(Received for publication, February 8, 1996; and in revised form, February 19, 1996)
From the
Nicotinic acid adenine dinucleotide phosphate
(NAADP) is a recently identified metabolite of
NADP
that is as potent as inositol trisphosphate
(IP
) and cyclic ADP-ribose (cADPR) in mobilizing
intracellular Ca
in sea urchin eggs and
microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H.
C.(1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and
Aarhus, R.(1995) J. Biol. Chem. 270, 2152-2157). The
mechanism of Ca
release activated by
NAADP
and the Ca
stores it acts on
are different from those of IP
and cADPR. In this study we
show that photolyzing caged NAADP
in intact sea urchin
eggs elicits long term Ca
oscillations. On the other
hand, uncaging threshold amounts of NAADP
produces
desensitization. In microsomes, this self-inactivation mechanism
exhibits concentration and time dependence. Binding studies show that
the NAADP
receptor is distinct from that of cADPR, and
at subthreshold concentrations, NAADP
can fully
inactivate subsequent binding to the receptor in a time-dependent
manner. Thus, the NAADP
-sensitive Ca
release process has novel regulatory characteristics, which are
distinguishable from Ca
release mediated by either
IP
or cADPR. This battery of release mechanisms may provide
the necessary versatility for cells to respond to diverse signals that
lead to Ca
mobilization.
Three independent Ca release mechanisms are
present in sea urchin eggs. In addition to IP
, (
)both NAD
and NADP
are
very effective in mobilizing internal Ca
(1) .
The Ca
release induced by NAD
shows
a characteristic initial delay due to conversion to
cADPR(1, 2, 3) . Cellular systems responsive
to cADPR are not limited to sea urchin egg, an invertebrate
cell(1, 2, 3) , but include various mammalian
cells (reviewed in (4) ), amphibian neurons(5) , and
plant vacuoles(6) . Accumulating evidence indicates the action
of cADPR is to dramatically increase the Ca
sensitivity of the Ca
-induced Ca
release mechanism in cells (5, 7, 8, 9) .
Unlike
NAD, the Ca
release induced by
NADP
shows no delay and is kinetically faster than
that induced by IP
(1) . Systematic analyses show
that the Ca
-releasing activity of NADP
comes from a contaminant present in commercial
NADP
(1, 10) . This contaminant can be
produced from NADP
by alkaline
treatment(1, 10) , and structural characterization
shows that it is NAADP
(10, 11) . The
NAADP
-sensitive Ca
release is likely
to be a new pathway for mobilizing internal Ca
since
it is unaffected by pharmacological agents of all known Ca
release mechanisms. It is insensitive to 8-amino-cADPR, a
specific antagonist of the cADPR receptor(12) , as well as
heparin, an antagonist of the IP
receptor(1, 10) . Antagonists of the ryanodine
receptor, high concentrations of Mg
(13) ,
procaine, and ruthenium red (11) , also have no effect on the
release. Fractionation studies show that the
NAADP
-sensitive stores distribute separately from
those sensitive to IP
and cADPR as well as from
mitochondria(1, 10) . Although the
NAADP
-sensitive mechanism is functionally distinct
from that mediated by cADPR, the same metabolic enzymes that produce
cADPR, ADP-ribosyl cyclase and CD38, can also catalyze the synthesis of
NAADP
from NADP
under appropriate
conditions(14) . In this study, we provide conclusive evidence
using a caged analog that NAADP
is active in live
cells. We demonstrate the presence of a specific receptor for
NAADP
in egg microsomes, and we describe a novel
self-regulating mechanism of NAADP
.
[P]NAADP
was synthesized by
phosphorylating [
P]NAD
with
NAD
kinase to
[
P]NADP
, which was then
converted to [
P]NAADP
using
ADP-ribosyl cyclase as described previously(14) .
NAD
kinase was isolated from sea urchin egg extracts (16) and further purified by a calmodulin affinity column. The
purification procedure separates the NAD
kinase from a
contaminating ATPase, which interferes with the phosphorylation of
NAD
.
To demonstrate conclusively that NAADP is
effective in live cells, caged NAADP
was synthesized
by derivatizing NAADP
with 2-nitrophenethyldiazoethane
using a procedure similar to that described for the synthesis of caged
cADPR(15) . Caged NAADP
had no
Ca
-releasing activity in egg homogenates and produced
no Ca
change when injected into intact eggs. Fig. 1A shows that photolyzing with a brief exposure to
UV light (340 nm) for 30 s produced a rapid elevation of intracellular
Ca
as indicated by a 30-fold increase in fluo 3
fluorescence. Thereafter, multiple Ca
oscillations
occurred over a period of more than 15 min. Similar Ca
oscillations were seen in 14 other eggs. Although the exact
patterns of oscillations differ between eggs, all 15 exhibited at least
two oscillations, and all of them underwent cortical reactions when
examined afterward (e.g. the black and white image in Fig. 1B). The Ca
changes cannot be
due to nonspecific UV exposure since control eggs loaded with a higher
concentration of caged ATP and exposed for much longer periods to UV
light produced no change in Ca
(15) . The
changes also cannot be due to injection artifacts since they were
induced by photolysis subsequent to injection. The use of caged
NAADP
, therefore, provides more convincing evidence
than the microinjection protocols used previously in showing that
NAADP
is effective in live
cells(10, 17) .
Figure 1:
Ca oscillations
induced by photolyzing caged NAADP
. A,
Ca
oscillations in a Lytechinus pictus egg
were monitored by fluo 3. Fluorescence intensity was normalized by the
initial value (F/Fo). The egg was microinjected with
a cellular concentration of 103 nM caged NAADP
and 57 µM fluo 3. The bar represents 30 s
of UV photolysis. B, at various time points indicated by the
numbers in A, images of the egg were recorded and
pseudo-colored to dramatize the intensity changes. The black and white
image is that of the egg at the end of the experiment showing the
formation of the fertilization envelope surrounding the egg (indicated
by the two arrowheads in the image). C, a different
egg injected with 92 nM caged NAADP
and 51
µM fluo 3. The uncaging period was reduced to 10.5 s,
which resulted in much smaller Ca
changes. The egg
became desensitized and did not respond to subsequent NAADP
release by 45.5 s of photolysis. The horizontal bars in
the figure represent the duration of
photolysis.
NAADP is also a
potent self-desensitizer of the Ca
release mechanism
as shown in Fig. 1C. By reducing the period of UV
uncaging to 10.5 s, a threshold concentration of NAADP
was generated, which induced a much smaller Ca
response. The egg, nevertheless, became totally desensitized
because after as long as 11 min later, photolysis for 45.5 s produced
no further change in Ca
. In 6 out of 8 eggs, an
initial photolysis period of 10-20 s produced a fluo 3 increase
of 2.8 ± 0.5 (±S.E.)-fold, and the eggs were strongly
desensitized since a subsequent photolysis of 45-231 s elicited a
fluo 3 increase of only 0.7 ± 0.3-fold.
The
self-desensitization can be best demonstrated in egg homogenates.
Addition of 4 nM NAADP to microsomes elicited
minimal Ca
release, but the microsomes failed to
respond to subsequent challenge by a saturating concentration of
NAADP
administered 2 min later (Fig. 2b). A higher subsequent Ca
response was seen if the concentration of NAADP
used in the pretreatment was reduced to 0.13 nM (Fig. 2a), but it was still less than control
without pretreatment (inset of Fig. 2). Indeed,
treating the microsomes for 2 min with as low as 0.5 nM NAADP
was sufficient to reduce the subsequent
Ca
response by half (inset of Fig. 2).
Figure 2:
Subthreshold concentrations of
NAADP effectively desensitize the subsequent response.
Egg homogenates were pretreated (1st NAADP) with various
concentrations of NAADP
and challenged 2 min later
with a second addition (2nd NAADP) of 1 µM NAADP
. Ca
release was monitored
by fluo 3 at a final concentration in the homogenates of 3
µM.
The self-desensitization is also concentration- and
time-dependent. Pretreatment of the homogenates with 1 nM NAADP for 1 min produced significant reduction of
the subsequent response to 0.97 µM NAADP
(Fig. 3A). After a 10-min pretreatment, the
microsomes became totally refractory, but the response to cADPR
remained normal as compared with its control without pretreatment. The
desensitization was faster and more complete the higher the
concentration of NAADP
used in the pretreatment (Fig. 3B). For a 10-min pretreatment, 0.25 nM NAADP
was sufficient to produce 50% inactivation.
Figure 3:
Time and concentration dependence of
self-inactivation. A, egg homogenates were pretreated with 1
nM NAADP and at 1 (1`), 3 (3`), 6 (6`), and 10 (10`) min later
challenged with 970 nM NAADP
. The response to
the second challenge progressively decreased with time. The first
trace shows a control without pretreatment. When indicated, cADPR
was added to a final concentration of 200 nM. B, egg
homogenates were pretreated with various concentrations of
NAADP
(indicated on the curves) for different
periods and subsequently challenged with 0.97 µM NAADP
. Ca
release induced by
the challenge is shown. No release was detected during the
pretreatment. Ca
release was monitored by fluo 3
(final concentration in the homogenates was 3 µM). In B, the relative fluorescence intensity changes of fluo 3 in
the homogenates were calibrated by adding known concentrations of
Ca
.
It is likely that the inactivation occurs at the receptor level. To
demonstrate specific binding,
[P]NAADP
was synthesized from
[
P]NADP
in the presence of
nicotinic acid using the Aplysia ADP-ribosyl cyclase as
described previously(14) . Fig. 4A shows the
specificity of the binding of
[
P]NAADP
to egg microsomes
purified by Percoll density centrifugation. The binding was inhibited
by nanomolar concentrations of NAADP
but was not
affected by 10 µM NAD
,
NAAD
, cADPR, or cyclic ADP-ribose
2`-phosphate(14) . A small inhibition of the binding was seen
with 10 µM NADP
. This is likely due to
the contaminating NAADP
in commercial NADP
preparations(1, 10) , since the inhibition can
be removed by purification of the sample by high pressure liquid
chromatography (data not shown). Under similar conditions as described
in Fig. 4, the binding reached steady state in 2.5 min at 4
°C. The saturation of the binding reaction was tested by varying
the concentration of radiolabeled NAADP
, and maximal
specific binding was attained with ligand concentrations higher than
about 10 nM (data not shown).
Figure 4:
Specific binding of NAADP to egg microsomes. A, specific binding to
Percoll-purified egg microsomes was determined by a filter assay.
Various competitors and [
P]NAADP
(1.3 nM, 17,800 cpm/0.1 ml of assay) were added
simultaneously. After a 30-min incubation on ice, the microsomes were
filtered and washed, and the radioactivity retained on the filter was
determined by liquid scintillation. B, microsomes were
incubated with [
P]NAADP
(2.1
nM, 30,600 cpm/0.1-ml assay) for the indicated periods on ice.
NAADP
(100 nM) was then added and the
microsomes filtered and washed 10 min later as described above. For the
zero time point, [
P]NAADP and 100 nM unlabeled NAADP
were added
simultaneously.
The competitive binding
studies shown in Fig. 4A were done with the competitors
and [P]NAADP
added
simultaneously. If the microsomes were pretreated with labeled
NAADP
(2 nM) for various periods before the
addition of 100 nM unlabeled NAADP
, the
displacement of [
P]NAADP
showed
a time-dependent decrease. After 5 min of pretreatment with the label
(2 nM), NAADP
no longer could displace the
binding (Fig. 4B). This is consistent with the
time-dependent inactivation of Ca
release shown in Fig. 3. It thus appears the binding of subthreshold
concentrations of the radioactive label (1 nM) to the
microsomes is sufficient to alter the binding site in such a manner
that it is no longer accessible to NAADP
added
afterward.
The self-inactivation mechanism described in this study
is novel in that it is complete and can be induced by remarkably low
concentrations of NAADP. It occurs in live cells and
in microsomes. To our knowledge, that subactivating concentrations of
ligand can induce desensitization of its receptor, possibly through
alteration of the receptor conformation, has not been described
previously. Equally novel is the effectiveness of NAADP
to release Ca
in live eggs. Photolyzing caged
NAADP
induces a 17.6 ± 2.9-fold (n = 15) increase in fluo 3 fluorescence (cf. Fig. 1), as compared with the 3-6-fold increase induced by
either microinjecting cADPR or photolyzing caged cADPR(15) . UV
photolysis simultaneously releases NAADP
from all
parts of the eggs, circumventing possible desensitization produced by
high local concentrations, which is likely to occur during
microinjection (10, 17) . With this procedure,
remarkable patterns of long term Ca
oscillations are
revealed. Because of its self-inactivating property, it is unlikely
that NAADP
itself is responsible for the long term
oscillations. The IP
- and/or cADPR-sensitive mechanism may
be involved. However, neither cADPR nor IP
alone has been
reported to be able to generate these types of oscillations in eggs.
This suggests that the global elevation of NAADP
may
specifically set off complex interactions between cADPR- and
IP
-sensitive Ca
stores, resulting in the
generation of the long term changes. Indeed, these changes are
reminiscent of the multiple Ca
oscillations occurring
after fertilization, which are correlated with various developmental
events(18) .