INTRODUCTION

Stimulation of many plasma membrane receptors increases the intracellular concentration of the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃).¹ In its best characterized activity, Ins(1,4,5)P₃ binds with high affinity to its receptor (InsP₃R), a Ca²⁺ channel that traverses the membranes enclosing intracellular Ca²⁺ stores (1). As a result of this interaction, the InsP₃R Ca²⁺ channel opens, and Ca²⁺ flows from the internal stores into the cytosol. Very often, however, Ins(1,4,5)P₃ not only releases intracellular Ca²⁺, it also stimulates the influx of Ca²⁺ across the plasma membrane. The cellular mechanism by which Ins(1,4,5)P₃ stimulates Ca²⁺ influx remains enigmatic. Although Ins(1,4,5)P₃ may directly regulate Ca²⁺ channels at the plasma membrane in T lymphocytes (2), most experimental data suggest that Ins(1,4,5)P₃ stimulates Ca²⁺ influx indirectly, by depleting the intracellular Ca²⁺ stores. We do not know how depletion of the Ca²⁺ store would translate into the opening of putative plasma membrane "Ca²⁺ release-activated Ca²⁺" (CRAC) channels (3). Perhaps a diffusible Ca²⁺ influx factor (CIF), whose chemical structure and mechanism of action remain to be elucidated (4-7), could bridge the gap between the Ca²⁺ stores and the plasma membrane. Alternatively, depletion of the Ca²⁺ stores may be associated with, but not required for, the development of Ca²⁺ influx. In the "conformational coupling" model, for example, the InsP₃R itself interacts with CRAC channels to stimulate Ca²⁺ 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 Ca²⁺ stores to stimulate Ca²⁺ influx.

Adenophostin A, a compound isolated from the culture broth of Penicillium brevicompactum, is the most potent known agonist at the InsP₃R. Its affinity for the InsP₃R is ^~100-fold greater than Ins(1,4,5)P₃ itself (9, 10). The adenophostin A molecule contains a glucose 3,4-bisphosphate and an adenosine 2-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)P₃-binding domain of the InsP₃R. The synthetic compound (2-hydroxyethyl)--D-glucopyranoside 2,3,4-trisphosphate (ADAN 1) (Fig. 1) possesses the glucose 3,4-bisphosphate but lacks the adenosine 2-phosphate moiety of adenophostin A. ADAN 1 binds to the InsP₃R with a much lower affinity than that of adenophostin A (11, 12). Thus, the adenosine 2-phosphate moiety appears to contribute significantly to the activity of adenophostin A. Because this moiety has no counterpart in the Ins(1,4,5)P₃ molecule, we hypothesized that it interacts with a region of the InsP₃R located outside the Ins(1,4,5)P₃-binding domain. If so, then stimulating the InsP₃R with adenophostin A could result in a distinctive Ca²⁺ signal that may uncover novel aspects of the InsP₃R function. To begin to test this hypothesis, we microinjected adenophostin A into Xenopus oocytes and compared the resulting Ca²⁺ signal with that induced by Ins(1,4,5)P₃. Our results show that in high concentrations, adenophostin A and Ins(1,4,5)P₃ produce a similar Ca²⁺ signal. However, in low concentrations, adenophostin A stimulates extracellular Ca²⁺-dependent Cl current (Ca²⁺ influx) without first releasing intracellular Ca²⁺.

EXPERIMENTAL PROCEDURES

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)P₃ 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)P₃. The injection volume was kept at 0.7 nl, as determined by measuring the diameter of a droplet microinjected under oil.

We assayed changes in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) by measuring Ca²⁺-activated Cl currents with the two-electrode voltage clamp technique as described before (13). This assay has been extensively validated using Ca²⁺-sensitive electrodes (14-16) and fluorescent Ca²⁺ indicators (13, 17-21). For most experiments, oocytes were initially stimulated in a bath solution containing 116 mM NaCl, 2 mM KCl, 6 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH 7.4. We assessed Ca²⁺ influx by measuring the change in Ca²⁺-gated Cl current induced by either lowering the Ca²⁺ (from 6 to 0.1 mM CaCl₂) or increasing the concentration of the inorganic ions Mn²⁺ (4 mM), Ni²⁺ (5 mM), or La³⁺ (5 mM) in the bath. Both Ni²⁺ and La³⁺ block Ca²⁺ channels. Although Mn²⁺ may go through Ca²⁺ influx channels, it does not activate the Ca²⁺-gated Cl channel in the oocyte (22). We preferred using the inorganic ions to reversibly inhibit the Ca²⁺-gated Cl current caused by Ca²⁺ influx (22-24) because the plasma membrane electrical resistance often decreases in low external [Ca²⁺], making some of the recordings difficult to interpret (24). The Ca²⁺-gated Cl current reflects [Ca²⁺]_i just beneath the cytoplasmic membrane; changes in [Ca²⁺]_i occurring deeper in the cell are not measured (25, 26). Thus, this assay inherently measures [Ca²⁺]_i in the cellular area most likely to be affected by Ca²⁺ influx. The assay also integrates the submembranous [Ca²⁺]_i changes across the oocyte's entire plasma membrane surface, thereby maximizing our ability to detect Ca²⁺ influx. Although the extracellular Ca²⁺-dependent Cl current assays Ca²⁺ influx indirectly, for clarity, we use the terms "extracellular Ca²⁺-dependent Cl current" and "Ca²⁺ influx" interchangeably.

RESULTS AND DISCUSSION

When injected in high concentration (10⁵ M in the pipette), adenophostin A causes a biphasic response; there is an initial, short lived increase in [Ca²⁺]_i followed by a slow increase (Fig. 2A, n = 16). This response pattern is similar to that generated by high concentrations of Ins(1,4,5)P₃ (10⁴ M in the pipette, n = 8; and see Ref. 22). Both components of the response to adenophostin A are due to an increase in [Ca²⁺]_i because they can be completely blocked by the Ca²⁺ chelator BAPTA (1 nmol, n = 6, flat tracings not shown). The initial transient increase of [Ca²⁺]_i caused by both agonists persists in the presence of extracellular Mn²⁺ (4 mM) (Fig. 2, C and D) or in low extracellular [Ca²⁺] (n = 5 for adenophostin A, n = 6 for Ins(1,4,5)P₃; see also Ref. 23) and represents the release of Ca²⁺ from the intracellular stores. In contrast, the slow increase in [Ca²⁺]_i can be blocked by adding either extracellular Mn²⁺ (4 mM, Fig. 2, A and B, n = 15 for adenophostin A, n = 3 and see Ref. 22 for Ins(1,4,5)P₃), extracellular Ni²⁺ (5 mM, n = 17 for adenophostin A, n = 5 for Ins(1,4,5)P₃) or extracellular La³⁺ (5 mM, n = 4 for adenophostin A, n = 6 for Ins(1,4,5)P₃). This slow increase in [Ca²⁺]_i can also be blocked by decreasing the extracellular [Ca²⁺] from 6 to 0.1 mM (n = 10 for adenophostin A; see Refs. 22 and 23 for Ins(1,4,5)P₃). These results indicate that the slow component of the response results from the influx of extracellular Ca²⁺. The current-voltage relationship of the Ins(1,4,5)P₃-induced (n = 7) and of the adenophostin-induced (n = 9) extracellular Ca²⁺-dependent currents was linear within the 90 to 10 mV range. Reversal potentials were similar for both agonists (23 ± 4 for Ins(1,4,5)P₃ and 27 ± 4 mV for adenophostin) and consistent with the extracellular Ca²⁺-dependent current being carried mainly by Cl ions (27, 28) (Fig. 3). While Ins(1,4,5)P₃-induced Ca²⁺ influx ends after 30-35 min. (22), with adenophostin A, the extracellular Ca²⁺-dependent Cl current continues for at least 1 h (n = 6). This prolonged Ca²⁺ influx probably reflects the poor metabolism of adenophostin A, since 1) adenophostin A resists inactivation by the Ins(1,4,5)P₃ 3-kinase and 5-phosphatase enzymes in vitro (9) and 2) poorly metabolized inositol phosphate cause prolonged Ca²⁺ influx in the oocyte (22, 23). Aside from the duration of Ca²⁺ influx, high concentrations of adenophostin A and Ins(1,4,5)P₃ produce similar Ca²⁺ signals.

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To determine if adenophostin A and Ins(1,4,5)P₃ released Ca²⁺ from the same intracellular pool, we injected Ins(1,4,5)P₃ and adenophostin A sequentially. As seen in Fig. 2C, Ins(1,4,5)P₃ (10⁴ M in the pipette) did not release Ca²⁺ when injected after adenophostin A (10⁴ M in the pipette, n = 8). Conversely, adenophostin A did not release Ca²⁺ when injected after Ins(1,4,5)P₃ (Fig. 2D, n = 3). This cross-desensitization suggests that adenophostin A releases Ca²⁺ from the oocyte's Ins(1,4,5)P₃-sensitive Ca²⁺ pools. Taken together, our data are consistent with adenophostin A stimulating the InsP₃R (10).

When we compared the functional potency of many inositol phosphates, we noticed that in low concentrations, they only released intracellular Ca²⁺; they did not cause Ca²⁺ influx (29, 30). Based on these results, we anticipated that when injected in threshold concentrations, adenophostin A would strictly release intracellular Ca²⁺. As the tracing in Fig. 4A shows, a low concentration of adenophostin A (10⁷ M in the pipette) caused a slow increase in Cl current. Contrary to what we had predicted, this current is caused by Ca²⁺ influx, since it can be blocked by either adding Mn²⁺ (n = 9) or Ni²⁺ (n = 7) to the extracellular bath or by decreasing bath [Ca²⁺] (n = 4). The absence of a fast initial component of the response suggested that threshold concentrations of adenophostin A did not release intracellular Ca²⁺. To test this possibility further, we injected adenophostin A in the continued presence of Mn²⁺ or Ni²⁺. As the example in Fig. 4B shows, adenophostin A did not elicit a response in the presence of Mn²⁺ in the bath, whereas subsequent removal of the Mn²⁺ confirmed that adenophostin had stimulated Ca²⁺ influx (n = 3). We also considered the possibility that we failed to observe intracellular Ca²⁺ release because it did not yield a sufficiently high [Ca²⁺]_i to stimulate the Ca²⁺-gated Cl channels. However, the smallest possible increase in [Ca²⁺]_i caused by an inositol phosphate is higher than the threshold [Ca²⁺]_i required to open the Cl channels (29, 31). Moreover, the absence of an initial release of intracellular Ca²⁺ implied that adenophostin A had not depleted the Ins(1,4,5)P₃-sensitive Ca²⁺ stores. To verify this prediction, we injected a low concentration of adenophostin A, blocked the resulting Ca²⁺ influx with Mn²⁺, and then injected Ins(1,4,5)P₃. As shown in Fig. 4C, injection of Ins(1,4,5)P₃ during adenophostin-stimulated Ca²⁺ influx causes a transient release in intracellular Ca²⁺ (n = 10). These results indicate that adenophostin A can stimulate Ca²⁺ influx without emptying the oocyte's Ins(1,4,5)P₃-sensitive Ca²⁺ stores. These results also argue against adenophostin A acting as an ATP inhibitor to cause Ca²⁺ influx: if adenophostin A inhibited the Ca²⁺ ATPases, then we would have expected the Ins(1,4,5)P₃-sensitive intracellular Ca²⁺ stores to be empty, and they were not. Furthermore, we could not cause Ca²⁺ influx with other purine compounds expected to inhibit ATPases, such as ADP (10 mM in the pipette, n = 5) or ATPS (10 mM in the pipette, n = 6).

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Given that adenophostin A has an extremely high affinity for the InsP₃R and that it cross-desensitizes with Ins(1,4,5)P₃ for the release of intracellular Ca²⁺, our working hypothesis was that adenophostin A acted through the InsP₃R to cause Ca²⁺ influx. This hypothesis predicted that adenophostin A and Ins(1,4,5)P₃ should stimulate a common Ca²⁺ entry pathway. To test this prediction, we first injected a high concentration of Ins(1,4,5)P₃ (10⁴ M in the pipette). When Ca²⁺ influx reached its maximum, we injected adenophostin A (10⁷ M). As shown in Fig. 5A, adenophostin A caused Ca²⁺ influx to return to the maximal value that had been reached with Ins(1,4,5)P₃ alone but not to exceed this value (n = 4). This lack of additivity is consistent with Ins(1,4,5)P₃ and adenophostin A ultimately stimulating a common Ca²⁺ entry pathway. Contrary to the lack of additivity for Ca²⁺ influx, the working hypothesis predicted that adenophostin A and Ins(1,4,5)P₃ would act additively to release intracellular Ca²⁺. Microinjections of adenophostin A in concentrations sufficient to cause Ca²⁺ influx, but below the threshold for Ca²⁺ release, lowered the threshold for Ins(1,4,5)P₃-induced Ca²⁺ release (Fig. 5B, n = 6). The working hypothesis also predicted that heparin, which prevents Ins(1,4,5)P₃ (32) and adenophostin A (9) from binding to the InsP₃R, should prevent adenophostin A from inducing Ca²⁺ influx. When we preinjected oocytes with heparin (10 mg/ml in pipette, 30-nl injection volume), adenophostin A (10⁷ M) no longer stimulated Ca²⁺ influx (n = 5, Fig. 5C). Although heparin could prevent Ca²⁺ influx through other mechanisms, our aggregate data nevertheless suggest that adenophostin A binds to the InsP₃R to stimulate Ca²⁺ influx.

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Despite evaluating 47 of the 64 possible positional isomers, we have not encountered an inositol phosphate that stimulates Ca²⁺ influx exclusively (30). Instead, all of the inositol phosphates, including Ins(1,4,5)P₃, released intracellular Ca²⁺ before causing Ca²⁺ influx (22). The most obvious structural difference between adenophostin A and Ins(1,4,5)P₃ is that the former possesses a large adenosine 2-phosphate moiety. We therefore asked if this moiety was responsible for adenophostin A's unique ability to preferentially stimulate Ca²⁺ influx. After establishing that D-glucose (n = 3) and glucose 1,6-diphosphate (n = 3) were inactive, we injected the polyphosphorylated D-glucose 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 (10⁴ M in the pipette, n = 6) caused a biphasic Ca²⁺ signal similar to that of adenophostin A (Fig. 6A). However, when injected in threshold concentrations (10⁶ M in the pipette), ADAN 1 did not behave like adenophostin A; it did not cause Ca²⁺ influx. Instead, ADAN 1 behaved like Ins(1,4,5)P₃, causing an oscillatory release of intracellular Ca²⁺ (Fig. 6B). The results with ADAN 1 suggest that the glucose 3,4-bisphosphate moiety of adenophostin A is sufficient to release intracellular Ca²⁺ but that the adenosine 2-phosphate moiety is required for adenophostin A to preferentially stimulate Ca²⁺ influx. However, we do not think that the adenosine 2-phosphate moiety is sufficient to stimulate Ca²⁺ influx, because the following compounds, which form an increasing part of the moiety, neither stimulated Ca²⁺ release nor elicited Ca²⁺ influx: D-ribose (n = 3), adenine (n = 3), adenosine (n = 4), and adenosine 2-phosphate (n = 6).

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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 Ca²⁺ influx (7) and can potentiate the Ins(1,4,5)P₃-induced release of intracellular Ca²⁺ (34) in the oocyte. Unlike adenophostin A however, CIF's Ca²⁺ influx-stimulating activity is not inhibited by heparin, and CIF does not release intracellular Ca²⁺ (7). Thus, if CIF and adenophostin A both interact with the InsP₃R, they may do so through different mechanisms. Also, CIF was found to be active when added extracellularly (6), and adenophostin A is not (n = 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 Ca²⁺ influx factor.

In summary, our results suggest that adenophostin A can stimulate Ca²⁺ influx without depleting the Ins(1,4,5)P₃-sensitive intracellular Ca²⁺ stores. Although definitive proof must await a specific InsP₃R binding inhibitor, our results also suggest that adenophostin A stimulates Ca²⁺ influx by binding to an InsP₃R. When considered along with the recent finding that overexpression of type 3 InsP₃R markedly increases the magnitude of Ca²⁺ influx without affecting the release of intracellular Ca²⁺ (35), our results raise the possibility that the InsP₃R influences Ca²⁺ influx through mechanisms that extend beyond its ability to release intracellular Ca²⁺.