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Nicotine is an addictive alkaloid in cigarette smoke and is responsible for tobacco dependence. It is important to consider the bloodto-liver transport of nicotine to understand the nicotine elimination from the body because most of the nicotine is converted to inactive metabolites by cytochrome P450 localized in the endoplasmic reticulum of the hepatocytes. In this study, the blood-to-liver transport of nicotine was investigated by means of an in vivo portal vein injection technique in rats, and the in vitro uptake by freshly isolated rat hepatocytes was used to clarify its mechanism. The results obtained showed that the in vivo blood-to-liver transport of [H]nicotine was significantly inhibited by 50 mM nicotine and pyrilamine, suggesting involvement of a carrier-mediated transport process in the blood-toliver transport of nicotine. The in vitro uptake study using freshly isolated rat hepatocytes showed a timeand concentration-dependent uptake of [H]nicotine with a Km value of 141 mM, and the uptake was increased under alkaline extracellular conditions. In addition, intracellular acidification caused an increase in [H]nicotine uptake, suggesting that the influx transport of nicotine is driven by an oppositely directed H gradient in hepatocytes. Moreover, [H]nicotine uptake was strongly inhibited in the presence of cationic drugs, such as pyrilamine, whereas only weak inhibitory effects were shown by substrates of typical organic cation transporters, such as tetraethylammonium, 1-methyl-4-phenylpyridinium, choline, and L-carnitine. In conclusion, a carrier-mediated system controlling the blood-to-liver transport of nicotine appears to be present on the sinusoidal membrane of hepatocytes. The pattern of inhibition and ion dependence is suggestive of an H/organic cation antiportermediated nicotine transport system. Introduction Smoking is an important risk factor in diseases such as cancer, cardiovascular disease, and chronic obstructive pulmonary disease. Nearly 6 million people die from smoking-related disease each year, and this number is expected to increase to more than 8 million by 2030 (Syed and Chaudhari, 2013), showing the great importance of smoking cessation to prevent increasingly severe health problems. Nicotine is a major alkaloid synthesized in Nicotiana tabacum and is responsible for tobacco dependence. In the brain, nicotine stimulates dopamine release from the neurons via nicotinic acetylcholine receptors and causes feelings of pleasure and reward (Dani and De Biasi, 2001). In addition, nicotine causes withdrawal symptoms, such as irritability and anxiety, as consequence of a reduction in its concentration in the circulating blood (Hughes et al., 1984). Because of these neural events associated with nicotine, smokers inhale cigarette smoke repeatedly to maintain the nicotine concentration in their circulating blood, suggesting that obtaining information about the modulation of the nicotine concentration in the circulating blood will help improve the effectiveness of smoking cessation therapy. Recently, our investigations revealed the involvement of a carriermediated transport process in the blood-to-brain transport of nicotine across the blood-brain barrier (BBB), based on the results obtained from in vivo and in vitro studies (Tega et al., 2013). In addition, carriermediated nicotine transport has also been reported in previous studies in Caco-2 cells and rat kidney (Fukada et al., 2002a,b), suggesting the important contribution of carrier-mediated transport to the tissue uptake of nicotine. In the liver, it has been reported that 70–80% nicotine in the circulating blood is metabolized to cotinine by cytochrome P450 (P450) (Benowitz and Jacob, 1994), showing that hepatic clearance plays an important role in nicotine elimination from the circulating blood. In humans, the genetic polymorphisms of CYP2A6, the hepatic enzyme involved in nicotine metabolism (Nakajima et al., 2000), alter pharmacokinetics of nicotine, and individuals with a higher activity of CYP2A6 tend to develop tobacco dependence (Ray et al., 2009). In an in vivo study in mice lacking the P450 enzyme involved in nicotine metabolism, the pharmacokinetic parameters for nicotine, such as its half-life in blood and the area under the blood concentration-time curve, were altered to increase the sensitivity to rewarding effects of nicotine (Li et al., 2013). These lines of evidence strongly suggest that the hepatic clearance of nicotine affects the pharmacological response to nicotine. In terms of the interaction of nicotine with P450, nicotine transport into intracellular space across the sinusoidal membrane of hepatocytes is essential for P450-mediated metabolism of nicotine, because P450 is mainly localized in the endoplasmic reticulum of hepatocytes. Therefore, clarification of the mechanism underlying nicotine uptake at the sinusoidal membrane of hepatocytes will provide helpful information about controlling the nicotine concentration in the circulating blood. In the present study, to examine the properties of the influx transport of nicotine by the liver, the nicotine transport was characterized using an in vivo portal vein injection technique called the liver uptake index method and freshly isolated rat hepatocytes. This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS). dx.doi.org/10.1124/dmd.114.061002. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: BBB, blood-brain barrier; OCT, organic cation transporter; OCTN, organic cation/carnitine transporter; P450, cytochrome P450. 89 http://dmd.aspetjournals.org/content/suppl/2014/10/28/dmd.114.061002.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods Animals and Reagents. Wistar rats (male, 150–200 g) were purchased from Japan SLC (Hamamatsu, Japan) and kept in a controlled environment. All experiments conformed to the provisions of the Animal Care Committee, University of Toyama. L-(2)-[N-Methyl-H]nicotine ([H]nicotine, 83.5 Ci/mmol) and [pyridinyl-5-H]pyrilamine ([H]pyrilamine, 20.0 Ci/mmol) were purchased from PerkinElmer (Boston, MA). n-[1-C]Butanol, ([C]n-butanol, 2 mCi/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). All other chemicals were commercial products of analytical grade. Liver Uptake Index Method and Cellular Uptake Study. The liver uptake index method and a cellular uptake study involving freshly isolated hepatocytes were used to investigate the in vivo and in vitro transport of nicotine in the liver, respectively. The details are described in the Supplemental Data. Statistical Analysis. The kinetic parameters are presented as the means6 S.D. Other data are presented as the means 6 S.E.M. To determine the significance of differences between two group means, an unpaired Student’s t test was used. To assess the statistical significance of differences among means of more than two groups, one-way analysis of variance followed by Dunnett’s test was used. Results and Discussion The in vivo hepatic uptake of [H]nicotine was 1.5-fold greater than that of [C]n-butanol, an internal reference. In the liver uptake index technique, the concentration of drugs in the space of Disse was assumed to be lower than that of the injectate by in part of mixing effect (Tsuji et al., 1990), and the inhibitors of high concentration (50 mM) were used. As a result, unlabeled nicotine and pyrilamine significantly reduced in vivo nicotine uptake in the liver, whereas tetraethylammonium and L-carnitine had little effect (Table 1). These results suggest that a pyrilamine-sensitive influx system is involved in blood-to-liver transport of nicotine. In the in vitro analysis, [H]nicotine uptake exhibited time and concentration dependence, with a Km of 141 mM, a Vmax of 1.78 nmol/(min×mg protein) and a Kd of 5.69 ml/(min×mg protein) that were estimated TABLE 1 Effect of inhibitors on in vivo [H]nicotine uptake by rat liver [H]Nicotine (1.8 mCi/rat) and [C]n-butanol (0.18 mCi/rat) dissolved in 200 ml RingerHEPES buffer (pH 7.4) were injected into the portal vein in the absence (control) or presence of inhibitors (50 mM). Each value represents the mean 6 S.E.M. (n = 3–5). Compounds LUI Percentage of Control