40% of the infants with PPHN treated with inhaled NO have an incomplete or unsustained response (16, 24). Furthermore, upon the acute withdrawal of inhaled NO, there can be a significant increase in the pulmonary vascular resistance, which may occur independently of the initial response to inhaled NO therapy. This phenomenon has been termed rebound pulmonary hypertension (3, 35). In sheep models, we have shown that inhaled NO exposure increases plasma endothelin-1 (ET-1) levels and decreases NOS activity (5, 34). ET-1 is a potent endothelially derived vasoconstricting peptide that exists in its nascent form as prepro-ET-1, a nonsecreted, physiologically inactive 203-amino-acid protein (21). Prepro-ET-1 is cleaved intracellularly in an incompletely understood step to pro-ET-1 or big ET-1, a 39-amino-acid peptide (21). Big ET-1 has been shown to have weak vasodilating effects when infused intravenously and is converted to the active 21-amino-acid peptide ET-1 by the tightly regulated enzyme endothelin-converting enzyme-1 (ECE-1; see Refs. 33 and 38). There are at least two isoforms of ECE-1 in the pulmonary vasculature, with ECE-1 the predominant form (11). It has been shown by multiple investigators that ET-1 and NO exert a paracrine regulation on each other (8, 33, 42). There have been several in vitro studies utilizing cultures of systemic vascular endothelial cells that demonstrated that increasing endogenous NO generation, through activation of NOS by various means, will lead to decreased ET-1 (8, 28). However, in vivo studies in sheep and pig models demon-strated that inhaled NO at a concentration of 3040 ppm produces increased circulating ET-1 levels (15, 34). These conflicting data prompted us to evaluate the effect of exogenous NO on cells cultured from the pulmonary arteries of sheep to examine the mechanism by which NO regulates the release on ET-1. Here we report that, in pulmonary vascular endothelial cells, NO decreases ET-1 secretion through activation of sGC and increased cellular generation of cGMP.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
For experimental treatments, 4-wk PAECs were transferred to serum-free DMEM-H16 media supplemented with antibiotics and antimycotics 12 h before the experiments to achieve cell cycle synchronization. All experiments were carried out using 500,000 cells of passage 610, unless otherwise specified. Cells were then treated (in a 3-ml volume) for 024 h with the long-acting NO donor DETA-NONOate (01 mM; Alexis Biochemical), unless otherwise specified, or with the NO-independent activator of sGC, YC-1 (30 µM; Calbiochem; see Ref. 4). Furthermore, cells were treated with the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM; Biomol; see Ref. 12) or the inhibitory cGMP stereoisomer of PKG, Rp-8-pCPT-cGMP (RcGMP; Biomol) at 2.5 and 5 µM, corresponding to 5 and 10 times the inhibitory constant (14) alone, before the addition of NO. In additional experiments, cells were treated with the long-acting cGMP analog Sp-8-pCPT-cGMP (Biomol), at concentrations ranging from 1.5 to 9 µM.
Western blot analysis. Cells were harvested using mammalian protein extraction reagent (Pierce) and then sonicated. Protein concentrations were estimated using Bradford reagent. Total protein (20 µg) was separated on a 420% SDS-polyacrylamide gel (Bio-Rad) and then electrotransferred to polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The gels were stained with 0.1% Coomassie blue and evaluated by densitometry to determine equal protein loading. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. After being blocked, the membranes were incubated at room temperature with the appropriate dilution of the antiserum of interest (1:1,000 for ECE-1, 1:500 for ECE-1
, 1:100 for sGC-
, and 1:1,000 for sGC-
). The ECE-1
antiserum was generated as previously described (34). The sGC antisera were a generous gift from Peter Yuen (University of Tennessee-Memphis). After being washed, membranes were hybridized with anti-rabbit horseradish peroxidase antibody, and the bands were visualized with chemiluminescence and analyzed using a Kodak Digital Science Image Station (NEN). Specificity was demonstrated using a mammalian expression vector containing full-length rat ECE-1
transiently transfected into COS-7 cells. This construct was generously provided by Dr. M. Yanagisawa (University of Texas Southwestern Medical Center, Dallas, TX).
Generation of ECE-1 antisera. A peptide was designed that was specific for ECE-1
. This peptide (CLGKKGPGLTVSLPL) corresponds to the NH2-terminal domain of ECE-1
, which can immunologically distinguish between the ECE-1 isoforms (8). The protein was purified and synthesized at >90% purity. The peptide was then conjugated, via addition of an NH2-terminal cysteine. Two female New Zealand White rabbits (12 wk of age and 2 kg in weight) were then injected with 200 µg conjugated peptide and 200 µg Fruend's complete adjuvant. This injection was repeated after 14, 28, 42, and 56 days with the exception that Fruend's incomplete adjuvant was used. Bleeds (15 ml) were collected at 42, 56, and 70 days. Aliquots of antisera were then stored at -20°C until used. Antibody generation was carried out commercially by Biosynthesis (Lewisville, TX). Specificity was demonstrated using a mammalian expression vector containing full-length rat ECE-1
transiently transfected into COS-7 cells. This construct was generously provided by Dr. Yanagisawa.
ET-1 determinations. Conditioned medium was collected at multiple time points and stored at -80°C until assayed. The medium was acidified with 0.1% trifluoroacetic acid and loaded in 3 x 18 C18 SepPak columns (Peninsula Laboratories) preactivated with 60% acetonitrile and 0.01% trifluoroacetic acid and equilibrated with 0.01% trifluoroacetic acid. The adsorbed material was eluted with 2 ml of 0.01% trifluoroacetic acid-60% acetonitrile. The eluant was dried in a Savant speed vacuum centrifuge, dissolved in assay buffer, stored at -20°C, or assayed immediately for immunoreactive ET-1. The ET-1 standard, 125I-labeled ET-1, anti-ET-1 antibody, and secondary antibody were purchased from Peninsula Laboratories. Cross-reactivity for measured human and bovine ET-1 antiserum is 100%, 7% for human ET-2 and ET-3, and <1% for bovine ET-2 and ET-3. Interassay and intra-assay variabilities were 8 and 4%, respectively. Each sample was run in duplicate. This assay was modified from a previously published procedure (43).
Cell proliferation assays. The 4-wk PAECs were seeded on 96-well plates (Costar) at 5,000 cells/well (
75% confluence) and allowed to adhere for at least 18 h. The initial number of viable cells was then determined to correct for differences in starting cell number between experiments and to monitor changes in cell number over time. The cells were transferred to serum-free media and treated with the long-acting NO donor DETA NONOate at various concentrations (6 µM-1 mM). After 24 h of treatment, the cell number was determined using the Cell Titer 96 AQueous One Solution kit (Promega) according to the manufacturer's instructions.
Determination of mitochondrial membrane potential integrity. Mitochondrial membrane potential has been analyzed previously using the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (17). This dye fluoresces red in its multimeric form in healthy mitochondria and is the active reagent in the DePsipher Mitochondrial Potential Assay kit (Trevigen). PAECs were seeded on 24-well plates and incubated with or without 500 µM DETA NONOate. The DePsipher reagent (25 µg/ml) was then added 24 h later and incubated for a further 20 min. Fluorescent images were captured using a CoolSnap digital camera, and the average fluorescent was quantified using Metamorph imaging software (Fryer).
cGMP quantification. PAECs were treated as described, washed with cold PBS, and then scraped into PBS and lysed by sonication. An aliquot was removed for protein quantification. Cold ethanol was added to a final concentration of 66% ethanol to desiccate the cells and elute cGMP. The cell lysate/ethanol solution was then centrifuged at 5,000 g for 15 min, and the pellet was discarded. The cell extract was then dried in a Savant speed vacuum at room temperature, resuspended in assay buffer, acetylated using acidified sodium acetate, and then used immediately for cGMP quantification by a commercially available ELISA kit (Cayman Chemical, Ann Arbor, MI). The antiserum has a specificity for acetylated cGMP of 100%, cGMP 9%, acetylated cAMP 0.05%, and cAMP <0.01%. The results were determined by colorimetry in a Labsystems Multiskan EX automated plate reader (Biotech) at 490 nM wavelength and compared with an acetylated cGMP standard curve. All samples were run in duplicate with two concentrations. The interassay and intra-assay variability was <10%.
Semiquantitative RT-PCR. Total RNA was collected using the Tri-Zol reagent, as previously described. After extraction with chloroform and purification with isopropanol and ethanol, total RNA was quantified by spectrophotometry. Next, total RNA (0.1 µg) was reverse transcribed and amplified using a single-step RT-PCR kit (Invitrogen). The following cycling conditions were used for the amplifications: 1 cycle at 50°C for 45 min (RT step) followed by a denaturation step at 94°C for 2 min, and a set of 1525 cycles of 94°C for 30 s, 55°C for 60 s, and 68°C for 90 s. Cycle curves were carried out for ET-1 and 18S (as a normalizer) to determine the linear amplification range. Based on these studies, the following cycle numbers were chosen: 15 cycles for 18S (internal control) and 25 cycles for ET-1. The following set of primers was used: ET-1 sense primer: 5'-GCCCTGAGTTCTTTTCCTGCTTGG-3'; ET-1 antisense primer: 3'-CCAAGGAGCTCCAGAAACAGC-3'; 18S sense primer: 5'-AGGGTTCGATTCCGGAGAGGG-3'; and 18S antisense primer: 5'-CATTCCAATTACAGGGCCTCG-3'.
Statistical analysis. ET-1 data over time were analyzed by ANOVA for repeated measures. Other data were analyzed by two-way ANOVA with Student Newman-Keuls post hoc testing. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To investigate whether the decrease in ET-1 was related to NO-induced activation of sGC, 4-wk PAECs were treated with YC-1 (30 µM), an NO-independent activator of sGC. This produced a significant decrease in secreted immunoreactive ET-1 at 24 h (515 ± 312 pg/ml, P < 0.05 vs. untreated, Fig. 4). This decrease was similar to that seen in cells treated with DETA NONOate (500 µM). Treatment with doses higher than 30 µM had no greater effect on ET-1 release (data not shown). Treatment of 4-wk PAECs with ODQ (10 µM), a heme site inhibitor of sGC, had no effect on the amount of ET-1 secreted (2,037 ± 454 vs. 2,743 ± 92 pg/ml for control cells). However, pretreatment of 4-wk PAECs with ODQ before adding DETA NONOate prevented the NO-induced decrease in secreted ET-1 (2,365 ± 230 pg/ml) compared with control values (2,743 ± 92 pg/ml, P < 0.05 vs. untreated; Fig. 4).
|
We next verified that the pharmacological regulation of sGC activity produced the expected changes in cellular cGMP levels. cGMP levels were significantly increased with activation of sGC by DETA NONOate (11.8 ± 0.13 pmol/µg protein, P < 0.05 vs. control) or YC-1 (12.9 ± 1.1 pmol/µg protein, P < 0.05 vs. control) compared with 4 ± 1.3 pmol/µg protein for control cells (Fig. 5A). Treatment with doses higher than 30 µM had no greater effect on cGMP levels (data not shown). Blocking sGC activity with ODQ (10 µM) did not alter basal cGMP levels. However, pretreatment of 4-wk PAECs with ODQ significantly reduced the NO-induced increase in intracellular cGMP (P < 0.05 vs. untreated; Fig. 5).
|
To determine if cGMP alone could alter ET-1 secretion, cells were treated with the long-acting cell-permeable cGMP analog Sp-8-pCPT-cGMP. The results obtained (Fig. 6) demonstrated a significant decrease in secreted ET-1 at concentrations of 3 µM (898 ± 316 pg/ml, P < 0.05 vs. untreated), 4.5 µM (1,040 ± 198 pg/ml, P < 0.05 vs. untreated), and 9 µM (790 ± 170 pg/ml, P < 0.05 vs. untreated) but not at 1.5 µM (2,445 ± 64 pg/ml) compared with untreated cells (2,743 ± 92 pg/ml). To determine if the mechanism for the regulation of ET-1 release occurred through PKG, cells were treated with the antagonistic stereoisomer of cGMP, Rp-CPT-8cGMP (RcGMP), before addition of the NO donor. Inhibition of PKG significantly, but only partially, blocked the effect of NO on ET-1 release (1,192 ± 166 pg/ml at a concentration 2.5 µM and 1,416 ± 156 pg/ml at concentration of 5 µM RcGMP, both P < 0.05 vs. NO alone; Fig. 6). Treatment with RcGMP alone had no effect on ET-1 release (data not shown).
|
To begin to investigate if NO decreases ET-1 release through changes in RNA levels, cells were again treated with DETA NONOate (500 µM), and total RNA was isolated and quantified by RT-PCR. The results obtained indicated that exposure to the NO donor produced a significant decrease in prepro-ET-1 mRNA to nearly undetectable levels after NO treatment (P < 0.05 vs. untreated; Fig. 7).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The endothelium is important in regulating vascular tone under a wide variety of physiological conditions and stresses. There are several important endothelially derived regulators of vascular tone, with NO and ET-1 among them. NO exerts its action in the vasculature through several distinct pathways. Important among them is the activation of sGC to increase intracellular cGMP, direct nitration and nitrosylation of proteins, and induction of reactive oxygen species (ROS; see Refs. 1 and 39). Exposure of the endothelium to pharmacological levels of NO undoubtedly alters the coordination of vasoactive agents, and the results are complex and may vary according to the vascular bed and developmental stage. Although the effect of NO on ET-1 release has been studied in a variety of systems, the mechanism by which NO regulates ET-1 production and release remains incompletely understood.
Previous studies with the use of primary cultures of systemic vascular endothelial cells isolated from adult animals have shown that stimulation of endogenous NO production with agonists will decrease secreted ET-1 (8). However, it is known that NO and ET-1 release is developmentally regulated and the response of the vasculature to these agents may change with developmental stage (26, 37). Therefore, in the context of pulmonary hypertension of the newborn, we felt it was important to investigate the response of pulmonary vascular endothelial cells of newborn and juvenile animals to begin to understand the changes seen in the pulmonary vasculature after treatment with exogenous NO. It has also been previously demonstrated, using human umbilical venous endothelial cells, that blocking NO production can increase ET-1 release (36). This increased ET-1 release was associated with an increase in ET-1 transcription (36). Conversely, treatment with the NO donor molsidomine reduced the hypoxia-induced increase in ET-1 release in the lungs of rats (7). Although the exact mechanism of the interaction was not resolved, these data indicate that NO and ET-1 have an inverse relationship and provide important information in the context of the regulation of the two vascular mediators. Similarly, the addition of an NO donor has been shown to reduce the hypoxia-dependent increase in ET-1 release in human umbilical vein endothelial cells (28). The response of the umbilical venous endothelial cells, which line vessels that constrict in the presence of normal oxygen tension and pH, may not be the same as that of the pulmonary vascular endothelial cells. However, the fact that the results obtained are similar to those obtained in our studies suggests that this may be a common regulatory pathway for the effect of NO on ET-1.
We have previously demonstrated that NO donors can increase ROS levels in pulmonary endothelial cells (9, 10), and other investigators have sought to evaluate the role of ROS on ET-1 release. The data obtained have indicated that increased superoxide production, but not increased NO or hydrogen peroxide, decreases ET-1 release via the inhibition of ECE-1 (32). The mechanism for this inhibitory effect was found to be through the displacement of the heavy metal zinc from the ECE-1 enzyme (32). Thus we investigated the effect of NO on ECE-1 expression. Our data indicated that exogenous NO had no effect on ECE-1 or ECE-1
expression, and, although we cannot rule out an effect on ECE-1 activity, this suggests that the mechanism by which NO exerts its effect on ET-1 is not via alterations in ECE-1 expression. In addition, studies with the use of primary cultures of coronary artery endothelial cells have shown that increased superoxide levels increased transcription of prepro-ET-1 mRNA and increased ET-1 release in the media (27). However, our results indicate that prepro-ET-1 mRNA levels and ET-1 release are both decreased by NO. The conflicting data obtained in these studies, although evaluated in different cell types, highlight the potential but complex role that ROS may play in the regulation of ET-1 gene expression and secretion and underscore the importance of understanding vascular-specific and cell-specific effects.
The data we present in these studies are in contrast to our previously published results where we found that 4-wk-old lambs exposed to 40 ppm inhaled NO for 24 h had a twofold increase in the circulating immunoreactive ET-1 levels (34). Thus it still remains unclear if this increase in ET-1 secretion is directly related to the use of inhaled NO rather than NO donors or may be the result of changes in cell-cell communication or alterations in biomechanical forces that exist in the whole animal situation. Important among biomechanical forces playing an active role in the whole animal is shear stress. It has previously been shown that exposure of human umbilical venous cells to shear stress increases ET-1 release and increases transcription of prepro-ET-1 mRNA after 24 h (36). Therefore, the effects of the increased blood flow in the pulmonary system we have observed (5) may increase shear stress in the animal exposed to inhaled NO and may account in part for the differences between our in vivo and in vitro data. In addition, the vessel is made up of more than one cell type, and it is possible that more "organotypic" cultures such as endothelial and smooth muscle cocultures may be required to more adequately evaluate the effect of NO on ET-1 expression and secretion. However, further studies will be required to examine these possibilities.
ET-1 is constitutively released by endothelial cells at a rate determined by the cellular or vascular milieu. Previous studies in capillary endothelial cells have shown that there is no intracellular store of the active 21-amino-acid peptide ET-1, although there is a small store of the 39-amino-acid precursor big ET-1 (25). This suggests that ET-1 secretion may be regulated predominantly at the level of prepro-ET-1 transcription. In addition, it has previously been shown that prepro-ET-1 mRNA has a short half-life in the cultured endothelial cells (<30 min; see Ref. 20). However, the transcriptional regulation of prepro-ET-1 mRNA and the role of NO in this regulation remain incompletely understood. Our data indicate that NO decreases ET-1 mRNA levels in 4-wk PAECs. Even allowing for the semiquantitative nature of our RT-PCR analysis, our data suggest that the decrease in secreted ET-1 is related to a decrease in transcription of the precursor, prepro-ET-1. The prepro-ET-1 promoter has putative binding sites for activator protein-1 (AP-1), c-myc, NF-B, and GATA-2 binding protein (6, 19, 30). Previous experiments in fibroblast cell lines have shown that c-myc at low concentrations will promote prepro-ET-1 transcription, while at higher concentrations it inhibits transcription to nearly undetectable levels (40). Furthermore, activation of the cis elements that comprise the AP-1 sequence, c-jun and c-fos, by phosphorylation and increased nuclear translocation will promote prepro-ET-1 transcription (20, 29, 31). In our current study, we have shown that activating sGC and increasing intracellular cGMP decrease ET-1 release and may decrease prepro-ET-1 mRNA. Previous studies in a neuroblastoma cell line demonstrated that increased intracellular cGMP through activating PKG significantly increases c-Myc expression and phosphorylation and increases cytosolic c-Jun. However, the increased c-Jun is not associated with an increase in phosphorylated c-Jun or nuclear translocation (2). Although these experiments were not carried out in endothelial cells, they provide evidence for a possible mechanism to explain the decreased ET-1 release seen in our system. Further studies will be needed to determine if in the pulmonary vascular endothelium increased cGMP regulates transcription of prepro-ET-1 through either an increase in phosphorylated c-myc or alteration in the activation of c-Jun.
In conclusion, our studies provide further mechanistic insight into the interaction between NO and ET-1. Moreover, our cell culture system indicates that the interactions between ET-1 and NO involve complex signaling pathways, and further studies will be required to determine how cGMP regulates ET-1 release in the pulmonary vasculature. It is our hope that, as we gain further insight into the complex interactions between NO and ET-1 in the endothelium, improved therapies for pulmonary hypertensive disorders can be developed.
![]() |
ACKNOWLEDGMENTS |
---|
GRANTS
This work was supported by grants from Forest Pharmaceutical (to L. K. Kelly) and National Heart, Lung, and Blood Institute Grants HL-67841, HL-60190, HL-72123, HD-39110, and HL-70061 (all to S. M. Black).
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|