Mutual Antagonism of Calcium Entry by Capacitative and Arachidonic Acid-mediated Calcium Entry Pathways*

Dali LuoDagger, Lisa M. Broad§, Gary St. J. Bird, and James W. Putney Jr.

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, January 12, 2001, and in revised form, March 22, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

In nonexcitable cells, the predominant mechanism for regulated entry of Ca2+ is capacitative calcium entry, whereby depletion of intracellular Ca2+ stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca2+ entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca2+ pumps, depletion of Ca2+ stores, or entry of Ca2+ through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca2+ overload.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

In nonexcitable cells, the major mechanism for receptor-regulated Ca2+ signaling involves the activation of polyphosphoinositide-specific phospholipase C, formation of inositol 1,4,5-trisphosphate (IP3),1 and release of intracellular stored Ca2+ by the activation of IP3 receptor/ion channels in the endoplasmic reticulum (1). The decline in Ca2+ content of the intracellular stores then secondarily signals the activation of plasma membrane calcium channels, a process known as capacitative calcium entry (2, 3). Recent studies have indicated that the channels in the plasma membrane may be members of the transient receptor potential family of channel proteins (4-7). The mechanism of coupling intracellular stores to the plasma membrane channels may involve direct interactions between the channel molecules and underlying IP3 receptors (8-11).

Although capacitative calcium entry seems to be present ubiquitously in receptor-regulated nonexcitable cells, other mechanisms for signaling entry have been found in specific cell types. Examples include receptor-gated channels (12), channels activated by second messengers such as cyclic nucleotides (13), phosphatidylinositol 3,4,5-trisphosphate (14), and arachidonic acid (15, 16) (for a review see Refs. 17 and 18). In the HEK293 cell line, capacitative and noncapacitative pathways co-exist (19, 20). In these cells, muscarinic receptor activation can lead to baseline [Ca2+]i spikes or [Ca2+]i oscillations. However, despite the discharge of stored Ca2+ during these spikes, the calcium entry necessary for maintaining continuous spiking seems to be noncapacitative (16). Also, in this cell line arachidonic acid induces a noncapacitative calcium entry; whether this response represents the noncapacitative mechanism seen with low concentrations of muscarinic agonists is controversial (21). Nonetheless, when exogenous arachidonic acid is used to activate entry in HEK293 cells, a significant discharge of stored calcium occurs, yet little if any capacitative entry is activated (see below). Collectively, these observations suggest some degree of interaction between the capacitative and noncapacitative arachidonic acid-mediated pathways. Thus, in the current study we have carried out experiments to examine the regulation of these two calcium entry mechanisms and specifically to determine in what ways the signaling pathways interact. Our findings indicate that although both pathways involve the release of a common intracellular calcium pool, the entry components of the two pathways operate in a mutually exclusive manner. This implies that the underlying signaling mechanisms may be able to interact in a way that prevents the occurrence of supramaximal activation of calcium entry, which could have potentially toxic effects on cell function.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Cell Culture-- Human embryonic kidney (HEK)-293 cells obtained from the ATCC were grown at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine in a humidified 95% air, 5% CO2 incubator. For Ca2+ measurements, cells were cultured to about 70% confluence, passaged onto glass coverslips, and used 24-48 h after plating.

Fluorescence Measurements-- Fluorescence measurements were made with Fura2-loaded single or groups of HEK293 cells as described previously (22). In brief, the coverslips with attached cells were mounted in a Teflon chamber and incubated in Dulbecco's modified Eagle's medium with 1 µM acetoxymethyl ester of Fura2 (Fura2/AM, Molecular Probes) at 37 °C in the dark for 25 min. Before [Ca2+]i measurements, the cells were washed three times and incubated for 30 min at room temperature (25 °C) in HEPES-buffered physiological saline solution (120 mM NaCl, 5.4 mM KCl, 0.8 mM Mg2SO4, 20 mM HEPES, 1.8 mM CaCl2, and 10 mM glucose, pH 7.4 (adjusted by NaOH)). Ca2+-free solutions contained no added CaCl2 in the HEPES-buffered physiological saline solution.

Fluorescence was monitored by placing the Teflon chamber with Fura2-loaded cells onto the stage of a Nikon Diaphot microscope (×40 Neofluor objective). The cells were excited alternatively by 340- and 380-nm wavelength light from a Deltascan D101 (Photon Technology International, Ltd.) light source equipped with a light-path chopper and dual excitation monochromators. Emission fluorescence intensity at 510 nm was recorded by a photomultiplier tube (Omega Optical). All experiments were conducted at room temperature (25 °C) and carried out within 2 h of loading for each coverslip. Changes in [Ca2+]i are reported for one single cell in oscillation experiments or a group of cells (6-10) in other protocols. The data are expressed as the ratio of Fura2 fluorescence due to excitation at 340 nm to that due to excitation at 380 nm.

In some experiments, [Ca2+]i was monitored on attached populations of cells in 96-well plates using a fluorometric imaging plate reader (FLIPR384, Molecular Devices, Sunnyvale, CA). When FLIPR384 was used, the cells (20,000 cells/well plated the day before use and cultured as above) were loaded with the single visible wavelength indicator, Fluo-4 (2 µM Fluo-4/AM for 45 min at 37 °C), excited at 488 nm, and emission-selected by a 510-570-nm bandpass filter. Detection was performed with a cooled charge-coupled device camera. Experiments were carried out at room temperature as described above.

Materials-- Arachidonic acid was obtained from BioMol. Carbachol and thapsigargin were purchased from Calbiochem. 2-Aminoethyoxydiphenyl borane (2-APB) was synthesized as described previously (23).

Statistics-- For some experiments, the average peak responses (the ratio of Fura2 fluorescence due to excitation at 340 nm to Fura2 fluorescence due to excitation at 380 nm) were calculated and expressed as mean ± S.E. for the indicated number (n) of experiments. Statistical significance was determined with the Student's t test (p < 0.05).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Thapsigargin and Arachidonic Acid Discharge the Same Intracellular Ca2+ Pool-- Treatment of HEK293 cells with either 1 µM thapsigargin or 30 µM arachidonic acid causes a release of intracellular stored Ca2+ as well as an increase in Ca2+ entry across the plasma membrane (Fig. 1). Yet, 1 µM Gd3+ completely blocks the thapsigargin-induced entry with little effect on the entry caused by arachidonic acid (Fig. 1). Thus, arachidonic acid seems to release Ca2+ stores, but unlike thapsigargin, arachidonic acid does not activate significant capacitative calcium entry. We carried out the experiment illustrated in Fig. 2 to determine whether thapsigargin and arachidonic acid cause the release of Ca2+ from the same intracellular pool. HEK293 cells were treated with either 30 µM arachidonic acid or 1 µM thapsigargin in the absence of extracellular Ca2+. In both cases a transient increase in [Ca2+]i was observed, indicative of the release of Ca2+ stores. When the arachidonic acid-treated cells were subsequently treated with 1 µM thapsigargin or when the thapsigargin-treated cells were subsequently treated with 30 µM arachidonic acid, no additional release of Ca2+ was observed. This indicates that these two reagents release Ca2+ from the same intracellular Ca2+ store, and the failure of arachidonic acid to activate capacitative calcium entry cannot be explained by its failure to release the pool of Ca2+ linked to calcium entry. The addition of the Ca2+ ionophore, ionomycin, to cells treated with both arachidonic acid and thapsigargin resulted in a further release of Ca2+, presumably from mitochondria or other cellular sites.


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Fig. 1.   Activation of Gd3+-sensitive capacitative calcium entry by thapsigargin and Gd3+-insensitive noncapacitative entry by arachidonic acid. HEK293 cells were treated with either 1 µM thapsigargin (A) or 30 µM arachidonic acid (B) as indicated in the absence of extracellular Ca2+. A transient rise in [Ca2+]i is indicative of intracellular Ca2+ release. The readdition of extracellular Ca2+ as indicated results in Ca2+ entry. The entry of Ca2+ in the thapsigargin-treated cells is capacitative and is blocked by 1 µM Gd3+ (dotted traces). The entry caused by arachidonic acid is noncapacitative and is largely insensitive to 1 µM Gd3+. The results are representative of 4-7 experiments. F340/F380, the ratio of Fura2 fluorescence due to excitation at 340 nm to Fura2 fluorescence due to excitation at 380 nm; AA, arachidonic acid.


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Fig. 2.   Thapsigargin (TG) and arachidonic acid (AA) release the same intracellular Ca2+ store. HEK293 cells incubated in the absence of extracellular Ca2+ were treated with either 30 µM arachidonic acid (solid trace) or 1 µM thapsigargin (dotted trace) (first arrow). A transient rise in [Ca2+]i indicates the release of intracellular Ca2+ stores. Subsequently, the arachidonic acid-treated cells were treated with thapsigargin, and the thapsigargin-treated cells were treated with arachidonic acid (second arrow). In both cases, the addition of the second reagent failed to cause a rise in [Ca2+]i, indicating that each agent releases the full Ca2+ store available to the other. Additional stores of Ca2+ could be released by the Ca2+ ionophore, ionomycin (third arrow). The experiment is representative of a total of four similar experiments.

Mutual Antagonism of the Capacitative and Arachidonic Acid-activated Calcium Entry Pathways-- One possible explanation for the failure of arachidonic acid to activate capacitative calcium entry is that arachidonic acid may act as an inhibitor of these channels or of some other step in the influx mechanism. There is precedence for this idea, because unsaturated fatty acids (24) and specifically arachidonic acid (25) have been shown to inhibit capacitative calcium entry in other systems. In the experiment shown in Fig. 3, HEK293 cells were treated with thapsigargin in the absence of extracellular Ca2+, and then Ca2+ was restored as indicated revealing a robust capacitative calcium entry. The subsequent addition of 30 µM arachidonic acid caused a transient inhibition of Ca2+ entry. This inhibition was not always transient, however, especially when lower concentrations of arachidonic acid were used. The reversal of the inhibition could reflect only transient inhibition of capacitative calcium entry or the development of an arachidonic acid-dependent noncapacitative calcium entry. Furthermore, this protocol does not provide information on possible interactions in the reverse direction, i.e. effects of the capacitative pathway on arachidonic acid-activated Ca2+ signaling. To address this issue, we designed experiments that would allow us to examine the effect of prior intracellular store depletion on the arachidonic acid response.


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Fig. 3.   Inhibition of capacitative calcium entry by arachidonic acid. HEK293 cells incubated in the absence of extracellular Ca2+ were treated with 1 µM thapsigargin (TG) as indicated. Restoration of extracellular Ca2+ reveals a robust capacitative calcium entry. The subsequent addition of arachidonic acid (AA) causes a transient inhibition of Ca2+ entry. The experiment depicted is representative of a total of four similar trials.

Pre-activation of Capacitative Calcium Entry Inhibits Arachidonic Acid-induced Ca2+ Entry-- To investigate the effects of activation of capacitative calcium entry on arachidonic acid-mediated Ca2+ entry, we carried out the experiment illustrated in Fig. 4. The membrane permeant IP3 receptor antagonist, 2-APB (23), has been shown to inhibit capacitative calcium entry, and there is evidence that it acts on the channels in a manner that interferes with the signaling mechanism (26). However, arachidonic acid-induced entry of Ca2+ is not inhibited by this reagent (see below; Ref. 21). HEK293 cells in the presence of extracellular Ca2+ were treated with 100 µM 2-APB as indicated in Fig. 4, and the subsequent addition of thapsigargin resulted in a transient response, indicating inhibition of capacitative calcium entry. When arachidonic acid was subsequently added, a robust [Ca2+]i rise was observed; because intracellular stores were released previously by thapsigargin, this indicates that under this experimental condition, arachidonic acid is capable of the activation of Ca2+ entry.


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Fig. 4.   Arachidonic acid induces Ca2+ entry when capacitative calcium entry is blocked by 2-APB. HEK293 cells incubated in the presence of extracellular Ca2+ were treated with 100 µM 2-APB as indicated. The subsequent addition of thapsigargin (TG, dotted trace) caused a transient rise in [Ca2+]i. The sustained Ca2+ entry was blocked by 2-APB. The addition of 30 µM arachidonic acid (AA) then activated sustained Ca2+ entry. This response was similar to that in control cells that were treated with 2-APB but not stimulated with thapsigargin (solid trace). This experiment is representative of a total of six similar experiments.

In the experiment shown in Fig. 5, we repeated the above protocol utilizing another reagent that selectively blocks capacitative calcium entry, 1 µM Gd3+ (27) (Fig. 1). However, while Gd3+ prevented Ca2+ entry in response to thapsigargin, arachidonic acid now failed to activate a [Ca2+]i signal.


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Fig. 5.   When capacitative calcium entry is blocked by Gd3+, arachidonic acid fails to induce Ca2+ entry. The protocol used was similar to Fig. 4 except that instead of 2-APB, the cells were first treated with the capacitative calcium entry channel blocker, Gd3+. Again, this resulted in a block of entry caused by thapsigargin (TG, dotted trace). Unlike the case for 2-APB, arachidonic acid (AA) now failed to activate a Ca2+ signal. However, if the cells were not treated with thapsigargin, arachidonic acid induced a rise in [Ca2+]i (solid trace). This experiment is representative of a total of seven similar experiments.

This latter result indicates that depletion of Ca2+ stores by thapsigargin in some manner inhibits the ability of arachidonic acid to activate the noncapacitative calcium entry pathway. The difference in the mechanism of action of 2-APB and Gd3+ is therefore critical to understanding the nature of this interaction. Although the action of 2-APB on capacitative calcium entry channels may not result from inhibition of inositol trisphosphate receptors as first proposed (28), there is nonetheless evidence that its mode of interaction may involve the interference with the coupling mechanism rather than by acting simply as a blocker of the channel pore (26). Gd3+, on the other hand, would be expected to block the channels directly. The known actions of thapsigargin are the inhibition of sarcoplasmic reticulum Ca2+ pumps, depletion of Ca2+ stores, and entry of Ca2+ through activated capacitative calcium entry channels (29). Neither inhibition of sarcoplasmic reticulum Ca2+ pumps nor depletion of Ca2+ stores can be responsible because 2-APB blocks the interaction (Fig. 5) without interfering with the ability of thapsigargin to deplete Ca2+ stores. The entry of Ca2+ cannot be important because this is blocked by Gd3+, yet this agent does not prevent the inhibition by thapsigargin of arachidonic acid-induced entry. We conclude that a 2-APB-sensitive step in the signaling pathway for capacitative calcium entry acts to inhibit the activation of noncapacitative calcium entry channels by arachidonic acid. The nature of this step is unknown. Clearly, we must first achieve a better understanding of the steps involved in signaling capacitative calcium entry to determine how this pathway interacts with and regulates the arachidonic acid pathway or other calcium signaling pathways. Note that despite the findings indicating that 2-APB does not block capacitative calcium entry by virtue of its action on inositol trisphosphate receptors, an inescapable conclusion from the current findings is that this reagent must be doing something more than simply acting as a channel blocker.

Although unlikely, an alternative interpretation of the findings in Figs. 4 and 5 is that somehow treatment with thapsigargin causes arachidonic acid-activated channels to become Gd3+-sensitive. To examine this possibility, we repeated the protocol of Figs. 4 and 5 utilizing both Gd3+ and 2-APB in combination. If thapsigargin causes arachidonic acid-regulated channels to become Gd3+-sensitive, the protocol should result in a block of the arachidonic acid-induced response. If 2-APB acts to prevent a store-depletion inhibitory effect on the channels, then arachidonic acid will give a normal response. The results of this experiment, shown in Fig. 6, show that the latter is indeed the case.


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Fig. 6.   Arachidonic acid induces Ca2+ entry when capacitative calcium entry is blocked by a combination of Gd3+ and 2-APB. The protocol used was similar to that for Figs. 4 and 5. As before, when capacitative calcium entry is blocked with 100 µM 2-APB, arachidonic acid (AA) induces a [Ca2+]i signal (dotted trace); when it is blocked with 1 µM Gd3+ (solid trace), it does not induce a [Ca2+]i signal. When a combination of both 2-APB and Gd3+ is used, the arachidonic acid response is not blocked. This experiment is representative of a total of four similar experiments.

A somewhat similar mutual antagonism has been described recently for the interaction between nicotinic- and purinergic-regulated cation channels (30). In this instance, the authors proposed close proximity between the interacting receptor/channel types. With respect to the current findings, there is evidence that arachidonic acid-regulated and capacitative calcium entry channels are located some undetermined distance apart in the plasma membrane (19). Also, in the previous study on ligand-gated channels, the inhibitory interactions resulted in a lack of additivity, but at least under the experimental conditions in our study the degree of cross inhibition was even more striking (Figs. 3 and 5).

What determines which of these pathways predominate when multiple upstream signals occur? The answer to this question may depend on a number of variables such as the stimulus strength of different initiating agonists. However, as shown in Fig. 1, when arachidonic acid is applied, store depletion occurs, but clearly the arachidonic acid-activated entry predominates. This would suggest that at least under this experimental protocol, the arachidonic acid pathway takes precedence.

In conclusion, the results of this study demonstrate for the first time a process of mutual antagonism between two distinct regulated Ca2+ entry pathways. The function of this mutual antagonism cannot be known for certain, but it is reasonable to suggest that this represents a mechanism to protect cells from the toxic effects of excessive [Ca2+]i loads. Thus, these two pathways exist as alternative pathways for Ca2+ signaling, and the process of mutual antagonism may ensure that they do not operate simultaneously.

    ACKNOWLEDGEMENTS

We thank Drs Elizabeth Murphy and Stephen Shears for constructive criticisms.

    FOOTNOTES

* 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 Present address: Dept. of Pharmacology, Harbin Medical University, Harbin 150086, Peoples Republic of China.

§ Present address: Eli Lilly and Company, Ltd., Lilly Research Center, Erl Wood Manor, Sunninghill Rd., Windlesham, Surrey GU20 6PH, England.

To whom correspondence should be addressed: Laboratory of Signal Transduction, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-7879; E-mail: putney@niehs.nih.gov.

Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100327200

    ABBREVIATIONS

The abbreviations used are: IP3, 1,4,5-trisphosphate; HEK, human embryonic kidney; 2-APB, 2-aminoethyoxydiphenyl borane.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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