J Biol Chem, Vol. 274, Issue 29, 20643-20649, July 16, 1999

Adenophostin A Induces Spatially Restricted Calcium Signaling in Xenopus laevis Oocytes^*

Gary St. J. Bird, Masaaki Takahashi^§, Kazuhiko Tanzawa^§, and James W. Putney Jr.^¶

From the  Calcium Regulation Section, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ^§ Biological Research Laboratories, Sankyo Company, Limited, Tokyo 140, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The activation of intracellular calcium release and calcium entry across the plasmalemma in response to intracellular application of inositol 2,4,5-trisphosphate and adenophostin A, two metabolically stable agonists for inositol 1,4,5-trisphosphate receptors, was investigated using Xenopus laevis oocytes and confocal imaging. Intracellular injection of inositol 2,4,5-trisphosphate induced a rapidly spreading calcium signal associated with regenerative calcium waves; the calcium signal filled the peripheral regions of the cell in 1-5 min. Injection of high concentrations of adenophostin A (250 nM) similarly induced rapidly spreading calcium signals. Injection of low concentrations of adenophostin A resulted in calcium signals that spread slowly (>1 h). With extremely low concentrations of adenophostin A (~10 pM), stable regions of Ca²⁺ release were observed that did not expand to peripheral regions. When the adenophostin A-induced calcium signal was restricted to central regions, compartmentalized calcium oscillations were sometimes observed. Restoration of extracellular calcium caused a rise in cytoplasmic calcium restricted to the region of adenophostin A-induced calcium mobilization. The limited diffusion of adenophostin A provides an opportunity to examine calcium signaling processes under spatially restricted conditions and provides insights into mechanisms of intracellular calcium oscillations and capacitative calcium entry.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Xenopus laevis oocytes have proven a useful model system for studying the inositol 1,4,5-trisphosphate ((1,4,5)IP₃)¹-mediated calcium signaling system, providing insights into mechanisms of calcium oscillations (1) and capacitative Ca²⁺ entry (2, 3). In many cell types, including Xenopus oocytes, activation of surface membrane receptors results in a biphasic Ca²⁺ response composed of an initial mobilization of internally stored Ca²⁺, followed by entry of extracellular Ca²⁺ (4). Much is known about the role of (1,4,5)IP₃ in mobilizing intracellular Ca²⁺, and a working model for the Ca²⁺ entry process is described by the capacitative model (5, 6) according to which the depletion of intracellular (1,4,5)IP₃-sensitive Ca²⁺ stores signals the activation of plasma membrane calcium channels.

Recently, a metabolite of Penicillium brevicompactum, adenophostin A, has been isolated and demonstrated to be an agonist for the (1,4,5)IP₃ receptor and to have a potency ~100-fold greater than that of (1,4,5)IP₃ (7). Interestingly, two studies performed in Xenopus oocytes (8, 9) suggested a site of action for adenophostin A in addition to that on intracellular Ca²⁺ stores. In both studies, low concentrations of adenophostin A (<10 nM) appeared to preferentially activate the Ca²⁺ entry process, although Hartzell et al. (9), unlike DeLisle et al. (8), could not find a clear dissociation between Ca²⁺ release and Ca²⁺ entry. In a subsequent report, Machaca and Hartzell (10) found that the diffusion of adenophostin A throughout the oocyte was considerably slower than that of IP₃ and concluded that this might explain the apparently diminished Ca²⁺ release signal.

In this study, we have used confocal microscopy to monitor directly spatial as well as temporal aspects of the effects of adenophostin A on intracellular Ca²⁺ in Xenopus oocytes using the calcium-sensitive dye Calcium Green. Our results confirm that diffusion of adenophostin A throughout the oocytes is slower than that of IP₃ and additionally indicate that low concentrations of adenophostin A cause a confined mobilization of intracellular Ca²⁺ that is capable of supporting spatially restricted Ca²⁺ oscillations and spatially restricted Ca²⁺ entry. This unique action of adenophostin A provides new insights into the role of IP₃ receptor binding in [Ca²⁺]_i oscillations and also into the spatial relationships between intracellular Ca²⁺ release and activation of capacitative calcium entry.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of Xenopus Oocytes

Adult albino female X. laevis (Xenopus One, Ann Arbor, MI) were anesthetized by hypothermia and decapitated. The ovaries were then removed and stored in ND96 buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 5 mM HEPES (pH 7.6)). Stage V and VI oocytes were manually dissected free from the ovaries and follicles and maintained in ND96 buffer at 18 °C until used.

Oocyte Microinjection

Various solutions (described below) were injected into oocytes via an oil-filled glass micropipette attached to a nanoliter injector (World Precision Instruments, Inc., Sarasota, FL). The injection volume could be varied from 4.6 to 50.6 nl, and final concentrations were calculated on the basis of an oocyte volume of 1 µl.

Calcium Measurement

Calcium Green Loading-- Prior to the experiment, defolliculated oocytes were microinjected with the Ca²⁺ indicator Calcium Green (~12.5 µM final concentration; Molecular Probes, Inc., Eugene OR). The oocytes were then incubated for at least 30-40 min at room temperature to allow equilibration of the dye throughout the cytoplasm. Oocytes were placed in a glass-bottomed microscope chamber (Bionique Testing Laboratories, Inc.) containing 1 ml of ND96 buffer and placed on the stage of an inverted Zeiss confocal microscope (LSM 410, Carl Zeiss Inc., Thornwood, NY). Oocytes were held in position by a microinjection pipette, and modifications of the bathing solution were made by dilution into the bath.

Fluorescence Measurements-- Measurements of intracellular calcium were performed with the inverted Zeiss LSM 410 confocal microscope equipped with a 10× objective (0.5 numerical aperture). The calcium-sensitive dye Calcium Green was excited by the 488-nm line from a krypton-argon laser (Omnichrome Model 643, Melles Griot, Carlsbad, CA), and the emission fluorescence monitored at 515 nm was selected by a band-pass filter. The pinhole aperture of the confocal microscope was set such that the fluorescence image represented an optical slice of ~10 µm (if not otherwise specified) at the bottom of the oocyte, i.e. just above the coverslip and just under the plasma membrane (Fig. 1). All fluorescence images (256 intensity levels, 256 × 256 pixels) were detected with an analog photomultiplier tube, and the resulting images were recorded directly onto videotape. Simultaneously, up to 10 regions of interest (ROIs) distributed across the image provided an intensity versus time graphic output. Subsequently, these fluorescence values were expressed as a fraction of the initial fluorescence intensity (F/F₀). During fluorescence data collection, each scan of a 256 × 256 image took 0.35 s, and the interval between each image scan was ~1.3 s.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Typical confocal images of Xenopus oocytes for detection of Calcium Green fluorescence. Xenopus oocytes, injected with Calcium Green (12.5 µM final concentration), were scanned by the confocal microscope in both the xy and z directions. The pinhole settings for collection of the emission fluorescence were set so that the resolution in the z direction, or "optical slice," was ~10 µm (10× objective, 0.5 numerical aperture). Upper panel, typical image of an oocyte used in experiments to collect spatial information in an xy plane. This xy image was obtained when focusing at the bottom of the oocyte, closest to the coverslip. Middle panel, with conditions optimal for detecting calcium-dependent changes in fluorescence in the xy plane, this image shows a typical confocal image collected in the z direction. Lower panel, the same z plane viewed in the middle panel was viewed again here, but under greater magnification (achieved with an electronic zoom function). Detection of fluorescence intensity in the z direction rapidly decreased toward the interior of the oocyte. Under optimal conditions for monitoring calcium changes in the xy images, signals were detectable to a maximum depth of ~20 µm into an oocyte that had a diameter of ~1000 µm.

Photolysis of Caged Compounds-- In some experiments, oocytes were co-injected with caged (1,4,5)IP₃ (~50 µM final concentration; Calbiochem) along with the calcium dye. Photocleavage of caged (1,4,5)IP₃ was achieved using the 351/364-nm lines of an argon ion laser (Coherent Enterprise Model 652, Coherent Inc., Santa Clara, CA), liberating (1,4,5)IP₃ into the cytoplasm. The area in which photolysis occurred was defined by use of a fast gated Uniblitz shutter (Vincent Associates, Rochester, NY) placed between the argon ion laser and the confocal microscope. By varying the duration the shutter was open (typically 5-10 ms), the area of photolysis could be varied in the xy plane.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In earlier studies (8-10), the actions of adenophostin A were compared with those of the physiological agonist for the IP₃ receptor, (1,4,5)IP₃. However, adenophostin A differs from (1,4,5)IP₃ in being resistant to inactivation by the 5-phosphatase and 3-kinase (7). Thus, for the majority of our studies, we chose to utilize a metabolism-resistant analog of (1,4,5)IP₃, (2,4,5)IP₃ (11), for comparison with adenophostin A. Fig. 2 shows the pattern of [Ca²⁺]_i signaling seen following injection of (2,4,5)IP₃ at a concentration of 1 µM (final estimated cytoplasmic concentration). This somewhat intermediate concentration of (2,4,5)IP₃ caused a spreading [Ca²⁺]_i signal that filled the field of view generally within 5 min. At this concentration, the [Ca²⁺]_i signal was usually associated with regenerative waves and spirals, as originally described by Lechleiter et al. (1).

View larger version (96K): [in this window] [in a new window]   Fig. 2.   Effect of (2,4,5)IP3 (1 µM) microinjected into an oocyte in the presence of extracellular calcium (10 mM). Oocytes, previously injected with Calcium Green (12.5 µM final concentration), were subsequently microinjected with a volume of a (2,4,5)IP3 solution so that its final concentration would be ~1 µM when diluted throughout the oocyte. These injections were performed in the presence of 10 mM Ca2+o. The panel of four images represents the time course of calcium changes in the oocyte following injection at t = 0, with the time indicated for each image. The boxes depicted in the bottom right panel illustrate selected ROIs, described in subsequent figures as central (black) or peripheral (white), information from which was used to generate graphs of the time-dependent fluorescence changes. Although the point of injection occurs at the top of the oocyte, distal to the xy image plane, calcium responses were observed in ~1 min. Within 5 min, calcium responses were observed throughout the oocyte, exhibiting characteristic calcium waves and spirals. This experiment is typical of 10 similar experiments.

Also shown in Fig. 2 is a typical arrangement of ROIs used for assessing the time course of [Ca²⁺]_i changes in the central and peripheral regions of the field of view. Fig. 3 illustrates examples of the time course of [Ca²⁺]_i changes in the peripheral and central regions of oocytes following the injection of four different concentrations of (2,4,5)IP₃. In these experiments, the oocytes were initially incubated in a Ca²⁺-deficient medium, and extracellular Ca²⁺ was subsequently restored to 10 mM to assess [Ca²⁺]_i signals due to influx. At the highest concentration (5 µM), diffusion throughout the oocyte was very rapid such that a rapid release occurred in the peripheral regions of the field of view seconds after appearing in the center. With 1 µM (2,4,5)IP₃, a somewhat greater delay was observed before the peripheral regions were activated, and as shown in Fig. 2, regenerative [Ca²⁺]_i oscillations were seen. With both 5 and 1 µM (2,4,5)IP₃, re-addition of Ca²⁺ externally resulted in a rapid increase in [Ca²⁺]_i indicative of Ca²⁺ entry, presumably due to the depletion of intracellular stores. With lower concentrations of (2,4,5)IP₃ (250 and 100 nM), release was seen only near the center of the field of view, and with these concentrations, the signal was seen only when the injection pipette was brought deep into the oocyte, close to the coverslip and the field of view. These smaller signals did not propagate to the periphery, and restoration of extracellular Ca²⁺ did not result in the activation of significant entry. The simplest interpretation of this result is that the lower concentrations of (2,4,5)IP₃ are insufficient to activate significant Ca²⁺ mobilization when finally diluted by diffusion throughout the cytoplasm. This is as not unexpected since the K_d for (2,4,5)IP₃ action on the IP₃ receptor is >1 µM (12).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Time course of the effect of (2,4,5)IP3 microinjected into oocytes in the absence and presence of extracellular calcium (10 mM). Oocytes, previously injected with Calcium Green (12.5 µM final concentration), were subsequently injected with different concentrations of (2,4,5)IP3 in the absence of extracellular calcium. The estimated final concentrations of (2,4,5)IP3 in the oocyte were 5 µM, 1 µM, 250 nM, and 100 nM, as indicated. The time course for the effects of (2,4,5)IP3 was detected in ROIs that were located either centrally (solid lines) or peripherally (dashed lines) in the xy plane (see Fig. 2). As indicated on each trace, extracellular calcium conditions were later restored to 10 mM. Similar results for each condition were observed in 5-12 oocytes.

A noticeable different picture was seen with adenophostin A. Fig. 4 illustrates a series of images depicting the spread of the [Ca²⁺]_i signal in an oocyte injected with adenophostin A to give a final concentration of 10 nM. This concentration of adenophostin A should, if anything, be somewhat more potent than 1 µM (2,4,5)IP₃, yet between 30 and 60 min was required for the [Ca²⁺]_i signal to fill the field of view. The full time courses of four peripheral and four central ROIs from this experiment and the experiment shown in Fig. 2 are given in Fig. 5.

View larger version (99K): [in this window] [in a new window]   Fig. 4.   Effect of adenophostin A (10 nM) microinjected into oocytes in the presence of 10 mM extracellular calcium. Oocytes, previously injected with Calcium Green (12.5 µM final concentration), were subsequently microinjected with a volume of an adenophostin A solution so that its final concentration would be ~10 nM when diluted throughout the oocyte (injections performed in the presence of 10 mM Ca2+o). The panel of four images represents the time course of calcium changes in the oocyte following adenophostin A injection at t = 0, the time point being indicated with each image. As in previous experiments, the point of injection is located at the top of the oocyte, distal to the image plane. Following injection, adenophostin A-induced calcium responses were first observed after a delay of >10 min and required >50 min before a sustained calcium response was observed throughout the oocyte. This experiment is typical of six similar experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Comparison of the time course of the effects of (2,4,5)IP3 (1 µM) and adenophostin A (10 nM) on intracellular calcium in oocytes. The time course of calcium-dependent fluorescence changes measured in Figs. 2 and 4 for (2,4,5)IP3 (1 µM final concentration) or adenophostin A (10 nM final concentration), respectively, are graphically depicted here. Each line represents a single ROI, each being positioned centrally (solid lines) or peripherally (dashed lines) as shown in Fig. 2.

Although somewhat larger than (2,4,5)IP₃, adenophostin A would not be expected to diffuse at such a relatively slow rate unless some structure specifically impedes its rate of diffusion. It seems likely that this structure is the IP₃ receptor itself. Thus, the concentrations of adenophostin A injected into the oocytes may be lower than the concentration of IP₃ receptors, and when adenophostin A molecules bind to these receptors with high affinity, their rate of diffusion through the cell is dependent to a large extent on the rather slow rate of receptor dissociation.² A prediction then is that higher concentrations of adenophostin A would diffuse much faster throughout the cell. Fig. 6 shows that with concentrations of 250 and 100 nM adenophostin A, the [Ca²⁺]_i signal reached the peripheral regions of the field of view much faster than with 10 nM (within 1 min or so). As for (2,4,5)IP₃, once the signal had filled the field of view, restoration of extracellular Ca²⁺ resulted in rapid influx of Ca²⁺. Fig. 7 summarizes the data on the influx of Ca²⁺ in oocytes activated by relatively high concentrations of (2,4,5)IP₃ (10 µM) and adenophostin A (250 nM). Under these conditions, the magnitude of the influx and its sensitivity to extracellular Ca²⁺ were indistinguishable with these two IP₃ receptor ligands.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of high concentrations of adenophostin A in the absence and presence of 10 mM extracellular calcium. Oocytes were injected with adenophostin A (AdA) such that the final intracellular concentration should be either 250 or 100 nM, concentrations >10-fold greater than that used in Fig. 4. Note that injections were performed either in the presence (10 mM; upper panel) or absence (middle and lower panels) of extracellular calcium. In the latter case, Ca2+ was restored to 10 mM where indicated. For each injection condition, calcium responses were recorded from ROIs in central (solid lines) and peripheral (dashed lines) regions. These traces are representative of 5, 12, and 4 experiments for each condition, respectively.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   Concentration effect curves for the effect of extracellular calcium on the sustained calcium entry elicited by either 10 µM (2,4,5)IP3 or 250 nM adenophostin A. Oocytes were injected with either (2,4,5)IP3 (10 µM) or adenophostin A (250 nM) in the absence of extracellular calcium. Oocytes were then exposed to a range of extracellular calcium conditions, and the subsequent rise in intracellular calcium (measured as F/F0) was monitored until a new, elevated steady state was achieved. This figure compares the dependence of the sustained calcium signal on extracellular calcium in oocytes injected with either (2,4,5)IP3 (10 µM; ) or adenophostin A (250 nM; ). Each data point represents the mean ± S.E. for four experiments.

We next investigated the effects of injecting very low concentrations of adenophostin A into oocytes. When injections were carried out with quantities of adenophostin A that would give cytoplasmic concentrations of ~10 pM, we saw no changes in [Ca²⁺]_i. However, when the pipette was brought very close to the coverslip and the field of view, a localized area of elevated [Ca²⁺]_i was observed. In contrast to the findings with low concentrations of (2,4,5)IP₃, this elevated region was relatively stable; the size of the region did not grow appreciably, and the elevated [Ca²⁺]_i level was maintained for up to 30 min. In some oocytes, [Ca²⁺]_i waves and spirals developed within this region (Fig. 8). These waves were similar in appearance to those seen in oocytes injected with 1 µM (2,4,5)IP₃, but the waves always extinguished when reaching the limiting periphery of the region of activation. The average wave velocities were 27.3 ± 2.5 µm/s (n = 3) for (2,4,5)IP₃ and 8.5 ± 0.6 µm/s (n = 7) for adenophostin A. The value for (2,4,5)IP₃ is within and that for adenophostin A is just below the range reported for agonist-dependent waves first described by Lechleiter et al. (1). Since wave velocity is dependent on the diffusional distance between release sites, the lower value for adenophostin A may indicate that the concentration of adenophostin A-bound receptors is lower than for (2,4,5)IP₃. We presume that the limited size of this activated region is determined by the limits of diffusion and action of adenophostin A. Thus, the restricted area of [Ca²⁺]_i waves underscores the absolute requirement for activated IP₃ receptors for propagation to continue. The presence of such waves under conditions whereby adenophostin appears essentially irreversibly affixed to the IP₃ receptor indicates that the affinity of the ligand for the IP₃ receptor, and specifically the rate of dissociation of the ligand from the IP₃ receptor, is not a determinant of wave generation or propagation.

View larger version (72K): [in this window] [in a new window]   Fig. 8.   Effects of a very low concentration (~10 pM) of adenophostin A in an oocyte. An oocyte was injected with an adenophostin A solution such that the final intracellular concentration would be ~10 pM (~1000-fold less than that used for Fig. 4) if distributed throughout the oocyte. Injections were performed in the absence of extracellular calcium. In some oocytes, spiraling waves were observed, but not in others. These images are from two different oocytes, captured at time points ~15 min after the injection of adenophostin A. These data are representative of eight different experiments.

We examined the activation of Ca²⁺ entry in oocytes injected with low concentrations of adenophostin A. Fig. 9 illustrates a result with an oocyte injected with 10 nM adenophostin A. With this concentration, spread of [Ca²⁺]_i was slow, but eventually filled the entire cell. Addition of external Ca²⁺ at a time when [Ca²⁺]_i was elevated in the central regions, but not yet in the peripheral regions, of the cell resulted in a further rise in [Ca²⁺]_i in the central regions, but no increase in the periphery. Thus, these localized responses to adenophostin A are apparently capable of activating Ca²⁺ entry.

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Time course of the effect of adenophostin A (10 nM) injection in the absence and presence of extracellular calcium (10 mM). In the absence of extracellular calcium, oocytes were injected with adenophostin A so that the final concentration was 10 nM. In the example shown, the changes in intracellular calcium were monitored in central (solid lines) and peripheral (dashed lines) regions. As observed in Figs. 4 and 5, the intracellular calcium changes developed slowly from the central regions. After a significant rise in intracellular calcium had occurred in the central (but not the peripheral) regions, the extracellular medium was switched to one containing 10 mM calcium. This increase in extracellular calcium had an immediate effect on the central regions where intracellular calcium release had already occurred, whereas significant changes in the peripheral regions occurred after a long delay (>10 min in this example). These data are typical of five different experiments.

There is considerable evidence that the endoplasmic reticulum of cells is at least somewhat continuous throughout the cell (13). Thus, we considered the possibility that despite the restricted localization of the sites of Ca²⁺ release within the oocyte, Ca²⁺ may become depleted from the lumen of the endoplasmic reticulum in the cellular periphery and that this depleted endoplasmic reticulum may contribute to the signaling of Ca²⁺ entry. To investigate this possibility, we injected an oocyte with caged (1,4,5)IP₃ along with Calcium Green. In the experiment shown in Fig. 10, after insertion of the microinjection pipette, the oocyte was briefly (5 ms) exposed to the UV laser, liberating (1,4,5)IP₃ in a small area of the cytoplasm (in the xy plane). This resulted in a rapid and transient release of intracellular Ca²⁺. Subsequently, the same oocyte was injected with adenophostin A to give a final concentration (if fully diluted) of ~10 pM. This quantity of adenophostin A gave a relatively stable localized region of elevated [Ca²⁺]_i. Exposure to the UV laser revealed that the release of Ca²⁺ in the peripheral regions appeared similar to that seen prior to adenophostin A injection. Following the discharge of Ca²⁺ by the photolytic release of (1,4,5)IP₃, the liberated Ca²⁺ was re-accumulated presumably following the time course of diffusion and metabolism of (1,4,5)IP₃. This indicates that when the action of adenophostin A is restricted to specific cellular regions, the endoplasmic reticulum Ca²⁺ stores outside of that region remain fully charged and functional.

View larger version (58K): [in this window] [in a new window]   Fig. 10.   Effect of uncaging (1,4,5)IP3 in oocytes on the adenophostin A-induced, spatially localized increase in intracellular calcium. In preparation for these experiments, oocytes were co-injected with Calcium Green (12.5 µM final concentration) and caged (1,4,5)IP3 (50 µM final concentration). Oocytes were maintained in the absence of extracellular calcium. The two upper panels demonstrate that exposing oocytes to a UV laser in a restricted area (using a fast gated shutter; see "Materials and Methods") results in a localized and reversible increase in intracellular calcium due to uncaging of (1,4,5)IP3. This same oocyte was then injected with a low concentration of adenophostin A (Adeno. A; ~10 pM), inducing a spatially localized increase in intracellular calcium. Subsequently, (1,4,5)IP3 was uncaged in areas where intracellular calcium was elevated as well as in areas that had not apparently responded to adenophostin A. These data are typical of eight separate experiments.

Fig. 11 illustrates an experiment in which Ca²⁺ entry was assessed in an oocyte injected with the lowest concentration of adenophostin A used (10 pM). In this particular cell, an initial central release of Ca²⁺ was followed by the appearance of [Ca²⁺]_i oscillations. Addition of 10 mM extracellular Ca²⁺ resulted in a sustained rise in [Ca²⁺]_i restricted to the region of the cell influenced by the adenophostin A injection. Elevation in [Ca²⁺]_i was seen in the cell periphery only in response to liberation of (1,4,5)IP₃ from its caged precursor.

View larger version (19K): [in this window] [in a new window]   Fig. 11.   Effect of changing extracellular calcium conditions on oocytes with spatially localized effects of adenophostin A. Shown is an F/F0 versus time plot of the experiment illustrated in Fig. 10, where each line represents an ROI either inside (solid line) or outside (dashed line) the area responding to adenophostin A. In addition, the effect of restoring extracellular calcium to 10 mM (as indicated) in these two areas is shown. Note that during the initial uncaging of (1,4,5)IP3, the central ROI lay outside of the area of photolysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results of this study demonstrate that adenophostin A has the unique ability to activate and regulate [Ca²⁺]_i signaling in precise, spatially restricted regions of the cell. The reason for this behavior is likely due to the high affinity of adenophostin A for the IP₃ receptor and its presumed slow rate of dissociation from the receptor. In a previous report on the binding of IP₃ to its receptor, Spät et al. (12) pointed out that the (1,4,5)IP₃ signaling system worked efficiently because the concentration of cellular receptors was less than the K_d of (1,4,5)IP₃ for its receptor. When the concentration of receptor is higher than the K_d of its ligand, nonlinear behavior can result, and the receptor becomes a reservoir for ligand, reducing its free concentration (14). This is likely the situation with low concentrations of adenophostin A, although adenophostin A at higher concentrations functions as a full and potent activator of the (1,4,5)IP₃ pathway (15, 16).

Utilizing Ca²⁺-dependent chloride currents as an indirect assay of [Ca²⁺]_i changes, both DeLisle et al. (8) and Hartzell et al. (9) observed activation of Ca²⁺ entry with low concentrations of adenophostin A (~100 pM (8) and ~50 pM (9)). DeLisle et al. saw no evidence of Ca²⁺ release with these concentrations, whereas Hartzell et al. observed small chloride currents attributable to intracellular release. Both groups of investigators concluded that an IP₃ receptor was involved in the action of adenophostin A, possibly through releasing from a small subset of the intracellular stores. In a more recent publication, Machaca and Hartzell (10) also observed that the spread of adenophostin A-induced [Ca²⁺]_i signals was somewhat slower than for (1,4,5)IP₃ and concluded that the slow release might diminish the magnitude of the associated chloride current. Our findings suggest an alternative explanation for the inability of adenophostin A to efficiently activate chloride currents associated with release. When chloride current is used as a measure of [Ca²⁺]_i, only changes in [Ca²⁺]_i immediately under the plasma membrane can be observed. Clearly, any influx of Ca²⁺ must occur across the plasma membrane, and thus, the use of a plasma membrane-associated marker, such as Ca²⁺-dependent chloride channels, will be a sensitive indicator of this mode of Ca²⁺ signaling. From the current results, it is clear that injections of small quantities of adenophostin A can induce a substantial degree of intracellular release of Ca²⁺, but that this release can occur at some distance from the plasma membrane and thus would not be seen by plasma membrane chloride channels. The opacity of the ooplasm, due to high concentrations of yolk proteins, makes it difficult to image deeply into the oocyte. Thus, to observe this spatially restricted action of adenophostin A, we injected close to the plasma membrane just above the coverslip. These regions of release presumably make contact with the plasma membrane where it sits on the coverslip. The Ca²⁺ influx observed in this situation may be derived from Ca²⁺ in the limited aqueous space between the oocyte and coverslip. Presumably with injections in more central regions of the cell, such as in the previous electrophysiological experiments, it would be possible to activate regions of Ca²⁺ release that make minimal contact with the plasma membrane of the oocyte. Such spatially restricted regions of Ca²⁺ depletion might signal to the plasma membrane to activate entry that would be detected as chloride current. In any event, it is obvious from the unusual behavior of adenophostin A that assessment of [Ca²⁺]_i changes near the plasma membrane may severely underestimate the extent of cellular Ca²⁺ discharge by this agent, and this may result in the apparent dissociation between release and entry observed in earlier studies.

In a recent report, the activity of a putative diffusible calcium influx factor, CIF, was described (17). This material was capable of diffusing through and activating calcium influx in regions associated with intact endoplasmic reticulum stores. In the current studies, if such a factor had been released from the regions of adenophostin A action, it would be expected to diffuse into and activate entry in the peripheral regions of the oocyte; however, no such signal was observed. Thus, the coupling of intracellular calcium release to calcium entry in the oocyte appears to depend more intimately on the proximity of IP₃-depleted calcium stores, perhaps because of the involvement of IP₃ receptors in conveying the signal to the plasma membrane (18).

Although adenophostin A may not have unique actions on the calcium entry pathway, its unusual kinetic behavior affords an opportunity to control experimentally the extent of Ca²⁺ signaling spatially within a single cell. For example, the spatially restricted calcium waves, such as those shown in Fig. 8, make it clear that wave propagation cannot extend beyond the region of action of an IP₃ receptor agonist. Since regenerative waves were frequently observed within this restricted region, it is clear that agonist affinity, and specifically the dissociation rate from the receptor, is not a factor in the propagation mechanism. This is somewhat surprising given the well documented inhibitory effect of Ca²⁺ on IP₃ receptor affinity and specifically the dissociation rate (19, 20). This unusual ability of adenophostin A to produce spatially restricted regions of activated calcium signaling may prove useful in other ways in future studies of the spatial and temporal aspects of Ca²⁺ signaling.

	ACKNOWLEDGEMENTS

We are indebted to Dr. James Lechleiter for advice on the techniques for injection of oocytes with inositol phosphates and confocal calcium imaging and to Jeff Reece for excellent technical support of the confocal facility. Useful critiques of this work were given by Drs. Lisa Broad and David Miller.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: NIEHS, NIH, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-1898; E-mail: putney@niehs.nih.gov.

² Machaca and Hartzell (10) came to a somewhat similar conclusion based on a reduction of the effective concentration of adenophostin A as a result of its binding to IP₃ receptors.

	ABBREVIATIONS

The abbreviations used are: (1,4,5)IP₃, inositol 1,4,5-trisphosphate; (2,4,5)IP₃, inositol 2,4,5-trisphosphate; IP₃, inositol trisphosphate; ROI, region of interest.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES