Signaling Pathways Underlying Muscarinic Receptor-induced [Ca2+]i Oscillations in HEK293 Cells*

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, August 17, 2000, and in revised form, October 24, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 µM) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 µM) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 µM carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 µM Gd3+, as well as 30-100 µM concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 µM 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 µM 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+ entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An increase in the level of intracellular free Ca2+ concentration ([Ca2+]i) plays a central role in signal transduction for a variety of cellular functions, including cellular secretion, muscle contraction, cell growth and differentiation, and apoptosis. Changes in [Ca2+]i in mammalian cells are mediated by mobilization of Ca2+ from internal Ca2+ stores and/or by entry of Ca2+ from the extracellular space. In many nonexcitable cells Ca2+ signaling by neurotransmitters or hormones is initiated through cell membrane receptors coupled to phospholipase C and the production of inositol 1,4,5-trisphosphate (IP3)1 (1). IP3 as a second messenger produces a biphasic Ca2+ signal, comprised of an initial Ca2+ release from endoplasmic reticulum (ER), followed by a sustained Ca2+ plateau due to Ca2+ entry across the plasma membrane. This Ca2+ entry usually results from the depletion of intracellular Ca2+ stores and in such instances is termed "capacitative Ca2+ entry" (2, 3). This mode of entry presumably involves store-operated Ca2+ channels in the plasma membrane. Although capacitative calcium entry has been documented in many different cell types, the signal by which store emptying activates store-operated Ca2+ channels remains uncertain (4, 5).

In addition to the sustained elevation of [Ca2+]i seen with high agonist concentrations, a more complex and subtle repetitive cycling of [Ca2+]i, known as [Ca2+]i spiking or [Ca2+]i oscillations, often results from lower concentrations of agonists in some cell types (1, 6, 7). The characteristics of [Ca2+]i oscillations vary widely among different cell types, and a single mechanism may be insufficient to account for the variety of observed responses (1, 7-9). Formation of IP3 and cyclical release of Ca2+ from IP3-sensitive stores may underlie the generation of oscillations induced by agonists (1, 10). However, a Ca2+-induced Ca2+ release pathway has been suggested in initiating oscillations by caffeine or other agents unrelated to IP3 generation (9, 11). Ca2+ influx from the external milieu is currently thought to be activated in such situations and appears to be needed to sustain [Ca2+]i oscillations (1, 7). However, the mechanism whereby Ca2+ entry is triggered during [Ca2+]i oscillations is not altogether clear. Some models suggest that capacitative calcium entry provides Ca2+ entry during oscillations (7, 12). More recently a novel, noncapacitative mechanism has been proposed that involves agonist-activated generation of arachidonic acid and arachidonic acid-induced Ca2+ entry (13-15).

Arachidonic acid is present in cell membranes esterified in phospholipids and can be released by phospholipase A2 (PLA2) in response to various extracellular stimuli (16, 17). Arachidonic acid can also be generated from diacylglycerol, a product of phospholipase C or phospholipase D activation, by action of diglyceride lipase (16). In recent years, an increasing number of reports have suggested that arachidonic acid directly modulates cellular responses, including Ca2+ signal transduction. As for IP3, Ca2+ release from ER and Ca2+ influx from the extracellular space induced by arachidonic acid have been demonstrated in a number of cell types (18-23). However, the mechanisms underlying [Ca2+]i changes in response to arachidonic acid are not clear.

In this study, we have used relatively specific pharmacological probes to analyze and compare capacitative, noncapacitative, and arachidonic acid-induced Ca2+ entry in HEK293 cells. We confirm earlier reports of a noncapacitative mechanism associated with [Ca2+]i oscillations in these cells. However, our findings indicate a possible role for the IP3 receptor in this signaling pathway and call into question the role of arachidonic acid, at least as a direct mediator of Ca2+ entry in this cell type.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Human embryonic kidney 293 (HEK293) cells obtained from the ATCC were grown at 37 °C in Dulbecco's 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 (24). In brief, coverslips with attached cells were mounted in a Teflon chamber and incubated in Dulbecco's 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, cells were washed three times and incubated for 30 min at room temperature (25 °C) in HEPES-buffered physiological saline solution (HPSS: NaCl, 120 mM; KCl, 5.4 mM; Mg2SO4, 0.8 mM; HEPES, 20 mM; CaCl2, 1.8 mM; and glucose, 10 mM; with pH 7.4 adjusted by NaOH). Ca2+-free solutions contained no added CaCl2 in the HPSS.

In preliminary experiments, we observed that [Ca2+]i oscillations were not reproducibly observed in cells loaded with 1 µM Fura2/AM, presumably due to excessive cytoplasmic Ca2+ buffering. Thus, for these experiments we used 100 nM Fura2/AM for loading and 1.5 mM extracellular CaCl2 as previously described (25).

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 (F340/F380).

Mn2+ Quench Measurements-- Mn2+ quench experiments were performed with a group of HEK293 cells in nominally Ca2+-free medium containing 0.1 or 2 mM MnCl2. Ftot, which is independent of [Ca2+]i responses (26), was obtained by a weighted summing of the fluorescence of 340 and 380 nm, and expressed as the percentage of the initial value in the absence of extracellular Mn2+.

Materials-- Arachidonic acid and 5,8,11,14-eicosatetraenoic acid were obtained from BioMol (Plymouth Meeting, PA). Carbachol, thapsigargin, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were purchased from Calbiochem (La Jolla, CA). 2-Aminoethyoxydiphenyl borane (2-APB) was synthesized as previously described (27).

Statistics-- For some experiments, average peak responses (F340/F380) 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[Ca2+]i Signaling Responses to Carbachol in HEK293 Cells-- In Ca2+-containing HPSS, 100 µM carbachol induced a large, somewhat transient increase in [Ca2+]i (F340/F380) followed by a slowly declining but generally sustained elevated level of [Ca2+]i (Fig. 1A). In nominally Ca2+-free medium, this same concentration of carbachol induced a transient increase in [Ca2+]i; following re-addition of Ca2+ to the medium, a second, sustained entry of Ca2+ was observed (Fig. 1B), indicating release of Ca2+ from internal sites and Ca2+ entry.



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Fig. 1.   Calcium release, calcium entry, and [Ca2+]i oscillations due to muscarinic receptor activation. Fura2-loaded HEK293 cells were activated as indicated by either 100 µM (A and B) or 5 µM (C and D) carbachol. In A, extracellular Ca2+ was present throughout the experiment. In B, Ca2+ was initially absent from the medium and was restored to 1.8 mM were indicated. In C and D, a single HEK293 cell was activated with 5 µM carbachol, and then the medium was removed with three consecutive washes, and the cell was allowed to recover for 25 min. Data collection was interrupted during this interval of washes and recovery, and is indicated by the arrow labeled W. Subsequently, the same cell was activated a second time with 5 µM carbachol. In D, prior to the second stimulation, the extracellular medium was changed to one containing 0.2 mM BAPTA and no added Ca2+. Similar results to the ones depicted were obtained in a total of five to eight experiments.

[Ca2+]i oscillations have been reported to be induced by 1 µM carbachol in HEK293 cells transfected with the M3 muscarinic receptors (28). However, in the current study in which wild type HEK293 cells were used, we failed to consistently produce repetitive transient responses of [Ca2+]i with 1-3 µM carbachol. When the cells were stimulated with 5 µM carbachol, about 50% of the tested cells (101 of 205) showed oscillatory [Ca2+]i responses at a frequency of ~0.5-1/min in the presence of 1.5 mM Ca2+ (Fig. 1C). These robust spikes could last up to 1 h, but the frequency progressively slowed with time. Because all cells did not oscillate and the frequency varied somewhat among the cells, which did oscillate, we adopted the protocol shown in Fig. 1C. In this protocol, a cell was stimulated for about 20 min, the carbachol was removed by three changes of incubation medium, and then, after an additional period of about 25 min (data acquisition was halted during this period), the same cell was again stimulated with 5 µM carbachol for an additional 20 min, generally under an altered experimental condition. As shown in Fig. 1C, in normal HPSS, the second stimulation always resulted in an oscillatory response that was similar to, although somewhat slower than the first.

This protocol was utilized for the experiment illustrated in Fig. 1D. In this experiment, 5 min before, and during the second exposure to carbachol, the cell was bathed in a nominally Ca2+-free medium containing 200 µM BAPTA. With this protocol, carbachol induced one or two spikes, but sustained oscillations were not observed. These results confirm that, in wild type HEK293 cells, as shown previously for cells transfected with the M3 muscarinic receptor, [Ca2+]i oscillations produced by a low concentration of carbachol depend on extracellular Ca2+, presumably indicating a role for Ca2+ influx.

Effects of Gd3+ on [Ca2+]i Responses to Thapsigargin and Carbachol in HEK293 Cells-- Gd3+ is a potent inhibitor of agonist-activated calcium entry (29) and has been shown to discriminate between capacitative and noncapacitative calcium entry (22). In experiments utilizing the same protocol as in Fig. 1B, the effects of Gd3+ on Ca2+ entry due to the SERCA inhibitor, thapsigargin, were determined. Thapsigargin depletes Ca2+ stores passively by virtue of its ability to inhibit the SERCA pumps on the endoplasmic reticulum, and the ensuing entry of Ca2+ is therefore assumed to be the very definition of capacitative calcium entry (3, 30). As shown in Fig. 2A, Gd3+ inhibited Ca2+ entry induced by the thapsigargin in a concentration-dependent manner, and with no significant effect on the Ca2+ release phase (Fig. 2A and results not shown). Gd3+ had no significant effect on basal [Ca2+]i (data not shown). The sensitivity of thapsigargin-induced capacitative calcium entry to inhibition by Gd3+ is similar to that reported by Broad et al. (22).



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Fig. 2.   Effect of Gd3+ on thapsigargin-activated Ca2+ entry. In A, the protocol was identical to that in Fig. 1B, except that 1 µM thapsigargin was utilized as agonist. In one of the traces (as indicated) 1 µM Gd3+ was added to the medium when Ca2+ was removed and was present throughout the re-addition of Ca2+. In B, the agonist was 100 µM carbachol. Results shown are representative of six (A) and five (B) independent determinations.

Similar experiments were carried out utilizing a maximal concentration of carbachol, and such an experiment is shown in Fig. 2B. Ca2+ entry due to maximal muscarinic receptor activation appeared similarly sensitive to inhibition by Gd3+, leading to the conclusion that the entry is largely or entirely capacitative.



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Fig. 3.   Effect of increasing concentrations of Gd3+ on [Ca2+]i oscillations induced by 5 µM carbachol. The top left panel depicts a control experiment carried out according to the protocol described for Fig. 1C. In subsequent panels, Gd3+, at the concentrations shown, was added during the interval indicated. Similar findings were obtained in a total of four to six experiments.

However, significantly different results were obtained when cells were stimulated to oscillate with the lower, 5 µM concentration of carbachol. As Fig. 3 illustrates, concentrations of 1, 10, 30, 100, or 500 µM Gd3+ produced little or no effect on the [Ca2+]i oscillations; only at the highest concentrations tested, 100 and 500 µM Gd3+, was there even partial suppression of the oscillatory frequency. The failure of even these very high concentrations of Gd3+ to block the oscillations was surprising. However, it is known that another lanthanide, La3+, can inhibit active membrane extrusion of Ca2+ at higher concentrations (31). We determined whether Gd3+ might have a similar action by examining the time course of the [Ca2+]i response to thapsigargin in the absence of extracellular Ca2+ and in the presence of varying concentrations of Gd3+. The decay of the [Ca2+]i response to thapsigargin under these conditions is due almost entirely to plasma membrane extrusion (32). As shown in Fig. 4, Gd3+ concentrations of 30 µM or greater caused an augmentation of the thapsigargin-induced [Ca2+]i signal and a slowing of its decay. Thus, at these higher concentrations, oscillations may continue due to "trapping" of intracellular Ca2+, despite an inhibition of Ca2+ entry. However, at 10 µM Gd3+, there was no significant augmentation of the response, indicating that the Ca2+ entry channels supporting the oscillations are truly less sensitive to Gd3+ than are capacitative calcium entry channels.



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Fig. 4.   Effects of Gd3+ on Ca2+ extrusion in HEK293 cells. To assess inhibitory actions of Gd3+ on Ca2+ extrusion, cells were activated by 1 µM thapsigargin (TG) in the absence of external Ca2+ and in the presence of the indicated concentrations of Gd3+. Concentrations of Gd3+ of 30 µM or greater delayed the decay of the thapsigargin transient, indicating a degree of inhibition of plasma membrane Ca2+ extrusion.

Arachidonic Acid-induced [Ca2+]i Signaling in HEK293 Cells-- Shuttleworth and his coworkers (13, 21, 28) have suggested that the noncapacitative calcium entry occurring in HEK293 cells and other cell types in response to low concentrations of muscarinic agonists is mediated by arachidonic acid, released from membrane lipids by phospholipase A2. Thus, we next examined the effects of Gd3+ on Ca2+ mobilization in HEK293 cells in response to arachidonic acid. In preliminary experiments, we found that between 30 and 300 µM arachidonic acid could reproducibly induce both Ca2+ release and Ca2+ entry in a concentration-dependent manner. However, arachidonic acid at concentrations > 100 µM occasionally resulted in [Ca2+]i levels that saturated the indicator likely due to a nonselective increase in membrane permeability (33). Concentrations in the range of 5 to 10 µM did not induce increases in [Ca2+]i in all cells. As shown in Fig. 5A, 30 µM arachidonic acid slowly increased the fluorescence ratio, and the response appeared to occur in two phases. In nominally Ca2+-free medium, arachidonic acid induced a transient [Ca2+]i rise followed by a sustained elevation of [Ca2+]i after restoration of Ca2+ to the medium (Fig. 5B), indicating that both Ca2+ release and Ca2+ entry are activated by arachidonic acid in HEK293 cells. To examine the possible involvement of metabolites of arachidonic acid in the [Ca2+]i responses, we employed 5,8,11,14-eicosatetraenoic acid, an inhibitor of cyclooxygenase, lipoxygenases, and cytochrome P450 arachidonic acid-metabolizing enzymes (34). 20 µM 5,8,11,14-eicosatetraenoic acid had no effect on either Ca2+ release or Ca2+ entry induced by 30 µM arachidonic acid, indicating that the [Ca2+]i changes induced by 30 µM arachidonic acid are unlikely to result from an arachidonic acid metabolite (data not shown). A similar conclusion was reached by Shuttleworth and Thompson based on a somewhat different strategy (28).



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Fig. 5.   Calcium signaling in HEK293 cells due to arachidonic acid and the effects of Gd3+. In A, a Fura2-loaded HEK293 cell was exposed to 30 µM arachidonic acid (AA) as indicated, resulting in a biphasic rise in [Ca2+]i. The protocol for B and C was as for Figs. 2 and 3. In C, summary data are included for both the entry (filled circles) and release (open circles) phases of the response to arachidonic acid. The data in C are means ± S.E. from six to seven experiments.

The pattern of [Ca2+]i signaling induced by arachidonic acid is reminiscent of that due to thapsigargin; a release of stored Ca2+ followed by activation of Ca2+ entry across the plasma membrane. Thus, we next examined the effects of Gd3+ on arachidonic acid-induced signaling, because this lanthanide appears to have relatively selective effects on store-operated or capacitative calcium entry. At a concentration of 1 µM, which completely blocked Ca2+ entry due to carbachol and thapsigargin, Gd3+ had no significant effect on Ca2+ entry in response to 30 µM arachidonic acid (Fig. 5, B and C). At concentrations of 3 and 10 µM, Gd3+ inhibited Ca2+ influx induced by 30 µM arachidonic acid with complete blockade at 10 µM. Surprisingly, 10 µM Gd3+ also caused a complete abolishment of arachidonic acid-induced Ca2+ release (Fig. 5, B and C). After complete inhibition with 10 µM Gd3+ of Ca2+ release due to 30 µM arachidonic acid in nominally Ca2+-free medium, a normal release of [Ca2+]i could be evoked on addition of 1 µM thapsigargin or 100 µM carbachol (not shown). These results indicate that arachidonic acid induces both Ca2+ release and Ca2+ entry in HEK293 cells, and both of these responses are sensitive to inhibition by Gd3+; however, this pathway is at least 10-fold less sensitive to Gd3+ than capacitative calcium entry.

Effects of 2-APB on [Ca2+]i Responses Induced by Carbachol, Thapsigargin, and Arachidonic Acid-- Recent studies have indicated that capacitative calcium entry involves interactions between IP3 receptors and the plasma membrane (35). One piece of evidence for this idea is the sensitivity of capacitative calcium entry to inhibition by 2-APB (36), a membrane-permeant inhibitor of the IP3 receptor (27). We next examined the actions of this reagent as a potential inhibitor of Ca2+ entry responses to agonists, to thapsigargin, and to arachidonic acid.

In unstimulated cells, and in the absence of extracellular Ca2+, 2-APB at 100 µM slightly augmented the baseline fluorescence ratio in about 80% of HEK293 cells tested (n = 48). The increment in the baseline was 8.6 ± 3.2% of that of Ca2+ release by 1 µM thapsigargin in nominally Ca2+-free medium (n = 9). A weak inhibitory effect on Ca2+-ATPase in the ER has been suggested to account for the rise of [Ca2+]i by high concentrations of 2-APB (27).

Utilizing a similar protocol as for the Gd3+ experiments, 2-APB produced a concentration-dependent inhibition of Ca2+ influx induced by 100 µM carbachol (Fig. 6, top) or 1 µM thapsigargin (Fig. 6, bottom) when Ca2+ was restored to the bath. Like Gd3+, 2-APB altered the Ca2+ entry phase with almost the same potency among the two agonists, with 30 µM 2-APB producing essentially complete block of Ca2+ entry for both modes of activation. However, 30 µM 2-APB attenuated the Ca2+ release peak induced by 100 µM carbachol only weakly, and this inhibition was still incomplete with 100 µM 2-APB (Fig. 6). With 100 µM 2-APB, an approximate 20% reduction of Ca2+ release due to 1 µM thapsigargin could also be seen (Fig. 6), which may be due to the inhibition of Ca2+-ATPase in the endoplasmic reticulum and a partial reduction of the size of the pool sensitive to thapsigargin.



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Fig. 6.   Effect of 2-APB on Ca2+ release and Ca2+ entry due to 100 µM carbachol. The protocol for the two panels is identical to that for Figs. 3B and 3C except that different concentrations of the membrane-permeant IP3 receptor inhibitor, 2-APB, were used. In the top panel, the agonist was 100 µM carbachol, and in the bottom panel the agonist was 1 µM thapsigargin. The results illustrate findings from six (top) and five (bottom) independent determinations.

2-APB at 100 µM, a concentration that caused complete inhibition of capacitative calcium entry, did not alter Ca2+ entry due to 30 µM arachidonic acid (Fig. 7). As for thapsigargin, 100 µM 2-APB caused a slight reduction of [Ca2+]i release in response to 30 µM arachidonic acid (Fig. 7 and results not shown).



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Fig. 7.   Effect of 2-APB on Ca2+ signaling due to 30 µM arachidonic acid (AA). 30 µM arachidonic acid was applied to a HEK293 cell when indicated, in the absence of external Ca2+, and Ca2+ restored to 1.8 mM during the indicated interval. The cells were pretreated with either 100 µM 2-APB (dashed line) or the solvent control, Me2SO (solid line). 100 µM 2-APB failed to inhibit arachidonic acid-induced Ca2+ entry in a total of six experiments.

These data, including the data obtained with Gd3+, provide evidence that the mechanisms by which arachidonic acid activates Ca2+ release and Ca2+ influx are different from those of the store-depleting agents, thapsigargin and carbachol. As first suggested by Shuttleworth, capacitative calcium entry appears not to be involved in Ca2+ entry due to arachidonic acid in HEK293 cells (21).

Effects of 2-APB on [Ca2+]i Oscillations and Ca2+ Entry in Response to Low Concentrations of Carbachol-- As shown in Fig. 8, 2-APB inhibited the repetitive transient [Ca2+]i responses in a concentration-dependent manner and 100 µM 2-APB completely blocked the sustained oscillatory response of HEK293 cells to 5 µM carbachol.



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Fig. 8.   Effect of 2-APB on [Ca2+]i oscillations due to 5 µM carbachol.. The protocol was as for Fig. 4. Sequential stimulations with 5 µM carbachol are shown in A, and the effects of increasing concentrations of 2-APB on the second stimulation are depicted in B-D. Similar findings were obtained in a total of five to six experiments.

The inhibition by 2-APB of the [Ca2+]i response to 5 µM carbachol was unexpected, because arachidonic acid-induced Ca2+ signaling was unaffected by this drug. However, we considered the possibility that this concentration of carbachol might induce a small influx of Ca2+ that is only detectable when amplified through calcium-induced calcium release, and this might depend on functional IP3 receptors. Therefore, to assess more directly the actions of 2-APB on Ca2+ entry during [Ca2+]i oscillations, we utilized Mn2+ quench measurements. Mn2+ enters cells through divalent cation channels, but quenches Fura2 fluorescence at all wavelengths (37). Thus, the activity of Ca2+ influx channels is reported by the rate of Mn2+ quench of Fura2. In the presence of 0.1 mM Mn2+, in nominally Ca2+-free medium, a resting rate of Mn2+ quench was seen in unstimulated cells, and this was blocked when the cells were pretreated with 100 µM 2-APB (Fig. 9A). 5 µM carbachol increased Mn2+ quench, and again the rate of quench in the presence of carbachol was completely blocked by 100 µM 2-APB (Fig. 9B). Because 2-APB essentially completely blocked even basal Mn2+ quench, we repeated the experiments with 2 mM Mn2+. Under these conditions, a basal rate of Mn2+ quench was seen in the presence of 100 µM 2-APB, but this was not further increased by 5 µM carbachol (Fig. 10); however, addition of 5 µM arachidonic acid induced a substantial increase in quench (Fig. 10A). These results demonstrate that 2-APB attenuated both the resting Mn2+ entry and divalent cation influx stimulated by 5 µM carbachol, but not that of exogenous arachidonic acid.



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Fig. 9.   Effects of 5 µM carbachol and 100 µM 2-APB on Mn2+ entry into HEK293 cells. The Ca2+-insensitive fluorescence (Ftot, see "Materials and Methods") of Fura2-loaded HEK293 cells was monitored. In control cells (A), addition of 0.1 mM Mn2+ to the medium causes an accelerated quench of Fura2 (solid line), and this effect is blocked by 2-APB (dotted line). Treatment of the cells with 5 µM carbachol (B) results in an enhanced rate of Fura2 quench (solid line) that is again completely blocked by 2-APB (dotted line). A total of seven experiments produced similar findings.



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Fig. 10.   Effects of 5 µM carbachol, 100 µM 2-APB, and 5 µM arachidonic acid (AA) on Mn2+ entry into HEK293 cells. As in Fig. 11, the Ca2+-insensitive fluorescence (Ftot, see "Materials and Methods") of Fura2-loaded HEK293 cells was monitored. In A, in cells treated with 100 µM 2-APB, addition of 2.0 mM Mn2+ to the medium causes an accelerated quench of Fura2, but subsequent addition of carbachol does not further increase the rate of quench (solid line). However, with this same protocol, arachidonic acid stimulates the rate of quench (dotted line). In B, addition of 2 mM Mn2+ to control, 2-APB-treated cells (solid line), or 2-APB-treated cells stimulated with 5 µM carbachol (dotted line) results in a similar increase in the rate of quench of Fura2. A total of four to five experiments produced similar findings.

These findings indicate that the signaling mechanism underlying noncapacitative calcium entry and [Ca2+]i oscillations may involve signals other than or in addition to arachidonic acid. There is previous pharmacological evidence for a role for PLA2; for example, the oscillations are inhibited by the PLA2 inhibitor, isotetrandrine (28). Data shown in Fig. 11 (A and B) essentially replicates the previous findings of Shuttleworth and Thompson (28), showing that isotetrandrine can block [Ca2+]i oscillations, and the addition of a low concentration of arachidonic acid can partially restore the response (in 7 of 14 cells tested). We found that 10 µM isotetrandrine completely blocked the oscillations in 12 of 21 cells tested, whereas 20 µM blocked completely in 3 of 5. However, like its close cousin, tetrandrine (38), isotetrandrine can also function as a calcium channel blocker. We thus tested the effects of isotetrandrine on the entry of Ca2+ directly activated by arachidonic acid. As illustrated in Fig. 12A, 10 µM isotetrandrine consistently inhibited the Ca2+ entry in response to arachidonic acid (3 of 3 with 10 µM isotetrandrine; 3 of 4 with 20 µM isotetrandrine). The inhibition is apparently not due to action as a nonspecific channel blocker, or to membrane depolarization, because isotetrandrine caused only slight inhibition of entry in response to thapsigargin (Fig. 12B). These findings call into question the validity of isotetrandrine as a specific tool to demonstrate PLA2 involvement. In addition, as discussed below, they may indicate that the effects and pharmacological sensitivity of exogenously added arachidonic acid do not faithfully reflect the behavior of arachidonic acid generated endogenously as a component of a physiological signaling cascade. Furthermore, the relative insensitivity of the thapsigargin-induced entry to isotetrandrine further supports the view that the entry driving the [Ca2+]i oscillations is not capacitative.



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Fig. 11.   Effects of isotetrandrine on carbachol-induced [Ca2+]i oscillations. The protocol for A was as in Fig. 1C. Addition of 10 µM isotetrandrine to the medium blocked the sustained oscillations due to carbachol (7 of 21 cells). In B, 10 µM isotetrandrine was added during the oscillations, and the oscillations ceased. Addition of 5 µM arachidonic acid restored a partial response in 7 of 14 cells tested.



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Fig. 12.   Effects of isotetrandrine on arachidonic acid- and thapsigargin-induced Ca2+ entry. In A, Ca2+ entry was activated by 70 µM arachidonic acid. Where indicated, 10 µM isotetrandrine was added. Similar results were obtained in a total of three experiments with 10 µM isotetrandrine, and in three of four experiments with 20 µM isotetrandrine. In B, 1 µM thapsigargin was used to activate capacitative calcium entry. The addition of 10 µM isotetrandrine induced only a slight diminution of Ca2+ entry, and further addition of 20 µM isotetrandrine inhibited to a slightly greater extent. Similar results were obtained in a total of five experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we initially established that Gd3+ and 2-APB appear to be relatively specific and potent inhibitors of capacitative calcium entry. Results shown here indicate that Ca2+ entry due to two different store-depleting agents, thapsigargin and carbachol, is sensitive to the inhibitory effects of Gd3+ and 2-APB in HEK293 cells. Gd3+ from 30 nM to 1 µM and 2-APB from 10 to 100 µM in a concentration-dependent manner inhibited this Ca2+ entry with similar potency for the two agonists (Fig. 6 and data not shown), indicating a similar mechanism for blocking Ca2+ entry, i.e. capacitative calcium entry activated by store depletion in HEK293 cells. At concentrations of 1 µM Gd3+ and 100 µM 2-APB, respectively, a complete abolishment of Ca2+ influx due to carbachol and thapsigargin was observed, consistent with previous reports in which a complete blockade of capacitative calcium entry induced by different store-depleting drugs could be obtained with 1 µM Gd3+ in rat A7r5 cells (22) and with 100 µM 2-APB in DDT1-MF2 cells, A7r5 cells, and HEK293 cells (36). These results clearly demonstrate that Gd3+ and 2-APB are both potent inhibitors of capacitative calcium entry. 2-APB has also been shown to modulate IP3 receptors (27), leading Ma et al. (36) to conclude that the IP3 receptor is somehow involved in the activation of capacitative calcium entry.

Arachidonic acid, an unsaturated fatty acid produced by the action of PLA2 or diacylglycerol lipase on membrane lipids, is mobilized in a variety of cell types by the actions of neurotransmitters and hormones. Arachidonic acid also induces Ca2+ fluxes (39-41) as well as a variety of Ca2+-dependent effects in cells, and thus has been suggested as a second messenger modulating Ca2+ signal transduction (13, 16, 17, 33). However, the mechanisms underlying Ca2+ signaling modulation due to arachidonic acid remain unclear. To better define the mechanisms of calcium signaling in response to arachidonic acid, we examined the effects of the two inhibitors of capacitative calcium entry, Gd3+ and 2-APB, on arachidonic acid-mediated Ca2+ signaling. Arachidonic acid at concentrations at or above 30 µM released Ca2+ from intracellular Ca2+ stores and induced Ca2+ entry (Fig. 5), consistent with findings in other cell lines (18, 19, 41). Interestingly, we found that 10 µM Gd3+ completely blocked Ca2+ release in response to arachidonic acid (Fig. 5, C and D), but did not affect release due to thapsigargin or carbachol (Fig. 6). The mechanism for this effect is not known. Direct interaction of Gd3+ with arachidonic acid seems unlikely, because the concentration of Gd3+ (10 µM) is less than that of arachidonic acid (30 µM).

Ca2+ entry induced by arachidonic acid is also attenuated by Gd3+ but only in concentrations in excess of 1 µM. As for the inhibition of Ca2+ release due to arachidonic acid, 10 µM Gd3+ was required to produce complete blockade of Ca2+ entry (Fig. 5, C and D). A similar result was reported for A7r5 cells (22). The mechanism by which Gd3+ inhibits both Ca2+ release and influx is not known, nor is it clear as to whether these two effects are even related. The significant point for the current study, however, is that 1 µM Gd3+, which is more than sufficient for complete inhibition of capacitative calcium entry, is without effect when arachidonic is used as an activator of Ca2+ mobilization.

In addition to Gd3+, 2-APB is emerging as a relatively specific inhibitor of capacitative calcium entry. Ma et al. (36) demonstrated that 2-APB blocked capacitative calcium entry in HEK293 cells, as well as the entry ascribed to the transfected Trp3 channel. In the latter case, previous work had shown that transfected Trp3 can be activated either through an interaction with subplasmalemmal IP3 receptors (35) or more directly by diacylglycerol (42). 2-APB blocked Trp3 channels when activated by phospholipase C-linked agonists, but not when activated by diacylglycerol. This finding led Ma et al. (36) to conclude that 2-APB was not acting as a channel-blocking drug and that its mechanism of action in the case of Trp3 channels, and probably also in the case of capacitative calcium entry, involved inhibition of IP3 receptors. These results have been considered strong evidence for the conformational coupling model (4, 43) for activation of capacitative calcium entry, because this model invokes an obligatory role for the IP3 receptor interacting with plasma membrane capacitative calcium entry channels (44). Clearly, 2-APB is one of the more specific inhibitors of capacitative calcium entry. It completely blocks capacitative calcium entry in concentrations that are without effect on voltage-operated calcium channels (27),2 and in the current study, we have found that it does not block channels activated by arachidonic acid (Figs. 7 and 10). To our knowledge no other organic antagonist of capacitative calcium entry channels shows this degree of specificity (see also Ref. 45). The drug has only one other known site of action, the IP3 receptor, and it is reasonable for the present to accept the interpretation of Ma et al. (36) that this action underlies its actions on calcium entry. However, we note, as was somewhat evident in the work of Ma et al., that Ca2+ entry appears to be more sensitive to inhibition by 2-APB than the intracellular, IP3-mediated release of Ca2+ (Fig. 6). Thus, it is possible that 2-APB may also have direct, albeit highly specific, actions on capacitative calcium entry channels, although this distinction is not critical to arguments based on its effects in this study.

Finally, with this background of clear and relatively specific actions of Gd3+ and 2-APB, we have utilized these reagents to evaluate the role of the capacitative calcium entry pathway in the complex Ca2+-signaling response giving rise to [Ca2+]i oscillations in HEK293 cells (28). In our study of wild type HEK293 cells, repetitive [Ca2+]i spikes could be induced by a relatively low concentration of carbachol, and the generation of this signaling pattern is dependent on extracellular Ca2+ (Fig. 1D). Although previous models have implicated a role for capacitative calcium entry in the maintenance of [Ca2+]i oscillations (7, 12), this view has been recently questioned by Shuttleworth (13). In the current study, the insensitivity of [Ca2+]i oscillations to Gd3+ provides clear pharmacological evidence that the entry pathway activated by low concentrations of carbachol is distinct from the capacitative pathway seen with higher concentrations of carbachol or with store depletion by the SERCA inhibitor, thapsigargin. There is considerable evidence that arachidonic acid can serve as an activator of a noncapacitative pathway in HEK293 cells (46). Consistent with this idea, arachidonic acid-induced entry of Ca2+ was relatively insensitive to inhibition by Gd3+, curiously, despite the ability of arachidonic acid to deplete intracellular Ca2+ stores.3 However, the association between arachidonic acid-induced entry and the entry associated with oscillations is lost when the effects of 2-APB are examined. 2-APB was capable of completely inhibiting both the [Ca2+]i oscillations and increased Mn2+ quench due to 5 µM carbachol but was without effect on arachidonic acid-induced entry of either Ca2+ or Mn2+.

With the evidence presently available, we cannot definitively determine the mechanisms of Ca2+ signaling that underlie [Ca2+]i oscillations in HEK293 cells. We suggest three possible alternatives that may be testable in the future by continued experimental work:

(i) The simplest explanation for our data is that the entry associated with [Ca2+]i oscillations is noncapacitative in nature, but involves some mode of activation other than arachidonic acid. At least some of the previous evidence for PLA2 involvement (28), based on inhibitory effects of the PLA2 inhibitor, isotetrandrine, is open to question (Fig. 12). Furthermore, regardless of the precise mechanism of action of 2-APB, its ability to distinguish clearly between arachidonic acid-induced entry and the Ca2+ entry associated with [Ca2+]i oscillations suggests that the arachidonic acid pathway is not involved.

(ii) These arguments notwithstanding, it is still possible, given other points of evidence that suggest an involvement of PLA2 and arachidonic acid, that PLA2 is involved in the oscillations. However, exogenously added arachidonic acid may act through a different mechanism than arachidonic acid generated by PLA2 in the cell, perhaps in the vicinity of Ca2+ channels.

(iii) Finally, we consider that it still is possible that the Ca2+ entry underlying oscillations is a store-operated entry, albeit involving different store-operated channels than those seen with maximal store depletion. Although only Gd3+-sensitive entry was observed in HEK293 cells following maximal depletion of Ca2+ stores, Gd3+-insensitive store-operated channels have been observed in other experimental systems (29). In the absence of extracellular Ca2+, at least one [Ca2+]i spike is always observed with 5 µM carbachol, suggesting that intracellular release is triggered independently of, and likely prior to entry. Also, the [Ca2+]i oscillations and Mn2+ entry due to 5 µM carbachol are both blocked by 2-APB, a drug with documented specificity for capacitative calcium channels. Thus, it is possible that, with minimal and short-lived Ca2+ discharge, a different population of capacitative calcium entry channels are activated with pharmacological distinctions and pharmacological similarities to the channels activated upon maximal store depletion.

The conclusion from this study is that the interplay among various Ca2+-signaling pathways that result in [Ca2+]i spikes and oscillations may be much more complex than originally envisioned. However, some cell types may utilize a simpler mechanism involving strictly phospholipase C, intracellular Ca2+ release, and capacitative calcium entry mechanisms. For example, in mouse lacrimal cells, which produce a characteristic sinusoidal pattern of [Ca2+]i oscillations (47), there are no Gd3+-insensitive responses seen at low agonist concentrations, and the cells appear completely insensitive to the addition of exogenous arachidonic acid.2 Clearly, additional work will be needed to understand the varied patterns and mechanisms that control the biologically important phenomenon known as [Ca2+]i oscillations.


    ACKNOWLEDGEMENTS

We are grateful to Elizabeth Murphy and Jerry Yakel who read the manuscript and provided helpful comments.


    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.

§ 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, November 28, 2000, DOI 10.1074/jbc.M007524200

2 D. Luo, L. M. Broad, G. St. J. Bird, and J. W. Putney, Jr., unpublished observations.

3 The failure of store depletion by arachidonic acid to activate capacitative calcium entry may be due to the previously documented ability of arachidonic acid to inhibit capacitative calcium entry (48).


    ABBREVIATIONS

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; PLA2, phospholipase A2; HPSS, HEPES-buffered physiological saline solution; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 2-APB, 2-aminoethyoxydiphenyl borane; SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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