Results and Discussion

The in vivo hepatic uptake of [³H]nicotine was 1.5-fold greater than that of [¹⁴C]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, [³H]nicotine uptake exhibited time and concentration dependence, with a K_m of 141 µM, a V_max of 1.78 nmol/(min⋅mg protein) and a K_d of 5.69 µl/(min⋅mg protein) that were estimated from the uptake data obtained at 1 minute (Fig. 1, A and B), suggesting the involvement of a carrier-mediated transport process in nicotine uptake by rat hepatocytes. The K_d was assumed to represent the nonsaturable process, including the nonspecific nicotine adsorption exhibited by the y intercept (Fig. 1A) and/or passive diffusion of nicotine. In human, nicotine concentration in the circulating blood is reported to rise to 179–370 nM after smoking (Gourlay and Benowitz, 1997). Under the concentration range, the contribution of saturable component for total uptake clearance is estimated as 69%, suggesting the major role of carrier-mediated transport in hepatic nicotine uptake.

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Fig. 1.

Nicotine uptake by rat hepatocytes. (A) Time-dependent uptake of [³H]nicotine (1 µCi/ml, 12 nM) was investigated for the indicated times. (B) Concentration-dependent uptake of [³H]nicotine was measured for 1 minute, and the K_m, V_max, and K_d were estimated as 141 ± 63 µM, 1.78 ± 0.71 nmol/(min⋅mg protein), and 5.69 ± 0.67 µl/(min⋅mg protein), respectively. The solid and dashed lines represent the overall and saturable transport, respectively. (C and D) Effect of Na⁺, membrane potential, and extracellular pH on [³H]nicotine uptake was examined for 1 minute. (E) Intracellular pH dependence on [³H]nicotine uptake was examined for 15 seconds. Acute treatment and pretreatment (Pre) with NH₄Cl were used to make the intracellular pH alkaline and acidic, respectively. (F) The cells were preincubated with or without 10 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) for 10 minutes, and subsequently [³H]nicotine uptake was measured for 15 seconds. These uptake studies were performed at 37°C. Each point or column represents the mean ± S.E.M. (n = 3–6). *P < 0.05; **P < 0.01, significantly different from control.

The study of ion-dependence suggests the extracellular Na⁺ and membrane potential independence of hepatic nicotine uptake because of the slight and insignificant changes in [³H]nicotine uptake in buffer where Na⁺ was replaced by Li⁺ and K⁺ (Fig. 1C). On the other hand, [³H]nicotine uptake was reduced and increased at an extracellular pH of 6.4 and 8.4, respectively (Fig. 1D). Intracellular acidification increased [³H]nicotine uptake, and intracellular alkalization and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a protonophore, reduced the uptake (Fig. 1, E and F), suggesting that hepatic nicotine transport is driven by an oppositely directed H⁺ gradient.

The results of the inhibition study suggest the involvement of a cation-sensitive transport system in hepatic nicotine transport, because the [³H]nicotine uptake was strongly inhibited by hydrophobic cationic drugs, such as pyrilamine, verapamil, propranolol, quinidine, amantadine, and clonidine (Table 2). As the organic cation transporters, OCTs, OCTNs, multidrug and toxin extrusion proteins, and plasma membrane monoamine transporters are known (Gründemann et al., 1994; Tamai et al., 1997; Kekuda et al., 1998; Wu et al., 1998; Engel and Wang, 2005; Hiasa et al., 2006); however, the substrates or inhibitors of organic cation transporters (OCTs, OCTNs, multidrug and toxin extrusion proteins, and plasma membrane monoamine transporters) and organic anion transporters, such as tetraethylammonium, l-carnitine, 1-methyl-4-phenylpyridinium, choline, and p-aminohippurate, slightly inhibited the uptake (Table 2), suggesting the involvement of a carrier-mediated transport process as distinct from the well-characterized transporters in hepatic nicotine transport.

Recently, the involvement of a putative pyrilamine transport system was suggested in the nicotine transport at the BBB (Okura et al., 2008; Tega et al., 2013), and the in vitro uptake study revealed that [³H]pyrilamine uptake by isolated rat hepatocytes was strongly inhibited by cationic drugs, such as nicotine, but not substrates of typical organic cation transporters (Table 2). Although [³H]pyrilamine uptake by isolated rat hepatocytes exhibited time and concentration dependence with a K_m1 of 0.928 µM, a V_max1 of 0.533 nmol/(min⋅mg protein), a K_m2 of 351 µM, and a V_max2 of 34.2 nmol/(min⋅mg protein) (Supplemental Fig. 1, A and B), these K_m values are inconsistent with the K_m (28 µM) of which pyrilamine uptake by TR-BBB13 cells, an in vitro rat BBB model (Okura et al., 2008). In addition, extracellular pH had no significant effect on [³H]pyrilamine uptake by isolated rat hepatocytes (Supplemental Fig. 1C) during the pH dependence shown in pyrilamine uptake by TR-BBB13 cells (Okura et al., 2008). These results indicate that the transport mechanism of pyrilamine in the liver is different from putative pyrilamine transport system at the BBB, suggesting that the transport mechanism, distinct from putative pyrilamine transport system at the BBB, contributes to the nicotine transport in the liver.

In a kinetic study, nicotine uptake by isolated rat hepatocytes in the presence of pyrilamine exhibited a K_m of 180 µM, a V_max of 0.468 nmol/(min⋅mg protein), and a K_d of 6.03 µl/(min⋅mg protein) (Supplemental Fig. 2) that suggested the noncompetitive inhibition by pyrilamine in the liver, because the V_max was significantly reduced in the presence of pyrilamine despite the closely similar K_m values. In our previous report, the competitive inhibition by pyrilamine was shown in nicotine uptake by TR-BBB13 cells (Tega et al., 2013), and these suggest that the nicotine uptake system in the liver is different from that of the BBB.

In conclusion, a carrier-mediated transport process involving the blood-to-liver transport of nicotine appears to be present on the sinusoidal membrane of the hepatocytes and is suggested to be driven by the oppositely directed H⁺ gradient. The inhibition study suggests that a novel transporter, highly sensitive to cationic drugs, contributes to nicotine transport by the liver. These findings provide helpful information to increase our understanding of the pharmacokinetics of nicotine.