1Departmento de Fisiología, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid; 2Instituto Reina Sofia de Investigación Nefrológicas, 28003 Madrid; and 3Sección de Nefrología, Hospital Príncipe de Asturias, Alcalá de Henares, 28871 Madrid, Spain
Submitted 18 December 2002 ; accepted in final form 11 June 2003
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ABSTRACT |
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nitric oxide; C-type natriuretic peptide; myosin light chain; cGMP-dependent protein kinase type I; endothelial cell barrier dysfunction
Several laboratories, including ours, have demonstrated that reactive oxygen species can induce endothelial cell contraction, which could be responsible for the increased endothelial permeability that often accompanies ischemia/reperfusion injuries (5, 17, 18, 20). Agents that activate guanylyl cyclases (GC) have been shown to relax smooth muscle cells; similarly, cGMP-elevating agents seem to attenuate oxidant-induced endothelial cell barrier dysfunction in some vascular beds (6, 10, 21, 24).
cGMP is a second messenger involved in many physiological processes such as smooth muscle tone, neural excitability, epithelial electrolyte transport, phototransduction in the retina, and cell proliferation (26). Despite the enormous importance of cGMP in cell physiology, little attention is given to the fact that its formation is not uniformly distributed within the cell. cGMP can be formed from GTP by the action of two distinct GCs: a soluble form (sGC) and a particulate membrane-bound form (pGC) (19), each of which is activated by different agonists. Nitric oxide (NO) and NO donors activate sGC, whereas pGC is a plasma membrane receptor for natriuretic peptides and related hormones. The pathways that control cGMP levels are complex due to the existence of several ubiquitously expressed phosphodiesterases (PDE), which hydrolyze cGMP (4). Some PDE are soluble, whereas others are plasma membrane bound. The intracellular actions of cGMP are primarily mediated by cGMP-dependent protein kinases (PKG), but several types of cyclic nucleotide-activated ion channels also appear to be involved (7, 16).
Given the separate sources of cGMP within the cell, it is possible to conceive a functional compartmentalization of cGMP due to localized elevation of these second messengers within the cell, i.e., membrane and cytosol. The purpose of this study was to examine the endothelial cell-relaxing activities of the cytosolic and particulated pools of cGMP in human endothelial cells exposed to hydrogen peroxide (H2O2) and to determine the mechanism by which cGMP inhibits contractility. We previously demonstrated that NO and natriuretic peptides are able to reverse the contraction induced by H2O2 in bovine endothelial cells, an effect that was mimicked by a cGMP analogue and mediated in part by PKG (17). The present study extends those results and addresses the hypothesis that cGMP generated via activation of soluble and particulate GCs may have differential activities in promoting endothelial cell relaxation. Our study will help to clarify how stimulation of different receptors that act via the same second messenger can elicit the appropriate functional response.
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MATERIALS AND METHODS |
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Plasmids. Flag-tagged PKG type I-regulatory region
(fcGK-I
R), which acts as a dominant negative mutant when overexpressed
in cells, was a kind gift from Dr. D. Browning
(3). pcDNA 3.1 plasmid,
obtained from Invitrogen (Carlsbad, CA), was used as a control plasmid in the
transfection experiments.
Cell culture. Human endothelial cells from umbilical vein (HUVEC) were obtained and cultured as described previously (8). Cells were seeded on dishes coated with 0.2% gelatin at 37°C in a humidified atmosphere of 95% O2-5% CO2. Individual clones were established and subcloned to obtain pure cell populations. Clones were characterized by their typical cobblestone morphology, by the presence of factor VIII-related antigen, and by the uniform uptake of fluorescent acetylated low-density lipoprotein, as described (13). Cells were fed every 2 days with E-199 medium supplemented with 20% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 20 mM HEPES, and 300 µg/ml endothelial cell growth factor. Cells were passaged when confluence was reached with trypsin-EDTA. Toxicity was evaluated in every experimental condition by the trypan blue dye exclusion method.
Measurement of planar cell surface area. Cells were grown at low density in 20-mm plates and studied before confluence. In every experiment, cells were washed twice, discarding the culture medium, and placed in buffer A (20 mM Tris, 130 mM NaCl, 5 mM KCl, 10 mM sodium acetate, 5 mM glucose, pH 7.45) containing 2.5 mM Ca2+ and maintained at room temperature. After 15 min of temperature equilibration, the experiments were started.
In the first group of experiments, the cells were preincubated with buffer, 1 µM sp-NONOate, or 0.1 µM CNP for 5 min. H2O2 (100 µM) was subsequently added to each treatment. Microphotographs were taken before H2O2 addition (time 0) and 30 min after this addition (time 30).
In the second group, cells were preincubated with buffer, 10 µM 8-Br-cGMP (cGMP analogue), sp-NONOate, or CNP. Five minutes later, H2O2 was added and the experiment was carried out as described previously.
In the third group of experiments, cells were preincubated with buffer, 1 µM ODQ (sGC inhibitor), or 1 µM Rp-cGMPs (PKG type I inhibitor) for 5 min. Then, sp-NONOate was added and cells were incubated for an additional 5 min, after which H2O2 was added. Microphotographs were taken as described previously, just before H2O2 addition and 30 min afterwards.
During each experiment, cells were observed under phase contrast with an inverted photomicroscope (Olympus IMT 2, Tokyo, Japan) with a x150 magnification. Photographs of the same cells were taken under the experimental conditions cited above. Every cell with a sharp margin suitable for the planimetric analysis was considered, and 6-12 cells were analyzed per photograph. Planar cell surface area (PCSA) was determined by computer aid planimetric techniques (17, 35). Measurements were performed by two different researchers in a blind fashion. The intraobserver and interobserver variations were 2 and 5%, respectively.
Measurement of cGMP synthesis by HUVEC. Cells were washed twice with buffer A. Cells were then preincubated in the same buffer containing 2.5 mM Ca2+ at room temperature. Reactions were started after addition of the reagents as indicated previously. At different intervals from 30 s to 30 min, the medium was aspirated and 1 ml of ice-cold ethanol was added to the plates, which were maintained at 4°C for 30 min. Cell extracts were centrifuged for 20 min at 2,000 g, the supernatant fraction was evaporated to dryness, and cGMP levels were determined with the use of a commercial [125I]cGMP radioimmunoassay kit as described (28). Protein concentration in the pellets was determined according to the Bradford method.
Measurement of myosin light chain phosphorylation. Phosphorylation of the myosin light chain (MLC) was determined after immunoprecipitation and protein separation by SDS-polyacrylamide gel electrophoresis, as reported previously (34). Briefly, after the cells were labeled with 50 µCi/ml of neutralized, carrier-free sodium [32P]orthophosphate (3 h, 37°C), incubations were performed under the conditions detailed elsewhere (see figure legends). Thereafter, the incubation medium was removed and cells were precipitated with ice-cold ethanol. After the proteins were solubilized with a pyrophosphate buffer (100 mM NaF, 8 mM sodium pyrophosphate, 250 mM NaCl, 5 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulphonyl fluoride, 50 µg/ml leupeptin, and 1% Nonidet P-40), the samples were centrifuged. The supernatants were collected and incubated with human antiplatelet myosin antibody at 4°C for 90 min, and Pansorbin was used to precipitate the immunolinked MLC. This fraction was separated by 12% SDS-polyacrylamide gel electrophoresis, and the gel was frozen and exposed to X-OMAT films. The phosphorylated MLC was identified on the autoradiographs, and the absorbance of the 20-kDa band was measured by densitometry. Results were calculated in arbitrary density units and corrected for the protein concentration in the sample.
Transient transfection experiments. HUVEC were plated at 65%
confluency on either 100-mm dishes or six-well plates. The cells were
transfected to express exogenous DNA using LipofectAMINE (Invitrogen)
according to the manufacturer's instructions. Subconfluent cell cultures were
transfected with 2 µg of the fcGK-IR plasmid, which expresses a
dominant negative form of PKG type I
. The pcDNA3.1 plasmid DNA was used
as a control. Transfection was performed for 6 h, after which regular medium
was added. Cells were treated with the corresponding reagents 24 h after
transfection.
Western blotting. HUVEC were washed briefly in PBS and solubilized in lysis buffer (10 mM Tris · HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 500 nM sodium orthovanadate, 50 nM sodium fluoride, 1 µg/ml pepstatin/leupeptin/aprotinin, 1 mM phenylmethylsulfonyl fluoride) for 40 min at 4°C. Lysates were spun down for 5 min and the supernatants were collected. Protein concentration was determined by the Bradford method. Proteins (100 µg) were separated in a 15% SDS-polyacrylamide gel overnight and transferred to a PDVF membrane (Polyscreen; Dupont). For protein detection, the membranes were incubated with a phosphomyosin light chain-specific antibody, generously supplied by Dr. J. Staddon (25), at a dilution of 1:500 at 4°C overnight. After washing, the blots were incubated with secondary antibody and ECL detection was performed using the manufacturer's instructions. The membranes were reprobed with anti-VASP and anti-P-VASP antibodies and developed as described above.
Statistical analysis. Every experimental condition was duplicated within each experiment, and each experiment was repeated at least three times. The data are expressed as means ± SE. Comparisons were made with analysis of variance followed by Dunnett's modification of the t-test whenever comparisons were made with a common control, whereas the unpaired two-tailed Student's test was used for other comparisons. The level of statistically significant difference was defined as P < 0.05.
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RESULTS |
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To examine the differential effects of cGMP on H2O2-induced endothelial cell contraction, HUVEC were preincubated for 5 min with exogenous NO from sp-NO (NO, 10-6 M), CNP (10-7 M), or vehicle in buffer A without IBMX, after which H2O2 (10-4 M) was added (time 0) and the incubation continued for another 30 min. Microphotographs were taken at times 0 and 30, and PCSA was measured. As shown in Fig. 1B, H2O2 (10-4 M, 30 min) induced a significant contraction of cultured HUVEC expressed as a reduction of the PCSA, which was abolished by preincubation with the NO donor. CNP preincubation also diminished the H2O2 contractile effect, but to a lesser extent than NO. Similar results were obtained when another NO donor (SNP, 10-6 M) was used instead of sp-NO (PCSA after SNP plus H2O2: 100.2 ± 2% of control value).
To determine whether a correlation exists between intracellular cGMP levels and the relaxing potencies of the GC agonists, we measured cGMP production under the same experimental conditions on which microphotograph experiments were carried out. To this aim, HUVEC were preincubated with NO and CNP with or without H2O2 as described above, in the absence of the PDE inhibitor IBMX. cGMP levels after NO or CNP stimulation varied both in magnitude and temporal pattern. Whereas the NO donor induced low cGMP levels that peaked at 2 min after the start of the experiment (Fig. 2A) and faded at 5 min, CNP-mediated cGMP production resulted in levels that were 10 times higher and remained elevated for at least 10 min after the start of the experiment, declining to control levels at 30 min probably due to PDE activity (Fig. 2B). H2O2 addition did not affect cGMP production induced by either NO or CNP, because cGMP produced in CNP/H2O2- and NO/H2O2-treated cells was comparable to those obtained in CNP- or NO-treated cells. cGMP determination was conducted after 30 min of NO or CNP treatment in the presence of IBMX (Fig. 2C). PDE inhibition caused a more pronounced difference between cGMP levels obtained after GC stimulation and the levels obtained via sGC, thus confirming that the decline in cGMP observed in Fig. 2, A and B, is due to PDE activity. Similar results were obtained using Zaprinast (10-6 M), a selective PDE type 5 (PDE-5) inhibitor (data not shown). In both cases, H2O2 did not have any effect on cGMP production when used in combination with CNP or NO.
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Taken together, these results show that the cGMP levels produced by CNP stimulation of pGC, which were one order of magnitude higher and remained elevated longer than the levels obtained by NO activation of sGC, did not correlate with the corresponding relaxing response elicited by this second messenger.
Recent findings indicate that H2O2 treatment of endothelial cells increases MLC phosphorylation, suggesting that endothelial contraction plays an important role in the oxidative stress-induced endothelial barrier dysfunction (18, 39). cGMP-dependent relaxation mechanisms involve MLC dephosphorylation via PKG activation (33). PKG-I is expressed in HUVEC in our experimental conditions (data not shown). To analyze whether this is the mechanism involved in our study, we labeled HUVEC with [32P]orthophosphate, as described in MATERIALS AND METHODS. H2O2 increased phosphate incorporation into MLC, an effect that was completely prevented by preincubation with NO. By contrast, preincubation with CNP was unable to significantly prevent MLC phosphorylation in response to H2O2 (Fig. 3).
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Effects of NO on endothelial cell relaxation depend on cGMP production. Because of the differences observed between NO- and CNP-relaxing potencies and cGMP production levels, we decided to test whether NO could produce its effects by means of an additional mechanism such as direct protein modification. As shown in Fig. 4, the effects of NO on PCSA were mimicked by the addition of a soluble cGMP analogue (8-Br-cGMP, 10-5 M) and were inhibited by treatment with a sGC inhibitor (ODQ, 10-6 M). In addition, a PKG-I inhibitor (Rp-cGMPs, 2.5 10-6 M) blocked the NO inhibitory effect on PCSA reduction obtained after HUVEC treatment with H2O2. This result suggests the involvement of the cGMP/PKG signaling pathway in the observed effects.
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To determine whether the NO effect on MLC phosphorylation was also dependent on the cGMP/PKG pathway, HUVEC were preincubated with the sGC antagonist (ODQ) or the PKG-I antagonist Rp-cGMPs. MLC phosphorylation levels were analyzed by [32P]orthophosphate cell labeling. As shown in Fig. 5, MLC phosphorylation induced by H2O2 was inhibited by NO and ODQ, whereas Rp-cGMPs treatment reversed the NO-induced effects. These results indicate that NO mediates HUVEC relaxation mainly through the activation of sGC followed by cGMP production and the activation of PKG.
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I isoform of PKG is involved in the differential
effects of NO/cGMP and CNP/cGMP on
H2O2-induced HUVEC
contraction. It has recently been shown that PKG-I
activates the
MLC phosphatase by phosphorylating its myosin-binding subunit, thereby
inhibiting MLC phosphorylation and contraction
(33). To clarify the mechanism
involved in the differential relaxing effect of NO-derived cGMP compared with
CNP-derived cGMP, we transfected HUVEC with a dominant negative form of PKG
type-I
(fcGK-I
R). The overexpression of this form of PKG, which
lacks the PKG catalytic subunit, is able to block cGMP-stimulated activity of
the endogenous kinase, having no basal kinase activity itself
(3).
Figure 6 shows MLC
phosphorylation of transfected cells using either fcGK-I
R or an empty
vector (pcDNA 3.1). In the pcDNA 3.1-transfected cells,
H2O2-induced MLC phosphorylation was completely
prevented by NO, whereas CNP produced only a moderate inhibition. By contrast,
fcGK-I
R transfection resulted in a complete reversal of the NO
inhibitory effect on MLC phosphorylation. However, the CNP effects were not
completely reversed by fcGK-I
R. The endogenous PKG activity of
transfected HUVEC was assessed by examining the phosphorylation status of its
vascular substrate, VASP, at serine (239)
(31).
Figure 6 shows an increase of
P-VASP levels in NO- or CNP-treated cells transfected with an empty plasmid.
By contrast, P-VASP levels are lower in cells transfected with a PKG negative
dominant construct, even in the presence of NO or CNP, which confirms the
biological activity of the transfected constructs.
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DISCUSSION |
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The concept of functional compartmentalization of second messengers is not new. One well-established example is the spatial control of [Ca2+] and cAMP signals (14, 38). However, parallels between those systems and cGMP have not been properly established. Zolle and coworkers (40) recently demonstrated that the activation of pGC inhibits Ca2+ extrusion via a plasma membrane Ca2+ ATPase, whereas the activation of sGC leads to an increase of Ca2+ uptake into the intracellular stores. The specific source of cGMP seems to be important for this effect. In our study, NO-derived cGMP produced a more intense relaxing effect than CNP-derived cGMP on endothelial cell contraction induced by H2O2. cGMP levels evoked by stimulation of sGC with NO or of pGC with CNP differ both in amplitude and duration. Whereas NO produced a typical pulse of cGMP during the first 5 min of incubation, which faded 15 min after the start of the experiment, CNP produced cGMP levels that were 10 times greater and remained elevated throughout the experiment. Despite the differences in cGMP production, there was no correlation with the observed cellular effect.
The first question that arises from this observation is whether the NO-induced effects are dependent on cGMP production. NO can act through cGMP-independent pathways to directly modify amino acid residues in several proteins and, thus, alter their function (32). In support of this hypothesis, Hart and coworkers (11) showed that NO effects on endothelial barrier dysfunction in porcine coronary artery endothelial cells stimulated by H2O2 were cGMP independent. In our study, incubation of HUVEC with inhibitors of either sGC or PKG and incubation with a cGMP analogue demonstrated that the NO-induced effects were mainly due to cGMP production rather than to a protein modification.
cGMP regulates cell responsiveness through PKG stimulation, which comprises
a major mechanism for cGMP action
(12). There are two reported
forms of PKG: a soluble type I PKG, which is expressed and present in
endothelial cells, smooth muscle cells, and neurons; and a membrane-bound type
II PKG (16). Two isoforms of
PKG type I (I and I
) are produced by alternate splicing of the
same gene and differ only in their amino terminus. Both PKG type I
and
I
isoforms are involved in the control of smooth muscle cell relaxation.
PKG type I
-dependent phosphorylation of inositol 1,4,5-triphosphate
receptor-associated G kinase (IRAG) decreases Ca2+ release from the
sarcoplasmic reticulum (1),
whereas PKG-I
phosphorylates the myosin-binding subunit of MLC
phosphatase, activating it and therefore inhibiting MLC phosphorylation and
contraction (33). The
similarities between the contractile apparatus in smooth muscle and
endothelial cells prompted us to investigate the role of the two PKG isoforms
in the differential effects of cGMP originated by the activation of the two
sets of GCs. Transfections with the dominant negative form of PKG-I
completely abrogated the responses elicited by NO on MLC phosphorylation
induced by H2O2. In this case, the responses to CNP
showed only partial inhibition. Therefore, these results suggest that
PKG-I
is involved in the transduction of NO/cGMP signaling rather than
the CNP/cGMP system. This could be simply due to a different spatial
confinement of GCs and PKG-I
. PKG-I
was originally described as
a cytosolic enzyme. However, under certain situations, it is partially
associated with the cytoskeleton
(31,
37). During oxidant injury,
there are several changes that promote endothelial cell contraction and
rearrangement of the actin cytoskeleton that compromises the endothelial
barrier function, producing tissular edema
(20). PKG-I
is able to
phosphorylate VASP, a protein member of the ENA/VASP family of proteins
involved in the regulation of the actin cytoskeleton, causing its detachment
from sites of focal adhesions. This could explain some of the effects of NO on
H2O2-induced endothelial cell contraction because focal
adhesions provide additional adhesive forces in the endothelial barrier
regulation (22).
In addition to MLC kinase, MLC phosphorylation in endothelial cells can be
induced by the Rho/Rho kinase pathway. PKG-I can phosphorylate Rho in
vitro and in vivo, causing its inhibition
(29). The role of a selective
inhibition of Rho kinase in our results awaits further investigation.
Besides the possible phosphorylation by PKG-I of different
substrates involved in cell relaxation, PKG-I
can control the level and
subcellular distribution of cGMP directly by regulating PDE activity. PDE-5 is
the major PDE that degrades cGMP, and PKG-I
can phosphorylate PDE-5
both in vivo and in vitro, activating it
(23,
27). Although the
physiological function for the phosphorylation and activation of PDE-5 has not
been analyzed in endothelial cells, a similar mechanism has been described for
platelets on NO sensitization
(30). Activation of PDE-5 may
then provide a negative feedback regulation of cGMP and PKG-I
, because
PDE-5 activation by PKG can also control the ability of PKG to phosphorylate
other substrates when the intracellular concentration of cGMP reaches a high
level, such as after CNP stimulation.
In summary, our results demonstrate that cGMP originating from separate sources within the endothelial cell plays a different role in the control of cell relaxation. Additional studies will be necessary to address whether these differences are due to a different subcellular location of the cGMP effectors, such as PKG and its substrates and cGMP degrading systems. This has potential implications for understanding the role of natriuretic peptides vs. NO in endothelial-dependent vascular relaxation and endothelial barrier function upon oxidant injury.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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.
* F. J. Rivero-Vilches and S. De Frutos contributed equally to this work.
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