Eur. J. Biohem. 247, 59664 (1997) FEBS 1997 Differenes in metabolism and isomerization of alltransretinoi aid and 9isretinoi aid between human endothelial ells and hepatoytes Mirian LANSINK, Ariette M. VAN BENNEKUM, William S. BLANER* and Teake KOOISTRA Gaubius Laboratory, TNOPG, Leiden, The Netherlands Institute of Human Nutrition, Columbia University, New York, USA (Reeived 18 April 1997) EJB 97 555/1 Retinoi aid stimulates the expression of tissuetype plasminogen ativator (tpa) in vasular endothelial ells in vitro and enhanes tpa levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoi aid onentrations are required to indue optimally tpa expression in vitro. These findings led us to study retinoi aid metabolism in ultured human endothelial ells. For omparison, these studies were also performed in the human hepatoma ell line, HepG2, and key experiments were repeated with human primary hepatoytes. Both hepatoyte ultures gave very similar results. Human endothelial ells were shown to possess an ative retinoi aid metabolizing apaity, whih is quantitatively omparable to that of hepatoytes, but different from that of hepatoytes in several qualitative aspets. Our results demonstrate that alltransretinoi aid is quikly metabolized by both endothelial ells and hepatoytes. Alltransretinoi aid indues its own metabolism in endothelial ells but not in hepatoytes. 9isRetinoi aid is degraded slowly by endothelial ells, whereas hepatoytes metabolize 9isretinoi aid very quikly. Furthermore, our data show that hepatoytes, but not endothelial ells, detetably isomerise alltransretinoi aid to 9isretinoi aid and vie versa. In both endothelial ells and hepatoytes alltransretinoi aid metabolism was inhibitable by the ytohrome P45 inhibitors liarozole (1 pm) and ketoonazole (1 pm), albeit to different extents and with different speifiities. In the presene of the most potent retinoi aid metabolism inhibitor in endothelial ells, liarozole, at least 1fold lower alltransretinoi aid onentrations were required than in the absene of the inhibitor to obtain the same indution of tpa. In onlusion, our results learly demonstrate that alltransretinoi aid and 9 4s retinoi aid are atively but differently metabolized and isomerised by human endothelid ells and hepatoytes. The rapid metabolism of retinoi aid explains the relatively high onentrations of retinoi aid required to indue tpa in ultured endothelial ells. Keywords: retinoi aid; metabolism; isomerization ; human endothelial ell ; human hepatoyte. Retinoi aid is an important physiologial regulator of plasma levels of tissuetype plastninogen ativator (tpa), a ruial enzyme in fibrin lot surveillane and in maintaining vasular pateny [l41. Analysis of tpa levels in the plasma and tissues of rats undergoing retinoi aid treatment showed an inrease in tpa levels by 5% [3, 41. However, plasma tpa levels in vitaminadefiient rats were found to be about threefold lower than ontrol values [4], and retinoi aid treatment of these animals restored plasma tpa ativity to ontrol levels (M. Lansink, unpublished results). These in vivo findings also illustrate that under normal physiologial onditions retinoi aid levels in plasma and tissues are suffiient to maintain almost maximal retinoiaiddependent tpa expression. tpa in the irulation originates mainly from the vasular endothelium [5]. We and others have demonstrated that the tpa stimulating effet of retinoi aid an be mimiked in ultured Correspondene to T. Kooistra, Gaubius Laboratory TNOPG, P.O. Box 2215, NL231 CE Leiden, The Netherlands Fax: +31 71 518 19 4. Abbreviations. tpa, tissuetype plasminogen ativator; RAR, retinoi aid reeptor; Me,SO, dimethylsulfoxide; DMEM, Dulbeo s modified Eagles medium: TIMOTA, alltrans7(1,1,3,3tetramethyl5 indanyl)3methylot2,4,6trienoi aid. human endothelial ells [3, 6, 71. The indution of tpa by retinoi aid in endothelial ells was shown to be mediated by retinoi aid reeptors (RAR) [8lo]. In line with this reeptormediated proess, we found retinol (vitamin A) to be only a weak induer of tpa synthesis [3]. During our studies on tpa regulation by retinoi aid in ultured human endothelial ells two outstanding questions emerged. Firstly, in our in vitro experiments 1 1 pm retinoi aid is required to indue optimally tpa synthesis [8], whereas in vivo plasma retinoi aid levels between 3.6 nm and 6.3 nm [ll, 121 apparently suffie to maintain nearly maximal tpa expression. The high retinoi aid onentrations neessary in endothelial ell ultures might be due to a rapid metabolism of retinoi aid by these ells, but this has not been doumented before. Considering the major endothelial surfae area, 72 m2 [I31 or 15 mz [14] in adults, an ative metabolism of retinoi aid by these ells would suggest that a substantial part of retinoi aid degradation in the body may our in the endothelium. Seondly, we found that in vitro both alltransretinoi aid and 9isretinoi aid are equipotent in stimulating tpa synthesis in ultured human endothelial ells [8]. However, in vivo plasma levels of 9isretinoi aid are muh lower than those of alltransretinoi aid : in humans 9isretinoi aid onentrations
Lansink et al. (Em J. Biohem. 247) 597 are below the detetion limit of 1 nm [15]. However, in some tissues like mouse liver and kidney 9isretinoi aid indeed has been shown to be present at signifiant levels [16]. This raises the question of whether 9isretinoi aid also auniulates in the endothelium, either by uptake from the plasma or by isomerization from alltransretinoi aid, and thus ontributes to the regulation of tpa in vivo. To address these questions we have studied the uptake and metabolism of alltransretinoi aid and 9isretinoi aid in ultured human endothelial ells. For omparison, we performed these experiments also in HepG2 ells and human hepatoytes, whih are reognized sites for retinoi aid metabolism (17191. MATERIALS AND METHODS Materials. Alltrans[ H]retinoi aid (5.3 53.9 Ci/mmol) was obtained from DuPont NEN. Alltransretinoi aid, dimethylsulfoxide (Me,SO), ketoonazole and metyrapone were purhased from Sigma. Liarozole was a kind gift from Dr J. P. van Wauwe, Janssen Researh Foundation, Beerse, Belgium. 9 isretinoi aid was kindly provided by Drs M. Klaus and C. Apfel (HoffmannLaRohe, Basel, Switzerland). Stok solutions of alltrunsretinoi aid and 9isretinoi aid (1 mm) were prepared in Me,SO and stored at 2 C. Stok solutions of test ompounds were either diluted with inubation medium to the final test onentrations immediately before the start of an experiment or were added to the inubation medium during an experiment at the time indiated. All experiments involving retinoids were arried out in subdued light and the tubes ontaining the retinoid solutions were overed with aluminium foil. The enzyme immunoassay kit for determination of human tpa antigen (Thrombonostika tpa) was obtained from Organon Teknika. All other materials used have been speified in the methods desribed or in the relating referenes. Cell ulture. Endothelial ells were isolated from human umbilial ord veins using the method of Jaffe et al. [2]. The ells were ultured in fibronetinoated 1m2 dishes in Dulbeo s modified Eagle s medium (DMEM) supplemented with 2 mm Hepes, ph 7.4, 1% (by vol.) heatinativated newborn alf serum, 1% (by vol.) human serum, 15 pg/ml endothelial ell growth supplement [21], 2 mm Lglutamine, 5 IU/ml heparin, 1 U/ml peniillin and 1 pglml streptomyin at 37 C in a 5% CO, atmosphere. The medium was replaed every 23 days. Subultures were obtained by trypsitdedta treatment of onfluent monolayers at a split ratio of 1 : 3. The ells were used for experiments at first or seond passage. The human hepatoma ell line HepG2 was ultured in unoated dishes in DMEM, supplemented with 2 mm Hepes, ph 7.4, 1% (by vol.) heatinativated fetal alf serum, 2 mm Lglutamine, 1 U/ml peniillin and 1 Fg/ml streptomyin at 37 C in a 5% CO, atmosphere. Cells of early passage (< 15) were used for our metabolism studies, sine ells at higher passage number gradually lost their retinoi aid metabolizing apaity. The proedure and onditions for the isolation and ulture of human hepatoytes have been desribed by Kooistra et al. [221. Experimental onditions for retinoi aid metabolism studies. For retinoi aid metabolism studies, human endothelial ells or HepG2 ells were grown to onflueny. In the ase of HepG2 ells, some ell aggregates were formed also. Primary ultures of human hepatoytes were used 24 h after seeding. The day before the experiment, the ulture medium was replaed by inubation medium, viz DMEM supplemented with 2 mm Hepes, ph 7.4, 1% (by vol.) human serum, 2 mm Lglutamine, 1 U/ml peniillin and 1 pg/ml streptomyin for endothelial ells; DMEM supplemented with 2 mm Hepes, ph 7.4, 1% (by vol.) heatinativated fetal alf serum, 2 mm Lglutamine, 1 U/ml peniillin and 1 pg/ml streptomyin for HepG2 ells; and DMEM supplemented with 2 mm Hepes, ph 7.4, 1% (by vol.) heatinativated fetal alf serum, 2 mm Lglutamine, 1 nm insulin, 5 nm dexamethasone, 1 U/ml peniillin and 1 pg/ml streptomyin, the standard ulture medium for primary human hepatoyte ultures [22]. At the start of an experiment, fresh inubation medium ontaining the appropriate test ompound or vehile was added. In experiments with the ytohrome P45 inhibitors liarozole, ketoonazole and metyrapone, the inhibitors were always present I h before the addition of retinoi aid. Data for the t = time points were obtained by olleting media immediately after addition of retinoi aid to the ells. For the other time points the media were olleted at the indiated time. Subsequently the ells were washed twie with 1 ml ieold NaCIP, (.15 M NaC1, 1 mm Na,HPO,, 1.5 mm KH,PO,, ph 7.4). The medium and both wash steps were ombined. Cells were olleted by sraping with a rubber polieman in 1 ml NaCI/P,. Cells and media were immediately frozen in liquid nitrogen and stored at 8 C until extration. Extration of retinoi aid metabolites from media and ells. All extration and analytial proedures were arried out in a darkened room using brown glass tubes to protet the retinoids from exposure to light. An internal standard onsisting of a known amount of TIMOTA [alltrans7(1,1,3,3tetramethyl Sindanyl)3methylota2,4,6trienoi aid], kindly provided by Dr A. Levin (HoffmannLaRohe In, Nutley, NJ), was added in 1 pl of ethanol to eah sample in order to monitor the reovery of retinoi aid during the extration and HPLC proedures [23]. Retinoids were extrated from the media and ells using a modifiation of the proedure desribed by Tang and Russell [12, 241. Briefly, the media and ell suspensions were extrated with 8 12 and 6 ml hlorofordmethanol (2: 1, by vol.), respetively. Next, the hloroform extrats were onentrated by evaporation under a gentle stream of N, to a final volume of about 1 ml. This retinoidontaining hloroform extrat was then applied to a 5 mg aminopropyl solidphase extration olumn (Baxter Labs In.) that had previously been equilibrated with hexane. Neutral lipids present in the extrat were eluted with 1 olumn volume hloroform/isopropanol (2: 1, by vol.) and disarded. Subsequently, the retinoids were eluted from the aminopropyl olumn with 2.5 olumn volumes of 2% (by vol.) aeti aid in diethyl ether. The aeti aid/diethyl ether eluates were olleted, evaporated to dryness under a gentle stream of N,, and redissolved in HPLC mobilephase (hexane/ aetonitrilelaeti aid, 99.5 :.4:.1, by vol.) for injetion onto the HPLC olumn. HPLC analysis. Retinoid levels were determined by normalphase HPLC employing two silia olumns linked in tandem. The silia olumns onsisted of a 3.9 mmx1so mm Waters 5 p Resolve (Waters Assoiates) and a 4.6 mmx 15 mm 3 p Supelosil LCSI (Supelo In). The first olumn was preeded by a Waters silia GuardPAK guard olumn. For hromatography, we employed an isorati system where the mobile phase onsisted of hexane/aetonitrile/aeti aid (99.5 :.4 :.1, by vol.) flowing at 1.8 ml/min. The mobile phase was made fresh daily and filtered and degassed immediately prior to use. The solvent was delivered by a Varian Star 91 pump (Varian Instruments). We routinely injeted 9 p1 sample onto the olumns using a Varian Star 995 Autosampler. Retinoi aid mass was deteted at 35nm using a SpetraPhysis Spetra 1 variable wavelength detetor (Spetra Physis In.). Alltr~ns[~H]retinoi aid was deteted and quantitated with an inline Berthold LB56C1 radioativity monitor (EG&G Berthold). All data shown are expressed as perentages of the amount alltransreti
598 Lansink et al. (Eul: J. Biohem. 247l noi aid or 9isretinoi aid present at time zero in media and ells and are orreted for extration effiieny, as assessed by the reovery of the internal standard TIMOTA. Mean (t SD) reoveries upon extration were 82.8r4.4 (n = 9) and 88.4? 2.3 (n = 6) for the media and ells, respetively. All data shown are for onfluent ells on 1m2 dishes inubated with 1 ml inubation medium. The amounts of ells in our experiments were determined to be about.7xlo" for endothelial ells, 2.OX1O6 for HepG2 ells and.8x16 for primary hepatoytes on 1 m'. The metabolism studies were usually performed with 1 pm alltransretinoi aid and amounts of retinoi aid were determined from absorbane profiles (wavelength = 35 nm) by omparing integrated peak areas with a standard urve relating peak areas to known masses of standard alltransretinoi aid. For some experiments, trae amounts (2 nm) alltr~ns[~h]retinoi aid were added to the 1 pm alltransretinoi aid ; the rate of disappearane of the radiolabeled alltransretinoi aid in ells and media was always idential to that of the unlabeled alltransretinoi aid. Sine determining the amount of retinoi aid by measuring radiaoativity is more sensitive than by measuring mass, metabolism studies with low retinoi aid onentrations (2 nm) were performed with alltrans ['Hlretinoi aid. Halflives of retinoids were alulated with regression analysis (Slide Write Plus, version 5.1, Advaned Graphihs Software In.). tpa synthesis experiments. For tpa synthesis experiments, onfluent ultures of human endothelial ells were used at first or seond passage, and ells were always refed with inubation medium the day before the experiment, viz DMEM supplemented with 2 mm Hepes ph 7.4, 1% (by vol.) human serum, 2 mm Lglutamine, 1 U/ml peniillin and 1 pg/ml streptomyin. Conditioned media were obtained by inubating ells at 37 C for 24 h with fresh inubation medium ontaining the appropriate onentration of alltransretinoi aid, 1 pm liarozole or stok solvent Me2S (final onentration.1 %, by vol.). Liarozole was always present 1 h before the addition of retinoi aid to the ells. Conditioned media were entrifuged for 5 min at 8 rpm in a Mirofuge entrifuge to remove ells and ellular debris and were stored at 2 C until use for tpa antigen determinations. RESULTS Metabolism of alltransretinoi aid and 9isretinoi aid in human endothelial ells and hepatoytes. Cultured human endothelial ells and HepG2 ells were inubated with 1 pm alltransretinoi aid or 9isretinoi aid, and after different time periods the amount of retinoi aid present in the media and ells was determined by HPLC analysis. As shown in Fig. 1A for endothelial ells, alltransretinoi aid rapidly disappeared from the medium, but only a minor part of it was reovered from the ells ; the amount of alltransretinoi aid assoiated with the ells reahed maximally 2.% of the added amount of alltransretinoi aid after 1 h of inubation and then rapidly dereased to levels below the detetion limit (2.6 pmol) after 12 h of inubation. The overall disappearane of alltransretinoi aid from the system (medium and ells together), showed a biphasi pattern (Fig. 1 A): an initial phase of about 8 h with a halflife of alltransretinoi aid of approximately 8 h and a seond phase during the next 16 h with a halflife of alltransretinoi aid of approximately 2 h. This biphasi elimination pattern ould reflet saturation kinetis, indution of retinoi aid metabolizing apaity or both. Evidene for saturation kinetis omes from retinoi aid elimination studies at a onentration of 2 nm alltransretinoi aid, where alltransretinoi A 1 8 6 4 2 lrh a, 5!!! a 1 m.1 4 8 12 16 \ 2 24 5.1 \!=% I%. 1 8 6 4 2 Fig. 1. Disappearane profile of alltransretinoi aid from the medium (solid line) and ellassoiated alltransretinoi aid (dotted line) in time in ultured human endothelial ells (A) or HepG2 ells (B). In the insert a semilogarithmi plot of the total amount of alltransretinoi aid (atra) present in media + ells as a funtion of time is shown. Cells were inubated with 1 pm alltransretinoi aid and at the indiated times ells and media were harvested and analyzed for retinoi aid levels as desribed in the methods setion. The data are expressed as perentages relative to the amount of alltransretinoi aid added at time point zero. Values shown are means t SE of three separate experiments. aid showed a rapid monophasi disappearane with a halflife of 1.4 h (Fig. 7A). To examine whether alltransretinoi aid at a onentration of 1 pm is apable of induing retinoi aid metabolizing apaity, we inubated ells for 24 h with or without 1 pm alltransretinoi aid, and then determined the atabolism of 2 nm alltrans['hlretinoi aid 4 h later. Whereas under ontrol onditions 362 12% (n = 3) of the alltrans['hi retinoi aid was left in the medium, the medium of ells pretreated with 1 pm alltransretinoi aid ontained only 12?4% (n = 3) of the added alltrans[3h]retinoi aid. These results indiate that both saturation kinetis and indued retinoi aid atabolism play a role in the elimination of 1 pm alltrunsretinoi aid. Alltransretinoi aid was relatively stable in the absene of ells : at the end of a 24 h inubation of 1 pm alltransretinoi aid in inubation medium at 37"C, 9% 1% (n = 6)
1 "..'.;..I, "t "1 /F..,.. J. Biohem. 247) 599 I 12 v n E. 1 E.2 8 '. 6 =f 4 [r 1 8 6 4 B ' ' 1 s I w 6 s W 2 4. v) Y 2 Q) h s v a : d, 41 \ Fig. 2. Disappearane profile of 9isretinoi aid from the medium (solid line) and ellassoiated 9isretinoi aid (dotted line) in time in ultured human endothelial ells (A) or HepG2 ells (B). In the insert a semilogarithmi plot of the total amount of 9isretinoi aid (9is RA) present in media + ells as a funtion of time is shown. Cells were inubated with 1 pm 9isretinoi aid and at the indiated times ells and media were harvested and analyzed for 9isretinoi aid levels as desribed in Materials and Methods. The data are expressed as perentages relative to the amount of 9isretinoi aid added at time point zero. Values shown are means f SE of three separate experiments. of alltransretinoi aid was still intat. Together these results demonstrate that ultured human endothelial ells ontain an ative alltransretinoi aid metabolizing apaity, whih is inreased by inubating the ells in the presene of 1 pm alltransretinoi aid. The data also suggest that the metabolizing apaity of endothelial ells is a limiting fator in the rate at whih alltransretinoi aid is metabolized. The overall disappearane rate of 1 pm alltransretinoi aid in HepG2 inubations is omparable to that during the initial phase of 1 pm alltransretinoi aid metabolism in endothelial ells, and showed a halflife of about 7 h (Fig. 1B). However, in ontrast to endothelial ells, HepG2 ells showed no inrease in alltransretinoi aid disappearane rate upon prolonged inubation (Fig. 1 B), and no differene in disappearane rate was observed for 1 pm and 2 nm alltransretinoi aid Fig. 3. Disappearane profiles. (A) Disappearane profile of alltransretinoi aid (atra) from the medium of primary human hepatoytes ultures (solid line) and formation of 9isretinoi aid (9is RA) and 13isretinoi aid (13is RA). Cells were inubated with 1 pm alltransretinoi aid and at the indiated times media were harvested and analyzed for alltransretinoi aid, 9isretinoi aid and 1 3isretinoi aid levels as desribed in Materials and Methods. The data are expressed as perentages relative to the amount of alltrunsretinoi aid added at time point zero. Values shown are means?ranges of two separate experiments. (B) Disappearane profile of 9isretinoi aid from the medium of primary human hepatoytes ultures (solid line) and formation of alltransretinoi aid and 13isretinoi aid. Cells were inubated with 1 pm 9isretinoi aid and at the indiated times media were harvested and analyzed for 9isretinoi aid, alltransretinoi aid and 13isretinoi aid levels as desribed in Materials and Methods. The data are expressed as perentages relative to the amount of 9isretinoi aid added at time point zero. Values shown are means 2 ranges of two separate experiments. (Fig. 7B). In omparison to endothelial ells, HepG2 ells ontained a high ellassoiated alltransretinoi aid fration, reahing maximally 25.7 % of the added amount of alltransretinoi aid after 2 h of inubation (Fig. 1 B). This reflets, at least partly, the larger ell volume and ell numbers of HepG2. 9isRetinoi aid (1 pm) is metabolized only slowly by ultured human endothelial ells showing a halflife of about 37 h over the entire inubation period of 24 h (Fig. 2A). No speifi aumulation of 9isretinoi aid in the endothelial ells was observed : the amount of 9isretinoi aid reovered from the ells varied between maximally 1.8% at 2 h and 1.2% at 24 h (Fig. 2A). In ontrast to endothelial ells, HepG2 ells metabolize 9isretinoi aid fast with a halflife of 3.4 h and at a higher rate than alltransretinoi aid (Fig. 2B). The amount of HepG2ellassoiated 9isretinoi aid reahed maximally 8.1 % after 1 h, and then rapidly delined to.1 % after 24 h. The lower levels of 9isretinoi aid relative to alltransretinoi aid reovered from HepG2 ells may be explained by more rapid metabolism of 9isretinoi aid and its onversion to alltransretinoi aid.
6 Lansink et al. (ELK J. Biohern. 247) C ULU 5 1 15 2 time (min) Fig. 4. HPLC hromatogram of extrat prepared from endothelium medium (panel 1) and HepG2 medium (panel 2), 8 hours after addition of 1 pm alltransretinoi aid and 2 nm alltr~ns[~h]retinoi aid to the ells. The hromatogram was obtained by detetion of the absorbane at 35 nm. Peak A, 13isretinoi aid; peak B, 9isretinoi aid; peak C, alltransretinoi aid; peak D, TIMOTA. Alltrunsretinoi aid, 13isretinoi aid and 9isretinoi aid were identified by oelution with known amounts of respetive standard solutions. For a small number of experiments, the metabolism of alltransretinoi aid and 9isretinoi aid was examined in primary ultures of human hepatoytes. Alltransretinoi aid (1 pm) and 9isretinoi aid (1 pm) were metabolized by primary hepatoytes at a similar rate to that found with HepG2 ells, with 9isretinoi aid being metabolized at a higher rate than alltransretinoi aid (Fig. 3). Similar to HepG2 ells, the maximal amount of ellassoiated alltrunsretinoi aid (9.6% t.2, n = 2) was higher than that of 9isretinoi aid (3.9%+.4, n = 2) 2 h after addition of the respetive retinoi aid isoforms. These results provide onfidene that the studies of retinoi aid metabolism in HepG2 ells are physiologially relevant. Isomerization of alltransretinoi aid and 9isretinoi aid in human endothelial ells and hepatoytes. To evaluate whether human endothelial ells are able to form 9isretinoi aid from alltransretinoi aid, the amount of 9isretinoi aid (and 13isretinoi aid, another isomer of alltransretinoi aid) in the medium and ells was determined at different time intervals after the addition of alltransretinoi aid (for a harateristi HPLC profile of medium see Fig. 4A). The total amounts of 9isretinoi aid and 13isretinoi aid reovered from the endothelial ells and medium together did not exeed the spontaneously formed amounts of 9isretinoi aid and 13isretinoi aid in medium inubated without ells, and aounted for 2.92 1.2% and 4.8% 1.5% (n = 6), respetively after 24 h inubation at 37 "C. Similarly, no elldependent isomerization of 9isretinoi aid to alltransretinoi aid was observed (data not shown). After 24 h inubation of medium ontaining 1 pm 9isretinoi aid under ellfree onditions at 37T, 4.4+.S% (n = 3) alltransretinoi aid was formed, whereas the amount of 13isretinoi aid was below detetion limits (n = 3). In ontrast to endothelial ells, when alltransretinoi aid was added to HepG2 ell ultures, both 9isretinoi aid and 13isretinoi aid were found to aumulate in both ells and media to levels whih were signifiantly greater than those ob Fig. 5. Formation of 9isretinoi aid (A) and 13isretinoi aid (B) from alltransretinoi aid by hepatoytes. HepC2 ells were inubated with 1 pm alltransretinoi aid and after different time points the retinoi aid isomers were determined in the media (solid lines) and in the ells (dotted lines). The total amount alltransretinoi aid at time zero in media and ells was set 1% and the data are expressed relative to this. The values represent the average (? SE) of three determinations. served when alltmnsretinoi aid was added to media with no ells present (see Fig. 4B for a harateristi HPLC profile for the medium). The amounts of 9isretinoi aid and 13isretinoi aid (expressed as perentage of the amount of alltransretinoi aid added at t = ) in the media and ells at different time points after addition of alltransretinoi aid are shown in Fig. 5. In the HepG2 ells the amount of 9isretinoi aid was maximally 1.2% after 1 h, remained high until 12 h and then started to deline (Fig. S A). The formed 9isretinoi aid also diffused from the ells into the medium, and reahed maximally a level of 4.7% at 8 h. In addition to the formation of 94sretinoi aid, HepG2 ells also generated 13isretinoi aid (Fig. SB). Starting at 4% 13isretinoi aid at time zero, the amount of 13isretinoi aid in the medium inreased to 5.4% at 8 h, and then slowly dereased to 2.1 % at 24 h. The amount of 13isretinoi aid in the ells peaked at 2.9% after 2 h and subsequently fell to.6% at 24 h. When 9isretinoi aid was added to HepG2 ell ultures, both alltransretinoi aid and 13isretinoi aid were found to aumulate, in a timedependent manner, in ells and media (Fig. 6). A maximal level of 5.4% alltransretinoi aid (of the amount of 9isretinoi aid added at t = ) in the ells was reahed 4 h after addition of 9isretinoi aid and in the medium a maximum amount of 8.9% was present at 4 h (Fig. 6A). Although at lower levels than alltrunsretinoi aid, 13isreti
z 1 8 6 4 2 ' Lansink et al. (Eul: J. Biohem. 247) 61 on... lia keto. mety n s v 1 1 a m m 1... \. 7.1 h U 2 1 U a * 8 U 6 5 4 a a 2 v). Y o m _ B r Fig. 6. Formation of alltraitsretinoi aid (A) and 13isretinoi aid (B) from 9isretinoi aid by hepatoytes. HepG2 ells were inubated with 1 pm 9isretinoi aid and at different time points amounts of the retinoi aid isomers were determined in the media (solid lines) and in the ells (dotted lines). The total amount of 9isretinoi aid at time zero in media and ells was set 1% and the data are expressed relative to this. The values represent the average (5 SE) of three determinations. on...,. la keto mety B Fig. 7. Effets of liarozole (lia), ketoonazole (keto), metyrapone (mety) and vehile (on),.1 % (by vol.) Me,SO, on the metabolism of alltransretinoi aid (atra) by ultured human endothelial ells (A) and HepG2 (B) ells. Cells were inubated with 2 nm alltrans [3H]retinoi aid and after the indiated times media and ells were olleted and analyzed as desribed. The inhibitors were added 1 h before inubation with alltrans[3h]retinoi aid. The disappearane of total alltransretinoi aid from media and ells is plotted on a semilogarithmi sale. The values shown represent the average of two inubations with ranges indiated by error bars. noi aid ould also be deteted in the ells and in the media (Fig. 6B). Comparison of the data in Figs 5 and 6 shows that a greater onversion of 9isretinoi aid to alltransretinoi aid ours, than vie versa. This is onsistent with what would be expeted thermodynamially, sine at equilibrium only a small part of retinoi aid (215%) is in the 94s isomeri form [25]. The isomerisation of both alltransretinoi aid and 9isretinoi aid by primary hepatoytes resembled that by HepG2 ells as shown in Fig. 3. Inhibition of retinoi aid metabolism by human endothelial ells and HepG2 ells by inhibitors of ytohrome P45. To assess whether the metabolism of alltransretinoi aid ours via a ytohrome P45(P45)dependent mehanism we studied the effet of various P45 inhibitors on the degradation of 2 nm alltrans['hlretinoi aid in human endothelial ells. We have used liarozole and ketoonazole, whih have been shown to inhibit retinoi aid metabolism in rats in vivo, and to blok 4hydroxylation of retinoi aid in vitro [26, 271, and metyrapone, whih is a more general P45 inhibitor [28]. We hose to perform these metaboli studies with 2 nm alltransretinoi aid, beause at this onentration retinoi aid shows a monophasi disappearane profile. Liarozole at a onentration of 1 pm is a very potent inhibitor of alltransretinoi aid degradation in endothelial ells, and inreases the halflife from 1.4 h to 17.2 h. In the presene of 1 FM ketoonazole, the halflife of alltransretinoi aid was 4.5 h, whereas with 1 pm metyrapone no inhibition of alltransretinoi aid metabolism was seen. Liarozole (1 pm) was also a very effetive inhibitor of 9isretinoi aid metabolism ; in the presene of liarozole 94 % 9isretinoi aid was left after 24 h inubation (data not shown) ompared with 65 % in the absene of the inhibitor. Different from endothelial ells, in HepG2 ells liarozole and ketoonazole were about equipotent in inhibiting alltransretinoi aid metabolism (Fig. 7 B), and the halflives of total alltransretinoi aid were inreased only about threefold, from 7.8 h under ontrol onditions to about 22.7 h and 18.9 h by 1 pm liarozole and 1 pm ketoonazole, respetively. Again, 1 pm metyrapone was a very weak inhibitor of alltransretinoi aid metabolism by HepG2 ells resulting in a halflife of 9 h. The metabolism of 9isretinoi aid was not inhibited by liarozole (data not shown). These results show that alltransretinoi aid and 9isretinoi aid metabolism in human endothelial ells and HepG2 ells an be inhibited by speifi ytohrome P45 inhibitors. The different effiay of the P45 inhibitors in the two ell types suggest that different P45 enzymes may be involved in retinoi aid metabolism in endothelial ells and HepG2 ells. Effets of P45 inhibitors on the indution of tpa by retinoi aid. To test the influene of alltransretinoi aid metabo
62 Lansink et al. (Eur: J. Biohem. 247) 3 T 9 8 7 6 Log onentration (M) Fig. 8. Effets of alltransretinoi aid alone (solid line) or in ombination with liarozole (dotted line) on tpa synthesis by ultured human endothelial ells. The ells were inubated for 24 h with differing onentrations of allfratisretinoi aid alone or in ombination with 1 pm liarozole, and onditioned media were analyzed for tpa antigen. The data are expressed as perentages relative to the ontrol value. The data shown are from one representative experiment of three performed and data are expressed as mean values of dupliate determinations with ranges indiated by error bars. lism on tpa indution, endothelial ells were inubated with different onentrations of alltransretinoi aid in the presene or absene of liarozole and tpa prodution was determined. In the presene of liarozole, at least 1fold lower alltransretinoi aid onentrations were required than in the absene of the inhibitor to obtain the same indution of tpa (Fig. 8). DISCUSSION Cirulating tpa plays a ruial role in fibrinlot surveillane and a longterm goal of our researh has been to establish whether manipulation of plasma retinoi aid levels an serve as an effetive means for elevating blood tpa levels and onsequently lessen the risk for thrombus formation. To this end in previous work, we and others have shown that retinoi aid is a regulator of tpa gene expression [3, 6, 71 and that tpa levels in the plasma and tissues of rats depend on their retinoi aid status [3, 41. In ell ulture experiments, we showed that media supplemented with alltransretinoi aid onentrations ranging between 1 pm and 1 pm are required to optimally indue tpa expression in human endothelial ells [S], whereas in vivo plasma alltransretinoi aid levels between 3.6 nm and 6.3 nm [Il, 121 apparently suffie to maintain high tpa expression. This disrepany between the in vitro and in vivo onentrations of retinoi aid required to indue tpa expression suggested to us that in vitro in vasular endothelial ells retinoi aid metabolism might be an important proess whih an modulate ellular responses towards retinoi aid. To gain understanding of this possibility, we arried out studies of alltransretinoi aid and 9isretinoi aid uptake and metabolism in primary ultures of human endothelial ells. To link our studies to other ell types to whih irulating retinoi aid will have extensive exposure and whih are thought to be importantly involved in retinoi aid metabolism [29], we arried out parallel studies in ultured primary human hepatoytes and the human HepG2 hepatoyte ell line. Four major findings emerged from our studies of retinoi aid uptake and metabolism by human endothelial ells and hepatoytes. First, alltransretinoi aid is taken up and rapidly metabolized by both human endothelial ells and hepatoytes. However, metabolism of alltransretinoi aid was found to be induible in endothelial ells whih had been preexposed to alltransretinoi aid. This indution of alltransretinoi aid metabolism was not observed in hepatoytes. Seondly, the uptake and rate of metabolism of 9isretinoi aid in endothelial ells was markedly different from that of alltransretinoi aid. Moreover, the rate of uptake and metabolism of 9isretinoi aid by hepatoytes was very different from that of endothelial ells. Third, through the use of a sensitive normalphase HPLC proedure for measuring alltransretinoi aid and 9isretinoi aid levels in the ultured ells and orresponding media, we were able to demonstrate that hepatoytes possess the apaity to atalyze the interonversion of alltransretinoi aid and 9isretinoi aid. Endothelial ells did not atalyze either the isomerization of alltransretinoi aid to its 9 4s isomer or the isomerization of 9isretinoi aid to the alltrans isomer. Finally, metabolism of alltransretinoi aid and 9isretinoi aid by both human endothelial ells and HepG2 ells an be inhibited by exposure of the ells to the ytohrome P45 inhibitors liarozole and ketoonazole. A surprising finding of this study is the ative role played by ultured human endothelial ells in metabolizing alltransretinoi aid, an ativity hitherto unknown. This rapid metabolism of alltransretinoi aid by ultured human endothelial ells probably explains the relatively high alltransretinoi aid levels whih are neessary to indue tpa expression in vitro ompared with in vivo, where a steadystate plasma level of alltransretinoi aid is present. In the presene of the most potent alltransretinoi aid metabolism inhibitor in endothelial ells, liarozole, at least 1fold lower alltransretinoi aid onentrations were required than in the absene of the inhibitor to obtain the same indution of tpa. This metabolism by endothelial ells may be an important fator in establishing plasma alltransretinoi aid levels, onsidering that the major endothelial surfae area in the adult human is estimated to be 7215 m2 113, 141, and is of obvious relevane for the pharmaologi use of alltransretinoi aid. It should be noted that alltransretinoi aid is initially a very effetive drug for treatment of aute promyeloyti leukemia (APL) but, with time, patients develop a tolerane for the alltransretinoi aid (for review see [31] and referenes therein). This tolerane has been attributed to an indution of alltransretinoi aid metabolism within the patient. It is interesting that for our shortterm ell ulture studies primary human endothelial ells but not human hepatoytes display an induible metabolism of alltransretinoi aid. Reently, White et al. desribed an induible retinoi aid 4hydroxylase, designated P45RAI [3]. It will be of interest to learn whether retinoi aid indues P45RAI in endothelial ells. Hepatoytes were found to differ from endothelial ells in their abilty to metabolize retinoi aid in two distint ways. First, hepatoytes metabolize 9isretinoi aid at a rate whih is muh greater than that observed in endothelial ells. The rate of 9isretinoi aid metabolism by hepatoytes is even greater than for alltransretinoi aid. Seond, hepatoytes but not endothelial ells are able to form 9isretinoi aid from alltransretinoi aid, and vie versa. Although our data do not provide us with insight as to whether this proess is enzymati or nonenzymati in nature, they are onsistent with the observation of Urbah and Rando [32] that thiol groups present in heatinativated hepati mirosomal membranes are able to bring about the isomerization of alltransretinoi aid to the 9is isomer. It would seem from these data and data from investigations of retinoi aid metabolism by human keratinoytes [33] and by human mammary arinoma ells 1341 that the abilities of ells to take up and metabolize retinoi aid are diverse and elltype dependent.
Lansink et al. (Eur: J. Biohem. 247) 63 It is well established that the P45 system is involved in the metabolism of retinoi aid [35] and that treatment of rats with P45 inhibitors suh as liarozole and ketoonazole bring about inreased blood and tissue levels of retinoi aid [27]. Liarozole has been shown to disrupt the metabolism of alltransretinoi aid at two levels : the onversion of alltransretinoi aid to alltran.s4hydroxyretinoi aid (whih is then oxidized to alltrans4keto retinoi aid) and the metabolism of alltrans 4keto retinoi aid [36]. Our studies with P45 inhibitors indiate the involvement of different metaboli routes and/or P45 isoenzymes in alltransretinoi aid and 9isretinoi aid metabolism in endothelial ells and HepG2 ells. We found that in endothelial ells liarozole (1 pm) inreased the halflife of alltransretinoi aid IZfold, ketoonazole (1 pm) threefold, while metyrapone (1 pm) had no effet. In ontrast, in HepG2 ells liarozole and ketoonazole are equipotent in inhibiting alltransretinoi aid metabolism and inreased the halflife of alltransretinoi aid within the ells by approximately threefold. The metabolism of 9isretinoi aid was almost ompletely inhibited by liarozole in endothelial ells, whereas in HepG2 ells liarozole had no effet on 9isretinoi aid metabolism. Experiments in rats have shown liarozole to be a muh more potent inhibitor of alltransretinoi aid metabolism than ketoonazole [27]. Administration of liarozole or ketoonazole enhaned endogenous plasma onentrations of alltransretinoi aid in rats from mostly undetetable values (less than.5 ng/ ml) to 2.5 ng/ml and 1.3 ng/ml, respetively [27]. Taken together with our observation that liarozole more markedly influenes endothelial ell metabolism of alltramretinoi aid than hepatoyte metabolism, it further substantiates our finding that extrahepati tissues inluding the endothelium may signifiantly ontribute to alltransretinoi aid metabolism in vivo. We have investigated the uptake and metabolism of alltransretinoi aid and 9isretinoi aid by two human ell types whih probably represent the two most abundant, and onsequently the two most frequently enountered ell types to whih irulating retinoi aid is exposed in the body. Our data suggest that the vasular endothelium plays a signifiant role in the metabolism of alltrunsretinoi aid within the body. This had not been previously suspeted. This observation regarding metabolism of alltransretinoi aid by endothelial ells also provides insight into, and raises questions about, how the body responds to the pharmaologial use of alltransretinoi aid for treatment of disease. Our data also show that the abilities of ells to take up and to metabolize alltransretinoi aid and 9isretinoi aid are elltype speifi. Finally, our data demonstrate that hepatoytes but not endothelial ells are able to atalyze the isomerization of alltransretinoi aid and 94sretinoi aid and again suggest that the metabolism of retinoi aid is different for different ell types. It seems lear from our studies of retinoi aid uptake and metabolism by ells in ulture and from studies in other isolated ell systems by others [34, 35, 371 that retinoi aid metabolism in the intat organism is very omplex. Thus, it would seem that not only is retinoi aid ation within ells omplex and elltype speifi but also that the metabolism of retinoi aid within ells is similarly omplex and elltype speifi. We gratefully thank Dr Hans M. G. Prinen and Elly C. M. de Wit for providing the primary human hepatoytes. 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