cAMP Mediated Upregulation of CYP2A5 in Mouse Hepatocytes Pirkko Viitala, Katja Posti, Aija Lindfors, Olavi Pelkonen, and Hannu Raunio 1 Department of Pharmacology and Toxicology, University of Oulu, POB 5000, FIN-90014 Oulu, Finland Received November 21, 2000 CYP2A5 is induced by a large number of chemicals including some cAMP modifiers. In a primary hepato- cyte model, stimulation of the cAMP signal transduc- tion pathway by glucagon and isoproterenol, acting via specific G-protein coupled plasma membrane re- ceptors, produced up to 17-fold increases in the marker activity of CYP2A5, coumarin 7-hydroxylase. In contrast, glucagon and isoproterenol caused no sig- nificant effects on two other major CYP forms, CYP2B10 and CYP1A1/2. Phenobarbital (PB) elicited a 3-fold increase in CYP2A5 expression (catalytic activ- ity and mRNA), while the cAMP and protein kinase A (PKA) stimulators dibutyryl-cAMP, forskolin and Sp-cAMPs caused up to 18-fold increases in the amount of CYP2A5 mRNA. Coadministration of PB and cAMP/ PKA stimulating agents produced an additive inducing effect. The expression of CYP2A5, but not CYP2B10 or CYP1A1/2, in DBA/2 mice displayed a marked circadian rhythm, the level of expression being highest in the evening. These results suggest that among xenobiotic metabolizing CYP enzymes, CYP2A5 is uniquely upregu- lated by cAMP, possibly having the physiological func- tion of priming the olfactory and digestive systems for nocturnal feeding. © 2001 Academic Press Key Words: CYP2A5; coumarin 7-hydroxylase, COH; cAMP mediated upregulation; mouse hepatocytes; phenobarbital, PB; protein kinase A, PKA; tyrosine aminotransferase, TAT. The cytochrome P450 (CYP) system has evolved to comprise a multitude of enzymes that metabolize vir- tually all xenobiotics, providing the organism protec- tion from foreign compounds. As an integral part of this protection mechanism, several CYP enzymes are inducible in response to xenobiotic exposure. Intracellular signal transduction pathways and phosphorylation events participate in CYP induction. For example, the Ah receptor-mediated induction of CYP1A1 by polycyclic aromatic hydrocarbons requires phosphorylation catalyzed by different protein kinases, especially protein kinase C (1). The cAMP signaling pathway plays a crucial role in the regulation of CYP enzymes catalyzing steroid hormone biosynthesis (2). Modulation of liver-specific gene expression has been correlated with increases in cAMP levels through the modulated expression of G-proteins, adenylyl cyclase, and protein kinase A (PKA) (3). The expression of several hepatic CYP forms has been shown to be neg- atively regulated by cAMP/PKA pathways. Activation of cAMP/PKA results in a reduction of PB induced CYP2B1/2 and CYP3A1 in rat primary hepatocytes (4), and a similar down-regulation occurs in PB induced hamster CYP3A31 (5) and rainbow trout CYP1A1 (6). In contrast to these observations, we reported previ- ously that enhancement of cAMP pathway results in a clear upregulation of mouse hepatocyte CYP2A5, and that this upregulation was additive upon coadminis- tration with PB (7). Phenobarbital (PB) is representative of a large group of structurally unrelated inducers and it preferentially induces CYP forms in the CYP1A, CYP2A, CYP2B, CYP2C and CYP3A subfamilies (8, 9).The mechanisms of CYP induction by PB have been obscure for a long time, but significant progress has recently been made in this field (10). PB increases CYP2A5 expression by a transcriptional activation, whereas pyrazole acts by stabilizing CYP2A5 mRNA (11, 12). The substrate specificity of CYP2A5 has been insuf- ficiently surveyed. In addition to coumarin, very few substances have been equivocally shown to be sub- strates of CYP2A5. However, the homologous human enzyme, CYP2A6, is able to metabolize a number of (naturally-occurring) procarcinogens and alkaloids, in- cluding nicotine and cotinine, but only a few pharma- ceuticals (13). This range of substrates implicates that the principal role of CYP2A5 may be in the metabolism of dietary components. The aim of the present study was to further elucidate the mechanisms involved in the cAMP-mediated CYP2A5 induction by employing various modifiers of 1 Current address: Department of Pharmacology and Toxicology, University of Kuopio, POB 1627, FIN-70211 Kuopio, Finland. Biochemical and Biophysical Research Communications 280, 761–767 (2001) doi:10.1006/bbrc.2000.4195, available online at http://www.idealibrary.com on 761 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.