(Received for publication, October 31, 1995; and in revised form, December 7, 1995)
From the
Cyclic adenosine diphosphate ribose (cADPR) is a potent
endogenous calcium-mobilizing agent synthesized from
-NAD
by ADP-ribosyl cyclases in sea urchin eggs
and in several mammalian cells (Galione, A., and White, A.(1994) Trends Cell Biol. 4, 431-436). Pharmacological studies
suggest that cADPR is an endogenous modulator of
Ca
-induced Ca
release mediated by
ryanodine-sensitive Ca
release channels. An
unresolved question is whether cADPR can act as a
Ca
-mobilizing intracellular messenger. We show that
exogenous application of nitric oxide (NO) mobilizes Ca
from intracellular stores in intact sea urchin eggs and that it
releases Ca
and elevates cADPR levels in egg
homogenates. 8-Amino-cADPR, a selective competitive antagonist of
cADPR-mediated Ca
release, and nicotinamide, an
inhibitor of ADP-ribosyl cyclase, inhibit the
Ca
-mobilizing actions of NO, while, heparin, a
competitive antagonist of the inositol 1,4,5-trisphosphate receptor,
did not affect NO-induced Ca
release. Since the
Ca
-mobilizing effects of NO can be mimicked by cGMP,
are inhibited by the cGMP-dependent-protein kinase inhibitor, R
-8-pCPT-cGMPS, and in egg homogenates show a
requirement for the guanylyl cyclase substrate, GTP, we suggest a novel
action of NO in mobilizing intracellular calcium from microsomal stores
via a signaling pathway involving cGMP and cADPR. These results suggest
that cADPR has the capacity to act as a Ca
-mobilizing
intracellular messenger.
Nitric oxide (NO) ()is now recognized as a signaling
molecule in many mammalian tissues where it has diverse functions as a
neurotransmitter as well as an agent mediating apoptosis (1, 2, 3, 4, 5, 6, 7) .
Although NO was first discovered as a mediator of vascular smooth
muscle relaxation, where it leads to a decrease in intracellular free
calcium
[Ca
]
(8) ,
recent reports in interstitial cells in the mammalian gut(9) ,
a macrophage line(10) , and pancreatic
cells (11) demonstrate that treatments with NO and NO donors elicit
increases in [Ca
]
.
These effects persist in the absence of extracellular calcium and can
be blocked by pretreatment with ryanodine(9, 11) ,
suggesting that NO may activate a signal transduction cascade, which
activates ryanodine-sensitive calcium release channels (RyRs). We have
studied this novel aspect of NO action in the sea urchin egg, since
Ca
release mechanisms have been extensively studied
in this system (12) and where multiple calcium mobilization
pathways have been shown and are amenable to detailed analysis. In the
sea urchin egg one Ca
release mechanism is gated by
the established second messenger, inositol 1,4,5-trisphosphate
(IP
), which is produced in response to the interaction of
many extracellular stimuli with cell surface receptors(13) .
Another involves the activation of ryanodine-sensitive calcium release
channels(14) . RyRs are present on intracellular calcium stores
in a wide range of cell types including sea urchin eggs(15) .
Here ryanodine receptors have been shown to be regulated by
cADPR(16) , a novel calcium-mobilizing metabolite that is
synthesized from
-NAD
by ADP-ribosyl
cyclases(12) . Accumulating evidence suggests that cADPR is a
widespread modulator of ryanodine receptor-mediated calcium release in
many different types of mammalian
cells(17, 18, 19, 20, 21, 22, 23, 24, 25, 26) as
well as in plants (27) .
A second messenger role for cADPR requires that it mediates the intracellular actions of hormones or neurotransmitters. We show that NO mobilizes calcium from intracellular stores in the sea urchin egg via a pathway in part involving cGMP and leading to the activation of the cADPR-sensitive calcium release mechanism.
Fig. 1shows that in single sea urchin eggs
microinjected with the Ca indicator fura-2,
application of exogenous NO dissolved in seawater (approximate final
concentration of 32 µM) caused an increase in
[Ca
]
. There was a latency of 17
± 3 s (n = 15; S.E.) before the initiation of
the [Ca
]
signal, which occurred
at a discrete locus and then spread across the egg as a rapid but
short-lived Ca
wave. The magnitude of NO-induced
calcium transients (800 ± 30 nM, n =
12; S.E.) was generally smaller than those elicited at fertilization (1
± 0.3 µM, n = 12; S.E.) and did not
result in the elevation of fertilization envelopes (0/24 eggs treated
with NO, 10-100 µM). Neither the magnitude (780
± 43 nM, n = 10; S.E.) nor the latency
for the calcium transient (18 ± 4 s, n = 10;
S.E.) was significantly affected by the removal of extracellular
calcium (Fig. 1, 5th column), indicating that it was
produced predominantly by release from intracellular Ca
stores.
Figure 1:
Images of changes in
intracellular Ca in sea urchin eggs in response to
addition of seawater containing NO. Digital ratio images measured with
the Ca
-sensitive dye fura-2 are displayed at
different times after exposure to a 50-µl aliquot of
350
µM NO to the egg chamber, resulting in an initial NO
concentration of approximately 30 µM. The images shown
were measured 3 s apart. The average egg diameter was approximately 100
µm. The first four columns from the left represent typical responses seen in four representative eggs
bathed in normal seawater, while the fifth column displays the
NO-induced Ca
signal pattern in an egg bathed in
Ca
-free seawater. Intracellular free Ca
was calibrated as described previously(38) . There was a
latency of at least 14 s before a calcium rise was detected, and the
calcium often increased in one region of the egg before spreading
across the cell (see columns 1, 2, and 5).
The increase in calcium was transient with a return to baseline, often
occurring within 10 s of its initiation. The approximate wave velocity
was of the order of 15 ± 3 µm/s (n = 12;
S.E.).
To confirm that NO was mobilizing Ca from intracellular stores we tested the effects of NO on
Ca
release in sea urchin egg homogenates. Fig. 2shows the simultaneous measurement of NO concentration
changes and transient Ca
release in sea urchin egg
homogenates stimulated with a bolus of NO-containing IM solution. There
was a rapid increase in NO concentration in the homogenate, which
reached a peak of approximately 5 µM as measured with a NO
electrode that declined over 150 s. The Ca
release
elicited by this stimulus occurred only after a latency of around 120
s. Fig. 3A shows the effect of varying the
concentration of NO (3-10 µM) in the presence of
-NAD
(50 µM) and GTP (250
µM). The magnitude of response increased with increasing
NO concentrations, whereas the latency was inversely dependent and was
as long as 180 s at lower NO concentrations. Using the sea urchin egg
microsomes as a bioassay for cADPR, we obtained a
concentration-response relationship for NO-induced cADPR production in
egg homogenates (Fig. 3A, inset).
Figure 2:
Time
course of changes in NO concentration and NO-induced Ca release in sea urchin egg homogenates. Application of a single
aliquot (10 µl) of a NO-containing IM solution (350
µM) to 5% egg homogenates (500 µl) increased the
homogenate concentration of gaseous NO immediately as measured with the
NO electrode. NO stimulated Ca
release from the same
egg homogenate at 17 °C with continuous stirring in the presence of
added
-NAD
(50 µM) and GTP (250
µM).
Figure 3:
NO-induced Ca release
from sea urchin egg homogenates. A, application of aliquots of
a NO-containing solution (350 µM) stimulated
Ca
release from 5% L. pictus egg
homogenates in a concentration-dependent manner in the presence of
added
-NAD
(50 µM) and GTP (250
µM), which was required for the effect. Inset,
concentration dependence of NO concentration on cADPR formation in egg
homogenates measured by its ability to release Ca
from 5% egg homogenates. L. pictus egg homogenates were
preincubated with
-NAD
(50 µM) and
GTP (250 µM) for 60 s prior to NO addition. Fluorescence
changes were translated to cADPR levels following calibration of the
bioassay with authentic cADPR as described under ``Experimental
Procedures.'' 100% cADPR synthesis represents the maximum
Ca
release response induced by 10.5 µM NO. B, the dependence of NO-induced Ca
release on supplementing egg homogenates with
-NAD
and GTP. Ca
release was
seen with treatments with NO (9 µM)/
-NAD
(50 µM) in the presence of 250 and 500 µM GTP. In the absence of either GTP or
-NAD
supplements, no NO-induced Ca
release was
detected. The effects of subsequent addition of cADPR (100 nM)
are also shown for each experiment. C, the reciprocal
relationship between Ca
release by different NO
concentrations (in the presence of 250 µM GTP/50
µM
-NAD
) (
) and subsequent
cADPR-induced Ca
release (
). D,
Ca
release in homogenates evoked by NO in the
presence of GTP (250 µM) and
-NAD
(50 µM) was blocked by 8-amino-cADPR (400
nM) as was release by cADPR (200 nM). R
-8-pCPT-cGMPS (200 µM) and
nicotinamide (5 mM) also blocked Ca
release
by NO but not by cADPR (200 nM). The data in each figure are
representative of at least three experiments performed on different
batches of egg homogenate.
The
mechanism of NO on Ca release was indirect since it
was unable to mobilize Ca
from purified microsomes
(data not shown), suggesting the requirement for cytosolic factors
present in crude homogenate. In addition it also required the presence
of
-NAD
and GTP. The dependence of both
-NAD
and GTP for the
Ca
-mobilizing effect of NO is shown in Fig. 3B. Addition of NO (9 µM) in the
absence of either
-NAD
or GTP to egg homogenates
alone caused no Ca
release. However, Ca
release by NO (9 µM) could be reconstituted in the
presence of both
-NAD
(50 µM) and
GTP (250 µM) (Fig. 3B). NO-induced release
displayed a latency of
100 s. Increasing the GTP concentration to
500 µM shortened the latency and increased the magnitude
of response (Fig. 3B). A rapid Ca
release by subsequent addition of cADPR (100 nM) with no
apparent delay could be achieved under all four conditions; however,
the magnitude of the cADPR response was diminished in proportion to
release obtained with NO. The effects of NO-induced Ca
release on the magnitude of subsequent release by either cADPR is
shown in Fig. 3C. The more Ca
released by NO reduces that triggered by cADPR (100 nM).
The pharmacology of NO-induced Ca
release is shown in Fig. 3D. NO-induced Ca
release was
antagonized by 8-amino-cADPR (400 nM), which also blocked
Ca
release by a subsequent addition of cADPR (200
nM) (Fig. 3D), suggesting that the effect of
NO was mediated by cADPR. Consistent with this result was that
nicotinamide, which inhibits
-NAD
conversion to
cADPR catalyzed by ADP-ribosyl cyclases, (
)abolished NO but
not cADPR-induced Ca
release (Fig. 3D). The cGMP-dependent protein kinase inhibitor, R
-8-pCPT-cGMPS (200 µM)(36) ,
also blocked Ca
release by NO but not by cADPR (Fig. 3D). These data suggest that NO-induced calcium
release requires the participation of cGMP-dependent protein kinases
and ADP-ribosyl cyclases, which may explain the requirement for cytosol
as well as GTP and
-NAD
. Since NO required the
cGMP precursor GTP for Ca
release and a
cGMP-dependent protein kinase inhibitor blocked the effects, we
examined the effects of cGMP on Ca
release in egg
homogenates. Previous studies have indicated that cGMP mobilizes
Ca
in sea urchin eggs (37) and in egg
homogenates(38) . cGMP alone does not have direct
Ca
mobilizing activity but has been reported to
enhance the synthesis of cADPR from
-NAD
(38) . Fig. 4shows that in
microsomal fractions derived from sea urchin eggs in the presence of
25% supernatant(32, 33) , treatment with cGMP leads to
the release of calcium from microsomes after a variable latency of a
number of seconds. The amplitude of
[Ca
]
release with cGMP was
dose-dependent, and the latency in the range of 300-600 s was
inversely dependent on cGMP concentration (Fig. 4A).
Mobilization of calcium was absolutely dependent on the presence of
-NAD
(38) and abolished (Fig. 4B) by the competitive cADPR antagonist,
8amino-cADPR(34) . R
-8-pCPT-cGMPS (200
µM) also completely abolished Ca
release
by cGMP (data not shown). Heparin (0.2 mg/ml), which blocks IP
receptors in sea urchin eggs (32, 38) and other
tissues, had no inhibitory effect on Ca
release in
response to cGMP, although it blocked release by IP
(1
µM) (Fig. 4C).
Figure 4:
cGMP-induced Ca release
from sea urchin egg microsomes and homogenates. A, comparison
between cADPR- and cGMP-induced Ca
release from sea
urchin egg microsomes (5%) in the presence of 25% supernatant (v/v).
cADPR caused an immediate Ca
release in a
dose-dependent manner. cGMP-induced Ca
release was
dose-dependent and only occurred in the presence of
-NAD
and showed a latency whose duration was
inversely dependent on cGMP concentration. B, the cADPR
receptor antagonist, 8-amino-cADPR (80 nM), completely
abolished cGMP-induced Ca
release. C,
IP
-induced Ca
release is immediate and
dose-dependent. Heparin (Hep, 0.2 mg/ml) abolished
Ca
release by IP
(1 µM) but
had no effect on cGMP-induced Ca
release. All figures
are representative of at least three
experiments.
We investigated whether
the Ca-mobilizing actions of NO in sea urchin eggs
were mediated by cGMP, since NO is well characterized as an activator
of soluble guanylate cyclase(7) . We measured intracellular
levels of cGMP in eggs and egg homogenates treated with NO. In
NO-treated eggs there was an approximate doubling in the intracellular
levels of cGMP (Fig. 5A). In egg homogenates NO
treatments lead to a concentration-dependent increase in the cGMP by
over 3-fold (Fig. 5B).
Figure 5:
NO increases cGMP levels in unfertilized
sea urchin eggs and egg homogenates. cGMP levels were measured using a
radioimmunoassay protocol (see ``Experimental Procedures''). A, nitric oxide was bubbled into degassed artificial seawater,
yielding a solution of 350 µM. 50 µl of this
solution was applied to sea urchin eggs (500 µl) suspended in
artificial seawater at t = 0 s. Incubations were then
continued at 22 °C. Levels of cGMP were measured at the times
indicated. Results are expressed as the mean of four separate
estimations ± S.D. B, cGMP levels were also measured in
5% egg homogenates before and after NO additions (350 nM to
105 µM) to homogenates. The initial concentrations of NO
in the homogenate were measured with a NO electrode. cGMP levels were
measured in unstimulated homogenates and 10 s after the addition of NO,
the time for peak Ca
release. Results are expressed
as the mean of six separate estimations ± S.D. for each NO
concentration.
To investigate the mechanism
of NO-induced Ca mobilization from intracellular
stores in intact sea urchin eggs, eggs were treated with
pharmacological agents that inhibit NO or cGMP effects or inhibit
cADPR-induced Ca
release mechanisms (Fig. 6).
Eggs microinjected with 8-amino-cADPR to a final concentration of 1
µM (Fig. 6, column 2) showed substantially
reduced NO-induced calcium increases in the egg (approximately 90%)
compared with the control (Fig. 6, column 1). In
experiments in Ca
-free medium the response in
8-amino-CADPR-injected eggs was completely abolished (data not shown). R
-8-pCPT-cGMPS (25 µM) also reduced
the NO-induced Ca
transient substantially (Fig. 6, column 3), suggesting a role of cGMP and
cGMP-dependent protein kinase in mediating Ca
mobilization by NO. Hemoglobin that scavenges NO and blocks
NO-mediated effects in mammalian systems (2) also blocked the
NO-induced Ca
signal in sea urchin eggs (column
4). However, intracellular injection of heparin (0.4 mg/ml, final
concentration) did not reduce the calcium transient in response to NO (column 5). There was a slight enhancement of the response (n = 5 eggs). One possibility is that heparin weakly
activates the RyR as has been reported for RyRs in lipid
bilayers(39) , and heparin has been shown to enhance
ryanodine-induced Ca
release in sea urchin
eggs(40) , although it did not augment Ca
release by cGMP in egg homogenates (Fig. 4C).
Figure 6:
Effects of cADPR antagonist
(8-amino-cADPR), G-kinase inhibitor (R-8-pCPT-cGMPS), hemoglobin, and heparin on the
NO-induced release of intracellular Ca
in intact sea
urchin eggs. Digital ratio images were measured at 22 °C, as
described in the legend for Fig. 1, and are representative of at
least five separate experiments. Column 1, an egg
microinjected with fura-2 with a 30 µM NO solution added
at t = 0, eliciting a control response. Column
2, as for column 1, except the egg was co-injected with 1
µM 8-amino-cADPR (approximate final concentration). Column 3, as for column 1, except the egg was
co-injected with 250 µM (approximate final concentration) R
-8-pCPT-cGMPS. Column 4, as for column
1, except 10 mg/ml hemoglobin was added to the bathing medium prior to
addition of a NO-containing solution. Column 5, as for column 1, except the egg was co-injected with 400 µg/ml
heparin.
cADPR has been identified as a potent
Ca-releasing agent through a Ca
release mechanism that is distinct from that regulated by
IP
(32) . In many systems, including sea urchin
eggs, cADPR appears to act as modulator of CICR through RyRs (41) . Although the number of cell types in which cADPR is an
effective Ca
-releasing agent continues to grow,
little is known about possible receptor mechanisms that may be coupled
to intracellular cADPR production.
In this investigation we
identified NO as an agonist that can mobilize intracellular
Ca by selectively activating a Ca
signaling pathway involving cADPR while having no effect on the
IP
receptor pathway. We have previously shown that cGMP can
enhance cADPR synthesis in sea urchin eggs and homogenates (38) and that this may underlie the
Ca
-mobilizing action of cGMP in this
cell(37, 42) . We have investigated whether the
guanylyl cyclase activator NO can also release Ca
from intracellular stores by activating the cADPR signaling
pathway. Surprisingly, for an agent that was first discovered as a
relaxant of smooth muscle(8) , NO was found to elicit a large
Ca
transient in intact sea urchin eggs loaded with
the intracellular Ca
reporter fura-2 (Fig. 1).
The sea urchin egg is rapidly becoming a useful system in which to
investigate Ca
mobilization since Ca
stores in these eggs express multiple Ca
release channels that participate in the fertilization
Ca
wave (43, 44) and the regulation
of these channels can be directly investigated in egg homogenates or
microsomal preparations(16) . The
Ca
-mobilizing action of NO could be reconstituted in
the egg homogenate system, greatly facilitating the analysis of its
mechanism of action. From homogenate experiments we have shown that the
NO-induced Ca
mobilization operates predominantly via
the cADPR rather than the IP
-sensitive
Ca
-release mechanism, since the effect of NO is
abolished by the cADPR antagonist, 8-amino-cADPR(34) . The
mechanism of NO action was indirect since it required additional
factors such as GTP and
-NAD
, the precursor for
cADPR synthesis. Since the NO effects are reduced by cGMP-dependent
protein kinase inhibitors and cGMP has been reported to stimulate
-NAD
metabolism to cADPR and
ADP-ribose(38) , a possible pathway for the
Ca
-mobilizing effects of NO is that NO activates a
soluble guanylate cyclase, and the resulting cGMP elevation (Fig. 5) activates a cGMP-dependent protein kinase, which then
phosphorylates ADP-ribosyl cyclase or a regulator of this enzyme,
resulting in an increase in cADPR levels. cADPR then binds to its
receptor(45) , leading to the opening of a RyR-like
Ca
channel in the endoplasmic reticulum (16, 42) resulting in a rise in
[Ca
]
(Fig. 7). However,
since the increases in cGMP in NO-stimulated eggs are modest (Fig. 5) compared with the concentrations of cGMP required to
mimic the effects of NO, we cannot exclude other actions of NO upon
cADPR synthesis such as direct ADP-ribosylation(46) , although
the requirement for the cGMP precursor GTP for NO-induced
Ca
release in homogenates and the ability of G-kinase
inhibitors to block NO effects in both intact eggs and homogenates may
favor a cGMP-dependent mechanism. The other possibility that NO/cGMP
sensitizes Ca
release through RyRs by endogenous
cADPR is unlikely since R
-8-pCPT-cGMPS or
nicotinamide blocks NO-induced Ca
release but does
not inhibit Ca
by exogenously added cADPR (Fig. 3D).
Figure 7:
Model for NO-induced Ca
mobilization in sea urchin eggs. NO activates a soluble guanylate
cyclase resulting in the conversion of GTP to cGMP. The resulting cGMP
elevation activates a cGMP-dependent protein kinase, which then
phosphorylates ADP-ribosyl cyclase or a regulator of the enzyme,
catalyzing the conversion of
-NAD
to cADPR. The
increase in cADPR levels results in the binding of cADPR to its
receptor leading to the opening of a RyR-like Ca
channel in the endoplasmic reticulum(16) , resulting in a
rise in [Ca
]
. IP
R, IP
receptor; NiAm, nicotinamide.
Since NO synthesis by constitutive NO
synthases is often calcium-dependent(1) , a NO-induced rise in
[Ca]
may serve to amplify NO
production as previously seen (9) and could also give rise to
regenerative Ca
waves seen in many single cells and
tissues(47) . Whether nitric oxide has a role in calcium
signaling at fertilization in the sea urchin egg remains to be
determined. Since the magnitude of the Ca
wave
elicited by the high concentrations of NO required to induce
Ca
release is insufficient to activate sea urchin
eggs, if such a mechanism is employed at fertilization it is likely to
be modulatory. One possible role of the NO-activated pathway being
investigated is that NO could be locally produced at the site of
sperm-egg fusion, which would rapidly diffuse across the entire cell.
This could lead to a global rise in cADPR, which could facilitate a
wave of CICR across the egg to activate it by sensitizing the
egg's CICR mechanism to activation by increases in
[Ca
]
.
The NO-stimulated
Ca mobilization pathway involving cADPR/RyRs might
augment the recently described effects of NO and cGMP in regulating
receptor-mediated Ca
influx across the plasma
membrane in other cells(48) , contribute to RyR-based
subsarcolemmal Ca
sparks, which have recently been
implicated in regulating relaxation of vascular smooth
muscle(49) , and be important in NO-induced changes in neuronal
plasticity(50) . Whether the recently described stimulation of
ADP-ribosyl cyclases in longitudinal smooth muscle by cholecystokinin (51) involves either NO or cGMP as intermediates remains to be
determined.