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Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells
Drug Metabolism and Pharmacokinetics 31 (2016) 99e101
Contents lists available at ScienceDirect
Drug Metabolism and Pharmacokinetics journal homepage: http://www.journals.elsevier.com/drug-metabolism-andpharmacokinetics
Note
Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells Yuma Tega a, 1, Chihiro Yuzurihara a, 1, Yoshiyuki Kubo a, Shin-ichi Akanuma a, Carsten Ehrhardt b, Ken-ichi Hosoya a, * a b
Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Research Institute, Trinity College Dublin, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2015 Received in revised form 9 November 2015 Accepted 27 November 2015 Available online 9 December 2015
Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 mM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and b2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with b2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation.
Keywords: Nicotine Drug transport Respiratory epithelium Inhalation biopharmaceutics Pulmonary drug disposition Bronchodilator
Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction The alkaloid, nicotine is a primary constituent of tobacco smoke, and is well-known as a potent addictive agent. Upon inhalation, nicotine is rapidly absorbed into the systemic circulation via the alveoli of the lung. Nicotine clearance was observed to be 2-fold enhanced by phenobarbital treatment in an ex vivo model of isolated and perfused rat lung [1]. The molecular mechanism of nicotine transport at the alveolar epithelial barrier, however, has not been fully identified. We previously demonstrated the involvement of a carriermediated process in nicotine transport at the rat bloodebrain barrier, inner blood-retinal barrier, and in the liver [2e4]. In
* Corresponding author. E-mail addresses: [email protected] (Y. Tega), [email protected] (C. Yuzurihara), [email protected] (Y. Kubo), akanumas@ pha.u-toyama.ac.jp (S.-i. Akanuma), [email protected] (C. Ehrhardt), hosoyak@pha. u-toyama.ac.jp (K.-i. Hosoya). 1 Tega Y and Yuzurihara C contributed equally to this work.
addition to our findings, it has been reported that carrier-mediated transport can occur in other tissues and species such as rabbit choroid plexus and human placenta [5,6]. The transporter protein involved in nicotine transport, however, has not yet been molecularly identified. Evidence is emerging for a significant contribution of organic cation transport systems in pulmonary drug absorption. So was it recently shown that organic cations such as verapamil, a Ca2þ channel blocker, and salbutamol, a b2-adrenergic agonist, are transported via carrier-mediated processes in human alveolar epithelial cells [7,8]. This led us to hypothesize that organic cation transporters are responsible for nicotine uptake across the human respiratory mucosa. Furthermore, the identification of the transport properties of nicotine will be helpful to improve our understanding of pulmonary drug disposition and possible interactions with tobacco smoking. In the present paper, we report the in vitro uptake study using human alveolar epithelial A549 cells to characterize nicotine transport across human alveolar epithelium.
http://dx.doi.org/10.1016/j.dmpk.2015.11.006 1347-4367/Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
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Y. Tega et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 99e101
2. Materials and methods 2.1. Reagents L-(-)-[N-methyl-3H]Nicotine ([3H]nicotine, 83.5 Ci/mmol) was purchased from PerkinElmer (Boston, MA). All other chemicals were analytical grade produced commercially.
2.2. Uptake study in A549 cells A549 (American Type Culture Collection, CCL-185) cells obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) were seeded at 1.0 105 cells/well in 24-well plate and cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL benzylpenicillin potassium, and 100 mg/mL streptomycin sulfate at 37 C. After 48 h from seeding, uptake experiments were performed as described previously [2,4]. In brief, cells were washed three times with extracellular fluid (ECF) buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 0.4 mM K2HPO4, 1.4 mM CaCl2, 1.2 mM MgSO4, 10 mM D-glucose, and 10 mM HEPES; pH 7.4), and [3H]nicotine (1 mCi/mL, 12 nM) dissolved in ECF buffer was applied to the cells at 37 C to initiate the uptake. [3H]Nicotine uptake was also studied in the absence or presence of inhibitors. To determine the influence of extracellular pH on nicotine uptake, ECF buffer was adjusted to different pH values (i.e. pH 6.4, 7.4, and 8.4). To terminate uptake, cells were rinsed three times with ice-cold buffer, and were solubilized with 1 N NaOH. Subsequently to neutralization of the solubilized solution with 1 N HCl, the radioactivity associated with cells was measured by liquid scintillation counting (LSC-7400, Hitachi Aloka Medical, Tokyo, Japan). Cellular protein content was determined by detergent-compatible protein assay (i.e. DC protein assay kit, Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. The [3H]nicotine uptake by A549 cells was expressed as shown in Eqn. (1). Cell/medium ratio (mL/mg protein) ¼ (3H-dpm in the cell per mg protein)/(3H-dpm in the medium per mL) (1) The kinetic parameters for nicotine uptake by A549 cells were obtained from the following Eqn. (2). V ¼ (Vmax C)/(Km þ C) þ Kd C
(2)
V, C, Vmax, Km, and Kd are the uptake rate, the substrate concentration, the maximum uptake rate, the Michaelis constant, nonsaturable transport clearance, respectively. The equation was fitted using the iterative nonlinear least-squares regression analysis program, MULTI. 2.3. Statistical analysis The kinetic parameters (Vmax, Km, and Kd) determined by leastsquares regression analysis were presented as the means ± S.D., and other data represent the means ± S.E.M. To determine the statistical difference in two unpaired group and more than two groups, Student's t-test and one-way ANOVA followed by Dunnett's post hoc test were executed, respectively. 3. Results and discussion [3H]Nicotine uptake by A549 cells increased linearly for approximately 30 s, before reaching a plateau after 1 min (Fig. 1A). Uptake was significantly decreased by 86% at 4 C compared to 37 C (Fig. 1B). In addition, uptake was concentration-dependent with a Km of 50.4 ± 18.3 mM, a Vmax of 4.11 ± 0.95 nmol/(min$mg protein), and a Kd of 7.56 ± 1.26 mL/(min$mg protein) (Fig. 1C). These results suggest that a carrier-mediated transport process is involved in nicotine uptake by A549 cells. The uptake clearance (Vmax/Km) of a saturable process was calculated as 81.5 mL/(min$mg protein) which is 10.8-fold greater than that of a non-saturable process (Kd), and the total uptake clearance was estimated as 89.1 mL/(min$mg protein). On the basis of these values, the contribution of a saturable process for total uptake clearance was determined as 92%. Nicotine concentration in the alveolar lining fluid after smoking one cigarette was estimated as 6e60 mM [9]. Under this concentration, the uptake clearance for a saturable process was calculated as 37.2e72.8 mL/(min$mg protein), and the contribution of a saturable process for total uptake clearance was determined as 83.1e90.6%. This suggests that a saturable process plays a major role in nicotine absorption via the alveolar epithelium in cigarette smoking. There were no significant differences in nicotine uptake by A549 cells between pH 7.4 and pH 8.4, and only a slight, yet significant, decrease in uptake was observed at pH 6.4 (Fig. 1D). In buccal mucosa, the absorption rate of nicotine was found to be affected by the pH value of tobacco smoke [10]. Nicotine in tobacco
Fig. 1. Nicotine uptake by A549 cells. (A) Time-course of [3H]nicotine uptake (1 mCi/mL, 12 nM) at 37 C. Each point represents the means ± S.E.M. (n ¼ 3). (B) Temperaturedependent uptake of [3H]nicotine (1 mCi/mL, 12 nM) at 37 C (control) and 4 C. Each column represents the means ± S.E.M. (n ¼ 3). (C) Concentration-dependent uptake of [3H]nicotine (1 mCi/mL, 12 nM) at 37 C for 30 s over the nicotine concentration range 10 mMe2000 mM. Data were subjected to Eadie-Scatchard and MichaeliseMenten (inset) analyses. The solid, dashed, and dotted line represent the overall, saturable, and non-saturable transport, respectively. Each point represents the means ± S.E.M. (n ¼ 3). (D) Effect of extracellular pH on [3H]nicotine uptake (1 mCi/mL, 12 nM) at 37 C for 30 s. Each column represents the means ± S.E.M. (n ¼ 9). *P < 0.05, **P < 0.01, significantly different from control.
Y. Tega et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 99e101 Table 1 Inhibitory effect of several compounds on the [3H]nicotine uptake by A549 cells. The uptake of [3H]nicotine (1 mCi/mL, 12 nM) was measured at 37 C for 30 s in the absence (control) or presence of compounds (1 mM). Each value represents the means ± S.E.M. (n ¼ 3e9). *P < 0.05, **P < 0.01, significantly different from control. Compounds
Percentage of control
Control Nicotine Verapamil Pyrilamine Propranolol p-Aminohippurate (PAH) Salbutamol Procaterol Tetraethylammonium (TEA) 1-Methyl-4-phenylpyridinium (MPPþ) L-Carnitine Choline
100 24.0 9.52 10.7 15.3 100 96.0 117 134 153 156 224
± ± ± ± ± ± ± ± ± ± ± ±
5 1.6** 1.41** 0.5** 0.4** 6 9.5 6 4 30* 35* 24**
smoke mainly exists in protonated form, since the pH of smoke from most cigarettes is acidic [11], and this protonation only allows poor absorption of nicotine across the buccal mucosa. Considering these pieces of evidence, extracellular pH is less significant in nicotine transport in pulmonary epithelium than that in buccal mucosa. Table 1 shows the inhibitory effect of several modulators of organic cation transporters. Verapamil, pyrilamine, and propranolol, which are all hydrophobic organic cations (log D > 0.76), strongly inhibited nicotine uptake by A549 cells by more than 85%. This suggests the involvement of organic cation transport system(s) in nicotine transport in alveolar epithelial cells. Organic cation transporter (OCT) 1, OCT3, and novel organic cation/ carnitine transporter (OCTN) 1, but not OCT2 and OCTN2, have been reported to be expressed in A549 cells by LC-MS/MS [12]. Verapamil and pyrilamine are able to interact with organic cation transporters such as OCT1 and OCTN1 [13,14]. However, tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPPþ), L-carnitine, and choline, which are typical substrates of organic cation transporters such as OCTs, OCTNs, plasma membrane monoamine transporter (PMAT), multidrug and toxin extrusion protein (MATE), and choline transporter (CHT), did not inhibit nicotine uptake. On the contrary, these compounds promoted nicotine uptake by A549 cells. These results suggest that neither OCTs, OCTNs, MATE, PMAT, nor CHT are responsible for nicotine uptake by A549 cells. As the mechanism involved in the uptake increase by compounds, it is considered to be the transcriptional-, translational-upregulation, and trans-stimulation of the transport system. Although the mechanism by which nicotine uptake in A549 cells is promoted remains unclear, it is unlikely that the transcriptional- and translational-upregulation is involved in the increase of nicotine uptake since the uptake time was very short (30 s). Compared with the specificity of nicotine uptake in other cell model such as brain capillary endothelial cells (TR-BBB13 cells), retinal capillary endothelial cells (TR-iBRB2 cells), and hepatocytes, the pH sensitivity of nicotine uptake in A549 cells was different, whereas the Km value of the high-affinity saturable process of nicotine uptake by A549 cells was similar to that reported in those other cell model (i.e. Km ¼ 92e492 mM) [2e4]. Although this difference is remains unclear, it may cause of difference between species. To fully understand this observed in the various cell types, it is essential to identify the molecule responsible for nicotine transport. Tobacco smoke is the primary cause of chronic obstructive pulmonary disease (COPD) in the developed world. Nevertheless, a substantial percentage of patients with moderate to severe COPD continue to smoke [15]. To examine, whether bronchodilators,
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which are the mainstay of COPD pharmacotherapy, interact with the nicotine influx transporter, inhibition studies with 1 mM salbutamol and procaterol were performed. Both bronchodilators had little effect on nicotine uptake by A549 cells, suggesting that inhalation of these drugs is unlikely to have an effect on nicotine disposition in the human lung. In conclusion, this is the first study to demonstrate that an influx system is responsible for nicotine transport at the human alveolar epithelial barrier. Moreover, it is unlikely to involve in drugedrug interaction with bronchodilators. The present findings provide important information to better understand pulmonary disposition of drug and other xenobiotics. Conflict of interest The authors declare there are no conflicts of interest. Acknowledgments This study was supported, in part, by a Grant-in-Aid for Scientific Research (B) [KAKENHI: 25293036] and Scientific Research (C) [KAKENHI: 26460193] from Japan Society for the Promotion of Science (JSPS), a Research Grant from Tamura Science and Technology Foundation, and a Clinical Investor Grant from Flight Attendant Medical Research Institute (FAMRI) [CIA130016]. References [1] Foth H, Looschen H, Neurath H, Kahl GF. Nicotine metabolism in isolated perfused lung and liver of phenobarbital- and benzoflavone-treated rats. Arch Toxicol 1991;65:68e72. [2] Tega Y, Akanuma S, Kubo Y, Terasaki T, Hosoya K. Blood-to-brain influx transport of nicotine at the rat bloodebrain barrier: involvement of a pyrilamine sensitive organic cation transport process. Neurochem Int 2013;62: 173e81. [3] Tega Y, Akanuma S, Kubo Y, Hosoya K. Involvement of the Hþ/organic cation antiporter in nicotine transport in rat liver. Drug Metab Dispos 2015;43: 89e92. [4] Tega Y, Kubo Y, Yuzurihara C, Akanuma S, Hosoya K. Carrier-mediated transport of nicotine across the inner blood-retinal barrier: involvement of a novel organic cation transporter driven by an outward H(þ) gradient. J Pharm Sci 2015;104:3069e75. [5] Zevin S, Schaner ME, Giacomini KM. Nicotine transport in a human choriocarcinoma cell line (JAR). J Pharm Sci 1998;87:702e6. [6] Spector R, Goldberg MJ. Active transport of nicotine by the isolated choroid plexus in vitro. J Neurochem 1982;38:594e6. [7] Salomon JJ, Hagos Y, Petzke S, Kühne A, Gausterer JC, Hosoya K, et al. Beta-2 adrenergic agonists are substrates and inhibitors of human organic cation transporter 1. Mol Pharm 2015;12:2633e41. [8] Salomon JJ, Ehrhardt C, Hosoya K. The verapamil transporter expressed in human alveolar epithelial cells (A549) does not interact with b2-receptor agonists. Drug Metab Pharmacokinet 2014;29:101e4. [9] Clunes LA, Bridges A, Alexis N, Tarran R. In vivo versus in vitro airway surface liquid nicotine levels following cigarette smoke exposure. J Anal Toxicol 2008;32:201e7. [10] Adrian CL, Olin HB, Dalhoff K, Jacobsen J. In vivo human buccal permeability of nicotine. Int J Pharm 2006;311:196e202. [11] Pankow JF, Tavakoli AD, Luo W, Isabelle LM. Percent free base nicotine in the tobacco smoke particulate matter of selected commercial and reference cigarettes. Chem Res Toxicol 2003;16:1014e8. [12] Sakamoto A, Matsumaru T, Yamamura N, Suzuki S, Uchida Y, Tachikawa M, et al. Drug transporter protein quantification of immortalized human lung cell lines derived from tracheobronchial epithelial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type IIlike cells (A549) by liquid chromatography-tandem mass spectrometry. J Pharm Sci 2015;104:3029e38. [13] Zhang L, Schaner ME, Giacomini KM. Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa). J Pharmacol Exp Ther 1998;286:354e61. [14] Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, et al. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pHdependent transport of organic cations. J Pharmacol Exp Ther 1999;289: 768e73. [15] Tashkin DP, Murray RP. Smoking cessation in chronic obstructive pulmonary disease. Respir Med 2009;103:963e74.
Contents lists available at ScienceDirect
Drug Metabolism and Pharmacokinetics journal homepage: http://www.journals.elsevier.com/drug-metabolism-andpharmacokinetics
Note
Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells Yuma Tega a, 1, Chihiro Yuzurihara a, 1, Yoshiyuki Kubo a, Shin-ichi Akanuma a, Carsten Ehrhardt b, Ken-ichi Hosoya a, * a b
Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Research Institute, Trinity College Dublin, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2015 Received in revised form 9 November 2015 Accepted 27 November 2015 Available online 9 December 2015
Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 mM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and b2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with b2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation.
Keywords: Nicotine Drug transport Respiratory epithelium Inhalation biopharmaceutics Pulmonary drug disposition Bronchodilator
Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction The alkaloid, nicotine is a primary constituent of tobacco smoke, and is well-known as a potent addictive agent. Upon inhalation, nicotine is rapidly absorbed into the systemic circulation via the alveoli of the lung. Nicotine clearance was observed to be 2-fold enhanced by phenobarbital treatment in an ex vivo model of isolated and perfused rat lung [1]. The molecular mechanism of nicotine transport at the alveolar epithelial barrier, however, has not been fully identified. We previously demonstrated the involvement of a carriermediated process in nicotine transport at the rat bloodebrain barrier, inner blood-retinal barrier, and in the liver [2e4]. In
* Corresponding author. E-mail addresses: [email protected] (Y. Tega), [email protected] (C. Yuzurihara), [email protected] (Y. Kubo), akanumas@ pha.u-toyama.ac.jp (S.-i. Akanuma), [email protected] (C. Ehrhardt), hosoyak@pha. u-toyama.ac.jp (K.-i. Hosoya). 1 Tega Y and Yuzurihara C contributed equally to this work.
addition to our findings, it has been reported that carrier-mediated transport can occur in other tissues and species such as rabbit choroid plexus and human placenta [5,6]. The transporter protein involved in nicotine transport, however, has not yet been molecularly identified. Evidence is emerging for a significant contribution of organic cation transport systems in pulmonary drug absorption. So was it recently shown that organic cations such as verapamil, a Ca2þ channel blocker, and salbutamol, a b2-adrenergic agonist, are transported via carrier-mediated processes in human alveolar epithelial cells [7,8]. This led us to hypothesize that organic cation transporters are responsible for nicotine uptake across the human respiratory mucosa. Furthermore, the identification of the transport properties of nicotine will be helpful to improve our understanding of pulmonary drug disposition and possible interactions with tobacco smoking. In the present paper, we report the in vitro uptake study using human alveolar epithelial A549 cells to characterize nicotine transport across human alveolar epithelium.
http://dx.doi.org/10.1016/j.dmpk.2015.11.006 1347-4367/Copyright © 2015, The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
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Y. Tega et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 99e101
2. Materials and methods 2.1. Reagents L-(-)-[N-methyl-3H]Nicotine ([3H]nicotine, 83.5 Ci/mmol) was purchased from PerkinElmer (Boston, MA). All other chemicals were analytical grade produced commercially.
2.2. Uptake study in A549 cells A549 (American Type Culture Collection, CCL-185) cells obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) were seeded at 1.0 105 cells/well in 24-well plate and cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL benzylpenicillin potassium, and 100 mg/mL streptomycin sulfate at 37 C. After 48 h from seeding, uptake experiments were performed as described previously [2,4]. In brief, cells were washed three times with extracellular fluid (ECF) buffer (122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 0.4 mM K2HPO4, 1.4 mM CaCl2, 1.2 mM MgSO4, 10 mM D-glucose, and 10 mM HEPES; pH 7.4), and [3H]nicotine (1 mCi/mL, 12 nM) dissolved in ECF buffer was applied to the cells at 37 C to initiate the uptake. [3H]Nicotine uptake was also studied in the absence or presence of inhibitors. To determine the influence of extracellular pH on nicotine uptake, ECF buffer was adjusted to different pH values (i.e. pH 6.4, 7.4, and 8.4). To terminate uptake, cells were rinsed three times with ice-cold buffer, and were solubilized with 1 N NaOH. Subsequently to neutralization of the solubilized solution with 1 N HCl, the radioactivity associated with cells was measured by liquid scintillation counting (LSC-7400, Hitachi Aloka Medical, Tokyo, Japan). Cellular protein content was determined by detergent-compatible protein assay (i.e. DC protein assay kit, Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. The [3H]nicotine uptake by A549 cells was expressed as shown in Eqn. (1). Cell/medium ratio (mL/mg protein) ¼ (3H-dpm in the cell per mg protein)/(3H-dpm in the medium per mL) (1) The kinetic parameters for nicotine uptake by A549 cells were obtained from the following Eqn. (2). V ¼ (Vmax C)/(Km þ C) þ Kd C
(2)
V, C, Vmax, Km, and Kd are the uptake rate, the substrate concentration, the maximum uptake rate, the Michaelis constant, nonsaturable transport clearance, respectively. The equation was fitted using the iterative nonlinear least-squares regression analysis program, MULTI. 2.3. Statistical analysis The kinetic parameters (Vmax, Km, and Kd) determined by leastsquares regression analysis were presented as the means ± S.D., and other data represent the means ± S.E.M. To determine the statistical difference in two unpaired group and more than two groups, Student's t-test and one-way ANOVA followed by Dunnett's post hoc test were executed, respectively. 3. Results and discussion [3H]Nicotine uptake by A549 cells increased linearly for approximately 30 s, before reaching a plateau after 1 min (Fig. 1A). Uptake was significantly decreased by 86% at 4 C compared to 37 C (Fig. 1B). In addition, uptake was concentration-dependent with a Km of 50.4 ± 18.3 mM, a Vmax of 4.11 ± 0.95 nmol/(min$mg protein), and a Kd of 7.56 ± 1.26 mL/(min$mg protein) (Fig. 1C). These results suggest that a carrier-mediated transport process is involved in nicotine uptake by A549 cells. The uptake clearance (Vmax/Km) of a saturable process was calculated as 81.5 mL/(min$mg protein) which is 10.8-fold greater than that of a non-saturable process (Kd), and the total uptake clearance was estimated as 89.1 mL/(min$mg protein). On the basis of these values, the contribution of a saturable process for total uptake clearance was determined as 92%. Nicotine concentration in the alveolar lining fluid after smoking one cigarette was estimated as 6e60 mM [9]. Under this concentration, the uptake clearance for a saturable process was calculated as 37.2e72.8 mL/(min$mg protein), and the contribution of a saturable process for total uptake clearance was determined as 83.1e90.6%. This suggests that a saturable process plays a major role in nicotine absorption via the alveolar epithelium in cigarette smoking. There were no significant differences in nicotine uptake by A549 cells between pH 7.4 and pH 8.4, and only a slight, yet significant, decrease in uptake was observed at pH 6.4 (Fig. 1D). In buccal mucosa, the absorption rate of nicotine was found to be affected by the pH value of tobacco smoke [10]. Nicotine in tobacco
Fig. 1. Nicotine uptake by A549 cells. (A) Time-course of [3H]nicotine uptake (1 mCi/mL, 12 nM) at 37 C. Each point represents the means ± S.E.M. (n ¼ 3). (B) Temperaturedependent uptake of [3H]nicotine (1 mCi/mL, 12 nM) at 37 C (control) and 4 C. Each column represents the means ± S.E.M. (n ¼ 3). (C) Concentration-dependent uptake of [3H]nicotine (1 mCi/mL, 12 nM) at 37 C for 30 s over the nicotine concentration range 10 mMe2000 mM. Data were subjected to Eadie-Scatchard and MichaeliseMenten (inset) analyses. The solid, dashed, and dotted line represent the overall, saturable, and non-saturable transport, respectively. Each point represents the means ± S.E.M. (n ¼ 3). (D) Effect of extracellular pH on [3H]nicotine uptake (1 mCi/mL, 12 nM) at 37 C for 30 s. Each column represents the means ± S.E.M. (n ¼ 9). *P < 0.05, **P < 0.01, significantly different from control.
Y. Tega et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 99e101 Table 1 Inhibitory effect of several compounds on the [3H]nicotine uptake by A549 cells. The uptake of [3H]nicotine (1 mCi/mL, 12 nM) was measured at 37 C for 30 s in the absence (control) or presence of compounds (1 mM). Each value represents the means ± S.E.M. (n ¼ 3e9). *P < 0.05, **P < 0.01, significantly different from control. Compounds
Percentage of control
Control Nicotine Verapamil Pyrilamine Propranolol p-Aminohippurate (PAH) Salbutamol Procaterol Tetraethylammonium (TEA) 1-Methyl-4-phenylpyridinium (MPPþ) L-Carnitine Choline
100 24.0 9.52 10.7 15.3 100 96.0 117 134 153 156 224
± ± ± ± ± ± ± ± ± ± ± ±
5 1.6** 1.41** 0.5** 0.4** 6 9.5 6 4 30* 35* 24**
smoke mainly exists in protonated form, since the pH of smoke from most cigarettes is acidic [11], and this protonation only allows poor absorption of nicotine across the buccal mucosa. Considering these pieces of evidence, extracellular pH is less significant in nicotine transport in pulmonary epithelium than that in buccal mucosa. Table 1 shows the inhibitory effect of several modulators of organic cation transporters. Verapamil, pyrilamine, and propranolol, which are all hydrophobic organic cations (log D > 0.76), strongly inhibited nicotine uptake by A549 cells by more than 85%. This suggests the involvement of organic cation transport system(s) in nicotine transport in alveolar epithelial cells. Organic cation transporter (OCT) 1, OCT3, and novel organic cation/ carnitine transporter (OCTN) 1, but not OCT2 and OCTN2, have been reported to be expressed in A549 cells by LC-MS/MS [12]. Verapamil and pyrilamine are able to interact with organic cation transporters such as OCT1 and OCTN1 [13,14]. However, tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPPþ), L-carnitine, and choline, which are typical substrates of organic cation transporters such as OCTs, OCTNs, plasma membrane monoamine transporter (PMAT), multidrug and toxin extrusion protein (MATE), and choline transporter (CHT), did not inhibit nicotine uptake. On the contrary, these compounds promoted nicotine uptake by A549 cells. These results suggest that neither OCTs, OCTNs, MATE, PMAT, nor CHT are responsible for nicotine uptake by A549 cells. As the mechanism involved in the uptake increase by compounds, it is considered to be the transcriptional-, translational-upregulation, and trans-stimulation of the transport system. Although the mechanism by which nicotine uptake in A549 cells is promoted remains unclear, it is unlikely that the transcriptional- and translational-upregulation is involved in the increase of nicotine uptake since the uptake time was very short (30 s). Compared with the specificity of nicotine uptake in other cell model such as brain capillary endothelial cells (TR-BBB13 cells), retinal capillary endothelial cells (TR-iBRB2 cells), and hepatocytes, the pH sensitivity of nicotine uptake in A549 cells was different, whereas the Km value of the high-affinity saturable process of nicotine uptake by A549 cells was similar to that reported in those other cell model (i.e. Km ¼ 92e492 mM) [2e4]. Although this difference is remains unclear, it may cause of difference between species. To fully understand this observed in the various cell types, it is essential to identify the molecule responsible for nicotine transport. Tobacco smoke is the primary cause of chronic obstructive pulmonary disease (COPD) in the developed world. Nevertheless, a substantial percentage of patients with moderate to severe COPD continue to smoke [15]. To examine, whether bronchodilators,
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which are the mainstay of COPD pharmacotherapy, interact with the nicotine influx transporter, inhibition studies with 1 mM salbutamol and procaterol were performed. Both bronchodilators had little effect on nicotine uptake by A549 cells, suggesting that inhalation of these drugs is unlikely to have an effect on nicotine disposition in the human lung. In conclusion, this is the first study to demonstrate that an influx system is responsible for nicotine transport at the human alveolar epithelial barrier. Moreover, it is unlikely to involve in drugedrug interaction with bronchodilators. The present findings provide important information to better understand pulmonary disposition of drug and other xenobiotics. Conflict of interest The authors declare there are no conflicts of interest. Acknowledgments This study was supported, in part, by a Grant-in-Aid for Scientific Research (B) [KAKENHI: 25293036] and Scientific Research (C) [KAKENHI: 26460193] from Japan Society for the Promotion of Science (JSPS), a Research Grant from Tamura Science and Technology Foundation, and a Clinical Investor Grant from Flight Attendant Medical Research Institute (FAMRI) [CIA130016]. References [1] Foth H, Looschen H, Neurath H, Kahl GF. Nicotine metabolism in isolated perfused lung and liver of phenobarbital- and benzoflavone-treated rats. Arch Toxicol 1991;65:68e72. [2] Tega Y, Akanuma S, Kubo Y, Terasaki T, Hosoya K. Blood-to-brain influx transport of nicotine at the rat bloodebrain barrier: involvement of a pyrilamine sensitive organic cation transport process. Neurochem Int 2013;62: 173e81. [3] Tega Y, Akanuma S, Kubo Y, Hosoya K. Involvement of the Hþ/organic cation antiporter in nicotine transport in rat liver. Drug Metab Dispos 2015;43: 89e92. [4] Tega Y, Kubo Y, Yuzurihara C, Akanuma S, Hosoya K. Carrier-mediated transport of nicotine across the inner blood-retinal barrier: involvement of a novel organic cation transporter driven by an outward H(þ) gradient. J Pharm Sci 2015;104:3069e75. [5] Zevin S, Schaner ME, Giacomini KM. Nicotine transport in a human choriocarcinoma cell line (JAR). J Pharm Sci 1998;87:702e6. [6] Spector R, Goldberg MJ. Active transport of nicotine by the isolated choroid plexus in vitro. J Neurochem 1982;38:594e6. [7] Salomon JJ, Hagos Y, Petzke S, Kühne A, Gausterer JC, Hosoya K, et al. Beta-2 adrenergic agonists are substrates and inhibitors of human organic cation transporter 1. Mol Pharm 2015;12:2633e41. [8] Salomon JJ, Ehrhardt C, Hosoya K. The verapamil transporter expressed in human alveolar epithelial cells (A549) does not interact with b2-receptor agonists. Drug Metab Pharmacokinet 2014;29:101e4. [9] Clunes LA, Bridges A, Alexis N, Tarran R. In vivo versus in vitro airway surface liquid nicotine levels following cigarette smoke exposure. J Anal Toxicol 2008;32:201e7. [10] Adrian CL, Olin HB, Dalhoff K, Jacobsen J. In vivo human buccal permeability of nicotine. Int J Pharm 2006;311:196e202. [11] Pankow JF, Tavakoli AD, Luo W, Isabelle LM. Percent free base nicotine in the tobacco smoke particulate matter of selected commercial and reference cigarettes. Chem Res Toxicol 2003;16:1014e8. [12] Sakamoto A, Matsumaru T, Yamamura N, Suzuki S, Uchida Y, Tachikawa M, et al. Drug transporter protein quantification of immortalized human lung cell lines derived from tracheobronchial epithelial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type IIlike cells (A549) by liquid chromatography-tandem mass spectrometry. J Pharm Sci 2015;104:3029e38. [13] Zhang L, Schaner ME, Giacomini KM. Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa). J Pharmacol Exp Ther 1998;286:354e61. [14] Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, et al. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pHdependent transport of organic cations. J Pharmacol Exp Ther 1999;289: 768e73. [15] Tashkin DP, Murray RP. Smoking cessation in chronic obstructive pulmonary disease. Respir Med 2009;103:963e74.