Download Angiotensin AT1-Receptors Depolarize Neonatal Spinal

J Neurophysiol
88: 2857–2863, 2002; 10.1152/jn.00978.2001.
Angiotensin AT1-Receptors Depolarize Neonatal Spinal Motoneurons
and Other Ventral Horn Neurons Via Two Different Conductances
MURAT OZ1 AND LEO P. RENAUD2
1
National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224; and 2Neurosciences,
Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario K1Y 4E9 Canada
Received 29 November 2001; accepted in final form 18 July 2002
roles also include modulation of cardiovascular reflexes (Phillips 1987; Sumners et al. 1994). In CNS, angiotensin-like
immunoreactive neurons and fibers as well as ANG II receptors, mainly of the AT1 type, have a wide but differential
distribution (see Gehlert et al. 1986; Lind et al. 1984; Mendelsohn et al. 1984). That the latter participate in regulating
neuronal excitability is inferred from electrophysiological observations of neuronal responsivity to exogenous ANG II (e.g.,
Bai and Renaud 1998; Ferguson and Washburn 1998; Li and
Guyenet 1996; Ono et al. 2001; Yang et al. 1992). These
features indicate considerable regional and cell-specific expression of ANG II receptors.
Binding studies and receptor-expression analyses also indicate plasticity within the central renin-angiotensin system. A
high level of expression during the first and second postnatal
week and change during ontogeny (Tsutsumi and Saavedra
1991) suggest a physiologically important role in CNS development. One of these sites may be in spinal cord, where ANG
II-immunoreactive fibers and receptors have been observed
(Gehlert et al. 1986; Lind et al. 1984; White et al. 1988) and
where ANG II has a depolarizing action on motoneurons
(Suzue et al. 1981) and lateral horn neurons (Lewis and Coote
1993). There are few details on mechanisms of ANG II action
on spinal neurons.
During patch-clamp recordings to evaluate functional evidence for peptide receptors on thoracolumbar neurons in neonatal spinal cord, we observed that a subpopulation of motoneurons and unidentified ventral horn neurons were
responsive to exogenous ANG II. We now report that these
neurons display ANG-II-induced membrane depolarization
and inward currents that engage two separate conductances: a
potassium conductance in motoneurons and/or a presumed
nonselective cationic conductance in interneurons.
METHODS
INTRODUCTION
In the peripheral circulation, angiotensin (ANG II) is not
only a powerful modulator of vasomotor activity but can act
through neurons in the subfornical organ, a circumventricular
organ, to initiate a behavioral response, i.e., drinking, a response dependent on a central renin-angiotensin system whose
The spinal cords of methoxyflurane-anesthetized Sprague-Dawley
rats of either sex (5–12 days old) were removed after a dorsal
thoracolumbar laminectomy and placed in ice-cold (4°C) artificial
cerebrospinal fluid (ACSF). ACSF was composed of (in mM) 127
NaCl, 26 NaHCO3, 3.1 KCl, 1.2 MgCl2, 2.4 CaCl2, and 10 D-glucose
(pH 7.35; osmolarity 290 –305 mosmol) and was gassed with 95%
O2-5% CO2. Transverse 350 – 450 ␮m sections from the Th7 to L5
Address for reprint requests: M. Oz, National Institute on Drug Abuse,
Intramural Research Program, 5500 Nathan Shock Dr., Baltimore, MD, 21224
(E-mail: [email protected]).
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.
www.jn.org
2857
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
Oz, Murat, and Leo P. Renaud. Angiotensin AT1-receptors depolarize neonatal spinal motoneurons and other ventral horn neurons via
two different conductances. J Neurophysiol 88: 2857–2863, 2002;
10.1152/jn.00978.2001. Angiotensin receptors are highly expressed in
neonatal spinal cord. To identify their influence on neuronal excitability,
we used patch-clamp recordings in spinal cord slices to assess responses
of neonatal rat (5–12 days) ventral horn neurons to bath-applied angiotensin II (ANG II; 1 ␮M). In 14/34 identified motoneurons tested under
current clamp, ANG II induced a slowly rising and prolonged membrane
depolarization, blockable with Losartan (n ⫽ 5) and (Sar1, Val5, Ala8)ANG II (Saralasin, n ⫽ 4) but not PD123319 (1 ␮M each; n ⫽ 4). Under
voltage clamp (VH ⫺65 mV), 7/22 motoneurons displayed an ANG-IIinduced tetrodotoxin-resistant inward current (⫺128 ⫾ 31 pA) with a
similar time course, an associated reduction in membrane conductance
and net current reversal at ⫺98.8 ⫾ 3.9 mV. Losartan-sensitive ANG II
responses were also evoked in 27/78 tested ventral horn “interneurons.”
By contrast with motoneurons, their ANG-II-induced inward current was
smaller (⫺39.9 ⫾ 5.2 pA) and analysis of their I-V plots revealed three
patterns. In eight cells, membrane conductance decreased with net inward
current reversing at ⫺103.8 ⫾ 4.1 mV. In seven cells, membrane conductance increased with net current reversing at ⫺37.9 ⫾ 3.6 mV. In 12
cells, I-V lines remained parallel with no reversal within the current range
tested. Intracellular dialysis with GTP-␥-S significantly prolonged the
ANG II effect in seven responsive interneurons and GDP-␤-S significantly reduced the ANG II response in four other cells. Peak inward
currents were significantly reduced in all 13 responding neurons recorded
in slices incubated in pertussis toxin (5 ␮g/ml) for 12–18 h or in 12
neurons perfused with N-ethylmaleimide. Of 29 interneurons sensitive to
pertussis toxin or N-ethylmaleimide treatment, 9 cells displayed a decrease in membrane conductance that reversed at ⫺101.3 ⫾ 3.8 mV. In
eight cells, membrane conductance increased and reversed at ⫺38.7 ⫾
3.4 mV. In 12 cells, the I-V lines remained parallel with no reversal within
the current range tested, suggesting that both conductances are modulated
by pertussis toxin-sensitive G proteins. These observations reveal a direct,
G-protein-mediated depolarizing action of ANG II on neonatal rat ventral
horn neurons. They also imply involvement of two distinct conductances
that are differentially distributed among different cell types.
2858
M. OZ AND L. P. RENAUD
RESULTS
Motoneurons
Data obtained from 56 motoneurons located in (within
Rexed laminae VIII and IX) displayed a mean resting membrane potential of ⫺71.4 ⫾ 1.9 mV and input resistance of
60.9 ⫾ 4.3 M⍀.
ANG-II-INDUCED POSTSYNAPTIC MEMBRANE DEPOLARIZATION
AND INWARD CURRENT. In 14/34 neurons tested while record-
ing in current-clamp mode, bath application of ANG II (1 ␮M;
30 s) initiated a slowly rising (60 –90 s to peak) membrane
depolarization that reached a plateau of 11.6 ⫾ 3.2 mV and
was sufficient to trigger a burst of action potentials in five cells
(Fig. 1A). Some desensitization seems likely since washout
intervals of 30 – 45 min were required to regain full recovery.
Application of an AT1 receptor antagonist Losartan, (1 ␮M;
n ⫽ 5 cells) was without effect on resting membrane properties
but completely blocked the ANG-II-induced membrane depolarizations. Similar results were achieved with Saralasin (1
␮M; n ⫽ 4 cells). However, an AT2 receptor antagonist
PD123319 (at 1 ␮M) (cf. Kang et al. 1992) was without effects
on ANG-II-induced depolarizations (Fig. 1B; n ⫽ 4 cells).
Under voltage clamp (VH ⫺65 mV) and in the presence of 1
␮M TTX, 1 ␮M ANG II applications to 22 motoneurons
yielded seven cells that responded with a slowly developing
postsynaptic response characterized by inward current (peak of
⫺128 ⫾ 31 pA) that slowly recovered over 8 –10 min (Fig.
2A). Comparison of instantaneous I-V plots before and at the
peak of the ANG II responses revealed net ANG II currents
(difference between control and peak ANG II effect) whose
slope indicated a 19.1% reduction in membrane conductance
(from 11.3 ⫾ 1.8 to 8.9 ⫾ 1.3 nS). The mean net ANG II
current reversed at ⫺98.8 ⫾ 3.9 mV, approximating the estimated equilibrium potential for potassium ions under these
J Neurophysiol • VOL
FIG. 1. Angiotensin II (ANG II) induces prolonged depolarizations through
activation of AT1-type receptors in motoneurons. A: whole cell current-clamp
recording from a spinal motoneuron recorded on postnatal day 8 (PN8; resting
membrane potential VR ⫺69 mV). In control artificial cerebrospinal fluid
(ACSF; top), the response to a 30-s ANG II application (horizontal bar) is a
slowly rising, prolonged and reversible membrane depolarization sufficient to
initiate a burst of action potentials. In the same cell, 35 min later, the ANG II
response is blocked by prior application of 1 ␮M AT1-type receptor antagonist
Losartan (bottom). B: histograms illustrate the effects of AT1-type receptor
antagonist Losartan (1 ␮M; n ⫽ 5), AT2-type receptor antagonist PD123319 (1
␮M; n ⫽ 5), and specific AT-receptor antagonist (Sar1, Val5, Ala8)-angiotensin
II (1 ␮M; n ⫽ 4) on ANG-II-induced membrane depolarizations (n ⫽ total of
14 cells). Asterisk: P ⬍ 0.05, compared with control values obtained without
pretreatments (paired Student’s t-test for each groups control values).
conditions (Fig. 2B). Increasing extracellular concentration of
K⫹ to 10 mM shifted the reversal potential to ⫺68 ⫾ 2.3 mV
(Fig. 2C; n ⫽ 3 cells) implying mediation via potassium
channels.
Interneurons
A population of 78 cells labeled as interneurons based on
failure of antidromic activation displayed a significantly less
negative resting membrane potential (⫺59.8 ⫾ 1.4 mV; P ⬍
0.05, Student’s t-test) and higher input resistance (179.8 ⫾
14.3 M⍀; P ⬍ 0.05, Student’s unpaired t-test) than motoneurons.
Recordings from
78 neurons in voltage-clamp mode in the presence of TTX
yielded 27 neurons that responded to ANG II (1 ␮M) with a
mean inward current of ⫺37.9 ⫾ 5.2 pA (Fig. 3A). Although
ANG-II-INDUCED POSTSYNAPTIC RESPONSES.
88 • NOVEMBER 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
segments were cut on a vibratome, equilibrated in ACSF at room
temperature, and continuously superfused at 4 – 6 ml/min in a recording chamber.
Using the blind whole cell patch-clamp technique, neurons were
recorded using Axopatch 1A or an Axopatch 1D amplifier (Axon
Instruments, Foster City, CA). Micropipettes were filled with (in mM)
130 K-Gluconate, 10 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, 1 EGTA,
1 GTP, and 2 Mg-ATP, adjusted to a pH of 7.3 with Tris buffer.
Lucifer yellow (1 mg/ml) was included for later visualization and
morphological identification using methods described earlier (Kolaj
and Renaud 1998). Corrections of the liquid-junction potentials (approximately ⫺10 mV) were performed off-line. Data was filtered
on-line at 2 kHz. Digidata 1200 interface and version 7 of pCLAMP
software were used on-line to generate clamp commands. Motoneurons were identified by their all-or-nonantidromic responses to ventral
stimulation applied with a concentric bipolar electrode (1–10 V,
duration 0.02 s) and/or by their morphology and evidence of an axon
projecting toward the ventral root. In this study, we applied the term
interneuron to any other neuron.
ANG II, a peptide receptor antagonist (Sar1, Val5, Ala8)-ANG II
(Saralasin), a nonpeptide AT2 receptor antagonist PD123319, GTP,
GTP-␥-S, GDP-␤-S, pertussis toxin, and tetrodotoxin were from
Sigma-RBI (St. Louis, MO). Agents were dissolved in ACSF at their
final concentrations, and delivered by bath application at a perfusion
rate of 4 – 6 ml/min. Losartan, a nonpeptide AT1 receptor antagonist,
was obtained from Merck (Rathway, NJ). For statistical evaluation,
we used paired or unpaired Student’s t-test as indicated in the text.
Results are presented as means ⫾ SE.
ANG II RECEPTORS IN NEONATAL VENTRAL SPINAL CORD
2859
different in magnitude from the response in motoneurons, the
time course of these ANG-II-induced current was virtually
identical (compare Figs. 2A and 3A). Similarly, responses to
ANG II were blocked by prior application of Saralasin (1 ␮M;
3/3 cells) and Losartan (1 ␮M; 4/4 cells), but not by PD123319
(1 ␮M, 4/4), all without effect on resting membrane properties
(Fig. 3A).
Analyses of voltage-current relationships obtained at the
peak of the responses for the population of cells indicated a
small decrease in membrane conductance (from a control value
of 4.9 ⫾ 0.8 to 4.6 ⫾ 1.2 nS; P ⬎ 0.05, paired t-test). However,
a comparison of the net ANG-II-induced currents for individual cells revealed three significantly differing patterns (Fig.
3B). One group of eight neurons demonstrated a net ANG-IIinduced conductance that decreased from a control of 4.5 ⫾
0.7 to 3.8 ⫾ 0.8 nS (15.6% decrease) and reversed close to
⫺100 mV (⫺102.6 ⫾ 4.3 mV). Thus the data from this group
resembled that from the motoneuron population, suggesting
mediated of the ANG II response via reduction in conductance
for potassium ions. By contrast, in another seven cells, the
slope of the net ANG II current reflected an increase in
conductance (from 4.8 ⫾ 0.6 to 5.3 ⫾ 1.2 nS, 11,3% increase)
with current reversal close to ⫺40 mV (⫺38.4 ⫾ 4.1 mV).
These values could reflect mediation of the ANG II response
via increase in a nonselective cationic conductance. In the
remaining 12 cells, the mean net ANG-II-induced current
displayed a slight reduction (only 3.6% decrease) in membrane
J Neurophysiol • VOL
conductance (from 5.1 ⫾ 1.2 to 4.9 ⫾ 1.1 nS), with no reversal
within the voltage range tested.
Because ANG II receptors belong to family of G-proteincoupled receptors, we first compared the control data (containing 1 mM GTP) with data obtained 5 min after establishment
of seals using pipettes that contained GTP-␥-S (0.4 mM), a
nonhydrolyzable derivative of GTP that activates G protein in
an irreversible manner (Gilman 1987). Of 18 cells recorded
and tested with ANG II, 7 cells responded. The maximum
amplitudes of their response (⫺38.7 ⫾ 6.1 pA) were not
significantly different from control, but the recovery phase was
greatly prolonged by the presence of GTP-␥-S in the pipette
(Fig. 4A). Of 7 cells treated with GTP-␥-S, membrane conductance increased in two cells (reversed close to EK⫹), decreased
in two cells (reversed close to ⫺40 mV), and was unchanged
in the remaining three cells (with no reversal potential detected
in the range of ⫺10 to ⫺120 mV). We also dialyzed four cells
with GDP-␤-S (1 mM for 10 min), a stable analogue of GDP
that competitively inhibits G protein binding by GTP (Gilman
1987). The data from four cells indicated that mean ANG-IIinduced inward current was significantly reduced (⫺17.4 ⫾ 3.7
vs. ⫺37.9 ⫾ 5.2 pA in 27 control cells; P ⬍ 0.05, Student’s
unpaired t-test, Fig. 4B).
Treatments with pertussis toxin (PTX) or N-ethylmaleimide
(NEM) have been reported to uncouple the receptors and Gi/o
G proteins (Gilman 1987; Shapiro et al. 1994). To evaluate the
role of PTX-sensitive G proteins in ANG-II-induced currents,
slices were incubated for 12–18 h either in ACSF or in ACSF
88 • NOVEMBER 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
FIG. 2. In motoneurons, ANG II receptor activation induces inward current, mediated via
suppression of a potassium conductance. A: in
ACSF containing TTX, a voltage-clamp trace
from a motoneuron (PN 11; 䡠 䡠 䡠 , holding current
VH ⫺65 mV) illustrates an ANG-II-induced
slowly rising and prolonged inward current. Below, current traces (left) in response to a series of
voltage pulses delivered before (control) and at
the peak of the ANG II response. The I-V plot
(right) was constructed from values taken at the
points indicated. B: I-V plot shows the data taken
from 7 cells responded to 1 ␮M ANG II. The net
ANG-II-induced current () determined by subtraction of these I-V values displays reversal
approximately ⫺100 mV. C: plots of net ANGII-induced currents from 3 motoneurons. In control ACSF (䊐), note a decrease in membrane
conductance with a reversal potential approximately ⫺100 mV. In ACSF containing 10 mM
potassium (■), note the shift in reversal potential
toward the Nernstian predicted equilibrium potential of ⫺68 mV.
2860
M. OZ AND L. P. RENAUD
containing PTX, and the magnitudes maximal ANG-II-induced
currents were compared. In the control cells, we also tested
bath-applied NEM (50 ␮M), a sulfhydryl-alkylating agent
shown to block G-protein-effector interactions by alkylating
the ␣-subunits of PTX-sensitive GTP-binding protein (Shapiro
et al. 1994; Viana and Hille 1996). The advantage of using
NEM was that it allowed us to examine ANG-II-induced
J Neurophysiol • VOL
DISCUSSION
The observation that exogenous ANG II induces membrane
depolarization and inward currents in a subpopulation of neonatal spinal ventral horn neurons, including identified motoneurons, implies the presence of functional angiotensin receptors. These receptors are of the AT1 subtype because
responses were sensitive to pretreatment with Losartan, a selective AT1 receptor antagonist, but not with PD123319, which
selectively blocks angiotensin AT2 type receptors.
Our analyses of I-V relationships suggest that more than one
membrane conductance underlies the depolarizing action of
ANG II. In both motoneurons and a subpopulation of unidentified neurons, the ANG-II-induced inward currents were con-
88 • NOVEMBER 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
FIG. 3. Activation of AT1-receptors induces TTX-resistant inward currents
in interneurons. A: In ACSF containing TTX (1 ␮M), current trace (VH ⫺65
mV) from an interneuron (PN 9 days) illustrates ANG -II-induced inward
current. Histograms (bottom) illustrate the effects of AT1-type receptor antagonist Losartan (1 ␮M; n ⫽ 4), AT2-type receptor antagonist PD123319 (1 ␮M;
n ⫽ 4), and specific AT-receptor antagonist (Sar1, Val5, Ala8)-angiotensin II (1
␮M; n ⫽ 3) compared with normalized controls values (n ⫽ total of 11 cells).
Asterisk, P ⬍ 0.05, compared with control values obtained without pretreatments (paired t-test compared with control values of each group). B: net
ANG-II-induced inward current analysis indicates 3 patterns in I-V relationships: current that reversed approximately ⫺100 mV (filled circles; n ⫽ 8
cells); current that reversed approximately ⫺39 mV (gray circles; n ⫽ 7 cells);
current that demonstrated no obvious reversal potential (open circles; n ⫽ 12
cells).
currents before and after inhibition of PTX-sensitive G proteins
within the same cell.
In control slices, mean ANG-II-induced inward currents in
13 of 29 cells tested were 38.4 ⫾ 4.1 pA. The mean ANG-IIinduced inward current was significantly reduced to 16.8 ⫾ 2.1
in PTX treated neurons (Fig. 4C, P ⬍ 0.05, Student’s unpaired
t-test). Further analysis of the net ANG-II-induced currents for
individual cells revealed three differing patterns (Fig. 5). One
group of four neurons demonstrated a net ANG-II-induced
conductance that decreased from a control of 4.4 ⫾ 0.5 to
3.7 ⫾ 0.6 nS and reversed close to ⫺100 mV (⫺101.2 ⫾ 4.1
mV). In another four cells, the slope of the net ANG-II-induced
current reflected an increase in conductance (from 4.6 ⫾ 0.6 to
5.1 ⫾ 1.1 nS) with current reversal close to ⫺40 mV (⫺39.4 ⫾
3.7 mV). In the remaining five cells, the mean net ANG-IIinduced current displayed a slight reduction in membrane
conductance (from 4.9 ⫾ 1.2 to 4.7 ⫾ 0.9 nS), with no reversal
within the voltage range tested.
In PTX-incubated slices, 16 of 34 neurons responded to
ANG II. Among these cells, one group of five neurons demonstrated a net ANG-II-induced conductance that decreased
from a control of 2.8 ⫾ 0.6 to 2.1 ⫾ 0.5 nS and reversed close
to ⫺100 mV (⫺102.9 ⫾ 3.4 mV). In another four cells, the
slope of the net ANG II current showed an increase in conductance (from 2.7 ⫾ 0.5 to 3.1 ⫾ 0.8 nS) with current reversal
close to ⫺40 mV (⫺37.7 ⫾ 2.8 mV). In the remaining seven
cells, the mean net ANG-II-induced current displayed a slight
reduction in membrane conductance (from 2.9 ⫾ 0.7 to 2.6 ⫾
0.8 nS), with no reversal within the voltage range tested.
In 12 of 13 cells in the control group described earlier, a
5-min exposure treatment with 50 ␮M NEM was seen to
reduce the ANG-II-induced inward current to 31.4% of control
values (12.2 ⫾ 3.6 vs. 38.1 ⫾ 4 pA in controls, P ⬍ 0.05,
Student’s paired t-test, Fig. 4C). Further analysis revealed that
all patterns of conductance were sensitive to NEM pretreatment (Fig. 5A-C). In four cells with current reversal close to
⫺100 mV, ANG-II-induced inward currents were significantly
reduced from 39.2 ⫾ 4.3 (controls) to 14.6 ⫾ 3.5 pA (P ⬍
0.05, Student’s paired t-test). In another four cells with current
reversal close to ⫺40 mV, ANG-II-induced inward currents
were significantly reduced from 36.9 ⫾ 4.1 (controls) to
11.6 ⫾ 3.2 pA (P ⬍ 0.05, Student’s paired t-test). In the
remaining four cells with no current reversal within the voltage
range tested, ANG-II-induced inward currents were significantly reduced from 40.2 ⫾ 4.3 (controls) to 12.9 ⫾ 3.4 pA
(P ⬍ 0.05, Student’s paired t-test).
ANG II RECEPTORS IN NEONATAL VENTRAL SPINAL CORD
2861
sistently associated with reduction in a membrane conductance
that reversed close to the potassium equilibrium potential and
shifted appropriately with the transmembrane potassium gradient. In these neurons, the linearity and reversal potential for
the net ANG-II-induced currents indicate involvement of a
voltage-independent potassium conductance that contributes to
resting membrane potential, often referred to as a leak conductance. Similar potassium-mediated responses to ANG II can be
seen in rat bulbospinal (Li and Guyenet 1996) and median
preoptic neurons (Bai and Renaud 1998) and hamster submandibular ganglion neurons (Ikegami et al. 2000). In rat adrenal
glomerulosa cells, ANG II suppresses a potassium conductance
FIG. 4. ANG-II-induced inward currents are mediated through pertussis
toxin-sensitive G proteins in interneurons. A: in voltage-clamp mode (VH ⫺65
mV) and in presence of tetrodotoxin, the top trace displays a control ANGII-induced current that recovers after several minutes. The bottom trace from
another cell dialyzed with GTP-␥-S (0.4 mM, for 10 min) illustrates inward
current that fails to recover. The plot below the current traces illustrates the
averaged data for the time course of the normalized inward current induced by
1 ␮M ANG II. The maximal currents were defined as the difference between
the resting currents and maximal amount of ANG II induced inward current.
Currents from the cells recorded with GTP are depicted as closed symbols
(control, n ⫽ 12), those recorded with GTP-␥-S as E (n ⫽ 5). B: ANG-IIinduced currents are mediated by pertussis toxin (PTX)-sensitive G proteins.
Histograms (left to right): normalized control ANG -II-induced current recorded after overnight incubation in ACSF (control, n ⫽ 13), in ACSF
containing PTX (5 ␮g/ml, n ⫽ 16), after 5 min incubation with 50 ␮M
N-ethylmaleimide (n ⫽ 12), and in cells recorded with electrodes containing
GDP-␤-S (1 mM for 10 min, n ⫽ 4). *, P ⬍ 0.05, compared with values
obtained without pretreatment (paired or unpaired Student’s t-test compared
with control values of each group).
J Neurophysiol • VOL
88 • NOVEMBER 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
FIG. 5. ANG-II-regulated conductances are sensitive to PTX and N-ethylmaleimide. In 13 control neurons, after overnight incubation in ACSF, net
ANG-II-induced inward current (● in A–C) analyses indicated 3 patterns in I-V
relationships: current that reversed at ⫺101.2 ⫾ 4.1 mV (A; n ⫽ 4 cells);
current that reversed at ⫺39.4 ⫾ 3.7 mV (B; n ⫽ 4 cells); and current that
demonstrated no obvious reversal potential (C; n ⫽ 5 cells). In the same
control cells, tested again after 30 – 45 min, incubation with 50 ␮M Nethylmaleimide (n ⫽ 12) for 5 min significantly reduced the current sizes in all
3 types of ANG II responses (E in A–C). In another 16 cells, recorded after
overnight incubation in ACSF containing PTX (5 ␮g/ml), current sizes were
significantly reduced compared with control neurons (Œ in A–C; n ⫽ 13). In 5
of 16 cells, currents were reversed at ⫺102.9 ⫾ 3.4 mV (A); in 4 cells, currents
were reversed at ⫺37.7 ⫾ 2.8 mV (B); in 7 cells, currents did not show an
apparent reversal potential (C).
2862
M. OZ AND L. P. RENAUD
J Neurophysiol • VOL
neonatal spinal cord neurons is important for their subsequent
development.
REFERENCES
BAI D AND RENAUD LP. ANG II AT1 receptors induce depolarization and
inward current in rat median preoptic neurons in vitro. J Neurophysiol 44:
R632–R639, 1998.
CZIRJAK G, FISCHER T, SPAT A, LESAGE F, AND ENYEDI P TASK. (TWIKrelated acid-sensitive K⫹ channel) is expressed in glomerulosa cells of rat
adrenal cortex and inhibited by angiotensin II. Mol Endocrinol 14: 863– 874,
2000.
CZIRJAK G AND ENYEDI P. Formation of functional heterodimers between the
TASK-1 and TASK-3 two-pore domain potassium channel subunits. J Biol
Chem 277: 5426 –5432, 2002
FERGUSON AV AND WASHBURN DLS. Angiotensin II: a peptidergic neurotransmitter in central autonomic pathways. Prog Neurobiol 54: 169 –192, 1998.
GEHLERT DR, SPETH RC, AND WAMSLEY JK. Distribution of [125]angiotensin
II binding sites in the rat brain: a quantative autoradiographic study. Neuroscience 18: 837– 856, 1986.
GILMAN AG. G proteins: transducers of receptor-generated signals. Annu Rev
Biochem 56: 615– 649, 1987.
IKEDA K, KINOSHITA M, IWASAKI Y, SHIOJIMA T, AND TAKAMIYA K. Neurotrophic effect of angiotensin, vasopressin and oxytocin on the ventral spinal
cord of the rat embryo. Int J Neurosci 48: 19 –23, 1989.
IKEGAMI H, YAMADA T, AND SUZUKI T. Angiotensin II-induced depolarizations
in hamster submandibular ganglion neurons. Bull Tokyo Dent Coll 41:
29 –34, 2000.
IWASAKI Y, KINOSHITA M, IKEDA K, SHIOJIMA T, KURIHARA T, AND APPEL SH.
Trophic effect of angiotensin II, vasopressin, and other peptides on the
cultured ventral spinal cord of rat embryo. J Neurol Sci 103: 151–155, 1991.
KANG J, SUMNERS C, AND POSNER P. Modulation of net outward current in
cultured neurons by angiotensin II involvement of AT1 and AT2 receptors.
Brain Res 580: 317–324, 1992.
KOLAJ M AND RENAUD LP. Vasopressin-induced currents in rat neonatal spinal
lateral horn neurons are G-protein mediated and involve two conductances.
J Neurophysiol 80: 1900 –1910, 1998.
KOLAJ M, SHEFCHYK SJ, AND RENAUD LP. Two conductances mediate thyrotropin-releasing hormone-induced depolarization of neonatal rat spinal
preganglionic and lateral horn neurons. J Neurophysiol 78: 1726 –1729,
1997.
LEWIS DI AND COOTE JH. Angiotensin II in the spinal cord of the rat and its
sympatho-excitatory effects. Brain Res 614: 1–9, 1993.
LI YW AND GUYENET PG. Angiotensin II decreases a resting K⫹ conductance
in rat bulbospinal neurons of the C1 area. Circ Res 78: 274 –282, 1996.
LIND RW, SWANSON LW, AND GANTEN D. Organization of angiotensin II
immunoreactive cells and fibers in the rat central nervous system. An
immunohistochemical study. Neuroendocrinology 40: 2–24, 1984.
LOTSHAW DP AND LI F. Angiotensin II activation of Ca2⫹-permeanant nonselective cation channels in rat adrenal glomerulosa cells. Am J Physiol Cell
Physiol 271: C1705–C1715, 1996.
MENDELSOHN FAO, QUIRION R, SAAVERDA JM, AGUILERA G, AND CATT KJ.
Autoradiographic localization of angiotensin II receptors in rat brain. Proc
Nat Acad Sci USA 81: 1575–1579 1984.
MILAS MA, KISS A, AND AGUILERA G. Developmental changes in brain angiotensin II receptors in the rat. Peptides 12: 723–737, 1991.
ONO K, HONDA E, AND INENAGA K. Angiotensin II induces inward currents in
subfornical organ neurons of rats. J Neuroendocrinology 13: 517–523 2001.
OZ M, KOLAJ M, AND RENAUD LP. Electrophysiological evidence for vasopressin V1 receptors on neonatal motoneurons, premotor and other ventral
horn neurons. J Neurophysiol 86: 1202–1210, 2001.
PALKOVITS M. Neuropeptide messenger plasticity in the CNS neurons following axotomy. Mol Neurobiol 10: 91–103, 1995.
PHILLIPS MI. Functions of angiotensin in the central nervous system. Annu Rev
Physiol 49: 413– 435, 1987.
REPPERT SM, SCHWARTZ WJ, AND UHL GR. Arginine vasopressin: a novel
peptide rhythm inn cerebrospinal fluid. Trends Neurosci 10: 76 – 80, 1987.
SHAPIRO MS, WOLLMUTH LP, AND HILLE B. Modulation of Ca2⫹ channels by
PTX-sensitive G proteins is blocked by N-ethylmaleimide in rat sympathetic
neurons. J Neurosci 14: 7109 –7116, 1994.
88 • NOVEMBER 2002 •
www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
that in brain stem motoneurons is attributed to a family of
two-pore domain pH-sensitive channels, named TASK-1 (Czirjak and Enyedi 2002; Czirjak et al. 2000; Talley and Bayliss
2002; Talley et al. 2000, 2002). Whether similar channels are
responsible for ANG-II-induced inward currents in neonatal
spinal motoneurons remains to be established.
By contrast, the ANG-II-induced inward currents in a population of interneurons were associated with an increase in
membrane conductance that reversed around ⫺40 mV, suggestive of a nonselective cationic conductance. This is similar to
the ANG-II-induced responses in rat supraoptic neurons where
net currents also reverse close to ⫺40 mV (Yang et al. 1992).
In a subpopulation of rat adrenal glomerulosa cells, net ANG II
currents reverse around ⫺10 mV (Lotshaw and Li 1996), and
in ANG-II-responsive subfornical organ neurons, a net current
that reverses closer to ⫺30 mV has been reported (Ono et al.
2001). Whether these differences reflect variations in underlying cationic conductances remains to be clarified, but they do
suggest cell specificity in coupling of ANG II receptors to ion
channels. Moreover, we speculate that in some neurons, ANG
II receptors can couple to both potassium and cationic conductances, in which situation the net ANG -II-induced conductance could be reflected in a nearly parallel shift, as observed
here in a subpopulation of ventral horn interneurons.
The binding of angiotensin to AT1 receptors can activate a
number of intracellular signaling pathways, mediated through
heterotrimeric G proteins (see Sumners et al. 1994 for review).
These may including Gq␣ to stimulate phosphoinositide hydrolysis, subsequent activation of protein kinase C and a rise in
intracellular calcium, Gi to inhibit adenylate cyclase, and a
G␤␥ /Ras/Raf-1 pathway that increases mitogen-activated protein (MAP) kinase activities. While it remains undefined as to
the identity of G proteins linking AT1 receptors to the conductances described above, the linkage must involve PTX-sensitive G proteins given the marked reduction in both types of
conductances after treatment with PTX or NEM (Fig. 5).
The present observations bear a striking resemblance to the
response of neonatal spinal neurons to other peptides including
thyrotropin-releasing hormone (Kolaj et al. 1997), vasopressin
(Kolaj and Renaud 1998; Oz et al., 2001), and substance P
(Yasuda et al., 2001). Two issues that derive from these observations include the sources for ligands for these receptors,
and the physiological roles that peptide receptors may have in
neuronal function in neonatal spinal cord. One source may
derive from the cerebrospinal fluid, which is known to contain
a variety of neuropeptides, including some that display a circadian rhythmicity in their concentrations (Reppert et al.
1987). Neuronal pathways may provide a source since angiotensin-like immunoreactivity has been observed in adult spinal
cord (Lind et al. 1984); however, information is lacking for
neonatal tissue. Whatever the origin, angiotensin receptors may
have specific functional links to neuronal development and
survival. This is suggested by their ability to undergo developmental changes (Milas et al. 1991), to participate in recovery
following axotomy (Palkovits 1995), and to modulate neurite
outgrowth from cultured embryonic neurons (Iwasaki et al.
1991; Sood et al. 1990) and by their neurotrophic action in
cultured explants of spinal cord (Ikeda et al. 1989; Yang et al.
2001). It is possible that their influence on excitability of
ANG II RECEPTORS IN NEONATAL VENTRAL SPINAL CORD
SOOD PP, RICHOUX JP, PANIGEL M, BOUHNIK J, AND WEGMANN R. Angiotensinogen in the developing rat fetal hindbrain and spinal cord from 18th to
20th day of gestation: an immunocytochemical study. Neuroscience 37:
517–522, 1990.
SUMNERS C, RAIZADA MK, KANG J, LU D, AND POSNER P. Receptor-mediated
effects of angiotensin II on neurons. Front Neuroendocrinol 15: 203–230, 1994.
SUZUE T, YANAIHARA N, AND OTSUKA M. Actions of vasopressin, gastrin
releasing peptide and other peptides on neurons of newborn rat spinal cord
in vitro. Neurosci Lett 26: 137–142, 1981.
TALLEY EM, LEI Q, SIROIS JE, AND BAYLISS DA. TASK-1, a two-pore domain
K⫹ channel, is modulated by multiple neurotransmitters in motoneurons.
Neuron 25: 399 – 410, 2000.
TALLEY EM, SOLORZANO G, LEI Q, KIM D, AND BAYLISS DA. CNS distribution
of members of the two-pore domain (KCNK) potassium channel family.
J Neurosci 21: 7491–7505, 2002.
TALLEY EM AND BAYLISS DA. Modulation of TASK-1 (Kcnk3) and TASK-3
(Kcnk9) potassium channels: volatile anesthetics and neurotransmitters
share a molecular site of action. J Biol Chem 277: 17733–17742, 2002.
2863
TSUTSUMI K AND SAAVEDRA JM. Characterization and development of angiotensin II receptor subtypes (AT1 and AT2) in rat brain. Am J Physiol
Regulatory Integrative Comp Physiol 261: R209 –R216, 1991.
VIANA F AND HILLE B. Modulation of high voltage activated calcium channels
by somatostatin in acutely isolated rat amygdaloid neurons. J Neurosci 16:
6000 – 6011, 1996.
WHITE SR, PENNER JD, SPETH RC, AND CHAN JY. Angiotensin II receptors in
the lumbar spinal cord of the rat. Brain Res 441: 195–201, 1988.
YANG CR, PHILLIPS MI, AND RENAUD LP. Angiotensin II receptor activation
depolarizes rat supraoptic neurons in vitro. Am J Physiol 263: R1333–
R1388, 1992.
YANG H, WANG X, AND RAIZADA MK. Characterization of signal transduction
pathway in neurotropic action of angiotensin II in brain neurons. Endocrinology 142: 3502–3511.
YASUDA K, ROBINSON DM, SELVARATNAM SR, WALSH CW, MCMORLAND
AJC, AND FUNK GD. Modulation of hypoglossal motoneuron excitability by
NK1 receptor activation in neonatal mice in vitro. J Physiol (Lond) 534:
447– 464, 2001.
Downloaded from http://jn.physiology.org/ by 10.220.32.247 on June 18, 2017
J Neurophysiol • VOL
88 • NOVEMBER 2002 •
www.jn.org