Modulation of Bradykinin Receptor Ligand Binding Affinity and Its Coupled G-proteins by Nitric Oxide*

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 ω-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]GTPγS 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 Gαi2, Gαi3, and Gαq/11 GTP binding activity by 93, 61, and 90%, respectively, whereas epinephrine-stimulated Gαs GTP binding activity was unaffected. These results suggest that NO can modulate BK signaling pathways by selectively inhibiting G-proteins of the Gi and Gqfamily.

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 transmembranespanning receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) (3,4). We have previously shown that bovine aortic endothelial cells contain predomi-nantly the type II kinin (BK 2 ) receptor that is coupled to G-proteins of the G i and G q family (5). Both G i and G q can activate phosphoinositide-specific phospholipase C, which mobilizes intracellular calcium via the hydrolysis of phosphatidylinositol 4,5-bisphosphate (5)(6)(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 G i proteins and phospholipase A 2 by BK leads to the production of arachidonic acids and leukotrienes, which are important in mediating the inflammatory response (9). In addition, stimulation of G i 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 G sadenylyl cyclase pathway, are often administered to alleviate anaphylactic reactions (12). Thus, factors that modulate BK 2 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)(14)(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 p21 ras 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 BK 2 receptor.
Cell Culture-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 g/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 (ϳ5 ϫ 10 6 ) 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 Studies-Partially purified membranes were prepared from control and GSNO-treated endothelial cells as described previously (5). Membranes (100 g) were added to 12 concentrations of [ 3 H]BK (1 pM to 10 nM) in a buffer containing Tris-HCl (100 mM, pH 7.4), MgCl 2 (5 mM), EDTA (0.6 mM), bacitracin (140 g/ml), Captopril (1 M), 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 BK 2 subtype (22). Nonspecific binding was determined in the presence of 10 M of HOE-140 (IC 50 of 0.1 M) and accounted for approximately 8% of total binding. The BK 2 receptor density (B max ) and affinity (K d ) were determined by the Ligand Program of Munson and Rodbard (23). All assays were performed three times in duplicate.
GTPase Assay-Membranes (30 g) 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 COOHterminal 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 [␥-32 P]GTP (0.5 M), GTP (2 M), MgCl 2 (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 g/ml), Captopril (1 M), leupeptin (100 g/ml), aprotinin (50 g/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 l of ice-cold 10% activated charcoal in 50 mM phosphoric acid. The mixture was then centrifuged for 10 min at 12,000 ϫ g at 4°C, and 300 l of the supernatant containing the liberated [ 32 P]P i was counted in a liquid scintillation counter. Nonspecific activity was determined in the presence of GTP␥S (10 M) 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.
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 [ 3 H]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 Ͼ95% of [ 3 H]arginine and retained Ͻ8% of [ 3 H]citrulline (24). Nonspecific activity was determined by [ 3 H]citrulline production in the presence of excess L-arginine (5 mM) and represented approximately 7% of total activity. Cell number (5 ϫ 10 6 /T-150 cm 2 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 Assay-The 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 M), 3-isobutyl-1-methylxanthine (1 mM), Captopril (10 M), NaCl (154 mM), KCl (5.6 mM), CaCl 2 (2.0 mM), MgCl 2 (1.0 mM), NaHCO 3 (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 ϫ 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 [ 125 I]cGMP and expressed as picomoles/10 6 cells. Each experiment was performed in triplicate with corresponding standard curve in acetate buffer. 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 l 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 ϫ 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 M), sodium phosphate (50 mM), NaCl (100 mM), Triton X-100 (1%), and SDS (0.1%). The final pellet containing the immunoprecipitated [ 35 S]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 M).

Immunoprecipitation of [ 35 S]GTP␥S-labeled G-proteins-Membrane
Data Analysis-Band intensities were analyzed densitometrically with the NIH Image program (26). All values are expressed as means Ϯ S.E. compared with controls and among separate experiments. EC 50 and IC 50 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.

RESULTS
Cell Culture-Relatively pure (Ͼ95%) 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 BK 2 Receptor Density-Untreated bovine aortic endothelial cell membranes contain 94 Ϯ 8 fmol/mg of BK 2 receptor with a K d of 0.48 Ϯ 0.07 nM. Treatment of endothelial cells with GSNO (10 -500 M) for 24 h did not affect total BK 2 receptor density (B max of 95 Ϯ 7 fmol/mg) or overall BK 2 receptor affinity (K d ϭ 0.52 Ϯ 0.1 nM) (p Ͼ 0.05 for both).
Effect of NO on G-protein Expression-In a concentration-dependent manner, treatment with GSNO did not significantly affect the amount of G␣ i2 , 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 M) 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 M) or LNMA (5 mM).
Effect of NO on BK 2 Receptor-G-protein Coupling-We have previously shown that the type II kinin (BK 2 ) receptor is the predominant BK receptor subtype in bovine aortic endothelial cells (22). Radioligand binding studies showed that untreated endothelial cell membranes contain two BK 2 receptor binding sites ( Fig. 2A). The high affinity agonist binding site that constitutes 32% of the total BK 2 receptor sites has a K d of 14 Ϯ 3 pM and a B max of 27 Ϯ 5 fmol/mg. The low affinity BK 2 binding site that constitutes 68% of the total BK 2 receptor sites has a K d of 480 Ϯ 42 pM and a B max of 67 Ϯ 6 fmol/mg. Treatment with increasing concentrations of GSNO (1, 10, 50, 100, 500, and 1000 M) for 24 h progressively decreased the amount of BK 2 receptor high affinity binding site (IC 50 value of 54 Ϯ 11 M) (Fig. 2B). Maximal decrease in BK 2 receptor high affinity binding site occurred at a GSNO concentration of 500 M, which converted 74% of BK 2 receptor high affinity agonist binding sites (20 fmol/mg) to low affinity binding sites (K d of 520 Ϯ 40 pM, B max of 87 Ϯ 5 fmol/mg) ( Fig. 2A). Complete conversion of BK 2 receptor high affinity agonist binding sites to low affinity sites (K d of 520 Ϯ 64 pM, B max of 94 Ϯ 5 fmol/mg) was observed in the presence of the nonhydrolyzable GTP analogue, GTP␥S (10 M).
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 Ϯ 2.0 pmol/min/mg) occurring at a BK concentration of 10 nM (Fig. 3A). The EC 50 value for BK-stimulated GTPase activity was 2.4 Ϯ 0.4 nM. When membranes from endothelial cells were treated with increasing concentrations of GSNO (10 -500 M), there was a progressive decrease in BKstimulated GTPase activity (Fig. 3B). The calculated IC 50 for GSNO by logarithmic extrapolation was 32 Ϯ 6 M. At a GSNO concentration of 500 M, a maximal 85% reduction in BKstimulated GTPase activity (2.3 Ϯ 0.7 pmol/min/mg) was observed (p Ͻ 0.01).
Untreated or control endothelial cells have a basal cGMP production of 0.24 Ϯ 0.08 pmol/10 6 cells. Stimulation with increasing concentrations of BK (0.1-100 nM) produced a progressive increase in cGMP levels with an EC 50 of 1. Treatment with LNMA (5 mM) caused a maximal 86% reduction in endothelial NO synthase activity (2.0 Ϯ 0.9 pmol/min/ 10 7 cells, p Ͻ 0.001) (Fig. 5). This inhibition of endothelial NO synthase activity by LNMA corresponded to a 61% increase in BK-stimulated GTPase activity (24 Ϯ 3.4 pmol/min/mg, p Ͻ 0.05), suggesting that endogenous endothelial NO production serves to tonically inhibit BK-stimulated G-protein activity.
Pretreatment with GSNO (500 M, 24 h) caused a 81 Ϯ 5% and 77 Ϯ 4% decrease in BK-stimulated GTPase and NO synthase activity, respectively (p Ͻ 0.001 for both) (Fig. 5). Comparable 80 Ϯ 5% and 75 Ϯ 5% decrease in BK-stimulated GTPase and NO synthase activity were observed when endothelial cells were treated with dibutyryl-cGMP (100 M, 24 h) (p Ͼ 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.
To determine whether GSNO inhibited BK-stimulated GTPase activity via direct uncoupling of BK 2 receptor from G i and G q , we performed BK-and epinephrine-stimulated GTPase assays on membranes from untreated (control) or GSNOtreated 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 BK 2 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.
Effects of NO on Agonist-stimulated GTP Binding Activity-Immunoprecipitation of [ 35 S]GTP␥S-labeled G-proteins with antisera directed against specific ␣ subunits demonstrated that treatment with 500 M of GSNO (EC 50 of 42 Ϯ 6 M) alone for 24 h produced a maximal increase in basal ␣ i2 , ␣ i3 , ␣ q/11 , and ␣ s GTP binding activity (18 Ϯ 2, 22 Ϯ 3, 24 Ϯ 2, and 26 Ϯ 3%, respectively). These findings indicate that both NO and cGMP stimulate the basal activities of all G-proteins but paradoxically inhibit only BK-stimulated G i and G q without affecting epinephrine-stimulated G s .

DISCUSSION
The findings in this study indicate that a brief 24-h exposure of endothelial cells to exogenous NO attenuates BK-stimulated G i and G q protein activity. The BK 2 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 BK 2 receptor or the amounts of G i , G q , or G s , 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 BK 2 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)(14)(15).
Inhibition of endogenous endothelial NO synthase activity with LNMA resulted in an increase in BK-stimulated GTPase activity, suggesting that constitutive endothelial NO produc-2 A. Miyamoto and J. K. Liao, unpublished observation. FIG. 6. A, basal (no stimulation) and BK (10 nM)-stimulated GTPase assay was performed on membranes obtained from endothelial cells untreated (Control) or pretreated with GSNO (500 M) or dibutyryl-cGMP (100 M) for 24 h. *, represents a significant difference compared with basal control. **, represents a significant difference between unstimulated (Basal) membranes from GSNO-and dibutyryl-cGMP-treated endothelial cells. B, BK (10 nM)or isoproterenol (ISO, 10 M)-stimulated GTPase activity in membranes from untreated (Control) and GSNO (500 M)-treated cells in the absence (None) or the presence of antibodies to the carboxyl terminus of ␣ i2 (P4), ␣ i3 (EC/2), ␣ q (QL), ␣ s (RM/1), or a combination of ␣ i2 , ␣ i3 , and ␣ q (ALL). *, represents a significant difference between control and GSNO-treated cells for a given antibody condition. **, represents a significant difference compared with no antibody treatment (None) for BK or isoproterenol.
tion can tonically and negatively regulate G i and G q activity. The relatively low GSNO concentration (i.e. IC 50 of 32 M) 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 G␣ i2 , G␣ i3 , and G␣ q/11 to BKstimulated GTPase activity are consistent with our previous study demonstrating that the endothelial BK 2 receptor is coupled predominantly to G␣ q/11 (5). The mechanism by which NO inhibits BK-stimulated G i and G q probably occurs through the activation of guanylyl cyclase because endogenous NO synthase activity and cGMP levels correlate inversely with BKstimulated G i and G q 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 p21 ras , via S-nitrosylation of these signaling molecules (17,18). Furthermore, S-nitrosylation of terminal cysteine residues of the neuronal heterotrimeric G-protein, G o renders G o 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 GTP␥S, 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 G i and G q family couple the BK 2 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 BK 2 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 allergyinduced 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 BK 2 receptor-coupled G-proteins. Because NO inhibits G i but not G s , it could also facilitate bronchial smooth muscle relaxation through its permissive action of the G s -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 G i and G q but not G s and whether these effects are mediated through cGMPdependent or redox-sensitive pathways.