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The Journal of Neuroscience, April 1, 1999, 19(7):2413-2423
Role of Phosphatidylinositol 3-Kinase in Angiotensin II
Regulation of Norepinephrine Neuromodulation in Brain Neurons of the
Spontaneously Hypertensive Rat
Hong
Yang and
Mohan K.
Raizada
Department of Physiology, College of Medicine, and University of
Florida Brain Institute, Gainesville, Florida 32610
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ABSTRACT |
Chronic stimulation of norepinephrine (NE) neuromodulation by
angiotensin II (Ang II) involves activation of the Ras RafMAP kinase
signal transduction pathway in Wistar Kyoto (WKY) rat brain neurons.
This pathway is only partially responsible for this heightened action
of Ang II in the spontaneously hypertensive rat (SHR) brain neurons. In
this study, we demonstrate that the MAP kinase-independent signaling
pathway in the SHR neuron involves activation of PI3-kinase and protein
kinase B (PKB/Akt). Ang II stimulated PI3-kinase activity in both WKY
and SHR brain neurons and was accompanied by its translocation from the
cytoplasmic to the nuclear compartment. Although the magnitude of
stimulation by Ang II was comparable, the stimulation was more
persistent in the SHR neuron compared with the WKY rat neuron.
Inhibition of PI3-kinase had no significant effect in the WKY rat
neuron. However, it caused a 40-50% attenuation of the Ang II-induced
increase in norepinephrine transporter (NET) and tyrosine hydroxylase
(TH) mRNAs and [3H]-NE uptake in the SHR neuron.
In contrast, inhibition of MAP kinase completely attenuated Ang II
stimulation of NET and TH mRNA levels in the WKY rat neuron, whereas it
caused only a 45% decrease in the SHR neuron. However, an additive
attenuation was observed when both kinases of the SHR neurons were
inhibited. Ang II also stimulated PKB/Akt activity in both WKY and SHR
neurons. This stimulation was 30% higher and lasted longer in the SHR
neuron compared with the WKY rat neuron. In conclusion, these
observations demonstrate an exclusive involvement of
PI3-kinase-PKB-dependent signaling pathway in a heightened NE
neuromodulatory action of Ang II in the SHR neuron. Thus, this study
offers an excellent potential for the development of new therapies for
the treatment of centrally mediated hypertension.
Key words:
PI3-kinase; protein kinase B; angiotensin; neuromodulation; spontaneously hypertensive rat; neuron
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INTRODUCTION |
The key in the central actions of
angiotensin II (Ang II) in the control of blood pressure (BP) is the
interaction of Ang II with the neuronal AT1 receptor
subtype in the cardioregulatory-relevant brain areas, such as
hypothalamic and brainstem nuclei (Saavedra 1992 ; Steckelings et
al., 1992; Timmermans et al., 1993; Raizada et al., 1994, 1998; Wright
and Harding, 1994). Physiological mechanisms associated with this
action are well understood and involve activation of sympathetic
pathways, increased vasopressin secretion, and dampening of
baroreceptor reflexes (Phillips, 1987; Saavedra, 1992; Steckelings et
al., 1992; Sumners and Raizada, 1993; Timmermans et al., 1993; Raizada
et al., 1994, 1998; Wright and Harding, 1994; Sumners et al., 1995).
Despite this, the cellular and molecular bases of these physiological
actions of brain Ang II in BP control are not clearly established.
Recent attempts to link Ang II activation of sympathetic pathways with
the stimulation of norepinephrine transporter (NET) activity and
transcriptional control of the synthesis and release of norepinephrine
(NE) have provided a major step forward toward our understanding of the
brain Ang II at a molecular level (Sumners and Raizada, 1993; Sumners
et al., 1995; Lu et al., 1996a; Yu et al., 1996a; Raizada et al.,
1998). These studies have established that enhanced or
chronic stimulation of NE neuromodulatory activity is a result of
transcriptional regulation of genes for NET, tyrosine hydroxylase (TH),
and dopamine  -hydroxylase (DH) in normotensive Wistar Kyoto (WKY)
rat neuron (Lu et al., 1996a; Yu et al., 1996a; Gelband et al., 1998).
This involves activation of the Ras-Raf-MAP kinase signaling pathway
(Lu et al., 1996b; Yang et al., 1996a; Gelband et al., 1998).
Activation of this cascade of kinases leads to an increased serum
response element (SRE) and activator protein-1 (AP-1) binding
activities that are associated with the stimulation of genes relevant
to the synthesis and release of catecholamines (Lu et al., 1996b;
Gelband et al., 1998).
Recent evidence implicates the brain Ang system in the development and
establishment of hypertension (Raizada et al., 1994, 1998; Sumners et
al., 1995). Thus, the cellular basis of the physiological actions of
brain Ang II could be important in the design of novel therapies for
treatment of the hypertensive state. This view is strengthened by the
studies with a genetic model for primary hypertension in humans,
spontaneously hypertensive rat (SHR), which have shown that the brain
Ang system is hyperactive as a result of an increased expression of
AT1 receptors (Phillips, 1987; Raizada et al., 1994, 1998;
Sumners et al., 1995). This is associated with an increased turnover
and synthesis of CA by Ang II in the SHR brain (Ding et al., 1990; Yang
et al., 1991). A similar hyperactivity of the brain Ang system has been
reported in a renin transgenic model of hypertension (Langheinrich et
al., 1996). Finally, interruption in the expression of brain Ang
hyperactivity by either pharmacological or genetic means normalizes BP
in the SHR and not in normotensive, WKY rats (Berecek et al., 1983;
Toney and Porter et al., 1993; Phillips et al., 1994). These studies
have lead to the hypothesis that a hyperactive brain Ang system is a
result of a heightened stimulation of signaling kinases by Ang II in
the SHR neuron.
Our group has established an in vitro neuronal cell culture
model from the hypothalamic-brainstem areas of WKY and SHR brains that
provides evidence in support of this hypothesis (Sumners and Raizada,
1993; Raizada et al., 1994, 1998; Sumners et al., 1995). These studies
have demonstrated that the in vivo expression of a
hyperactive brain Ang system is maintained in the neuronal cultures of
the SHR. This includes increased expression of AT1 receptors and increased Ang II stimulation of NE neuromodulation in the
SHR neuron compared with the WKY rat neuron (Raizada et al., 1993; Lu
et al., 1996a; Gelband et al., 1998). These alterations are specific
for the AT1 receptor system, because the AT2
receptor and its signaling pathway are not altered (Sumners et al.,
1995; Raizada et al., 1998). Thus, the in vitro neuronal
cell culture system provides an excellent model for comparing the
AT1 receptor-mediated signal transduction mechanism and
determining the basis for heightened neuromodulatory actions of Ang II
in the SHR neuron.
In previous studies we focused our attention on the RasRafMAP
kinase pathway and postulated that an increased NE neuromodulation would be attributable to increased Ang II stimulation of this signaling
system in the SHR neuron. However, our data demonstrated that although
completely responsible for Ang II action in the WKY rat neuron, the
RasRafMAP kinase pathway is only a partial player in the SHR neuron
(Yang and Raizada, 1998). These observations led us to propose the
presence of a MAP kinase-independent signaling pathway that must
account for an increased NE neuromodulation effect of Ang II in the SHR
neuron (Yang and Raizada, 1998). We hypothesized that the PI3-kinase
signaling system could be such a pathway in the SHR. The rationale for
this conclusion included the following: calcineurin/nuclear factor of
activated T cells or protein kinase A and protein tyrosine kinase did
not appear to participate in this signaling (Yang and Raizada, 1998);
some G-protein-coupled receptors are capable of coupling to the
PI3-kinase signaling system (Lopez-Ilasaca et al., 1997; Hazeki et al.,
1998; Leopoldt et al., 1998); and Ang II has recently been shown to stimulate PI3-kinase activity in vascular smooth muscle cells (Saward
and Zahradka, 1997). These cells are one of the major targets of
peripheral actions of Ang II (Griendling et al., 1993). Because neurons
are the primary target for the brain actions of Ang II, it was of
obvious significance to explore this hypothesis.
Observations presented in this study demonstrate that although Ang II
stimulates PI3-kinase in both WKY and SHR neurons, its effect on the
SHR neuron is more persistent and longer lasting. The activation
accounts for MAP kinase-independent signaling in Ang II stimulation of
NE neuromodulation in the SHR. Our data also establish that this effect
involves activation of PKB/Akt, whose stimulation by Ang II is more
prolonged and significantly higher in the SHR neuron.
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MATERIALS AND METHODS |
Materials
WKY rats and SHRs (1 d old) were obtained from our breeding
colony, which originated from Harlan Sprague Dawley (Indianapolis, IN).
Blood pressure for breeder WKY rats was an average of 117 ± 5 mmHg, whereas it was 205 ± 8 mmHg for the SHRs. DMEM,
plasma-derived horse serum (PDHS), and 1× crystallized trypsin were
from Central Biomedia (Irwin, MO). [ -32P]-ATP (3000 Ci/mmol), [-32P]-dCTP (3000 Ci/mmol),
[3H]-NE, and chemiluminescence assay reagents were
from DuPont/NEN (Boston, MA). Nitrocellulose membranes were from Micron
Separations (Westboro, MA). Ang II, Wortmannin, and
polyethyleneimine-cellulose thin-layer plates were purchased from Sigma
(St. Louis, MO). Losartan potassium was a gift from DuPont/Merck
(Wilmington, DE). PD123319 was from RBI (Natick, MA). Superscript Rnase
H reverse transcriptase and deoxynucleotide
mixture were from Life Technologies (Grant Island, NY). Dynal beads and
other reagents for poly(A+) RNA isolation were from
Dynal (Lake Success, NY). Oligo-dT and Taq DNA
polymerase were purchased from Promega (Madison, WI). Anti-PI3-kinase
p85 and PKB/Akt were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). All other reagents were purchased from Fisher Scientific
(Pittsburgh, PA) and were the highest quality available.
Primers for TH, NET, and -actin were synthesized in the DNA
synthesis facility of the Interdisciplinary Center for Biotechnology for Research, University of Florida (Gainesville, FL). The sequences of
these primers have been published previously (Lu et al., 1996a; Yu et
al., 1996a).
Methods
Preparation of hypothalamus-brainstem neuronal cultures
from WKY and SHR brains. Neuronal cultures were prepared
essentially as described previously (Raizada et al., 1984, 1993, 1994).
Briefly, hypothalamus-brainstem of 1-d-old WKY and SHR brains, which
contained the paraventricular nucleus, the supraoptic, anterior,
lateral, posterior dorsomedial and ventromedial nuclei, the medulla
oblongata, and the pons, were dissected, and brain cells were
dissociated by trypsin. Cells were plated on
poly-L-lysine-precoated tissue-culture dishes in DMEM
containing 10% PDHS. Culture dishes of 35 mm diameter (3 × 106 cells) or 100 mm diameter (1.5 × 107 cells) were prepared for experiments. Cultures
were treated with 1% cytosine arabinoside for 3 d followed by
establishment of cultures for 10 d before their use in the
experiments. These cultures have been shown to contain 90-95%
neuronal cells and 5-10% astroglial cells (Raizada et al., 1984,
1993, 1994). Properties of neuronal cultures from the WKY brain are
comparable with the SHR brain in regard to neuronal cells per dish,
total protein per dish, catecholamines present, and many other
biochemical criteria. Thus, they have been used extensively by our
in vitro model to study hyperactivity of the brain
AT1 receptor function and its interaction with
catecholamines in the SHR brain (Raizada et al., 1984, 1993, 1994).
PI3-kinase assay. PI3-kinase activity in extracts from
neuronal cultures and in hypothalamic brain areas was measured
essentially as described previously (Kaplan et al., 1986, 1987).
Briefly, neuronal cell lysates were prepared in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 50 mM NaF, 1% NP-40, 1 mM EDTA, 1 mM
Na3VO4, 1 mM PMSF, 2 µg/ml
aprotinin, 2 µg/ml leupeptin). After centrifugation at 12,000 × g for 10 min at 4°C, lysates were used for
immunoprecipitation with anti-PI3-kinase p85 antibody essentially as
described previously (Kaplan et al., 1986, 1987). Immunoprecipitates
were washed twice in lysis buffer and twice in 0.5 M LiCl,
100 mM HEPES, pH 7.5, and once with 10 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA. They
were then incubated in PI3-kinase reaction buffer [20 mM HEPES, pH 7.4, 30 mM MgCl2, 50 µM ATP, 200 µM adenosine, 200 µg of PI,
10 µCi of [-32P] ATP (3000 Ci/mmol)] for 20 min at
room temperature. Reactions were stopped by the addition of 100 µl of
1.0N HCl and 200 µl of a 1:1 mixture of chloroform/methanol, and
lipids in the organic phase were resolved on TLC plates developed in
chloroform/methanol/H2O/25% NH4OH (43:38:7:5).
Radioactive phosphatidylinositol 3-monophosphate (PIP) products were
visualized by autoradiography and quantitated (Lu et al., 1996b; Yang
et al., 1996a).
Measurement of protein kinase B (PKB/Akt) activity. PKB
activity was measured by a previously published method (Franke et al.,
1995; Stokoe et al., 1997). In brief, the protocol was as follows.
Neuronal cultures were extracted in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10%
glycerol, 1% NP-40, 10 mM NaF, 1 mM
Na3VO4, 1 mM
Na4O7P2, 2 µM
leupeptin, 2 µM aprotinin, and 1 mM PMSF.
Lysates were centrifuged for 10 min at 12,000 × g. The
PKB protein was immunoprecipitated from cell-free extracts (200 µg
protein) with the use of PKB-specific polyclonal antibody. Immune
complexes were collected with protein A/G plus-agarose and washed three
times with lysis buffer, and once with kinase assay buffer (20 mM HEPES, pH 7.4, 1 mM DTT, 10 mM
MnCl2, 10 mM MgCl2).
In vitro kinase assays were performed at 30°C in a 50 µl
reaction volume of kinase assay buffer containing 5 µM
ATP, 100 µg/ml histone 2B (H2B), and 10 µCi of
[-32P] ATP per sample. The reactions were stopped with
5× SDS-PAGE sample buffer, and reaction products were separated by
SDS-PAGE gel and dried for autoradiography. Phosphorylated H2B
was quantitated by ultraviolet products Imagestore 5000 system (Lu et
al., 1996b; Yang et al., 1996a).
Western blotting for PI3-kinase and PKB/Akt. Neuronal
cell-free lysates were prepared as described above for the enzyme
assay. Lysates were electrophoresed on 10% SDS/PAGE, and proteins were transferred to nitrocellulose membranes. Membranes were blocked by
using 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) for 1 hr followed by
incubation for 1 hr at room temperature with anti-PI3-kinase p85
antibody or anti-PKB antibody as described previously (Yang et al.,
1996a). Protein-bound primary antibody was detected by using
horseradish peroxidase-labeled secondary antibody and enhanced by
chemiluminescence assay reagent (Lu et al., 1996b; Yang et al., 1996a).
The bands recognized by the primary antibody were visualized by
autoradiography and quantitated essentially as described previously (Lu
et al., 1996b; Yang et al., 1996a).
Preparation and cytoplasmic and nuclear extracts from neuronal
cultures. Neuronal cultures from WKY and SHR that had been established on 100-mm-diameter culture dishes were used to prepare cytoplasmic and nuclear fractions to determine Ang II-induced redistribution of PI3-kinase and PKB immunoreactivity. The protocol was
established previously (Lu et al., 1996b; Yang et al., 1997).
Measurement of AP-1 binding activity by gel shift assay. The
protocol was essentially the same as described previously (Lu et al.,
1996b). In brief, 3 pmol of each oligonucleotide was first labeled with
[-32P] ATP at the 5' end by 10 min incubation at
37°C with 4 µ of T4-polynucleotide kinase in 10 µl
solution containing 70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, and 10 µCi of [-32P] ATP. The reaction was stopped by the
addition of 1 µl of 0.5 M EDTA, and the volume was
expanded to 100 µl with 1× gel-shift assay buffer.
[32P]-labeled oligonucleotides (0.1 µCi) were
mixed with 5 µg of nuclear extract protein containing 1× gel-shift
assay buffer, 100 mg of salmon sperm DNA, 2 µg of DNA duplex poly
(dl-dc), and 10 µg of BSA in a final volume of 10 µl. The mixture
was incubated at room temperature for 15 min and electrophoresed on a
5% PAGE gel in 20 mM Tris-acetate buffer containing 5 nM EDTA, pH 8.0, for 90 min at a constant 200 V. The gel
was decasted, wrapped in a plastic bag, and exposed and analyzed
by an Imagestore system (Ultra Violet Products, San Gabriel, CA). The
densities of shifted bands on the gel representing the
oligonucleotide-protein complexes were quantitated by the SW 5000 Gel
Analysis program (Lu et al., 1996b; Yang et al., 1996a).
Measurements of norepinephrine neuromodulation activity.
Uptake of [3H]-NE and levels of NET and TH mRNAs
were used as parameters of the effects of Ang II on NE neuromodulation.
The effect of Ang II on specific uptake of [3H]-NE
by neuronal cells was determined as described previously (Lu et al.,
1996a). Levels of NET, TH, and -actin mRNAs were measured and
quantitated essentially as described elsewhere (Lu et al., 1996b, 1998;
Yang et al., 1996a).
Measurement of the effects of Ang II on PI3-kinase in vivo.
Adult male WKY rats and SHRs were housed singly after being fitted with
an indwelling cannula (10 mm long, 23 gauge stainless steel) stereotaxically aimed to end in or just above the lumen of the right
lateral ventricle and firmly fixed to the skull with jeweler's screws
and dental acrylic (Yang et al., 1996b). Rats were allowed to recover
for 1 week before the experiments. Injections of Ang II were made
through an 11 mm gauge injector needle attached to a 35 mm syringe.
Five microliters of either PBS, pH 7.4, or PBS containing 10 ng of Ang
II were injected into each rat. Hypothalami were dissected, and tissues
were processed for the measurement of PI3-kinase activity as described above.
Experimental groups and data analysis. Each experiment for
the effect of Ang II on the activation of PI3-kinase was conducted in
triplicate culture dishes, and cells in each dish were derived from
multiple brains of 1-d-old rats. WKY and SHR brain neurons were used in
parallel for each experiment, and both samples were run on the same gel
to minimize experimental variations. Triplicate hypothalami were used
to determine the effect of Ang II on PI3-kinase in vivo.
Each experiment was repeated at least three times, unless indicated
otherwise. [3H]-NE uptake and release were
measured with the use of the same number of cultures in triplicate
dishes. For the analysis of mRNA levels, triplicate culture dishes were
used for each data point, and poly(A+) RNA was
pooled. Each experiment was repeated three times unless indicated
otherwise. Densities of PCR bands were quantified, and data were
presented as relative absorbance of the mean ± SE derived from
normalization with -actin for equal loading (Lu et al., 1996b, 1998;
Yang et al., 1996a). Comparisons between the control and experimental
groups were made using t test with the use of statistical software.
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RESULTS |
Effects of Ang II on PI3-kinase in WKY and SHR neurons
A time-dependent stimulation of PI3-kinase activity was observed
when WKY rat brain neurons were incubated with 100 nM Ang II. The increase was transient. A significant stimulation was seen as
early as 1 min; it reached maximal levels of ~3.6-fold within 5 min.
By 30 min the activity returned to control levels (Fig.
1A). The stimulation
was mediated by the interaction of Ang II with the AT1
receptor subtype, because it was completely blocked by 10 µM losartan but not by PD 123319, an AT2
receptor subtype antagonist (Fig. 1B). The
stimulation was also Ang II concentration-dependent, and 100 nM of Ang II provided a maximal stimulation of 3.8-fold
(Fig. 1C). The effect of Ang II on PI3-kinase activity
between WKY and SHR neurons was compared, because neurons from SHR
express two- to fourfold higher levels of AT1 receptors and
a parallel increase in NE neuromodulation (Lu et al., 1996a; Yu et al.,
1996a; Raizada et al., 1998). Despite these increases, the stimulation
of PI3-kinase by Ang II was comparable (~3.6-fold) in both strains of
neurons (Fig. 1). However, the time course of inactivation after Ang II
stimulation was significantly delayed in the SHR neuron. As a result,
PI3-kinase activity remained maximally stimulated at 30 min in the SHR,
whereas it returned to control levels in the WKY rat neuron (Fig.
1A). In fact, the enzyme activity was approximately
twofold higher even at 60 min in the SHR neuron compared with the WKY
rat neuron.
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Figure 1.
A, Effect of Ang II on PI3-kinase
activity in WKY and SHR brain neurons. Time course: neuronal cultures
of WKY and SHR were incubated with 100 nM Ang II for the
indicated time periods. PI3 kinase activity was measured after
immunoprecipitation with the PI3-kinase p85 subunit-specific antibody
essentially as described in Materials and Methods. Top,
Representative autoradiogram of a TLC plate. Bottom,
Quantitation of radioactive spots corresponding to PIP, mean ± SE
(n = 3 experiments). * Significantly different
from zero time (p < 0.01). # Significantly
different from Ang II-treated WKY rat neuron
(p < 0.01). B, Effect of Ang
receptor antagonists. WKY and SHR neuronal cultures were incubated
without (Control) or with (2, 4, 6) 100 nM Ang II for 10 min in the absence
or presence of 10 µM losartan (Los) or PD
123319 (PD). PI3-kinase activity was determined as
described in Materials and Methods. Top, Representative
autoradiogram. Bottom, Quantitation of radioactive spot,
mean ± SE (n = 3). * Significantly different
from control (p < 0.0l). C,
Ang II concentration. Incubation and assay conditions with indicated
concentration of Ang II for 10 min were essentially as described in
A. Top, Representative autoradiogram.
Bottom, Data from three experiments ± SE.
* Significantly different from no Ang II (p < 0.05).
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The effect of Ang II on PI3-kinase activity was performed in intact
hypothalamus from adult WKY rats and SHRs to validate that the effects
observed in primary neuronal cultures are not an artifact of culture
conditions. Figure 2 shows that similar to neuronal cultures, Ang II caused a three- to fourfold stimulation of
PI3-kinase in 15 min, and the levels of this stimulation were comparable in WKY and SHR hypothalamus. In addition, the stimulation was persistent even at 30 min in the SHR, whereas it was completely reversed in the WKY rat hypothalamus (Fig. 2). Ang II stimulation of
PI3-kinase activity in neuronal cultures as well as in intact hypothalamus of both WKY and SHR did not alter the immunoreactive PI3-kinase levels (Fig. 3).
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Figure 2.
Effect of Ang II on PI3-kinase activity in
hypothalami of adult WKY rats and SHRs. WKY rats and SHRs were
cannulated and subjected to Ang II injection and isolation of
hypothalamic blocks. Tissues were homogenized and used for the
measurement of PI3-kinase activity as described in Materials and
Methods. Top, Representative autoradiogram.
Bottom, Quantitation of PIP spots from three animals.
Data are mean ± SE (n = 3). * Significantly
different from zero time (p < 0.01).
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Figure 3.
Effect of Ang II on immunoreactive PI3-kinase in
WKY and SHR brain neurons (in vitro) and in hypothalami
(in vivo). Lysates from neuronal cultures and
hypothalamic blocks of adult WKY and SHR neurons were prepared, and
PI3-kinase immunoreactivity was precipitated with the use of p85
PI3-kinase-specific antibody. Western blotting was performed as
described in Materials and Methods.
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Intracellular distribution of PI3 kinase was studied with the use of
immunoblot analysis in Ang II-treated neurons, and nuclear and
cytoplasmic fractions were isolated from Ang II-treated neurons and
subjected to immunoblotting with the use of p85 antibody. Ang II
caused a time-dependent decrease in immunoreactive PI3-kinase in
cytoplasmic fractions of both WKY and SHR neurons (Fig.
4A). A 54% decrease
was observed in 30 min. This was associated with a time-dependent
increase in the immunoreactivity in the nuclear functions of Ang
II-treated WKY and SHR neurons (Fig. 4B).
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Figure 4.
Ang II-induced nuclear translocation of PI3-kinase
in WKY and SHR neurons. Neuronal cultures of WKY rats and SHRs were
incubated without or with 100 nM Ang II for indicated time
periods. Cytoplasmic and nuclear fractions were isolated and subjected
to Western analysis as described in Materials and Methods:
(A) cytoplasmic fractions and
(B) nuclear fractions. Whole-cell lysates from
control and Ang II-treated cells were also run in separate experiments
to determine levels of the enzyme. A, B,
Top, Representative autoradiograms;
bottom, quantitation of immunoreactive bands. Mean ± SE (n = 3). * Significantly different from
control (p < 0.05).
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Role of PI3-kinase in Ang II regulation of NE neuromodulation
We studied the effect of PI3-kinase inhibition by Wortmannin on
Ang II stimulation of NE neuromodulation in WKY and SHR neurons. We
postulated that MAP kinase-independent NE neuromodulation may involve
PI3-kinase in the SHR neurons. Neuronal cultures were preincubated
with 100 nM Wortmannin for 30 min. This resulted in a
complete inhibition of PI3-kinase activity in both strain of neurons
(Fig. 5). Ang II caused threefold and
sixfold increases in NET mRNA levels in the WKY and the SHR neurons,
respectively (Fig. 6A).
This observation is consistent with our earlier report demonstrating an
increase of AT1 receptors in this strain (Raizada et al.,
1993, 1994). Treatment of the WKY neuron with 100 nM
Wortmannin had no effect on Ang II stimulation of NET mRNA levels. In
contrast, Wortmannin caused a 50% inhibition of NET mRNA in the SHR
neuron (Fig. 6A). Interestingly, the level of
stimulation of PI3-kinase by Ang II in Wortmannin-treated SHR neurons
was comparable to the Wortmannin-treated WKY neurons. Similar
observations were noted for the effects of Ang II and Wortmannin on TH
mRNA and [3H]-NE uptake between WKY and SHR
neurons (Fig. 6B,C). For example, Ang II stimulation
of TH mRNA levels and [3H]-NE uptake were
significantly higher in the SHR neuron. Also, Wortmannin partially
inhibited Ang II stimulation of these activities in the SHR neuron and
not in the WKY neuron. These data indicated that PI3-kinase plays a
minimal role in Ang II regulation of NE neuromodulation in the
WKY rat neuron. However, it is partially responsible for NE
neuromodulatory effects of Ang II in the SHR neuron. This difference in
the ability of Wortmannin to selectively inhibit Ang II stimulation of
NE neuromodulation in the SHR neuron is not a result of a differential
inhibition of PI3-kinase, because Wortmannin inhibition of this enzyme
was comparable in both strains of neurons. These observations led us to
hypothesize that the RasRafMAP kinase signaling pathway is involved
in the response of Ang II on the WKY rat neuron, whereas both
RasRafMAP kinase and PI3-kinase signaling pathways are responsible
for the neuromodulatory actions of Ang II in the SHR neuron.
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Figure 5.
Effect of Wortmannin on Ang II stimulation of
PI3-kinase activity in WKY and SHR neurons. Neuronal cultures of WKY
rats and SHRs were incubated with 100 nM Ang II for 15 min
in the absence and presence of 100 nM Wortmannin.
PI3-kinase activity was determined as described in Materials and
Methods. A, Representative autoradiogram.
B, Mean ± SE (n = 3).
* Significantly different from untreated cells
(p < 0.01). ** Significantly different
from Ang II-treated cells (p < 0.01).
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Figure 6.
Neuronal cultures of WKY rats and SHRs were
incubated with 100 nM Ang II in the absence or presence of
100 nM Wortmannin for 4 hr at 37°C. Levels of NET mRNA
(A), TH mRNA (B), and
[3H]-NE uptake (C) were
measured essentially as described in Materials and Methods. A,
B, Top, Representative autoradiograms; bottom,
mean data from three experiments ± SE. * Significantly different
from control (p < 0.01). ** Significantly
different from Ang II-treated neurons (p < 0.05). # Significantly different from Ang II-treated WKY rat neurons
(p < 0.01).
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Next, we performed experiments with the use of both Wortmannin and PD
98059, a MAP kinase-kinase inhibitor, to provide evidence in support of
the above hypothesis. Previous studies have established that PD 98059, at 50 µM, caused a complete attenuation of the Ang II
stimulation of NET and TH mRNAs in the WKY rat neuron, whereas a
similar treatment caused only a 43-50% inhibition in the SHR neuron
(Yang and Raizada, 1998). This was despite the fact that PD 98059 completely and equally attenuated Ang II-induced activation of MAP
kinase in both strains of neurons (Yang and Raizada, 1998). In contrast
to PD 98059, Wortmannin showed no effect in the WKY rat neuron and
partially inhibited Ang II stimulation of NET and TH mRNA levels in the
SHR neuron. Interestingly, inclusion of both PD 98059 and Wortmannin
completely attenuated Ang II's stimulatory effects on NET and TH mRNA
levels in the SHR neuron (Fig. 7). This
combination failed to further lower the stimulation significantly in
the WKY rat neuron.
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Figure 7.
Effects of Wortmannin and PD 98059 on NE
neuromodulation in WKY and SHR neurons. WKY rat and SHR neurons were
treated without or with 100 nM Ang II
(AngII) for 4 hr at 37°C in the absence or
presence of 100 nM Wortmannin (Wort) or 50 µM PD 98059 (PD). mRNA levels for NET
(A) and TH (B) were
measured essentially as described in Materials and Methods.
Top, Representative autoradiograms;
bottom, mean ± SE (n = 3).
* Significantly different from control (p < 0.01). ** Significantly different from Ang II-treated neurons
(p < 0.05). # Significantly different from
Ang II-treated WKY rat neurons (p < 0.01).
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Our previous studies have demonstrated that enhanced
neuromodulatory actions of Ang II are associated with the stimulation of MAP kinase, its translocation into the nucleus, and the activation of AP-1 binding activity, which is involved in the transcription of
NET, TH, and DH genes in the WKY rat neuron (Lu et al., 1996b). On
the basis of this information and our above data on a selective effect
of PI3-kinase in the SHR neuron, we decided to compare the effect of
PI3-kinase inhibition on Ang II stimulation of AP-1 binding activity in
both strains of neurons. Figure 8
shows that Ang II caused a fivefold increase in the AP-1 binding
activity in the nuclear fractions of the WKY rat neuron. The
stimulation in the nuclear fractions of the Ang II-treated SHR neuron
was 50% higher compared with WKY rat neuron. Wortmannin caused a 60% decrease in the AP-1 binding activity in the SHR neuron, whereas it had
no effect on the WKY rat neuron.
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Figure 8.
Effect of Wortmannin on Ang II stimulation of AP-1
binding activity in the nuclear fractions of WKY and SHR neurons.
Nuclear extracts of control, Ang II-treated (100 nM) or Ang
II + Wortmannin-treated WKY rat and SHR neurons were subjected to gel
shift analysis to determine the levels of AP-1 binding activity
essentially as described in Materials and Methods.
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Involvement of PI3-kinase in Ang II stimulation of PKB/Akt in WKY
rat and SHR neurons
PKB/Akt is an established downstream signaling kinase in the
propagation of PI3-kinase-mediated signals (Franke et al., 1997; Marte
and Downward, 1997). Activation of PKB/Akt is associated with its
translocation from the cytoplasmic to nuclear compartment, where it
acts as a transcription factor to influence the expression of various
genes (Andjelkovic et al., 1997; Alessi and Cohen, 1998). In view of
our above observations, our next objective was to determine whether the
differential effect of Ang II on the SHR neuron was caused by the
effects of Ang II on PKB/Akt activation. Ang II caused a
time-dependent increase in PKB/Akt activity in both strains of neurons
(Fig. 9A). There were two
differences in the nature of stimulation between the WKY and SHR
neurons: (1) the stimulation persisted for longer time periods in the
SHR neuron, and (2) maximal stimulation of PKB/AKt activity was
30-40% higher in the SHR neuron compared with the WKY rat neuron. The stimulation was Ang II concentration-dependent in both cell types, although the SHR neuron was 30% more responsive at each dose of Ang II
(Fig. 9B). Losartan completely blocked the Ang II
stimulation of PKB/Akt activity, whereas PD 123319 had little effect
(Fig. 9C).
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Figure 9.
Effect of Ang II on protein kinase B activity in
WKY and SHR neurons. A, Time course: neuronal culture of
WKY rat and SHR were incubated with 100 nM Ang II for the
indicated time at 37°C. PKB activity in neuronal extract was
determined essentially as described in Materials and Methods.
Top, Representative autoradiogram;
bottom, mean ± SE (n = 3).
* Significantly different from zero time (p < 0.05). # Significantly different from the WKY
(p < 0.05). B, Ang II
concentrations: neuronal cultures of WKY rat and SHR were incubated
with the indicated concentrations of Ang II for 10 min at 37°C before
determination of PKB activity. Top, Representative
autoradiogram; bottom, mean ± SE
(n = 3). * Significantly different from control
(p < 0.05). # Significantly different from
the WKY (p < 0.05). C,
Effect of Ang receptor antagonists: experimental conditions were
essentially as described in the legend to Figure
10B. WKY rat and SHR neuronal cultures were
incubated without (C) or with 100 nM
Ang II in the absence or presence of 10 µM losartan
(Los) or PD123319 (PD).
Top, Representative autoradiogram;
bottom, mean ± SE (n = 3).
* Significantly different from control (p < 0.05). ** Significantly different from Ang II treatment
(p < 0.05).
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The stimulation of PKB/Akt activity by Ang II appeared to be mediated
by activation of PI3-kinase and not by MAP kinase. Incubation of WKY
and SHR neurons with 100 nM Wortmannin completely
attenuated Ang II stimulation of PKB/Akt activity, whereas 50 µM PD 98059 had little effect on this stimulation (Fig.
10). The levels of PKB/Akt in the
cytoplasmic and nuclear fractions were compared to determine whether
Ang II treatment causes nuclear translocation of this enzyme.
Stimulation with Ang II caused a time-dependent decrease in the
cytoplasmic PKB/Akt. Its levels were 60% lower in the cytoplasmic
fraction of the WKY rat neuron after 30 min of Ang II treatment (Fig.
11A). This decrease
was associated with increases in its levels in the nuclear fraction
(Fig. 11B). An approximately twofold increase was
seen 30 min after Ang II treatment in the WKY rat neuron. Comparison of
data showed that the levels of PKB/Akt immunoreactivity in the nucleus
and fold translocation by Ang II in the SHR neuron were comparable with
the WKY rat neuron (Fig. 11). This would suggest that the
translocated PKB/Akt activity in the nuclear fraction of the SHR neuron
would be significantly higher than in the WKY rat neurons, because
the activity of this enzyme per unit of protein is higher in this
strain of cells.
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Figure 10.
Effects of PD 98059 and Wortmannin on PKB
activity in WKY and SHR neurons. WKY rat and SHR neurons were treated
without or with 100 nM Ang II (AngII)
for 10 min at 37°C in the absence or presence of 50 µM
PD 98059 (PD) or 100 nM Wortmannin
(Wort). Top, Representative
autoradiogram; bottom, mean ± SE
(n = 3) * Significantly different from control
(p < 0.05). ** Significantly different
from Ang II-treated neurons (p < 0.05).
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Figure 11.
Ang II-induced nuclear translocation of PKB in
WKY and SHR neurons. Neuronal cultures of WKY and SHR were incubated
without or with 100 nM Ang II for the indicated time
periods. Cytoplasmic and nuclear fractions were isolated and subjected
to Western analysis as described in Materials and Methods.
A, Cytoplasmic fractions; B, nuclear
fractions. A, B, Top, Representative autoradiograms;
bottom, quantitation of immunoreactive bands. Mean ± SE (n = 3). * Significantly different from
control (p < 0.01).
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DISCUSSION |
The most significant observation of this study is the exclusive
involvement of PI3-kinase and PKB/Akt activities in Ang II-induced regulation of NE neuromodulation in the SHR neuron. Thus, the PI3-kinase-PKB signaling system constitutes the MAP kinase-independent signal transduction mechanism for a heightened Ang II stimulation of
NET, TH, and DH gene transcription and [3H]-NE
uptake in the SHR neuron previously postulated by us (Yang and Raizada,
1998). Diagram 1 summarizes our observations. RasRafMAP kinase
appears to be the sole signal transduction pathway for NE
neuromodulatory action in the WKY rat neuron. This is primarily based
on our earlier observations that inhibition of MAP kinase activity,
either by PD 98059 or by MAP kinase-specific antisense oligonucleotides, completely attenuates Ang II stimulation of NET, TH,
and DH mRNAs and [3H]-NE uptake (Yang et al.,
1996a; Gelband et al., 1998). In addition, a complete attenuation is
seen in Ang II activation of SRE and AP-1 binding activities, a
downstream regulatory step in the transcriptional control of
CA-synthesizing genes (Lu et al., 1996b). AT1 receptor levels and its transcription are two- to fourfold higher in the SHR
neuron compared with the WKY rat neuron, a normotensive control (Raizada et al., 1993, 1998). This increase is associated with a two-
to fourfold heightened response of the SHR neuron to Ang II in the
stimulation of NE neuromodulation (Lu et al., 1996a; Yu et al., 1996a;
Yang and Raizada, 1998). Despite this, the MAP kinase signaling pathway
is only partially responsible. The heightened response appears to be
caused by the coupling of the SHR AT1 receptor to the
PI3-kinase-PKB signaling pathway. Thus, neuromodulation mediated by
AT1 receptors in the SHR neuron is linked to both MAP
kinase and PI3-kinase signaling systems, whereas it is linked to only
the MAP kinase system in the WKY rat neuron. This conclusion is further
supported by our data, which indicate that PI3-kinase is not involved
in the regulation of MAP kinase activity in brain neurons, in contrast
to the evidence in other systems (Karnitz et al., 1995; Nishioka et
al., 1995).
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Diagram 1.
Summary of signaling mechanisms of
AT1 receptor-mediated neuromodulation in WKY and SHR
neurons.
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These observations are highly significant because (1) they constitute
the first example in which the PI3-kinase-PKB signaling system
has been linked to the NE neuromodulatory activity. The involvement of
PI3-kinase in the regulation of growth, differentiation, and cellular
metabolic actions of many hormones is well established (Soltoff et al.,
1992; Kapeller and Cantley, 1994), but its ability to regulate
catecholamine turnover, synthesis, and release is a unique function and
links this enzyme to the regulation of neurotransmission. The
observations establish that (2) the AT1 receptor is coupled to the PI3-kinase-neuromodulation cascade only in the SHR neuron. This
is highly relevant from a pathophysiological perspective and may turn
out to have important pharmacological implications in the control of
centrally regulated hypertension. For example, members of the
PI3-kinase signaling cascade could serve as highly specific targets for
pharmacological and/or gene therapy, because the pathway is only active
in the SHR. Our data further support this implication. Hypothalamic
nuclei in the adult brain contain functional AT1 receptors,
which are predominantly responsible for Ang II-mediated NE
neuromodulation leading to is effects on blood pressure (Saavedra,
1992; Steckelings et al., 1992; Timmermans et al., 1993). In addition,
AT1 receptor expression in SHR hypothalamus is increased
similar to that observed in vitro (Raizada et al., 1998).
The present observation that Ang II stimulates PI3-kinase in the
hypothalamus of both WKY and SHR, with more sustained stimulation in
the SHR, is also consistent with the in vitro studies and
their relevance to the in vivo situation. Despite a strong
correlation between in vitro and in vivo data on
the effects of Ang II on the NE system, some degree of caution must
observed when be in vitro data are extrapolated to the adult
animal. This is particularly relevant in view of the fact that neuronal
phenotypes may be different in vitro and in situ.
In addition, although in vitro effects of Ang II on
catecholamic systems are fairly well studied (Raizada et al., 1998),
the nature of these interactions in situ is not established
and may be distinct.
This study raises an important question concerning the mechanism by
which the PI3-kinase pathway is uniquely linked to neuromodulation only in the SHR. Currently there is no direct evidence that
examines this issue. However, we propose that a persistent
stimulation of both PI3-kinase and PKB/Akt and a significantly higher
stimulation of PKB/Akt in the SHR neuron may be sufficient to shift the
signaling equilibrium toward this specificity. It is likely that
increased activation of PKB/Akt by persistently stimulated PI3-kinase
could lead to an increased nuclear translocation of PKB/Akt-specific activity. This increase in the nuclear PKB/Akt activity may participate in a transcriptional control of catecholamine-relevant genes. In the
future, the availability of improved techniques to measure nuclear
PKB/Akt activity in neuronal cultures would provide evidence to support
or refute this view.
Many other possibilities concerning the mechanism, however, cannot be
ruled out at the present time. For example, the signaling steps
downstream from the PKB/Akt activation may be different between the two
strains of neurons. This hypothesis would suggest that Ang II
stimulation of PI3-kinase cascade in the WKY rat neuron may be involved
in other distinct functions. Preliminary data, in fact, support this
view. Ang II stimulates plasminogen activator inhibitor-1 (PAI-1)
synthesis in brain neurons (Yu et al., 1996b). This stimulation, which
is at the level of PAI-1 gene transcription, has been linked to the
trophic actions of Ang II in neuronal cultures (Rydzewski et al.,
1993). Ang II stimulation of PAI-1 mRNA was exclusively attenuated by
Wortmannin in the WKY rat neuron. PI3-kinase inhibition did not have
such an effect on the stimulation of PAI-1 in the SHR neuron. From
these data, although preliminary, it is tempting to speculate that the
PI3-kinase cascade is involved in trophic actions of Ang II in the WKY neuron.
The presence of two distinct AT1 receptor subtypes in the
SHR neuron, one coupled to MAP kinase and the other to PI3-kinase, can
also explain these observations. Although no pharmacological difference
between the AT1 receptors of WKY and SHR neurons have been
noted (Sumners and Raizada, 1993; Raizada et al., 1994, 1998; Sumners
et al., 1995), the proposal needs a critical evaluation. This is
especially relevant because the AT1 receptor gene
expression is higher in the SHR neuron and because two subtypes of the
AT1 receptor, AT1A and AT1B,
have been reported in the neurons (Sumners and Raizada, 1993). Finally,
coupling of the AT1 receptor to G-proteins may be different
in the two strains of neurons. We have proposed that
G  subunits of Gq may be involved in the
NE neuromodulatory actions of the AT1 receptor (Gelband et
al., 1998). This is consistent with the evidence that
G subunits play an important role in the regulation
of this enzyme (Hazeki et al., 1998; Leopoldt et al., 1998). Whatever
the precise mechanism for this diversity of Ang II actions may be, the
observations pinpoint a unique signaling cascade involving PI3-kinase
in NE neuromodulatory actions of Ang II in the SHR neuron.
| |
FOOTNOTES |
Received Nov. 11, 1998; revised Dec. 12, 1998; accepted Jan. 14, 1999.
This study was supported by National Institutes of Health Grant
HL33610. Hong Yang is a postdoctoral fellow of the American Heart
Association, Florida Affiliate. We thank Ling Liu and Hung Dang for
technical assistance.
Correspondence should be addressed to Dr. Mohan K. Raizada, Department
of Physiology, College of Medicine, University of Florida, P.O. Box
100274, Gainesville, FL 32610.
| |
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
