Abstract
Various hormonal and monoaminergic systems play determinant roles in the regulation of several cytochromes P450 (P450s) in the liver. Growth hormone (GH), prolactin, and insulin are involved in P450 regulation, and their release is under dopaminergic control. This study focused on the role of D2-dopaminergic systems in the regulation of the major drug-metabolizing P450s, i.e., CYP3A, CYP2C, and CYP2D. Blockade of D2-dopaminergic receptors with either sulpiride (SULP) or 4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol (L-741,626) markedly down-regulated CYP3A1/2, CYP2C11, and CYP2D1 expression in rat liver. This suppressive effect appeared to be mediated by the insulin/phosphatidylinositol 3-kinase/Akt/FOXO1 signaling pathway. Furthermore, inactivation of the GH/STAT5b signaling pathway appeared to play a role in D2-dopaminergic receptor-mediated down-regulating effects on these P450s. SULP suppressed plasma GH levels, with subsequently reduced activation of STAT5b, which is the major GH pulse-activated transcription factor and has up-regulating effects on various P450s in hepatic tissue. Levels of prolactin, which exerts down-regulating control on P450s, were increased by SULP, which may contribute to SULP-mediated effects. Finally, it appears that SULP-induced inactivation of the cAMP/protein kinase A/cAMP-response element-binding protein signaling pathway, which is a critical regulator of pregnane X receptor and hepatocyte nuclear factor 1α, and inactivation of the c-Jun N-terminal kinase contribute to SULP-induced down-regulation of the aforementioned P450s. Taken together, the present data provide evidence that drugs acting as D2-dopaminergic receptor antagonists might interfere with several major signaling pathways involved in the regulation of CYP3A, CYP2C, and CYP2D, which are critical enzymes in drug metabolism, thus affecting the effectiveness of the majority of prescribed drugs and the toxicity and carcinogenic potency of a plethora of toxicants and carcinogens.
Introduction
Cytochromes P450 (P450s) play major roles in the hepatic metabolism of xenobiotics such as drugs, carcinogens, and toxic agents. Metabolism can activate prodrugs to pharmacologically more-active metabolites, inactivate pharmacologically active compounds, or activate precarcinogens, thus triggering the formation of several types of tumors in humans (Gonzalez and Gelboin, 1994; Ingelman-Sundberg, 2004). P450s also catalyze the biotransformation of endogenous compounds, such as steroids and fatty acids (Spatzenegger and Jaeger, 1995; Guengerich, 2003). Inhibition of P450 isoforms that metabolize clinically used drugs may lead to increased levels of drug substrate in the body and may cause adverse effects, especially when drugs with low therapeutic indices are used (Spatzenegger and Jaeger, 1995). Conversely, up-regulation of critical enzymes may accelerate the metabolism of drug substrate, thus leading to reduced pharmacological outcomes for the drug (Konstandi et al., 2005; Daskalopoulos et al., 2012). It is important, particularly for multidrug therapies, to be aware of the possibilities of drug-drug interactions that might lead to reduced or increased drug efficacy, severe toxicity, and/or tumorigenesis (Gonzalez and Gelboin, 1994; Guengerich, 2003; Konstandi et al., 2004; Pelkonen et al., 2008).
Some of the most important P450 enzymes for hepatic phase I drug metabolism are members of the CYP3A, CYP2C, and CYP2D subfamilies (Ingelman-Sundberg, 2004). These cytochromes are responsible for the biotransformation of more than 90% of the most widely prescribed drugs (Guengerich, 2003).
Central and peripheral catecholaminergic systems have been determined to regulate P450 expression (Konstandi et al., 1998, 2004; Rendic and Guengerich, 2010; Daskalopoulos et al., 2012). In addition, previous studies clearly indicated critical roles for hormones, such as growth hormone (GH) (Waxman and O'Connor, 2006), thyroid hormones (Takahashi et al., 2010), and insulin (Kim and Novak, 2007), in the regulation of various hepatic P450s (Daskalopoulos et al., 2012).
Diabetic rats display increased hepatic CYP2A1, CYP2C6/7, CYP2E1, CYP3A2, and CYP4A3 expression, which can be restored to normal levels with insulin administration (Shimojo et al., 1993). Insulin exerts many of its effects through the PI3K/Akt signaling pathway, which regulates the expression of several genes, including various P450 isoforms (Kim and Novak, 2007). Among upstream signaling factors regulating the release of insulin in response to increased plasma glucose levels, dopamine plays a significant role; stimulation of D2-dopaminergic receptors in pancreatic β-cells suppresses insulin release (Rubí et al., 2005). In accordance with this finding and other earlier studies, we showed that blockade of D2-dopaminergic receptors stimulated insulin release and markedly down-regulated CYP2E1 expression (Konstandi et al., 2008). It is possible that the insulin/PI3K/Akt signaling pathway plays a determinant role in this regulation (Woodcroft et al., 2002).
A broad spectrum of drugs prescribed to treat a variety of disease states, including schizophrenia, bipolar disorder, depression, and Parkinson's disease, exert their effects mainly through D2-dopaminergic receptors and related signaling pathways (Beaulieu and Gainetdinov, 2011). Given its central role in the regulation of insulin release, it is conceivable that drugs acting through this pathway could alter the expression of various drug-metabolizing P450 enzymes, thus affecting the pharmacodynamic characteristics and toxicity of drug substrates during pharmacotherapy (Gonzalez and Yu, 2006).
The aim of this study was to assess the role of D2-dopaminergic receptor-linked signaling pathways in the regulation of the P450s CYP3A, CYP2C, and CYP2D. For this purpose, rats were treated with the D2-dopaminergic receptor antagonist sulpiride (Konstandi et al., 2008), a typical antipsychotic drug that is used mainly for the treatment of psychosis associated with schizophrenia or major depressive disorder (Maitre et al., 1994). The direct effects of sulpiride on hepatocytes were assessed in vitro with primary hepatocyte cultures. Blockade of the D2-dopaminergic receptor signaling pathways caused strong reductions in the expression of CYP3A, CYP2C, and CYP2D. Given the fact that the vast majority of prescribed drugs are metabolized by these P450s, their down-regulation might lead to strongly increased blood levels of drug substrates during multidrug therapy and to drug-drug interactions with potentially detrimental side effects.
Materials and Methods
Animals
All experimental animals used in this study (adult male Wistar Kuo/Io/rr rats, 2–3 months of age) were inbred in the animal facility of the University of Ioannina (Ioannina, Greece) and were housed in groups of three or four in plastic cages, with standard rodent chow and water available ad libitum. The animals were maintained on a 12-h light/dark cycle (lights on at 6:00 AM) and were adapted to handling over a period of 5 to 7 days before the experiment. All procedures involving animals were reviewed and approved by the local ethics committee, and they conformed to the European Commission ethical standards for the care and use of laboratory animals (Directive 86/609-EEC).
In Vivo Assessment of Roles of D2-Dopaminergic Receptor-Related Pathways in Regulation of Hepatic Drug Metabolism
Control animals (group I) received normal saline solution administered subcutaneously, twice daily, for 4 consecutive days. Animals in group II received sulpiride (SULP) (2 μg/kg b.wt.; Sigma-Aldrich, St. Louis, MO) administered subcutaneously, twice daily, for 4 consecutive days. Rats in group III received the highly selective D2-dopaminergic receptor antagonist 4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol (L-741,626) (PubChem SID 50104688; 1.5 mg/kg b.wt.; Sigma-Aldrich) administered intraperitoneally, twice daily, for 4 consecutive days. All animals were euthanized 1 h after the last injection.
Isolation of Microsomes
Microsomal fractions were prepared from liver samples, which were homogenized in ice-cold homogenization buffer (0.15 M KCl, 10 mM K2EDTA, 1 mM dithiothreitol, pH 7.4). The homogenates were centrifuged for 20 min at 14,075g (4οC). The upper phase was transferred into new vials and centrifuged for 60 min at 96,552g (4οC). The microsomal pellet was resuspended in ice-cold homogenization buffer, homogenized, and centrifuged for 45 min at 96,552g (4οC). The washed microsomal pellet was resuspended in ice-cold storage buffer (K2HPO4/KH2PO4, pH 7.4, 1 mM K2EDTA, 0.1 mM dithiothreitol, 20% glycerol) and stored at −80οC until used in assays (Lang et al., 1981).
Primary Hepatocyte Cultures
Primary hepatocytes were isolated and used in cultures according to the method described by Klaunig et al. (1981). In brief, primary hepatocytes were isolated from rats (250–300 g) by using a two-step collagenase perfusion method. The hepatocytes were suspended in Williams' medium E (Invitrogen, Carlsbad, CA) containing 1% l-glutamine and 1% penicillin/streptomycin. The cells were counted in a Neubauer cell chamber and were plated at a density of 1 × 105 cells per well in collagen type I-coated dishes (diameter, 3.8 cm; BioCoat cell environment; BD Biosciences, San Jose, CA). The viability of the isolated hepatocytes was checked through trypan blue dye (0.4%) exclusion, and only cell preparations with viability rates of more than 85% just before plating were used. Hepatocytes were cultured at 37°C for 24 h in an atmosphere of humidified 5% CO2, which allowed them to adhere to the wells. Time- and dose-response experiments were initiated 24 h later. Primary hepatocyte cultures were treated with either SULP (1–25 μM) or insulin (INS), at different doses ranging from 1 to 100 μM, in combination with wortmannin, an inhibitor of the PI3K/Akt signaling pathway. The cAMP/PKA inhibitor 5-isoquinolinesulfonamide (H-89) (10 μM, 24 h) and the JNK inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) (10 μM, 24 h) were added to the primary hepatocyte cultures 30 min before SULP. Time-response experiments were conducted with drug treatments of primary hepatocytes ranging between 4 and 36 h.
Enzyme Activity Assays
Bufuralol 1′-Hydroxylation.
Bufuralol 1′-hydroxylation reflects CYP2D activity (Matsunaga et al., 1990). Liver microsomal protein (40 μg) was preincubated at 37°C for 5 min in a 200-μl reaction mixture containing potassium phosphate (100 mM, pH 7.4), in the presence of 50 μM bufuralol substrate. The reaction was initiated with NADPH (0.5 mM) at 37°C and lasted 7.5 min; it was terminated with 20 μl of perchloric acid (60%). After centrifugation for 10 min at 14,075g, the concentration of 1′-hydroxybufuralol (the main metabolite of bufuralol) in the supernatant (100 μl) was determined through HPLC, with fluorescence detection at 252 and 302 nm. A reverse-phase Luna C18 column (5 μm, 150 × 3 mm; Phenomenex, Torrance, CA) was used. The mobile phase contained a 30% acetonitrile (ACN)/70% perchlorate buffer (20 mM, pH 2.5) mixture, and the sample was eluted with a flow rate of 1 ml/min for 15 min.
The positive control reaction for bufuralol 1′-hydroxylation was performed by using recombinant rat CYP2D1 and CYP2D2 plus P450 reductase BD Supersomes (BD Gentest, Woburn, MA). Approximately 200 μl of potassium phosphate buffer (0.1 M, pH 7.4) containing the substrate bufuralol (50 μM) were preincubated at 37°C for 2 min. Recombinant P450 (50 μM) and NADPH (1 mM) were then added, and the mixture was incubated at 37°C for 30 min. The reaction was terminated with 20 μl of ACN; the samples were left undisturbed on wet ice for 10 min and then were injected into the HPLC system for analysis.
Testosterone Hydroxylation.
CYP3A1/2, CYP2C11, and CYP2A1 catalyze testosterone hydroxylation in rat liver. The activities studied were CYP3A1/2-dependent 6β-testosterone hydroxylation and CYP2C11-dependent 2α- and 16α-testosterone hydroxylation (Murray et al., 2001). Liver microsomal proteins (1 mg) were incubated at 37°C for 7.5 min in 500 μl of a mixture containing potassium phosphate (50 mM, pH 7.4), in the presence of 200 μM testosterone (the substrate was diluted in 5 μl of ACN). The reaction was initiated with NADPH (1 mM) and was terminated through the addition of 2 ml of a chloroform/methanol (2:1) mixture at 4°C. All incubations were performed in conditions under which linearity with time was established. After extraction, centrifugation, and separation, the organic phase was removed and evaporated under nitrogen. The residues from evaporation were dissolved in 200 μl of ACN, and aliquots of 100 μl were injected into the HPLC system. Testosterone metabolites were resolved on a reverse-phase Zorbax C18 column (5 μm, 150 × 4.6 mm; Agilent Technologies, Santa Clara, CA), with elution for 30 min with mobile phase A, containing 10% ACN and 0.5% acetic acid (flow rate, 0.8 ml/min), followed by elution for 35 min with mobile phase B, containing 36% ACN and 0.5% acetic acid.
For the positive control for 6β-testosterone hydroxylation, rat CYP3A1 and CYP3A2 Supersomes containing P450 reductase and cytochrome b5 (BD Gentest) were used. For the positive control for 16α-testosterone hydroxylation, rat CYP2C11 plus P450 reductase plus cytochrome b5 Supersomes were used (BD Gentest). As described above, approximately 200 μl of potassium phosphate buffer (0.1 M, pH 7.4) containing the substrate testosterone (5 μM) were preincubated at 37°C for 2 min. The recombinant P450 (50 μM) and NADPH (1 mM) were then added, and the mixture was incubated at 37°C for 30 min. The reaction was terminated with 20 μl of ACN, the samples were left undisturbed on wet ice for 10 min, and the samples were stored at −20°C for 24 h before analysis.
Ketoconazole (Sigma-Aldrich), a CYP3A inhibitor, was used for the negative control for CYP3A-dependent 6β-testosterone hydroxylation. Sulfaphenazole (Sigma-Aldrich), a CYP2C11 inhibitor, was used for the negative control for CYP2C-dependent 2α- and16α-testosterone hydroxylation. In brief, 200 μl of potassium phosphate buffer (0.1 M, pH 7.4) containing testosterone as substrate (5 μM), the P450 inhibitor (1 μM), and microsomes (0.1 mg) were preincubated at 37°C for 2 min. NADPH (1 mM) was added, and the mixture was incubated at 37°C for 30 min (when sulfaphenazole was used as the inhibitor) or 5 min (when ketoconazole was used as the inhibitor). The reaction was terminated with 200 μl of ACN (sulfaphenazole) or 200 μl of methanol (ketoconazole), the samples were kept on wet ice for 5 min, and then the samples were centrifuged for 15 min at 100,000g (4°C). The supernatant was collected and maintained at −20°C for 24 h. The following day, the samples were centrifuged for 10 min at 10,000g (4°C), for protein precipitation, and were analyzed through HPLC.
Western Blot Analyses
Immunoblot analysis of P450, STAT5b, and FOXO1 apoprotein levels was performed by using microsomes and nuclear extracts and cytosol from liver samples, respectively. For the preparation of nuclear extracts and cytosol, a nuclear and cytosolic extraction kit (Thermo Fisher Scientific, Waltham, MA) was used. The levels of phosphorylated CREB, JNK, Akt, and p70S6K were determined through Western blot analysis of total cellular proteins, which were extracted from the liver by using RIPA buffer supplemented with protease inhibitors, phenylmethylsulfonyl fluoride (10 μM), β-glycerophosphate (50 μM), and NaF (50 μM). Protein concentrations were determined with the bovine serum albumin assay (Thermo Fisher Scientific). Proteins were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with the following antibodies: rat polyclonal CYP3A1-, CYP3A2-, CYP2C11-, and CYP2D1-specific IgGs, mouse monoclonal total STAT5a/b-specific IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit monoclonal phosphorylated (Tyr694) STAT5b-specific IgG (Cell Signaling Technology, Danvers, MA). Rabbit polyclonal phosphorylated (Ser133) CREB-1-specific IgG, rabbit polyclonal total JNK2-specific IgG, mouse monoclonal phosphorylated (Thr183 and Tyr185) JNK-specific IgG (Santa Cruz Biotechnology), rabbit polyclonal phosphorylated (Thr389) p70S6K- and total p70S6K-specific IgGs (Cell Signaling Technology), rabbit polyclonal phosphorylated (Ser256) FOXO1- and total FOXO1-specific IgGs (Santa Cruz Biotechnology), and rabbit polyclonal phosphorylated (Ser473) Akt- and total Akt-specific IgGs (Santa Cruz Biotechnology) were also used. Secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) were used, and proteins were detected by using an enhanced chemiluminescence detection kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Immunoblotting with glyceraldehyde 3-phosphate dehydrogenase- or β-actin-specific antibody (Santa Cruz Biotechnology) and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody was used as a loading control.
Quantitative Real-Time Polymerase Chain Reaction Assays
For the isolation of total RNA from liver tissue and primary hepatocytes, the TRIzol reagent (Invitrogen) was used according to the manufacturer's protocol. The concentration of total RNA was determined with a spectrophotometric method. Quantitative, real-time, reverse transcription-polymerase chain reaction (PCR) assays were performed with cDNA generated from 1 μg of total RNA with a SuperScript II reverse transcriptase kit (Invitrogen). The sequences for the forward and reverse gene-specific primers used are shown in Table 1. SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was used for the real-time reactions, which were performed by using a C1000 Touch thermal cycler with a real-time detection system (Bio-Rad Laboratories, Hercules, CA). Relative mRNA expression levels were normalized to β-actin levels (QuantiTect primer assay; QIAGEN, Valencia, CA), and values were quantified by using the comparative threshold cycle method.
Hormone Level Determinations
Serum corticosterone concentrations were measured by using a corticosterone RIA kit (Coat-A-Count kit; Diagnostic Products, Los Angeles, CA). The detection limit was approximately 5.7 ng/ml, and the intra-assay coefficient of variation was 4.0%. GH serum levels were assessed with a rat growth hormone RIA kit (Millipore Corp., Billerica, MA). The detection limit was 0.5 ng/ml, and the intra-assay coefficient of variation was 10%. PRL serum levels were measured with a rat prolactin RIA kit (MP Biomedicals, Solon, OH), and the detection limit was 0.5 ng/ml. Serum thyroid hormone concentrations were measured with triiodothyronine, thyroxin, and thyroid-stimulating hormone kits (Dynatest, Brahms, Germany); the normal ranges were 80 to 200 ng/dl for triiodothyronine levels, 4.5 to 12 μg/dl for thyroxin levels, and 0.4 to 4 mg/ml for thyroid-stimulating hormone levels. Insulin levels were measured with a rat enzyme-linked immunosorbent assay kit (Mercodia, Uppsala, Sweden). The detection limit was 3.3 ng/ml, and the intra-assay coefficient of variation was 3.1%. Blood glucose levels were measured with a commercially available kit (Merck, Darmstadt, Germany), by using the glucose oxidase technique (Trinder, 1969).
Statistical Analyses
Data were analyzed by using one-way analysis of variance followed by multiple comparisons with Bonferroni's and Tukey's least honest significant difference methods, and results are presented as mean ± S.E. In all cases, p values of ≤0.05 were considered significant.
Results
Involvement of D2-Dopaminergic Receptor-Linked Pathways in CYP3A Regulation.
In vivo administration of the D2-dopaminergic receptor antagonist SULP resulted in down-regulation of hepatic CYP3A1/2 expression. CYP3A-dependent 6β-testosterone hydroxylation and CYP3A1/2 apoprotein and mRNA levels were detected at markedly lower levels in SULP-treated rats, compared with control rats (Fig. 1). The highly selective D2-dopaminergic receptor antagonist L-741,626 also repressed CYP3A1/2 expression at the mRNA, apoprotein, and activity levels (Fig. 1).
In contrast, in vitro experiments using primary hepatocyte cultures demonstrated that D2-dopaminergic receptor blockade with SULP markedly increased CYP3A1 and CYP3A2 mRNA levels (Fig. 2A). This increase was not mediated by the JNK-, cAMP/PKA-, or PI3K-related pathways, inasmuch as neither the JNK inhibitor SP600125, the PKA inhibitor H-89, nor the PI3K inhibitor wortmannin (WORT) prevented it (Fig. 2A). Incubation of primary hepatocytes with INS strongly suppressed CYP3A1/2 expression, and this suppressive effect was completely reversed by WORT (Fig. 2B).
Involvement of D2-Dopaminergic Receptor-Linked Pathways in CYP2C Regulation.
In vivo administration of SULP markedly suppressed CYP2C11 expression in the liver of rats, as assessed at the enzyme activity level (2α- and 16α-testosterone hydroxylation) and the apoprotein and mRNA levels (Fig. 3). L-741,626 also repressed CYP2C11 expression at the mRNA, apoprotein, and 2α- and 16α-testosterone hydroxylation levels (Fig. 3). However, SULP significantly increased CYP2C11 expression in vitro (Fig. 2A), an effect that was not prevented by SP600125, H-89, or WORT (Fig. 2A). As in the case of CYP3A, treatment of primary hepatocytes with INS markedly decreased CYP2C11 mRNA transcript levels, an effect that was completely blocked by WORT (Fig. 2B).
Involvement of D2-Dopaminergic Receptor-Linked Pathways in CYP2D Regulation.
Blockade of D2-dopaminergic receptors with SULP markedly decreased CYP2D-dependent 1′-bufuralol hydroxylation and CYP2D1 mRNA transcript and apoprotein levels, compared with control values (Fig. 4). L-741,626 also repressed CYP2D1/2 expression (Fig. 4). Similar to findings for CYP3A and CYP2C, treatment of primary hepatocytes with SULP resulted in up-regulation of CYP2D1 and CYP2D2 (Fig. 2A), and this up-regulation was not prevented by SP600125, H-89, or WORT (Fig. 2A). In contrast, INS strongly suppressed CYP2D1 and CYP2D2 expression, an effect that was completely blocked by WORT (Fig. 2B).
Positive and Negative Control of P450-Dependent Activities.
Incubation of liver microsomes with ketoconazole markedly suppressed CYP3A-catalyzed 6β-testosterone hydroxylation, compared with that in microsomes incubated with vehicle (Supplemental Fig. 1A). Sulfaphenazole also significantly suppressed 2α- and 16α-testosterone hydroxylation (Supplemental Fig. 1, B and C). In contrast, incubation of liver microsomes with SULP did not modify these activities (Supplemental Fig. 1, A–C).
Incubation of recombinant CYP3A1 with SULP increased 6β-testosterone hydroxylation levels, whereas SULP had no effect on recombinant CYP3A2-catalyzed activity (Supplemental Fig. 1D). The D2-dopaminergic receptor antagonist markedly repressed recombinant CYP2C11-catalyzed 2α- and 16α-testosterone hydroxylation (Supplemental Fig. 1, E and F), whereas SULP had no effect on recombinant CYP2D1- or recombinant CYP2D2-catalyzed bufuralol 1′-hydroxylation (Supplemental Fig. 1, G and H).
Assessment of Effects of D2-Dopaminergic Receptor Blockade on Hormone Levels.
Treatment of rats with SULP strongly suppressed plasma GH, triiodothyronine, thyroxin, and corticosterone concentrations (Table 2). In contrast, SULP markedly increased plasma PRL levels (Table 2). It is of importance to this study that the drug also increased plasma insulin levels, which was followed by reduced plasma glucose concentrations (Table 2).
Assessment of D2-Dopaminergic Receptor-Mediated Effects on PI3K/Akt/FOXO1 Pathway.
The D2-dopaminergic receptor antagonist SULP markedly increased the phosphorylation of Akt in the liver of rats (Fig. 5A). The phosphorylation of p70S6K, a downstream element in the PI3K/Akt pathway, was also strongly increased after SULP treatment (Fig. 5A). SULP strongly decreased the levels of phosphorylated FOXO1 in the nucleus of hepatocytes (Fig. 5B), whereas levels were markedly increased in the cytosol (Fig. 5C). It should be noted that SULP up-regulated eNOS and iNOS (mean ± S.E.: iNOS, control versus SULP, 1.0 ± 0.13- versus 1.8 ± 0.22-fold induction, n = 10, p < 0.01; eNOS, 1.0 ± 0.12- versus 2.0 ± 0.14-fold induction, n = 10, p < 0.01). L-741,626 had effects similar to those of SULP on the PI3K/Akt/FOXO1 signal transduction pathway (Fig. 6).
Assessment of D2-Dopaminergic Receptor-Mediated Effects on STAT5b Activation.
It is well established that GH and the GH pulse-activated transcription factor STAT5b (Holloway et al., 2006) play major roles in the regulation of several P450s (Waxman and Holloway, 2009). Treatment of rats with SULP strongly suppressed STAT5b activation, as assessed at the nuclear and cytosolic STAT5b phosphorylation (Tyr694) levels (Fig. 5, B and C). Similarly, L-741,626 markedly reduced STAT5b activation in the nucleus and cytoplasm, compared with control values (Fig. 6, B and C).
Assessment of D2-Dopaminergic Receptor-Mediated Effects on Nuclear Transcription Factors Involved in P450 Regulation.
Constitutive androstane receptor (CAR), pregnane X receptor (PXR), retinoic X receptor α (RXRα), and HNF1α mRNA levels were detected at markedly lower levels in the livers of SULP- and L-741,626-treated rats, compared with control rats (Fig. 7, A–D). No significant change in hepatic HNF4α expression was observed after SULP treatment, whereas L-741,626 suppressed expression (Fig. 7E). CREB phosphorylation, a downstream element in the cAMP/PKA signaling pathway, and JNK phosphorylation were suppressed by both SULP and L-741,626 (Figs. 5A and 6A).
In vitro experiments using primary hepatocyte cultures showed that treatment of the cells with SULP increased CAR mRNA transcript levels. This increase was profoundly mediated by the JNK-, cAMP/PKA-, and PI3K-linked signaling pathways, inasmuch as it was prevented by the corresponding inhibitors (i.e., SP600125, H-89, and WORT) (Fig. 7A). RXRα mRNA levels also were increased after treatment of the hepatocytes with SULP, and this increase was mediated mainly by the JNK-linked pathway (Fig. 7C). PXR, HNF1α, and HNF4α mRNA expression levels were not affected by direct exposure of hepatocytes to SULP (Fig. 7, B, D, and E).
Discussion
An increasing body of evidence suggests that central and peripheral catecholaminergic systems play an important role in the regulation of drug-metabolizing enzymes, including the P450s (Konstandi et al., 2004, 2005, 2006; Daskalopoulos et al., 2012). In accordance with this, the data in the present study clearly indicated a critical role for the dopaminergic pathways in P450 regulation. The investigation focused on the role of the D2-dopaminergic receptor-related pathways, targets of many therapeutically important drugs prescribed for treatment of several neurodegenerative and psychopathological disorders, including Parkinson's disease, depression, and psychosis (Craig and Lin, 1981; Bonci and Hopf, 2005; Kabbani et al., 2012). These drugs, acting as either D2-dopaminergic receptor agonists or antagonists, directly affect the dopamine receptor system by mimicking, blocking, or altering the sensitivity to dopamine, thus altering the functional output of dopaminergic systems (Cooper et al., 1996).
Pharmacological blockade of D2-dopaminergic receptors using SULP, a selective D2 receptor antagonist and typical antipsychotic drug, resulted in strong down-regulation of CYP3A1/2, CYP2C11, and CYP2D1 (but not CYP2D2). The involvement of D2-dopaminergic receptors in the aforementioned P450 regulation was confirmed by using the highly selective D2-dopaminergic receptor antagonist L-741,626. The SULP-induced down-regulation appeared to be indirect, inasmuch as treatment of primary hepatocytes with SULP had an opposite effect. We hypothesize that the SULP-induced down-regulation of these P450s is the outcome of combined effects of the drug on central and peripheral aminergic and hormonal systems, which overrides the drug's direct up-regulating effect on hepatocytes. In terms of CYP2C11, it is of interest to note that in vitro experiments with recombinant CYP2C11 revealed a direct down-regulating effect of the drug on this P450.
The role of insulin in D2 receptor-mediated P450 down-regulation appears to be crucial. This hypothesis is based on the notion that release of insulin, which exerts negative regulatory control on several P450s (Yoshida et al., 1996; Woodcroft and Novak, 1997, 1999) is under dopaminergic control through pancreatic β-cell D2 receptors (Rubí et al., 2005) (Scheme 1).
We showed previously that blockade of D2-dopaminergic receptors stimulated the release of insulin (Konstandi et al., 2008). The current in vitro experiments with primary hepatocytes clearly indicated that insulin down-regulated CYP3A1/2, CYP2C11, and CYP2D1 through the PI3K/Akt signaling pathway, inasmuch as pretreatment of hepatocytes with wortmannin, a PI3K inhibitor, completely reversed the suppressive effect of insulin. Further investigations indicated that treatment of rats with SULP increased plasma insulin levels, followed by activation of the PI3K/Akt signaling pathway, an event associated with phosphorylation of FOXO1 in the nucleus, subsequent translocation of FOXO1 into the cytoplasm, and termination of P450 transcription (Kodama et al., 2004; Kim and Novak, 2007). The Akt/p70S6K pathway does not seem to contribute to the SULP-induced suppressive effects on the aforementioned P450s, because activation of p70S6K is connected with increased gene transcription (Kim and Novak, 2007). The activation of JNK was markedly weaker in the liver of SULP-treated rats. Taking into account the accumulating evidence that Akt- and JNK-linked pathways have opposing effects on FOXO, with Akt preventing FOXO nuclear localization and inhibiting its activity but JNK increasing FOXO activity by promoting import into the nucleus (Brunet et al., 1999; Essers et al., 2004; Wang et al., 2005), we hypothesize that the SULP-induced down-regulating effects on P450s involving FOXO1 are the result of the drug's combined effects on the Akt and JNK signaling pathways.
FOXO1 is part of a complex cross-talk mechanism that includes the nuclear receptors CAR and PXR (Kodama et al., 2004). These nuclear receptors, along with RXR, HNF1α, and HNF4α (Liddle et al., 1998; Wiwi and Waxman, 2004), are known to regulate the most important hepatic P450 genes, including members of the CYP3A and CYP2C subfamilies (Dvorak and Pavek, 2010). SULP also down-regulated the expression of CAR, PXR, RXRα, and HNF1α, which indicates that the drug's suppressive effects on CYP3A1/2 and CYP2C11 might be mediated by these nuclear factors.
Moreover, SULP up-regulated both eNOS and iNOS in the liver. It is well established that both of these enzymes exert negative regulatory control on several P450s (Hara and Adachi, 2002). Possible contributions of eNOS and iNOS to the down-regulation of CYP3A, CYP2C, and CYP2D cannot be excluded. Finally, SULP-induced suppression of hepatic CREB phosphorylation may contribute to down-regulation of the aforementioned P450s, because it is well established that the cAMP/PKA/CREB signaling pathway controls the expression of PXR and HNF1α (Soutoglou et al., 2000; Kodama et al., 2007).
It is possible that both GH and PRL play roles in the down-regulation of these P450s by SULP. SULP reduced plasma GH levels, with subsequent reduced phosphorylation of STAT5b, which is the major GH pulse-activated transcription factor and is involved in the regulation of various P450s in the liver (Waxman and O'Connor, 2006; Waxman and Holloway, 2009). In contrast, PRL levels were increased by SULP, and it is well established that PRL exerts down-regulating control on several P450 isoforms (Yamazoe et al., 1987; Fitzgerald and Dinan, 2008).
The SULP-induced perturbation of the aforementioned signaling pathways might also be a result of off-target drug effects, including interactions of SULP with G protein-coupled receptors, a hypothesis that likely merits further investigation (Kristiansen, 2004; Theodoropoulou et al., 2008). In conclusion, the present data indicate several possible mechanisms that might play roles in the down-regulation of CYP3A, CYP2C, and CYP2D by SULP. It is apparent that the in vivo effects of SULP on P450s represent the result of the drug's combined effects mainly on the GH/STAT5b, PRL, cAMP/PKA/CREB, PI3K/Akt/FOXO1, JNK, and eNOS/iNOS signaling pathways. The use of the highly selective D2-dopaminergic receptor antagonist L-741,626 confirmed the involvement of D2 receptor-linked pathways in the modulation of the aforementioned signaling pathways, which mediate the down-regulation of CYP3A, CYP2C, and CYP2D. However, more-detailed studies are required to determine their respective roles in down-regulation.
On the basis of the data described above and previous reports (Konstandi et al., 2008), a question arises regarding the extent to which pharmacotherapy with D2-dopaminergic receptor antagonists or agonists may influence the expression of CYP3A, CYP2C, and CYP2D and thus the metabolism of the majority of prescribed drugs. The possibility that drugs acting as D2-dopaminergic receptor antagonists or agonists might modify the function of major signal transduction pathways involved in the regulation of P450s with crucial importance in the metabolism of numerous prescribed drugs, toxicants, and carcinogens, thus drastically affecting the outcomes of pharmacotherapy, drug toxicity, and carcinogenicity, leads to some very interesting prospects. For instance, several previous studies reported lower relative risks of cancer incidence among patients with psychosis who were receiving drug therapy, usually including D2-dopaminergic receptor antagonists (Rassidakis et al., 1973; Rice, 1979; Craig and Lin, 1981; Mortensen, 1989). Could this finding be attributed to reduced expression of CYP3A, CYP2C, and CYP2D? Or should the strong SULP-mediated down-regulation of CYP2D be taken into account in assessments of the effectiveness of pharmacotherapy and possible drug-drug interactions among patients who receive antidepressant or antipsychotic therapy, given the fact that the majority of these drugs are metabolized by CYP2D (Wójcikowski and Daniel, 2009)? Furthermore, it is worth noting that the present findings shed more light on the regulation of CYP2D, which has been considered to be resistant to direct hormonal regulation and is known as “uninducible” P450.
Authorship Contributions
Participated in research design: Konstandi.
Conducted experiments: Daskalopoulos, Malliou, and Konstandi.
Contributed new reagents or analytic tools: Lang, Marselos, and Konstandi.
Performed data analysis: Daskalopoulos and Konstandi.
Wrote or contributed to the writing of the manuscript: Daskalopoulos, Lang, Marselos, and Konstandi.
Acknowledgments
We thank Drs. Ronald Wolf and Susumu Imaoka for their kind offer of the rat CYP3A1-, CYP3A2-, CYP2C11-, and CYP2D1-specific antibodies. We thank Dr. Mats Heinderstadt for his valuable support in the HPLC-based determination of P450-dependent activities. We are thankful to Olga Tsoumani for her valuable technical assistance.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the European Social Fund and Greek Ministry of Development Reinforcement Programme of Human Research Manpower [Grant 03ΕΔ/957] and by the European Union European Regional Development Fund and Greek national funds through the Thessaly-Mainland Greece and Epirus 2007–2013 Operational Program of the National Strategic Reference Framework [Grant 346985/80753].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- P450
- cytochrome P450
- ACN
- acetonitrile
- CAR
- constitutive androstane receptor
- HPLC
- high-performance liquid chromatography
- PKA
- protein kinase A
- PCR
- polymerase chain reaction
- RIA
- radioimmunoassay
- eNOS
- endothelial nitric oxide synthase
- iNOS
- inducible nitric oxide synthase
- CREB
- cAMP-response element-binding protein
- GH
- growth hormone
- HNF
- hepatocyte nuclear factor
- INS
- insulin
- JNK
- c-Jun N-terminal kinase
- PI3K
- phosphatidylinositol 3-kinase
- PRL
- prolactin
- PXR
- pregnane X receptor
- RXR
- retinoic X receptor
- SULP
- sulpiride
- WORT
- wortmannin
- H-89
- 5-isoquinolinesulfonamide
- SP600125
- anthra[1,9-cd]pyrazol-6(2H)-one
- L-741,626
- 4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol.
- Received March 9, 2012.
- Accepted July 6, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics