 |
INTRODUCTION |
Cyclic ADP-ribose
(cADPR),1 first found in sea
urchin eggs, mobilizes Ca2+ by a mechanism independent of
the inositol 1,4,5-trisphosphate (IP3) pathway (1)
and may act on the Ca2+-induced Ca2+ release
mechanism as an endogenous modulator (2-8). That both cADPR and its
synthetic enzyme are ubiquitously present in mammalian and invertebrate
tissues (9, 10) suggests that cADPR is a type of cellular
Ca2+-mobilizing messenger, playing a role in
Ca2+ signaling in a variety of cells. The best
characterized mediator for the signal is IP3 (11), and the
possibility has been raised that cADPR is a different class of
mediator. How cells differentiate between these signals is a question
of fundamental importance. One possibility is that they act on their
respective receptors in different Ca2+ pools. The major
purpose of the present study is to determine their contributions to
overall Ca2+ signaling. We started by depicting the precise
concentration response curves for Ca2+ mobilization with or
without cADPR. This could be achieved by using CD38 knockout mice
because the majority of cADPR has been ascribed to the activity of
ADP-ribosyl cyclase of CD38 (12). By estimating the change in the
cellular content of cADPR and monitoring the muscarinic
Ca2+ response in pancreatic acinar cells, either from
normal or CD38 knockout mice (13), we have successfully separated the
CD38- and therefore cADPR-dependent component from the
inositol phosphate-sensitive component. Thus, this paper provides
insight into the mechanism of cADPR-dependent
Ca2+ mobilization mediated by CD38 activities, which is
independent of IP3-dependent Ca2+
mobilization in muscarinic acetylcholine (ACh) receptor signaling.
 |
EXPERIMENTAL PROCEDURES |
Mice--
Mice lacking CD38 were generated by homologous
recombination. The generation and genotyping of the mice have been
described in detail previously (13). The mice used for each experiment were derived from ICR background and were from the same litter or the same family.
Cell Preparation--
Fragments of pancreatic tissue from both
normal ICR (CD38+/+) and CD38 knockout ICR mice
(CD38
/
) were excised and treated with enzymes at
37 °C. Single pancreatic acinar cells used for the fluorescence
and/or whole-cell current measurements were prepared using collagenase
(200 units/ml; Wako, Osaka, Japan) for 3 min and trypsin (0.5 mg/ml;
Sigma type XI) for 2 min and then with the same collagenase for 1 min,
similar to the procedure described previously (14). The pancreatic
acini used for the measurements of the cell content of cADPR and
IP3 were prepared using collagenase (1000 units/ml) alone
for 10 min.
Experimental Solutions and Materials--
We used a
Ca2+-free solution to avoid any contribution of external
Ca2+ to the signaling in pancreatic acinar cells. The
Ca2+-free solution contained (in mM): 140 NaCl,
4.2 KCl, 1.13 MgCl2, 10 glucose, 10 HEPES (pH 7.2 adjusted
with NaOH). Fluorescence experiments were carried out using a solution
containing 0.5 mM EGTA. Pretreatment of the cells with
ryanodine (Calbiochem) was performed at 37 °C for 5 min using the
Ca2+-free solution (15). The reagent was removed by washing
the cells several times with the experimental solution before the fluorescence measurements (16).
Fura-2 Loading and Fluorescence Measurements--
The single
pancreatic acinar cells were incubated with 1 µM
fura-2/AM (Dojin Chemical Institute, Kumamoto, Japan) for 40 min at
37 °C and then washed several times with the normal solution and
kept at room temperature until use. The cells, attached to a glass
coverslip, were placed in a small chamber (400 µl) mounted on the
stage of an inverted microscope and perfused continuously (0.5 ml/min)
with a stream of the experimental solution. This arrangement permitted
a rapid exchange of the bathing solution (17). The fluorescence was
measured by an epifluorescence inverted microscope system described
previously (18, 19). Single acinar cells were alternately illuminated
at excitation wavelengths of 340 and 380 nm by a rotating sector mirror
at 3-ms
1-s intervals. The emission was monitored at 510 nm. Cytosolic
Ca2+ concentration ([Ca2+]i) changes
were monitored as changes in the fluorescence ratio for excitation at
340 and 380 nm
(F340/F380). The
[Ca2+]i was determined from the ratio of
fluorescence traces at the two excitation wavelengths (340/380 nm)
(20).
Whole-cell Injection of cADPR, Ca2+, and
IP3--
The single pancreatic acinar cells were subjected
to standard whole-cell patch-clamp recordings for the injection of
reagents (IP3, cADPR, and Ca2+), in a manner
similar to the procedure described previously (21, 22). Patch pipettes
were pulled from plain hematocrit glass capillaries by a two-stage
puller (PP-83, Narishige, Tokyo) coated with beeswax. The current
signals were amplified by a List EPC-7 amplifier (List Electronics,
Darmstadt, Germany), appropriately low pass filtered (200-500 Hz) and
displayed on an oscilloscope screen and a digital chart recorder. The
cells were immersed with external solution containing (mM):
145 NaCl, 4 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES at
pH 7.2 (by NaOH). Patch pipettes were filled with a solution (pipette
solution) containing (mM): 144 KCl, 2 MgCl2,
0.1 EGTA, 10 HEPES at pH 7.2 (by NaOH). For loading of the reagents, 20 µM cADPR (Molecular Probes), 20 µM
IP3 (Wako, Osaka), or 200 µM
CaCl2 were included in the pipette solution. In the last
case, EGTA was eliminated from the pipette solution. The pipette
resistance when filled with the pipette solution was 3-5
megaohms. The establishment of the whole-cell recordings was monitored with repetitive voltage pulses superimposed on the holding potential, and it appeared as a sudden increase of capacitative current
during the application of a train of short suction onto the patch
pipette. The pulses were interrupted soon after the current responses
started. The series resistance of the whole-cell recordings was 10-15
megaohms, which was measured with a trimmer system of the amplifier
after the current response evoked by the reagent recovered to the
resting level. All the experiments were carried out at room temperature
(24 °C).
Western Blot Analysis--
Proteins were extracted with 9 volumes of 100 mM Tris-HCl (pH 7.6), 1 mM EDTA,
1% (v/v) Triton X-100 supplemented with CompleteTM (one
table/50 ml, Roche Diagnostics GmbH, Mannheim, Germany) from mouse
tissues. The proteins (50 µg) were separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated at room temperature for 1 h with an anti-CD38 polyclonal antibody raised against a peptide fragment of mouse CD38 (residues 283-301, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The concentration of
antibody was 0.4 µg/ml with 5% skim milk. After rinsing, the membrane was further incubated at room temperature for 1 h with a
secondary antibody labeled with horseradish peroxidase and developed using an ECL detection system (Amersham Pharmacia Biotech) as described
(6, 22).
Measurement of cADPR by Radioimmunoassay--
The cADPR content
was measured in either the presence or absence of ACh as described
(23). Briefly, the suspension of enzymatically dispersed pancreatic
acini (about 20 mg wet weight/ml) in 1-ml test tubes was incubated for
60 s (with or without ACh), the supernatant was discarded after
centrifugation at 600 × g for 1 min at 4 °C, and
the preparation was then stocked at
80 °C. The acinar preparation was homogenized in 9 volumes of perchloric acid solution (2.5%, v/v).
The homogenates were stored at
80 °C and later defrosted and
centrifuged for 10 min at 13,000 × g. The supernatant
(330-350 µl) of the homogenates was then mixed with 150 µl of a
suspension of Norit A (27 mg/ml in H2O, Nacalai tesque,
Kyoto, Japan). After a 30-min incubation at 37 °C, the samples were
again centrifuged, and the supernatant was discarded. The pellet was
washed three times with 1.0 ml of H2O, resuspended in a
pyridine-ethanol-H2O mixture (10:50:40, v/v/v), and
incubated for 120 min at 37 °C. After a further centrifugation, the
supernatant was collected and evaporated (Speedvac, Savant Instrument
Inc., Farmingdale, NY). The evaporated materials eluted from Norit A
charcoal were resuspended with 50 µl of H2O and then
incubated at 25 °C for 2 h with bovine alkaline phosphatase
(Sigma) and venom phosphodiesterase (Worthington Biochemical) at final
concentrations of 50 and 2 units/ml, respectively, in 100 mM imidazole-HCl (pH 7.5), 2 mM MgCl2, 100 mM NaCl, and 400 mM KCl.
This enzyme treatment was effective for degrading nucleotides that
weakly cross-react with the anti-cADPR antibody (24). The reaction was
terminated by adding a solution of trichloroacetic acid (at a final
concentration of 4%, w/v) and kept on ice for 20 min. A clear
supernatant was obtained after centrifugation at 13,000 × g for 10 min. An aliquot (10-20 µl) of the supernatant
was immediately neutralized with a solution of 2 M Tris
base and subjected to the procedure of cADPR measurement. As a control,
another aliquot was heated at 95 °C for 10 min before being
neutralized and analyzed by the radioimmunoassay. This heat treatment
converted cADPR to ADP-ribose, resulting in no cross-reaction with the
anti-cADPR antibody. The immunoreactivity of all the samples was
abolished by the heat treatment. The recovery of cADPR, monitored by
the recovery of [3H]cADPR added in each homogenate, was
74.2 ± 2.92% (n = 8). Correction was introduced
for the recovery of cADPR.
Measurement of IP3--
The cellular content of
IP3 was estimated with a
D-myo-inositol
1,4,5-[3H]trisphosphate assay system (Amersham
Pharmacia Biotech) either in the presence or absence of ACh. The
suspension of enzymatically dispersed pancreatic acini (about 20 mg wet
weight/ml) in 1-ml test tubes was incubated for 60 s (with or
without ACh), the supernatant was discarded after centrifugation at
600 × g for 1 min at 4 °C, and the preparation was
then stocked at
80 °C. The cells were homogenized in 200 ml of
ice-cold 10% (v/v) perchloric acid with an ultrasonic processor
(Astrason®, Heat Systems, Farmingdale, NY) for 10 s.
Then, the samples were neutralized by the addition of 1.5 M
KOH, 60 mM HEPES. After sedimentation of KClO4
by centrifugation at 2000 × g for 15 min at 4 °C,
the supernatant was used for the IP3 measurement. The assay
was performed according to the manufacturer's instructions.
Statistics--
Statistical significance was analyzed with
Student's t test. Data are expressed as means ± S.E.
 |
RESULTS |
The CD38 Expression, cADPR Content, and the Changes of cADPR in
Response to ACh in Pancreatic Acinar Cells from Wild
(CD38+/+) and Knockout Mice
(CD38
/
)--
By Western blot analysis using a
polyclonal antibody against mouse CD38, we detected no CD38 expression
in the pancreatic acinar cells from CD38 knockout mice, but its
expression was detected from those of the wild type mice (Fig.
1A). In addition, by
radioimmunoassay, we scarcely detected cADPR in the cells from the
knockout mice, but it was detected in the wild type mice (Fig.
1B). Thus, the majority of the cellular cADPR content was
caused by the activity of the ADP-ribosyl cyclase of CD38.

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Fig. 1.
CD38 expression and cADPR content in
CD38+/+ and CD38 / pancreas.
A, each pancreatic gland homogenate (50 µg of
protein/lane) was subjected to Western blot analysis using anti-mouse
CD38 antibody. The arrow indicates the position of CD38.
Molecular mass markers are shown at the left in kDa.
B, cADPR extracted from pancreata was measured by
radioimmunoassay using an antibody against cADPR in each of 4 animals.
Vertical bars indicate S.E. The statistical analysis was
performed using Student's t test.
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|
Changes in Cellular Content of cADPR and IP3 in
Response to ACh--
Fig. 2 shows the
changes in the cellular content of cADPR (Fig. 2A) and
IP3 (Fig. 2B) in response to ACh ranging from 40 nM to 4 µM. The cADPR content was increased
by the ACh stimulation in the acini from wild mice but not in those
from the knockout mice. In contrast, the IP3 content was
increased similarly in both types of acini. However, we were not
certain that the method employed to measure the cADPR and
IP3 contents was sensitive enough to detect the difference
in content at different ACh concentrations. The difference is shown in
later sections in terms of changes in the cellular Ca2+
concentration ([Ca2+]i) by fura-2
microfluorimetry.

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Fig. 2.
Changes in cellular content of cADPR
(A) and IP3
(B) in response to ACh.
Pancreatic acini of CD38+/+ and CD38 / mice
were incubated in increasing concentrations of ACh, and the cellular
contents of IP3 and cADPR were measured. A,
cADPR in the cells was extracted, concentrated, and measured by
radioimmunoassay using an antibody against cADPR. The cADPR content was
calculated by accounting for the recovery of cADPR in the extraction
and concentration procedures. The control values of cADPR in
CD38 / and CD38+/+ cells were 0.248 ± 0.248 fmol/mg protein and 0.413 ± 0.240 fmol/mg protein,
respectively. Asterisk (*) indicates significant difference
from the value with 0 mM ACh at p < 0.05. There were no significant differences among the values with 0.04-4
µM ACh. B, IP3 in the cells was
measured. The control IP3 values in CD38 /
and CD38+/+ cells were 4.09 ± 2.65 fmol/mg protein
and 3.42 ± 1.46 fmol/mg protein, respectively. n = 4 for each point. Vertical bars indicate S.E.
Asterisk (*) and symbol (#) indicate significant
difference from the values with 0 mM ACh at
p < 0.05 (*) and < 0.01 (**) in
CD38 / and at p < 0.01 (##) in
CD38+/+, acinar cells, respectively. There were no
significant differences among the values with 0.04-4 µM
ACh.
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|
Effects of Ca2+, cADPR, and IP3 on
Ca2+ Responses of Pancreatic Acinar Cells from
CD38+/+ and CD38
/
Mice--
Pancreatic
acinar cells possess two types of Ca2+ activated ion
channels (25). One is selective for monovalent cations (26, 27) and the
other for Cl
, and both are poorly dependent on membrane
potentials between
60 mV and 60 mV but highly dependent on
[Ca2+]i (25, 28). Thus, increases and decreases
of [Ca2+]i should be reflected by the activity of
these channels. To demonstrate the presence of normal Ca2+
release mechanisms responsible for IP3 (28, 29),
Ca2+-induced Ca2+ release (30, 31), and
cADPR-mediated signaling, we injected these reagents directly into the
single cells with the whole-cell recording technique and monitored the
Ca2+-dependent current activities
(Ca2+ responses). Here, at a holding potential of
40 mV
achieved by the whole-cell recordings,
Ca2+-dependent current responses were expected
to emerge as large inward currents carried by both Cl
and
monovalent cations. Shortly after the establishment of the whole-cell
recordings (delay of 1-3 s) when the pipette included IP3,
cADPR, or Ca2+, we observed a large deflection of the
inward current at the membrane potential of
40 mV (Fig.
3, 3/3 in each reagent injections). However, without the reagents there were no responses (3/3). That all
the reagents successfully induced Ca2+ responses in the
single acinar cells from both the wild and knockout mice indicated that
the Ca2+ releasing machinery was well preserved in both
types of cells.

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Fig. 3.
Activation of Ca2+ release
mechanisms by patch-clamp whole-cell injection of Ca2+,
cADPR, and IP3 into single CD38+/+ and
CD38 / pancreatic acinar cells. Single pancreatic
acinar cells of CD38+/+ and CD38 / mice were
subjected to whole-cell patch-clamp recordings. 200 µM
Ca2+ (A), 20 µM cADPR
(B), or 20 µM IP3 (C)
was included in the pipette solution. The establishment of whole-cell
recordings was monitored with repetitive voltage pulses that were
interrupted soon after the responses started. The holding potential
(pipette potential) was 40 mV. Arrows indicate the start
of the injection.
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|
Close inspection of the records revealed that there might have been
differences between the responses of the wild type and knockout cells
to cADPR and/or IP3. That is, there was a longer response
to cADPR and a shorter response to IP3 in the knockout cells than in those of the wild type. The reduction in cADPR hydrolase activity in the knockout pancreatic acinar cells (13) may explain their
prolonged response to cADPR. The cooperative action of cADPR for
enhancing IP3-induced Ca2+ responses (32) may
explain the shortened response to IP3 because cADPR was
eliminated in the knockout cells. However, further study is needed to
determine the precise mechanisms of the reagent sensitivity.
ACh-evoked Ca2+ Responses from Single Pancreatic Acinar
Cells of Wild (CD38+/+) and Knockout Mice
(CD38
/
)--
Based on the above results, we employed
fura-2 microfluorimetry and compared the ACh-induced pancreatic
Ca2+ responses of the wild (CD38+/+) and CD38
knockout mice (CD38
/
) in a wide range of ACh
concentrations from 10 to 20,000 nM. We used a
Ca2+-free solution containing 0.5 mM EGTA to
avoid any contribution of external Ca2+ to the signaling in
pancreatic acinar cells in the later experiments with fura-2
microfluorimetry. Table I summarizes the
results in terms of the peak magnitude, the onset time after the
application of ACh, and the frequency of the periodic responses. The
real traces of the responses are shown in Fig.
4 for 40 and 400 nM ACh.
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Table I
Magnitude, frequency, and onset of Ca2+ release induced by ACh
in single pancreatic acinar cells of CD38+/+ and
CD38 / mice
Each value is expressed as means ± S.E. Magnitude was measured by
subtracting the prestimulated level of [Ca2+]i from
the peak response.
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Fig. 4.
ACh-evoked Ca2+ responses from
single CD38+/+ and CD38 / pancreatic acinar
cells. Increases in the cellular Ca2+ concentration
were monitored by fura-2 fluorescence measurement. External
Ca2+ was eliminated with EGTA (no added Ca2+
plus 0.5 mM EGTA). Traces in A and
B show each pair of three examples (a-c)
stimulated with 40 nM and 400 nM ACh,
respectively. Traces in the left column represent
responses from CD38+/+ and the right column from
CD38 / . Cytosolic Ca2+ oscillations were
severely impaired in CD38 / cells at 40 nM
ACh. Arrows indicate the start of ACh stimulation.
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|
The threshold concentration of ACh that induced a detectable
Ca2+ response was 10 nM, and the response was a
transient deflection with no significant difference observed in the two
types of pancreatic acinar cells. A difference appeared at 40 nM ACh. Immediately after the application of 40 nM ACh, the cells from CD38+/+ mice showed a
repetitive Ca2+ response (oscillations) during the ACh
application, lasting for 2-3 min. In contrast, that of
CD38
/
was sporadic with the same ACh
stimulation (Fig. 4A). However, at 400 nM ACh,
the difference between the responses from CD38+/+ and
CD38
/
mice became less than that at 40 nM
ACh, and both responses showed large phasic increases in the fura-2
signals (Fig. 4B).
The numerical data of the [Ca2+]i increases in
Table I were plotted against the ACh concentration (Ca2+
response curve) in Fig. 5, A
and B. The magnitude response curve from the knockout mice
(CD38
/
) was smoothly graded with the increasing ACh
concentrations. However, that of CD38+/+ showed two
prominent phases separated at the concentration of 400 nM
ACh. This feature was also seen in the Ca2+ index (18),
which reflects the mixed information of both the magnitude and
frequency of the response. Both response curves were replotted in Fig.
5, C and D, where the Ca2+ response
from the knockout mice (CD38
/
) was subtracted from that
of the wild type (CD38+/+) at each ACh concentration. Two
prominent peaks were seen in the diagrams at the values of 40 and 4000 nM, indicating that two CD38-dependent phases,
in other words, two cADPR-dependent phases, separated at
400 nM, are present in the ACh-induced release of internal
Ca2+. One was induced by low ACh concentrations up to 400 nM (the first cADPR-dependent component) and
the other by high concentrations of ACh over 400 nM (the
second cADPR-dependent component).

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Fig. 5.
Concentration-response curve for the change
in [Ca2+]i evoked by ACh in CD38+/+
and CD38 / pancreatic acinar cells. A,
the peak magnitude of evoked [Ca2+]i increases
was plotted against the ACh concentration. B, a normalized
estimate of the mean increase of [Ca2+]i, the
Ca2+ index, was plotted against the ACh concentration.
Ca2+ index was calculated by integrating the area covered
by the trace of fluorescence intensity from the start to 200 s
after the response. *** and ** represent p < 0.001 and < 0.01 versus CD38+/+, respectively.
, response from CD38+/+; , response from
CD38 / pancreatic acinar cells. C and
D, the difference between the response from
CD38+/+ and that of CD38 / shown in
A and B ( minus ).
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Effect of Ryanodine on the ACh-induced Ca2+ Release in
Single Pancreatic Acinar Cells from Wild Type (CD38+/+) and
Knockout (CD38
/
) Mice--
Because cADPR has been
postulated to be an endogenous modulator of the ryanodine-sensitive
Ca2+ release mechanism (33-35), we next examined the
effect of ryanodine on the pancreatic ACh-induced Ca2+
responses from normal cells. The results were compared with the Ca2+ responses from the knockout cells.
Fig. 6 shows the fura-2 signals in both
types of cells treated either with or without ryanodine (500 µM) prior to the ACh stimulation (40 or 400 nM). The 40 nM ACh stimulation, without ryanodine, induced repetitive and sporadic transient responses in the
wild and knockout mice, respectively, as described in Fig. 4A. In contrast, with ryanodine, the same stimulation
induced a sporadic transient response and no response in the wild and knockout mice, respectively. Comparing these records, we noticed that
the response to 40 nM ACh stimulation in the wild type
cells with ryanodine resembled that of knockout mice without ryanodine (Fig. 6A). The diagram in Fig. 6C
summarizes the effects of ryanodine on the ACh-induced Ca2+
responses in pancreatic acinar cells from the wild mice, showing that
ryanodine abolished the first cADPR-dependent component
but not the second component.

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Fig. 6.
Effects of ryanodine on ACh-induced
Ca2+ responses in CD38+/+ and
CD38 / pancreatic acinar cells. Traces of
Ca2+ increases evoked by 40 nM (A)
or 400 nM ACh (B) in single pancreatic acinar
cells from CD38+/+ (left column) and
CD38 / (right column) mice. A-a
and B-a, control; A-b and B-b, after
500 µM ryanodine treatment. Arrows indicate
the start of ACh stimulation. C, diagram of ryanodine effect
on ACh-induced Ca2+ responses in the wild
(CD38+/+) type cells. The peak magnitude of the evoked
[Ca2+]i increases was plotted against the ACh
concentration. Open circles ( ) represent the wild
(CD38+/+) control response, and open triangles
( ) are the wild with 500 µM ryanodine. D,
diagram of ryanodine effect on ACh-induced Ca2+ responses
in the knockout (CD38 / ) type cells. Filled
circles ( ) represent the knockout control cells
(CD38 / ) and filled triangles ( ) those
with 500 µM ryanodine. *** and ** represent the
significance level p < 0.001 and < 0.01, respectively, for the test with ryanodine versus
control.
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|
The complex effects of ryanodine on the knockout cells depended on the
ACh concentration. That is, at concentrations of ACh from 10 to 400 nM, we could not detect any significant effect of ryanodine
on the magnitude of the Ca2+ response in knockout cells
(Fig. 6D). In contrast, it enhanced the response at
concentrations of ACh over 400 nM. It was noteworthy that
the respective concentrations of ACh corresponded to the first and
second cADPR-dependent components normally present in the
wild type cells.
The effect of ryanodine at concentrations of ACh corresponding to the
first cADPR-dependent component could be interpreted as
indicating either that ryanodine treatment depletes the first cADPR-sensitive Ca2+ pool or that it inhibits the
Ca2+ release from the pool. Either interpretation could be
deduced from the result that ryanodine eliminated the first
cADPR-dependent component from the wild cells and was
without effect on the knockout cells. However, we prefer the first
interpretation because the basal [Ca2+]i level
was higher in the ryanodine-treated cells than that in both the
wild and knockout cells without the treatment. The basal
[Ca2+]i without ryanodine was 143.2 ± 3.2 nM (n = 269) in the wild cells and
151.3 ± 3.1 nM (n = 292) in the
knockout cells. With ryanodine it was 234.6 ± 4.6 nM
(n = 68) in the wild cells and 247.5 ± 2.6 nM (n = 87) in the knockout cells.
 |
DISCUSSION |
Comparing the ACh-induced Ca2+ responses in pancreatic
acinar cells from normal mice (CD38+/+ wild type: normal
cells) with those of CD38 knockout mice (CD38
/
type: KO
cells), we distinguished the CD38- and therefore
cADPR-dependent component from the overall muscarinic
Ca2+ signaling. The major findings are that: 1) ACh
stimulation increased the cellular content of cADPR in the normal cells
but not in KO cells; 2) the Ca2+ response curve in the
normal cells was separated into two phases at 400 nM ACh;
3) in contrast, the Ca2+ response curve from KO cells was a
smoothly graded one, and it lacked the two components inducible by ACh
at concentrations below 400 nM (the first
cADPR-dependent component) and over 400 nM (the second cADPR-dependent component) usually present in normal
cells; 4) the first cADPR-dependent component contributed
to the generation of repetitive Ca2+ spikes; 5) ryanodine
treatment eliminated the first cADPR-dependent component
(Fig. 6).
Physiological Significance of cADPR-dependent
Ca2+ Release--
IP3 and cADPR are
established Ca2+-mobilizing messengers that activate
internal IP3 and ryanodine receptors, respectively
(36-38). Because we scarcely detected cADPR formation in KO cells, the majority of Ca2+ release in these cells could arise from
Ca2+ pools insensitive to cADPR but sensitive to
IP3 and/or yet unknown messengers. Thus, the
IP3-sensitive pool obviously plays an important role in
pancreatic Ca2+ responses, and it steadily contributes to
the Ca2+ release in a wide range of ACh concentrations as
evidenced by the smoothly graded response curve from KO cells in Fig.
5A. The superposition of cADPR on IP3 and/or yet
unknown messengers manifested two prominences on the curve, suggesting
the presence of two subdivisions of cADPR-sensitive Ca2+
pools (the first and second cADPR-dependent components
described above). The first cADPR-dependent
Ca2+ pool is strictly sensitive to ryanodine, but the
second one is rather resistant as evidenced by Fig. 6C.
A large part of the Ca2+ release is from the first
cADPR-dependent pool at concentrations of ACh up to 40 nM, as shown in Table I and Fig. 4. The contribution of
this pool promotes the sharp repetitive spikes (Fig. 4A),
which could be advantageous for the fine control of
Ca2+-dependent cell function (39). That the
repetitive spikes were severely impaired in the knockout mice suggests
that they depend on cADPR formation. Thus, it could be that cADPR is
one of the crucial messengers for maintaining the repetitive
Ca2+ spikes. However, we cannot exclude the possible
contribution of IP3. It has been reported in other cells
that IP3 is generated in an oscillatory manner (40). We do
not know the detailed pattern of cADPR and IP3 formation in
our cells; however it is likely that the ACh stimulation generates both
messengers in a repetitive or oscillatory fashion. The synchronized
formation of these two messengers may generate various patterns of
repetitive Ca2+ spikes under cooperation with increased
cellular Ca2+, which is ascribed mainly to the first
cADPR-dependent Ca2+ pool. The critical
concentration of ACh that switched the mode of the Ca2+
response from the repetitive spike to the phasic deflection was 400 nM (Table I, "Frequency"). Based on the above findings,
it could be that the messenger formation is no longer repetitive but
rather phasic over this concentration of ACh. The significance of the
second cADPR-dependent Ca2+ pool could be that
it serves as the resource of the phasic release of
Ca2+.
Ryanodine was able to induce Ca2+ release at an exceedingly
high rate (Fig. 6D) with concentrations of ACh over 400 nM in KO cells. This may have resulted from a complex
mechanism that involves enhanced sensitivity to ryanodine in the second
cADPR-sensitive Ca2+ pool. Such an enhancement may have
arisen from the constant lack of cADPR in the KO cells (Fig.
1B).
CD38 in the Ca2+ Signaling of Muscarinic Receptor
Stimulation--
The presence of CD38 in the Ca2+ release
mechanism is of particular importance. The CD38-mediated
Ca2+ signaling may be associated directly with the receptor
stimulation (Fig. 2A). This signaling pathway may be
separated from that of IP3 since both the formation of
cADPR (Fig. 2A) and its contribution to the Ca2+
release (Fig. 5A) are large at 40 nM ACh, at
which concentration the contribution of IP3 is still low.
It is likely that muscarinic stimulation activates both signaling
pathways in parallel and that they contribute synergistically to the
overall Ca2+ signaling in pancreatic acinar cells. However,
the precise underlying mechanism of cADPR formation after the
muscarinic ACh stimulation remains unknown.