Adenophostin A and Inositol 1,4,5-Trisphosphate Differentially Activate Clminus Currents in Xenopus Oocytes Because of Disparate Ca2+ Release Kinetics*

Khaled Machaca and H. Criss HartzellDagger

From the Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322

    ABSTRACT
Top
Abstract
Introduction
References

Depletion of endoplasmic reticulum Ca2+ stores induces Ca2+ entry from the extracellular space by a process termed "store-operated Ca2+ entry" (SOCE). It has been suggested that the novel fungal metabolite adenophostin-A may be able to stimulate Ca2+ entry without stimulating Ca2+ release from stores. To test this idea further, we compared Ca2+ release, SOCE, and the stimulation of Ca2+-activated Cl- currents in Xenopus oocytes in response to inositol 1,4,5-trisphosphate (IP3) and adenophostin-A injection. IP3 stimulated an outward Cl- current, ICl1-S, in response to Ca2+ release from stores followed by an inward current, ICl2, in response to SOCE. In contrast, low concentrations of adenophostins (AdAs) activated ICl2 without activating ICl1-S, consistent with the suggestion that AdA can activate Ca2+ entry without stimulating Ca2+ release. However, when Ca2+ entry has been stimulated by AdA, Ca2+ stores are largely depleted of Ca2+, as assessed by the inability of ionomycin to release additional Ca2+. The Ca2+ release stimulated by AdA, however, was 7 times slower than the release stimulated by IP3, which could explain the minimal activation of ICl1-S; when Ca2+ is released slowly, the threshold level required for ICl1-S activation is not attained.

    INTRODUCTION
Top
Abstract
Introduction
References

Ca2+ signals regulate many cellular processes including cell growth, fertilization, gene transcription, and apoptosis (1). Increases in cytosolic Ca2+ levels are produced both by Ca2+ released from internal stores and Ca2+ influxed from the extracellular space. A major pathway for Ca2+ mobilization from internal stores is through inositol 1,4,5-trisphosphate receptors (IP3R)1 after stimulation of G-protein- or tyrosine kinase-coupled plasma membrane receptors linked to phospholipase C (2-4). The decrease in the Ca2+ content of the internal store then stimulates Ca2+ entry through plasma membrane store-operated Ca2+ channels (SOCs) by a process called store-operated Ca2+ entry (SOCE) (5, 6). The mechanism by which a reduction in the content of store Ca2+ results in opening of SOCs remains unknown, but there are two major hypotheses. The conformational coupling hypothesis suggests that there is direct physical contact between IP3Rs and SOCs such that conformational changes in the IP3R occurring upon Ca2+ depletion of the internal store can affect the opening of SOCs (7, 8). The diffusible messenger hypothesis suggests that the Ca2+ store (endoplasmic reticulum) produces a diffusible messenger that opens SOCs (9, 10).

Recently, a novel family of compounds called adenophostins (AdAs), which are structurally distinct from IP3, have been isolated from cultures of the fungus Penicillium brevicompactum (11, 12). The AdAs are 10-100-fold more potent than IP3 in opening IP3Rs (13) and are capable of activating all three IP3R subtypes (13-15). Recently, Hartzell et al. (16) and DeLisle et al. (17) have shown that in Xenopus oocytes, low concentrations of AdA stimulate Ca2+-activated Cl- currents that are activated by Ca2+ influx more than Cl- currents that are activated by Ca2+ released from stores. Based on these observations, DeLisle et al. (17) suggested that AdA may be capable of activating store-operated Ca2+ entry without first stimulating Ca2+ release from stores. This is significant because it suggests that AdA may share structural features with the putative diffusible Ca2+ entry signal released by Ca2+-depleted endoplasmic reticulum.

Ca2+-activated Cl- currents have been used for many years as real time indicators of sub-plasmalemmal Ca2+ in Xenopus oocytes (18-24), but clearly Cl- currents are only indirect indicators of Ca2+ concentration. Consequently, conclusions about cytosolic Ca2+ concentration derived from these measurements are subject to different interpretations. We have recently found that there are two Ca2+-activated Cl- currents in the oocyte that are selectively activated by Ca2+ released from stores and by Ca2+ influx (24). The Ca2+ release-activated Cl- current (ICl1-S) has an outwardly rectifying steady-state current-voltage relationship, whereas the Ca2+ influx-activated Cl- current (ICl2) has an inwardly rectifying steady-state current-voltage relationship (24). This means that Ca2+-activated Cl- currents measured at constant negative membrane potentials, as was done in the experiments of DeLisle et al. (17), are relatively insensitive indicators of Ca2+ released from stores. In our experiments (16), we measured ICl1-S as an outward current at positive membrane potentials and ICl2 as an inward current at negative membrane potentials to more clearly differentiate between Ca2+ influx and Ca2+ release and to increase the sensitivity of detection of Ca2+ release. Using this protocol, IP3 activated both ICl1-S ("Ca2+ release") and ICl2 ("Ca2+ influx"), but low concentrations of AdA often activated only a tiny amount of ICl1-S, even though ICl2 was robustly activated. But, because we could not find a concentration of AdA which could activate ICl2 without activating some small amount of ICl1-S, we concluded that AdA did not activate SOCE independently of Ca2+ release from stores. We hypothesized that AdA activated relatively little ICl1-S either because AdA released Ca2+ from stores very slowly or that AdA released Ca2+ from a subpopulation of stores which was tightly coupled to SOCs.

The purpose of this paper was to examine further the mechanisms of AdA regulation of Ca2+-activated Cl- currents using confocal scanning microscopy of oocytes loaded with fluorescent Ca2+ indicators and two-microelectrode voltage clamp. Here we show that activation of SOCE following injection of low concentrations of AdA depends upon depletion of intracellular Ca2+ stores. However, at low AdA concentrations the kinetics of Ca2+ release from stores was >7-fold slower than that observed with IP3. This slower mode of Ca2+ release is apparently not effective in activating ICl1-S. Therefore, different kinetics of Ca2+ release can differentially affect Cl- current activation.

    EXPERIMENTAL PROCEDURES

Isolation of Xenopus Oocytes-- Stage V-VI oocytes were harvested from adult albino or normal Xenopus laevis females (Xenopus I) as described by Dascal (18). Xenopus were anesthetized by immersion in Tricaine (1.5 g/liter). Ovarian follicles were removed and digested in normal Ringer with no added calcium, containing 2 mg/ml collagenase type IA (Sigma Chemical Co., St. Louis, MO), for 2 h at room temperature. The oocytes were extensively rinsed with normal Ringer, placed in L-15 medium (Life Technologies, Inc., Gaithersburg, MD) and stored at 18 °C. Oocytes were usually used within 1-5 days after isolation.

Imaging and Electrophysiological Methods-- Xenopusoocytes were injected with 9 nl Ca-green-1 coupled to 70kd dextran (333 µM) for a final calculated oocyte concentration of ~3 µM, and voltage-clamped with two-microelectrodes using a GeneClamp 500 (Axon Instruments, Foster City, CA). Electrodes were filled with 3 M KCl and had resistances of 1-4MOmega . Oocyte resting potentials were between -20 mV and -50 mV. Typically, the membrane was held at 0 mV and stepped to +40 mV for 1.5 s every 15 s to monitor ICl1-S. Every 2.25 min, a 1.5-s duration pulse to -140 mV followed by a 1.5-s duration pulse to +40 mV was given to monitor ICl2 and ICl1-T, respectively. Images (256 × 256 pixels) were acquired 500 ms after the onset of each voltage pulse using a Zeiss LSM 410 confocal box fitted to a Zeiss Axiovert 100TV inverted microscope using a Zeiss 10× objective (0.5 numerical aperature). The confocal aperture was set at the maximal opening, resulting in a focal section 1267 × 1267 × 35 µm. Image data was analyzed using the LSM 410 software or NIH image 1.60 on a Mac IIfx. Current data was analyzed on a Pentium PC using Origin 5.0 (Microcal Software, Northampton, MA). For plots of Ca2+ fluorescence, the fluorescence intensity of the entire confocal section was averaged and expressed as a ratio of the background fluorescence taken before IP3 injection. Experiments were performed at room temperature (22-26 °C). Normal Ringer solution contained 123 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.8 mM MgCl2, 10 mM HEPES, pH 7.4; Ca2+-free Ringer solution was the same except that CaCl2 was omitted and, MgCl2 was increased to 5 mM.

Oocytes were injected with IP3 using a Nanoject automatic oocyte injector (Drummond Scientific Co., Broomall, PA). The injection pipette was pulled from glass capillary tubing in a manner similar to the recording electrodes and then broken so that it had a beveled tip with an inside diameter <20 µm. Solutions of IP3 or AdA were prepared in Chelex resin-treated H2O. The Ca2+ concentration in this solution was not buffered, but injection of H2O produced no change in Ca-green fluorescence or membrane current. Levels of the IP3R were lowered by injection of 60 ng of the IP3R antisense primer (AACTAGACATCTTGTCTGACATTGCTGCAG) one day before the experiment as described by Kume et al. (25). The reverse sense primer (CTGCAGCAATGTCAGACAAGATGTCTAGTT) was injected at the same level as a control.

    RESULTS

Ca2+ Transient and Cl- Currents Activated by High Concentrations of IP3-- The protocol used to measure Ca2+-activated Cl- currents in Xenopus oocytes in response to IP3 or AdA injection while simultaneously measuring cytosolic Ca2+ with confocal microscopy and Ca-green dextran is shown in Fig. 1. About 30 min after injection of Ca-green dextran, the oocytes were voltage-clamped at 0 mV and stepped to +40 mV every 15 s to monitor ICl1-S. ICl1-S (current at the end of the +40 mV pulse, Fig. 1b) is an outward current at depolarizing potentials that is activated quickly (~10 s) after IP3 injection by Ca2+ released from intracellular stores (16, 24, 26). In addition, once every 2.25 min, the oocyte was also stepped to -140 mV to monitor ICl2 and then to +40 mV to monitor ICl1-T. ICl2 (current at the end of the -140 mV pulse, Fig. 1c) is an inward current that is activated by Ca2+ entry through SOCs driven by the negative membrane potential. ICl1-T is a transient outward current (peak outward current during the +40 mV pulse, Fig. 1c) that was activated by a depolarizing pulse preceded by a hyperpolarizing pulse to stimulate Ca2+ influx. The -140 mV pulse was given only once every 2.25 min to minimize Ca2+ influx (and store refilling) during the experiment. For a more detailed discussion of the Cl- currents see Hartzell and co-workers (24, 27, 28).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of large amounts of IP3 on Cl- currents and Ca2+ fluorescence in Xenopus oocyte. The voltage protocol was designed to minimize the amount of Ca2+ influx while still allowing the visualization of the time-dependent activation of the Cl- channels after Ca2+ influx. The cell was stepped to +40 mV for 1.5 s from a holding potential of 0 mV every 15 s for 9 consecutive episodes. In the 10th episode, the cell was stepped to -140 mV then to +40 mV for 1.5 s each. Therefore, every 10th pulse elicited ICl2 and ICl1-T, whereas the intervening pulses elicited ICl1-S. Cells were bathed in normal Ringer. The oocyte was injected with Ca-green-1 coupled to 70-kDa dextran 30 min before the experiment. The oocyte was voltage-clamped with two microelectrodes, and 23 nl of 1 mM IP3 was injected at the arrow. At the end of the experiment, the oocyte was exposed to Ca2+-free Ringer containing 14 µM ionomycin (Ionom.) to assess the Ca2+ content remaining in the stores. a, summary of Cl- current amplitudes before and after IP3 injection. Filled squares, ICl1-S; open circles, ICl2; and open triangles, ICl1-T. b, current traces corresponding to the +40 mV pulses labeled 1 and 2 in a. The voltage protocol used is shown at the top. ICl1-S was measured as the outward current at the end of the +40 mV pulse. c, current traces corresponding to the -140 mV/+40 mV pulse combination labeled 3 and 4 in a. The voltage protocol used is shown at the top. ICl2 was measured as the inward current at the end of the -140 mV pulse. ICl1-T was measured as the peak time-dependent outward current during the +40 mV pulse after the -140 mV pulse. d, Ca2+ fluorescence measured simultaneously with the Cl- currents. Ca2+ levels were measured by Ca-green-1 fluorescence at +40 mV during the +40 mV pulses from the 0-mV holding potential (FCa+40 (filled squares) and at -140 mV (FCa-140 (open circles)) every 10th pulse. Ca2+ fluorescence levels were measured from the entire focal section and normalized to Ca2+-dependent fluorescence before IP3 injection. At the end of the experiment, the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0 Ca2+) to release any residual Ca2+ from stores (see Fig. 6). This cell is representative of 13 cells.

Fig. 1, a-c, shows the response of Cl- currents after injection of large amounts of IP3 (estimated intra-oocyte concentration ~20 µM). When saturating levels of IP3 were injected, ICl1-S (filled squares) was activated immediately. As the stores became depleted of Ca2+ and SOCE developed, ICl1-T (open triangles) and ICl2 (open circles) were activated. Injection of IP3 caused a large increase in Ca2+ fluorescence at all potentials (Fig. 1d) because of Ca2+ release from stores. Before the peak fluorescence was reached, the fluorescence was the same at all potentials, but afterward the fluorescence during the -140 mV pulse became greater than the fluorescence during the +40 mV pulse. The difference between the fluorescence at -140 mV and +40 mV is the voltage-dependent Ca2+ fluorescence, which we have shown is related to Ca2+ entry through SOCs (28).

Ca2+ Waves Stimulated by AdA Are Very Slow-- Injection of large amounts of AdA (estimated intra-oocyte concentration ~2 µM; note that AdA is 10-100 times more potent than IP3 (13)) produced rather similar effects on the Cl- currents to those produced by IP3 (Fig. 2, a-c). There was a striking difference, however, in the kinetics of the Ca2+ fluorescence change produced by IP3 and by AdA. The Ca2+ fluorescence did not begin to increase for several min and peaked ~8 min after injection of AdA (Fig. 2d). By comparison, after IP3 injection, the Ca2+ fluorescence peaked in less than 2 min (Fig. 1d).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of large amounts of AdA on Cl- currents and Ca2+ fluorescence in Xenopus oocyte. The conditions were identical to Fig. 1 except that 23 nl of 100 µM AdA was injected at the arrow. a, summary of Cl- current amplitudes before and after AdA injection. Filled squares, ICl1-S; open circles, ICl2; and open triangles, ICl1-Tb, current traces corresponding to the +40 mV pulses labeled 1 and 2 in a. c, current traces corresponding to the -140 mV/+40 mV pulse combination labeled 3 and 4 in a. d, Ca2+ fluorescence during the +40 mV pulses from the 0 mV holding potential (FCa+40 (filled squares) and at -140 mV (FCa-140 (open circles)) every 10th pulse. At the end of the experiment the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0 Ca2+) to release any residual Ca2+ from stores (see Fig. 6). Note that ICl-1T in this cell does not completely inactivate when the cell is switched to Ca2+-free Ringer, because not all of the Ca2+ in the bath had been removed by the time the pulse that stimulated ICl1-T occurred. Typically, when the cell is switched to Ca2+-free Ringer after AdA injection, ICl1-T and ICl2 completely inactivated as after IP3 injection (Fig. 1a). This cell is representative of six cells.

It may seem surprising in Fig. 2 that the Ca2+ wave peaked so much more slowly than ICl1-S. It should be noted that the 1-mm diameter oocyte is on the stage of an inverted microscope and that the injection takes place at the top, whereas the confocal image plane is <30 µm from the bottom. Cl- currents, which are measured from the entire surface of the oocyte, increase as soon as Ca2+ is released from stores near the injection site. However, the slow increase in Ca2+ fluorescence partly reflects the very slow transit time of the Ca2+ wave from the injection site to the confocal image plane ~1 mm away. There is some variability in the lag period between AdA injection and the increase in Ca2+ fluorescence. This variability is most likely related to the depth and position of the injection pipette in the oocyte.

The Ca2+ waves induced by injection of smaller amounts of AdA moved even more slowly. In Fig. 3a, typical traces of Ca2+ fluorescence at +40 mV in response to injection of large amounts of IP3 (~20 µM, filled squares), large amounts of AdA (~2 µM, open circles), and small amounts of AdA (~5 nM, open triangles) are superimposed. In the case of low concentrations of adenophostin, the time-to-peak of the Ca2+ fluorescence was ~20 min. Fig. 3b shows averages of the time-to-peak of the Ca2+ fluorescence to these injections. The time-to-peak for large concentrations of AdA was >2 times slower than for large concentrations of IP3, and the time-to-peak for small concentrations of AdA was >7 times slower than for large concentrations of IP3. It was not possible to measure the time-to-peak for small IP3 concentrations because small IP3 concentrations produced oscillating Ca2+ waves that exhibited no clear peak. The slowness of the Ca2+ wave is illustrated in a different way in the images in Fig. 3c. After injection of AdA, the spread of the Ca2+ fluorescence is very slow relative to the spread of the IP3-induced wave of Ca2+ release.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.   Comparison of the velocity of Ca2+ waves in response to IP3 and AdA injection. a, Ca2+ fluorescence at +40 mV in response to injection of 23 nl of 1 mM IP3 (filled circles), 23 nl of 100 µM AdA (open circles), and 10 nl of 0.5 µM AdA (open triangles). b, time-to-peak of Ca2+ waves measured from time of injection of 23 nl of 1 mM IP3 (High IP3, n = 6), 23 nl of 100 µM AdA (High AdA, n = 5), and 10 nl of 0.5 µM AdA (Low AdA, n = 7). The speed of the Ca2+ release wave after IP3 or AdA injection was estimated by calculating the time required for the Ca2+-dependent fluorescence to reach its maximal value. It was not possible to perform the same analysis on cells injected with low levels of IP3, because in many instances, such IP3 injections lead to repetitive Ca2+ waves that oscillate and not a single wave that sweeps through the entire oocytes as observed when the oocyte is injected with high IP3 levels or AdA. The speed of the wave was significantly slower (p < 0.006) between high IP3 and AdA injections and between high and low AdA injections. c, images of Ca-green-1 fluorescence in response to injection of 23 nl of 1 mM IP3 (High IP3) and 10 nl of 0.5 µM AdA (Low AdA). The times in min at which the confocal images were taken are indicated in the top left corner of each image. IP3 or AdA were injected at time 0.

These data confirm our earlier suggestion that AdA causes release of Ca2+ from stores much more slowly than IP3 does. These findings support the idea that small concentrations of AdA do not stimulate ICl1-S because slow release of Ca2+ from stores does not elevate Ca2+ in the vicinity of the Cl- channels to an activating level. This could occur if efflux and/or local Ca2+ buffering removes free Ca2+ as rapidly as it is released, so that an effective Ca2+ concentration is not attained.

Small Concentrations of AdA Completely Deplete Ca2+ Stores-- Although Fig. 3 shows that low concentrations of AdA release Ca2+ from stores, the question remains whether the stimulation of Ca2+ entry by low concentrations of AdA is because of depletion of stores. For example, the AdA-stimulated Ca2+ release might be so slow that the stores refill. To examine this question, we measured the effects of low concentrations of AdA (~5 nM) that did not activate ICl1-S on Ca2+ store depletion. Fig. 4 shows the results of a typical experiment. Injection of 10 nl of 0.5 µM AdA did not detectably stimulate ICl1-S (Fig. 4, a-c), but both ICl1-T and ICl2 developed robustly. ICl1-T and ICl2 were dependent on extracellular Ca2+, and their activation corresponded to the activation of SOCE (16). Ca2+ fluorescence began to increase about 5 min after AdA injection and continued to increase for 20 min (Fig. 4d). Voltage-dependent Ca2+ fluorescence (open circles), which reflects SOCE, developed shortly after Ca2+ release and remained at a high level for the duration of the experiment. To test whether stores were depleted of Ca2+, ionomycin in Ca2+-free Ringer was applied to release Ca2+ from any remaining stores. Ionomycin had only a very small effect on ICl1-S and had no effect on the Ca2+ fluorescence at +40 mV. This showed that the stores had been virtually completely depleted of Ca2+ by AdA.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of small amount of AdA on Cl- currents and Ca2+ fluorescence in Xenopus oocyte. The conditions were identical to Fig. 1 except that 10 nl of 0.5 µM AdA was injected at the arrow. a, summary of Cl- current amplitudes before and after AdA injection. Filled squares, ICl1-S; open circles, ICl2; and open triangles, ICl1-T. b, current traces corresponding to the +40 mV pulses labeled 1 and 2 in a. c, current traces corresponding to the -140 mV/+40 mV pulse combination labeled 3 and 4 in a and d. Ca2+ fluorescence during the +40 mV pulses from the 0-mV holding potential (FCa+40 (filled squares)) and at -140 mV (FCa-140 (open circles)) every 10th pulse. At the end of the experiment, the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0 Ca2+) to release any residual Ca2+ from stores (see Fig. 6). This cell is representative of eight cells.

This result contrasts to that observed when small amounts of IP3 were injected (Fig. 5). Concentrations of IP3 that stimulated Ca2+ influx, as determined by the presence of voltage-dependent Ca2+ fluorescence and activation of ICl1-T and ICl2, inevitably stimulated ICl1-S. In some cells, as in Fig. 5, the increase in ICl1-S was not accompanied by a significant increase in Ca2+ fluorescence, because the IP3 effect was local and did not propagate into the region of the oocyte that was imaged. Both voltage-dependent Ca2+ fluorescence and ICl1-T and ICl2 eventually declined to base line. Application of ionomycin at the end of the experiment evoked a large increase in Ca2+ fluorescence and in ICl1-S, showing that the stores were not completely depleted of Ca2+.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of small amounts of IP3 on Cl- currents and Ca2+ fluorescence in Xenopus oocyte. The conditions were identical to Fig. 1 except that 10 nl of 10 µM IP3 was injected at the arrow. a, summary of Cl- current amplitudes before and after IP3 injection. Filled squares, ICl1-S; open circles, ICl2; and open triangles, ICl1-T. b, current traces corresponding to the +40 mV pulses labeled 1, 2, and 5 in a. c, current traces corresponding to the -140 mV/+40 mV pulse combination labeled 3 and 4 in a. d, Ca2+ fluorescence during the +40 mV pulses from the 0-mV holding potential (FCa+40 (filled squares)) and at -140 mV (FCa-140 (open circles)) every 10th pulse. At the end of the experiment, the oocyte was exposed to 14 µM ionomycin in calcium-free Ringer (Ionom./0 Ca2+) to release any residual Ca2+ from stores (see Fig. 6). This cell is representative of 14 cells.

To obtain a more quantitative measure of the extent of store depletion after injection of IP3 or AdA, we calculated the ratio of ionomycin-induced Ca2+ release to IP3- or AdA-induced Ca2+ release. This ratio gives a measure of the level of residual Ca2+ in intracellular stores after IP3R agonist injection. The results from these experiments are shown in Fig. 6. Injections of high IP3, high AdA, or low AdA all left the stores largely depleted of Ca2+. In contrast, low IP3 concentrations were less effective in depleting the stores. These data show that concentrations of AdA that did not noticeably activate ICl1-S were capable of depleting intracellular Ca2+ stores to similar levels as high concentrations of IP3.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 6.   The extent of store depletion after IP3 or AdA injection. The relative Ca2+ content remaining in the internal stores after injection of IP3 or AdA was assessed in Figs. 1, 2, 4, and 5 by exposure of the oocyte to 14 µM ionomycin. The relative amount of Ca2+ remaining in the store was expressed as the ratio of the peak amplitude of the Ca2+ fluorescence at +40 mV produced by ionomycin exposure to the peak Ca2+ fluorescence at +40 mV produced by IP3 or AdA injection. Hi IP3, 23 nl of 1 mM IP3; Hi AdA, 23 nl of 10 µM AdA; Lo IP3, 10 nl of 10 µM IP3; Lo AdA, 10 nl of 0.5 µM AdA. The bars show mean ratio ±S.E. The number of cells used for this analysis is indicated on top of each bar. The level of Ca2+ in intracellular stores after high IP3, high AdA, or low AdA treatments was not significantly different (p > 0.32). However, all three treatments were significantly different (p < 0.025) than injection of low concentrations of IP3.

Thus, we conclude that AdA stimulates SOCE as a consequence of depletion of internal Ca2+ stores and not by some direct effect on SOCs. Furthermore, previous conclusions, based on Ca2+-activated Cl- current activation, which suggested that low concentrations of AdA stimulate SOCE without releasing Ca2+ from stores (17), can be explained by the observation that slow release of Ca2+ from stores is often insufficient to activate ICl1-S.

Effect of AdA on SOCE Requires Active IP3R-- If this conclusion is correct, the effects of AdA on SOCE should depend on the ability of the IP3R to release Ca2+. Thus, treatments that suppress IP3R function should inhibit the effects of AdA injections. We suppressed IP3R function either by injecting the competitive inhibitor heparin (Fig. 7) or by reducing IP3R expression by injection of antisense oligonucleotides to the Xenopus IP3R (Fig. 8).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Heparin blocks both IP3- and AdA-dependent store depletion. a-b,cells were injected with 10 nl of 0.5 µM AdA alone (a; n = 5) or preinjected with 92 nl of 100 mg/ml heparin before AdA injection (b; n = 6). c-d, cells were injected with 10 nl of 50 µM IP3 alone (c; n = 5) or preinjected with 92 nl of 100 mg/ml heparin before IP3 injection (d; n = 3). The site of injection is indicated by the arrow. Cl- currents are measured as described in Fig. 1.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 8.   Lowering IP3R levels blocks AdA and IP3 induced SOCE. Oocytes were injected with 60 ng of sense (a and c) or antisense (b and d) IP3R oligonucleotides (25) and incubated for 1-2 days. Oocytes were injected with 10 nl of 0.5 µM AdA (a and b) or with 23 nl of 10 µM IP3 (c and d) as indicated by the arrow in each panel. Filled squares, ICl1-S; open circles, ICl2; and open triangles, ICl1-T. n = 6-7 oocytes/panel.

Injection of heparin to block the IP3R significantly reduced ICl2 and ICl1-T currents induced by small AdA injections (Fig. 7, a-b). In a similar fashion, heparin blocked the Cl- current response induced by IP3 (Fig. 7, c-d). Reducing IP3R levels by antisense oligonucleotides as described previously by others (25, 29) also reduced the effects of IP3 and AdA treatments on ICl1-T and ICl2 (Fig. 8). The effects of antisense treatment were less pronounced than the effects of heparin, but it was clear that antisense had a significant effect. Note that although antisense treatment inhibited ICl2 and ICl1-T in response to IP3 injection, there was no decrease in levels of ICl1-S (Fig. 8d). Actually, ICl1-S was slightly potentiated as compared with sense-injected cells (Fig. 8c). This observation could be explained if one assumes there are two distinct subpopulations of IP3 receptors with differential turnover rates of the IP3R. If we postulate the existence of subsets of stores, one close to the ICl1-S Cl- channels containing IP3Rs with a very slow turnover rate and a second located further from the Cl- channels containing an IP3R population that turns over rapidly, then injection of antisense IP3 oligonucleotides will reduce the levels of IP3Rs in the latter subset faster, resulting in sufficient Ca2+ release after IP3 injection to activate ICl1-S but insufficient release from most of the stores to induce significant SOCE.

    DISCUSSION

In many cell types, release of Ca2+ from endoplasmic reticulum stores stimulates Ca2+ influx into the cytosol from the extracellular space through SOCs by a process termed SOCE. The mechanisms by which release of Ca2+ from stores stimulates SOCE is unknown, but one hypothesis states that the endoplasmic reticulum releases a diffusible chemical messenger that opens SOCs. The search for such a calcium influx factor has so far not been very fruitful, and the putative calcium influx factors that have been discovered have not found universal acceptance (9, 10). When it was suggested that AdA could stimulate Ca2+ influx without stimulating Ca2+ release from stores (17), some hope was raised that clues to the structure of calcium influx factors would be learned from AdA. The suggestion that AdA could stimulate Ca2+ entry without depleting Ca2+ from stores was based on the observation that low concentrations of AdA did not stimulate Ca2+-activated Cl- currents in the absence of extracellular Ca2+ and therefore did not release Ca2+ from stores but did stimulate Ca2+-activated Cl- currents in the presence of Ca2+ influx. The present studies using Ca2+ imaging demonstrate, however, that even very low amounts of AdA (calculated oocyte concentration ~5 nM) released Ca2+ from stores. Under these conditions, even though ICl1-S was not activated, the stores were completely depleted of Ca2+ as demonstrated by the inability of ionomycin to increase Ca2+ fluorescence. We believe that the Ca2+ released from stores by low concentrations of AdA is unable to activate ICl1-S because of its significantly slower rate of Ca2+ release (~7 times slower than IP3).

How do the kinetics of Ca2+ release from stores determine the response of the Cl- channels? It has been suggested that ICl1-S responds to the rate-of-change of cytosolic Ca2+ (20) because the peak activation of ICl1-S corresponds to the maximum rate of change of cytosolic Ca2+ and because the amplitude of ICl1-S does not correlate with the steady-state levels of cytosolic Ca2+. However, we have shown (27) that the turn-off of ICl1-S is not explained by inactivation of the current as previously suggested (20). Furthermore, we have found that the Ca2+ concentration measured by cytosolic Ca2+ dyes (such as Ca2+-green dextran) does not reflect the concentration of Ca2+ just below the plasma membrane (measured by lipophilic Ca2+ dyes such as Ca-green C18) (28). We have presented evidence that the subplasmalemmal Ca2+ concentration changes much more quickly than does the Ca2+ concentration deeper in the cytosol because plasma membrane Ca2+ efflux systems can rapidly clear Ca2+ from the subplasmalemmal space. Consequently, we would predict that the subplasmalemmal Ca2+ concentration would depend on the relative rates of Ca2+ release from stores and cytosolic Ca2+ buffering and Ca2+ efflux from the oocyte. If Ca2+ release is slow, the concentration of Ca2+ in the subplasmalemmal space may not rise sufficiently to activate Ca2+-activated Cl- channels.

The different kinetics of Ca2+ release produced by AdA and IP3 are probably related to differences between AdA and IP3 activation of IP3Rs. First, the apparent diffusion coefficient of AdA or IP3 in the cytosol will depend on the fraction of molecules (kappa ) that are bound to the IP3R at any one time (Dobs = D/kappa ). Because AdA has a 100-fold higher affinity for the IP3R than IP3 does, AdA diffusion will be slower because a larger fraction of the total AdA (compared with IP3) will be bound to IP3Rs. Second, AdA exhibits a higher cooperativity in activating IP3Rs than IP3 does. Hirota et al. 1995 (13) have shown that IP3 has a Hill coefficient of 1.8 for Ca2+ release by the type 1 IP3R, whereas the Hill coefficient for AdA was 3.9. This implies that at least 2 molecules of IP3 and 4 molecules of AdA are needed to open an IP3R. This factor will also contribute to the slow movement of the Ca2+ release wave in response to small amounts of AdA. Accordingly, the elementary Ca2+ release events ("Ca2+ puffs") induced by AdA have been shown by Marchant and Parker (30) to be smaller and faster than those induced by IP3. Because Ca2+ waves are initiated by the summation of Ca2+ puffs, the smaller and faster puffs induced by AdA may contribute to the slower propagation of the AdA wave. However, the mechanisms by which AdA releases Ca2+ from stores remains to be fully elucidated.

Although low concentrations of AdA evoke a slow release of Ca2+ from stores and little or no ICl1-S, high concentrations release Ca2+ only about 2-fold more slowly than IP3 and also evoke significant ICl1-S. This finding that AdA activates different ICl1-S responses depending upon the kinetics of Ca2+ release from intracellular stores is interesting because it provides another example of how the temporal features of a Ca2+ signal contribute to its physiological consequences. Different receptors can induce different Ca2+ release kinetics depending on factors including spatial localization of the receptor and/or IP3-sensitive stores or the activation of different PLC isoforms (31-36). These different release kinetics can then play an important role in determining which effectors are activated.

    ACKNOWLEDGEMENTS

We thank Dr. K. Tanzawa and Sankyo Co. Ltd. for the adenophostin-A, Alyson Ellingson and Elizabeth Lytle for superb technical assistance, and Raya for inspiration.

    FOOTNOTES

* Supported by National Institutes of Health Grants HL21195 and GM55276.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.

Dagger To whom correspondence should be addressed. Tel.: 404-727-0444; Fax: 404-727-6256; Criss{at}cellbio.emory.edu.

    ABBREVIATIONS

The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptors; SOC, store-operated Ca2+ channels; SOCE, store-operated Ca2+ entry; AdA, adenophostins.

    REFERENCES
Top
Abstract
Introduction
References
  1. Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve]
  2. Clapham, D. (1995) Cell 80, 259-268[Medline] [Order article via Infotrieve]
  3. Rana, R. S., and Hokin, L. E. (1990) Physiol. Rev. 70, 115-164[Free Full Text]
  4. Mikoshiba, K. (1997) Curr. Opin. Neurobiol. 7, 339-345[CrossRef][Medline] [Order article via Infotrieve]
  5. Barish, M. E., and Baud, C. (1984) J. Physiol. 352, 243-263[Abstract]
  6. Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624[Medline] [Order article via Infotrieve]
  7. Irvine, R. F. (1990) FEBS Lett. 263, 5-9[CrossRef][Medline] [Order article via Infotrieve]
  8. Berridge, M. J. (1995) Biochem. J. 312, 1-11[Medline] [Order article via Infotrieve]
  9. Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814[CrossRef][Medline] [Order article via Infotrieve]
  10. Gilon, P., St, J., Bird, G., Bian, X., Yakel, J. L., and Putney, J. W., Jr. (1995) J. Biol. Chem. 270, 8050-8055[Abstract/Free Full Text]
  11. Takahashi, M., Tanzawa, K., and Takahashi, S. (1994) J. Biol. Chem. 269, 369-372[Abstract/Free Full Text]
  12. Takahashi, M., Kagasaki, T., Hosoya, T., and Takahashi, S. (1993) J. Antibiot. (Tokyo) 46, 1643-1647[Medline] [Order article via Infotrieve]
  13. Hirota, J., Michikawa, T., Miyawaki, A., Takahashi, M., Tanzawa, K., Okura, I., Furuichi, T., and Mikoshiba, K. (1995) FEBS Lett. 368, 248-252[CrossRef][Medline] [Order article via Infotrieve]
  14. Marchant, J. S., Beecroft, M. D., Riley, A. M., Jenkins, D. J., Marwood, R. D., Taylor, C. W., and Potter, B. V. L. (1997) Biochemistry 36, 12780-12790[CrossRef][Medline] [Order article via Infotrieve]
  15. Missiaen, L., Parys, J. B., Sienaert, I., Maes, K., Kunzelann, K., Takahashi, M., Tanzawa, K., and De Smedt, H. (1998) J. Biol. Chem. 273, 8983-8986[Abstract/Free Full Text]
  16. Hartzell, H. C., Machaca, K., and Hirayama, Y. (1997) Mol. Pharmacol. 51, 683-692[Abstract/Free Full Text]
  17. DeLisle, S., Marksberry, E. W., Bonnett, C., Jenkins, D. J., Potter, B. V. L., Takahashi, M., and Tanzawa, K. (1997) J. Biol. Chem. 272, 9956-9961[Abstract/Free Full Text]
  18. Dascal, N. (1987) CRC Crit. Rev. Biochem. 22, 317-387[Medline] [Order article via Infotrieve]
  19. Parker, I., and Miledi, R. (1987) Proc. R. Soc. Lond. Ser. B Biol. Sci. 232, 59-70[Medline] [Order article via Infotrieve]
  20. Parker, I., and Yao, Y. (1994) Cell Calcium 15, 276-288[Medline] [Order article via Infotrieve]
  21. Gillo, B., Lass, Y., Nadler, E., and Oron, Y. (1987) J. Physiol. 392, 349-361[Abstract]
  22. Grygorczyk, R., Feighner, S. D., Adam, M., Liu, K. K., LeCouter, J. E., Dashkevicz, M. P., Hreniuk, D. L., Rydberg, E. H., and Arena, J. P. (1996) J. Neurosci. Methods 67, 19-25[CrossRef][Medline] [Order article via Infotrieve]
  23. Boton, R., Dascal, N., Gillo, B., and Lass, Y. (1987) J. Physiol. 408, 511-534[Abstract]
  24. Hartzell, H. C. (1996) J. Gen. Physiol. 108, 157-175[Abstract]
  25. Kume, S., Muto, A., Aruga, J., Nakagawa, T., Michikawa, T., Furuichi, T., Nakade, S., Okano, H., and Mikoshiba, K. (1993) Cell 73, 555-570[Medline] [Order article via Infotrieve]
  26. Machaca, K., and Hartzell, H. C. (1998) Biophys. J. 74, 1286-1295[Abstract/Free Full Text]
  27. Kuruma, A., and Hartzell, H. C. (1999) Am. J. Physiol. 276, C161-C175[Medline] [Order article via Infotrieve]
  28. Machaca, K., and Hartzell, H. C. (1999) J. Gen. Physiol., in press
  29. Kobrinsky, E., Ondrias, K., and Marks, A. R. (1995) Dev. Biol. 172, 531-540[CrossRef][Medline] [Order article via Infotrieve]
  30. Marchant, J. S., and Parker, I. (1998) Biochem. J. 334, 505-509[Medline] [Order article via Infotrieve]
  31. Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993) Cell Calcium 14, 746-757[Medline] [Order article via Infotrieve]
  32. Thorn, P., Lawrie, A. M., Smith, P. M, Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668[Medline] [Order article via Infotrieve]
  33. Xu, X, Zeng, W., Diaz, J., and Muallem, S. (1996) J. Biol. Chem. 271, 24684-24690[Abstract/Free Full Text]
  34. Nathanson, M. H., Padfield, P. J., O'Sullivan, A. J., Burgstahler, A. D., and Jamieson, J. D. (1992) J. Biol. Chem. 267, 18118-18121[Abstract/Free Full Text]
  35. Smrcka, A. V., and Sternweis, P. C. (1993) J. Biol. Chem. 268, 9667-9674[Abstract/Free Full Text]
  36. Camps, M., Carozzi, A., Schnabel, P., Scheer, A., Parker, P. J., and Gierschlik, P. (1992) Nature 360, 684-686[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.