(Received for publication, March 29, 1995; and in revised form, July 11, 1995)
From the
We have previously shown that agonist-induced Ca mobilization in intestinal longitudinal muscle is mediated by
ryanodine-sensitive, inositol 1,4,5-trisphosphate-insensitive
sarcoplasmic Ca
channels. Ca
release via these channels is triggered by agonist-stimulated
Ca
influx and results in Ca
-induced
Ca
release. The present study examined whether cyclic
ADP-ribose (cADPR) is synthesized in response to stimulation of
longitudinal muscle by agonists and modulates the activity of
Ca
release channels. Cyclic ADPR bound with high
affinity to dispersed longitudinal muscle cells (IC
1.9
nM) and induced Ca
release (EC
3.8 nM), increase in
[Ca
]
(EC
2.0 nM), and contraction (EC
1.1
nM); cADPR had no effect on circular muscle cells. The effects
of cADPR were blocked by ruthenium red, dantrolene, and the specific
antagonist, 8-amino-cADPR, and were augmented by caffeine but not
affected by heparin. The binding of cADPR and its ability to stimulate
Ca
release were dependent on the concentration of
Ca
. Cyclic ADPR was capable of stimulating
Ca
release at subthreshold Ca
concentrations (25-100 nM) and of enhancing
Ca
-induced Ca
release. Longitudinal
muscle extracts incubated with
-NAD
produced a
time-dependent increase in Ca
-mobilizing activity
identified as authentic cADPR by blockade of Ca
release with 8-amino-cADPR and ruthenium red.
Ca
mobilizing activity was increased by
cholecystokinin octapeptide (CCK-8) in a concentration-dependent
fashion. The increase induced by CCK-8 was suppressed by the CCK-A
antagonist, L364,718, nifedipine, and guanyl-5`-yl thiophosphate. The
study shows that ADP-ribosyl cyclase can be stimulated by agonists and
that cADPR can act as an endogenous modulator of
Ca
-induced Ca
release.
Cyclic adenosine diphosphoribose (cADPR) ()was
originally identified as a Ca
-mobilizing agent by its
ability to release intracellular Ca
in sea urchin
eggs(1, 2) . More recent studies suggest that cADPR
can release intracellular Ca
in a variety of
mammalian cells including sensory neurons(3) , cardiac
myocytes(4) , pituitary cells(5) ,
cells of
pancreatic islets(6) , and pancreatic acinar cells(7) .
An isoform of ADP-ribosyl cyclase, the enzyme responsible for cADPR
synthesis, has been purified from the ovotestis of the marine mollusc, Aplysia californica(8) , and subsequently cloned (9) . A homologous enzyme with dual ADP-ribosyl cyclase and
cADPR hydrolase activities is widely expressed in the plasma membrane
of mammalian cells (10) and is similar or identical to the
human leukocyte antigen CD38(11) . CD38 has been cloned from
human insulinoma (12) and rat pancreatic islets(13) ;
its expression in COS1 cells leads to cADPR-sensitive Ca
release(14) .
The ability of cADPR to release
Ca is blocked by procaine, ruthenium red, and high
concentrations of ryanodine (15, 16) and enhanced by
caffeine and divalent cations(17) , lending support to the
notion that cADPR activates ryanodine receptor/Ca
release channels. However, cADP ribose may not bind directly to
ryanodine receptors but to accessory proteins, probably calmodulin,
that may couple cADPR to channel activation(18) . Consistent
with this notion, 8-amino-cADP ribose, which acts as a selective cADPR
antagonist, does not block caffeine- or ryanodine-induced
Ca
release(19) .
A functional role for
cADPR as a modulator of ryanodine receptor/Ca release
channels has not been established in many tissues where the enzymatic
machinery for its synthesis is present(10, 20) .
Convincing evidence, however, exists for its role as a
Ca
-mobilizing messenger in sea urchin eggs (1, 2) and in
cells of pancreatic
islets(6) . IP
and cADPR are produced during
fertilization of sea urchin eggs where they activate distinct
Ca
channels jointly responsible for Ca
waves(21, 22) . In intact islet
cells,
where exogenous cADPR has been shown to induce Ca
release and insulin secretion, glucose stimulates cADPR synthesis
and Ca
influx via voltage-sensitive Ca
channels; the increase in cADPR and cytosolic Ca
act synergistically to stimulate Ca
release via
ryanodine-sensitive, IP
-insensitive Ca
channels(6) . The sensitivity to cADPR is not retained in the
RINmf5 cell line or in
cells from ob/ob mice(23) .
Agonist-induced Ca mobilization in intestinal
longitudinal smooth muscle cells exhibits a striking similarity to
glucose-induced Ca
mobilization in intact islet
cells. Ca
mobilization in intestinal longitudinal
muscle is mediated by Ca
influx via voltage-sensitive
Ca
channels, which triggers Ca
release via ryanodine-sensitive, IP
-insensitive
Ca
channels(24) . The mechanism differs from
that in adjacent circular muscle, which is mediated by Ca
release via IP
-sensitive, ryanodine-insensitive
Ca
channels(25) . In the present study, we
have explored the possibility that cADPR is synthesized in response to
stimulation of longitudinal muscle cells by contractile agonists (e.g. CCK-8) and acts as a modulator of Ca
release from ryanodine-sensitive Ca
stores.
cADPR bound with high-affinity, stimulated Ca
release
and contraction, and enhanced Ca
-induced
Ca
release in longitudinal but not circular muscle
cells. Cyclic ADPR was synthesized by longitudinal muscle cells only,
and its synthesis was increased in a concentration-dependent fashion by
treatment of the cells with the contractile agonist, CCK-8. The results
provide the first evidence of a messenger role for cADPR in
agonist-mediated Ca
mobilization.
Muscle cells were permeabilized in some
experiments by incubation for 10 min with 35 µg/ml saponin in a
medium containing 50 nM Ca as described
previously(24, 25) . The medium consisted of 20 mM NaCl, 100 mM KCl, 1 mM MgSO
, 25
mM NaHCO
, 0.18 mM CaCl
, 1
mM EGTA, and 1% bovine serum albumin. The cells were washed
free of saponin by centrifugation at 150
g and
resuspended in the same medium with 1.5 mM ATP and
ATP-regenerating system (5 mM creatine phosphate and 10
units/ml creatine phosphokinase). A HEPES-buffered permeabilization
medium was used in experiments involving measurement of cytosolic
Ca
.
In other experiments, muscle cells were
transiently permeabilized by incubation for 20 min at 31 °C with
TransPort reagent (15 µl/ml) in Ca
-free
medium as described previously(26, 27) . The resealed
cells retained their length (105 ± 2 µm), excluded trypan
blue (98 ± 1%), and did not contract upon addition of 2 mM Ca
. Cells treated with GDP
S during
permeabilization contracted with KCl (30 mM) but not with
CCK-8.
The supernatants were incubated at 37 °C in
the presence of 3 mM -NAD
, the preferred
substrate for ADP-ribosyl cyclase. Samples (20 µl) were obtained
prior to addition of
-NAD
, immediately after
addition of
-NAD
, and at intervals thereafter for
up to 60 min. The samples were assayed for their ability to mobilize
Ca
from nonmitochondrial Ca
stores
using permeabilized longitudinal muscle cells as a bioassay system.
Ca
mobilization was measured by fura2 fluorescence
and by Ca
release from cells preloaded with
Ca
. The presence of cADPR was identified
using 8-amino-cADPR, a selective antagonist of cADPR(19) , and
confirmed with ruthenium red, a blocker of ryanodine-sensitive
Ca
channels.
Figure 1:
Binding of
[H]cADPR to permeabilized intestinal muscle
cells. Upper panel, time course of
[
H]cADPR binding. Permeabilized longitudinal and
circular intestinal muscle cells (5
10
cells in 0.5
ml) were incubated with 1 nM [
H]cADPR
(specific activity, 68 Ci/mmol) at 21 °C for various time
intervals. Bound and free radioligand were separated by filtration, and
nonspecific binding (15 ± 6% of total binding) was determined in
the presence of 1 µM unlabeled cADPR. Inset,
Ca
dependence of [
H]cADPR
binding in longitudinal muscle cells. Results are expressed in
fmol/10
cells. Measurements were made in triplicate; values
are means ± S.E. of three experiments. Lower panel, inhibition of [
H]cADPR binding by unlabeled
cADPR. Permeabilized longitudinal muscle cells were incubated for 10
min with 1 nM [
H]cADPR in the presence
of increasing concentrations of unlabeled cADPR. Results are expressed
as a percent of specific binding (IC
1.9 ± 0.5
nM). Measurements were made in triplicate; values are means
± S.E. of three experiments.
[H]cADPR
binding was inhibited in a concentration-dependent fashion by unlabeled
cADPR with an IC
of 1.9 ± 0.5 nM (Fig. 1). Binding was also inhibited 70 ± 1% (p < 0.001) by 10 µM ryanodine and 54 ± 6% (p < 0.001) by 1 µM ruthenium red but was not
affected by 10 µM IP
(2 ± 3%; not
significant).
cADPR stimulated Ca
release from
longitudinal but not circular muscle cells.
Ca
release was rapid, attained a maximum (37 ± 4% decrease in
steady-state
Ca
cell content) within 15
s and was followed by slow re-uptake of Ca
(Fig. 2). cADPR-induced
Ca
release was concentration-dependent (EC
3.8 ±
1.4 nM) (Fig. 2) and was inhibited by ruthenium red and
dantrolene and was augmented by caffeine in a concentration-dependent
fashion (Table 1). Maximal release was similar to that induced by
CCK-8 and ryanodine in longitudinal muscle cells(24) . In
contrast, as previously shown(23, 24) , a maximally
effective concentration of IP
(10 µM)
stimulated
Ca
release from circular but
not longitudinal muscle cells (data not shown).
Figure 2:
Ca release induced by
cADPR in permeabilized intestinal circular (open circles) and
longitudinal (closed circles) muscle cells. Upper panel, time course of cADPR-induced
Ca
release. Permeabilized muscle cells were suspended in a medium
containing 50 nM Ca
,
Ca
(10 µCi/ml), 10 µM antimycin, and ATP regenerating system.
Ca
uptake was initiated with 1.5 mM ATP and attained a steady state within 60 min (2.41 ± 0.08
and 2.43 ± 0.12 nmol/10
in circular and longitudinal
muscle cells). Results are expressed as percent of steady-state
Ca
cell content. Measurements were made
in triplicate; values represent means ± S.E. of three to six
experiments. Lower panel, concentration dependence of
cADPR-induced Ca
release. Measurements were made 15 s
after addition of cADPR (EC
3.8 ± 1.4 nM).
Results are expressed as a percent of the maximal decrease in
steady-state
Ca
cell content induced by
10 µM ryanodine. Measurements were made in triplicate;
values are means ± S.E. of three to six
experiments.
Figure 3:
Concentration-dependent contraction and
increase in [Ca] induced by cADPR in
intestinal muscle cells. Upper panel, permeabilized muscle
cells were loaded with fura2 in 50 nM Ca
for
measurement of fluorescence as described under ``Experimental
Procedures.'' Results are expressed as increase in
[Ca
] above ambient level (50 nM).
Values are means ± S.E. of five experiments. Lower panel, muscle cells were treated with cADPR for 15 s, and the reaction
was terminated with 1% acrolein. Cell length was measured by scanning
micrometry and contraction expressed as percent decrease in cell length
from control (control cell length in longitudinal and circular muscle
cells, 108 ± 2 and 109 ± 1 µm, respectively). Values
are means ± S.E. of four experiments.
In permeabilized longitudinal muscle
cells treated with 10 µM ryanodine or 1 µM
thapsigargin so as to deplete intracellular Ca
stores, cADPR lost its ability to cause contraction (99 ± 2 and
99 ± 4% inhibition with ryanodine and thapsigargin). Treatment
with 10 µg/ml heparin for 10 min had no effect on contraction
induced by cADPR (data not shown).
Figure 4:
Potentiation of
Ca-induced Ca
release in
longitudinal muscle cells by cADPR. Cells were loaded with
Ca
, and Ca
release was
measured as described in the legend to Fig. 2. The cells were
exposed to discrete changes in Ca
concentration,
alone or in the presence of 1 nM cADPR. cADPR elicited
significant Ca
release at subthreshold Ca
concentrations (25-100 nM) and potentiated
Ca
release at higher Ca
concentrations. Measurements were made in triplicate; values are
means ± S.E. of seven experiments.**, p < 0.01;
*, p < 0.05 from control Ca
release
induced by Ca
alone.
Figure 5:
Ca release induced by
extracts of longitudinal muscle cells incubated with
-NAD
. Upper panel, extracts from
longitudinal (closed circles) or circular (open
circles) muscle cells were incubated for various intervals with 3
mM
-NAD
. The extracts were assayed for
their ability to cause an increase in [Ca
]
in permeabilized longitudinal muscle cells loaded with fura2. No
increase in [Ca
] was induced by extracts
prior to incubation with
-NAD
or immediately
after addition of
-NAD
(zero time). Measurements
were made in duplicate; values are means ± S.E. of four
experiments.**, p < 0.01; *, p < 0.05. Lower panel, extracts were obtained from control longitudinal
muscle cells and cells pretreated with 1 nM CCK-8. Various
amounts of extract (mg of protein) were incubated for 10 min with 3
mM
-NAD
and assayed for their ability to
increase [Ca
] in fura2-loaded longitudinal
muscle cells in the presence (open circles) or absence (closed circles) of 8-amino-cADPR. Measurements were made in
duplicate; values are means ± S.E. of four experiments.**, p < 0.01; *, p < 0.05 for the difference between
extracts from CCK-treated and untreated
cells.
Figure 6:
Effect of ruthenium red and heparin on
Ca release induced by longitudinal muscle cell
extracts. Extracts were obtained from control and CCK-treated cells as
described in the legend to Fig. 5and incubated for 10 min with
3 mM
-NAD
. The ability of the extracts
to stimulate Ca
release was measured in fura2-loaded (upper panel) and in
Ca
-loaded
longitudinal muscle cells (lower panel). Measurements were
made in duplicate; values are means ± S.E. of five
experiments.**, p < 0.01; *, p < 0.05, for the
difference from control.
Ca-mobilizing activity in longitudinal muscle
extracts was also assayed by measurement of
Ca
release. The addition of extracts incubated with
-NAD
for 10 min to permeabilized longitudinal
muscle cells loaded with
Ca
caused
prompt
Ca
release that was maximal
within 15 s. The addition of extracts obtained from cells that had
first been treated with various concentrations of CCK-8 elicited a
significantly greater increase in
Ca
release, which was proportional to the concentration of CCK-8 (Fig. 7). Ca
release induced by extracts from
cells pretreated with 1 nM CCK-8 was 79 ± 10% of
Ca
release induced by a maximally effective
concentration of authentic cADPR (1 µM). This
concentration of CCK-8 elicits maximal Ca
release and
contraction in longitudinal muscle cells(24) . Ca
release induced by extracts obtained from CCK-treated and
untreated muscle cells was virtually abolished by ruthenium red (1
µM) but was not affected by heparin (10 µg/ml) (Fig. 6).
Figure 7:
Ca release induced by
longitudinal muscle cell extracts obtained from cells treated with
various concentrations of CCK-8. Extracts were obtained from control
longitudinal muscle cells and from cells treated for 30 s with various
concentrations of CCK-8. Ca
release induced by
extracts incubated for 10 min with
-NAD
was
measured in permeabilized longitudinal muscle cells loaded with
Ca
. Results are expressed in percent of
maximum Ca
release induced by 1 µM authentic cADPR. Measurements were made in triplicate; values are
means ± S.E. of three to five
experiments.
The ability of CCK-8 to cause an increase in the
Ca-mobilizing activity of longitudinal muscle
extracts was blocked when the cells were treated with either 1
µM L364,718, a selective CCK-A receptor antagonist, or
with 10 µM nifedipine (Fig. 8). Basal
Ca
-mobilizing activity (i.e. activity of
extract from cells not treated with CCK-8) was not affected by
nifedipine or L364,718. In transiently permeabilized longitudinal
muscle cells incubated with 100 µM GDP
S and then
resealed, CCK-8 also failed to cause an increase in
Ca
-mobilizing activity (Fig. 8); basal
Ca
-mobilizing activity was not affected.
Figure 8:
Inhibitory effect of CCK-A receptor
antagonist (L364,718), nifedipine, and GDPS on
Ca
-mobilizing activity stimulated by CCK-8 in
longitudinal muscle. Lower panel, longitudinal muscle cells
were treated for 30 s with 1 nM CCK-8 alone or in combination
with either 1 µM L364,718 or 10 µM nifedipine. Cell extracts were incubated for 10 min with 3 mM
-NAD
and then tested for their ability to
induce Ca
release from permeabilized longitudinal
muscle cells loaded with
Ca
. L364,718
had no effect on basal Ca
mobilizing activity.
Measurements were made in triplicate; values are means ± S.E. of
eight experiments.**, p < 0.01 for the difference between
extracts from CCK-treated and untreated cells. Upper panel, longitudinal muscle cells were transiently permeabilized in the
presence of 100 µM GDP
S and then resealed as
described under ``Experimental Procedures.'' After treatment
of the cells for 30 s with 1 nM CCK-8, extracts were prepared
and incubated for 10 min with 3 mM
-NAD
and assayed for their ability to release Ca
.
Measurements were made in triplicate; values are means ± S.E. of
four experiments. *, p < 0.05 for the difference between
extracts from CCK-treated and untreated
cells.
The study provides evidence for agonist-mediated stimulation
of ADP-ribosyl cyclase and for a functional role of cADPR as a
Ca-mobilizing messenger in intestinal longitudinal
muscle cells. The evidence is based the ability of authentic cADPR and
longitudinal muscle cell extracts to activate Ca
release channels in this cell type.
Previous studies had shown
that agonist-induced Ca mobilization in longitudinal
muscle cells is mediated by Ca
influx, which triggers
Ca
release from IP
-insensitive,
ryanodine-sensitive Ca
stores(24) . The
initial Ca
influx is mediated by G protein-coupled
activation of phospholipase A
and generation of arachidonic
acid(30, 31) . In contrast, Ca
mobilization in adjacent intestinal circular muscle cells is mediated
by IP
-dependent Ca
release(24, 25) . In the present study,
authentic cADPR was shown to bind with high-affinity to permeabilized
longitudinal muscle cells, release Ca
from
nonmitochondrial Ca
stores, increase cytosolic
Ca
, and induce contraction in a
concentration-dependent fashion. Ca
release induced
by cADPR was inhibited by the competitive inhibitor, 8-amino cADPR (19) and by the ryanodine receptor/Ca
channel
blockers, ruthenium red and dantrolene, and augmented by caffeine, but
it was not affected by heparin. The pattern is identical to that
elicited by ryanodine in this cell type (24) and is
characteristic of Ca
release via sarcoplasmic,
ryanodine-sensitive Ca
channels(32, 33, 34) . In contrast,
cADPR did not bind to permeabilized circular muscle cells or induce
Ca
release and contraction.
Both the binding of
cADPR and its ability to stimulate Ca release in
longitudinal muscle were dependent on the concentration of
Ca
. cADPR binding and cADPR-induced Ca
release increased to a maximum at 500 nM Ca
and declined at higher concentrations. A
similar enhancement in binding and cADPR-induced Ca
release at submicromolar concentrations of Ca
was previously reported in cardiac muscle microsomes (4) and sea urchin eggs(17) . cADPR was capable of
stimulating Ca
release at subthreshold concentrations
of Ca
(25-100 nM) and of enhancing
Ca
-induced Ca
release at higher
concentrations. The properties of cADPR in longitudinal muscle cells
are thus consistent with its ability to activate ryanodine
receptor/Ca
release channels as well as enhance
Ca
-induced Ca
release by these
channels.
The sensitivity of longitudinal muscle cells to cADPR made
them suitable for use as a bioassay system to measure ADP-ribosyl
cyclase activity in the basal state and after stimulation by agonists.
The usefulness of the assay is underscored by the fact that the
EC (<4 nM) for cADPR-induced Ca
release in these cells was lower than that for Ca
release from microsomes of other cell types (17 nM in
sea urchin eggs (15, 17) ; and 100-200 nM in pituitary cells(5) ,
islet cells(6) ,
cardiac myocytes(4) , and neurons(3) ). Extracts of
longitudinal muscle cells immediately upon addition of
-NAD
did not exhibit
Ca
-mobilizing activity, implying that the activity
was not attributable to
-NAD
.
Ca
-mobilizing activity developed within 5 min of
addition of
-NAD
in line with similar results in
sea urchin eggs (10) and pituitary cells(5) . Extracts
of longitudinal muscle cells incubated for 10 min with
-NAD
increased [Ca
]
and induced
Ca
release upon addition to
permeabilized longitudinal muscle cells. The decline in Ca
mobilizing activity after 10 min probably reflects concurrent ADP
hydrolase activity. Extracts prepared from cells that had first been
treated with CCK-8 for 30 s elicited significantly greater increase in
[Ca
] and
Ca
release in proportion to the concentration of CCK. The increase
in Ca
release induced by CCK-8 was blocked by the
CCK-A receptor antagonist, L364,718. The
Ca
-mobilizing activity in extracts from unstimulated
and CCK-stimulated muscle cells was identified as authentic cADPR by
the inhibitory effects of 8-amino cADPR and ruthenium red. No
Ca
-mobilizing activity was obtained from circular
muscle cells treated with
-NAD
with or without
CCK-8.
The ability of CCK-8 to stimulate ADP-ribosyl cyclase in
longitudinal muscle cells appeared to depend on Ca influx, since blockade of G protein-mediated Ca
influx with GDP
S or nifedipine abolished the increment in
Ca
-mobilizing activity induced by
CCK-8(24, 25, 27) . A sequence whereby
Ca
influx regulates cADPR synthesis does not preclude
additional G protein-dependent activation of ADP-ribosyl cyclase
similar to that recently demonstrated for activation of membrane-bound
nitric oxide synthase in gastric smooth muscle(26) . The
Ca
requirement, however, suggests that cADPR acts to
modulate Ca
-induced Ca
release
rather than initiate Ca
release.
In conclusion,
this study provides evidence that cADPR could act as an
agonist-stimulated Ca-mobilizing messenger in
intestinal longitudinal muscle cells on a par with IP
in
intestinal circular muscle cells(25) . Either messenger is
capable of inducing Ca
release and enhancing
Ca
-induced Ca
release in its target
muscle cell(24, 25) . Whereas only one messenger
operates in intestinal longitudinal (cADPR) and circular
(IP
) muscle cells, both messengers operate at discrete
sites in other cell types (e.g. sea urchin
eggs(35, 36, 37) , pancreatic acinar
cells(7) , and cerebellar neurons(38) ) where they are
jointly responsible for the spatio-temporal patterns of
Ca
mobilization.