 |
INTRODUCTION |
It is well established that
mammalian brain contains angiotensin II (Ang II) type 2 (AT2) receptors, and that these sites are more
prevalent in neonates (Lenkei et al. 1996
; Millan
et al. 1991
; Nuyt et al. 1999
; Tsutsumi
and Saavedra 1991
). A number of studies have suggested that
these brain AT2 receptors are involved in a
variety of different physiological and pathological processes (Gallinat et al. 2000
). For example, the fact that they
are present in neonates has led to the suggestion that
AT2 receptors are involved in development and
differentiation (Cook et al. 1991
; Millan et al.
1991
). Support for this has come from studies that have
determined that stimulation of AT2 receptors
causes neurite outgrowth in undifferentiated neuroblastoma × glioma cells (Laflamme et al. 1996
) and plays a role in
apoptosis of pheochromocytoma PC12W cells and neurons cultured from
newborn rat brain (Shenoy et al. 1999
; Yamada et
al. 1996
). Studies from mutant mice that lack the
AT2 receptor gene have revealed that the knockout
animals exhibit decreased exploratory behavior, spontaneous movements, an impaired drinking response to water deprivation, and lower basal
body temperature (Hein et al. 1995
; Ichiki et al.
1995
). In fact, more recent studies in these mutant mice
suggest an important role for brain AT2 receptors
in stress-induced hyperthermia (Watanabe et al. 1999
). A
pathological role for CNS AT2 receptors has been indicated by studies that show that global ischemia elicits a transient
increase in AT2 receptor mRNA in rat brain
(Makino et al. 1996
) and that glutamate-induced toxicity
of cultured cortical neurons is associated with increased expression of
AT2 receptor mRNA and increased
AT2 receptor binding (Shibata et al.
1998
). Thus AT2 receptors may be involved
in the central control of certain behaviors and physiological
responses, and in the pathological processes that result from ischemia
in the brain. It is also clear that stimulation of brain
AT2 receptors elicits physiological effects at
the fundamental level of changes in neuronal firing rate and membrane
ionic currents. For example, in situ electrophysiological recordings
have indicated that Ang II elicits an AT2
receptor-mediated excitation of inferior olivary neurons
(Ambuhl et al. 1992
) and depresses glutamate-induced
depolarization in locus coeruleus (Xiong and Marshall
1994
). Our studies using neurons cultured from neonatal rat
brain stem and hypothalamus have attempted to define the ionic basis of
such AT2 receptor-mediated changes in neuronal
activity. We have determined that Ang II elicits an
AT2 receptor-mediated increase in neuronal
delayed rectifier K+ current
(IKV) (Kang et al.
1994
), and that this effect is mediated mostly through
activation of phospholipase A2
(PLA2) and generation of arachidonic acid (AA)
(Zhu et al. 1998
). AA itself is a well-known modulator
of neuronal K+ current and channels
(Premkumar et al. 1990
; Schweitzer et al. 1990
; Zona et al. 1993
). However, AA is rapidly
metabolized via three major groups of enzymes within cells, namely the
lipoxygenases (LO), cyclooxygenases (COX), and cytochrome p450
monooxygenases, and it is clear that AA metabolites produced from each
of these pathways have the ability to modulate K+
channels in various tissues (Arima et al. 1997
;
Duerson et al. 1996
; Harder et al. 1997
;
Roman and Alonso-Galicia 1999
; Twitchell et al.
1997
; Yu 1995
). In particular, one study has
indicated that a p450 metabolite of AA mediates the actions of Ang II,
via AT2 receptors, in vascular tissue
(Arima et al. 1997
). Thus it was possible that the
actions of Ang II on IKV involved AA
itself or AA metabolites, or a combination of these factors. Our
preliminary studies have suggested that the stimulation of
IKV by Ang II involves generation of
LO, but not COX metabolites of AA (Zhu et al. 1998
). Thus in the present study, we have investigated the role of AA metabolism in the Ang II-induced increase in neuronal
IKV, in particular the role of LO
metabolites. The results indicate that the AT2
receptor-mediated stimulation of neuronal
IKV by Ang II is partially
mediated through 12-LO metabolite of AA, but does not involve 5-LO
metabolites nor p450 monooxygenase metabolites of AA. However, the data
leave open the possibility of a further signaling mechanism that is
responsible for the residual action of Ang II on
IKV.
 |
METHODS |
Materials
Newborn Sprague-Dawley rats were obtained from our breeding
colony, which originated from Charles River Farms (Wilmington, MA).
Dulbecco's modified Eagle's medium (DMEM) and TRIzol reagent were
obtained from GIBCO-BRL (Gaithersburg, MD). Plasma-derived horse serum
(PDHS) was from Central Biomedia (Irwin, MO). Renaissance enhanced
chemiluminescence (ECL) kits were purchased from Dupont-NEN (Boston,
MA). Losartan potassium (Los) was generously provided by W. Henckler,
Merck (Rahway, NJ). PD123,319 (PD), and 5,6,7-trihydroxyflavone (baicalein) were purchased from Research Biochemicals International (Natick, MA). Tetrodotoxin (TTX) was purchased from Calbiochem (La
Jolla, CA). Gene-Amp RT-PCR kits and all reagents for RT-PCR were
purchased from Perkin Elmer Biotechnologies (Norwalk, CT). MK 886 and
cinnamyl-3,4-dihydroxy-
-cyanocinnamate (CDC) were from
Biomol (Plymouth Meeting, PA). Leukocyte 12-LO standard was from
Oxford Biomedical (Oxford, MI). Ang II, sodium GTP, cadmium chloride
(CdCl2), HEPES, ethylene glycol-bis
(
-aminoethyl ether)-N,N,N',N'-tetracetic acid (EGTA),
dipotassium ATP, peroxidase-conjugated affinity purified goat
anti-rabbit IgG, 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 17-octadecynoic acid (17-ODYA), Leukotriene B4,
Leukotriene C4, and 12-(S)-hydroxy-(5Z, 8Z, 10E,
14Z)-eicosatetraenoic acid [12-(S)-HETE] were purchased from Sigma
Chemical (St. Louis, MO). All other chemicals were purchased from
Fisher Scientific (Pittsburgh, PA). Oligonucleotide primers for the rat
AT2 receptor gene (Mukoyama et al.
1993
), the rat brain cytosolic PLA2 gene
(Owada et al. 1994
) and the 12-LO gene (Watanabe
et al. 1993
) were synthesized by Gemini Biotech (Alachua, FL).
The sequences of these primers are as follows: 1)
AT2 receptor gene: sense,
5'-TGGTGTATGGCTTGTCTGTC-3'; antisense,
5'-CACACTACGGAGCTTCTGTT-3', 2)
PLA2 gene: sense, 5'-GCTCCACATGGTACATGTCA-3'; antisense, 5'-CCTCAAGCTACTCAAGGTCG-3', 3) 12-LO: sense,
5'-ACCTGATCTCAGAGAGAGGCT-3'; antisense,
5'-TCAGTGATCTCTCGACACCAG-3'.
Preparation of cultured neurons
Neuronal co-cultures were prepared from the hypothalamus and
brain stem of newborn Sprague-Dawley rats exactly as described previously (Sumners et al. 1991
). Cultures were grown on
35-mm plastic culture dishes in DMEM/10% PDHS for 10-14 days, at
which time they consisted of 90% neurons and 10% astrocyte glia and microglia, as determined by immunofluorescent staining (Sumners et al. 1991
).
Electrophysiological recordings
Macroscopic K+ current was recorded using
the whole cell configuration of the patch-clamp technique as detailed
previously (Hamill et al. 1981
; Kang et al.
1994
). Experiments were performed at room temperature
(22-23°C) using an Axopatch 1D amplifier and Digidata 1200 B
interface (Axon Instruments, Burlingame, CA). Neurons were bathed in a
modified Tyrode's solution containing (in mM) 137 NaCl, 5.4 KCl, 2 MgSO4, 1.35 CaCl2, 0.3 NaH2PO4, 0.3 CdCl2, 10 dextrose, 10 HEPES, and 0.0015 TTX, pH
7.4 (NaOH). The patch electrodes had resistances of 3-4 M
when
filled with an internal pipette solution containing (in mM) 130 KCl, 2 MgCl2, 0.25 CaCl2, 1.0 ATP,
8 dextrose, 0.1 GTP, 10 HEPES, and 5 EGTA, pH 7.2 (KOH). For whole cell
recordings, cell capacitance was canceled electronically, and the
series resistance (<10 M
) was compensated for by 75-80%. Data
acquisition and analysis were performed using pCLAMP 6.04. Whole cell
currents were digitized at 3 kHz and filtered at 1 kHz (
3 dB
frequency filter). Standard recording conditions for
K+ current were achieved by stepping from a
holding potential of
80 to +10 mV for 100 ms every 10 s. Under
these recording conditions, both the
IKV and A-type
K+ current were obtained. Therefore the tracings
shown here should reflect both Kv and A-type currents, except that not
all of the neurons used here contain the latter current. As a result of
this, the current measurements from which mean current densities were derived were made at the end of the test pulse, at which time they
reflect only IKV (Kang et al.
1994
). In some recordings IKV was measured directly by stepping from holding potential of
40 to +10
mV for 100 ms. Under these recording conditions
IA is completely inactivated. Current
density was derived by dividing transmembrane current (pA) by membrane
capacitance (pF), which was calculated as detailed by us recently
(Zhu et al. 1999
). The average cell capacitance for
neurons used in this study was 33.7 ± 15.5 pF (mean ± SE;
n = 34 neurons).
Selective stimulation of neuronal AT2 receptors
causes an increase in IKV (Zhu
et al. 1998
), whereas selective stimulation of neuronal
AT1 receptors causes a decrease in neuronal
IKV (Zhu et al. 1999
).
Some of the neurons in the cultures used here contain both
AT1 and AT2 receptors
(Gelband et al. 1997
). Since the aim of the present
studies was to measure AT2 receptor-mediated
effects of Ang II on IKV, all
electrophysiological recordings were performed in the presence of 1 µM Los to block AT1 receptors. Los did not alter basal IKV.
Extraction of total RNA and reverse transcriptase-polymerase
chain reaction (RT-PCR)
Growth media were removed from neuronal cultures that were then
washed once with ice-cold Tyrode's solution, pH 7.4. After this,
neurons were lysed in TRIzol reagent (0.5 ml/dish), and total RNA was
extracted as detailed previously (Huang et al. 1997
). The overall procedures for RT-PCR of the AT2
receptor, PLA2, and 12-LO from single neurons
were as detailed by us previously (Zhu et al. 1998
). In
brief, following recordings of IKV,
the neuronal intracellular contents were drawn into the tip of the
patch pipette using negative pressure, and the tip was broken off
inside the RT-PCR tube. The volume of intracellular contents and patch
pipette solution in the broken tip was adjusted to 8 µl for the
RT-PCR, which was performed using Gene-Amp RT-PCR kits. The RT was
performed at 25°C for 10 min, followed by 42°C for 60 min, 99°C
for 5 min, and 4°C for 10 min. A first PCR was performed at 94°C
for 4 min, followed by 38 cycles at 94°C for 45 s, 55°C for 1 min (for AT2 receptor), or 63°C for 1 min
40 s (for PLA2 and 12-LO), and 72°C for 2 min. Final extension was achieved at 72°C for 10 min and 4°C for 30 min. A second PCR was performed (on 20 µl of the 1st PCR products) at
94°C for 4 min, followed by 36 cycles (for AT2 receptor) or 30 cycles (for PLA2 or 12-LO) at
94°C for 45 s, 55°C for 1 min (for AT2
receptor) or 63°C for 1 min 40 s (for PLA2 or 12-LO), and 72°C for 2 min. After final extension at 72°C for 10 min and 4°C for 30 min, the PCR products were electrophoresed on a
2% agarose gel containing 1 µg/ml ethidium bromide. Using these
conditions, we observed the production of a 495 bp
AT2 receptor-specific DNA, a 263 bp
PLA2-specific DNA and a 295 bp12-LO-specific DNA, which correspond to the AT2 receptor,
PLA2, and 12-LO mRNAs, respectively. In all
situations, exclusion of either RNA or MuLV reverse transcriptase resulted in no visible bands following gel electrophoresis.
Western blot analysis
Detection of 12-LO protein in control cultured neurons and in
rat hypothalamus and brain stem was performed by Western blot analysis
using a polyclonal peptide antibody to the leukocyte type of 12-LO
(Natarajan et al. 1996
). All procedures, including extraction of total cell protein, were as detailed previously (Gelband et al. 1999
).
Drug and antibody applications
Ang II and drugs were dissolved in the appropriate solvent,
followed by dilution in superfusate solution, patch pipette solution, or DMEM, depending on the route of administration. Intracellular application of anti-12-LO antibodies and 12-(S)-HETE were achieved by
injection through the patch pipette as detailed by us previously (Zhu et al. 1999
). In brief, a sidearm pipette holder is
attached to the head stage of the Axopatch. One side arm is used to
supply suction for seal formation, and the second side arm is used to advance a very fine polyethylene catheter (PE-50) down the inside of
the patch pipette. Control measurements of K+
current are made 5 min after the whole cell configuration is established in a given neuron. After this, the peptide solution (5 µl) is injected into the tip of the recording electrode via the PE-50
tube. From the pipette tip, the anti-12-LO antibodies are allowed to
diffuse into the neuron, and measurements of K+
current are made 4 min later, at which time a stable peak response is
obtained. Care is take not to overperfuse the neuron, and this is
monitored electrically via the Axopatch and on the TV monitor. Thus the
concentrations of anti-12-LO antibodies that are given in the results
refer to the amounts that are injected at the pipette tip, and so are
likely higher than the amounts that reach the site of action.
Experimental groups and data analysis
Electrophysiological analyses were performed with the use of
multiple 35-mm dishes of cultured neurons. Comparisons were made with
the use of a one-way ANOVA followed by Newman-Keuls test to assess
statistical significance.
 |
RESULTS |
The present study is focused on which particular LO isozyme
mediates the stimulation of IKV by Ang
II, and whether other AA metabolic pathways (e.g., cytochrome p450
monooxygenase) are involved in this response. Thus the first series of
experiments were performed to confirm that the Ang II-induced
stimulation of IKV involved AA
metabolism. The data presented in Fig. 1
indicate that the stimulation of IKV
by Ang II was attenuated by superfusion of 10 µM ETYA, which is an
analogue of AA and a general inhibitor of AA metabolism (Collins
and Davies 1998
; Muller et al. 1998
; Schmitt and Meves 1995
). Full reversal of this Ang II
effect was obtained with subsequent superfusion of 1 µM PD123,319, a
selective AT2 receptor blocker. PD123,319 (1 µM) alone did not alter baseline IKV
(data not shown), in concert with our previous findings (Kang et
al. 1993
). Our data also indicate that superfusion of ETYA (10 µM) alone did not alter neuronal IKV
(Fig. 1). Thus the data from the first set of experiments confirm our
previous findings that the Ang II stimulation of neuronal
IKV involves AA metabolism. In the
next series of experiments, we examined the roles of LO isozymes (5-LO
and 12-LO) in this Ang II response, with the use of selective
inhibitors of each isozyme. Treatment of cultured neurons with 10 µM
MK 886, a selective inhibitor of 5-LO activating protein (FLAP)
(Lammers et al. 1996
), had no significant effects on the
stimulation of IKV by Ang II (Fig.
2). In contrast, pretreatment of cultures
with selective 12-LO inhibitors significantly attenuated the Ang
II-induced increase in IKV. For
example, inclusion of specific anti-12-LO antibodies (Kim et al.
1995
) in the pipette solution reduced the effects of Ang II on
IKV (Fig.
3A). Inclusion of normal
rabbit IgG in the pipette solution did not alter the stimulation of
IKV produced by Ang II (not shown). Similar
inhibitory effects were observed following treatment of cultured
neurons with the selective 12-LO inhibitors CDC (200 nM; Fig.
3A) (Wen et al. 1996
) or baicalein (1-10
µM) (Nadler et al. 1987
). In the latter case,
baicalein at 1 and 10 µM reduced the stimulatory effects of Ang II on
neuronal IKV by 28.2 ± 2.4% and
60 ± 5.2%, respectively (n = 5 neurons in each
case). Analysis of IKV under recording
conditions in which IA was inactivated
produced similar results, i.e., Ang II produced a significant increase
in IKV that was attenuated 40-80% by
200 nM CDC (Fig. 3B). The role of 12-LO was further
demonstrated by experiments in which the stimulatory action of Ang II
on neuronal IKV was partially reversed
by acute administration of anti-12-LO antibodies via intracellular
application (Fig. 4). Full reversal of
the Ang II effect was obtained with subsequent superfusion of 1 µM
PD123,319 (Fig. 4). Control recordings of neuronal
IKV in the presence of the 5-LO and
12-LO inhibitors (including 12-LO antibodies) were not significantly
different from the recordings of neuronal
IKV made in untreated neurons (Figs. 2
and 3). In addition, higher concentrations of the 12-LO inhibitors
produced no further attenuation of Ang II-modulated IKV (data not shown). These data
therefore indicate that 12-LO is involved in the
AT2 receptor-mediated increase in neuronal IKV by Ang II. This idea is supported
by experiments that indicate that intracellular application of the
12-LO metabolite 12-(S)-HETE (1 µM), the receptors for which are
primarily intracellular (Herbertsson et al. 1999
),
elicits a significant increase in neuronal
IKV (Fig. 5). By contrast, superfusion of either of
the 5-LO metabolites leukotriene B4
(LTB4; 10 nM) or leukotriene
C4 (LTC4; 1 µM), the receptors for which are G-protein coupled sites located on the plasma
membrane (Gaudreau et al. 1998
; Geirsson et al.
1998
), produced no significant changes in neuronal
IKV (Fig. 5). To further establish a
role for 12-LO metabolites of AA in the Ang II-induced increase in
neuronal IKV, cultured neurons were
superfused with AA (50 µM) in the absence or presence of 5-LO or
12-LO inhibitors. The data presented in Fig.
6 indicate that superfusion of AA elicits a significant increase in neuronal
IKV, an effect that is attenuated by
pretreatment of cells with the 12-LO inhibitor baicalein (1 µM), but
that is not altered by 5-LO activating protein inhibitor MK 886 (10 µM). Collectively, the present data and our previous studies
(Zhu et al. 1998
) indicate that the
AT2 receptor-mediated stimulation of neuronal
IKV involves a pathway that includes
activation of PLA2 and of 12-LO. To further
establish these signaling molecules as mediators of the Ang II-induced
increase in neuronal IKV, it is
necessary to demonstrate their presence within the Ang II-responsive neurons. The Western immunoblot data presented in Fig.
7 (top) demonstrate the
presence of a 72-kDa 12-LO in cultured neurons, similar to that found
in rat hypothalamus and brain stem and reported previously
(Watanabe et al. 1993
). Single-cell RT-PCR studies indicate that AT2 receptor,
PLA2, and 12-LO mRNAs are present within a single
neuron that had responded to Ang II with an increase in
IKV (Fig. 7, bottom). Thus
the immunoblot and RT-PCR data provide strong support for a role of
PLA2 and 12-LO in the Ang II-induced stimulation
of neuronal IKV.

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Fig. 1.
Stimulation of neuronal voltage-dependent delayed rectifier
K+ current (IKV) by angiotensin
II (Ang II) is attenuated by 5, 8, 11, 14-eicosatetraynoic acid (ETYA).
K+ was recorded during 100-ms voltage steps from a holding
potential of 80 to +10 mV. A: representative time
course showing the effects of superfusion of 10 µM ETYA on the
stimulation of IKV produced by superfusion
of 100 nM Ang II. Once the effects of ETYA had stabilized, 1 µM
PD123,319 was added to the superfusate. B: bar graphs
are means ± SE of IKV current
densities recorded during the application of Ang II, ETYA, and
PD123,319. Data are from 5 neurons. * P < 0.05 compared with control; ** P < 0.05 compared with
Ang II alone. C: effects of superfused ETYA (10 µM) on
K+ current in untreated neurons. Control (CON) recordings
were made before the application of ETYA. Bar graphs show means ± SE of IKV current densities obtained in each
treatment situation. Data are from 5 neurons.
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Fig. 2.
Stimulation of neuronal IKV by Ang II is not
altered by MK 886. K+ current was recorded as described in
Fig. 1. Top: from left to right,
representative current tracings showing the effects of superfused Ang
II on K+ current in untreated neurons, or in neurons
pretreated with the selective 5-lipoxygenase (5-LO) inhibitor MK 886 (10 µM, added to the DMEM and to the superfusate). Control (CON)
recordings were made before the application of Ang II, and all
recordings were made in the presence of 1 µM Los to block
AT1 receptors. Bottom: bar graphs showing
the means ± SE of IKV current
densities obtained in both treatment situations. Sample sizes were 13 and 4 neurons for the untreated and MK 886 groups, respectively.
* P < 0.05 compared with the respective
control.
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Fig. 3.
Ang II-induced stimulation of neuronal
IKV: attenuation by selective inhibition of
12-LO. A: treatment of neurons with the selective 12-LO
inhibitors attenuates the stimulation of IKV
produced by Ang II. K+ current was recorded as described in
Fig. 1. Top: from left to right
are representative current tracings showing the effects of superfused
Ang II (100 nM) on K+ current in untreated neurons or in
neurons treated with the 12-LO inhibitor
cinnamyl-3,4-dihydroxy- -cyanocinnamate (CDC; 200 nM for 30 min and
added to superfusate) or with anti-12-LO antibodies (1:1,000 dilution
into pipette solution). Control (CON) recordings were made before the
application of Ang II, and all recordings were made in the presence of
1 µM Los to block AT1 receptors. Bottom:
bar graphs showing means ± SE of IKV
current densities obtained in the above treatment situations and in
untreated neurons. Sample sizes were 17, 4, and 7 neurons for the
untreated CDC and anti-12-LO groups, respectively.
* P < 0.05 compared with respective control
treatment. ** P < 0.05 compared with Ang II in
untreated group. B: treatment of neurons with the
selective 12-LO inhibitor CDC reduces the increase of the
IKV elicited by Ang II. K+
current was recorded during 100-ms voltage steps from a holding
potential of 40 to +10 mV. Top: from left
to right are representative current tracings showing the
effects of superfused Ang II (100 nM) or K+ current in
untreated neurons or in neurons treated with CDC as in
A. Control (CON) recordings were made before the
application of Ang II, and all recordings were made in the presence of
Los. Bottom: bar graph showing means ± SE of
IKV current densities obtained under these
treatment and recording conditions. Sample sizes were 3 and 4 neurons
for the untreated and CDC groups, respectively.
* P < 0.05 compared with the respective control
treatment.
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Fig. 4.
Effects of acute treatment with anti-12-LO antibodies on Ang
II-stimulated increases in IKV.
K+ current was recorded as described in Fig. 1.
Top: representative time course showing the effects of
intracellular application of anti-12-LO antibodies (1:1,000) on the
stimulation of neuronal IKV produced by
superfusion of 100 nM Ang II. Once the effects of a 12-LO had
stabilized, 1 µM PD123,319 was added to the superfusate.
Bottom: bar graphs are means ± SE of
IKV current densities recorded during the
application of Ang II, 12-LO, and PD123,319. Data are from 3 neurons.
* P < 0.05 compared with control.
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Fig. 5.
Effects of LO metabolites of arachidonic acid (AA) on neuronal
IKV. K+ current was recorded as
in Fig. 1. Bar graphs show the means ± SE of
IKV current densities recorded as a percent
of control IKV. Control (CON) recordings
were made before the application of LO metabolites following
superfusion of LTB4 (10 nM), LTC4 (1 µM), or
intracellular application of 12-(S)-HETE (1 µM).
Sample sizes were 7, 3, and 8 neurons for the LTB4,
LTC4, and 12 (S) HETE groups, respectively.
* P < 0.05 compared with respective control.
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Fig. 6.
Stimulation of neuronal IKV by AA is
attenuated by baicalein. K+ current was recorded as in Fig.
1. Neurons were superfused with control solution (CON; superfusate) or
AA (50 µM) in the absence (untreated) or presence of MK 886 (10 µM
added to the DMEM for 30 min, and to the superfusate) or baicalein (1 µM added to the DMEM for 20 h, and to the superfusate). Bar
graphs are means ± SE of IKV current
densities obtained in each treatment situation. Sample sizes were 5, 5, and 6 neurons for the untreated, MK886, and baicalein groups,
respectively. * P < 0.05 compared with respective
controls. ** P < 0.05 compared with AA alone (no
baicalein).
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Fig. 7.
Localization of 12-LO, PLA2, and AT2 receptors
within cultured neurons. Top: representative Western
immunoblot showing the presence of 12-LO within cultured neurons. Data
were obtained using a polyclonal anti-12-LO antibody as detailed in
METHODS. Lane 1, 12-LO standard (5 µg);
lane 2, blank; lane 3, cultured neurons;
lane 4, cultured astroglia; lane 5, rat
hypothalamus and brain stem. Bottom:
presence of 12-LO, PLA2, and
AT2 receptor mRNAs within the Ang II-responsive
neurons. Left: current tracing from a representative neuron
showing the increase in K+ current
(AT2 receptor mediated) produced by Ang II (100 nM) in the presence of 1 µM Los. K+ current was
recorded as in Fig. 1. After this recording the neuron was prepared for
single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) as
detailed in METHODS. Right: ethidium
bromide-stained gel showing RT-PCR DNA products that correspond to the
12-LO, PLA2, and AT2
receptor mRNAs. Leftmost lane is 100 bp ladder. Lanes 1, 2, and 3 are the AT2 receptor,
PLA2, and 12-LO mRNAs, respectively, from the
responsive neuron shown at left.
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|
Since activation of 12-LO is only partially responsible for the
stimulation of IKV by Ang II, and COX
is not involved in this Ang II action (Zhu et al. 1998
),
in further experiments we investigated the involvement of p450
monooxygenases. Treatment of cultured neurons with the selective
cytochrome p450 monooxygenase inhibitor 17-ODYA (10 µM) did not alter
the increase in neuronal IKV produced by either Ang II or AA (Fig. 8).

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Fig. 8.
Stimulation of neuronal IKV by Ang II
or AA is not altered by 17-ODYA. K+ current was
recorded as described in Fig. 1. Neurons were superfused with control
solution (superfusate; CON), Ang II (100 nM), or AA (50 µM) in the
absence (untreated) or presence of the selective cytochrome p450
monooxygenase inhibitor 17-ODYA (10 µM, added to DMEM and to the
superfusate). Top: representative current tracings showing
the effects of Ang II and AA in each treatment situation. CON
recordings were made before the application of Ang II or AA, and Ang II
experiments were performed in the presence of 1 µM Los to block
AT1 receptors. Bottom: bar graphs
showing means ± SE IKV of
current densities obtained in each treatment situation. Sample sizes
were 14 and 5 neurons for the untreated and 17-ODYA groups (Ang II
superfusions), respectively. Sample sizes were 5 and 5 neurons for the
untreated and 17-ODYA groups (AA superfusions), respectively.
* P < 0.05 compared with the respective control.
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|
 |
DISCUSSION |
The physiological and behavioral effects that are stimulated by
Ang II binding to its neuronal AT2 receptors in
brain involve modulation of membrane ionic currents and firing rate. We
have utilized neurons cultured from newborn rat brain stem and
hypothalamus to investigate the AT2
receptor-mediated actions of Ang II on neuronal activity and on the
underlying changes in membrane ionic currents. Our studies indicate
that stimulation of neuronal AT2 receptors
elicits an increase in neuronal firing rate (Zhu et al.
2000
) via shortening of action potential duration, and this is
consistent with our previous studies that indicate that Ang II
increases both neuronal Kv and A-type K+ current
(Kang et al. 1993
). We have examined the intracellular mechanisms through which Ang II stimulates an increase in neuronal IKV. Our data indicate that Ang II
elicits an AT2 receptor-mediated increase in
PLA2 activity and generation of AA, and that this mechanism is mostly responsible for the observed increase in neuronal IKV (Zhu et al. 1998
).
Since AA and AA metabolites are able to modulate the activity of
K+ channels (Arima et al. 1997
;
Colbert and Pan 1999
; Duerson et al.
1996
; Harder et al. 1997
; Premkumar et
al. 1990
; Schweitzer et al. 1990
;
Twitchell et al. 1997
; Zona et al. 1993
),
IKV, the stimulation of neuronal
IKV by Ang II potentially involved
these intracellular factors. Previous studies from our group gave an indication that this effect of Ang II involved LO, but not COX, metabolites of AA (Zhu et al. 1998
). Results from the
present study have confirmed this and have shown that the increase in neuronal IKV produced by Ang II is
partially mediated through generation of 12-LO metabolites of AA.
Furthermore, it appears that 5-LO metabolites and p450 monooxygenase
metabolites of AA are not involved in this response. However, it should
be emphasized that the 12-LO metabolic pathway is, according to our
data, only partially responsible for this Ang II action. This leaves
open the possibility that other pathways are involved in the Ang II effect on IKV. An obvious candidate is
AA itself, since it has been shown to directly modulate
K+ channels. It is also possible that other
intracellular mechanisms separate from the
PLA2/AA pathway are involved. For example, we cannot exclude the possibility that the Ang II-induced stimulation of
neuronal IKV also involves direct
membrane-delimited coupling of the AT2
receptor/Gi complex to the specific delayed rectifier K+ channel that is involved. A further
possibility is that an alternate intracellular signaling pathway may be
involved. For example, it has been determined that stimulation of
AT2 receptors in PC12W pheochromocytoma cells
increases the synthesis of the sphingolipid ceramide (Gallinat
et al. 1999
; Lehtonen et al. 1999
). Considering that ceramide increases IKV in
cortical cultured neurons (Yu et al. 1999
), it is
possible that the Ang II-induced increase in IKV seen in the present study also
involves this sphingolipid. However, our preliminary studies indicate
that short term (1-30 min) incubation of cultured neurons with Ang II
does not alter cellular ceramide levels, nor does ceramide application
alter neuronal IKV (E. M. Richards, M. Zhu, and C. Sumners, unpublished data). The identity of
the other signaling component(s) that is involved in the stimulation of
IKV by Ang II, in particular the possible direct action of AA at the Kv channel, will be the subject of
further investigation once the identity of the channel that is involved
has been ascertained.
The present studies have raised a number of interesting questions
concerning the mechanisms by which 12-LO metabolites of AA might alter
IKV. For example, in previous studies
we determined that the Ang II- and AA-induced stimulations of neuronal
IKV are completely abolished by
selective inhibition of serine/threonine phosphatase type 2A (PP-2A)
(Kang et al. 1995
; Zhu et al. 1998
). This
data, along with the observation that the Ang II stimulation of
PLA2 in cultured neurons is not altered by PP-2A
inhibitors (Zhu et al. 1998
), suggests that PP-2A is
downstream of the PLA2/AA/12 HETE pathway. It is
well known that K+ channels can be modulated by
phosphorylation and dephosphorylation of amino acid residues within the
subunit proteins (Breitwieser 1996
; Levitan
1999
). Thus one possibility is that the 12-LO metabolites of AA
or AA itself influence IKV via
activation of PP-2A, which in turn modulate K+
channel activity. There is certainly a precedent for this since AA has
been shown to stimulate the activity of PP-5, a novel serine/threonine phosphatase from brain (Skinner et al. 1997
). A further
possibility is that the 12-LO metabolites of AA modulate the
K+ channel subunits directly, and that PP-2A has
a permissive role over channel activation/inactivation. The actual
mechanism of AA/12-HETE/PP-2A modulation of
IKV will only be deciphered once certain fundamental questions have been answered. For example, do
12-HETE and/or AA stimulate PP-2A activity in cultured neurons? How
does PP-2A modulate IKV: directly via
dephosphorylation of channel subunits, or indirectly via activation of
a kinase?
A final point concerns the fact that similar intracellular signaling
pathways have been proposed for the modulation of
K+ currents by calcium and somatostatin in
neurons and pituitary tumor cells, respectively (Duerson et al.
1996
; Yu 1995
). Thus it is possible that a
common series of intracellular events
(PLA2/AA/12-LO metabolites of AA/PP-2A) are
responsible for the modulation of K+ currents by
different agents in different cell types. This commonality of signaling
pathways might help to explain the fact that the AT2 receptor knockout mice did not exhibit more
dramatic changes in physiological or behavioral responses; i.e.,
perhaps other factors that utilize the same intracellular signaling
mechanisms were able to compensate for the lack of Ang II stimulation
of AT2 receptors in these animals.
This work was supported by National Institutes of Health Grants
HL-49130 (C. Sumners and C. H. Gelband) and DK-39721 (J. L. Nadler and R. Natarajan).
Address for reprint requests: C. Sumners, Dept. of Physiology,
College of Medicine, University of Florida, Box 100274, Gainesville, FL
32610 (E-mail: csumners{at}phys.med.ufl.edu).