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-Subunits and the Phosphoinositide 3-Kinase Pathway
Max Planck Institute of Psychiatry D-80804 Munich, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunits, insensitive to pertussis toxin and independent of
protein kinase A (PKA). In contrast, the catecholamine-evoked GR
enhancement was strongly reduced by wortmannin, suggesting a critical
role for phosphoinositide 3-kinase (PI3-K). In agreement, epinephrine
directly activated PI3-K in vivo. Similarly, stimulation of
tyrosine kinase receptors coupled to PI3-K activation, e.g.
receptors for insulin-like growth factor I (IGF-I) or fibroblast growth
factor (FGF), increased GR transactivation. Further analysis indicated
that G protein-coupled receptor (GPCR) and tyrosine kinase receptor
signals converge on PI3-K through separate mechanisms. Blockade of GR
enhancement by wortmannin was partially overcome by expression of the
downstream-acting protein kinase B (PKB/Akt). Collectively, our
findings demonstrate that GPCRs can regulate GR transactivation by
stimulating PI3-K. This novel cross-talk may provide new insights into
the molecular processes of learning and memory and the treatment of
stress-related disorders. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The GR is a member of the steroid receptor family and is closely related to the mineralocorticoid, progesterone, androgen, and estrogen receptors. After glucocorticoid binding, the activated GR is released from its cytoplasmatic protein complex and translocates into the nucleus. There, it binds to glucocorticoid-responsive elements (GREs) located in the regulatory region of target genes and modulates their activity by interacting with the transcriptional machinery (3). Depending on the nature and location of the GREs and, in addition, on the presence of coregulators, the GR displays transactivation or transrepression. Furthermore, cross-talk between the GR and other transcription factors has been observed (3, 4).
Ligand-independent modulation of steroid receptor function in a cell type-specific manner is well known for the estrogen, progesterone, and androgen receptors with regard to growth factors, cyclins, cAMP, or other signals. In contrast, a huge number of studies agreed that the GR can be modulated by such stimuli only after ligand activation (for review see Refs. 5, 6). However, the mechanisms by which these signal transduction pathways modify GR function are less well understood. An advanced insight into these processes appears mandatory to improve our understanding of various physiological and pathophysiological conditions related to GR function and dysfunction.
A number of studies indicate that adrenergic and glucocorticoid signaling is intimately interwoven in the brain (2, 7). In particular, memory consolidation requires activation of both glucocorticoid- and norepinephrine-dependent pathways, and both kinds of stress hormones have been implicated in psychiatric disorders (8, 9). Despite this increased understanding at the systemic level, no evidence for a direct interaction of the two stress hormone pathways within neuronal cells has been reported so far. Hence, we set out to examine whether, at the cellular level, neuronal GR activity is modulated by catecholamines, and if so, to identify the relevant pathway.
The catecholamines epinephrine and norepinephrine bind to G
protein-coupled receptors (GPCRs) in the cell membrane, leading to
dissociation of the G protein 
and ß subunits. In addition to
the well known signaling of GPCRs via G, the interest in biological
effects mediated by Gß has increased continuously during recent
years. In particular, ß-subunits from inhibitory
Gi proteins have been demonstrated to interact
with specific isoforms of phosphoinositide-3 kinase (PI3-K) and to
stimulate the mitogen-activated protein kinase (MAPK) pathway (10, 11).
In this study, we report that (nor)epinephrine, acting via ß2-adrenergic receptors, potentiates GR transactivation in hippocampal cells. Our data reveal a novel cross-talk between endogenous adrenergic receptors and GRs in neuronal cells via a pathway depending on PI3-K.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, open circles).
Interestingly, when transfected cells were costimulated with
dexamethasone and epinephrine, a marked increase in
glucocorticoid-induced reporter activity was observed while the
catecholamine alone was ineffective. A similar enhancement was obtained
with the natural ligand corticosterone, which displays lower affinity
for the GR (Fig. 1A).
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by
presenting the data as per cent of maximal induction in the presence or
absence of epinephrine. The left-shift caused by epinephrine reflects
an enhanced effectiveness of available glucocorticoids, reducing the
ED50 from 26 nM to 12 nM.
Remarkably, catecholamine regulation was strongest at low doses of
glucocorticoid, with a 5-fold increase of the induction by 3
nM dexamethasone alone (Fig. 1B, inset). The
extent of potentiation gradually diminished at higher dexamethasone
concentrations, but epinephrine still doubled GR transactivation at 100
nM of glucocorticoids and beyond. Hence, in
neuronal cells epinephrine strengthens GR transactivation potency
predominantly at low doses of GR agonists.
Epinephrine concentrations as low as 30 nM were sufficient
for GR enhancement (data not shown). Moreover, treatment of HT22 cells
with glucocorticoids and norepinephrine instead of epinephrine caused a
similar enhancement of GR transactivation (Fig. 1C). Potentiation could
be blocked by the GR antagonist RU 38486, demonstrating specificity of
the transcriptional response (Fig. 1C). Furthermore, enhancement could
be prevented by addition of the ß-adrenergic antagonist propranolol
and also by ICI 118551, suggesting that GR enhancement was mediated by
ß2-adrenergic receptors (AR) (Fig. 1C).
To examine whether enhanced GR transactivation occurred through the
GREs or, alternatively, through unrelated regulatory elements in the
MMTV promoter, we used a minimal thymidine kinase (TK) promoter as
control. Neither stimulation with dexamethasone nor epinephrine
increased reporter gene activity in HT22 cells. However, after
insertion of two GREs adjacent to the regulatory region, this promoter
displayed both glucocorticoid induction and potentiation by epinephrine
(Fig. 1D). We conclude that the effect of epinephrine on enhanced GR
transactivation requires a GRE and is not confined to the MMTV promoter
context.
Enhanced GR function might be due to an increase in receptor
concentration under catecholamine treatment. However, we found no
significant change in GR transcript or protein levels after 6 h or
24 h of epinephrine treatment (Fig. 1E). Likewise, the number of
ligand binding sites and the affinity constant were unaltered after
incubation of HT22 cells with epinephrine for 0, 4, 8, or 24
h (not shown). Therefore, higher transactivation occurred despite
unaltered GR binding parameters.
Binding to the GRE is an important step before induction of
glucocorticoid-responsive genes. In nontransfected HT22 cells,
electrophoretic mobility shift assay (EMSA) revealed that specific DNA
binding of the GR is significantly stronger in extracts of cells
stimulated with epinephrine than in those of untreated cells (Fig. 1F, lanes 1 and 2; quantification depicted in right panel).
Specific GR-DNA complexes disappeared in the presence of an excess of
cold competitor GRE oligonucleotides (Fig. 1F, lane 3). As a negative
control, the same excess of cold mutated GRE oligonucleotides did not
compete with complex formation (lane 4), demonstrating specificity of
the complexes. These results suggest that adrenergic receptor
activation potentiates GR function, at least in part, by improving DNA
binding.
Enhanced GR Transactivation by Epinephrine Is Mediated via G
Protein ß-Subunits
ß2-ARs are Gs
protein-coupled receptors leading to activation of protein kinase A
(PKA) (14). To investigate the role of PKA in enhanced GR
transactivation, we treated transfected HT22 cells with both
dexamethasone, epinephrine, and specific inhibitors of PKA.
Cotransfection of an expression vector encoding a PKA inhibitor (PKI)
peptide or treatment with the inactive cAMP-analog Rp-cAMP did not
reduce enhanced GR transactivation, suggesting that GR potentiation is
not mediated by PKA in HT22 cells (Fig. 2A). Of note, pretreatment with H-89
further increased GR function (Fig. 2A), possibly due to effects of
H-89 unrelated to PKA (15).
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s has been
reported to switch to Gi as a consequence of
receptor phosphorylation by PKA (16). In light of the above results,
such a PKA-mediated switch from Gs to
Gi proteins appeared unlikely to contribute to
enhanced GR transactivation. To further exclude the involvement of
Gi proteins, we investigated the effect of
pertussis toxin (Ptx) on GR enhancement. HT22 cells preincubated with
Ptx showed no reduction of GR enhancement after costimulation with
dexamethasone and epinephrine (Fig. 2A, inset, right bar).
As an additional control, cells prestimulated with epinephrine for up
to 90 min were treated with vehicle or Ptx before the addition of
dexamethasone. Blockade of Gi signaling by Ptx
after prestimulation with epinephrine did not reduce the GR response
either (Fig. 2A, inset). Finally, Ptx alone did not modulate
reporter gene induction (not shown). Together, these findings
demonstrate that 1) blockade of the cAMP-PKA pathway does not prevent
enhanced GR transactivation by ß2-adrenergic
receptors; and 2) differential coupling of
ß2-AR to Gi proteins is
not required for potentiation of GR transactivation.
Within recent years, increasing attention has been paid to the cellular
actions of Gß-subunits upon GPCR activation (17). Whereas the
biological functions of ß-subunits released from inhibitory
Gi have been described in some detail,
e.g. activation of PI3-K and MAPK (for review see Ref. 18),
much less is known about the role of ß signaling in the case of
receptors coupled to stimulatory Gs. In view
of our experiments, we reasoned that the stimulatory effect of
epinephrine on the GR may be relayed by ß-subunits in response to
stimulation by ß2-ARs. To further test this
hypothesis, we asked whether catecholamine treatment could be replaced
by cotransfection of free Gß-subunits. In fact, expression of Gß
and G cDNAs could substitute for epinephrine stimulation (Fig. 2B).
In contrast, coexpression of Gß in conjunction with a mutated
G- that fails to associate with the membrane
(10) did not increase GR transactivation, indicating that
Gß-subunits are critical for enhanced GR transactivation by
epinephrine (Fig. 2B). Collectively, these data strongly suggest that
ß2-adrenergic signaling to the GR is transduced
via Gß-subunits released from Gs.
To examine whether GR enhancement was restricted to
ß2-AR, we ectopically expressed other GPCRs in
HT22 cells. Costimulation with dexamethasone and dopamine, pituitary
adenylate cyclase- activating peptide (PACAP), or LH did not
influence GR transactivation in HT22 cells (not shown). In contrast,
these ligands strongly enhanced GR activity when their cognate
receptors were cotransfected, indicating that the observed effect on
the GR is not specific to ß2-ARs but also
extends to other GPCRs coupled to Gs (Fig. 2C).
PI3-K Relays Signaling to the GR
Several recent studies reported a cross-talk between
Gß-subunits released from Gi and the
PI3-K cascade (11, 19). We asked whether PI3-K might also play a
role in the Gs-mediated effect of epinephrine
on GR transactivation. To test this hypothesis, we treated
cotransfected HT22 cells with wortmannin, a potent and specific
inhibitor of PI3-K (20). In fact, wortmannin decreased the enhancement
of GR transactivation by epinephrine in a dose-dependent manner (Fig. 3A) while it was ineffective alone.
Importantly, this reduction was also obtained with LY 294002, a
structurally unrelated inhibitor of PI3-K, demonstrating specificity of
the blockade (Fig. 3A). Inhibition by wortmannin was maximal at 100
nM, preventing approximately 80% of the
epinephrine-mediated GR potentiation. This suggests a major role of
PI3-K in GR enhancement.
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). Remarkably, cotreatment with both the catecholamine and
either growth factor further enhanced the GR response in an additive
manner, indicating that both signals were transduced through separate
pathways (Fig. 3B).
The family of PI3-Ks comprises various isoforms, and those belonging to
class I are composed of a heterodimer consisting of a regulatory and a
catalytic subunit. Different isoforms of each subunit have been
isolated, and their patterns of expression and modes of activation show
some variation (21). The crucial role of PI3-K was further underlined
by results obtained from cotransfecting a constitutively active
catalytic subunit (p110*) of PI3-K into HT22 cells. In agreement with
above data, expression of this kinase enhanced ligand-dependent GR
transactivation by 2-fold in the absence of both IGF-I and bFGF (Fig. 3B). Consequently, PI3-K activation is necessary and sufficient for
mediating growth factor-dependent GR potentiation and, additionally,
represents a major pathway for catecholamine-mediated enhancement of GR
transactivation in HT22 cells.
One of the PI3-K-regulatory subunits, p85, binds to phosphotyrosine
residues, e.g. on receptor tyrosine kinases (RTK) upon
growth factor stimulation, and recruits the catalytic subunit p110 to
the membrane (21). To investigate whether phosphotyrosine binding of
p85 was also required for ß-induced signaling, we cotransfected
HT22 cells with a dominant negative form of p85 (
p85) lacking the
binding site for p110. p85 can still bind to phosphotyrosine
residues, but it can no longer recruit p110. In agreement with this
prediction, the IGF-I-induced GR potentiation could be nearly
completely blocked by cotransfection of p85 but not wild-type p85
(Fig. 3C). In contrast, neither p85 nor wild-type p85 significantly
affected the enhancement conferred by epinephrine (Fig. 3C). We
conclude that epinephrine-induced ß signaling does not depend on
phosphotyrosine binding of the PI3-K-regulatory subunit p85 but appears
to lead to direct activation of p110. This confirms the notion that
growth factor and ß signaling occur via distinct mechanisms
leading to stimulation of PI3-K.
Epinephrine Activates the PI3-K Cascade
To further confirm that epinephrine leads to activation of PI3-K
in vivo, we treated nontransfected HT22 cells with vehicle
or epinephrine under serum-free conditions and measured the activity of
PI3-K immunoprecipitated by anti-p85-Ab. As shown in Fig. 4A, incubation with epinephrine
stimulated the catalytic activity of PI3-K by 2.1-fold. As a control,
PI3-K activity was also enhanced by 2-fold after incubation with IGF-I
(not shown). These results corroborate our transfection studies and
clearly demonstrate a cross-talk between adrenergic receptors and PI3-K
in neuronal cells.
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). Next, we tested whether expression of
PKB* could overcome the impaired epinephrine-mediated GR potentiation
under wortmannin treatment. In fact, cotransfection of PKB* reversed
nearly half of the wortmannin-mediated inhibition of GR enhancement,
while SGK and p70S6K were ineffective (Fig. 4C).
These findings strongly suggest a role for PKB in epinephrine signaling
to the GR. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunits.
Interestingly, we further identified the phosphoinositide-3-phosphate
signal transduction cascade as a major determinant downstream of
ß2-ARs in HT22 cells. Our findings demonstrate
that activation of PI3-K is an important step in GR enhancement. First,
wortmannin, as well as the structurally unrelated PI3-K inhibitor LY
294002, blocked increased GR transactivation by epinephrine. Second,
expression of PI3-K’s constitutively active catalytic subunit p110*
augmented GR activity. Third, epinephrine directly stimulated PI3-K
activity in neuronal cells. Our data point to a new mode of cross-talk
between Gs-coupled receptors and PI3-K via
Gß-subunits in a neuronal cell line. In this view, our findings
further exemplify the complexity of cross-talk between GPCRs and
downstream kinases (18). Inhibition of GR potentiation by wortmannin
could be partially overcome by PKB, which acts downstream of PI3-K.
Hence, ß2-adrenergic stimulation of GR in HT22
cells is predominantly transferred by a pathway under the control of
PI3-K (Fig. 5). In agreement with these
findings, growth factor-mediated GR potentiation in HT22 cells
could be completely blocked by wortmannin, again establishing the
crucial role of PI3-K.
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and PI3-K (P. Schmidt,
F. Holsboer, and D. Spengler, unpublished observation). Elucidation of
this additional pathway for GR enhancement will require further
study.
Activation by G protein ß-subunits has been
shown for PI3-K in vivo and for PI3-Kß in
vitro (11, 23). However, only PI3-Kß is widely expressed in
various tissues (21). Our results on PI3-K activation by epinephrine
in vivo strongly suggest a role for PI3-Kß in HT22 cells,
since PI3-K does not immunoprecipitate with the regulatory subunit
p85 (21). The additional increase in GR transactivation after
costimulation with growth factors may be mediated by another PI3-K
isoform recruited to tyrosine-phosphorylated residues, i.e.
or 
. If PI3-Kß mediated both signals, a synergistic rather
than additive effect would be expected (23, 24). In agreement with the
observation that the regulatory subunit p85 is required for growth
factor signaling but does not interfere with Gß signaling (24),
our results using a dominant negative form of p85 support the
possibility of two different mechanisms leading to PI3-K activation. In
particular, they demonstrate that epinephrine signaling does not
proceed via tyrosine phosphorylation of growth factor receptors in HT22
cells (25).
PI3-K can activate several downstream targets including PKB, PKC, MAPK, SGK, and p70S6K (22). We found that, with respect to these PI3-K targets, only PKB stimulated GR function in HT22 cells. Furthermore, PKB can, in part, restore the wortmannin-inhibited effect of catecholamines on GR function, suggesting an involvement of this kinase in GR enhancement. PKB has been reported to increase GR function in PC12 cells by antagonizing the inhibitory action of glycogen synthase kinase-3 (GSK-3) (26). In HT22 cells, however, coexpression of GSK-3 did not inhibit enhanced GR transactivation by epinephrine. Likewise, expression of a dominant negative form of GSK-3 did not improve GR function (P. Schmidt, F. Holsboer, and D. Spengler, unpublished observations). Therefore, our results gave no evidence for an involvement of GSK-3 in GR transactivation in HT22 cells, but they strongly support a critical role of the PI3-K target PKB in relaying adrenergic signaling to the GR.
Consistent with a number of previous studies, epinephrine was unable to activate the GR in the absence of glucocorticoids. In this respect, the GR seems to behave differently from most other steroid hormone receptors (5, 6). The strict ligand dependency of the epinephrine-induced GR potentiation in our model system distinguishes this regulation from a recent report on ligand-independent activation of the GR by ß2-adrenergic agonists in fibroblasts (27).
Interestingly, the highest enhancement by epinephrine was achieved at low concentrations of glucocorticoids and led to a 5-fold increase of GR transactivation compared with dexamethasone alone. Hence, the strongest potentiation of GR function occurred at glucocorticoid concentrations comparable to in vivo baseline conditions. Furthermore, we detected an equivalent enhancement of GR transactivation by epinephrine in the presence of the natural ligand corticosterone. The fact that the ED50 values observed were somewhat higher than expected from studies based on receptor overexpression may be due to the limited, but physiological, levels of GR expression in HT22 cells. Moreover, in this cell line the effect of a given concentration of glucocorticoids could be nearly doubled by blocking the membrane P-glycoprotein, which extrudes several lipophilic molecules from the cytoplasm, with verapamil (28). This was the case for both cortisol and dexamethasone (28), and we could reproduce this finding for corticosterone (data not shown).
The observed cross-talk between catecholamines and steroids might play a role under various physiological conditions. For instance, enhanced recruitment of the GR under low glucocorticoid levels may facilitate glucocorticoid-dependent memory storage (8). Furthermore, our findings might represent a molecular mechanism for fine tuning the neuronal stress response by (nor)epinephrine (2). Notably, epinephrine reduced the dose of glucocorticoids necessary for half-maximal GR activity by more than 50%, which might also be of relevance for stress-related disorders. Long-term treatment with antidepressants or lithium has been shown to influence GPCR and PI3-K signaling at various levels and might help to restore negative feedback regulation by glucocorticoids in depressed patients (9, 29).
The fact that both catecholamines and polypeptide growth factors
enhance GR transactivation seems to indicate redundancy in this
process. However, the complexity of these signaling systems suggests
that distinct biological responses are achieved based on
context-dependent activity profiles and cell type-specific expression
patterns. In addition, epinephrine and IGF-I probably act via different
isoforms of PI3-K, which may ensure selectivity. Thus, despite
overlapping effects on GR transactivation, either stimulus may modulate
glucocorticoid signaling in the nervous system in a specific and
context-dependent manner. The interaction between growth factors and
glucocorticoids may, for instance, play a role in neurodevelopment and
neuronal plasticity of the hippocampus (30). Moreover, additional
physiological functions in tissues other than the hippocampus might be
suggested by our finding of GR enhancement by different
Gs- coupled receptors.
In summary, this study reveals that adrenergic stress hormones enhance glucocorticoid activity in hippocampus-derived cells via the phosphoinositide pathway. Our findings demonstrate a novel cross-talk between GPCRs, PI3-K, and the GR and suggest a critical role for PKB in regulating steroid receptor function. These results imply a model of GR activity in which constant levels of steroid receptor and ligand may bring forth a broad range of activity in neuronal cells. This notion of GPCR-dependent regulation of GR function appears significant in the light of both physiological and pathophysiological processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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p85 (31) have been subcloned as
BamHI-EcoRI fragments into the vector pRK7
harboring a cytomegalovirus (CMV) promoter (32). Construction of PKI
(33), Gß (34), G- (10), constitutively
active p110* (35), SGK (36, 37), p70S6K (38), PKB
(39), constitutively active PKB* (40), and GSK-3 constructs (41) has
been described.
Cell Culture and Transient Transfections
HT22 cells were grown in DMEM supplemented with 10% FCS
(Life Technologies, Inc., Karlsruhe, Germany). Cells
(3 x 106) were transfected with reporter
plasmid [1 µg MMTV-Luc, TK-Luc, or TK-GRE2-Luc
(42)] and, where indicated, with expression plasmids using an
electroporation system (BTX, Inc., San Diego, CA). The
ß-galactosidase expression vector pRK7-Gal [0.1 µg (32)] and
carrier DNA (pGEM4, Promega Corp., Madison, WI) up to a
final amount of 6 µg were included in each experiment.
Electroporated cells were replated in DMEM supplemented with 10%
charcoal-stripped, steroid-free FCS, except where indicated, and
incubated with substances as described in the figure legends. Unless
indicated otherwise, concentrations of 10 nM dexamethasone
and 30 µM epinephrine were used. After 24 h, cell
extracts were assayed for luciferase activity, and results were
normalized by ß-galactosidase activity as described previously (42).
ICI 118551 and PD 98059 were purchased from Tocris (Ballwin, MO)
and IGF-I and bFGF from R&D Systems (Wiesbaden, Germany). Wortmannin,
LH, and PACAP-27 were obtained from Calbiochem
(Schwalbach, Germany). All other substances were from
Sigma (Deisenhofen, Germany). Statistical evaluation was
performed using Student’s t test.
Northern Blot Analysis
Cells were treated with dexamethasone (300 nM),
epinephrine (30 µM), or both and total RNA was harvested
using Trizol Reagent (Life Technologies, Inc.). Blotting
was performed with 20 µg RNA by capillary transfer. As a probe, a
fragment of the hGR cDNA was used (42). Signals were quantified using a
digital image analysis system (BAS reader, Fuji Photo Film Co., Ltd.).
Western Blot Analysis
Total cellular lysates (50 µg) from cells pretreated with
vehicle or dexamethasone (10 nM) and epinephrine (30
µM) were subjected to SDS-PAGE and blotted as described
(43). Detection of GR was performed with polyclonal rabbit GR primary
antibody (PA1–512, Affinity BioReagents, Inc. Golden, CO)
and a secondary antibody coupled to horseradish peroxidase
(Amersham Pharmacia Biotech, Freiburg, Germany) using
enhanced chemiluminescence (Roche Molecular Biochemicals, Mannheim, Germany).
Electrophoretic Mobility Shift Assay
Annealed GRE-oligonucleotides
(5'-GGAGCTTAGAACACAGTGTTCTCTAGAG-3' and
5'-GGAGTCCTCTAGAGAACACTGTGTTCTA-3') were labeled with
32P-dCTP (Amersham Pharmacia Biotech) using Klenow fragment (New England Biolabs, Inc., Schwalbach, Germany). HT22 cells were homogenized in
ice-cold buffer containing 20 mM Tris-HCl pH 7.5, 600
mM KCl, 20% glycerol, and 2 mM dithiothreitol
(DTT) and centrifuged for 45 min (100,000x g, 4 C). Samples
(20 µl) contained 10 mM HEPES, pH 7.9, 4%
Ficoll, 1 mM DTT, 1 µg poly[dIdC], and 1 µl
cell extract (10 µg protein). A 150-fold excess of unlabeled
competitor GRE or mutated oligonucleotide was included in parallel
samples to confirm specificity of the GR-DNA complex. After 10 min at 0
C, 0.1 ng 32P-labeled GRE was added and
incubation was continued for 15 min at 25 C. DNA-protein complexes were
resolved on a 4% polyacrylamide gel in 0.5x Tris-borate-EDTA
(TBE), and autoradiographs were quantified with a digital image
analysis system.
PI3-K Assay
HT22 cells grown to 70% confluence were treated for 1 min with
epinephrine or vehicle before lysis in the presence of sodium
orthovanadate. Precleared lysates were immunoprecipitated with
anti-PI3K-p85 (No. 06–195, Upstate Biotechnology, Inc.,
Lake Placid, NY) according to the manufacturer’s instructions.
Immunoprecipitates were resuspended three times in washing buffer (500
mM LiCl, 100 mM Tris-HCl, pH 7.4, 2
mM sodium orthovanadate) and twice in equilibration buffer
(100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1
mM sodium orthovanadate). Equal concentrations of PI3-K as
determined by immunoblotting were used for PI3-K assays. Reactions were
started by adding 12.5 mM MgCl2 and 5
µM ATP in the presence of 10 µg phosphatidylinositol
and 830 nM 32P-dATP (3,000
Ci/mmol, Amersham Pharmacia Biotech). Incubation was
stopped after 20 min with 1 M HCl and samples were
extracted using chloroform-methanol (1:1). Products were separated by
TLC (silica gel 60, Merck Eurolab, Ismaning, Germany) in
chloroform-methanol-H2O- ammonium hydroxide
(43:38:7:5) and dried plates were autoradiographed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 14, 2000. Revision received December 1, 2000. Accepted for publication December 5, 2000.
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V. Korz and J. U. Frey Stress-Related Modulation of Hippocampal Long-Term Potentiation in Rats: Involvement of Adrenal Steroid Receptors J. Neurosci., August 13, 2003; 23(19): 7281 - 7287. [Abstract] [Full Text] [PDF]
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 R. H. STRAUB, C. GUNZLER, L. E. MILLER, M. CUTOLO, J. SCHOLMERICH, and S. SCHILL Anti-inflammatory cooperativity of corticosteroids and norepinephrine in rheumatoid arthritis synovial tissue in vivo and in vitro FASEB J, July 1, 2002; 16(9): 993 - 1000. [Abstract] [Full Text] [PDF]
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,
P < 0.05 compared with IGF-I + dexamethasone.
C, Epinephrine does not require phosphotyrosine binding
of p85. Cells were cotransfected with wild-type p85, the dominant
negative form 
