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Am J Physiol Heart Circ Physiol 281: H1946-H1954, 2001;
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Vol. 281, Issue 5, H1946-H1954, November 2001

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, induces apoptosis of endothelial cells

Patricia K. Tithof1, Mona Elgayyar1, Hildegard M. Schuller2, Maryann Barnhill2, and Richard Andrews3

1 Department of Comparative Medicine and 2 Department of Pathology, University of Tennessee College of Veterinary Medicine, and 3 Graduate School of Medicine, University of Tennessee, Knoxville, Tennessee 37996-4500


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Smoking causes endothelial cell (EC) injury; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury are understood. The nitrosated derivative of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been implicated in the carcinogenic effects of tobacco; however, the effects of NNK on the cardiovascular system are largely unknown. NNK binds to beta 1- and beta 2-adrenergic receptors. Because beta -adrenergic receptor activation causes arachidonic acid (AA) release and cellular injury, we postulated that NNK causes EC injury by a mechanism that involves beta -adrenergic-mediated release of AA. NNK stimulated [3H]AA release from ECs, and this effect was mediated by both beta 1- and beta 2-adrenergic receptors because pretreatment with atenolol or ICI 118,551 inhibited the response. NNK also induced EC apoptosis, as measured by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and annexin V staining. NNK-mediated apoptosis was attenuated by pretreatment with atenolol or ICI 118,551. Furthermore, depletion of cellular AA by incubation with eicosapentaenoic acid abolished the apoptotic effect of NNK. These data suggest that NNK causes EC apoptosis by a mechanism that involves beta 1- and beta 2-adrenergic receptor-mediated release of AA.

endothelial cell; atherosclerosis; tobacco; nitrosamine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CIGARETTE SMOKING is an important risk factor for the development of atherosclerotic vascular disease; however, the identity of causative components in cigarette smoke, as well as the molecular means by which they induce lesions, remain ill defined. Augmentation of the atherogenic process by cigarette smoking likely involves endothelial cell (EC) injury, a critical and early event in the development of atherosclerosis (26). Evidence for this conclusion is based on studies that demonstrated an increase in EC death and loss into the circulation in smokers compared with nonsmokers (34).

Nicotine may be involved in smoking-induced EC injury because exposure to nicotine in vivo induces death of ECs and accelerates the atherogenic process in laboratory animals (26). However, similar results have not been demonstrated in vitro, suggesting that the effects of nicotine on EC integrity may be indirect (26). Nicotine activates the sympathetic nervous system and causes catecholamine release, and this effect may be responsible for the EC injury that is observed in vivo but not in vitro. An alternative, but previously unexplored, explanation for these results is that nitrosated derivatives of nicotine are responsible for the effects of smoking on ECs.

Tobacco-specific N-nitrosamines, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), cause cancer in all laboratory animals and are believed to contribute significantly to the cancer burden observed in smokers (21). However, the effects of these nicotine derivatives on the cardiovascular system have received little attention. The most potent and most abundant N-nitrosamine in tobacco smoke is NNK, which reproducibly induces adenocarcinoma of the lung in mice, rats, and hamsters (21). We (36) demonstrated in recent studies that NNK binds to beta 1- and beta 2-adrenergic receptors transfected into CHO cells. The affinity of NNK for beta 1- and beta 2-adrenergic receptors is ~1,000-fold greater than the affinity for norepinephrine. Furthermore, NNK, in the nanomolar range, induced proliferation of human adenocarcinoma cells by a mechanism that involved beta 1- and beta 2-adrenergic receptor-stimulated release of arachidonic acid (AA) (36). This is the first report of binding of NNK to beta -adrenergic receptors and represents a novel mechanism of NNK-induced tumorigenesis.

The fact that NNK binds to both beta 1- and beta 2-adrenergic receptors implicates it as a potential promoter of atherosclerosis and coronary artery disease in smokers. It is well known from both human and animal studies that activation of the beta -adrenergic system exacerbates atherosclerosis (24). Direct experimental evidence for this conclusion is based on at least 19 animal studies that evaluated the effects of beta -adrenergic blockade on atherogenesis. In 17 of these studies, beta -antagonists retarded the development of atherosclerosis as measured by effects on lesion size, severity, and EC injury. For example, treatment of rabbits with a beta -agonist resulted in a significant increase in EC death compared with untreated controls. Cotreatment with the beta 1-antagonist metoprolol abolished beta -adrenergic receptor-mediated EC injury (29). Although these studies demonstrated a role for the beta -adrenergic system in EC injury, the mechanism of injury (i.e., apoptosis vs. necrosis) was not elucidated.

Recent studies (12, 13, 17, 19, 20) have indicated that apoptotic cell death plays an important role in the atherogenic process. Oxidized low-density lipoproteins (LDLs) induce atherosclerosis by a mechanism that involves EC apoptosis (12, 13), and plaque rupture in advanced atherosclerosis has been associated with an increase in apoptosis of several cell types contained within the lesion (12, 17). Interestingly, beta -adrenergic activation has been associated with an increase in apoptosis of several cell types, including cardiac myocytes (9, 10, 16, 23, 32, 37). However, beta -receptor-mediated apoptosis of ECs has not been evaluated despite the known role of apoptosis in atherogenesis.

The purpose of the present study was to test the hypothesis that the tobacco-specific nitrosamine NNK causes EC apoptosis by a mechanism that involves beta -adrenergic receptor activation. Furthermore, because activation of the AA cascade is a downstream effect of beta -receptor activation (6, 31, 35), an additional aim of this study was to investigate the role of arachidonate in NNK-induced EC programmed cell death.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture

Fresh porcine aortas were treated with 0.05% collagenase, and porcine aortic ECs (PAECs) were harvested. Cells were grown in DMEM-F12 medium containing 10% fetal bovine serum, penicillin, and streptomycin and maintained at 37°C in an atmosphere of 5% CO2-95% O2. The medium was changed every 2 days until confluence was reached. PAECs were identified by their morphology and by positive staining for factor VIII. At 100% confluence, PAECs were harvested by trypsinization and seeded into six-well plates, tissue culture slide chambers, or 25-cm3 flasks.

Determination of [3H]AA Release from Prelabeled PAECs

PAECs were grown in six-well plates to 75% confluence and labeled for 22-24 h with 0.25 µCi/ml [3H]AA as described previously (40). At the end of the incubation period, an aliquot of medium was subjected to scintillation counting to determine cellular uptake of radiolabel: uptake was routinely ~70% of added [3H]AA. PAECs were washed two times and resuspended in Hanks' balanced salt solution (HBSS) containing 0.1% BSA and equilibrated for 45 min. Cumulative release of [3H]AA was measured in cells stimulated for 60 min with various concentrations of NNK (0, 10-8, 10-7, 10-6, and 10-5 M). To determine the kinetics of release of arachidonate, the accumulation of [3H]AA in the medium was determined in cells stimulated for various times (0, 1, 5, 10, and 60 min) with NNK (1 µM). To determine the role of beta -adrenergic receptors in NNK-induced AA release, cells were pretreated for 20 min with the beta 1-antagonist atenolol (10 µM) or the beta 2-antagonist ICI 118,551 (10 µM).

Determination of Cytotoxicity

Apoptosis assays. Identification of apoptosis in PAECs was performed by terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) using the Boehringer-Mannheim In Situ Cell Death detection kit. PAECs (2 × 105 cells/well) were grown in tissue culture slide chambers and treated with NNK or vehicle (PBS) for various times. After treatments, the cells were fixed in 4% paraformaldehyde, blocked with hydrogen peroxide in methanol-distilled deionized H2O, washed, permeabilized, and treated with a mixture containing TdT and fluorescein-labeled nucleotides (dUTPs). The incorporation of fluorescein-conjugated dUTP into DNA was determined by incubation of slides with anti-fluorescein antibody conjugated with horseradish peroxidase, followed by counterstaining with 3,3'-diaminobenzidine. TUNEL-positive cells were identified by brown nuclear stain and altered nuclear morphology. To verify the activity of TdT, a positive control using DNase to induce strand breaks was employed. The percentage of TUNEL-positive cells was calculated by dividing the number of TUNEL-positive cells by the total number of cells (400 cells counted). To determine the kinetics of NNK-induced apoptosis, cells were treated with NNK (1 µM) for 0, 4, 8, and 12 h. To determine the role of beta -adrenergic receptors in NNK-induced apoptosis, cells were pretreated for 20 min with atenolol (10 µM) or ICI 118,551 (10 µM) before treatment with NNK. To examine the role of arachidonate in NNK-induced apoptosis, TUNEL staining was performed in cells depleted of AA by incubation for 24 h with the omega -3 fatty acid eicosapentaenoic acid (EPA; 100 µM). Preliminary experiments indicated that incubation with 100 µM EPA for 24 h decreased uptake of [3H]AA to <15% of the total added radioactivity (cells routinely take up 65-80% of total added [3H]AA in the absence of EPA).

To verify the results of TUNEL assays, fluorescence microscopy was performed after staining of cells with annexin V-FITC and propridium iodide (PI; PharMingen; San Diego, CA). Annexin V is a phospholipid-binding protein that has high affinity for phosphatidylserine. Annexin V binds only to apoptotic cells with externalized phosphatidylserine and necrotic cells with leaky membranes. Counterstaining with PI allows for differentiation of necrotic and apoptotic cells. Cells were cultured to 90% confluence in tissue slide chambers and exposed to NNK or vehicle for 8 h. At the end of the treatment period, cells were washed two times with cold PBS, exposed for 20 min in the dark to annexin V-FITC (5 µl) and PI (10 µl), and examined by fluorescence microscopy.

The apoptotic response was also verified by electron microscopy. After treatment with NNK (1 µM) or vehicle for 6 h, cells were trypsinized, resuspended in control medium, centrifuged, and then fixed in 2% glutaraldehyde-0.1 M cacodylate buffer. The fixed cells were washed three times in cacodylate buffer, and the pellets were cut into small cubes and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer. After three washes with cacodylate buffer, the cells were dehydrated by passage through graded concentrations of ethyl alcohol followed by sequential treatment with Epon-ethyl alcohol (1:4 vol/vol), Epon-ethyl alcohol (1:2 vol/vol), Epon-ethyl alcohol (1:4 vol/vol), and 100% Epon. Polymerization was at 50°C overnight. Ultrathin sections were cut with a DuPont diamond knife, stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope.

Lactate dehydrogenase assays. To rule out a primary necrotic effect of NNK on PAECs, lactate dehydrogenase (LDH) assays were performed in cells treated with NNK for 4, 8, or 12 h. The activity of LDH in cell-free supernatant fluids was measured as described previously (40). Annexin V-PI staining was also used as a measure of necrosis.

Data Analysis

Data are presented as means ± SE. Data were analyzed by analysis of variance, and group means were compared using the Student-Newman-Keuls test. Appropriate transformations were performed on all data that did not follow a normal distribution (e.g., percentage data). If transformation failed to normalize the data, nonparametric statistics (Mann-Whitney rank sum test) were used. For all studies, the criterion for statistical significance was P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Adrenergic Receptor-Mediated Release of [3H]AA in PAECs Exposed to NNK

Treatment of PAECs with NNK caused a concentration-dependent release of [3H]AA; however, no significant [3H]AA release was observed in the absence of NNK (Fig. 1A). The kinetics of NNK-elicited [3H]AA release are shown in Fig. 1B. NNK caused [3H]AA release within 10 min of treatment, and this response was significantly greater than the response obtained in the absence of NNK (Fig. 1B). Pretreatment with the beta 1-antagonist atenolol (10 µM) abolished the NNK-mediated [3H]AA release (Fig. 2). Similar results were observed in cells pretreated with the beta 2-antagonist ICI 118,551 (Fig. 2).


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  		Fig. 1.   A: concentration-dependent release of [3H]arachidonic acid from prelabeled porcine aortic endothelial cells (PAECs) exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). PAECs were prelabeled with [3H]arachidonic acid as described in MATERIALS AND METHODS and treated for 1 h with various concentrations of NNK or vehicle. Cumulative release of [3H]arachidonate into the extracellular medium was measured, and data are expressed as a percentage of total cellular radioactivity (dpm, disintegrations per minute). aSignificantly different from respective value obtained in the presence of vehicle, n = 6. B: time course of cumulative release of [3H]arachidonic acid from PAECs exposed to NNK. PAECs were exposed to 1 µM NNK or vehicle for various times, and release of [3H]arachidonic acid into the medium was measured as described in MATERIALS AND METHODS. Data are expressed as a percentage of total cellular radioactivity. aSignificantly different from respective value obtained in the presence of vehicle, n = 6.



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  		Fig. 2.   The inhibitory effect of beta -adrenergic antagonists on release of [3H]arachidonic acid from PAECs exposed to NNK. Radiolabeled PAECs were pretreated with the beta 1-adrenergic antagonist atenolol (10 µM) or the beta 2-adrenergic antagonist ICI 118,551 (10 µM) for 10 min and then exposed to 1 µM NNK or vehicle for 1 h. Cumulative release of [3H]arachidonate was measured as described in MATERIALS AND METHODS. aSignificantly different from respective value obtained in the presence of vehicle; bsignificantly different from respective value obtained in the absence of inhibitor, n = 6.

Induction of Apoptosis in PAECs Exposed to NNK

NNK caused a time-dependent increase in the number of TUNEL-positive cells at a concentration that caused significant [3H]AA release (Figs. 3 and 4). The percentage of TUNEL-positive cells increased from 3.7 ± 0.9% at 4 h to 37.2 ± 2.2% at 8 h after treatment with NNK. No significant increase in TUNEL staining was observed in vehicle-treated controls (Figs. 3 and 4).


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  		Fig. 3.   Detection of NNK-induced apoptosis in PAECs by terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). PAECs were treated for various times with NNK or vehicle, and in situ DNA end-labeling was determined as described in MATERIALS AND METHODS. A: cells treated with vehicle for 8 h. B: cells treated with NNK (1 µM) for 4 h. C: cells treated with NNK (1 µM) for 8 h. D: cells pretreated with atenolol (10 µM) for 20 min and then exposed to NNK (1 µM) for 8 h. E: cells pretreated with ICI 118,551 (10 µM) for 20 min and then exposed to NNK (1 µM) for 8 h. F: cells depleted of arachidonic acid by incubation with eicosapentaenoic acid (EPA; 100 µM) for 24 h as described and then treated with NNK (1 µM) for 8 h.



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  		Fig. 4.   NNK induces time-dependent apoptosis of PAECs. PAECs were treated for various times with NNK or vehicle as described in Fig. 3. The data shown are a summary of the morphometric data from the experiments shown in Fig. 3 and represent the number of apoptotic cells/400 cells counted. aSignificantly different from control at same time point, n = 6.

Similar results were obtained with NNK-treated cells stained with PI and annexin V-FITC and analyzed by fluorescence microscopy (Fig. 5). After 8 h of treatment with NNK, there was an increase in the number of annexin V-FITC-positive cells (Fig. 5B) compared with control (Fig. 5A); however, no cells stained positively for PI at this time point. Electron microscopic evaluation of endothelial cells treated for 6 h with NNK revealed the presence of morphological characteristics consistent with early apoptosis. These included condensation of chromatin at the nuclear periphery and vacuolization of the cytoplasm (Fig. 6).


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  		Fig. 5.   Detection of NNK-induced apoptosis by fluorescence microscopic evaluation of PAECs stained with annexin V-FITC and propridium iodide. Cells were cultured for 8 h with or without NNK (1 µM), stained with propridium iodide and/or annexin V-FITC, and analyzed by fluorescence microscopy as described in MATERIALS AND METHODS. A: photomicrograph of vehicle-treated cells stained with annexin V-FITC and propridium iodide. There is virtually no staining of vehicle-treated cells with either annexin V or propridium iodide. B: After treatment with NNK for 8 h, PAECs stained positively with annexin V-FITC but not propridium iodide. The data are representative of 3 separate experiments.



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  		Fig. 6.   Transmission electron microscopy of cultured PAECs exposed for 6 h to vehicle (A) or 1 µM NNK (B). Control cells show normal architecture with diffuse heterochromatin. In contrast, NNK-treated cells show condensation of chromatin at the periphery and vacuolization of the cytoplasm indicative of early apoptosis. An aqueous uranyl acetate and lead citrate stain was used; magnification, ×14,000.

To investigate the role of beta -adrenergic receptors in NNK-induced apoptosis, TUNEL assays were performed in the presence and absence of atenolol or ICI 118,551. As can be seen in Figs. 3, D and E, and 7, both atenolol and ICI 118,551 significantly decreased NNK-induced apoptosis.


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  		Fig. 7.   Inhibition of NNK-induced apoptosis by beta 1- and beta 2-adrenergic antagonists. NNK-induced apoptosis was detected as described in Fig. 3. Pretreatment with the beta 1-adrenergic antagonist atenolol or the beta 2-adrenergic antagonist ICI 118,551 significantly attenuated apoptosis induced by NNK. The data shown are a summary of the morphometric data from the experiments shown in Fig. 3 and represent the percentage of TUNEL-positive cells/400 cells counted. aSignificantly different from response obtained in the presence of vehicle; bsignificantly different from response obtained in the absence of inhibitor, n = 6.

To investigate the role of arachidonate in apoptosis caused by NNK, experiments were conducted with cells that had been depleted of AA by incubation for 24 h with 100 µM EPA (in preliminary experiments, this concentration of EPA inhibited [3H]AA uptake to <15% of total added [3H]AA). The number of TUNEL-positive cells was significantly decreased in PAECs treated with EPA and exposed to NNK compared with the response obtained in the presence of NNK alone (Figs. 3 and 8).


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  		Fig. 8.   Inhibition of NNK-induced apoptosis by depletion of intracellular arachidonic acid with EPA. PAECs were incubated for 24 h with 100 µM EPA and then treated for 12 h with 1 µM NNK as described in MATERIALS AND METHODS. The data shown are a summary of the morphometric data from the experiments shown in Fig. 3 and represent the percentage of TUNEL-positive cells/400 cells counted. aSignificantly different from response obtained in the presence of vehicle; bsignificantly different from response obtained in the absence of EPA, n = 3.

Extracellular release of the cytosolic enzyme LDH was measured to rule out a primary necrotic effect of NNK. Release of LDH after 8 h was not different in NNK-treated cells (11.6 ± 0.3%) compared with control (10.4 ± 0.2%). These results were substantiated by fluorescence microscopic examination of annexin V-PI-stained cells. PAECs treated with NNK for 8 h stained positively for annexin V but not PI, indicating a primary apoptotic effect of NNK (Fig. 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NNK caused a concentration-dependent release of AA from prelabeled PAECs. The concentration of NNK that caused significant release of [3H]AA (1 µM) also caused significant programmed cell death, as assessed by TUNEL, annexin V-PI staining, and electron microscopy. NNK-induced [3H]AA release preceded the development of apoptosis. Significant release of [3H]AA in response to NNK was observed within 10 min after treatment, and significant apoptosis was observed between 4 and 8 h after treatment. These data support the hypothesis that NNK causes apoptosis of ECs by a mechanism that involves release of AA.

The conclusion that NNK-induced AA release and apoptosis are linked is further substantiated by experiments with the omega -3 fatty acid EPA. Because EPA competes with omega -6 fatty acids for incorporation into membrane phospholipids, incubation of PAECs with EPA causes depletion of intracellular arachidonate (11). In the present study, EPA, at a concentration that significantly reduced the uptake of [3H]AA by PAECs, inhibited apoptosis caused by NNK. These results provide strong evidence that NNK causes apoptosis by a mechanism that involves the AA cascade. The mechanism by which EPA afforded protection against NNK-induced apoptosis may involve other effects of EPA besides depletion of arachidonate. EPA not only competes with omega -6 fatty acids for incorporation into membrane phospholipids but also competes with arachidonate for binding to prostaglandin H synthase. In addition, omega -3 fatty acids directly inhibit the activity of prostaglandin H synthase, phospholipases, tumor necrosis factor, and interleukin 1 (1, 14, 15). All of these activities, either directly or indirectly, involve the AA cascade. Thus the results of this study suggest strongly that the two events, AA release and apoptosis, are linked.

Phospholipase-mediated release of AA has been shown to induce apoptosis in several different cell types, including ECs (2, 8, 18, 20, 25, 27, 28, 33, 38, 41). Various mechanisms are involved in the process by which the AA cascade causes apoptosis. These include membrane alterations induced by various phospholipase isoforms (2, 25, 33). Externalization of phosphatidylserine to the outer leaflet renders it susceptible to cleavage by secretory phospholipase A2, and this effect has been shown to be important in the apoptotic process (25). Alternatively, AA or other long-chain fatty acids released as a result of phospholipase activation may activate downstream signaling pathways involved in the cell death process. These pathways include c-Jun NH2-terminal kinase, caspase-3, and the sphingomyelinase/ceramide pathways (8, 28, 41). Finally, AA can be converted to various metabolites that promote cell death (20, 22, 27). These include prostaglandin J2, hydroperoxides, or cytochrome P-450 metabolites of AA (20, 22, 27). The mechanism by which activation of the AA cascade by NNK resulted in apoptosis was not addressed in this study. Future studies will focus on characterizing the phospholipase isoform(s) activated by NNK and their specific roles in the apoptotic response. In addition, experiments will be performed to determine whether this response involves AA itself or its metabolites.

The results of this study also provide evidence that beta -adrenergic receptor activation is involved in NNK-induced AA release and apoptosis. Pretreatment with atenolol (beta 1-antagonist) or ICI 118,551 (beta 2-antagonist) significantly inhibited both apoptosis and AA release from NNK-treated cells, suggesting the involvement of both beta 1- and beta 2-adrenergic receptors in these responses.

The protection afforded by beta -adrenergic antagonists against EC injury is well documented in vivo (24, 29). Psychosocial stress or high-cholesterol diets induced EC injury and atherosclerosis in the rabbit and monkey by a mechanism that involved beta -adrenergic receptor activation of ECs. These studies, however, did not address the mechanism of injury (i.e., necrosis vs. apoptosis). To our knowledge, this is the first report describing beta -adrenergic receptor-induced apoptosis of ECs and suggests that EC injury induced by stress may involve apoptosis.

beta -Adrenergic receptor-mediated apoptosis has been reported in other cell types (9, 10, 16, 23, 32, 37), but intracellular signaling mechanisms vary depending on the cell type studied. beta -Adrenergic receptor-dependent apoptosis of cardiac myocytes involves adenylate cyclase (9) and calcineurin (32); however, in thymocytes, this effect is mediated by Lck, a member of the Src family of tyrosine kinases (16). No previous studies have linked beta -adrenergic activation, AA release, and apoptosis even though the AA cascade plays an important role in regulation of apoptosis (2, 8, 18, 25, 33, 38) and AA release is a well-characterized downstream effect of beta -adrenergic receptor activation (6, 31, 35). Because the AA cascade modulates the activity of adenylate cyclase (35) and the Src family of tyrosine kinases (39), these enzymes may also have a role in NNK-induced apoptosis of ECs. Future studies will address this possibility.

The roles of specific beta -adrenergic receptors in apoptosis also vary according to cell type. Programmed cell death of rat ventricular myocytes is mediated by beta 1-adrenergic receptors (42), and beta 2-receptor activation results in inhibition of apoptosis (9, 10). In contrast, apoptosis of eosinophils requires beta 2- but not beta 1-adrenergic receptor activation (23). Results from the present study suggest that both beta 1- and beta 2-adrenergic receptors are involved in the programmed cell death of ECs. These variations in the roles of specific receptor subtypes in apoptosis may reflect differences among these cell types in downstream signal transduction mechanisms. beta 1- and beta 2-adrenergic receptors on ECs may be linked to the same proapoptotic signal transduction pathways, whereas these receptors may be associated with opposing pathways in cardiac myocytes. beta 1-Adrenergic receptor activation of rat ventricular myocytes results in adenylate cyclase-mediated apoptosis; however, beta 2-adrenergic receptors are coupled to a Gi-mediated signaling pathway that opposes the actions of adenylate cyclase and inhibits apoptosis (42).

NNK-induced apoptosis of ECs may be important in the atherogenic effect of tobacco smoke; however, there are several alternative pathways by which derivatives of nicotine such as NNK may promote atherosclerosis. An increasing body of evidence implicates oxidative stress in the pathogenesis of atherosclerosis (7). A causal relationship exists between oxygen radical formation in the vessel wall and key events that occur during atherosclerosis, including EC injury, inflammation, plaque rupture, thrombosis, and tissue infarction (7). Moreover, oxidation of LDLs occurs under conditions of oxidative stress, and oxidized LDL is highly atherogenic (3). Cigarette smoking is an important risk factor for the development of oxidative stress (7), and nicotine as well as its derivative, NNK, are important components of cigarette smoke that promote this effect (4). The possibility that NNK promotes EC dysfunction by inducing oxidative stress was not addressed in this study but may be important in the pathogenesis of NNK-induced EC apoptosis. Furthermore, NNK may promote other key events in the atherosclerotic process by mechanisms that involve oxidative stress and/or beta -adrenergic activity.

NNK is the most abundant carcinogen in cigarette smoke (4), and it binds to beta -adrenergic receptors with ~1,000-fold greater affinity than endogenous ligands (36). In this study, extremely low concentrations of NNK stimulated the release of AA and caused subsequent EC apoptosis in vitro. Thus the effective concentration of NNK was well within the range of levels of NNK that may be achieved in smokers. Furthermore, the concentration of NNK used in this study was 1,000-fold less than the concentration required to induce k-ras mutations in vitro, a well-documented carcinogenic effect of this compound (4). In addition, this study did not investigate whether NNK or its active metabolites were responsible for apoptosis of ECs in vitro. It is possible that the effects observed were due to active metabolites such as keto acid, keto aldehyde, or keto alcohol derivatives of this compound. Studies are underway to measure and to investigate the role of these metabolites in EC apoptosis.

The results of the present study may have important clinical ramifications in light of recent epidemiological data (30). The Honolulu Heart Program initiated in 1968 followed a cohort of 8,006 Japanese-American men aged 45-65 yr. The results of this study demonstrated clearly that mortality and morbidity due to coronary heart disease was significantly less (50%) in heavy smokers who consumed a diet high in omega -3 fatty acids found in fish compared with smokers who consumed a low-fish diet. Similar protection was not observed in nonsmokers. Because omega -3 fatty acids are known to inhibit the AA cascade, these data suggest that cigarette smoking promotes coronary artery disease by a mechanism that involves this pathway. The present study demonstrates clearly that NNK, a compound found in cigarette smoke, causes EC death by a mechanism that involves arachidonate. Depletion of cellular arachidonate by the omega -3 fatty acid EPA afforded protection against NNK-induced EC apoptosis. Thus this study provides a plausible cellular mechanism to explain the interaction between smoking and fish intake demonstrated by the Honolulu Heart Program.

The interaction between NNK and beta -adrenergic receptors also has potential clinical ramifications. The Heart Attack Primary Prevention in Hypertension Trial (HAPPHY Trial) was conducted to determine the role of beta -antagonists in prevention of coronary heart disease. Patients receiving beta -antagonist therapy demonstrated significantly less mortality and less coronary heart disease than patients treated with diuretics (P < 0.06). This protective effect of beta -blockers was particularly pronounced in smokers (P < 0.013), suggesting that smoking augments coronary heart disease by a mechanism that involves the beta -adrenergic pathway (24). Because NNK causes beta -adrenergic receptor-mediated apoptosis of ECs, it may be one potential component of cigarette smoke responsible for the interaction between smoking and the beta -adrenergic system. Thus the results of this study also provide potential cellular mechanisms to explain the results of the HAPPHY Trial. Identification of specific components of cigarettes that alter pathways known to be important in atherogenesis will likely provide potential means for developing safer cigarettes and developing effective preventative and/or therapeutic strategies in smokers.


    ACKNOWLEDGEMENTS

We thank Wanda Aycock for expert clerical assistance.


    FOOTNOTES

This work was supported by American Heart Association Grant 01602645.

Address for reprint requests and other correspondence: P. K. Tithof, Dept. of Comparative Medicine, College of Veterinary Medicine, Univ. of Tennessee, Knoxville, TN 37996-4500 (E-mail: ptithof{at}utk.edu).

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.

Received 15 September 2000; accepted in final form 13 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(5):H1946-H1954
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