Ca2+ Entry Induced by Cyclic ADP-ribose in Intact T-Lymphocytes*

(Received for publication, December 5, 1996, and in revised form, January 24, 1997)

Andreas H. Guse Dagger §, Ingeborg Berg Dagger , Cristina P. da Silva Dagger , Barry V. L. Potter par ** and Georg W. Mayr Dagger

From the Dagger  University of Hamburg, Institute of Physiological Chemistry, Department of Enzyme Chemistry, Grindelallee 117, D-20146 Hamburg, Germany and the par  University of Bath, School of Pharmacy and Pharmacology, Claverton Down, Bath BA2 7AY, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cyclic ADP-ribose (cADPr) is a potent Ca2+-mobilizing natural compound (Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615) which has been shown to release Ca2+ from an intracellular store of permeabilized T-lymphocytes (Guse, A. H., Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Microinjection of cADPr into intact single T lymphocytes dose dependently induced repetitive but irregular Ca2+ spikes which were almost completely dependent on the presence of extracellular Ca2+. The Ca2+ spikes induced by cADPr could be blocked either by co-injection of cADPr with the specific antagonist 8-NH2-cADPr, by omission of Ca2+ from the medium, or by superfusion of the cells with Zn2+ or SK-F 96365. Ratiometric digital Ca2+ imaging revealed that single Ca2+ spikes were initiated at several sites ("hot spots") close to the plasma membrane. These hot spots then rapidly formed a circular zone of high Ca2+ concentration below the plasma membrane which subsequently propagated like a closing optical diaphragm into the center of the cell. Taken together these data indicate a role for cADPr in Ca2+ entry in T-lymphocytes.


INTRODUCTION

Intracellular Ca2+ signaling is one of the major events transducing extracellular signals into many different types of living cells. In Jurkat T-lymphocytes it is well accepted that D-myo-inositol 1,4,5-trisphosphate (IP3)1 releases Ca2+ from an intracellular store located in the endoplasmic reticulum via its specific receptor (1-3). In addition, in T-lymphocytes a sustained long-lasting Ca2+ entry can be observed in response to stimulation of the T cell receptor-CD3 complex (4-6). This Ca2+ entry is necessary for clonal expansion of T cells and therefore essential for a functional immune response. One of the basic mechanisms underlying Ca2+ entry in electrically non-excitable cells appears to be the "capacitative" mechanism (reviewed in Refs. 7 and 8). The central idea of the capacitative mechanism is that a decrease in intraluminal Ca2+, e.g. as a result of IP3-induced Ca2+ release, in turn leads to Ca2+ entry. It is less clear, however, how this information from the intracellular stores is then transduced to the plasma membrane to activate opening of Ca2+ channels (reviewed in Refs. 7 and 8).

In addition to IP3, cyclic ADP-ribose (cADPr) has been shown to be a potent natural ligand for mobilization of Ca2+ from intracellular stores in sea urchin eggs (9). Now, there is increasing evidence that in addition to some other higher eukaryotic cell systems (10-15), cADPr is also involved in intracellular Ca2+ signaling in T-lymphocytes. We and others have recently demonstrated cADPr-induced Ca2+ release from a non-thapsigargin sensitive intracellular store of T-lymphocyte cell lines (16, 17). These stores were isolated from mouse T-lymphoma cells as light-density membrane vesicles carrying ryanodine receptors being different from the endoplasmic reticulum and localized close to the plasma membrane (17). In addition, the expression of brain-type ryanodine receptors also in human Jurkat T cells was observed (18), although in certain Jurkat subclones the expression of ryanodine receptors was not detectable (19).

To determine the function of cADPr in Ca2+ signaling in intact T cells, especially a potential link between a decrease in the intraluminal Ca2+ concentration in the cADPr-sensitive Ca2+ stores and Ca2+ entry, we microinjected cADPr into intact cells while recording digital Ca2+ images of Fura2-loaded cells.


EXPERIMENTAL PROCEDURES

Materials

cADPr, 8-NH2-cADPr, and D-myo-inositol 1,4,6-trisphosphorothioate were synthesized as described (20-22), purified by anion-exchange chromatography on Q-Sepharose, and used as their triethylammonium salts. Purity of ligands was assessed by 1H and 31P NMR spectroscopy, mass spectrometry and, when appropriate, high performance liquid chromatography.

Digital Ca2+ Imaging and Analysis of Data

Jurkat T-lymphocytes were cultured in RPMI 1640 medium supplemented with fetal bovine calf serum (10%, v/v), penicillin (100 units/ml), and streptomycin (50 µg/ml). The cells were loaded with Fura2/AM as described (3). For parallel microinjection and Ca2+ imaging experiments, the T cells had to be firmly attached to thin (0.2 mm) glass coverslips (to keep them in a defined position while forcing them with the microinjection pipette), but to stay inactivated in terms of Ca2+ signaling. To achieve this, several different coating methods for the glass surface were tested, e.g. bovine serum albumin at 0.1, 0.5, or 1.0 mg/ml, agarose at 0.01, 0.05, 0.1, or 0.25 mg/ml, GelRite (Roth, Karlsruhe, Germany) at 0.05 or 0.1 mg/ml and poly-L-lysine at 0.01, 0.05, or 0.1 mg/ml. Many of these conditions resulted in activation of Ca2+ signals, except low concentration of bovine serum albumin and poly-L-lysine. Of these different approaches, only the following combination of coatings proved to be the most useful: the glass coverslips were coated first with bovine serum albumin (5 mg/ml), and then with poly-L-lysine (0.1 mg/ml). Then, a small chamber (80 µl volume) was fixed onto the coverslip and the Fura2-loaded cells were added and superfused with a buffer containing 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes, 1 mM NaH2PO4, 5.5 mM glucose, pH 7.4. The [Ca2+]i of the single, adherent T-lymphocytes was analyzed with a digital ratiometric imaging station (PhotoMed GmbH/Photon Technology, Wedel, Germany). The coated coverslips with cells were mounted on the stage of an inverted Axiovert 100 fluorescence microscope (Zeiss, Oberkochen, Germany). The excitation light source beam was split using an optical chopper, then passed through either a 340- or 380-nm optical filter, and guided into the microscope via fiber optics. The fluorescence intensity was filtered at 510 nm and then monitored using a CCD camera at a resolution of 525 × 487 pixels (type C2400-77, Hamamatsu, Garching, Germany). The data sampling rate usually was 1 ratio/5 s, in some experiments 1 ratio/s. Re-analysis of Ca2+ image data was carried out using the so-called region-of-interest function of the ImageMaster software (PhotoMed GmbH/Photon Technology, Wedel, Germany). Region of images were set either to cover the whole cell or subregions of the cell. The numerical median ratios and the corresponding free Ca2+ concentrations were calculated by the software using external calibration.

Microinjection

Microinjections were done with an Eppendorf system (transjector type 5246, micromanipulator type 5171; Eppendorf-Netheler-Hinz, Hamburg, Germany) using Femtotips II as pipettes. The compounds to be microinjected were diluted to their final concentrations in intracellular buffer (20 mM Hepes, 110 mM KCl, 2 mM MgCl2, 5 mM KH2PO4, 10 mM NaCl, pH 7.2) and filtered (0.2 µm) directly before filling into the Femtotips. Injections were made using the semiautomatic mode of the Eppendorf system at a pipette angle of 45° and the following instrumental settings: injection pressure 80 hPa, compensatory pressure 60 hPa, injection time 0.5 to 1 s, and velocity of the pipette 700 µm/s. Under such conditions the injection volume was 1-1.5% of the cell volume as measured by microinjection of a fluorescent compound (Fura2-free acid) and subsequent determination of its quantity in a spectrofluorimeter at 360 nm (excitation) and 500 nm (emission).


RESULTS

Ca2+ Signaling Induced by Microinjection of cADPr

In response to microinjection of cADPr, the free intracellular Ca2+ concentration ([Ca2+]i) increased in an oscillatory manner; either two to several irregular Ca2+ spikes or a combination of spikes and sustained elevated Ca2+ levels were observed (Fig. 1, B, C, and D). The effect of cADPr was dose-dependent showing no increases in [Ca2+]i when intracellular buffer was injected, and increasing responses when cADPr in the pipette was elevated stepwise to 100 µM (Fig. 1). During every injection about 1-1.5% of the cell volume was injected. We assumed a fast dilution of cADPr in the cell being comparable to the diffusion reported for IP3 in cytosolic extracts of Xenopus laevis (23). Considering the delay between injection and onset of the signal (Fig. 1, B, C, and D), a dilution factor of at least 50 must be assumed when estimating the effective intracellular concentration. Thus, at 1 µM cADPr in the pipette, which was the threshold concentration (Fig. 1, B and F), the intracellular concentration of cADPr should be about 20 nM. Ca2+ spikes with amplitudes up to 1.5 µM [Ca2+]i were observed at a pipette concentration of 10 µM cADPr, amounting to an effective concentration of about 200 nM cADPr in the cell (Fig. 1, C and G). At 100 µM cADPr in the pipette (about 2 µM cADPr in the cell) usually 1 or 2 fast and large spikes were observed followed by a decline to an elevated level which was maintained for a relatively long period of time (Fig. 1, D and H). A pipette concentration of 100 µM cADPr as compared with 10 µM resulted in a considerably faster onset of the Ca2+ signal (compare Fig. 1, G and H); however, later after microinjection, e.g. after 600 s, high Ca2+ spikes were still observed at 10 µM, but not at 100 µM cADPr in the pipette (Fig. 1, C and D). At a pipette concentration of 1 mM cADPr (data not shown), the Ca2+ signals were very similar to the ones obtained at 100 µM indicating saturation of the dose-response relationship.


Fig. 1. Ca2+ signals in single intact T-lymphocytes microinjected with cADPr. Jurkat T-lymphocytes were loaded with Fura2/AM and [Ca2+]i was measured as detailed under "Experimental Procedures." During re-analysis of the image data, regions of interest were set to cover the whole cell; data obtained from these regions of interest are plotted against time. Cells were injected as described under "Experimental Procedures" in the presence of 1 mM extracellular Ca2+ with: A, intracellular buffer (number of cells displayed, n = 3), or increasing concentrations of cADPr as indicated (B-D, n = 4-10). Averages from these measurements are shown in E-H. Arrows indicate the time point of microinjection.
[View Larger Version of this Image (37K GIF file)]


Ca2+ Signaling Induced by cADPr and IP3 Can Be Blocked Specifically

The effect of cADPr was specific, because it could be blocked by co-injection of a 10-fold excess of the specific antagonist 8-NH2-cADPr (Refs. 24 and 25; Fig. 2, A and B). For comparison, IP3 was also microinjected. The resulting Ca2+ spike pattern was somewhat more regular and usually not as long-lasting as compared with cADPr (Fig. 2C). Specificity was demonstrated by inhibition of the effect by co-injection with a 10-fold excess of the partial antagonist D-myo-inositol 1,4,6-phosphorothioate (Fig. 2D).


Fig. 2. Specific antagonists inhibit both cADPr- and IP3-induced Ca2+ signaling. [Ca2+]i was measured in Fura2-loaded Jurkat T-lymphocytes using the digital ratiometric Ca2+ imaging system. The cells were then microinjected with ligands as described under "Experimental Procedures." Data are presented as typical tracings from 1 individual cell. During reanalysis of the image data, regions of interest were set to cover the whole cell; data obtained from these regions of interest are plotted against time. Shown are (n = number of experiments): A, microinjection of cADPr (10 µM) in the presence of extracellular Ca2+ (n = 30); B, co-injection of cADPr (10 µM) with a 10-fold excess of the antagonist 8-NH2-cADPr (n = 5); C, microinjection of IP3 (4 µM) in the presence of 1 mM extracellular Ca2+ (n = 15); D, co-injection of IP3 (4 µM) with a 10-fold excess of the partial antagonist D-myo-inositol 1,4,6-trisphosphorothioate in the presence of 1 mM extracellular Ca2+ (n = 7). Arrows mark the time point of microinjection.
[View Larger Version of this Image (26K GIF file)]


Ca2+ Entry Induced by cADPr

Since in most of the experiments, microinjection of cADPr did not result in a single Ca2+ spike, but in longer lasting trains of spikes (Figs. 1, 2A, and 3A), the involvement of Ca2+ entry in these sustained signals was investigated. Such cADPr-induced Ca2+ signals were nearly completely abolished when the cells were superfused with extracellular buffer containing no Ca2+ (Fig. 3B). Moreover, when cells superfused with 1 mM Ca2+ and microinjected with 10 µM cADPr were challenged with Zn2+ (1 mM) after the first Ca2+ spike, further spikes were completely inhibited (Fig. 3C). The effect of Zn2+ could be washed out (data not shown), indicating that Zn2+ acted by blocking Ca2+ entry at the plasma membrane level. Further evidence for Ca2+ entry in response to microinjected cADPr was obtained by blocking the Ca2+ signals with the drug SK-F 96365 (Ref. 26; Fig. 3D). The IP3-induced Ca2+ spikes also were largely dependent on the presence of external Ca2+ (data not shown).


Fig. 3. Extracellular Ca2+ is necessary for cADPr-induced Ca2+ signaling. [Ca2+]i was measured in Fura2-loaded Jurkat T-lymphocytes using the digital ratiometric Ca2+ imaging system. The cells were then microinjected with ligands as described under "Experimental Procedures." Data are presented as typical tracings from 1 individual cell. During reanalysis of the image data, regions of interest were set to cover the whole cell; data obtained from these regions of interest are plotted against time. Shown are (n = number of experiments): A, microinjection of cADPr (10 µM) in the presence of extracellular Ca2+ (n = 30); B, microinjection of cADPr (10 µM) in the absence of extracellular Ca2+ (n = 5); C, microinjection of cADPr (10 µM) in the presence of extracellular Ca2+ (1 mM), Zn2+ (1 mM) was added for the time span indicated (n = 7); D, microinjection of cADPr (10 µM) in the presence of extracellular Ca2+ (1 mM), the antagonist SK-F 96365 (15 µM) was added where indicated (n = 4). Arrows mark the time point of microinjection.
[View Larger Version of this Image (34K GIF file)]


Spatial Development of Ca2+ Spikes Induced by cADPr

The spatial development of a single Ca2+ spike in response to microinjection of cADPr is characterized by (i) a rapid increase of [Ca2+]i to a slightly elevated level within 5 to 10 s throughout the cell (Fig. 4, upper panel, image 1-3), (ii) a slowly generated wave of high [Ca2+]i initiating from distinct sites ("hot spots") which then formed a circular zone close to the plasma membrane (Fig. 4, upper panel, images 3 and 4) and propagating like a closing optical diaphragm into the center of the cell (Fig. 4, upper panel, images 4-6). At the top of the spike very high [Ca2+]i were observed in the central part of the cell where the nucleus is located (Fig. 4, upper panel, image 6). At a slightly slower velocity, Ca2+ was removed from the central part of the cell reaching a level that was slightly elevated (Fig. 4, lower panel, images 7-9). This level in the central part of the cell was then maintained for some 100 s, while Ca2+ oscillations were observed during this period of time in the subplasmalemmal cytoplasm (Fig. 4, lower panel, images 10 and 11). Finally, the cytoplasm also nearly reached basal Ca2+ levels (Fig. 4, lower panel, image 12), before the next Ca2+ spike appeared (data not shown). Such cADPr-induced Ca2+ waves propagating at high [Ca2+] throughout the whole cell including the large nucleus of T-lymphocytes were not seen in all cases; in a number of cells Ca2+ signals of high amplitude developed mainly in the cytoplasm reaching at the top of the spike a status comparable to image 4 in Fig. 4. Then [Ca2+]i in this circular zone returned to basal concentrations. However, irrespective of whether cADPr might have been injected into the cytoplasm or in the nucleus, Ca2+ waves were always initiated in the subplasmalemmal space pointing toward a crucial role of Ca2+ influx in both cases.


Fig. 4. Ca2+ wave induced by microinjection of cADPr. A single Fura2-loaded Jurkat T cell was microinjected with cADPr (10 µM) in the presence of 1 mM extracellular Ca2+ resulting in a train of Ca2+ spikes out of which the first spike is shown in detail. During reanalysis of the image data, three different regions of interest (ROI) (A-C) have been located inside the cell to display [Ca2+]i between the plasma membrane (ROIA) and the nucleus (ROIC). Pseudocolor ratiometric images taken at individual time points as indicated by the numbered arrows are displayed in an upper panel (showing the development of the Ca2+ signal) and in a lower panel (showing the decrease of the Ca2+ signal). Note that the Ca2+ signal started in a circular manner (image 2) propagating like a closing optical diaphragm to a very high intracellular concentration except a region close to the plasma membrane (image 6). The sampling rate of each ratio image was 5 s.
[View Larger Version of this Image (60K GIF file)]



DISCUSSION

The mechanism of Ca2+ entry in T-lymphocytes as well as in other electrically non-excitable cells is not well understood. The trigger to switch on the capacitative Ca2+ entry has been reported to be likely the depletion of the IP3-sensitive intracellular Ca2+ pool in T cells (4-6). The nature of the subsequent signal to stimulate Ca2+ entry itself is discussed to be either the soluble calcium-influx factor CIF, protein-protein interaction between the IP3-receptor and the Ca2+ entry channel(s), or a G-protein-mediated process (reviewed in Refs. 7 and 8). We confirmed the potential role of IP3 in this process by showing that microinjection of this second messenger induced a train of Ca2+ signals which depended on extracellular Ca2+ (Fig. 2C).

As a novel finding we now add cADPr as a compound activating Ca2+ entry in response to microinjection. During our experiments in nominally Ca2+-free medium only very small Ca2+ signals were observed regardless whether cADPr or IP3 was microinjected. However, these Ca2+ signals were more pronounced at the site of microinjection, as compared with the signals averaged from the whole cell. Our data indicate that both ligands induced Ca2+ release from their distinct target Ca2+ stores, as has been clearly demonstrated for IP3 and cADPr in permeabilized cells (16) and in vesicular membrane subfractions separated by density gradient centrifugation (17). The relatively small and local Ca2+ release induced by both cADPr and IP3 is also explained by the dilution of cADPr and IP3 within the cell after microinjection (pipette concentrations 10 and 4 µM; assumed intracellular effective concentrations about 200 and 80 nM). In the presence of extracellular Ca2+, the small Ca2+ release then was followed for both ligands, IP3 and cADPr, by a secondary Ca2+ entry of much higher magnitude. This interpretation is in agreement with recent reports showing fast abrogation of Ca2+ spikes induced by extracellular stimuli by omission of extracellular Ca2+ in individual T cells (27-29). Also, the type of Ca2+ wave (Fig. 4) argues for Ca2+ influx since the wave started in a circular manner close to the plasma membrane all around the cell and propagated like a closing optical diaphragm into the center of the cell.

The mechanism by which cADPr stimulated Ca2+ entry is not yet clear. However, at least three models are possible: first, microinjected cADPr released Ca2+ from its target Ca2+ store, namely membrane vesicles which are located close to surface receptor-capped structures (17). This primary event then led to activation of a Ca2+ entry mechanism similar to the capacitative mechanism. The fact that SK-F 96365 inhibited both cADPr-and IP3-mediated Ca2+ entry2 may argue for this possibility. Second, cADPr may have opened directly Ca2+ channels in the plasma membrane. This possibility appears to be less likely, since no such action of cADPr has ever been described. Furthermore, this hypothetical cADPr-responsive Ca2+ channel then must have the same pharmacological properties, e.g. inhibition by 8-NH2-cADPr, which also is not very likely. As a third possibility, cADPr may have activated IP3 receptors. However, all experiments carried out in permeabilized T cells suggest that cADPr acts completely independent of IP3 receptors (16).

In conclusion, we add as a novel observation that Ca2+ release by cADPr can induce Ca2+ entry by a mechanism obviously being analogous to the capacitative mechanism induced by microinjection of IP3. Thus, cADPr, an intracellular ligand which is modulated in its activity indirectly by the T cell receptor-CD3 complex (30), may represent a new intracellular tool to control Ca2+ entry in T cells. The recent discovery and molecular cloning of a whole family of human homologues of the Drosophila photoreceptor trp-channel points toward diversity in the structural basis for Ca2+ entry (31) and opens up the possibility of identifying the Ca2+ entry channel involved in cADPr-mediated Ca2+ signaling in the future.


FOOTNOTES

*   This work was supported in part by Deutsche Forschungsgemeinschaft Grants Gu 360/2-1, Gu 360/2-2, and Gu 360/2-3 (to A. H. G. and G. W. M.), the Medical Research Council, and the British-German Academic Research Communication Program (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.
§   To whom correspondence should be addressed: Institut für Physiologische Chemie, Abt. Enzymchemie, Grindelallee 117, D-20146 Hamburg, Germany. Tel.: 49-40-4123-4276; Fax: 49-40-4123-4275; E-mail: guse{at}uke.uni-hamburg.de.
   Supported by Junta Nacional de Investigacao Cientifica e Technologica (Portugal, Praxis XXI-BPD/4228/94).
**   A Lister Institute Research Professor.
1   The abbreviations used are: IP3, D-myo-inositol 1,4,5-trisphosphate; cADPr, cyclic ADP-ribose; [Ca2+]i, free intracellular Ca2+ concentration.
2   A. H. Guse and I. Berg, unpublished data.

ACKNOWLEDGEMENTS

We thank G. A. Ashamu and S. J. Mills (Bath) for synthesis of ligands and Karin Müller (Hamburg) for expert technical assistance.


REFERENCES

  1. Guse, A. H., Roth, E., and Emmrich, F. (1992) Biochem. J. 288, 489-495 [Medline] [Order article via Infotrieve]
  2. McDonald, T. V., Premack, B. A., and Gardner, P. (1993) J. Biol. Chem. 268, 3889-3896 [Abstract/Free Full Text]
  3. Guse, A. H., Roth, E., and Emmrich, F. (1993) Biochem. J. 291, 447-451 [Medline] [Order article via Infotrieve]
  4. Gouy, H., Cefai, D., Christensen, S. B., Debré, P., and Bismuth, G. (1990) Eur. J. Immunol. 20, 2269-2275 [Medline] [Order article via Infotrieve]
  5. Zweifach, A., and Lewis, R. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6295-6299 [Abstract]
  6. Premack, B. A., McDonald, T. V., and Gardner, P. (1994) J. Immunol. 152, 5226-5240 [Abstract/Free Full Text]
  7. Berridge, M. J. (1995) Biochem. J. 312, 1-11 [Medline] [Order article via Infotrieve]
  8. Favre, C. J., Nüsse, O., Lew, D. P., and Krause, K.-H. (1996) J. Lab. Clin. Med. 128, 1-9
  9. Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615 [Abstract/Free Full Text]
  10. Gerasimenko, O. V., Gerasimenko, J. V., Tepikin, A. V., and Petersen, O. H. (1995) Cell 80, 439-444 [Medline] [Order article via Infotrieve]
  11. White, A. M., Watson, S. P., and Galione, A. (1993) FEBS Lett. 318, 259-263 [CrossRef][Medline] [Order article via Infotrieve]
  12. Morrissette, J., Heisermann, G., Cleary, J., Ruoho, A., and Coronado, R. (1993) FEBS Lett. 330, 270-274 [CrossRef][Medline] [Order article via Infotrieve]
  13. Koshiyama, H., Lee, H. C., and Tashjian, A. H., Jr. (1991) J. Biol. Chem. 266, 16985-16988 [Abstract/Free Full Text]
  14. Mészáros, L. G., Bak, J., and Chu, A. (1993) Nature 364, 76-79 [CrossRef][Medline] [Order article via Infotrieve]
  15. Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373 [Medline] [Order article via Infotrieve]
  16. Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359 [Abstract]
  17. Bourguignon, L. Y. W., Chu, A., Jin, H., and Brandt, N. R. (1995) J. Biol. Chem. 270, 17917-17922 [Abstract/Free Full Text]
  18. Hakamata, Y., Nishimura, S., Nakai, J., Nakashima, Y., Kita, T., and Imoto, K. (1994) FEBS Lett. 352, 206-210 [CrossRef][Medline] [Order article via Infotrieve]
  19. Bennett, D. L., Cheek, T. R., Berridge, M. J., De Smedt, H., Parys, J. B., Missiaen, L., and Bootman, M. D. (1996) J. Biol. Chem. 271, 6356-6362 [Abstract/Free Full Text]
  20. Ashamu, G. A., Galione, A., and Potter, B. V. L. (1995) J. Chem. Soc. Chem. Commun. 1359-1360
  21. Ashamu, G. A., Galione, A., and Potter, B. V. L. (1995) J. Chem. Soc. Chem. Commun. Corrigendum 1929
  22. Mills, S. J., Riley, A. M., Murphy, C. T., Bullock, A. J., Westwick, J., and Potter, B. V. L. (1995) Bioorg. Med. Chem. Lett. 5, 203-208 [CrossRef]
  23. Albritton, N. L., Meyer, T., and Stryer, L. (1992) Science 258, 1812-1815 [Medline] [Order article via Infotrieve]
  24. Lee, H. C., Aarhus, R., and Walseth, T. F. (1993) Science 261, 352-355 [Medline] [Order article via Infotrieve]
  25. Walseth, T. F., and Lee, H. C. (1993) Biochim. Biophys. Acta 1178, 235-242 [Medline] [Order article via Infotrieve]
  26. Merritt, J. E., Armstrong, W. P., Benham, C. D., Hallam, T. J., Jacob, R., Jaxa-Chamiec, A., Leigh, B. K., McCarthy, S. A., Moores, K. E., and Rink, T. J. (1990) Biochem. J. 271, 515-522 [Medline] [Order article via Infotrieve]
  27. Donnadieu, E., Cefai, D., Tan, Y. P., Paresys, G., Bismuth, G., and Trautmann, A. (1992) J. Immunol. 148, 2643-2653 [Abstract/Free Full Text]
  28. Donnadieu, E., Bismuth, G., and Trautmann, A. (1992) J. Biol. Chem. 267, 25864-25872 [Abstract/Free Full Text]
  29. Dolmetsch, R. E., and Lewis, R. S. (1994) J. Gen. Physiol. 103, 365-388 [Abstract]
  30. Guse, A. H., da Silva, C. P., Weber, K., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1996) J. Biol. Chem. 271, 23946-23953 [Abstract/Free Full Text]
  31. Zhu, X., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E., and Birnbaumer, L. (1996) Cell 85, 661-671 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.