INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

T CELL ACTIVATION and interleukin (IL)-2 production are essential for appropriate functioning of the immune system (1, 2, 35). The activation of T cells is effected primarily via stimulation of T cell antigen receptor (TCR). Two of the TCR polypeptide chains forming a heterodimer are important in recognizing the antigen; the others, collectively called CD3, are involved in receptor assembly and signal transmission (2, 36). The stimulation of TCR, in vivo, results from its interactions with antigen-presenting cells (1, 2, 35, 36). In vitro, TCR stimulation could be achieved with lectins or specific antibodies directed against the CD3 complex (1, 2, 36) and gives rise to a series of intracellular responses eventually leading to T cell IL-2 production (1, 2, 4, 12, 35, 36). The intracellular responses include the activation of protein tyrosine kinases and phospholipase C- (1, 2, 4) with subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol. The IP₃-mediated release of Ca²⁺ from intracellular stores is followed by Ca²⁺ influx through the plasma membrane causing a sustained elevation in intracellular Ca²⁺ concentration ([Ca²⁺]_i; see Refs. 1, 2, 4, 5, 12, 14, 37). The increase in [Ca²⁺]_i sustained for several hours is considered critical for T cell activation and IL-2 production (2, 4, 5, 21, 37).

An increased production and release of PGE₂ after burn and septic injuries has been correlated with a decrease in T cell IL-2 production and proliferation (8, 18, 20, 33). The role of PGE₂ in the suppression of T cell IL-2 production and proliferation is borne out by direct effects of PGE₂ on T cells (6, 7, 24, 26, 28, 34). In previous studies, we showed that PGE₂-mediated suppression of T cell functions is associated with an attenuation in T cell Ca²⁺ signaling as assessed in T cell suspensions (8, 10).

In the present study, we evaluated the effects of PGE₂ on [Ca²⁺]_i responses in individual T cells using the Ca²⁺ imaging technique. These assessments allowed us to ascertain variability in the [Ca²⁺]_i responses within the T cell population. We examined also the effects of PGE₂ on release of Ca²⁺ from intracellular stores and its influx through the plasma membrane. Additionally, we ascertained the effect of intracellular cAMP, the second messenger generated in the action of PGE₂, in the modulation of T cell [Ca²⁺]_i (3, 17, 23, 34).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals and reagents. Male Sprague-Dawley rats (200-225 g) were obtained from Harlan Sprague Dawley (Indianapolis, IN). PGE₂, dibutyryl-cAMP (DBcAMP), EGTA, and concanavalin A (Con A) were purchased from Sigma Chemical (St. Louis, MO). Fura 2-AM was purchased from Molecular Probes (Eugene, Oregon). Thapsigargin was obtained from Calbiochem-Novabiochem International (San Diego, CA). Ficoll-Paque and nylon wool fiber were obtained, respectively, from Pharmacia Sweden and Polysciences (Warrington, PA). All other cell culture reagents were purchased from GIBCO-BRL (Grand Island, NY).

T cell preparation. Anesthetized rats were killed, and their spleens were removed. Spleens were minced to dissociate cells, and suspensions of single cells were prepared in Hanks' balanced salt solution (HBSS). Red blood cells present in these cell suspensions were removed using Ficoll-Paque. For the enrichment of T cells, splenocytes were passed through a nylon wool column preequilibrated with HBSS containing 10 mM HEPES, 5% FCS, and 50 µg/ml gentamicin. After 45-60 min of incubation of the columns with cells at 37°C, T cells were obtained by eluting the columns with 20-25 ml of warm HBSS. Details of the T cell preparation procedure have been reported previously (8, 10, 11).

Measurements of single cell [Ca²⁺]_i using Ca²⁺ imaging. T cell suspensions were loaded with 10 µM fura 2-AM for 1 h at room temperature as described earlier (8, 10). Approximately 100 µl of the cell suspension were placed on a coverslip and examined under a ×40 objective of an inverted microscope (Nikon). The cells were exposed to alternating 340- and 380-nm excitation wavelengths, and emission of fura 2 was collected through a 505-nm band-pass filter. Images were obtained using a cooled-CCD-Camera (Sensys; Photometrics) and image acquisition system (Universal Imaging). Images were corrected for background fluorescence, separated into to ratios, and analyzed using Metafluor software (Universal Imaging; see Ref. 25). [Ca²⁺]_i was estimated by calibrating the imaging system with mixtures of solutions of known Ca²⁺ and fura 2 concentrations (27).

Fluorometric measurements of [Ca²⁺]_i in T cell suspension. Fura 2-loaded cells were transferred to a cuvette, and fluorescence signals were recorded using a Hitachi Spectrophotofluorometer (model F-2000) at excitation wavelengths of 340 and 380 nm and emission at 510 nm. Details of the fluorometric techniques for determining [Ca²⁺]_i have been described elsewhere (8, 10). EGTA (3 mM) was used in some experiments to lower extracellular [Ca²⁺]_i to 150 nM. IP₃-mediated Ca²⁺ release in T cells was carried out after permeabilizing the cells with saponin (150 µg/ml), as described earlier (15). [Ca²⁺]_i were recorded after calibration of the fluorescent signals using Ca²⁺ standard solutions (27). Results were digitized and imported into a statistical analysis program (Statistical package for Social Sciences Software Program, version 2.0; SigmaStat, Chicago, IL) for quantitative analyses. Normalized [Ca²⁺]_i responses shown in some figures were calculated in the following two steps: 1) the lowest basal [Ca²⁺]_i was subtracted from all of the [Ca²⁺]_i values, and 2) the resulting [Ca²⁺]_i values were divided by maximum [Ca²⁺]_i value. This allowed us to hold the maximum [Ca²⁺]_i equal to one and then express remaining [Ca²⁺]_i values as fractions of one. Integrated [Ca²⁺]_i were determined by calculating the area under the [Ca²⁺]_i response curve; the integrated [Ca²⁺]_i values were pooled separately for control and experimental groups and were presented as means ± SE. Unless mentioned, integrated [Ca²⁺]_i in T cells were calculated over the period of 300 s starting from the time of concanavalin A (Con A) stimulation. In the present study, we determined the effects of various Con A concentrations on T cell [Ca²⁺]_i elevation and found T cell stimulation to be maximal at the 100-µg/ml concentration. Therefore, we employed the 100-µg/ml concentration in this series of experiments. We used 10 µM PGE₂ in all PGE₂-related experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

[Ca²⁺]_i images in individual T cells before and ~150 s after their stimulation with the mitogen (Con A) are shown in Fig. 1. In unstimulated conditions, almost all cells appeared blue, a color corresponding to 20-60 nM [Ca²⁺]_i as shown on the pseudocolor scale bar (Fig. 1A). Similarly, unstimulated T cells incubated with PGE₂ also exhibited a color that corresponds to 20-60 nM [Ca²⁺]_i (Fig. 1C). Addition of Con A caused an increase in [Ca²⁺]_i to different levels in different cells. Such [Ca²⁺]_i increases ranged from 90 to 200 nM (Fig. 1B). The stimulation of cells with Con A after PGE₂ treatment failed to cause an increase in [Ca²⁺]_i (Fig. 1D). As can be seen from Fig. 2A, 85 ± 5.2% of unstimulated T cells had [Ca²⁺]_i 50 nM, which is likely the basal [Ca²⁺]_i. After stimulation with Con A, 40 ± 8.7% of the cells maintained [Ca²⁺]_i 50 nM, suggesting that the remaining ~60% of the basal state cells were activated by Con A to attain [Ca²⁺]_i 50 nM. Con A apparently caused increases in the number of cells with [Ca²⁺]_i at 100, 150, and 200 nM. PGE₂ did not have a demonstrable effect on [Ca²⁺]_i distribution before stimulation of cells with Con A (Fig. 2B). In the PGE₂-treated group, [Ca²⁺]_i was 50 nM in 88 ± 0.9% of the unstimulated cells and 72 ± 9% of Con A-stimulated cells, suggesting that Con A caused increases in [Ca²⁺]_i in only ~16% of the PGE₂-treated cells. This level of stimulation by Con A is apparently less than that observed in the untreated T cells.

View larger version (80K): [in this window] [in a new window]   Fig. 1.   Pseudocolor representation of intracellular Ca2+ concentration ([Ca2+]i) level in fura 2-loaded T cells. A: T cells without concanavalin A (Con A) stimulation. B: T cells ~150 s after Con A stimulation. C: PGE2-treated T cells without stimulation. D: PGE2-treated T cells ~150 s after Con A stimulation. Treated T cells were incubated with 10 µM PGE2 for ~60 min before [Ca2+]i measurements.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Distribution of [Ca2+]i in T cells before and after stimulation with Con A. A: T cells without PGE2 treatment. B: T cells with PGE2 treatment. T cells from each of 5 rats were divided into 2 batches; cells of 1 batch were incubated with PGE2 (see legend to Fig. 1). Approximately 200 cells from 1 batch were first imaged for [Ca2+]i before they were stimulated with Con A and then were reimaged after Con A. A similar number of cells from the batch treated with PGE2 were also imaged before and after Con A. Image analyses yielded numbers of cells with a given [Ca2+]i that ranged from 20 to 250 nM. The number of cells with a given [Ca2+]i was determined in the cell population from each rat before and ~150 s after stimulation with Con A. For the percentage determination, a scatter graph (y-axis showing [Ca2+]i for each of the ~200 cells, nos. 1-200, represented on the x-axis) of [Ca2+]i in these cells was generated using imaging software (Metamorph; UIC, Westchester, PA). Scatter graph was analyzed visually by counting cells in each of the several [Ca2+]i ranges of 25-50 nM, 51-100 nM, 101-150 nM, 151-200 nM, and 201-250 nM and determining percentage of cells in each concentration range (of the total ~200 cells). Percentage of cells in the various concentration ranges was again determined after exposing these cells to Con A. Similarly, [Ca2+]i were determined in the same 200 cells 150 s after Con A stimulation, and [Ca2+]i values in different categories were grouped as described above. The percentage of cells in a category was determined by calculating number of cells in that category per total number of T cells analyzed. Percent responsiveness in a population was determined by calculating the difference in the responses observed before and after stimulation of T cells with Con A. Values shown represent means ± SE from 5 rats.

The loss of [Ca²⁺]_i response to mitogen in PGE₂-treated T cells could result from a decrease either in Ca²⁺ release from intracellular stores, from a decrease in Ca²⁺ influx, or both. We first determined the kinetics and relative contributions of these two mechanisms in T cell suspensions without added PGE₂. Without the exposure of cells to EGTA, the stimulation by Con A resulted in a rapid increase in [Ca²⁺]_i followed by a slow recovery (over an ~200-s duration) that was incomplete and then by a second slowly rising phase of the [Ca²⁺]_i (Fig. 3A). In the presence of 3 mM EGTA, the kinetics and the magnitude of the initial rise in [Ca²⁺]_i were not different from those observed in the absence of EGTA. However, the subsequent decline in [Ca²⁺]_i was more rapid and was not followed by the second rise in [Ca²⁺]_i (Fig. 3B). The second rise in [Ca²⁺]_i in the absence of EGTA was presumably due to Ca²⁺ influx (Fig. 3C). We compared the areas under the [Ca²⁺]_i curves to estimate integrated [Ca²⁺]_i responses (over the period of 300 s starting from time of Con A addition to the cells) in the absence and presence of EGTA. Con A caused an increase in integrated [Ca²⁺]_i (3.51 ± 0.22 × 10⁴ nM/s, mean ± SE from 8 different animals) in the absence of EGTA that was significantly higher (P < 0.05) than that obtained in the presence of EGTA (1.07 ± 0.05 × 10⁴ nM/s). The difference between the two [Ca²⁺]_i responses (~2.44 × 10⁴ nM/s) probably represented the magnitude of Ca²⁺ influx from the extracellular space in cells stimulated with Con A. The integrated responses indicate that, during the initial 300 s of Con A simulation of control T cells, ~40% of the total [Ca²⁺]_i originated from the intracellular stores and ~60% originated from the extracellular pool. These data showed also that the Con A-mediated initial rapid rise in [Ca²⁺]_i in control T cells was primarily due to release of Ca²⁺ from the intracellular stores and that Ca²⁺ influx was initiated after peak [Ca²⁺]_i was achieved. This finding provided us with the rationale for examining the effect of PGE₂ on Ca²⁺ influx by applying it to T cells at the time of peak [Ca²⁺]_i response to the mitogen. PGE₂ addition at the peak [Ca²⁺]_i (attained at ~100 s after Con A stimulation) increased the rate of the subsequent decline in [Ca²⁺]_i relative to that observed in the absence of PGE₂ (Fig. 4). Furthermore, we found that [Ca²⁺]_i elevation in the presence of PGE₂ returned to the basal level in ~300 s, whereas, in the presence of 3 mM EGTA, this took ~200 s. Because the rate of [Ca²⁺]_i decline with PGE₂ was still slower than that which occurred in the presence of EGTA, it can be surmised that PGE₂ causes a net decrease in cytosolic [Ca²⁺]_i accumulation by decreasing Ca²⁺ influx.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Representative traces from spectrophotofluorometric assessments of Con A-induced [Ca2+]i in T cells in the presence and absence of 3 mM EGTA. A: [Ca2+]i before and after stimulation of T cells with Con A. B: Con A-induced [Ca2+]i response in T cells in the presence of 3 mM EGTA. C: composite of normalized [Ca2+]i responses in A and B. Details of normalized [Ca2+]i are given in EXPERIMENTAL PROCEDURES.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Representative traces from spectrophotofluorometric assessments showing the effects of PGE2 or EGTA on the Con A-mediated [Ca2+]i response in T cells. Traces are typical of results obtained from cells of 8 different animals. Normalized [Ca2+]i response was calculated as described in EXPERIMENTAL PROCEDURES.

We also examined the effects of the second messenger of PGE₂, cAMP, by applying DBcAMP to T cells at the peak [Ca²⁺]_i response to Con A. Like PGE₂, DBcAMP augmented the rate of decline of [Ca²⁺]_i. The [Ca²⁺]_i decline to basal level in the presence of DBcAMP occurred over a time period comparable to that observed with PGE₂ (~540 s after Con A). To compare the effects of PGE₂ with that of DBcAMP on Ca²⁺ influx, we integrated the area under the [Ca²⁺]_i response curve from 340 s after Con A addition to the time of return of [Ca²⁺]_i to near basal levels, a time interval during which the [Ca²⁺]_i elevation was presumably due to influx alone (Fig. 3C). The average integrated [Ca²⁺]_i response values obtained from T cells of eight different animals are shown in Fig. 5. In the absence of PGE₂ or DBcAMP, the integrated [Ca²⁺]_i in T cells after their stimulation with Con A was found to be 2.15 ± 0.35 × 10⁴ nM/s. This [Ca²⁺]_i response was significantly (P < 0.01) suppressed when the cells were treated with PGE₂ or DBcAMP after Con A stimulation.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of PGE2 (10 µM) and dibutyryl-cAMP (DBcAMP, 1 mM) on Con A-induced T cell integrated [Ca2+]i response during the time interval that presumably allowed Ca2+ influx, for the most part. The time period selected (~340-540 s after the addition of Con A) began at the presumed end of the release of intracellular Ca2+ and ended at the return of [Ca2+]i response to near basal levels. Integrated [Ca2+]i were estimated as described in EXPERIMENTAL PROCEDURES. Data (means ± SE) represent measurements in T cells from 8 different animals. * P < 0.01, Con A vs. Con A + PGE2 or Con A + DBcAMP. ** P < 0.05, Con A + PGE2 vs. Con A + DBcAMP by ANOVA.

A more definitive PGE₂ effect on Ca²⁺ influx is indicated by data in Fig. 6. Thapsigargin, which presumably inhibited Ca²⁺ uptake into the intracellular reservoir and caused depletion of the reservoir, allowed for the monitoring of Ca²⁺ influx after CaCl₂ was added to the extracellular compartment. Figure 6 shows a cessation of the Ca²⁺ influx with the reintroduction of additional quantities of EGTA. PGE₂ addition after CaCl₂ was evidently effective in attenuating the influx of Ca²⁺.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Representative traces from spectrophotofluorometric assessments showing the effects of PGE2 (10 µM) on Ca2+ entry in T cells when there was no refilling of the intracellular stores. Thapsigargin (100 nM) was used to inhibit Ca2+-ATPase pump. EGTA (3 mM) and CaCl2 (3 mM) were used, respectively, to chelate and replenish Ca2+ in the medium. Traces are typical of T cells from 5 different animals.

To assess the potential effect of PGE₂ on intracellular Ca²⁺ release, 10 µM PGE₂ was first added to T cells followed by the addition of 3 mM EGTA 100 s later. Con A was then added when a stable T cell [Ca²⁺]_i was established, ~100 s after EGTA. T cell [Ca²⁺]_i changes after these additions are shown in Fig. 7, A and B. In these experiments, we noticed a higher basal [Ca²⁺]_i in T cells (Fig. 7, A and B). The [Ca²⁺]_i was reduced to ~70 nM after adding 3 mM EGTA. As shown in Fig. 7, Con A-induced Ca²⁺ release in the presence of EGTA and PGE₂ (0.94 ± 0.16 × 10⁴ nM/s, mean integrated [Ca²⁺]_i ± SE values obtained from 6 different animals) was not significantly different from that observed in T cells exposed to EGTA alone (0.95 ± 0.15 × 10⁴ nM/s, mean integrated [Ca²⁺]_i ± SE values obtained from 6 different animals). In some experiments, we used IP₃ to directly stimulate the IP₃-sensitive Ca²⁺ reservoirs in T cells with or without pretreatment with PGE₂. Cells were first permeabilized with saponin (150 µg/ml) for ~40-60 min and then were stimulated with IP₃ after their exposure to PGE₂. After establishing a stable [Ca²⁺]_i with 3 mM EGTA, addition of 50 µM IP₃ in the absence of PGE₂ induced an elevation in [Ca²⁺]_i from ~60 to ~140 nM (Fig. 7C). The IP₃-induced Ca²⁺ release in the presence of PGE₂ did not appear to be different from that in its absence (Fig. 7D). These experiments suggested that Con A stimulation of T cells ~100 s after their exposure to PGE₂ did not have a significant effect on intracellular Ca²⁺ release. Furthermore, T cell release of Ca²⁺ by IP₃ was also not affected when PGE₂ was added to T cells 100 s before IP₃. These results are in contrast to our previous findings of the effect of 2 h of exposure of T cells to PGE₂ (10). In the previous studies, we showed a suppression in the initial rise in [Ca²]_i elevation in T cells preincubated with PGE₂ for 2 h. As we found in this study, the initial rise is primarily due to Ca²⁺ release from intracellular stores. A suppression in the initial Ca²⁺ rise in T cells incubated with PGE₂ for 2 h could also result from a decrease in Ca²⁺ release from intracellular stores. To elucidate the effects of long-term exposure (~2 h pretreatment) of PGE₂ on T cell intracellular Ca²⁺ release, we have now examined Ca²⁺ release in the presence of EGTA (Fig. 8). We found that Con A-mediated [Ca²⁺]_i elevation in the presence of 3 mM EGTA was significantly (P < 0.01) suppressed in the T cells preincubated with PGE₂ (0.81 ± 0.05 × 10⁴ nM/s, mean integrated [Ca²⁺]_i ± SE values obtained from 6 different animals) compared with T cells incubated without PGE₂ (1.21 ± 0.08 × 10⁴ nM/s, mean integrated [Ca²⁺]_i ± SE values obtained from 6 different animals). These data indicated that a prolonged incubation of T cells with PGE₂ led to a decrease in both Ca²⁺ influx and release from intracellular stores.

View larger version (1716K): [in this window] [in a new window]   Fig. 7.   Representative traces from spectrophotofluorometric assessments showing the effects of PGE2 on Ca2+ release from intracellular reservoir after stimulation with Con A (A and B) or inositol 1,4,5-trisphosphate (IP3; C and D). For IP3-mediated release, T cells were permeabilized by incubating with saponin (150 µg/ml) for 30-45 min at room temperature. Traces are typical of results obtained from cells of 6 different animals.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Representative traces from spectrophotofluorometric assessments showing effects of PGE2 on Con A-mediated [Ca2+]i elevation in T cells in the presence of 3 mM EGTA. T cells were incubated with and without PGE2 (10 µM) for 2 h before [Ca2+]i measurements at 37°C. Traces are typical of results obtained from cells of 6 different animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role of PGE₂ as an immunosuppressant has been amply established (8, 18, 20, 33). Specifically, PGE₂ is known to suppress T cell IL-2 production and proliferation (6, 7, 8, 10, 18, 20, 23, 24, 34). A number of studies have shown burn/sepsis injury-related alterations in T cell IL-2 transcriptional regulation (6, 24, 28). Previous studies from our laboratory have shown an impairment in T cell early signaling events, including intracellular Ca²⁺ mobilization in sepsis (8, 9). We showed also that the treatment of septic animals with the PGE₂ blocker indomethacin prevented the sepsis-related suppression in T cell Ca²⁺ signaling and the proliferative responses (8, 10). Similar changes in Ca²⁺ signaling were implicated in trauma-induced T cell functional disturbances (20). On the other hand, Faist et al. (16) suggested a primary role of protein kinase C (PKC) in trauma-related suppression of T cell proliferation. The role of PKC in PGE₂-mediated T cell proliferative disturbances was also supported by the studies of Chouaib et al. (7). They suggested that, although PGE₂ affected both Ca²⁺ mobilization and PKC activation, the restitution of Ca²⁺ signal partially restored T cell proliferation, whereas PKC activation via tetradecanoylphorbol 13-acetate (TPA) completely restored the T cell response. This is understandable because, although the activation of some PKC isoforms is dependent on Ca²⁺, certain other isoforms are independent of Ca²⁺. TPA stimulation would be expected to have near-maximal stimulation of both Ca²⁺-dependent and -independent PKC, leading to an abrogation of the PGE₂-mediated suppression. Stimulation of T cells with mitogen or antigen results in a cascade of intracellular events, including elevation in [Ca²⁺]_i and PKC activation (1, 2, 4, 33). Recent studies have suggested that the sustained elevation in [Ca²⁺]_i for several hours is critical for T cell IL-2 production and subsequent proliferation (2, 4, 21, 37).

Recent studies have shown that anti-CD3 or mitogen stimulation of T cells induced Ca²⁺ signals of diverse magnitudes in a population of T cells and that only 20-30% of the cells responded to such stimuli (19). These findings would imply that effects of PGE₂ on T cell Ca²⁺ signaling are likely mediated through the action of PGE₂ action on the 20-30% anti-CD3 or mitogen-sensitive cell population. Our previous assessments of Ca²⁺ signaling in T cell suspensions had also indicated variations in responsiveness within the T cell population. In the present study, Ca²⁺ imaging in individual T cells indicated that the addition of Con A to control T cells elevated [Ca²⁺]_i in ~50-60% of the cells present in the microscopic field. The difference in our finding and the earlier study indicating 20-30% responsive cells could be due to the different sources of the T cells studied. The mitogen-responsive control rat T cell population was comprised of ~43% cells showing a 2-fold increase in their [Ca²⁺]_i and 15-20% cells with a 5- to 10-fold increase. Clearly, PGE₂ suppressed [Ca²⁺]_i in all responsive T cells, regardless of the level of their responsiveness to the mitogen. Based on the color distribution in unstimulated T cells, there seems to be a tendency of decrease in the basal [Ca²⁺]_i in PGE₂-treated T cells (Fig. 1C) compared with unstimulated T cells incubated in the absence of PGE₂ (Fig. 1A). However, this decrease was not demonstrable when the data were pooled from five different animals (Fig. 2).

Previous studies suggested that the PGE₂-related decrease in T cell Ca²⁺ signaling could be due to a PGE₂-mediated inhibition of PIP₂ hydrolysis and the subsequent IP₃ production (3, 7, 17, 21-23). These studies implied that the decrease in IP₃ production could lead to a decreased release of Ca²⁺ from the intracellular reservoir. However, Gouy et al. (17) found that, despite a decrease in IP₃ production occurring after treatment of T cells with cholera toxin, there was no change in Ca²⁺ release from the intracellular reservoir. Our present study demonstrated a decrease in Ca²⁺ release from intracellular stores when T cells were preincubated with PGE₂ for 2 h, although the treatment of T cells with PGE₂ immediately before their stimulation with Con A or at peak [Ca²⁺]_i response to Con A failed to affect the capability of intracellular stores to release Ca²⁺ or the sensitivity of the release mechanism to IP₃. These findings suggest that, although prolonged exposure of T cells to PGE₂ (2 h) before cross-linking of the TCR with Con A could attenuate Con A-mediated formation of IP₃, such IP₃ generation may not be compromised immediately after PGE₂ application to T cells. We speculate that the trigger for Ca²⁺ release from intracellular stores may be affected only after the prolonged PGE₂ exposure.

Previous studies have shown that, although Ca²⁺ influx is important in the maintenance of the sustained elevation of T cell [Ca²⁺]_i (2, 5, 14, 21, 37), all of the Ca²⁺ entering the cell may not contribute to the [Ca²⁺]_i elevation. Some of the Ca²⁺ are presumably either extruded via a Ca²⁺ efflux mechanism or pumped into the intracellular Ca²⁺ stores (4, 14, 29). An alteration in any of these mechanisms could also affect the sustained Ca²⁺ elevation. As pointed out in RESULTS, the influx of Ca²⁺ was observed (Fig. 6) in T cell depleted of the intracellular Ca²⁺ store. The influx of Ca²⁺ in store-depleted cells represents a store-dependent influx pathway referred to as the "capacitive Ca²⁺ entry" (31, 32). A number of recent studies suggested that capacitive Ca²⁺ entry in various cell systems, including the T cells, is activated by the depletion of the Ca²⁺ store and is terminated by subsequent refilling (13, 29, 30, 38). We evaluated the effects of PGE₂ on Ca²⁺ entry in T cells after their exposure to thapsigargin in the presence of 3 mM EGTA. In the presence of EGTA, thapsigargin allowed for the depletion of the Ca²⁺ stores and prevented their refilling. The results in Fig. 6 showing the elevation in [Ca²⁺]_i after restitution of Ca²⁺ in the extracellular medium suggest capacitive Ca²⁺ entry in rat T cells. These studies are consistent with earlier observations in human transformed Jurkat T cells and other cell systems (13, 31). The elevation in T cell [Ca²⁺]_i in the presence of thapsigargin was presumably due to entry from extracellular medium, as reintroduction of EGTA led to return of [Ca²⁺]_i to basal levels. Similar to EGTA, addition of PGE₂ also produced a marked suppression in [Ca²⁺]_i elevation. These results supported the hypothesis that the PGE₂-mediated decrease in sustained elevation could also result from an attenuation in a capacitive Ca²⁺ entry. The decrease in [Ca²⁺]_i elevation due to a decrease in Ca²⁺ influx and its release from intracellular stores is likely a major contributor to PGE₂-mediated suppression of T cell responses.