Nitric oxide acts independently of cGMP to modulate
capacitative Ca2+ entry in mouse
parotid acini
Eileen L.
Watson1,2,
Kerry L.
Jacobson1,
Jean C.
Singh1, and
Sabrina M.
Ott1
Departments of 1 Oral Biology
and 2 Pharmacology, University
of Washington, Seattle, Washington 98195
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ABSTRACT |
Carbachol- and
thapsigargin-induced changes in cGMP accumulation were highly dependent
on extracellular Ca2+ in mouse
parotid acini. Inhibition of nitric oxide synthase (NOS) and soluble
guanylate cyclase (sGC) resulted in complete inhibition of
agonist-induced cGMP levels. NOS inhibitors reduced agonist-induced Ca2+ release and capacitative
Ca2+ entry, whereas the inhibition
of sGC had no effect. The effects of NOS inhibition were not reversed
by 8-bromo-cGMP. The NO donor GEA-3162 increased cGMP levels blocked by
the inhibition of sGC. GEA-3162-induced increases in
Ca2+ release from
ryanodine-sensitive stores and enhanced capacitative Ca2+ entry, both of which were
unaffected by inhibitors of sGC but reduced by NOS
inhibitors. Results support a role for NO, independent of
cGMP, in agonist-mediated Ca2+
release and Ca2+ entry. Data
suggest that agonist-induced Ca2+
influx activates a Ca2+-dependent
NOS, leading to the production of NO and the release of
Ca2+ from ryanodine-sensitive
stores, providing a feedback loop by which store-depleted
Ca2+ channels are activated.
carbachol; thapsigargin; GEA-3162; nitric oxide synthase
inhibitors; calcium ion release
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INTRODUCTION |
INTRACELLULAR CALCIUM PLAYS a fundamental role in
linking receptor stimulation to enzyme secretion in exocrine cells. In
nonexcitable cells, the rapid rise in intracellular
Ca2+ concentration
([Ca2+]i)
is due to the release of Ca2+ from
intracellular stores. This is followed by an influx of
Ca2+ from the extracellular
medium, which lasts for minutes. The depletion of intracellular
Ca2+ pools appears to be
sufficient to induce capacitative
Ca2+ entry (33). An important
question relating to Ca2+
signaling has been how the depletion of intracellular
Ca2+ stores leads to increased
Ca2+ entry. Until recently, the
proposed model has been that capacitative Ca2+ entry is due to the
generation of an intracellular mediator(s). Candidates include tyrosine
kinase or phosphatase (6), Ca2+
influx factor (34), and a GTP-binding protein (4, 12, 47). In addition,
Ca2+ entry has been proposed to be
activated by cGMP in several cell types including pancreatic acinar
cells (2, 17, 30, 31, 48, 49), submandibular cells (49), colonic
epithelial cells (5), pituitary
GH3 cells (43), and NIE-115
neuroblastoma cells (19), but not in other cell types (3, 7). Further, debate as to the role of cGMP in capacitative
Ca2+ entry in the same cell type,
e.g., in pancreatic acinar cells (15), still remains. In cells showing
a positive correlation between cGMP and
Ca2+ entry, the effects of cGMP
appear to occur via a signaling pathway involving NO (5, 10, 17, 19).
The NO produced after activation of a
Ca2+-dependent NOS increases cGMP
via activation of a soluble guanylate cyclase (22). In addition to the
mediator hypothesis, an alternative model, in which
D-myo-inositol
1,4,5-trisphosphate (IP3)
receptors in Ca2+ stores are
coupled to store-operated channels and
Ca2+ release-activated
Ca2+ current, has been proposed
(24).
NO is an important messenger with complex cellular effects. From a
recent review by Clementi (9), it is clear that NO has profound effects
on Ca2+ homeostasis. NO is
involved in the regulation of voltage-dependent Ca2+ channels (29) and
voltage-independent, store-operated
Ca2+ channels (2, 48), modulation
of IP3-induced intracellular Ca2+ release (25),
Ca2+ release from ryanodine stores
(32, 39, 44), regulation of Ca2+
influx (2, 5, 10, 17, 19, 28, 48, 49), and IP3 and cyclic ADP-ribose
generation (45). Many of these actions appear to be mediated via cGMP
through activation of a G-kinase (9) or phosphodiesterase (29).
Recently, NO has been shown to produce effects that are independent of
cGMP, e.g., direct activation of ryanodine receptors (RyRs) via
nitrosylation of regulatory thiols (37, 46).
The goal of the present study was to determine the role of NO in
capacitative Ca2+ entry and the
underlying molecular mechanism(s) involved. To test the relationship
between NO and capacitative Ca2+
entry, we examined the effects of carbachol and the endoplasmic reticulum Ca2+-ATPase inhibitor
thapsigargin on Ca2+
entry in the absence and presence of the nitric oxide synthase (NOS)
inhibitors
NG-nitro-L-arginine
(L-NNA) and 7-nitroindazole
(7-NI) (1). To determine the mechanism of NO, we examined
1) the effects of the soluble
guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione (LY-83583)
(35) and
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (14) as well as the NOS inhibitor 7-NI on agonist- and NO
donor-induced Ca2+ release and
capacitative Ca2+ entry and
2) the effects of NO on
Ca2+ release via
ryanodine-sensitive Ca2+ stores
and on [3H]ryanodine
binding to isolated microsomes. Results suggest that NO, acting
independently of cGMP, is involved in capacitative Ca2+ entry by releasing
Ca2+ from ryanodine-sensitive
Ca2+ stores in mouse parotid cells.
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MATERIALS AND METHODS |
Materials.
Materials were obtained as follows: hyaluronidase, carbachol,
8-bromo-cGMP, BSA, IBMX, dithiothreitol (DTT), and HEPES were from
Sigma (St. Louis, MO); thapsigargin was from Calbiochem (La Jolla, CA);
collagenase type CLS2 was from Worthington (Freehold, NJ); cGMP RIA
kits were from New England Nuclear (Boston, MA); fura 2-AM was from
Molecular Probes (Eugene, OR); ODQ was from Tocris (Ballwin, MO);
LY-83583 was from Biomol Research Laboratories (Plymouth Meeting, PA);
L-NNA and 7-NI were from RBI
(Natick, MA); and 1,2,3,4-oxatriazolium
5-amino-3-(3,4-dichlorophenyl)-chloride (GEA-3162) was from Alexis (San
Diego, CA). 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. (41). 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 HEPES, 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 to 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 containing 1% BSA and rested for 30 min at 37°C with
continuous gassing.
Measurement of
[Ca2+]i in
intact cells.
Acini were suspended 1:50 (wt/vol) in KHB containing 0.176 mg/ml
ascorbic acid and 0.2% BSA, pH 7.4, and loaded with fura 2-AM at 3.3 µg/ml of cell suspension for 30 min at 37°C with continuous gassing (5% CO2-95%
O2) and shaking. Fura 2-AM was
prepared at 1 mg/ml in DMSO just before use. Loaded cells were washed
three times in the 0.2% BSA-KHB 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 or without Ca2+,
diluted 1:10 in fresh buffer, and placed in
ultraviolet-grade fluorometric cuvettes (Spectrocel) for
[Ca2+]i
measurements.
[Ca2+]i
was calculated from the equation of Grynkiewicz et al. (16), where the
dissociation constant (Kd) = 224 mM.
A Filterscan spectrofluorometer system equipped with a magnetic stirrer
and constant-temperature cuvette holder from Photon Technology
International (South Brunswick, NJ) was used for the
[Ca2+]i measurements.
Cyclic nucleotide measurements.
cGMP levels in intact mouse parotid acini suspended at ~1:300
(wt/vol) in KHB, pH 7.4, containing 0.1% BSA were measured as described previously (41). Incubations were terminated by the addition
of an equal volume of ice-cold 10% TCA. cGMP levels were determined by
the RIA procedure of Steiner et al. (36). Samples were acetylated for
cGMP determinations as described by Harper and Brooker (18). Results
were calculated as femtomoles of cGMP per milligram of protein.
Microsomal membrane preparation.
Microsomal membranes were isolated at 4°C from parotid acinar cells
by fractionation of a 10% (wet wt/vol) homogenate of the cells by
using differential centrifugation in isomolar sucrose as described
previously (11). Acini were suspended in a solution containing 250 mM
sucrose, 10 mM HEPES, 2 mM EDTA buffer (pH 7.4), and the following
protease inhibitors: leupeptin (1 µg/ml), pepstatin A (0.7 µg/ml),
and phenylmethylsulfonyl fluoride (PMSF; 0.1 mM) and homogenized by 10 complete passes in a glass mortar with a motor-driven Teflon
pestle. The homogenate was centrifuged for 5 min at 250 g. Homogenization of the pellet was
repeated, and the pooled 250 g
supernatants were centrifuged for 20 min at 10,000 g. The 10,000 g supernatant was centrifuged for 1 h
at 100,000 g, and the resulting
microsomal fraction (pellet) was separated from the soluble fraction
(supernatant), held overnight submerged in suspension buffer (588 mM
sucrose, 50 mM KCl, 20 mM MOPS buffer, pH 6.8, containing 1 µg/ml
leupeptin and 0.7 µg/ml pepstatin A) at 4°C, and resuspended the
next day at a protein concentration of 5-11 mg/ml by gentle
homogenization in the same buffer for immediate use or storage at
80°C.
[3H]ryanodine binding assay.
[3H]ryanodine binding
to mouse parotid acinar cell membranes was performed as described
previously (11) except for the binding temperature and duration.
Briefly, membrane samples were incubated in binding buffer consisting
of 0.5 M KCl, 100 µM CaCl2, 20 mM HEPES (pH 7.4), and protease inhibitors aprotinin (0.5 mg/ml), leupeptin (1 µg/ml), and pepstatin A (0.7 µg/ml) with the protease substrate BSA (100 µg/ml), with or without GEA-3162 for 2 h at 37°C. The IC50 value for
GEA-3162 was obtained from concentration-response experiments assessing
the binding of
[3H]ryanodine added at
a nonsaturating concentration of 6 nM. The assay was terminated by
rapid dilution of the sample with 4 ml of wash buffer containing 0.5 M
KCl, 20 mM HEPES (pH 7.4), and 100 µM
Ca2+ and passage of the sample
through a Whatman GF/F glass fiber filter, followed immediately by
three 4-ml washes of the filter with the same buffer; all procedures
were completed within 1 min. The filters were dried overnight and
placed in vials containing scintillant, and the bound
[3H]ryanodine was
measured by liquid scintillation counting with a Packard Tri-Carb
2200CA analyzer. Specific bound
[3H]ryanodine was
calculated by subtracting nonspecific binding, measured in the presence
of 10 µM unlabeled ryanodine.
Protein was determined by the Hartree (20)-modified method of Lowry et
al. (26).
Data analysis.
cGMP data and
[Ca2+]i
determinations involving NOS inhibitors and GEA-3162 are presented as
means ± SE. Statistical analysis was performed by using Student's
t-test
(P < 0.05).
The IC50 for GEA-3162 inhibition
of [3H]ryanodine
binding was determined by linear analysis of the log-logit
transformation of concentration-response curves. Binding constants
Kd and maximum binding capacity
(Bmax) values for
[3H]ryanodine with and
without GEA-3162 were derived by curvilinear analysis using the
computer program RADLIG, subroutine EBDA (Elsevier-BIOSOFT), and were
depicted graphically by a Rosenthal (Scatchard) plot and linear
analysis. Observed differences in RyR concentration and affinity
constants in parotid membranes treated or not treated with
GEA-3162 were tested for significance
(P < 0.05) by using the computer
program LIGAND and F statistics. Values reported represent the means ± SE of experiments performed in duplicate.
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RESULTS |
Role of Ca2+
release and influx in muscarine- and thapsigargin-induced increases in
cGMP accumulation.
Previous studies have shown that activation of muscarinic receptors
leads to increases in cGMP levels in mouse parotid acini (40, 41).
However, the contribution of Ca2+
influx and release from intracellular stores to cGMP accumulation in
these cells was not examined. As shown in Fig.
1A, carbachol (10 µM)
increased cGMP accumulation significantly in the presence of 1.28 mM
extracellular Ca2+
(trace a). cGMP levels increased
from 1,178 ± 225 to 3,794 ± 290 fmol/mg protein within 0.75 min
and declined slightly by 5 min. In a nominally
Ca2+-free medium, however,
carbachol-induced cGMP accumulation represented only 12% of the cGMP
produced in a Ca2+-replete buffer;
cGMP increased from 695 ± 55 to 1,016 ± 74 fmol/mg protein
within 0.25 min, then declined rapidly to baseline
(trace b). The percent increase in
cGMP accumulation in the absence of extracellular
Ca2+ was only slightly increased
by a higher concentration of carbachol (1 mM), i.e., from 12 to 15%;
IBMX (100 µM) had little effect on carbachol (10 µM)-induced cGMP
levels, i.e., maximum cGMP levels were increased by 6% (data not
shown).

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Fig. 1.
Time course of carbachol (10 µM)-induced changes in cGMP accumulation
(A) and intracellular
Ca2+ concentration
([Ca2+]i)
(B) in mouse parotid acini incubated
in a 1.28 mM Ca2+-containing
buffer (trace a) or a nominally
Ca2+-free Krebs-Henseleit
bicarbonate (KHB) buffer (trace b).
Results are representative of 4 experiments.
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To further assess the role of Ca2+
in cGMP accumulation, we used the
Ca2+-ATPase inhibitor
thapsigargin, which, unlike carbachol, acts independently of the
phospholipase C pathway. Like carbachol, thapsigargin increased cGMP
levels significantly in the presence of 1.28 mM extracellular
Ca2+ (Fig.
2A);
cGMP levels increased by 418% from 1,197 ± 99 to 6,201 ± 434 fmol/mg protein in the presence of 1.28 mM extracellular Ca2+ (trace
a). In a nominally
Ca2+-free medium, cGMP
accumulation increased by 73.2% from 741 ± 54 to 1,284 ± 21 fmol/mg protein and represented 11.8% of the cGMP generated in a
Ca2+-replete buffer
(trace b). Thus, like carbachol,
Ca2+ influx was a major
contributor to cGMP accumulation. The inclusion of IBMX (100 µM) in
the medium had little effect on cGMP levels (data not shown). Unlike
what was found for carbachol, maximum cGMP accumulation, induced by
thapsigargin in the presence of 1.28 mM
Ca2+, was reached at 3 min.

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Fig. 2.
Time course of thapsigargin (2 µM)-induced changes in cGMP
accumulation (A) and
[Ca2+]i
(B) in mouse parotid acini incubated
in a 1.28 mM Ca2+-containing
buffer (trace a) or a nominally
Ca2+-free KHB buffer
(trace b). Results are
representative of 4 experiments.
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Effects of inhibitors of the NO/cGMP pathway on stimulated cGMP
levels and Ca2+.
As shown in Table 1, NOS inhibitors
L-NNA (2 mM) and 7-NI (200 µM)
and soluble guanylate cyclase inhibitors LY-83583 (30 µM) and ODQ
(3-10 µM) completely inhibited carbachol- and
thapsigargin-stimulated cGMP production. In parallel studies, using the
Ca2+-free/Ca2+
reintroduction protocol described previously (42), these inhibitors were employed to determine the role of NO and cGMP in
agonist-induced Ca2+ release and
capacitative Ca2+ entry. For the
NOS inhibitor studies, L-NNA (2 mM) and 7-NI (200 µM) were preincubated with acini for 10 and 2 min,
respectively, before the addition of agonists. In a nominally
Ca2+-free buffer,
L-NNA (2 mM) reduced carbachol-
and thapsigargin-induced Ca2+
release by 31.0 ± 6.4 and 36.0%
(n = 2), respectively
(Fig. 3, A
and B, trace
b). After the reintroduction of 1.28 mM
Ca2+, capacitative
Ca2+ entry was reduced by 39.5 ± 4.8 and 31.7 ± 3.5%, respectively. Trace
c represents the control. Similarly, 7-NI (200 µM)
reduced carbachol- and thapsigargin-induced
Ca2+ release by 42.8 ± 5.7 and
44.3 ± 6.7%, respectively (P < 0.05), and capacitative Ca2+ entry
by 44.2 ± 3.0 and 54.3 ± 4.3%, respectively (P < 0.05) (Fig. 4, A and
B, trace b). Trace c represents the
control.

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Fig. 3.
Effects of nitric oxide synthase (NOS) inhibitor
NG-nitro-L-arginine
(L-NNA) on carbachol-
(A) and thapsigargin
(B)-induced
Ca2+ release and capacitative
Ca2+ entry in mouse parotid acini.
Trace a: carbachol (10 µM) or
thapsigargin (2 µM) was added at 180 s to acini incubated in a
nominally Ca2+-free KHB buffer;
this was followed by reintroduction of 1.28 mM
Ca2+ at 350 s
(A) or 420 s
(B). Trace
b: L-NNA (2 mM)
was added to acini for 10 min before addition of agonist.
Trace c (control): acini were
incubated with L-NNA (2 mM)
alone; this was followed by reintroduction of 1.28 mM
Ca2+. Results are representative
of 4 experiments.
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Fig. 4.
Effect of NOS inhibitor 7-nitroindazole (7-NI) on carbachol-
(A) and thapsigargin
(B)-induced
Ca2+ release and capacitative
Ca2+ entry in mouse parotid acini.
Trace a: carbachol (10 µM) or
thapsigargin (2 µM) was added at 180 s to acini incubated in a
nominally Ca2+-free KHB buffer;
this was followed by reintroduction of 1.28 mM
Ca2+ at 360 s
(A) or 420 s
(B). Trace
b: 7-NI (200 µM) was added to acini 2 min before
addition of agonist. Trace c
(control): acini were incubated with 7-NI (200 µM) alone; this was
followed by reintroduction of 1.28 mM
Ca2+. Results are representative
of 4 experiments.
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To determine whether cGMP could reverse the effects of NOS inhibition,
we examined the effect of 8-bromo-cGMP (0.5 mM) on thapsigargin-induced Ca2+ release
and Ca2+ entry in the presence of
7-NI (200 µM). As shown in Fig.
5A, 8-bromo-cGMP added 10 min before the addition of thapsigargin (2 µM)
did not reverse 7-NI inhibition of either thapsigargin-induced Ca2+ release or capacitative
Ca2+ entry (trace
c). ODQ (3 µM), an inhibitor of NO-stimulated
guanylate cyclase (14), had no effect on thapsigargin-induced responses (Fig. 5B, trace
b). There was little increase in
Ca2+ entry in control acini (data
not shown; refer to Figs. 4, A and B, and 8). LY-83583 was not used in
these studies because it reduced GEA-3162-induced cGMP accumulation by
only 37% (Table 1). The inability of LY-83583 to completely inhibit
GEA-3162-induced cGMP accumulation may be because part of its action is
to block NOS activity (27) as well as that of soluble guanylate
cyclase; blocking NOS activity would not interfere with the actions of NO donors.

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Fig. 5.
Effects of 8-bromo-cGMP on 7-NI inhibition of thapsigargin-induced
Ca2+ release and capacitative
Ca2+ entry
(A), and effects of ODQ on
thapsigargin-induced responses (B)
in mouse parotid acini. A,
trace a: thapsigargin (2 µM) was
added at 180 s to acini incubated in a nominally
Ca2+-free KHB buffer; this was
followed by reintroduction of 1.28 mM
Ca2+ at 400 s.
Trace b: 7-NI (200 µM) was added 2 min before addition of thapsigargin. Trace
c: 8-bromo-cGMP (500 µM) was added 8 min before 7-NI.
B, trace
a: thapsigargin (2 µM) was added at 180 s; this was
followed by reintroduction of 1.28 mM
Ca2+ at 420 s.
Trace b: ODQ (3 µM) was added 10 min
before addition of thapsigargin. Results are representative of 3 experiments.
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Effects of NO donors on cGMP levels and
[Ca2+]i.
To further determine the role of the NO/cGMP pathway in capacitative
Ca2+ entry, we first examined the
ability of exogenous NO, in the form of the NO-releasing compound
GEA-3162, to increase cGMP levels. GEA-3162 has been characterized by
its ability to generate nitrate and nitrite in aqueous solution in a
time-dependent manner and to increase cGMP levels (23). The production
of nitrites and nitrates was much slower with GEA-3162 than with the NO
donor S-nitroso-N-acetylpenicillamine,
which may be due to fast decomposition of GEA-3162 in
aqueous solution (23). An advantage of using GEA-3162 is that, in
contrast to other NO donors, such as
3-morpholinosydnonimine, it produces negligible amounts
of peroxynitrite (ONOO
)
(21), which has cellular effects that previously may have been
attributed to NO.
cGMP accumulation in the presence of GEA-3162 (100 µM) was rapidly
and significantly increased (Fig.
6A), as
was shown previously by Kankaanranta et al. (23); at 0.5 min, cGMP
levels increased from 877 ± 386 to 31,246 ± 410 fmol/mg
protein. These effects were independent of extracellular
[Ca2+]i
(data not shown). By 5 min, cGMP levels were reduced to 11,433 ± 376 fmol/mg protein. ODQ (10 µM) reduced GEA-3162-induced cGMP accumulation by ~90% (Table 1), whereas 7-NI and
L-NNA were without effect (Table
1).

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Fig. 6.
Time course of GEA-3162 on cGMP accumulation
(A) and
[Ca2+]i
(B) in mouse parotid acini. For cGMP
determinations GEA-3162 (100 µM) was added at time
0 to acini incubated in a 1.28 mM
Ca2+-containing KHB buffer. For
the
[Ca2+]i
determinations GEA-3162 (100 µM) was added to acini incubated in a
1.28 mM Ca2+-containing KHB buffer
(trace a) or a nominally
Ca2+-free KHB buffer
(trace b). Trace
c (control): acini incubated without GEA-3162. Results
are representative of 5 experiments.
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In other studies, GEA-3162 was used to assess the effects of NO on
[Ca2+]i.
In contrast to the rapid effects of GEA-3162 on cGMP accumulation, GEA-3162 (100 µM) produced a slow but significant increase in [Ca2+]i in
a 1.28 mM Ca2+-containing KHB
buffer;
[Ca2+]i
increased from 80 to 250 nM over 15 min (Fig.
6B, trace
a), and the increase was similar to the maximal
increases produced by carbachol and thapsigargin (see Figs.
1B and
2B). A slow increase in
[Ca2+]i by
GEA-3162 has been previously reported by Favre et al. (13) for
DDT1MF-2 cells and may be related
to the fact that GEA-3162, although lipophilic, releases nitrates and
nitrites slowly in aqueous solutions (23). The differences noted in the
time required for production of cGMP accumulation and increases of
[Ca2+]i
suggest that the release of low levels of nitrites and nitrates by
GEA-3162 is sufficient to rapidly induce cGMP accumulation, whereas
greater amounts of nitrites and nitrates are required for observing
significant changes in
[Ca2+]i.
Further, data are also consistent with the premise that the effects of
GEA-3162 on
[Ca2+]i
are independent of cGMP.
To determine whether the NO-induced
Ca2+ response was due to
Ca2+ release from intracellular
stores or to Ca2+ influx,
experiments were conducted with a nominally
Ca2+-free buffer. The response to
GEA-3162 in a nominally Ca2+-free
buffer was ~40% of that found in the presence of 1.28 mM Ca2+; the average of four
experiments was 45.6 ± 6.0% (P < 0.05). As shown in Fig. 6B
(trace b),
[Ca2+]i
increased from 50 to 150 nM. Because 7-NI was found to reduce carbachol- and thapsigargin-induced
Ca2+ release, in addition to
capacitative Ca2+ entry, the data
suggested that NOS may be involved in the agonist-induced release of
Ca2+ from intracellular stores.
Therefore, because GEA-3162 released Ca2+ from intracellular stores, we
determined whether 7-NI would also inhibit this response. 7-NI (200 µM) added 2 min before GEA-3162, significantly reduced the effects of
GEA-3162 on Ca2+ release
(P < 0.05; Fig.
7, trace
b). The average of four experiments was 77.2 ± 5.4% compared with 42.8 ± 5.7 and 44.3 ± 6.7% for carbachol and thapsigargin, respectively (Fig. 4,
A and
B, trace
b), confirming an involvement of NOS in this process.

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Fig. 7.
Effects of 7-NI on GEA-3162-induced
Ca2+ release.
Trace a: GEA-3162 (100 µM) was added
at 60 s to acini incubated in a nominally
Ca2+-free KHB buffer.
Trace b: 7-NI (200 µM) was added 2 min before addition of GEA-3162. Trace
c (control): acini incubated with 7-NI (200 µM)
alone. Results are representative of 3 experiments.
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To assess whether GEA-3162-induced
Ca2+ release activates
capacitative Ca2+ entry, acini
were treated with GEA-3162 before the reintroduction of 1.28 mM
Ca2+. As shown in Fig.
8, GEA-3162 produced a gradual increase in [Ca2+]i in
a nominally Ca2+-free KHB buffer
(trace a). The reintroduction of
1.28 mM Ca2+ caused a rapid,
significant increase in
[Ca2+]i
that was followed by a slower, sustained rise in
[Ca2+]i
(trace a); there was little increase
in Ca2+ entry in control acini
(trace c). ODQ (3 µM) had no
effects on GEA-3162-induced Ca2+
release or capacitative Ca2+ entry
(trace b), supporting the premise
that NO acts independently of cGMP to produce these effects.

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Fig. 8.
Effects of GEA-3162 on Ca2+
release and capacitative Ca2+
entry in mouse parotid acini in absence and presence of ODQ. Baseline
was recorded to 60 s. At this time, DMSO (trace
a) or ODQ (3 µM) (trace
b) was added for 10 min (but not recorded), and then
GEA-3162 (100 µM) was added to acini incubated in a nominally
Ca2+-free KHB buffer for a total
of 15 min (5 min of which were not recorded).
Ca2+ (1.28 mM) was reintroduced at
660 s (trace a).
Trace c (control): acini incubated
with ODQ alone. Results are representative of 3 experiments.
|
|
Effects of NO on RyRs.
Because RyRs have been characterized in mouse parotid cells (11) and NO
has been reported to release Ca2+
from ryanodine-sensitive stores (10, 32), we examined the effects of
GEA-3162 on RyRs by determining 1)
the effects of ryanodine on GEA-3162-induced
Ca2+ release and capacitative
Ca2+ entry and
2) the binding of radiolabeled
[3H]ryanodine to
microsomal vesicles. As shown in Fig.
9A
(trace b), incubation of acini for 1 h with 200 µM ryanodine reduced GEA-3162 (100 µM)-induced
Ca2+ release, which was more
easily seen in the reduction in capacitative Ca2+ entry (~27%). This low
degree of inhibition may be related to the difficulty of ryanodine to
penetrate acini or the difficulty in achieving conditions that are used
to favor binding in cell-free systems. Because of the low level of
inhibition in intact cells, further studies were conducted in vitro to
directly determine whether NO interacts with the RyR. As
shown in Fig. 9B, in a cell-free system, GEA-3162 inhibited, in a concentration-dependent manner, [3H]ryanodine binding
to microsomes incubated for 2 h at 37°C; the IC50 for two experiments was 20.7 µM (values were 20.5 and 20.9 µM). The incubation of microsomes at
earlier time periods produced similar results; however, the inhibition
was less pronounced (data not shown). At a concentration shown to
decrease occupancy by 50%, GEA-3162 had no significant effect
(P > 0.05) on
Kd; values were
7.0 ± 0.5 and 9.4 ± 1.2 nM in the absence and presence of GEA-3162, respectively. However,
Bmax was reduced significantly (P < 0.01) by 34% (Fig.
10). Values were 332 ± 11 and 231 ± 24 fmol/mg protein in the absence and presence of GEA-3162,
respectively. As shown in Fig. 9B
(inset), the inhibition of
[3H]ryanodine binding
by GEA-3162 was significantly diminished in the presence of the
sulfhydryl-reducing agent DTT (1 mM).


View larger version (40K):
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|
Fig. 9.
Effect of ryanodine on GEA-3162-induced
Ca2+ release and capacitative
Ca2+ entry in mouse parotid acini
(A) and effects of increasing
concentrations of GEA-3162 on
[3H]ryanodine binding
to acinar cell microsomal membranes
(B).
A: acini were incubated in nominally
Ca2+-free KHB buffer for 1 h
before addition of GEA-3162 (100 µM). Baseline (after 1-h incubation)
was recorded to 60 s; GEA-3162 (100 µM) was added for a total of 15 min (5 min not recorded); Ca2+ was
reintroduced at 660 s (trace a).
Ryanodine (200 µM) was present throughout the 1-h incubation
(trace b). Trace
c (control): acini incubated with ryanodine alone. Data
are representative of 3 experiments.
B: mouse parotid microsomal membranes
(100 µg protein) were incubated for 2 h with 0-3,000 µM
GEA-3162 at 37°C. Inset: acini
were incubated in absence or presence of dithiothreitol (DTT; 1 mM) for
2 min before addition of GEA-3162. See MATERIALS AND
METHODS for further details. Data represent 2 experiments performed in duplicate.
|
|

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|
Fig. 10.
Rosenthal (Scatchard) plot of equilibrium binding of 0.3-30 nM
[3H]ryanodine to
acinar cell microsomal membranes in absence and presence of GEA-3162
(20.7 µM). See MATERIALS AND METHODS
for further details. Values are means ± SE from 4 experiments
performed in duplicate.
Kd, dissociation
constant; Bmax, maximum binding
capacity; r, correlation
coefficient.
|
|
 |
DISCUSSION |
The link that communicates the filling state of intracellular
Ca2+ stores to the plasma membrane
has been the focus of a number of recent studies. Although there is
sufficient data to implicate NO as a key player in the generation of
cGMP in exocrine cells, as well as other cell types (17, 38, 48), and
to support a role for cGMP in capacitative
Ca2+ entry (2, 5, 17, 19, 30, 31,
38, 48, 49), these studies have been challenged by negative findings
from other laboratories (3, 7, 15). If capacitative
Ca2+ entry is mediated by cGMP, it
would be expected that Ca2+
released from intracellular stores would increase cGMP levels, cGMP
analogues would mimic the effects of agonists on
Ca2+ entry, and agonist-mediated
Ca2+ entry would be inhibited by
agents that block cGMP accumulation. In pancreatic acinar cells, Gilon
et al. (15) showed that cGMP was not produced in the absence of
extracellular Ca2+ and concluded
that cGMP could not be a mediator of
Ca2+ entry. In contrast, other
data from pancreatic acinar cells support the view that
Ca2+ released from intracellular
stores is sufficient to activate NOS, leading to increases in cGMP
levels (31, 48).
Data from mouse parotid acini clearly indicate that capacitative
Ca2+ entry plays the major role in
the activation of NOS leading to cGMP accumulation. The location of
NOS, i.e., neuronal NOS (nNOS), identified in another
exocrine cell, i.e., the submandibular cell (49), close to the plasma
membrane where Ca2+ channels
reside supports the greater role of
Ca2+ influx in cGMP accumulation.
Our data also suggest that Ca2+
released from intracellular stores may contribute, at least partially, to the increase in agonist-induced cGMP levels and, under similar conditions used by Gilon et al. (15), in pancreatic acini. However, we
did not find any evidence to support a role for cGMP in capacitative Ca2+ entry; cGMP analogues failed
to increase
[Ca2+]i or
reverse the effects of NOS inhibitors on
Ca2+ entry. We did find, however,
that NOS inhibitors L-NNA and
7-NI blocked, to some extent, agonist-induced
Ca2+ entry in mouse parotid acini,
as they were reported to have blocked entry in pancreatic acini
(15). These inhibitors have been widely used to study the
role of cGMP in various cellular processes including capacitative
Ca2+ entry.
L-NNA is less selective in that
it inhibits more than one NOS isoform. 7-NI was used because of its
specificity for nNOS, which has been reported to be present in plasma
membranes of submandibular acinar cells (46). Because 7-NI is a
selective inhibitor of nNOS, data would suggest that nNOS plays an
important role in capacitative
Ca2+ entry in mouse parotid acini.
LY-83583 was also able to partially block thapsigargin-induced
capacitative Ca2+ entry in Jurkat
T lymphocytes (3), which did not respond to cGMP. The fact that
LY-83583 has been found to inhibit nNOS (27), an isoform shown to be
present in high levels in secretory cells (49), suggests
that NO rather than cGMP is involved in capacitative Ca2+ entry in these cell types.
Similar effects of NO on capacitative Ca2+ entry have also been reported
for endothelial cells (39). This conclusion is further supported by
data from mouse parotid acini showing that
Ca2+ entry, induced in the
presence of the NO donor GEA-3162, is not inhibited by the specific
guanylate cyclase inhibitor ODQ. ODQ has been reported to have no
effects on particulate guanylate or adenylyl cyclases, it does not
interfere with the steps leading to NO synthesis, it does not block
actions of NO that are unrelated to guanylate cyclase activation, and
it is the first inhibitor to act on the NO receptor soluble guanylate
cyclase (14).
Although it is difficult to explain the differences in the effects of
NO and cGMP on capacitative Ca2+
entry in the same cell type, i.e., pancreatic acinar cells, Xu et al.
(49) recently suggested that a contributing factor may be the state of
the cells. Gilon et al. (15) reported only a 1.2- to 1.4-fold
stimulation of cGMP by carbachol and thapsigargin compared with the
higher levels of cGMP reported by Xu et al. (48) and Gukovskaya and
Pandol (17). This may account for the observed differences in
pancreatic cells; however, it is clear that cGMP is not involved in
Ca2+ entry in the mouse parotid
acini even when the degree of increase in cGMP induced by carbachol and
thapsigargin is comparable to that observed in pancreatic cells (49).
One possibility, as suggested by Bischof et al. (7), to explain an
effect of cGMP on capacitative
Ca2+ entry in gastrointestinal
cells, but not in HEK-293 and HEK-293/NOS cells, is that some key
component is missing in parotid cells that is present in colonic and
pancreatic cells. However, it is more likely that the
effects of cGMP on capacitative
Ca2+ influx may be tissue
specific. This would account for differences in the effects of cGMP on
Ca2+ entry observed in parotid vs.
pancreatic acinar cells, as well as differences between different
salivary cells, i.e., parotid and submandibular cells (49). These
differences could be explained on the basis that NO has independent
effects as well as cGMP-dependent effects.
Of particular importance are questions relating to the mechanism(s) by
which NO is involved in capacitative
Ca2+ entry. As discussed above, it
is clear that cGMP is not involved. Data also do not support a direct
role of NO on capacitative Ca2+
entry. The data do suggest, however, that capacitative
Ca2+ entry is primarily
responsible for activation of NOS and that once activated, the NO
produced acts to release Ca2+ from
ryanodine stores, setting up a positive feedback loop by which
store-operated Ca2+ channels are
activated. This conclusion is supported by
1) data showing that in a nominally
Ca2+-free KHB buffer, the NO donor
GEA-3162 releases significant amounts of
Ca2+ from intracellular stores
leading to increases in Ca2+
influx when Ca2+ is reintroduced,
2) studies showing that RyRs are
present in mouse parotid acini (11) and that ryanodine blocks the
effects of NO on Ca2+ release, and
3)
[3H]ryanodine-binding
studies showing that NO directly interacts with the
Ca2+ release protein/ryanodine
receptor in mouse parotid acini. Previous studies have shown that
nitrosothiol formation underlies the direct modifying effects of NO on
a number of channels including the ryanodine channel (37, 46). Further,
Favre et al. (13) reported that NO donor GEA-3162-induced
Ca2+ entry is activated by
S-nitrosylation. Data showing the
reversal of GEA-3162-induced inhibition of
[3H]ryanodine binding
by the sulfhydryl-reducing agent DTT is consistent with
S-nitrosylation of the RyR in mouse
parotid acini. The finding that GEA-3162 alters
Bmax without a change in
Kd is consistent with studies by Stoyanovsky et al. (37), who used NO-related compounds
to activate skeletal RyRs. On the basis of these studies, as well as
our studies, it is suggested that NO produced by GEA-3162 directly
interacts with a site on the Ca2+
release protein/RyR (which activates the channel) and prevents the
binding of ryanodine to this site.
Release of Ca2+ from intracellular
stores by NO is consistent with similar findings reported for other
cell types including endothelial cells (39), rat pancreatic
-cells
(44), and interstitial cells from canine colon (32), and with the
ability of ryanodine to block NO-induced
Ca2+ release (Fig.
9A, trace
b) (32, 44). It is unlikely that NO
releases Ca2+ from
IP3-sensitive
Ca2+ stores, as NO has been
reported to inhibit the IP3
receptor (8).
In addition to NOS activation by capacitative
Ca2+ entry, NOS also appears to be
activated by Ca2+ released from
intracellular stores. The finding that the NOS inhibitor 7-NI reduces
agonist- and GEA-3162-induced Ca2+
release in a nominally Ca2+-free
buffer is consistent with an involvement of NOS in the release process.
Data suggest that NOS activated by intracellularly released Ca2+ produces NO, which causes a
further release of stored Ca2+.
Thus the direct interaction of NO with RyRs may serve as an important
signaling mechanism to modulate rather than to mediate capacitative
Ca2+ entry in mouse parotid acini.
It is clear that the cellular effects of NO are complex and depend on
the cell type and perhaps the levels of NO. As suggested by Clementi
(9), NO appears to be part of an on/off switch mechanism devoted to the
fine tuning of the opening of store-operated
Ca2+ channels.
 |
ACKNOWLEDGEMENTS |
We thank Dennis H. DiJulio for his assistance in data analysis.
 |
FOOTNOTES |
This work was supported by National Institute of Dental Research Grant
DE-05249.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. L. Watson,
Dept. of Oral Biology, Box 357132, Univ. of Washington, Seattle,
WA (E-mail: ewatson{at}u.washington.edu).
Received 23 December 1998; accepted in final form 28 April 1999.
 |
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