(Received for publication, July 23, 1996, and in revised form, November 1, 1996)
From the Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455, § Molecular Probes, Inc., Eugene, Oregon 97402, and ¶ 3M Company, St. Paul, Minnesota 55144-1000
Nicotinic acid adenine dinucleotide phosphate
(NAADP) is a metabolite of NADP with Ca2+ mobilizing
activity. The Ca2+ release mechanism activated by NAADP as
well as the Ca2+ stores that it acts on are different from
those activated by either cyclic ADP-ribose or inositol
1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to
demonstrate unambiguously that NAADP can mobilize Ca2+
stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high
pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the
2-phosphate. This is confirmed by 31P NMR spectroscopy,
showing that the 2
-phosphate of the caged compound exhibits an altered
chemical shift of
2.6 ppm as compared with 2.3 ppm determined for the
2
-phosphate of NAADP. Caged NAADP had no Ca2+ releasing
activity at a concentration as high as 1 µM when tested on sea urchin egg microsomes. After photolysis, it released
Ca2+, was effective in nanomolar range, and was
indistinguishable from authentic NAADP. The regeneration of NAADP after
photolysis was also confirmed by HPLC analyses. The analog is
particularly susceptible to UV and can be efficiently photolyzed using
a spectrofluorimeter. To demonstrate its utility in live cells, caged
NAADP was microinjected into sea urchin eggs. Photolysis effectively
regenerated NAADP and activated Ca2+ oscillations in the
eggs. Removal of external Ca2+ did not prevent the
Ca2+ oscillations but only delayed the second
Ca2+ peak by about 45 s, indicating that the
oscillations are due to release from internal stores and not caused by
Ca2+ influx. A mechanism based on sensitization of the
Ca2+ release by Ca2+ loading is proposed to
account for the Ca2+ oscillation observed.
In addition to inositol trisphosphate, two other independent mechanisms for mobilizing internal Ca2+ stores have been identified in sea urchin eggs. Cyclic ADP-ribose (cADPR)1 and nicotinic acid adenine dinucleotide phosphate (NAADP) are Ca2+-mobilizing metabolites derived, respectively, from NAD (1-3) and NADP (4). Two modes of action of cADPR have been documented (reviewed in Ref. 5). In one case it can function as a modulator of the Ca2+-induced Ca2+ release mechanism and, synergistically with calmodulin, increase the sensitivity of the release mechanism to divalent cations by several orders of magnitude (6-9). Alternatively, it can also function as a Ca2+ messenger. Nitric oxide, through elevating intracellular cGMP levels, can activate the synthesizing enzyme of cADPR, resulting in an increase in cellular cADPR and mobilization of Ca2+ stores (10, 11). Since the Ca2+ releasing activity of cADPR was first described in sea urchin eggs, a variety of mammalian, amphibian, and plant cells have been shown to be responsive to cADPR, indicating its general relevance (1) (reviewed in Ref. 12).
NAADP is not a cyclic molecule; instead, it is formed by replacing the nicotinamide group of NADP with nicotinic acid (4). The Ca2+ release mechanism activated by NAADP has many characteristics of an independent signaling pathway. In sea urchin eggs, NAADP is by far the most effective Ca2+ release agonist and is active at nanomolar concentrations (1, 4). Heparin, an antagonist of the IP3-receptor, has no effect on the NAADP mechanism (1, 4, 13), and cell fractionation studies show that the NAADP-sensitive Ca2+ stores can be separated from those responsive to cADPR and IP3 (4). The NAADP-dependent Ca2+ release is not inhibited by high concentrations of Mg2+ (14), 8-amino-cADPR, an antagonist of the cADPR-receptor (4, 15), and does not require calmodulin (4). These properties distinguish it from the cADPR-dependent pathway. NAADP is likely to be operating through a distinct receptor. Specific binding of 32P-NAADP to sea urchin egg microsomes has been demonstrated, and cADPR has no effect on the binding (16). One novel property of the NAADP mechanism is that, at subthreshold concentrations, NAADP can completely inactivate the release system such that subsequent challenge with a maximal concentration of NAADP is ineffective (16, 17). Ligand binding studies show that the self-inactivation occurs at the level of the receptor (16). This novel property is not seen in either cADPR- or IP3-dependent Ca2+ release and is likely to be the first description of such a process in receptor-mediated function. Although the structures and functions of cADPR and NAADP are totally distinct, the two Ca2+ agonists can, in fact, be synthesized by the same enzymes (18). Both ADP-ribosyl cyclase and CD38, a lymphocyte antigen which is also a bifunctional enzyme involved in the synthesis of cADPR (reviewed in Ref. 19), can catalyze the exchange of the nicotinamide in NADP with nicotinic acid (18). The base exchange reaction dominates at acidic pH, while at neutral and alkaline pH, both enzymes preferentially cyclize NADP to produce cyclic ADP-ribose phosphate (18). In this study, we describe the synthesis of caged NAADP and the usefulness of the analog in investigating the Ca2+ release mechanism of NAADP in microsomes as well as in live cells.
NAADP was synthesized by incubating NADP (1 mM) at pH 5.0 with the Aplysia ADP-ribosyl cyclase (25 ng/ml) in the presence of 30 mM nicotinic acid for several hours at 20-23 °C and purified by HPLC using an AG MP-1 column as described previously (18).
The caging reagent 1-(2-nitrophenyl)diazoethane (NPE), originally described by Walker et al. (20) for the synthesis of caged ATP and related nucleotides and more recently used to prepare NPE-cADPR (21), was used to prepare NPE-NAADP. NAADP has three phosphate groups and one carboxylate, all of which are potentially reactive with the caging reagent, provided they are protonated during the caging reaction. The caging reaction was done at two different pH values; first at pH 4.5 and then at pH 1.3. A solution of NAADP (1.0 mg, 1.3 µmol) in water (0.5 ml) was prepared at room temperature; the pH was 4.5. A solution of the caging diazoethane reagent (13 µmol) in ether (0.5 ml) was added, and the resulting biphasic mixture stirred vigorously for 2 h in darkness, during which the ether layer changed from amber to light yellow. The ether layer was drawn off, and a fresh solution of the caging reagent was added. After stirring for another 3 h, the amber ether layer was drawn off. Thin layer chromatographic analysis indicated complete conversion of starting NAADP (RF 0.13, methanol:choroform:water:acetic acid, 13:10:3.5:0.2, silica gel plates) to a single less polar product (RF 0.87). The aqueous layer was extracted with ether (2 × l.5 ml), then applied directly to a column of Sephadex LH-20 (2 × 6 cm). The product was eluted by gravity with water and lyophilized, yielding 0.5 mg of a pale yellow powder. This caged compound was found to be not very susceptible to photolysis and was not characterized further.
Reducing the pH of the caging reaction to 1.3 resulted in a caged product much more photolabile. NAADP (24 mg, 32 µmol) was dissolved in 2.5 ml of water and the solution was adjusted to pH 1.3 by addition of dilute aqueous HCl. The diazoethane reagent (0.2 mmol) in 3 ml ether was added and stirred vigorously for 8 h in darkness as described above. The reagent was replaced twice and stirred each time for 3 h. TLC analysis indicated no remaining NAADP and two major products with RF values of 0.87 and 0.46. The least polar product (RF 0.87) co-eluted on TLC with the higher pH product described above. After the aqueous layer was extracted with ether (2 × 3 ml), the more polar product (RF 0.46) was separated on a column of Sephadex LH-20 (2 × 8 cm). A pale yellow powder of 8.6 mg (32% yield) of the caged product was obtained, which was found to be much more photolabile than the higher pH product described above. This product was further characterized and used in this study. Photolysis with a hand-held UV lamp showed significant conversion to free NAADP, as judged by TLC analysis.
Ca2+ Release in Homogenates and Intact EggsHomogenates of sea urchin egg (Strongylocentrotus purpuratus) were prepared as described previously (21). Frozen egg homogenates (25%) were thawed at 17 °C for 20 min and diluted to 5% with a medium containing 250 mM N-methylglucamine, 250 mM potassium gluconate, 20 mM Hepes, 1 mM MgCl2, 2 units/ml creatine kinase, 8 mM phosphocreatine, 0.5 mM ATP, and 3 µM fluo-3, pH 7.2, adjusted with acetic acid. The homogenates were diluted to 2.5% and finally 1.25% with the medium described and were incubated at 17 °C for 1 h between dilutions. Ca2+ release was measured spectrofluorimetrically in 1.25% homogenates with an excitation wavelength of 485 nm and emission wavelength of 535 nm. The measurements were done in a cuvette maintained at 17 °C, and the homogenates were continuously stirred. The volume of homogenate used was 0.2 ml, and additions were usually made in 2-µl volumes.
Lytechinus pictus eggs were used for the microinjection experiments. The procedures for microinjection by pressure were as described previously (21). Ca2+ changes in the injected eggs were measured using fluo-3. Samples were dissolved in the injection buffer containing 0.5 M KCl, 0.1 mM EGTA, 10 mM Hepes, pH 6.7. The injected volume was about 1% of the egg. Eggs were attached to the bottom of a protamine sulfate-coated culture dish and incubated with artificial seawater (ASW) containing 460 mM NaCl, 27 mM MgCl2, 28 mM MgSO4, 10 mM CaCl2, 10 mM KCl, 2.5 mM NaHCO3, pH 8.0. In some experiments, ASW was changed to Ca2+-free seawater (0CaSW) after microinjection using gravity perfusion. 0CaSW had the same composition as ASW except without CaCl2, and the concentration of NaCl was increased to 470 mM. In most experiments, the 0CaSW was supplemented with 0.2-1 mM EGTA. All media were kept at 17 °C.
PhotolysisActivation of caged cADPR in egg homogenates was achieved in a Hitachi spectrofluorimeter (S-2000) by alternating the excitation wavelength every 2 s between 350 nm for photolysis and 485 nm for monitoring fluo-3 fluorescence.
In some experiments, photolysis with UV light (1015 quanta/s) for 1 min was done in a Rayonet photochemical reactor (Southern New England Ultraviolet Co.) at 0-4 °C.
The UV photolysis and fluorescence measurement of individual eggs were done using the InCa2+ imaging system (Intracellular Imaging Inc., Cincinnati, OH). Excitation was provided by a 300-Watt xenon lamp equipped with filters for 340- and 485-nm light. During photolysis, the excitation light was alternated between the uncaging (340 nm) and the monitoring (485 nm) wavelengths, both of which were reflected by a BCECF Sp dichroic filter toward the objective. The fluo-3 fluorescence was selected by a long pass filter with a 500-nm cutoff and monitored by a CCD camera. Fluo-3 fluorescence was measured every 4 s. Photolysis was performed for 3.5 s between measurements. As the xenon lamp aged, the intensity of UV light decreased. In some experiments, to compensate for the diminished UV intensity, the 340-nm filter was removed during photolysis.
HPLC AnalysesHPLC separation was done with columns packed with AG MP-1 resin (Bio-Rad) and eluted with a nonlinear gradient of trifluoroacetic acid similar to that described previously (15). The final purification of the caged NAADP was achieved using a 0.5 × 5-cm Mono Q column (Pharmacia Biotech Inc.). The product was eluted using a gradient of water (solvent A) and 1 M triethylamine bicarbonate (solvent B, pH 8.8): 0-12 min, 0% B; linearly increased to 20% B from 12 to 16 min, linearly increased to 30% B from 16 to 36 min, linearly increased to 100% from 36 to 37 min and held at 100% B for 3 min before returning to 0% B.
Enzymatic Cleavage and Phosphate MeasurementsCaged NAADP, NAADP, and photolyzed caged NAADP, all at 28 µM, were incubated with 2 units/ml alkaline phosphatase (Sigma), or 3.5 units/ml nucleotide pyrophosphatase (Sigma), or both enzymes together, in the presence of 10 mM MgCl2 and 80 mM triethylamine bicarbonate, pH 8.8, for 20 min at 37 °C. The total volume of the reaction mixture was 10 µl. The phosphate released by the enzymes was measured by adding 0.1 ml of the Malachite Green reagent (22) and 10 µl of 34% sodium citrate. Absorbance at 660 nm was measured and compared with sodium phosphate standards.
31P NMR AnalysesSpectra were collected at an observation frequency of 161.9 MHz using a Varian UNITY plus 400 FT-NMR spectrometer which was equipped with a computer switchable Nalorac 4N400-5+ 5 mm 4-nucleus probe (1H, 19F, 13C, 31P). All samples were dissolved in a D2O solution buffered at pH 7.5 with 10 mM Hepes. All spectra were acquired at an ambient room temperature of 22-23 °C. A phosphoric acid (H3PO4) solution in methanol in a 2-mm coaxial tube was used as external 31P NMR standard for zero chemical shift calibration for all spectra. Negative chemical shifts are upfield of (lower frequency than) H3PO4. The acquisition conditions used for all the spectra include spectral width of 48543 Hz; acquisition time of 0.338 s; number of acquisition data points of 32768; pulsed flip angles ranging from 20 to 50°, depending on the experiment (PW90 was about 50 µs); relaxation delay of 3-4 s, depending on the experiment; and number of transients ranging from 400 to 30,000, depending on the experiment. All 31P NMR spectra were proton-decoupled, with the decoupler gated on only during the acquisition period of the free induction decay. Data processing included zero-filling the free induction decays to 65536 data points and using an exponential weighting apodization function with a line broadening of 2.5 Hz to improve the signal-to-noise ratio in the transformed spectrum.
We have previously synthesized caged cADPR that is particularly photolabile. Effective photolysis can be accomplished with standard spectrofluorimeters or epifluorescence setups, and no specialized equipment is required (21). NAADP represented a special challenge since it has three phosphate groups as well as a carboxyl group which are all reactive toward the NPE reagent (20). Since it is known that the NPE reagent reacts mainly with protonated groups, the caging reaction was performed at either pH 4.5 or 1.3 so that different ionizable groups could be sampled. The caged product obtained at the higher pH value was found to be difficult to photolyze. Even after prolonged exposure to a hand-held UV lamp, no detectable NAADP was produced as judged by TLC analysis. In sea urchin egg homogenate, the caged material was inactive as a Ca2+ mobilizer, but prolonged photolysis only slowly and weakly generated free NAADP (data not shown). The product was not characterized further, but the poor photolysis properties are characteristic of carboxylates caged as NPE esters (23).
We next reduced the pH of the reaction to 1.3, a condition at which all four groups would be reactive. We did not have prior knowledge of which phosphate group would be the most appropriate for caging and which specific groups should be protected from being caged. Therefore, the strategy was not to focus on a particular group, but instead, to separate the mixture of products by chromotography and analyze each fraction using sea urchin egg homogenates as a bioassay for Ca2+ release (1). The product was expected not to release Ca2+ on its own but to be easily photolyzed using a spectrofluorimeter and to regenerate the Ca2+ release activity of NAADP.
TLC analyses of the caged products produced at pH 1.3 showed two main
spots with RF values of 0.87 and 0.46, respectively. The more polar products (RF 0.46) were separated
using a Sephadex column (see "Experimental Procedures"). Fig. 1
(top) shows a HPLC chromatograph of the caged
products analyzed on an AG MP-1 column. It was found to contain several
components. All three peaks were collected and tested for
Ca2+ release activity before and after photolysis. Only the
smallest peak, indicated with an asterisk, satisfied our
criteria. The product was purified one more time on an AG MP-1 column
followed by twice on a Mono-Q column. Fig. 1 (bottom) shows
the purified product eluted as a single peak on the second Mono-Q
column. Starting from 5 mg of the product mixture, about 50 µg of
purified caged NAADP was obtained.
Structural Determination
To determine which of the phosphates
is caged, we used nucleotide pyrophosphatase to cleave the
pyrophosphate linkage of the molecule and alkaline phosphatase to
release the phosphate groups as inorganic phosphate. Alkaline
phosphatase should also cleave the 2-phosphate group if it is not
caged. The Pi released was measured using the Malachite
Green method (22). Fig. 2 shows that treatment with
alkaline phosphatase released about 1 mol of Pi per mol of
NAADP (open bars), which corresponds to the 2
-phosphate of
the molecule. Nucleotide pyrophosphatase cleaved the molecule but did
not release any Pi by itself. After nucleotide
pyrophosphatase treatment, all the phosphate groups of the cleaved
molecule should be susceptible to alkaline phosphatase. Indeed,
treatment with both enyzmes (MIX) produced about 3 mol of
Pi/mol of NAADP, as expected. In contrast to NAADP,
treatment of caged NAADP (black bars) with alkaline
phosphatase produced no Pi, indicating that the
2
-phosphate is caged. Treatment with the combined enzymes released the
two phosphates forming the pyrophosphate linkage. The pattern of
Pi released from the photolyzed caged NAADP (gray bars) following the enzyme treatments is the same as that of
NAADP. These results are consistent with the caged group being attached to the 2
-phosphate of NAADP.
The structural assignment is further confirmed by 31P NMR
analyses. Fig. 3 compares the 31P NMR
spectra of two standards, NAADP and nicotinic acid adenine dinucleotide
(NAAD), with that of the purified caged NAADP. The chemical shift was
calibrated with an external phosphoric acid sample that had its
chemical shift set to zero. The spectrum of NAADP shows two peaks at
2.3 ppm and 12.2 ppm. The negative parts/million peak represents the
diphosphate of NAADP since its integrated area is about twice (2.09)
that of the other peak. The two phosphorus atoms of the diphosphate are
chemically equivalent and both are expected to have the same chemical
shift. This assignment is supported by the spectrum of NAAD. The
compound lacks the 2
-phosphate but contains the same diphosphate, and
its spectrum shows only one peak at
12.2 ppm, a chemical shift value
very similar to the diphosphate peak of NAADP. The spectrum of the
caged NAADP also shows two peaks. The diphosphate peak has a chemical
shift of
12.4, very similar to the diphosphate peaks of both NAADP
and NAAD. The graphical integration shown above the spectrum indicates the area of the diphosphate peak is 1.78 times that of the 2
-phosphate peak. The chemical shift of the 2
-phosphate of the caged compound is
changed to
2.6 ppm from 2.3 ppm of the 2
-phosphate of NAADP. This
dramatic shift of the 2
-phosphate is consistent with the caging group
being attached there. A noticeable feature of the spectrum of caged
NAADP is that the peaks are broader than that of NAADP. The reason for
the broadening is not known but could be due to reduced mobility of the
caged compound in solution. The hydrophobicity of the cage group could
promote intermolecular association especially at the relatively high
concentration (
4 mg/ml) needed for obtaining the 31P NMR
spectrum. In any case, results from specific enzymatic cleavage and
31P NMR analyses are consistent with the caging group being
on the 2
-phosphate. The structure of caged NAADP is shown in Fig.
4.
Photolysis
Fig. 5B shows that
addition of caged NAADP from 90 to 900 nM to egg
homogenates produced no Ca2+ release. The small jumps of
fluo-3 fluorescence at high concentrations of caged NAADP were due to
addition artifacts (e.g. small Ca2+
contamination in the samples), since they were present even when the
release mechanism was totally inactivated (tracing labeled 8 in Fig. 5A). A novel property of the
NAADP-sensitive Ca2+ release is that the release mechanism
can be totally inactivated by pretreatment with a subthreshold
concentration of NAADP as low as 1 nM (16, 17). The
multistep procedure described above for the purification of caged NAADP
was designed to ensure that the contaminating NAADP is below the
self-inactivating levels. Fig. 5 also shows the results of testing the
inactivating effect of caged NAADP. Egg homogenates were pretreated
with 1 nM NAADP and subsequently challenged with a
maximal concentration (40 nM) (Fig. 5A). After 2 min of pretreatment, the response to 40 nM NAADP was
substantially reduced (tracing labeled 2
) as compared to
without pretreatment (tracing labeled 0
) and was totally
eliminated after 8 min of pretreatment (tracing labeled 8
).
Pretreatment of the homogenates with 90 nM caged NAADP for
2 min produced very little inactivation (Fig. 5B). At 540 nM caged NAADP, the extent of inactivation following the
pretreatment was similar to that effected by 1 nM of NAADP
(comparing Fig. 5A, 2
, with Fig. 5B, 540 nM). This extent of inactivation
by the caged compound can be accounted for if the sample is
contaminated with about 0.1-0.2% NAADP.
This appears to be the case since freshly purified samples of the caged
compound at 200 nM exhibited essentially no inactivation as
shown in Fig. 6. Even at 900 nM of caged
NAADP, the inactivation due to pretreatment was only about 30-40%.
Results in Fig. 2 indicate NAADP is very sensitive to alkaline
phosphatase while the caged compound is not. Treatment of the 900 nM sample with alkaline phosphatase (+APase),
indeed, essentially removed all the inactivation. Results described in
Figs. 5 and 6 thus show that caged NAADP is biologically inactive. It
does not release Ca2+ nor does it induce inactivation.
Fig. 7 shows that photolysis of caged NAADP regenerates
NAADP. The samples, before and after photolysis, were analyzed by HPLC.
The retention time of the photolyzed product was the same as that of
NAADP, which was shifted as compared with caged NAADP. The photolyzed
product was effective in releasing Ca2+ as shown in Fig.
8 and, in fact, its concentration dependence was
indistinguishable from that of NAADP. Also shown in Fig. 8 is that
caged NAADP, before photolysis, had no Ca2+ releasing
activity at concentrations as high as 1 µM. Further evidence that the photolyzed product is NAADP is provided by its desensitization of the NAADP-dependent Ca2+
release in egg homogenates. The results are shown in the
inset of Fig. 8. At 28 nM, the photolyzed
product induced rapid Ca2+ release. The egg homogenate
became totally desensitized such that subsequent addition of 80 nM NAADP, after the Ca2+ was resequestered, did
not produce any release. Homogenates desensitized to prior exposure to
80 nM NAADP also did not respond to the photolyzed product.
This cross-desensitization indicates the photolyzed product is indeed,
NAADP.
Ca2+ Release Activity
Fig. 9 shows
that NAADP can be regenerated from caged NAADP using a
spectrofluorimeter. The excitation wavelength was alternated between
350 nm for photolysis and 485 nm for monitoring the fluorescence of the
Ca2+ indicator, fluo-3. Addition of caged NAADP to the egg
homogenates with the alternating UV excitation turned on resulted in
Ca2+ release after a brief delay (Fig. 9A). The
delay was more prominent at the lower concentrations. Comparison of the
Ca2+ release activity with that induced by NAADP itself
shows that about 1% of the caged NAADP added was photolyzed. This low
efficiency is due to the relative weak UV excitation light of the
spectrofluorimeter. Fig. 9B compares the
concentration-response of NAADP and caged NAADP with or without UV
photolysis. Because NAADP is effective in releasing Ca2+ at
nanomolar concentrations, the low efficiency of photolysis by the
spectrofluorimeter does not hamper its use in this setting. A common
problem in measuring Ca2+ release from a suspension of
permeabilized cells or cell-free assays, such as that shown in Fig. 9,
is distinguishing Ca2+ release from Ca2+
contamination in the samples. The caged analog should be useful since
it is not biologically active until photolysis, which can be
accomplished conveniently by simply alternating the excitation wavelength in a spectrofluorimeter.
The caged analog is also useful in single cell measurements. We have
previously shown that photolyzing caged NAADP loaded into sea urchin
eggs can induce Ca2+ oscillations (16). In some cases,
these Ca2+ oscillations persist for more than 30 min. The
possibility that Ca2+ influx may be involved in generating
these oscillations was investigated by removing external
Ca2+. We focused on the first two Ca2+
oscillations that occur within 6-7 min after photolysis. Fig. 10 shows that removal of external Ca2+ does
not prevent the Ca2+ change induced by photolysis nor does
it inhibit the subsequent Ca2+ oscillation that occurs
spontaneously. The main effect of removal of external Ca2+
is a delay of the occurrence of the second Ca2+ peak. This
delay separates the second peak farther from the first peak and makes
it appear more prominent in the case of 0CaSW. Table I
summarizes the results from 16 eggs in 0CaSW and 17 eggs in ASW. Both
the magnitude of the two Ca2+ peaks and the time of the
first Ca2+ peak are independent of external
Ca2+. These results show that the internal stores are the
main source of the Ca2+ changes induced by photolyzing
caged NAADP as well as the subsequent Ca2+ oscillation. It
should be noted that in another five eggs in 0CaSW, photolysis induced
no change in internal Ca2+. It is likely that these eggs
had suffered damage during microinjection. The leakage of EGTA into the
eggs could have buffered the Ca2+ changes. In ASW the
second peak occurred at 115.7 ± 6.6 s after the start of
photolysis. In 0CaSW, the second peak occurred at 161.5 ± 12.6 s, a 45-s delay. The exact mechanism of how removal external
Ca2+ can delay the Ca2+ oscillation remains to
be elucidated. One possibility is proposed in the "Discussion."
|
As described in the introduction, the Ca2+ release mechanism activated by NAADP has many properties of a signaling pathway. In this study, we describe two other properties of NAADP that strengthen its signaling role. First, it is highly effective in live cells, which can be unambiguously demonstrated using the caged analog. Indeed, loading of eggs with about 200 nM caged NAADP was more than sufficient (Table I), a concentration which is 10-fold lower than that required for caged cADPR (21). Removal of external Ca2+ did not inhibit the Ca2+ change induced by photolyzing caged NAADP in live eggs, indicating the source of Ca2+ is from internal stores.
Second, NAADP can be degraded effectively by phosphatases, such as nucleotide pyrophosphatase and alkaline phosphatase. An effective enzymatic system for removal is a hallmark of a signaling molecule, whose action needs to be terminated once its function is completed. The degradation pathway is particularly important for NAADP because of its potent self-inactivation property. The general presence of phosphatases in cells should ensure that NAADP is degraded.
Photolysis of caged NAADP in live eggs produces not only a single Ca2+ transient but also spontaneous Ca2+ oscillations that last for more than 30 min (16). The oscillation is not abolished by removal of external Ca2+, indicating that the Ca2+ also comes from internal stores (Fig. 10). The exact mechanism is not known. One possibility is that the Ca2+ released from the NAADP stores is sequestered by cADPR-sensitive stores, overloading the latter and triggering spontaneous release. It has previously been shown that Ca2+ overloading not only can trigger spontaneous release in egg homogenates, but also can sensitize the stores to cADPR by 50-fold or more (24). The sensitization could conceivably enable the basal concentration of cADPR endogenously present in eggs to activate further release, generating the second Ca2+ peak seen in Fig. 10. If external Ca2+ is present, influx could hasten the overloading and/or sensitization of the cADPR stores and speed up the occurrence of the second Ca2+ peak as shown in Fig. 10. The overloading mechanism could also account for the ineffectiveness of either cADPR or IP3 in triggering Ca2+ oscillation, since both agonists release from the same stores (16, 25), and the majority of the Ca2+ released by either of them would likely be sequestered by the NAADP-sensitive stores, which may not have the same type of Ca2+ release mechanism that is sensitive to intravesicular Ca2+. Whether this overloading mechanism is in fact operative in eggs remains to be established, but it does provide rationalization of how the interplay of various Ca2+ stores can result in generation of Ca2+ oscillation.
The synthesis of caged NAADP described in this study produces only
modest yield. This is not a major problem for biological use because of
the incredible potency of NAADP in inducing Ca2+ release.
The main reason for adopting the nonselective strategy we have used is
that there was no a priori knowledge of which of the four
acidic groups should be caged. The results described in this study
establish that the caging group attached to the 2-phosphate is the
most appropriate for photolysis. Improvement on the yield can now be
rationally designed by protecting the other groups before the caging
reaction.
We thank Richard Graeff for help in the phosphate measurements and critical reading of the manuscript and Timothy F. Walseth for the use of the photochemical reactor.