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Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461
Address all correspondence and requests for reprints to: Michael Ansonoff, Department of Neuroscience, F113, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: ansonoff{at}aecom.yu.edu
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Abstract |
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, ß, and 
PKC isoforms combined, or the
PKC isoform alone, did not detect differences between hormone-treated
and control OVX female rats. Therefore, estrogen treatment increased
PKC catalytic activity in the POA of OVX female rats but not in the
HYP. However, the increased PKC catalytic activity was not correlated
with detectable changes in the level of the , ß, or PKC
isoforms or in the density of phorbol ester binding sites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1B-adrenergic receptor binding
sites and messenger RNA (mRNA) in the HYP-POA (4, 5). Stimulation of
1B-adrenergic receptors activates phospholipase C (6),
which cleaves phosphoinositol 2-phosphate into inositol
1,4,5-triphosphate and diacylglycerol, which in turn, activates protein
kinase C (PKC) (7, 8). Presumably, PKC goes on to phosphorylate and
activate proteins required for reproductive behavior and gonadotropin
release. Cross-talk has been shown to occur between PKC and estrogen receptors. In mammary, ovarian, and pituitary tissue, estrogen up-regulates PKC protein and mRNA expression (9, 10, 11, 12, 13, 14, 15). In these cells, PKC activation augments the ability of estradiol to promote estrogen receptor-mediated increases in gene transcription. In some cases, PKC produces ligand-independent activation of estrogen receptor-induced gene transcription (13, 16, 17). Therefore, present studies examine whether estrogen regulates PKC in the HYP-POA.
We used assays of PKC catalytic activity and measurement of PKC augmentation of cAMP synthesis in brain slices to determine whether estrogen replacement in OVX female rats changes PKC activity in the POA or HYP. We report that estrogen elevates PKC catalytic activity in the POA of OVX female rats. Subsequent phorbol ester binding and immunoblot studies demonstrate that estrogen enhancement of PKC activity does not result from increased density of phorbol ester binding sites or protein levels of classical PKC isoforms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drug treatment
After incubation of the slices, to equilibrate their cAMP level,
they were treated with vehicle or drug for 20 min. Each slice received
either vehicle, agonist alone, 1 µM
phorbol-12,13-dibutyrate (PDB) alone, or agonist and 1 µM
PDB dissolved in aCSF. Agonist treatments were either 10
µM forskolin (FOR), 100 µM adenosine (ADE),
or 10 µM isoproterenol (ISO). Each experiment used tissue
from two control and two EB-treated animals, and each experiment was
repeated at least four times.
cAMP determination
After drug treatment, the slices were disrupted by sonication in
5% ice-cold trichloroacetic acid. The supernatant containing cAMP and
the pellet containing tissue protein were separated by centrifugation.
The supernatant was acidified with 1 N HCl and extracted
four times with hydrated ether. The samples were dried by
lyophilization and analyzed for cAMP concentration using a modified
Gilman cAMP assay (18). The protein concentration was determined by
resuspending the pellet in 2 N NaOH and assaying by a
modified Lowry assay (19). All cAMP values were expressed as pmoles
cAMP per milligram protein.
PKC extraction
OVX female Sprague-Dawley rats (150–175 g) were injected sc
twice with either 0.1 ml peanut oil (control) or 2 µg of EB dissolved
in 0.1 ml peanut oil at 24 and 48 h before decapitation. Animals
were decapitated, and the brains were removed and washed with PBS. The
HYP-POA, frontal cortex (CTX), pituitary or HYP, and POA separately
were dissected and homogenized in a dounce homogenizer in extraction
buffer [20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5
mM EGTA, 0.5% Triton X-100, and 25 mg/ml each aprotinin
and leupeptin]. The HYP-POA was dissected with an anterior cut
approximately 2 mm anterior to the optic chiasm, a posterior cut just
caudal to the mammillary bodies, lateral cuts at the hypothalamic
fissures, and a final cut 4 mm dorsal to the ventral surface. The HYP
and POA were divided by cutting the HYP-POA block just posterior to the
optic chiasm. The homogenate was incubated on ice for 30 min, then
centrifuged for 2 min at 10,000 x g. The supernatant
was loaded onto a previously poured diethylaminoethyl-cellulose column.
The column was made by suspending 0.5 g Whatman DE 52 in 1 ml
washing buffer [20 mM Tris (pH 7.5), 0.5 mM
EDTA, 0.5 mM EGTA, and 10 mM
ß-mercaptoethanol]. Columns were washed with 2 ml washing buffer and
stored in a refrigerator until use. The loaded column was washed with 8
ml washing buffer. After washing, PKC was extracted from the column
with 5 ml extraction buffer [20 mM Tris (pH 7.5), 0.5
mM EDTA, 0.5 mM EGTA, 10 mM
ß-mercaptoethanol, and 0.2 M NaCl]. In total, 5 ml
extraction buffer was recovered. The protein concentration in the
column eluate was determined using a modified Lowry assay.
PKC assay
Protein extracts were assayed for PKC activity using a kit (Life
Technologies, Gaithersburg, MD) (20). The assay was linear between 2
and 8 µg protein (data not shown). Samples were assayed for 5 min at
30 C in a mixture containing approximately 5 µg protein extract, 50
µM Ac-MBP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) (an acetylated synthetic peptide
corresponding to amino acids 4–14 of myelin basic protein, which is a
general PKC substrate), 20 µM ATP, 1 mM
CaCl2, 20 mM MgCl2, 4
mM Tris (pH 7.5), 0.1 µCi 32P ATP, 10
µM phorbol 12-myristate 13-acetate, 280 µg/ml
phosphatidyl serine, and Triton X-100 mixed micelles. Nonspecific
activity was determined in the same manner as total activity, except
protein extract was preincubated for 20 min with 20 mM PKC
(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (a synthetic peptide corresponding to the pseudosubstrate
region of PKC that specifically inhibits PKC) at room temperature.
Four replicates were done of the specific activity and two of the
nonspecific activity. After the enzyme activation and incubation, half
of the sample was spotted onto phosphocellulose paper and washed in 1%
phosphoric acid. Phosphocellulose spotted discs were counted in a
scintillation counter. Total PKC activity was determined by subtracting
PKC activity in the presence of inhibitor peptide from total phorbol
12-myristate 13-acetate-stimulated activity.
[3H] PDB binding
Tissue protein was extracted in the same manner as for the PKC
activity assay, from the HYP or POA combined from three animals or from
the CTX from a single animal. Binding conditions were derived from a
combination of several protocols (21, 22, 23, 24, 25, 26, 27, 28, 29). Final concentrations of
reagents were 20 mM Tris-HCl (pH 7.5), 500 µg/ml bovine
-globulin, 0.5 mM CaCl2, 5 mM
MgCl2, 10 µg/ml phosphatidylserine, and 40 µg/ml
phosphatidylcholine. Before the binding reaction, the components of the
binding buffer were vigorously vortexed and sonicated. Binding buffer,
protein extract (
15 µg for CTX; > 100 µg for HYP and POA), and
[3H] PDB (Amersham Life Sciences, Arlington Heights, IL;
17.2 Ci/mmol), at seven concentrations ranging from 0.5–20
nM, with or without 100 µM cold PDB, were
added in triplicate to glass tubes (0.25 ml final vol). The tubes were
incubated at room temperature for 1 h. Mixtures were then filtered
three times with PBS through a cell harvester onto GF/B filters, which
were presoaked for 1 h in 0.3% polyethyleneimine. Filters were
placed into vials, filled with scintillation fluid, and counted in a
liquid scintillation counter. Data were plotted and analyzed by means
of Scatchard analysis using the computer program EBDA.
Immunoblots
Protein was prepared by dounce homogenization of tissue (HYP,
POA, or combined HYP-POA) in 1% SDS, 1 mM sodium vanadate,
and 10 mM Tris-HCl, pH 7.4. After homogenization, tubes
were boiled for 10 min and microwaved for 10–15 sec. Samples were spun
for 5 min at 10,000 x g to remove insoluble material.
Protein concentrations were determined by a modified Lowry assay.
Twenty-five micrograms of protein, in duplicate, were applied to 12.5%
SDS-polyacrylamide minigels and resolved at 150 volts for 1.5 h.
Proteins were transferred electrophorectically to nitrocellulose
membranes at 100 A for 1 h. Membranes were then blocked for
0.5 h in 5% nonfat dry milk in Tris-buffered saline (TBS).
Membranes were incubated for 1 h at 37 C with primary antibody.
Primary antibodies used were ß PKC rabbit polyclonal antibody
(Life Technologies; 1:500 dilution in 5% milk and 0.1% goat serum),
PKC mouse monoclonal antibody (Transduction Laboratories,
Lexington, KY; 1:250 dilution in 5% milk and 0.1% goat serum) and
ß-tubulin type I and II mouse monoclonal antibody (Sigma, St. Louis,
MO; 1:500 dilution in 5% milk and 0.1% goat serum). After four washes
with 0.1% Tween 20 in TBS and two washes in 1x TBS, blots were
incubated at room temperature for 1 h with horseradish
peroxidase-conjugated antirabbit or antimouse secondary antibodies
(Boehringer Mannheim, Indianapolis, IN; 1:1000 dilution in 5% milk and
0.1% goat serum), where appropriate. Blots were then washed in the
same manner as for the primary antibody. Peroxidase activity was
visualized by means of chemiluminescence (Renaissance, New England
Nuclear, Boston, MA).
Blots were exposed to film, at two different times, to obtain signals within the linear range of the film and the densitometer. Standard curves were generated for each antibody, using 5–50 µg whole rat brain protein extract, and these produced linearly increasing signals over the entire range of starting protein. Twenty-five micrograms of protein extract was chosen for semiquantitative analysis, because signal reliably fell within the linear range of the film. Membranes were blotted for ß-tubulin after initial blotting for PKC isoforms. ß-tubulin blots were used as a control to verify that protein load was comparable among samples. Data were expressed as a ratio of OD of the PKC isoform signal divided by OD of the ß-tubulin signal. ODs were obtained by scanning autoradiograms on a Molecular Dynamics densitometer and analyzing the image using Molecular Dynamics ImageQuant v3.03 (Molecular Dynamics, Sunnyvale, CA).
Statistics
PKC activity data in control and hormone-treated extracts were
analyzed using Student’s t tests for independent samples.
PDB binding parameters were also analyzed using Student’s t
tests for independent samples. Data obtained from cAMP experiments were
analyzed using two-way ANOVA, with drug treatment and hormone as the
two factors. Significant differences between means were determined for
main effects using Tukey’s test and least-square means for
interactions. Differences were considered significant if
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). PKC catalytic activity was
higher in extracts from CTX than from the combined HYP-POA, and very
low in pituitary (data not shown). To test whether estradiol directly
modified PKC catalytic activity, 100 nM estradiol or 100
nM EB was added to combined HYP-POA protein extracts from
OVX control rats before assaying PKC catalytic activity. In
vitro addition of estradiol or EB had no effect on PKC catalytic
activity in combined HYP-POA extracts (Table 1).
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). In HYP
extracts, no difference was seen between extracts from control and
EB-treated animals (Fig. 1B).
Effects of EB treatment on PKC potentiation of cAMP
accumulation
Activation of PKC can increase cAMP synthesis by potentiating both
receptor-dependent and receptor-independent activity of adenylyl
cyclase (AC). This effect of PKC occurs in the HYP and POA of female
rats (6, 7, 8). This experiment used the phorbol ester PDB to examine
whether brain slices from the HYP or POA of EB-treated animals show
greater PKC potentiation of AC than slices from control OVX females. As
expected, in EB-treated and control slices, PDB alone had no effect on
cAMP accumulation (Fig. 2). The
receptor-independent activator of AC, FOR, stimulated cAMP accumulation
(P < 0.001 vs. basal). PDB potentiated
FOR-stimulated cAMP accumulation in control and EB-treated slices
(P < 0.01 vs. FOR alone). Finally (as
expected, on the basis of PKC catalytic activity assays), EB treatment
increased the ability of PKC to potentiate FOR-stimulated cAMP
accumulation in POA slices (P < 0.05; FOR+PDB in EB
vs. FOR+PDB in oil; Fig. 2A) but not in HYP slices.
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). PDB binding affinity and the density of PDB binding sites
were the same in POA, HYP, and CTX extracts from OVX control and
EB-treated animals (Fig. 3; Table 3). The number of PDB binding sites was
highest in CTX, in agreement with published literature (26, 28, 29, 30).
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, ß, and isoforms, were done on combined HYP-POA tissue.
EB treatment for 48 h had no effect on PKC immunoreactivity for
the combined , ß, and isoforms (Fig. 4A). Immunoblots with a monoclonal PKC
antibody recognizing only the isoform were done on separate HYP and
POA extracts. The isoform is highly expressed in brain, so we
wished to verify that a hormone-dependent change in PKC
immunoreactivity was not missed in the initial blots because they were
done on combined HYP-POA using a PKC antibody that recognizes multiple
isoforms. EB treatment for 48 h did not affect PKC
immunoreactivity in the HYP (data not shown) or the POA (Fig. 4B).
Immunoblots with a monoclonal PKC antibody specific for the 
isoform
were done on POA extracts. PKC immunoreactivity in the POA did not
increase after EB treatment (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen effects on G-protein-coupled receptors may explain the
discrepancy between the results on receptor-independent and
receptor-dependent activation of AC. Previously, we showed that
ß-adrenergic receptors were uncoupled from G-proteins in the HYP and
POA of estrogen-treated animals (31). Therefore, activation of AC by
ß-adrenergic receptors was suppressed in estrogen-treated
animals. The present data confirmed that activation of ß-adrenergic
receptors with ISO increased cAMP accumulation in control but not in
EB-treated slices (Table 2). That there was no difference between cAMP
levels of estrogen-exposed and control slices after treatment with ISO
and PDB, despite the absence of an ISO response in hormone-treated
slices, suggests that PKC potentiation of receptor-dependent AC
activity may have increased in slices from estrogen-treated animals.
Estrogen treatment did not suppress ADE receptor activation of AC or
modify PKC potentiation of the ADE response. This may suggest that
estrogen does not regulate PKC in cells expressing ADE receptors.
In an attempt to identify which PKC isoforms are regulated by estrogen,
we carried out PDB binding and immunoblots for specific PKC isoforms.
Neither PDB binding affinity nor the density of PDB binding sites
differed in any brain region as a function of estrogen treatment.
Semiquantitative immunoblots, using antibodies recognizing either the
classical (, ß, and ) PKC isoforms or the PKC isoform
specifically, detected no differences in POA extracts from animals with
and without estrogen treatment. These results suggest that either the
protein levels of the phorbol ester-sensitive PKC isoforms are not
elevated by estrogen or that PDB binding and PKC immunoblots are not
sensitive enough methods to detect small differences in protein
expression.
Some information about the identity of PKC isoforms affected by
estrogen can be deduced from the methods used to evaluate PKC catalytic
activity. The PKC activity assay used a substrate that is
preferentially phosphorylated by the classical (, ß, and ) and
novel (
, , 
, µ, and 
) PKC isoforms (21, 28). The assay
also measured phorbol ester-stimulated kinase activity, which would
predominantly measure the classical and novel PKC isoforms. Moreover,
we used PDB, a phorbol ester, to activate PKC in the cAMP studies.
Therefore, estrogen regulation of PKC catalytic activity in the POA is
likely to involve phorbol ester-sensitive PKC isoforms. Estrogen could
modulate PKC catalytic activity by influencing PKC phosphorylation
state or PKC helper proteins, such as receptor for activated C-kinase
(32, 33, 34, 35), rather than by increasing protein expression.
Estrogen elevation of PKC catalytic activity in the POA may have
implications for noradrenergic actions that are vital for gonadotropin
release and reproductive behavior (1, 2, 3). PKC is a downstream mediator
of 1B-adrenergic receptor signaling in the POA and
elsewhere (2, 6, 7, 8, 36). We have already shown that
1B-adrenergic receptor binding and mRNA are elevated in
the HYP-POA of estrogen-treated female rats (4, 5). Estrogen treatment
of OVX female rats thus elevates signaling through the
1B-adrenergic pathway in the HYP-POA. Another lab has
shown increased phospholipase C- mRNA in the rat uterus and
ventromedial HYP after estrogen treatment (37). Because PKC is a
mediator of 1B-adrenergic signaling, increased PKC
catalytic activity would further potentiate any response caused by
activation of 1B-adrenergic receptors.
Another consequence of elevated PKC activity may be desensitization of
other G-protein-coupled receptors. PKC can promote heterologous
desensitization, which is the phosphorylation and subsequent uncoupling
of a G-protein-coupled receptor from its associated G-protein (38, 39).
PKC phosphorylation can also elevate G-protein-coupled receptor kinase
activity, which is responsible for homologous desensitization (38). Our
laboratory showed that estrogen treatment of OVX female rats uncouples
both 2- and ß-adrenergic receptors from G-proteins in
the HYP-POA (31, 40). Thus, increased PKC catalytic activity, by
promoting either heterologous or homologous desensitization, could be
involved in these processes.
Elevation of PKC catalytic activity could also have a wide range of consequences on gene transcription in cells of the POA. In mammary and ovarian cells, PKC potentiates the ability of liganded estrogen receptor to augment gene transcription by phosphorylation of the estrogen receptor (41, 42, 43, 44, 45). Both PKC and estrogen receptor can also augment transcription by increasing the phosphorylation or protein level of transcription factors such as c-fos and c-jun. (42, 43, 46, 47, 48). Therefore, PKC may contribute to estrogen facilitation of reproductive function by amplifying estrogen actions on target genes (49, 50).
In summary, estrogen elevated PKC catalytic activity in the POA, an estrogen-responsive brain region, but not in the HYP. This elevation of PKC catalytic activity seemed to have functional relevance, in that the ability of PKC to potentiate AC activity is also increased by estrogen. Finally, immunoblots and PDB binding were unable to detect a change in PKC protein expression in the POA of estrogen-treated rats. Therefore, future studies will need to examine whether the increased PKC catalytic activity in estrogen-treated rats is caused by increased expression of one or more PKC isoforms or an alteration in the activity of the PKC isoforms already present.
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Acknowledgments |
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Footnotes |
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Received December 4, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 but not ß or 2
noradrenergic receptors in hypothalamus of female rats. Neurochem Int 16:1–9
1b-adrenoceptor mRNA in female rat
hypothalamus-preoptic area. J Neuroendocrinol 8:449–455[CrossRef][Medline]
1- and ß-adrenergic
intercommunication in rat hypothalamic slices. J Neurochem 56:628–635[CrossRef][Medline]
protein kinase C during
rat ovarian differentiation. Biochim Biophys Acta 1179:260–270[Medline]
protein kinase C in the rat ovary: estrogen regulation and
localization. Endocrinology 135:1669–1678[Abstract]
protein and mRNA levels in the
rabbit corpus luteum. Mol Cell Endocrinol 122:213–221[CrossRef][Medline]
messenger ribonucleic acid in the rat
uterus and hypothalamus. Endocrinology 133:99–1042 -adrenoceptor in
the hypothalamus of female rats: identification as the
2D subtype. Mol Pharmacol 45:509–516[Abstract]
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