Capacitative Ca2+ entry is
involved in cAMP synthesis in mouse parotid acini
Eileen L.
Watson1,2,
Zhiliang
Wu2,
Kerry L.
Jacobson1,
Daniel R.
Storm2,
Jean C.
Singh1, and
Sabrina M.
Ott1
1 Departments of Oral Biology
and 2 Pharmacology, University of
Washington, Seattle, Washington 98195
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ABSTRACT |
Muscarinic
receptor interaction leading to augmentation of
isoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca2+ (28). The
effectiveness of capacitative Ca2+
entry and intracellular Ca2+
release on this response was determined in time course studies by using
three independent tools to manipulate the free intracellular Ca2+ concentration: the muscarinic
agonist carbachol, thapsigargin, and ionomycin. Time course studies
revealed that Ca2+ release from
intracellular stores by carbachol produced an early rapid increase
(0.25-0.5 min) in stimulated cAMP levels, whereas capacitative
Ca2+ entry resulted in a sustained
increase in stimulated cAMP levels that was blocked by
La3+. Capacitative
Ca2+ entry, alone, was involved in
thapsigargin and ionomycin augmentation of stimulated cAMP
accumulation. The inability of phosphodiesterase inhibitors,
3-isobutyl-1-methylxanthine and milrinone, to prevent agonist
augmentation of cAMP levels, as well as the finding that the type VIII
adenylyl cyclase (ACVIII) is expressed in parotid acini, suggests that
capacitative Ca2+ entry augments
stimulated cAMP accumulation, at least in part, via activation of this
adenylyl cyclase isoenzyme.
thapsigargin; carbachol; ionomycin; phosphodiesterase; intracellular calcium ion stores; adenylyl cyclase; adenosine
3',5'-cyclic monophosphate
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INTRODUCTION |
MUSCARINIC RECEPTORS COUPLE to biochemical effector
systems, causing stimulation of phosphoinositide (PI) turnover,
inhibition of adenylyl cyclase (AC), activation of guanylate cyclase,
and regulation of ion channels (19). In addition, these receptors are
also linked to activation of phospholipase
A2 (13) and phospholipase D (12)
and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (28, 30). In the mouse parotid gland, muscarinic
receptor interaction has been shown to augment forskolin and
isoproterenol-stimulated cAMP accumulation by mechanisms involving Ca2+ (28, 30). Because direct
coupling between muscarinic receptors and stimulation of AC was not
observed in membranes from these cells, the response has been
interpreted to be the result of cross talk between the PI and AC
systems. This is supported by data that show that
Ca2+, generated on muscarinic
receptor interaction, stimulates AC (21, 30) and enhances the effects
of forskolin on cyclase activity in isolated membranes (30). Other
studies indicate that the interactions between the cAMP and
Ca2+ pathways in parotid cells are
more complex. In the presence of isoproterenol, muscarinic receptor
interaction leads to biphasic effects on cAMP accumulation, i.e.,
augmentation and inhibition that is dependent on agonist concentration.
These responses are linked to the M3 receptor subtype in parotid acinar
cells (27). Inhibition of stimulated cAMP accumulation appears to
involve the activation of enzymes that degrade cAMP (28).
The goal of the present study was to determine the contribution of
capacitative Ca2+ entry and
Ca2+ release from intracellular
stores in muscarinic augmentation of isoproterenol-stimulated cAMP
accumulation in mouse parotid acini. To explore this relationship,
three independent tools were used to manipulate free intracellular
Ca2+ concentration
([Ca2+]i):
the muscarinic agonist carbachol, the microsomal
Ca2+-ATPase inhibitor
thapsigargin, and the Ca2+
ionophore ionomycin. In parallel time course studies, changes in
capacitative Ca2+ entry and
Ca2+ release were correlated with
changes in cAMP accumulation. Phosphodiesterase (PDE) inhibitors were
utilized to determine the involvement of the enzymes that synthesize
and degrade cAMP. Time course studies reveal that
Ca2+ release from intracellular
stores by carbachol is sufficient to augment stimulated cAMP levels at
early time periods (0.25-0.5 min), whereas capacitative
Ca2+ entry is required for the
sustained increase in stimulated cAMP levels. Capacitative
Ca2+ entry, alone, is involved in
thapsigargin and ionomycin augmentation of stimulated cAMP
accumulation. Failure of PDE inhibitors to reverse agonist augmentation
of stimulated cAMP accumulation and the finding that ACVIII is
expressed in mouse parotid acini suggest that capacitative
Ca2+ entry augments stimulated
cAMP accumulation, at least in part, via activation of this AC
isoenzyme.
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MATERIALS AND METHODS |
Materials were obtained as follows: hyaluronidase, carbachol,
isoproterenol, La3+, bovine serum
albumin (BSA), ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 3-(N-morpholino)propanesulfonic acid (MOPS),
and 3-isobutyl-1-methylxanthine (IBMX) were from Sigma
Chemical (St. Louis, MO); cAMP radioimmunoassay kits were from Incstar
(Stillwater, MN); collagenase type CLS2 was from Worthington (Freehold,
NJ); thapsigargin and ionomycin were from Calbiochem (La Jolla, CA);
acetoxymethyl ester of fura 2 (fura 2-AM) was from Molecular Probes
(Eugene, Oregon); milrinone was from Biomol (Plymouth Meeting, PA); the
vertical gel electrophoresis apparatus, PosiBlot pressure
blotter, and ultraviolet Stratalinker were from Stratagene (La Jolla,
CA); and oligo(dT) cellulose columns were from Pharmacia LKB
Biotechnology (Piscataway, NJ). All other reagents were of analytical
grade or higher.
Preparation of parotid acini. Small
groups of isolated mouse parotid cells (acini) were prepared as
described previously by Watson et al. (28) with modification. Briefly,
parotid glands from male Swiss Webster mice (27-30 g) were removed
quickly, trimmed, and minced in a siliconized dish in Krebs-Henseleit
bicarbonate solution (KHB), pH 7.4, containing 0.9 mM
Mg2+ and 1.28 mM
Ca2+, 30 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, 90 U/ml collagenase (CLS2), and 1 mg/ml hyaluronidase. Enzyme
digestion was conducted in a rotary water bath at 37°C for 60 min
under continuous 5% CO2-95%
O2 gassing. After the first 40 min
of digestion, the suspension was pipetted up and down 12 times with a
10-ml plastic pipette. This was repeated two more times at ~5-min
intervals. The pH during the dispersion was maintained at 7.2-7.4.
After digestion, the cells were centrifuged at 50 g for 2 min, washed with buffer (KHB
minus enzymes with 4% BSA, pH 7.4), filtered through two layers of
nylon, and washed two additional times. Cells were suspended in KHB
minus enzyme buffer containing 1% BSA and were rested for 30 min at
37°C with continuous gassing.
Cyclic nucleotide measurements. cAMP
levels were measured in intact mouse parotid acini suspended 1:300
(wt/vol) in KHB buffer, pH 7.4, containing 0.1% BSA as described
previously (30). For experiments in which
La3+
(LaCl3) was used, a phosphate
and bicarbonate-free buffer was used. Cell suspensions (1,500 µl)
were incubated with agonists for varying times up to 5 min. Incubations
were terminated by addition of an equal volume of ice-cold 10%
trichloroacetic acid. cAMP was determined by the radioimmunoassay
procedure of Steiner et al. (24). Results were calculated as picomoles
of cAMP per milligram of protein. Protein was determined by the method
of Lowry et al. (17).
Measurement of
[Ca2+]i
in intact cells.
Acini were suspended 1:50 (wt/vol) in KHB buffer containing 0.176 mg/ml
ascorbic acid and 0.1% BSA, pH 7.4, and were loaded with fura 2-AM at
3.3 µg/ml cell suspension for 30 min at 37°C with continuous
gassing (95% O2-5%
CO2) and shaking. Fura 2-AM was
prepared at 1 mg/ml in dimethyl sulfoxide just before use. Loaded cells
were washed three times in the 0.1% BSA/KHB buffer containing ascorbic
acid, resuspended at 1:50 (wt/vol), and maintained at 24°C with
gassing and shaking. After a 20-min incubation period, an aliquot was
washed twice in the above buffer with and without Ca2+ (diluted 1:10) and placed in
ultraviolet grade fluorometric cuvettes (Spectrocel) for
[Ca2+]i
measurements. For experiments in which
LaCl3 was used, a phosphate- and
bicarbonate-free buffer (3) was prepared. A 2-min temperature equilibration period was observed before commencing the experiment. Cells were used within a 2.5-h period.
[Ca2+]i
was calculated using the equation of Grynkiewicz et al. (11), where
dissociation constant
(Kd) = 224 nM. A Filterscan spectrofluorometer system equipped with a
magnetic stirrer and constant temperature cuvette holder from Photon
Technology International (S. Brunswick, NJ) was used for the
[Ca2+]i
measurements.
Northern analysis. Total RNA was
isolated from frozen parotid tissues by the acid guanidinium
thiocyanate-phenol-chloroform extraction method (7).
Poly(A)+-selected RNA was isolated
from total RNA using type III oligo(dT) cellulose (Collaborative
Research, Bedford, MA) and was analyzed on a formaldehyde-1.2% agarose
gel in MOPS buffer (1). mRNA was transferred onto Nytran membranes
(Schleicher & Schuell, Keene, NH) in 10× saline sodium citrate
(SSC) for 16-20 h, cross-linked at 80°C for 1 h, and
prehybridized at 42°C in a hybridization buffer containing 50%
formamide, 5× SSC, 1× Denhardt's solution, and 250 µg/ml
denatured salmon sperm DNA for 4 h.
[
-32P]dCTP
random-primed cDNA probes generated from the first 771 nucleotides of
the coding region and 329 nucleotides of the 5'-untranslated region of the ACVIII were used for Northern analysis. These probes were
hybridized to the immobilized RNA for 16-20 h at 42°C in the
hybridization buffer. The membrane was washed in 2× SSC/0.5% SDS
for 10 min at room temperature and once for 5 min at 65°C. It was
then washed three times for 20 min in 1× SSC/0.5% SDS at 65°C before autoradiography. Phosphorimager quantitation was
performed by exposing the radioactive blot to a phosphor screen. After
appropriate exposure, the screens were scanned using the phosphorimager
model 400S (Molecular Dynamics, Sunnyvale, CA).
Data analysis. Data are presented as
means ± SE. Statistical analysis was performed using a paired
Student's t-test.
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RESULTS |
Relationship between muscarinic augmentation of
isoproterenol-stimulated cAMP accumulation and
[Ca2+]i.
In previous studies, we found that muscarinic receptor interaction
produced a twofold increase in cAMP levels in the presence of the PDE
inhibitor IBMX (30). Data also showed that
Ca2+ stimulation of cAMP levels is
evident without PDE inhibition, provided that the enzyme is stimulated
with another effector (30). In the presence of isoproterenol, carbachol
produced a biphasic effect on cAMP accumulation, i.e., augmentation at
low concentrations of carbachol (0.1-10 µM) and a decrease in
cAMP levels at carbachol concentrations >10 µM; the former was
reported to involve Ca2+ and the
latter was found to be independent of
Ca2+ and due to activation of a
PDE isoenzyme(s) (28).
To examine the contribution of
Ca2+ entry and
Ca2+ release from intracellular
stores in muscarinic augmentation of stimulated cAMP accumulation, the
time course of effects of carbachol (10 µM) on isoproterenol (0.1 µM)-stimulated cAMP levels was examined. The rationale for conducting
time course experiments was based on previous studies that were
designed to determine the mechanism involved in carbachol inhibition of
cAMP accumulation. In these time course studies, two phases of cAMP
accumulation were revealed: an early rapid (0.25-0.5 min) increase
in cAMP that was subsequently followed by inhibition of stimulated cAMP
accumulation (28). As shown in Fig.
1A,
under conditions where carbachol augmented stimulated cAMP
accumulation, an early rapid increase (0.25-0.5 min) in cAMP
levels was also noted. This was followed by a sustained increase in
cAMP levels measured over a 5-min period. Removal of extracellular
Ca2+ did not decrease the early
rapid rise in cAMP levels but resulted in a reduction of the sustained
phase of cAMP accumulation to levels produced by isoproterenol alone
(Fig. 1B). Pretreatment of acini
with 2 µM thapsigargin for 10 min before the addition of carbachol
plus isoproterenol significantly reduced the early rapid phase of cAMP
accumulation as described previously (28), suggesting that this phase
was due to the release of Ca2+
from intracellular stores.

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Fig. 1.
Time course of cAMP accumulation in mouse parotid acini stimulated by
isoproterenol (Iso, 0.1 µM) and isoproterenol + carbachol (Carb, 10 µM) in a Ca2+ (1.28 mM)-containing Krebs-Henseleit bicarbonate (KHB) buffer
(A); and a
Ca2+-free KHB buffer containing
0.5 mM EGTA (B). Results are
representative of at least 10 experiments.
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To assess whether capacitative
Ca2+ entry was involved in the
sustained phase of cAMP accumulation, we first determined whether capacitative Ca2+ entry was
operative in mouse parotid acini. Fura 2-AM-loaded acini were suspended
in KHB buffer containing 1.28 mM
Ca2+ for 1 min, followed by the
addition of carbachol (10 µM). Carbachol produced a rapid increase in
[Ca2+]i,
from a resting value of 80 to 300 nM. Addition of 1.5 mM EGTA rapidly
reduced
[Ca2+]i
to resting levels; reintroduction of
Ca2+ caused a rapid rise in
Ca2+ entry that was concentration
dependent (Fig. 2). These data are consistent with capacitative entry as proposed by Putney (23). Because
it was important to examine the role of both
Ca2+ release and capacitative
Ca2+ entry in cAMP accumulation,
further studies were conducted in a
Ca2+-free KHB buffer using the
Ca2+-free/Ca2+
reintroduction protocol commonly used to determine capacitative Ca2+ entry (8). Acini were
suspended in a Ca2+-free KHB
buffer containing 0.5 mM EGTA for 1 min, and, as shown in Fig.
3A,
carbachol (10 µM) produced a rapid increase in
[Ca2+]i,
which returned to basal levels after 3 min. This increase in
[Ca2+]i
represents the emptying of intracellular
Ca2+ pools. Reintroduction of
Ca2+ (1.28 mM) to the buffer
induced a rapid and sustained rise in [Ca2+]i
due to capacitative Ca2+ entry
that was blocked by La3+ (50 µM).

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Fig. 2.
Effects of carbachol on free intracellular
Ca2+ concentration
([Ca2+]i)
in mouse parotid acini in a buffer containing
Ca2+ (1.28 mM) with 1.5 mM EGTA at
180 s; 3.0 mM (a), 1.28 mM
(b), and 0.5 mM
(c)
Ca2+ were added at 300 s. Results
are representative of 4 experiments.
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Fig. 3.
Effects of carbachol on
[Ca2+]i
and stimulated cAMP accumulation in mouse parotid acini in a
Ca2+-free KHB buffer.
A, top: carbachol (10 µM) was added
at 60 s and 1.28 mM Ca2+ was added
at 360 s (trace a). In
trace b,
La3+ (50 µM) was added 2 min
before addition of Ca2+.
A, bottom (control): acini were
incubated with 50 µM
La3+ at 240 s, and 1.28 mM
Ca2+ was added at 360 s.
B: isoproterenol (0.1 µM) + carbachol (10 µM) were added for 1 min in absence of
Ca2+, followed by reintroduction
of 1.28 mM Ca2+
(trace a), no addition of
Ca2+ (trace
b), or reintroduction of 1.28 mM
Ca2+ in presence of
La3+ (50 µM;
trace c). Results are representative
of 3 experiments.
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In parallel cAMP time course experiments (Fig.
3B), the
Ca2+-free/Ca2+
reintroduction protocol was also used to evaluate the role of Ca2+ release and capacitative
Ca2+ entry in carbachol
augmentation of stimulated cAMP accumulation. These experiments allowed
examination of both the early rapid and sustained phases of cAMP
accumulation during the initial rapid and sustained increases in
[Ca2+]i.
In a Ca2+-free KHB buffer
containing 0.5 mM EGTA, the simultaneous addition of carbachol plus
isoproterenol produced an early rapid rise in cAMP (see Fig.
3B). This was followed by a decrease
in cAMP levels over 5 min to levels observed for isoproterenol alone
(see Fig. 1B). As stated above, this
inhibition of cAMP accumulation is due to activation of a PDE isoenzyme
(28). Reintroduction of 1.28 mM
Ca2+ to the incubation buffer at 1 min, however, reversed the inhibition and caused a significant
sustained increase in cAMP accumulation. The sustained increase in cAMP
levels produced by carbachol was blocked by
La3+ (50 µM), confirming that
capacitative Ca2+ entry was
required for this phase of cAMP accumulation.
Relationship between thapsigargin augmentation of
isoproterenol-stimulated cAMP accumulation and
[Ca2+]i.
Thapsigargin was considered a useful tool for examining the
relationship between Ca2+ entry
and release on stimulated cAMP accumulation because it depletes
intracellular Ca2+ pools
independently of receptor activation and PI production (26). Any
potential effects of protein kinase C (PKC) and 
-subunits of G
proteins would thus be eliminated. Time course studies, presented in
Fig. 4, show that thapsigargin (2 µM)
mimicked the effects of carbachol on the sustained phase of cAMP
accumulation. The thapsigargin response was slower in onset than that
produced by carbachol, and an early rapid increase (0.25-0.5 min)
in cAMP was not observed.

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Fig. 4.
Time course of cAMP accumulation in mouse parotid acini stimulated by
isoproterenol (0.1 µM) and isoproterenol + thapsigargin (Thaps, 2 µM) in a Ca2+ (1.28 mM)-containing KHB buffer. Results are representative of at least 10 experiments.
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To further assess whether capacitative
Ca2+ entry was involved in
thapsigargin augmentation of the sustained phase of cAMP accumulation, the effects of thapsigargin on
[Ca2+]i
were compared with effects on cAMP accumulation using the
Ca2+-free/Ca2+
reintroduction method. As shown in Fig.
5A, in a
Ca2+-free KHB buffer containing
0.5 mM EGTA, thapsigargin (2 µM) produced a transient increase in
[Ca2+]i,
from 60 to 120 nM, which slowly declined to resting levels after 3 min.
Reintroduction of 1.28 mM Ca2+
caused a dramatic increase in
[Ca2+]i
that was blocked by La3+ (50 µM). In parallel cAMP time course experiments, the simultaneous addition of isoproterenol plus thapsigargin (2 µM) in a
Ca2+-free KHB buffer containing
0.5 mM EGTA did not increase cAMP at any of the time periods examined
(Fig. 5B); cAMP levels were similar
to those observed with isoproterenol alone. On the introduction of 1.28 mM Ca2+ to the incubation buffer
at 1 min, however, there was a significant sustained increase in cAMP
accumulation that was blocked by
La3+ (50 µM).

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Fig. 5.
Effects of thapsigargin on
[Ca2+]i
and stimulated cAMP accumulation in mouse parotid acini in a
Ca2+-free KHB buffer.
A: thapsigargin (2 µM) was added at
60 s and 1.28 mM Ca2+ was added at
480 s (trace a). In
trace b,
La3+ (50 µM) was added 2 min
before addition of 1.28 mM Ca2+.
B: isoproterenol (0.1 µM) + thapsigargin (2 µM) were added for 1 min in absence of
Ca2+, followed by reintroduction
of 1.28 mM Ca2+
(trace a), or no addition of
Ca2+ (trace
b), or reintroduction of 1.28 mM
Ca2+ in presence of
La3+ (50 µM;
trace c). Results are representative
of 3 experiments.
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Relationship between ionomycin augmentation of
isoproterenol-stimulated cAMP accumulation and
[Ca2+]i.
The Ca2+ ionophore ionomycin was
also used to manipulate
[Ca2+]i
and, like thapsigargin, acts independently of receptor activation and
inositol trisphosphate formation. Ionomycin was used at a concentration
that is somewhat selective for intracellular membranes (18). Data
presented in Fig. 6 show that, in a KHB
buffer containing 1.28 mM Ca2+,
ionomycin (0.5 µM) mimicked the effects of carbachol on cAMP accumulation. The ionomycin response was similar in onset to that produced by carbachol.

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Fig. 6.
Time course of cAMP accumulation in mouse parotid acini stimulated by
isoproterenol (0.1 µM) and by isoproterenol + ionomycin (Iono, 0.5 µM) in a Ca2+ (1.28 mM)-containing KHB buffer. Results are representative of 5 experiments.
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The relationships between Ca2+
release, capacitative Ca2+ entry,
and cAMP accumulation are shown in Fig. 7.
In a Ca2+-free KHB buffer
containing 0.5 mM EGTA, ionomcycin, like thapsigargin, produced a
transient increase in
[Ca2+]i,
from 70 to 118 nM, which slowly declined to resting levels (Fig.
7A). Reintroduction of 1.28 mM
Ca2+ at 8 min caused a dramatic
increase in
[Ca2+]i
that was blocked by La3+ (50 µM). In parallel cAMP time course experiments, the addition of
isoproterenol plus ionomycin in a
Ca2+-free KHB buffer containing
0.5 mM EGTA did not increase cAMP at any of the time periods examined
(Fig. 7B); cAMP levels were similar
to those observed with isoproterenol alone. On reintroduction of 1.28 mM Ca2+ at 1 min, however, there
was a significant sustained increase in cAMP accumulation that was
blocked by La3+ (50 µM).

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Fig. 7.
Effects of ionomycin on
[Ca2+]i
and stimulated cAMP accumulation in mouse parotid acini in a
Ca2+-free KHB buffer.
A: ionomycin (0.5 µM) was added at
60 s and 1.28 mM Ca2+ was added at
480 s (trace a). In
trace b,
La3+ (50 µM) was added 2 min
before addition of 1.28 mM Ca2+.
B: isoproterenol (0.1 µM) + ionomycin (0.5 µM) were added for 1 min in absence of
Ca2+, followed by reintroduction
of 1.28 mM Ca2+
(trace a), no addition of
Ca2+ (trace
b), or reintroduction of 1.28 mM
Ca2+ in presence of
La3+ (50 µM;
trace c). Results are representative
of 3 experiments.
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Involvement of enzymes that synthesize and degrade
cAMP in muscarinic augmentation of stimulated cAMP
levels. Because the above augmentation studies were
conducted in the absence of a PDE inhibitor, the steady-state levels of
cAMP measured represent the net effect of changes in the enzymes that
both synthesize and degrade cAMP. Further experiments were designed to
evaluate whether augmentation of stimulated cAMP accumulation results
from inhibition of a PDE isoenzyme or activation of AC isoenzyme(s). For the studies presented, acini were treated with and without the
nonspecific PDE inhibitor IBMX (100 µM) and milrinone (10 µM) for
10 min, in the absence and presence of 1.28 mM
Ca2+, before the addition of
isoproterenol plus carbachol, or isoproterenol plus thapsigargin. The
rationale for using milrinone, a specific inhibitor of the guanosine
3',5'-cyclic monophosphate (cGMP)-inhibited PDE (cGI) (20),
was based on previous findings supporting a role for cGMP in muscarinic
augmentation of forskolin-stimulated cAMP accumulation (30).
It was expected that, if augmentation was due to inhibition of a PDE
isoenzyme, then carbachol would not augment isoproterenol-stimulated cAMP levels in the presence of IBMX. As shown in Fig.
8A, time course studies showed that, in the absence of IBMX and in a
Ca2+-free buffer, carbachol plus
isoproterenol-stimulated cAMP accumulation was less than in
Ca2+-containing KHB buffer, due to
activation of a PDE isoenzyme (see Ref. 28; also see Fig. 1); cAMP
values were 112.0 and 220.6 pmol/mg protein, respectively, at 5 min.
When IBMX (100 µM) was added to the
Ca2+-free KHB buffer, cAMP levels
increased dramatically to 1,089.6 pmol/mg protein at 5 min. Values
obtained with isoproterenol alone in the absence and presence of IBMX
were 141.0 ± 18 and 830.5 ± 33 pmol/mg protein, respectively
(Table 1).

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Fig. 8.
Time course of effects of 3-isobutyl-1-methylxanthine (IBMX; 100 µM)
on carbachol (A) and thapsigargin
(B) augmentation of
isoproterenol-stimulated cAMP accumulation. Acini were preincubated
with the phosphodiesterase inhibitor for 10 min, in absence and
presence of 1.28 mM Ca2+, before
addition of isoproterenol (0.1 µM) + carbachol (10 µM) or
isoproterenol + thapsigargin (2 µM). Results are representative of 5 experiments.
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Table 1.
Effects of the phosphodiesterase inhibitors IBMX and milrinone on
augmentation of stimulated cAMP accumulation in mouse parotid acini
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In a Ca2+-containing buffer
containing IBMX (100 µM), isoproterenol plus carbachol further
increased cAMP levels to 1,726.4 pmol/mg protein. cAMP levels in the
presence of isoproterenol alone were 130.2 ± 17 and 902.9 ± 56 pmol/mg protein, respectively (Table 1). The effects of IBMX on
thapsigargin and ionomycin augmentation of stimulated cAMP levels in
the absence and presence of Ca2+
were similar to those produced by carbachol (Fig.
8B and Table 1).
If cGMP were involved in muscarinic augmentation of stimulated cAMP
levels by activating cGI, then it would be expected that milrinone
would mimic the effects of carbachol in augmenting stimulated cAMP
levels. Incubation of acini for 10 min with milrinone (10 µM) did not
modify isoproterenol-stimulated cAMP accumulation. At 5 min,
isoproterenol-stimulated values were 132.2 ± 18.4 and 129.0 ± 14.5 pmol/mg protein in the absence and presence of milrinone, respectively. Also, as shown in Table 1, preincubation of acini with
milrinone had no effect on carbachol augmentation of
isoproterenol-stimulated cAMP levels in a
Ca2+-free or
Ca2+-containing buffer (Table 1).
Of the three ACs stimulated by
Ca2+ in vitro (2, 5, 25), ACI and
ACVIII have been reported to be exclusively regulated by capacitative
Ca2+ entry (9, 10). It was noted
that in HEK-293 cells transfected with ACVIII, carbachol-induced
Ca2+ release was also found to
stimulate AC (10) in a manner and time frame similar to those observed
in mouse parotid acini. Furthermore, the stimulatory effect on AC was
dependent on emptying of Ca2+
stores, as thapsigargin eliminated the response. Based on these findings, experiments were conducted to determine whether ACVIII is
expressed in mouse parotid glands. In Fig.
9,
poly(A)+-enriched mouse parotid
and neocortex RNA were analyzed by blot hybridization using a cDNA
probe specific for ACVIII. Two distinct type VIII messages of 5.5 and
4.4 kilobases were observed for both tissues.

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Fig. 9.
Northern blot analysis of type VIII adenylyl cyclase mRNA in mouse
parotid gland (P) and neocortex (N). RNA (2 µg) was loaded in each
lane and Northern blot analysis was carried out as described in
MATERIALS AND METHODS. Kb,
kilobase.
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DISCUSSION |
In this study, the time course of stimulated cAMP accumulation was
examined to provide a useful approach for determining the importance of
capacitative Ca2+ entry and
Ca2+ release from intracellular
stores in agonist augmentation of cAMP accumulation in mouse parotid
acini. The important findings of this study were that
1) capacitative
Ca2+ entry is sufficient to
produce the sustained effects of carbachol, thapsigargin, and ionomycin
on stimulated cAMP accumulation; 2) Ca2+ release from intracellular
stores, although insufficient to affect cAMP levels by thapsigargin and
ionomycin, appears to be required for the early rapid phase of
muscarinic augmentation of stimulated cAMP accumulation; and
3)
Ca2+ appears to play a role in
promoting AC synthesis.
An involvement of capacitative
Ca2+ entry in cAMP metabolism has
been reported for C6-2B glioma cells (3), SH-Sy5y human neuroblastoma cells (14), and HEK-293 cells transfected with ACI and
ACVIII (9, 10, 31). In C6-2B glioma cells, thapsigargin, ionomycin, and UTP, agents that affect intracellular
Ca2+ release by different
mechanisms, increased Ca2+ entry,
which was associated with inhibition of stimulated cAMP synthesis (3),
whereas, in HEK-293 cells transfected with ACI and ACVIII, capacitative
Ca2+ entry was associated with
augmentation of stimulated cAMP synthesis (9, 10). Capacitative
Ca2+ entry also appears to be
associated with augmentation of isoproterenol-stimulated cAMP synthesis
in mouse parotid acini, as augmentation was observed in the presence of
IBMX at concentrations sufficient to inhibit soluble PDE activity.
Of interest was the finding that, although thapsigargin and/or
ionomycin did not affect stimulated cAMP accumulation in mouse parotid
acini, or cAMP levels in HEK-293 cells transfected with ACI and ACVIII
in a Ca2+-free buffer (10, 31),
carbachol was found to augment stimulated cAMP accumulation at early
time points in mouse parotid acini and in HEK cells expressing ACVIII
(10). In addition, carbachol alone also increased cAMP levels at early
time periods in SH-Sy5y cells under conditions where
Ca2+ influx was eliminated (14).
Time course studies of cAMP accumulation also revealed that the
presence of a PDE inhibitor was required for the detection of
carbachol-stimulated cAMP levels at early time periods in HEK-293 cells
expressing ACVIII and in SH-Sy5y and mouse parotid acini.
Carbachol (1 mM), in the presence of IBMX (1 mM), increased cAMP levels
by ~40% at 0.5 min in SH-Sy5y cells (14) and by 70% at 1 min in
HEK-293 cells incubated with 0.5 mM IBMX. In mouse parotid
acini, in the presence of IBMX (100 µM), carbachol (10 µM)
increased cAMP levels twofold (unpublished observations).
To date, nine different isoforms of adenylate cyclase have been cloned;
their existence suggests that they may be differentially regulated. The
enzymes exhibit type-specific stimulatory and inhibitory regulation by
G protein
- and 
-subunits,
Ca2+, calmodulin, forskolin,
P-site inhibitors, and PKC (15, 22, 37). A number of the members of the
family of ACs can be regulated by alterations in intracellular
Ca2+. Of these, ACI, ACIII, and
ACVIII are stimulated by Ca2+ in
vitro (2, 6, 25). In vivo, ACI and ACVIII are stimulated and ACIII is
inhibited by Ca2+ (2, 5, 33, 35).
Capacitative Ca2+ entry has been
shown to be sufficient for stimulation of ACI and ACVIII (9, 10). Our
present finding of ACVIII expression in mouse parotid acini, combined
with previous findings that Ca2+
stimulates AC and augments the effects of forskolin on cyclase activity
in membrane fractions and intact cells (21, 30), is consistent with
results obtained in HEK-293 cells expressing ACVIII (2, 10).
Furthermore, Ca2+ in combination
with agonists acting via the G protein subunit
s produced a synergistic
stimulation of cAMP accumulation in parotid cells and in HEK cells
expressing ACVIII. Although ACVIII has only been reported to be present
in brain (2), this represents only one study in which a limited number
of tissues, excluding parotid, were examined. Furthermore, recent
studies using multiple primers and both polymerase chain reaction and
Northern blot analysis show that ACVIII is present in non-brain
tissues, including heart and kidney (L. Muglia, personal
communication). Although both ACVIII and ACI can be stimulated by
capacitative Ca2+ entry (9, 10,
31) and ACI can be activated in a similar fashion to ACVIII (5, 33), it
is unlikely that ACI is also expressed in mouse parotid acini and that
it plays a role in cAMP metabolism, since this AC isoform appears to be
specifically expressed in neural tissues (36).
The finding that isoproterenol stimulates cAMP in mouse parotid acini
but has little or no effect on cAMP levels in HEK-293 cells transfected
with either ACI or ACVIII (2, 33) is paradoxical, as
Gs
clearly stimulates AC and
produces a synergistic effect with
Ca2+/calmodulin in isolated
membrane fractions (2). The fact that isoproterenol alone stimulates AC
activity in mouse parotid acini suggests that another AC isoenzyme is
involved in regulated cAMP synthesis. A likely candidate is ACIII, as
this isoenzyme has been shown to be activated by isoproterenol without
increasing intracellular Ca2+ in
HEK-293 cells expressing ACIII (10, 34).
The ability of carbachol, but not thapsigargin and ionomycin, to
augment stimulated cAMP accumulation at early time periods in mouse
parotid acini, SH-Sy5y (14), and HEK-293 cells expressing ACVIII (10)
suggests that the release of
Ca2+ per se from
intracellular stores is insufficient to affect cAMP levels but appears
to be required for this response. This is supported by the present data
as well as data from HEK-293 cells expressing ACVIII (10), showing that
pretreatment of cells with thapsigargin eliminated this effect.
The fact that 
-subunits of G proteins as well as
PKC, generated as a consequence of receptor interaction, have specific
effects on AC isoenzymes (15, 16, 31) suggests that AC isoenzymes other
than ACVIII may contribute to the augmentation effects of carbachol on
stimulated cAMP accumulation at early time periods in a
Ca2+-free buffer. To date, there
is no evidence of 
-subunit activation of AC isoenymes that are
known to be stimulated by Ca2+.
On the other hand, there is support for an involvement of PKC in enzyme
activation. Watson et al. (32) showed that treatment of HEK-293 cells,
overexpressing ACVII, with phorbol ester resulted in a synergistic
increase in
-adrenergic-stimulated cAMP accumulation. This increase
was noted at early time periods and peaked by 4 min. Phorbol esters
have also been found to enhance forskolin responsiveness of ACI in
HEK-293 cells (4). Because we reported previously that PKC can
stimulate AC and augment the effects of isoproterenol in isolated
membrane fractions (29), it is suggested that ACVII or ACI may be
involved in the early rapid phase of stimulated cAMP accumulation. The
observation that increases in intracellular
Ca2+ failed to result in increases
in cAMP levels in ACVII-expressing cells (31) is consistent with data
showing that intracellular Ca2+
release alone is insufficient to activate cAMP in mouse parotid acini.
Activation of the type II isoform by PKC, reported to occur more
gradually in HEK-293 cells with peak activity at 20 min, however, would
not be consistent with the time frame of cAMP accumulation noted herein
for mouse parotid acini. Further studies are required to determine
whether these AC isoenzymes are present in mouse parotid cells.
In summary, by using identical protocols to examine the relationship
between agonist-induced capacitative
Ca2+ entry and augmentation of
stimulated cAMP accumulation, time course studies revealed that the two
events are coincident. Data also suggest that augmentation is due to
increased cAMP synthesis rather than to decreased PDE activity and may
involve multiple AC isoenzymes. The lack of effect of thapsigargin and
ionomycin on cAMP levels in a
Ca2+-free media, despite a
substantial release of intracellular
Ca2+, is consistent with the
hypothesis that Ca2+-stimulated
ACs are compartmentalized in the same domain as are capacitative
Ca2+ entry channels and thus are
functionally colocalized for cAMP synthesis to be regulated by
Ca2+ (3, 10).
 |
ACKNOWLEDGEMENTS |
We thank Scott Young for helpful comments.
 |
FOOTNOTES |
This work was supported by National Institute of Dental Research Grant
DE-05249.
Address for reprint requests: E. L. Watson, Dept. of Oral Biology. Box
357132, Univ. of Washington, Seattle, WA 98195-7132.
Received 17 December 1996; accepted in final form 15 December
1997.
 |
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