© 1999 American Heart Association, Inc.
Original Contribution |
Proadrenomedullin N-Terminal 20 Peptide Hyperpolarizes the Membrane by Activating an Inwardly Rectifying K+ Current in Differentiated PC12 Cells
From the Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan.
Correspondence to Koji Takano, MD, PhD, Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112-8688, Japan.
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Abstract |
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Abstract—The mechanism of proadrenomedullin N-terminal 20 peptide (PAMP)–induced inhibition of catecholamine release from adrenergic nerve was investigated in nerve growth factor–treated PC12 cells that have differentiated characteristics somewhat similar to noradrenergic neurons. The effect of PAMP on the excitability of these cells was investigated with the use of perforated whole-cell clamp. PAMP hyperpolarized the membrane by increasing a K+ conductance in a dose-dependent manner. The current-voltage relationship (I-V) relationship of the PAMP-induced K+ conductance exhibited inward-going rectification. The activation was abolished by microinjecting GDPßS into the cells or pretreating the cells with pertussis toxin. These results indicate that a pertussis toxin–sensitive G protein is involved in the signal transduction. The PAMP-induced activation of the K+ conductance was attenuated by microinjecting antibody against the carboxyl terminus of G
i3, but it was not
influenced by microinjecting antibody against the common carboxyl
termini of Gi1 and Gi2, which indicated
that the G protein coupling the PAMP receptor to the inwardly
rectifying K+ current is Gi3. The
PAMP-induced hyperpolarization may inhibit the
catecholamine release from the neurons by attenuating the
action potential frequency.
Key Words: channel • PC12 cell • hypertension
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proadrenomedullin N-terminal 20 peptide (PAMP) is a newly identified peptide1 2 that has a hypotensive effect in vivo.3 This peptide is part of the amino terminus of the precursor peptide proadrenomedullin and is cleaved from this precursor by proteolysis.1 2 This peptide is found in plasma and tissues including adrenal medulla, right atrium, kidney, and brain.4 This distribution indicates that it may play a physiological role in the control of circulation. Recently, PAMP was found to induce hypotension through the inhibition of catecholamine release from peripheral sympathetic nerve endings and adrenal medulla.3 5 The inhibition of catecholamine release was not mediated through the interaction with nicotinic or
2
receptors, which suggested a direct action of PAMP on
catecholamine-releasing cells. Because many
neurotransmitters modulate neural activity by changing the membrane
potential, we examined whether PAMP modulates membrane excitability by
changing the membrane potential in nerve growth factor (NGF)–treated
PC12 cells, which express some
electrophysiological characteristics common
to noradrenergic sympathetic neurons.6 We
found that PAMP hyperpolarized these cells by activating an inwardly
rectifying K+ current. We have also attempted to
identify the G-protein subtype involved in coupling the PAMP receptor
and the inwardly rectifying K+ current. |
Materials and Methods |
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Cell Culture
PC12 cells were maintained in DMEM that contained 10% FCS. Cells were cultured in humidified air that contained 5% CO2 at 37°C. Cells were subcultured every week, and the medium was changed every 4 days. For electrophysiological experiments, cells were seeded on 35-mm plastic dishes and cultured in DMEM that contained 10% FCS and 2.5S NGF (0.75 nmol/L) for 7 days. We selected cells with the axon outgrowth for electrophysiology because this morphology indicates that the cell has differentiated into sympathetic neuronlike cells.6
Electrophysiology
The perforated whole-cell clamp technique7 was used
in most of the experiments. The standard patch electrode solution
contained (in mmol/L) K aspartate 95, KCl 47.5,
MgCl2 1, EGTA TMA salt 0.1, and HEPES 10 (TMA, pH
7.2). The standard external solution was (in mmol/L) NaCl 128, KCl
5, MgCl2 1, CaCl2 2.5, and
HEPES 10 (Na+ salt, pH 7.4). The 20
mmol/L K+ solution and 40 mmol/L
K+ solutions were made by the substitution of
NaCl in the standard extracellular solution with iso-osmotic KCl.
Na+-free extracellular solution was made by
replacing Na+ in standard solution with
iso-osmotic TMA+. Na+-free
solution with 300 µmol/L BaCl2 was made by
isoosmotically replacing TMACl with 300 µmol/L
BaCl2. In the experiments that investigated the
involvement of second messengers, the conventional whole-cell clamp
technique was used. The patch pipette solution for the conventional
whole-cell experiments contained (in mmol/L) K aspartate 95, KCl
47.5, MgCl2 1, CaCl2 0.013,
EGTA TMA salt 2.0, ATP (Na+ salt) 2, GTP (Mg
salt) 0.1, and HEPES 10 (TMA salt, pH 7.2). The calculated pCa
(pCa=-[log Ca2+]) of the pipette solution
was 6.8.
During the experiments, the extracellular solution was supplied
continuously by a peristaltic pump. Selected pharmacological agents
were applied by changing the superfusate. The liquid junction
potentials between the standard extracellular solution and other
solutions used (internal and external) were measured with a 3 mol/L KCl
electrode as a reference, and all the data were corrected for the
liquid junctional potential (-8 to -2 mV). A EPC-7 amplifier (List)
was used to record the membrane current and potential. All
experiments were performed at room temperature (22°C to 25°C).
Glass capillaries of 1.5-mm diameter that contained an inner filament
were used to make patch electrodes. The resistance of the patch
electrodes was between 5 and 8 M
. For the perforated whole-cell
clamp experiments, a fresh stock solution of nystatin was made in DMSO
(540 mmol/L) daily. Shortly before recording, the stock
solution was diluted with the patch electrode solution (final nystatin
concentration, 0.22 mmol/L). Details of the perforated whole-cell
clamp technique have been reported elsewhere.8
Current-clamp recordings were started after the series
resistance fell to <50 M. Voltage-clamp recordings were
made after the series resistance fell to <10 M. Because the
amplitude of the current was less than 200 pA, the errors caused by the
series resistance were ignored.
Microinjection of GDPßS and Antibodies
Guanosine 5' O-2-thiodiphosphate (GDPßS) as
injected into the cell by microinjection. The details of the method for
microinjection have been reported elsewhere.9 GDPßS
was dissolved in 50 mmol/L KCl at concentration of 100
mmol/L. For the microinjection, 2 kinds of polyclonal antibodies
against the carboxyl terminus peptides of Gi
were used. They were (1)
anti-Gi1/Gi2 antibody
(2.8 mg/mL, affinity purified; No. 371723, Calbiochem), which was
developed against a peptide that represented amino acids
345 to 354 of the common carboxyl termini of
Gi1 and
Gi210 11 12 13 ; (2)
anti-Gi3 antibody (4.2 mg/mL, affinity
purified; No. 371729, Calbiochem), which was developed against a
peptide that represented amino acids 345 to 354 of the
carboxyl terminus of Gi3 and which is
monospecific to Gi3.12 13 The
antibodies were diluted x200 by the internal solution used for
perforated patch. The solution was microinjected through
microcapillaries (Femtotips, Eppendorf) by pressure injection (110 hPa,
0.1 second). At the time of injection, a slight swelling of the cell
was observed. The volume of the injected solution was 
100 fl,
which was estimated by the decrease of the solution after multiple
injections. Only cells with input resistance of >1 G after the
microinjection were used for the
electrophysiological experiment.
Drugs
Nystatin was obtained from Sigma; pertussis toxin (PTX) and NGF
(2.5S) from Funakoshi Chemicals; and GDPßS from
Boehringer Mannheim. cAMP, GMP, IP3, and
PMA were obtained from Calbiochem, and BAPTA from Molecular Probe. PAMP
was kindly supplied by Dr Kenji Kangawa at National
Cardiovascular Center Research Institute (Osaka,
Japan). A scrambled peptide of PAMP was synthesized and purified by
HPLC (>95%) by Sawady Co Ltd (Tokyo, Japan). The amino acid sequence
of the scrambled peptide was
AFRSSWQNKKSRTAKDRLLW-NH2.
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Results |
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PAMP-Induced Hyperpolarization
Figure 1A
shows a membrane potential
record under the current clamp in the standard extracellular
solution from an NGF-treated PC12 cell. This cell had a resting
potential of -60 mV, and application of PAMP (100 nmol/L)
hyperpolarized the membrane by 10 mV. The membrane potential
partially recovered slowly by washing out PAMP from the extracellular
solution. Hyperpolarization was observed in all the
cells examined (n=11), and the amplitude of
hyperpolarization by PAMP (100 nmol/L) was 10±2 mV
(mean±SD, n=11). To investigate the ionic mechanism of PAMP-induced
hyperpolarization, voltage clamp experiments were
performed. Figure 1B shows a current record under the
voltage clamp at the holding potential of -48 mV. Membrane conductance
was monitored by applying a 60-mV hyperpolarizing pulse step (0.8
seconds) every 20 seconds. Application of PAMP (100 nmol/L) induced an
outward current accompanied by an increase in membrane conductance in
each of the 20 cells investigated. When the cell was superfused with a
scrambled peptide of PAMP (1 µmol/L), there was no change in the
membrane current.
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Ionic Mechanism of PAMP-Induced Outward Current
To determine which ions are involved in the PAMP-induced outward
current, the reversal potential of the PAMP-induced current was
investigated. Figure 2A shows the basal
membrane currents under the voltage clamp in the standard extracellular
solution. The holding potential was -48 mV, and test potentials were
-78, -88, -98, -108, -118, and -128 mV. The I-V
relationship of the control current is plotted in Figure 2B
(closed circle). The basal current showed a distinct inward
rectification. To analyze components of the basal membrane
conductance, the extracellular solution was changed to
Na+-free (TMA+-substituted)
solution. The membrane conductance was decreased in the
Na+-free extracellular solution [Na(-)] but
some residual conductance remained. To determine whether
K+ conductance sensitive to low concentration of
BaCl2 was involved in the residual membrane
conductance, the extracellular solution was changed to
Na+-free extracellular solution containing
300 µmol/L BaCl2. The membrane current did
not change by BaCl2
([Na(-)]–+ BaCl2).
These data indicated that the basal current consists of a conductance
dependent on extracellular Na+ and a residual
conductance that is insensitive to 300 µmol/L
BaCl2. The I-V relationships of the
membrane current in Na+-free solution (open
circle) and Na+-free with 300 µmol/L
BaCl2 (open triangle) are plotted in Figure 2B.
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), in Na+-free
extracellular solution (
), and Na+-free with low
concentration (300 mmol/L) of BaCl2 (
).
Figure 3A shows the membrane currents
under the voltage clamp before and after the application of PAMP (10
nmol/L). The holding potential and the test potentials are the same.
The PAMP-induced current ("subtracted" in Figure 3A) was
obtained by subtracting the control currents from those after the
application of PAMP. Application of PAMP (10 nmol/L) changed the
membrane current at -78 mV to the outward direction and changed the
membrane currents to the inward direction at potentials more
hyperpolarized than -88 mV. The amplitude of the PAMP-induced current
was more prominent at hyperpolarized potentials than depolarized
potentials. The reversal potential, where there is no effect on the
membrane current, was about -90 mV, which was close to the equilibrium
potential of K+. This suggests that the
PAMP-induced current is a K+ current. To
determine whether the PAMP-induced current was carried by
K+, reversal potentials of the PAMP-induced
current at various extracellular K+ concentration
were measured. Figure 3B shows the membrane currents before
(cont) and after (PAMP) the application of PAMP (10 nmol/L) in the
extracellular solution containing 40 mmol/L
K+. The membrane currents shifted to the inward
direction at -124 mV but did not shift at -34 mV by the application
of PAMP, which indicated that the reversal potential of the
PAMP-induced conductance shifted with the change of extracellular
K+ concentration. These data suggest that
PAMP-induced current is a K+ current with inward
rectification. We investigated the effect of low concentration of
BaCl2 on the effect of PAMP on the membrane
currents. Figure 3C shows the membrane currents before (cont)
and after (PAMP) the application of PAMP (10 nmol/L) when the
extracellular solution contained 300 µmol/L
BaCl2. The effect of PAMP was abolished at both
the outward limb and inward limb of the current.
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Figure 4A shows the current-potential
(I-V) relationship of the PAMP-induced current at 2
extracellular K+ concentrations (5 and 40
mmol/L). The I-V relationships exhibited inward
rectification. The reversal potential of the PAMP-induced current was
-90 mV in 5 mmol/L K+ extracellular
solution and -35 mV in 40 mmol/L K+
extracellular solution. Figure 4B shows the reversal potentials
at various extracellular K+ concentrations
plotted against the extracellular K+
concentration. The reversal potentials were -90±3 mV (mean±SD, n=5)
in 5 mmol/L K+, -49±3 mV (n=5) in 20
mmol/L K+, and -30±4 mV (n=5) in 40 mmol/L
K+. These experimental data that describe the
reversal potentials were close to the equilibrium potentials calculated
by the Nernst equation, which indicated that the PAMP-induced current
is carried by the K+ ion. These data suggest that
the PAMP activated an inwardly rectifying
K+ current. Figure 4C shows that PAMP
induced the K+ conductance in a
concentration-dependent manner.
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Because the outward limb of the inwardly rectifying
K+ current regulates membrane potential at a
voltage more depolarized than -90 mV, the effect of PAMP on the
membrane current at the depolarized potentials was investigated. Figure 5A shows the effect of PAMP on the
membrane currents at test potentials to -48, -58, -68, -78, and
-88 mV. The membrane current shifted to the outward direction by 1
nmol/L PAMP (PAMP 1 nmol/L), and the shift was more prominent at 10
nmol/L PAMP (PAMP 10 nmol/L) compared with the control current (cont)
at potentials more depolarized than -88 mV. The response was
reversible when PAMP was washed out from the superfusate
(wash). The PAMP-induced conductance was calculated at the outward limb
of the I-V curve (between -48 and -88 mV), and the
concentration dependence was examined. Figure 5F shows the
result, which indicates that PAMP-induced conductance at the outward
limb of the I-V curve was also concentration dependent.
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Signal Transduction of PAMP-Induced Activation of the Inwardly
Rectifying K+ Current
To investigate the signal transduction of PAMP response, we
microinjected GDPßS, a nonhydrolyzable GDP analogue into the cells
and applied PAMP (10 nmol/L). As is shown in Figure 6A, the
membrane currents were not changed by PAMP (n=6). When the cells were
pretreated with 100 ng/mL PTX for 24 hours, application of PAMP (10
nmol/L) did not change the membrane currents (Figure 6B, n=7).
These data indicate that the activation of the inwardly rectifying
K+ current by PAMP was mediated by a
PTX-sensitive G protein. To identify the PTX-sensitive G-protein
subtype, the antibody against the common carboxyl terminal amino acid
sequence of Gi1 and
Gi2 (anti-Gi1+2) or
antibody against the carboxyl terminal amino acid sequence of
Gi3 (anti-Gi3) was
microinjected into the cells. Figure 6C shows that PAMP induced
a membrane current in a cell microinjected with
anti-Gi1+2 antibody. On the other hand, PAMP
did not induce a membrane current in a cell microinjected with
anti-Gi3 antibody (Figure 6D). To
confirm that this attenuation of the PAMP-induced response is due to
the binding of the injected antibody to the respective G protein, we
neutralized anti-Gi3 by mixing it with an
excess amount of the antigen peptide and injected it into the cells.
Application of PAMP on the cell injected with the neutralized
anti-Gi3 activated the membrane
conductance (Figure 6E), which indicated that the attenuation of
PAMP response was due to the binding of injected antibody to the
respective G protein. Figure 6E shows the summary of these
experiments. Anti-Gi3 attenuated PAMP-induced
response. These data indicate that the G protein that mediates the
PAMP-induced activation of the inwardly rectifying
K+ current was Gi3. The
signal transduction mechanism was investigated further with the
conventional whole-cell clamp technique. The PAMP-induced activation of
the inwardly rectifying K+ current was observed
and was comparable to that observed with the perforated whole-cell
clamp technique (Figure 6F, column A). The involvement of
several second messengers was investigated by introducing these second
messengers in the cell through the patch pipette. The pipette was
filled with an intracellular solution that contained either cAMP
(2 mmol/L), GMP (2 mmol/L), or IP3
(1 mmol/L). To investigate the involvement of PKC, the cell was
treated with a phorbol ester, PMA (1 µmol/L), for 30 minutes. To
evaluate the involvement of intracellular Ca2+,
the intracellular Ca2+ concentration was reduced
by chelating with BAPTA. EGTA in the pipette solution was eliminated,
and K aspartate was replaced iso-osmotically with a high concentration
of BAPTA (20 mmol/L). The PAMP-induced conductance was calculated
by the same method in Figure 3C
. As is shown in Figure 6F, the PAMP-induced conductances were not significantly changed
in cells introduced with cAMP, GMP, or IP3, or in
cells treated with PMA. The PAMP-induced conductance was not
significantly changed in cells where intracellular
Ca2+ was chelated low by a high concentration of
BAPTA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAMP is a newly identified peptide that has a hypotensive action. One mechanism that underlies this hypotensive effect is the inhibition of catecholamine secretion from sympathetic nerve terminals.3 The inhibition of catecholamine secretion by PAMP is not mediated by interfering with nicotinic or
2 receptors, which suggests a direct effect of
PAMP on sympathetic nerve terminals. In this paper, we investigated the direct effect of PAMP on NGF-treated PC12 cells. We used PC12 cells that showed prominent axon outgrowth since these characteristics were consistent with those of differentiated PC12 cells that have characteristics similar to noradrenergic sympathetic neurons.6 In the previous study, we analyzed the voltage-gated Ca2+ currents (VGCC) of these NGF-treated PC12 cells and found that these cells with prominent axon outgrowth express N-type currents as a major component of the VGCC.9 This is also characteristic to PC12 cells differentiated into sympathetic neuronlike cells.14 15 16 17
PAMP hyperpolarized the membrane by inducing an outward current due to
the activation of an inwardly rectifying K+
conductance. The activation of the K+ conductance
was significant, reversible, and concentration dependent at the outward
limb of the inwardly rectifying K+ current that
regulates membrane potential at voltage more depolarized than -88 mV.
The activation of the K+ current by PAMP was
concentration dependent and was observed at the concentration of 1
nmol/L at which PAMP decreased significantly the
catecholamine secretion from sympathetic nerve
endings.3 As is shown in Figure 2B, the basal
current showed a steep inward rectification. The basal membrane current
consisted of a conductance dependent on extracellular
Na+ and a residual conductance that is
insensitive to 300 µmol/L BaCl2.
PAMP-induced current was abolished in extracellular solution that
contained 300 µmol/L BaCl2, which is
inconsistent with the observation that the PAMP-induced current
is an inwardly rectifying K+ current. This also
suggests that the PAMP-induced inwardly rectifying
K+ conductance was not induced by augmenting the
basal inwardly rectifying K+ conductance because
the residual membrane conductance in Na+-free
extracellular solution was not sensitive to 300 µmol/L
BaCl2.
The latency of the PAMP-induced outward current in Figure 1B was
slow in part because it took 2 minutes to change the solution with
the perfusion system we used. Other apparatus, such as puff
application of the peptide, will be necessary to obtain the accurate
latency of the response.
In a physiological situation in which the sympathetic neurons are firing because of excitatory inputs, hyperpolarization by PAMP results in the inhibition of action potential firing or decrease in firing frequency. The inhibition of action potentials reduces Ca2+ influx through the VGCC and thereby decreases [Ca2+]i. Because Ca2+ influx through N-type Ca2+ channels is closely related to the catecholamine secretion,18 19 the inhibition of Ca2+ influx through N-type Ca2+ channels could explain the PAMP-induced inhibition of catecholamine secretion.
Microinjection of GDPßS into the cells or PTX treatment
abolished the action of PAMP, which indicates that a PTX-sensitive G
protein is involved in the response.20 By microinjecting
antibody against the carboxyl terminal sequence of G protein
subunit, this G protein was determined to be Gi3.
Several neurotransmitters and neuromodulators, such as somatostatin and
dopamine, also activate inwardly rectifying
K+ current through a PTX-sensitive G
protein.21 The signal transduction mechanism was
investigated further with the conventional whole-cell clamp technique.
PAMP-induced conductances were not significantly changed in cells
introduced with cAMP, GMP, or IP3 or in cells
treated with PMA. The PAMP-induced conductance was not significantly
changed in cells in which intracellular Ca2+ was
chelated low by high concentration of BAPTA. These data suggest that
the signal transduction mechanism does not involve cAMP, GMP,
IP3, PKC, or intracellular
Ca2+.
In our previous study,9 PAMP inhibited N-type
Ca2+ current at concentrations higher than 1
nmol/L. Because PAMP significantly decreased catecholamine
secretion from the sympathetic nerve endings at concentrations >1
nmol/L,3 the effect of PAMP on the VGCC and on the
inwardly rectifying K+ current may have
physiological significance. The concentration
dependence of the inhibition of N-type Ca2+
current was similar to that of the PAMP-induced activation of the
inwardly rectifying K+ current. Because the
outward limb of the inwardly rectifying K+
current is responsible for the regulation of membrane potential, the
PAMP-induced conductance was calculated at the outward limb of the
I-V curve. As is shown in Figure 5, PAMP-induced
conductance at the outward limb of the I-V curve was also
concentration dependent and was comparable to the concentration
dependence of the PAMP-induced inhibition of the VGCC and PAMP-induced
inhibition of catecholamine secretion. The
inhibitory effect appears to be mediated by a PTX-sensitive
G protein. The inhibition of the N-type Ca2+
channels and hyperpolarization by PAMP
cooperatively inhibit the Ca2+ influx through the
VGCC and reduce [Ca2+]i
and thus decrease the catecholamine secretion. The
inhibition of VGCC together with the activation of an inwardly
rectifying K+ current through some PTX-sensitive
G proteins are modulations of ionic channels shared by other
inhibitory neurotransmitters, such as somatostatin and
dopamine.21 22 23 24 These characteristics of ion channel
modulation by PAMP may nominate PAMP as a candidate for
inhibitory neuromodulator or neurotransmitter.
The use of NGF-treated PC12 cells instead of primary-cultured sympathetic neurons offer the advantage that we can analyze the biochemical effect of PAMP, such as changes in the second messenger levels, with the use of numerous cells. We are planning to investigate these effects of PAMP and compare the signal transduction mechanism to that of PAMP-induced modulation of the ion channels in future studies.
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Acknowledgments |
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This work was supported by grants from the Uehara Memorial Foundation and the Ministry of Health and Welfare of Japan.
Received November 11, 1998; accepted December 4, 1998.
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