From the
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
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)
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
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.
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.
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
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).
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.
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
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
Further support for
specific involvement of the cAMP pathway, and more significantly, PKA,
in modulating PB induction arose from results obtained with
(R
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
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
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
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
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)-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.
(
)
underlies the basis for many effects of cyclic nucleotides
on cellular function
(5) .
-, 17
-, and
21
-hydroxylase (CYP11B1, 17, and 21)
genes
(35, 36) , very little is known regarding its
potential control of other CYP genes.
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 Me
SO (final concentration of
Me
SO 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).
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.
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.
)-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
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-mediated
Induction of CYP1A1, 1A2, and mEH
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.
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.
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) .
)-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.
)-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) .
)-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).
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) .
-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) .
-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) .
/f
) Zucker
rat
(84) .
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.
)-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.