©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chromatographic Resolution of an Intracellular Calcium Influx Factor from Thapsigargin-activated Jurkat Cells
EVIDENCE FOR MULTIPLE ACTIVITIES INFLUENCING CALCIUM ELEVATION IN XENOPUS OOCYTES (*)

Hak Yong Kim , David Thomas (§) , Michael R. Hanley (¶)

From the (1) Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616-8635

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Acid extracts of thapsigargin-stimulated Jurkat cells revealed both intracellular and extracellular activities stimulating Ca-dependent Clcurrents on Xenopus laevis oocytes. Chromatographic fractionation of these extracts on gel filtration separated two active fractions of Mapproximately 600 and 400. Moreover, the M600 fraction exhibited both intracellular and extracellular activities. However, the intracellular activity was absent from extracts of unstimulated Jurkat cells, suggesting its production was stimulated by thapsigargin. The further purification of this fraction by high performance thin layer chromatography resolved a single fraction which was active only on microinjection and which required calcium entry for activation of current responses. These results suggest that a single authentic calcium influx factor can be resolved by purification from confounding activities detected in crude acid extracts.


INTRODUCTION

Cytosolic Cais elevated through multiple mechanisms, including release from stores mediated by InsP() or ryanodine receptor channels (1) . Following release, the depletion of intracellular Castores induces Cainflux across the plasma membrane. This calcium influx pathway has been termed ``capacitative calcium entry'' (2) . However, the mechanism which couples the depletion of the stores to Cainflux across the plasma membrane is unclear. Recently, it was proposed that depletion of Castores might release a novel diffusible messenger (calcium influx factor (CIF)) which opens a specific class of calcium channels (3, 4) .

We recently reported that activated Jurkat cells produce an intracellular-acting CIF activity measured indirectly as a Ca-dependent Clcurrent in Xenopus oocytes (5) . Here we establish that there are different factors which act extracellularly or intracellularly. The partially purified CIF reported here acts exclusively intracellularly to induce the characteristic indicator of elevated cytosolic calcium changes in oocytes, the Ca-dependent Clcurrent. These results suggest that acid extracts from Jurkat cells are a complex mixture of agents active on calcium metabolism, but an authentic CIF regulating the capacitative Caentry pathway can be selectively purified away from both intracellular discharge and extracellular calcium-elevating activities.


EXPERIMENTAL PROCEDURES

Materials

Thapsigargin (TG) was purchased from LC Services (Woburn, MA). Hanks' balanced salt solution (HBSS) and L-15 medium were from Life Technologies, Inc. RPMI 1640 was from BioWhitaker (Walkersville, MD). Bio-Gel P-2 gel was from Bio-Rad. Microcon-30 was from Amicon (Beverly, MA). Sep-Pak cartridge (C) was from Millipore (Milford, MA). HPTLC plate (200-µm layer) was from Whatman.

Cell Culture

Jurkat T lymphocytes 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.

Extract Preparation

Crude extracts were prepared from resting and stimulated Jurkat cells as described previously with minor modification (5) . Briefly, cells were washed three times with HBSS containing 20 mM HEPES. Jurkat cells were stimulated with 1 µM TG for 15 min at 25 °C. Cells were centrifuged for 5 min at 200 g, and the pellet was resuspended in 0.85 ml of HBSS containing 20 mM HEPES. The suspension was extracted with 0.15 M HCl and was incubated for 30 min at 25 °C. The extract was clarified by centrifugation for 10 min at 400 g, and the supernatant was neutralized by addition of 10 M NaOH. After neutralization, 10 mM BaClwas added to the extract for precipitation of vicinal phosphate compounds, including inositol polyphosphates. Insoluble material was removed from extracts by centrifugation for 5 min at 12,000 g. The supernatant was lyophilized, and the residue was extracted with methanol with continuous mixing for 15 min at 25 °C. The methanol extract (0.8 ml) was loaded on the Sep-Pak Ccartridge for removal of hydrophobic material and was rinsed with methanol (0.8 ml, flow rate: 0.25 ml/min). The methanol eluants were combined (1.6 ml), dried under Ngas (30 °C), and resuspended in 0.1 M acetic acid (0.25 ml). The reconstituted extract was clarified by centrifugal ultrafiltration through a Microcon-30 filter. The filtrate is enriched for materials of M< 30,000, whereas the retentate is material M> 30,000. The filtrate was loaded on the gel filtration column for further purification.

Gel Filtration Chromatography

The filtrate was loaded onto a Bio-Gel P-2 column (0.7 27 cm), equilibrated with 0.1 M acetic acid, and was eluted by the same buffer (7 ml/h), collecting fractions of 0.5 ml. Activity was assayed by measuring Ca-dependent Clcurrent in Xenopus oocytes under voltage clamp as described previously (5, 6) . Fraction 15 from TG-stimulated cell extracts has both extracellular and intracellular activities and is termed ``Peak 1.'' Fractions 18 and 19 have only intracellular activity, and were pooled to form ``Peak 2.''

HPTLC

Peak 1 was applied to a 200-µm HPTLC plate and developed with chloroform/methanol/acetic acid/water (20/15/8/4, v/v). Peak 1 was resolved into at least five bands using UV detection. Each identified band was scraped from the plate, extracted with 0.1 M acetic acid, and lyophilized. Each fraction was then reconstituted in 50 µl of buffer (10 mM HEPES, pH 7.0) and activity tested extracellularly and intracellularly by measuring Ca-dependent Clcurrent in Xenopus oocytes.

Oocyte Injections and Voltage Clamp Recording

Each fraction was tested for activity by measuring Ca-dependent Clcurrents under voltage clamp conditions. Xenopus laevis 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 L-15 medium (diluted 1:1 with 30 mM HEPES buffer, pH 7.4, containing 0.25% chicken ovalbumin, 1 mML-glutamine, and 50 µg/ml gentamycin). Conventional two-electrode voltage clamping was performed as described previously (5, 6) . Oocyte injections were performed using the Picospritzer (General Valve Corp, Fairfield, NJ) pressure injection apparatus. Oocytes were voltage-clamped at 60 mV in OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl, 1 mM MgCl, 1 mM NaHPO, 5 mM HEPES, pH 7.4) and were injected with 10 nl of each chromatographic fraction. 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

We have shown previously that acidic extracts from TG-stimulated Jurkat lymphocytes have intracellular activity in activating a calcium influx pathway (5) . We now report that these extracts also exhibit extracellular activity on oocytes in stimulating the Ca-dependent Clcurrent. This extracellular response was, however, unlike the intracellular activity, in that extracellular responses have no dependence on extracellular calcium (Fig. 1). This is similar to the earlier report of extracellular CIF activity (3) , but the dramatic difference in sensitivity to extracellular calcium removal suggested that these intracellular and extracellular activities were not the same and were therefore attributable to distinct and independent factors.


Figure 1: The extracellular response induced by Jurkat extract does not require extracellular calcium. Extracts prepared as described previously (5) from thapsigargin-stimulated Jurkat lymphocytes were diluted 1:2 in Ca-free OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl, 1 mM NaHPO, 5 mM HEPES, pH 7.4) supplemented with 1 mM EGTA, and 5 µl was applied to the oocyte bath (300 µl). To remove extracellular Ca( dotted trace) oocytes were perfused for 2 min in nominally Ca-free OR2 supplemented with 1 mM EGTA before application of extract. The responses were representative of three similar experiments from different batches of oocytes.



Accordingly, in order to purify intracellularly acting CIF activity from Jurkat cell extracts, we have introduced a sequence of purification steps: 1) Sep-Pak reverse-phase columns, 2) Microcon-30 ultrafiltration, 3) Bio-Gel P-2 gel filtration, and 4) HPTLC.

The activities were not retained on reverse phase cartridges or by a high molecular weight cut-off filter. The first separation of the activities into distinct fractions was observed on gel filtration (Fig. 2). Two active fractions were obtained. The first, called Peak 1, with an estimated molecular weight of 600, exhibited both extracellular and intracellular activities. The second, called Peak 2, with an estimated molecular weight of 400, exhibited exclusively intracellular activity (Fig. 2 B).


Figure 2: Thapsigargin stimulation specifically augments the intracellular activity of high molecular weight gel filtration fraction. Fractionation of unstimulated ( A) and TG-stimulated ( B) cell extracts on gel filtration chromatography. Bio-Gel P-2 columns (0.7 27 cm) were equilibrated and eluted with 0.1 M acetic acid. Fractions (0.5 ml) were collected at a flow rate of 7 ml/h. The extracellular ( open circles) and intracellular ( crosses) activities (nA) are shown as Ca-dependent Clcurrent in Xenopus oocytes, as described under ``Experimental Procedures.''



The fractionation of extracts from unstimulated Jurkat cells (Fig. 2 A) was compared to that from TG-stimulated cells. Peak 2, which is only active by intracellular injection, was unaffected by TG stimulation. Although the extracellular activity of Peak 1 was also unaffected by TG stimulation, the intracellular activity of Peak 1 was specifically augmented (Fig. 2 A). These results suggest Peak 1 is heterogenous and contains two distinct factors, the intracellular, but not the extracellular, activity being produced by depletion of Castores. Therefore, the intracellularly active component in Peak 1 is a candidate for an authentic CIF.

In order to compare the intracellular activity of Peak 1 to the previously reported properties of crude acid extracts (5) , the effect of external calcium removal was examined. Using calcium-free medium (containing 1 mM EGTA) only 40% of the current could be blocked, indicating the remaining response was due to calcium discharge. This incomplete external calcium dependence of the current response was unlike that of crude acid extracts and was confirmed using extracellular medium containing 0.5 mM Nias a Caentry channel blocker (7) . Under these conditions, the response was also blunted by about 40% (Fig. 3). These results suggested that the intracellular activity of Peak 1 contained factors influencing both calcium entry as well as calcium mobilization from stores. The calcium discharge activity can be ascribed to a contaminant in that it is completely lost by a 1:4 dilution. Using such diluted extracts, the response to microinjection is not reduced, but shows complete extracellular calcium dependence (data not shown).


Figure 3: Gel filtration Peak 1 activates Ca-dependent Cl currents in Xenopus oocytes by microinjection. Peak 1 (10 nl) prepared as described under ``Experimental Procedures,'' induced chloride current by intracellular injection (2300 ± 365 nA, n = 25). Removal of extracellular Ca(inclusion of 1 mM EGTA to nominally Cafree medium, dotted trace) blocked about 40% of maximum current ( n = 4). Addition of 0.5 mM Nito OR2 medium ( dashed trace) also blocked about 40% of maximum current ( n = 4). The current shown is carried by chloride ions as assessed by its appropriate reversal potential (25 mV) and blockade by the selective inhibitor niflumic acid (1 mM, see Ref. 4). Injection of BAPTA (1 mM final concentration) eradicates all current activity, indicating that the responses are completely Ca-dependent.



Thus, in order to resolve authentic CIF from contaminating discharge activities, Peak 1 was further fractionated using HPTLC. Peak 1 was indeed heterogeneous and separated into at least five bands detectable under UV light. Two bands exhibited significant intracellular activity, but the extracellular activity of Peak 1 was resolved from both of these bands (data not shown). Thus, the intracellular and extracellular activities of Peak 1 are completely separated by this procedure.

One HPTLC-purified band ( R= 0.57) elicited current responses, exclusively on microinjection and not on external application, that were eliminated by extracellular free calcium removal or addition of extracellular NiCl(Fig. 4). In unstimulated cells, this band is completely absent. These results suggest that this HPTLC-enriched fraction contains authentic CIF.


Figure 4: Dependence on Ca entry of CIF partially purified by HPTLC. Microinjection (10 nl) of HPTLC-purified fraction ( R = 0.57), prepared as described under ``Experimental Procedures'' induced current (1776 ± 379 nA, n = 10). Removal of extracellular Ca(Ca-free OR2 containing 1 mM EGTA) ( dotted trace, n = 8) or addition of 0.5 mM Ni( dashed trace, n = 9) inhibited current elicited by microinjection.




DISCUSSION

In previous work, we have reported that a novel CIF can be identified by intracellular injection of oocytes with extracts from TG-activated cells. However, the issue of the possible extracellular activity of CIF remained unresolved. The results reported here demonstrate that extracellular activity can be detected by external application of CIF-containing extracts to oocytes, but that it is attributable to a factor distinct from an authentic CIF. Moreover, the acid extracts are surprisingly complex in the diversity of distinct components active on calcium elevation.

We have identified two size fractions on gel filtration which are active. The smaller size fraction, of approximately 400, is an intriguing and unexpected activity in that it induces calcium discharge by an InsP-independent mechanism (data not shown). However, this has not been characterized further, other than its activity is unaffected by alkaline phosphatase treatment.

The larger size fraction, of approximately 600 daltons, contains distinct and separable extracellular and intracellular factors. Only the latter, resolved by HPTLC, is a candidate for authentic CIF based on 1) its increased levels in cell extracts following thapsigargin treatment and 2) its distinctive profile of biological activity, eliciting calcium entry, but not discharge, exclusively by intracellular application.

Thus, extracts thought to contain a single active component have, by this analysis, proven to have at least three distinct factors regulating calcium-dependent currents from external or intracellular sites of action in Xenopus oocytes. This introduces a significant complication in efforts to identify CIF activities in crude extracts.


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 an National Institutes of Health training grant in Molecular and Cellular Biology.

To whom correspondence should be addressed: Dept. Biological Chemistry, School of Medicine, University of California, Davis, CA 95616-8635. Tel.: 916-752-8332; Fax: 916-752-3516; E-mail: mrhanley@ucdavis.edu.

The abbreviations used are: InsP, Inositol 1,4,5-trisphosphate; TG, thapsigargin; CIF, calcium influx factor; HBSS, Hanks' balanced salt solution; BAPTA, 1,2-bis(2-aminophenoxy)ethane- N, N, N`, N`-tetraacetic acid; HPTLC, high performance thin layer chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Brett Premack (Department of Molecular Pharmacology, Stanford University School of Medicine) for helpful discussions and his critical advice on the experiments described in this manuscript. We also gratefully acknowledge Dr. Rich Nuccitelli (Section of Molecular and Cellular Biology, University of California, Davis) for usage of the X. laevis oocyte colony.


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