©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cAMP-associated Inhibition of Phenobarbital-inducible Cytochrome P450 Gene Expression in Primary Rat Hepatocyte Cultures (*)

Jaspreet S. Sidhu , Curtis J. Omiecinski (§)

From the (1) Department of Environmental Health, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effects of elevated intracellular cyclic adenosine monophosphate (cAMP) in regulating phenobarbital (PB)-inducible gene expression in primary rat hepatocyte cultures were investigated. Cells were exposed to various concentrations (0.1-100 µM) of cAMP analogs and/or activators of intracellular cAMP-dependent pathways. Effects of these treatments were assessed either using a 1-h pulse prior to PB (100 µM) exposure or in conjunction with PB during a 24-h exposure period. PB-inducible responses were measured in hepatocytes by hybridization to cytochrome P450 (CYP) CYP2B1, CYP2B2, and CYP3A1 mRNAs. The cAMP analogs, 8-bromo-cAMP, 8-(4-chlorophenylthio)-cAMP, dibutyryl cAMP,and (S)-5,6-DCl-cBiMPS ((S)-5,6-dichloro-1--D-ribofuranosylbenzimidazole - 3 ` ,5 ` - monophosphorothioate), and the activators of adenylate cyclase, forskolin and glucagon, dramatically inhibited PB-mediated induction of CYP2B1 and CYP2B2 in a concentration-dependent manner. A similar inhibition of PB-induced CYP3A1 mRNA levels was effected by the cAMP analogs and glucagon. The phosphodiesterase inhibitors isobutylmethylxanthine and RO 201724 potentiated the cAMP responses. Increasing the concentration of PB (0.05-1.00 mM) did not alleviate the cAMP-mediated repression. A requirement for protein kinase A (PKA) was demonstrated by the use of (S)-cAMPS, a highly specific activator of PKA, whereas the inactive diastereoisomer, (R)-cAMPS, was ineffective in modulating PB induction. The response to cAMP was specific since elevated intracellular cAMP levels did not perturb -naphtholflavone-mediated induction of CYP1A1, CYP1A2, microsomal epoxide hydrolase, or dexamethasone-mediated induction of CYP3A1 gene expression. Nor did elevated intracellular cAMP modulate the liver-selective albumin gene expression levels. The results of the present study demonstrated striking inhibition of PB-mediated CYP gene induction by cAMP and PKA activators, indicating a negative regulatory role for the cAMP signal transduction pathway on PB gene induction.


INTRODUCTION

In the liver, as with other organs, a battery of extracellular signals regulate various intracellular processes via a complex cascade of receptors, transducers, and second messengers (1, 2) . The most prolific of these second messengers, cyclic nucleotides (adenosine 2`,3`-cyclic monophosphate, cAMP, and guanosine 3`,5`-cyclic monophosphate (cGMP)), remain the best characterized (3, 4) . The resulting activation of cyclic nucleotide-dependent protein kinases (PK)() underlies the basis for many effects of cyclic nucleotides on cellular function (5) .

The liver is considered the major organ involved in the biotransformation of a myriad of structurally and chemically diverse compounds of both endogenous and exogenous nature (6) . A multigene family (7, 8) of hemeproteins, the cytochromes P450 (CYP), is the principal enzyme system catalyzing oxidative biotransformation reactions. Of the 12 gene families characterized thus far in mammals (8), certain members are inducible in the presence of specific agents (6-9). While the mechanisms involved in the regulation of various constitutive and inducible CYP genes have been well characterized (8, 9) , relatively little is known regarding the induction process mediated by a major prototypic drug, phenobarbital (PB). PB and other PB-like agonists are known to exhibit a number of pleiotropic effects in liver, including gene induction (10) , tumor promotion (11) , and perturbation of gap-junctional intercellular communication (12) . With respect to induction, PB agonists in vivo induce CYP genes at the transcriptional level (8, 10) . Several recent studies have identified a critical role for specific signal transduction pathways in the control of dioxin-inducible CYP gene expression. Notably, protein kinase C-mediated phosphorylation (13, 14) appears to be a determinant in the regulation of dioxin-inducible CYP gene expression. Similar signal transduction pathways have not been established with respect to the regulation of PB-inducible CYP genes.

A number of early in vivo studies reported that biotransformation activity in rat liver was depressed by catecholamines, e.g. epinephrine (15, 16) . Subsequent reports also suggested that P450-mediated biotransformation activities associated with PB-inducible isozymes were depressed by agents that elevated cAMP levels in liver (17) . However, these data are difficult to interpret since the animals were treated with chronic and high doses of the respective agents (15, 16, 18) and the resulting activities that were monitored were either only partially (15, 16, 18) or indirectly altered (19) . In addition, certain studies specifically monitored cAMP modulation of only constitutive biotransformation activity (15, 16, 19, 20, 21) . Therefore, potential concerns with the interpretation of these in vivo studies include the nonspecific nature of the end points being ascertained and whether the injected cAMP analogs were effecting hepatic processes directly or through an indirect consequence of other hormonal interactions.

In vivo, phenobarbital has been shown to stimulate phosphorylation of acidic nuclear proteins (22) . Other investigators concluded that an early event following the in vivo administration of prototypic inducers such as PB was a significant activation of cAMP and PKA (23, 24, 25) . These results appeared to imply that cAMP and resulting PKA-mediated phosphorylation events might be involved in PB-induced physiological alterations and perhaps gene transcriptional activation. In general, cAMP-mediated induction of other liver-selective genes, e.g. tyrosine aminotransferase (TAT), involves activation via PKA-mediated phosphorylation of specific nuclear transcription factors (26, 27, 28) .

More recent studies in this line have focused primarily on post-translational modulation of preexisting pools of PB-induced P450s by cAMP analogs. For example, inhibition by cAMP/PKA of functional purified P450 activity (29) , or P450-associated activities in freshly isolated hepatocytes (30, 31, 32) has been reported. In addition, Banhegyi et al.(33) and Berry and Skett (34) demonstrated that elevated intracellular cAMP resulted in decreased aminopyrine oxidation, p-nitrophenol conjugation, and P450-mediated steroid metabolism in both freshly isolated hepatocytes and short term cultures. Thus, the general consensus of these latter investigations implies that post-translational modification of certain P450 proteins by PKA-mediated phosphorylation may serve as an intracellular control mechanism regulating biotransformation activity (29, 31) . While cAMP has been associated with the induction of adrenal P450 11-, 17-, and 21-hydroxylase (CYP11B1, 17, and 21) genes (35, 36) , very little is known regarding its potential control of other CYP genes.

Further delineation of molecular mechanisms involved in the regulation of PB-inducible CYP genes has thus far been hampered by difficulties associated with the maintenance of the prototypic induction response in vitro. Recently, we have established a primary rat hepatocyte culture system which faithfully reproduces the PB induction response observed in vivo(37, 38, 39) . In this study we used this system to examine the interactions of cAMP-dependent processes on PB induction. We report that elevated intracellular cAMP concentrations results in a dramatic inhibition of PB-mediated induction of CYP2B1, CYP2B2 and CYP3A1 gene expression.


MATERIALS AND METHODS

Cell Culture Materials

All cell culture media were obtained from Life Technologies, Inc. Matrigel and ITS+ (insulin, transferrin, selenium, bovine serum albumin, and linoleic acid) were obtained from Collaborative Biomedical Products (Bedford, MA). Tissue-culture treated plastic dishes were obtained from Nunc (Naperville, IL). Forskolin, 8-bromo-cAMP, N,O-dibutyryl cAMP, 8-(4-chlorophenylthio)-cAMP, 8-bromo-cGMP, dibutyryl cGMP, and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma. (S)-5,6-DCl-cBiMPS and RO 201724 were obtained from Biomol (Plymouth Meeting, PA). (R)-cAMPS and (S)-cAMPS were obtained from Research Biochemicals International (Natick, MA). -Naphthoflavone (NF) was obtained from Aldrich. Collagenase (type 1) was obtained from Worthington. Type I collagen, dexamethasone, and glucagon were obtained from Sigma, as were all other unspecified chemicals (of the highest grade possible). The non-isotopic cAMP ELISA kit is in a prototypic stage of development and was generously provided by Life Technologies, Inc.

Isolation and Culture of Hepatocytes

Rat hepatocytes were isolated by a modification of the two-step collagenase perfusion in situ(40) and cultured with a modification of the protocol described previously (37) . Briefly, cells were isolated from non-induced rat liver and resuspended in serum-free Williams' E medium as described previously (37) . The dexamethasone concentration, however, was reduced to 100 nM during the isolation and attachment period (38, 39) . Hepatocytes were plated at 1 10 cells/ml in 3.5 ml of complete Williams' E medium on 60-mm tissue culture-treated plastic dishes coated with collagen type 1 (37). Experimental observations were repeated with uncoated tissue-culture treated plastic dishes as recently reported (38) . No differences in treatment responsiveness were observed between collagen-coated or uncoated dishes. Unless otherwise stated, after the attachment period of 3 h, the dexamethasone concentration was reduced to 25 nM for the subsequent culture period (38, 39) . Medium changes were conducted thereafter on a daily basis.

Matrigel Overlay

A dilute concentration (233 µg/ml, final concentration) of ECM (Matrigel) was added (38, 39) as an overlay at 4 h after plating following initial dilution of ECM to a concentration of 5 mg/ml (37) with cell culture medium.

Gene Induction Treatments

Glucagon (0.2 mg/ml) and all cAMP analogs (100 mM) examined were dissolved in tissue-culture grade water as stock solutions and stored as aliquots at -20 °C. Forskolin, IBMX, and RO 201724 were dissolved in MeSO as stock solutions (100 mM, 500 mM, and 500 mM, respectively) and also stored at -20 °C. Cells were cultured for 48 h prior to addition of drugs. Cells were treated with the various activators and analogs of cAMP/PKA for 60 min prior to the addition of PB (0.9% saline). Treatment with cAMP was either discontinued at this point (60-min pulse) or continued in the absence or presence of PB. Unless otherwise stated the PB concentration was 100 µM. Where indicated, similar treatments were conducted with -naphthoflavone (NF). NF was added (22 µM) in MeSO (final concentration of MeSO was 0.05% v/v). Dexamethasone was added (1 µM) in absolute ethanol (final concentration of ethanol was 0.001% v/v). Unless otherwise stated, all inducer treatments were conducted for 24 h, at which point total RNA was isolated. Representative data are shown from multiple studies.

Slot-blot Analysis

Total RNA was isolated (41) from cells pooled from two to three dishes for each treatment, and 5 µg was evaluated by slot-blot analysis and oligonucleotide hybridizations as described previously (37, 38, 39, 42) . An oligonucleotide specific for rat liver TAT was designed as follows: 5`-CTTCAGGGTCTGTAGGCAGG-3`. This probe hybridized to the expected mRNA band migration of 2.3 kilobase pairs (43) as determined by Northern analysis.

cDNA Probes and Hybridization Conditions

All cDNA probes employed in the present study were as described previously (38, 39) . Briefly, a cDNA probe specific for CYP1A1 consisted of a 326-base pair, exon 9 fragment provided by Dr. James Whitlock, Jr. (University of California, Stanford, CA). cDNA probes specific for CYP1A2 and microsomal epoxide hydrolase (mEH) consisted of PCR products which were derived from rat liver cDNAs (38, 39, 42) . Hybridizations were performed essentially as described (44) , except that hybridizations were conducted at 60 °C in the absence of formamide (38, 39) .

ELISA Determination of Intracellular cAMP

A non-isotopic immunoassay system (ELISA) was used in the detection of intracellular cAMP in cells stimulated with various concentrations of forskolin (µM) and glucagon (nM). Stimulation was conducted in the presence or absence of 100 µM IBMX. Cells were stimulated for the indicated times and cell extracts were prepared as follows; cells were washed twice with phosphate-buffered saline and then lysed and scraped in 1 ml of ice-cold 70% ethanol. Cell debris was pelleted at 10,000 g and the resulting supernatant subsequently lyophilized, resuspended in 500 µl of 0.1 M sodium citrate buffer, pH 6.1, and then assayed for cAMP as per supplier's instructions. Protein concentration was determined with bovine serum albumin as standard using a commercial kit (BCA protein assay reagent, Pierce).


RESULTS

Effect of Elevated Intracellular cAMP on PB-mediated Induction

The level of intracellular cAMP, and therefore the activation of cAMP-PK, was modulated in primary rat hepatocytes through the use of a series of analogs of cAMP. After a period of 48 h in culture and immediately prior to initiating PB induction, hepatocytes were exposed for 60 min to increasing concentrations of 8-bromo-cAMP, N,O-dibutyryl cAMP (dibutyryl cAMP), 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), (S)-5,6-1--D-ribofurano-sylbenzimidazole-3`,5`-monophosphorothioate ((S)-5,6-DCl-cBiMPS). After this preincubation period, the treatment was either discontinued (60-min pulse only), or maintained in the absence (Control, C) or presence of phenobarbital (PB, 100 µM) for another 24 h. Total RNA was isolated, and results of RNA slot-blot hybridization are presented. Treatments were also examined by Northern blot analysis to verify the specificity of each of the hybridization probes. In addition, mRNA responses were selectively confirmed by Western immunoblot analysis (data not shown). All studies were repeated at least three times, and representative data are presented.

As shown in Fig. 1A for the dibutyryl analog, PB-mediated induction of CYP2B1, 2B2, and 3A1 mRNAs was inhibited dramatically by this agent in a concentration-dependent manner. In contrast, the same treatment resulted in a dose-dependent induction of the liver and parenchymal cell-selective TAT gene. Indeed, the induction of TAT appeared to inversely parallel the equivalent repression of the PB-inducible CYP genes. In addition, we examined mEH gene expression, a gene that is constitutively expressed in hepatocytes and induced severalfold by PB (10). As demonstrated in Fig. 1A, elevating cAMP did not modulate the constitutive expression level of mEH but effectively repressed its PB-mediated induction. Elevated cAMP concentrations did not modulate the expression of serum albumin, another liver-selective but PB-non-responsive gene.


Figure 1: Effect of dibutyryl cAMP (panel A) and/or CPT-cAMP (panel B) treatment on PB induction of CYP2B1, CYP2B2, and CYP3A1 mRNAs in primary rat hepatocytes. Primary rat hepatocytes were treated with increasing concentrations of cAMP (µM) for 60 min prior to, and then continuously in the absence (Control, C) or presence of PB (100 µM) for another 24 h. As demonstrated previously, the 100 µM concentration of PB is optimal for induction of CYP2B1/2 but not CYP3A1 gene expression (38, 39, 49). Conditions favoring more selective CYP3A1 induction are achieved using higher concentrations of PB as presented in Fig. 7. Total RNA was isolated and evaluated by slot-blot analysis as stated under ``Materials and Methods.'' In addition, TAT, mEH, and albumin gene expression levels are represented. Ribosomal 18 S RNA hybridization levels were used as normalization standards.



Similar results were obtained when substituting 8-CPT-cAMP for the dibutyryl cAMP analog (Fig. 1B). The 8-CPT-cAMP analog demonstrated dramatically higher potency as a repressor of PB induction, e.g. a concentration of 0.5 µM completely abolished PB-inducible CYP2B1 expression. It is interesting to note that in general, much greater concentrations of these analogs are typically used in the literature than were employed in the present study.

Effects of other cAMP analogs also were evaluated, and the results are presented in Fig. 2. Of the analogs tested, 8-CPT-cAMP was the most potent inhibitor of the PB induction response. This agent exhibited similarly high potency as a TAT gene inducer but exerted no effect on serum albumin mRNA levels (Fig. 1B). These results were qualitatively similar to those obtained with dibutyryl cAMP (Fig. 1A). The derivatized analog (S)-5,6-DCl-cBiMPS exhibits greater specificity and intracellular stability than other cAMP analogs (45) , and also exerted a highly potent inhibition of CYP2B1, CYP2B2, and CYP3A1 induction (Fig. 2, and data not shown). Thus, these results are essentially in line with the expected variations in potency of the analogs as PKA activators, arising from their inherent differences in lipophilicity, membrane permeability, and intracellular specificity (45).


Figure 2: Comparison of elevated intracellular cAMP treatment on PB-mediated induction of CYP2B1. Primary rat hepatocytes were treated with increasing concentrations (µM) of 8-Bromo-cAMP (panel A), dibutyryl cAMP (panelB), 8-CPT-cAMP (panel C), and (S)-5,6-DCl-BiMPS (panel D) for 60 min prior to, and then continuously in the presence of PB (100 µM) for another 24 h. Total RNA was isolated and evaluated by slot-blot analysis as stated under ``Materials and Methods.'' Autoradiographic data were quantified by whole band analysis and normalized to 18 S ribosomal rRNA hybridization data. Normalized signal values were then expressed relative to PB induction responses in the absence of cAMP analog treatment and are expressed as percentage of maximal response relative to PB treatment alone. Each bar represents the mean of three separate experiments.



In separate experiments, we also examined the effect of pulsatile treatments with the cAMP agents, adding the compounds for 60 min, prior to their removal and subsequent addition of PB. The same concentration ranges of the cAMP derivatives were tested as in the preceding experiments. The 60-min pulse treatment was again highly effective in repressing the PB induction responses of all marker genes (data not shown). In addition, similar PB-inhibitory potential was realized when the cAMP stimulus was applied either in conjunction with PB or 60 min after PB treatment was initiated (data not shown).

Effect of Elevated Intracellular cGMP on PB-mediated Induction

We examined whether the cAMP-mediated negative modulation of PB induction could also be reproduced with elevated concentrations of intracellular cGMP. As shown in Fig. 3A, only the highest concentration of dibutyryl cGMP used was marginally effective at inhibiting PB induction of the CYP2B1 gene. This compound was also without effect on CYP3A1, TAT, mEH, or albumin gene expression. The 8-bromo derivative of cGMP was tested in separate experiments and was similarly ineffective in modulating the PB induction response (data not shown).


Figure 3: Panel A, effect of dibutyryl cGMP treatment on PB induction of CYP2B1, and CYP3A1 mRNA expression in primary rat hepatocytes. Primary rat hepatocytes were treated with increasing concentrations of dibutyryl cGMP (µM) for 60 min prior to, and then continuously in the absence (Control, C) or presence of PB (100 µM) for another 24 h. Total RNA was isolated and evaluated by slot-blot analysis as stated under ``Materials and Methods.'' In addition, TAT, mEH, and albumin gene expression levels are presented. Ribosomal 18 S RNA hybridization levels were used as normalization standards. PanelB, effect of dibutyryl cAMP treatment on dexamethasone induction of CYP2B1 and CYP3A1 mRNA expression in primary rat hepatocytes. Treatments were as described for panelA.



Effect of Elevated Intracellular cAMP on Dexamethasone-mediated Induction of CYP3A1 Gene Expression

Unlike PB, dexamethasone alone does not induce the CYP2B gene subfamily but is a potent inducer of glucocorticoid-inducible CYP genes, e.g. CYP3A1 (39) . Hepatocytes were treated for 60 min with various concentrations of cAMP analogs, immediately prior to additions of dexamethasone (dibutyryl cAMP results are shown in Fig. 3B). Following cAMP exposure, treatments were continued either in the absence or presence of dexamethasone (1 µM) for 24 h. Dexamethasone addition led to a dramatic induction of CYP3A1 gene expression but did not induce CYP2B1 mRNA levels. At all concentrations tested, increased cAMP levels had no effect on the dexamethasone-inducible CYP3A1 response.

Effect of Elevated Intracellular cAMP on NF-mediated Induction of CYP1A1, 1A2, and mEH

To further investigate the specificity of the effect of cAMP on gene induction, we tested whether elevated intracellular cAMP levels would modulate another prototypic induction response, polycyclic aromatic hydrocarbon-inducible CYP1A1/2 gene expression. As shown in Fig. 4, NF (22 µM) treatment resulted in striking induction of CYP1A1, 1A2, and mEH mRNA levels. However, using the treatment schemes described above, exposure to 8-CPT-cAMP resulted in no modulation of NF-inducible CYP gene expression. Nor did the cAMP treatments affect NF induction of mEH. Substituting the CPT analog with either 8-bromo derivative, dibutyryl cAMP, or forskolin or glucagon resulted in a similar lack of effect on polycyclic aromatic hydrocarbon induction (data not shown).


Figure 4: Effect of 8-CPT-cAMP treatment on NF induction of CYP1A1, and CYP1A2 mRNA expression in primary rat hepatocytes. Primary rat hepatocytes were treated with increasing concentrations of 8-CPT-cAMP (µM) for 60 min prior to and then continuously in the absence (Control, C) or presence of NF (22 µM) for another 24 h. Total RNA was isolated and evaluated by slot-blot analysis as stated under ``Materials and Methods.'' In addition, TAT, mEH, and albumin gene expression levels are presented. Ribosomal 18 S RNA hybridization levels were used as normalization standards.



Effect of Adenylate Cyclase Stimulation on PB Induction

The data presented thus far were obtained by elevating intracellular cAMP concentrations with synthetic analogs of cAMP. To support these results, we also examined the effects of elevated cAMP levels achieved via forskolin and glucagon-mediated stimulation of adenylate cyclase receptor activity.

As determined by a specific ELISA procedure, both adenylate cyclase activators led to a dramatic and time-dependent elevation in intracellular cAMP levels (Fig. 5A). The stability of the cAMP generated was clearly modulated by the inclusion of IBMX (100 µM), a potent and nonspecific inhibitor of intracellular PDE-associated activity (46) . The data presented in Fig. 5A were generated subsequent to prior analysis of dose-response studies conducted with each agent (data not shown).


Figure 5: Panel A, comparison of forskolin and glucagon on time course of cAMP stimulation in primary rat hepatocytes. Primary rat hepatocytes were treated with forskolin (100 µM) or glucagon (100 nM) in the absence or presence of IBMX (100 µM) for the indicated time period (min). The graph also shows untreated cells (CONT) or cells treated with IBMX (100 µM) alone. Cyclic AMP was extracted and determined by ELISA as described under ``Materials and Methods.'' Panels B and C, comparison of elevated intracellular cAMP treatment via activation of adenylate cyclase by forskolin and glucagon on PB-mediated induction of CYP2B1. Primary rat hepatocytes were treated with various concentrations of forskolin (panel B) or glucagon (panel C) for 60 min prior to, and then continuously in the presence of PB (100 µM) for another 24 h. Total RNA was isolated and evaluated by slot-blot analysis. Autoradiographic data were quantified by whole band analysis and normalized to corresponding 18 S ribosomal RNA data. Normalized signal values were then related to PB induction levels occurring in the absence of analog treatment, and expressed as percentage of maximal response relative to no analog treatment. Each bar represents the mean of three separate experiments.



Also in Fig. 5, densitometric data are presented obtained from slot-blot analysis of CYP2B1 mRNA expression profiles. Similar to the effects noted previously for the cAMP analogs, elevating intracellular cAMP levels through adenylate cyclase activation led to a concentration-dependent repression of PB-inducible gene expression. A 2.5 µM concentration of forskolin (Fig. 5B) resulted in a 50% inhibition of CYP2B1 induction, while glucagon (Fig. 5C) elicited an even more dramatic PB-repressive response and did so at concentrations 3 orders of magnitude less than for forskolin. Highly similar results were obtained when examining induction patterns of the CYP2B2 gene (data not shown). In contrast, only glucagon elicited a similar repression of PB-inducible CYP3A1 expression. Forskolin, alone or in the presence of PB, caused a very potent induction of CYP3A1 mRNA levels (data not shown). The exact mechanism of forskolin-mediated induction of CYP3A1 gene expression is currently under investigation but appears to be an indirect, i.e. non-cAMP mediated, event.()

Effect of Dibutyryl cAMP and Phosphodiesterase Inhibition on PB-mediated Induction of CYP2B1 and CYP3A1 Genes

It was important in these studies to assess whether the effects of cAMP on PB induction were directly tied to the physiological actions of cAMP itself, or to a chemical effect resulting from some portion of the cAMP structure, e.g. a PDE-mediated hydrolysis product. The primary hydrolysis product of cAMP is 5`-AMP, and indirectly the native adenosine moiety. Thus, if hydrolysis products were mediating repression of PB induction (21) , inhibition of intracellular PDE activity should reduce the effect. As demonstrated in Fig. 5A, incorporation of classical inhibitors of intracellular PDEs of either a nonspecific (IBMX) or specific (RO 201724) nature permitted potentiation of the intracellular cAMP signal (47) . As shown in Fig. 6, no suppression of the PB inhibition effect produced by dibutyryl cAMP was achieved in the presence of either IBMX or RO 201724. In contrast, both PDE inhibitors actually potentiated the cAMP-mediated inhibition of PB induction of CYP2B1 and 3A1 (and 2B2; data not shown). This was evidenced by the lower cAMP dose required to effect inhibition. In parallel, the dose of cAMP required to induce TAT was also reduced in the presence of PDE inhibition.


Figure 6: Effect of dibutyryl cAMP and PDE inhibitors on PB-mediated induction of CYP2B1, and CYP3A1 mRNA expression in primary rat hepatocytes. Primary rat hepatocytes were treated with increasing concentrations of dibutyryl cAMP in the absence or presence of RO 201724 (100 µM) or IBMX (100 µM) for 60 min prior to and then continuously in the presence of phenobarbital (500 µM). Total RNA was isolated and evaluated by slot-blot analysis. In addition, TAT, mEH, and albumin gene expression levels are presented. Corresponding ribosomal 18 S RNA hybridization levels were used to normalize treatment effects on CYP gene induction.



To support these observations we conducted separate experiments with phenylisopropyladenosine, a very potent adenosine analog with elevated affinity for the A adenosine receptor (48) , and with 5`-AMP, the secondary PDE-associated hydrolysis product. Neither agent was effective in modulating PB induction (data not shown). Thus, the overriding conclusion from these results is that cAMP itself, and not individual hydrolysis products or nonspecific chemical effects, is responsible for inhibiting PB-induced CYP2B1, 2B2, and 3A1 gene expression.

Effect of PB Concentration on cAMP-mediated Modulation of PB Induction of CYP2B1, 2B2, and 3A1 Gene Expression

We next considered whether the observed modulation of PB induction by elevated intracellular cAMP could be attributed to a nonspecific antagonism between cAMP and PB, for example at a potential PB-binding receptor or equivalent intracellular site. Consequently, we examined cAMP modulation in the presence of a range of PB concentrations shown previously to be permissive for CYP2B/3A gene induction.

The preceding studies were conducted using a PB concentration of 100 µM, optimal for CYP2B gene induction in hepatocytes (38, 39) . By increasing the concentration of PB relative to that of cAMP, we sought to delineate any potential direct competition/antagonism associated with the cAMP-mediated effects on PB induction. As shown in Fig. 7, cAMP-mediated inhibition of PB induction was not alleviated by increasing the dose of PB relative to cAMP (for CYP2B and CYP3A). At a concentration of 10 µM, dibutyryl cAMP completely inhibited PB-mediated induction of CYP2B1 (and CYP2B2; data not shown), irrespective of the PB concentration. Since the dose of PB required to maximally induce CYP3A1 is substantially greater than that for CYP2B (38, 39, 49), maximal cAMP inhibition of CYP3A1 induction was particularly pronounced at the highest dose of PB used (1 mM). However, dibutyryl cAMP also visibly inhibited PB-inducible CYP3A1 expression even at lower PB concentrations, i.e. less than or equivalent to 100 µM. In addition, 10 µM dibutyryl cAMP concentrations effectively inhibited PB induction of the mEH gene, even at 1 mM PB. Elevated PB or cAMP concentrations were without apparent effect on albumin gene expression. Taken together, these results establish that the inhibition of PB-inducible CYP2B, CYP3A, and mEH gene expression by cAMP is not mediated by either hydrolysis products or nonspecific antagonism. The results similarly are consistent with the idea that cAMP-mediated inhibition of PB-inducible gene expression is not likely mediated through a nonspecific displacement or antagonism interaction occurring at some common, but still undefined, intracellular binding site.


Figure 7: Comparison of increasing PB concentration on elevated cAMP-mediated modulation of PB induction. Primary rat hepatocytes were treated with increasing concentrations of dibutyryl cAMP (0-100 µM) for 60 min prior to and then continuously in the presence of PB (0.05-1.00 mM) for another 24 h. Total RNA was isolated and evaluated by slot-blot analysis. In addition, TAT, mEH, and albumin gene expression levels are presented. Corresponding ribosomal 18 S hybridization RNA levels were used to normalize treatment effects on CYP gene induction.



Role of Protein Kinase A Activation/Inhibition in the Modulation of PB Induction of CYP2B1, 2B2, and 3A1 Gene Expression

Since the principal intracellular effects of cAMP are mediated via its activation of cAMP-dependent PKA, we examined the relative activation and inhibition of this pathway with respect to PB-inducible CYP gene expression. To effect this modulation, we employed the R and S diastereoisomers of adenosine 3`, 5`-cyclic monophosphorothioate ((R)- and (S)-cAMPS, respectively). Both diastereoisomers share the distinction of being highly membrane-permeable and resistant toward cAMP-PDE activity (50) . They differ in that the (R)-cAMPS is a potent inhibitor (51) , while (S)-cAMPS is, in contrast, a potent and selective activator of PKA (52) . In addition, (S)-cAMPS exhibits greater specificity and affinity than forskolin and the cAMP analogs, e.g. dibutyryl cAMP, without exhibiting any of their side effects (45) .

The results of these analyses are shown in Fig. 8. Hepatocytes were treated with diastereoisomer concentrations ranging from 0.05-100 µM, 60 min prior to and then in the presence of 100 µM PB for another 24 h. From the data, it is clear that (R)-cAMPS did not inhibit the PB induction of CYP2B1, CYP3A1, or mEH (and CYP2B2, data not shown). Nor did (R)-cAMPS induce TAT gene expression. In contrast, (S)-cAMPS dramatically blocked PB induction of CYP2B1, CYP3A1, and mEH (and CYP2B2, data not shown). At a concentration of 2.5 µM, a complete inhibition of PB-inducible gene expression was evident by this diastereoisomer. This concentration is consistent with the K (1.8 µM) associated with this agent for PKA activation (45) . At concentrations inhibitory to PB induction, (S)-cAMPS was highly effective in inducing the TAT gene. Neither the R or S diastereoisomers modulated liver-selective albumin gene expression.


Figure 8: Effect of specific inhibition and activation of protein kinase A on PB induction of CYP2B1 and CYP3A1 mRNAs in primary rat hepatocytes. Primary rat hepatocytes were treated with increasing concentrations of (R)-cAMPS and (S)-cAMPS for 60 min prior to, and then continuously in the presence of PB (100 µM) for another 24 h. Total RNA was isolated and evaluated by slot-blot analysis. In addition, TAT, mEH, and albumin gene expression levels are represented. Corresponding ribosomal 18 S RNA hybridization levels were used to normalize treatment effects on CYP gene induction.




DISCUSSION

We have characterized a primary rat hepatocyte culture system that is highly permissive for differentiated hepatocyte function and PB induction, features that are greatly enhanced by the application of an overlay of ECM (37, 38, 39, 53) . These in vitro culture conditions have the potential of facilitating interpretation of otherwise complex in vivo pharmacokinetics issues. Given the conflicting evidence for the role of signal transduction intermediates in modulating PB-inducible biotransformation activity in vivo(18, 23, 24, 25) , in the present investigation we used the hepatocyte culture system to investigate the mechanistic control exerted by intracellular cAMP in the regulation of PB-inducible gene expression. The results obtained demonstrate that cAMP effects a dramatic modulation of the PB induction response.

In a concentration-dependent manner, elevated intracellular cAMP levels, achieved through the use of membrane-permeable cAMP analogs, resulted in virtually complete inhibition of PB-mediated induction of CYP2B1, 2B2, 3A1, and mEH gene expression. Some concern has been expressed previously regarding the specificity of one of these cAMP derivatives, the 8-CPT analog, indicating that although this analog exhibited a high degree of lipophilicity and stability to cAMP-associated PDE, it also activated cGMP-associated protein kinase with the same affinity as that for PKA (45) . However, through the use of membrane-permeable derivatives of cGMP, 8-bromo- and dibutyryl cGMP, we demonstrated that cGMP additions had either extremely marginal or no effects on PB-inducible gene expression. In addition, Sandberg et al.(45) reported that the derivatized analog (S)-5,6-DCl-cBiMPS exhibited >300-fold lower affinity for cGMP-PK than the 8-CPT analog, as well as possessing a greater affinity for PKA. Likewise, (S)-5,6-DCl-cBiMPS is highly resistant to PDE-mediated hydrolysis (45) . Results obtained in this study using the latter analog also strongly supported the specific involvement of the cAMP-mediated inhibition pathway. Furthermore, the potentiation of cAMP-mediated inhibition of PB induction by PDE inhibitors and the additional lack of effect of adenosine and/or 5`-AMP argue against the possibility that the PB repression is due to hydrolysis products of cAMP (21) . Along these lines, we also considered whether cAMP-mediated inhibition of PB induction could be simply a nonspecific antagonism or competition between cAMP and PB for a common intracellular binding site. However, our data demonstrated that elevating the PB concentration relative to cAMP levels did not alleviate the cAMP-mediated inhibition of CYP2B1, 2B2, or 3A1 gene induction. Evidence for a common receptor involved in the binding of PB and PB-like agonists has not been forthcoming. On the contrary, we have reported recently that if such a receptor does exist it does not exhibit agonist enantioselectivity (54) .

Further support for specific involvement of the cAMP pathway, and more significantly, PKA, in modulating PB induction arose from results obtained with (R)-cAMPS and (S)-cAMPS. As for the cAMP analogs, when hepatocytes were treated with (S)-cAMPS, marked inhibition of PB-mediated induction of CYP2B1, 2B2, 3A1, and mEH was achieved. The inhibition occurred at a concentration approximately equivalent to this agent's K for PKA (1.8 µM, Ref. 45). In a parallel fashion, (S)-cAMPS was a potent inducer of TAT gene expression, a gene known to be expressed in the parenchymal cell population of hepatocytes and highly inducible by cAMP (26, 28) . We used the induction of TAT gene expression as a positive control measure to demonstrate the efficacy of intracellular cAMP activation. Each analog of cAMP examined was a potent inducer of TAT. However, no evidence of TAT activation or inhibition of PB induction, was demonstrated using (R)-cAMPS, a diastereoisomer characterized as a potent inhibitor of PKA (51) and thus expected to produce effects opposite that for (S)-cAMPS (52).

Striking inhibition of PB induction also was noted when intracellular cAMP levels were modulated via adenylate cyclase receptor activation. Treatment of hepatocytes with either the diterpene, forskolin, or the physiological hormone, glucagon, resulted in a dramatic stimulation of intracellular cAMP levels. The stimulation was enhanced approximately 3-fold by the inclusion of 100 µM IBMX (and RO 201724; data not shown). The resulting cAMP signal, although transient in nature, was stable over a period of 4 h relative to either control or IBMX-only treated cells. The transient signal returned to basal levels over a 24-h period post-treatment. In a recent study, Bjornsson et al.(55) reported a similar stimulation of intracellular cAMP by forskolin in primary rat hepatocytes. Indeed, the absolute levels of cAMP in the latter investigation were almost identical to those obtained in the current report and likewise were enhanced through inhibition of PDE activity. It is noteworthy that, although the cAMP signal generated as a result of forskolin and/or glucagon stimulation peaks quite rapidly, a dramatic inhibition of the subsequent PB induction response was still manifested. Other investigators have reported similar observations of cAMP transients regulating diverse downstream physiological phenomena. For example, despite the rapid elevation of intracellular cAMP levels within 20 min following stimulation with forskolin, marked physiological effects were still maintained over 24 h subsequently (55) . Consistent with these latter effects, it appears that rapid initial changes in cAMP and resulting PKA activation effectively interfere downline with the PB-associated signaling pathway in a relatively persistent manner, as noted additionally with our experiments using 1-h pulse exposures to cAMP analogs. Recently, Graves et al.(56) reported that in arterial smooth muscle cells, transient elevations of PKA activity resulted in a correspondingly striking inhibition of mitogen-activated protein kinase activity. These results suggest that cAMP and PKA may in turn act to ``cross-talk'' with other pathways involved in PB-associated signaling.

Effects of elevated cAMP levels also were assessed for other prototypic gene inducers. We examined dexamethasone-inducible CYP3A1 and NF-inducible CYP1A1, 1A2, and mEH expression but obtained no evidence of any cAMP modulation for any of these respective genes or inducing agents. Thus, it would appear that cAMP, via PKA activation, exerts effects selectively on PB-inducible gene responsiveness. Recently, Eliasson et al.(57) reported that PKA-mediated phosphorylation of dexamethasone-induced CYP3A1 caused an enhanced degradation of 3A1 protein. These investigators employed hepatocytes isolated from dexamethasone-induced rat liver and placed in suspension or very short term culture. The latter study provides evidence for additional and separate post-translational influences of cAMP on certain P450s (29, 31) .

In contrast to results presented here, there exist conflicting reports in the literature regarding effects of cAMP on CYP gene expression. In certain in vivo studies, a stimulation of intracellular cAMP and PKA activity was achieved rapidly following intraperitoneal PB injection (23, 24) . An additional study suggested that the elevation of liver cAMP levels and associated PKA activation following PB injection was actually a prerequisite for both the liver hypertrophy response (25) and the induction of mixed-function monooxygenases (23) . Indeed, Blankenship and Bresnick (22) reported that PB induction stimulated the phosphorylation of acidic nuclear proteins in rat liver nuclei. The latter finding and those from the studies by Byus et al. (23), Costa et al.(24) , and Manen et al.(25) suggested that cAMP and thus PKA cooperate in the PB-mediated induction of monooxygenase gene expression, a conclusion contrary to that obtained here. These earlier investigations were attempting to ascertain effects of cAMP-linked processes in whole animal systems, where other hormonal factors can combine to complicate the interpretation of pharmacological end points. Thus, clear conclusions were difficult to obtain from many of these studies. However, even with in vitro approaches, disparate results have been published. For example, Giger and Meyer (58) also reported that glucagon and dibutyryl cAMP enhanced the induction of -aminolevulinate synthase and cytochrome P450 by PB in primary cultures of chick hepatocytes. Since the basic induction phenomenon relative to PB-mediated CYP gene induction is conserved between the rat and chick (59) , these results appear to contradict our findings. This is despite good agreement between the respective investigations regarding the concentrations of glucagon and/or dibutyryl cAMP used. In other work, Saad et al.(60) recently demonstrated that glucagon (100 nM) differentially repressed a battery of PB-inducible testosterone-hydroxylase activities in PB-induced rat hepatocytes. The activities were monitored subsequent to 96 h of exposure to PB (0.75 mM) and glucagon. In contrast, Canepa et al.(61, 62) reported that cAMP actually potentiates the induction by PB of -aminolevulinate synthase, ferrochelatase, and cytochrome P450 biosynthesis. These latter effects were observed with either cAMP analogs, forskolin, or epinephrine. In addition, the authors noted that inhibition of adenylate cyclase, or activation of PDE activity, both acted to inhibit PB-mediated induction of the assessed activities (61) . Several differences in protocol exist however between these and the current study. Most of the previous in vitro work was conducted in freshly isolated cell suspensions. Results from such incubations can be highly artifactual due to the limited life span of the cells in this environment, together with the high degree of heterogeneity of viable and dying cells. These artifacts are generally reduced in primary cultured cells, especially when the cultures display a degree of functional differentiation equivalent to the in vivo phenotype (37, 38, 39) .

Recently, we reported that a combination of optimal dexamethasone concentrations together with an overlay of ECM greatly enhanced the differentiated responsiveness of hepatocytes in primary monolayer culture (39) . Indeed, various investigators have suggested that ECM effects its response by facilitating or ordering intracellular structure, which in turn confers an increased responsiveness to trophic hormones (63, 64) . In a similar light, Dym et al.(65) reported that rat Sertoli cells grown on ECM (Matrigel or laminin) acquired the hormonal responsiveness to follicle-stimulating hormone manifested in vivo. ECM dramatically increased G-protein levels and adenylate cyclase activity in response to follicle-stimulating hormone and forskolin relative to cells grown on non-ECM substrata. Similarly, other researchers, using cultured bovine and human adrenocortical cells, have found that responsiveness to cholera toxin/insulin-like growth factor-1, and hence cAMP-mediated inducibility of the steroid hydroxylase enzymes CYP21 and 11B1, was preferentially maintained only when cells were cultured on a substratum of Matrigel (35) , whereas the 17-hydroxylase (CYP17) was unaffected. Thus, all of these results appear consistent with the assertion that more highly differentiated morphology and function of cells is maintained in ECM cultures, similar to those used in the current investigation with primary hepatocytes (37, 38, 39) .

Various pharmacological interactions between cAMP and PB-like drugs have been reported. In liver, PB is a potent tumor promoter (11) and in this capacity is known to interfere with intercellular communication. The suspected mechanism involves disruption of gap-junction function and/or interference with gap-junctional protein expression (12) . On the other hand, cAMP is known to up-regulate hepatocyte intercellular communication. Saez et al.(66) demonstrated that addition of 8-bromo-cAMP (1.00 mM) for 16 h to hepatocytes in culture assisted in maintaining spherical shape of the cells, likely associated with a stabilization of gap-junctional fidelity. In addition, the authors reported that agents which alter intracellular cAMP concentration affect intracellular communication by changing the stability of mRNAs encoding specific gap-junction proteins. Subsequent studies have established that PKA-mediated phosphorylation of the major liver gap-junction protein, connexin 32, resulted in an elevation of gap-junction function due to increased channel permeability (67, 68) . A recent report by Klaunig et al.(69) demonstrated that in mouse hepatocytes, 8-bromo-cAMP (0.5 mM) reversed the inhibition of intercellular communication exerted by PB and other tumor promoters. Thus, it would appear that cAMP and PB (and perhaps other tumor promoters) exert opposite and perhaps antagonistic control mechanisms on cellular communication. In support of this concept, the potent antitumor agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide was shown to competitively inhibit cAMP-PDE in rat liver and, in turn, to amplify hormonal-mediated cAMP stimulation in hepatocytes and hepatoma cells (70). It remains to be ascertained whether the negative modulation by cAMP of PB responsiveness in primary rat hepatocytes is intimately linked to the regulation of gap-junctional fidelity.

Interactions between PB and cAMP also have been reported for the central nervous system. Kuo et al.(71) reported that in mouse brain, long term administration of PB resulted in an inhibition of cerebellar PKA activity. Indeed, intravenous injection of dibutyryl cAMP in rats has been shown to induce seizures and, subsequently, to counteract the pharmacological efficacy of anti-epileptic drugs such as PB and phenytoin (72) . In fact, PB and phenytoin inhibited the ouabain-mediated release of cAMP in rat brain cortex slices (73) . Nistico observed a similar effect in chick brain in vivo by showing that PB and similar drugs antagonized the convulsant effect of injected dibutyryl cAMP (74) . In an earlier report, Weiner et al.(20) reported that coinjection with dibutyryl cAMP or various agents that stimulated cAMP release actually extended the hypnotic/sleep time period induced by hexobarbital. The authors attributed this effect to be due to the antagonism by cAMP of liver P450-mediated metabolism of hexobarbital. Recent studies by Ishibashi et al.(75) and Ormandy and Jope (76) have demonstrated that certain pharmacologically active depressants inhibit cAMP stimulation in brain tissue by impairing G-protein coupling and that the latter activity actually forms the basis of their anti-seizure properties.

There are some interesting contrasts and parallels to be drawn between cAMP-mediated inhibition of PB-inducible CYP gene expression and the corresponding interactions of cAMP with other liver-selective genes. For example, TAT gene expression is inducible by both glucocorticoids and cAMP (26, 28) and is repressed by insulin (77) . Two selective 5`-gene enhancer elements appear to be involved in the induction responses, at -2.5 and -3.6 kilobase pairs, respectively (28) . These hormonally responsive elements in turn synergize with liver-enriched transcription factors, including HNF-3 and HNF-4. Of interest, insulin also opposes the inductive effects of cAMP and dexamethasone on the expression of the phosphoenolpyruvate carboxykinase gene (PEPCK; Ref. 78). The inhibitory effect of insulin has been identified within a 15-base pair insulin responsive negative element in the 5`-flanking region of the PEPCK gene (79, 80) . In contrast, insulin-mediated transcriptional-activation of glucokinase gene expression is repressed by cAMP, even in the presence of insulin (81) . Similar to PB-inducible CYP2B1, glucokinase expression is silent in the absence of insulin. In other tissues, the expression of the insulin-responsive glucose transporter gene, GLUT4 (82) , also is repressed by cAMP. cAMP-mediated repression has recently been identified with chimeric gene transfection experiments and shown to reside between -469 and -78 base pairs of the transcription start site of GLUT4 (83) . Thus, based on the results reported in the present study, the possibility exists that cAMP also modulates PB induction through a similarly acting negative element. Perhaps cAMP functions to stimulate the phosphorylation, or dephosphorylation, of a transcription factor interacting in cis with PB-inducible genes. In any event, since PB induction of CYP2B gene expression occurs directly via transcriptional activation (10, 84) , it appears likely that the cAMP/PKA-mediated repression response also targets the CYP2B transcriptional machinery.

Although the molecular mechanisms underlying the repressive effects of growth hormone on PB induction in vitro(85) and in vivo(86) have not been explained, the effects of certain physiological factors such as glucagon and epinephrine release on biotransformation activity in vivo(15, 16) are likely mediated via a cAMP signal (17) . It is of interest that PB induction potential is depressed approximately 3-fold in primary hepatocytes isolated from the livers of spontaneously diabetic (f/f) Zucker rat (84) .

The data reported here clearly demonstrate a potent repressive effect of elevated cAMP levels on transcriptional activation of PB-inducible gene expression and have potential physiological relevance based on the discussion above. Results of preliminary studies with protein kinase C activation/down-regulation do not support an involvement for this signaling pathway in mediating the PB induction response. Future studies are therefore directed toward the identification of potential intracellular sites for cAMP/PKA-mediated phosphorylation and examination of the interplay of these events with transduction of the PB-mediated signal.


FOOTNOTES

*
This study was supported by United States Public Health Service Grant GM32281 (to C. J. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Environmental Health, XD-41, University of Washington, Seattle, WA 98195. Tel.: 206-543-1700; Fax: 206-685-4696; E-mail: cjo@u.washington.edu.

The abbreviations used are: PK, protein kinase; PKA, protein kinase A; CYP, cytochrome P450; PB, phenobarbital; TAT, tyrosine aminotransferase; IBMX, 3-isobutyl-1-methylxanthine; (S)-5,6-DCl-cBiMPS, (S)-5,6-dichloro-1--D-ribofuranosylbenzimidazole-3`,5`-monophosphorothioate; (R)-cAMPS, phosphorothioate stereoisomer of cAMP; (S)-cAMPS, phosphorothioate stereoisomer of cAMP; PDE, phosphodiesterase; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; dibutyryl cAMP, N,O-dibutyryl cAMP; NF, -naphthoflavone; mEH, microsomal epoxide hydrolase; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; PEPCK, phosphoenolpyruvate carboxykinase.

J. S. Sidhu and C. J. Omiecinski, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent technical assistance of Richard Ramsden and Dr. Fred Farin in the design of oligonucleotides and cDNAs used in this study. We also thank Dr. Lee Graves for helpful comments and for reading this manuscript.


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