From the
Catalytic subunits of mammalian adenylyl cyclases have been
proposed to contain 12 transmembrane domains, a property shared with
some voltage-sensitive ion channels. Here we report that adenylyl
cyclase activity in cerebellar neurons is synergistically stimulated by
depolarizing agents and
Molecular mechanisms underlying synaptic plasticity have been
intensely studied because of their potential importance for learning
and memory (reviewed in Frank and Greenberg(1994) and Stevens(1994)).
Several regulatory systems have been implicated in neuroplasticity
including the cAMP signal transduction system (Kandel and Schwartz,
1982; Chavez-Noriega and Stevens, 1992, 1994; Chetkovich and Sweatt,
1993; Frey et al., 1993; Weisskopf et al., 1994; Wu et al., 1995). cAMP-dependent protein kinases regulate several
neuronal functions including ion channel activity, neurotransmitter
synthesis, synaptic transmission, and gene expression (reviewed in
Krebs and Beavo(1979), Nestler and Greengard(1983), and Nairn et
al.(1985)). Evidence from invertebrates (Kandel and Schwartz,
1982; Dudai, 1988; Livingston, 1985; Levin et al., 1992) and
mammalian brain (Xia et al., 1991, 1993; Wu et al.,
1995) indicates that adenylyl cyclases may be important for some forms
of synaptic plasticity. For example, transgenic mice lacking the
Ca
Brain adenylyl cyclases are
regulated by neurotransmitter receptors coupled to the enzymes through
the G regulatory proteins, G
On the basis of hydropathy plots, the adenylyl cyclases are proposed
to contain 12 transmembrane sequences and two large cytoplasmic domains
(Krupinski et al., 1989; Gao and Gilman, 1991). Although
topographical similarities between mammalian adenylyl cyclases and
voltage-sensitive ion channels raised the possibility that they may
have ion channel activity, none of these enzymes have been reported to
function as ion channels or membrane transport systems. In this study,
we report that adenylyl cyclase activity in primary cultured neurons is
synergistically stimulated by membrane depolarization and various
activators of adenylyl cyclase including
Because changes in osmolarity have been reported to affect adenylyl
cyclase activity in other types of cells (Watson, 1990), we evaluated
the effect of increased osmolarity on adenylyl cyclase activity in
cultured neurons using 60 or 120 mM sucrose ().
Sucrose at 120 mM did not affect basal adenylyl cyclase
activity or its sensitivity to isoproterenol in the absence of
Ca
Adenylyl cyclases are regulated by a number of
physiologically important messengers, including neurotransmitters,
intracellular Ca
In the
presence of extracellular Ca
Veratridine and
other depolarizing agents have been reported to increase the formation
of cAMP in brain slices by elevation of intracellular Ca
The voltage-sensitive protein subunit of the
adenylyl cyclase system was not identified, although the evidence most
strongly implicated the catalytic subunit. Since cholera toxin
activation was synergistic with depolarizing agents, it seems likely
that either G
The voltage-sensitive adenylyl
cyclase(s) present in neurons were not identified; however, type I
adenylyl cyclase was eliminated from consideration using neurons from
type I adenylyl cyclase mutant mice. Although individual adenylyl
cyclases can be expressed in several cell lines including HEK-293
cells, these non-neuronal cell lines cannot be used to study the
voltage sensitivity of individual adenylyl cyclases because it is not
possible to vary the membrane potential over a range comparable with
that of neurons. Analysis of the amino acid sequences of the cloned
mammalian adenylyl cyclases suggests that they all may contain two
tandem arrays of six transmembrane helices separated by a cytoplasmic
loop (Krupinski et al., 1989). The principle subunits of
voltage-gated ion channels contain four groups of six probable
transmembrane
There are several mechanisms
that may account for the phenomenon described in this study.
Voltage-dependent conformational changes in the catalytic subunit of
the enzyme may enhance stimulation by activated G
It is becoming increasingly evident
that specific adenylyl cyclases in the brain are synergistically
regulated by combinations of signals. For example, type I adenylyl
cyclase is not stimulated by G
To the best of our
knowledge, this is the first report describing a voltage-sensitive
enzyme. The discovery that adenylyl cyclase activity in neurons may be
sensitive to membrane potential is a new regulatory mechanism that has
important implications for neuron function. Depolarization of neuronal
membranes caused by various physiological stimuli coupled with receptor
activation can synergistically stimulate adenylyl cyclase activity and
generate exceptionally high intracellular cAMP. This regulatory
mechanism may be important for some forms of neuroplasticity and other
neuromodulatory events.
We thank Dr. William A. Catterall, Dr. Niel Nathanson,
and Scott Wong for advice and discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptor activation. This
phenomenon is Ca
-independent and not attributable to
Ca
-stimulated adenylyl cyclase activity. Cholera
toxin and forskolin also synergistically stimulate adenylyl cyclase
activity in combination with depolarizing agents. We hypothesize that
conformational changes in the catalytic subunit of the enzymes caused
by changes in the membrane potential may enhance stimulation of
adenylyl cyclases by the guanylyl nucleotide stimulatory protein. This
novel mechanism for regulation of adenylyl cyclases generates robust
cAMP signals that may contribute to various neuromodulatory events
including some forms of neuroplasticity.
-stimulated type I adenylyl cyclase are deficient
in spatial memory and show altered LTP in the CA1 region of the
hippocampus (Wu et al., 1995).
and G
(reviewed in
Ross and Gilman(1980)) and by intracellular Ca
(reviewed in Cheung and Storm (1982)). Clones for eight distinct
adenylyl cyclases have been published (Krupinski et al., 1989,
1992; Feinstein et al., 1991; Bakalyar and Reed, 1990; Gao
& Gilman, 1991; Ishikawa et al., 1992; Katsushika et
al., 1992; Yoshimura and Cooper, 1992; Cali et al.,
1994), all of which are expressed in mammalian brain. Although these
enzymes share sequence homology, they contain hypervariable regions and
exhibit different regulatory properties. For example, type I (Tang et al., 1991; Choi et al., 1992b), type III (Choi et al., 1992a), and type VIII adenylyl cyclases (Cali et
al., 1994) are stimulated by Ca
and calmodulin
whereas type II, IV, V, VI, and VII are not. Synergistic regulation of
adenylyl cyclase activity in neurons by neurotransmitters and
Ca
may play an important role in synaptic plasticity
by coupling of the Ca
and cAMP regulatory systems
(Xia et al., 1991; Choi et al., 1993; Wayman et
al., 1994; Impey et al., 1994; Wu et al., 1995).
-adrenergic agonists.
Primary Neuron Cultures
Rat or mouse pups
(postnatal day 5-8) were used for cerebellar neuron cultures, and
hippocampal neurons were obtained from 2-day-old pups. The cerebellum
or hippocampus was removed and placed into high glucose
Dulbecco's modified Eagle's medium in a 100-mm culture dish
at room temperature. The tissue was transferred to a 15-ml conical tube
containing 5 ml of prewarmed trypsin/EDTA solution (0.25% trypsin and 1
mM EDTA) and incubated at 37 °C for 20 min with agitation.
At the end of incubation, the issue was allowed to settle, the
supernatant was discarded, and the trypsin treatment was repeated. The
trypsinized tissue was then resuspended in 10 ml of Dulbecco's
modified Eagle's medium containing 10% bovine calf serum and 100
units/ml penicillin G and 100 µg/ml streptomycin sulfate. The
tissue was triturated by gentle pipetting. The tissue debris was
allowed to settle, the supernatant containing dissociated neurons was
recovered, and cell viability was examined using trypan blue exclusion.
The cells were plated onto poly-L-lysine-coated 35-mm wells in
a 6-well plate at a density of 6 million cells/well. 2 days after
plating, the growth medium was supplemented with cytosine
-arabinofuroside to a final concentration of 10 µM to
inhibit the growth of non- neuronal cells. On the 8th day after
plating, neurons were treated with various adenylyl cyclase activators
and used for cAMP accumulation assays.
cAMP Accumulation
Changes in intracellular cAMP
levels were measured by determining the ratio of
[H]cAMP to total ATP, ADP, and AMP pool in
[
H]adenine-loaded cells as described by Wong et al.(1991). The growth medium of the cells was supplemented
with [
H]adenine (5 µCi/ml), and cells were
incubated for 2 h. Growth medium was removed, and cells were washed
twice with Krebs-Ringer-Hepes (KRH)
(
)buffer
(128 mM NaCl, 5 mM KCl, 1 mM NaHPO
10 mM glucose, 20 mM Hepes, pH 7.40, 1.2 mM MgSO
, and 2.7 mM CaCl
). The cells
were preincubated with KRH buffer for 30 min, and effectors were added
to KRH buffer and incubated an additional 30 min. When cAMP
accumulations were examined in the absence of Ca
,
CaCl
was omitted in the KRH buffer, and it was supplemented
with 2.0 mM EGTA and 15 uM BAPTA/AM. Veratridine
depolarizations were carried out in Ca
-free buffer in
which the NaCl was increased to 150 mM. Antagonism of
veratridine-stimulated Na
channel activity was
accomplished by pretreatment with 1 µM tetrodotoxin
overnight in culture medium. All cAMP accumulation assays were done in
the presence of 1.0 mM isobutylmethylxanthine, an inhibitor of
cAMP phosphodiesterase activity. After treating neurons with various
effectors, the buffer was removed, and the reaction was stopped by
adding 1 ml of 5% trichloroacetic acid containing 1 µM cAMP. Following 30 min of incubation at room temperature,
acid-soluble nucleotides were separated by ion-exchange chromatography
as described (Salomon et al., 1974).
Ca
Neurons were subcultured onto
poly-L-lysine-coated, 4-chambered NUNC dishes. Within 72 h
after subculturing, cells were rinsed once with KRH or
CaImaging Using
Fura-2
-free KRH buffer and then loaded with 4 µM Fura-2 at 37 °C in the dark. After 40 min of loading with
Fura-2, cells were rinsed twice with KRH or Ca
-free
KRH buffer and allowed to sit for 30 min. Ca
imaging
was carried out in either a Ca
-containing KRH buffer
or a Ca
-free KRH buffer supplemented with 2 mM EGTA and 15 uM BAPTA/AM using a Nikon Diaphote inverted
microscope. The four-chambered coverglass (Lab-Tek, Nunc) was
epi-illuminated through a 20
objective at 340 and 380 nm using a
filter wheel and a 75-W xenon lamp at 25 °C. Emitted fluorescence
was collected by the 20
objective and filtered through a 510-nm
band pass filter. Fluorescence was subsequently magnified with a
2
lens, and an image was obtained with an intensified CCD
camera. The ratios of 340/380 were obtained every 8 s up to 20 min with
no observable photo-bleaching. Control of the camera and filter wheel
and the rate of sampling, data collection, data display, and analysis
was done with the software Image-1/FL (Universal Imaging Corp.).
Synergistic Stimulation of Intracellular cAMP
Levels in Cerebellar Neurons by KCl and Isoproterenol Is
Ca
Our objective was to
determine if adenylyl cyclase activity in neurons is affected by
changes in the membrane potential. This question was initially
addressed by treatment of cultured neurons with KCl, which depolarizes
neuronal membranes (Di Virgilio et al., 1987). Neurons were
chosen for this study because they have excitable membranes that can be
depolarized by several agents. Depolarization of primary cultured
neurons from rat cerebellum with 60 mM KCl in the presence of
2.7 mM extracellular CaCl-independent
caused a 2.0 ±
0.1-fold increase in intracellular cAMP (Fig. 1A),
presumably because of the presence of type I adenylyl cyclase or other
Ca
-sensitive adenylyl cyclases (Xia et al.,
1991; Wu et al., 1995). Isoproterenol stimulated cAMP
accumulation 3.1 ± 0.2-fold, and the combination of
isoproterenol and KCl was synergistic, elevating cAMP 11.2 ±
1.1-fold. Because these experiments were carried out in the presence of
cyclic nucleotide phosphodiesterase inhibitors, the increases in
intracellular cAMP were due to stimulation of adenylyl cyclase activity
rather than inhibition of phosphodiesterases. Nifedipine, an L-type
voltage-sensitive Ca
channel antagonist, completely
inhibited cAMP increases stimulated by KCl alone. However, synergistic
stimulation of adenylyl cyclase activity by KCl and isoproterenol was
not affected by nifedipine, suggesting that activation of
voltage-sensitive Ca
channels may not be required for
this phenomenon and that it may be Ca
-independent (Fig. 1B).
Figure 1:
Effect of KCl and isoproterenol on cAMP
production in primary cultured neurons from rat cerebellum. Rat
cerebellar neurons were isolated and treated with 60 mM KCl,
10 µM isoproterenol (Iso), or both, for 30 min
without (A) or with (B) nifedipine as described under
``Experimental Procedures.'' When present, nifedipine was at
5 µM. Relative cAMP accumulations were determined as
described under ``Experimental Procedures.'' Reported values
are the averages of triplicate determinations ±
S.D.
To address the role of intracellular
Ca for synergistic stimulation of adenylyl cyclase by
KCl and isoproterenol, the experiments described above were repeated
under conditions that inhibited intracellular Ca
increases (no extracellular Ca
, 2.0 mM EGTA, and 15 µM BAPTA/AM). Under
Ca
-free conditions, KCl alone did not stimulate
intracellular cAMP in cerebellar neurons, but isoproterenol did (Fig. 2A). Synergistic stimulation of cAMP formation by
KCl and isoproterenol was consistently seen in eight independent
experiments using different preparations of cerebellar neurons. Under
Ca
-free conditions, isoproterenol stimulation was 3.3
± 0.5-fold, whereas the combination of isoproterenol and 60
mM KCl stimulated 12.1 ± 2.0-fold. Similar results were
obtained with cultured hippocampal neurons; isoproterenol stimulated
6.6 ± 0.3-fold, KCl did not increase cAMP, and the combination
of isoproterenol and 60 mM KCl stimulated cAMP accumulation
18.1 ± 0.6-fold (Fig. 2B). Substitution of 60
mM KCl with 60 mM of NaCl (total NaCl increased from
128 mM to 188 mM) did not increase isoproterenol
stimulation of cAMP levels.
Figure 2:
Effect
of KCl and isoproterenol on cAMP production in primary cultured neurons
from rat cerebellum and hippocampus in the absence of
Ca. Rat cerebellar (A) or hippocampal (B) neurons were isolated and treated with 60 mM KCl,
10 µM isoproterenol, or both, KCl for 30 min under
Ca
-free conditions as described under
``Experimental Procedures.'' Increases in intracellular
Ca
were inhibited by treatment of neurons in
Ca
-free KRH buffer containing 2.0 mM EGTA
and 15 µM BAPTA/AM. Intracellular cAMP was determined as
described under ``Experimental Procedures.'' Reported values
are the averages of triplicate determinations ±
S.D.
To verify that KCl did not actually
increase intracellular Ca under
Ca
-free conditions, cultured cerebellar neurons were
treated with 60 mM KCl in the presence or the absence of
extracellular Ca
and imaged for changes in
intracellular Ca
using Fura-2 (Fig. 3). In the
presence of extracellular Ca
, 60 mM KCl and
10 µM isoproterenol caused a significant increase in
intracellular Ca
that persisted for greater than 15
min. Under Ca
-free conditions (no extracellular
Ca
, 2.0 mM EGTA, and 15 µM
BAPTA/AM), no increase in intracellular Ca
was
detectable when neurons were treated with 60 mM KCl and 10
µM isoproterenol. These data strongly support the
conclusion that synergistic stimulation of adenylyl cyclase activity by
KCl and isoproterenol was not dependent upon increases in intracellular
Ca
. Under Ca
-free conditions, KCl
or combinations of KCl and isoproterenol did not increase intracellular
Ca
.
Figure 3:
KCl
depolarization of cultured neurons does not in-crease intracellular
Ca under Ca
-free conditions. Rat
cerebellar neurons were isolated and treated with 60 mM KCl
and 10 µM isoproterenol in the presence or the absence of
Ca
as described under ``Experimental
Procedures.'' Relative increases in intracellular Ca
(relative fluorescence ratio, 340/380) were monitored using
Fura-2-loaded cells.
The synergism between isoproterenol and KCl
was dependent upon the concentrations of both reagents (Fig. 4).
In the presence of 60 mM KCl, half-maximal stimulation of
intracellular cAMP occurred between 0.1 and 1.0 µM isoproterenol and was completely blocked by the -adrenergic
antagonist propranolol. KCl as low as 15 mM significantly
enhanced isoproterenol stimulation of cAMP accumulation.
K
SO
, a depolarizing agent that does not cause
cell swelling, also synergistically increased intracellular cAMP with
isoproterenol (Fig. 4C). These data indicate that
adenylyl cyclase activity in cultured neurons is synergistically
activated by stimulation of
-adrenergic receptors and KCl without
increases in intracellular Ca
.
Figure 4:
Isoproterenol, KCl, and
KSO
concentration dependence for synergistic
stimulation of adenylyl cyclase in cerebellar neurons. Rat cerebellar
neurons were isolated and treated with 60 mM KCl and varying
concentrations of isoproterenol (A), 10 µM
isoproterenol and varying concentrations of KCl (B), or 10
µM isoproterenol and varying concentrations of
K
SO
(C) for 30 min under
Ca
-free conditions, and intracellular cAMP was
determined as described under ``Experimental Procedures.''
When present, propranolol was at 10 µM. Data are the
averages of triplicate determinations ±
S.D.
Increases in cAMP
stimulated by KCl plus isoproterenol were reversibly dependent upon the
presence of KCl (). When neurons were pretreated with KCl
for 30 min, washed, and then assayed for cAMP accumulation in the
presence of isoproterenol, cAMP levels were identical to those seen
without pretreatment with KCl. Furthermore, isoproterenol stimulation
of adenylyl cyclase activity in membranes prepared from neurons was not
increased when the neurons were pretreated with 60 mM KCl
prior to membrane isolation (data not shown). Changes in the adenylyl
cyclase system caused by depolarization or isoproterenol alone were
readily reversible, and synergistic stimulation required the
simultaneous presence of both agents. Apparently, membrane
depolarization does not cause stable covalent modifications of the
enzyme that enhance stimulation by -adrenergic receptors.
. Similar results were obtained with 120 mM glucose when NaCl in the growth media was increased by 60 mM or when the growth media were diluted to decrease osmolarity (data
not shown). Furthermore, K
SO
, a depolarizing
agent that does not cause cell swelling, also gave synergistic
stimulation of cAMP levels when applied with isoproterenol (Fig. 4C). Therefore, it is unlikely that changes in
osmolarity or cell swelling contributed to the changes in adenylyl
cyclase activity caused by combinations of KCl and isoproterenol.
Synergistic Stimulation of Intracellular cAMP Levels in
Cerebellar Neurons by KCl and Isoproterenol Is Not Due to Type I
Adenylyl Cyclase
The major Ca-stimulated
adenylyl cyclase in rat and mouse cerebellum is type I adenylyl cyclase
(Xia et al., 1991; Wu et al., 1995). Because type I
adenylyl cyclase responds synergistically to combinations of activators
(Wayman et al., 1994) and is one of the major forms of
adenylyl cyclase present in cerebellar neurons, the increases in
intracellular cAMP caused by KCl and
-adrenergic receptor
stimulation might be due to this enzyme. Recently, we disrupted the
gene for type I adenylyl cyclase in mice and reported that
Ca
-stimulated adenylyl cyclase activity in the
cerebellar neurons from the mutant mice is significantly reduced (Wu et al., 1995). Cultured cerebellar neurons from wild type and
mutant mice lacking type I adenylyl cyclase were analyzed for
sensitivity to KCl and isoproterenol. In the presence of
Ca
, cerebellar neurons from wild type and type I
adenylyl cyclase mutant mice showed synergistic stimulation of adenylyl
cyclase activity by KCl and isoproterenol, but neurons from mutant mice
showed little cAMP increase in response to KCl alone (Fig. 5A). Isoproterenol-stimulated cAMP increases were
not depressed in type I adenylyl cyclase mutant neurons because type I
adenylyl cyclase is not stimulated by G
-coupled receptors in vivo (Wayman et al., 1994). In the absence of
Ca
, KCl and isoproterenol synergistically stimulated
cAMP in neurons from mutant mice, indicating that type I adenylyl
cyclase did not contribute to this process (Fig. 5B).
Figure 5:
KCl and
isoproterenol synergistically stimulate cAMP in neurons from type I
adenylyl cyclase mutant mice. Cerebellar neurons from wild type and
mutant mice lacking type I adenylyl cyclase were treated with 60
mM KCl, 10 uM isoproterenol, or both, for 30 min in
the presence (A) or the absence (B) of Ca as described under ``Experimental Procedures.'' Data
are the averages of triplicate determinations ±
S.D.
Adenylyl Cyclase Activity in Cultured Neurons Is
Synergistically Stimulated by Veratridine and Isoproterenol
The
data described above strongly suggested that one or more adenylyl
cyclases in cerebellar neurons may be sensitive to the membrane
potential and that the phenomenon is Ca-independent.
We hypothesize that membrane depolarization may cause conformational
changes in the adenylyl cyclase system that enhance stimulation by
activated G
or other effectors. If this hypothesis is
valid, treatment of neurons with other depolarizing agents and
isoproterenol in the absence of Ca
should
synergistically elevate cAMP. Veratridine is a sodium channel agonist
that depolarizes neuronal membranes in the presence of extracellular
Na
by promoting the influx of Na
(Catterall, 1974). In the absence of Ca
,
veratridine at concentrations up to 200 µM had no effect
on intracellular cAMP in cerebellar neurons (Fig. 6A).
Combinations of veratridine and isoproterenol, however, synergistically
stimulated intracellular cAMP. Half-maximal stimulation was at 100
µM veratridine, consistent with the K
of this drug for Na
channels (Catterall,
1974). Stimulation of intracellular cAMP by veratridine and
isoproterenol was partially inhibited by tetrodotoxin, a sodium channel
antagonist (Narahashi et al., 1964).
Figure 6:
Depolarization of membranes with
veratridine or TEA/4AP synergistically stimulates adenylyl cyclase
activity in cerebellar neurons. A, stimulation of cAMP
accumulation in cerebellar neurons in the presence of increasing
concentrations of veratridine. When present, tetrodotoxin was at 1.0
µM. B, stimulation of cAMP accumulations by
combinations of isoproterenol and depolarization using TEA/4AP. When
present, KCl was at 60 mM, TEA was at 10 mM, 4AP was
at 10 mM, and isoproterenol was at 10 µM.
Intracellular cAMP was determined as described under
``Experimental Procedures.'' Data are the averages of
triplicate determinations ± S.D.
Excitable cells can
also be depolarized using tetraethylammonium (TEA) and 4-aminopyridine
(4AP), agents that depolarize neurons and prolong the action potential
(Barrett et al., 1988). Like KCl and veratridine, TEA/4AP had
no effect on cAMP in the absence of Ca; however,
synergistic stimulation of cAMP was seen in combination with
isoproterenol (Fig. 6B). Intracellular cAMP levels
stimulated by TEA/4AP plus isoproterenol were comparable with those
caused by KCl and isoproterenol. Thus, three different depolarizing
agents enhanced isoproterenol stimulation of adenylyl cyclase activity
under Ca
-free conditions consistent with the proposal
that the membrane potential may regulate sensitivity of adenylyl
cyclases to
-adrenergic agonists.
KCl Enhances Cholera Toxin- and Forskolin-stimulated
Adenylyl Cyclase Activities
-Adrenergic stimulation of
adenylyl cyclases requires three protein components; the catalytic
subunit, the guanylyl nucleotide stimulatory complex G
, and
the
-adrenergic receptor (May et al., 1985). Each of
these proteins is associated with the cytoplasmic membrane and could,
in principle, be a voltage-sensitive subunit of the adenylyl cyclase
system. In an attempt to identify the voltage-sensitive component of
the enzymes, we examined the cholera toxin and forskolin sensitivity of
adenylyl cyclase activity with 60 mM KCl. Cholera toxin
stimulates adenylyl cyclases by catalyzing the ADP-ribosylation of the
subunit of G
, thereby inhibiting its intrinsic GTPase
activity (Cassel and Selinger, 1977; Moss and Vaughan, 1977). Treatment
of cerebellar neurons with cholera toxin alone in the absence of
Ca
increased intracellular cAMP 5.0 ± 0.2-fold (Fig. 7A). A combination of cholera toxin treatment and
KCl depolarization enhanced cAMP levels 12.1 ± 0.5-fold. Similar
results were obtained when neurons were treated with cholera toxin and
TEA/4AP (data not shown). These data suggested that stimulation of the
catalytic activity by cholera toxin-activated G
was
sensitive to the membrane potential.
Figure 7:
Cholera toxin or forskolin synergistically
stimulate adenylyl cyclase activity with KCl depolarization. A, cerebellar neurons were pretreated with cholera toxin (1.0
µg/ml) for 24 h and then assayed for cAMP accumulation at varying
concentrations of KCl in Ca-free KRH buffer as
described under ``Experimental Procedures.'' B,
cerebellar neurons were treated with 60 mM KCl with varying
concentrations of forskolin and were assayed for cAMP in free
Ca
-free KRH buffer as described under
``Experimental Procedures.'' Data are the averages of
triplicate determinations ± S.D.
Forskolin activates adenylyl
cyclases by direct interaction with the catalytic subunit, and purified
adenylyl cyclase catalytic subunits are stimulated by forskolin in the
absence of G-coupling proteins or receptors (Seamon and Daly, 1981).
Forskolin at 50 µM increased intracellular cAMP
approximately 10-fold relative to untreated controls (Fig. 7B). In Ca-free buffer,
combinations of forskolin and KCl synergistically stimulated adenylyl
cyclase activities. For example, 50 µM forskolin and 60
mM KCl stimulated cAMP 73.5-fold ± 4.2-fold,
demonstrating that forskolin activation of the enzyme is
voltage-sensitive. The effect of depolarization on forskolin-stimulated
adenylyl cyclase activity strongly suggests that the voltage-sensitive
component of the adenylyl cyclase system is the catalytic subunit.
, and hormones (reviewed in Tang and
Gilman(1992), Choi et al.(1993), and Pieroni et
al.(1993)). Hydropathy analysis of the amino acid sequence of
mammalian adenylyl cyclases indicates that these enzymes have general
structural similarity to voltage-sensitive ion channels. Therefore, it
was of interest to determine whether adenylyl cyclase activity in
cultured neurons is voltage-sensitive. Our data indicate that
cerebellar and hippocampal neurons contain voltage-sensitive adenylyl
cyclase activity that responds synergistically to depolarization and
various effectors including
-adrenergic agonists.
, adenylyl cyclase
activity in cerebellar and hippocampal neurons was stimulated by KCl
depolarization, consistent with the presence of type I adenylyl cyclase
in rat cerebellum and hippocampus (Xia et al., 1991; Wu et
al., 1995) and type VIII adenylyl cyclase in hippocampus (Cali et al., 1994). Furthermore, Ca
-dependent KCl
stimulation of adenylyl cyclase activity in mouse cerebellar neurons
was greatly diminished in neurons from type I adenylyl cyclase mutant
mice. In the absence of Ca
, adenylyl cyclase activity
in neurons was not directly stimulated by depolarizing agents. However,
combinations of depolarizing agents with isoproterenol, cholera toxin,
or forskolin synergistically stimulated adenylyl cyclase in the absence
of increased intracellular Ca
. In addition, neurons
from mutant mice lacking type I adenylyl cyclase also showed
synergistic stimulation of cAMP by KCl and isoproterenol. The fact that
neurons from type I adenylyl cyclase still showed synergistic
stimulation of adenylyl cyclase activity by KCl and isoproterenol
supports the general conclusion that this phenomenon is not due to a
Ca
-stimulated adenylyl cyclase.
and stimulation of neurotransmitter release (Shimizu et
al., 1970). It is unlikely that the phenomenon described in this
study was due to the release of neurotransmitters because
Ca
is generally critical for neurotransmitter
release. If veratridine and KCl stimulated neurotransmitter release
under Ca
-free conditions, one would expect
stimulation of adenylyl cyclases activity by depolarizing agents alone.
KCl, K
SO
, veratridine, or TEA/4AP had no effect
on intracellular cAMP unless paired with other activators of adenylyl
cyclase. There are four possible mechanisms that might explain the
effect of KCl on adenylyl cyclase activity: increases in intracellular
Ca
, cell swelling, specific chemical effects of KCl
on adenylyl cyclases, or changes in membrane depolarization. The
phenomenon was Ca
-independent, and it was not due to
cell swelling. Several distinct depolarizing agents with different
chemical properties synergistically stimulated adenylyl cyclase
activity with isoproterenol. Therefore, the phenomenon described in
this paper is most likely due to synergistic stimulation of adenylyl
cyclase activity by changes in membrane potential coupled with adenylyl
cyclase activators.
and/or the catalytic subunit may be sensitive
to the membrane potential. Although the
subunit of G
is membrane-associated, membrane attachment is through
palmitylation of Cys-3 (Degtyarev et al., 1993; Wedegaertner et al., 1993) and is less likely to be sensitive to the
membrane potential than the catalytic subunit(s). Although forskolin
and
/
synergistically stimulate some adenylyl cyclases, there
is no evidence that membrane depolarization releases
/
. The
fact that depolarizing agents and forskolin synergistically stimulated
adenylyl cyclase activity is consistent with the conclusion that the
catalytic subunit is probably the voltage-sensitive component of the
adenylyl cyclase system in neurons.
-helices surrounding a central pore (reviewed in
Catterall(1994)). The S4 transmembrane domain of voltage-sensitive ion
channels, which is thought be responsible for the voltage sensitivity
of these proteins, has charged amino acids at every third residue.
Adenylyl cyclases do contain charged amino acids within putative
transmembrane domains that may impart voltage sensitivity. For example,
several of the adenylyl cyclases contain charged amino acids within the
1st, 6th, 7th, and 12th transmembrane domains. The membrane topology of
mammalian adenylyl cyclases has not been experimentally defined, and
identification of transmembrane domains and voltage-sensitive elements
will require more extensive analysis.
or
forskolin. It is also possible that conformational changes caused by
membrane depolarization may allow phosphorylation of the enzyme by
specific protein kinases, which increases sensitivity to activated
G
. Although our data does not distinguish between these two
mechanisms, stimulation by combinations of KCl and isoproterenol
required the simultaneous presence of both agents. Pretreatment of
neurons with KCl followed by washout did not increase stimulation by
subsequent addition of isoproterenol. Thus it seems unlikely that
membrane depolarization led to stable covalent modifications of the
catalytic subunit that enhanced stimulation by various effectors.
Alternatively, specific adenylyl cyclases may be in close proximity
with voltage-sensitive proteins that influence their sensitivity to
activated G
. Regardless of the exact mechanism, the data
presented in this study describe a new mechanism for the regulation of
adenylyl cyclase activity in neurons that is distinct from all others
documented in the literature.
-coupled receptors in
vivo unless the enzyme is also activated by Ca
and calmodulin (Wayman et al., 1994). The
/
complex from G proteins stimulates G
-activated type II
adenylyl cyclase and type IV adenylyl cyclase (Tang and Gilman, 1991),
providing another mechanism by which adenylyl cyclases can function as
coincidence detectors (Bourne and Nicoll, 1993). The data in this
report identify another mechanism for cross-talk and integration of
signals by adenylyl cyclases. Prolonged or robust cAMP signals are
particularly important for cAMP-mediated stimulated transcription,
which may be crucial for some forms of synaptic plasticity in
vertebrates, or for positive feedback regulation of Ca
ion channels (Chetkovich et al., 1991; Frey et
al., 1993; Bacskai et al., 1993; Impey et al.,
1994; Weisskopf et al., 1994).
Table: Synergistic stimulation of adenylyl
cyclase activity by KCl and isoproterenol is reversibly dependent upon
KCl depolarization
Table: Increased
osmolarity does not affect adenylyl cyclase activity in primary
cultured neurons from the cerebellum
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.