Lung cancer is the leading cause of death in the United States, and it demonstrates a strong etiological association with smoking. The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) reproducibly induces pulmonary adenocarcinomas (ACs) in laboratory rodents and is considered an important contributing factor to the high lung cancer burden observed in smokers. It has been shown that the development of NNK-induced ACs in mice is reduced by inhibitors of cyclooxygenase and lipoxygenase and that the growth of human AC cell lines is regulated by β-adrenergic receptors. On the basis of structural similarities of NNK with classic β-adrenergic agonists, we tested the hypothesis that NNK stimulates the growth of human AC cells via agonist-binding to β-adrenergic receptors, resulting in the release of arachidonic acid (AA). In support of this hypothesis, radioreceptor assays with transfected CHO cell lines stably expressing the human β1- or β2-adrenergic receptor demonstrated high affinity binding of NNK to each of these receptors. Two human AC cell lines expressed β1- and β2-adrenergic receptors by reverse transcription-PCR and responded to NNK with the release of AA and an increase in DNA synthesis. β-Adrenergic antagonists completely blocked the release of AA and increase in DNA synthesis. The cyclooxygenase inhibitor aspirin and the 5-lipoxygenase inhibitor MK-886 both partially inhibited DNA synthesis in response to NNK. Our findings identify the direct interaction of NNK with β-adrenergic, AA-dependent pathways as a novel mechanism of action which may significantly contribute to the high cancer-causing potential of this nitrosamine. Moreover, NNK may also contribute to the development of smoking-related nonneoplastic disease via this mechanism.

Lung cancer is the leading cause of cancer death in the United States, with a mortality rate of >95% within 1 year of diagnosis (1, 2). AC3 is the most common type of lung cancer (∼60%; Ref. 3), and it is highly resistant to current therapeutic strategies.

Smoking is the single most extensively documented risk factor for all histological types of lung cancer, including AC (4). Among the numerous toxic and carcinogenic agents contained in tobacco products, the nicotine-derived NNK is the most potent carcinogen in laboratory animals and has, therefore, been implicated as a major cause of tobacco-associated lung cancer (5). NNK reproducibly induces a high incidence of AC in laboratory rodents (6). In analogy to the human disease (7, 8), these experimentally induced ACs express activating point mutations in the Ki-ras gene that are thought to be caused by the formation of promutagenic DNA adducts from reactive NNK metabolites (9, 10, 11, 12).

We have shown that cell lines derived from human pulmonary AC express β-adrenergic receptors and that classic agonists for these receptors stimulate DNA synthesis in these cells (13). Moreover, recent studies in a mouse model have demonstrated that inhibitors of 5-lipoxygenase and cox-1 and cox-2 reduced the incidence and multiplicity of NNK-induced ACs (14, 15, 16), whereas studies in human lung tumors have revealed high levels of cox-2 protein in well-differentiated ACs (17). Although the predominant signal transduction pathway activated by β-adrenergic receptors in most mammalian cells involves the activation of adenylate cyclase and cyclic 3′,5′-AMP (18, 19), it has been shown that, in some cell systems, binding of agonist to this receptor family can result in the release of AA (20, 21, 22). AA itself is an important second messenger that plays a role in the regulation of cellular proliferation in smooth muscle cells through the activation of several signaling pathways, which include mitogen-activated protein kinases and protein kinase C (23, 24, 25). Alternatively, inhibition of metabolism of AA may increase AA concentrations, and this may be important in the inhibition of cell proliferation. In a previous study, nonsteroidal anti-inflammatory agents were shown to inhibit colon tumorigenesis by increasing the levels of free AA, which, in turn, stimulated the production of ceramides, a known mediator of apoptosis (26). Moreover, AA serves as a precursor for the generation of biologically active eicosanoids, including prostaglandins, thromboxanes, and leukotrienes (27, 28). The formation of prostaglandins and thromboxanes involves metabolism of AA by members of the cox family, whereas the generation of leukotrienes is the result of AA metabolism by 5-lipoxygenases (28).

Because of structural similarities of NNK with classic β-adrenergic agonists (Ref. 19; Fig. 1), we hypothesized that this carcinogenic nitrosamine may be an agonist for this receptor family and may activate an AA-dependent mitogenic signal transduction pathway via ligand binding to these receptors in AC cells. In support of this hypothesis, NNK bound with high affinity to stably expressed human β1- or β2-adrenergic receptors in two transfected CHO cell lines and caused the release of AA via ligand binding to these receptors in AC cells, resulting in cox- and lipoxygenase-dependent stimulation of DNA synthesis.

Cell Culture.

The human AC cell lines NCI-H322 and NCI-H441 were purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI supplemented with fetal bovine serum (10%, v/v), l-glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37°C in an atmosphere of 5% CO2. A CHO cell line (Rex 50) transfected with the human β1 adrenoreceptor gene and stably expressing human β1 receptors and a CHO cell line transfected with the human β2 gene and stably expressing human β2-adrenergic receptors were kindly provided to us by Dr. R. J. Lefkowitz (Duke University Medical Center, Durham, NC). These cell lines were maintained in Ham’s F-12 medium supplemented with fetal bovine serum (10%, v/v), l-glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37°C in an atmosphere of 5% CO2.

Radioreceptor Assay.

CHO cells or AC cells at 75% confluency were washed twice with PBS, scraped off the culture vessels, resuspended in PBS, and collected in ice-cold lysis buffer [5 mm Tris-5 mm EDTA (pH 7.4)] containing the protease inhibitors benzamidine, soybean trypsin inhibitor, and leupeptin (Sigma Chemical Co., St. Louis, MO), all at 10 μg/ml. Crude membrane preparations were generated by centrifugation of the cell suspension (10 min at 4°C at 19,000 rpm), followed by removal of the supernatant and recentrifugation (12,000 rpm for 10 s) after the addition of fresh lysis buffer (29). The resulting membranes were resuspended in 75 mm Tris, 5 mm MgCl2, and 2 mm EDTA (pH 7.4) buffer containing protease inhibitors. Radioreceptor assays (30) were conducted in which ascending concentrations of nonradioactive ligands (NNK, epinephrine, norepinephrine, atenolol, and ICI118,551) competed with the β-radioligand [125I]CYP (at the concentrations indicated in the figure legends; 2200 Ci/mmol; NEN, Boston, MA), under steady-state conditions (incubation for 45 min at room temperature), established by saturation binding assays with the radioligand at various temperatures and for various incubation times. Each assay consisted of 25 μl of competing ligand or water, 25 μl of [125I]CYP, and 200 μl of crude membrane preparation (25 μg of protein per assay tube; Ref. 31) and reaction buffer containing protease inhibitors. Ascorbic acid (1 mm) was added to prevent oxidative breakdown of the catecholamines (32), 5-hydroxytryptamine (10 μm) was added to prevent binding of the radioligand to serotonin receptors (33), pargyline (10 μm) was added to inhibit the metabolic conversion of the catecholamines by monoamine oxidase (29), and 1-amino-benzotriazole (10 μm) was added to inhibit the metabolic conversion of NNK by cytochrome P450 (5, 11). Nonspecific binding was determined by incubations in the presence of alprenolol (1 mm; RBI, Natick, MA). The reaction was terminated by the addition of 2 ml of ice-cold Tris buffer (10 mm) and collection of bound radioactivity on Whatman GF/C filters by vacuum filtration (Brandel cell harvester). Following three washes with Tris buffer in the harvester, radioactivity bound to the filters was determined with a gamma counter (Packard, Meriden, CT).The binding data were analyzed by nonlinear regression for single-site or two-site isotherms using a computer program (Ref. 34; Prism/GraphPad for the Macintosh).

RT-PCR.

RNA was isolated from NCI-H322 or NCI-H441 cells using guanidine isothiocyanate-cesium chloride ultracentrifugation (35). Concentration of the RNA was determined by absorbance at 260 nm.

For the RT reaction, 2 μg of RQ1-treated RNA and 1 μg of oligo(dT)12–18 primers (Life Technologies, Inc., Grand Island, NY) in nuclease-free water were heated to 82°C for 3 min and then placed on ice. To this solution was added 0.5 mm dNTPs, 10 mm DTT, 40 units of RNasin RNase inhibitor (Promega, Madison, WI), 200 units of M-MLV reverse transcriptase (Life Technologies, Inc.), and 10× buffer [100 mm Tris-HCl (pH 8.3), 500 mm KCl, and 15 mm MgCl2], in a final volume of 20 μl. The reaction mixture was incubated at 37°C for 1 h, followed by heat inactivation for 10 min at 92°C. A negative control reaction was performed without the M-MLV.

The PCR was performed with 5 μl of the RT reaction, which was mixed with 0.2 mm dNTPs, 5 μl of 10× PCR buffer [100 mm Tris-HCl (pH 8.3), 500 mm KCl, and 15 mm MgCl2], 1.25 units of SuperTaq polymerase (Ambion, Austin, TX), 5% DMSO, a primer pair for cyclophilin used as an internal control (75 or 125 nm; Ambion), primers for the human β1 or β2-adrenergic receptors (250 or 500 nm) and nuclease-free water in a final volume of 50 μl. The β1-adrenergic receptor primers (forward, 5′-caagtgctgcgacttcgtcacc-3′; and reverse, 5′-gccgaggaaacggcgctc-3′) amplified a 159-bp fragment (36). The PCR conditions for the β1 primers were: 1 cycle of 2 min at 94°C; 35 cycles of 94°C for 45 s, 55°C for 45 s, and 74°C for 45 s; and a final extension for 5 min at 74°C. The β2-adrenergic receptor primers (forward, 5′-acgcagcaaagggacgag-3′; and reverse, 5′-cacaccatcagaatgatcac-3′) amplified a 401-bp fragment (37). The PCR conditions for the β2 primers were: 1 cycle of 2 min at 94°C; 37 cycles of 94°C for 60 s, 56°C for 60 s, and 72°C for 60 s; and a final extension for 5 min at 72°C. Reactions were run on a MJ Research (Watertown, MA) PTC-200 thermal cycler.

One-half of the PCR (25 μl) was run on a 1.5% agarose (Life Technologies, Inc.) gel for 2.15 h at 75 V. A 100-bp DNA ladder (Life Technologies, Inc.) was run on the same gel. The gel was imaged by ethidium bromide staining using a UVP (Upland, CA) GDS 7500 or an Ultra Lum (Paramount, CA) TUI-5000 gel documentation system.

The PCR fragments were sequenced using the forward primers used to amplify the fragment by RT-PCR with the ABI Terminator Cycle Sequencing reaction kit on an ABI 373 DNA sequencer (Perkin-Elmer, Foster City, CA). Sequences were entered into DNASIS software (Hitachi, South San Francisco, CA). The sequences using the forward primers were compared with the sequence of human β1-adrenergic receptor (GenBank accession no. J03019, bases 747–887) or β2-adrenergic receptor (GenBank accession no. M15169, bases 1677–2060).

Determination of AA Release from Prelabeled AC Cells.

The release of AA by AC cells was determined as described previously (38). Briefly, NCI-H322 or NCI-H441 cells were seeded into six-well plates (105 cells/well) in complete RPMI. When they had reached 75% confluency (3 days later), the cells were incubated with [3H]AA (0.25 μCi/ml; specific activity, 200–240 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) for 24 h. Following two washes with medium containing 0.1% BSA, cells were incubated in medium with 0.1% BSA for 45 min. BSA was added to the medium to trap released fatty acids, thus inhibiting subsequent metabolism and reacylation. Accordingly, the radioactivity in the supernatant reflected cumulative deacylation of [3H]AA from phospholipid pools. Preliminary experiments on AA release over time in response to NNK (1 μm) established that maximum release occurred after 2 min of incubation (data not shown). This time interval was then chosen to study concentration dependence of this effect as well as modulation of the AA response by preincubation (10 min) with the β1-adrenergic antagonist atenolol or the β2-adrenergic antagonist ICI118,551. Following incubation, the medium was removed and placed into scintillation vials with scintillation cocktail (Microscint, 20 ml). The remaining monolayers were dissociated by 10× trypsin-EDTA (1 ml) and placed into scintillation vials with scintillation cocktail (20 ml). Radioactivity was determined by liquid scintillation spectrophotometry (Top Count; Packard). Released AA was expressed as percent of total incorporated cellular AA. Statistical analysis of data was performed by ANOVA, and group means were compared using the Student-Newman-Keul test. Appropriate transformations were performed on all data that did not follow a normal distribution.

[3H]Thymidine Incorporation Assays.

Cells were seeded into 96-well plates (5 × 103 cells/well, triplicate wells per treatment group) in complete RPMI and allowed to settle for 4 h. [3H]Thymidine (76 Ci/mmol, 0.5 Ci/well; Amersham) and NNK (at the concentrations listed in the figure legends) were then added. The β-adrenergic antagonist propranolol, the cox-inhibitor aspirin, or the 5-lipoxygenase-inhibitor MK-886 was added immediately prior to NNK at the concentrations specified in the figure legends. Following an incubation period of 24 or 48 h (as specified in the figure legends) in an atmosphere of 5% CO2 at 37°, the cells were washed twice with PBS, followed by 0.1 n NaOH. Scintillation fluid (Microscint) was then added to the wells, and radioactivity was measured with a microplate scintillation and luminescence counter (Top Count). Assays under identical conditions but using numbers of viable cells by hemocytometer after trypan blue dye exclusion stain as end point were conducted in parallel. Statistical evaluation of data were by nonparametric ANOVA, Mann-Whitney U test, or unpaired Student’s t test.

Radioreceptor assays with CHO cell line Rex 50, which stably expresses the human β1-adrenoreceptor, showed that NNK competed with high affinity for β1-binding sites against [125I]CYP (Fig. 2). Under the assay conditions used here, the affinity of NNK (EC50 = 5.8 nm) to this receptor was higher than that of the site-selective β1-antagonist atenolol (EC50 = 14.0 nm) or the physiological agonist norepinephrine (EC50 3.49 μm). Similarly, NNK competed with high affinity for β2-adrenergic binding sites against [125]CYP in CHO cell line NBR29 transfected with the human β2 adrenoreceptor gene (Fig. 3). The affinity of NNK (EC50 = 128 nm) to this receptor was higher than that of the site-selective β2 antagonist ICI118,551 (EC50 = 2.91 μm) or the physiological agonist epinephrine (EC50 = 278 μm). Because the affinity of NNK to the β1 receptor was about 22 times higher than its affinity to the β2 receptor, it is expected that, in the presence of both receptor types, NNK will preferentially bind to the β1 receptor. NNK also competed with high affinity with [125I]CYP for β-adrenergic binding sites in the AC cells but yielded a shallower curve, suggesting the presence of more than one binding site (see Fig. 5, inset). Accordingly, atenolol and ICI118,551, each of which is highly site-selective for only one type of β-adrenergic receptor (atenolol for β1, ICI118,551 for β2 receptors) and binds to other β receptors only at relatively high concentrations, yielded a clearly biphasic binding curve in AC cells (see Fig. 5, inset). Analysis of the binding data by nonlinear regression for a two-site binding isotherm identified 60% of the receptors present as β1 and 40% as β2 receptors. Predictably, the nitrosamine NNN did not bind to β1- or β2-adrenergic receptors. This finding is in accord with structural characteristics of NNN, which is formed from nicotine by nitrosation of the pyrrolidine ring (Fig. 1) and lacks the aliphatic side chain containing a nitrogen atom formed by ring-opening of the pyrrolidine ring and nitrosation during the formation of NNK from nicotine (Fig. 1).

In accordance with our receptor binding data, the human AC cell lines NCI-H322 and NCI-H441 both expressed mRNA for β1 and β2-adrenergic receptors by RT-PCR, with β1 mRNA yielding the more prominent band (Fig. 4). The PCR fragments amplified by the human β1 primers in the AC cells were 100% identical to the published sequence (GenBank accession no. J03019, bases 747–887). The PCR fragments amplified by the human β2 primers in AC cells were 100% identical to the published sequence (GenBank accession no. M15169, bases 1677–2060). These findings are in accordance with published data generated in radioreceptor assays, which had revealed the presence of β-adrenergic receptors in these cell lines (13).

NNK caused a concentration-dependent release of AA in both AC cell lines (Fig. 5). AA release in response to 10 nm NNK was significantly inhibited by preincubation with the β1 antagonist atenolol (1 μm; P < 0.005) or the β2 antagonist ICI118,551 (1 μm, P < 0.005). These relatively high antagonist concentrations were used to counteract the exceptionally high affinity of NNK to β1 and β2 receptors. As the selectivity of the antagonists may have been negatively affected in these assays, further studies with lower NNK concentrations are required to establish the role of each receptor type in dose-response curves for each antagonist. Collectively, these findings identify NNK as a high-affinity agonist for β1- and β2-adrenergic receptors, with activation of the AA cascade as the downstream effector in human pulmonary AC cells. Further studies are clearly warranted to delineate more clearly the role of each β-adrenergic receptor, to identify which products are formed from AA in these cells, and to determine how these products may affect intracellular signaling events.

Analysis of DNA synthesis in AC cells by the incorporation of [3H]thymidine as well as cell counts by hemocytometer demonstrated a significant (P < 0.001) stimulation by NNK (10 nm; Fig. 6). This effect was completely inhibited by the broad-spectrum β-adrenergic antagonist propranolol (1 μm, P < 0.001) and partially inhibited by the cox-inhibitor aspirin (100 μm; P < 0.05) and the lipoxygenase-inhibitor MK-886 (10 μm; P < 0.001 Fig. 6). These data suggest that binding of agonist NNK to β1- and β2-adrenegic receptors in AC cells activated an AA-dependent mitogenic signal transduction cascade that involved both cox- and lipoxygenase-dependent messengers. The observed NNK-induced increase in cell numbers were consistently greater than the increase in DNA synthesis, suggesting that mechanisms in addition to stimulation of DNA synthesis (e.g., inhibition of apoptosis) may be involved. Assays using a lower concentration of NNK (1 nm) in the presence of atenolol or ICI118,551 ranging in concentration from 1 nm to 1 μm identified atenolol as the more potent inhibitor of cell proliferation (Fig. 6, inset). Future studies will need to clarify whether the observed difference in antagonist efficacy is a reflection of the relative predominance of β1-adrenergic receptors in AC cells or whether they are caused by the generation of different AA products.

Our findings implicate binding of NNK as an agonist to β1- and β2-adrenergic receptors and the resulting activation of the AA cascade as cellular events contributing to the development of pulmonary AC in smokers. This interpretation is in accordance with recent reports on the activation of a ras and Src tyrosine kinase-dependent mitogen-activated protein kinase pathway by β2-adrenergic receptors in fibroblasts (39). Because reactive NNK metabolites cause activating point mutations in the ras gene (9, 10), an additional activation of ras by binding of NNK as an agonist to β-adrenergic receptors likely potentiates the mitogenic response. Further studies are clearly needed to dissect the complex signaling events involved.

Our data suggest that β-adrenergic antagonists may prevent the continuous growth stimulation that contributes to the development of this histological lung cancer type in smokers chronically exposed to NNK, whereas inhibitors of cox or lipoxygenase may be only partially effective. However, these enzyme inhibitors may have additional cancer-preventive effects by reducing the metabolic activation of NNK. This interpretation is supported by recent reports that have shown the metabolic activation of NNK by members of the cox or lipoxygenase families (15, 16, 4, 40). Broad-spectrum β-adrenergic antagonists such as propranolol or alprenolol as well as β1 antagonists such as atenolol are widely used for the therapy of hypertension (41) and atherosclerosis (42, 43). Moreover, the broad-spectrum cox-inhibitor aspirin as well as β-blockers are widely used for the therapy and prevention of heart attacks (43, 44). Because these cardiovascular diseases are among the many adverse health effects caused by smoking (45, 46), epidemiological data to determine the chemopreventive effects of these agents on AC development should be readily available.

Apart from the obvious significance of our findings for the genesis and potential cancer intervention of pulmonary AC, the fact that NNK is a high-affinity agonist for β1-and β2-adrenergic receptors additionally implicates this tobacco-specific nitrosamine in the etiology of smoking-related cardiovascular disease. This nonneoplastic disease complex has traditionally been attributed primarily to an increased release of catecholamines in response to nicotine (22, 46, 47). On the other hand, it has been shown that smoking reduces the efficacy of β-blockers as antihypertensive agents (46). In light of our data, a plausible explanation for this phenomenon is that, in smokers, these agents must compete with NNK for β-adrenergic binding sites. Further studies are clearly needed to address this important issue.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

Supported by USPHS Grant RO1CA51211 from the National Cancer Institute (to H. M. S.).

            
3

The abbreviations used are: AC, adenocarcinoma; NNK, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; cox, cyclooxygenase; AA, arachidonic acid; [125I]CYP, [125I]iodocyanopindolol; RT, reverse transcription; M-MLV, Moloney murine leukemia virus; NNN, N′-nitrosonornicotine.

Fig. 1.

Nicotine is a tertiary amine consisting of a pyridine and a pyrrolidine ring. NNN is formed by nitrosation of the pyrrolydine ring, whereas the formation of NNK involves nitrosation under ring opening of the pyrrolidine ring (11, 12, 48). The catecholamines epinephrine and norepinephrine are comprised of a catechol ring with an aliphatic side chain containing a nitrogen atom, and they are agonists for all α- and β-adrenergic receptors (18). The intact catechol ring is a requirement for α- but not β-adrenergic agonists. Increasing the steric bulk of the N-substituents, as in isoproterenol, increases the selectivity for β-adrenergic receptors (18). The heterocyclic pyridine ring of NNK resembles the catechol ring stereologically. Similar to classic β-adrenergic agonists such as isoproterenol, NNK contains a nitrogen atom in an aliphatic side chain, the bulk of which in isoproterenol is increased by two methyl groups or in NNK by the N-nitroso group and a methyl group.

Fig. 1.

Nicotine is a tertiary amine consisting of a pyridine and a pyrrolidine ring. NNN is formed by nitrosation of the pyrrolydine ring, whereas the formation of NNK involves nitrosation under ring opening of the pyrrolidine ring (11, 12, 48). The catecholamines epinephrine and norepinephrine are comprised of a catechol ring with an aliphatic side chain containing a nitrogen atom, and they are agonists for all α- and β-adrenergic receptors (18). The intact catechol ring is a requirement for α- but not β-adrenergic agonists. Increasing the steric bulk of the N-substituents, as in isoproterenol, increases the selectivity for β-adrenergic receptors (18). The heterocyclic pyridine ring of NNK resembles the catechol ring stereologically. Similar to classic β-adrenergic agonists such as isoproterenol, NNK contains a nitrogen atom in an aliphatic side chain, the bulk of which in isoproterenol is increased by two methyl groups or in NNK by the N-nitroso group and a methyl group.

Close modal
Fig. 2.

Results of radioreceptor assays with cell membrane fractions from CHO cell line Rex 50 transfected with the human β1-adrenenergic receptor gene and stably expressing human β1-adrenergic receptors. Ascending concentrations of NNK, the physiological agonist with preference for β1 receptors, norepinephrine, or the site-selective β1 antagonist, atenolol, competed for β1 binding sites with [125I]CYP (100 pm). The affinity of NNK to this receptor was 2.5 times greater than that of atenolol and 602 times greater than the affinity of norepinephrine (EC50s, calculated by nonlinear regression for single site-binding isotherms, were: NNK, 5.8 nm; atenolol, 14.0 nm; and norepinephrine, 3.49 μm).

Fig. 2.

Results of radioreceptor assays with cell membrane fractions from CHO cell line Rex 50 transfected with the human β1-adrenenergic receptor gene and stably expressing human β1-adrenergic receptors. Ascending concentrations of NNK, the physiological agonist with preference for β1 receptors, norepinephrine, or the site-selective β1 antagonist, atenolol, competed for β1 binding sites with [125I]CYP (100 pm). The affinity of NNK to this receptor was 2.5 times greater than that of atenolol and 602 times greater than the affinity of norepinephrine (EC50s, calculated by nonlinear regression for single site-binding isotherms, were: NNK, 5.8 nm; atenolol, 14.0 nm; and norepinephrine, 3.49 μm).

Close modal
Fig. 3.

Results of radioreceptor assays with cell membrane fractions of CHO cell line NBR29 transfected with the human β2-adrenergic receptor gene and stably expressing human β2-adrenergic receptors. Ascending concentrations of NNK, the physiological agonist with preference for β2 receptors, epinephrine, or the site-selective β2 antagonist, ICI118,551, competed for β2 binding sites with [125I]CYP (100 pm). The affinity of NNK to this receptor was 22.7 times greater than that of ICI118,551 and 2172 times greater than the affinity of epinephrine (EC50s, calculated by nonlinear regression for single site- binding isotherms, were: NNK, 128 nm; ICI118,551, 2.91 μm; and epinephrine, 278 μm).

Fig. 3.

Results of radioreceptor assays with cell membrane fractions of CHO cell line NBR29 transfected with the human β2-adrenergic receptor gene and stably expressing human β2-adrenergic receptors. Ascending concentrations of NNK, the physiological agonist with preference for β2 receptors, epinephrine, or the site-selective β2 antagonist, ICI118,551, competed for β2 binding sites with [125I]CYP (100 pm). The affinity of NNK to this receptor was 22.7 times greater than that of ICI118,551 and 2172 times greater than the affinity of epinephrine (EC50s, calculated by nonlinear regression for single site- binding isotherms, were: NNK, 128 nm; ICI118,551, 2.91 μm; and epinephrine, 278 μm).

Close modal
Fig. 4.

Expression of mRNA for β1- and β2-adrenergic receptors in the human AC cell lines NCI-H322 and NCI-H441 by RT-PCR. The β1 primers amplified a 159-bp fragment, whereas the β2 primers amplified a 401-bp fragment. Lane 1, NCI-H322 with β2 primers; Lane 2, NCI-H441 with β2 primers; Lane 3, NCI-H441 with β2 and cylophylin primer; Lane 4, NCI-H441 with cylophylin primer alone; Lane 5, NCI-H322 negative control without M-MLV reverse transcriptase; Lane 6, NCI-H322 with β1 primers; Lane 7, NCI-H441 with β1 primers; Lane 8, NCI-H322 with β1 and cylophylin primer; Lane 9, NCI-H322 negative control without M-MLV reverse transcriptase; Lane M, a 100-bp DNA ladder.

Fig. 4.

Expression of mRNA for β1- and β2-adrenergic receptors in the human AC cell lines NCI-H322 and NCI-H441 by RT-PCR. The β1 primers amplified a 159-bp fragment, whereas the β2 primers amplified a 401-bp fragment. Lane 1, NCI-H322 with β2 primers; Lane 2, NCI-H441 with β2 primers; Lane 3, NCI-H441 with β2 and cylophylin primer; Lane 4, NCI-H441 with cylophylin primer alone; Lane 5, NCI-H322 negative control without M-MLV reverse transcriptase; Lane 6, NCI-H322 with β1 primers; Lane 7, NCI-H441 with β1 primers; Lane 8, NCI-H322 with β1 and cylophylin primer; Lane 9, NCI-H322 negative control without M-MLV reverse transcriptase; Lane M, a 100-bp DNA ladder.

Close modal
Fig. 5.

Results of AA release assay in NCI-H322 cells. NNK caused a concentration-dependent release of AA with a maximum release observed with 10 nm NNK. The response to 10 nm NNK was significantly (P < 0.005) inhibited by atenolol (1 μm) and ICI118,551 (1 μm), indicating that the release of AA was mediated by ligand binding of NNK to β1- and β2-adrenergic receptors. Similar results were obtained with NCI-H441 cells. Inset, results of radioreceptor assays in AC cell line NCI-H322. Because one objective of these assays was to assess the relative proportions of β1- and β2-adrenergic receptors in these cells, these assays were conducted with the saturation concentration of [125I]CYP (300 pm) to ensure complete β receptor occupancy with radioligand. In accordance with our findings in the transfected CHO cell lines (Figs. 2 and 3), which identified NNK as a high-affinity ligand for both β1- and β2-adrenergic receptors, NNK yielded a shallow binding curve well to the left of the curves generated by atenolol or ICI118,551, suggestive of more than one high-affinity binding site for NNK. By contrast, atenolol and ICI118,551, each of which is highly site selective for only one of these receptors (atenolol, β1; ICI118,551, β2) generated clearly biphasic curves. Analysis of the binding data by nonlinear regression for two-site binding isotherms revealed that the β1-selective antagonist atenolol bound with high affinity to 60 ± 3.0% and with low affinity to the remaining receptors occupied by the radioligand, whereas the β2-selective antagonist ICI118,551 bound with high affinity to 40 ± 4% and with low affinity to the remaining receptors occupied by [125I]CYP.

Fig. 5.

Results of AA release assay in NCI-H322 cells. NNK caused a concentration-dependent release of AA with a maximum release observed with 10 nm NNK. The response to 10 nm NNK was significantly (P < 0.005) inhibited by atenolol (1 μm) and ICI118,551 (1 μm), indicating that the release of AA was mediated by ligand binding of NNK to β1- and β2-adrenergic receptors. Similar results were obtained with NCI-H441 cells. Inset, results of radioreceptor assays in AC cell line NCI-H322. Because one objective of these assays was to assess the relative proportions of β1- and β2-adrenergic receptors in these cells, these assays were conducted with the saturation concentration of [125I]CYP (300 pm) to ensure complete β receptor occupancy with radioligand. In accordance with our findings in the transfected CHO cell lines (Figs. 2 and 3), which identified NNK as a high-affinity ligand for both β1- and β2-adrenergic receptors, NNK yielded a shallow binding curve well to the left of the curves generated by atenolol or ICI118,551, suggestive of more than one high-affinity binding site for NNK. By contrast, atenolol and ICI118,551, each of which is highly site selective for only one of these receptors (atenolol, β1; ICI118,551, β2) generated clearly biphasic curves. Analysis of the binding data by nonlinear regression for two-site binding isotherms revealed that the β1-selective antagonist atenolol bound with high affinity to 60 ± 3.0% and with low affinity to the remaining receptors occupied by the radioligand, whereas the β2-selective antagonist ICI118,551 bound with high affinity to 40 ± 4% and with low affinity to the remaining receptors occupied by [125I]CYP.

Close modal
Fig. 6.

Results of [3H]thymidine incorporation assay in NCI-H322 cells. The NNK concentration (10 nm), which had yielded maximum release of AA (Fig. 5), significantly stimulated DNA synthesis. This effect was partially inhibited by the cox-inhibitor aspirin and the lipoxygenase-inhibitor MK-886 and was completely blocked by the broad-spectrum antagonist of β-adrenergic receptors, propranolol. Inset, analysis of viable cell numbers after trypan blue dye exclusion stain by hemocytometer following a 48-h incubation period of NCI-H441 cells with a low concentration (1 nm) of NNK and the effect of atenolol or ICI118,551 (1–1000 nm) on NNK-induced increase in cell number. The β1 antagonist atenolol more potently inhibited the response to NNK, possibly reflecting the relative predominance of β1-adrenergic receptors in AC cells. Columns, means of triplicate samples per treatment group; bars, SD.

Fig. 6.

Results of [3H]thymidine incorporation assay in NCI-H322 cells. The NNK concentration (10 nm), which had yielded maximum release of AA (Fig. 5), significantly stimulated DNA synthesis. This effect was partially inhibited by the cox-inhibitor aspirin and the lipoxygenase-inhibitor MK-886 and was completely blocked by the broad-spectrum antagonist of β-adrenergic receptors, propranolol. Inset, analysis of viable cell numbers after trypan blue dye exclusion stain by hemocytometer following a 48-h incubation period of NCI-H441 cells with a low concentration (1 nm) of NNK and the effect of atenolol or ICI118,551 (1–1000 nm) on NNK-induced increase in cell number. The β1 antagonist atenolol more potently inhibited the response to NNK, possibly reflecting the relative predominance of β1-adrenergic receptors in AC cells. Columns, means of triplicate samples per treatment group; bars, SD.

Close modal

We gratefully acknowledge the invaluable help of Dr. R. J. Lefkowitz (Duke University Medical Center), who provided the transfected CHO cell lines and advised us on radioreceptor assays. We also thank Dr. D. A. Schwinn (Duke University Medical Center) for her advice on the RT-PCR assays and Dr. N. Quigley (University of Tennessee, Sequencing Laboratory) for his assistance with the sequencing.

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