Adenophostin A Can Stimulate Ca2+ Influx without Depleting the Inositol 1,4,5-Trisphosphate-sensitive Ca2+ Stores in the Xenopus Oocyte*

(Received for publication, December 2, 1996, and in revised form, February 3, 1997)

Sylvain DeLisle Dagger §, Erik W. Marksberry Dagger , Carl Bonnett Dagger , David J. Jenkins , Barry V. L. Potter , Masaaki Takahashi ** and Kazuhiko Tanzawa **

From the Dagger  Veterans Administration Medical Center, Department of Internal Medicine, and Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52240, the  School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom, and ** Biological Research Laboratories, Sankyo Co. Ltd., Tokyo 108, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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.


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)-alpha -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+.


Fig. 1. Molecular structure of adenophostin A (A), Ins(1,4,5)P3 (B), and ADAN 1 (C). All three compounds possess two adjacent phosphate-substituted ring carbons (a D-threo-bisphosphate arrangement), with an adjacent hydroxyl-substituted carbon at the equivalent of ring position 6 of Ins(1,4,5)P3. This grouping is also found in all of the inositol phosphate positional isomers that are highly potent at releasing intracellular Ca2+.
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EXPERIMENTAL PROCEDURES

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)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.

Electrophysiology

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 (10-5 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 (10-4 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.


Fig. 2. Adenophostin A releases Ca2+ from Ins(1,4,5)P3-sensitive intracellular stores. On each tracing of this report, Ca2+-gated Cl- 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 [Ca2+]i. Injection (arrow) of adenophostin A (10-5 M in the pipette) (A) or Ins(1,4,5)P3 (10-4 M in the pipette) (B) causes a transient increase in [Ca2+]i, which reflects the release of intracellular Ca2+ (see "Results and Discussion"), followed by a slow increase in [Ca2+]i, which can be blocked by adding Mn2+ to the bath (bar) and therefore represents Ca2+ influx. C, following an injection of adenophostin A (left arrow), Ins(1,4,5)P3 no longer releases intracellular Ca2+ (right arrow). D, the converse experiment is also true, illustrating that Ins(1,4,5)P3 and adenophostin A cross-desensitize for the release of intracellular Ca2+. Note that these experiments are performed in the continued presence of Mn2+ (bar) to block Ca2+ influx.
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Fig. 3. Whole cell current-voltage relationship for the Ni2+- or Mn2+-inhibitable current induced by adenophostin A (closed circles, each representing the average ± S.E. of nine cells) or Ins(1,4,5)P3 (open circles, each representing the average ± S.E. of seven cells).
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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 (10-4 M in the pipette) did not release Ca2+ when injected after adenophostin A (10-4 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 (10-7 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 ATPgamma S (10 mM in the pipette, n = 6).


Fig. 4. Adenophostin A stimulates Ca2+ influx without emptying the Ins(1,4,5)P3-sensitive Ca2+ stores. A, injection of a low concentration of adenophostin A (10-7 M in the pipette) causes a slow increase in [Ca2+]i that can be blocked by adding extracellular Mn2+ (bar). Note the absence of an initial transient release of intracellular Ca2+. B, adenophostin A (10-7 M in the pipette) (arrow) does not increase [Ca2+]i until Mn2+ is removed from the extracellular bath (bar interruption). C, experiment similar to A except for an injection of Ins(1,4,5)P3 (right arrow) performed during the course of adenophostin A-induced Ca2+ influx. Ins(1,4,5)P3 causes a transient increase in [Ca2+]i that is due to the release of intracellular Ca2+, since it occurs despite the presence of extracellular Mn2+ (bar).
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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 (10-4 M in the pipette). When Ca2+ influx reached its maximum, we injected adenophostin A (10-7 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 (10-7 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.


Fig. 5. Evidence that supports adenophostin A acting at the InsP3R to stimulate Ca2+ influx. A, an injection of adenophostin A (right arrow, 10-7 M in the pipette) does not cause Ca2+ influx to increase beyond that induced by Ins(1,4,5)P3 (left arrow, 10-4 M in the pipette). B, an injection of a low concentration of Ins(1,4,5)P3 (10-7 M in the pipette) that had not released Ca2+ initially (left arrow) did so (right arrow) when performed after a subthreshold injection of adenophostin (10-6 M in the pipette, center arrow). Because the second Ins(1,4,5)P3 injection was performed after a time period long enough to allow full metabolism of the initially injected Ins(1,4,5)P3 (30), this tracing represents additivity between adenophostin A and Ins(1,4,5)P3. Note that Ni2+ in the bath (5 mM) prevents adenophostin A from causing Ca2+ influx. C, injection of adenophostin A (10-6 M in the pipette; arrow) in a cell preinjected with heparin did not cause Mn2+-inhibitable current (bar). D, adenophostin A was inactive when added extracellularly (10-6 M in the bath; left arrow) but caused Mn2+-inhibitable current (under the bar) when injected into the cell (10-7 M in the pipette; right arrow).
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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 (10-4 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 (10-6 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).


Fig. 6. Ca2+-signaling activity of ADAN 1. A, injection of ADAN 1 (10-4 M in the pipette) (arrow) results in transient intracellular Ca2+ release, followed by Mn2+-inhibitable current (under the bar). B, injection of threshold concentration of ADAN 1 (10-6 M in the pipette; right arrow) causes prolonged oscillations in [Ca2+]i. These oscillations represent periodic release in intracellular Ca2+, since they are not blocked by extracellular Mn2+ (bar).
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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+.


FOOTNOTES

*   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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   An Established Investigator at the American Heart Association. To whom correspondence should be addressed: Departments of Medicine and Physiology, University of Maryland, School of Medicine, 3B-185, VA Medical Center, 10 N. Greene Street, Baltimore, MD 21201. Tel.: 410-605-7000 (ext. 6449); Fax: 410-605-7957; E-mail: sdelisle{at}umabnet.ab.umd.edu.
1   The abbreviations used are: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; InsP3R, Ins(1,4,5)P3 receptor; CRAC, Ca2+ release-activated Ca2+; CIF, Ca2+ influx factor; ADAN 1, (2-hydroxyethyl)-alpha -Dglucopyranoside 2',3,4-trisphosphate; ATPgamma S, adenosine 5'-O(thiotriphosphate).

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