Ryanodine receptor expression is associated with intracellular
Ca2+ release in rat parotid acinar
cells
Xuejun
Zhang1,
Jiayu
Wen1,
Keshore R.
Bidasee2,
Henry R.
Besch Jr.2, and
Ronald P.
Rubin1
1 Department of Pharmacology
and Toxicology, School of Medicine and Biomedical Sciences,
State University of New York at Buffalo, Buffalo, New York
14214; and 2 Department of
Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202-5120
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ABSTRACT |
The ryanodine
receptor mediates intracellular
Ca2+ mobilization in muscle and
nerve, but its physiological role in nonexcitable cells is less well
defined. Like adenosine 3',5'-cyclic monophosphate and
inositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) and
ADP (1-25 µM) produced a concentration-dependent rise in
cytosolic Ca2+ in permeabilized
rat parotid acinar cells. Adenosine and AMP were less effective.
Ryanodine markedly depressed the
Ca2+-mobilizing action of the
adenine nucleotides and forskolin in permeabilized cells and was
likewise effective in depressing the action of forskolin in intact
cells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced by
calmodulin and depressed by W-7, a calmodulin inhibitor. A
fluorescently labeled ligand,
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression and
distribution of ryanodine receptors. In addition, ryanodine receptor
expression was detected in rat parotid cells with a sequence highly
homologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of a
ryanodine-sensitive intracellular
Ca2+ store in rat parotid cells
that shares many of the characteristics of stores in muscle and nerve
and may mediate Ca2+-induced
Ca2+ release or a modified form of
this process.
cyclic adenosine diphosphate-ribose; adenosine
3',5'-cyclic monophosphate
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INTRODUCTION |
IN NONEXCITABLE CELLS,
Ca2+ signaling depends on
Ca2+ influx and a regenerative
release of Ca2+ from intracellular
stores. The inositol 1,4,5-trisphosphate
(IP3) receptor
(IP3R) and the ryanodine receptor
(RyR) modulate the two channels responsible for mobilizing
intracellular Ca2+ (3).
IP3 is generated from
phospholipase C-mediated breakdown of membrane-bound
polyphosphoinositides. Ryanodine binds to a family of receptors (RyR)
that are subtypes characterized for skeletal muscle (type 1, RyR1), cardiac muscle (type 2, RyR2), and a widely distributed
brain isoform (type 3, RyR3)
(27). Unlike the IP3R, a natural
ligand(s) that regulates the RyR
Ca2+-mobilizing mechanism has not
been identified, although cyclic ADP-ribose (cADPR), a naturally
occurring NAD+ metabolite, has
been proposed to serve as an endogenous messenger (16).
A major portion of the work on the RyR has been carried out on skeletal
or cardiac muscle preparations, although specific ryanodine-binding
sites localized to nonexcitable cells and cell lines have also been
identified (2, 5, 29). In single nonmuscle cells the RyR, along with
the IP3R, is thought to be responsible for Ca2+ spiking,
which is presumed to reflect
Ca2+-induced
Ca2+ release (CICR) (3). This
process involves the influx of extracellular Ca2+, which induces
Ca2+ release from the sarcoplasmic
reticulum (SR) or endoplasmic reticulum (ER) to cause cellular
activation (7, 8).
Our previous studies demonstrated that adenosine
3',5'-cyclic monophosphate (cAMP) produced by
-adrenergic receptor activation potentiates the
-receptor-mediated rise in intracellular
Ca2+ in intact rat parotid acinar
cells (19) and mobilizes Ca2+ from
permeabilized cells (23). The findings that the
Ca2+ response to cAMP was
sensitive to ryanodine and insensitive to heparin, a blocker of the
IP3R, provided preliminary
evidence for the presence of a
Ca2+-release channel that is not
regulated by IP3 (23). To probe further the possibility that the parotid cell possesses an RyR Ca2+ release channel for
regulating Ca2+, we utilized a
number of physiological and nonphysiological ligands, including cyclic
nucleotides, adenine nucleotides, ryanodine, calmodulin (CaM), and
caffeine, which regulate the RyR in excitable cells (27). In addition,
the expression and distribution of the RyR were examined by reverse
transcriptase polymerase chain reaction (RT-PCR) and by monitoring of
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid succinimidyl ester (BODIPY)-ryanodine fluorescence. The data support the idea of an RyR in rat parotid cells that resembles the RyR
of a ryanodine-sensitive Ca2+
release channel that exists in muscle and nerve.
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MATERIALS AND METHODS |
Materials.
Adenine nucleotides, adenosine, forskolin,
3-isobutyl-1-methylxanthine (IBMX), and
IP3 were purchased from Sigma
Chemical (St. Louis, MO). Fura 2, fura 2-acetoxymethyl ester, and
BODIPY 409/503 SE were obtained from Molecular Probes (Eugene, OR).
cADPR, ryanodine, CaM, and W-7 were purchased from Calbiochem (San
Diego, CA).
Preparation of permeabilized cells.
Rat parotid acinar cells were prepared and isolated as previously
described (23). For permeabilization, rat acinar cells (8-10 × 106 cells/ml) were washed
twice with a cytosol-like medium containing 140 mM KCl, 20 mM NaCl, 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3 mM ATP, 10 mM phosphocreatine, and 10 U/ml creatine phosphokinase (pH 7.2). After permeabilization with saponin at room
temperature (23), the cells were centrifuged and resuspended in
high-K+ buffer containing 5 µM
oligomycin, 10 µM antimycin, and 6 mM ATP.
Measurement of
Ca2+ release.
Ca2+ levels were determined in
permeabilized cells preloaded with fura 2, as previously described
(23). Fluorescence was measured in a spectrophotometer (model RF-5000,
Shimadzu). This single-excitation-wavelength instrument yielded values
comparable to those obtained with a dual-excitation-wavelength
instrument. Stimulated Ca2+
release is defined as the maximal concentration of
Ca2+
(
[Ca2+]) determined
after the addition of stimulating agent minus the concentration of
Ca2+ measured in the absence or
presence of any pretreatment, including ryanodine. Basal
Ca2+ release from untreated
permeabilized cells averaged 366 ± 14 nM
(n = 66). In a few experiments,
Ca2+ mobilization was measured in
intact parotid cells after exposure to 5 µM fura 2-acetoxymethyl
ester for 30 min (19).
Fluorescence microscopic analysis.
Confocal fluorescence microscopy was carried out on intact parotid
cells utilizing a new fluorescent ryanodine derivative prepared by
coupling an activated ester of BODIPY 493/503 SE to glycyl ryanodine
using the method previously described (12). The 50% inhibitory
concentration (IC50) for the
resultant BODIPY-ryanodine was evaluated from competition binding
assays, as described elsewhere (11). Cells were incubated in total
darkness at 37°C in HEPES-buffered Krebs-Ringer solution (pH 7.4)
with various concentrations of BODIPY-ryanodine. At the end of the
incubation period, the cells were washed twice with Krebs-Ringer
solution to remove unbound ligand and placed on a slide under a
coverslip. The cells were initially viewed using epifluorescence optics
of a Nikon upright Optiphot microscope, and confocal imaging was
performed with a laser scanning microscopy system (model MRC-1024,
Bio-Rad) configured with a Nikon microscope and a krypton-argon laser
(488 nm). A ×40 oil immersion objective was first employed to
give overall views, then zoom ×2 optics were used to provide a
higher-magnification view. The system was operated by a Compaq Pentium
100 computer, and photographs were processed using a disublimation
printer (model NP-1600, Codonic). Concentration and time course
analysis indicated that fluorescence intensity reached a plateau after
a 3-h exposure to 200 nM BODIPY-ryanodine (data not shown). Therefore,
these parameters were chosen for our studies. Displacement experiments to determine binding specificity were carried out by incubating cells
with 200 nM BODIPY-ryanodine + 2 µM ryanodine for 3 h.
mRNA analysis and RT-PCR.
mRNA was isolated from rat parotid acinar cells and brain using TRIzol
reagent (GIBCO Life Technologies, Grand Island, NY). First-strand cDNA
was synthesized using a SuperScript II ribonuclease H+ RT
kit (GIBCO). Although rat sequences are not available in the GenBank,
rat RyR type-specific primers were designed on the basis of the
comparative sequence of the novel mouse brain
RyR1 and the partial sequence of
rat skeletal muscle RyR1, which
was graciously provided by Dr. Deborah Bennett (Cambridge University,
Cambridge, UK). The sequences of the primers were as follows:
5'-GGTGGCCTTCAACTTCTTCL-3' (sense) and
5'-ACTTGCTCTTGTTGGTCTCCG-3' (anitsense). By use of these
primers, RyR cDNAs were amplified by PCR with a thermocycler (model
2400, Perkin Elmer) for 45 cycles with 1 unit of
Pfu polymerase. Reactions were carried
out as follows: denaturation for 1 min at 95°C, annealing for 2 min
at 60°C, and extension for 2.5 min at 74°C. An aliquot of each
PCR reaction was subjected to electrophoresis through a 2% agarose
gel, and DNA was visualized by ethidium bromide staining. The DNA
product was purified and sequenced in both directions by the CAMBI
Nucleic Acid Facility (DNA Sequencing Service, State University of New
York at Buffalo).
Statistical analysis.
Values are means ± SE. Comparisons between control and experimental
samples were made using two-tailed unpaired Student's t-test. Differences were accepted as
significant at P < 0.05.
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RESULTS |
Effects of forskolin.
Forskolin, which raises parotid cell cAMP levels by directly activating
adenylyl cyclase (6), produced a concentration-dependent enhancement of
Ca2+ release from
saponin-permeabilized parotid acinar cells (Fig. 1). The
Ca2+-mobilizing action of
forskolin was enhanced by preincubating cells with the
phosphodiesterase inhibitor IBMX (Fig. 1). These findings demonstrate
that Ca2+ release from permeable
cells can be regulated by endogenously generated cAMP levels as well as
by the addition of cAMP (23). However, cAMP was much less efficacious
in this regard than IP3 (Fig. 1)
(23). Like cAMP, IBMX enhanced the
Ca2+ response to
IP3 (Fig. 1). This potentiation
appears to involve sensitization of the
IP3R by protein kinase A-dependent
phosphorylation (23).

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Fig. 1.
Concentration-dependent increase in forskolin (FSK)-stimulated
Ca2+ release from fura 2-loaded
permeabilized parotid cells in presence (+) and absence ( ) of
3-isobutyl-1-methylxanthine (IBMX, 200 µM). Response to a submaximal
concentration of inositol 1,4,5-trisphosphate (IP3) is also presented for
comparison. Values (means ± SE of 5 experiments)
represent peak Ca2+ release
measured after ~1 min with basal values subtracted
( [Ca2+]).
* Significantly different from corresponding control group (P < 0.05).
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Effects of cADPR on
Ca2+ release.
Another cyclic nucleotide, cADPR, which has been proposed as an
endogenous regulator of CICR mediated by RyRs (16), produced a
concentration-dependent rise in
Ca2+ release. The time course of a
representative experiment is shown in Fig.
2, and a quantitative assessment is
provided in Fig. 3. The stepwise nature of
the Ca2+ mobilized by cADPR, like
that of cAMP, is consistent with a quantal (all-or-none) release
mechanism (Fig. 2) (23). Graded effects were observed at 0.3-5
µM (Figs. 2 and 3). A higher concentration of cADPR (25 µM)
produced no further elevation in
Ca2+ release (data not shown).

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Fig. 2.
Concentration-dependent increase in
Ca2+ release produced by cyclic
ADP-ribose (cADPR). Trace is representative of 12 traces obtained from
11 independent preparations.
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Fig. 3.
Ryanodine inhibition of cADPR-stimulated
Ca2+ release. Permeabilized
parotid cells were pretreated with 10 µM ryanodine or vehicle for 5 min before addition of increasing concentrations of cADPR. Vertical
bars represent values for peak
Ca2+ mobilization obtained in
presence and absence of ryanodine. Values are means ± SE of 4 independent experiments. Ryanodine (10 µM) alone increased basal
Ca2+ release by 34.2 ± 8.5 nM.
* Significantly different from corresponding control group
(P < 0.05).
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Effects of other adenine-containing compounds.
The Ca2+-mobilizing action of
cADPR was shared by other adenine-containing compounds. Exposure to
1-25 µM ADP produced a concentration-dependent rise in
Ca2+ mobilization (Fig.
4A). At
25-500 µM, the adenine nucleoside adenosine also elicited a
concentration-dependent elevation in Ca2+ release (Fig.
4B). By contrast, consistent
responses to AMP were observed only at
500 µM (data not shown).

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Fig. 4.
Effects of ADP (A) and adenosine
(B) on
Ca2+ release. Peak
Ca2+ release was measured after 30 s. Values are means ± SE of 4 independent experiments.
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A comparison of the relative potencies of adenine-containing compounds
in promoting Ca2+ mobilization is
provided in Table 1. The data show that
cADPR was most potent in this regard and ADP was somewhat less potent, although it was greater than adenosine, cAMP, or AMP. It is also of
interest that cADPR was ~200-fold more potent than cAMP and 10-fold
more potent than IP3 (Table 1).
Effects of ryanodine.
To investigate potential mechanisms whereby intracellular
Ca2+ release may be activated, the
effects of ryanodine were examined. Ryanodine (10 µM) attenuated
Ca2+ release elicited by the
adenylate cyclase activator forskolin in permeabilized cells (Fig.
5A),
thus indicating that ryanodine can block the response to endogenously
generated cAMP, just as it markedly depressed the response to added
cAMP (23). Experiments were also carried out to determine whether
ryanodine could block the stimulatory effects of forskolin in intact
parotid cells. Figure 5B shows that
forskolin was able to elicit a concentration-dependent stimulation of
Ca2+ mobilization that was reduced
~50% by ryanodine. These latter findings confirm earlier work
demonstrating that cAMP modulates Ca2+ homeostasis in intact parotid
cells (19) and provide a physiological basis for the actions of
ryanodine in permeabilized cells.

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Fig. 5.
Inhibition of forskolin-stimulated
Ca2+ release by ryanodine.
A: permeabilized cells were pretreated
with 10 µM ryanodine or vehicle for 5 min before addition of
increasing concentrations of forskolin.
B: intact cells were pretreated with
50 µM ryanodine for 30 min before addition of forskolin. Values are
means ± SE of at least 4 determinations from 3-4 independent
experiments with basal values subtracted. Ryanodine (10 µM) alone
increased basal Ca2+
release in permeabilized cells by 36 ± 5 nM. In intact
cells, basal Ca2+ release averaged
86 ± 9 and 205 ± 15 nM in absence and presence of 50 µM
ryanodine, respectively. * Significantly different from corresponding control group (P < 0.05).
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Ryanodine also depressed the
Ca2+-mobilizing action of cADPR in
permeabilized cells (Fig. 3). Even at the maximal stimulating concentration of cADPR (3 µM), ryanodine attenuated
Ca2+ release by ~70%. Ryanodine
(10 µM) also blocked Ca2+
mobilization induced by ADP and adenosine (data not shown). However, the blockade was statistically significant only at the lowest concentration of adenosine employed (25 µM), thus suggesting the competitive nature of the blockade.
In excitable and nonexcitable cells, ryanodine at relatively low
concentrations (10 nM-10 µM) is reported to cause activation of
the Ca2+ release channel, whereas,
at >10 µM, ryanodine eventually blocks channel activation (24, 30).
In permeabilized parotid cells, 2-100 µM ryanodine produced only
a modest graded enhancement of Ca2+ release within the limited
time frame of the experiments (data not shown). No blockade of channel
activation was observed, despite the fact that ryanodine attenuated
cADPR- and forskolin-stimulated Ca2+ release (cf. Figs. 3 and 5).
Further studies are underway to evaluate this phenomenon.
Role of CaM in
Ca2+
mobilization.
Pretreatment of permeabilized parotid cells with CaM enhanced
Ca2+ release induced by cADPR
(Fig.
6A).
With 1 µM cADPR, CaM augmented Ca2+ mobilization by twofold. The
addition of 5 µM CaM alone resulted in a 25% rise in
Ca2+ levels (data not shown),
which is probably the result of the addition of a small amount of
contaminating Ca2+ contained in
the CaM buffer. In addition, W-7, a CaM inhibitor, reduced the
Ca2+ response to increasing
concentrations of cADPR (Fig. 6B).
Thus, in the presence of 70 µM W-7, the response to 1 and 5 µM
cADPR was diminished by >50%.

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Fig. 6.
Effects of calmodulin (CaM) and W-7 on cADPR-induced
Ca2+ release. Permeabilized
parotid cells were pretreated with 5 µM CaM (A) or 70 µM W-7
(B) for 5 min before addition of
increasing concentrations of cADPR. Peak
Ca2+ release was obtained in
presence and absence of CaM or W-7. Values are means ± SE of 4 independent experiments. Addition of CaM and W-7 in absence of cADPR
increased basal Ca2+ release by
57.6 ± 7.5 and 71.3 ± 3.4 nM, respectively.
* Significantly different from corresponding control group
(P < 0.05).
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Effects of caffeine.
Caffeine (1 mM), a pharmacological activator of the RyR (5, 26, 30),
produced only a modest elevation of cytosolic Ca2+ (~20%). Caffeine also
attenuated Ca2+ release induced by
cADPR by ~50% (Fig.
7A). By
contrast, the ability of IP3 to
raise Ca2+ levels was augmented
(Fig. 7A). These actions of caffeine
mimicked those of forskolin in the ability to depress the
Ca2+ response to cADPR and enhance
the response to IP3 (Fig.
7B).

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Fig. 7.
Effects of caffeine and forskolin on cADPR- and
IP3-mediated
Ca2+ release. Permeabilized
parotid cells were pretreated with 1 mM caffeine
(A) or 2 µM forskolin
(B) for 5 min before addition of cADPR or IP3. Peak
Ca2+ release was obtained in
presence and absence of caffeine or forskolin. Values are means ± SE of 4 independent experiments. Caffeine and forskolin alone increased
basal Ca2+ release by 34.0 ± 5.3 and 18.6 ± 2.3 nM, respectively. * Significantly different from corresponding control group
(P < 0.05).
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Detection of ryanodine-binding sites.
Figure 8 shows that BODIPY-ryanodine
competitively inhibited binding of
[3H]ryanodine to
rabbit muscle SR vesicles with an
IC50 of 100 nM. The fluorescently
labeled ligand was subsequently employed to study the expression and
distribution of the RyR in parotid cells. Intact cells were utilized
for this study, since the
high-Mg2+-low-Ca2+
medium used for permeabilized cells markedly reduces the affinity of
ryanodine for specific binding sites (21). Confocal microscopy revealed
that cells exposed to BODIPY-ryanodine displayed a granular fluorescence throughout the cell body that was excluded from the nucleus and plasma membrane (Fig.
9A). The
localization of the fluorescence was consistent with the presence of
binding sites within the ER. Under our experimental conditions, 89 ± 7% of the cells exposed to BODIPY-ryanodine exhibited
fluorescence (mean of 8 images from 3 different preparations).
Competition experiments were also performed to determine whether
BODIPY-ryanodine binding was specific. Figure
9B shows that fluorescence intensity
was markedly decreased in cells incubated with BODIPY-ryanodine + 2 µM ryanodine. Less than 10% of control cells exhibited significant fluorescence. These results document the existence of specific binding
sites for ryanodine in rat parotid acinar cells.

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Fig. 8.
Displacement by
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic
acid succinimidyl ester (BODIPY)-ryanodine of
[3H]ryanodine from
ryanodine receptor Ca2+ release
channel of rabbit skeletal muscle junctional sarcoplasmic reticulum
vesicles. Values for BODIPY-ryanodine experiments are means ± SE
for at least 3 separate assays, each done in duplicate, using 2 different membrane preparations. Displacement of
[3H]ryanodine by
unlabeled ryanodine is shown for comparison and represents 19 separate
assays using 6 different preparations. [Ryanoid], ryanoid
concentration.
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Fig. 9.
Fluorescence localization of ryanodine-binding sites in rat parotid
acinar cells. Representative confocal images of cells labeled with
BODIPY-ryanodine (A) or
BODIPY-ryanodine + ryanodine (B) are
shown.
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Expression of RyR mRNA.
The pharmacological and fluorescence experiments demonstrating the
presence of RyR in rat parotid cells provided the impetus for extending
this study to identify the type of RyR expressed in these cells. After
primers were designed according to the partial sequence of rat skeletal
muscle RyR1 or novel mouse brain
RyR1 (see
MATERIALS AND METHODS), the
amplified mRNA from rat parotid cells failed to yield detectable PCR
product. However, by use of primer pairs according to a sequence common
to rat skeletal muscle RyR1 and
mouse brain RyR1, the amplified
cDNA from parotid and brain mRNA yielded a band of the expected size of
293 base pairs in parotid and brain (Fig.
10A).

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Fig. 10.
A: reverse transcriptase-polymerase
chain reaction (RT-PCR) detection of ryanodine receptors (RyR) in rat
parotid cells and brain. RT-PCR analysis with specific primers common
to novel mouse brain type 1 RyR
(RyR1) and rat skeletal muscle
RyR1, showing their expression in
rat parotid cells (lane 1) and brain
(lane 2). cDNA template
concentration for PCR amplification of rat parotid cells was 10 times
higher than that of brain. Molecular weight marker (M) is
X174/Hae III.
B: comparison of nucleotide sequence for putative RyR in rat parotid (RPR) with novel mouse brain
RyR1 (MBR).
Common
regions.
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The PCR product from rat cells analyzed by DNA sequencing was highly
homologous to rat skeletal muscle
RyR1 (98%) and mouse brain
RyR1 (94%; Fig.
10B). Like the novel mouse brain
RyR1 (1), the PCR product from rat
parotid cells also has a high degree of homology to rabbit
RyR1 (88%).
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DISCUSSION |
The present study utilized three complementary techniques
(pharmacological, fluorescent, and molecular) to demonstrate that parotid cells contain an RyR that mediates intracellular
Ca2+ release. The genesis of this
work emanated from an earlier study that showed that the ability of
cAMP to induce intracellular Ca2+
release involves an RyR-mediated mechanism (23). Further
experimentation revealed that the ability of cAMP to activate the RyR
is shared not only by another cyclic nucleotide, cADPR, but also by a
number of other adenine-containing compounds. Analysis of the
concentration-response data for a series of adenine-containing
compounds demonstrated a wide disparity in
Ca2+-releasing activity. However,
most significantly, apart from cADPR, the rank order of potency of
these compounds (ADP > adenosine > cAMP > AMP; Table 1) was
remarkably similar to that reported in muscle preparations (33).
Adenine appears to be the preferred ligand for the nucleotide-binding
site on the RyR in parotid cells and muscle preparations, since GTP and
guanosine 3',5'-cyclic monophosphate are ineffective in
both systems (21, 23), and the putative nucleotide-binding site
possesses a much higher affinity for adenine nucleotides (33).
The fact that cADPR proved to be the most potent of the
adenine-containing compounds examined is also consistent with findings in other model systems where it mobilizes intracellular
Ca2+ in the nanomolar
concentration range (18). The cADPR concentration for half-maximal
stimulation of Ca2+ release from
permeabilized parotid cells (0.6 µM) compares favorably with values
derived from isolated cardiac SR (0.15 µM) and brain microsomes (0.11 µM) (22). The ability of cADPR to potentiate CICR in sea urchin eggs
(15) and activate the cardiac RyR (5) bears on the possibility that
cADPR represents a physiologically relevant second messenger, although
the physiological relevance of the cADPR effect on cardiac RyR is
controversial (25). Its relative potency in parotid cells supports this
supposition, as does the finding that this cyclic nucleotide elicits
Ca2+ spiking in intact exocrine
cells (28). However, additional experimentation is needed to determine
whether parotid cells are capable of synthesizing cADPR and regulating
its cellular levels during periods of
Ca2+ mobilization.
The additional result that the potency of cADPR was >10-fold greater
than that of IP3 but ~3-fold
less efficacious may be a consequence of a less efficient coupling
mechanism for cADPR or the fact that RyR are less concentrated in the
ER of parotid cells. Although receptor density was not determined in
this study, the latter alternative gains favor by the knowledge that
the density of RyR in the submicrosomal fraction of intestinal smooth
muscle is 10-fold less than that of
IP3 receptors (32).
The ability of ryanodine to block the
Ca2+-mobilizing action of cADPR in
parotid cells, as well as the actions of other adenine nucleotides, is
in agreement with previous work that demonstrated that cADPR releases
Ca2+ from cardiac SR and rat brain
microsomes by a mechanism that is sensitive to inhibition by ryanodine
(22, 25, 31). Furthermore, the fact that ryanodine was able to block
the Ca2+-releasing action of
forskolin in intact cells documents the physiological relevance of the
findings in permeabilized cells. On the other hand, the characteristic
property of ryanodine to open the RyR channel in low concentrations and
block the channel in higher concentrations (14, 24) at functionally
separate sites (11) was not observed in parotid cells. The observation
that CaM potentiates cADPR-induced
Ca2+ mobilization from parotid
cells is also in accord with the finding that CaM has a potentiating
effect on CICR in skinned muscle fibers at submicromolar
Ca2+ concentrations, although CaM
does inhibit CICR at higher Ca2+
concentrations (13). The apparent discrepancy that CaM serves an
apparent permissive role in parotid cells but is required for cADPR
action in sea urchin egg homogenates (17) may be resolved by the fact
that CaM is tightly associated with parotid membranes (4), thereby
enabling permeabilized parotid cells to retain sufficient CaM to
sustain the activation of Ca2+
release.
Further insight into the mechanism by which adenine nucleotides
regulate the Ca2+ release channel
of parotid cells is gleaned from the knowledge that cADPR, ADP, cAMP,
and adenosine promote Ca2+
mobilization from permeabilized cells incubated in a medium that contains millimolar concentrations of ATP and an ATP-regenerating system. This medium, which also contains several mitochondrial inhibitors, is utilized for permeabilized parotid cells to alleviate any influence of intact cells. Under such experimental conditions the
adenine moiety probably exerts its
Ca2+-releasing action at a locus
other than the ATP-binding site on the RyR. In this context, it is
known that two or more nucleotide-binding sites exist in each RyR
monomer with distinct affinities (33). The physiological implications
of the present studies may pertain to the ability of adenine
nucleotides, as well as CaM, to serve modulatory roles by sensitizing
the mechanism that allows Ca2+
influx to activate Ca2+ release,
i.e., CICR. Such a model has been proposed for CICR in muscle (5, 26).
Caffeine has been widely employed to activate CICR in excitable cells
(26, 30), and the property of adenine nucleotide-induced Ca2+ release from muscle
preparations is shared by caffeine (5). However, attempts to duplicate
with caffeine the stimulatory effects of the adenine nucleotides in
permeabilized parotid cells yielded inconclusive results. The effects
of IBMX that we observed in parotid cells appear to be due to
phosphodiesterase inhibition to raise cAMP levels, rather than a direct
action on a caffeine-sensitive Ca2+ pool (26). Thus caffeine
behaved like forskolin in attenuating intracellular
Ca2+ release induced by cADPR,
presumably because cAMP and cADPR activate and thereby deplete the same
intracellular Ca2+ pool. Moreover,
the ability of caffeine to enhance the sensitivity of the
Ca2+-releasing activity of
IP3 was shared by forskolin and
exogenous cAMP (cf. Fig. 7 and Ref. 23). The fact that caffeine lacks the ability to mobilize Ca2+ in
certain nonexcitable cells supports the assertion that caffeine cannot
be used to demonstrate the presence of musclelike RyR in nonexcitable
cells (2, 20). However, Foskett and Wong (9) reported that caffeine can
induce ryanodine-sensitive Ca2+
oscillations in intact parotid cells depleted of their
IP3-sensitive store by
thapsigargin. Thus other experimental strategies are required to
ascertain whether parotid cells manifest a caffeine-sensitive Ca2+ store with properties
comparable to those found in excitable cells.
Because ryanodine binds specifically to the
Ca2+-gated
Ca2+ release channels in the SR
(or ER) of cells (5), the validity of the pharmacological studies could
be confirmed by using a specific ligand (ryanodine) derivatized with a
fluorophore to identify the distribution of ryanodine receptors in
parotid acinar cells. The granular distribution of the fluorescence
within the cell body indicated that these receptors were clustered
within a discrete cytoplasmic domain, perhaps representing the ER. The
binding of BODIPY-ryanodine was time dependent and specific, in that it
was displaced by 10-fold excess ryanodine. The additional fact that binding was attained at nanomolar concentrations of BODIPY-ryanodine, which were comparable to its IC50
value for skeletal muscle receptors (100 nM), is consistent with a
high-affinity binding site. Thus these experiments provide a second
line of evidence that demonstrates the location of RyR in parotid
acinar cells.
In addition, molecular techniques were employed to identify the type of
RyR present in parotid cells. Using appropriate RyR primers, we
succeeded in demonstrating the expression of an RyR in rat acinar cells
that is highly homologous to a rat skeletal muscle
RyR1 (98%), novel mouse brain
RyR1 (94%), and rabbit skeletal muscle RyR1 (88%). A brain RyR
type has previously been identified in human Jurkat T lymphocytes,
which, like the RyR of the rat parotid, is ryanodine sensitive and
caffeine insensitive (10). In examining the DNA sequences of rat types
1 and 2 RyR; mouse types 1, 2, and 3 RyR; novel brain type 1 RyR; and
rabbit types 1, 2, and 3 RyR, it is of interest to note that the most
highly conserved region is in the COOH terminus, which is the
Ca2+-binding domain. Thus the RyR
channel of rat parotid cells exhibits properties that are similar to
those previously described in excitable and nonexcitable cells.
Our earlier work utilizing intact parotid cells revealed that the dual
activation of the cAMP- and
Ca2+-phosphoinositide pathways by
norepinephrine, the physiological neurotransmitter, is associated with
an elevated Ca2+ response and
augmented amylase secretion (19). The present and a previous study (23)
utilized permeabilized cells to define the nature and biochemical basis
of the coupling between cAMP- and
IP3-mediated
Ca2+ release. On the basis of
these findings, we conclude that parotid cells possess a minimum of two
types of intracellular Ca2+
channels. One channel type is activated by
IP3; the other, which comprises
the RyR, is regulated by ryanodine, adenine nucleotides, cyclic
nucleotides, and CaM. Furthermore, regenerative increases in
intracellular Ca2+ during
agonist-induced IP3 generation may
be mediated by RyR-mediated CICR or a modified form of this process.
The identification of a second basic process involved in intracellular
Ca2+ release in parotid acinar
cells presages new insights into what appears to be a highly regulated
system for Ca2+ signaling.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Robert Summers (Dept. of Anatomical Sciences
and Cell Biology, University of Buffalo) for expert assistance and
advice in conducting the experiments employing confocal microscopy.
 |
FOOTNOTES |
This work was funded by National Institute of Dental Research Grant
DE-059654 and by the Showalter Trust.
Address for reprint requests: R. P. Rubin, Dept. of Pharmacology and
Toxicology, School of Medicine and Biomedical Sciences, State
University of New York at Buffalo, 102 Farber Hall, Buffalo, NY 14214.
Received 17 December 1996; accepted in final form 21 May 1997.
 |
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