Thrombin-, bradykinin-, and arachidonic acid-induced Ca2+ signaling in Ehrlich ascites tumor cells

Nanna Koschmieder Jørgensen, Stine Falsig Petersen, and Else Kay Hoffmann

Biochemical Department, August Krogh Institute, DK-2100 Copenhagen, Denmark

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Stimulation of single Ehrlich ascites tumor cells with agonists (bradykinin, thrombin) and with arachidonic acid (AA) induces increases in the free intracellular Ca2+ concentration ([Ca2+]i) in the presence and absence of extracellular Ca2+, measured using the Ca2+-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increases in [Ca2+]i, unlike addition of the same agonist twice. Bradykinin and thrombin have additive effects on [Ca2+]i in Ca2+-free medium. The phosphoinositidase C inhibitor U-73122 inhibits the agonist-induced increases in [Ca2+]i, whereas ryanodine has no effect. Pretreatment of cells in Ca2+-free medium with thapsigargin abolishes the bradykinin-induced increase in [Ca2+]i but not the response to thrombin. The AA-induced response is not inhibited by U-73122 and cannot be mimicked by the inactive structural analog trifluoromethylarachidonyl ketone. Pretreatment of the cells with 50 µM AA (but not with 10 µM AA) abolishes the agonist-induced increase in [Ca2+]i. Thus bradykinin, thrombin, and AA induce increases in [Ca2+]i in Ehrlich cells due to Ca2+ entry and release from intracellular stores. Thrombin causes release of Ca2+ from an intracellular store that is insensitive to bradykinin and is not depleted by thapsigargin but is depleted by AA.

thapsigargin; intracellular stores; U-73122; U-73343; ryanodine

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CHANGES IN FREE intracellular Ca2+ concentration ([Ca2+]i) play an important role in a variety of cellular signaling pathways. Many hormones and other agonists exert their effects via an increase in [Ca2+]i. In this investigation, we study changes in [Ca2+]i in single cells and cell suspensions of Ehrlich ascites tumor cells after stimulation with the agonists bradykinin and thrombin as well as with the fatty acid arachidonic acid (AA).

The nonapeptide hormone bradykinin belongs to the group of kinins, a family of peptide mediators released after proteolytic cleavage of the precursor proteins (kininogens) after, for example, pathophysiological stimuli such as inflammation and tissue trauma. Bradykinin has a wide range of effects in both neuronal and nonneuronal tissues (see Ref. 11) and has been reported to be able to activate phosphoinositidase C (PIC) via the widely distributed, G protein-linked B2 receptors, leading to an increase in D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and in [Ca2+]i. Activation of phospholipase A2 (PLA2) and phospholipase D (PLD) as well as stimulation of cAMP production, the latter perhaps secondary to an increased prostaglandin production, have likewise been reported to occur after activation of B2 receptors (see Ref. 19). The stimulation of PLA2 may be secondary to activation of PIC and PLD, as suggested for Madin-Darby canine kidney cells (24). The less common B1 receptors have been proposed to be linked to production of nitric oxide or prostaglandins (see Ref. 19). Bradykinin-mediated responses may be inactivated via degradation of bradykinin by proteolytic enzymes. Furthermore, downregulation of the receptors via aggregation and internalization as well as desensitization, perhaps involving cGMP-dependent phosphorylation, may be important (see Ref. 11).

The serine protease thrombin is a potent physiological agonist and plays a key role in hemostasis. Thrombin is thus important for converting fibrinogen into fibrin and for stimulation of aggregation and secretion in platelets and can elicit mitogenic responses from vascular smooth muscle cells. In addition, thrombin has been shown to affect regulation of neurite outgrowth from neuronal cells and initiation of resorption of bone cells. Thrombin is reported to bind to a specific, G protein-coupled receptor, activating the receptor by proteolytic cleavage. Receptor activation causes stimulation of PIC. Activation of PLD and PLA2 has also been reported; this could be downstream of activation of PIC (see Ref. 17). Furthermore, thrombin has been described as activating mitogen-activated protein kinase (MAPK; Refs. 4, 2; see Ref. 17 and references therein) and a range of different nonreceptor tyrosine kinases, including the Src family, focal adhesion kinase, and a member of the Janus family (JAK2). Also, stimulation of phosphoinositol 3-kinase and stimulation of Ras and Ras-related proteins have been reported (see Ref. 17 and references therein). The thrombin receptor rapidly inactivates via desensitization, probably due to phosphorylation. Downregulation via internalization has also been demonstrated (see Ref. 17).

The polyunsaturated fatty acid AA (20:4) has double bonds at carbons 5, 8, 11, and 14 and is present in cellular membranes as part of the phospholipids, mainly in phosphatidylcholine and phosphatidylethanolamine but also in the less common phosphoinositol lipids. AA can be released from membrane lipids after stimulation with, for example, hormones, neurotransmitters, or antigens, via activation of PIC, PLD, or PLA1 or more commonly via activation of PLA2 (see Refs. 16, 26). The released AA can be reincorporated into membrane lipids or converted into metabolites (eicosanoids; see Ref. 26). An increased production of AA due to activation of a Ca2+-dependent, cytosolic PLA2 has been reported to occur after exposure of Ehrlich cells to hyposmotic medium (46), and AA metabolites, especially leukotriene D4, have been proposed to play a role in the signaling involved in volume regulation after cell swelling in Ehrlich cells (Refs. 23, 28; see Ref. 26).

In addition to its important role as a precursor for eicosanoid production, AA has been suggested to have more direct cellular effects. AA has thus been shown to activate ion channels either directly or via nonspecific effects on membrane lipids. Examples include potassium channels in smooth muscle cells (36) and the volume-activated taurine channel in Ehrlich cells (Ref. 27; see Ref. 33). Inhibitory effects of AA on membrane transport have also been reported, such as inhibition of the swelling-activated Cl- channel in Ehrlich cells (25, 27), of Cl- channels in airway epithelium (1, 22), and of a Ca2+ channel in smooth muscle cells (35) [see Meves et al. (33)]. AA has been reported to enhance PIC activity in cells and synaptosomes and to activate protein kinase C (PKC). In contrast, an inhibitory effect of AA on PLA2 has been reported (see Refs. 16, 33). Also, direct stimulation of protein tyrosine phosphorylation by AA, perhaps through nonspecific perturbation of membrane structures, has been described (5). AA has also been shown to modulate cellular Ca2+ signaling (32, 47, 50), to cause release of Ca2+ from intracellular stores, and in some cases to activate Ca2+ influx (37, 40, 48). Whether the release of Ca2+ occurs secondarily to Ins(1,4,5)P3 production is not clear, and AA has been suggested to directly affect a site on the endoplasmic reticulum membrane. Inhibition of the Ins(1,4,5)P3-dependent current through the Ins(1,4,5)P3 receptor by AA has, however, also been suggested (44). Furthermore, the AA-mediated influx of Ca2+ may be regulated by mechanisms other than the AA-mediated Ca2+ release (37). Both inhibitory and stimulatory direct effects of AA on Ca2+ channels have been described (see Ref. 33, and above). The effective concentrations of AA range from 1 µM to 1 mM (37, 47, 48, 50).

In Ehrlich cells, the agonists bradykinin and thrombin have previously been shown to induce an increase in [Ca2+]i in cell suspension measurements (41, 21, 23). In the present investigation, the effects of the agonists bradykinin and thrombin on [Ca2+]i in Ehrlich cells are studied further and at the single-cell level. The aims are to elucidate 1) the effect of multiple stimulation with the agonists, 2) the effect of simultaneous addition of the agonists, and 3) the possible involvement of PIC, ryanodine receptors, or thapsigargin-sensitive Ca2+ stores in the agonist-induced Ca2+ signaling. In addition, 4) the effect of AA on [Ca2+]i in Ehrlich cells and 5) the effect of pretreatment with AA on the thrombin- and bradykinin-induced increase in [Ca2+]i are studied.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents

All reagents and agonists were analytical grade and were obtained from Sigma unless otherwise indicated. Fura 2-AM and fura 2 pentapotassium salt were obtained from Molecular Probes (Leiden, The Netherlands). AA and trifluoromethylarachidonyl ketone (AACOCF3) were obtained from Cascade Biochemicals (Berkshire, UK). Thapsigargin and ryanodine were obtained from Alamone Labs (Jerusalem, Israel). Heparin was obtained from Løvens Kemiske Fabrik (Ballerup, Denmark). U-73122 and U-73343 were obtained from Biomol Research Laboratories. For stock solutions of the agonists and of AA, bradykinin was dissolved in deionized distilled H2O at 1 mM, thrombin was added from a 1,000 U/ml stock solution in deionized distilled H2O, AA was dissolved at 50 mM in 96% ethanol, and AACOCF3 was added from a 28 mM stock in 96% ethanol.

Cell Suspension

Ehrlich mouse ascites tumor cells (hyperdiploid strain) were maintained in NMRI mice by weekly intraperitoneal transplantation into fresh mice. The Ehrlich cells were harvested as described by Hoffmann et al. (20). Briefly, the mice were killed by cervical dislocation, and the cells were harvested in standard medium containing heparin (2.5 U/ml) and washed with heparin-free standard medium by gentle centrifugation. The cells were suspended at the desired cytocrit, typically 0.4%, in standard medium and incubated for ~30 min before the start of the experiments. Loading with the Ca2+-sensitive fluorescent probe fura 2 (see Loading of Ehrlich Cells With Fura 2) was initiated during this period. All experiments were conducted at 37°C. The maximal final concentration of DMSO in this study was 0.2%, and the maximal concentration of ethanol was ~0.5%. At these concentrations, the solvents had no detectable effect on [Ca2+]i (not illustrated).

Media

Standard medium (300 mosM) contained (in mM) 150 Na+, 5 K+, 1 Mg2+, 1 Ca2+, 150 Cl-, 1 SO2-4, 1 HPO2-4, 3 MOPS, 3 TES, and 5 HEPES (pH 7.4). Ca2+-free medium was the same as standard medium, except that CaCl2 was omitted and EGTA (2 mM) was included. High-K+ calibration media contained 10 µM fura 2 pentapotassium salt and (in mM) 158 K+, 158 Cl-, 1 Mg2+, 1 SO2-4, 1 HPO2-4, 3 MOPS, 3 TES, and 5 HEPES (pH 7.40); the Ca2+ concentration was either 1 mM, obtained by adding 1 mM CaCl2, or zero, obtained by adding 2 mM EGTA.

Loading of Ehrlich Cells With Fura 2

The Ehrlich cells were loaded with fura 2 as described by Jørgensen et al. (23), by incubation of the cell suspension at cytocrit 0.4%, in standard medium containing 0.2% (wt/vol) BSA and 2 µM fura 2-AM, for 20 min at 37°C in a shaking water bath. Excess fura 2 was removed by washing the cells once in standard medium with 0.2% (wt/vol) BSA and once in the experimental medium. The cells were resuspended in the experimental medium at cytocrit 0.3% (single-cell studies) or 5% (cell suspension experiments) and kept under gentle stirring at room temperature (~20°C) after loading. The cells used for the single-cell experiments were selected for showing a bright appearance in phase contrast, uniformly distributed fluorescence, and stable fluorescence intensity. Unloaded cells possessed no detectable (single-cell experiments) or negligible (cell suspension experiments) autofluorescence under the experimental conditions used in this study. Cells or cell suspensions showing high or unstable resting levels of [Ca2+]i were discarded (see also Ref. 23).

Fluorescence Measurements of [Ca2+]i

Cell suspensions. [Ca2+]i was estimated in fura 2-loaded cells in suspension using a Perkin-Elmer R 100A luminescence spectrometer as described in Jørgensen et al. (23). Briefly, the fura 2-loaded cells were diluted with experimental medium to a final cytocrit of 0.5% and transferred to polystyrene cuvettes (Elkay Ultra-VU). The cells were stirred using Teflon-coated magnets, and the cuvette housing was thermostatically controlled at 37°C. The excitation wavelengths were alternated between 340 and 380 nm under computer control. Emission was detected at 510 nm. Excitation and emission slit widths were 5 nm. Background correction was performed as described in Jørgensen et al. (23).

Single cells. [Ca2+]i was measured in single cells using fluorescence microscopy as described in Jørgensen et al. (23). Briefly, the fluorescence emission from fura 2-loaded single cells was detected using a Zeiss Axiovert 100 fluorescence microscope with a ×40 oil immersion objective (1.30 numerical aperture; Akrostigmat, UV). The excitation light source was a 75-W xenon lamp. A BPB 380/20 filter and a K12 filter were inserted in the excitation light path, and a shutter was used to control illumination. The dual excitation wavelengths were selected by filters (BP 340/10 and BP 380/10). The light was collected by an intensified charge-coupled device camera (CCD72 with a GenIIsys intensifier, Dage-MTI). On average, six frames were collected at each excitation wavelength, and the fluorescence ratio image (340-nm fluorescence divided by 380-nm fluorescence) was calculated after background subtraction on a pixel-to-pixel basis by the image analysis software (Image1/Fluor, Universal Imaging). Experiments were performed at 37°C.

The cells were stimulated either by addition of a large volume (typically addition of 2 ml to the 2 ml already present in the experimental chamber) or by direct addition of a more concentrated stock solution by pipette (typically addition of 10 µl), as described in Ref. 23.

Estimation of [Ca2+]i From Fura 2 Measurements

The measured 340 nm-to-380 nm ratio values were converted into values of [Ca2+]i by the use of in vitro calibration as described in Ref. 23, using the equation (from Ref. 18)
[Ca<SUP>2+</SUP>] = <IT>K</IT><SUB>d</SUB>[(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)](S<SUB>f380</SUB>/S<SUB>b380</SUB>) (1)
where Kd is the dissociation constant (224 nM; see Ref. 18), R is the measured 340 nm-to-380 nm fluorescence ratio, and Rmax and Rmin are R at a saturating Ca2+ concentration and in Ca2+-free medium (containing 2 mM EGTA), respectively. Sf380 and Sb380 are measured from the fluorescence intensity of fura 2 at 380-nm excitation in solutions containing zero Ca2+ and a saturating Ca2+ concentration, respectively (18). Rmax, Rmin, and Sf380/Sb380 have previously been estimated at 9.1, 0.3, and 7.2, respectively, in single-cell experiments. In cell suspension experiments, the values are 20.6, 0.8, and 9.3, respectively (23), or, for a newer xenon lamp, 25.7, 0.8, and 8.3, respectively (S. Pedersen, personal communication).

Statistical Evaluation

The values are given as means ± SD or as means ± SE, when independent experiments are compared, as indicated. The number of observations (n) is indicated in parentheses. Statistical significance is evaluated by the use of Student's t-test, except for single-cell data, as the F-test prohibits the use of Student's t-test on these data. Cells showing a maximal increase in [Ca2+]i after stimulation with agonists or AA in the absence of inhibitors (see RESULTS) less than the SD of unstimulated cells were not included. All data are from a minimum of three independent experiments.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Bradykinin and Thrombin on [Ca2+]i in Single Ehrlich Cells

Stimulation of single Ehrlich cells with the agonists bradykinin or thrombin results in transient increases in [Ca2+]i. This is shown in Fig. 1, A and B, in which [Ca2+]i is followed over time in single fura 2-loaded Ehrlich cells after addition of maximal doses of bradykinin (added by pipette, final concentration 5 µM) or thrombin (added by pipette, final concentration 5 U/ml), respectively, in Ca2+-containing standard medium. The means and ranges of the maximal increase in [Ca2+]i, i.e., the difference between the peak value of [Ca2+]i after stimulation of single Ehrlich cells with bradykinin or thrombin and the average prestimulatory level of [Ca2+]i, are given in Table 1.


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Fig. 1.   Effect of bradykinin or thrombin on free intracellular Ca2+ concentration ([Ca2+]i) in single-cell experiments. Ehrlich cells were loaded with fura 2 as described in MATERIALS AND METHODS, followed by incubation for 10-60 min in standard medium (1 mM Ca2+). Experiments were conducted in standard medium (1 mM Ca2+). [Ca2+]i was followed with time in single cells, using fluorescence microscopy and digital image processing. A: bradykinin (added by pipette, final concentration 5 µM) was added as indicated by arrow. Data are from 1 cell, representative of 45 cells from 6 experiments. B: thrombin (added by pipette, final concentration 5 U/ml) was added as indicated by arrow. Data are from 1 cell, representative of 25 cells from 6 experiments giving similar results. Means ± SD for maximal increase in [Ca2+]i are given in Table 1.

                              
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Table 1.   Quantification of sequential bradykinin- and thrombin-induced increases in [Ca2+]i

When Ehrlich cells in suspension are stimulated with bradykinin (10 µM) followed by thrombin (10 U/ml), the cells are able to respond to both agonists with sequential increases in [Ca2+]i, as illustrated by the data in Table 1. Table 1 also shows that the bradykinin-induced maximal increase in [Ca2+]i is significantly (P < 0.0001) reduced by removal of external Ca2+ (Ca2+-free medium, 2 mM EGTA) and by a previous stimulation with thrombin in Ca2+-containing standard medium (P < 0.01) or in Ca2+-free medium (2 mM EGTA; P < 0.05). That the response is reduced, but not abolished, on removal of extracellular Ca2+ indicates that the bradykinin-induced increase in [Ca2+]i is due to both Ca2+ entry and release of Ca2+ from intracellular stores. In contrast, the thrombin-induced maximal increase in [Ca2+]i is not significantly reduced in Ca2+-free medium (2 mM EGTA) compared with Ca2+-containing standard medium. A previous stimulation with bradykinin does not inhibit the thrombin-induced maximal increase in [Ca2+]i in Ca2+-containing medium: if anything, the increase is enhanced. In Ca2+-free medium (2 mM EGTA), the response to thrombin is reduced by prestimulation with bradykinin (P < 0.05).

These data from cell suspensions show that the cells are able to respond with an increase in [Ca2+]i when stimulated with more than one agonist. Similar experiments were performed on the single-cell level to clarify whether the individual Ehrlich cells are able to respond with more than one increase in [Ca2+]i after sequential addition of the two agonists.

Figure 2A shows the effect of stimulation of single Ehrlich cells with bradykinin (added by pipette, final concentration 5 µM) followed by thrombin (added by pipette, final concentration 5 U/ml) in standard medium (1 mM Ca2+). Figure 2A demonstrates that sequential addition of the agonists leads to sequential increases in [Ca2+]i in the individual cells. Similar results were obtained when thrombin was added before bradykinin (data not shown). Figure 2B shows the effect of sequential addition of the agonists in Ca2+-free medium (2 mM EGTA) and illustrates that addition of thrombin (added by pipette, final concentration 5 U/ml) followed by bradykinin (added by pipette, final concentration 5 mM) results in increases in [Ca2+]i also after the second agonist addition, even in the absence of extracellular Ca2+. This indicates that preincubation of Ehrlich cells in the Ca2+-free medium and stimulation with one of the agonists does not lead to depletion of all intracellular Ca2+ stores. Similar results were obtained when bradykinin was added before thrombin (not illustrated). Figure 2C illustrates that stimulation of the same cells twice with thrombin does not cause any detectable increase in [Ca2+]i after the second agonist addition in Ca2+-free medium [3 experiments in Ca2+-free medium (2 mM EGTA); 3 experiments in standard medium (1 mM Ca2+)]. Similar results were obtained with bradykinin (data not shown). This could be due to saturation of the receptors, since the agonists were not removed after stimulation and were applied in maximal concentrations. Desensitization of the receptors could likewise be involved (see Introduction). The average agonist-induced maximal increase in [Ca2+]i in single cells measured in Ca2+-containing or Ca2+-free medium are given in Table 1.


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Fig. 2.   Effect of sequential addition of bradykinin and thrombin on [Ca2+]i in single-cell experiments. Fura 2-loaded cells were incubated in either standard medium (1 mM Ca2+) for 10-60 min or Ca2+-free medium (2 mM EGTA) for 10-30 min before experiments, as indicated. [Ca2+]i was followed with time in 1 cell, using fluorescence microscopy and digital image processing. Bradykinin (added by pipette, final concentration 5 µM) and thrombin (added by pipette, final concentration 5 U/ml) were added as indicated by arrows. A: data for 1 cell from 1 experiment in Ca2+-containing standard medium, representative of 11 cells from 3 similar experiments. B: data from 1 cell from 1 experiment in Ca2+-free medium, representative of 15 cells from 6 experiments giving similar results. C: data from 1 cell from 1 experiment in Ca2+-free medium, representative of 16 cells from 3 experiments giving similar results. Means ± SD for maximal increase in [Ca2+]i are given in Table 1.

In some cells, the transient increase in [Ca2+]i after stimulation of Ehrlich cells with bradykinin is, in Ca2+-containing medium, followed by a sustained increase in [Ca2+]i (see Fig. 2A). In other cells, this sustained phase is very small or absent (Fig. 1A). In cell suspension experiments, such a sustained elevation of [Ca2+]i is generally observed after bradykinin addition in Ca2+-containing medium. This sustained phase with elevated [Ca2+]i is likely to be caused by influx of Ca2+ from the extracellular medium, since it is not detectable in Ca2+-free medium (not illustrated), supporting the finding that the bradykinin-induced maximal increase in [Ca2+]i is significantly reduced in the absence of extracellular Ca2+. Such a phase with elevated [Ca2+]i is not clearly discernible after stimulation with thrombin in the single-cell studies (Figs. 1B and 2A) but is occasionally (in 3 of 6 experiments) seen in cell suspension measurements. This could indicate that thrombin causes Ca2+ entry as well as release, even though no significant reduction of the thrombin-induced maximal increase in [Ca2+]i is found after removal of extracellular Ca2+.

Effect of Inhibition of PIC on the Thrombin-Induced Increase in [Ca2+]i

To elucidate the possible role of PIC for the bradykinin- and thrombin-induced increases in [Ca2+]i, the effect of the PIC inhibitor U-73122 (3, 42) was investigated. Figure 3A illustrates the increase in [Ca2+]i in suspensions of Ehrlich cells in Ca2+-containing standard medium after addition of bradykinin (10 µM) followed by thrombin (10 U/ml), in the presence of the inactive structural analog of U-73122, U-73343 (10 µM) (42). As seen in Fig. 3 and in Table 2, preincubation with U-73343 (5 min) has no significant effect on either the bradykinin-induced maximal increase in [Ca2+]i (n = 4, paired t-test) or the thrombin-induced change in [Ca2+]i (n = 4, paired t-test). Figure 3B and Table 2 show that addition of U-73122 (10 µM) significantly (P < 0.05 for bradykinin and P < 0.01 for thrombin, n = 4, paired t-test) inhibits both the bradykinin- and the thrombin-induced increases in [Ca2+]i, compared with parallel control experiments in Ca2+-containing standard medium in the absence of inhibitor. In the presence of U-73122, the maximal increase in [Ca2+]i, calculated relative to values from parallel control experiments without the inhibitor, was reduced to 0.5 ± 0.1 and 0.3 ± 0.1 (means ± SE; n = 4) after addition of bradykinin and thrombin, respectively. It should be noted that addition of U-73343 or U-73122 causes an increase in the resting level of [Ca2+]i in the Ca2+-containing medium, as seen in Fig. 3. This increase is not detectable in Ca2+-free medium (2 mM EGTA; see Fig. 3, C and D) and is thus suggested to be due to Ca2+ entry. Figure 3, C and D, illustrates the effects of U-73344 and U-73122 on the bradykinin- and thrombin-induced increases in [Ca2+]i in Ca2+-free medium (2 mM EGTA).


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Fig. 3.   Effect of the phosphoinositidase C (PIC) inhibitor U-73122 on bradykinin- and thrombin-induced increases in [Ca2+]i in cell suspension measurements. Fura 2-loaded Ehrlich cells were treated as described for Table 1, and [Ca2+]i was followed with time in standard medium (1 mM Ca2+) or Ca2+-free medium (2 mM EGTA) in cell suspensions, using a fluorescence spectrophotometer. Bradykinin (10 µM) and thrombin (10 U/ml) were added as indicated by arrows. A: cells were suspended in Ca2+-containing standard medium and pretreated with U-73343 (10 µM) for 5 min, as indicated by arrow. Data are from 1 experiment, representative of 4 experiments. B: cells were suspended in Ca2+-containing standard medium and pretreated with U-73122 (10 mM) for 5 min, as indicated by arrow. Data are from 1 experiment, representative of 4 experiments. C: cells were suspended in Ca2+-free medium (2 mM EGTA) and pretreated with U-73343 (10 µM) for 5 min, as indicated by arrow. Data are from 1 experiment, representative of 3 experiments. D: cells were suspended in Ca2+-free medium (2 mM EGTA) and pretreated with U-73122 (10 µM) for 5 min, as indicated by arrow. Data are from 1 experiment, representative of 3 experiments.

                              
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Table 2.   Effect of the phosphoinositidase C inhibitor U-73122 on agonist-induced increase in [Ca2+]i

Figure 3 and the data in Table 2 show that in the absence of extracellular Ca2+ both the bradykinin- and thrombin-induced increases in [Ca2+]i are completely inhibited by U-73122 (see Fig. 3D; n = 3). U-73343 has no significant effect on the bradykinin-induced response (paired t-test, n = 3), whereas the thrombin-induced maximal increase in [Ca2+]i is reduced in the presence of U-73343 (P < 0.05, paired t-test, 3 sets of experiments).

These observations indicate that the bradykinin- and thrombin-induced release of Ca2+ from intracellular stores is likely to be mediated by activation of the PIC signaling pathways, whereas agonist-induced Ca2+ entry observed in the presence of external Ca2+ could be partially mediated via activation of PIC and partially via a PIC-independent influx pathway.

Inhibition of Ryanodine Receptors With Ryanodine Has No Effect on Agonist-Induced Increase in [Ca2+]i

The ryanodine receptors are structurally and functionally related to the Ins(1,4,5)P3 receptors. The plant alkaloid ryanodine binds to the intracellular Ca2+ release channel of the ryanodine receptor family and inhibits release of Ca2+ via the ryanodine receptors in concentrations between 10 and 300 µM (see Ref. 8). Preincubation of a suspension of Ehrlich cells with ryanodine has no significant inhibitory effect on the bradykinin-induced (n = 4, paired t-test) or thrombin-induced (n = 4, paired t-test) increases in [Ca2+]i compared with parallel control experiments. This indicates that the bradykinin- and thrombin-induced increases in [Ca2+]i in Ehrlich cells are not likely to be due to mobilization of Ca2+ via ryanodine receptors (data not shown).

Effect of Thapsigargin on Bradykinin- and Thrombin-Induced Increases in [Ca2+]i

Thapsigargin is a selective inhibitor of the sarco(endo)plasmic reticulum type of Ca2+-ATPase and has been shown to inhibit reuptake of Ca2+ into the Ins(1,4,5,)P3-sensitive intracellular Ca2+ store (45), thus leading to depletion of the store. The effect of addition of thapsigargin on single Ehrlich cells in Ca2+-free medium (2 mM EGTA) is illustrated in Fig. 4. It is seen that addition of thapsigargin (2 µM) in Ca2+-free medium causes a slow increase, followed by a gradual decrease, in [Ca2+]i. The increase in [Ca2+]i is due to the depletion of the intracellular Ca2+ store, whereas the gradual decrease most likely is caused by the absence of Ca2+ in the medium, which prohibits Ca2+ entry, in combination with Ca2+ extrusion by the cell. A small, slow decrease in [Ca2+]i is often observed in cells in Ca2+-free medium. Stimulation with bradykinin (added by pipette, final concentration 5 µM) results in no detectable change in [Ca2+]i in thapsigargin-treated cells in Ca2+-free medium (2 mM EGTA), as shown in Fig. 4. In contrast, addition of thrombin (added by pipette, final concentration 5 U/ml) is able to induce a significant, transient increase in [Ca2+]i even after addition of thapsigargin, and bradykinin, in Ca2+-free medium (2 mM EGTA).


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Fig. 4.   Effect of thapsigargin on bradykinin- and thrombin-induced increase in [Ca2+]i in single-cell experiment. Ehrlich cells were treated as described for Fig. 1, except that Ca2+-free medium (2 mM EGTA) was used. [Ca2+]i was followed in 1 cell, using fluorescence microscopy and digital image processing. Thapsigargin (TG; added as a large volume, final concentration 2 µM), bradykinin (added by pipette, final concentration 5 µM), and thrombin (added by pipette, final concentration 5 U/ml) were added as indicated by arrows. Data are from 1 cell, representative of a total of 33 cells from 5 experiments. No detectable increase in [Ca2+]i was seen after stimulation with bradykinin in any of 33 cells. Similar results were obtained in 29 cells from 6 experiments in which bradykinin was added following thapsigargin and after thrombin addition. Means for maximal increase in [Ca2+]i are given in RESULTS. In a few very recent experiments, increase in [Ca2+]i after addition of thrombin to thapsigargin-treated cells in Ca2+-free medium has not been detected. It thus seems that properties of cells have changed with respect to thapsigargin-insensitive stores.

The thrombin-induced maximal increase in [Ca2+]i after pretreatment with thapsigargin and bradykinin in Ca2+-free medium (2 mM EGTA) is estimated at 228 ± 198 nM (mean ± SD; 33 cells, 5 experiments). This is in the same range as the maximal increase in [Ca2+]i measured in single cells in Ca2+-free medium (2 mM EGTA) with thrombin as the first stimulus (379 nM) in the absence of thapsigargin. Similar results were obtained when thrombin was added after thapsigargin, and before bradykinin, in Ca2+-free medium (2 mM EGTA), when the thrombin-induced increase in [Ca2+]i is estimated to be 124 ± 70 nM (mean ± SD; 29 cells, 6 experiments). Thus stimulation with thrombin, unlike bradykinin, is able to release Ca2+ from an intracellular store that is not depleted by pretreatment with thapsigargin. These experiments were performed on stable cells after >50 passages in mice. It should be noted, however, that this increase in [Ca2+]i after addition of thrombin to thapsigargin-treated cells in Ca2+-free medium has not been detected in a few very recent experiments performed on cells that have undergone fewer than five passages in mice, either in single cells or in cell suspension experiments. It thus seems that the properties of the cells have somewhat changed with respect to the thapsigargin-insensitive stores.

Additive Effect of Simultaneous Addition of Thrombin and Bradykinin

If bradykinin (10 µM) and thrombin (10 U/ml) are added simultaneously to Ehrlich cells in suspension, the maximal increase in [Ca2+]i in Ca2+-free medium (2 mM EGTA) is significantly increased compared with the response after stimulation with bradykinin alone (P < 0.001, n = 6, paired t-test) or thrombin alone (P < 0.01, n = 6, paired t-test). This enhanced increase in [Ca2+]i after simultaneous addition of the agonists to Ehrlich cells suspended in Ca2+-free medium is illustrated in Fig. 5.


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Fig. 5.   Additive effect of simultaneous addition of bradykinin and thrombin in cell suspension experiments. Fura 2-loaded Ehrlich cells were preincubated for 10-80 min in Ca2+-free medium (2 mM EGTA), and [Ca2+]i was followed with time in cells suspended in Ca2+-free medium (2 mM EGTA), using a fluorescence spectrophotometer. Bradykinin (10 µM), thrombin (10 U/ml), or both agonists simultaneously (Bra + Thr) were added. Data are means ± SE from 6 sets of paired experiments. d[Ca2+]i max, maximal increase in [Ca2+]i.

In Ca2+-free medium, the maximal increase in [Ca2+]i after stimulation with both agonists simultaneously (10 U/ml thrombin and 10 µM bradykinin) is enhanced compared with that after addition of either agonist alone. The maximal increase in [Ca2+]i after addition of both agonists simultaneously is 2.5 ± 2 relative to the value from parallel control experiments with addition of bradykinin alone or 1.4 ± 0.1 relative to the value from parallel control experiments with addition of thrombin alone (means ± SE, n = 6). The maximal increase in [Ca2+]i after stimulation with both agonists simultaneously (10 U/ml thrombin and 10 µM bradykinin) is 115 ± 2 nM, compared with 38 ± 2 nM after stimulation with bradykinin (10 µM) alone and 89 ± 16 nM after stimulation with thrombin (10 U/ml) alone (means ± SE, n = 6).

AA-Induced Changes in [Ca2+]i

The fatty acid AA is able to induce an increase in [Ca2+]i in Ehrlich cells in suspension. Figure 6 shows the effect of AA (50 µM) on [Ca2+]i in cells suspended in Ca2+-free medium (2 mM EGTA). The data illustrate that AA induces an increase in [Ca2+]i, followed by a phase in which [Ca2+]i is elevated and then by a slow decline toward the resting level.


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Fig. 6.   Arachidonic acid (AA)-induced increase in [Ca2+]i in cell suspension experiment. Cells were loaded with fura 2 and preincubated in Ca2+-free medium (2 mM EGTA) for 10-60 min before experiments. At time 0, cells (cytocrit 5%) were diluted 10-fold into Ca2+-free medium (2 mM EGTA) in cuvette, and [Ca2+]i was followed with time in cell suspension. AA (50 µM), bradykinin (10 µM), and thrombin (10 U/ml) were added as indicated by arrows. Data are from 1 experiment, representative of a total of 5 experiments. Mean increase in [Ca2+]i after addition of 50 µM AA is given in RESULTS.

The maximal increase in [Ca2+]i after addition of AA in Ca2+-free medium is 47 ± 6 nM (mean ± SE, n = 6). When AA (50 µM) is added to Ehrlich cells suspended in Ca2+-containing standard medium, a sustained increase in [Ca2+]i is observed, with no or only a slight decrease within the timescale of these experiments (<7.5 min after addition), indicating that an influx of Ca2+ is likely to be involved. The maximal increase in [Ca2+]i after addition of AA in Ca2+-containing medium is 125 ± 74 (mean ± SE; n = 3). There is, however, no significant difference in the maximal increase in [Ca2+]i after addition of AA in Ca2+-free medium compared with Ca2+-containing medium.

Neither bradykinin (10 µM) nor thrombin (10 U/ml), when added after AA, is able to induce further increases in [Ca2+]i in Ehrlich cells in suspension, as shown for both agonists in Fig. 6 and for thrombin in Fig. 7B. Interestingly, when a lower concentration of AA (10 µM) is applied to Ehrlich cells in Ca2+-free medium (2 mM EGTA), a different pattern is seen. As illustrated in Fig. 7, the lower concentration of AA causes a slow gradual increase in [Ca2+]i, and a normal transient increase in [Ca2+]i can be detected after addition of thrombin (10 U/ml) to the AA-treated cells. The maximal increase in [Ca2+]i after stimulation with thrombin (10 U/ml) in the presence of AA (10 µM) is in Ca2+-free medium (2 mM EGTA; 70 ± 8 nM, mean ± SE, n = 3) and is thus not significantly reduced compared with the value obtained for thrombin as the second agonist in Ca2+-free medium (2 mM EGTA; see Table 1). This could indicate that the inhibition of the thrombin-induced increase in [Ca2+]i in cells pretreated with AA (50 µM) may be due to emptying of a specific intracellular Ca2+ store. It cannot be excluded, however, that the higher concentration of AA could have specific or nonspecific inhibitory effects on other steps in the thrombin signaling pathway (see DISCUSSION).


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Fig. 7.   Effect of AA on thrombin-induced increase in [Ca2+]i in cell suspension experiment. Cells were treated as described for Fig. 6, and [Ca2+]i was followed with time in cells suspended in Ca2+-free medium (2 mM EGTA). AA (10 µM, A; 50 µM, B) and thrombin (10 U/ml) were added as indicated by arrows. Data are from 1 experiment, representative of 3 similar sets of experiments.

We were not able to obtain a concentration-response curve, since the higher concentrations of AA interfere significantly with the fura 2 measurements due to the higher absorbance of AA at 340 nm than at 380 nm (data not shown). It is noteworthy that the concentrations of AA indicated here are the added concentrations, since the solubility of AA, and thus the actual concentration of AA, has not been determined.

AACOCF3 is an analog of AA in which the COOH group of AA is replaced by a COCF3 group. After longer preincubation times (in Ehrlich cells, 155-160 min; see Ref. 46), AACOCF3 is an inhibitor of the cytosolic 85-kDa PLA2 (43). We investigated the effect of AACOCF3 (50 µM) on [Ca2+]i and on the AA-induced increase in [Ca2+]i. In five experiments performed on cells suspended in Ca2+-free medium (2 mM EGTA), AACOCF3 caused no detectable increase in [Ca2+]i (results not shown). AA (50 µM) still caused a normal increase in [Ca2+]i when added after AACOCF3. In the presence of AACOCF3 (50 µM), the maximal increase in [Ca2+]i after addition of AA (50 µM) is 51 ± 13 nM (mean ± SE; n = 5), which is not significantly different from the AA-induced maximal increase in [Ca2+]i in the absence of AACOCF3 (see above). This could indicate that the effect of AA on [Ca2+]i is not due to nonspecific effects of addition of a fatty acid. The cells used in the present investigation had been cultured for a longer period (>50 passages) in mice. New cells (<50 passages) did not show the difference between AA and AACOCF3 (n = 3; data not shown). The presence of AACOCF3 (50 µM), unlike AA, does not prevent increases in [Ca2+]i after subsequent stimulation with the agonists bradykinin or thrombin [3 experiments in Ca2+-free medium (2 mM EGTA) with no AACOCF3-induced increase; data not shown], indicating that the inhibition by AA could be due to effects on Ca2+ stores or to a specific effect on other steps in the agonist-induced signaling pathways.

Effect of Inhibition of PIC on AA-Induced Increase in [Ca2+]i

The maximal increase in [Ca2+]i after addition of AA (50 µM) to Ehrlich cells suspended in Ca2+-free medium (2 mM EGTA) in the presence of the PIC inhibitor U-73122 is 32 ± 8 nM (mean ± SE; n = 3). This is not significantly different from that seen in the presence of the inactive analog U-73343, which is 52 ± 22 nM (mean ± SE; n = 3). Likewise, U-73122 does not significantly inhibit the AA-induced increase in [Ca2+]i compared with controls performed in the absence of U-73343. This could indicate that the effect of AA is not due to activation of the PIC signaling pathway.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bradykinin- and Thrombin-Induced Increases in [Ca2+]i in Ehrlich Cells

Stimulation of single Ehrlich cells with bradykinin or thrombin results in a transient increase in [Ca2+]i (Figs. 1 and 2). This observation is in agreement with earlier findings in Ehrlich cells in suspension, as well as with data from other cell types (see Introduction and below for references). A transient increase in [Ca2+]i is observed also in the absence of extracellular Ca2+, both in single cells and in cell suspensions (Fig. 2, B and C, and Table 1), indicating that bradykinin and thrombin are able to release Ca2+ from intracellular stores. Because stimulation with bradykinin in some experiments or cells causes a sustained phase with elevated [Ca2+]i, sensitive to removal of external Ca2+, this agonist is likely to stimulate an influx of Ca2+. This is supported by the significant reduction of the maximal increase in [Ca2+]i on removal of extracellular Ca2+ (Table 1) and is in agreement with the previous observation that bradykinin could elicit an increase in [Ca2+]i in thapsigargin-treated cells in Ca2+-containing medium (38). In the present study, we find that bradykinin does not cause any detectable increase in [Ca2+]i in thapsigargin-treated Ehrlich cells in Ca2+-free medium (Fig. 4), pointing toward an involvement of Ca2+ entry in the bradykinin response in the presence of thapsigargin and extracellular Ca2+. That stimulation with bradykinin can lead to both release of Ca2+ from intracellular stores and activation of Ca2+ influx has been reported for several mammalian cell types (Refs. 6, 39, 49; see Ref. 19). In a study on human gingival fibroblasts, however, it was reported that the bradykinin-induced increase in [Ca2+]i was independent of extracellular Ca2+ (30). In contrast, the thrombin-induced maximal increase in [Ca2+]i is not reduced in the absence of external Ca2+ (see Table 1), although a sustained phase indicating involvement of Ca2+ entry was observed in some experiments in cell suspensions. This indicates that thrombin primarily causes release of Ca2+ from intracellular stores in Ehrlich cells. In osteosarcoma cells, thrombin causes a G protein-mediated stimulation of Ins(1,4,5)P3 production, as well as an increase in [Ca2+]i that is suggested not to involve influx of Ca2+ (2). Release of Ca2+ from intracellular stores in combination with activation of Ca2+ influx has been reported after stimulation with thrombin in human platelets (9) and in endothelial cell lines (15, 31).

Sequential addition of bradykinin and thrombin leads to sequential increases in [Ca2+]i, both in cell suspensions (Table 1) and in single-cell studies (Fig. 2). The bradykinin-induced maximal increase in [Ca2+]i is reduced by a previous stimulation with thrombin in both Ca2+-containing and Ca2+-free media, whereas the response to thrombin is reduced by a previous stimulation with bradykinin in Ca2+-free medium but not in Ca2+-containing medium. This shows that addition of one agonist does not prevent the response to the other agonist by depleting intracellular Ca2+ stores or inactivating intracellular signaling pathways. The reduction of the response to the second agonist in Ca2+-free medium could, however, be due to partial depletion of intracellular Ca2+ stores in the absence of Ca2+ entry. The reason for the reduced response to bradykinin after a previous stimulation with thrombin in Ca2+-containing medium is not clear.

The increase in [Ca2+]i elicited by addition of bradykinin or thrombin appears to depend on activation of PIC in Ehrlich cells. Stimulation of PIC is well known to cause increases in Ins(1,4,5)P3 and [Ca2+]i. An increase in Ins(1,4,5)P3 after addition of thrombin has previously been demonstrated in Ehrlich cells (41). This is supported by the present data showing that the PIC-inhibitor U-73122 significantly inhibits the bradykinin- and thrombin-induced increases in [Ca2+]i in Ca2+-containing medium (Fig. 3) and completely abolishes the increases in Ca2+-free medium. It should be noted, however, that the inactive structural analog of U-73122, U-73343, has a slight inhibitory effect on the thrombin-induced increases in [Ca2+]i in Ca2+-free medium. Results obtained using U-73122 should be interpreted with caution, due to the reported nonspecific effects of this compound, such as stimulation of release of Ca2+ from internal stores in rabbit pancreatic acinar cells (51), perhaps by inhibition of the internal Ca2+-ATPase, as reported in a study on rat liver microsomes (10). An earlier study on Ehrlich cells in Ca2+-containing medium, however, showed no effect of U-73343 on the bradykinin-induced increase in [Ca2+]i, whereas U-73122 strongly inhibited the response. The leukotriene D4-induced increase in Ins(1,4,5)P3 was likewise inhibited by U-73122 (38). Pretreatment of Ehrlich cells in suspension with ryanodine (100 µM) did not cause any significant inhibition of the bradykinin- or thrombin-induced increase in [Ca2+]i. This finding is in agreement with a study on Ehrlich cells by Gamberucci et al. (13), from which it was concluded that ryanodine receptors are unlikely to be present in this cell type, based on the lack of effect of caffeine and on Western blot analysis of microsomal membranes.

The data presented in this study show that thrombin is able to cause an increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-free medium (2 mM EGTA), even after treatment with thapsigargin (see Fig. 4). This could indicate that stimulation with thrombin, unlike bradykinin, can induce release of Ca2+ from an intracellular store ("thrombin-sensitive store") that is different from the thapsigargin-depleted, "Ins(1,4,5)P3-sensitive" store. It has not been tested directly whether the thapsigargin treatment used in the present study is sufficient to deplete the Ins(1,4,5)P3-sensitive store. However, ATP (S. F. Pedersen, S. Pedersen, N. K. Jørgensen, I. H. Lambert, and E. K. Hoffmann, unpublished observation) and bradykinin are not able to induce any detectable increase in [Ca2+]i after thapsigargin treatment in Ca2+-free medium, and, furthermore, data presented by Gamberucci et al. (13) for 1-10 µM thapsigargin indicate that the thapsigargin-sensitive store corresponds to the Ins(1,4,5)P3-sensitive store in Ehrlich cells. Simultaneous addition of bradykinin and thrombin to Ehrlich cells suspended in Ca2+-free medium (2 mM EGTA) causes an enhanced increase in [Ca2+]i compared with controls to which either agonist alone was added (Fig. 5). This finding could indicate that the two agonists elicit an increase in [Ca2+]i by activation of partly different signaling pathways and/or release Ca2+ from different intracellular stores, supporting the notion that thrombin can release Ca2+ from an intracellular store not "sensitive" to bradykinin. The mechanism(s) behind the differences between bradykinin and thrombin with respect to mobilization of Ca2+ from intracellular stores are currently not clear; however, as mentioned in the introduction, thrombin is known to activate different G proteins as well as a range of signaling pathways including MAPK and nonreceptor tyrosine kinases. Also, thrombin-induced stimulation of phosphoinositol 3-kinase and stimulation of Ras and Ras-related proteins have been reported (see Introduction for references). A possible involvement of protein phosphorylation in regulation of Ca2+-influx as well as regulation of the filling state of intracellular Ca2+ stores has been suggested in a study on rabbit platelets, in which phorbol ester treatment was found to inhibit the thapsigargin-induced increase in [Ca2+]i in Ca2+-free medium (34). Similarly, protein tyrosine phosphorylation was found to play a role in the regulation of Ca2+ entry in human endothelial cells (12). It is thus possible that thrombin could activate intracellular signaling pathways, including changes in phosphorylation patterns, that are not affected by bradykinin.

AA-Induced Increases in [Ca2+]i

Addition of AA can evoke an increase in [Ca2+]i in Ehrlich cells, both in the presence and in the absence of extracellular Ca2+. In Ca2+-containing medium, AA generally induces a sustained elevated level of [Ca2+]i (not illustrated), whereas in Ca2+-free medium an increase in [Ca2+]i followed by a slow downregulation toward the resting level is observed (Figs. 6 and 7). This indicates that part of the AA-induced increase in [Ca2+]i is dependent on Ca2+ entry. A similar response, i.e., a transient increase followed by a sustained phase of elevated [Ca2+]i in Ca2+-containing medium but not in Ca2+-free medium, was seen after AA addition in a study on HL-60 cells (37). As described in the introduction, effects of AA (in concentrations ranging from 1 µM to 1 mM) on Ca2+ signaling have been demonstrated in other cell types (see introduction for references). AA has also been suggested to activate protein tyrosine phosphorylation (5); as described above, this could have effects on both Ca2+ entry and store refilling. The AA-induced increase in [Ca2+]i is not inhibited by U-73122, indicating that the response is not dependent on PIC activation. It is conceivable that AA can cause directly both release of Ca2+ from intracellular stores and Ca2+ entry, but involvement of a metabolite cannot be excluded.

Pretreatment with AA (50 µM), but not with AACOCF3 (not shown), inhibits the bradykinin- and thrombin-induced increase in [Ca2+]i (see Figs. 6 and 7 for thrombin), but a lower concentration of AA (10 µM) does not significantly reduce the response to thrombin (Fig. 7). These data, taken together with the lack of inhibition by AACOCF3, could indicate that AA in Ehrlich cells is able not only to mobilize the Ins(1,4,5,)P3-sensitive intracellular Ca2+ store but also to deplete the thrombin-sensitive store and that this is the mechanism behind the inhibition of the thrombin-induced increase in [Ca2+]i by AA. More direct effects of AA on the thrombin-induced signaling events can, however, not be excluded.

Both bradykinin and thrombin addition can cause formation of AA by activation of PLA2 (see Introduction). It is, however, not likely that such an agonist-induced AA formation would produce concentrations in the range of 50 µM. Furthermore, both the bradykinin- and the thrombin-induced increases in [Ca2+]i are abolished in the presence of U-73122 in Ca2+-free medium (2 mM EGTA), whereas the AA-induced response is unaffected by this inhibitor. This argues against the notion that bradykinin and, more importantly in this context, thrombin could exert their effects by stimulating AA production.

The nature of the thrombin-sensitive store, which is not sensitive to ryanodine or to thapsigargin, is not clear. In this context, it is interesting to note that an intracellular Ca2+ store that is different from the Ins(1,4,5)P3-sensitive store and from the cyclic adenosine diphosphoribose-sensitive store (corresponding to the ryanodine-sensitive store) has been reported in sea urchin eggs. This store was found to be sensitive to nicotinic acid adenine dinucleotide phosphate (7, 29, 14). This again demonstrates the complex nature of intracellular Ca2+ signaling.

In summary, the agonists bradykinin and thrombin induce increases in [Ca2+]i in Ehrlich cells, due to release of Ca2+ from intracellular stores. In addition, bradykinin elicits Ca2+ entry; whether thrombin induces Ca2+ entry is not clear. The response to the agonists is inhibited by inhibition of PIC but is not inhibited by ryanodine. Sequential addition of the agonists, in contrast to addition of the same agonist twice, elicits sequential increases in [Ca2+]i. Bradykinin and thrombin have additive effects on [Ca2+]i in Ca2+-free medium. Pretreatment with thapsigargin in Ca2+-free medium abolishes the increase in [Ca2+]i after addition of bradykinin but not after stimulation with thrombin. AA induces an increase in [Ca2+]i, both in the presence and absence of external Ca2+. The AA-induced Ca2+ response is not sensitive to inhibition of PIC. Pretreatment with high, but not low, concentrations of AA abolishes the agonist-induced increase in [Ca2+]i. Thrombin thus leads to release of Ca2+ from an internal store that is insensitive to bradykinin and is not depleted by thapsigargin but is depleted by AA.

    ACKNOWLEDGEMENTS

We thank Birte J. Hansen for excellent technical assistance.

    FOOTNOTES

This work was supported by the Danish Natural Sciences Research Council.

Address for reprint requests: N. K. Jørgensen, Dept. of Medical Physiology, Panum Institute, Blegdamsvej 3, 12.5, DK-2200 Copenhagen N, Denmark.

Received 30 September 1997; accepted in final form 16 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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