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POSTNATAL DEVELOPMENT OF ORGANIC CATION TRANSPORT IN THE RAT LIVER
Pharmacological Research, Vol. 37, No. 2, 1998
POSTNATAL DEVELOPMENT OF ORGANIC CATION TRANSPORT IN THE RAT LIVER 1 ´ F. MARTELU , M.J. MARTINS1 , C. CALHAU, C. HIPOLITO-REIS and I. AZEVEDO
Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto and 1Department of Biochemistry, Faculty of Medicine, 4200 Porto, Portugal Accepted 23 December 1997
Previous studies have demonstrated that the small permanently charged organic cation 1-methyl-4-phenylpyridinium ŽMPPq. is avidly taken up by rat hepatocytes. The aim of this study was to characterise the postnatal development of hepatic uptake of organic cations, using the model compound MPPq. Accumulation of w 3 HxMPPq by liver slices obtained from rats ranging from 1 day to 7 weeks was measured, and the effect of a series of compounds on w 3 HxMPPq uptake was examined. The accumulation of w 3 HxMPPq by liver slices was similar in adult Ž87.5" 19.9 pmol gyl ; n s 7. and neonatal rats Ž110.6" 11.5 pmol gyl ; n s 15.. Verapamil, quinidine Ž100 m M. and progesterone Ž200 m M. produced very marked reductions on w 3 HxMPPq uptake at all ages, and the inhibitory effect of verapamil and quinidine was maximum in livers from 1-day-old rats. Bilirubin Ž200 m M. significantly reduced w 3 HxMPPq uptake by liver slices from 1 day, 1 week and 7-week-old rats. However, w 3 HxMPPq accumulation was reduced by cimetidine, vinblastine and daunomycin Ž100 m M. in 1-day-old rats, but the effect of these drugs disappeared as the animals age increased. These findings demonstrate that hepatic organic cation uptake capacity is remarkably high shortly after birth and suggest that at least two distinct uptake mechanisms are involved in this process. These uptake systems are the type I hepatic transporter of organic cations, active from birth to adulthood, and P-glycoprotein, active only in very young rats. Q 1998 The Italian Pharmacological Society KEY
WORDS:
postnatal, development, liver, transport, MPPq.
INTRODUCTION Biological membranes prevent transmembrane diffusion of the majority of organic molecules that bear net charges at physiological pH. As charged compounds, organic cations must use more or less specific membrane-bound transport systems to be imported to or exported from cells or organisms. Thus, specific membrane-bound transport systems largely affect the distribution and elimination of these compounds. The liver plays a very important role in the removal, metabolism and excretion of organic cations of both natural and synthetic origin. Indeed, detoxification of organic cations from the circulation is usually accomplished by the kidney w1x and the liver w2x. The aim of this work was the characterisation of the postnatal development of hepatic uptake of orU
Corresponding author.
1043]6618r98r020131]06r$25.00r0rfr970283
ganic cations. Two reasons led us to investigate this subject. Firstly, although the characteristics of the mechanisms responsible for the hepatic uptake of organic cations have been extensively studied w3, 4x, to our knowledge little is known about the ontogenic development of organic cation transport in the liver w2x. Secondly, this investigation could help us to better characterise the transporterŽs. involved in hepatic uptake of organic cations. In the kidney, for instance, there is a differential development of organic cation transport and MDR Žmultidrug-resistance gene. expression with age w5x. 1-Methyl-4-phenylpyridinium ŽMPPq. is a low molecular weight organic cation, recently shown to be avidly taken up and accumulated by cultured rat hepatocytes and by freshly isolated rat hepatocytes w6]8x. The uptake of this compound by cultured rat hepatocytes was shown to occur through OCT1, a recently cloned renal transporter for organic cations that seems to be identical to the type I hepatic transporter of organic cations w7, 9x. Moreover, MPPq uptake by freshly isolated hepatocytes was Q1998 The Italian Pharmacological Society
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found to occur not only through OCT1, but also through a P-glycoprotein transporter w8x. We have measured the uptake of the organic cation MPPq by liver slices obtained from rats aged from newborn to 7 weeks. The liver slices model, chosen because of technical difficulties in the preparation of isolated hepatocytes from 1-day-old rats, has been thoroughly validated Žsee w4x.. The uptake and metabolism of noradrenaline, an organic cation structurally similar to MPPq, was found to be very similar in rat liver slices and isolated hepatocytes w10x. The uptake of MPPq by liver slices from rats with different ages was characterised by examining the effect of a series of compounds, including substratesrinhibitors of P-glycoprotein.
w 3 HxMPPq 200 nM. In another series of experiments, slices were preincubated for 10 min in Krebs]Henseleit medium followed by an incubation with w 3 Hxsorbitol Ž200 nM. for 5 min. At the end of the incubation period, slices were removed, rapidly washed in ice-cold Krebs]Henseleit solution, blotted, weighed and placed in 2 ml perchloric acid 0.2 M. Radioactivity was measured by liquid scintillation counting. The accumulation of w 3 HxMPPq in liver slices Žexpressed as pmol gy1 tissue. was corrected for the amount of w 3 HxMPPq calculated to be present in the w 3 Hxsorbitol Žextracellular . space. When the effect of drugs was tested, these compounds were present during both the preincubation and incubation periods.
MATERIALS AND METHODS
Water and protein content of the li¨ er
Materials
w 3 HxMPPq Ž N-w methyl- 3 Hx-4-phenylpyridinium acetate; spec. act. 84.0 Ci mmoly1 ., w 3 Hxsorbitol ŽD-w1- 3 Hx N-sorbitol; spec. act. 12.9 Ci mmoly1 . ŽNew England Nuclear Chemicals, Dreieich, Germany.; bilirubin ŽMerck, Darmstadt, Germany.; MPPq Ž1methyl-4-phenylpyridinium iodide; Research Biochemicals International, Natick, MA, USA.; verapamil hydrochloride ŽKnoll AG, Ludwigshafen, Germany.; cimetidine, daunomycin hydrochloride, progesterone, quinidine sulfate, vinblastine sulfate ŽSigma, St. Louis, MO, USA.. Progesterone was dissolved in ethanol at a final concentration in the incubation media of 86 mM.
Uptake measurements Wistar rats ŽBioterio ´ do Instituto Gulbenkian de . were bred in house and Ciencia, Oeiras, Portugal ˆ animals were kept under controlled environmental conditions Ž12 h lightrdark cycle and room temperature 248C.. Up to the age of 20 days, animals were kept with their mother, the environmental conditions being those described above. Food and tap water were allowed ad libitum. Liver was harvested from rats at 24 h, 1 week, 2 weeks and 7 weeks of age. For 7-week-old rats Žyoung adults., only males were used. The rats were killed by decapitation under pentobarbital anaesthesia and the liver was rapidly removed through an abdominal midline incision and rinsed free from blood with saline Ž0.9% NaCl.. Liver slices Ž20]100 mg weight and 0.5 mm thickness; prepared with a surgical blade. were incubated in 2 ml Krebs]Henseleit medium, containing Žin mM.: NaCl, 137; KCl, 5.37; NaHCO3 , 25; Žq.-glucose, 11; KH 2 PO4 , 1.18; MgSO4 , 0.57; CaCl 2 , 2.51; ascorbic acid, 0.3 and Na 2 EDTA, 0.04 ŽpH 7.4. at 378C, under continuous shaking and gassing with 95% O 2 and 5% CO 2 . After a 10-min preincubation period, the slices were incubated for 5 min with
For determination of the water content of the liver, tissue fragments were collected, weighed and dried at 908C. After a 24-h period, the fragments were weighed again and the water content was determined by subtraction. For determination of hepatic protein content, liver fragments were collected, weighed and placed in physiologic salt solution. The liver was homogenised in saline Ž0.9% NaCl. with a Thomas teflon homogeniser, while continuously kept on ice. The protein content was determined by the method of Bradford w11x, using human serum albumin as standard, and results were expressed as mg protein gy1 tissue.
Li¨ er morphology Tissue fragments were collected immediately after killing and immersed overnight in 2.5% glutaraldehyde in 0.1 M cacodylate buffer ŽpH 7.3. at 48C. Tissues were then washed in cacodylate buffer with 7% sucrose at 48C, post-fixed in a 1% solution of osmium tetroxide in 0.1 M cacodylate buffer ŽpH 7.3. at 48C for 2 h, dehydrated with ethanol and propylene oxide and embedded in Epon 812. Semi-thin sections Ž1 m m. stained with 1% toluidine blue in 1% borax were examined by light microscopy.
Calculations and statistics
Results are expressed as arithmetic means Ž"SEM.. n indicates the number of experiments. Statistical significance of the difference between various groups was evaluated by one-way analysis of variance ŽANOVA test. followed by the Newman]Keuls test. For comparison between two groups, Student’s t-test was used. Differences were considered to be significant at P- 0.05.
RESULTS
Water and protein content of the li¨ er These parameters were determined in 1-day-old
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and 7-week-old rats. Hepatic water content of 1-dayold rats Ž73.6" 1.2%; n s 4. was significantly higher than the hepatic water content of 7-week-old rats Ž62.5" 1.2%; n s 3. Ž P- 0.05.. Also, there was a difference in the protein content of the liver from 1-day-old rats Ž108.1" 2.7 mg protein gy1 tissue; n s 4. as compared with the liver from 7-week-old rats Ž131.7" 2.7 mg protein gy1 tissue; n s 3. Ž P0.05..
[ 3H ]Sorbitol space
The w 3 Hxsorbitol Žextracellular . space of rat liver slices did not differ significantly for the various age groups and was found to be 0.243" 0.03, 0.283" 0.05, 0.294" 0.02 and 0.234" 0.05 ml gy1 for 1-dayold, 1-week-old, 2-week-old and 7-week-old rats, respectively Ž n s 4..
Uptake of [ 3H ]MPP q by rat li¨ er slices In order to characterise the postnatal development of hepatic w 3 HxMPPq uptake, liver slices obtained from rats of 1 day, 1 week, 2 weeks and 7 weeks of age were incubated for 5 min with 200 nM w 3 HxMPPq. As shown in Fig. 1, the accumulation of w 3 HxMPPq by the liver was quantitatively similar in the adult Ž87.5" 19.9 pmol gy1 tissue; n s 7. and the neonatal animal Ž110.61" 11.5 pmol gy1 tissue; n s 15.. Moreover, if accumulation of w 3 HxMPPq is expressed in terms of the protein content of the liver, there appears to exist a higher capacity to accumulate the compound in 1-day-old Ž1023.1" 106.4 pmol gy1 protein; n s 15. than in adult rat liver Ž664.4" 151.1 pmol gy1 protein; n s 7.. However, this difference did not reach statistical significance Ž Ps 0.069..
Fig. 1. Accumulation of w 3 HxMPPq by rat liver slices at various times after birth. Rats of 1 day Ž n s 15., 1 week Ž n s 6., 2 weeks Ž n s 8. and 7 weeks of age Ž n s 7. were used. Slices were incubated at 378C with 200 nM w 3 HxMPPq for 5 min. Results Žpmol gy1 . are expressed as arithmetic mean " SEM.
The effects of some potent inhibitors of w 3 HxMPPq uptake by rat freshly isolated hepatocytes w8x were examined. Verapamil Ž100 m M., progesterone Ž200 m M. and quinidine Ž100 m M. markedly reduced w 3 HxMPPq uptake at all ages. The inhibitory effect of verapamil and quinidine on w 3 HxMPPq uptake was maximum in livers from 1-day-old rats Žin the presence of verapamil, the uptake of w 3 HxMPPq was 3.5" 3.1% of controls; in the presence of quinidine, the uptake of w 3 HxMPPq was 3.7" 2.1% of controls., and as the animals age increased these drugs tended to have a less pronounced effect on w 3 HxMPPq accumulation Žfor quinidine, the inhibition produced in liver from 1-day-old rats was significantly different from that produced in liver from the 7-week-old rats Ž Ps 0.001... Bilirubin Ž200 m M. produced a signifi-
Fig. 2. Effect of drugs on w 3 HxMPPq uptake by rat liver slices. Rats of 1 day, 1 week, 2 weeks and 7 weeks of age were used. Slices were incubated at 378C with 200 nM w 3 HxMPPq for 5 min, in the absence or presence of 100 m M verapamil ŽVER; n s 4]5., 200 m M bilirubin ŽBIL; n s 4]6., 200 m M progesterone ŽPRO; n s 4]5., 100 m M quinidine ŽQUI; n s 4]5., 100 m M cimetidine ŽCIM; n s 4]6., 100 m M vinblastine ŽVIN; n s 4]6. or 100 m M daunomycin ŽDAU; n s 3]5.. w 3 HxMPPq uptake by control rat liver slices amounted to 110.6" 11.5 pmol gy1 ŽI, 1-day-old rats; n s 15., 130.1" 23.1 .. rr ... 1-week-old rats; n s 6., 101.7" 14.1 pmol gy1 ŽI, pmol gy1 ŽI, 2-week-old rats; n s 8. and 87.5q 19.9 pmol gy1 ŽB, 7-week-old rats; n s 8.. Results are shown as percentage of control Žarithmetic mean " SEM.. U Significantly different from controls, P- 0.05..
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cant reduction in w 3 HxMPPq accumulation from day 1 onwards, with the exception of the 2-week-old rats, in which no significant inhibitory effect was observed ŽFig. 2.. On the other hand, w 3 HxMPPq accumulation was reduced by cimetidine Ž100 m M., vinblastine Ž100 m M. and daunomycin Ž100 m M. in rats of 1 day of age, but the effect of these drugs disappeared as the animals age increased. In rats of 2 weeks of age or more, none of these compounds had an inhibitory effect on the uptake of w 3 HxMPPq ŽFig. 2..
Morphological study In the livers of 7-week-old rats Žyoung adults., hepatocytes with voluminous cytoplasm and round nuclei, rich in euchromatin and nucleolus, were aggregated in long rows, constituting a very high fractional volume of the tissue wFig. 3ŽA.x. The livers obtained from newborn rats showed a very different aspect: the hepatocytes, with a less homogeneous cytoplasm, constituted a much smaller fractional volume of the liver tissue and dispersed among the hepatocytes, frequent islets of hematopoietic cells could be seen wFig. 3ŽB.x.
DISCUSSION The liver plays a very important role in the elimination of endogenous and exogenous organic cations
Pharmacological Research, Vol. 37, No. 2, 1998
from the blood stream. The removal of these compounds from the circulation firstly involves their uptake by hepatocytes, followed by metabolism andror excretion into the bile or blood. As organic cations are charged compounds at physiological pH, they must use membrane-bound transport systems to be taken up into hepatocytes. The mechanisms involved in the uptake of organic cations by hepatocytes have been extensively studied in the past and several studies point to the existence of multiple transport systems for organic cations in the sinusoidal membrane of hepatocytes with overlapping substrate specificities. Recent reviews on this subject propose at least six different mechanisms: sodiumdriven choline and thiamine transporters, a Nmethyl-nicotinamiderproton antiport, adsorptive endocytosis and two transporters for cationic drugs Žtype I and type II.. The type I uptake system accepts relatively small monovalent organic cations and the type II system accepts mostly bivalent organic cations with bulky ring structures w3, 4x. Recently, we have characterised the uptake of w 3 HxMPPq by cultured rat hepatocytes and by freshly isolated rat hepatocytes w6]8x. The uptake of this compound by cultured rat hepatocytes was shown to occur through OCT1, a recently cloned renal transporter for organic cations which seems to correspond to the type I hepatic transporter of organic cations w7, 9x. Moreover, w 3 HxMPPq uptake by freshly
Fig. 3. Light micrographs of liver sections from a 7-week-old rat ŽA. and a 1-day-old rat ŽB.. Whereas in A hepatocytes Žh. are contiguous, forming rows Žarrowheads., and constitute a very high fractional volume of the tissue, in newborn rat liver ŽB. hepatocytes Žh. are separated by abundant islets of hematopoietic cells Žarrows.. Bars s 10 m M.
Pharmacological Research, Vol. 37, No. 2, 1998
isolated hepatocytes was found to occur not only through OCT1, but also through a P-glycoprotein transporter w8x. P-Glycoprotein, or the multi-drug resistance gene ŽMDR. product, was first described as a transmembrane protein in tumour cells conferring on them resistance to a broad range of cytotoxic compounds w12x. Subsequent investigation showed that P-glycoprotein is not a unique entity but instead a family of related transporters, existing also in normal tissues such as the kidney, intestine and liver w13, 14x. In the liver, P-glycoprotein transporters are confined to the apical Žbile canalicular . membrane, where they are known to excrete organic cations Žacridine orange, vincristine and daunomycin. and phospholipids into the bile w15]18x. The aim of our study was the characterisation of the postnatal development of hepatic uptake of organic cations, using MPPq as a model compound. Two reasons led us to undertake this study. Firstly, the postnatal development of this hepatic transport mechanism has not been, to our knowledge, studied. Secondly, the putative differential development of organic cation transport and MDR gene expression in the liver, similarly to what happens in the kidney w5x, could help us to better identify the transporterŽs. involved. Our results showed that uptake of w 3 HxMPPq by rat liver slices was quantitatively similar in adult and neonatal rats. This high capacity of neonatal rat liver is in contrast to what is observed in the kidney, where the organic cation transport system is not fully developed at birth in dogs, pigs or mice and the ability to secrete organic cations is acquired during early neonatal life, gradually developing to adult capacity w1, 5, 19x. In a recent study, it was found that uptake of classical substrates for renal secretion into kidney slices developed gradually in neonatal mice, reaching adult capacity only after 4]6 weeks w5x. The apparent development of the uptake of organic cations shortly after birth is in contrast to the well known immaturity of the biliary excretion of a number of compounds in the newborn w2x. A functional biliary excretion of organic cations depends on a functional hepatic uptake at the sinusoidal membrane of hepatocytes, hepatic metabolism and biliary excretion at the canalicular membrane of hepatocytes. Although very little is known about development of the biliary excretion of organic cations, it is possible that an active hepatic uptake of organic cations in the newborn rat is not associated with an active biliary excretion of these compounds, either because of an immaturity of the enzymatic machinery that metabolises such compounds andror of the excretion mechanisms. The analysis of the effects of the different drugs tested here suggests that w 3 HxMPPq uptake by newborn and adult rat liver is not mediated by the same
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mechanisms. w 3 HxMPPq uptake by newborn liver slices was inhibited by verapamil, quinidine, cimetidine, vinblastine and daunomycin. On the other hand, uptake of the same compound by adult liver slices was inhibited by verapamil and quinidine, albeit less potently than in livers from newbom rats, and was not inhibited by cimetidine, vinblastine or daunomycin. Thus, the classic P-glycoprotein substrates, vinblastine and daunomycin, inhibited w 3 HxMPPq uptake in the younger rats, but were devoid of effect in livers from older Ž2]7-week-old. rats, whereas compounds that are simultaneously substrates for P-glycoprotein and for the type I hepatic transporter of organic cations, such as verapamil and quinidine, had a less marked but still significant effect in livers from adult rats. Taken together, these results indicate that w 3 HxMPPq transport through P-glycoprotein plays a more important role in 1-day-old than in adult rats. It is important to note that both the type I hepatic transporter of organic cations and P-glycoprotein accept organic cations as substrates, and that for this reason, the effect of organic cations on w 3 HxMPPq uptake must be regarded carefully w4, 12x. The maintenance of an inhibitory effect of small molecular weight organic cations such as verapamil and quinidine at an age when vinblastine and daunomycin have no effect, however, suggests the involvement of the type I hepatic transporter of organic cations w7, 8x. Bilirubin and progesterone strongly reduced w 3 HxMPPq accumulation in liver slices of newborn Ž1-day-old. and young adult Ž7-week-old. rats. However, the effects of these drugs on w 3 HxMPPq uptake must be interpreted with caution, because bilirubin and progesterone are known inhibitors of P-glycoprotein w12x, and they are also taken up by hepatocytes through other specific transport mechanisms w2x. As previously noted, P-glycoprotein is generally described as an efflux pump, which actively exports compounds from the cells w12x. However, it was recently proposed that w 3 HxMPPq is taken up by freshly isolated rat hepatocytes through, at least partially, P-glycoprotein. Moreover, it was suggested that reversal of transport through P-glycoprotein was due to the loss of cell polarity of hepatocytes in suspension. This loss of polarity, with free diffusion of P-glycoprotein over the whole cell membrane, could eventually separate the transporter from regulatory molecules such as alkaline phosphatase, thus modifying its activity w8x. The observation of a significant reduction of w 3 HxMPPq uptake into liver slices from newborn rats by substratesrinhibitors of P-glycoprotein prompted us to investigate the liver morphology in these animals. The results obtained are most interesting: in neonatal liver, hepatocytes represent only a fraction of total cell content, being separated by frequent islets of hematopoietic cells. This observa-
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tion is in agreement with the well known fact that the liver is the most important hematopoietic organ in the fetal rat but that this hepatic function is gradually lost after birth w20x. Besides the presence of a high number of hematopoietic cell clusters in the newborn rat liver there is a lack of organisation of hepatocytes in rows and lobules. Newborn rat hepatocytes are, thus, at least in part non-organised and thus presumably non-polarised. It is not surprising, therefore, that in newborn rat liver slices hepatocytes exhibit the same behaviour as isolated hepatocytes with regard to P-glycoprotein activity. With time, liver maturation and organisation this P-glycoprotein importing activity is lost. The fact that, in spite of the much lesser number of hepatocytes in newborn rat liver as compared with adult liver, w 3 HxMPPq uptake is quantitatively similar in rats with ages ranging from 1 day to 7 weeks has at least two possible explanations: Ž1. neonatal rat hepatocytes possess a higher capacity to take up w 3 HxMPPq than adult rat hepatocytes, andror Ž2. other cell types present in abundance in the neonatal rat liver are capable of taking up w 3 HxMPPq. Our results support the first explanation, as we have shown that newborn rat hepatocytes take up w 3 HxMPPq through P-glycoprotein as well as the type I hepatic uptake of organic cations, which is the only system active in adult hepatocytes. The second explanation, namely that all or some hematopoietic cells take up w 3 HxMPPq, will be addressed in a future study. In conclusion, this study characterises the postnatal development of the hepatic uptake of the small organic cation w 3 HxMPPq. The uptake of this organic cation is very active at birth, and two distinct uptake mechanisms seem to be involved in that process: the type I hepatic transporter of organic cations, active from birth to adulthood, and P-glycoprotein, active only in very young rats. ACKNOWLEDGEMENTS
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This work was supported by JNICT ŽPRAXIS r2r2.1rSAUr1251r95.. 17.
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nisms of membrane transport. Biochim Biophys Acta 1995; 1241: 215]68. Dutt A, Priebe TS, Teeter LD, Kuo MT, Nelson JA. Postnatal development of organic cation transport and MDR gene expression in mouse kidney. J Pharmacol Exp Ther 1992; 261: 1222]30. Martel F, Russ H, Azevedo I, Schomig E. Uptake and accumulation of N-methyl-4-phenylpyridinium ŽMPPq. by rat hepatocytes. ŽAbstract.. Br J Pharmacol 1995; 114: 262P. Martel F, Vetter T, Russ H, Grundemann D, Azevedo ¨ I, Koepsell H, Schomig E. Transport of small organic ¨ cations in the rat liver. The role of the organic cation transporter OCT1. Naunyn-Schmiedeberg’s Arch Pharmacol 1996; 354: 320]6. Martel F, Martins MJ, Hipolito-Reis C, Azevedo I. ´ Inward transport of w 3 HxMPPq in isolated rat hepatocytes: putative involvement of a P-glycoprotein transporter. Br J Pharmacol 1996; 119: 1519]24. Grundemann D, Gorboulev V, Gambaryan S, Veyhl ¨ M, Koepsell H. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 1994; 372: 549]52. Martel F, Azevedo I, Osswald W. Uptake and metabolism of w 3 Hxadrenaline and w 3 Hxnoradrenaline by isolated hepatocytes and liver slices of the rat. Naunyn-Schmiedeberg’s Arch Pharmacol 1993; 348: 450]7. Bradford M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248]54. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Re¨ Biochem 1993; 62: 385]427. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I. Expression of a multidrug-resistance gene in human tumours and tissues. Proc Natl Acad Sci USA 1987; 84: 265]9. Buschman E, Lepage P, Gros P. P-glycoprotein homologues. In: Goldstein LJ, Ozols RF, eds. Anticancer Drug Resistance: Advances in Molecular and Clinical Research. London: Kluger Academic Publishers, 1994: pp. 17]39. Kamimoto Y, Gatmaitan Z, Hsu J, Arias IM. The function of Gp 170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles. J Biol Chem 1989; 264: 11693]8. Watanabe T, Miyauchi S, Sawada Y, Iga T, Hanano M, Inaba M, Sugiyama Y. Kinetic analysis of hepatobiliary transport of vincristine in perfused rat liver. Possible roles of P-glycoprotein in biliary excretion of vincristine. J Hepatol 1992; 16: 77]88. Smit JJM, Schinkel AH, Oude Elferink RPJ, Groen AK, Wagenaar E, Van Deemter L, Mol CAAM, Ottenhoff R, van derLogt NM, Van Roon MA, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993; 75: 451]62. Thalhammer T, Stapf V, Gajdzik L, Graf J. Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport. Eur J Pharmacol (En¨ iron Toxicol Pharmacol Sect) 1994; 270: 213]20. Rennick BR, Hamilton B, Evans R. Development of renal tubular transport of TEA and PAH in the puppy and piglet. Am J Physiol 1961; 201: 743]6. Ratnoff OD. Blood components. In: Berne RM, Levy MN, eds. Physiology. St. Louis, MO: Mosby Year Book, 1993: pp. 327]38.
POSTNATAL DEVELOPMENT OF ORGANIC CATION TRANSPORT IN THE RAT LIVER 1 ´ F. MARTELU , M.J. MARTINS1 , C. CALHAU, C. HIPOLITO-REIS and I. AZEVEDO
Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto and 1Department of Biochemistry, Faculty of Medicine, 4200 Porto, Portugal Accepted 23 December 1997
Previous studies have demonstrated that the small permanently charged organic cation 1-methyl-4-phenylpyridinium ŽMPPq. is avidly taken up by rat hepatocytes. The aim of this study was to characterise the postnatal development of hepatic uptake of organic cations, using the model compound MPPq. Accumulation of w 3 HxMPPq by liver slices obtained from rats ranging from 1 day to 7 weeks was measured, and the effect of a series of compounds on w 3 HxMPPq uptake was examined. The accumulation of w 3 HxMPPq by liver slices was similar in adult Ž87.5" 19.9 pmol gyl ; n s 7. and neonatal rats Ž110.6" 11.5 pmol gyl ; n s 15.. Verapamil, quinidine Ž100 m M. and progesterone Ž200 m M. produced very marked reductions on w 3 HxMPPq uptake at all ages, and the inhibitory effect of verapamil and quinidine was maximum in livers from 1-day-old rats. Bilirubin Ž200 m M. significantly reduced w 3 HxMPPq uptake by liver slices from 1 day, 1 week and 7-week-old rats. However, w 3 HxMPPq accumulation was reduced by cimetidine, vinblastine and daunomycin Ž100 m M. in 1-day-old rats, but the effect of these drugs disappeared as the animals age increased. These findings demonstrate that hepatic organic cation uptake capacity is remarkably high shortly after birth and suggest that at least two distinct uptake mechanisms are involved in this process. These uptake systems are the type I hepatic transporter of organic cations, active from birth to adulthood, and P-glycoprotein, active only in very young rats. Q 1998 The Italian Pharmacological Society KEY
WORDS:
postnatal, development, liver, transport, MPPq.
INTRODUCTION Biological membranes prevent transmembrane diffusion of the majority of organic molecules that bear net charges at physiological pH. As charged compounds, organic cations must use more or less specific membrane-bound transport systems to be imported to or exported from cells or organisms. Thus, specific membrane-bound transport systems largely affect the distribution and elimination of these compounds. The liver plays a very important role in the removal, metabolism and excretion of organic cations of both natural and synthetic origin. Indeed, detoxification of organic cations from the circulation is usually accomplished by the kidney w1x and the liver w2x. The aim of this work was the characterisation of the postnatal development of hepatic uptake of orU
Corresponding author.
1043]6618r98r020131]06r$25.00r0rfr970283
ganic cations. Two reasons led us to investigate this subject. Firstly, although the characteristics of the mechanisms responsible for the hepatic uptake of organic cations have been extensively studied w3, 4x, to our knowledge little is known about the ontogenic development of organic cation transport in the liver w2x. Secondly, this investigation could help us to better characterise the transporterŽs. involved in hepatic uptake of organic cations. In the kidney, for instance, there is a differential development of organic cation transport and MDR Žmultidrug-resistance gene. expression with age w5x. 1-Methyl-4-phenylpyridinium ŽMPPq. is a low molecular weight organic cation, recently shown to be avidly taken up and accumulated by cultured rat hepatocytes and by freshly isolated rat hepatocytes w6]8x. The uptake of this compound by cultured rat hepatocytes was shown to occur through OCT1, a recently cloned renal transporter for organic cations that seems to be identical to the type I hepatic transporter of organic cations w7, 9x. Moreover, MPPq uptake by freshly isolated hepatocytes was Q1998 The Italian Pharmacological Society
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found to occur not only through OCT1, but also through a P-glycoprotein transporter w8x. We have measured the uptake of the organic cation MPPq by liver slices obtained from rats aged from newborn to 7 weeks. The liver slices model, chosen because of technical difficulties in the preparation of isolated hepatocytes from 1-day-old rats, has been thoroughly validated Žsee w4x.. The uptake and metabolism of noradrenaline, an organic cation structurally similar to MPPq, was found to be very similar in rat liver slices and isolated hepatocytes w10x. The uptake of MPPq by liver slices from rats with different ages was characterised by examining the effect of a series of compounds, including substratesrinhibitors of P-glycoprotein.
w 3 HxMPPq 200 nM. In another series of experiments, slices were preincubated for 10 min in Krebs]Henseleit medium followed by an incubation with w 3 Hxsorbitol Ž200 nM. for 5 min. At the end of the incubation period, slices were removed, rapidly washed in ice-cold Krebs]Henseleit solution, blotted, weighed and placed in 2 ml perchloric acid 0.2 M. Radioactivity was measured by liquid scintillation counting. The accumulation of w 3 HxMPPq in liver slices Žexpressed as pmol gy1 tissue. was corrected for the amount of w 3 HxMPPq calculated to be present in the w 3 Hxsorbitol Žextracellular . space. When the effect of drugs was tested, these compounds were present during both the preincubation and incubation periods.
MATERIALS AND METHODS
Water and protein content of the li¨ er
Materials
w 3 HxMPPq Ž N-w methyl- 3 Hx-4-phenylpyridinium acetate; spec. act. 84.0 Ci mmoly1 ., w 3 Hxsorbitol ŽD-w1- 3 Hx N-sorbitol; spec. act. 12.9 Ci mmoly1 . ŽNew England Nuclear Chemicals, Dreieich, Germany.; bilirubin ŽMerck, Darmstadt, Germany.; MPPq Ž1methyl-4-phenylpyridinium iodide; Research Biochemicals International, Natick, MA, USA.; verapamil hydrochloride ŽKnoll AG, Ludwigshafen, Germany.; cimetidine, daunomycin hydrochloride, progesterone, quinidine sulfate, vinblastine sulfate ŽSigma, St. Louis, MO, USA.. Progesterone was dissolved in ethanol at a final concentration in the incubation media of 86 mM.
Uptake measurements Wistar rats ŽBioterio ´ do Instituto Gulbenkian de . were bred in house and Ciencia, Oeiras, Portugal ˆ animals were kept under controlled environmental conditions Ž12 h lightrdark cycle and room temperature 248C.. Up to the age of 20 days, animals were kept with their mother, the environmental conditions being those described above. Food and tap water were allowed ad libitum. Liver was harvested from rats at 24 h, 1 week, 2 weeks and 7 weeks of age. For 7-week-old rats Žyoung adults., only males were used. The rats were killed by decapitation under pentobarbital anaesthesia and the liver was rapidly removed through an abdominal midline incision and rinsed free from blood with saline Ž0.9% NaCl.. Liver slices Ž20]100 mg weight and 0.5 mm thickness; prepared with a surgical blade. were incubated in 2 ml Krebs]Henseleit medium, containing Žin mM.: NaCl, 137; KCl, 5.37; NaHCO3 , 25; Žq.-glucose, 11; KH 2 PO4 , 1.18; MgSO4 , 0.57; CaCl 2 , 2.51; ascorbic acid, 0.3 and Na 2 EDTA, 0.04 ŽpH 7.4. at 378C, under continuous shaking and gassing with 95% O 2 and 5% CO 2 . After a 10-min preincubation period, the slices were incubated for 5 min with
For determination of the water content of the liver, tissue fragments were collected, weighed and dried at 908C. After a 24-h period, the fragments were weighed again and the water content was determined by subtraction. For determination of hepatic protein content, liver fragments were collected, weighed and placed in physiologic salt solution. The liver was homogenised in saline Ž0.9% NaCl. with a Thomas teflon homogeniser, while continuously kept on ice. The protein content was determined by the method of Bradford w11x, using human serum albumin as standard, and results were expressed as mg protein gy1 tissue.
Li¨ er morphology Tissue fragments were collected immediately after killing and immersed overnight in 2.5% glutaraldehyde in 0.1 M cacodylate buffer ŽpH 7.3. at 48C. Tissues were then washed in cacodylate buffer with 7% sucrose at 48C, post-fixed in a 1% solution of osmium tetroxide in 0.1 M cacodylate buffer ŽpH 7.3. at 48C for 2 h, dehydrated with ethanol and propylene oxide and embedded in Epon 812. Semi-thin sections Ž1 m m. stained with 1% toluidine blue in 1% borax were examined by light microscopy.
Calculations and statistics
Results are expressed as arithmetic means Ž"SEM.. n indicates the number of experiments. Statistical significance of the difference between various groups was evaluated by one-way analysis of variance ŽANOVA test. followed by the Newman]Keuls test. For comparison between two groups, Student’s t-test was used. Differences were considered to be significant at P- 0.05.
RESULTS
Water and protein content of the li¨ er These parameters were determined in 1-day-old
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and 7-week-old rats. Hepatic water content of 1-dayold rats Ž73.6" 1.2%; n s 4. was significantly higher than the hepatic water content of 7-week-old rats Ž62.5" 1.2%; n s 3. Ž P- 0.05.. Also, there was a difference in the protein content of the liver from 1-day-old rats Ž108.1" 2.7 mg protein gy1 tissue; n s 4. as compared with the liver from 7-week-old rats Ž131.7" 2.7 mg protein gy1 tissue; n s 3. Ž P0.05..
[ 3H ]Sorbitol space
The w 3 Hxsorbitol Žextracellular . space of rat liver slices did not differ significantly for the various age groups and was found to be 0.243" 0.03, 0.283" 0.05, 0.294" 0.02 and 0.234" 0.05 ml gy1 for 1-dayold, 1-week-old, 2-week-old and 7-week-old rats, respectively Ž n s 4..
Uptake of [ 3H ]MPP q by rat li¨ er slices In order to characterise the postnatal development of hepatic w 3 HxMPPq uptake, liver slices obtained from rats of 1 day, 1 week, 2 weeks and 7 weeks of age were incubated for 5 min with 200 nM w 3 HxMPPq. As shown in Fig. 1, the accumulation of w 3 HxMPPq by the liver was quantitatively similar in the adult Ž87.5" 19.9 pmol gy1 tissue; n s 7. and the neonatal animal Ž110.61" 11.5 pmol gy1 tissue; n s 15.. Moreover, if accumulation of w 3 HxMPPq is expressed in terms of the protein content of the liver, there appears to exist a higher capacity to accumulate the compound in 1-day-old Ž1023.1" 106.4 pmol gy1 protein; n s 15. than in adult rat liver Ž664.4" 151.1 pmol gy1 protein; n s 7.. However, this difference did not reach statistical significance Ž Ps 0.069..
Fig. 1. Accumulation of w 3 HxMPPq by rat liver slices at various times after birth. Rats of 1 day Ž n s 15., 1 week Ž n s 6., 2 weeks Ž n s 8. and 7 weeks of age Ž n s 7. were used. Slices were incubated at 378C with 200 nM w 3 HxMPPq for 5 min. Results Žpmol gy1 . are expressed as arithmetic mean " SEM.
The effects of some potent inhibitors of w 3 HxMPPq uptake by rat freshly isolated hepatocytes w8x were examined. Verapamil Ž100 m M., progesterone Ž200 m M. and quinidine Ž100 m M. markedly reduced w 3 HxMPPq uptake at all ages. The inhibitory effect of verapamil and quinidine on w 3 HxMPPq uptake was maximum in livers from 1-day-old rats Žin the presence of verapamil, the uptake of w 3 HxMPPq was 3.5" 3.1% of controls; in the presence of quinidine, the uptake of w 3 HxMPPq was 3.7" 2.1% of controls., and as the animals age increased these drugs tended to have a less pronounced effect on w 3 HxMPPq accumulation Žfor quinidine, the inhibition produced in liver from 1-day-old rats was significantly different from that produced in liver from the 7-week-old rats Ž Ps 0.001... Bilirubin Ž200 m M. produced a signifi-
Fig. 2. Effect of drugs on w 3 HxMPPq uptake by rat liver slices. Rats of 1 day, 1 week, 2 weeks and 7 weeks of age were used. Slices were incubated at 378C with 200 nM w 3 HxMPPq for 5 min, in the absence or presence of 100 m M verapamil ŽVER; n s 4]5., 200 m M bilirubin ŽBIL; n s 4]6., 200 m M progesterone ŽPRO; n s 4]5., 100 m M quinidine ŽQUI; n s 4]5., 100 m M cimetidine ŽCIM; n s 4]6., 100 m M vinblastine ŽVIN; n s 4]6. or 100 m M daunomycin ŽDAU; n s 3]5.. w 3 HxMPPq uptake by control rat liver slices amounted to 110.6" 11.5 pmol gy1 ŽI, 1-day-old rats; n s 15., 130.1" 23.1 .. rr ... 1-week-old rats; n s 6., 101.7" 14.1 pmol gy1 ŽI, pmol gy1 ŽI, 2-week-old rats; n s 8. and 87.5q 19.9 pmol gy1 ŽB, 7-week-old rats; n s 8.. Results are shown as percentage of control Žarithmetic mean " SEM.. U Significantly different from controls, P- 0.05..
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cant reduction in w 3 HxMPPq accumulation from day 1 onwards, with the exception of the 2-week-old rats, in which no significant inhibitory effect was observed ŽFig. 2.. On the other hand, w 3 HxMPPq accumulation was reduced by cimetidine Ž100 m M., vinblastine Ž100 m M. and daunomycin Ž100 m M. in rats of 1 day of age, but the effect of these drugs disappeared as the animals age increased. In rats of 2 weeks of age or more, none of these compounds had an inhibitory effect on the uptake of w 3 HxMPPq ŽFig. 2..
Morphological study In the livers of 7-week-old rats Žyoung adults., hepatocytes with voluminous cytoplasm and round nuclei, rich in euchromatin and nucleolus, were aggregated in long rows, constituting a very high fractional volume of the tissue wFig. 3ŽA.x. The livers obtained from newborn rats showed a very different aspect: the hepatocytes, with a less homogeneous cytoplasm, constituted a much smaller fractional volume of the liver tissue and dispersed among the hepatocytes, frequent islets of hematopoietic cells could be seen wFig. 3ŽB.x.
DISCUSSION The liver plays a very important role in the elimination of endogenous and exogenous organic cations
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from the blood stream. The removal of these compounds from the circulation firstly involves their uptake by hepatocytes, followed by metabolism andror excretion into the bile or blood. As organic cations are charged compounds at physiological pH, they must use membrane-bound transport systems to be taken up into hepatocytes. The mechanisms involved in the uptake of organic cations by hepatocytes have been extensively studied in the past and several studies point to the existence of multiple transport systems for organic cations in the sinusoidal membrane of hepatocytes with overlapping substrate specificities. Recent reviews on this subject propose at least six different mechanisms: sodiumdriven choline and thiamine transporters, a Nmethyl-nicotinamiderproton antiport, adsorptive endocytosis and two transporters for cationic drugs Žtype I and type II.. The type I uptake system accepts relatively small monovalent organic cations and the type II system accepts mostly bivalent organic cations with bulky ring structures w3, 4x. Recently, we have characterised the uptake of w 3 HxMPPq by cultured rat hepatocytes and by freshly isolated rat hepatocytes w6]8x. The uptake of this compound by cultured rat hepatocytes was shown to occur through OCT1, a recently cloned renal transporter for organic cations which seems to correspond to the type I hepatic transporter of organic cations w7, 9x. Moreover, w 3 HxMPPq uptake by freshly
Fig. 3. Light micrographs of liver sections from a 7-week-old rat ŽA. and a 1-day-old rat ŽB.. Whereas in A hepatocytes Žh. are contiguous, forming rows Žarrowheads., and constitute a very high fractional volume of the tissue, in newborn rat liver ŽB. hepatocytes Žh. are separated by abundant islets of hematopoietic cells Žarrows.. Bars s 10 m M.
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isolated hepatocytes was found to occur not only through OCT1, but also through a P-glycoprotein transporter w8x. P-Glycoprotein, or the multi-drug resistance gene ŽMDR. product, was first described as a transmembrane protein in tumour cells conferring on them resistance to a broad range of cytotoxic compounds w12x. Subsequent investigation showed that P-glycoprotein is not a unique entity but instead a family of related transporters, existing also in normal tissues such as the kidney, intestine and liver w13, 14x. In the liver, P-glycoprotein transporters are confined to the apical Žbile canalicular . membrane, where they are known to excrete organic cations Žacridine orange, vincristine and daunomycin. and phospholipids into the bile w15]18x. The aim of our study was the characterisation of the postnatal development of hepatic uptake of organic cations, using MPPq as a model compound. Two reasons led us to undertake this study. Firstly, the postnatal development of this hepatic transport mechanism has not been, to our knowledge, studied. Secondly, the putative differential development of organic cation transport and MDR gene expression in the liver, similarly to what happens in the kidney w5x, could help us to better identify the transporterŽs. involved. Our results showed that uptake of w 3 HxMPPq by rat liver slices was quantitatively similar in adult and neonatal rats. This high capacity of neonatal rat liver is in contrast to what is observed in the kidney, where the organic cation transport system is not fully developed at birth in dogs, pigs or mice and the ability to secrete organic cations is acquired during early neonatal life, gradually developing to adult capacity w1, 5, 19x. In a recent study, it was found that uptake of classical substrates for renal secretion into kidney slices developed gradually in neonatal mice, reaching adult capacity only after 4]6 weeks w5x. The apparent development of the uptake of organic cations shortly after birth is in contrast to the well known immaturity of the biliary excretion of a number of compounds in the newborn w2x. A functional biliary excretion of organic cations depends on a functional hepatic uptake at the sinusoidal membrane of hepatocytes, hepatic metabolism and biliary excretion at the canalicular membrane of hepatocytes. Although very little is known about development of the biliary excretion of organic cations, it is possible that an active hepatic uptake of organic cations in the newborn rat is not associated with an active biliary excretion of these compounds, either because of an immaturity of the enzymatic machinery that metabolises such compounds andror of the excretion mechanisms. The analysis of the effects of the different drugs tested here suggests that w 3 HxMPPq uptake by newborn and adult rat liver is not mediated by the same
135
mechanisms. w 3 HxMPPq uptake by newborn liver slices was inhibited by verapamil, quinidine, cimetidine, vinblastine and daunomycin. On the other hand, uptake of the same compound by adult liver slices was inhibited by verapamil and quinidine, albeit less potently than in livers from newbom rats, and was not inhibited by cimetidine, vinblastine or daunomycin. Thus, the classic P-glycoprotein substrates, vinblastine and daunomycin, inhibited w 3 HxMPPq uptake in the younger rats, but were devoid of effect in livers from older Ž2]7-week-old. rats, whereas compounds that are simultaneously substrates for P-glycoprotein and for the type I hepatic transporter of organic cations, such as verapamil and quinidine, had a less marked but still significant effect in livers from adult rats. Taken together, these results indicate that w 3 HxMPPq transport through P-glycoprotein plays a more important role in 1-day-old than in adult rats. It is important to note that both the type I hepatic transporter of organic cations and P-glycoprotein accept organic cations as substrates, and that for this reason, the effect of organic cations on w 3 HxMPPq uptake must be regarded carefully w4, 12x. The maintenance of an inhibitory effect of small molecular weight organic cations such as verapamil and quinidine at an age when vinblastine and daunomycin have no effect, however, suggests the involvement of the type I hepatic transporter of organic cations w7, 8x. Bilirubin and progesterone strongly reduced w 3 HxMPPq accumulation in liver slices of newborn Ž1-day-old. and young adult Ž7-week-old. rats. However, the effects of these drugs on w 3 HxMPPq uptake must be interpreted with caution, because bilirubin and progesterone are known inhibitors of P-glycoprotein w12x, and they are also taken up by hepatocytes through other specific transport mechanisms w2x. As previously noted, P-glycoprotein is generally described as an efflux pump, which actively exports compounds from the cells w12x. However, it was recently proposed that w 3 HxMPPq is taken up by freshly isolated rat hepatocytes through, at least partially, P-glycoprotein. Moreover, it was suggested that reversal of transport through P-glycoprotein was due to the loss of cell polarity of hepatocytes in suspension. This loss of polarity, with free diffusion of P-glycoprotein over the whole cell membrane, could eventually separate the transporter from regulatory molecules such as alkaline phosphatase, thus modifying its activity w8x. The observation of a significant reduction of w 3 HxMPPq uptake into liver slices from newborn rats by substratesrinhibitors of P-glycoprotein prompted us to investigate the liver morphology in these animals. The results obtained are most interesting: in neonatal liver, hepatocytes represent only a fraction of total cell content, being separated by frequent islets of hematopoietic cells. This observa-
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tion is in agreement with the well known fact that the liver is the most important hematopoietic organ in the fetal rat but that this hepatic function is gradually lost after birth w20x. Besides the presence of a high number of hematopoietic cell clusters in the newborn rat liver there is a lack of organisation of hepatocytes in rows and lobules. Newborn rat hepatocytes are, thus, at least in part non-organised and thus presumably non-polarised. It is not surprising, therefore, that in newborn rat liver slices hepatocytes exhibit the same behaviour as isolated hepatocytes with regard to P-glycoprotein activity. With time, liver maturation and organisation this P-glycoprotein importing activity is lost. The fact that, in spite of the much lesser number of hepatocytes in newborn rat liver as compared with adult liver, w 3 HxMPPq uptake is quantitatively similar in rats with ages ranging from 1 day to 7 weeks has at least two possible explanations: Ž1. neonatal rat hepatocytes possess a higher capacity to take up w 3 HxMPPq than adult rat hepatocytes, andror Ž2. other cell types present in abundance in the neonatal rat liver are capable of taking up w 3 HxMPPq. Our results support the first explanation, as we have shown that newborn rat hepatocytes take up w 3 HxMPPq through P-glycoprotein as well as the type I hepatic uptake of organic cations, which is the only system active in adult hepatocytes. The second explanation, namely that all or some hematopoietic cells take up w 3 HxMPPq, will be addressed in a future study. In conclusion, this study characterises the postnatal development of the hepatic uptake of the small organic cation w 3 HxMPPq. The uptake of this organic cation is very active at birth, and two distinct uptake mechanisms seem to be involved in that process: the type I hepatic transporter of organic cations, active from birth to adulthood, and P-glycoprotein, active only in very young rats. ACKNOWLEDGEMENTS
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This work was supported by JNICT ŽPRAXIS r2r2.1rSAUr1251r95.. 17.
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