Abstract
We have suggested that adenine-related compounds are allosteric inhibitors of UGT in rat liver microsomes (RLM) treated with detergent. To clarify whether the same occurs with a pore-forming peptide, alamethicin, the effects of adenine-related compounds on 4-metylumbelliferone (4-MU) glucuronidation were examined using RLM and human liver microsomes (HLM). ATP inhibited 4-MU glucuronidation when polyoxyethylene cetyl alcohol ether (Brij-58)-treated RLM were used (IC50 = approximately 70 μM). However, alamethicin-treated RLM exhibited a lower susceptibility (IC50 = approximately 460 μM) than Brij-58-treated RLM. A similar phenomenon was observed when pooled HLM were used. Then, the endogenous ATP content of RLM was determined in the presence and absence of alamethicin or detergent, and although no ATP remained in the microsomal pellets after Brij-58 treatment, more than half of the microsomal ATP remained even after treatment with alamethicin. Furthermore, the Vmax in the absence of an adenine-related compound was approximately three times higher in Brij-58-treated than in alamethicin-treated RLM. The difference in the inhibitory potency observed was due to the difference in remaining endogenous ATP and the accessibility of exogenous ATP to the luminal side of the endoplasmic reticulum (ER), where the active site of UDP-glucuronosyltransferase (UGT) is located. Gefitinib (Iressa), a protein tyrosine kinase inhibitor, markedly inhibited human UGT1A9 activity. It is interesting to note that AMP antagonized Gefitinib-provoked inhibition of UGT1A9, and ATP exhibited an additive inhibitory effect at a lower concentration. Therefore, Gefitinib inhibits UGT1A9 at the common ATP-binding site shared with ATP and AMP. Releasing adenine nucleotide from the ER is suggested to be one of the mechanisms that explain the “latency” of UGT.
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science [Research Nos. 19590147, 21590164].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- UGT
- UDP-glucuronosyltransferase
- GlcUA
- UDP-glucuronic acid
- ER
- endoplasmic reticulum
- GRP
- glucose-regulated protein
- 4-MU
- 4-metylumbelliferone
- RLM
- rat liver microsomes
- HLM
- human liver microsomes
- EGFR
- epidermal growth factor receptor
- 4-MUG
- 4-methylumbelliferyl-β-d-glucuronide
- Brij-58
- polyoxyethylene cetyl alcohol ether
- PDI
- protein disulfide isomerase
- 3D
- three-dimensional.
- Received May 22, 2012.
- Accepted July 30, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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