Department of Physiology, College of Medicine, and University of Florida Brain Institute, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Zhu, Mingyan,
Craig H. Gelband,
Philip Posner, and
Colin Sumners.
Angiotensin II Decreases Neuronal Delayed Rectifier Potassium
Current: Role of Calcium/Calmodulin-Dependent Protein Kinase II.
J. Neurophysiol. 82: 1560-1568, 1999.
Angiotensin II (Ang II) acts at specific receptors located on neurons
in the hypothalamus and brain stem to elicit alterations in blood
pressure, fluid intake, and hormone secretion. These actions of Ang II
are mediated via Ang II type 1 (AT1) receptors and involve
modulation of membrane ionic currents and neuronal activity. In
previous studies we utilized neurons cultured from the hypothalamus and
brain stem of newborn rats to investigate the AT1
receptor-mediated effects of Ang II on neuronal K+
currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to
activation of protein kinase C (PKC), specifically PKC. However, the
data also indicated that another Ca2+-dependent mechanism
was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a
known modulator of K+ currents in neurons, we investigated
the role of this enzyme in the AT1 receptor-mediated
reduction of neuronal Kv current by Ang II. The reduction of neuronal
Kv current by Ang II was attenuated by selective inhibition of either
calmodulin or CaM KII and was mimicked by intracellular application of
activated (autothiophosphorylated) CaM KII
. Concurrent inhibition of
CaM KII and PKC completely abolished the reduction of neuronal Kv by
Ang II. Consistent with these findings is the demonstration that Ang II
increases CaM KII activity in neuronal cultures, as evidenced by
increased levels of autophosphorylated CaM KII
subunit. Last,
single-cell reverse transcriptase (RT)-PCR analysis revealed the
presence of AT1 receptor-, CaM KII
-, and PKC
subunit
mRNAs in neurons that responded to Ang II with a decrease in Kv
current. The present data indicate that the AT1
receptor-mediated reduction of neuronal Kv current by Ang II involves
a Ca2+/calmodulin/CaM KII pathway, in addition to the
previously documented involvement of PKC.
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INTRODUCTION |
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The octapeptide angiotensin II (Ang II) has
various physiological effects mediated by the brain, including
stimulation of increased blood pressure, water and sodium intake,
vasopressin secretion, and modulation of baroreflex function
(Campagnole-Santos et al. 1988; Phillips and
Sumners 1998
). These actions of Ang II are mediated by specific
Ang II type 1 (AT1) receptors that are located on neurons
in the hypothalamus and brain stem (Hogarty et al. 1992
;
Koepke et al. 1990
; Qadri et al. 1994
).
Consistent with these physiological actions are electrophysiological
studies that have demonstrated that Ang II elicits specific
AT1 receptor-mediated changes in neuronal activity in the
hypothalamus and brain stem. For example, selective activation of
AT1 receptors elicits increases in neuronal firing rate in
the paraventricular nucleus, the subfornical organ, the supraoptic
nucleus, and the rostral ventrolateral medulla (Ambuhl et al.
1992
; Li and Ferguson 1993
; Li and
Guyenet 1995
; Yang et al. 1992
). Similar to the
in vivo situation, neurons cultured from the hypothalamus and brain
stem of newborn rats contain AT1 receptors (Gelband
et al. 1997
; Raizada et al. 1993
). In previous studies, we have used these cultured neurons to investigate the AT1 receptor-mediated effects of Ang II on neuronal
K+ and Ca2+ currents and the
intracellular signaling pathways that are involved. The rationale for
this approach was that changes in these currents form the basis of
changes in neuronal firing rate and ultimately of behavioral and
physiological effects that are stimulated by Ang II. We determined that
Ang II, via AT1 receptors, increases neuronal firing rate
(Wang et al. 1997a
). Consistent with this, we have
determined that Ang II elicits an AT1 receptor-mediated stimulation of total neuronal calcium current
(ICa) and decreases neuronal delayed
rectifier K+ current (Kv) and transient (A-type)
K+ current (Gelband et al. 1999
;
Sumners et al. 1996
; Wang et al. 1997b
).
These inhibitory effects of Ang II on Kv current involve a
G
q/11 protein, stimulation of phosphoinositide (PI)
hydrolysis and activation of protein kinase C (PKC), specifically
Ca2+-dependent PKC
(Pan et al. 1999
;
Sumners et al. 1996
; Zhu et al. 1997
).
However, our investigations indicated that PKC is only partially
responsible for modulation of these K+ currents, and that
another Ca2+-dependent mechanism is also involved
(Sumners et al. 1996
; Zhu et al. 1997
).
Our present studies are aimed at defining the identity of this
Ca2+-dependent pathway. These investigations have centered
around calcium/calmodulin-dependent protein kinase II (CaM KII)
(Braun and Schulman 1995a
; Colbran et al.
1989
), for two major reasons. First, several studies have
indicated that this enzyme is involved in the AT1
receptor-mediated actions of Ang II in certain cell types
(Abraham et al. 1996
; Muthalif et al.
1998
; Pezzi et al. 1996
). Second, even though
there is no background literature indicating that CaM KII can modulate
neuronal Kv current, this enzyme is a known modulator of neuronal
A-type- and calcium-activated K+ currents (Ikeuchi
et al. 1996
; Pedarzani and Storm 1996
;
Roeper et al. 1997
; Sakakibara et al.
1986
; Yamamoto et al. 1997
). The data presented
here indicate that Ang II, via AT1 receptors, stimulates increases in neuronal intracellular calcium
[Ca2+]i and CaM KII activity, the latter
indicated by increased levels of autophosphorylated CaM KII
subunit.
In addition, the data also indicate that the inhibition of neuronal Kv
current by Ang II involves a Ca2+/calmodulin/CaM KII
signaling pathway as well as the previously documented PKC pathway.
These studies provide the first indication that CaM KII is able to
modulate neuronal Kv current.
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METHODS |
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Materials
Newborn Sprague-Dawley rats were obtained from our breeding
colony, which originated from Charles River Farms (Wilmington, MA).
Dulbeccos modified Eagle's medium (DMEM) and TRIzol reagent were
obtained from GIBCO-BRL (Gaithersburg, MD). Plasma derived horse serum
(PDHS) came 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 & Co. (Rahway, NJ). PD 123,319 (PD), Calphostin C, KN-93, KN-92,
W-7, and CaM KII (281-302) peptide were purchased from Research
Biochemicals International (Natick, MA). Tetrodotoxin (TTX) was
purchased from Calbiochem (La Jolla, CA). Monoclonal anti-CaM KII
antibody was obtained from Transduction Laboratories (Lexington, KY).
Anti-ACTIVE CaM KII pAb was obtained from Promega (Madison, WI).
Gene-Amp reverse transcriptase-polymerase chain reaction (RT-PCR) kits
and all reagents for RT-PCR were purchased from Perkin Elmer
Biotechnologies (Norwalk, CT). Autothiophosphorylated CaM KII was
kindly provided by Dr. T. R. Soderling (University of Washington).
Ang II, sodium GTP, HEPES, cadmium chloride
(CdCl2), fura-2/AM, ethylene glycol-bis
(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA),
bovine serum albumin, dipotassium ATP, and peroxidase-conjugated affinity purified goat anti-rabbit IgG were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA) and were of analytic grade or
higher. Oligonucleotide primers for the AT1A
receptor (Kakar et al. 1992
), CaM KII
(Lin et
al. 1987
), and PKC
(Ono et al. 1988
) genes
were synthesized in the DNA core facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. The sequences
of these primers are as follows:
AT1A receptor
Sense, 5 prime-CCATGCCATCTGTAATCCAC-3 prime
Antisense, 5 prime-AAGGCCACATGAACTGACTC-3 prime
CaM KII subunit
Sense, 5 prime-GACTCCATGACAGCATCTC-3 prime
Antisense, 5 prime-CATCTGGTGACACTGTAGC-3 prime
PKC
Sense, 5 prime-GGTGTCTCAGAGCTACTCAA-3 prime
Antisense, 5 prime-TGAGGAAGCTGAAGTCAGAG-3 prime
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. 1994
).
[Ca 2+]i analyses
Analysis of [Ca
2+]i in cultured neurons
was achieved using imaging fluorescence microscopy in cells preloaded
with 5 µM fura-2/AM, as detailed by us previously (Wang et al.
1996).
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 A
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 60 ms every 10 s. Under
these recording conditions, both the Kv current 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 50 ms after the initiation of the test pulse, at
which time they reflect only Kv current (Kang et al.
1994
). Current density was derived by dividing transmembrane
current (pA) by membrane capacitance (pF). Membrane capacitance was
calculated by the equation C = Ac/
V, where C
is capacitance, Ac is the area of
capacitative current, and
V is the voltage step.
Ac was obtained by the equation
Ac = IoRC, which is from
Ic =
Io × e
t/RC, where
I is the maximal capacitative current and RC is
the time constant, equal to the time when
Io is 36%. The average cell
capacitance for neurons used in this study was 36.7 ± 10.4 pF
(mean ± SE; n = 94 neurons; range, 3.7-70 pF).
Selective stimulation of neuronal AT1 receptors
causes a decrease in Kv current (Sumners et al. 1996),
whereas selective stimulation of neuronal AT2
receptors causes an increase in neuronal Kv current (Zhu et al.
1998
). Some of the neurons in the cultures used here contain
both AT1 and AT2 receptors
(Gelband et al. 1997
). Because the aim of the present
studies was to measure AT1 receptor-mediated effects of Ang II on Kv current, all electrophysiological recordings were performed in the presence of 1 µM PD123,319 to block
AT2 receptors. PD123,319 did not alter basal Kv current.
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). For the experiments using cells from the whole dish, RT-PCR of the
AT1A receptor, CaM KII
, and PKC
were
performed using Gene-Amp RT-PCR kits essentially as described
previously (Huang et al. 1997
). In brief, PCR was
performed at 95°C for 4 min, followed by 38 cycles at 95°C for
45 s, 61°C for 90 s, and 72°C for 120 s. After final
extension at 72°C for 10 min, PCR products were subjected to
electrophoresis on a 2% agarose gel containing 1 µg/ml ethidium
bromide. Using these conditions, we observed the production of a 169 bp
AT1A receptor-specific DNA, a 167 bp CaM KII
-specific DNA, and a 280 bp PKC
-specific DNA from the PCR. These correspond to the AT1A receptor and PKC
mRNAs, respectively.
For the experiments on single neurons, RT-PCR of the
AT1A receptor, CaM KII, and PKC
was
performed as detailed by us previously (Zhu et al.
1998
). In brief, following recordings of Kv current, 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. A first PCR was performed
exactly as described above for neurons from the whole dish. A second
PCR was performed (on 20 µl of the 1st PCR products) at 95°C for 4 min followed by 32 cycles at 95°C for 45 s, 61°C for 90 s, and 72°C for 120 s. After final extension at 72°C for 10 min, the PCR products were electrophoresed as above. Using these
conditions for single-cell RT-PCR, we observed the production of 169 bp
AT1A receptor-, 167 bp CaM KII
-, and 280 bp
PCK
-specific DNAs, similar to the bands obtained from the whole dish
of neurons. In all situations, exclusion of either RNA or MuLV reverse
transcriptase resulted in no visible bands after gel electrophoresis.
Analysis of CaM KII subunit proteins
The presence of CaM KII and CaM KII
subunit proteins in
control neuronal cultures was determined by Western Blot analysis using
a monoclonal anti-CaM KII antibody (1:250 dilution). Extraction of
total cellular protein, SDS polyacrylamide gel electrophoresis, and
immunoblotting were performed exactly as detailed previously (Kopnisky et al. 1997
). These procedures yielded bands
of ~50 and ~60 kDa, corresponding to CaM KII
and CaM KII
subunits, respectively.
The presence of autophosphorylated CaM KII subunit protein in
control or Ang II-treated neuronal cultures was determined by Western
Blot analysis using an anti-ACTIVE CaM KII pAb. This antibody
preferentially detects CaM KII that is phosphorylated on threonine 286 (pT286) of the
-subunit. Total cellular
protein was isolated from neuronal cultures as follows. Cells were
washed twice with ice-cold PBS (pH 7.2), and 200 µl of ice-cold lysis
buffer (1% NP-40, 10% glycerol, 150 mM NaCl, 20 mM Tris-HCl, 1 mM
phenylmethyl-sulfonyl fluoride, and 2.5 mg/µl each of aprotinin,
leupeptin, antipain, and chymostatin) was added to each dish. Samples
were centrifuged at 4°C and 350 × g for 5 min, and
the supernatant was removed to a clean tube. A small aliquot was used
for analysis of protein concentration (Bradford 1976
),
and the remainder was stored at
70°C. Proteins were separated by
size on a 10% SDS polyacrylamide gel using the system of
Laemmli (1970)
, and transferred to nitrocellulose
(Bioblot, Costar) at 100 V for 1 h in Towbin-SDS transfer buffer
(25 mM Tris 192 mM glycine, 20 mM methanol, and 0.01% SDS). After
transfer the blot was washed once in PBS with 0.05% Tween (PBST) for
10 min. The membrane was blocked in PBST and 1% bovine serum albumin overnight at 37°C. Next, the membrane was incubated for 1 h at room temperature with a 1:5,000 dilution of anti-ACTIVE CaM KII pAb in
PBST and 0.1% bovine serum albumin. After this, the membrane was
washed three times in PBST, and was then incubated for 1 h at room
temperature with a 1:16,000 dilution of peroxidase-conjugated affinity
purified goat anti-rabbit IgG in PBST and 0.1% bovine serum albumin.
The membrane was then washed three times in PBST at room temperature.
Detection of the resulting antigen-antibody complex was performed using
the Renaissance ECL kit, according to the manufacturer's directions,
and visualized by exposure to Kodak film (Bio Max light) for 90 s.
Drug 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. Solvent controls were performed for each protocol. Intracellular application of CaM KII
(281-302) peptide and of autothiophosphorylated CaM KII were
achieved as detailed by us previously (Zhu et al. 1997
). In brief, a sidearm pipette holder is attached to the head stage of the
Axopatch. One side arm is used to apply 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 Kv 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 CaM KII
or CaM KII
(281-302) are allowed to diffuse into the neuron, and measurements of
Kv current are made 4 min later, at which time a stable peak response
is obtained. Care is taken not to overperfuse the neuron, and this is
monitored electrically via the Axopatch and on the TV monitor. Thus the
concentrations of CaM KII
and CaM KII (281-302) that are given in
RESULTS indicate the amounts that were injected at the
pipette tip and 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 neuronal cultures. Comparisons were made with the use of a one-way ANOVA followed by Newman-Keuls test to assess statistical significance.
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RESULTS |
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In previous studies, we have determined that the Ang II-induced
decrease in neuronal Kv current is partially mediated via activation of
a Ca2+-dependent PKC (Pan et al.
1999; Sumners et al. 1996
; Zhu et al. 1997
). These results also indicated that the inhibitory effect of Ang II on neuronal Kv current was totally abolished by concurrent inhibition of PKC and chelation of
[Ca2+]i. Thus it appeared
that aside from PKC, an additional Ca2+-dependent
mechanism was responsible for mediating the decrease in neuronal Kv
current produced by Ang II. Because CaM KII is a known modulator of
certain neuronal K+ currents and is activated by
AT1 receptor stimulation in certain cell types,
in the present studies we decided to investigate the possible role of a
Ca2+/calmodulin/CaM KII pathway in the Ang
II-induced decrease in neuronal Kv current.
The first series of experiments were performed to establish the effect
of Ang II on [Ca2+]i in
cultured neurons. The data presented in Fig.
1 demonstrate that superfusion of
cultures with normal Tyrode's solution (containing 2 mM
CaCl2) in the presence of 100 nM Ang II produces
an increase in [Ca2+]i in
a representative neuron. The increase in
[Ca2+]i elicited by Ang
II was abolished by co-superfusion of the AT1 receptor antagonist Los (1 µM; data not shown). The mean resting [Ca2+]i, mean Ang
II-stimulated peak
[Ca2+]i, and mean
steady-state/plateau
[Ca2+]i were 116 ± 13, 622 ± 24, and 142 ± 12 nM, respectively
(n = 4 neurons). These findings are consistent with our
previous demonstration that Ang II elicits on AT1
receptor-mediated increase in PI hydrolysis in neuronal cultures
(Sumners et al. 1996). Increases in
[Ca2+]i will lead to
activation of a number of Ca2+-dependent
signaling molecules, including calmodulin, which subsequently activates
CaM KII. Thus in the next series of experiments we investigated whether
calmodulin was involved in the Ang II-induced decrease in neuronal Kv
current. Superfusion of cultures with Ang II (100 nM) caused a
significant reduction in neuronal Kv current that was completely
inhibited by 1 µM Los (Fig. 2), in
agreement with our previous studies (Sumners et al.
1996
). Treatment of cultures with a calmodulin antagonist W-7
(10 µM) significantly attenuated the AT1
receptor-mediated inhibition of neuronal Kv current by Ang II (Fig.
2). Higher concentrations of W-7 (20-50 µM) produced no greater
attenuation of this Ang II effect (data not shown). Control recordings
of neuronal Kv in the presence of W-7 were not significantly different
from control recordings in untreated neurons (Fig. 2). Thus the data in
Fig. 2 indicate that calmodulin is involved in the
AT1 receptor-mediated inhibition of neuronal Kv
current by Ang II.
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Calmodulin (in combination with Ca2+) activates
CaM KII, so the next aim was to investigate the role of this kinase in
the reduction of neuronal Kv current produced by Ang II. However, it
was first important to determine whether Ang II stimulates CaM KII
activity in neuronal cultures. Immunoblot analyses using a specific
anti-CaM KII antibody revealed the presence of an ~50 kDa CaM KII
subunit and an ~60-kDa CaM KII
subunit in neuronal cultures (Fig.
3A). This is consistent with
the picture in rat brain, where these are the predominant CaM KII
isoforms (Braun and Schulman 1995a
). Incubation of
neuronal cultures with 100 nM Ang II (in the presence of 1 µM
PD123,319 to block AT2 receptors), elicited an increase in
the levels of phosphorylated CaM KII
subunit protein (Fig. 3B). Because the glia within these cultures do not
contain AT1 receptors (Sumners, unpublished results), the
present findings are indicative of an AT1
receptor-mediated increase in neuronal CaM KII activity. Treatment of
cultures with CaM KII inhibitors significantly attenuated the
AT1 receptor-mediated inhibition of neuronal Kv current by
Ang II. For example, inclusion of CaM KII inhibitory peptide [CaM KII
(281-302) 2 µM (Braun and Schulman 1995b
;
Smith et al. 1992
)] in the pipette solution or
pretreatment of cultures with the CaM KII inhibitor KN-93 (10 µM; 10 min) (Sumi et al. 1991
) caused a significant attenuation
of Ang II-inhibited Kv current (Fig. 4).
By contrast, pretreatment of cultures with KN-92 (10 µM; 10 min), an
inactive analogue of KN-93, did not alter this Ang II effect on
neuronal Kv current (Fig. 4). The role of CaM KII was further
demonstrated by experiments in which the inhibitory effect of Ang II on
neuronal Kv current was partially reversed by acute administration of 2 µM CaM KII (281-302) via intracellular application (Fig.
5). Full reversal of the inhibitory effect of Ang II on neuronal Kv current was obtained with subsequent superfusion of 1 µM Los (Fig. 5). Control recordings of neuronal Kv
current in the presence of these CaM KII inhibitors were not significantly different from control recordings in untreated neurons (Fig. 4). In addition, higher concentrations of these CaM KII inhibitors (e.g., 20 µM KN-93) produced no further attenuation of Ang
II-modulated Kv current (data not shown). The data presented in Figs.
4 and 5 therefore indicate that CaM KII is involved in the
AT1 receptor-mediated inhibition of neuronal Kv current by Ang II. If this is the case, it follows that selective activation of
CaM KII may mimic the effects of Ang II on neuronal Kv current. To
simulate activation of CaM KII, we utilized activated
(autothiophosphorylated) CaM KII
. This has been used previously by
others to modulate neuronal K+ currents (Lledo et
al. 1995
). Intracellular application of 200 nM
autothiophosphorylated CaM KII
produced a significant decrease in
neuronal Kv current of 7.8 ± 1.4% (n = 3 neurons, P < 0.005) compared with control
recordings (see representative tracings in Fig.
6). In contrast, similar application of
boiled (denatured) autothiophosphorylated CaM KII (200 nM) did not
significantly alter neuronal Kv current (decrease of 1.3 ± 0.6%,
n = 5 neurons). Although small, the reduction of
neuronal Kv current produced by the autothiophosphorylated CaM KII
is reasonable. This is explained because the maximal reduction of Kv
current produced by Ang II is ~20% (Fig.
7), and our present data indicate that this effect of Ang II is only partially due to activation of CaM KII
(Figs. 4 and 5).
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The Ang II-induced reduction of neuronal Kv current is partially
mediated by PKC (Sumners et al. 1996). Considering that
the present data also indicate a role for CaM KII in this response, we
tested the effects of co-inhibition of PKC and CaM KII on the reduction
of neuronal Kv current produced by Ang II. Treatment of cultures with
the PKC inhibitor Calphostin C (Cal; 750 nM, 30 min) produced a partial
inhibition of Ang II's effects on Kv current (Fig. 7), in agreement
with our previous data (Sumners et al. 1996
). In
cultures that had been co-treated with Cal (750 nM) and KN-93 (10 µM), Ang II failed to produce a significant reduction in neuronal Kv
current (Fig. 7). Collectively, the present studies suggest that the
Ang II-induced reduction of neuronal Kv current via AT1
receptors is mediated via dual intracellular messenger pathways, namely
PKC and CaM KII. To further establish these signaling molecules as
mediators of the Ang II-induced decrease in neuronal Kv current, it is
necessary to demonstrate their presence within the responsive neurons.
The RT-PCR data presented in Fig. 8
demonstrate the presence of AT1 receptor, PKC
, and CaM
KII
mRNAs in a whole dish of neuronal cultures. Further, the data indicate the presence of these mRNAs in a single neuron that responded to Ang II with an AT1 receptor-mediated reduction of Kv
current (Fig. 8). Thus these RT-PCR data provide strong support for a role of PKC and CaM KII in the Ang II-induced decrease in neuronal Kv
current.
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DISCUSSION |
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The physiological and behavioral events that are stimulated by Ang
II binding to its AT1 receptors in brain involve
depolarization and activation of specific neuronal pathways. In
previous studies, we have utilized primary neuronal cultures from
newborn rat hypothalamus and brain stem to investigate the effects of
Ang II on changes in neuronal activity and the underlying ionic
currents. Our data indicated that Ang II acting via
AT1 receptors increases neuronal firing rate
(Wang et al. 1997a), increases total
Ca2+ current and reduces Kv and A-type
K+ currents (Gelband et al. 1999
;
Sumners et al. 1996
; Wang et al. 1997b
).
In further studies, we investigated the intracellular signaling
pathways that mediate the effects of Ang II on these currents. The Ang
II-induced decrease in neuronal Kv current involves a signaling
pathway that begins with G
q/11-mediated
activation of phospholipase C (PLC) and increased PI hydrolysis
(Sumners et al. 1996
). The products of PI
hydrolysis, diacylglycerol and inositol 1,4,5-triphosphate
(IP3), activate PKC and increase intracellular Ca2+, respectively (Berridge
1997
). Because
[Ca2+]i transients are
usually short in duration, the Ang II-stimulated increase in
[Ca2+]i probably sets off
a cascade of biochemical events that involves a number of
Ca2+-dependent kinases. Our data have indicated
that a calcium-dependent PKC isozyme, PKC
, is partially responsible
for the Ang II-induced decrease in neuronal Kv current (Pan et
al. 1999
; Sumners et al. 1996
). However, our
data also indicated that another Ca2+-dependent
mechanism was responsible for the residual inhibitory action of Ang II
on neuronal Kv current. The present studies clearly indicate that this
other Ca2+-dependent mechanism involves
activation of CaM KII, because the inhibitory effect of Ang II on
neuronal Kv current is attenuated by a calmodulin antagonist (Fig. 2),
or by inhibition of CaM KII (Figs. 4 and 5). Furthermore, the
inhibitory effect of Ang II on neuronal Kv current is mimicked by
intracellular application of activated (autothiophosphorylated) CaM
KII
(Fig. 6). The demonstration that Ang II activates CaM KII in
neuronal cultures (Fig. 3) also supports the conclusion that this
enzyme mediates the actions of Ang II on neuronal Kv current.
Concurrent inhibition of CaM KII and PKC resulted in no significant
inhibitory effect of Ang II on neuronal Kv current (Fig. 7).
Collectively, the data from our present and previous studies indicate
that the reduction of neuronal Kv current elicited by Ang II results
from Gq/11/PLC- mediated activation of two
distinct Ca2+-dependent/enzymes, CaM KII and PKC.
Importantly, single-cell RT-PCR data also demonstrate the presence of
these signaling components within the same (Ang II-responsive) neuron.
This is essential, because it might be argued that superfusion of Ang
II activates neuronal AT1 receptor at one locus,
causing release of a paracine factor, which diffuses to the recording
neuron and inhibits Kv current (via CaM KII and PKC).
The fact that Ang II reduces neuronal Kv current via a dual modulatory
pathway has a number of important implications. Because activation of
both PKC and CaM KII is needed for the full inhibitory action of Ang II
on Kv current, it is possible that the magnitude of Ang II's action
can be modified by pathways or factors that interrupt the activation of
these enzymes. For example, inhibition of either PKC or CaM KII would
blunt, but not eliminate the effect of Ang II on Kv current. By
contrast, a factor that inhibits the stimulation of PLC by Ang II would
abolish the actions of this peptide on neuronal Kv current. Therefore
these may be potential mechanisms through which other
neurotransmitters/hormones can modify the physiological and behavioral
actions of Ang II in the brain. Other important points concern the
mechanisms through which Kv current is modulated by PKC and CaM KII. It
is well-known from studies in other systems that both PKC and CaM KII
can modulate neuronal K+ currents (Doerner
et al. 1988; Grega et al. 1987
; Ikeuchi
et al. 1996
; Pedarzani and Storm 1996
;
Roeper et al. 1997
; Shearman et al.
1989
), although few of these studies have addressed the molecular mechanisms involved. There is now a great deal of evidence that phosphorylation/dephosphorylation of K+
channel proteins is important in the regulation of their activity, and
in the regulation of K+ currents (Levitan
1994
). With respect to AT1 receptor
activation, we have determined that the reduction in neuronal Kv
currents is due to inhibition of the Kv2.2 K+
channel subunit (Gelband et al. 1999
). Inspection of the
amino acid sequence of the rat Kv2.2 channel reveals the presence of multiple consensus PKC (S/T-X-R/K) and CaM KII (R-X-X-S/T)
phosphorylation sites within the cytoplasmic domains (Chandy and
Gutman 1995
; Hwang et al. 1992
; Luneau et
al. 1991
; Rettig et al. 1992
). Therefore considering that Ang II stimulates both PKC (Sumners et al.
1996
) and CaM KII activities in cultured neurons, it is
reasonable to speculate that the reduction in Kv current caused by Ang
II is mediated via phosphorylation of Kv2.2 by these enzymes. It is also possible that through selective phosphorylation events, PKC and
CaM KII can inhibit neuronal Kv current via modulation of different
biophysical properties of the underlying K+
channels (e.g., channel open and closed times, activation and inactivation kinetics, time of 1st latency, etc.). The relationships between channel phosphorylation via PKC and CaM KII, channel activity, biophysical properties, and changes in Kv current will be the subject
of our future studies.
Clearly, many questions remain to be answered. For example, do the
observed changes in neuronal Kv current include direct channel
phosphorylation by PKC and CaM KII? Or, are the effects of these
serine/threonine kinases mediated via activation of other enzymes
(e.g., tyrosine kinases) that subsequently modulate channel activity
via phosphorylation? The latter situation is a realistic possibility
because in preliminary studies we have shown that the tyrosine kinase
inhibitor genistein attenuates Ang II-induced decreases in neuronal Kv
current (unpublished observations). Other questions concern the
functional or cellular role of the reduction in neuronal Kv current
produced by Ang II. It is established that some of the physiological
and behavioral actions of Ang II in the brain involve modulation of
central noradrenergic neurons (Sumners et al. 1994).
Indeed, our previous studies indicate that AT1
receptors are present on noradrenergic neurons in the cultures used
here, and that stimulation of these receptors results in an increase in
neuronal firing rate and release of norepinephrine (Gelband et
al. 1997
; Richards et al. 1999
; Wang et
al. 1997a
). Thus it is tempting to speculate that the reduction
in Kv current caused by Ang II via PKC and CaM KII contributes to
membrane depolarization and ultimately, the release of norepinephrine.
This speculation will be the subject of our further studies.
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ACKNOWLEDGMENTS |
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The authors thank J. Moore for technical assistance and M. Fancey for typing this manuscript.
This work was supported by National Institutes of Health Grants HL-49130 and NS-19441.
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FOOTNOTES |
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Address for reprint requests: C. Sumners, Dept. of Physiology, Box 100274, 1600 SW Archer Rd., Gainesville, FL 32610.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 23 March 1999; accepted in final form 25 May 1999.
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REFERENCES |
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