Adenophostin A can stimulate Ca2+ influx without depleting the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in the Xenopus oocyte.

Adenophostin A possesses the highest known affinity for the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor (InsP3R). The compound shares with Ins(1,4,5)P3 those structural elements essential for binding to the InsP3R. However, its adenosine 2'-phosphate moiety has no counterpart in the Ins(1,4,5)P3 molecule. To determine whether its unique structure conferred a distinctive biological activity, we characterized the adenophostin-induced Ca2+ signal in Xenopus oocytes using the Ca2+-gated Cl- current assay. In high concentrations, adenophostin A released Ca2+ from Ins(1,4, 5)P3-sensitive stores and stimulated a Cl- current that depended upon the presence of extracellular Ca2+. We used this Cl- current as a marker of Ca2+ influx. In low concentrations, however, adenophostin A stimulated Ca2+ influx exclusively. In contrast, Ins(1,4,5)P3 and (2-hydroxyethyl)-alpha-D-glucopyranoside 2',3, 4-trisphosphate, an adenophostin A mimic lacking most of the adenosine moiety, always released intracellular Ca2+ before causing Ca2+ influx. Ins(1,4,5)P3 could still release Ca2+ during adenophostin A-induced Ca2+ influx, confirming that the Ins(1,4, 5)P3-sensitive intracellular Ca2+ stores had not been emptied. Adenophostin- and Ins(1,4,5)P3-induced Ca2+ influx were not additive, suggesting that both agonists stimulated a common Ca2+ entry pathway. Heparin, which blocks binding to the InsP3R, prevented adenophostin-induced Ca2+ influx. These data indicate that adenophostin A can stimulate the influx of Ca2+ across the plasma membrane without inevitably emptying the Ins(1,4,5)P3-sensitive intracellular Ca2+ stores.

Stimulation of many plasma membrane receptors increases the intracellular concentration of the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ). 1 In its best characterized activity, Ins(1,4,5)P 3 binds with high affinity to its receptor (InsP 3 R), a Ca 2ϩ channel that traverses the membranes enclosing intracellular Ca 2ϩ stores (1). As a result of this interaction, the InsP 3 R Ca 2ϩ channel opens, and Ca 2ϩ flows from the internal stores into the cytosol. Very often, however, Ins(1,4,5)P 3 not only releases intracellular Ca 2ϩ , it also stimulates the influx of Ca 2ϩ across the plasma membrane. The cellular mechanism by which Ins(1,4,5)P 3 stimulates Ca 2ϩ influx remains enigmatic. Although Ins(1,4,5)P 3 may directly regulate Ca 2ϩ channels at the plasma membrane in T lymphocytes (2), most experimental data suggest that Ins(1,4,5)P 3 stimulates Ca 2ϩ influx indirectly, by depleting the intracellular Ca 2ϩ stores. We do not know how depletion of the Ca 2ϩ store would translate into the opening of putative plasma membrane "Ca 2ϩ releaseactivated Ca 2ϩ " (CRAC) channels (3). Perhaps a diffusible Ca 2ϩ influx factor (CIF), whose chemical structure and mechanism of action remain to be elucidated (4 -7), could bridge the gap between the Ca 2ϩ stores and the plasma membrane. Alternatively, depletion of the Ca 2ϩ stores may be associated with, but not required for, the development of Ca 2ϩ influx. In the "conformational coupling" model, for example, the InsP 3 R itself interacts with CRAC channels to stimulate Ca 2ϩ influx (8). In this work, we used the compound adenophostin A and a novel synthetic analogue thereof to investigate if it is essential to deplete the Ca 2ϩ stores to stimulate Ca 2ϩ influx.
Adenophostin A, a compound isolated from the culture broth of Penicillium brevicompactum, is the most potent known agonist at the InsP 3 R. Its affinity for the InsP 3 R is ϳ 100-fold greater than Ins(1,4,5)P 3 itself (9, 10). The adenophostin A molecule contains a glucose 3,4-bisphosphate and an adenosine 2Ј-phosphate moiety (Fig. 1). The glucose 3,4-bisphosphate moiety includes the structural elements common to all of the highly potent inositol phosphate positional isomers (Fig. 1) and thus probably targets the Ins(1,4,5)P 3 -binding domain of the InsP 3 R. The synthetic compound (2-hydroxyethyl)-␣-D-glucopyranoside 2Ј,3,4-trisphosphate (ADAN 1) (Fig. 1) possesses the glucose 3,4-bisphosphate but lacks the adenosine 2Ј-phosphate moiety of adenophostin A. ADAN 1 binds to the InsP 3 R with a much lower affinity than that of adenophostin A (11,12). Thus, the adenosine 2Ј-phosphate moiety appears to contribute significantly to the activity of adenophostin A. Because this moiety has no counterpart in the Ins(1,4,5)P 3 molecule, we hypothesized that it interacts with a region of the InsP 3 R located * This work was supported by grants from the American Heart Association, the American Lung Association, and the U.S. Department of Veterans Affairs (to S. D.) and by the Biotechnology and Biological Sciences Research Council (Intracellular Signaling Program) and The Wellcome Trust, Programme Grant No. 045491 (to B. V. L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18

Materials-Adenophostin
A was isolated (10) and ADAN 1 was synthesized (11) as described before. The purity of adenophostin A was more than 90% by high pressure liquid chromatography analysis, and no contaminating signal was observed by NMR spectrometry. ADAN 1 was purified by ion exchange chromatography and was used as the triethylammonium salt. Ins(1,4,5)P 3 was obtained from Calbiochem. BAPTA was from Molecular Probes (Eugene, OR). All other chemicals were from Sigma. Oocyte harvesting, micropipette calibration, and cytosolic microinjections were performed as described previously (13). Injection pipettes were back-filled with adenophostin A, ADAN 1, or Ins(1,4,5)P 3 . The injection volume was kept at 0.7 nl, as determined by measuring the diameter of a droplet microinjected under oil.
Electrophysiology-We assayed changes in free cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) by measuring Ca 2ϩ -activated Cl Ϫ currents with the two-electrode voltage clamp technique as described before (13). This assay has been extensively validated using Ca 2ϩ -sensitive electrodes (14 -16) and fluorescent Ca 2ϩ indicators (13,(17)(18)(19)(20)(21). For most experiments, oocytes were initially stimulated in a bath solution containing 116 mM NaCl, 2 mM KCl, 6 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4. We assessed Ca 2ϩ influx by measuring the change in Ca 2ϩ -gated Cl Ϫ current induced by either lowering the Ca 2ϩ (from 6 to 0.1 mM CaCl 2 ) or increasing the concentration of the inorganic ions Mn 2ϩ (4 mM), Ni 2ϩ (5 mM), or La 3ϩ (5 mM) in the bath. Both Ni 2ϩ and La 3ϩ block Ca 2ϩ channels. Although Mn 2ϩ may go through Ca 2ϩ influx channels, it does not activate the Ca 2ϩ -gated Cl Ϫ channel in the oocyte (22). We preferred using the inorganic ions to reversibly inhibit the Ca 2ϩ -gated Cl Ϫ current caused by Ca 2ϩ influx (22)(23)(24) because the plasma membrane electrical resistance often decreases in low external [Ca 2ϩ ], making some of the recordings difficult to interpret (24). The Ca 2ϩ -gated Cl Ϫ current reflects [Ca 2ϩ ] i just beneath the cytoplasmic membrane; changes in [Ca 2ϩ ] i occurring deeper in the cell are not measured (25,26). Thus, this assay inherently measures [Ca 2ϩ ] i in the cellular area most likely to be affected by Ca 2ϩ influx. The assay also integrates the submembranous [Ca 2ϩ ] i changes across the oocyte's entire plasma membrane surface, thereby maximizing our ability to detect Ca 2ϩ influx. Although the extracellular Ca 2ϩ -dependent Cl Ϫ current assays Ca 2ϩ influx indirectly, for clarity, we use the terms "extracellular Ca 2ϩdependent Cl Ϫ current" and "Ca 2ϩ influx" interchangeably.
When we compared the functional potency of many inositol phosphates, we noticed that in low concentrations, they only released intracellular Ca 2ϩ ; they did not cause Ca 2ϩ influx (29,30). Based on these results, we anticipated that when injected in threshold concentrations, adenophostin A would strictly release intracellular Ca 2ϩ . As the tracing in Fig. 4A shows, a low concentration of adenophostin A (10 Ϫ7 M in the pipette) caused a slow increase in Cl Ϫ current. Contrary to what we had pre-

FIG. 2. Adenophostin A releases Ca 2؉ from Ins(1,4,5)P 3 -sensitive intracellular stores.
On each tracing of this report, Ca 2ϩ -gated Cl Ϫ current (y axis) is expressed as a function of time (x axis). For clarity, the parallel bars delineate the portion of the tracing that has been blanked to remove the artifacts caused by removal and reinsertion of the microinjection pipettes. Inward current (downward deflection) represents an increase in [Ca 2ϩ ] i . Injection (arrow) of adenophostin A (10 Ϫ5 M in the pipette) (A) or Ins(1,4,5)P 3 (10 Ϫ4 M in the pipette) (B) causes a transient increase in [Ca 2ϩ ] i , which reflects the release of intracellular Ca 2ϩ (see "Results and Discussion"), followed by a slow increase in [Ca 2ϩ ] i , which can be blocked by adding Mn 2ϩ to the bath (bar) and therefore represents Ca 2ϩ influx. C, following an injection of adenophostin A (left arrow), Ins(1,4,5)P 3 no longer releases intracellular Ca 2ϩ (right arrow). D, the converse experiment is also true, illustrating that Ins(1,4,5)P 3 and adenophostin A cross-desensitize for the release of intracellular Ca 2ϩ . Note that these experiments are performed in the continued presence of Mn 2ϩ (bar) to block Ca 2ϩ influx. dicted, this current is caused by Ca 2ϩ influx, since it can be blocked by either adding Mn 2ϩ (n ϭ 9) or Ni 2ϩ (n ϭ 7) to the extracellular bath or by decreasing bath [Ca 2ϩ ] (n ϭ 4). The absence of a fast initial component of the response suggested that threshold concentrations of adenophostin A did not release intracellular Ca 2ϩ . To test this possibility further, we injected adenophostin A in the continued presence of Mn 2ϩ or Ni 2ϩ . As the example in Fig. 4B shows, adenophostin A did not elicit a response in the presence of Mn 2ϩ in the bath, whereas subsequent removal of the Mn 2ϩ confirmed that adenophostin had stimulated Ca 2ϩ influx (n ϭ 3). We also considered the possibility that we failed to observe intracellular Ca 2ϩ release because it did not yield a sufficiently high [Ca 2ϩ ] i to stimulate the Ca 2ϩ -gated Cl Ϫ channels. However, the smallest possible increase in [Ca 2ϩ ] i caused by an inositol phosphate is higher than the threshold [Ca 2ϩ ] i required to open the Cl Ϫ channels (29,31). Moreover, the absence of an initial release of intracellular Ca 2ϩ implied that adenophostin A had not depleted the Ins(1,4,5)P 3 -sensitive Ca 2ϩ stores. To verify this prediction, we injected a low concentration of adenophostin A, blocked the resulting Ca 2ϩ influx with Mn 2ϩ , and then injected Ins(1,4,5)P 3 . As shown in Fig. 4C, injection of Ins(1,4,5)P 3 during adenophostin-stimulated Ca 2ϩ influx causes a transient release in intracellular Ca 2ϩ (n ϭ 10). These results indicate that adenophostin A can stimulate Ca 2ϩ influx without emptying the oocyte's Ins(1,4,5)P 3 -sensitive Ca 2ϩ stores. These results also argue against adenophostin A acting as an ATP inhibitor to cause Ca 2ϩ influx: if adenophostin A inhibited the Ca 2ϩ ATPases, then we would have expected the Ins(1,4,5)P 3sensitive intracellular Ca 2ϩ stores to be empty, and they were not. Furthermore, we could not cause Ca 2ϩ influx with other purine compounds expected to inhibit ATPases, such as ADP (10 mM in the pipette, n ϭ 5) or ATP␥S (10 mM in the pipette, n ϭ 6).
Given that adenophostin A has an extremely high affinity for the InsP 3 R and that it cross-desensitizes with Ins(1,4,5)P 3 for the release of intracellular Ca 2ϩ , our working hypothesis was that adenophostin A acted through the InsP 3 R to cause Ca 2ϩ influx. This hypothesis predicted that adenophostin A and Ins(1,4,5)P 3 should stimulate a common Ca 2ϩ entry pathway. To test this prediction, we first injected a high concentration of Ins(1,4,5)P 3 (10 Ϫ4 M in the pipette). When Ca 2ϩ influx reached its maximum, we injected adenophostin A (10 Ϫ7 M). As shown in Fig. 5A, adenophostin A caused Ca 2ϩ influx to return to the maximal value that had been reached with Ins(1,4,5)P 3 alone but not to exceed this value (n ϭ 4). This lack of additivity is consistent with Ins(1,4,5)P 3 and adenophostin A ultimately stimulating a common Ca 2ϩ entry pathway. Contrary to the lack of additivity for Ca 2ϩ influx, the working hypothesis predicted that adenophostin A and Ins(1,4,5)P 3 would act additively to release intracellular Ca 2ϩ . Microinjections of adenophostin A in concentrations sufficient to cause Ca 2ϩ influx, but below the threshold for Ca 2ϩ release, lowered the threshold for Ins(1,4,5)P 3 -induced Ca 2ϩ release (Fig. 5B, n ϭ 6). The working hypothesis also predicted that heparin, which prevents Ins(1,4,5)P 3 (32) and adenophostin A (9) from binding to the InsP 3 R, should prevent adenophostin A from inducing Ca 2ϩ influx. When we preinjected oocytes with heparin (10 mg/ml in pipette, 30-nl injection volume), adenophostin A (10 Ϫ7 M) no longer stimulated Ca 2ϩ influx (n ϭ 5, Fig. 5C). Although heparin could prevent Ca 2ϩ influx through other mechanisms, our aggregate data nevertheless suggest that adenophostin A binds to the InsP 3 R to stimulate Ca 2ϩ influx.
Despite evaluating 47 of the 64 possible positional isomers, we have not encountered an inositol phosphate that stimulates Ca 2ϩ influx exclusively (30). Instead, all of the inositol phosphates, including Ins(1,4,5)P 3 , released intracellular Ca 2ϩ before causing Ca 2ϩ influx (22). The most obvious structural difference between adenophostin A and Ins(1,4,5)P 3 is that the former possesses a large adenosine 2Ј-phosphate moiety. We therefore asked if this moiety was responsible for adenophostin A's unique ability to preferentially stimulate Ca 2ϩ influx. After establishing that D-glucose (n ϭ 3) and glucose 1,6-diphosphate (n ϭ 3) were inactive, we injected the polyphosphorylated Dglucose derivative ADAN 1 (11), which also possesses the glucose 3,4-bisphosphate moiety of adenophostin A (Fig. 1C). When injected in high concentrations, ADAN 1 (10 Ϫ4 M in the pipette, n ϭ 6) caused a biphasic Ca 2ϩ signal similar to that of adenophostin A (Fig. 6A). However, when injected in threshold concentrations (10 Ϫ6 M in the pipette), ADAN 1 did not behave like adenophostin A; it did not cause Ca 2ϩ influx. Instead, ADAN 1 behaved like Ins(1,4,5)P 3 , causing an oscillatory release of intracellular Ca 2ϩ (Fig. 6B). The results with ADAN 1 suggest that the glucose 3,4-bisphosphate moiety of adenophostin A is sufficient to release intracellular Ca 2ϩ but that the adenosine 2Ј-phosphate moiety is required for adenophostin A to preferentially stimulate Ca 2ϩ influx. However, we do not think that the adenosine 2Ј-phosphate moiety is sufficient to FIG. 5. Evidence that supports adenophostin A acting at the InsP 3 R to stimulate Ca 2؉ influx. A, an injection of adenophostin A (right arrow, 10 Ϫ7 M in the pipette) does not cause Ca 2ϩ influx to increase beyond that induced by Ins(1,4,5)P 3 (left arrow, 10 Ϫ4 M in the pipette). B, an injection of a low concentration of Ins(1,4,5)P 3 (10 Ϫ7 M in the pipette) that had not released Ca 2ϩ initially (left arrow) did so (right arrow) when performed after a subthreshold injection of adenophostin (10 Ϫ6 M in the pipette, center arrow). Because the second Ins(1,4,5)P 3 injection was performed after a time period long enough to allow full metabolism of the initially injected Ins(1,4,5)P 3 (30), this tracing represents additivity between adenophostin A and Ins(1,4,5)P 3 . Note that Ni 2ϩ in the bath (5 mM) prevents adenophostin A from causing Ca 2ϩ influx. C, injection of adenophostin A (10 Ϫ6 M in the pipette; arrow) in a cell preinjected with heparin did not cause Mn 2ϩ -inhibitable current (bar). D, adenophostin A was inactive when added extracellularly (10 Ϫ6 M in the bath; left arrow) but caused Mn 2ϩ -inhibitable current (under the bar) when injected into the cell (10 Ϫ7 M in the pipette; right arrow). stimulate Ca 2ϩ influx, because the following compounds, which form an increasing part of the moiety, neither stimulated Ca 2ϩ release nor elicited Ca 2ϩ influx: D-ribose (n ϭ 3), adenine (n ϭ 3), adenosine (n ϭ 4), and adenosine 2Ј-phosphate (n ϭ 6).
Being a small phosphorylated polar compound, adenophostin A discloses some of the key properties attributed to the CIF partially purified from Jurkat cells (6, 7, 33). Like adenophostin A, CIF stimulates Ca 2ϩ influx (7) and can potentiate the Ins(1,4,5)P 3 -induced release of intracellular Ca 2ϩ (34) in the oocyte. Unlike adenophostin A however, CIF's Ca 2ϩ influxstimulating activity is not inhibited by heparin, and CIF does not release intracellular Ca 2ϩ (7). Thus, if CIF and adenophostin A both interact with the InsP 3 R, they may do so through different mechanisms. Also, CIF was found to be active when added extracellularly (6), and adenophostin A is not (n ϭ 6, Fig.  5D). Although there are potential explanations for each of the observed functional differences between the two compounds, only the purification of CIF will determine if adenophostin A is a prototypical Ca 2ϩ influx factor.
In summary, our results suggest that adenophostin A can stimulate Ca 2ϩ influx without depleting the Ins(1,4,5)P 3 -sensitive intracellular Ca 2ϩ stores. Although definitive proof must await a specific InsP 3 R binding inhibitor, our results also suggest that adenophostin A stimulates Ca 2ϩ influx by binding to an InsP 3 R. When considered along with the recent finding that overexpression of type 3 InsP 3 R markedly increases the magnitude of Ca 2ϩ influx without affecting the release of intracellular Ca 2ϩ (35), our results raise the possibility that the InsP 3 R influences Ca 2ϩ influx through mechanisms that extend beyond its ability to release intracellular Ca 2ϩ .