©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ca-mobilizing Actions of a Jurkat Cell Extract on Mammalian Cells and Xenopus laevis Oocytes (*)

(Received for publication, December 22, 1994; and in revised form, January 24, 1995)

Patrick Gilon (§) Gary St. J. Bird Xiaopeng Bian Jerry L. Yakel James W. Putney Jr. (¶)

From the Calcium Regulation Section, Laboratory of Cellular and Molecular Pharmacology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Randriamampita and Tsien (Randriamampita, C., and Tsien, R. Y.(1993) Nature 364, 809-814) suggested that an acid-extracted fraction from a Jurkat cell line contains a messenger responsible for the coupling of calcium entry to the depletion of intracellular stores, i.e. capacitative calcium entry. We found that the extract, prepared as described by Randriamampita and Tsien, caused Ca entry in 1321N1 astrocytoma cells which was not blocked by the D-myo-1,4,5-trisphosphate-receptor antagonist, heparin. In contrast to astrocytoma cells, when applied to mouse lacrimal acinar cells and rat hepatocytes the Jurkat extract always caused the release of intracellular Ca, followed by Ca entry across the plasma membrane. This activity of the extract on lacrimal cells was blocked by either intracellular injection of heparin or extracellular atropine. Similarly prepared lacrimal cell extracts gave Ca responses when applied to astrocytoma cells or lacrimal cells which were similar to those for Jurkat-derived extract. However, extracts from hepatocytes had no effect. In most Xenopus oocytes, the Jurkat extract had no effect, while in a few oocytes, the extract gave a [Ca] response similar to that seen in lacrimal cells, that is, release of Ca followed by Ca entry. We conclude that the actions of the Jurkat cell extract are not consistent with its containing the long sought messenger for capacitative calcium entry. It is likely that this fraction contains a number of factors that mediate Ca response in different cell types, possibly through receptor-mediated mechanisms.


INTRODUCTION

Activation of surface membrane receptors in many cell types results in a complex, biphasic Ca response composed of an initial mobilization of internally stored Ca, followed by entry of extracellular Ca. An early event following receptor activation is the well characterized generation of the putative second messenger, D-myo-inositol 1,4,5-trisphosphate ((1,4,5)IP)(3), (^1)which is responsible for the mobilization of internally stored Ca(1) . Considerable evidence suggests that depletion of the agonist- and IP(3)-sensitive intracellular Ca-pool then leads to the activation of the secondary Ca entry phase of the response, a process which has been termed capacitative calcium entry(2, 3) . Although the nature of the linkage between the depletion of internal Ca pools and the entry of Ca is unclear, recent reports have suggested that internal pool depletion leads to the generation of a soluble messenger that signals the activation of the entry process(4, 5) . In addition, there is evidence for a GTP-dependent step in regulating this entry process(6, 7) . Recently, Randriamampita and Tsien (5) reported the isolation of an acid extracted fraction from Jurkat T-cells that contained a factor that could specifically activate capacitative Ca entry, a factor which they termed CIF (calcium influx factor).

In the studies reported here, we have isolated an acid-extracted fraction from Jurkat T-cells and other mammalian cell types, and examined the effects of these extracts on calcium signaling in various mammalian cells, including astrocytoma cells, and in Xenopus laevis oocytes. The activity of the Jurkat extract on astrocytoma cells was similar to that described for CIF, confirming observations of Randriamampita and Tsien. However, the effects of the Jurkat extract on other cell types and the effects of extracts isolated from cells other than Jurkat cells are not consistent with its purported role as a mediator of capacitative calcium entry. Rather, our findings suggest that such crude extracts contain a number of factors that mediate Ca response in different cell types, possibly through receptor-mediated mechanisms.


MATERIALS AND METHODS

Jurkat Cell Culture and Extraction

Cells of the T-lymphocyte line, Jurkat, were grown in suspension at 37 °C in 5% CO(2), in RPMI 1640 medium supplemented with glutamine (2 mM), 10% fetal bovine serum, and gentamicin (50 mg/µl). The cells were passed at dilution of 1:15 once or twice a week to maintain maximal density of 2-3 times 10^6 cells/ml.

Following the procedures described by Randriamampita and Tsien(5) , we isolated an acid-extracted fraction from the Jurkat cells. The cells, 0.4-0.6 ml packed cell volume, were washed in nominally Ca-free HPSS. After centrifugation (180 times g, 4 min), the cell pellet was resuspended in 700 µl of the same HPSS buffer, acidified to pH 1 with approximately 150 µl of 1 M HCl, and kept on ice for 20 min. The suspension was then centrifuged (180 times g for 10 min), and the supernatant was retained and restored to pH 7.3 with 1 M NaOH. The final volume of the extract at this stage was approximately 1 ml. When necessary, the extract was further incubated for 20 min with 2 units/ml hexokinase (Sigma) to remove ATP, thereby avoiding any possible effects through activation of purinergic receptors. In the experiments described below, 200 µl of the Jurkat extract plus 400 µl of HPSS (final [Ca] was either 1.8 mM or nominally Ca-free) were added to the cells tested.

One modification to this procedure was in the preparation of an extract for oocytes. The procedure was essentially as described above, except that the initial cell pellet was brought up in water to minimize the osmolality of the extract. With this procedure, the osmolality of the extract was approximately 275 mOsM and retained Ca-mobilizing activity when assayed on astrocytoma cells. The extract was diluted to an osmolality of 200 mOsM before being applied to oocytes.

The distribution of calcium signaling activity between soluble and particulate fractions of stimulated and nonstimulated Jurkat cells was determined as described by Randriamampita and Tsien(5) . Packed cells (0.4-0.6 ml) were resuspended in 60 mls of nominally Ca-free HPSS supplemented with 0.5 mM EGTA. The cells were then treated with or without phytohemagglutinin (PHA; 20 µg/ml) for 10 min. After stimulation, the suspension was centrifuged (180 times g, 4 min), and the pellet was resuspended in 700 µl of nominally Ca-free HPSS containing saponin (2000 µg/ml) and incubated for 5 min to permeabilize the plasma membrane. Permeabilization was monitored by trypan blue dye exclusion. The suspension was centrifuged (180 times g for 10 min) and the supernatant retained, while the pellet was resuspended in an equal volume of nominally Ca-free HPSS. Both fractions were acidified to pH 1 with 1 M HCl and kept on ice for 20 min. Each fraction was then centrifuged, and the supernatants were passed through a reverse-phase octadecyl silica cartridge (SPICE C18 cartridge, Rainin) to remove saponin. The extracts were neutralized, and ATP was removed with hexokinase treatment, as described above. The fraction derived from the supernatant after saponin treatment was considered the soluble fraction, and the fraction from the resulting pellet was the particulate fraction.

1321N1 Astrocytoma Cell Culture

1321N1 Astrocytoma cells were maintained at 37 °C under 5% CO(2) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 5 mM glutamine. After 3 days of culture, the cells were passed at a dilution of 1:2. For Ca measurements on astrocytoma cells, the cells were plated on glass coverslips at least 2 days before use. The cells were then loaded with fura-2 as described below, except that the incubation was with 1 mM fura-2/AM for 30 min at 37 °C.

Lacrimal Cell Isolation

Mouse lacrimal acinar cells were prepared essentially as described by Parod et al.(8) . Briefly, the excised glands from 5 mice (male CD-1; 30-40 g) were finely minced and treated for 1 min with 0.25 mg/10 ml trypsin (Sigma). The trypsin was then removed by centrifugation, followed by a 5-min incubation of the tissue fragments with 2 mg/10 ml soybean trypsin inhibitor (Sigma), in the presence of 2.5 mM EGTA. Finally, the acinar cells were isolated after treating the tissue with 4 mg/10 ml collagenase (Boehringer Mannheim) for 10 min. Viability of the isolated cells was >95% based on trypan blue exclusion. All enzyme solutions were prepared in DMEM. Following isolation, the acinar cells were washed and resuspended in sterile DMEM containing 10% fetal bovine serum, 5 mM glutamine, 50 units/ml penicillin, and 50 units/ml streptomycin. The cells were then allowed to attach to glass coverslips coated with Matrigel (Collaborative Biomedical Products, Bedford, MA). Acinar cells were incubated on the glass coverslips for at least 3 h before use.

Fura-2 Loading

The attached cells were mounted in a Teflon chamber (Bionique Testing Labs., Inc., Saranac Lake, NY) and incubated with 0.5 µM fura-2/AM (Molecular Probes, Inc., Eugene, OR) for 30 min at room temperature. The cells were then washed and bathed in HPSS at room temperature for at least 30 min before Ca measurements were made.

Fluorescence Measurements

The fluorescence of the fura-2-loaded cells was monitored with a photomultiplier-based system, mounted on a Nikon Diaphot microscope equipped with a Nikon 40times (1.3 NA) Neofluor objective. The fluorescence light source was provided by a Deltascan D101 (Photon Technol. Intl., Monmouth Junction, NJ), equipped with a light path chopper and dual excitation monochromators. The light path chopper enabled rapid interchange between two excitation wavelengths (340 and 380 nm), and a photomultiplier tube monitored the emission fluorescence at 510 nm, selected by a barrier filter (Omega Optical Inc., Brattleboro, VT). All experiments were carried out at 24 °C. Calibration and calculation of [Ca](i) were carried out as described previously(9) .

Cell Microinjection

Mouse lacrimal cells were microinjected essentially as described before(9) . An injection medium (IM) consisting of 27 mM K(2)HPO(4), 8 mM Na(2)HPO(4), 26 mM KH(2)PO(4), pH 7.2, and 2 mM fura-2 (acid) was pressure injected into cells via a glass micropipette attached to a WPI PV830 Picopump (World Precision Instruments, New Haven, CT). Prior to microinjection, lacrimal cells were loaded with fura-2 by incubation with fura-2/AM so that [Ca](i) levels could be monitored prior to and during the microinjection procedure.

X. laevis Oocyte Preparation, Membrane Current, and Fluorescence Measurement

X. laevis oocytes (stages V-VI) were treated with collagenase (collagenase type B; Boehringer Mannheim, 3 mg/ml) in a Ca-free OR-2 solution (NaCl 82.5 mM, KCl 2 mM, MgCl(2) 1 mM, HEPES 5 mM, pH 7.55) for 2 h. After washing in ND96 (NaCl 96 mM, KCl 2 mM, CaCl(2) 1.8 mM, MgCl(2) 1 mM, HEPES 5 mM, pH 7.55) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, and 50 µg/ml gentamicin, they were maintained for up to 7 days in this solution. All of the experiments were performed at 22 °C in either nominally Ca-free ND96 or in ND96 containing 5 mM CaCl(2). In some experiments, 50 nl of the Jurkat extract (water preparation, no dilution) or oocyte intracellular medium (OIM: KH(2)PO(4) 1 mM, KCL 125 mM, NaCl 10 mM, NaHCO(3) 2 mM, pH 7.3) containing(2, 4, 5) IP(3) (Life Technologies, Inc.) were injected via a glass micropipette attached to a WPI Nanopump A140 (World Precision Instruments, New Haven, CT) or a Drummond Nanoinjector (Drummond Scientific Co., Broomall, PA).

For [Ca](i) measurements, the oocytes were injected with 50 nl of OIM containing 2 mM fura-2 free acid (Molecular Probes) 30 min prior to the beginning of the experiment. The fluorescence of the fura-2-loaded cells was monitored as described except that a Nikon 20times Neofluor objective was used.

For membrane current measurements, two electrodes (filled with 3 M KCl, resistance leq 1 megaohms) were inserted into oocytes and connected to a voltage-clamp amplifier (GeneClamp 500, Axon Instruments, Foster City, CA). The membrane potential was held at -60 mV, and recordings were sampled at 5 Hz and low pass filtered at 100 Hz.

All figures show either means ± S.E. or representative results from at least three independent experiments.


RESULTS

Effects of Jurkat Cell Extracts on 1321N1 Astrocytoma Cells

In fura-2 loaded astrocytoma cells, both the muscarinic agonist, acetyl-methacholine (MeCh), and the intracellular Ca-ATPase inhibitor, thapsigargin, elevate intracellular Ca in a similar fashion. In the absence of extracellular Ca, both agents cause a transient release of intracellular Ca, followed by a sustained elevated intracellular Ca on restoring extracellular [Ca] to 1.8 mM (Fig. 1, a and b). The addition of the Jurkat extract to astrocytoma cells also results in an elevation of intracellular Ca, usually accompanied by irregular oscillations (Fig. 1c), but 5/34 of the cells observed displayed a transient release of intracellular Ca in the absence of extracellular Ca (data not shown). Thus, in confirmation of the observations of Randriamampita and Tsien(5) , in astrocytoma cells the Jurkat extract appears mainly to act by stimulating Ca entry. To determine whether the effects of the extract might be mediated through the actions of(1, 4, 5) IP(3), we applied the extract to astrocytoma cells injected with the (1, 4, 5) IP(3) receptor antagonist, heparin. As shown in Fig. 2, although heparin-injected astrocytoma cells failed to respond to MeCh, the same astrocytoma cell was still able to respond to the Jurkat extract.


Figure 1: Ca responses in 1321N1-astrocytoma cells. Single, fura-2-loaded astrocytoma cells were incubated in the absence of extracellular Ca, and then treated with a, 100 µM MeCh; b, 2 mM thapsigargin; or c, Jurkat extract. Extracellular Ca (1.8 mM) was restored to the cell at the time indicated. Typically, astrocytoma cells responded to both MeCh (15 cells) and thapsigargin (11 cells) with mobilization of intracellular Ca and a sustained elevation of cytoplasmic Ca on restoring extracellular Ca to 1.8 mM. The Jurkat extract mainly caused Ca entry in 1321N1 astrocytoma cells when extracellular Ca was restored (29/34 cells), but 5/34 of the cells responded with an initial release of Ca from internal stores in the absence of extracellular Ca (not shown).




Figure 2: The(1, 4, 5) IP(3)-receptor antagonist, heparin, blocks MeCh but not Jurkat extract responses in 1321N1 astrocytoma cells. Single, fura-2 loaded astrocytoma cells were microinjected with heparin (200 mg/ml, pipette concentration) as described under ``Materials and Methods.'' The astrocytoma cell was first exposed to a, 100 mM MeCh, and then to b, Jurkat extract. Whereas the actions of the muscarinic agonist MeCh were blocked, Jurkat extract-activated Ca entry in astrocytoma cells was not affected by intracellular application of the(1, 4, 5) IP(3)-receptor antagonist, heparin. The data are representative traces from three similar experiments.



By using the responsiveness of astrocytoma cells as an assay, we evaluated the subcellular distribution in Jurkat cells of this calcium entry stimulating activity. Following the method described by Randriamampita and Tsien(5) , the soluble fraction was derived from the supernatant following the centrifugation of saponin treated Jurkat cells (see ``Materials and Methods''), and the remaining pellet yielded the particulate fraction. As shown in Fig. 3, in nonstimulated Jurkat cells, the calcium entry stimulating activity appeared exclusively in the particulate fraction. Upon stimulating the cells with PHA (20 µg/ml), activity appeared in the soluble fraction (Fig. 3), although with little apparent loss of activity from the organelle fraction. Although this redistribution of activity is qualitatively consistent with the data of Randriamampita and Tsien (5) , these authors reported a quantitative redistribution of activity from a particulate to a soluble fraction which we were never able to achieve. Thus, the findings to this point largely reproduce those of Randriamampita and Tsien (5) and indicate that the extract we have prepared contains the active principle which Randriamampita and Tsien designated as CIF.


Figure 3: Subcellular distribution of calcium signaling activity isolated from either resting or PHA-stimulated Jurkat cells. Cytoplasmic and particulate fractions of Jurkat cells were prepared, as described under ``Materials and Methods.'' The traces on the left demonstrate the effect on astrocytoma cells of a soluble and particulate fraction of non-stimulated Jurkat cells. The traces on the right demonstrate the effect on astrocytoma cells of the same fractions prepared from PHA-stimulated Jurkat cells. The activity, assayed on astrocytoma cells, was entirely in the particulate fraction of resting Jurkat cells, and stimulation of the cells with PHA resulted in the appearance of activity in the soluble fraction. Note that dilution of the extracts yielded appreciably less Ca signaling activity, indicating that the material was assayed at a less than supramaximal concentration. The data are representative traces from five independent preparations.



Effects of the Jurkat Extract on Hepatocytes, Lacrimal Acinar Cells, and X. laevis Oocytes

We next examined the activity of the Jurkat extract on other cell types that exhibit capacitative Ca entry. In hepatocytes and mouse lacrimal cells the extract evoked a response pattern that was quite different from that seen in astrocytoma cells. As shown in Fig. 4for lacrimal cells, the extract induced a clear intracellular mobilization of Ca, as well as a sustained entry of Ca. The extract also produced a similar biphasic [Ca](i) response in hepatocytes (7/7 cells; data not shown). Injection of lacrimal cells with the (1, 4, 5) IP(3)-receptor antagonist, heparin (Fig. 4), or extracellular application of the muscarinic receptor antagonist, atropine (1 µM; data not shown) completely blocked the [Ca](i) response to the Jurkat extract. Note that the extract usually evoked sinusoidal Ca oscillations in lacrimal cells, a response pattern similar to that reported for low concentrations of MeCh on this cell type(10) . This response pattern and the effects of heparin and atropine strongly suggest that the extract contains a compound with muscarinic agonist activity. The response in hepatocytes was not blocked by atropine (hepatocytes do not have muscarinic receptors), but the extract may be activating endogenous receptors in these cells as well. The muscarinic-like activity (assayed in lacrimal cells) was found exclusively within the soluble fraction from both stimulated and nonstimulated Jurkat cells (data not shown), indicating that this is a different principle from that eliciting the Ca entry responses in astrocytoma cells. (^2)Further, it appears that when this muscarinic agonist activity is blocked, lacrimal cells do not respond to the calcium entry-stimulating principle designated as CIF by Randriamampita and Tsien(5) .


Figure 4: Jurkat extract effects on mouse lacrimal acinar cells. A single, fura-2-loaded mouse lacrimal cell was incubated in the absence of extracellular Ca, and then treated with the Jurkat extract. When [Ca]returned to basal levels, extracellular Ca (1.8 mM) was restored to the cell. The same protocol was also followed on a lacrimal cell injected with the (1, 4, 5) IP(3)-receptor antagonist, heparin (200 mg/ml, pipette concentration). Under these conditions, the response to the Jurkat extract was completely blocked. The data shown are representative traces from three similar experiments.



We considered that the failure of hepatocytes and lacrimal cells to respond to CIF might reflect the absence of the principle in these cell types. We thus applied the isolation procedure described by Randriamampita and Tsien (5) to these cell types, assaying the subsequent activities on astrocytoma cells and lacrimal cells. An acid extracted fraction from rat hepatocytes displayed no activity when applied to either astrocytoma cells or lacrimal cells (three experiments, data not shown). However, an extract isolated from mouse lacrimal cells induced a response in astrocytoma cells similar to that induced by the Jurkat extract (Fig. 5a). As found for the Jurkat extract, the lacrimal extract induced a muscarinic-like activity when applied to mouse lacrimal cells (Fig. 5b).


Figure 5: A lacrimal cell derived extract exhibited effects similar to those produced by the Jurkat extract on 1321N1-astrocytoma cells and lacrimal cells. The extraction procedure for Jurkat cells was also performed on lacrimal acinar cells, and the activity of the resulting extract was assayed on astrocytoma cells (a) and lacrimal cells (b). The response to the lacrimal extract on both cell types was essentially the same as that observed for the Jurkat extract. The extract was added to astrocytoma cells in the continued presence of external Ca, while for the lacrimal cells, the extract was added in the absence of external Ca, and external Ca was then restored as indicated. The experiments shown are representative of similar findings in three independent experiments.



We next investigated the actions of the Jurkat extract in oocytes of X. laevis. Previous studies with this cell type have provided some of the most compelling evidence for the existence of a diffusible messenger for capacitative calcium entry(4) . (1, 4, 5) IP(3), or metabolically stable analogues of (1, 4, 5) IP(3), are known to activate Ca entry in this cell type(11, 12) . In confirmation of these findings, Fig. 6shows that intracellular injection of the nonmetabolizable(1, 4, 5) IP(3) analogue(2, 4, 5) IP(3)(9) , caused, in all oocytes, an intracellular Ca release, followed by a sustained elevation of [Ca](i) on restoring extracellular Ca. By contrast, most of the oocytes tested (9/14) were insensitive to the Jurkat extract (Fig. 6a). The remainder responded to the extract with a [Ca](i) response involving both the release of internal Ca as well as entry of extracellular Ca (Fig. 6b). In addition, a small percentage of oocytes (5/19) responded to MeCh with a typical biphasic mobilization of Ca. Some oocytes were tested for their responsiveness to both MeCh and the Jurkat extract, and only MeCh-responsive oocytes were responsive to the extract (three cells responsive to both; three cells responsive to neither) (Fig. 6b). However, in the presence of 10 µM atropine, no oocytes responded to either MeCh or the extract. Thus, the [Ca](i) responses to extracellularly applied Jurkat cell extract seen in a few oocytes apparently represents activation of muscarinic receptors as seen in lacrimal acinar cells. Intracellular injection of the Jurkat extract in oocytes perifused with a medium containing 5 mM Ca gave no [Ca](i) response (n = 6).


Figure 6: Effects of extracellular application of the Jurkat extract on [Ca] in Xenopus oocytes. Fura-2-injected oocytes were initially bathed in a Ca-free medium. After testing their response to 10 µM MeCh, they were perifused with a medium containing 5 mM Ca. A small transient rise in [Ca] was sometimes seen on addition of Ca to unstimulated oocytes. Thereafter, the Jurkat extract (JE) was applied in a medium lacking Ca and then in the presence of 5 mM Ca. At the end of the experiment, intracellular release and entry of Ca was activated by injection of (2, 4, 5) IP(3). As illustrated by these two representative traces, the small percentage of oocytes sensitive to the Jurkat extract was also sensitive to MeCh. The data shown are representative traces from three experiments (for a and b) carried out with this protocol; however, the majority of oocytes were not responsive to MeCh (see text).



In the course of studying the effects of the Jurkat extract on Ca signaling in Xenopus oocytes, we also examined the effects of the extract on membrane currents because in this cell type, an endogenous Ca-dependent chloride current is often used as an indicator of [Ca](i) changes(13, 14) . As shown in Fig. 7, intracellular injection of(2, 4, 5) IP(3) produced a large transient chloride current in a Ca-free medium which was reactivated on restoring extracellular Ca. Application of the extract in a Ca-free medium produced a large and sustained inward current (n = 32; Fig. 7, a and b), which occurred in oocytes insensitive to 10 µM MeCh and which was resistant to 10 µM atropine. This current did not result from Ca mobilization since it also developed after the intracellular Ca pools were depleted by (2, 4, 5) IP(3) injection (Fig. 7b). Furthermore, the extract current was totally insensitive to 500 µM niflumic acid, a potent blocker of the endogenous Ca-activated chloride current(15) . Voltage ramps between -80 and +80 mV (2-s duration) indicated that the current was relatively linear with slight inward rectification at negative potentials and with a reversal potential of -16.5 ± 0.3 mV (n = 3; Fig. 8). The extract-induced current was largely reduced in a medium containing 1 mM Ca (n = 3) and completely abolished in a medium containing 5 mM Ca (n = 13; Fig. 7a). Intracellular injection of the extract (approximately diluted 10-fold after injection in the oocyte) did not affect the current (n = 23), whereas the same oocyte displayed a large current response to extracellular application of the extract diluted to a similar extent (n = 4; Fig. 7b). Thus the Jurkat extract activated, in all oocytes, a current response that was independent of any change in [Ca](i), and that was blocked by extracellular Ca.


Figure 7: Effects of intracellular and extracellular application of the Jurkat cell extract on the voltage-clamped current in Xenopus oocytes. The oocytes were initially bathed in a Ca-free medium. Thereafter, they were perifused with a medium containing 5 mM Ca, 10 µM MeCh or Jurkat extract (JE) where indicated, and they were injected with(2, 4, 5) IP(3) or the extract as indicated. Intracellular application of (2, 4, 5) IP(3) always induced a large Ca entry on restoring Ca which was reflected by an increase in the Ca-dependent chloride current. By contrast, the extract was without effect in the presence of 5 mM Ca, whereas it induced a large sustained current in a Ca-free medium (a). This current could not be fully reversed by washing the extract with a Ca-free medium, whereas it returned quickly to basal level upon readdition of 5 mM Ca to the medium (a). Intracellular injection of the extract (final dilution 10-fold) did not activate any current response, whereas extracellular application of the extract diluted to a similar extent produced a large current in a Ca-free medium, even when intracellular Ca pools were emptied following(2, 4, 5) IP(3) injection (b). The data shown are representative traces from 13 (a) and 4 (b) similar experiments.




Figure 8: Current-voltage relationship of the responses activated by the Jurkat extract () and Ca injection (bullet) in Xenopus oocytes bathed in a Ca-free medium. The currents of the two traces were normalized by dividing all points by the amplitude of the current at +80 mV. The current activated by the Jurkat extract at +80 mV averaged 6.22 ± 0.19 µA and that activated by Ca injection was 2.25 ± 0.4 µA. Each trace was generated from voltage ramps (2-s duration) obtained in three oocytes.




DISCUSSION

In this study, we have followed on the work of Randriamampita and Tsien (5) who reported the presence of a Ca entry-stimulating principle in acidic extracts of Jurkat cells, and who concluded that this principle may be the long sought messenger for capacitative calcium entry. We confirmed some of the observations of Randriamampita and Tsien in that extracellular application of an acid extract of Jurkat cells to astrocytoma cells induced a calcium entry and, at least in the majority of cells, this was not accompanied by a release of intracellular Ca. We also confirmed that stimulation of the Jurkat cells with PHA caused a release of this Ca entry-stimulating principle into the soluble fraction of Jurkat cells. Finally, we found that the actions of the Jurkat extract on astrocytoma cells were not blocked by the intracellular application of the(1, 4, 5) IP(3) receptor antagonist, heparin. All of these observations indicate that we have prepared an extract containing the active principle which Randriamampita and Tsien (5) called CIF. Further, they are consistent with the interpretation given by Randriamampita and Tsien that the calcium entry stimulating principle in the Jurkat extract is the second messenger for capacitative calcium entry.

However, when we investigated further the properties of this extract in astrocytoma cells and other cell types, our findings cast doubt on this interpretation. In three other cell types known to utilize the capacitative Ca mechanism, lacrimal acinar cells, hepatocytes, and X. laevis oocytes (only a minority responded at all), Jurkat extracts did not induce Ca entry alone, but rather activated a biphasic Ca mobilization pattern which appeared to result from activation of surface membrane receptors; when these receptor responses were blocked, the Jurkat extract was inactive in these cell types. This despite the fact that lacrimal cells appeared to contain the same active principle(s) as found in Jurkat cells. Further, in at least one cell type in which capacitative Ca entry is known to occur, the rat hepatocyte(16, 17) , no Ca signaling activity could be extracted. In a previous communication(18) , we have pointed out that the temporal pattern of [Ca](i) signaling induced by Jurkat extracts in astrocytoma cells is quite different from that induced by MeCh or thapsigargin, agents presumably acting through the true messenger for capacitative Ca entry. This difference is also apparent in the experiments reported here, wherein MeCh and thapsigargin induced sustained [Ca](i) signals, while, as also reported by Randriamampita and Tsien(5) , the extract tended to induce irregular oscillations. In addition, we found that the Ca entry-stimulating activity in the Jurkat extract was difficult to remove by washing astrocytoma cells; removal of MeCh, which presumably acts through the true messenger for calcium entry, resulted in rapid reversal of Ca entry(18) .

It is important to note that the Jurkat cell extract was found to contain not a single Ca signaling principle, but a number of activities which affected not only Ca movements across the cell membrane, but also surface membrane receptors, as well as membrane channels for ions other than Ca. Further, these activities were expressed in a cell type-specific manner. Given the complex signaling and paracrine functions of the T-lymphocytes from which the Jurkat line is derived, it is perhaps not surprising that these cells contain many substances with diverse biological activities. However, we conclude from our data that it is probably incorrect to attribute to any of these the role of intracellular messenger for capacitative Ca entry.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Chargé de Recherches of the Fonds National de la Recherche Scientifique, Brussels.

To whom correspondence should be addressed: NIEHS/NIH, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-3298.

(^1)
The abbreviations used are: (1, 4, 5) IP(3), D-myo-inositol 1,4,5-trisphosphate (the inositol phosphates are abbreviated according to the ``Chilton convention''(19) ); CIF, calcium influx factor; PHA, phytohemagglutinin; DMEM, Dulbecco's modified Eagle's medium; MeCh, methacholine.

(^2)
We were at first puzzled as to why the astrocytoma cells, which possess muscarinic receptors, did not respond to the Jurkat extract with a muscarinic-like response, as seen with the lacrimal cells. However, based on previous studies(10) , we estimate that the Jurkat extract response in lacrimal cells was equivalent to a concentration of methacholine of between 0.5 and 1.0 mM. Astrocytoma cells were considerable less sensitive to MeCh than lacrimal cells and required concentrations between 10 to 100 mM to produce a Ca response. As some 15% or so of the astrocytoma cells did in fact show an intracellular Ca release response, we suspect that this is due to muscarinic receptor activation, but as this response was weak and infrequent, we did not examine this further.


ACKNOWLEDGEMENTS

We are thankful to Drs. David Armstrong and Carmen Louzao who provided insightful comments during the preparation of this manuscript.


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