NAADP activates a Ca2+ current that is ... - The FASEB Journal

The FASEB Journal express article 10.1096/fj.03-0178fje. Published online August 15, 2003.

NAADP activates a Ca2+ current that is dependent on Factin cytoskeleton Francesco Moccia, Dmitri Lim, Gilda A. Nusco, Emanuela Ercolano, and Luigia Santella Laboratory of Cell Biology, Stazione Zoologica “Anton Dohrn,” Villa Comunale I-80121, Naples, Italy Corresponding author: Luigia Santella, Laboratory of Cell Biology, Stazione Zoologica “Anton Dohrn,” Villa Comunale I-80121, Naples, Italy. E-mail: [email protected] ABSTRACT Nicotinic acid adenine dinucleotide phosphate (NAADP) is involved in the Ca2+ response observed at fertilization in several species, including starfish. In this study, we have employed Ca2+ imaging and the single-electrode voltage-clamp technique to investigate whether the NAADP-mediated Ca2+ entry discovered in our laboratory in starfish oocytes was underlain by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of preinjected NAADP evoked a cortical Ca2+ flash that was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+-free sea water. Under voltage-clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+ but not of Na+. The current was affected by preincubation with verapamil, SK&F 96356, and thapsigargin but not by preinjection of heparin, 8-NH2- cyclic ADP-ribose, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin-A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+-permeable membrane current that requires an intact F-actin cytoskeleton as other Ca2+-permeable currents, such as ICRAC and IARC. Key words: Ca2+ imaging • electrophysiology • nicotinic acid adenine dinucleotide phosphate

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alcium ions represent an intracellular messenger responsible for the regulation of a number of cellular functions, ranging from fertilization to cell death (1). An increase in intracellular free Ca2+ concentration ([Ca2+]i) is triggered by both Ca2+ release from internal stores, such as sarco/endoplasmic reticulum, and Ca2+ influx from extracellular space (1, 2). Ca2+ mobilization from sarco/endoplasmic reticulum is mainly mediated by ligand-operated ionic channels, namely the inositol 1,4,5-trisphospate (InsP3) and ryanodine receptors that open upon binding of their specific modulators, InsP3 and cyclic ADP-ribose (cADPr), respectively (1). Ca2+ entry may occur through Ca2+-permeable plasma membrane channels that are activated by a number of stimuli, including membrane depolarization, store depletion, and extracellular and intracellular messengers, such as InsP3, arachidonic acid, and ADP-ribose (1–6). A rise in [Ca2+]i can be evoked also by the newly discovered second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in a variety of cell types, such as sea urchin eggs (7, 8), starfish (9, 10) and ascidian oocytes (11), pancreatic acinar cells (12, 13), human T-lymphocytes

(14), and pancreatic human β-cells (15). In most of these cells, NAADP only releases Ca2+ from an internal store, which is pharmacologically and physically distinct from InsP3- and cADPrsensitive pathways (7, 11, 12, 16), presumably the lysosomes (17) or the secretory vesicles (18). Furthermore, NAADP-induced Ca2+ release is neither inhibited nor potentiated by intracellular Ca2+ (19), but it can be self-inactivated by a previous challenge with NAADP itself (8, 12, 13). By contrast, original observations in starfish mature oocytes (i.e., oocytes exposed for 50 min to the maturing hormone 1-methyladenine) have shown that the Ca2+ response to NAADP consists of a cortical flash that is absent in Ca2+-free sea water and that spreads throughout the cytoplasm as a Ca2+ wave (9, 10). Evidently, the flash is due to Ca2+ influx from the extracellular space. In agreement with this observation, NAADP-elicited Ca2+ entry is inhibited by SK&F 96365, an inhibitor of receptor-operated calcium entry, and by the L-type calcium channel blocker verapamil (9, 10). In immature oocytes (i.e., oocytes not challenged with 1-methyladenine) as well, the Ca2+ response to NAADP is dependent on external Ca2+, but the Ca2+ wave has not yet been visualized (9). As extracellular Ca2+ was necessary for NAADP-induced Ca2+ signaling, a membrane current was likely to underlie it, although its existence has not yet been assessed. Conflicting results have been instead provided in intact sea urchin eggs, where the Ca2+ response to NAADP has been first claimed to be independent on extracellular Ca2+ (8) and, then, to involve a cortical flash produced by Ca2+ inflow (20). Whatever the source of the initial NAADP-elicited Ca2+ increase, both in mature and immature starfish oocytes (9, 10) and in intact sea urchin eggs (8) or pancreatic acinar cells (12, 13), it is then amplified by InsP3 and cADPr/ryanodine receptors through a mechanism of Ca2+-induced Ca2+ release (CICR). A mounting body of evidence is now emerging on the role played by the F-actin cytoskeleton in the regulation of the intracellular and of the plasma membrane Ca2+-permeable channels. In general, one-half of the actin in the cell is monomeric (G-actin) and the rest is polymerized into filaments (F-actin; 21). Impairment of cytoskeleton by actin depolymerizing agents inhibits hormone-induced Ca2+ release from InsP3- and ryanodine-sensitive stores in NIH 3T3 cells (22), AR4-2J cells (23), and hepatocytes (24). Moreover, actin depolymerization has been reported to reduce Ca2+ influx through voltage-gated Ca2+ channels (25), while store-operated Ca2+ influx through transient receptor potential (TRP) ion channels is affected by both actin polymerization and depolymerization (24, 26). Finally, actin depolymerization itself may elicit oscillations in [Ca2+]i in Astropecten auranciacus mature oocytes (27). So far, however, no evidence has been reported about the involvement of actin cytoskeleton in the Ca2+ response to NAADP. Because NAADP is emerging as a key messenger in the regulation of several cell functions (28), it would be of interest to investigate whether actin filaments may also modulate NAADP-evoked Ca2+ signaling. In the present study, we have employed both Ca2+ imaging and the single-electrode voltageclamp technique to assess whether NAADP-evoked increase in [Ca2+]i in immature oocytes from Asterina pectinifera was underlain by a Ca2+-permeable membrane current. We have further investigated the requirement of an intact actin cytoskeleton for the Ca2+ response to NAADP. Our results are the first demonstration of the activation of a Ca2+-mediated current by NAADP that displays biophysical properties similar to those exhibited by the Ca2+-permeable current, Ca2+ release-activated Ca2+ current (ICRAC; ref 29), and arachidonic acid-activated current (IARC; ref 30). Moreover, we have provided evidence that Ca2+ influx through NAADP-elicited current is strongly affected by both actin depolymerization and polymerization.

MATERIALS AND METHODS Preparation of oocytes Starfish (A. pectinifera) were collected during the breeding season in the Mutsu Bay (Aomori, Japan) and kept in circulating sea water (16°C). Fully grown prophase-arrested oocytes (~200 µm in diameter) containing a large nucleus were dissected from the ovaries in filtered artificial sea water (NSW; 460 mM NaCl, 10.1 mM KCl, 9.2 mM CaCl2, 35.9 mM MgCl2, 17.5 mM MgSO4, and 2.5 mM NaHCO3, pH 8.0), washed several times with it, and kept for 30 min before use. Microinjections and photolysis of caged NAADP and Ca2+ imaging The calcium fluorescent dye Oregon Green 488 BAPTA-1 coupled to a 10-kDa dextran (OGBD) was injected at a concentration of 5 mg/ml in the injection buffer (IB; 450 mM potassium chloride, 10 mM HEPES, pH 7.4) into the cytoplasm of immature oocytes. Caged NAADP (100 µM in the pipette) was injected in the cytoplasm of an oocyte 10-15 min before uncaging. As the volume of the injected substances corresponded to 1-2% of the oocyte volume, the final concentrations of OGBD and caged NAADP were 50-100 mg/ml and 1-2 µM, respectively. Cytosolic Ca2+ changes were measured by using a cooled CCD camera (MicroMax, Princeton Instruments, Inc., Trenton, NJ) mounted on a Zeiss Axiovert 200 microscope with a PlanNeofluar 20x/0.50 objective. Fluorescence images were processed with a MetaMorph Imaging System software (Universal Imaging Corporation, West Chester, PA). To exclude variations of fluorescence intensity, the signals were corrected for variations in dye concentration by normalizing fluorescence against baseline fluorescence. Experiments were carried out at room temperature (20-23°C). Electrophysiological measurements All records were obtained with a single electrode to avoid leak currents caused by the insertion of a second electrode, as already shown by Miyazaki and Hirai (31). The oocytes were impaled with borosilicate glass micropipettes (filament type, 1.2 mm, Clark Electromedical) filled with 450 mM KCl and had a resistance of 15-20 MΩ. An AxoClamp 2B (Axon Instruments, Union City, CA) amplifier, operating at a sampling rate of 10-15 kHz in the discontinuous singleelectrode voltage-clamp mode, optimized for the RC characteristics of the cells (32), was used to voltage clamp the cells. To check the leak current and the space clamp conditions, before the uncaging of NAADP, the I-V relation was measured by applying 1 s voltage steps from –100 mV to +50 mV from a holding potential of –70 mV. Only cells that displayed a resting membrane potential more negative then –60 mV and that exhibited a clear inward rectification at potentials lower than –70 mV, a transient inward current between –50 and –20 mV, and a transient outward current at positive potentials (data not shown), as previously shown by Miyazaki et al. (33), were subsequently exposed to ultraviolet (UV) light. The oocytes were always bathed in NSW. To investigate the Na+ permeability of the membrane current, 495 mM NaCl was replaced by an equimolar amount of choline chloride. For experiments in absence of external Ca2+, the oocytes were transferred to a solution containing (CaFSW): 500 mM NaCl, 8 mM KCl, 12 mM MgCl2, 2.5 mM NaHCO3, and 2 mM EGTA, pH 8.0 titrated with NaOH. Experiments were carried out at room temperature (20-23°C).

Statistics Pooled data were given as mean ± SE, and the statistical significance was evaluated by the Student’s t test for unpaired observations. P < 0.05 was considered statistically significant. The duration of the current activated by NAADP was evaluated as the time during which its amplitude remained above half the peak. Reagents OGBD, caged NAADP, and BAPTA were obtained by Molecular Probes (Eugene, OR). NPEGTA was purchased by Calbiochem (La Jolla, CA). All other chemicals were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, MO). RESULTS NAADP-induced Ca2+ wave in immature oocytes Immature oocytes were preinjected with caged NAADP (100 µM) and OGBD (5 mg/ml), as previously shown (10). The uncaging reaction was performed 5-15 min after the injection, when caged NAADP had presumably spread to the entire cytoplasm, by exposing the oocytes to UV light for 15 s. A cooled CCD camera monitored the following Ca2+ response. The photolysis of NAADP triggered a uniform fluorescent ring, which enveloped the entire cortical region of the oocyte (cortical flash; see the third fluorescent image in Fig. 1A), with a latency of 10.2 ± 1.8 s (n=8). The cortical flash then spread from the cortex to the whole oocyte, as a centripetally directed Ca2+ wave, at a rate of 153.7 ± 26.4 µm/sec (n=7; see fluorescent images in Fig. 1A). This pattern of propagation of the NAADP-induced Ca2+ sweep was similar to that previously observed in mature oocytes (10). The graph of the relative fluorescence of the Ca2+ indicator provides a numerical equivalent of the colors shown in the fluorescent images in Fig. 1A. The Ca2+ response to NAADP reached a peak of 0.63 ± 0.11 arbitrary units (n=8) and decayed in ~10-20 min (blue trace in Fig. 1C). We have previously shown that the NAADP-elicited Ca2+ signal was strongly reduced in absence of extracellular Ca2+ (9). In agreement with this observation, no increase in [Ca2+]i was detected upon uncaging NAADP in CaFSW (Fig. 1B and red trace in Fig. 1C). The small Ca2+ rise observed in absence of external Ca2+ was an artifact due to the exposure to UV of the Ca2+ indicator, as evidenced by the green trace in Fig. 1C. Taken together, these results demonstrate that the Ca2+ response in immature oocytes of Asterina pectinifera was largely due to extracellular Ca2+, thus confirming the results we have previously obtained with a photomultiplier system (9). However, the intracellular mobilization of Ca2+ by NAADP cannot be ruled out, as the Ca2+ imaging method we employed may not have enough sensitivity to detect small and highly localized Ca2+ release close to the plasma membrane. NAADP activates a Ca2+-permeable membrane current To investigate whether NAADP-elicited Ca2+ influx was produced by a membrane current, immature oocytes were impaled with a single microelectrode 3-15 min after the injection of caged NAADP. The resting membrane potential was measured in the current-clamp mode and ranged from –62 mV to –90 mV, the mean value being –69.9 ± 1.2 mV (n=36), which is not different from that previously reported by others (31). In oocytes held at –70 mV, photolysis of

NAADP induced the activation of a membrane current with a latency of 2.04 ± 0.06 s (n=9; Fig. 2A, upper trace). The current reached the peak in 12.6 ± 4.2 s (n=10) and lasted for 3-16 min, the mean value being 8.28 ± 2.53 min (n=8). The variability in time-to-peak (ttp), duration, and amplitude (see below) of NAADP-evoked membrane current was probably due to a declining responsiveness to NAADP toward the end of the breeding season, as already reported for the fertilization current in oocytes from Mediaster aequalis (34). The peak and the speed of the Ca2+ wave induced by NAADP also changed during the breeding season (see below). No current was stimulated in control oocytes that had not been preinjected with NAADP (Fig. 2C, trace a). The current-voltage (I-V) relation of the NAADP-activated current was calculated by measuring the peak of the responses elicited in oocytes clamped at –70, –40, –10, +20, and +50 mV (Fig. 2A). The I-V relation showed a strong inward rectification and reversed at potentials more positive than +50 mV (Fig. 2B). These features resemble those of the pure Ca2+-permeable currents, ICRAC (29), the most Ca2+-selective conductance in the family of TRP channels (35), and IARC (5, 30) but differ from those of the current activated by Ca2+ released from InsP3- and cADPr/ryanodinesensitive intracellular stores in starfish oocytes (36). Indeed, this current is permeable to Na+, but not to Ca2+, reverses at a significantly less positive potential (≈+20 mV), and rapidly inactivates. The NAADP-evoked membrane current became smaller and smaller upon subsequent uncagings of NAADP (n=3; Fig. 2C, trace b), a property that could be explained by the desensitization of NAADP receptors by the previous challenge with NAADP (12, 13). The same result was obtained when the Ca2+ sweep triggered by NAADP was measured (n=4; data not shown). Removal of extracellular Ca2+ abolished the current induced by NAADP (Fig. 3C), whereas replacement of external Na+ with an equimolar amount of choline did not affect it (Fig. 3B). Furthermore, the membrane current activated by NAADP was significantly reduced after 15 min of pretreatment of the oocytes with the Ca2+ channels inhibitors SK&F 96356 (10 µM) and verapamil (100 µM; Fig. 4). This finding is in agreement with the previously reported inhibition of the Ca2+ wave exerted by both blockers in starfish oocytes at the doses employed in the present study (10). Although rather high, the concentration of verapamil is in the range of that inhibiting the response to NAADP also in homogenates from sea urchin eggs (37) and heart microsomes (38). Taken together, these results demonstrate that NAADP elicits a Ca2+-mediated current in starfish oocytes, which may trigger the Ca2+ wave detected by the CCD camera. Heparin and 8-NH2-cADPr do not affect the membrane current It has previously been shown that the Ca2+ increase evoked by NAADP is amplified by InsP3 and cADPr/ryanodine receptors through a CICR process (9). However, as shown in detail in Fig. 4, preinjection of heparin (0.5 mg/ml final concentration), an inhibitor of InsP3 receptors; 8-NH2cADPr (4 µM final concentration), an antagonist of cADPr; or both did not significantly affect the membrane current elicited by NAADP. It is worth noting that the concentrations of heparin and 8-NH2-cADPr we employed have been previously shown to fully block the response to InsP3 and cADPr, respectively (9, 36, 39). These findings, therefore, suggest that the activation of the Ca2+-mediated current by NAADP does not require the recruitment of InsP3 and cADPr/ryanodine receptors.

Role of the intracellular Ca2+ in the stimulation of the membrane current evoked by NAADP Although the current triggered by NAADP displayed biophysical properties different from those exhibited by the Ca2+-activated current in starfish oocytes and was not affected by the block of InsP3- and cADPr/ryanodine-sensitive stores, preincubation of oocytes with thapsigargin (5 µM) significantly reduced both its amplitude and duration current in the three oocytes analyzed (Fig. 4). Furthermore, preinjection of the Ca2+ chelator BAPTA (20 µM final concentration) prevented the onset of the current in five out of five oocytes (Fig. 5C). To further confirm the difference between the currents induced by NAADP and Ca2+, respectively, the increase in [Ca2+]i obtained by photoliberating Ca2+ from NP-EGTA induced a transient inward current in 3 out 11 oocytes, which was different from that triggered by NAADP (Fig. 5B, bottom trace) but similar to the one elicited by cADPr and InsP3 (36). To explain the discrepancy between this finding and the requirement of cytosolic Ca2+ to induce the NAADP-dependent Ca2+ current, we suggest that NAADP triggers a highly localized, cortical Ca2+ increase from a thapsigargin-sensitive store that is tightly coupled to the NAADP-dependent membrane channels. The activation of the latter would, then, require both the submembrane Ca2+ pulse and NAADP itself (see Discussion). Impairment of actin cytoskeleton reduces the Ca2+ response to NAADP As the maintenance of the filamentous actin cytoskeleton is required for the activation of several Ca2+-permeable ionic channels (24–26), oocytes were incubated for 1 h with the cell-permeant toxin latrunculin-A (3 µM), which inhibits actin filament polymerization by binding to G-actin monomers (26). The photoliberation of NAADP in latrunculin-A-treated oocytes activated a cortical Ca2+ flash (Fig. 6A) with a latency of 8.46 ± 1.2 s (n=5), which was not significantly different from that measured in control cells (6.60±0.54 s, n=7). By contrast, the peak of Ca2+ response to NAADP in latrunculin-A-treated oocytes was 0.18 ± 0.03 arbitrary units (n=5), a value that was significantly smaller than that measured in control cells (0.41±0.05 arbitrary units, n=5; Fig. 6C). In agreement with this finding, the velocity of the Ca2+ wave elicited by NAADP in latrunculin-A-treated oocytes was significantly slower that in control cells, whereas their values were 256.53 ± 58.30 µm/sec (n=7) and 334.1 ± 76.63 µm/sec (n=5), respectively. This feature is evident in the relative fluorescent images in Fig. 6A, which show that, in an oocyte exposed to latrunculin-A, the Ca2+ wave was not completely closed 44 s after the uncaging, whereas in untreated cells it had already traveled throughout all the oocyte after 30 s (for a comparison, see fluorescent images in Fig. 1A). In according with these observations, the NAADP-activated current was inhibited by the addition of latrunculin-A (compare Fig. 7A vs. Fig. 7B). Indeed, the ttp, the amplitude, and the duration of the current in control cells were equal to 3.38 ± 0.37 s (n=5), -0.75 ± 0.13 nA (n=5), and 7.18 ± 1.02 min (n=5), respectively, while in latrunculin-A-treated cells their values were 5.36 ± 2.40 s (n=5), -0.13 ± 0.05 nA (n=5), and 1.32 ± 0.61 min (n=5). In a subsequent set of experiments, the cell-permeant drug jasplakinolide was used to induce actin polymerization and stabilize F-actin (26). The incubation of the oocytes for 20 min with jasplakinolide (12 µM) did not delay the onset of the cortical Ca2+ ring but reduced both the amplitude of the global Ca2+ elevation (0.19±0.06 arbitrary units, n=4) and the speed of the intracellular Ca2+ wave (130.50±23.52 µm/sec, n=4; Fig. 6B). In accordance with this result, the pretreatment of the oocytes with jasplakinolide (12 µM) significantly reduced the ttp, the amplitude, and the duration of the membrane current elicited by NAADP (Fig. 7A and C).

Indeed, the values of these parameters in jasplakinolide-treated cells were 6.99 ± 2.49 min (n=5), -0.17 ± 0.05 nA (n=5), and 0.5 ± 0.21 min (n=5), respectively. These findings, therefore, demonstrate that an intact F-actin cytoskeleton is required by NAADP to trigger the intracellular Ca2+ sweep by activating Ca2+ entry from extracellular space. DISCUSSION The results presented in this study demonstrate for the first time that 1) NAADP activates a Ca2+permeable membrane current that, in turn, triggers an intracellular Ca2+ wave in immature starfish oocytes; and 2) the Ca2+ response to NAADP requires an intact F-actin cytoskeleton. The lack of any detectable Ca2+ response in the absence of external Ca2+ shows that the increase in [Ca2+]i induced by NAADP is largely dependent by Ca2+ influx from the extracellular space, confirming our previous findings obtained with a photomultiplier system (9). In the presence of external Ca2+, NAADP stimulates a cortical Ca2+ flash, which rapidly propagates throughout the entire cytoplasm. This pattern is similar to that originally observed in mature starfish oocytes (10) and suggests that the cellular mechanisms underlying the response to NAADP are maintained during the maturation process after hormonal stimulation. Ca2+ entry initiates the NAADP-induced Ca2+ wave also in T-lymphocytes (14), while in other cell types, such as pancreatic acinar cells (12, 13) and ascidian oocytes (11), NAADP only mobilizes Ca2+ from an intracellular store different from that sensitive to InsP3 and cADPr (16). In sea urchin eggs homogenates, this store has recently been suggested to be the lysosomes (17), while it has been located in insulin-containing secretory vesicles in living pancreatic β-cells (18). Conflicting data have instead been reported in intact sea urchin eggs, where one group initially ruled out the contribution of Ca2+ inflow (8) but then claimed it in a later study (20). Although the findings described in this contribution have clearly shown that the source of Ca2+ linked to the NAADP response is extracellular, we cannot rule out the possibility that a small and highly localized Ca2+ release, undetectable by the method we have used, occurred in close proximity to the plasma membrane. This feature would explain the finding that both thapsigargin and BAPTA affected the Ca2+ response to NAADP (see below). The Ca2+-mediated membrane current activated by NAADP described in our study could underlie Ca2+ entry also in T-lymphocytes (14) and sea urchin eggs (20). This current does not display a significative permeability to Na+ in NSW, exhibits a strong inward rectification, and reverses at potentials more positive than +50 mV. These properties strongly resemble those of ICRAC (29) and IARC (5, 30), allowing us to suggest that the NAADP-dependent current may be considered a novel member in the family of the second messenger-activated, Ca2+-permeable currents. Further work will assess whether the NAADP-induced current shares other biophysical properties with ICRAC and IARC, such as the dependence of peak amplitude on extracellular Ca2+ concentration and the increase in monovalent cations permeability in divalent ions-free external solution (5, 29). By contrast, the biophysical properties of the NAADP-elicited current are strikingly different from those of the nonselective cationic current triggered by Ca2+ release from cADPr/ryanodine and InsP3-dependent stores in the same preparation (36). In support of this observation, the Ca2+-gated current in starfish oocytes is permeable to Na+, but not to Ca2+, in NSW; exhibits an almost linear I-V relationship reverting at a less positive membrane potential (≈+20 mV); and, finally, rapidly inactivates despite the prolonged increase in [Ca2+]i. These findings make it unlikely that the NAADP-dependent current is merely by a Ca2+-gated one

activated after Ca2+ discharge from NAADP-sensitive intracellular stores. Moreover, the possibility that the current is activated by NAADP after depletion of intracellular stores is ruled out by considering that starfish oocytes seem to lack any type of store-operated Ca2+ influx, as stores emptying by thapsigargin (5 µM) do not produce any detectable Ca2+ influx (Lim and Santella, unpublished observations). Taken together, our results could be explained by suggesting that NAADP produces a highly localized Ca2+ release (see above) from cortical, thapsigargin-sensitive NAADP-dependent stores that are tightly coupled to Ca2+-permeable membrane channel, the activation of which strictly requires both the submembrane Ca2+ pulse and NAADP. According to this model, then, NAADP acts on both the cortical stores and membrane channels, a feature that would account for both the Ca2+ sensitivity of the NAADPtriggered current and its difference from the pure Ca2+-activated nonselective cationic current. It could be argued that the intracellular stores activated by NAADP are usually not affected by thapsigargin. Recent evidence, however, clearly demonstrates that this drug reduces or abolishes the NAADP-dependent Ca2+ release at the frog neuromuscolar junction (40) and in intact pancreatic acinar cells (41). The lack of effect of both heparin and 8-NH2-cADPr on the Ca2+-mediated membrane current induced by NAADP suggests that NAADP did not act on intracellular stores sensitive to InsP3 and cADPr and that Ca2+ mobilization from these sites was not involved in the onset of the current. This result is at variance with that obtained in pancreatic acinar cells (12, 13), where chloride and monovalent cation currents are activated after the release of Ca2+ from NAADPsensitive intracellular stores. The global Ca2+ wave elicited by NAADP in starfish oocytes, however, was strongly reduced in cells preinjected with both heparin and 8-NH2-cADPr (9). It is conceivable that Ca2+ influx triggered by NAADP is intracellularly amplified by InsP3 and cADPr/ryanodine receptors by a CICR mechanism. A similar pattern of propagation of the Ca2+ sweep evoked by NAADP has been described in rat pancreatic acinar cells (12, 13) and sea urchin eggs (8) but not in human T-lymphocytes (14) and pancreatic β-cells (15). The Ca2+ current and the following Ca2+ wave induced by NAADP were dramatically reduced after impairment of F-actin cytoskeleton. Dynamic assembly and disassembly of submembraneous actin cytoskeleton have both been shown to affect Ca2+ release from InsP3 and ryanodine receptors (21, 23, 26, 42, 43) and to modulate voltage-gated and store-operated Ca2+ channels (24–26, 44, 45). Indeed, F-actin depolymerization decreased the amplitude of voltagegated Ca2+ currents (24, 45), whereas actin filament stabilization increased it (45). This result was explained by an augmented or reduced mechanical strain imposed by the cytoskeleton to the Ca2+ channel protein by actin polymerization and depolymerization, respectively (46). Due to the inhibitory effect of both disruption and stabilization of actin filaments on the Ca2+ current, however, this mechanism is unlikely to be involved in the response to NAADP in starfish oocytes. On the other hand, both latrunculin-A and jasplakinolide reduced store-operated Ca2+ influx in human platelets (26) by impairing the coupling between endoplasmic reticulum and plasma membrane, which is dependent on actin cytoskeleton and underlies capacitative Ca2+ entry in these cells (26). Although this explanation does not fit the effects of actin manipulation on NAADP-activated current, our results indicate that it is affected by any change in the turnover rate of actin filaments. This inhibition is probably not due to the impairment of NAADP binding to its receptors, as the latency of the response does not significantly change in latrunculin-A- and jasplakinolide-treated oocytes. The relatively long delay, as compared with ionotropic receptors (6), between the uncaging of NAADP and the onset of the membrane current indicates that one

or more intermediate steps occur between the two events. As suggested above, one of these steps may consist in the Ca2+ release from cortical stores and in the following activation of the current by both NAADP and Ca2+ or, more likely, a Ca2+-dependent protein. On the basis of our findings, we speculate that the tight coupling among these processes requires a dynamic turnover of actin filaments, whose impairment by cytoskeleton manipulation strongly affects both the Ca2+ current and the Ca2+ wave. It remains to be elucidated whether the NAADP receptor in starfish oocytes is the same 120-kDa receptor that has been solubilized from homogenates of sea urchin eggs (47). The activation of NAADP receptors is the trigger for the Ca2+ wave observed at fertilization of Asterina pectinifera oocytes (10), a result that has recently been confirmed also in sea urchin eggs (20). A further confirmation of the role played by NAADP at fertilization comes from the finding that sea urchin spermatozoa contain micromolar concentrations of NAADP (48). The Ca2+ response to NAADP in mature starfish oocytes, i.e., oocytes that have been challenged with the maturing hormone 1-methyladenine for ~50 min before sperm addition, still consists in Ca2+ influx, which is then amplified by InsP3 and ryanodine receptors by CICR (9). It is, therefore, likely that the Ca2+-permeable current activated by NAADP in starfish oocytes is involved in the fertilization potential, the cortical flash, and the Ca2+ wave observed at fertilization of starfish oocytes. ACKNOWLEDGMENTS We are grateful to E. Carafoli for helpful suggestions and discussions. We thank Dr. Keiichiro Kyozuka (Asamushi Marine Biological Station, Japan) for providing us with the starfish and the Marine Resources Service (Stazione Zoologica, Naples) for maintaining them. This research was supported by Italian Ministry of Scientific Research (PRIN 2000). REFERENCES 1.

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Fig. 1

Figure 1. Ca2+ wave induced by NAADP in starfish oocytes. The Ca2+ response to NAADP (100 µM) was monitored by employing a cooled CCD camera and shown as relative fluorescence images taken at different times after the uncaging. A) Ca2+ response in the cortical region occurred immediately after the irradiation and spread within 15 s to the center of the cell. B) No increase in [Ca2+]i was detected in oocytes incubated for 1 min in CaFSW. C) Graph of the relative fluorescence of the Ca2+ increase after the liberation of NAADP shows the numerical equivalent of the colors in the fluorescent images measured in NSW (blue trace) and CaFSW (red and green traces). The red trace shows the response of an oocyte that had not been preinjected with NAADP. The UV flash was given at the time indicated by the arrow.

Fig. 2

Figure 2. NAADP activates a membrane current in starfish oocytes. A) Membrane currents evoked by NAADP (100 µM) at different holding potentials. Each trace was obtained on the same day by different oocytes extracted by the same animal. The UV flash was given at the time indicated by the arrow. B) I-V relation of the peak current induced by NAADP (n=3-4 for each potential). C) Trace a, the current was elicited when oocytes were exposed to UV without previous injection of NAADP; trace b, the membrane current became smaller and smaller upon subsequent uncagings of NAADP. The UV flash was given at the time indicated by the arrow.

Fig. 3

Figure 3. NAADP-triggered current is permeable to Ca2+ but not to Na+. Membrane currents stimulated by NAADP (100 µM) in presence of 10 mM Ca2+ (NSW; A) and 5 mM Na+ (B) in the extracellular solution. No current was visible when NAADP was photoliberated in absence of extracellular Ca2+. The UV flash was given at the time indicated by the arrow.

Fig. 4

Figure 4. Histograms of the amplitude (A), latency (B), ttp (C), and duration (D) of the membrane currents elicited by NAADP following the designated treatments. Each experiment has been performed in 3-4 different cells. Single and double asterisks are significant difference from the control in thapsigargin- and SK&F- or verapamil-treated cells, respectively. Note that the parameters in the control currents change during the breeding season as mentioned in the text.

Fig. 5

Figure 5. Effect of intracellular Ca2+ on NAADP-evoked current. A) Ca2+ current activated by NAADP (100 µM) in NSW. The UV flash was given at the time indicated by the arrow. B) Photoliberating Ca2+ by NP-EGTA (10 µM) did not elicit a membrane current similar to that induced by NAADP. The cells were exposed to UV light throughout the entire recording. C) Preinjection of BAPTA (20 µM) prevented the activation of the current by NAADP (100 µM). The UV flash was given at the time indicated by the arrow.

Fig. 6

Figure 6. Latrunculin-A and jasplakinolide impaired the Ca2+ wave elicited by NAADP. The Ca2+ response to NAADP (100 µM) in the cortical region propagated to the center of the oocytes with a delay after incubation in latrunculin-A (A) and jasplakinolide (B). The pattern of the Ca2+ sweep measured in control cells was similar to that reported in Fig. 1A. C) Graph of the relative fluorescence of the Ca2+ rise upon NAADP uncaging shows the numerical equivalent of the colors in the fluorescent images in latrunculin-A (red trace) and after jasplakinolide incubation (green trace). The UV flash was given at the time indicated by the arrow.

Fig. 7

Figure 7. Latrunculin-A and jasplakinolide reduced the membrane current activated by NAADP. Membrane currents elicited by NAADP (100 µM) in control oocytes (A) and in oocytes incubated in presence of latrunculin-A (3 µM; B) and jasplakinolide (12 µM; C). The UV flash was given at the time indicated by the arrow. Note the reduction in both the amplitude and the duration of the current exerted by latrunculin-A and jasplakinolide.