Abstract
P-glycoprotein (Pgp) is expressed in various normal tissues and plays an important role in drug absorption and disposition. In addition, it is supposed that alterations in the expression levels of Pgp are involved in the inter- and intraindividual variability of pharmacokinetics of many drugs. Since pharmacokinetic properties of various drugs are altered in patients with thyroid disorders, we examined the expression of Pgp and mdr1a/1b mRNA in the kidney, liver, jejunum, and ileum from euthyroid and hyperthyroid rats. Western blot analysis revealed that Pgp expression was markedly increased in the kidney and liver of hyperthyroid rats. In contrast, it was slightly increased in the jejunum and ileum. mdr1a/1b mRNA levels were significantly increased in the kidney of hyperthyroid rats. However, they were not increased in the liver as well as in the jejunum and ileum of hyperthyroid rats. Expression levels of bile salt export pump and mdr2 mRNA were also unchanged in hyperthyroid rat liver. Taken together, these findings suggest that thyroid hormone induces Pgp expression in a tissue-selective manner, and that the modulation of mdr1a/1b mRNA expression in the hyperthyroid state varies among tissues.
P-glycoprotein (Pgp) is expressed in various tissues such as brain, liver, kidney, and intestine (Cordon-Cardo et al., 1990; Brady et al., 2002) and plays an important role in defining the pharmacokinetics of many drugs. Pgp functions as a drug efflux pump and exports hydrophobic, bulky drugs such as anticancer agents, cardiac glycosides, β-blockers, calcium channel blockers, and immunosuppressants. Since Pgp has a broad substrate recognition, the concomitant administration of drugs often causes drug interactions by inhibiting Pgp-mediated transport (Yu, 1999). For example, inhibition of digoxin transport in cultured epithelial cell lines expressing Pgp by various drugs such as quinidine (Tanigawara et al., 1992; Fromm et al., 1999), verapamil (Tanigawara et al., 1992), and cyclosporin A (Okamura et al., 1993) has been reported. We have also demonstrated that the renal clearance of digoxin was decreased in patients receiving a concomitant administration of clarithromycin and, accordingly, the plasma concentration of digoxin was increased (Wakasugi et al., 1998). The mechanism of this interaction was explained by the inhibition of Pgp-mediated tubular secretion of digoxin. On the other hand, recent studies have demonstrated that changes in the expression levels of Pgp affect the pharmacokinetic properties of Pgp substrates. Greiner et al. (1999) reported that rifampin administration induced Pgp expression in the small intestine and reduced the plasma concentration of orally administered digoxin, suggesting that alterations in the expression levels of Pgp are closely involved in the inter- and intraindividual variability of pharmacokinetics of Pgp substrates.
Thyroid hormone is secreted from the thyroid gland to maintain normal growth and development, normal body temperature, and normal energy levels. Most of its effects appear to be mediated by the activation of nuclear receptors that lead to increased expression of mRNA and subsequent protein synthesis. Disorders of the thyroid gland are among the most common endocrine disorders, and are known as hyperthyroidism and hypothyroidism. It was reported that pharmacokinetic properties of various drugs were altered in patients with thyroid disorders (Shenfield, 1981; O'Connor and Feely, 1987). As for Pgp substrates, plasma concentrations of digoxin were decreased in patients with hyperthyroidism as compared with euthyroid patients. Such an altered pharmacokinetics of digoxin in hyperthyroid patients has been explained by an increase in renal clearance (Lawrence et al., 1977) and volume of distribution (Shenfield et al., 1977; Shenfield, 1981). The reason for the increased renal clearance of digoxin is considered to be a facilitation of tubular secretion (Bonelli et al., 1978); however, the mechanisms underlying the altered pharmacokinetics of digoxin in thyroid disease have not been fully elucidated.
Previous studies have shown that thyroid hormone regulates the expression levels of various membrane transporters such as the fructose transporter GLUT5 (Matosin-Matekalo et al., 1999), the peptide transporter PEPT1 (Ashida et al., 2002), Na+/K+-ATPase (Giannella et al., 1993), and the Na+/H+ exchanger NHE1 (Li et al., 2002). Therefore, we hypothesized that the alteration in the plasma concentration of digoxin in patients with thyroid disorders might be due to changes in Pgp expression by thyroid hormone. In the present study, to elucidate the influence of a hyperthyroid state on the expression of Pgp in various tissues, we investigated Pgp and mdr1a/1b mRNA levels in liver, kidney, jejunum, and ileum from euthyroid and hyperthyroid rats.
Materials and Methods
Materials.l-Thyroxine (T4) was purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal antibody C219 and anti-villin polyclonal antibody were obtained from CIS Bio International (Gif-sur-Yvette, France) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. All other chemicals used were of the highest purity available.
Animals. The animal experiments were performed in accordance with the Guideline for Animal Experiments of Kyoto University. Eight-week-old male Wistar rats were housed in a temperature- and humidity-controlled room and fed rat chow ad libitum. Hyperthyroidism was induced by adding T4 (12 mg/l) to the drinking water for 21 days as previously described (Ashida et al., 2004). After treatment, the kidney, liver, jejunum, and ileum were excised. Blood was also collected for measurement of plasma level of T3. Plasma T3 levels in euthyroid and hyperthyroid rats were measured by an Enzyme Immuno Assay method (IMx; Dainabot, Tokyo, Japan). Plasma T3 levels in euthyroid and hyperthyroid rats were 0.32 ± 0.02 and 2.40 ± 0.18 ng/ml, respectively (mean ± S.E., n = 6). In addition, hyperthyroid rats lost an average of 0.055 kg in response to T4 treatment (data not shown).
Western Blot Analysis. Isolation of crude plasma membrane fractions from each tissue and Western blot analysis were performed as described previously (Ogihara et al., 1996). Monoclonal antibody C219 and anti-villin polyclonal antibody were used as primary antibodies.
RNA Isolation, Semiquantitative Reverse Transcription-Polymerase Chain Reaction (PCR), and Competitive PCR. RNA isolation, reverse transcription, and competitive PCR procedures were performed as described previously (Masuda et al., 2000) with some modifications. The specific primer sets (5 μM) used were as follows: for rat mdr1a primers, 5′-GATGGAATTGATAATGTGGACA-3′ and 5′-AAGGATCAGGAACAATAAA-3′; for rat mdr1b primers, 5′-GAAATAATGCTTATGAATCCCAAA G-3′ and 5′-GGTTTCATGGTCGTCGTCTCTTGA-3′ (Zhang et al., 1996); for rat bile salt export pump (bsep) primers, 5′-GAGGTTACTTAATAGCCTACG-3′ and 5′-CATCTATCATCACAGTTCCC-3′; and for rat mdr2 primers, 5′-AAGAATTTGAAGTTGAGCTAAGTGA-3′ and 5′-TGGTTTCCACATCCAGC CTAT-3′. For the detection of mdr1a and mdr1b, semilogarithmic serial dilutions of mimic competitor DNA from 50 to 0.01 amol were added.
Statistical Analysis. Data were analyzed statistically using the nonpaired t test. Probability values of less than 5% were considered significant.
Results and Discussion
To investigate the effect of thyroid hormone on the expression of Pgp, we examined the Pgp expression by Western blotting. As shown in Fig. 1, the expression of Pgp was remarkably increased in crude membranes of hyperthyroid rat kidney and liver (1.9-fold and 2.3-fold, respectively) as compared with those of euthyroid rats. In contrast, hyperthyroidism caused a slight increase in the expression of Pgp in rat jejunum and ileum (1.1-fold and 1.4-fold, respectively). These results suggest that the differences in the behavior of thyroid hormone in each tissue are involved in tissue selectivity of Pgp induction. Similarly, it was reported that MDR1 mRNA was elevated in a dexamethasone-treated human hepatoma cell line but not in a nonhepatoma cell line, suggesting that the hormonal regulation of mdr gene expression is gene- and cell type-specific (Zhao et al., 1993). Their report supports our results suggesting that the regulation of Pgp expression by thyroid hormone was possibly tissue-specific.
We then examined the expression of mdr1a and mdr1b mRNA using competitive PCR in each tissue. As shown in Fig. 2, the expression of both mdr1a and mdr1b mRNA was significantly increased in hyperthyroid rat kidney as compared with the control. Since the action of thyroid hormone appears to be mediated by the activation of nuclear receptors, which leads to increased levels of mRNA and subsequent protein synthesis (Ribeiro et al., 1995), it is likely that the induction of Pgp expression in the hyperthyroid kidney is mediated by the increased transcription of mdr1a/1b mRNA. On the other hand, hyperthyroidism caused a significant decrease in the expression of mdr1b mRNA in the liver but did not affect the expression of mdr1a mRNA. Since it was reported that C219 antibody reacted with not only Pgp but bsep and mdr2 (Childs et al., 1995) and bsep and mdr2 are expressed in the liver, we examined whether the immunoreactive protein bands obtained using C219 antibody reflected the induction of these transporters in hyperthyroid liver. As shown in Fig. 3, however, the expression of bsep and mdr2 mRNA did not change in hyperthyroid rat liver. Although the precise mechanism is not clear at this stage, thyroid hormone may regulate Pgp expression via nontranscriptional control in the liver. Further studies are needed to elucidate the regulation of Pgp and MDR1 by thyroid hormone in the liver.
Siegmund et al. (2002) examined the effect of levothyroxine administration on human intestinal Pgp expression. They demonstrated that duodenal MDR1 mRNA expression and immunoreactive Pgp were increased by levothyroxine administration, although the increase in MDR1 mRNA was not significant. However, changes in the expression of Pgp were not associated with major alterations in the pharmacokinetics of talinolol, a substrate of Pgp. In addition, the effect of thyroid hormone on the expression of Pgp in other tissues was not elucidated. In the present study, we observed a significant increase in Pgp expression in the kidney and liver (Fig. 2) and a slight increase in the intestine (Fig. 4) in hyperthyroid rats. Our results suggest that the decrease in the serum concentration of digoxin in hyperthyroidism is attributable to the increased expression of Pgp in the kidney and/or liver. Further studies are needed to assess the pharmacokinetics of Pgp substrates in hyperthyroid rats.
For obvious ethical reasons, Siegmund et al. (2002) administered levothyroxine in doses that do not cause thyrotoxicosis. In the present study, the expression of Pgp was only slightly increased in rat intestinal tissues, although we used hyperthyroid rats. It is known that the gene encoding Pgp differs between humans and rats. Pgp is encoded by MDR1 in humans and by mdr1a and mdr1b in rats. Therefore, it is supposed that the mechanisms by which thyroid hormone induces Pgp expression differ between species, and Pgp expression in human intestinal tissues may increase dramatically under thyrotoxicosis.
In conclusion, thyroid hormone induces Pgp expression in a tissue-selective manner. In addition, the modulation of mdr1a/1b mRNA expression in the hyperthyroid state varied among tissues. These results provide useful information for elucidating the drug interaction and pharmacokinetic variability in thyrotoxicosis.
Footnotes
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This work was supported in part by 21st Century COE Program “Knowledge Information Infrastructure for Genome Science,” by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by The Nakatomi Foundation. N.N. is supported as a Teaching Assistant by 21st Century Center of Excellence (COE) Program “Knowledge Information Infrastructure for Genome Science.”
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.004770.
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ABBREVIATIONS: Pgp, P-glycoprotein; T4, l-thyroxine; bsep, bile salt export pump; PCR, polymerase chain reaction.
- Received March 19, 2005.
- Accepted August 3, 2005.
- The American Society for Pharmacology and Experimental Therapeutics