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The Journal of Neuroscience, July 1, 2000, 20(13):4776-4785
Bidirectional Modulation of Exocytosis by Angiotensin II Involves
Multiple G-Protein-Regulated Transduction Pathways in Chromaffin
Cells
Anja G.
Teschemacher and
Elizabeth
P.
Seward
Department of Pharmacology, School of Medical Sciences, University
of Bristol, Bristol BS3 1TD, United Kingdom
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ABSTRACT |
Angiotensin II (AngII) receptors couple to a multitude of different
types of G-proteins resulting in activation of numerous signaling
pathways. In this study we examined the consequences of this
promiscuous G-protein coupling on secretion. Chromaffin cells were
voltage-clamped at 
80 mV in perforated-patch configuration, and
Ca2+-dependent exocytosis was evoked with brief
voltage steps to +20 mV. Vesicle fusion was monitored by changes in
membrane capacitance (
Cm), and
released catecholamine was detected with single-cell amperometry.
Ca2+ signaling was studied by recording
voltage-dependent Ca2+ currents
(ICa) and by measuring intracellular
Ca2+
([Ca2+]i) with fura-2 AM.
AngII inhibited ICa (IC50 = 0.3 nM) in a voltage-dependent, pertussis toxin
(PTX)-sensitive manner consistent with Gi/o-protein coupling to Ca2+ channels.
Cm was modulated bi-directionally;
subnanomolar AngII inhibited depolarization-evoked exocytosis, whereas
higher concentrations, in spite of ICa
inhibition, potentiated Cm fivefold
(EC50 = 3.4 nM). Potentiation of
exocytosis by AngII involved activation of phospholipase C (PLC) and
Ca2+ mobilization from internal stores. PTX
treatment did not affect AngII-dependent Ca2+
mobilization or facilitation of exocytosis. However, protein kinase C
(PKC) inhibitors decreased the facilitatory effects but not the
inhibitory effects of AngII on stimulus-secretion coupling. The AngII
type 1 receptor (AT1R) antagonist losartan blocked both inhibition and
facilitation of secretion by AngII. The results of this study show that
activation of multiple types of G-proteins and transduction pathways by
a single neuromodulator acting through one receptor type can produce
concentration-dependent, bi-directional regulation of exocytosis.
Key words:
angiotensin II; G-protein-coupled receptor; calcium
channels; intracellular calcium stores; protein kinase C; phospholipase
C; pertussis toxin; exocytosis; chromaffin cells
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INTRODUCTION |
Modulation of neurotransmitter
release and hormone secretion via activation of G-protein-coupled
receptors (GPCRs) is a key element in information processing within an
organism. To understand how secretion is modulated, it is important to
characterize the mechanisms underlying GPCR regulation of ion channels,
Ca2+ signaling, and the exocytotic
machinery. Given the divergence and convergence of downstream signaling
cascades modulated by G-protein subunits (Gudermann et al., 1996
), the
effects of a single neuromodulator on regulated exocytosis may be
complex and variable depending on stimulus conditions.
GPCRs have been postulated to modulate neurotransmission either through
an effect on membrane excitability and
Ca2+ signaling or via second
messenger-mediated changes in the activity of the proteins controlling
exocytosis (Wu and Saggau, 1997; Miller, 1998). The inaccessibility of
most mammalian nerve terminals makes direct investigation of
stimulus-secretion coupling and its modulation difficult. However, such
studies can be performed on neuroendocrine cells where the
Ca2+ signals regulating exocytosis may be
controlled and monitored and vesicle fusion assessed directly using
membrane capacitance measurements and amperometry (Neher, 1998). With
such an approach, previous studies have shown that activity-dependent
changes in exocytosis are mediated by Ca2+
and protein kinase C (PKC) (Smith et al., 1998). The aim of this study
was to examine the mechanism(s) underlying GPCR modulation of
exocytosis. We studied the effects of angiotensin II (AngII) on
secretion because (1) it is well established that this peptide modulates catecholamine release from neurons and chromaffin cells (Feldberg and Lewis, 1964; Bottari et al., 1993), and (2) AngII type 1 receptors (AT1Rs) are coupled to a plethora of different types of
G-protein (Richards et al., 1999). How AT1R signaling pathways modulate
exocytosis is unknown.
We combined voltage-clamp, Cm,
ratiometric fluorescence, and electrochemical techniques on single
cells to examine directly the effects of AngII on stimulus-secretion
coupling in adrenal chromaffin cells. We show that subnanomolar
AngII inhibits Ca2+ influx and vesicle
fusion via a Gi/o-dependent pathway. At higher concentrations, the inhibitory effects of AngII are surmounted, producing an increase in exocytosis. The facilitatory effects of AngII
are pertussis toxin (PTX)-insensitive and are associated with
activation of phospholipase C (PLC), store-dependent rise in
intracellular Ca2+ concentration
([Ca2+]i), and
activation of PKC. The results of this study show that GPCR regulation
of multiple signaling pathways may produce antagonistic modulation of
neurotransmitter release.
Some preliminary data have been published previously in abstract form
(Teschemacher et al., 1998).
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MATERIALS AND METHODS |
Chromaffin cell culture. Chromaffin cells were
dissociated as described previously (Seward and Nowycky, 1996). Adrenal
glands from 18- to 24-month-old cows were obtained from a local
abattoir and were retrogradely perfused at 25 ml/min for 30 min at
37°C with the digestive enzymes 0.03% collagenase type 2 (Worthington Biochemical, Lakewood, NJ) and 0.001% DNase I (Boehringer
Mannheim, Indianapolis, IN) added to a Locke's solution consisting of
(in mM): 154.2 NaCl, 2.6 KCl, 2.2 K2HPO4, 0.85 KH2PO4, 10 glucose, 5 HEPES; 0.0005% Phenol Red (Life Technologies, Paisley, UK); pH
adjusted to 7.2 with NaOH. After surgical removal of the cortex, the
medulla was dissected and dissociated with fresh enzyme solution for 30 min at 37°C. After this incubation, cells were transferred to
Earle's balanced salt solution (Life Technologies), centrifuged twice
at 50 × g for 15 min, and resuspended in DMEM (Life
Technologies) supplemented with 44 mM
NaHCO3 and 15 mM HEPES,
10% fetal calf serum (Life Technologies), 0.5 mM
glutamine, and 0.01% penicillin-streptomycin solution. Cells were
plated on glass coverslips coated with matrigel (Becton Dickinson
Labware, Bedford, MA) at an approximate density of 800 cells/mm2. The medium was replaced 24 hr
after plating, and cells were maintained for up to 7 d in a
humidified atmosphere of 95% O2/5% CO2 at 37°C. Some cultures were treated with
250 ng/ml PTX (Sigma, Poole, UK) at 37°C for 24 hr.
Electrophysiology. A coverslip carrying chromaffin cells was
placed in a microperfusion chamber on the stage of an inverted phase-contrast Axiovert 100 microscope equipped with a 40×
oil-immersion objective with a numerical aperture of 1.3 (Zeiss, Jena,
Germany). Cells were continuously superfused at 1.5 ml/min with an
external solution consisting of (in mM): 140 NaCl, 2 KCl,
0.5 NaHCO3, 1 MgCl2, 2.5 CaCl2, 10 D-glucose, 10 HEPES; pH
adjusted to 7.3 with NaOH. Ionic currents were recorded in
perforated-patch-clamp configurations using borosilicate glass
electrodes coated with Sylgard 184 (Dow Corning, Midland, MI) and
fire-polished to a resistance of 1-2 M
. Electrodes were filled with
a solution consisting of (in mM): 145 Cs-glutamate
(Calbiochem, Nottingham, UK), 9.5 NaCl, 0.3 BAPTA (Molecular Probes,
Eugene, OR), and 10 HEPES; pH adjusted to 7.3 with CsOH (ICN
Biomedicals, Aurora, OH). For perforated-patch recording experiments,
gramicidin D (Sigma) at a final concentration of 65 µg/ml [with
0.9% dimethylsulfoxide (DMSO) as solvent] was added. Series
resistance was <12 M and compensated (typically >70%)
electronically using a patch-clamp amplifier (Axopatch 200B; Axon
Instruments, Foster City, CA). Voltage protocol generation and data
acquisition were performed using custom data acquisition software
(kindly provided by Dr. A. P. Fox, University of Chicago) running
on a Pentium computer equipped with a Digidata 1200 acquisition board
(Axon Instruments). Current traces were low-pass-filtered at 5 kHz
using the four-pole Bessel filter supplied with the amplifier and
digitized at 10 kHz. Current traces were corrected off-line for linear
leakage current (typically <10 pA, at 80 mV) using the P4 method.
Chromaffin cells were voltage-clamped at 80 mV, and
Cm was sampled with a resolution of 12 msec using a software-based phase-tracking method as described
previously (Seward et al., 1995). Exocytosis was evoked every 25 sec
with a voltage step to +20 mV of a fixed duration, which elicited a
reproducible Cm of 50-100 fF (see
below). Cm sampling was resumed 40 msec after the stimulus to exclude gating charge artifacts (Horrigan
and Bookman, 1994). Data were stored on the computer hard drive and analyzed off-line (Axobasic; Excel, Microsoft; Origin, Microcal). Unless otherwise indicated, Ca2+ influx
was quantified by integrating ICa,
omitting the first 2 msec, which were contaminated by
Na+ currents.
Cm was measured relative to a 100 fF calibration signal that was routinely switched in and out of the
circuit during the course of a recording. All experiments were
performed at ambient temperature (21-25°C).
[Ca2+]i measurements. Cells
were preloaded with Ca2+ indicator by
incubating for 25 min at 37°C in DMEM medium containing 5 µM fura-2 AM (Molecular Probes) followed by washing with
fresh DMEM and incubating for a further 15 min. Chromaffin cells were
alternately illuminated at 340 and 380 nm using a monochromator (TILL
Photonics, Martinsried, Germany) controlled by the
Cm data acquisition software. Emission
>430 nm was collected with a photomultiplier tube (TILL Photonics) and
sampled every 12 msec. Data were stored on PC and ratios of 340/380 nm
were calculated off-line (Axobasic-written software; Excel, Microsoft).
Calibration of fura-2 AM was performed by the method of Grynkiewicz et
al. (1985). Rmin and
Rmax and Sf2/Sb2
were obtained by permeabilizing chromaffin cells with 10 µM ionomycin or 3 µM
digitonin in the presence of 10 mM EGTA or 20 mM Ca2+, respectively.
Electrochemical catecholamine detection. Catecholamine
release was detected by single-cell amperometry according to the method described by Schulte and Chow (1998). Carbon fiber probes (5 µM diameter; ALA Scientific Instruments, New York, NY)
were charged to +800 mV and brought into contact with the plasma
membrane of chromaffin cells that were voltage-clamped in
perforated-patch-clamp configuration. Currents were measured and
low-pass-filtered at 3 kHz using a VA-10 amplifier (npi electronics,
Hamm, Germany) and digitized at 5 kHz with commercial software (pCLAMP,
Axon Instruments) running on a second Pentium computer equipped with a
Digidata 1200 data acquisition board (Axon Instruments). At the end of
each experiment, the quality of the carbon fiber electrode and the
ability of a chromaffin cell to produce amperometric signals were
verified by application of high concentrations of nicotine or by
rupturing the cell membrane. Amperometric events were analyzed by
software developed by S. Kasparov (University of Bristol). Individual
events with a rapid rise-time (<0.5 pA/msec) and integrated charge >30 fC were automatically detected by the software and summed
for the duration (25 sec) of the corresponding capacitance trace.
Drug application. All drugs were added to the superfusing
external solution. Because of the prominent desensitization of
responses at >1 nM AngII, unless stated otherwise, only
one agonist application was made per coverslip. The specific nonpeptide
AngII receptor antagonists losartan (kind donation from Merck, Sharp & Dohme, Hertfordshire, UK) and PD 123,319 (RBI, Natick, MA) were applied for 2 min before and during AngII treatment. All drugs were made up as
stock solutions and stored in aliquots at 20°C. A fresh aliquot was
used for each experiment and diluted at least 1000-fold. U-73122,
U-73443 (BIOMOL">Biomol, Plymouth Meeting, PA), Calphostin C (BIOMOL">Biomol), and
bisindolylmaleimide (BIS; Calbiochem, Nottingham, UK) were solved in
DMSO. Cyclopiazonic acid (CPA; Calbiochem) was dissolved in chloroform.
Because of the transient nature of responses to AngII, percentile
changes of parameters under investigation were obtained by relating
maximal deflections after drug application to the average of four
measurements immediately preceding treatment. All results are presented
as mean ± SEM. Unless stated otherwise in the text, changes were
tested with Student's paired t test. Statistical
significance was accepted at a level of p < 0.05. A
total of 135 chromaffin cells from 35 cultures were used in this study.
Of these, seven recordings from three cultures were excluded because
they failed to show any response to AngII.
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RESULTS |
AngII inhibition of ICa in
chromaffin cells
Ca2+ entry through voltage-operated
Ca2+ channels (VOCCs) regulates
depolarization-evoked exocytosis in neurons and chromaffin cells. GPCRs
are known to modulate VOCCs either through a well characterized,
ubiquitous membrane-delimited pathway or through an undefined
second-messenger mediated pathway(s) (Hille, 1994; Dolphin, 1998). To
gain insight into the mechanisms underlying modulation of exocytosis by
AT1Rs, in our first series of experiments we investigated the effect of
AngII on VOCCs in chromaffin cells. All of our studies were performed
using the perforated-patch configuration to avoid dialysis of
agonist-induced changes in diffusable second messengers and rundown of
ICa and exocytosis (Seward and
Nowycky, 1996). Furthermore, we used Ca2+
as the divalent cation to retain normal
Ca2+ homeostasis pathways and to maintain
the function of Ca2+-dependent proteins
important in cell signaling and exocytosis (Seward et al., 1996).
Superfusion of AngII for 2-3 min produced a reversible inhibition of
ICa (Fig.
1A,B).
The concentration-response curve for AngII inhibition of
Ca2+ entry (determined by integration of
ICa; see Materials and Methods) had an
IC50 of 0.28 nM, a maximum
of 39 ± 3% at 10 nM, and a Hill slope of
1.18 (Fig. 1C). At 100 nM, the
inhibition of ICa was reduced to
30 ± 3% (n = 16). In eight cells, a second
application of 100 nM AngII after 10 min wash
failed to inhibit ICa, suggesting that
AT1Rs in chromaffin cells undergo rapid and profound desensitization similar to that reported previously (Wang et al., 1994; Oppermann et
al., 1996). Characterization of this desensitization is beyond the
scope of the present study and was not pursued further; in all
subsequent experiments, responses to only the first application of
AngII are reported.
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Figure 1.
AngII inhibition of ICa
in bovine adrenal chromaffin cells. A, Superimposed
current traces evoked by 60 msec voltage steps to +20 mV in a cell
clamped at 80 mV in perforated-patch configuration. Currents were
recorded immediately before (control) and during superfusion of AngII
(1 nM) as indicated. The transient current observed
during the first 3 msec of the voltage step is attributable to
activation of Na+ channels; this is followed by a
more sustained inward current resulting from activation of VOCCs. AngII
inhibition of ICa decreased during the
voltage step. B, Time course of the inhibition of
Ca2+ influx during superfusion with AngII (indicated
by bar above the data). Data are from a single cell.
Ca2+ influx was calculated by integration of
ICa. C,
Concentration-response curve for AngII inhibition of
Ca2+ influx. Each point is the mean percentage
inhibition of Ca2+ entry ± SEM for the number
of cells indicated. Solid line through the data
represents the best fit of pooled data (0.01-10 nM) with
the Hill equation, giving an IC50 of 0.28 nM
and Hill coefficient of 1.18. D, Large depolarizing
prepulses reversed the inhibition of ICa by
AngII. The top trace is a schematic representation of
the voltage protocol used to evoke the superimposed currents shown
below before (control) and during superfusion
with 10 nM AngII.
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Inhibition of neuronal VOCCs by G
-subunits via a
"membrane-delimited" pathway is voltage dependent and typified by a
slowing of activation kinetics and time-dependent recovery during
depolarization (Dolphin, 1998). The inhibition of
ICa produced by 1 nM AngII in chromaffin cells was found to
decrease from 36.0 ± 3.2% at the peak to 25.6 ± 2.3%
during a 30 msec depolarization to +20 mV (n = 5).
Application of a large depolarizing prepulse to +120 mV for 40 msec
before the test pulse reduced the AngII inhibition of
ICa from 40.8 ± 3.6 to 2.6 ± 4.7% (10 nM; n = 4) (Fig.
1D). Treatment of cells with PTX abolished the
effects of AngII on ICa (see Fig.
8C). No evidence for a voltage-independent inhibition of
ICa by AngII as described for
sympathetic neurons (Shapiro et al., 1994) nor any facilitation of
ICa as described for sensory neurons
(Bacal and Kunze, 1994) was observed in chromaffin cells. The subtype
of receptor mediating the effects of AngII on VOCCs was determined
using specific antagonists (Hunyady et al., 1996). The AT1R antagonist
losartan (10 µM) abolished
ICa inhibition by AngII (100 nM, n = 8), whereas the specific
AngII type 2 receptor antagonist PD123,319 (10 µM, n = 5) did not (see Fig.
8C). Taken together, these results show that in chromaffin
cells AngII acts via a Gi/o-protein coupled to
AT1Rs to inhibit VOCCs through a voltage-dependent mechanism.
Bidirectional, concentration-dependent modulation of exocytosis
by AngII
Cm is proportional to cell
surface area and is increasingly used as a method for monitoring
exocytosis with exquisite resolution. Fusion and endocytosis of
vesicles are observed as increases and decreases in
Cm, respectively. To examine the
effects of AngII on secretion, cells were stimulated every 25 sec with
voltage steps to +20 mV from a holding potential of 80 mV. Before
drug application, the step duration was adjusted to between 30 and 60 msec to achieve a reproducible Cm
of 50-100 fF. This stimulus paradigm avoids depletion of the readily
releasable pool (RRP) (Smith et al., 1998) and activity-dependent
changes in exocytotic efficiency (Engisch et al., 1997). Application of
1 nM AngII produced concomitant inhibition of
Ca2+ entry (30 ± 2%) and
Cm (42 ± 9%;
n = 6) (Fig.
2A); however, the
inhibition of exocytosis by AngII did not produce a significant change
in exocytotic efficiency (Cm/
Ca2+ ions) (92 ± 6% of control,
n = 6), suggesting that at this concentration AngII
does not alter the Ca2+ dependence of
exocytosis. At higher AngII concentrations (100 nM), Cm
increases were no longer inhibited but potentiated to 524 ± 118%
of control (n = 11) despite inhibition of
Ca2+ entry by 31 ± 3% (Fig.
2B,C). The exocytotic efficiency
was facilitated to 819 ± 291% of control (n = 11) (Table 1). The facilitatory effect of
AngII on secretion was transient, and its onset either coincided with
(n = 8) or was delayed by up to 1 min with respect to
ICa inhibition (n = 3) (Fig.
2C). At the intermediate concentration of 10 nM, the effect of AngII on
Cm was highly variable from cell to
cell, resulting in either inhibition (40% of cells) or potentiation
(60% of cells) (Fig. 2D).
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Figure 2.
Bimodal concentration-dependent modulation of
stimulus-evoked exocytosis by AngII. A, Inhibition of
stimulus-evoked exocytosis by low concentrations of AngII. Superimposed
ICa and corresponding
Cm traces evoked by 30 msec voltage steps
to +20 mV before (control) and during application
of AngII (1 nM). B, In the same cell,
subsequent application of AngII (100 nM) potentiated
Cm (top right) while still
depressing ICa. The traces marked
wash were recorded 10 min after AngII (1 nM) treatment had terminated and serve as controls for the
AngII (100 nM) measurements. C, Diary plots
of the effect of 100 nM AngII on normalized
Cm and Ca2+ entry as
measured by integrating ICa (data plotted
are mean ± SEM for 8 cells). Note that the potentiation of
Cm and inhibition of
Ca2+ influx are not maintained throughout the 3 min
period of agonist application (indicated by bar).
D, Concentration dependence of
Cm modulation by AngII. Each
point represents the mean ± SEM for the number of
experiments shown below each point; asterisks indicate
significant difference from control (p < 0.05; Student's paired t test).
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Amperometric evidence for catecholamine secretion
Previous studies have shown that AngII increases catecholamine
release from populations of chromaffin cells (Bunn and Marley, 1989;
O'Sullivan and Burgoyne, 1989), and it is likely that the Cm increases observed in this study
result from fusion of catecholamine-containing vesicles. To confirm
this and to obtain some indirect spatial information on the effects of
AngII on stimulus-evoked exocytosis, we combined
Cm measurements with single-cell
amperometry. The carbon fiber electrodes that were used had tip
diameters of ~5 µm so that single vesicle release events of
catecholamine were only detected when the fiber was in close proximity
to a release site (Robinson et al., 1995). We had no means of
determining where the release sites were and found empirically that the
probability of obtaining synchronous amperometric spikes and
depolarization-evoked Cm under
control conditions was low. Thus for a 100 fF
Cm increase, the average integrated
charge was 4.4 ± 3.2 pC (n = 3 cells, 10 depolarizations per cell). Increasing stimulus strength to evoke a
Cm of 300-400 fF did not increase
the number of events detected with amperometry, consistent with
previous reports that secretion remains highly localized (Schroeder et
al., 1994; Robinson et al., 1995). In the presence of AngII (100 nM), previously silent sites became active, with
both asynchronous unitary events and synchronous release events being
observed (Fig.
3A,C).
The mean amperometric charge recorded in the presence of AngII was
32 ± 17 pC, which represents a 1051 ± 565% increase over
control (Fig. 3B). These results confirm that the
potentiation of Cm observed after
application of high concentrations of AngII are caused by increased
exocytosis of catecholamine-containing vesicles and furthermore suggest
that AngII increases the number of active release sites.
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Figure 3.
AngII increased catecholamine secretion detected
by single-cell amperometry. A, Data shown are from a
single cell showing simultaneous recording of
Cm (top traces) and
amperometric current (bottom traces) before and during
superfusion of AngII (indicated by bar). Exocytosis was
evoked at the start of each trace with a 60 msec voltage step to +20 mV
from a holding potential of 80 mV (indicated by the
gap in the Cm trace;
asterisks indicate 100 fF calibration steps). In the
absence of the agonist, the voltage stimulus elicited a
Cm of 60 fF, but no amperometric spikes
were detected. Thirty seconds into AngII application (middle
trace), amperometric spikes that were not synchronized with
stimulus-evoked Cm were observed. A
subsequent voltage stimulus evoked a Cm
potentiated by 400% (top right) and corresponding large
amperometric current response (below). B,
Mean data from three experiments similar to those illustrated in
A. Data plotted are mean ± SEM integrated
amperometric charge recorded over 30 sec intervals for the duration of
the experiment. AngII (100 nM) application is indicated by
the bar. C, Amperometric response that
was recorded in synchrony with the potentiated
Cm during application of AngII. Note that
several spikes can be distinguished. Data are from the same cell
illustrated in A shown on an expanded time scale.
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Ca2+ mobilization is required for
AngII-dependent facilitation of exocytosis
Previous studies have shown that AngII activates PLC, leading to
formation of inositol phosphates and a rise in
[Ca2+]i in bovine
chromaffin cells (Plevin and Boarder, 1988; Bunn and Marley, 1989;
O'Sullivan et al., 1989). To examine the relationship between
AngII-induced Ca2+ signaling and
stimulus-evoked exocytosis, we combined recording of
ICa and
Cm with fura-2 AM measurements of
[Ca2+]i. Under
control conditions, the mean basal
[Ca2+]i in
chromaffin cells held at 80 mV was 218 ± 22 nM (n = 11), which is comparable
to that reported in other studies using constant, low-frequency voltage
stimulation (Smith et al., 1998). AngII (100 nM)
increased basal
[Ca2+]i to
502 ± 66 nM (n = 11),
corresponding to a mean increase of 235 ± 28%. The rise in
[Ca2+]i peaked
within 30-60 sec of AngII reaching the cell and subsequently declined
despite the continued presence of agonist (Fig.
4A,B). In all cells the rise in
[Ca2+]i induced by
100 nM AngII was associated with a profound but transient facilitation of exocytosis (Fig.
4A,B). We observed a strong
correlation between the percentage increase in
Cm and the percentage increase in
[Ca2+]i relative
to control (R2 = 0.87) (Fig.
4C). In contrast, there was a poor correlation (R2 = 0.29) between the
percentage increase in exocytosis and the peak
[Ca2+]i recorded
in the presence of AngII.
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Figure 4.
AngII potentiation of
Cm was correlated to a rise in
[Ca2+]i. A,
Simultaneous recording of Cm
(top) and [Ca2+]i
(bottom) in a single chromaffin cell voltage-clamped to
80 mV. [Ca2+]i was measured
with fura-2 AM. Values plotted are the ratios of emitted fluorescence
at excitation wavelengths 340 and 380 nm. Cm
and [Ca2+]i measurements were
interrupted (indicated by arrows) to apply 40 msec
voltage steps to +20 mV. Forty-five seconds into application of AngII
(indicated by bar above the trace), a profound
potentiation of Cm and corresponding rise
in [Ca2+]i are observed. AngII
increased basal [Ca2+]i from 219 to a
maximum of 1060 nM. B, Time course of AngII
potentiation of stimulus-evoked secretion. Cells were stimulated every
25 sec with voltage steps to +20 mV from a holding potential of 80
mV. Mean ± SEM changes in
[Ca2+]i before and
Cm after voltage steps are plotted
against time (n = 8). Time of drug application is
indicated by the horizontal bar and hatched
lines. Note that the increase in
[Ca2+]i preceded
Cm potentiation. C, Pooled
data from 11 cells showing the correlation between rise in
[Ca2+]i (% of
control) and potentiation of
Cm (% of control)
caused by AngII (100 nM). Solid line through
the data was fit by linear regression
(R2 = 0.87).
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In addition to facilitation of stimulus-evoked exocytosis, in 52% of
cells examined (n = 21), the rising phase of the
[Ca2+]i increase
produced by AngII was followed by a Cm
increase, in the absence of voltage stimulation (Fig.
5A). Because this increase in
Cm occurred at the holding potential
of 80 mV, we will refer to it as depolarization-independent
exocytosis. In cells in which AngII induced depolarization-independent
exocytosis, the potentiation of subsequent stimulus-evoked exocytosis
was greatest (range 466-2681%; n = 7), indicating
that depolarization-independent exocytosis does not deplete the cell of
releasable vesicles. Depolarization-independent exocytosis was observed
in cells in which (1) the
[Ca2+]i rise
reached significantly higher levels (608 ± 72 nM) than in other cells (398 ± 53 nM; unpaired t test), and (2) the
average holding current increased in the presence of AngII from 8.5
to 10.5 pA (Fig. 5B). The small amplitude of this current
is consistent with previously characterized store-operated
Ca2+ entry currents that trigger
voltage-independent exocytosis in chromaffin cells (Fomina and Nowycky,
1999). After removal of Ca2+ from the
external solution for 1.5 min, the rise in
[Ca2+]i produced
by AngII (430 ± 119%, n = 6) or caffeine (50 mM; n = 9) at a concentration
shown previously to deplete stores in chromaffin cells (Cheek et al.,
1993b) failed to trigger depolarization-independent exocytosis. This is
in agreement with previous studies that also found that external
Ca2+ was necessary for AngII-induced
catecholamine release (Bunn and Marley, 1989; O'Sullivan and Burgoyne,
1989; Cheek et al., 1993a). Collectively, the results suggest that
agonist-stimulated Ca2+ entry across the
plasma membrane in addition to Ca2+
release from internal stores is required to trigger vesicle fusion in
the absence of membrane depolarization.
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Figure 5.
AngII-induced voltage-independent exocytosis is
associated with an increased leak current. A shows an
example of a chromaffin cell clamped to 80 mV in which AngII (100 nM) produced a spontaneous increase
Cm (top trace) 20 sec after
agonist application. For comparison, Cm
evoked by a voltage step is shown at the start of the trace (indicated
by arrow). Changes in
[Ca2+]i recorded in the same cell are
shown below. AngII-induced voltage-independent exocytosis followed an
increase in [Ca2+]i from 296 to 1064 nM. B, Diary plots of the mean ± SEM
holding current recorded in the presence of AngII (100 nM).
Filled circles show data from 11 cells in which
voltage-independent exocytosis was observed. Open
circles show data from 10 cells in which AngII failed to
stimulate voltage-independent exocytosis. Comparison of the change in
holding current of cells exhibiting voltage-independent exocytosis in
the presence of AngII with those that did not is highly significant
(unpaired Student's t test, p < 0.05).
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To determine the role of Ca2+ released
from internal stores in facilitation of exocytosis, cells were treated
with CPA (3 µM), a blocker of neuronal endoplasmic
Ca2+-ATPases (Sandler and Barbara, 1999).
Before application of AngII, depletion of the stores was ensured and
tested with two to three applications of caffeine (50 mM,
1.5 min). Depletion of internal stores abolished the AngII-induced rise
in basal [Ca2+]i
(122 ± 3% control) and reduced the facilitation of
stimulus-evoked exocytosis (174 ± 17% of control;
n = 3) (Fig. 6, Table 1).
After washout of CPA and store refilling, reapplication of caffeine increased basal
[Ca2+]i to
232 ± 12% of control and potentiated stimulus-evoked
Cm to 226 ± 93% of control
(n = 3) (Fig. 6). An equivalent rise in basal
[Ca2+]i induced by
AngII facilitated Cm by ~500%
(Fig. 4C), suggesting that increased
[Ca2+]i was not
the only mechanism responsible for facilitation of exocytosis after
activation of AngII receptors.
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Figure 6.
Ca2+ release from
internal stores is required for AngII-dependent facilitation of
exocytosis. Traces represent simultaneous measurements
of Cm (top) and
[Ca2+]i (bottom)
recorded in a single cell voltage-clamped to 80 mV. Increases in
[Ca2+]i and exocytosis were evoked
every 25 sec with voltage steps to +20 mV (indicated by
double-headed arrows). Data shown are on an expanded
time scale and show the first 10 sec of recording after each voltage
step. Drugs present during the voltage step are indicated above each
trace. Continuous application of CPA for 18 min
attenuated AngII (100 nM)-dependent increases in
[Ca2+]i and stimulus-evoked
Cm. Before AngII, store depletion
was tested at 5 min intervals by application of caffeine (50 mM, 1.5 min; data not shown). On the far right can be seen
that after washout of CPA for 3 min, stores refilled, and a subsequent
application of caffeine (50 mM) elicited a rise in
[Ca2+]i.
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In addition to generating inositol phosphates, activation of PLC by
AngII generates the second messenger sn-1,2-diacylglycerol (Tuominen et al., 1993). The role of PLC in mediating the effects of
AngII (100 nM) on stimulus-secretion coupling was
examined by treating cells for 7 min with either the PLC inhibitor
U-73122 (1 µM) or its inactive isomer U-73443
(1 µM). In the presence of the active isomer,
both the AngII-induced rise in basal
[Ca2+]i and
facilitation of stimulus-evoked exocytosis were significantly decreased
to 154 ± 44 and 79 ± 18% of control (n = 5), respectively. By contrast, U-73443 had no significant effects on
AngII-induced increases in basal
[Ca2+]i (282 ± 92%) or on stimulus-evoked exocytosis (627 ± 263%;
n = 3) (Table 1). These studies support the need for
generation of a Ca2+-mobilizing second
messenger and activation of PLC in agonist-induced facilitation of exocytosis.
Role of PKC in AngII-dependent facilitation of
stimulus-evoked exocytosis
AngII-stimulated sn-1,2-diacylglycerol production may
facilitate exocytosis through activation of PKC and/or Doc2
-Munc13 interactions (Terbush et al., 1988; Hori et al., 1999). The role of PKC
in mediating the effects of AngII on stimulus-evoked exocytosis was
examined by treating cells with either Calphostin C (100 nM for 7 min) or BIS (500 nM >20 min) before application of AngII. These
two inhibitors were selected because of their different modes of
action. Calphostin C inhibits PKC and possibly other recently
identified signaling molecules by competing with diacylglycerol for the
regulatory C1 binding site (Mellor and Parker, 1998). BIS, on the other
hand, at nanomolar concentrations acts as a highly selective
competitive inhibitor for the ATP-binding site of PKC (Toullec et al.,
1991). We observed that BIS reduced the potentiation of
Cm by AngII from 524 ± 118%
(n = 11) to 215 ± 90% (n = 7)
(Fig.
7A,B,
Table 1), suggesting that PKC is involved in agonist-dependent
facilitation of exocytosis. Consistent with this, Calphostin C was
found to abolish the AngII-induced potentiation of stimulus-evoked
exocytosis (104 ± 36% of control; n = 7) (Fig. 7B, Table 1). In contrast to BIS, Calphostin C also
attenuated the AngII-induced rise in
[Ca2+]i (118 ± 17%). We do not believe that the effects of Calphostin C on
[Ca2+]i were
attributable simply to toxicity, because neither Calphostin C nor BIS
blocked the AngII inhibition of ICa
(Fig. 7C). Instead, Calphostin C may be depleting stores
through an inhibitory effect on diacylglycerol-regulated
Ca2+ entry pathways (Hofmann et al.,
1999).
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Figure 7.
PKC is partially responsible for AngII-dependent
facilitation of exocytosis. A, Left,
Superimposed traces are from a single cell and show the effect of PMA
(50 nM) and AngII (100 nM) on voltage-evoked
Cm. Right, Data from
another cell, in which the PKC inhibitor BIS was applied for 20 min
before addition of PMA (50 nM) and AngII (100 nM). B, Diary plots showing the effect of
AngII (100 nM) on voltage-evoked
Cm recorded in cells treated with BIS for
20 min (filled triangles; n = 5) or Calphostin C (open triangles;
n = 7). Data plotted are the mean ± SEM.
C, Diary plots showing the effect of AngII on
voltage-dependent Ca2+ entry in the same sets of
cells as shown in B. Treatment with the PKC inhibitors
did not affect AngII inhibition of VOCCs but significantly attenuated
the facilitation of stimulus-evoked exocytosis.
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For comparison, in some experiments we used PMA (50 nM) to
directly activate diacylglycerol-regulated proteins. In agreement with
previous reports, PMA facilitated exocytosis to 442 ± 100% of
control (n = 5) but, in contrast to AngII, without
significantly affecting ICa (94 ± 3% control) (Fig. 7A, Table 1). The facilitatory effects
of PMA on Cm were abolished by
Calphostin C (n = 5) and reduced by BIS to 228 ± 46% of control (n = 5) (Fig. 7A).
Collectively, the results from these experiments suggest that AngII
facilitates depolarization-evoked exocytosis through activation of PLC,
raising [Ca2+]i
and activation of PKC.
AT1Rs couple to multiple G-proteins to produce bimodal regulation
of exocytosis
Results from both binding and cloning experiments suggest that
bovine chromaffin cells express only a single type of AngII receptor
with the pharmacological properties of an AT1R (Marley et al., 1989).
In heterologous expression systems, AT1Rs have been found to couple to
multiple G-proteins (Shibata et al., 1996). Thus in our final set of
experiments we wished to determine whether AT1R activation of multiple
G-proteins could produce bimodal regulation of exocytosis. Consistent
with this hypothesis, we observed that all of the effects of AngII on
stimulus-secretion coupling were blocked by the AT1R antagonist
losartan (Fig.
8C-F).
Furthermore, uncoupling of receptors from
Gi/o-proteins with PTX abolished the inhibitory
effects of AngII on ICa and exocytosis
but not the facilitatory effects (Fig. 8). In PTX-treated cells, the
concentration-response curve for AngII modulation of exocytosis became
unimodal, and thus the EC50 for facilitation
could be determined and was found to be 3.4 nM
(compare Figs. 2D and 8B).
Moreover, the maximum facilitation of stimulus-evoked
Cm observed with 100 nM AngII was increased to 785 ± 246%
(n = 7) compared with the 524 ± 118% potentiation observed in untreated cells. These results suggest that
AT1Rs in chromaffin cells are coupled via distinct G-proteins to
multiple signal transduction cascades to produce opposing effects on
ICa, cytosolic
[Ca2+], and exocytosis.
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Figure 8.
AT1Rs activate multiple G-proteins and divergent
transduction pathways to inhibit and facilitate exocytosis.
A, PTX treatment does not block the facilitatory effects
of AngII (100 nM) on stimulus-secretion coupling. Data from
a single cell showing Cm
(top) evoked by voltage steps to +20 mV from a holding
potential of 80 mV given every 25 sec,
[Ca2+]i (middle)
measured immediately before each voltage step, and integrated
Ca2+ influx (bottom) in response to
each stimulus plotted against time. AngII (indicated by
bar) increased basal
[Ca2+]i from 230 to 670 nM
and potentiated Cm but did not inhibit
Ca2+ influx. B,
Concentration-response curve of Cm
potentiation by AngII in cells treated with PTX. Each
point represents the mean ± SEM for the number of
experiments indicated. The solid line drawn through the
data represents the best fit with the Hill equation of pooled data with
an EC50 3.4 nM and Hill coefficient 1.30. C-F, Summary of the pharmacology of the inhibitory and
facilitatory effects of AngII on stimulus-secretion coupling in
chromaffin cells. Data values are the means ± SEM for the number
of cells indicated above each bar. The AT1R antagonist
losartan but not the AT2R antagonist PD123,319 abolished both the
inhibitory and facilitatory effects of AngII; PTX treatment abolished
only the inhibitory effects. C, Drug effects on
voltage-stimulated integrated Ca2+ entry. PTX
abolished ICa inhibition. D,
Drug effects on the inhibitory effects of AngII (1 nM) on
exocytosis. E, Drug effects on AngII-induced (100 nM) changes in [Ca2+]i.
F, Drug effects on AngII (100 nM)-dependent
facilitation of exocytosis.
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DISCUSSION |
The results of this study show that activation of multiple
G-proteins and transduction pathways by a single neuromodulator acting
through one receptor type can produce concentration-dependent, bidirectional regulation of exocytosis. Metabotropic glutamate receptors have also been shown to switch between facilitation and
inhibition of synaptic transmission; however, in this case coupling of
the receptor to Gi/o- and
Gq-proteins is regulated by desensitization
(Rodriguez-Moreno et al., 1998). This does not appear to be the
mechanism underlying AngII bimodal regulation of secretion, because the
inhibitory effects of the Gi/o-protein-coupled receptor on VOCCs and the facilitatory effects on exocytosis mediated by the Gq-protein-coupled receptor could be seen
simultaneously with high concentrations of agonist. Thus it would
appear that coupling of a single type of receptor to different signal
transduction pathways not only serves to coordinate short-term and
long-term changes in neuronal function, but may also allow neurons to
adapt their secretory output in response to fluctuating levels of agonist.
Transduction pathway mediating AT1R inhibition of
depolarization-dependent exocytosis
Inhibition of transmitter release by other G-protein-coupled
receptors is generally thought to involve either changes in membrane excitability and Ca2+ signaling or a
direct effect on some component of the release machinery (Hille, 1994;
Wu and Saggau, 1997; Miller, 1998). In this study we showed that low
concentrations of AngII induced a parallel inhibition of
ICa and exocytosis. The inhibition of ICa displayed the characteristic
voltage sensitivity commonly associated with GPCR inhibition of
neuronal VOCCs by a membrane-delimited pathway involving
G-subunits (Dolphin, 1998). PTX abolished the inhibition of
ICa and exocytosis by AngII without
affecting AT1R coupling to PLC, Ca2+
mobilization, and facilitation of exocytosis. Inhibition of PKC, on the
other hand, did not prevent AngII modulation of
ICa. Taken together, the results
suggest that G-subunits from Gi/o-coupled AT1Rs inhibit ICa in chromaffin cells,
but G-subunits associated with
Ca2+-mobilizing AT1Rs do not. Our results
are consistent with the observation that -subunits associated
with non-PTX-sensitive G-proteins have a low affinity for VOCCs (Garcia
et al., 1998). Further restraints on the transduction pathway involved
in inhibition of stimulus-evoked exocytosis may result from
compartmentalization of the Gi/o- and
Gq/11-coupled AT1Rs to different poles of the cell, with only the former being in a position to regulate VOCCs through the membrane-delimited pathway. GPCRs and voltage-dependent evoked rises in
[Ca2+]i have
indeed been shown to occur in discreet areas of the cell (Robinson et
al., 1996).
There are two Ca2+-sensitive processes
that contribute to exocytosis and could therefore be affected by
inhibition of ICa: a high-affinity
step that regulates the number of vesicles in the RRP and a
low-affinity step that controls vesicle fusion (Neher, 1998). The size
of the RRP is directly correlated to
[Ca2+]i (Heinemann
et al., 1993), whereas depolarization-evoked vesicle fusion is
determined by the integrated Ca2+ entry
through VOCCs (Engisch and Nowycky, 1996; Seward and Nowycky, 1996). At
low concentrations, AngII decreased Ca2+
entry through VOCCs and exocytosis without significantly affecting the
resting [Ca2+]i.
Therefore, inhibition of vesicle fusion rather than the filling state
of the RRP is the most likely mechanism underlying inhibition of
secretion. The fusion machinery itself appeared unaffected by activated
Gi/o-proteins because exocytotic efficiency was
unaffected by low concentrations of AngII. This is consistent with our
previous studies in chromaffin cells on another
Gi/o-protein-coupled receptor and with studies on
a central glutamatergic synapse and support the hypothesis that
depression of exocytosis observed after activation of PTX-sensitive
G-proteins is fully accounted for by inhibition of
Ca2+ influx through VOCCs (Takahashi et
al., 1996; Powell et al., 2000).
Mechanism of exocytotic facilitation by AngII
At concentrations of 10 nM or higher, the inhibitory
effect of AngII on secretion was overcome, and exocytosis was
facilitated. Potentiation of secretion involved activation of PLC,
Ca2+ mobilization, and PKC and was
independent of the inhibitory pathway. It is well known that
GPCR-regulated PLC-(1-4) isoenzymes may be activated by
-subunits of Gq-proteins or with less potency by G-subunits of numerous G-proteins, including
Gi/o (Morris and Scarlata, 1997). The
transduction pathway involved in facilitation of exocytosis did not
involve the Gi/o-coupled AT1R because PTX had no
significant effects on AngII-induced Ca2+
mobilization, nor did it attenuate potentiation of exocytosis. Therefore, the AngII receptors mediating facilitation likely correspond to the Gq-protein-coupled AT1Rs described
previously (Plevin and Boarder, 1988).
The mechanism underlying agonist-dependent facilitation of exocytosis
was found to be dependent on a rise in
[Ca2+]i. Results
from this and other studies showed that the rise in [Ca2+]i was
attributable to release from intracellular stores and influx across the
plasma membrane (Cheek et al., 1993a). Elevated
[Ca2+]i has been
shown to enhance Cm by increasing
the RRP (von Ruden and Neher, 1993; Smith et al., 1998). Interestingly,
in this study we found that caffeine-induced rises in
[Ca2+]i were not
as effective as AngII in potentiating stimulus-evoked exocytosis. The discrepancy may arise from differences in the location
of the two Ca2+ signals with regard to the
secretory apparatus because the agonist would preferentially activate
IP3-sensitive stores whereas caffeine activates
ryanodine-sensitive stores (Berridge, 1998). Chromaffin cells, like
neurons, are known to possess independent
IP3-sensitive and caffeine-sensitive stores that
are localized to different compartments of the cell (Cheek et al.,
1991, 1993a,b; Koizumi et al., 1999).
Activation of PLC by AT1R will also lead to production of
diacylglycerol, which regulates at least two families of proteins known
to modulate exocytosis directly, namely PKC and Munc-13 (Hori et al.,
1999), as well as noncapacitative Ca2+
entry pathways (Hofmann et al., 1999). Evidence in support of a role
for PKC in AngII facilitation of stimulus-evoked exocytosis was shown
by the use of two different inhibitors. The PKC-dependent facilitation
of exocytosis appeared to require a rise in
Ca2+ because it was not observed in
CPA-treated cells. This is consistent with the known properties of
conventional PKC isoforms, which require both diacylglycerol and
Ca2+ for activation (Newton and Johnson,
1998). Note, however, that at concentrations of BIS that are reported
to be selective for PKC, AngII-induced facilitation was not abolished,
indicating that Ca2+-dependent proteins
other than PKC are also involved. Activity-dependent potentiation of
secretion is also reported to be mediated by
Ca2+ and PKC and to be quantitatively
comparable to the potentiation observed with PMA (Smith et al., 1998).
We found that PMA was less effective than AngII in facilitating
exocytosis, suggesting that different or additional effectors are
involved in agonist- versus activity-dependent facilitation.
We can conclude from these studies that activation of
Ca2+-mobilizing AT1Rs will facilitate
exocytosis through both Ca2+- and
PKC-dependent mechanisms. At present the molecular targets in the
secretory pathway that are subject to modulation are unknown. However,
considering what is known about compartmentalization of signaling
molecules and the processes underlying stimulus-evoked exocytosis,
several possibilities arise. Ca2+ released
from IP3-sensitive stores may act locally to
produce actin disassembly through activation of
Ca2+-sensitive actin-severing proteins and
thereby promote vesicle recruitment from the reserve pool to the
RRP (Zhang et al., 1995). Additionally, store-released
Ca2+ may diffuse toward the plasma
membrane and sum with incoming Ca2+ to
promote exocytosis. Activation of Ca2+
entry at the plasma membrane may trigger fusion of vesicles docked close to the receptor-operated calcium channels or promote vesicle priming through effects on Ca2+-binding
proteins such as DOC2, Rabphilin, or CAPS (Benfenati et al., 1999;
Elhamdani et al., 1999). Additionally, generation of diacylglycerol at
the plasma membrane may increase vesicle docking and priming by
activation of PKC and (1) phosphorylation of cytoskeletal proteins
controlling vesicle recruitment to the RRP (Vitale et al., 1995) and/or
(2) phosphorylation of proteins that regulate SNARE complex
formation (Turner et al., 1999). Diacylglycerol may also activate
Munc-13 directly to regulate SNARE complex formation (Hori et al.,
1999). Interestingly, a pathway facilitating exocytosis composed of
Gq, PLC-, and UNC-13 has recently been
described in Caenorhabditis elegans (Lackner et al., 1999).
Molecular studies coupled with high-resolution imaging will be needed
to discern which of these mechanisms is involved in AngII-dependent
facilitation and whether the same signaling cascades are activated by
other Ca2+-mobilizing GPCRs.
Physiological significance and implications of bimodal regulation
of secretion
AngII is produced both in the blood and, independently, in the
adrenal (Bottari et al., 1993). Subnanomolar levels of circulating AngII are reached in certain physiological states such as dehydration (Belles et al., 1988). Our results suggest that they could act to
inhibit catecholamine secretion as part of a regulatory feedback loop.
The concentrations of AngII required to increase catecholamine release
(EC50 3.4 nM) are less likely to be
reached in plasma under steady-state physiological conditions but would
be generated locally in the adrenal and contribute to clinical
conditions such as hypertension (Francis, 1988) or responses to severe
hemorrhage (Gupta et al., 1995; Ponchon and Elghozi, 1997).
Coupling of AT1Rs to multiple G-proteins has been reported previously
(Richards et al., 1999) and is relatively common among GPCRs (Gudermann
et al., 1996). Earlier studies have identified and characterized the
selectivity of receptor G-protein-effector coupling or the diversity
of effectors regulated by a single receptor subtype (Hille, 1994;
Delmas et al., 1998). AT1R activation of multiple transduction pathways
is known to be necessary for coordination of short-term and long-term
changes in neuronal function (Richards et al., 1999). In this study we
have shown that one function of AT1R coupling to diverse G-proteins is
to produce bimodal regulation of exocytosis, thereby allowing a
chromaffin cell to adapt rapidly its secretory output to fluctuating
levels of AngII. A similarly complex regulation of hormone release
by AngII has been reported in anterior pituitary cells (Enjalbert et
al., 1986; Crawford et al., 1992) and adrenal glomerulosa cells (Kojima
et al., 1986). Thus the potential for bimodal regulation of secretion
may be a general feature of AT1Rs and possibly other members of the
GPCR superfamily.
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FOOTNOTES |
Received Feb. 3, 2000; revised April 3, 2000; accepted April 11, 2000.
This study was supported by the Wellcome Trust. We gratefully
acknowledge the help of Prof. R. H. Chow and Dr. A. Schulte in
setting up the amperometry experiments and Dr. S. Kasparov for analysis
software, as well as Drs. M. Nowycky, G. Henderson, A. D. Powell,
and J. M. Trifaro for helpful discussion of this manuscript.
Correspondence should be addressed to Elizabeth P. Seward, Department
of Pharmacology, University Walk, Bristol BS3 1TD, U.K. E-mail:
Liz.Seward{at}bristol.ac.uk.
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TOP
ABSTRACT
INTRODUCTION
