Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-5218
Submitted 30 September 2002 ; accepted in final form 16 May 2003
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ABSTRACT |
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isolated rat lung; nitric oxide; pulmonary hypertension; 8-bromoguanosine 3',5'-cyclic monophosphate; KT-5823; Rp--phenyl-1, N2-etheno-8-bromoguanosine
3',5'-cyclic monophosphorothioate
NO leads to pulmonary vascular smooth muscle (VSM) relaxation by stimulating soluble guanylyl cyclase (sGC) (7). The subsequent elevation in cGMP activates protein kinase G-1 (PKG-1), causing relaxation through a variety of mechanisms involving decreased VSM intracellular calcium and desensitization of the contractile apparatus to calcium (24). Interestingly, CH appears to elevate pulmonary sGC activity, protein, and mRNA expression (22), despite evidence for attenuated NO-mediated vasodilation (19). The reason for this discrepancy is unclear but may be accounted for by altered activity and/or expression of cellular targets of cGMP. Consistent with this possibility are findings from our laboratory that responses to the membrane-permeable cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) are similarly impaired following CH (19). Therefore, we hypothesized that attenuated cGMP-dependent pulmonary vasodilation following CH is mediated by downregulation of VSM PKG-1 expression and/or activity. To test this hypothesis, we examined vasodilatory responses to 8-BrcGMP in the presence or absence of PKG inhibition in lungs isolated from control and CH rats. In addition, expression, localization, and activity of pulmonary PKG-1 were assessed by Western blot analysis, immunohistochemistry, and a PKG activity assay, respectively.
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METHODS |
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Experimental Groups
Sprague-Dawley rats (200-250 g, Harlan Industries) were divided into two
groups for each experiment. Animals designated for exposure to CH were housed
in a hypobaric chamber with barometric pressure maintained at 380 mmHg
for 4 wk. The chamber was opened three times per week to provide animals with
fresh food, water, and bedding. On the day of experimentation, rats were
removed from the hypobaric chamber and immediately placed in a Plexiglas
chamber continuously flushed with a 12% O2-88% N2 gas
mixture to reproduce inspired PO2 (
70 mmHg) within
the hypobaric chamber. Age-matched control animals were housed at ambient
barometric pressure (
630 mmHg). All animals were maintained on a 12:12-h
light-dark cycle.
CH-Induced Right Ventricular Hypertrophy and Polycythemia
Blood samples were obtained by direct cardiac puncture at the time of lung isolation for measurement of hematocrit. Right ventricular (RV) hypertrophy was assessed as an index of CH-induced pulmonary hypertension, as previously described (19, 33, 34). In brief, after isolation of the heart, the atria and major vessels were removed from the ventricles. The RV was dissected from the left ventricle and septum (LV + S), and each was weighed. The degree of RV hypertrophy is expressed as the ratio of RV to total ventricle weight (T).
Isolated Lung Preparation
Rats from each group were anesthetized with pentobarbital sodium (52 mg
ip), and the lungs were isolated for recirculating perfusion with a
physiological saline solution (PSS) as previously described
(19,
33,
34). After the trachea was
cannulated with a 17-gauge needle stub, the lungs were ventilated with a
Harvard positive-pressure rodent ventilator (model 683) at a frequency of 55
breaths/min and a tidal volume of 2.5 ml with a warmed and humidified gas
mixture (6% CO2 in room air). Inspiratory pressure was set at 9
cmH2O, and positive end-expiratory pressure was set at 3
cmH2O. After a median sternotomy, heparin (100 units) was injected
directly into the RV, and the pulmonary artery was cannulated with a 13-gauge
needle stub. The preparation was immediately perfused with PSS containing (in
mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8
CaCl2, and 5.5 glucose, with 4% (wt/vol) albumin, 30 µM
meclofenamate, and 300 µM
N-nitro-L-arginine (L-NNA)
(all from Sigma) at 0.8 ml/min with a Masterflex microprocessor pump drive
(model 7524-10). Meclofenamate and L-NNA were added to acutely
minimize the potential complicating influences of endogenous prostaglandins
and NO on vascular reactivity. We have previously demonstrated that the dose
of L-NNA employed is effective in inhibiting EDNO-dependent
pulmonary vasodilation in the isolated perfused rat lung
(32,
34). This dose of
meclofenamate is approximately threefold higher than that previously shown to
provide effective inhibition of prostaglandin synthesis in this preparation
(12).
The LV was cannulated with a plastic tube (4-mm outer diameter), and the heart and lungs were removed en bloc and suspended in a humidified chamber maintained at 38°C. The perfusion rate was gradually increased to 30 ml · min-1 · kg body wt-1 and maintained at this rate for the duration of the experiment. Twenty milliliters of perfusate were washed through the lungs and discarded before recirculation was initiated with the remaining 30 or 40 ml, depending on the protocol (see Isolated Lung Experiments). Experiments were performed with lungs in zone 3 conditions, achieved by elevating the perfusate reservoir until pulmonary venous pressure (Pv) was 3-4 mmHg. Pulmonary arterial pressure (Pa) and Pv were measured with Spectramed model P23XL pressure transducers and recorded on a Gould RS 3400 chart recorder. Data were stored and processed with a computer-based data acquisition/analysis system (Advanced-CODAS, Dataq Instruments).
Isolated Lung Experiments
Effects of PKG inhibition on cGMP-mediated vasodilation. The
contribution of PKG in mediating vasodilatory responses to the
membrane-permeable cGMP analog 8-BrcGMP (1 µM, Sigma) was assessed in each
group of lungs using either the competitive PKG inhibitor
Rp--phenyl-1, N2-etheno-8-bromoguanosine
3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS,
Ki = 0.03 µM; Alexis) or the highly specific PKG
inhibitor KT-5823 (Ki = 0.234 µM, Alexis).
Rp-8-Br-PET-cGMPS (30 µM) or its vehicle (saline) was added to the
recirculating reservoir (40 ml) immediately after lung isolation and was
present throughout the experiment. This dose of Rp-8-Br-PET-cGMPS has
been previously employed by other investigators to inhibit PKG
(10,
14). In separate sets of lungs
from control and CH rats, KT-5823 (10 µM) or its vehicle (DMSO) was added
to the recirculating reservoir (30 ml) immediately after lung isolation and
was present throughout the experiment. This dose of KT-5823 has been
demonstrated by other investigators to inhibit PKG in this preparation
(13). After a 30-min
equilibration, U-46619 was added to obtain a stable vasoconstrictor response
(
10 mmHg). 8-BrcGMP (1 µM) was administered to assess vasodilatory
responses in the presence or absence of PKG inhibition. As previously
described, (19) 8-BrcGMP
produces slowly developing and progressive vasodilatory responses in lungs
from control rats; therefore, assessments of reactivity were made 20 min after
administration. In contrast, responses to 8-BrcGMP in lungs from CH rats were
stable during this period.
Vasodilatory response to the Rp diastereomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate. To determine whether potential diffusional limitations associated with arterial remodeling limit access of 8-BrcGMP to VSM in lungs from CH rats, vasodilatory responses to the more membrane-permeable analog the Rp diastereomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) (6) were assessed in control and CH rats. After attainment of a stable vasoconstrictor response to U-46619, 8-cCPT-cGMP (1 µM) was administered. A double occlusion maneuver was employed to allow calculation of segmental vascular resistances as described previously (30-33).
PKG-1 Western Blot
To examine whether attenuated reactivity to cGMP is associated with altered
lung PKG-1 expression, we performed Western blots to determine the level of
PKG-1 protein (both and
) in lungs from both control and CH
rats. Lungs were snap-frozen in liquid N2 at the completion of
isolated lung experiments and homogenized in 10 mM Tris · HCl
homogenization buffer containing 255 mM sucrose, 2 mM EDTA, 12 µM
leupeptin, 1 µM pepstatin A, 0.3 µM aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. Samples were centrifuged at 10,000 g
for 10 min at 4°C to remove insoluble debris. The supernatant was
collected, and sample protein concentrations were determined by the Bradford
method (Bio-Rad Protein Assay). Control experiments were conducted with
different concentrations of protein to ensure linearity of the densitometry
curve. A molecular-weight standard (Bio-Rad) was added to each gel, and the
optimal concentration of sample protein (15 µg/lane) was separated by
SDS-PAGE (7.5% Tris · HCl gels, Bio-Rad) and transferred to
polyvinylidene difluoride membranes. Blots were blocked overnight at 4°C
with 5% nonfat milk, 3% bovine serum albumin, and 0.05% Tween 20 (Bio-Rad) in
Tris-buffered saline containing 10 mM Tris · HCl and 50 mM NaCl (pH
7.5). The blots were then incubated for 1 h at room temperature with a rabbit
polyclonal antibody for PKG-1
and -
(1:8,000) (Stressgen). For
immunochemical labeling, blots were incubated for 2 h at room temperature with
goat anti-rabbit IgG-horseradish peroxidase (1:7,500, Stressgen). After
chemiluminescence labeling (ECL, Amersham), we detected PKG bands by exposing
the blots to chemiluminescence-sensitive film (Kodak). We stained membranes
with Coomassie brilliant blue to confirm equal protein loading in all lanes.
Quantification of the bands was accomplished by densitometric analysis of
scanned images (Sigma-Gel software, SPSS). All reagents were purchased from
Sigma unless otherwise noted.
PKG-1 Immunohistochemistry
To examine the site of altered PKG-1 expression following CH, we anesthetized animals from each experimental group with pentobarbital sodium (52 mg ip) for subsequent immunohistochemical analysis similar to that previously described (30, 33). The lungs were isolated as described in Isolated Lung Experiments and perfused (60 ml · min-1 · kg body wt-1) with 250 ml of PSS containing 4% (wt/vol) albumin (Sigma) and 10-4 M papaverine to maximally dilate and flush the circulation of blood. The lungs were then perfused with 250 ml fixative [0.2 M phosphate-buffered saline (PBS; 0.05 M Na2HPO4 dibasic and 0.14 M NaCl; pH 7.4) with 4% paraformaldehyde and 10-4 M papaverine]. During the perfusion with PSS and fixative, Pv was maintained at 12 mmHg. Previous work from our laboratory (11) suggests that maximal recruitment and thus maximal vascular surface area are achieved at this Pv. In addition, the lungs were inflated via the trachea to a pressure of 23 cmH2O with fixative at the same time that the lungs were perfused. The trachea was ligated with 2-0 silk, and the lungs were immersed in fixative for 30 min. A transverse section (2-3 mm thick) of tissue from the hilar level of the left cranial lobe was removed and placed in fixative for 3.5 h and rinsed with PBS. Sections were then dehydrated in increasing concentrations of ethanol, with a final dehydration in xylene, and then mounted in paraffin.
Transverse sections of the left lobe were cut (5 µm thick) at the hilar
level and mounted onto Superfrost Plus slides (Fisher Scientific). Sections
were rehydrated, and antibody-antigen binding was enhanced by an antigen
retrieval method in which sections were boiled 5 min in a 10 mM citrate
buffer, pH 6.0 (0.1 M citric acid and 0.1 M sodium citrate). Sections were
allowed to cool, treated with 0.33% H2O2 to inhibit
endogenous peroxidases, and then incubated with normal goat serum (15%)
followed by incubation for 24 h at 4°C with a polyclonal antibody for
PKG-1 and -
(1:5,000, Stressgen) in PBS containing 0.3% Triton
X-100 (Sigma). Immunocytochemical labeling was demonstrated by incubation with
biotinylated goat anti-rabbit IgG (1:400, Vector Laboratories) followed by
incubation with an avidin biotinylated peroxidase complex (1:200, ABC Elite
Kit; Vector Laboratories). Immunoprecipitation of the antigen peroxidase
conjugate was achieved by treatment of sections with a diaminobenzidine
peroxidase substrate labeling kit with the addition of nickel chloride (Vector
Laboratories) for 3 min. Sections were washed in deionized water, dehydrated,
cleared, and mounted with Permount. Control sections were prepared by
incubation with rabbit serum instead of primary antibody for assessment of
specific staining. Serial sections were stained for elastin and counterstained
with van Gieson solution (Sigma Accustain Elastic Stain) for accurate
identification of arteries and airways and for measurement of external vessel
and bronchiole diameters. Arteries were identified by the presence of an
internal elastic lamina. Arteries, bronchioles, and alveoli on which
densitometry measurements were assessed were first identified in the
elastic-stained tissue and subsequently identified in the adjacent
PKG-1-stained sections for analysis of staining intensity, thus providing an
unbiased method for selection of structures for assessment of PKG-1
immunoreactivity. Vessels and airways that were sectioned at oblique angles
were excluded from analysis. A total of 190 arteries from three rats/group
were analyzed. All measurements were made using green wavelength illumination
with a x20 objective. Images were generated with a Dage-MTI CCD-72 video
camera from a Nikon Optiphot microscope and processed with MetaMorph Imaging
System software (Universal Imaging).
PKG-1 staining intensity is expressed in optical density (OD) units according to the following calculation (40): OD = -log10(GLSpecimen)/(GLWhite), where GLSpecimen is the gray level of the stained image and GLWhite is the gray level of the image obtained from an area of the microscope slide absent of tissue. Dividing the numerator by GLWhite compensates for any uneven field illumination and for the OD contributed by the glass slide, Permount, and coverslip. Specific staining is defined as the difference in staining intensity of sections incubated with primary antibody vs. rabbit serum (negative control). PKG-1 staining intensity for each structure was calculated as the average specific staining OD. OD was calibrated by use of a stepped OD filter (Edmund Scientific).
PKG Activity Assay
PKG activity was determined as described by other investigators
(10). PKG activity was based
on the phosphorylation of BPDEtide, a peptide sequence derived from the
phosphorylation sequence of phosphodiesterase (PDE)-5. PKG incorporates
32P from ATP onto BPDEtide. Freshly frozen lungs from each group
were homogenized on ice in a buffer containing (in mM): 50 Tris · HCl
(pH 7.4), 10 EDTA, 2 dithiothreitol, 1 IBMX, 0.3 L-NNA, and 0.03
meclofenamate. Samples were centrifuged at 10,000 g for 10 min at
4°C to remove insoluble debris. The supernatant was collected, and sample
protein concentrations were determined by the Bradford method (Bio-Rad Protein
Assay). Control experiments were conducted with different concentrations of
protein to ensure linearity of the counts per min (cpm) curve. The assay
mixture in a total volume of 40 µl contained 50 mM Tris · HCl (pH
7.4), 20 mM MgCl2, 0.1 mM IBMX, 150 µM BPDEtide, 1 µM PKA
inhibitor (5-24 synthetic peptide inhibitor of PKA), and 0.2 mM
-32P]ATP (
400 cpm/pmol). The reactions were started by
the addition of 5 µg of protein to an assay mixture. Each sample was
assayed in the presence or absence of 5 µM 8-BrcGMP and in the presence or
absence of the PKG inhibitors Rp-8-Br-PET-cGMPS (30 µM) or KT-5823
(10 µM). The reaction mixture was incubated at 30°C for 10 min and
terminated by spotting the 40 µl onto P-81 phosphocellulose filters and
immediately placed in ice-cold phosphoric acid (75 mM). The filters were
washed, placed in 5 ml of scintillation fluid, and counted by using a Packard
Liquid Scintillation Analyzer (Tri-Carb 2100TR). Counts obtained were
corrected for nonspecific binding of [32P]ATP in the absence of
peptide substrate.
Calculations and Statistics
Total pulmonary vascular resistance in isolated, perfused lungs was
calculated as the difference between Pa and Pv divided
by flow (30 ml · min-1 · kg body
wt-1). Pulmonary arterial resistance was calculated as
the difference between Pa and pulmonary capillary pressure
(Pc) divided by flow. Similarly, pulmonary venous resistance was
calculated as the difference between Pc and Pv divided
by flow. Vasodilatory responses were calculated as percent reversal of
U-46619-induced vasoconstriction for the total pulmonary vasculature as well
as for arterial and venous segments. All data are expressed as means ±
SE, and values of n refer to the number of animals in each group. For
optical density measurements, all arteries, bronchioles, and alveoli analyzed
for each animal within the size ranges indicated were averaged to obtain a
single n value. Where appropriate, a t-test, two-way ANOVA,
or two-way repeated-measures ANOVA was used to make comparisons. If
differences were detected by ANOVA, individual groups were compared with the
Student-Newman-Keuls test. A probability of P 0.05 was accepted
as significant for all comparisons.
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RESULTS |
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RV weight was significantly greater in CH rats (0.249 ± 0.060 g, n = 19) compared with controls (0.147 ± 0.006 g, n = 19), whereas LV + S weight did not differ between groups (CH = 0.464 ± 0.041 g, control = 0.513 ± 0.014 g). RV/T ratios were consequently greater in CH rats (0.326 ± 0.001, n = 19) compared with control rats (0.219 ± 0.001, n = 19), thus demonstrating RV hypertrophy indicative of pulmonary hypertension. Further, CH rats exhibited polycythemia as indicated by a significantly greater hematocrit (61 ± 1%, n = 19) compared with control rats (43 ± 0%, n = 19).
Isolated Lung Experiments
Baseline vascular resistances and responses to U-46619. Consistent with previous reports (32-34) baseline vascular resistances were significantly greater in lungs from CH rats (0.138 ± 0.011 mmHg·ml-1·min·kg, n = 19) compared with controls (0.075 ± 0.006 mmHg· ml-1·min·kg, n = 19). Because the pulmonary circulation exhibits no detectable tone in this preparation (31), these data provide functional evidence for CH-induced vascular remodeling. U-46619 produced similar increases in resistance between CH (0.509 ± 0.051 mmHg·ml-1· min·kg) and control (0.479 ± 0.026 mmHg·ml-1·min·kg) groups. However, we observed a slight but significant difference in the concentration of U-46619 required to elicit comparable vasoconstriction between control (102 ± 5 nM) and CH (77 ± 8 nM) groups.
Effects of PKG inhibition on cGMP-mediated vasodilation. Vasodilatory responses to the phosphodiesterase-resistant cGMP analog 8-BrcGMP (1 µM) were largely attenuated in the presence of the PKG inhibitors, Rp-8-Br-PET-cGMPS (30 µM, Fig. 1A) and KT-5823 (10 µM, Fig. 1B) in each group. These findings support a role for PKG in mediating 8-BrcGMP-induced vasodilation in lungs from both control and CH rats.
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Vasodilatory responses to 8-pCPT-cGMP. Similar to 8-BrcGMP responses, total vasodilatory responses to the more membrane-permeable cGMP analog 8-pCPT-cGMP (1 µM) were attenuated following CH (Fig. 2). This attenuation was present in both the arterial and venous segments of the pulmonary vasculature.
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PKG-1 and -
Western Blot
Immunoreactive PKG-1 was detected as a single band of 75 kDa in lungs
from control and CH rats (Fig.
3A). PKG-1 protein levels were approximately twofold
greater in lungs from CH rats compared with those of control rats.
(Fig. 3B).
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PKG Immunohistochemistry
PKG-1 immunoreactivity in pulmonary arteries from control and CH rats is illustrated in Fig. 4. PKG-1 staining was localized primarily in vascular and bronchial smooth muscle and vascular endothelium, with no detectable staining observed in bronchial epithelium. PKG-1 staining was more intense in smooth muscle of pulmonary arteries from CH rats compared with those of control animals. Further, PKG-1 staining intensity varied inversely with arterial diameter in CH rats (Fig. 5A). PKG-1 immunoreactivity was similarly less in arteries >100 µm compared with smaller vessels in control rats. There were no differences between CH rats and control rats in PKG-1 immunoreactivity detected in bronchioles and alveoli (Fig. 5B).
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PKG Activity Assay
PKG activity was increased in the presence of 8-BrcGMP (5 µM) and attenuated by the addition of the PKG inhibitors KT-5823 (10 µM) or Rp-8-Br-PET-cGMPS (30 µM) in lung homogenates from both control and CH rats (Fig. 6). Consistent with our findings of PKG-1 upregulation (Figs. 3, 4, 5), basal and stimulated PKG activity was greater in lungs from CH rats compared with controls.
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DISCUSSION |
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Previous studies from our laboratory (32-34) and others (18, 26, 28, 36, 37) suggest that CH augments EDNO-dependent pulmonary vasodilation, a response associated with upregulation of pulmonary eNOS mRNA and protein levels (18, 21, 26, 33, 34, 39, 46), as well as increased synthesis of NO (18, 26, 44). However, despite this enhanced reactivity to EDNO-mediated agonists, we have recently reported that CH attenuates pulmonary vasodilatory responsiveness to both exogenous NO (19) and to the membrane-permeable cGMP analog 8-BrcGMP (19). The reason for this discrepancy between EDNO- and exogenous NO-mediated reactivity in lungs from CH rats is not clear, although it is possible that elevated NO synthesis following CH associated with eNOS upregulation is sufficient to mask diminished VSM reactivity to NO.
The mechanism(s) by which CH interferes with NO-dependent pulmonary vasodilation is not well understood, although several possibilities exist. In many vascular beds, activation of VSM sGC by NO leads to cGMP synthesis and subsequent stimulation of PKG-1 (1, 7, 17, 24). PKG-1 induces VSM relaxation through various mechanisms involving a decrease in intracellular calcium and desensitization of the contractile apparatus to calcium (24). Previous findings from our laboratory support a role for sGC in mediating NO-dependent pulmonary vasodilation in both control and CH rats (19). However, CH-induced attenuation of NO-dependent reactivity does not appear to be a function of decreased sGC expression or activity (19). Furthermore, recent studies by Li et al. (22) have demonstrated increases in pulmonary arterial sGC protein and mRNA levels, as well as increased sGC activity in whole lung tissue from male rats following 21 days of hypoxic exposure (22). These observations appear to be at odds with diminished NO-mediated vasodilation observed in lungs from CH rats, suggesting that CH may interfere with the expression and/or activity of intra-cellular targets of cGMP, or possibly increase the rate of cGMP degradation. cGMP hydrolysis is regulated primarily by cGMP-specific phosphodiesterase (PDE-5) in pulmonary VSM (8, 9, 25). Furthermore, there is evidence suggesting that pulmonary PDE-5 expression and activity are augmented in CH-induced pulmonary hypertensive rats (25, 27) and in 4-wk-old pulmonary hypertensive lambs (3) compared with control animals. Consistent with these results, we have recently demonstrated that PDE-5 inhibitors augment vasodilatory responses to exogenous NO in lungs from CH rats, while minimally influencing reactivity in controls. However, decreased reactivity to NO was maintained in CH lungs following PDE-5 inhibition (19). These findings, together with the observation that vasodilatory responses to the PDE-5-resistant analog of cGMP 8-BrcGMP were also attenuated following CH (19), led us to the hypothesis that the impaired NO/cGMP vasodilatory response following CH is due to decreased PKG-1 expression/activity.
PKG has been shown to be involved in NO-dependent relaxation in both ovine
pulmonary arteries (10) and
pulmonary veins (14). This is
consistent with the current study in that we effectively inhibited pulmonary
vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats with
mechanistically distinct inhibitors of PKG-1. Rp-8-Br-PET-cGMPS is
the most potent and selective PKG inhibitor amongst the
(Rp)-phosphorothioate cGMP analogs and antagonizes signaling by
binding to PKG 1 or -
and prohibiting the conformational change
of the enzyme required for its activation
(5). In contrast, KT-5823, one
of several K-252b derivatives that mediate inhibition by competing with ATP,
is highly selective for PKG. However, at high concentrations, KT-5823 may also
inhibit PKC (20).
Although our findings support a role for PKG in mediating 8-BrcGMP-induced
pulmonary vasodilation, the attenuated vasodilatory responsiveness following
CH does not appear to be mediated by decreased expression of PKG-1. Rather, we
found that pulmonary levels of PKG-1 are elevated following CH and,
furthermore, that this upregulation appears to be localized to the
vasculature. Although PKG-1 staining was additionally present in bronchial
smooth muscle and alveoli, there was no detectable difference in staining
intensity between control and CH lung tissue. Zhan et al.
(47) have similarly localized
PKG-1 to pulmonary vascular and bronchial smooth muscle. However, in
contrast to our present findings, PKG-1
was also found to be expressed
in ciliated airway epithelial cells
(47). The reason for this
discrepancy is not clear but may be due to differences in protocols or
antibodies used. The mechanism by which PKG-1 expression is upregulated
following CH is not known. Although the increase in whole lung PKG-1 levels
apparent by Western blotting could be due in part to VSM proliferation
associated with arterial remodeling, the increase in staining intensity
observed by immunohistochemical analysis is suggestive of increased
concentrations of PKG-1 in the arterial wall. Potentially, PKG-1 expression
may be stimulated in response to pulmonary hypertension or the vascular
remodeling process and may serve as a compensatory response to CH-induced
pulmonary hypertension. Alternatively, expression of PKG-1 could be regulated
directly by hypoxia, if the PKG-1 promoter contains a hypoxia-response
element.
On the basis of previous results from our laboratory demonstrating impaired vasodilatory responses to 8-BrcGMP following CH (19), the current finding that CH upregulates PKG-1 in pulmonary arteries is unexpected. Although it is possible that the activity of PKG-1 is decreased following CH despite increased expression, our current observations that lung PKG activity is augmented following CH do not support this possibility. However, a potential limitation of this assay is that it may not be indicative of PKG activity at the level of the vasculature, in that whole lung PKG activity was measured. Nonetheless, this increase in PKG activity is consistent with increased vascular PKG-1 expression as assessed by quantitative immunohistochemistry. Alternatively, some investigators (13, 29) have reported that PKG activity may not be essential to the NO/cGMP vasodilatory response. Fouty et al. (13) have suggested that the inhibitory influence of NO on vascular tone is dependent on cGMP formation in the pulmonary circulation of both normotensive and hypertensive rats but is not dependent on PKG activation. They demonstrated that the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the NOS inhibitor L-NNA augment hypoxic vasoconstriction but that the PKG inhibitors H-8, Rp-8-pCPT-cGMPS, and KT-5823 have no effect on perfusion pressure in isolated saline-perfused lungs. The reason for the discrepancy between the current study and that of Fouty et al. (13) is not clear. However, 8-substituted cGMP analogs such as 8-BrcGMP used in the current study may exhibit greater selectivity for PKG activation compared with native cGMP (41). Indeed, consistent with the current study, Fouty and colleagues (13) demonstrate that the PKG inhibitor H-8 significantly, albeit modestly, inhibited 8-BrcGMP-mediated vasodilation in isolated lungs. Moreover, Pauvert et al. (29) demonstrated that NO-mediated dilation in rat main pulmonary arterial rings involves cGMP but appears to be independent of PKG activation. These investigators show that the sGC inhibitor ODQ abolished sodium nitroprusside relaxation in ATP-preconstricted rings, but PKG inhibition with 1 µM KT-5823 did not. Our present findings that the selective PKG inhibitors KT-5823 and Rp-8-Br-PET-cGMPS did not consistently provide complete inhibition of 8-BrcGMP-induced vasodilation are consistent with PKG-independent vasodilatory influences of cGMP, although it is alternatively possible that diffusional limitations or otherwise incomplete inhibition of enzyme activity explains this effect. Consistent with the latter possibility, unpublished observations from our laboratory indicate that abluminal administration of either KT-5823 (10 µM) or ODQ (50 µM) provides complete blockade of reactivity to the NO donor spermine NONOate in isolated, pressurized, small pulmonary arteries from both control and CH rats.
Other potential targets of cGMP include cyclic nucleotide-gated cation channels (2, 23), cGMP-inhibitable cAMP phosphodiesterase (PDE-3) (15, 42, 45), and PKA (16, 38). Modulation of cyclic nucleotide-gated cation channels by direct binding of cGMP has been described in retinal cells, olfactory cells, and renal epithelium, although whether direct cGMP regulation of ion channels occurs in smooth muscle is unknown. The relative contribution of the cAMP/PKA pathway to NO-dependent pulmonary responses is also not well understood. However, several studies suggest that cGMP does not cause smooth muscle relaxation indirectly by increasing cAMP (4, 10, 14). Furthermore, the membrane-permeable cGMP analog 8-BrcGMP, used in this study, does not activate cGMP-binding cyclic nucleotide phosphodiesterases (23, 43), suggesting cGMP is unlikely acting through cAMP/PKA. Nevertheless, it is apparent from the present study that PKG contributes in large part to 8-BrcGMP-mediated pulmonary vasodilation in both control and CH groups.
Our finding that CH-induced attenuation of cGMP-mediated pulmonary vasodilation is paradoxically associated with increased lung PKG-1 expression and activity could additionally be explained by potential diffusional limitations associated with arterial remodeling that limit access of 8-BrcGMP to VSM in lungs from CH rats. However, this is not likely, since we have observed similar attenuated vasodilatory responsiveness to the more membrane-permeable cGMP analog 8-pCPT-cGMP in lungs from CH rats vs. control animals. Furthermore, we found that CH leads to a comparable reduction of reactivity to 8-pCPT-cGMP within the venous circulation, which exhibits no detectable remodeling following CH in these animals (34). Finally, impaired cGMP-mediated responsiveness following CH could be due to altered Ca2+ sequestration, influx, or efflux mechanisms; to a change in sensitivity of the contractile apparatus to Ca2+ (24); or rather to alterations in endothelial modulation of vascular tone. For example, locally produced vasoactive factors, including endothelin-1 and reactive oxygen or nitrogen species, may provide a basal vasoconstrictor influence or otherwise modify VSM function in the pulmonary circulation of CH rats to diminish vasoreactivity to cGMP. Further research is necessary to determine these possibilities.
In summary, we have investigated the possibility that the attenuated cGMP-dependent pulmonary vasodilatory response observed after CH is a function of decreased PKG-1 expression/activity. Our findings suggest that whereas PKG contributes to 8-BrcGMP-dependent pulmonary vasodilation, altered reactivity to NO following CH is not likely a function of decreased pulmonary PKG-1 expression. Rather, CH appears to increase PKG-1 expression selectively within pulmonary VSM and this correlates with increased basal and stimulated PKG activity in CH lungs.
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DISCLOSURES |
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T. C. Resta is a Parker B. Francis Fellow in Pulmonary Research.
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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.
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REFERENCES |
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