AJP - Lung Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 290: L919-L930, 2006. First published December 9, 2005; doi:10.1152/ajplung.00434.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/L919    most recent
00434.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moldobaeva, A.
Right arrow Articles by Pearse, D. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moldobaeva, A.
Right arrow Articles by Pearse, D. B.

Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells

Aigul Moldobaeva,1 Laura E. Welsh-Servinsky,1 Larissa A. Shimoda,1 R. Scott Stephens,1 Alexander D. Verin,1 Rubin M. Tuder,2 and David B. Pearse1

1Division of Pulmonary and Critical Care Medicine, Department of Medicine, and 2Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Submitted 11 October 2005 ; accepted in final form 2 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)I, human pulmonary artery endothelial cells (HPAEC) lacking PKGI expression were infected with a recombinant adenovirus encoding PKGIbeta (Ad.PKG) and compared with uninfected and control-infected (Ad.betagal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H2O2 (250 µM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.betagal, expressed PKGI protein and demonstrated Ser239 and Ser157 phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.betagal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 µM) caused a PKGI-independent decrease in permeability (8.2 ± 0.6%). In all three groups, H2O2 (250 µM) caused a similar ~35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca2+ concentration ([Ca2+]i). In uninfected and Ad.betagal-infected HPAEC, pretreatment with CPT-cGMP (100 µM) partially blocked the increased permeability induced by H2O2. In Ad.PKG-infected HPAEC, CPT-cGMP (50 µM) prevented the H2O2-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca2+]i caused by H2O2 similarly (23%) in Ad.betagal- and Ad.PKG-infected HPAEC, indicating a PKGI-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKGI-independent process, whereas the ability of cGMP to prevent H2O2-induced barrier dysfunction was predominantly mediated by PKGI through a Ca2+-independent mechanism.

cAMP; pulmonary edema; acute lung injury; hydrogen peroxide

PULMONARY ENDOTHELIAL BARRIER function is an important determinant of fluid and protein movement across the pulmonary vasculature. Membrane-permeant analogs of cGMP have been shown to enhance basal pulmonary endothelial barrier function in vitro (14, 35) and protect against the barrier-disrupting effects of reactive oxygen species (ROS) both in vitro (35, 44) and in vivo (26, 34, 36, 37). The precise mechanisms behind these effects are unclear because cGMP has several intracellular targets in endothelial cells, including membrane ion channels (39, 52, 54), phosphodiesterases (10, 45), and cGMP-dependent protein kinase G (PKG), a serine/threonine kinase family member (31). Mammalian PKG exists in two forms, PKGI and PKGII, derived from separate genes (31). PKGI, which exists as two splice variants, PKGI{alpha} and PKGIbeta, is the predominant type in vascular tissue (31). Vascular smooth muscle cells express both splice variants, but the distribution of PKGI in endothelial cells is unclear (31). The PKGIbeta form was thought to predominate in rat pulmonary microvascular endothelium (9).

In systemic endothelial cells, a role for PKGI in cGMP-mediated barrier function enhancement has been suggested based on a correlation between the effectiveness of cGMP and the presence of PKGI protein expression across several endothelial monolayers of different origin (11) or the inhibitory effects of Rp-8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-CPT-cGMPS), a PKG antagonist (51). The exact contribution of PKGI remains unclear, however, because a cause-and-effect relationship was not established within a single endothelial cell type (11) and micromolar concentrations of Rp-CPT-cGMPS can also block cAMP-dependent protein kinase A (PKA; Ref. 43).

Less is known about the role of PKGI in pulmonary endothelium. Membrane-permeant analogs of cGMP had little effect on permeability in porcine (15) or human (HPAEC) (49) pulmonary artery endothelial cells, but the presence of PKGI expression was not examined. Rat (9) and bovine (35) pulmonary endothelial cells have been shown to express PKGI, but the role of PKG in cGMP-mediated protection of ROS-induced pulmonary endothelial barrier dysfunction was not specifically examined.

The goal of the present study was to determine the contribution of PKGI to the barrier-enhancing properties of cGMP analogs in pulmonary endothelial cells. Defining the predominant downstream target of cGMP responsible for this effect will help determine the molecular mechanisms involved and possibly explain the variable response of cGMP-elevating agents on endothelial permeability (15, 47, 49). In preliminary experiments, we determined that commercially acquired HPAEC did not express PKGI. In contrast, human lung microvascular endothelial cells (HLMVEC) demonstrated PKGI expression that varied by lot. Although pulmonary capillary endothelial cells are potentially more relevant to mechanisms of lung edema, the variable PKGI expression found in cultured HLMVEC made them more difficult to study. Moreover, we previously found (35) that bovine pulmonary vascular conduit and capillary endothelial cells both expressed PKGI and responded in a similar manner to 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP), suggesting the presence of similar downstream pathways. In the current study, we therefore compared the effects of cGMP treatment on HPAEC before and after adenovirus-mediated gene transfer of PKGI. We report that the barrier-enhancing effects of cGMP in HPAEC before H2O2 exposure are PKGI independent, whereas the ability of cGMP to attenuate H2O2-mediated barrier dysfunction is largely mediated by activation of PKGI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell Culture Conditions and Reagents

HPAEC (Clonetics, Walkersville, MD; Cell Applications, San Diego, CA) were obtained at third passage and studied between passages 4 and 5. Human fetal pulmonary artery endothelial cells (HFPAEC) were obtained at second passage (ScienCell Research Laboratories, San Diego, CA) and studied between passages 3 and 5. All experiments were performed in at least three different cell lots. Endothelial cell culture basal medium (EBM-2) with growth supplements was obtained from Clonetics. Cells were cultured in completed medium containing 2% FBS, endothelial growth supplement, antibiotics, and antifungals (penicillin 10,000 U/ml, streptomycin 10 µg/ml, and amphotericin B 25 µg/ml). Endothelial cells were incubated at 37°C in a humidified atmosphere in a 5% CO2 air incubator and grown to monolayers with typical cobblestone morphology. Cells from each primary flask were detached with 0.05% trypsin, resuspended in fresh culture medium, and passaged into eight-well electrical cell-substrate impedance sensing system (ECIS) electrode arrays for electrical resistance determinations or 12-well plates (on 18-mm no. 1D coverslips; Fisher Scientific) for fluorescence microscopy. Nonstabilized 30% H2O2 was purchased from Fisher Chemical. CPT-cGMP and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) were purchased from Biolog Life Science Institute (La Jolla, CA). All agents were diluted in DMEM (GIBCO BRL) to achieve the desired concentration. Texas red-X phalloidin was purchased from Molecular Probes (Eugene, OR). The following antibodies were commercially obtained: anti-PKGI antibody (Stressgen Bioreagents, Victoria, BC, Canada), anti-vasodilator-stimulated phosphoprotein (VASP) and anti-Pser239-VASP (Alexis), anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG antibody (Stratagene), and anti-VE-cadherin antibody (BD Biosciences-Transduction Laboratories, Lexington, KY).

Adenoviral Infection

Recombinant adenoviruses containing the full-length human cDNA for PKGIbeta with an amino-terminal FLAG epitope (4), with or without an additional expression cassette for green fluorescent protein (GFP) (17), were the generous gift of Dr. K. D. Bloch (Harvard University, Boston, MA) along with a similarly prepared control adenovirus containing the cDNA for beta-galactosidase and GFP. An additional control Ad.betagal-FLAG without GFP was the generous gift of Dr. Michael Crowe (Johns Hopkins University, Baltimore, MD). The viral stock solutions were as follows: Ad.PKG-FLAG-GFP, 1.7 x 1011 plaque-forming units (PFU)/ml; Ad.betagal-FLAG-GFP, 5.0 x 109 PFU/ml; Ad.PKG-FLAG, 3 x 1010 PFU/ml; Ad.betagal-FLAG, 1 x 1012 PFU/ml.

Permeability. Cells were grown to 80% confluence on gelatin-coated microelectrode plates as previously described (35). Medium was removed, and 200 µl of fresh, complete medium containing 1 µl/ml stock virus was added and incubated for 60 min at 37°C with 5% CO2. After 60 min, 200 µl of complete medium was added to each well, bringing the total volume up to 400 µl. Cells were incubated overnight at 37°C with 5% CO2. Ad.PKG-FLAG-GFP and Ad.betaGal-FLAG-GFP viruses were used in the permeability experiments, and infection efficiency was determined by fluorescent microscopy.

Intracellular Ca2+ concentration measurements. Cells were grown on 25-mm coverslips coated with 1% gelatin-PBS to 5% confluence. Medium was replaced with addition of 1 ml of fresh, complete medium containing 1 µl/ml of stock virus to wells. The cells were incubated for 60 min at 37°C with 5% CO2. After 60 min, 1 ml of complete medium was added to each dish, bringing the volume up to 2 ml, and incubated overnight at 37°C with 5% CO2. Ad.PKG-FLAG and Ad.betagal-FLAG viruses were used for the calcium experiments because GFP appeared to interfere with the measurement of intracellular Ca2+ concentration ([Ca2+]i). The infection efficiency was determined by immunofluorescent staining of anti-FLAG antibody (Stratagene) and anti-PKG antibody (Stressgen).

Measurement of Transendothelial Cell Electrical Resistance

Measurement of transendothelial resistance (TER) was performed with ECIS (Applied BioPhysics, Troy, NY) as previously described (35). Briefly, HPAEC were grown to confluence on gold microelectrodes in series with a larger gold counter electrode connected to an amplifier. Current was applied across the electrodes by a 4,000-Hz AC voltage source with amplitude of 1 V in series with a resistance of 1 M{Omega} to approximate a constant current source (~1 µA). The in-phase and out-of-phase voltages between the electrodes were monitored and converted to TER. TER was monitored for 30 min to establish a baseline resistance. Resistance data were normalized to the initial baseline resistance measured just before the addition of the first drug and plotted as a normalized TER. The effects of CPT-cGMP (1, 10, 50, and 100 µM), H2O2 (250 µM), and the combination of CPT-cGMP pretreatment followed by H2O2 were examined in uninfected HPAEC and in HPAEC infected with Ad.PKG or Ad.betagal. In parallel experiments, the effect of CPT-cAMP (1, 10, 50, and 100 µM) on basal TER was compared with CPT-cGMP to control for the effects of viral infection and expression of GFP.

Endothelial Cell Fractionation

Endothelial cells were fractionated into detergent-soluble and -insoluble fractions as previously described (48).

Gel Electrophoresis and Immunoblot Analysis

The immunoblots for PKG, VASP, Pser239-VASP, and PKA were performed as previously described (35). Briefly, total homogenates of cell lysates (200 µl) were solubilized in 1% beta-mercaptoethanol and subjected to SDS-PAGE on an 8% gel. The separated proteins were transferred to nitrocellulose membrane (BA-S 83, 0.2 µm), and the membrane was probed with primary antibody at a 1:1,000 dilution. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Bio-Rad) used at 1:3,000 dilution. Bands were visualized with enhanced chemiluminescence reagents (Amersham) and exposure of the blot to X-ray film (BioMax MR, Kodak). The gels were then stripped and reprobed for actin to confirm equal loading. Densitometric quantification was performed with Un-Scan-It Gel Automated Digitizing System software, version 5.1 (Silk Scientific, Orem, UT).

Fluorescence Microscopy

Endothelial cells grown on gelatinized coverslips were incubated with H2O2 (250 µM) for 20 min with or without a 30-min preincubation with 50 or 100 µM CPT-cGMP. The cells were then fixed in 4% paraformaldehyde for 10 min, washed three times with Tris-buffered saline solution containing 0.1% Tween 20 (TBS-T, Sigma), permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked with 2% BSA in PBS for 30 min. Actin filaments were visualized by staining cells with Texas red-conjugated phalloidin for 1 h at room temperature. A second 1-h incubation containing VE-cadherin antibody (BD Biosciences-Transduction Laboratories) at a 1:200 dilution was completed. After three washes with TBS-T, the coverslips were mounted with a Slow Fade kit (Molecular Probes). Images were acquired with an Olympus IX51 inverted fluorescent microscope, using a x60 oil objective and a Cooke digital camera.

Measurement of [Ca2+]i

Coverslips containing cells were placed in a laminar flow cell chamber perfused with Krebs solution [Krebs Ringer bicarbonate (KRB)] containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.1 glucose, and 1.2 KH2PO4, gassed with 16% O2, 5% CO2, balance N2 to maintain a pH of 7.4. [Ca2+]i was measured in cells incubated with 5 µM fura-2 AM, a membrane-permeant (acetoxymethyl ester) form of FURA-2, for 60 min at 37°C under an atmosphere of 21% O2-5% CO2. The cells were washed with KRB for 15 min at 37°C to remove extracellular dye and allow complete deesterification of cytosolic dye. Ratiometric measurement of fluorescence from fura-2 was performed on a workstation from Intracellular Imaging (Cincinnati, OH) containing a fluorescence microscope (Nikon TS 100), an excitation filter changer, and an imaging system. The collimated light beam from a 75-W xenon arc lamp was filtered by interference filters and focused onto the cells under examination with a x20 fluorescence objective (Super Fluor 20, Nikon). Measurement of [Ca2+]i was achieved by rapidly alternating between 340- and 380-nm excitation wavelengths and measuring the resulting 510-nm emission from fura-2 with a cooled charge-coupled device camera. The protocols were executed and data analyzed online with the InCyt software package (Intracellular Instruments). Images were collected every 12 s, and background fluorescence was subtracted. [Ca2+]i was estimated from fluorescence ratio data by linear regression using a calibration curve generated from calibration solutions with [Ca2+] ranging from 0 to 610 nM (Molecular Probes). Baseline [Ca2+]i was defined as the value measured just before adding CPT-cGMP or H2O2. Peak [Ca2+]i was the highest value after H2O2 administration.

Lung Immunohistochemistry

Human lung sections from pathological specimens obtained from three patients undergoing lung resection for cancer were examined for PKGI expression after approval by the Johns Hopkins Medicine Institutional Review Board. To detect PKGI expression, lung sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol. Antigen retrieval was performed with Dako citrate retrieval buffer (DakoCytomation, Carpinteria, CA), followed by a 10-min exposure to 1% H2O2 to quench endogenous peroxides. Avidin and biotin blocking were accomplished with commercially available blocking solutions (Vector Laboratories, Burlingame, CA). Nonspecific binding was blocked with normal goat serum for 60 min at room temperature. Immunostaining of PKGI was performed with rabbit anti-PKGI antibody (Stressgen Bioreagents) at 1:800 dilution. Staining was detected with biotinylated goat anti-rabbit secondary antibody at 1:400 dilution followed by application of Vectastain elite avidin-biotin complex reagent and diaminobenzidine tetrahydrochloride with NiCl (Vector Laboratories, Burlingame, CA).

Statistics

The effects of cyclic nucleotides and H2O2 on the time course of TER and [Ca2+]i were analyzed by two-way split-plot ANOVA. The effect of CPT-cGMP and H2O2 on VASP phosphorylation was analyzed by randomized two-way ANOVA. The effect of viral infection on baseline TER was analyzed by a randomized one-way ANOVA. When significant variance ratios were obtained, least significant differences were calculated to allow comparison of individual means. Values presented in the text are means ± SE. Differences were considered significant when P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PKGI Expression in Cultured HPAEC, HFPAEC, and Adult Human Lung

Figure 1A, top, shows an immunoblot of HPAEC and HFPAEC whole cell homogenates probed with antibody to PKGI. No PKGI expression was detected in any of the lots of HPAEC obtained from either vendor. One of the known targets of activated PKGI in endothelial cells is VASP, a protein found in both focal adhesions and intercellular junctions (11). PKGI preferentially phosphorylates VASP Ser239, whereas PKA prefers Ser157, although neither site is exclusively linked to a single kinase (42). Thus the observation that CPT-cGMP pretreatment did not result in phosphorylation of VASP at Ser239 (Fig. 1A, bottom) was further confirmatory evidence for the absence of functional PKGI (28). In contrast, analysis of third-passage HFPAEC demonstrated an 80-kDa PKGI-immunoreactive protein (Fig. 1A, top) and the presence of Pser239-VASP after activation with CPT-cGMP (Fig. 1A, bottom), confirming the presence of PKGI expression and activity, respectively. Interestingly, both expression and activity of PKG were reproducibly lost in HFPAEC with the fourth passage.


Figure 1
View larger version (60K):
[in this window]
[in a new window]
  		Fig. 1. A: Western blots of cGMP-dependent protein kinase (PKG)I and Pser239-vasodilator-stimulated phosphoprotein (VASP) in adult human pulmonary artery endothelial cells (HPAEC) from 2 different commercial sources (lanes 1–3, 4th passage, Cell Applications; lanes 4–6, 5th passage, Clonetics) and human fetal pulmonary artery endothelial cells (HFPAEC) (lanes 7–9, 3rd passage; lanes 10–12, 4th passage) after a 20-min treatment with diluent (Control), 100 µM 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP) (cGMP), or 250 µM H2O2. Blots are representative of 3 experiments in separate lots of HPAEC and HFPAEC. B: PKGI immunohistochemistry in adult human lung with longitudinal section of conduit pulmonary artery (black arrow, PKGI staining in endothelium. C: human lung processed with nonspecific IgG antibody shown for comparison.

 
To confirm that the absence of PKG expression in HPAEC was a cell culture-dependent phenomenon similar to that in HFPAEC and observations in a variety of other cell types in culture (4, 28), sections of intact adult human lungs were examined for endothelial expression of PKGI. Figure 1B shows a conduit pulmonary artery cut in cross section with evidence of PKGI expression in both endothelium and smooth muscle. PKGI staining was present in conduit pulmonary arteries of all sizes as well as pulmonary capillary endothelial cells (not shown). Lung tissue stained with a control primary antibody was negative for PKGI-immunoreactive protein staining (Fig. 1C).

Effect of Ad.PKG and Ad.betagal Infection on PKGI Expression and Function in HPAEC

The infection efficiency of Ad.PKG and Ad.betagal in HPAEC was >90%. Ad.PKG infection of HPAEC resulted in the expression of PKGI (Fig. 2, top). Most of this expression was found in the soluble fraction. There was no detectable PKGI expression in the HPAEC infected with Ad.betagal in either cellular fraction. The expression of PKGI was accompanied by significant phosphorylation of VASP Ser 239 and Ser157 after treatment with CPT-cGMP (Figs. 2 and 3). For example, 50 µM CPT-cGMP increased Pser239-VASP by 1.9 ± 0.4-fold and Pser157-VASP by 2.4 ± 0.9-fold in Ad.PKG-infected HPAEC (P < 0.05). In contrast, CPT-cGMP had no effect on VASP phosphorylation in the Ad.betagal-infected cells at either drug concentration (Fig. 3). H2O2 alone had no effect on Pser239-VASP but significantly increased Pser157-VASP in Ad.PKG-infected cells compared with control but not compared with H2O2-treated Ad.betagal-infected cells.


Figure 2
View larger version (42K):
[in this window]
[in a new window]
  		Fig. 2. Western blots of PKGI, Pser239-VASP, Pser157-VASP, and VASP in HPAEC infected with Ad.betagal or Ad.PKG and exposed to 250 µM H2O2 with and without pretreatment with either 50 or 100 µM CPT-cGMP (cGMP). HPAEC were separated into soluble and insoluble fractions.

 

Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3. Effect of 50 or 100 µM CPT-cGMP (cGMP), 250 µM H2O2, and the combination of CPT-cGMP and H2O2 on expression of Pser239-VASP (A) and Pser157-VASP (B) in Ad.betagal- or Ad.PKG-infected HPAEC. Values are means ± SE of fold increases over control determined from densitometry of immunoblots (n = 4–5 separate experiments). *P < 0.05 compared with Ad.betagal by ANOVA interaction. #P < 0.05 compared with control.

 
Effect of PKGI Expression and Activation Before and After H2O2 Exposure on HPAEC Monolayer TER

Adenoviral infection caused a significant increase in basal TER measured before the addition of cyclic nucleotide analogs or H2O2 compared with uninfected HPAEC (Fig. 4). There was no difference between Ad.PKG and Ad.betagal. Figure 5, A, C, and E, shows the time course of normalized TER after administration of increasing concentrations of CPT-cGMP in uninfected, Ad.betagal-infected, and Ad.PKG-infected HPAEC. All three cell groups responded in a similar fashion, with a significant increase in TER after 50 or 100 µM CPT-cGMP. For example, the maximum percent change in TER caused by 100 µM CPT-cGMP in uninfected, Ad.betagal-infected, and Ad.PKG-infected HPAEC averaged 7.9 ± 1.8%, 7.3 ± 2.4%, and 9.3 ± 1.3%, respectively. Administration of the same concentrations of CPT-cAMP (Fig. 5, B, D, and F) resulted in larger increases in TER compared with CPT-cGMP (100 µM CPT-cAMP increased TER by 23 ± 2.4%), with a statistically significant increase after every concentration >1 µM CPT-cAMP. Similar to the results for CPT-cGMP, there were no differences in response across the three groups of HPAEC.


Figure 4
View larger version (24K):
[in this window]
[in a new window]
  		Fig. 4. Comparison of transendothelial resistance (TER) in uninfected HPAEC and HPAEC monolayers infected with Ad.betagal or Ad.PKG (n = 72–93 monolayers). Values are means ± SE. *P < 0.0001 compared with uninfected HPAEC.

 

Figure 5
View larger version (45K):
[in this window]
[in a new window]
  		Fig. 5. Time course of normalized TER after treatment with increasing concentrations of CPT-cGMP (A, C, and E) or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; B, D, and F) in uninfected HPAEC (A and B), HPAEC infected with Ad.betagal (C and D), and HPAEC infected with Ad.PKG (E and F) (n = 3–9 monolayers per treatment group). Values are means ± SE. *P < 0.03 compared with corresponding diluent control group by ANOVA interaction. Time-course comparisons of the same drug treatment between uninfected, Ad.betagal-infected, and Ad.PKG-infected HPAEC were not significant (P > 0.05).

 
H2O2 caused a reversible decrease in TER in uninfected, Ad.betagal-infected, and Ad.PKG-infected HPAEC (Fig. 6). Pretreatment with 100 µM CPT-cGMP resulted in a small but significant 30% attenuation of the H2O2 response in uninfected HPAEC (P < 0.05); 50 µM CPT-cGMP had no significant effect. The results in the Ad.betagal-infected HPAEC were similar in that 100 µM CPT-cGMP caused a statistically significant 33% inhibition of the H2O2-induced decrease in TER (P < 0.05). In both of these treatment groups, however, the HPAEC monolayers pretreated with CPT-cGMP still had H2O2-induced decreases in TER that were significantly greater than those with the diluent control. As shown in Fig. 6C, however, the decrease in TER caused by H2O2 in the HPAEC expressing PKG was completely prevented by pretreatment with either concentration of CPT-cGMP (P = 0.01).


Figure 6
View larger version (32K):
[in this window]
[in a new window]
  		Fig. 6. Effect of H2O2 (250 µM) with and without CPT-cGMP (50 or 100 µM) pretreatment on the time course of normalized TER in uninfected HPAEC (A), HPAEC infected with Ad.betagal (B), or HPAEC infected with Ad.PKG (C). TER is normalized to time point just before administration of CPT-cGMP or cGMP diluent (n = 3–6 monolayers per group). Values are means ± SE. *P < 0.05 vs. corresponding diluent control by ANOVA interaction. #P < 0.05 vs. corresponding value in H2O2-treated group. +P < 0.05 vs. all other treatment groups.

 
Effect of PKGI Expression on H2O2-Induced Actin Cytoskeletal Rearrangement and Junctional VE-Cadherin in HPAEC.

In Ad.betagal-infected HPAEC (Fig. 7, A–D), H2O2 caused cell elongation and increased F-actin stress fiber formation, intercellular gaps, and loss of intercellular VE-cadherin staining (Fig. 7B) compared with diluent-treated HPAEC (Fig. 7A). Pretreatment with either concentration of CPT-cGMP in Ad.betagal-infected HPAEC had little effect on these H2O2-induced changes (Fig. 7, C and D). H2O2 treatment also caused actin cytoskeletal rearrangement with intercellular gap formation and focal loss of VE-cadherin staining in Ad.PKG-infected HPAEC (Fig. 7F). Unlike the Ad.betagal-infected HPAEC, however, pretreatment of Ad.PKG-infected HPAEC with either concentration of CPT-cGMP attenuated the H2O2-induced cytoskeletal changes. Specifically, the Ad.PKG-infected HPAEC maintained their cobblestone shape, dense peripheral actin band with less actin stress fiber formation, and continuous junctional VE-cadherin staining (Fig. 7, G and H) despite H2O2 exposure.


Figure 7
View larger version (108K):
[in this window]
[in a new window]
  		Fig. 7. Composite immunofluorescent images of HPAEC monolayers infected with Ad.betagal (A–D) or Ad.PKG (E–H) and treated with diluent (Control; A and E), 250 µM H2O2 (B and F), 50 µM CPT-cGMP (cGMP) + 250 µM H2O2 (C and G), and 100 µM CPT-cGMP + 250 µM H2O2 (D and H). Adenoviral infection is signified by production of GFP. The F-actin cytoskeleton is stained with Texas red-conjugated phalloidin. VE-cadherin staining is in blue. Magnified views of areas encompassed by white rectangles highlight the status of VE-cadherin staining in intercellular junctions. Representative images from 3 separate experiments are shown.

 
Effect of PKGI Expression on H2O2-Induced Changes in [Ca2+]i

As shown in Fig. 8, B, D, and F, and summarized in Fig. 9A, CPT-cGMP had no significant effect on baseline [Ca2+]i in any of the three groups of HPAEC. H2O2 alone produced a rapid increase in [Ca2+]i in uninfected HPAEC from 119 ± 9 nM to a peak of 192 ± 15 nM followed by a decline to a plateau of 148 ± 13 nM (Figs. 8A and 9B). Pretreatment with CPT-cGMP had no significant effect on the peak [Ca2+]i after H2O2 or the final [Ca2+]i before H2O2 was removed (Figs. 8B and 9B).


Figure 8
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 8. Time course of mean intracellular Ca2+ concentration ([Ca2+]i) in uninfected HPAEC (n = 35–55 cells; A and B) and in HPAEC infected with Ad.betagal (n = 84 cells; C and D) or Ad.PKG (n = 93–97 cells; E and F). Bars indicate exposure time to reagents.

 

Figure 9
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 9. A: effect of CPT-cGMP (50 µM) pretreatment on the basal [Ca2+]i in uninfected (n = 35 cells), Ad.betagal-infected (n = 84 cells) or Ad.PKG-infected (n = 93 cells) HPAEC. B: effect of CPT-cGMP (50 µM) pretreatment on the peak and plateau [Ca2+]i after exposure to 250 µM H2O2 in uninfected HPAEC (n = 35–55 cells). C and D: effect of CPT-cGMP (50 µM) pretreatment on the peak [Ca2+]i after exposure to 250 µM H2O2 in HPAEC infected with Ad.betagal (n = 84 cells; C) or Ad.PKG (n = 93–97 cells; D). Values are means ± SE. *P < 0.05 vs. corresponding value by ANOVA interaction.

 
Ad.betagal infection enhanced the effect of H2O2 on [Ca2+]i compared with uninfected HPAEC (P < 0.05), resulting in a peak [Ca2+]i of 327 ± 18 nM followed by a decrease to 214 ± 8 nM measured just before H2O2 washout (Figs. 8C and 9C). Unlike uninfected HPAEC, pretreatment with CPT-cGMP in Ad.betagal-infected HPAEC (Figs. 8D and 9C) significantly decreased the H2O2-induced [Ca2+]i peak compared with H2O2 alone (257 ± 12 vs. 327 ± 17 nM, respectively) but had no effect on the sustained phase of [Ca2+]i after H2O2.

The results in Ad.PKG-infected HPAEC were identical to those found in the Ad.betagal-infected HPAEC. Specifically, both the magnitude of the peak [Ca2+]i after H2O2 (Figs. 8E and 9D) and the ~23% attenuating effect of CPT-cGMP pretreatment on the peak but not the plateau [Ca2+]i (Figs. 8F and 9D) were not statistically different from the Ad.betagal-infected HPAEC results.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A large body of evidence demonstrates that generation of endogenous endothelial cGMP or administration of membrane-permeant cGMP analogs attenuates the endothelial barrier dysfunction caused by ROS in the systemic circulation (2, 3, 20, 23) and in the intact lung (26, 34, 36, 37). Consistent with the results in intact lungs, we (35) recently found that CPT-cGMP decreased basal permeability (increased TER) and eliminated the increased permeability (decreased TER) caused by H2O2 in bovine lung pulmonary artery and microvascular endothelial monolayers. The cGMP-induced protective effects on endothelial barrier function were accompanied by inhibition of actin cytoskeletal rearrangement and attenuation of both peak and plateau increases in [Ca2+]i after H2O2 (35). Although we demonstrated that both bovine lung endothelial subtypes expressed PKGI, the role of PKGI as a mediator of the cGMP-mediated protection was not determined in that study.

Other investigators have shown cGMP-mediated barrier-enhancing effects in porcine (18, 44), bovine (27, 33), and human (11) aortic and human brain (51) endothelial cell monolayers. In contrast, Westendorp et al. (49) and Gupta and coworkers (15) found that 8-bromoguanosine 3',5'-cyclic monophosphate (1 mM) had no effect on basal endothelial permeability in human umbilical vein endothelial cells (HUVEC) (49), HPAEC (49), or porcine pulmonary artery endothelial cells (15) and little to no effect on the increased permeability following thrombin (49) or H2O2 (15). Draijer et al. (11) confirmed the lack of a cGMP-mediated effect in HUVEC and reported that neither cultured nor primarily isolated HUVEC expressed PKGI. Given the results of Draijer et al. (11), we wondered whether the previous negative results in HPAEC (49) could also be due to the absence of PKGI expression.

Consistent with this possibility, HPAEC obtained at the fourth or fifth passage in culture failed to express PKGI or demonstrate VASP phosphorylation after exposure to CPT-cGMP (Fig. 1A). In contrast, HFPAEC obtained at an earlier passage expressed PKGI protein and activity, as shown by VASP Ser239 phosphorylation after activation with CPT-cGMP, but this capacity was lost at the fourth passage in culture (Fig. 1A). In vitro loss of PKGI expression is a well-known phenomenon in cultured pulmonary artery smooth muscle cells (4, 8), T lymphocytes (13), and aortic endothelial cells (11). The presence of pulmonary artery endothelial PKGI staining in adult human lung (Fig. 1B) suggests that in situ HPAEC normally express PKGI. The mechanism behind the loss of PKGI expression in cultured cells is unknown.

To examine the role of PKGI as the downstream mediator of the barrier-enhancing effects of cGMP in pulmonary endothelial cells, we restored PKGI expression in HPAEC with adenoviral gene transfer of PKGIbeta and compared the results to both uninfected and control-infected HPAEC. As shown in Fig. 2, the HPAEC infected with the control adenovirus Ad.betagal did not express PKGI or demonstrate Pser239-VASP after treatment with CPT-cGMP in either soluble or insoluble cell fractions. We examined both fractions because PKGI was shown to bind to cytoskeletal proteins in aortic endothelial cells (30) and neutrophils (53). In contrast, HPAEC infected with Ad.PKG exhibited significant VASP phosphorylation at both Ser239 and Ser157 after activation with either concentration of CPT-cGMP. In some systems, cGMP has been shown to signal through PKA either by inhibition of phosphodiesterase 3 and increased cAMP or direct activation of PKA by cGMP (28). Comparison of VASP phosphorylation by densitometric analysis in Fig. 3 ruled this out and confirmed the specificity of ≤100 µM CPT-cGMP for PKGI activation in HPAEC because there was no effect of CPT-cGMP in Ad.betagal-infected cells.

Treatment with H2O2 alone had no significant effect on Pser239-VASP content in either group, whereas Pser157-VASP was increased after H2O2 alone in the Ad.PKG-infected HPAEC compared with their baseline level of phosphorylation (Fig. 3B). There was also a trend for a H2O2-induced increase in Pser157-VASP in Ad.betagal-infected HPAEC, although this did not reach statistical significance. These results are potentially consistent with recent data showing PKC-mediated activation of PKGI (19) as well as a direct PKC-mediated phosphorylation of VASP Ser157 (5). H2O2 is known to cause PKC activation in pulmonary artery endothelial cells (40). A direct PKC-mediated phosphorylation of VASP Ser157 (5) may explain the small but consistent increase in Pser157-VASP in H2O2-exposed Ad.betagal-infected cells, whereas the combination of both effects may have resulted in the statistically significant increase in Pser157-VASP in Ad.PKG-infected cells after H2O2. Additional experiments in the presence of PKC inhibition will be necessary to confirm this possibility.

We were surprised to find that adenoviral infection caused a significant increase in baseline TER compared with uninfected HPAEC that did not differ between Ad.betagal- and Ad.PKG-infected cells (Fig. 4). Ad.betagal infection of human dermal microvascular endothelial cells caused a dose-dependent trend toward barrier enhancement (29), but control adenoviral infection in porcine (50) or bovine (6) pulmonary artery cells had no effect on baseline permeability. The mechanism of this effect in adenovirus-infected HPAEC is unknown but underscores the need to include a control adenoviral infection when interpreting the effect of gene transfer on endothelial barrier function.

We (35) and others (14, 18, 51) previously reported a cGMP-induced decrease in basal endothelial monolayer permeability. This effect does not appear to be mediated through PKGI, given the similar increase in TER from increasing CPT-cGMP concentrations across the three groups of HPAEC (Fig. 5). Given that adenoviral infection caused a nonspecific increase in baseline TER, we wondered whether the infection or the expression of GFP could have limited the ability of monolayer permeability to maximally respond to a barrier-enhancing stimulus. To address this possibility, additional control experiments were carried out with the membrane-permeant analog of cAMP, CPT-cAMP. Exposure to the same concentration range of the cAMP analog caused a significantly greater increase in TER compared with CPT-cGMP that again was not statistically different across the three cell groups (Fig. 5, B, D, and F). These results indicate that the increase in TER from CPT-cGMP was not artifactually limited by an unrecognized effect of adenovirus infection or GFP expression.

The mechanism of the PKGI-independent increase in TER from cGMP is unknown. cGMP can directly interact with cyclic nucleotide-gated ion channels in endothelial cells (52), which could alter barrier function through changes in membrane potential or basal [Ca2+]i. We previously found (39) that cGMP caused a PKGI-independent inhibition of an inwardly rectifying K+ channel in bovine pulmonary endothelial cells that resulted in a depolarization of the resting membrane potential. The specific channel that may be involved in the effect of cGMP on baseline permeability is unknown, but a change in basal [Ca2+]i did not appear to be involved (Fig. 9A). A similar cGMP-mediated, calcium-independent decrease in basal permeability was previously reported in aortic endothelial monolayers (18).

Unlike the effect of CPT-cGMP on baseline permeability, the protective effect of CPT-cGMP on the barrier disruption caused by H2O2 was a combination of both PKGI-dependent and -independent actions. The PKGI-independent effect was observed in the uninfected and Ad.betagal-infected HPAEC monolayers because 100 µM CPT-cGMP produced a small but statistically significant attenuation of the decrease in TER caused by H2O2 (Fig. 6, A and B). Either CPT-cGMP concentration, however, completely eliminated the H2O2-induced decrease in TER in Ad.PKG-infected cells, suggesting potent barrier protection mediated by the presence of activated PKGI (Fig. 6C). The magnitude of this cGMP-mediated protective effect is similar to our previous observations (35) in bovine pulmonary artery and lung microvascular endothelial cells, which, unlike HPAEC, retained the ability to express PKGI in culture.

The PKGI-dependent endothelial barrier protection was manifest under the microscope by a marked attenuation of the actin cytoskeletal rearrangement, intercellular gapping, and loss of junctional VE-cadherin staining caused by H2O2 (Fig. 7). Similar to the TER results, maintenance of near-normal cellular morphology after H2O2 exposure was achieved at the lower CPT-cGMP concentration (Fig. 7G). Neither CPT-cGMP concentration affected the adherens junction disruption caused by H2O2 in Ad.betagal-infected HPAEC (Fig. 7, B–D). Internalization of junctional VE-cadherin is well known to occur in association with ROS-mediated endothelial barrier dysfunction (1, 21, 22, 46). Although the precise mechanism is unclear, recent evidence suggests that H2O2-mediated activation of focal adhesion kinase (46) may cause excessive protein tyrosine phosphorylation of key junctional and focal adhesion proteins including paxillin, beta-catenin, and VE-cadherin (22, 46). As a result, VE-cadherin undergoes internalization and cell-cell adhesion is disrupted.

The mechanism behind the PKGI-dependent protective effects on ROS-mediated endothelial barrier dysfunction and cytoskeletal rearrangement is unknown because little is known about the downstream protein targets of endothelial PKGI. Diwan et al. (9) found six proteins phosphorylated by PKGI in rat lung microvascular endothelial cells but only identified one, the cytoskeletal protein VASP. VASP is located in cell-cell junctions and cell-matrix focal adhesions connecting junction proteins to cytoskeletal F-actin microfilaments, but its function is largely unknown. Unphosphorylated VASP causes polymerization and bundling of F-actin filaments, leading to F-actin stress fiber formation and cell membrane ruffling (38). Phosphorylation of VASP by PKGI inhibits actin binding and reverses these effects (16). VASP phosphorylation has been suggested as a possible mechanism behind the endothelial barrier-enhancing effects of PKGI (11) and PKA (7).

Other known targets of interest in endothelial cells that could modulate barrier function include nitric oxide synthase and 6-pyruvoyltetrahydropterin synthase (31). Both are stimulated by PKGI, the former to generate NO and the latter tetrahydrobiopterin, an essential cofactor for the generation of NO by nitric oxide synthase. Endothelial PKGI has also been shown to inactivate a store-operated Ca2+ channel (24, 25) that could play a role in ROS-mediated barrier dysfunction (32). Extensive work in smooth muscle cells (31) has identified other PKGI targets that serve to inhibit increases in [Ca2+]i from inositol 1,4,5-trisphosphate receptor-coupled stores and decrease sensitivity to Ca2+ (such as inhibition of Rho kinase through phosphorylation of RhoA) that could apply to endothelial barrier function.

As shown in Fig. 8, and previously by others (41), one of the earliest events in pulmonary endothelial cells after H2O2 exposure was an increase in [Ca2+]i. The H2O2-induced increase in [Ca2+]i occurred from a combination of internal Ca2+ store release followed by extracellular Ca2+ influx down a membrane potential-dependent electrochemical gradient (41). The increased [Ca2+]i was required for H2O2-induced barrier dysfunction and cytoskeletal rearrangement in bovine lung microvascular endothelial cells (40, 41).

We previously showed (35) in bovine pulmonary artery and lung microvascular endothelial cells that CPT-cGMP markedly attenuated the peak and plateau [Ca2+]i caused by H2O2 but did not determine whether these effects were mediated through PKGI. The effects of CPT-cGMP on the changes in [Ca2+]i caused by H2O2 in the current study are much less significant, being limited to an inhibition of peak [Ca2+]i in the adenovirus-infected HPAEC without any effect on basal or plateau [Ca2+]i (Figs. 8 and 9). For unclear reasons, there was no effect of CPT-cGMP in the uninfected HPAEC, again suggesting that adenoviral infection had an effect on membrane function independent of the gene being transferred. The effect on peak [Ca2+]i in the infected cells was PKGI independent, occurring to the same degree in both Ad.PKG- and Ad.betagal-infected HPAEC (Figs. 8 and 9). Similar to the PKGI-independent effect of CPT-cGMP on basal permeability, the inhibitory effect of CPT-cGMP on peak [Ca2+]i after H2O2 could be compatible with either activation of a cyclic nucleotide-gated cation channel (52) or inhibition of a delayed rectifying K+ channel (39). Interestingly, the cGMP-mediated inhibition of agonist-induced [Ca2+]i transients observed in smooth muscle cells from normal mice was lost in PKGI knockout mice and only restored via infection with PKGI{alpha}, not PKGIbeta (12). If mechanisms of PKGI-mediated [Ca2+]i regulation are similar in endothelial cells, this finding could suggest that the more robust inhibitory effect of CPT-cGMP on changes in [Ca2+]i previously observed in bovine pulmonary endothelium (35) occurred because they expressed PKGI{alpha} rather than PKGIbeta. Additional work will be necessary to characterize the specific PKGI splice variants in pulmonary endothelial cells. Nevertheless, our current results have demonstrated a significant endothelial barrier-protective effect of PKGI that appears to be independent of changes in [Ca2+]i. Future experiments will focus on the potential role of PKGI to modulate Ca2+ sensitivity after ROS exposure or critical pathways downstream from Ca2+ signaling involved in the alteration of endothelial barrier function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-67189 and HL-075236.


   ACKNOWLEDGMENTS
 
We thank Dr. Kenneth D. Bloch for critically reviewing the manuscript.

Present address of A. D. Verin: Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, IL.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. B. Pearse, Division of Pulmonary and Critical Care Medicine, Hopkins Bayview Medical Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (e-mail: dpearse{at}jhmi.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alexander JS, Alexander BC, Eppihimer LA, Goodyear N, Haque R, Davis CP, Kalogeris TJ, Carden DL, Zhu YN, and Kevil CG. Inflammatory mediators induce sequestration of VE-cadherin in cultured human endothelial cells. Inflammation 24: 99–113, 2000.[CrossRef][Web of Science][Medline]
  2. Bilzer M, Jaeschke H, Vollmar AM, Paumgartner G, and Gerbes AL. Prevention of Kupffer cell-induced oxidant injury in rat liver by atrial natriuretic peptide. Am J Physiol Gastrointest Liver Physiol 276: G1137–G1144, 1999.[Abstract/Free Full Text]
  3. Bilzer M, Witthaut R, Paumgartner G, and Gerbes AL. Prevention of ischemia/reperfusion injury in the rat liver by atrial natriuretic peptide. Gastroenterology 106: 143–151, 1994.[Web of Science][Medline]
  4. Chiche JD, Schlutsmeyer SM, Bloch DB, de la Monte SM, Roberts JD Jr, Filippov G, Janssens SP, Rosenzweig A, and Bloch KD. Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem 273: 34263–34271, 1998.[Abstract/Free Full Text]
  5. Chitaley K, Chen L, Galler A, Walter U, Daum G, and Clowes AW. Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C. FEBS Lett 556: 211–215, 2004.[CrossRef][Web of Science][Medline]
  6. Clements RT, Minnear FL, Singer HA, Keller RS, and Vincent PA. RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability. Am J Physiol Lung Cell Mol Physiol 288: L294–L306, 2005.[Abstract/Free Full Text]
  7. Comerford KM, Lawrence DW, Synnestvedt K, Levi BP, and Colgan SP. Role of vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional permeability. FASEB J 16: 583–585, 2002.[Free Full Text]
  8. Cornwell TL and Lincoln TM. Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells. Reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J Biol Chem 264: 1146–1155, 1989.[Abstract/Free Full Text]
  9. Diwan AH, Thompson WJ, Lee AK, and Strada SJ. Cyclic GMP-dependent protein kinase activity in rat pulmonary microvascular endothelial cells. Biochem Biophys Res Commun 202: 728–735, 1994.[CrossRef][Web of Science][Medline]
  10. Draijer R, Atsma DE, van der Laarse A, and van Hinsbergh VW. cGMP and nitric oxide modulate thrombin-induced endothelial permeability. Regulation via different pathways in human aortic and umbilical vein endothelial cells. Circ Res 76: 199–208, 1995.[Abstract/Free Full Text]
  11. Draijer R, Vaandrager AB, Nolte C, de Jonge HR, Walter U, and van Hinsbergh VW. Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circ Res 77: 897–905, 1995.[Abstract/Free Full Text]
  12. Feil R, Gappa N, Rutz M, Schlossmann J, Rose CR, Konnerth A, Brummer S, Kuhbandner S, and Hofmann F. Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms. Circ Res 90: 1080–1086, 2002.[Abstract/Free Full Text]
  13. Fischer TA, Palmetshofer A, Gambaryan S, Butt E, Jassoy C, Walter U, Sopper S, and Lohmann SM. Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells. J Biol Chem 276: 5967–5974, 2000.[Medline]
  14. Gainor JP, Morton CA, Roberts JT, Vincent PA, and Minnear FL. Platelet-conditioned medium increases endothelial electrical resistance independently of cAMP/PKA and cGMP/PKG. Am J Physiol Heart Circ Physiol 281: H1992–H2001, 2001.[Abstract/Free Full Text]
  15. Gupta MP, Ober MD, Patterson C, Al Hassani M, Natarajan V, and Hart CM. Nitric oxide attenuates H2O2-induced endothelial barrier dysfunction: mechanisms of protection. Am J Physiol Lung Cell Mol Physiol 280: L116–L126, 2001.[Abstract/Free Full Text]
  16. Harbeck B, Huttelmaier S, Schluter K, Jockusch BM, and Illenberger S. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 275: 30817–30825, 2000.[Abstract/Free Full Text]
  17. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, and Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95: 2509–2514, 1998.[Abstract/Free Full Text]
  18. Holschermann H, Noll T, Hempel A, and Piper HM. Dual role of cGMP in modulation of macromolecule permeability of aortic endothelial cells. Am J Physiol Heart Circ Physiol 272: H91–H98, 1997.[Abstract/Free Full Text]
  19. Hou Y, Lascola J, Dulin NO, Ye RD, and Browning DD. Activation of cGMP-dependent protein kinase by protein kinase C. J Biol Chem 278: 16706–16712, 2003.[Abstract/Free Full Text]
  20. Inserte J, Garcia-Dorado D, Agullo L, Paniagua A, and Soler-Soler J. Urodilatin limits acute reperfusion injury in the isolated rat heart. Cardiovasc Res 45: 351–359, 2000.[Abstract/Free Full Text]
  21. Kevil CG, Ohno N, Gute DC, Okayama N, Robinson SA, Chaney E, and Alexander JS. Role of cadherin internalization in hydrogen peroxide-mediated endothelial permeability. Free Radic Biol Med 24: 1015–1022, 1998.[CrossRef][Web of Science][Medline]
  22. Kevil CG, Okayama N, and Alexander JS. H2O2-mediated permeability. II. Importance of tyrosine phosphatase and kinase activity. Am J Physiol Cell Physiol 281: C1940–C1947, 2001.[Abstract/Free Full Text]
  23. Kubes P. Nitric oxide affects microvascular permeability in the intact and inflamed vasculature. Microcirculation 2: 235–244, 1995.[Medline]
  24. Kwan HY, Huang Y, and Yao X. Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism. J Biol Chem 275: 6758–6763, 2000.[Abstract/Free Full Text]
  25. Kwan HY, Huang Y, and Yao X. Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G. Proc Natl Acad Sci USA 101: 2625–2630, 2004.[Abstract/Free Full Text]
  26. Lofton CE, Baron DA, Heffner JE, Currie MG, and Newman WH. Atrial natriuretic peptide inhibits oxidant-induced increases in endothelial permeability. J Mol Cell Cardiol 23: 919–927, 1991.[CrossRef][Web of Science][Medline]
  27. Lofton CE, Newman WH, and Currie MG. Atrial natriuretic peptide regulation of endothelial permeability is mediated by cGMP. Biochem Biophys Res Commun 172: 793–799, 1990.[CrossRef][Web of Science][Medline]
  28. Lohmann SM and Walter U. Tracking functions of cGMP-dependent protein kinases (cGK). Front Biosci 10: 1313–1328, 2005.[Web of Science][Medline]
  29. Lum H, Jaffe HA, Schulz IT, Masood A, RayChaudhury A, and Green RD. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction. Am J Physiol Cell Physiol 277: C580–C588, 1999.[Abstract/Free Full Text]
  30. MacMillan-Crow LA, Murphy-Ullrich JE, and Lincoln TM. Identification and possible localization of cGMP-dependent protein kinase in bovine aortic endothelial cells. Biochem Biophys Res Commun 201: 531–537, 1994.[CrossRef][Web of Science][Medline]
  31. Munzel T, Feil R, Mulsch A, Lohmann SM, Hofmann F, and Walter U. Physiology and pathophysiology of vascular signaling controlled by cyclic guanosine 3',5'-cyclic monophosphate-dependent protein kinase. Circulation 108: 2172–2183, 2003.[Free Full Text]
  32. Norwood N, Moore TM, Dean DA, Bhattacharjee R, Li M, and Stevens T. Store-operated calcium entry and increased endothelial cell permeability. Am J Physiol Lung Cell Mol Physiol 279: L815–L824, 2000.[Abstract/Free Full Text]
  33. Oliver JA. Endothelium-derived relaxing factor contributes to the regulation of endothelial permeability. J Cell Physiol 151: 506–511, 1992.[CrossRef][Web of Science][Medline]
  34. Pearse DB and Becker PM. Effect of time and vascular pressure on permeability and cyclic nucleotides in ischemic lungs. Am J Physiol Heart Circ Physiol 279: H2077–H2084, 2000.[Abstract/Free Full Text]
  35. Pearse DB, Shimoda LA, Verin AD, Bogatcheva N, Moon C, Ronnett GV, Welsh LE, and Becker PM. Effect of cGMP on lung microvascular endothelial barrier dysfunction following hydrogen peroxide. Endothelium 10: 309–317, 2003.[Web of Science][Medline]
  36. Pearse DB, Wagner EM, and Permutt S. Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs. J Appl Physiol 86: 123–132, 1999.[Abstract/Free Full Text]
  37. Pinsky DJ, Naka Y, Chowdhury NC, Liao H, Oz MC, Michler RE, Kubaszewski E, Malinski T, and Stern DM. The nitric oxide/cyclic GMP pathway in organ transplantation: critical role in successful lung preservation. Proc Natl Acad Sci USA 91: 12086–12090, 1994.[Abstract/Free Full Text]
  38. Price CJ and Brindle NP. Vasodilator-stimulated phosphoprotein is involved in stress-fiber and membrane ruffle formation in endothelial cells. Arterioscler Thromb Vasc Biol 20: 2051–2056, 2000.[Abstract/Free Full Text]
  39. Shimoda LA, Welsh LE, and Pearse DB. Inhibition of inwardly rectifying K+ channels by cGMP in pulmonary vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 283: L297–L304, 2002.[Abstract/Free Full Text]
  40. Siflinger-Birnboim A, Goligorsky MS, Del Vecchio PJ, and Malik AB. Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability. Lab Invest 67: 24–30, 1992.[Web of Science][Medline]
  41. Siflinger-Birnboim A, Lum H, Del Vecchio PJ, and Malik AE. Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 270: L973–L978, 1996.[Abstract/Free Full Text]
  42. Smolenski A, Bachmann C, Reinhard K, Honig-Liedl P, Jarchau T, Hoschuetzky H, and Walter U. Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody. J Biol Chem 273: 20029–20035, 1998.[Abstract/Free Full Text]
  43. Smolenski A, Burkhardt AM, Eigenthaler M, Butt E, Gambaryan S, Lohmann SM, and Walter U. Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects. Naunyn Schmiedebergs Arch Pharmacol 358: 134–139, 1998.[CrossRef][Web of Science][Medline]
  44. Suttorp N, Hippenstiel S, Fuhrmann M, Krull M, and Podzuweit T. Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability. Am J Physiol Cell Physiol 270: C778–C785, 1996.[Abstract/Free Full Text]
  45. Thomas MK, Francis SH, and Corbin JD. Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase. J Biol Chem 265: 14964–14970, 1990.[Abstract/Free Full Text]
  46. Usatyuk PV and Natarajan V. Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesion kinase and adherens junction proteins. Am J Physiol Lung Cell Mol Physiol 289: L999–L1010, 2005.[Abstract/Free Full Text]
  47. Van Hinsbergh VW and van Nieuw Amerongen GP. Intracellular signalling involved in modulating human endothelial barrier function. J Anat 200: 549–560, 2002.[CrossRef][Web of Science][Medline]
  48. Verin AD, Cooke C, Herenyiova M, Patterson CE, and Garcia JG. Role of Ca2+/calmodulin-dependent phosphatase 2B in thrombin-induced endothelial cell contractile responses. Am J Physiol Lung Cell Mol Physiol 275: L788–L799, 1998.[Abstract/Free Full Text]
  49. Westendorp RG, Draijer R, Meinders AE, and van Hinsbergh VW. Cyclic-GMP-mediated decrease in permeability of human umbilical and pulmonary artery endothelial cell monolayers. J Vasc Res 31: 42–51, 1994.[CrossRef][Web of Science][Medline]
  50. Wojciak-Stothard B, Tsang LYF, and Haworth SG. Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 288: L749–L760, 2005.[Abstract/Free Full Text]
  51. Wong D, Dorovini-Zis K, and Vincent SR. Cytokines, nitric oxide, and cGMP modulate the permeability of an in vitro model of the human blood-brain barrier. Exp Neurol 190: 446–455, 2004.[CrossRef][Web of Science][Medline]
  52. Wu S, Moore TM, Brough GH, Whitt SR, Chinkers M, Li M, and Stevens T. Cyclic nucleotide-gated channels mediate membrane depolarization following activation of store-operated calcium entry in endothelial cells. J Biol Chem 275: 18887–18896, 2000.[Abstract/Free Full Text]
  53. Wyatt TA, Lincoln TM, and Pryzwansky KB. Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils. J Biol Chem 266: 21274–21280, 1991.[Abstract/Free Full Text]
  54. Zhang J, Xia SL, Block ER, and Patel JM. NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells. Am J Physiol Cell Physiol 283: C1080–C1089, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. P. Schmidt, M. Damarla, O. Rentsendorj, L. E. Servinsky, B. Zhu, A. Moldobaeva, A. Gonzalez, P. M. Hassoun, and D. B. Pearse
Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice
Am J Physiol Lung Cell Mol Physiol, December 1, 2008; 295(6): L1056 - L1065.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. M. Dodd-o, M. L. Hristopoulos, K. Kibler, J. Gutkowska, S. Mukaddam-Daher, A. Gonzalez, L. E. Welsh-Servinsky, and D. B. Pearse
The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse
Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L714 - L723.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
O. Rentsendorj, T. Mirzapoiazova, D. Adyshev, L. E. Servinsky, T. Renne, A. D. Verin, and D. B. Pearse
Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function
Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L686 - L697.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
W.-L. Yeh, D.-Y. Lu, C.-J. Lin, H.-C. Liou, and W.-M. Fu
Inhibition of Hypoxia-Induced Increase of Blood-Brain Barrier Permeability by YC-1 through the Antagonism of HIF-1{alpha} Accumulation and VEGF Expression
Mol. Pharmacol., August 1, 2007; 72(2): 440 - 449.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
G. Boerrigter, L. C. Costello-Boerrigter, A. Cataliotti, H. Lapp, J.-P. Stasch, and J. C. Burnett Jr
Targeting Heme-Oxidized Soluble Guanylate Cyclase in Experimental Heart Failure
Hypertension, May 1, 2007; 49(5): 1128 - 1133.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. Glynos, A. Kotanidou, S. E. Orfanos, Z. Zhou, D. C. M. Simoes, C. Magkou, C. Roussos, and A. Papapetropoulos
Soluble guanylyl cyclase expression is reduced in LPS-induced lung injury
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1448 - R1455.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
A. W. Orr, R. Stockton, M. B. Simmers, J. M. Sanders, I. J. Sarembock, B. R. Blackman, and M. A. Schwartz
Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis
J. Cell Biol., February 26, 2007; 176(5): 719 - 727.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/L919    most recent
00434.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moldobaeva, A.
Right arrow Articles by Pearse, D. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moldobaeva, A.
Right arrow Articles by Pearse, D. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.