©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evaluation of Calcium Influx Factors from Stimulated Jurkat T-lymphocytes by Microinjection into Xenopus Oocytes(*)

(Received for publication, December 6, 1994; and in revised form, January 23, 1995)

David Thomas (§) Michael R. Hanley (¶)

From the Department of Biological Chemistry, University of California School of Medicine, Davis, California 95616-8635

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Acid extracts of thapsigargin-activated Jurkat cells have been shown to have intracellular activity in inducing a dose-dependent rapid chloride current upon microinjection in Xenopus laevis oocytes. The extracts act by elevation of calcium through calcium entry. The factor(s) responsible for this activity have been termed calcium influx factor (CIF) and have been found to be small, relatively polar molecules (<1000 daltons) whose activity is abolished by alkaline phosphatase treatment and potentiated by co-injection of okadaic acid (a protein phosphatase inhibitor). CIF is produced in a time-dependent manner following thapsigargin treatment of Jurkat cells, being first elevated above basal levels by 2 min. Intracellular CIF activity is completely absent from NG115-401L neuronal cells, which lack capacitative entry. On this basis, it appears that Jurkat cells, activated by stimuli that deplete internal calcium stores, produce one or more CIF activities acting intracellularly, and Xenopus oocytes may be a powerful tool to purify and characterize CIFs.


INTRODUCTION

Receptors that mobilize calcium through production of InsP(3)(^1)frequently exhibit two phases in the elevation of cytosolic [Ca], an initial transient peak due to release from stores and a sustained entry through surface channels(1) . The latter response has been termed ``capacitative entry,'' and its underlying mechanism is unknown(1, 2) . However, the trigger for activation of capacitative calcium entry is widely held to be depletion of intracellular calcium stores(3) . Recently, it was proposed that coupling between the stores and the surface might be via a novel diffusible messenger, which was identified in acid extracts of Jurkat T-lymphocytes(4) . However, this activity was exclusively characterized by extracellular application. The intracellular activity, if any, of such extracts has not been evaluated. Here, we provide evidence that activated, but not resting, Jurkat cells have a calcium influx activity acting intracellularly when assayed in Xenopus oocytes.


EXPERIMENTAL PROCEDURES

Materials

Thapsigargin (TG), InsP(3), and okadaic acid were obtained from LC Services (Woburn, MA). Fura-2/AM was from Molecular Probes (Eugene, OR). Calf Intestinal alkaline phosphatase was from Boehringer Mannheim. Oleoyl lysophosphatidic acid (LPA), low molecular weight heparin, cyclopiazonic acid (CPA), and type 1A collagenase were from Sigma. Hanks' balanced salt solution (HBSS), L-15 medium, and phytohemagglutinin (PHA) were from Life Technologies, Inc. RPMI 1640 and Dulbecco's modified Eagle's medium were from BioWhitaker (Walkersville, MD).

Preparation of Cell Extracts

Crude extracts were prepared from resting and stimulated Jurkat and NG115-401L cells using a modification of a procedure previously described(4) . Typically, 5 times 10^7 cells were used for preparation of extracts. Cells were washed (3 times 20 ml of HBSS) and resuspended (10 ml of HBSS containing 20 mM HEPES pH 7.4). Jurkat cells and NG115-401L cells were stimulated with TG (1 µM in HBSS + 20 mM HEPES, 10 min, continuous mixing, 25 °C). In some experiments, Jurkat cells were stimulated with PHA (added according to manufacturer's recommendations, 5 min) or CPA (20 µM, 10 min). Following stimulation, cells were centrifuged (5 min, 200 times g) and the pellet was resuspended in 0.85 ml of HBSS. The suspension was extracted by addition of HCl (1 M, 0.15 ml, 30 min) with continuous mixing (25 °C). The extract was clarified by centrifugation (10 min, 400 times g) and the supernatants neutralized by addition of NaOH (10 M). Following neutralization, BaCl(2) was added from a stock 1 M solution to the extract (final concentration, 10 mM) to precipitate vicinal phosphate compounds, including inositol polyphosphates. Insoluble material was removed from the extracts by centrifugation (5 min, 12,000 [times g). The supernatant was lyophilized, and the freeze-dried residue was extracted with 1 ml of methanol (15 min, continuous mixing, 25 °C). The methanol extract was dried (80 °C) and subsequently reconstituted (0.1 ml, 10 mM HEPES pH 7.4) for storage and testing. Routinely, 10 nl of this reconstituted extract was injected into oocytes, corresponding to material from 5000 original cells.

Oocyte Injections and Voltage Clamp Recording

Xenopuslaevis oocytes were obtained by ovarectomy. Follicular cells were removed from oocytes by treating with collagenase (2 mg/ml) for 1 h and 45 min, followed by rolling oocytes on plastic Petri dishes. Defolliculated oocytes were maintained in modified L15 medium (diluted 1:1 with a buffer containing 0.25% chicken ovalbumin, 30 mM HEPES, 1 mML-glutamine, and 50 µg/ml gentamycin, pH 7.4). Conventional two-electrode voltage clamping was performed as described previously(5) . For recording of currents, oocytes were immersed and continuously perfused in OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 1.0 mM CaCl(2), 1.0 mM MgCl(2), 1.0 mM Na(2)HPO(4), 5 mM HEPES pH 7.4). Oocyte injections were performed using the Picospritzer (General Valve Corp., Fairfield, NJ) pressure injection apparatus. Briefly, oocytes voltage-clamped at -60 mV were injected with 10 nl of extracts or InsP(3). Injection volumes were calibrated by measuring the diameter of injected droplets in air. To remove extracellular Ca oocytes were perfused for 2 min in nominally Ca-free OR2 supplemented with 1 mM EGTA before injecting extracts. Currents were digitized using the Tl-1 A/D board (Axon Instruments Inc., Foster City, CA) in combination with the current analysis program SCAN (Dagan Corp., Minneapolis, MN).


RESULTS

In order to test the intracellular calcium-elevating activity of Jurkat cell extracts, which have previously been shown to have extracellular activity on mammalian cells(4) , we have exploited unique advantages of the Xenopus oocyte. First, Xenopus oocytes are large cells that can be readily and rapidly microinjected with small amounts of cell extracts. Second, the well characterized Ca-activated chloride current is a rapid electrogenic measure of calcium elevation. Third, the powerful calcium stores-depleting reagent, TG, has no direct calcium-elevating activity in Xenopus oocytes (Fig. 1A; also see (7) ), thus permitting its use for stimulating target mammalian cells.


Figure 1: Extracts from thapsigargin-stimulated Jurkat cells, but not thapsigargin itself, activate large Ca-dependent chloride currents by an InsP(3)-independent mechanism in Xenopus oocytes. A, responses to InsP(3) (IP) injection (1 pmol, dottedtrace), InsP(3) co-injected with thapsigargin (1 µM), and thapsigargin alone are shown. Direct responses to InsP(3) were 520 nA ± 115 nA (n = 10), whereas injecting 1 µM thapsigargin into oocytes always failed to induce responses (n = 10). Including thapsigargin in the InsP(3) injection pipette had no significant effect on InsP(3)-induced responses (610 nA ± 100, n = 5). B, microinjection of 10 nl of extract prepared as described under ``Experimental Procedures'' consistently induced large chloride currents (2120 ± 445 nA, n = 35). Removal of extracellular Ca (inclusion of 1 mM EGTA to nominally Ca-free OR2 medium) completely abolished the response (n = 5). C, microinjection of diluted extracts demonstrates a dose dependence for oocyte responses with the activity becoming undetectable at a 1:10 dilution. D, injection of approximately 500 µg/ml heparin into oocytes (dottedtrace) failed to block responses induced by extract injection (n = 4). Jurkat T cells were maintained in suspension in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mML-glutamine, and penicillin (100 units)/streptomycin (100 µg/ml). Jurkat cells were passaged by 1:10 dilution every 4 days.



Acid extracts prepared from TG-stimulated Jurkat lymphocytes rapidly induced large current responses (approx2 µA) when injected into Xenopus oocytes (Fig. 1B). Removal of extracellular calcium abolished current responses, suggesting absolute dependence on calcium influx (Fig. 1B). On this basis, the extracts contain a ``calcium influx factor'' (CIF), acting from the inside. The activity in the extract exhibited dose sensitivity as shown by serial dilution (Fig. 1C). Extract responses could not be blocked by prior heparin injection (Fig. 1D), demonstrating that extracts induce Ca elevation in a non-InsP(3)-dependent manner.

To obtain additional evidence that the Jurkat extract was not releasing Ca from internal stores, extract-induced current responses were tested for cross-desensitization with receptor-induced current responses elicited by endogenous LPA receptors(5) . Extract responses did not desensitize oocyte responses to subsequent exposure to LPA. Similarly, oocytes first exposed to LPA did not lose their ability to respond to the microinjected Jurkat extract (data not shown). This clearly suggests that Ca was not released from internal stores by Jurkat cell extracts, because both calcium-mobilizing receptor-activated current responses and direct InsP(3) injections exhibited complete, long lived, and reciprocal desensitization under these conditions in the Xenopus oocyte(8) .

Using the magnitude of the current response as a measure of the level of production of CIF activity, the time course for CIF appearance following TG stimulation was determined. Extracts prepared at intervals following TG stimulation showed a 4-fold enhancement of activity that was first observed between 1 and 2 min, which was then maintained (Fig. 2). This suggests a latency for production of CIF following TG treatment, which correlates exactly with the time to activation of the capacitative entry channel following TG treatment in the same cell type, Jurkat lymphocytes(9) . These experiments also detected intracellular CIF activity in resting cells, in agreement with the previous report(4) , although the levels were greatly enhanced by TG treatment, unlike earlier results.


Figure 2: Time-dependent appearance of extract CIF activity. Extracts from thapsigargin-stimulated Jurkat lymphocytes were taken at 0, 30, 60, 120, and 600 s. Thapsigargin stimulations were quenched at the indicated times by rapid immersion in liquid nitrogen, and extracts from each time point were prepared as described under ``Experimental Procedures.'' Responses were measured by microinjection into Xenopus oocytes as described under ``Experimental Procedures.'' Results are representative of three similar determinations.



To rule out that the activity observed was due to TG itself, rather than calcium depletion, Jurkat cells were activated by other calcium-depleting treatments. The mitogenic lectin, PHA, increased intracellular CIF activity but to a lower level than that elicited by TG (Fig. 3A). Treating Jurkat cells with the structurally distinct Ca-ATPase inhibitor cyclopiazonic acid (10) also generated extracts with activity comparable with the level produced by TG stimulation (Fig. 3A, dottedtrace).


Figure 3: Extract activity is stimulus-specific, abolished by alkaline phosphatase treatment and potentiated by the protein phosphatase inhibitor okadaic acid. A, Jurkat lymphocytes were stimulated as described under ``Experimental Procedures'' with PHA, CPA, or TG. Acid extracts were prepared and tested for activity by oocyte injection. PHA-induced current was 676 ± 221 nA, CPA (dottedtrace) was 1786 ± 340 nA, and TG-induced current was 1876 ± 435 nA. PHA- and CPA-stimulated extracts were duplicated and gave similar current responses in different oocyte batches. B, extracts from TG-stimulated Jurkat lymphocytes were incubated with 10 units of alkaline phosphatase (10 units/100 µl of extract) for 20 min at 37 °C. Alkaline phosphatase-treated extracts were then microinjected into oocytes to test for activity. No attempt was made to remove the alkaline phosphatase because injecting equivalent doses of the enzyme caused no response in oocytes. The response is representative of at least three alkaline phosphatase treatments of different extracts. C, extracts were diluted 1:8 with injection medium (10 mM HEPES pH 7.4) to achieve small reproducible responses (111 ± 34 nA; note the different scale for C). Okadaic acid was included in the injection pipette (final estimated concentration, 5 nM), where it potentiated the diluted extract response (826 ± 122 nA, n = 3). Injection of okadaic acid alone at these levels and up to 1 µM (estimated final concentration in oocyte cytoplasm) did not induce a response.



As a preliminary chemical characterization, the intracellular CIF activity was heat-stable (70-95 °C, 20 min), did not bind to fatty acid-free bovine serum albumin, and migrated as a small molecule of less than 1000 daltons on gel filtration. (^2)The intracellular CIF activity of Jurkat extracts was completely abolished by alkaline phosphatase treatment (Fig. 3B). These properties are similar to those reported earlier, but intracellular CIF activity was not retained on reverse-phase C18 columns, unlike extracellular activity(4) , indicating the possibility of a chemical distinction between extracellular and intracellular CIF activities.

Previously, it was reported that the protein phosphatase inhibitor okadaic acid prolonged capacitative Ca influx in Xenopus oocytes(11) . Strikingly, co-injection of low doses of okadaic acid with Jurkat cell extracts gave an immediate and dramatic enhancement of the current response (Fig. 3C).

The cell specificity of production of intracellular activity was evaluated using other mammalian cell lines. U937 monocytes showed TG-induced production of intracellular CIF activity (data not shown). In contrast, a neuronal cell line, NG115-401L, when stimulated by TG did not produce detectable CIF activity. This is important inasmuch as the NG115-401L cell line has been shown to lack capacitative entry, because these cells exhibit only a transient calcium elevation upon TG treatment, arising exclusively from discharge of calcium stores(12) . This difference in extracts from stimulated Jurkat and U937 cells or NG115-401L cells suggests that the latter lack the capacity to produce CIF acting intracellularly, which may correlate with the absence of a capacitative Ca entry pathway in this cell type. These results are summarized in Fig. 4. These experiments also constitute crucial controls indicating that intracellular CIF activity is not attributable to trivial consequences, such as pH changes or stretch-activated currents, of cell extraction or oocyte microinjection assay procedures.


Figure 4: Cell specificity of extract CIF production. A, extracts obtained from TG-stimulated Jurkat lymphocytes elicit large (>2,000 nA) Ca-dependent chloride currents (n = 35). Inset shows typical response when TG (1 µM) is added to a population of Jurkat lymphocytes loaded with the Ca-sensitive dye Fura-2/AM. B, extracts obtained from the NG115-401L cell line fail to induce responses in oocytes. Acid extracts were prepared as for stimulated Jurkat cells in cell number-matched preparations and tested for their ability to induce oocyte responses by intracellular injection. Results were confirmed by duplicate NG115-401L cell extract preparations. Inset shows typical responses when TG (1 µM) is added to populations of NG115-401L cells loaded with Fura-2/AM. NG115-401L neuronal cells were maintained in monolayer culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mML-glutamine and penicillin (100 units)/streptomycin (100 µg/ml). NG115-401L cells were passaged at 1:10 ratios every 3 days by trypsinization. Fura-2 loading and measurements followed standard procedures(6) .




DISCUSSION

Receptors activating intracellular calcium discharge through the production of InsP(3) have been repeatedly correlated with triggering a second sustained phase of calcium entry, which has been termed ``capacitative entry.'' This calcium entry pathway is distinct from known voltage-sensitive calcium channels in that it is initiated by loss of calcium from stores(3, 13) , implying a coupling mechanism between calcium stores and the channel itself. There are several proposals for the mechanism of capacitative entry, including physical conformational coupling (14) or biochemical pathways involving cytochrome P450(15) , guanylate cyclase(16) , tyrosine kinases(17) , sphingolipid metabolism(18) , GTP-binding proteins(19) , or a novel diffusible messenger(4) . In arguing for a novel messenger, Randriamampita and Tsien (4) presented evidence for a calcium entry activity extracted from Jurkat cells, but the assay and characterization of this activity were based upon extracellular application. Here, we have tested similar Jurkat cell extracts for intracellular activation of calcium entry in Xenopus oocytes. Extracts are here shown to contain one or more factors acting intracellularly with properties consistent with functions in depletion-activated calcium entry.

This factor can be described correctly as a CIF, using the term first applied to an extracellular activity(4) , on the basis that activated Ca-dependent chloride currents elicited by microinjection are absolutely dependent on extracellular calcium. Consequently, the activity of this extract is acting intracellularly to elicit an inward calcium flux. We have tested extracellular activities of crude and fractionated cell extracts on Xenopus oocytes as well, and a distinct factor acting extracellularly has been identified that can be purified away from the intracellular factor.^2 Thus, the simplest interpretation is that two or more active components contribute to the CIF activities of mammalian cell extracts. Indeed, it is unclear what the relationship of the intracellular CIF activity is to the extracellular CIF activity reported earlier. We would suggest that ``CIF'' should be considered an operational term of a functional activity, by analogy with the conventional use of Endothelium-Dependent Relaxation Factor (``EDRF'') for an activity that may be attributable to nitric oxide in some, but not necessarily all, cases. Accordingly, CIF should not be presumed to be an explicit extract component at this stage, since several substances may have this activity. Indeed, the CIF described here is not identical to that described earlier on the basis of extracellular actions.

Intracellular CIF activity is abolished by alkaline phosphatase treatment, suggesting it is a phosphomonoester, and its behavior on gel filtration indicates it is a small molecule of M(r) < 1000. These properties are shared with the extracellular CIF activity described earlier, but the intracellular activity behaves on reverse phase chromatography as a polar molecule (data not shown). On this basis, the activity described here would not cross membranes and be active by external application. Intracellular CIF is unlikely to be cyclic ADP-ribose, sphingosine-1-P, lysophosphatidic acid, or an inositol polyphosphate in that none of these substances mimic extract properties and actions in oocytes.

Intracellular CIF shows time-dependent production following TG treatment. Thus, it appears to be synthesized in response to stores depletion. Intracellular CIF is correlated with capacitative Ca entry in that it cannot be measured, and may be completely absent, in extracts from a cell type (NG115-401L cells) that lacks TG-stimulated capacitative entry. These results imply an interesting possibility that non-excitable cells may preferentially express a capacitative entry pathway, whereas excitable cells may not.

Earlier experiments using Xenopus oocytes, which established the possibility of a diffusible messenger activating capacitative entry, demonstrated potentiation of calcium entry by the protein phosphatase inhibitor okadaic acid(11) . A comparable potentiation was observed here upon co-injection of low doses of okadaic acid with Jurkat extracts. The speed, potency, and selectivity of the potentiation are consistent with okadaic acid acting on its identified protein phosphatase targets. This implies a serine/threonine kinase/phosphatase system in CIF regulation of capacitative entry channels.

In summary, these results show that Jurkat cells contain one or more substances with intracellular CIF activity. Moreover, Xenopus oocytes can be used as a simple and flexible assay for purification of such factors.


FOOTNOTES

*
This research was supported in part by grants from the National Institutes of Health and the Council for Tobacco Research. 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.

§
Supported by a National Institutes of Health training grant in molecular and cellular biology.

To whom correspondence should be addressed. Tel.: 916-752-8332; Fax: 916-752-3516; mrhanley{at}ucdavis.edu.

(^1)
The abbreviations used are: InsP(3), inositol 1,4,5-trisphosphate; TG, thapsigargin; CPA, cyclopiazonic acid; PHA, phytohemagglutinin; CIF, calcium influx factor; HBSS, Hanks' balanced salt solution; LPA, oleoyl lysophosphatidic acid.

(^2)
H-Y. Kim, D. Thomas, and M. R. Hanley, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Brett Premack (Molecular Pharmacology, Stanford University School of Medicine) for critical advice and provision of U937 cells and Peter Cala (Human Physiology, UC Davis School of Medicine) for use of the Hitachi fluorimeter.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.