Diphenhydramine transport by pH-dependent tertiary amine transport system in Caco-2 cells

Hiroshi Mizuuchi, Toshiya Katsura, Kayoko Ashida, Yukiya Hashimoto, and Ken-Ichi Inui

Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Substrate specificity and pH dependence of the transport system for diphenhydramine were investigated in Caco-2 cell monolayers. Diphenhydramine uptake was not affected by any typical substrate for the renal organic cation transport system except procainamide. Along with procainamide, tertiary amine compounds with N-dimethyl or N-diethyl moieties in their structures inhibited the diphenhydramine uptake. Moreover, accumulation of diphenhydramine was stimulated by preloading the Caco-2 cells with these tertiary amines (trans-stimulation effect), indicating the existence of the specific transport system for tertiary amines with N-dimethyl or N-diethyl moieties. Efflux of diphenhydramine from monolayers was enhanced by medium acidification. In addition, intracellular acidification resulted in marked stimulation of diphenhydramine accumulation. ATP depletion of the cells caused an enhancement of diphenhydramine accumulation, suggesting the involvement of an active secretory pathway. However, P-glycoprotein did not mediate the diphenhydramine transport. These findings indicate that a novel pH-dependent tertiary amine transport system that recognizes N-dimethyl or N-diethyl moieties is involved in diphenhydramine transport in Caco-2 cells.

organic cation; intestinal absorption; intestinal secretion; hydrogen/organic cation antiport system


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTESTINAL ABSORPTION mechanisms of organic cations have been observed through the passive diffusion of nonionized compounds according to the pH partition theory. Although most organic cations are present in an ionized form over the pH range in the gastrointestinal tract, several organic cations are known to be well absorbed from the small intestine after oral administration. Recent studies have suggested the contribution of specific transport systems to the intestinal absorption of organic cations (15, 25, 27). In these studies, saturable transport of organic cations was demonstrated. The mechanisms of organic cation transport have been extensively investigated in the kidney and liver (8, 13, 34); however, those in the intestine are not well understood. On the other hand, it has been suggested that the intestinal epithelia function as an absorptive barrier for drug absorption (4, 14, 17). Intestinal secretion of organic cations was first demonstrated in isolated guinea pig intestinal mucosa (30, 31). It was shown that P-glycoprotein localized in the intestinal brush-border membrane is involved in the active intestinal secretion of organic cations (5). A guanidine/H+ exchanger was characterized in intestinal brush-border membrane vesicles from rabbit (20). It has been suggested that the guanidine/H+ exchanger functioned as an efflux system for the organic cations. Furthermore, it was demonstrated that ciprofloxacin, a fluoroquinolone, was secreted by another secretory system distinct from both the guanidine/H+ exchanger and P-glycoprotein (3). However, the details of these intestinal transport systems for organic cations are not well understood at present.

Recently, we have demonstrated that diphenhydramine, an antihistamine, is accumulated by the pH-dependent transport system in Caco-2 cells (21). Diphenhydramine accumulation was temperature dependent, saturable, and shown to be decreased at lower extracellular pH. Diphenhydramine accumulation was not inhibited by tetraethylammonium or biological amines and/or neurotransmitters such as histamine, serotonin, dopamine, and choline. However, cellular accumulation of diphenhydramine was affected by cis- and trans-interaction with another structurally similar organic cation, chlorpheniramine. Therefore, it was suggested that a specific transport system for diphenhydramine is present in Caco-2 cells. The purpose of the present study was to elucidate further the substrate specificity and pH dependence of this transport system. Our findings indicate that diphenhydramine transport in Caco-2 cells is mediated by a novel pH-dependent tertiary amine transport system that recognizes N-dimethyl or N-diethyl moieties.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Diphenhydramine hydrochloride was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). dl-Chlorpheniramine maleate, cimetidine, 2-deoxy-D-glucose, 2-dimethylaminoethanol, dimethylaminoethylchloride hydrochloride, dimethylaminopropylchloride hydrochloride, 2,4-dinitrophenol, guanidine hydrochloride, sodium azide, tetrabutylammonium bromide, tetraethylammonium bromide, triethanolamine hydrochloride, and triethylamine hydrochloride were obtained from Nacalai Tesque (Kyoto, Japan). FCCP was purchased from Fluka (Buchs, Switzerland). 1-Methyl-4-phenylpyridinium (MPP), N1-methylnicotinamide (NMN), procainamide hydrochloride, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and BCECF-AM were obtained from Sigma Chemical (St. Louis, MO). All other chemicals were of the highest purity available.

Cell culture. Caco-2 cells at passage 18 obtained from the American Type Culture Collection (ATCC HTB 37) were maintained by serial passage in plastic culture dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) as described previously (9, 19). For uptake or efflux studies, 60-mm plastic dishes were inoculated with 5 × 105 cells in 5 ml of the complete culture medium. The medium consisted of DMEM (GIBCO Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Whittaker Bioproducts, Walkersville, MD) and 1% nonessential amino acids (GIBCO) without antibiotics. The cells were grown in an atmosphere of 5% CO2-95% air at 37°C and given fresh medium every three or four days.

LLC-GA5-COL150 cells, stably transfected with human MDR1 cDNA (29, 32), and LLC-PK1 cells (ATCC, CRL 1392) as host cells were maintained by serial passage in plastic culture dishes as previously described (10). For transport studies, LLC-GA5-COL150 and LLC-PK1 cells were seeded on polycarbonate membrane filters (3-µm pores, 4.71 cm2 growth area) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a cell density of 5 × 105 cells/cm2. Each Transwell culture chamber was placed in a 35-mm well of a tissue culture plate with 2.6 ml of the outside medium (basolateral side) and 1.5 ml of the inside medium (apical side). The cell monolayers were fed fresh complete medium every 2 days and were used on the 6th day for the transport experiments.

Measurement of diphenhydramine accumulation and transport. The composition of the incubation medium was as follows (in mM): 145 NaCl, 3 KCl, 1 CaCl2, 0.5 MgCl2, 5 D-glucose, and 5 MES (pH 6.0) or HEPES (pH 7.4). The accumulation of diphenhydramine by monolayers grown in 60-mm plastic culture dishes was determined as previously described (21). To evaluate the efflux of diphenhydramine, the monolayers were preloaded with 1 mM diphenhydramine (pH 7.4) for 30 min. After removing the medium, the monolayers were washed once with ice-cold incubation medium and then incubated with incubation medium for the specified periods. Thereafter, remaining intracellular diphenhydramine was measured.

The transepithelial transport of diphenhydramine across LLC-GA5-COL150 and LLC-PK1 cells was measured using monolayer cultures grown in the Transwell chambers. After the removal of the culture medium from both sides of the monolayers, the cell monolayers were preincubated for 10 min at 37°C with 2 ml of incubation medium in both sides. At the end of preincubation, the medium was immediately removed, the incubation medium containing diphenhydramine was added to the basolateral side, and 2 ml of incubation medium (without drug) was added to the apical side. The incubation proceeded for specified periods of time at 37°C. To measure transepithelial transport, the incubation medium in the opposite side was collected periodically. The collected samples were diluted fourfold with 0.01 N HCl/methanol (1:1) and analyzed by HPLC as described in Analytical methods.

Measurement of intracellular pH. Intracellular pH of Caco-2 cells was measured using the fluorescent probe BCECF. Caco-2 cells grown on plastic dishes were loaded with 5 µM BCECF-AM for 30 min at 37°C. Then the cells were washed twice with incubation medium, trypsinized, washed three more times, and then resuspended in the incubation medium (1 × 106 cells/ml). Fluorescence ratio of BCECF (excitation, 440 and 490 nm; emission, 520 nm) was monitored with a spectrofluorophotometer (RF-5000; Shimadzu, Kyoto, Japan) equipped with a stirred cuvette, thermostated to 37°C. The emission intensity at each excitation wavelength was averaged over a 1-s interval, and the ratios were taken every 2 s. After stabilization of the baseline, drug solutions (100 µl) were added to the BCECF-loaded cell suspensions (2 ml) and initial changes in fluorescence ratio for 1 min after addition of the drug were monitored. Intracellular pH was calibrated by external measurements using BCECF-free acid in cell-free incubation medium titrated to different pH values between 6.6 and 7.8. The ratios plotted against pH resulted in a highly linear correlation, with correlation coefficients >0.999 (data not shown). Under our experimental conditions, a resting intracellular pH of 6.865 ± 0.012 (n = 21) was obtained.

Analytical methods. Diphenhydramine was assayed using a high-performance liquid chromatograph (LC-10A; Shimadzu) equipped with a UV spectrophotometric detector (SPD-6A; Shimadzu) and an integrator (Chromatopac C-R1A, Shimadzu) as previously described (21). The protein content of the cell monolayers solubilized in 1 ml of 1 N NaOH was determined by the method of Bradford (2) using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) with bovine gamma -globulin as a standard.

Statistical analysis. Data were analyzed statistically by unpaired t-test or one-way ANOVA followed by Scheffé's test when multiple comparisons were needed. Probability values <5% were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of substrates of renal organic cation transport system on diphenhydramine accumulation in Caco-2 monolayers. To clarify the substrate specificity of the diphenhydramine transport system, the effect of various organic cations on diphenhydramine accumulation was first examined. Figure 1 shows the effect of substrates of the renal organic cation transport system on diphenhydramine accumulation. Among compounds tested, only procainamide inhibited the diphenhydramine accumulation in a concentration-dependent manner, whereas NMN, MPP, and cimetidine had no effect. Furthermore, we confirmed that procainamide accumulation in Caco-2 cells was inhibited by diphenhydramine (data not shown).


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Fig. 1.   Effect of substrates for renal organic cation transport system on diphenhydramine accumulation in Caco-2 cells. Caco-2 cells were incubated for 5 min at 37°C with incubation medium (pH 7.4) containing diphenhydramine (1 mM) in the absence (control) or presence (5, 10, or 20 mM) of other organic cations. Thereafter, accumulation of diphenhydramine was measured. A: procainamide; B: N1-methylnicotinamide; C: 1-methyl-4-phenylpyridinium; D: cimetidine. Values are means ± SE of 3 monolayers. ** P < 0.01 vs. control.

Effect of various tertiary amine compounds on diphenhydramine accumulation in Caco-2 monolayers. Because diphenhydramine, chlorpheniramine, and procainamide are tertiary amines, we then examined the effect of various tertiary amine compounds on diphenhydramine accumulation in Caco-2 cells. As shown in Fig. 2, diphenhydramine accumulation was inhibited by tertiary amine compounds that contain N-dimethyl or N-diethyl moieties in their structures but not by 2-dimethylaminoethanol and triethanolamine. Quaternary amines such as tetraethylammonium and tetrabutylammonium were not effective even though they had N-diethyl or N-dibutyl moieties (data not shown). Furthermore, as shown in Fig. 3, diphenhydramine uptake was stimulated when monolayers were preloaded with dimethylaminoethylchloride or triethylamine (trans-stimulation effect). On the other hand, the uptake was not stimulated by an N-diethanol compound, triethanolamine.


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Fig. 2.   Effect of various tertiary amine compounds on diphenhydramine accumulation in Caco-2 cells. Caco-2 cells were incubated for 1 min at 37°C with incubation medium (pH 7.4) containing diphenhydramine (1 mM) in absence (control) or presence of various tertiary amines (5 mM). Thereafter, accumulation of diphenhydramine was measured. Values are means ± SE of 3 monolayers. ** P < 0.01 vs. control.



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Fig. 3.   Trans-stimulation effect of tertiary amines on diphenhydramine uptake by Caco-2 cells. Caco-2 cells were preincubated for 30 min at 37°C with incubation medium (pH 7.4) in absence (control) or presence (10, 20, or 40 mM) of tertiary amines. After removing medium, monolayers were incubated with 1 mM diphenhydramine for 1 min at 37°C. Thereafter, accumulation of diphenhydramine was measured. A: dimethylaminoethylchloride; B: triethylamine; C: triethanolamine. Values are means ± SE of 3 monolayers. ** P < 0.01 vs. control.

Efflux of diphenhydramine from Caco-2 monolayers. Because diphenhydramine accumulation by Caco-2 cells was pH dependent, the effect of extracellular pH on efflux of diphenhydramine from the monolayers was examined. Diphenhydramine efflux was measured after preloading by means of a 30-min incubation with 1 mM diphenhydramine (pH 7.4). Figure 4 shows the time course of the amount of diphenhydramine remaining in the monolayers. The efflux rate of diphenhydramine was more facilitated under the extracellular pH 6.0 than that of 7.4. 


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Fig. 4.   Effect of medium pH on diphenhydramine efflux from Caco-2 monolayers. After 30-min incubation at 37°C with incubation medium (pH 7.4) containing 1 mM diphenhydramine, monolayers were incubated with incubation medium at pH 7.4 (open circle ) or 6.0 (). Amounts of diphenhydramine remaining in monolayers after incubation were determined. Values are means ± SE of 3 monolayers.

Effect of intracellular pH on diphenhydramine accumulation. We then examined the effect of alterations in intracellular pH on diphenhydramine accumulation in Caco-2 cells. Intracellular pH was manipulated by treatment of the cells with ammonium chloride. When ammonium chloride was added to the preincubation medium and then removed (pretreatment), intracellular pH fell. On the contrary, exposure of the cells to ammonium chloride (acute treatment) causes rapid alkalization of intracellular pH (24). As shown in Fig. 5, intracellular acidification by pretreatment resulted in marked stimulation of diphenhydramine accumulation. In contrast, diphenhydramine accumulation was decreased by intracellular alkalization.


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Fig. 5.   Effect of intracellular pH on diphenhydramine uptake by Caco-2 cells. Caco-2 cells were preincubated with incubation medium (pH 7.4) in absence (control, Acute NH4Cl) or presence (Pre NH4Cl) of 30 mM NH4Cl for 20 min. Medium was removed, and cells were incubated for 5 min at 37°C with incubation medium (pH 7.4) containing 1 mM diphenhydramine in absence (control, Pre NH4Cl) or presence (Acute NH4Cl) of 30 mM NH4Cl. Values are means ± SE of 3 monolayers.

Effect of various tertiary amines on intracellular pH. Effect of various tertiary amines on intracellular pH in Caco-2 cells was then examined because tertiary amines could alter intracellular pH via nonionic diffusion of unionized species. Such intracellular pH changes in turn might modulate diphenhydramine accumulation via nonionic diffusion. Tertiary amines were added to the BCECF-loaded cell suspensions, and intracellular alkalization was calculated. As shown in Table 1, addition of chlorpheniramine or ammonium chloride resulted in rapid and marked alkalization of intracellular pH. However, dimethylaminoethylchloride, which considerably inhibited the diphenhydramine uptake (Fig. 2), had little effect on intracellular pH.

                              
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Table 1.   Effect of tertiary amines on intracellular pH in Caco-2 cells

Metabolic energy requirement of diphenhydramine uptake. To determine the requirement of metabolic energy for the diphenhydramine uptake by Caco-2 cells, the effects of various metabolic inhibitors were examined. Caco-2 cells were pretreated with sodium azide and 2-deoxyglucose, dinitrophenol, or FCCP, and ATP-depleted conditions were generated. As shown in Fig. 6, diphenhydramine accumulation was apparently increased, rather than decreased, under all ATP-depleted conditions examined.


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Fig. 6.   Effect of ATP depletion on diphenhydramine uptake by Caco-2 cells. Caco-2 cells were incubated for 1 min at 37°C with diphenhydramine (1 mM) under ATP-depleted conditions. ATP-depleted conditions were generated as follows: A: Caco-2 cells were preincubated for 20 min at 37°C with incubation medium containing 10 mM sodium azide (NaN3) and 20 mM 2-deoxy-D-glucose without D-glucose. B: Caco-2 cells were preincubated for 10 min with incubation medium containing 0.1 or 1 mM 2,4-dinitrophenol (DNP). C: Caco-2 cells were incubated for 10 min at 37°C with 10 µM FCCP. Values are means ± SE of 3 monolayers. ** P < 0.01 vs. control.

Role of P-glycoprotein in diphenhydramine accumulation. Because diphenhydramine accumulation was enhanced under ATP-depleted conditions, we then examined whether diphenhydramine was transported by P-glycoprotein, an active efflux pump. When Caco-2 monolayers were incubated with 10 µM cyclosporin A, a typical inhibitor/substrate of P-glycoprotein (1, 22), diphenhydramine accumulation was not changed (control, 0.60 ± 0.03; with cyclosporin A, 0.55 ± 0.02 nmol · mg protein-1 · 15 min-1, mean ± SE of 3 monolayers). Furthermore, the basolateral-to-apical transepithelial transport of diphenhydramine in LLC-GA5-COL150 cells that overexpress P-glycoprotein was not different from LLC-PK1 cells (Fig. 7).


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Fig. 7.   Transport of diphenhydramine across LLC-PK1 and LLC-GA5-COL150 cell monolayers. Diphenhydramine (100 µM) was added to medium of basolateral side of monolayers of LLC-PK1 (open circle ) or LLC-GA5-COL150 () cells, and amounts of diphenhydramine appearing in apical side were measured. Values are means ± SE of 3 monolayers.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Compared with the kidney and liver, there is little information regarding the mechanisms of organic cation transport in the intestine (13, 34). We have recently shown that diphenhydramine, an antihistamine, is accumulated by a pH-dependent transport system in Caco-2 cells (21). It was demonstrated that diphenhydramine accumulation was not inhibited by biological amines and/or neurotransmitters such as histamine, serotonin, dopamine, and choline. In addition, tetraethylammonium and cimetidine had no effect on diphenhydramine accumulation. On the other hand, diphenhydramine accumulation was inhibited by chlorpheniramine and was stimulated when Caco-2 cells were preloaded with chlorpheniramine (trans-stimulation effect). Therefore, it was suggested that a specific transport system for diphenhydramine and chlorpheniramine is present in Caco-2 cells. In the present study, we further investigated the substrate specificity and pH dependence of this transport system.

Diphenhydramine uptake was inhibited only by procainamide among the typical substrates for the renal organic cation transport system (Fig. 1). This finding suggests that substrate specificity was different between the renal organic cation transport system and the transport system for diphenhydramine. Since diphenhydramine, chlorpheniramine, and procainamide are tertiary amines, we then examined the effect of various tertiary and quaternary amines on diphenhydramine accumulation. Among compounds tested, tertiary amine compounds with N-dimethyl or N-diethyl moieties in their structures, such as dimethylaminoethylchloride, dimethylaminopropylchloride, and triethylamine, inhibited the accumulation of diphenhydramine. Diphenhydramine, chlorpheniramine, and procainamide also contain N-dimethyl or N-diethyl moieties. On the contrary, triethanolamine and quaternary amines had no effect on diphenhydramine accumulation. Furthermore, diphenhydramine uptake was trans-stimulated by preloading the cells with dimethylaminoethylchloride or triethylamine but not with triethanolamine. Thus these results clearly indicate that this transport system could recognize the N-dimethyl or N-diethyl moieties in these tertiary amine compounds.

Diphenhydramine accumulation in Caco-2 cells is pH dependent and shown to be decreased at the lower pH (21). Following the analogy of the organic cation transport system (H+/organic cation antiport system) in the kidney, pH-dependent diphenhydramine transport could be interpreted as an H+/diphenhydramine antiport mechanism. To clarify this assumption, we examined the effect of intracellular pH on diphenhydramine accumulation. As clearly shown in Fig. 5, diphenhydramine accumulation was stimulated by acidifying the intracellular pH. In contrast, diphenhydramine accumulation was decreased by intracellular alkalization. Moreover, efflux of diphenhydramine from the cells was facilitated under an extracellular pH of 6.0. These results suggest that an outward H+ gradient serves as a driving force for diphenhydramine transport, i.e., H+/diphenhydramine antiport. However, it is possible that pH-dependent transport of diphenhydramine might be explained by nonionic diffusion of the unionized form. Cis-inhibition and trans-stimulation effects of various tertiary amines on diphenhydramine accumulation could also be explained by the ability of tertiary amines to alter intracellular pH via nonionic diffusion as ammonium chloride did (Fig. 5). Indeed, acute treatment of Caco-2 cells with tertiary amines such as chlorpheniramine and dimethylaminopropylchloride caused rapid intracellular alkalization. However, dimethylaminoethylchloride, which inhibited the diphenhydramine accumulation considerably, did not induce rapid intracellular alkalization. Thus it is likely that inhibition of diphenhydramine uptake by tertiary amines is not due to their ability to alter intracellular pH via nonionic diffusion but due to their direct interaction with the diphenhydramine transport system. It is possible that pH-dependent transport of diphenhydramine can be explained at least in part by nonionic diffusion; however, our results suggest that the specific transport system for tertiary amines is involved in diphenhydramine transport in Caco-2 cells.

It is supposed that physiological function of this transport system is secreting tertiary amines with N-dimethyl or N-diethyl moieties to the luminal side of the intestine followed by exchanging the tertiary amine for the extracellular H+. Because luminal pH is more acidic than intracellular pH in the intestine, an inward H+ gradient is helpful for secretion via this exchange system. However, it is possible that diphenhydramine transport is regulated not by H+ gradient but by pH itself. Therefore, the apparent pH dependence of diphenhydramine transport could be explained by its optimal pH. In fact, the renal H+/organic cation antiport system is sensitive to pH values (12, 18). Accordingly, from the data obtained from the cell culture model, it is difficult to conclude that an H+ gradient is the driving force for tertiary amine transport. Further studies using isolated membrane vesicles are necessary to determine the driving force.

Recently, the thiamine/H+ exchange system was studied in rat intestinal brush-border membrane vesicles (16). It was suggested that the thiamine/H+ exchange system allows the intestinal absorption of thiamine, though inwardly directed H+ gradient is generated in the intestine. Moreover, it was also reported that procainamide might be accumulated in the porcine renal epithelial cell line, LLC-PK1, via the apical organic cation/H+ exchanger (24). Therefore, it is supposed that diphenhydramine is accumulated via a pH-dependent tertiary amine transport system in Caco-2 monolayers.

Diphenhydramine accumulation in Caco-2 cells was enhanced under ATP-depleted conditions (Fig. 6). This finding suggests that diphenhydramine is actively secreted to the luminal side. Indeed, the basolateral-to-apical transepithelial transport of diphenhydramine across Caco-2 cell monolayers grown on permeable support as well as the efflux of diphenhydramine from the cells to the apical side is higher than that in the opposite direction (H. Mizuuchi, T. Katsura, Y. Hashimoto, and K. Inui, unpublished observations). It is well known that an active efflux pump, P-glycoprotein, was expressed in Caco-2 cells as well as in the intestine (5, 7). This efflux pump was reported to secrete some lipophilic organic cations and reported to prevent absorption of cationic drugs (5-7, 11, 23, 25). In the present study, however, diphenhydramine accumulation was not altered by cyclosporin A, a typical inhibitor of P-glycoprotein. In addition, transport characteristics were not different between P-glycoprotein stably expressed cell line LLC-GA5-COL150 and the LLC-PK1 host cells. Previously, we have demonstrated that the extensive secretory transport of substrates of P-glycoprotein in LLC-GA5-COL150 monolayers compared with the host cell (10, 28). Thus it seems unlikely that diphenhydramine is transported via P-glycoprotein. Alternatively, it is possible that stimulation of diphenhydramine uptake under ATP-depleted conditions is due to intracellular acidification by the treatment of metabolic inhibitors. Because it was demonstrated that FCCP and DNP acidify the cytosol when added to intact cells, diphenhydramine accumulation could be stimulated by an imposed outward H+ gradient via the pH-dependent tertiary amine transport system. Further studies are needed to clarify whether diphenhydramine is secreted by another active efflux pump.

Recently, a novel organic cation transporter, OCTN1, was cloned, and its functional properties were characterized (26, 33). It was suggested that this transporter might function as the organic cation/H+ exchanger, although direct coupling of H+ and organic cations has not been demonstrated. Furthermore, it was suggested that transport activity of OCTN1 was regulated not only by the pH levels but also by ATP. In the present study, ATP depletion caused the enhancement of diphenhydramine accumulation. Therefore, it is possible that ATP might affect the activity of the diphenhydramine transport by the novel pH-dependent tertiary amine transport system.

In summary, our findings suggest that diphenhydramine is transported by a novel pH-dependent tertiary amine transport system in Caco-2 cells. This transport system specifically recognizes the N-dimethyl or N-diethyl moieties in tertiary amine compounds and may function not only as a secretory pathway but also as an absorptive pathway for the tertiary amine compounds, such as diphenhydramine.


    ACKNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a Grant-in-Aid from the Uehara Memorial Foundation.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. Inui, Dept. of Pharmacy, Kyoto Univ. Hospital, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: inui{at}kuhp.kyoto-u.ac.jp).

Received 9 August 1999; accepted in final form 2 December 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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