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J Biol Chem, Vol. 274, Issue 37, 26337-26343, September 10, 1999

Nitric Oxide Modulates ₂-Adrenergic Receptor Palmitoylation and Signaling^*

Lynda Adam, Michel Bouvier^§, and Teresa L. Z. Jones^¶

From the Département de Biochimie and Le Groupe de Recherche sur le Système Nerveux Autonome, Université de Montréal, Montréal, Quebec H3T 1J4, Canada and ^¶ Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To determine whether nitric oxide (NO) modulates the -adrenergic signaling pathway, we treated cells expressing ₂-adrenergic receptors (₂AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4-oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to -adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the -adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and G_s, respectively. Treatment with the NO donor increased the guanyl nucleotide-sensitive high affinity constant for the agonist, isoproterenol, thus suggesting that it reduced functional coupling between the receptor and G_s. Stimulation of endogenous NO production by lipopolysaccharide in RAW 264.7 macrophages also caused a significant increase in the EC₅₀ for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [³H]palmitate into the ₂AR. Signaling through the nonpalmitoylated, Gly³⁴¹₂AR mutant was unchanged by SIN-1 treatment. Given the link between ₂AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the ₂AR resulting in decreased signaling through the ₂AR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric oxide (NO)¹ is a biologic signal involved in vasodilatation, neurotransmission, and immune defense (1, 2). It is an unstable gas that diffuses across membranes, lacks specific receptors, and is rapidly inactivated by a chemical reaction. Its synthesis by the enzyme NO synthase (NOS) from arginine and oxygen is finely regulated and can be achieved through both calcium-dependent and calcium-independent pathways (3). Both the calcium-dependent endothelial NOS and the calcium-independent inducible NOS (iNOS or NOS2) have been shown to play an important role in the control of vascular tone in normal and inflammatory conditions (4-8), respectively. NO can modulate the action of various vasoactive hormones and transmitters (7). In particular, NO-mediated decreases in -adrenergic responsiveness have been described (6, 9). However, the biochemical processes underlying this effect remain largely unexplored.

At the cellular level, the best characterized effect of NO is the activation of guanylate cyclase by the formation of a heme-NO complex that enhances the catalytic activity of the enzyme and increases cGMP production (10). Although several of the effects of NO have been attributed to cGMP formation, NO also leads to nitrosylation of thiol groups on cysteine residues (3, 11) that may contribute to the diverse physiological actions of this gaseous second messenger. Indeed, nitrosylation has been suggested to modulate protein function as a consequence of conformational changes (3, 11), facilitation of ADP-ribosylation (12), and inhibition of protein palmitoylation (13).

The ₂AR and its cognate G protein (G_s) both undergo palmitoylation on cysteine residues. This post-translational modification is dynamically regulated on the receptor and G_s upon -adrenergic stimulation (14, 15). Moreover, it has been proposed that regulation of the palmitoylation state of these proteins may have important effects on their biological activity (16-18). The aims of the present study were to determine if NO can directly modulate the signaling efficiency of the -adrenergic-G_s-adenylyl cyclase signaling pathway and to assess if regulation of the palmitoylation state of the receptor or G_s could play a role in such a regulatory process. We found that NO reduces the potency of a -adrenergic agonist to stimulate adenylyl cyclase and promotes depalmitoylation of the ₂AR.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Cell culture supplies were from Biofluids, Rockville, MD; COS7 cells were from American Type Culture Collection; [9,10-³H]palmitate (60 Ci/mmol) and [8-³H]adenine (18 Ci/mmol) were from American Radiolabeled Chemicals; pluronic acid was from Life Technologies, Inc.; SIN-1 was from Biomol and Alexis Corp.; GEA and N^G-monomethyl-L-arginine (L-NMMA) were from Alexis Corp.; 8-bromo-cGMP, norepinephrine, and epinephrine were from Research Biochemical International; forskolin, isoproterenol, terbutaline, procaterol, hemoglobin-Ao, IBMX, alumina, and lipopolysaccharide (LPS) were from Sigma; ₂-macroglobulin was from Roche Molecular Biochemicals; protein A-Sepharose CL-4B was from Amersham Pharmacia Biotech; cholera toxin was from List Biologicals; and AG 50W-X4 (Dowex) resin was from Bio-Rad.

Construction of Mutated ₂AR cDNA and Cell Transfection-- The wild type ₂AR was inserted in the modified pcDNA3, with an RSV promoter, at BamHI and EcoRI sites. The generation of pcDNA3-RSV-Gly³⁴¹₂AR, in which cysteine 341 was replaced by a glycine, was constructed by site-directed mutagenesis using wild type pcDNA3-RSV-₂AR (19). Positive clones were confirmed by dideoxy sequencing. The wild type ₂AR and the mutant were stably transfected in human embryonic kidney 293 (HEK293) cells line using the calcium phosphate precipitation method (20). Neomycin-resistant cells were selected by culturing in medium containing G418 (450 µg/ml) (Life Technologies, Inc.). Resistant clones were then screened for ₂AR expression by radioligand assays using ¹²⁵I-labeled iodocyanopindolol (CYP) as the ligand. COS, NIH 3T3, and HEK293 cells were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Macrophage RAW 264.7 cells were grown in DMEM with 10% FBS without antibiotic. Sf9 cells were grown at 27 °C in Grace's insect medium supplemented with 10% FBS (10%) and pluronic acid to prevent cell tearing due to agitation. They were infected with recombinant baculovirus encoding the wild type human ₂AR at a density of 1 × 10⁶ to 2 × 10⁶ cells per ml for 48 h with multiplicities of infection varying from 2 to 6 (21).

Metabolic Labeling and Cell Fractionation-- For metabolic labeling with [³H]palmitate, COS7 and HEK293 cells were incubated in serum-free medium for 2 and 1 h, respectively. The cells were then labeled with [³H]palmitate (200 to 500 µCi/ml) for 1 h. They were rinsed twice in cold phosphate-buffered saline (PBS), pH 7.4, to stop the labeling and centrifuged at 2000 × g for 10 min at 4 °C. Cell pellets were then stored at 70 °C. HEK293 cells were thawed in cold lysis buffer (20 mM Tris-HCl, 5 mM EDTA, pH 7.4) containing a protease inhibitor mixture (5 µg/ml leupeptin, 5 µg/ml trypsin inhibitor, and 10 µg/ml benzamidine), disrupted by sonication, and the lysates centrifuged 5 min at 500 × g at 4 °C. The supernatants were then centrifuged at 45,000 × g for 20 min at 4 °C. Pelleted membranes were solubilized for 90 min at 4 °C in 10 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4, containing 0.3% n-dodecyl maltoside and the above protease inhibitor mixture. COS cells were thawed in buffer composed of 5 mM Hepes, pH 7.4, 50 mM mannitol, 100 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM EDTA, 0.7 µg/ml pepstatin, and 10 milliunits/ml ₂-macroglobulin. They were homogenized by passage through a 25-gauge needle 15 times. The cell lysate was centrifuged at 3000 × g for 3 min. The supernatant was centrifuged at 125,000 × g for 1 h at 4 °C, and the resulting pellet was resuspended in homogenization buffer. Protein concentrations of the cellular fractions were determined by the Bio-Rad Protein Assay Dye kit with IgG as the standard or the method of Bradford (22), using bovine serum albumin as the standard.

Treatment with NO Donors-- 3-Morpholinosydnonimine (SIN-1) and 1,2,3,4-oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)-chloride (GEA) were used to generate NO. Cells were incubated with 1 mM SIN-1 or 1 mM GEA for the indicated time. When in aqueous solution, SIN-1 degrades spontaneously to SIN-1A and subsequently to SIN-1C, with NO being generated in the latter step (23). At 37 °C, the rate constants governing these steps are 0.11 and 0.04 min¹, respectively (23), and the half-life of NO is about 10 s (24). NO-depleted SIN-1 was thus generated by keeping a 100 mM solution of SIN-1 at room temperature or at 37 °C for at least 24 h.

Immunoprecipitation of Gs-- The polyclonal, affinity purified RM antibody, which recognizes the carboxyl-terminal decapeptide of Gs, was used (25). Immunoprecipitation was performed on equal amounts of the particulate fraction (0.5-1 mg of total protein) in a solubilization buffer consisting of 50 mM Tris-HCl, pH 7.5 (25 °C), 150 mM NaCl, 1% (w/v) Triton X-100, 0.2% (w/v) SDS, and 1 mM EDTA. The immunoprecipitates were recovered by incubation with protein A-Sepharose, washed, and prepared for SDS-PAGE (23). Fixed gels were treated for 45 min with En³Hance (NEN Life Science Products), dried, and exposed to XAR-2 film (Kodak) at 70 °C.

Purification of ₂AR-- Alprenolol-Sepharose affinity purification matrix was synthesized according to the method of Benovic et al. (26). This matrix was used to purify the ₂AR as previously reported (27) using n-dodecyl-maltoside as the detergent instead of digitonin. The affinity purified preparations were concentrated using Centriprep and Centricon cartridges (Amicon), and the amount of ₂AR in each sample was determined by soluble radioligand binding assays using [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) as described (28). Samples with equal numbers of receptor were then transferred into sample buffer, and SDS-PAGE was conducted under nonreducing conditions. Fixed gels were incubated in 1 M salicylic acid for 45 min, dried, and exposed to Dupont Reflection^TM film at 80 °C.

Radioligand Binding Assays-- Radioligand binding assays were conducted essentially as described in Bouvier et al. (26) using 10 µl of membrane suspension (5 µg) in a total volume of 0.5 ml of 75 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 2 mM EDTA. Competition binding assays with isoproterenol were conducted using 50 pM [¹²⁵I]CYP as the radioligand. The concentration of the -adrenergic agonist was varied from 0 to 100 µM in the presence or absence of 100 µM Gpp(NH)p. For determination of receptor number, the assay was performed in triplicate with a nearly saturating concentration of [¹²⁵I]CYP (200 pM) in the presence or absence of 10 µM alprenolol to define specific binding.

Intracellular cAMP Accumulation Assay-- Approximately 2-6 × 10⁵ cells/well of COS, NIH 3T3, or HEK293 cells were incubated in 24-well plates with 2 µCi/ml [³H]adenine in complete DMEM for 24 h. The cells were washed once with PBS and incubated for 15 min in Hepes/DMEM with 0.5 mM IBMX. They were then treated for 45 min with -adrenergic agonists, forskolin, cholera toxin, 8-bromo-cGMP or vehicle in Hepes/DMEM with 0.5 mM IBMX. The reaction was stopped following aspiration of the medium by adding 1 ml of 5% trichloroacetic acid or 1 ml of 0.2% (w/v) SDS, 50 mM Tris, pH 7.4. One mM of unlabeled cAMP was added to decrease enzymatic degradation of [³H]cAMP. The cAMP was then separated by sequential chromatography over Dowex and Alumina columns as described previously (29). The cAMP accumulation was expressed as ([³H]cAMP cpm/([³H]cAMP cpm + [³H]ATP cpm)) × 10³).

Amperometric Detection of Isoproterenol-- To rule out the possibility that treatment with NO donors could promote oxidation of the -adrenergic agonist, the concentration of isoproterenol before and after SIN-1 treatment was assessed by amperometry (30, 31). Isoproterenol (10 µM) was incubated for 45 min at 37 °C in PBS with 1 mM SIN-1. The mixture was then separated by cation-exchange chromatography using a Spherisorb-OD52, 10 cm × 4.6 mm column, and a Waters HPLC system. Isoproterenol was detected using a Coulochem II (ESA); amperometric system set at +0.6 V. Calibration was carried out using known concentrations of isoproterenol. Since the amperometric method is based on the recording of current produced by voltage-dependent oxidation, only the non-oxidized isoproterenol can be detected (30, 32) thus allowing the assessment of NO-promoted oxidation.

LPS Treatment and Nitrite Measurement-- Macrophages (RAW 264.7) were treated with LPS (1 µg/ml) for 24 h to induce nitric oxide synthase and thus stimulate endogenous NO production. Nitrite (NO₂) was then used as an indicator of NO synthesis and measured in the culture media as described previously (33). Briefly, 400 µl of Griess solution (1 part of 0.1% naphthylethylenediamine dihydrochloride and 1 part of 1% sulfanilamide in 5% phosphoric acid) was added to 400 µl of cell medium. The formation of azochromophore was detected by measuring the absorbance of the reaction mixture at 550 nm (A₅₅₀). Standard curves were generated using known concentrations of sodium nitrite in culture medium.

Data Analysis-- Palmitoylation levels were estimated by densitometric analysis of the fluorograms using an LKB 2202 UltroScan laser densitometer or an AGFA/ARCUS II scanner and NIH Image 1.61. The Allfit (34) software was used to calculate the EC₅₀ from the dose-response curves. Radioligand binding data were analyzed using the computer program GraphPAD Prism.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cAMP Accumulation Studies-- To determine if NO can modulate -adrenergic signaling, the effect of the NO donor 3-morpholinosydnonimine (SIN-1) on isoproterenol-stimulated adenylyl cyclase activity was assessed in COS cells. As shown in Fig. 1A, treatment with 1 mM SIN-1 for 45 min decreased the potency of the -adrenergic agonist from an EC₅₀ of 1.7 ± 0.4 µM in control condition to 19.8 ± 6.0 µM in the presence of the NO donor. The isoproterenol potency returned to control values within 30 min following removal of the NO donor indicating that the effect was readily reversible (data not shown). To rule out the possible contribution of SIN-1C, the stable degradation product of SIN-1, a NO-depleted SIN-1C solution obtained by incubation of SIN-1 at 37 °C for 48 h, was used. Such solution was without effect on the -adrenergic-stimulated cAMP production (data not shown) thus indicating that the released NO, rather than accumulated SIN-1 C, caused the rightward shift in the isoproterenol dose-response curve. This is also supported by the observation that treatment with another NO donor, 1,2,3,4-oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)chloride (GEA, 1 mM), for 45 min also inhibited receptor-stimulated cAMP production (Fig. 1B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effects of SIN-1 on isoproterenol-stimulated cAMP production in COS cells. A, COS cells were incubated with 2 µCi/ml [3H]adenine overnight and treated for 45 min with 0.5 mM IBMX and different concentrations of isoproterenol in the presence and absence of 1 mM SIN-1. Following the incubation, cells were harvested, lysed, and cAMP separated using Dowex and alumina columns. cAMP accumulation was calculated as the [3H]cAMP cpm/([3H]ATP cpm + [3H]cAMP cpm) × 103). Values represent the mean ± S.E. of three independent experiments performed in triplicate. B, NIH 3T3 cells were treated with 10 µM isoproterenol (ISO) alone or with 1 mM SIN-1 and 1 mM GEA. cAMP accumulation was determined as described above. The values shown are the mean ± S.E. of three independent experiments. CON, control.

As shown in Fig. 1A, the SIN-1-induced rightward shift in the cAMP accumulation dose-response curve was such that the response evoked by 10 µM isoproterenol in control cells was almost abolished by the SIN-1 treatment. Therefore, further studies aimed at characterizing the effect of the NO donor in other cell types were carried out using this concentration of isoproterenol. SIN-1 blocked the stimulating effect of 10 µM isoproterenol on cAMP production in HEK293, COS, and NIH 3T3 cells (Fig. 2) indicating that the effect was not cell type-specific. This effect was not unique to isoproterenol since SIN-1 treatment substantially decreased the responses to all -adrenergic agonists tested (epinephrine, norepinephrine, isoproterenol, and procaterol) (Fig. 2C).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effects of SIN-1 on adenylyl cyclase activation in COS, HEK293, and NIH 3T3 cells. The intracellular cAMP assay was performed in triplicate on cells exposed to the agents listed below for 45 min as described in the legend to Fig. 1. A, HEK293 cells were treated or not with 10 µM isoproterenol (ISO) or 1 mM SIN-1. The mean of three independent experiments ± S.E. is shown. B, COS cells were treated with 10 µM isoproterenol (ISO) or 1 µg/ml cholera toxin (CTX) in the presence or absence of 1 mM SIN-1. The mean ± S.E. of four independent experiments is shown. C, NIH 3T3 cells were treated with 10 µM isoproterenol (ISO), 10 µM norepinephrine (NE), 10 µM procaterol (PRO), 10 µM epinephrine (Epi), and 10 µM forskolin (FORS) in the presence or absence of 1 mM SIN-1. CON, control. The mean ± S.E. of three independent experiments is shown.

In contrast to the dramatic effect on the receptor-stimulated cAMP production, SIN-1 was without effect on either forskolin or cholera toxin-stimulated activity (Fig. 2, B and C) indicating that neither the adenylyl cyclase catalytic activity nor G_s-stimulated activity are affected. SIN-1 was also without effect on cAMP accumulation when concentrations of forskolin that gave submaximal cAMP responses were used (data not shown).

NO can increase the hydrolysis of cAMP by activating phosphodiesterases through a cGMP pathway (9). However, the blunting effect of NO on the response to isoproterenol is unlikely to be due to increased degradation of cAMP since a nonspecific phosphodiesterase inhibitor, IBMX, was present in all these experiments. In addition, treatment with 1 mM 8-bromo-cGMP did not alter cAMP production in response to isoproterenol (0.63 for isoproterenol alone and 0.76 with 1 mM 8-bromo-cGMP, the values are the mean of two independent experiments, calculated as [³H]cAMP cpm/([³H]ATP cpm + [³H]cAMP cpm) × 10³). Finally, if the effect of NO resulted from an activator of the phosphodiesterase, reduction in the cholera- and forskolin-stimulated adenylyl cyclase would have also been observed.

Assay for Oxidized Isoproterenol-- Since it has been previously suggested that NO can promote oxidation of catecholamines (11, 35), we tested whether NO could cause the oxidative degradation of isoproterenol thus reducing its effective concentration. Starting from a concentration of 10 µM isoproterenol, incubation for 1 h at 37 °C with or without 1 mM SIN-1 led to a loss of 12 ± 3 and 8 ± 2%, respectively, of the agonist as assessed by high performance liquid chromatography (HPLC) coupled to electrochemical detection. Since amperometry only detects the non-oxidized form of the agonist, these data indicate that incubation with SIN-1 did not promote any significant oxidation of the -adrenergic agonist and cannot account for the biological effect observed. In addition, the inhibitory effect on SIN-1 on the isoproterenol-stimulated cAMP accumulation was also observed in the presence of the anti-oxidant, ascorbate (10 µM).

Agonist Affinity Studies-- The ability of ₂AR to mediate agonist stimulation of adenylyl cyclase activity has been correlated with the propensity of the receptor to adopt a guanyl nucleotide-sensitive high affinity state that is thought to reflect the formation of a ligand-receptor-G_s ternary complex (36). In order to test if NO could mediate its action by affecting the coupling of the ₂AR to G_s, the agonist binding properties of the receptor were tested in membranes derived from HEK293-₂AR cells treated or not with 1 mM SIN-1. As shown in Fig. 3, competition of [¹²⁵I]CYP binding with isoproterenol was biphasic and the curve best resolved by a two-affinity state model. The guanyl nucleotide sensitivity of the high affinity state is illustrated by the observation that upon addition of Gpp(NH)p the curve is best fitted to a single low affinity state model. Both high and low affinity states for agonist were observed in membranes derived from cells treated with SIN-1, but the high affinity constant (K_i(H)) was found to be significantly increased with SIN-1 treatment compared with control cells (Table I). In contrast, the SIN-1 treatment did not affect the low affinity value (K_i(L)). Similarly, the total number of binding sites detected by [¹²⁵I]CYP was not changed by the treatment. Neither the high nor the low affinities for isoproterenol were affected by treatment of the cells with SIN-1 depleted of NO (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effects of SIN-1 on the agonist binding characteristics of the 2AR in HEK293 cells. Isoproterenol competition of [125I]CYP binding was assessed in membrane preparations derived from HEK293-2 AR cells that had been treated for 1 h with or without 1 mM SIN-1. The binding assay was carried out in the presence or absence of 100 µM Gpp(NH)p. The curves shown are computer-generated fits of data obtained in a representative experiment, using the program GraphPad Prism. Con, control.

View this table: [in this window] [in a new window]   Table I Effect of SIN-1 on the isoproterenol binding parameters of the -AR in HEK293 cells The binding assay was performed as described in Fig. 3 legend. The estimates were obtained using the GraphPad Prism software. The values are the mean ± S.E. , number of independent experiments.

Effect of Endogenous NO Production in Macrophage Cells-- Experiments described above used chemical NO donors. To determine if endogenously produced NO could also have an effect on receptor-stimulated cAMP production in a more physiologically relevant system, a mouse macrophage cell line (RAW 264.7) naturally expressing the -adrenergic receptor was used. As expected, treatment of these cells with lipopolysaccharide (LPS) led to the activation of iNOS as indicated by the marked increase in nitrite accumulation promoted by the treatment (Fig. 4A). In agreement with the results obtained using a NO donor, LPS treatment caused a significant (p < 0.05) increase in the EC₅₀ for isoproterenol-stimulated cAMP production (156 ± 64 versus 20 ± 6 nM) (Fig. 4B). Both the nitrite production and the rightward shift of the isoproterenol-stimulated cAMP accumulation were blocked by the NO synthase inhibitor L-NMMA (Fig. 4) suggesting that the endogenous production of NO is responsible for the decrease potency of isoproterenol. Neither receptor number nor forskolin-stimulated cAMP production was affected by LPS (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of LPS-stimulated NO production on cAMP accumulation in RAW 264.7 macrophages. To induce NO synthase, macrophages were incubated with 1 µg/ml LPS in the presence or absence of 1 mM NG-methyl-L-arginine (L-NMMA) for at least 18 h. A, nitrite levels in the medium were measured as described under "Experimental Procedures." B, intracellular cAMP accumulation was assessed following a 45-min stimulation with isoproterenol (1 × 1010 -1 × 105) as described in the legend to Fig. 1. The cAMP accumulation is expressed as the fold stimulation over the cAMP accumulation without isoproterenol in each of the conditions. Data represents the mean ± S.E. of 3-8 independent experiments. The curves shown are computer-generated fits using the program GraphPad Prism. Con, control.

Palmitoylation of G_s and ₂AR in the Presence of SIN-1-- A large body of evidence suggests that NO can modulate the activity of various proteins by directly interacting with accessible thiol groups within the protein to form nitrosocysteine (13, 37, 38). In one case, it was shown that such a mechanism could promote depalmitoylation (13). Since both ₂AR and G_s are palmitoylated and this modification can affect their function, we investigated whether NO could influence the palmitoylation state of these proteins.

The effect of NO on G_s palmitoylation was assessed by incubating COS cells with [³H]palmitic acid in the presence or absence of SIN-1. Immunoprecipitation of G_s, using the affinity purified RM antibody (25), revealed the incorporation of [³H]palmitate into both the short and long forms of the  subunit (Fig. 5A). As previously reported (15), activation of the G protein via receptor stimulation with isoproterenol or directly with cholera toxin increased palmitate incorporation in both forms of G. The presence of SIN-1 during the metabolic labeling was without effect on the extent of [³H]palmitate incorporation under basal conditions or following cholera toxin stimulation but blocked the stimulatory influence of 10 µM isoproterenol (Fig. 5, A and B). The effect of SIN-1 on isoproterenol-stimulated palmitate incorporation was blocked by hemoglobin, which binds and inactivates NO (37) (Fig. 5C). Also, SIN-1 depleted of NO was without effect on the isoproterenol-stimulated palmitoylation level (Fig. 5C). These results indicate that NO blunted only the agonist-promoted palmitoylation of G_s suggesting that NO did not directly inhibit G_s palmitoylation but acted to decrease its activation by the receptor.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effect of SIN-1 on the basal and isoproterenol-stimulated incorporation of [3H]palmitate into Gs subunits. COS cells were incubated with 500 µCi/ml [3H]palmitic acid for 1 h in the presence or absence of 1 mM SIN-1, 10 µM isoproterenol (ISO), 1 µg/ml of cholera toxin (CTX), and 1 mM SIN-1 and 75 µM hemoglobin (hgb) or NO-depleted SIN-1 obtained by storage overnight at room temperature (ON SIN-1). After radiolabeling, the cells were homogenized and separated into particulate and soluble fractions by centrifugation. A and C, Gs were immunoprecipitated from 1 mg of total protein solubilized from the particulate fractions with an affinity purified antibody. The proteins were separated by SDS-PAGE and prepared for fluorography. The films were exposed for 4 weeks at 70 °C. The long and short forms of Gs, resulting from alternative splicing, migrate at 45 and 42 kDa, respectively. B, densitometric analysis of 3H-labeled Gs subunit bands on fluorographs are presented. The densitometry measurements are expressed as a percentage of the basal condition without SIN-1 treatment. Results are the mean ± the S.E. for 3-5 independent experiments.

Similarly to what was observed for G_s, isoproterenol greatly enhanced the incorporation of the [³H]palmitate into the ₂AR (Fig. 6, A and B), and the presence of 1 mM SIN-1 during the metabolic labeling with [³H]palmitate blunted this increase. This action of SIN-1 was not mimicked by a SIN-1 solution depleted of NO (Fig. 6A). The low level of receptor palmitoylation observed under basal conditions in HEK293 cells made it difficult to assess whether SIN-1 could influence the palmitoylation state of the nonstimulated ₂AR. Sf9 cells infected with recombinant baculovirus encoding the human ₂AR were used to verify the influence of SIN-1 on the basal incorporation of [³H]palmitate into the receptor because the basal level of palmitoylation was easier to detect. Sf9 cells were metabolically labeled with [³H]palmitate for 1 h in the presence or absence of 1 mM SIN-1. The presence of SIN-1 promoted a reduction of the basal palmitate incorporation into the receptor (Fig. 6, C and D). This result indicates that, contrary to what is observed for G_s, NO can modify the palmitoylation state of the receptor in an agonist-independent manner.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect of SIN-1 on basal and isoproterenol-stimulated incorporation of [3H]palmitate into the 2AR. HEK293 stably transfected with the 2AR (A) or Sf9 cells infected with recombinant baculovirus encoding the 2AR (C) were metabolically labeled with [3H]palmitate for 1 h with 1 mM SIN-1, 10 µM isoproterenol, or NO-depleted SIN-1 as indicated. Purified receptors were resolved by SDS-PAGE, and incorporation of [3H]palmitate was detected by fluorography. Equal amounts of purified receptor were loaded into each lane. The fluorographs shown were exposed for 3-4 weeks. B and D, densitometric analyses of 3H-labeled 2AR bands on fluorographs are presented. The densitometry measurements are expressed as a percentage of the basal condition and represent the mean ± S.E. of 3-4 independent experiments.

Effect of SIN-1 Treatment on cAMP Accumulation Stimulated by a Nonpalmitoylated ₂AR Mutant-- To test more formally the hypothesis that the blunting effect of SIN-1 on the -adrenergic signaling results, at least in part, from the NO-mediated depalmitoylation of the ₂AR, the effect of SIN-1 treatment was assessed on a mutant ₂AR lacking its palmitoylation site (Gly³⁴¹₂AR). As shown in Fig. 7 and as previously reported in other cell types (16, 39, 40), HEK293 cells expressing the nonpalmitoylated Gly³⁴¹₂AR showed a reduced isoproterenol-stimulated adenylyl cyclase activity when compared with cells expressing the wild type receptor despite a modestly higher number of receptors (9 pmol/mg for the wild type versus 12 pmol/mg for Gly³⁴¹₂AR). Treatment with SIN-1 that resulted in a 60% decrease in cAMP production in response to isoproterenol in WT ₂AR-expressing cells did not reduce the cAMP accumulation in response to the same stimuli in Gly³⁴¹₂AR-expressing cells (Fig. 7). This observation is consistent with the hypothesis that palmitoylation of the receptor plays an important role in the modulatory action of NO.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effect of SIN-1 treatment on cAMP accumulation stimulated by a nonpalmitoylated 2AR mutant. HEK293 cells expressing either the human wild type 2AR (WT) or the nonpalmitoylated mutant, Gly3412AR, were treated with 1 µM isoproterenol in the presence or absence of 1 mM SIN-1 for 20 min. cAMP accumulation was determined as described under "Experimental Procedures." The WT 2AR and Gly3412AR cells expressed 9 and 12 pmol of receptor per mg of protein, respectively. The values shown are the mean ± S.E. of three independent experiments. The cAMP response to isoproterenol in the cells transfected with the WT 2AR was 6-fold greater than isoproterenol stimulation of nontransfected cells and 10-fold greater than basal cAMP accumulation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study demonstrates that NO produced either using chemical NO donors or following activation of iNOS by LPS significantly inhibits -adrenergic-stimulated adenylyl cyclase in cultured cells thus offering a potential mechanism for some of the physiological actions of NO. Indeed, several studies have suggested that NO could act as a functional inhibitor of -adrenergic signaling events. For example, acetylcholine-mediated inhibition of the -adrenergic-stimulated L-type calcium current in the heart has been shown to involve NO synthesis (41). Also, inhibition of NOS by L-NMMA in rat heart has been shown to potentiate the positive inotropic response to isoproterenol suggesting that NO can dampen the -adrenergic stimulation of the contractile response (7). Similarly, interleukin 1 and LPS, which are present during septic shock, can inhibit -adrenergic-stimulated cardiac contraction through a NO-dependent mechanism (42, 43). Until now, however, the molecular basis for such antagonistic action of NO on -adrenergic responses remained unexplored.

The data reported here suggest that the effect of NO on the -adrenergic-stimulated adenylyl cyclase activity occurs upstream of the G protein since neither forskolin- nor cholera toxin-stimulated activities were affected. It follows that NO acts either by changing the effective concentration of agonist or by modulating coupling between the receptor and G_s. NO-induced nitration of catecholamine through an oxidative process has been previously described (32, 35) and thus could be responsible for a reduction in the effective concentration of -adrenergic agonist present in the assay. This hypothesis is, however, very unlikely since no significant reduction of the non-oxidized isoproterenol could be observed following NO treatment. Moreover, the addition of the anti-oxidant, ascorbate, which efficiently inhibits catecholamine nitration (32), was without effect on the inhibitory action of NO on the -adrenergic-stimulated adenylyl cyclase activity observed in this study. Finally, agonist binding analysis revealed a NO-mediated augmentation of the high but not of the low affinity constant for isoproterenol. Given that the low (K_L)/high (K_H) affinity constant ratio for agonist is an index of the receptor-G protein coupling state (44), these data suggest that NO promotes uncoupling of the ₂AR from G_s, and this uncoupling is responsible for the blunted -adrenergic-stimulated adenylyl cyclase activity. A similar uncoupling between another G protein-coupled receptor, the bradykinin receptor, and its cognate G proteins, G_q and G_i, has also been observed following NO donor treatment (45). These effects were mimicked by a cGMP analogue suggesting that activation of the guanylate cyclase may be involved. However, this mechanism cannot be invoked for the -adrenergic uncoupling observed in our study since treatment with 8-bromo-cGMP did not alter cAMP production in response to isoproterenol.

The observation that SIN-1 did not affect basal cAMP production is consistent with a number of previous reports (4, 46, 47) that fail to detect changes in basal adenylyl cyclase activity upon NO treatment. However, an increase in basal cAMP production has been observed in kidney and anal sphincter cells following NO stimulation (48, 49). The discrepancy among these studies may be explained by the finding that NO can directly stimulate the GTPase of both G_s and G_i (50). The relative contribution of these two G proteins to the basal adenylyl cyclase activity in a specific cell type may therefore determine the ultimate effect of NO on this activity. In any case, no effect of NO donor was observed on the basal cAMP accumulation in any of the cell types examined in the present study.

The inhibitory effect of NO on the -adrenergic signaling efficacy was also illustrated by the reduced -adrenergic-stimulated palmitoylation of G_s observed upon SIN-1 treatment. This contrasted with the absence of effect of NO on either basal or cholera toxin-stimulated palmitate incorporation indicating that neither the intrinsic activity of G_s nor the palmitoylation process itself were affected by the gaseous second messenger.

As for G proteins, the palmitoylation state of the ₂-adrenergic receptor has been shown to be regulated by agonist occupancy and activation (13, 15). Therefore, the inhibitory effect of NO on the agonist-stimulated level of receptor palmitoylation could also be a reflection of the decreased ability of the receptor to be activated and thus a consequence of the blunted AR responsiveness. However, in contrast to what was observed for G_s, the basal palmitoylation level of the ₂AR was also found to be affected by NO. This raises the possibility of a direct action of NO on the palmitoylation status of the receptor. A direct effect of NO on the palmitoylation state has been previously suggested. Palmitoylation of SNAP-25 (a synaptic protein) and GAP-43 (a growth cone protein) was shown to be inhibited by NO through a cGMP-independent pathway(s) in PC12 cells and dorsal root ganglion neurons (13). In that study, the inhibition of protein acylation was accompanied by slower neurite growth, growth cone collapse, and retraction of dorsal root ganglion neurons suggesting that NO-mediated regulation of palmitoylation may have had functional consequences.

NO-mediated depalmitoylation of the ₂AR could also have functional consequences. Inhibition of signaling occurs for several G protein-coupled receptors, including the endothelin A and B (51, 52), D₁-dopamine (53), and m2 muscarinic receptors (54), by mutation of their palmitoylation sites. Likewise for the ₂AR, inhibition of receptor palmitoylation by site-directed mutagenesis of its unique palmitoylation site (cysteine 341) significantly decreases the ability of the receptor to interact with G_s and to stimulate adenylyl cyclase activity (16, 39, 40). Our hypothesis that NO-promoted depalmitoylation of the ₂AR leads to receptor uncoupling and decreased efficacy of -adrenergic signaling is strengthened by our findings that SIN-1 had no effect on signaling through the nonpalmitoylated Gly³⁴¹₂AR. Although one cannot exclude that NO may be able to act at several steps in G protein signaling, its major effect in this system is likely to be the result of depalmitoylation of the receptor.

The identity of the active species that mediate biological actions promoted by NO production is always a delicate issue. Indeed, NO can react with oxygen in aqueous solution to generate at least seven nitrogen oxide species that can exist simultaneously (NO, OONO, NO₂, (NO)₂, N₂O₃, NO₂, and NO₃) (55). Depending on the relative concentrations of reactants and targets, reactive intermediates can cross-react with each other and with a number of biological molecules, leading to an array of possible reaction pathways and products. The use of specific NO donors can favor the formation of a subset of possible nitrogen oxide species. For example, SIN-1 releases NO and SIN-1C but also superoxide anion (O₂) that can react with NO to accelerate the formation of peroxynitrite (O₂ + NO=OONO) (55) that could be toxic. However, the toxicity that could result from the formation of peroxynitrite, when using SIN-1, is most likely not responsible for the effects observed in this study since both endogenous NO production and another NO donor, GEA, that do not promote superoxide anion formation (56) had similar effects on -adrenergic responsiveness.

In summary, our study demonstrates that NO inhibits -adrenergic-stimulated cAMP production through functional uncoupling of the receptor from G_s. This attenuated -adrenergic responsiveness was accompanied by an inhibition of receptor palmitoylation and was only seen for the wild type receptor that undergoes palmitoylation. Our data suggest that the action of NO to regulate -adrenergic signaling occurs through modulation of receptor palmitoylation. More studies are needed to determine whether NO has similar effects on other receptors and whether other agents act as physiologic regulators of palmitoylation. We may find that changes in protein palmitoylation is a common means for cross-talk among signaling pathways.

	ACKNOWLEDGEMENTS

We thank Drs. Suzanne Mumby, Joshua Zimmerberg, and Pierre Moreau for helpful advice on the manuscript; Dr. Paul K. Goldsmith for the RM antibodies; and Marthe Parent and Dr. Jacques DeChamplain for their HPLC technical assistance with the isoproterenol measurement. Macrophage RAW 264.7 cells were provided by Dr. Philipe Gros.

	FOOTNOTES

^* This work was supported in part by grants from Heart and Stroke Foundation of Canada and the Medical Research Council of Canada (MRCC).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.

Recipient of a studentship from the MRCC.

^§ Recipient of an MRCC scientist award.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10/Room 8C101, Bethesda, MD 20892-1752. Tel.: 301-496-8711; Fax: 301-496-0200; E-mail: TLZJ@helix.nih.gov.

	ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; ₂AR, ₂-adrenergic receptor; G protein, guanine nucleotide-binding protein; G_s, G protein associated with adenylyl cyclase stimulation; DMEM, Dulbecco's modified Eagle's media; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; SIN-1, 3-morpholinosydnonimine; GEA, 1,2,3,4-oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)-chloride; IBMX, 1-methyl-3-isobutylxanthine; CYP, cyanopindolol; LPS, lipopolysaccharide; HPLC, high pressure liquid chromatography; WT, wild type; FBS, fetal bovine serum; Gpp(NH)p, guanosine 5'-(,-imido)triphosphate; HEK, human embryonic kidney; L-NMMA, N^G-monomethyl-L-arginine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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