(Received for publication, March 3, 1997, and in revised form, May 20, 1997)
From the Cardiovascular Division, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachussets 02115
To determine whether nitric oxide (NO) can
modulate bradykinin (BK) signaling pathways, we treated endothelial
cells with an NO donor, S-nitrosoglutathione (GSNO), to
determine its effect(s) on G-proteins (Gi and
Gq) that are coupled to the type II kinin (BK2)
receptor. Radioligand binding assays and Western analyses showed that
GSNO (10-500 µM, 0-72 h) did not alter the expression of BK2 receptor, Gi, or Gq.
However, GSNO caused a 6-fold increase in basal cGMP production and
decreased high affinity BK bindings sites and GTPase activity by 74 and
85%, respectively. The cGMP analogue, dibutyryl-cGMP, also inhibited
BK-stimulated GTPase activity by 74% suggesting that some of the
effects of NO may be mediated through activation of guanylyl cyclase.
The NO synthase inhibitor,
N-monomethyl-L-arginine,
inhibited endogenous NO synthase activity and cGMP production by 91 and
76%, respectively, but increased BK-stimulated GTPase activity by
61%. To determine which G-proteins are affected by NO, we performed
GTP binding assays with [35S]GTP
S followed by
immunoprecipitation with specific G-protein antisera. Both GSNO and
dibutyryl-cGMP increased basal G-protein GTP binding activities by
18-26%. However, GSNO decreased BK-stimulated G
i2,
G
i3, and G
q/11 GTP binding activity by
93, 61, and 90%, respectively, whereas epinephrine-stimulated
G
s GTP binding activity was unaffected. These results
suggest that NO can modulate BK signaling pathways by selectively
inhibiting G-proteins of the Gi and Gq
family.
The vasoactive nonapeptide, bradykinin (BK),1 is released during immune hypersensitivity reactions and contributes to the inflammatory process by modulating endothelial cell permeability, vascular tone, and neutrophil chemotaxis (1, 2). The cellular effects of BK are mediated by seven transmembrane-spanning receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) (3, 4). We have previously shown that bovine aortic endothelial cells contain predominantly the type II kinin (BK2) receptor that is coupled to G-proteins of the Gi and Gq family (5). Both Gi and Gq can activate phosphoinositide-specific phospholipase C, which mobilizes intracellular calcium via the hydrolysis of phosphatidylinositol 4,5-bisphosphate (5-7). This intracellular calcium signal is necessary for many of the vascular responses elicited by BK including the release of endothelial-derived nitric oxide (NO) (8).
The stimulation of Gi proteins and phospholipase
A2 by BK leads to the production of arachidonic acids and
leukotrienes, which are important in mediating the inflammatory
response (9). In addition, stimulation of Gi proteins by BK
can potentially decrease cAMP production via inhibition of adenylyl
cyclase activity (10). The cAMP-dependent pathway
serves to counteract many of the clinical symptoms associated with
immune hypersensitivity reactions (11). Indeed, -adrenergic receptor
agonists such as epinephrine, which activates the
Gs-adenylyl cyclase pathway, are often administered to
alleviate anaphylactic reactions (12). Thus, factors that modulate
BK2 receptor-coupled G-proteins may influence the course and outcome of BK-mediated inflammatory processes.
Sustained high levels of NO are produced during inflammatory conditions by cytokine-inducible type II NO synthase in resident and nonresident vascular cells (13-15). Although both BK and NO are released during immune hypersensitivity reactions, the effects of NO on BK-mediated responses are not known. Recent studies suggest that exogenous NO donors can activate mitogen-activated protein kinase pathways and stimulate p21ras via S-nitrosylation of these signaling molecules (16, 17). A similar mechanism has been proposed for the activation of heterotrimeric G-proteins by NO in peripheral blood mononuclear cells (18). Although these studies demonstrated activation of basal heterotrimeric G-protein activity by NO, it is not known which G-proteins are affected and how NO affects agonist-stimulated G-protein activity.
Because BK and NO are important inflammatory mediators, the effects of NO on BK-mediated responses may have important clinical implications. The purpose of this study, therefore, is to determine whether NO can regulate BK signaling pathways via its effects on G-proteins that are coupled to the BK2 receptor.
All standard culture reagents were obtained from
JRH Bioscience. Bradykinin, HEPES, L-arginine, ascorbic
acid, creatinine phosphate, phosphocreatine kinase,
phenylmethylsulfonyl fluoride, leupeptin, aprotinin, bacitracin,
1,10-phenanthroline, triethanolamine HCl, dithiothreitol, bovine serum
albumin (BSA), ATP, GDP, GTP, sodium nitrite, and glutathione were
purchased from Sigma. CaptoprilTM was obtained from E. R. Squibb & Sons, Inc. (Princeton, NJ). HOE-140 (a BK2 receptor
antagonist) was obtained from Hoechst Marion Roussel, Inc. (Cincinnati,
OH). The NO synthase inhibitor,
N-monomethyl-L-arginine
(LNMA), was purchased from Calbiochem (San Diego, CA).
S-Nitrosoglutathione (GSNO) was synthesized as described (19). The radioisotopes, [3H]arginine (40.5 Ci/mmol),
[3H]BK (121.6 Ci/mmol), [
-32P]GTP (30 Ci/mmol), and [35S]GTP
S (1250 Ci/mmol), and the
polyclonal rabbit antisera to G
i3 (EC/2),
G
q/11 (QL), and G
s (RM/1) were supplied
by NEN Life Science Products. [125I]cGMP (>2500 Ci/g)
was obtained from Biomedical Technologies Inc. (Stoughton, MA). The
polyclonal rabbit antiserum P4 was raised against a purified
decapeptide corresponding to the COOH-terminal regions of
G
i2 (Research Genetics, Inc., Huntsville, AL), and its
specificity has been previously verified (20). Protein molecular weight
markers were purchased from Life Technologies, Inc. The chemiluminescence detection kit (ECL) was obtained from Amersham Corp.
The polyvinylidene difluoride transfer membrane (pore size, 0.2 µm)
was purchased from Bio-Rad.
Bovine aortic endothelial cells were isolated and cultured in a growth medium containing Dulbecco's modified Eagle's medium, 5 mM L-glutamine (Life Technologies, Inc.), 10% fetal calf serum (Hyclone, Logan, UT), and antibiotic mixture of 100 units/ml penicillin/100 µg/ml streptomycin/250 ng/ml Fungizone as described previously (5). They were characterized by morphology using phase-contrast microscopy (Nikon, Optiphot 200) and by staining for Factor VIII-related antigens (21). All passages were performed with a disposable cell scraper (Costar Inc., Cambridge, MA), and only endothelial cells of less than 6 passages were used. Confluent endothelial cells (~5 × 106) were treated with various concentrations of GSNO, dibutyryl-cGMP, and LNMA for the indicated time intervals. Treatment with GSNO was renewed every 12 h.
Radioligand Binding StudiesPartially purified membranes were prepared from control and GSNO-treated endothelial cells as described previously (5). Membranes (100 µg) were added to 12 concentrations of [3H]BK (1 pM to 10 nM) in a buffer containing Tris-HCl (100 mM, pH 7.4), MgCl2 (5 mM), EDTA (0.6 mM), bacitracin (140 µg/ml), CaptoprilTM (1 µM), 1 mM dithiothreitol, 1 mM 1,10-phenanthroline, and 0.1% BSA in a total volume of 0.1 ml. The assay mixture was incubated at 4 °C for 90 min with gentle shaking. All reaction tubes and filters were pretreated overnight with 0.1% BSA and 0.1% polyethyleneimine, respectively, to decrease nonspecific binding. The assays were terminated by vacuum filtration on Whatman GF/C filters. Each filter was counted for 2 min in a liquid scintillation counter (Beckman LS 1800). Bovine aortic endothelial cell membrane contain only one kinin receptor, the BK2 subtype (22). Nonspecific binding was determined in the presence of 10 µM of HOE-140 (IC50 of 0.1 µM) and accounted for approximately 8% of total binding. The BK2 receptor density (Bmax) and affinity (Kd) were determined by the Ligand Program of Munson and Rodbard (23). All assays were performed three times in duplicate.
Western BlottingMembrane proteins (25 µg) and molecular weight markers were separated by SDS/polyacrylamide gel electrophoresis (10% running, 4% stacking gel) as described previously (5, 22). The proteins were electrophoretically transferred onto polyvinylidene difluoride membranes and incubated overnight at 4 °C with blocking solution (5% nonfat dry milk and 0.1% Tween 20 in PBS) prior to the addition of the following dilutions of specific rabbit polyclonal antisera: P4 (1:400), EC/2 (1:1000), QL (1:1000), and RM/1 (1:1000). The membranes were washed twice with PBS buffer containing 0.1% Tween 20 and then treated with donkey anti-rabbit horseradish peroxidase antibody (1:4000) (Amersham Corp.). Radiographic chemiluminescence was performed several times at 23 °C, and the appropriate exposures were subjected to densitometric analysis.
GTPase AssayMembranes (30 µg) from endothelial cells
treated with the indicated conditions for 24 h were incubated for
90 min at 22 °C in the presence or the absence of the indicated
specific COOH-terminal antisera prior to GTPase assay. Preliminary
studies revealed that maximal inhibition of receptor-G-protein coupling
was achieved by the antisera at the following dilutions: P4 (1:50),
EC/2 (1:50), QL (1:50), and RM/1 (1:100). The assay was initiated by
the addition of BK (10 nM) to the reaction mixture
consisting of [-32P]GTP (0.5 µM), GTP (2 µM), MgCl2 (5 mM), EGTA (0.1 mM), NaCl (50 mM), creatine phosphate (4 mM), phosphocreatine kinase (5 units), ATP (0.1 mM), dithiothreitol (1 mM), bacitracin (140 µg/ml), CaptoprilTM (1 µM), leupeptin (100 µg/ml),
aprotinin (50 µg/ml), 1,10-phenanthroline (1 mM), BSA
(0.2%), and triethanolamine HCl (50 mM, pH 7.4) in a total
volume of 0.1 ml. The reaction was allowed to proceed for 20 min at
22 °C and terminated with 500 µl of ice-cold 10% activated
charcoal in 50 mM phosphoric acid. The mixture was then
centrifuged for 10 min at 12,000 × g at 4 °C, and
300 µl of the supernatant containing the liberated
[32P]Pi was counted in a liquid scintillation
counter. Nonspecific activity was determined in the presence of GTP
S
(10 µM) and represented between 4 and 15% of total
activity. BK-stimulated GTPase activity was calculated as the
difference between total and nonspecific activity and expressed as
mol/min/mg of membrane protein. Assays were performed in duplicate with
less than 10% variation.
Endothelial NO production was measured by [3H]arginine to [3H]citrulline conversion as described previously (24). Briefly, confluent endothelial cells were treated with LNMA (5 mM), GSNO (0.5 mM), or dibutyryl-cGMP (0.1 mM) for 24 h. The culture medium was removed, and the cells were washed twice with PBS and placed in 10 ml of Krebs-Ringer buffer containing NaCl (118 mM), KCl (4.7 mM), CaCl2 (2.5 mM), MgSO4 (1.2 mM), KH2PO4 (1.2 mM), NaHCO3 (25 mM), and glucose (11 mM), pH 7.4. After 5 min, [3H]arginine (10 µCi) and L-arginine (10 µM) were added to the buffer for 10 min followed by stimulation with BK (10 nM) for an additional 10 min at 37 °C. The assay was terminated with ice-cold PBS containing L-arginine (5 mM) and EDTA (5 mM).
The supernatant was removed, and the cells were scraped and lysed by a probe sonicator (Model W185F, Ultrasonics, Inc., Plainview, NY). Approximately 4 ml of this cellular extract and supernatant was applied to a column containing 2 ml of Dowex 50WX-8 resin (pre-equilibrated with NaOH) followed by elution of [3H]citrulline with 2 ml of water. A sample of the elutant (1 ml) was counted for 2 min in a liquid scintillation counter (Beckman LS 1800). The Dowex columns extracted >95% of [3H]arginine and retained <8% of [3H]citrulline (24). Nonspecific activity was determined by [3H]citrulline production in the presence of excess L-arginine (5 mM) and represented approximately 7% of total activity. Cell number (5 × 106/T-150 cm2 culture flask) was determined using duplicate sets of confluent endothelial cells in another flask under corresponding treatment conditions followed by trypsinization and counting on a dispersion grid.
cGMP AssayThe basal and BK-stimulated intracellular cGMP production were determined by cGMP radioimmunoassay as described previously with some modifications (25). Briefly, confluent endothelial cells grown in 35-mm dishes were treated with LNMA (5 mM) or GSNO (0.5 mM) for 24 h. The medium was removed, and the cells were washed twice with PBS followed by incubation at 37 °C for 30 min in a buffer containing indomethacin (10 µM), 3-isobutyl-1-methylxanthine (1 mM), CaptoprilTM (10 µM), NaCl (154 mM), KCl (5.6 mM), CaCl2 (2.0 mM), MgCl2 (1.0 mM), NaHCO3 (3.6 mM), glucose (5.6 mM), and HEPES (10 mM, pH 7.4). The endothelial cells were then stimulated with BK (10 nM) for 5 min, the medium was rapidly removed, and the reaction was terminated with 1 ml of trichloroacetic acid (10%). Cells were disrupted by a probe sonicator and centrifuged for 10 min at 3000 × g. The supernatant was extracted twice with three volumes of water-saturated ether prior to lyophilization and resuspension in a sodium acetate buffer (50 mM, pH 6.2). The cGMP production was determined by a radioimmunoassay kit (Biomedical Technologies Inc., Stoughton, MA) using [125I]cGMP and expressed as picomoles/106 cells. Each experiment was performed in triplicate with corresponding standard curve in acetate buffer.
Immunoprecipitation of [35S]GTPMembrane proteins (30 µg) from control and
GSNO-treated endothelial cells were incubated for 30 min at 30 °C in
a buffer containing [35S]GTPS (20 nM), GTP
(2 µM), MgCl2 (5 mM), EGTA (0.1 mM), NaCl (50 mM), creatine phosphate (4 mM), phosphocreatine kinase (5 units), ATP (0.1 mM), dithiothreitol (1 mM), bacitracin (140 µg/ml), captopril (1 µM), leupeptin (100 µg/ml),
aprotinin (50 µg/ml), 1,10-phenanthroline (1 mM), BSA
(0.2%), and triethanolamine HCl (50 mM, pH 7.4). The assay
was initiated by the addition of BK (10 nM) and terminated
after 30 min with unlabeled GTP
S (100 µM). Samples
were then resuspended in 100 µl of immunoprecipitation buffer
containing Triton X-100 (1%), SDS (0.1%), NaCl (150 mM), EDTA (5 mM), Tris-HCl (25 mM, pH 7.4),
leupeptin (10 µg), aprotinin (10 µg), and phenylmethylsulfonyl
fluoride (2 mM). The following G-protein antisera with
their corresponding final dilutions were added to the mixture:
i2 (P4, 1:20),
i3 (EC/2, 1:100),
q/11 (QL, 1:100), and
s (RM/1,
1:100).
The samples were allowed to incubate for 16 h at 4 °C with
gentle mixing. The antibody-G-protein complexes were then incubated with 50 µl of protein A-Sepharose (1 mg/ml, Pharmacia Biotech Inc.)
for 2 h at 4 °C, and the precipitate was collected by
centrifugation at 12,000 × g for 10 min. Preliminary
studies indicated that all i2,3,
q/11,
and
s were completely precipitated by this procedure because Western blot analysis of the supernatant with the P4, EC/2, QL,
and RM/1 antisera did not reveal the presence of 40-41 kDa proteins.
The pellets were washed three times in a buffer containing HEPES (50 mM, pH 7.4), NaF (100 µM), sodium phosphate (50 mM), NaCl (100 mM), Triton X-100 (1%), and
SDS (0.1%). The final pellet containing the immunoprecipitated
[35S]GTP
S-labeled G-protein was counted in a liquid
scintillation counter (LS 1800, Beckman Instruments, Inc., Fullerton,
CA). Nonspecific activity was determined in the presence of unlabeled
GTP
S (100 µM).
Band intensities were analyzed densitometrically with the NIH Image program (26). All values are expressed as means ± S.E. compared with controls and among separate experiments. EC50 and IC50 values were calculated by linear or logarithmic extrapolation. Paired and unpaired Student's t tests were employed to determine any significant changes in values. A significant difference was taken for p values less than 0.05.
Relatively pure (>95%) bovine aortic endothelial cell cultures were confirmed by morphologic features and immunofluorescent staining with Factor VIII antibodies (results not shown). There were no observable adverse effects of GSNO, LNMA, or dibutyryl-cGMP on cell number, morphology, or immunofluorescent staining.
Effect of NO on BK2 Receptor DensityUntreated bovine aortic endothelial cell membranes contain 94 ± 8 fmol/mg of BK2 receptor with a Kd of 0.48 ± 0.07 nM. Treatment of endothelial cells with GSNO (10-500 µM) for 24 h did not affect total BK2 receptor density (Bmax of 95 ± 7 fmol/mg) or overall BK2 receptor affinity (Kd = 0.52 ± 0.1 nM) (p > 0.05 for both).
Effect of NO on G-protein ExpressionIn a
concentration-dependent manner, treatment with GSNO did not
significantly affect the amount of Gi2,
G
i3, and G
q/11 after 24 h as
determined by densitometric analysis of band intensities on three
separate Western blots (Fig.
1A). Similarly, in a
time-dependent manner, GSNO (500 µM) had no
effect on G
i2, G
i3, and
G
q/11 protein levels for up to 72 h (Fig.
1B). The P4 (
i2), EC/2 (
i3), and QL (
q/11) antisera were quite specific because
recognition of their respective
subunits could be blocked only in
the presence of excess decapeptides from which they were derived (5,
21). Treatment with the NO synthase inhibitor, LNMA (5 mM),
also did not affect the amount of any G-protein
subunits. In
addition, the amount of
s and common
subunit as
determined by the RM/1 and SW/1 antisera, respectively, was also
unaffected by GSNO (500 µM) or LNMA (5 mM).
Effect of NO on BK2 Receptor-G-protein Coupling
We have previously shown that the type II kinin
(BK2) receptor is the predominant BK receptor subtype in
bovine aortic endothelial cells (22). Radioligand binding studies
showed that untreated endothelial cell membranes contain two
BK2 receptor binding sites (Fig.
2A). The high affinity agonist
binding site that constitutes 32% of the total BK2
receptor sites has a Kd of 14 ± 3 pM and a Bmax of 27 ± 5 fmol/mg. The low affinity BK2 binding site that constitutes
68% of the total BK2 receptor sites has a
Kd of 480 ± 42 pM and a
Bmax of 67 ± 6 fmol/mg. Treatment with
increasing concentrations of GSNO (1, 10, 50, 100, 500, and 1000 µM) for 24 h progressively decreased the amount of
BK2 receptor high affinity binding site (IC50
value of 54 ± 11 µM) (Fig. 2B). Maximal
decrease in BK2 receptor high affinity binding site
occurred at a GSNO concentration of 500 µM, which
converted 74% of BK2 receptor high affinity agonist
binding sites (20 fmol/mg) to low affinity binding sites
(Kd of 520 ± 40 pM,
Bmax of 87 ± 5 fmol/mg) (Fig.
2A). Complete conversion of BK2 receptor high affinity agonist binding sites to low affinity sites
(Kd of 520 ± 64 pM,
Bmax of 94 ± 5 fmol/mg) was observed in
the presence of the nonhydrolyzable GTP analogue, GTPS (10 µM).
Effect of NO on BK-stimulated GTPase Activity and cGMP Production
In a concentration-dependent manner,
stimulation of endothelial cell membranes with of BK (0.1-100
nM) produced a progressive increase in GTPase activity with
maximal activity (15.0 ± 2.0 pmol/min/mg) occurring at a BK
concentration of 10 nM (Fig.
3A). The EC50
value for BK-stimulated GTPase activity was 2.4 ± 0.4 nM. When membranes from endothelial cells were treated with
increasing concentrations of GSNO (10-500 µM), there was
a progressive decrease in BK-stimulated GTPase activity (Fig.
3B). The calculated IC50 for GSNO by logarithmic
extrapolation was 32 ± 6 µM. At a GSNO concentration of 500 µM, a maximal 85% reduction in
BK-stimulated GTPase activity (2.3 ± 0.7 pmol/min/mg) was
observed (p < 0.01).
Untreated or control endothelial cells have a basal cGMP production of
0.24 ± 0.08 pmol/106 cells. Stimulation with
increasing concentrations of BK (0.1-100 nM) produced a
progressive increase in cGMP levels with an EC50 of
1.9 ± 0.3 nM and a maximal 10.3-fold increase in cGMP
production at a BK concentration of 10 nM (2.5 ± 0.3 pmol/106 cells, p < 0.001) (Fig.
4A). Inhibition of endothelial
NO synthase by 5 mM of LNMA (IC50 of 0.8 ± 0.1 mM) resulted in 63 and 92% reductions in the
corresponding basal and BK-stimulated cGMP levels (0.09 ± 0.04 and 0.21 ± 0.07 pmol/106 cells, respectively)
(p < 0.05 for both) (Fig. 4B). Endothelial cells treated with 500 µM of GSNO (EC50 of
44 ± 6 µM) showed a maximal 6-fold increase in
basal cGMP levels after 24 h (0.24 ± 0.08 pmol/106 cells to 1.5 ± 0.2 pmol/106
cells, p < 0.01). However, stimulation with BK (10 nM) did not result in any further increase in cGMP
production from basal levels in GSNO-treated cells (1.8 ± 0.2 pmol/106 cells, p > 0.05). Compared with
untreated or control cells, GSNO-treated cells showed a 28% decrease
in BK-stimulated cGMP production (2.5 ± 0.3 pmol/106
cells versus 1.8 ± 0.2 pmol/106 cells,
p < 0.05).
Effect of NO and cGMP on BK-stimulated GTPase and NO Synthase Activity
To determine whether endogenous NO, GSNO, or
dibutyryl-cGMP can regulate BK-stimulated G-protein and NO synthase
activity, endothelial cells were treated with 5 mM of LNMA
(EC50 of 0.6 ± 0.1 mM), 500 µM of GSNO (EC50 of 45 ± 6 µM), or 100 µM of dibutyryl-cGMP (EC50 of 23 ± 5 µM) for 24 h.
Control endothelial cells have a BK-stimulated GTPase and NO synthase
activity of 15 ± 2.0 pmol/min/mg and 23 ± 2.1 pmol/min/107 cells, which represent a 3- and 11-fold
increase from basal GTPase and NO synthase activity, respectively (Fig.
5).
Treatment with LNMA (5 mM) caused a maximal 86% reduction in endothelial NO synthase activity (2.0 ± 0.9 pmol/min/107 cells, p < 0.001) (Fig. 5). This inhibition of endothelial NO synthase activity by LNMA corresponded to a 61% increase in BK-stimulated GTPase activity (24 ± 3.4 pmol/min/mg, p < 0.05), suggesting that endogenous endothelial NO production serves to tonically inhibit BK-stimulated G-protein activity.
Pretreatment with GSNO (500 µM, 24 h) caused a 81 ± 5% and 77 ± 4% decrease in BK-stimulated GTPase and NO synthase activity, respectively (p < 0.001 for both) (Fig. 5). Comparable 80 ± 5% and 75 ± 5% decrease in BK-stimulated GTPase and NO synthase activity were observed when endothelial cells were treated with dibutyryl-cGMP (100 µM, 24 h) (p > 0.05 for both values when compared with GSNO treatment). These findings suggest that the mechanism by which endogenous and exogenous NO exerts its inhibitory effects on BK-stimulated G-proteins and NO synthase is mediated through cGMP-dependent pathways.
Effect of NO and cGMP on BK-stimulated G-protein ActivityIn
control (untreated) endothelial cells, basal GTPase activity was
4.8 ± 0.5 pmol/min/mg. Stimulation with BK (10 nM)
produced a maximal 3.1-fold increase in GTPase activity (15 ± 3.7 pmol/min/mg, p < 0.01) (Fig.
6A). Treatment with 500 µM of GSNO (EC50 of 45 ± 6 µM) caused a greater increase in basal GTPase activity
than treatment with 100 µM of dibutyryl-cGMP
(EC50 of 25 ± 5 µM) (7.2 ± 0.6 versus 6.0 ± 0.4 pmol/min/mg, p < 0.05). However, treatment with either GSNO or dibutyryl-cGMP completely
inhibited BK-stimulated GTPase activities to their corresponding basal
levels (6.8 ± 0.5 and 6.5 ± 0.7 pmol/min/mg, respectively,
p < 0.05).
To determine whether GSNO inhibited BK-stimulated GTPase activity via
direct uncoupling of BK2 receptor from Gi and
Gq, we performed BK- and epinephrine-stimulated GTPase
assays on membranes from untreated (control) or GSNO-treated cells in
the presence of antibodies directed against the carboxyl terminus of
specific G-protein subunits. We have previously shown that 1:50
dilutions of P4, EC/2, and QL antisera maximally and specifically
uncouple
i2,
i3, and
q/11
from the BK2 receptor in bovine aortic endothelial cells,
respectively (5, 21). Furthermore, the effects of these antibodies can
be reversed only in the presence of excess peptides from which the
antibodies were generated.
In control or untreated cells, BK-stimulated GTPase activity was
decreased by 42 ± 3, 52 ± 6, and 66 ± 6% in the
presence of P4 (i2), EC/2 (
i3), and QL
(
q/11), respectively (p < 0.05 for all)
(Fig. 6B). The combination of P4, EC/2, and QL reduced BK-stimulated GTPase activity by 95 ± 3% (p < 0.01). The RM/1 antibody (
s) had no effect on
BK-stimulated GTPase activity but did decrease isoproterenol (10 µM)-stimulated GTPase activity by 79 ± 4% in
control cells (p < 0.01). In cells treated with GSNO
(500 µM, 24 h), BK-stimulated GTPase activity was
reduced by 78 ± 3% (p < 0.01), whereas
isoproterenol-stimulated GTPase activity was completely unaffected
(p > 0.05). The addition of P4 or QL antibody to
membranes from GSNO-treated cells did not cause any further decrease in
BK-stimulated GTPase activity (79 ± 2 and 81 ± 3%
decreases, respectively, p > 0.05), whereas the addition of EC/2 or the combination of all three antibodies did produce
a further decrease in BK-stimulated GTPase activity (84 ± 2 and
96 ± 2%, respectively, p < 0.05). These
findings indicate that treatment with GSNO produced similar inhibitory
effects on BK-stimulated GTPase activity as the combination of P4,
EC/2, and QL antibodies. The addition of RM/1 antisera inhibited
isoproterenol-stimulated GTPase activity in both control and
GSNO-treated cells (79 ± 4 and 80 ± 3% decreases,
respectively, p < 0.05).
Immunoprecipitation of
[35S]GTPS-labeled G-proteins with antisera directed
against specific
subunits demonstrated that treatment with 500 µM of GSNO (EC50 of 42 ± 6 µM) alone for 24 h produced a maximal increase in
basal
i2,
i3,
q/11, and
s GTP binding activity (18 ± 2, 22 ± 3, 24 ± 2, and 26 ± 3%, respectively). These findings
indicate that both NO and cGMP stimulate the basal activities of all
G-proteins but paradoxically inhibit only BK-stimulated Gi
and Gq without affecting epinephrine-stimulated
Gs.
In untreated endothelial cell membranes, i2,
i3, and
q/11 accounted for 27% (2.8 ± 0.1 fmol/min/mg or 3880 ± 125 cpm), 29% (3.0 ± 0.5 fmol/min/mg or 4160 ± 693 cpm), and 44% (4.5 ± 0.6 fmol/mg/min or 6240 ± 832 cpm) of BK-stimulated
subunit GTP
binding activities, respectively (Fig.
7A). In membranes pretreated with GSNO (500 µM) for 24 h, BK-stimulated
i2 GTP binding activity was reduced by 93% (0.2 ± 0.1 fmol/min/mg or 280 ± 130 cpm, p < 0.01).
Similarly, treatment with GSNO (0.5 mM) for 24 h
resulted in 61 and 90% decreases in BK-stimulated
i3
(1.2 ± 0.2 fmol/min/mg or 1660 ± 277 cpm, p < 0.05) and
q/11 (0.4 ± 0.1 fmol/mg/min or
5540 ± 139 cpm, p < 0.01) GTP binding activity,
respectively.
Treatment with GSNO (500 µM) for 24 h, however, did
not significantly affect epinephrine-stimulated s GTP
binding activity (Fig. 7B). In untreated endothelial cell
membranes, epinephrine (0.1 mM) produced a 3-fold increase
in
s GTP binding activity from 2.3 ± 0.3 fmol/mg/min (3120 ± 360 cpm) to 6.9 ± 0.5 fmol/mg/min (9490 ± 624 cpm) (p < 0.005). Treatment with
GSNO (0.5 mM) alone for 24 h caused a 30% increase in
basal
s GTP binding activity (3.0 ± 0.4 fmol/mg/min or 4090 ± 513 cpm, p < 0.05). In
membranes treated with GSNO, stimulation with epinephrine did not
produce a significant change in
s GTP binding activity
compared with that of untreated membranes (6.8 ± 0.8 fmol/min/mg
or 9480 ± 1070 cpm, p > 0.05).
The findings in this study indicate that a brief 24-h exposure of
endothelial cells to exogenous NO attenuates BK-stimulated Gi and Gq protein activity. The BK2
receptor-G-protein coupling was inhibited by GSNO treatment as
demonstrated by reductions in BK-stimulated high affinity binding sites
and GTPase activity. This inhibitory effect of NO was relatively
specific because epinephrine-stimulated s GTP binding
activity was relatively unaffected. There were no observable changes in
the density of BK2 receptor or the amounts of
Gi, Gq, or Gs, suggesting that NO
inhibited G-protein function rather than expression. These findings,
therefore, suggest that NO can preferentially inhibit the function of
G-proteins that are coupled to the BK2 receptor in
endothelial cells.
In this study, GSNO was selected as the NO donor because of its relatively long half-life compared with other shorter acting NO donors such as sodium nitroprusside and 3-morpholinosydnonimine (27). In addition, sodium nitroprusside and 3-morpholinosydnonimine can also release cyanide and superoxide anion in addition to NO and therefore are relatively more toxic than GSNO at comparable concentrations (28). Furthermore, the precursors of GNSO, sodium nitrite and glutathione, have no effect on BK-stimulated G-protein function at GSNO concentrations comparable with those used in this study.2 Because the level of NO encountered during inflammatory conditions are in the micromolar range consistent with the activation of inducible NO synthase from macrophages and smooth muscle cells, the amount of NO released from GSNO under our experimental conditions would be comparable with the relatively high levels of endogenous NO produced during inflammation (13-15).
Inhibition of endogenous endothelial NO synthase activity with LNMA resulted in an increase in BK-stimulated GTPase activity, suggesting that constitutive endothelial NO production can tonically and negatively regulate Gi and Gq activity. The relatively low GSNO concentration (i.e. IC50 of 32 µM) required to inhibit BK-stimulated G-protein activity makes it likely that endogenous NO can physiologically modulate the sensitivity of specific G-proteins to ligand stimulation. Indeed, recent studies have demonstrated that endothelial NO synthase is located within close proximity to putative G-proteins in the caveolae of plasma membranes (29).
The relative contributions of Gi2, G
i3,
and G
q/11 to BK-stimulated GTPase activity are
consistent with our previous study demonstrating that the endothelial
BK2 receptor is coupled predominantly to
G
q/11 (5). The mechanism by which NO inhibits
BK-stimulated Gi and Gq probably occurs through
the activation of guanylyl cyclase because endogenous NO synthase
activity and cGMP levels correlate inversely with BK-stimulated
Gi and Gq activities. Furthermore, the
permeable cGMP analogue, dibutyryl-cGMP, produced similar levels of
inhibition on BK-stimulated G-protein activity as NO donors.
Although both GSNO and cGMP analogues attenuated BK-stimulated G-protein activity, we found that they also nonspecifically increased basal G-protein activity by approximately 20%. The direct activation of heterotrimeric G-proteins by NO donors, however, may not occur exclusively via the stimulation of cGMP production because treatment with dibutyryl-cGMP produced a lower level of G-protein activation compared with that of GSNO. Indeed, previous studies in peripheral blood mononuclear cells showed that NO donors can directly activate heterotrimeric G-proteins and p21ras, via S-nitrosylation of these signaling molecules (17, 18). Furthermore, S-nitrosylation of terminal cysteine residues of the neuronal heterotrimeric G-protein, Go renders Go less susceptible to ADP-ribosylation by pertussis toxin (30). It remains to be determined, however, whether S-nitrosylation of G-proteins has any affect on agonist-stimulated G-protein activity.
The ability of NO to modulate BK receptor ligand binding affinity via
effects on specific G-protein activities could have important
biochemical and physiological consequences. Because both NO and
bradykinin are released under certain inflammatory conditions, NO may
function as an important autocrine and paracrine inhibitor of
BK-mediated processes including the release of BK-stimulated NO from
vascular endothelial cells. The conversion of high to low affinity BK
receptor sites by NO is similar to the effects of nonhydrolyzable GTP
analogues such as GTPS, which uncouples the BK receptor from its
G-proteins (5, 20-22). Thus, it is conceivable that NO may modify
critical cysteine residues on
i and
q but
not
s, which are important in regulating GTP binding and
hydrolysis. Alternatively, we cannot exclude the possibility that NO
affects
subunits whose association with the
subunit is
required to generate the formation of high affinity BK ligand binding
sites (31). However, the role of
subunit in mediating the
inhibitory effects of NO is less likely given that specific antibodies
to the carboxyl terminus of
subunits produce similar inhibitory
effects as NO. Finally, it is possible that NO may directly modify the
BK receptor but not the
2-adrenergic receptor, particularly in the region of the third cytoplasmic loop and carboxyl terminus, which are known to interact with G-proteins (32). It remains
to be determined, however, whether such modifications, if any, could
alter BK receptor-G-protein coupling.
Many BK-mediated inflammatory processes such as mucous hypersecretion
and smooth muscle contraction occur via phosphatidylinositol 4,5-bisphosphate hydrolysis and elevation of intracellular calcium (33,
34). We have previously shown that the G-proteins of the Gi
and Gq family couple the BK2 receptor to the
stimulation of phospholipase C and generation of inositol
1,4,5-trisphophate in endothelial cells (5). Thus, the findings of this
study suggest that NO may counteract many of the inflammatory responses elicited by BK through inhibition of BK2 receptor-coupled
G-proteins. Interestingly, a recent study indicates that NO can also
inhibit growth factor-mediated phospholipase C activation via a
cGMP-dependent protein kinase I pathway (35). Thus,
NO-induced increases in intracellular cGMP levels may not only modulate
BK signaling pathways at the level of heterotrimeric G-proteins but
also may affect downstream effectors such as the and
isoforms
of phospholipase C.
Clinically, NO may have a bronchoprotective role in allergy-induced asthma, in part, by alleviating BK-mediated bronchoconstriction (36, 37). NO causes bronchial smooth muscle relaxation through direct stimulation of soluble guanylyl cyclase (38). Furthermore, NO may block BK-mediated inflammatory responses and bronchial smooth muscle contraction by inhibiting BK2 receptor-coupled G-proteins. Because NO inhibits Gi but not Gs, it could also facilitate bronchial smooth muscle relaxation through its permissive action of the Gs-adenylyl cyclase pathway. Indeed, a recent randomized double-blind placebo-controlled trial showed that bronchoconstriction after BK inhalation is attenuated by endogenous NO production in the bronchial airways (39).
In summary, we have identified a potentially important effect of NO on BK signaling pathways. Our findings indicate that NO can attenuate BK receptor ligand binding affinity and its coupled G-proteins via cGMP-dependent pathway(s). It remains to be determined how NO actually inhibits Gi and Gq but not Gs and whether these effects are mediated through cGMP-dependent or redox-sensitive pathways.