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We previously evaluated the renal excretion mechanism of quinidine, which is a tertiary amine compound, using porcine kidney epithelial LLC-PK 1 cells and P-glycoprotein (P-gp)-expressed LLC-GA5-COL150 cells.1) The transepithelial transport of quinidine in the basolateral-to-apical direction in LLC-PK 1 cells was similar to that in the opposite direction. In contrast, quinidine was transported actively in the basolateral-to-apical direction in LLC-GA5-COL150 cells. The results suggested that P-gp is mainly responsible for the tubular secretion of quinidine in the kidney. 1) We also evaluated the intestinal absorption mechanism of quinidine using human intestinal epithelial Caco-2 cells.2) The temperaturedependent uptake of quinidine in Caco-2 cells grown on a plastic dish was increased by alkalization of the apical medium, and was inhibited by diphenhydramine and imipramine. The results suggested that a cation transport system was involved in the influx of quinidine at the apical membrane in intestinal epithelial cells. 2)Procainamide, another tertiary amine compound with a pK a value of 9.23, is classified as a type IA antiarrhythmic drug that works by decreasing conduction velocity, and prolonging tissue refractoriness.3) More than 80% of orally administered procainamide is absorbed from the intestine in humans.4) The Kp (octanol/buffer at pH 7.4) value of procainamide is about 0.1, and approximately half of the dose is excreted in the urine as unchanged drug.3-6) However, the mechanisms responsible for the membrane transport of procainamide in intestinal and renal epithelial cells are still unclear.In the present study, the transport characteristics of procainamide in LLC-PK 1 cells were compared with those of quinidine. In addition, we evaluated whether the transport system for quinidine is present in another intestinal cell line, LS180, as well as Caco-2. We also investigated whether the transport system of procainamide in LS180 cells is the same as that of quinidine. Cell Culture and Preparation of Monolayers LLC-PK 1 cells at passage 197 and LS180 cells at passage 38 were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.). These cells were maintained by serial passage in plastic dishes with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Biowest Inc., Nuaille, France) in an atmosphere of 5% CO 2 -95% air at 37°C. MATERIALS AND METHODS MaterialsLLC-PK 1 cells were seeded at a density of 5ϫ10 5 cells/ cm 2 on a 1.12 cm 2 porous membrane (3 mm pore size) in a polyester membrane Transwell ® -Clear insert (Costar, Cambridge, MA, U.S.A.) to evaluate the transcellular transport of cationic drugs. The seeded cells were maintained for 6 d to prepare differentiated cell monolayers. The maturity of the monolayer was judged by transepithelial electrical resistance (TEER). TEER was measured using a Millicell-ERS resistance system (Millipore, Bedford, MA, U.S.A.). LLC-PK 1 cell monolayers whose TEER was above 60 W · cm 2 were used to The aim of the present study was t...

Background Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats. Methods Fifty-one male Sprague–Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. The mean pulmonary artery pressure (mPAP), the right ventricle weight-to-left ventricle + septum weight ratio (RV/LV + S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed. Levels of the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB proteins in lung tissue were measured using Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed. Results mPAP [35.1 ± 1.7 mmHg (MCT) (n = 9) vs. 16.6 ± 0.7 (control) (n = 9) (P < 0.05); 29.1 ± 1.5 mmHg (CH) (n = 10) vs. 17.5 ± 0.5 (control) (n = 10) (P < 0.05)], RV/LV + S [0.40 ± 0.01 (MCT) (n = 18) vs. 0.24 ± 0.01 (control) (n = 10) (P < 0.05); 0.41 ± 0.03 (CH) (n = 13) vs. 0.27 ± 0.06 (control) (n = 10) (P < 0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1 ± 1.1 mmHg (n = 11) (P < 0.05), RV/LV + S 0.30 ± 0.01 (n = 14) (P < 0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and increased in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats. Conclusions We found model differences in the effect of CLZ on PH development. CLZ might exert a preventive effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might depend on the PH etiology.

Background: Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats.Methods: Fifty-one male Sprague-Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. Mean PAP (mPAP), the right ventricle weight-to-left ventricle+septum weight ratio (RV/LV+S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed.Protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB in lung tissue was measured by Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed.Results: mPAP [35.1±1.7 mmHg (MCT) (n=9) vs.16.6±0.7 (control) (n=9) (p<0.05); 29.1±1.5 mmHg (CH) (n=10) vs. 17.5±0.5 (control) (n=10) (p<0.05)], RV/LV+S [0.40±0.01 (MCT) (n=18) vs. 0.24±0.01 (control) (n=10) (p<0.05); 0.41±0.03 (CH) (n=13) vs. 0.27±0.06 (control) (n=10) (p<0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1±1.1 mmHg (n=11) (p<0.05), RV/LV+S 0.30±0.01 (n=14) (p<0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and high in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats.Conclusion: We found model differences in the effect of CLZ on PH development. CLZ might have a preventable effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might be dependent on PH etiology.