Abstract
We investigated the uptake of imipramine (IMP) in highly purified lysosomes from rat liver and its inhibition by a variety of basic drugs in vitro. The uptake of [3H]IMP into lysosomes peaked in less than 20 s, showing little temperature dependency or countertransport phenomena. It was accelerated by increase of extralysosomal pH, stimulated by Mg2+-ATP in KCl buffer, and suppressed by acidic ionophores. However, the uptake of [3H]IMP in lysosomes was approximately 140-fold higher than the value expected from the pH-partition theory. IMP and other weak lipophilic bases like chlorpromazine and propranolol raised the intralysosomal pH, and their potency was stronger than that of NH4Cl, a typical pH-perturbing weak base. A variety of basic drugs inhibited the uptakes of [3H]IMP and [14C]methylamine into lysosomes, their 50% inhibitory concentrations (IC50) being almost the same for [3H]IMP and [14C]methylamine uptake (r = 0.842). A high correlation (r = 0.946) was observed between the IC50 values (for the inhibition of [3H]IMP uptake) and the lipophilicity (Poct values). These results suggest that the accumulation of lipophilic basic drugs is driven primarily by the transmembrane pH difference (pH-partition theory) but with the involvement of some additional mechanism(s) related to drug lipophilicity, possibly binding (partition or adsorption) to lipophilic substance(s) and/or aggregation within lysosomes. Based on this idea, we have established a model that described and successfully simulated the weak base-induced pH increase, the accumulation of a lipophilic weak base (IMP), and the inhibition of accumulation of IMP by lipophilic basic drugs.
Footnotes
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Send reprint requests to: Shoji Ohkuma, Department of Molecular and Cellular Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa, Ishikawa 920-0934, Japan. E-mail: ohkuma{at}kenroku.kanazawa-u.ac.jp
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↵FN1 This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan. This work was performed partly to fulfill a Ph.D. dissertation (J.I.) submitted to the Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan (1999) and was presented at the Annual Meeting of the Japanese Society for the Study of Xenobiotics, Hamamatsu, Japan (October, 1999).
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Received for publication January 14, 2000.
- Abbreviations:
- IMP
- imipramine
- AMA
- amantadine
- ATR
- atropine
- BAF
- bafilomycin A1
- CPZ
- chlorpromazine
- CQ
- chloroquine
- ΔpH
- transmembrane pH gradient
- DTZ
- diltiazem
- FD
- fluorescein isothiocyanate-dextran
- MeNH2
- methylamine
- NIG
- nigericin
- Poct
- octanol-water partition coefficient of the nonionized drug
- QN
- quinine
- PPR
- propranolol
- TFP
- trifluoperazine
- TMAH
- tetramethylammonium hydroxide
- V-ATPase
- vacuolar-type H+-ATPase
- VP
- verapamil
- Accepted May 22, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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