(Received for publication, June 21, 1994; and in revised form, October 25, 1994)
From the
In non-differentiated NG108-15 cells, both angiotensin II
(Ang II) (100 nM) and CGP 42112 (100 nM) decreased
the T-type calcium current amplitude by 24 ± 2% and 21 ±
3%, respectively. cGMP is not a mediator of the Ang II effect, since
loading of cells with 50 µM cGMP did not prevent the
inhibitory effects of Ang II. The effects of Ang II involves a
non-identified GTPase activity since incubation with GDPS (3
mM) completely reversed the inhibitory effect of Ang II while
GTP
S mimicked its effect. However, Ang II binding was not affected
by GTP
S, and the effect of Ang II was not modified in pertussis
toxin-treated cells. The inhibitory effect of Ang II on the T-type
Ca
current involves a phosphotyrosine phosphatase
activity since sodium orthovanadate prevented the effects of Ang II,
although microcystin-LR, a selective Ser/Thr phosphatase 1 and 2A
inhibitor, did not modify the effect of Ang II. These results provide
the first evidence of a modulation of membrane conductance by Ang II
through the AT
receptor and demonstrate the involvement of
a phosphotyrosine phosphatase and a G protein in the AT
transduction mechanism in NG108-15 cells. Moreover, our
data suggest that phosphotyrosine phosphatase activation is proximal to
receptor occupation, since sodium orthovanadate inhibits both GTPase
activity and T-type current blockage induced by Ang II or CGP 42112,
while GTP
S inhibition of the T-type calcium current is not
impaired.
Pharmacological studies have clearly identified two classes of
angiotensin II (Ang II) ()receptors. The AT
receptor is closely associated with cardiovascular regulation,
fluid volume homeostasis, and cellular growth(1, 2) .
Activation of the AT
receptor is linked to phospholipase C
activation and Ca
influx, effects which are mediated
by G proteins(1, 2) . The AT
receptors
have been identified in many fetal tissues, including brain(3, 4, 5, 6, 7, 8) and cell
lines of neuronal origin(9, 10, 11) . In
contrast with the AT
receptor, no physiological function
has yet been attributed to the AT
receptor, and its
transduction signaling pathway(s) is a much debated
question(1, 2) . Stimulation of the AT
receptor does not stimulate inositol phosphate accumulation,
release of Ca
from intracellular stores, cAMP
production, or arachidonic release (10, 11, 12) . Ang II binding on AT
receptors has been shown to decrease intracellular cGMP
levels(13, 14, 15) . Two mechanisms have been
proposed as mediators of this decrease: a phosphodiesterase activation
through calcium entry (16) or the inhibition of a particulate
guanylate cyclase through phosphotyrosine phosphatase (PTPase)
activity(1, 14, 17) . However, this
AT
-mediated cGMP decrease is not observed in all
studies(10, 11, 18) . Results regarding
AT
modification of cellular phosphotyrosine patterns are
even more controversial; some authors observe no change in tyrosine
phosphorylation (10) while others describe an increased
phosphorylation of tyrosine residues(19, 20) , and
Bottari et al.(14) and Brechler et al.(17) report the stimulation of a PTPase activity. The
angiotensin AT
receptor does not interact with guanine
nucleotide binding proteins (G proteins) in PC12W cells (21) ,
in ovarian granulosa cells(18) , nor in fetal skin or fetal
skeletal muscle(22) . Nevertheless, binding experiments using
rat brain suggest that two subclasses of AT
receptors can
be distinguished: AT
for receptors that are sensitive to
guanine nucleotides and pertussis toxin (PTX) and AT
for
receptors that are not(6) . Substantial differences in binding
of [
I]CGP 42112 between brain and adrenal
AT
receptors have also been reported(23) .
Recently, the AT
receptor was cloned by two different
groups(24, 25) . Both found that the AT
receptor belongs to the seven-transmembrane domain receptor class
with a low homology (32-34%) with the AT
receptor.
We previously reported the Ang II modulation of the T-type calcium
current in non-differentiated NG108-15 cells expressing only the
angiotensin AT receptor type(26) . In the present
study, we show that, in this cell line, the inhibitory effect of Ang II
on the T-type Ca
current by Ang II is not dependent
on intracellular cGMP concentration, is not PTX-sensitive, but is
abolished by GDP
S and involves a phosphotyrosine phosphatase
activity. Moreover, to correlate the effects of Ang II on T-type
current with signaling pathways, we demonstrate in the same
experimental conditions that Ang II decreases the level of
phosphotyrosine proteins and stimulates a membraneous GTPase activity.
It could be concluded that the signaling pathway of the AT
receptor involves a PTPase and yet unknown G protein, which
regulates the T-type current.
Figure 1:
Effect of Ang II and of the
AT selective ligand CGP 42112 on the
T-type calcium current. A and B, effect of Ang II in
control medium. A, plot of the peak current versus time; Ang II (100 nM) was applied as indicated by the darkline. B, currents recorded before (1) and after (2) Ang II addition. C and D, effect of CGP 42112 in control medium. C, plot of
the peak current versus time; CGP 42112 (100 nM) was
applied as indicated by the darkline. D,
currents recorded before (1) and after (2) CGP 42112
addition. E and F, effect of 50 µM cGMP
added into the pipette solution. E, plot of the peak current versus time; CGP 42112 (100 nM) was applied as
indicated by the darkline. F, currents
recorded before (1) and after (2) CGP 42112 addition.
Intracellular buffering of the cGMP concentration did not affect the
T-type Ca
current modulation by CGP 42112. 200 ms
depolarizing steps were delivered every 20 s from -80 to
-25 mV.
Figure 2:
Effects of GDPS, GTP
S, and PTX
on the effect of Ang II on the T-type calcium current. A,
replacement of GTP by GDP
S (3 mM) into the standard
intracellular solution; plot of the peak current versus time
is shown. B, currents before (1) and after (2) the addition of Ang II (100 nM) as indicated in panelA. C, replacement of GTP by GTP
S
(3 mM) into the standard intracellular solution. The current
amplitude decreased slowly as soon as the patch was established and
reached a steady state after 3 min. At the plateau level, addition of
Ang II (100 nM) induced a further decrease of the current
amplitude. D, control current trace (1) at the
plateau level in presence of GTP
S (2) and after Ang II
addition(3) . E and F, effect of Pertussis
toxin treatment on CGP 42112 modulation of the T-type calcium current. E, plot of the peak current versus time; F,
currents before (1) and after (2) the addition of CGP
42112 (100 nM) as indicated for panelC; 200
ms depolarizations from -80 to -25 mV were delivered every
20 s.
Figure 3:
A G
protein is involved in the AT effect. A,
competition for Sar
Ile
angiotensin II binding
(where Sar is saralasin). Plasma membranes from NG108-15 cells
(50 µg) were incubated with 0.2 nM
I-labeled
Sar
Ile
-Ang II for 15 min at 37 °C in the
presence of increasing concentrations of Ang II alone (
) or with
100 µM GTP
S (
). Results represented the mean
± S.E. from three different experiments, each conducted in
triplicate incubations. B, effects of Ang II (100 nM)
and CGP 42112 (100 nM) on the low K
GTPase activity in NG108-15 cells. Pharmacological
effects have been studied by the addition of DUP 753 (1
µM), a specific antagonist of the AT
receptor
subtype, and PD 123310 (10 µM), a specific antagonist of
the AT
receptor subtype. Involvement of phosphatase
activity has been studied by the addition of sodium orthovanadate
(Na
VO
, 100 µM), a tyrosine
phosphatase antagonist, and okadaic acid (OKA, 1 mM),
a Ser/Thr phosphatase antagonist. GTPase activity was determined by
measuring the release of
P from
[
-
P]GTP using 20 µg of membrane protein
prepared from NG108-15 cells as described under ``Materials
and Methods.'' The low K
GTPase
activity was measured after subtracting the amount of hydrolysis that
remained in the presence of 50 µM GTP (high K
GTPase activity) from that measured
with 0.03 µM [
-
P]GTP. The high
basal K
activity was not affected by
addition of Ang II or CGP 42112.
, control (C);
&cjs2113;, Ang II (100 nM); &cjs2112;, CGP (100 nM).
Results are the mean ± S.E. of triplicate determinations from
one experiment representative of three.**, p < 0.001; *, p < 0.05 (difference compared to control
values).
Figure 4:
Involvement of a protein tyrosine
phosphatase in AT receptor signaling pathway. A,
20 nM microcystin-LR (a specific Ser/Thr phosphatase 1 and 2A
inhibitor) was added into the standard intracellular solution before
application of Ang II. This addition did not alter the Ang II-mediated
inhibition of the T-type Ca
current. B, 50
µM sodium orthovanadate (a specific tyrosine phosphatase
inhibitor) added into the intracellular solution before application of
Ang II abolished the Ang II-mediated inhibition of the T-type
Ca
current. C, preincubation of cells in a
solution containing sodium pervanadate (0.2 mM, 5 min) did not
affect the blocking effect of intracellular GTP
S. At the plateau
level, addition of Ang II did not produce any further blocking
effect.
The lack of additional effect of Ang II once the plateau
is reached following GTPS-induced blockage indicates that with
sodium pervanadate preincubation, Ang II does not produce any further
decrease of the T current because the AT
receptor is
uncoupled from the G protein by sodium pervanadate inhibition of the
PTPase (Fig. 4C). Furthermore, without sodium
pervanadate preincubation, Ang II is able to further increase the
blocking effect of GTP
S because the signaling pathway is intact (Fig. 2, C and D). These results indicate that
PTPase activation is proximal to the AT
receptor
activation.
The AT receptor has recently
been cloned and belongs to the class of the seven-transmembrane domain
receptors (24, 25) along with somatostatin SSTR1 and
dopamine D
receptors(39, 40) . Using cDNA
libraries from different sources (PC 12W and whole rat fetus), the two
groups (24, 25) found similar amino acid sequences but
opposite results concerning G protein involvement and phosphotyrosine
phosphatase implication. The binding of ligand to the two cloned
AT
receptors is not affected by
GTP
S(24, 25) . However, sensitivity to PTX and
GDP
S indicates that one of these receptors involves a G protein in
its signaling pathway(20, 25) . This discrepancy could
be explained by the presence of two subclasses of AT
receptors(6) . Comparatively, we found that the native
AT
receptor from NG108-15 cells demonstrates
GTP
S-insensitive binding of Ang II and involvement of a GTPase
activity in the transduction mechanism (Fig. 3).
Using the
cell line PC12 W, Kambayashi et al.(25) and Takahashi et al.(20) demonstrated that the AT receptor is linked to the inhibition of a PTPase via a G protein
sensitive to pertussis toxin. However, in the same cell line, Bottari et al.(14) and Brechler et al.(17) demonstrated a stimulation of PTPase activity, which
is independent of a G protein activation. When NG108-15 cells
were treated overnight with lisinopril to block endogenous Ang II
production(38, 41) , we observed that Ang II decreases
the amount of tyrosine phosphorylation of several proteins, which may
be explained by stimulation of endogenous PTPase activity. This effect
is abolished by co-incubation with PD 123319, indicating its
specificity toward the AT
receptor. These results and those
of patch clamp experiments support the conclusion that a PTPase
activity is involved in the inhibitory effect of Ang II on the T-type
Ca
current, since orthovanadate and a PTPase
monoclonal antibody reverse the effect of Ang II on this inhibition.
Moreover, patch clamp and GTPase experiments demonstrate that a G
protein is involved in this inhibition and that its activation is
blocked by a tyrosine phosphatase antagonist. Although the signaling
pathway needs to be determined more accurately, our results do allow us
to propose the following time sequence. Activation of the AT
receptor is followed by activation of a PTPase, which is
necessary for the coupling between a G protein and the T-type channel.
In the present work, the exact substrate of PTPase has not been
determined, but we can postulate that the coupling between the AT
receptor and the G protein is dependent upon the phosphorylated
state of one of the components of the signaling pathway, i.e. the AT
receptor itself, the G protein, or an unknown
accessory protein. Modulation of the coupling could thus be obtained by
a balance between phosphatase and kinase activities. Association of a
PTPase activity with activation of several seven-transmembrane domain
receptors has been described either via a stimulation for somatostatin
receptor(39, 42) , dopaminergic D
receptor(34) , and AT
receptor (14, 17, this
paper) or via an inhibition(19, 20, 25) .
These effects may be direct (39, 40) or mediated via a
PTX-sensitive G protein(34, 42) .
In summary, the
present observations demonstrate that in non-differentiated
NG108-15 cells, which exclusively express AT receptors, Ang II (100 nM) and CGP 42112 (100
nM) both decrease the T-type calcium current and that this
effect is mediated by the involvement of a tyrosine phosphatase and a
yet unknown G protein. Considering the abundance of T-type
Ca
channels in neurons from fetal brain(43) ,
the crucial role of Ca
in neuronal differentiation,
and the abundance of AT
receptors during this period, it
could be postulated that Ang II, via the AT
receptor,
modulates the Ca
channel regulating pacemaker
activity in neuronal cells. These modulations may have a profound
effect on neuronal orientation, guidance, and
differentiation(44) .