©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sphingolipid-gated Ca Release from Intracellular Stores of Endothelial Cells Is Mediated by a Novel Ca-permeable Channel (*)

(Received for publication, December 12, 1994; and in revised form, January 6, 1995)

Stella Kim (1) Vipul Lakhani (1) Debra J. Costa (1) Ala I. Sharara (2) J. Gregory Fitz (2) Li-Wen Huang (1) Kevin G. Peters (1) L. Allen Kindman (1) (3)(§)

From the  (1)Cardiology Division, the (2)Gastroenterology Division, and the (3)Program in Molecular Medicine, Department of Medicine and the Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sphingolipid-gated Ca signaling is mediated through Ca-permeable channels. In this report, we characterize the properties of the channel in a human endothelial cell line (EA.hy926). Ca release from intracellular stores is not antagonized by nifedipine, conotoxin G-VIa, or heparin. To further characterize the molecular properties of the channel, we developed a novel assay to directly measure efflux of Ca from intracellular stores of permeabilized Xenopus oocytes. Following size fractionation by sucrose gradient, poly(A) RNA from EA.hy926 cells is microinjected into oocytes of Xenopuslaevis. We find that the mRNA encoding Ca release activity is 1.5-2.0 kilobases in length. The sphingolipid-gated Ca-permeable channel is thus likely to be a novel Ca-permeable channel distinct from other characterized intracellular Ca channels such as the ryanodyne receptor and the inositol 1,4,5-trisphosphate receptor. The method described here provides a new approach to further characterizing this channel and other intracellular Ca channels.


INTRODUCTION

Calcium is a ubiquitous intracellular messenger. Changes in intracellular Ca result when extracellular ligands interact with specific plasma membrane receptors. Second messengers are elaborated, which then induce release of Ca from intracellular Ca stores. Three such messengers have been identified. Inositol trisphosphate and cADP-ribose (ryanodine receptor) activate specific channels that share several features in common (for review, see (1) ). Sphingosine has recently been identified as a third member of the intracellular Ca messenger family (2, 3, 4, 5) . A unique feature of sphingolipid-mediated signaling is that in addition to its direct intracellular actions, sphinglipids act across intact plasma membranes, providing potential for intercellular signaling at a distance(4) . Sphingosine 1-phosphate is a putative ligand for this intracellular channel(6) . We(7) , and others(5, 8) have used sphingosylphosphoryl choline as a surrogate for the direct acting agonist. We have recently reported the properties of this intracellular sphingolipid-gated Ca-permeable channel in the rat basophilic leukemia cell(7) . This channel mediates release of Ca from intracellular stores in response to sphingosylphosphorylcholine (SPC). (^1)The properties of the channel are novel. It has a slope conductance of 160 picosiemens with 53 mM Ba as the charge carrier and is not antagonized by heparin, nifedipine, conotoxin GVIa, Ni, or La. The open probability is biphasic with a maximum between -10 and -20 mV.

In this report, we describe the properties of sphingolipid-gated Ca release from a human umbilical vein endothelial cell line (EA.hy926)(9) . Through the use of a novel strategy for the molecular characterization of intracellular Ca channels, we establish the approximate molecular size of transcripts encoding this novel activity. By doing so, we provide new structural information about this novel activity, further distinguishing it from the inositol 1,4,5-trisphosphate receptor and the ryanodine receptor.


EXPERIMENTAL PROCEDURES

Materials

ATP, MgCl(2), SPC, heparin, nifedipine, conotoxin GVIa, and saponin were obtained from Sigma. Fluo-3 was from Molecular Probes, and digitonin, A23187, and inositol 1,4,5-trisphosphate were from Calbiochem.

Cells

The human endothelial cell line EA.hy926 was originally described by Edgell and co-workers(9) . It is an immortal cell line that forms gap junctions between adjacent cells, and expresses factor VIII antigen. It is grown in DMEM supplemented with 5% fetal bovine serum and hypoxanthine/aminopterin/thymidine, as described(9) .

Ca Release Assays

Ca release from saponin-permeabilized cells was performed as described previously, using the fluorescent Ca indicator fluo-3(7, 10) .

Oocyte Holder

A custom designed oocyte holder was machined by the Electronics Instrument Shop at Duke University (Fig. 1). The holder was designed to insert into standard 1 cm times 1 cm polystyrene optical cuvettes. The holder consists of a polyethylene mesh screen (Spectra/Mesh, 146376) supported by an acrylic framework. The unit rests on the lip of the cuvette and is suspended 9 mm above the bottom of the cuvette, providing adequate clearance for a Cell Stirrer stir bar (Bel-Art). Free access into the interior of the cuvette is provided for the addition of reagents through a 7.9-mm diameter hole in the top of the holder. The time required for complete mixing of added reagents is less than 8 s. This was determined by monitoring the change in fluorescence signal following the addition of 20-200 pmol of Ca.


Figure 1: Schematic view of oocyte holder. A, without cuvette; B, in a standard optical cuvette accompanied by a Cell Stirrer stir bar.



Preparation of Oocytes

Xenopus oocytes were obtained from female Xenopus laevis as previous described(11) . The oocytes were prepared by collagenase treatment, and healthy stage 6 oocytes were selected. The oocytes were kept at 19 °C in modified Barth's solution consisting of 88 mM NaCl, 1 mM KCl, 0.205 mM CaCl(2), 0.165 mM Ca(NO(3))(2), 0.82 mM MgSO(4), 2.4 mM NaHCO(3), 10 mM HEPES, pH 7.4, supplemented with 1 mg/ml Ficoll, 1 mg/ml bovine serum albumin, 1 times penicillin (10 µg/ml), streptomycin (0.1 mg/ml), and 50 µg/ml gentamicin for up to 4 days before use. Use of X.laevis was approved by the Duke University Institutional Animal Care and Use Committee.

Oocyte Permeabilization

Oocytes were permeabilized as described previously(12) . Digitonin was prepared by boiling at 90 °C for 10 min followed by filtration through Whatman No. 1 paper. Groups of up to 20 X. oocytes were washed 3 times with 20 mM HEPES-KOH, 5 mM NaCl, 130 mM KCl, pH 7.4, and placed in 5 ml of a permeabilization medium consisting of 20 mM HEPES-KOH, 5 mM NaCl, 130 mM KCl, 1 uM fluo-3, pH 7.4, 1 mM ATP, 2 mM MgCl, and 10 µM digitonin. Cells were incubated at 4 °C for 30 min under slow horizontal shaking (Belly Dancer, Stovall Life Science). The cells were then transferred to fresh permeabilization solution without digitonin and incubated for another 15 min under the same conditions. The permeabilization of these oocytes was confirmed by trypan blue staining.

Isolation of mRNA from EA.hy926 Endothelial Cells

Messenger RNA from the EA.hy926 endothelial cell line was isolated from 25 confluent T-75 flasks using the guanidinium isothiocyanate method (13) (RNAzol, Tel-test). Poly(A) RNA was obtained using oligo(dT) cellulose chromatography(14) . Poly(A) mRNA was size-selected on a 5-20% sucrose gradient(15) .


RESULTS

Concentration Dependence in Endothelial Cells

To establish the sensitivity of the EA.hy926 endothelial cell line to SPC, we first tested a range of concentrations from 10 to 100 µM. The maximal rate of Ca efflux was at 40 µM SPC. Higher concentrations are inhibitory. At all concentrations, Ca efflux was triphasic (Fig. 2). A fast burst of Ca release is followed by a slow response, which then accelerates. The rate of the initial fast burst exceeds the time resolution of the system, which is limited by the speed that the solution in the cuvette is mixed. However, the concentration dependence for SPC of all three responses (fast, slow, and accelerated) is similar (Fig. 2, b and c). SPC appears to inhibit Ca efflux at concentrations greater than 40 µM. A similar concentration dependence was observed in rat basophilic leukemia cells(7) . There, we presented evidence indicating that the slow rate resulted from differential access of the Ca stores, as microsomes prepared from rat basophilic leukemia cells did not demonstrate biphasic release. The initial rapid burst may represent release of Ca from stores immediately adjacent to the plasma membrane.


Figure 2: SPC-gated Ca release from EA.hy926 human endothelial cells. Cells at 37 °C are in HEPES (20 mM), NaCl (5 mM), KCl (130 mM), MgCl(2) (2 mM), ATP (0.4 mM), saponin (2.5 µg/ml), and fluo-3 (1 µM). Ca release is normalized. At the indicated time, 20 µM SPC is added. This is followed by a second addition of 20 µM SPC, 2 µM inositol 1,4,5-trisphosphate, and finally 10 µM A23187. a, data for 40 µM SPC. An initial fast release is followed by a slow phase, which then accelerates. The initial fast release exceeds the time resolution of the system. b, dose response for fast Ca release phase. Because release of Ca exceeds the time resolution of the system, data are presented as total pmol of Ca released during this phase only. c, dose-response curve for slow and accelerated responses. Errorbars are standard error of the mean. For both b and c, each point represents a total of six determinations (two independent experiments performed on three separate days).



Ca Channel Inhibitors Do Not Antagonize SPC-induced Ca Release

We examined the effects of a panel of Ca channel blockers in antagonizing the release of Ca by SPC from endothelial cells. Nifedipine, 10-100 µM, conotoxin GVIa, 0.1-10 µM, and heparin, 50 µg, did not block the release of Ca by 20 µM SPC. This pharmacologic profile is similar to that observed previously from rat basophilic leukemia cells(7) .

Ca Release by Sphingosylphosphorylcholine Can Be Transfected by Injection of mRNA

To determine whether SPC sensitivity could be transfected into Xenopus oocytes, we isolated poly(A) RNA from EA.hy926 cells, and microinjected 50 ng into Xenopus oocytes. Following injection of mRNA, we observed that 60% of the ionophore-sensitive Ca was now released by SPC (Fig. 3). Control RNA (a 7.5-kb marker mRNA from Life Technologies, Inc.) did not elicit a response. Hence, sensitivity of the EA.hy926 cell to SPC can be transfected to oocytes. To determine the molecular size of the sphingolipid-gated Ca-permeable channel from endothelial cells, poly(A) mRNA was size fractionated using a sucrose gradient(15) . Approximately 50 ng of mRNA from each fraction was than microinjected into Xenopus oocytes. The effect on SPC-sensitive Ca efflux was examined 48 h after injection, following digitonin-permeabilization of the oocyte (Fig. 4). SPC-mediated Ca release is conferred by a single peak of mRNA. Size characterization using denaturing agarose gel electrophoresis(14) , against known standards (Life Technologies, Inc.) reveals the peak to have a mean molecular size of 1.5-2 kb. Thus, it is unlikely that SPC gates Ca release through ryanodine or inositol 1,4,5-trisphosphate-gated channels, which are encoded by transcripts of 16 kb (16) and >8 kb(17) , respectively.


Figure 3: Sphingolipid sensitivity transferred to Xenopus oocytes by poly(A) RNA. Xenopus oocytes were injected either with 50 ng of poly(A) RNA isolated from EA.hy926 cells or with diethylpyrocarbonate treated water. Two days later, oocytes were permeabilized, washed, and assayed for Ca efflux as described under ``Methods.'' At the indicated times, additions of 40 µM SPC and 10 µM A23187 were made. In these tracings, two oocytes were used for each determination.




Figure 4: Sucrose gradient of EA.hy926 mRNA. A sucrose gradient of 300 µg of poly(A) mRNA was performed as described under ``Experimental Procedures.'' Oocytes were injected with 50 ng each of mRNA. Oocytes were scored positive if Ca release in mRNA-injected oocytes was twice that of water-injected controls. The percent of the mRNA-injected oocytes scoring positive for SPC-gated Ca release (leftaxis) and the RNA concentration of the fraction (rightaxis) are plotted against the gradient fraction number. When compared with RNA standards (Life Technologies, Inc.) on a denaturing agarose gel, the peak fractions 6-8 have a size of 1.5-2 kb. Numbers in parentheses are total number of oocytes injected with RNA from each fraction.




DISCUSSION

Sphingolipid-gated Ca signaling is a recently revealed mechanism by which cells can respond to extracellular messages with an intracellular release of Ca(2, 3, 4, 5) . This new mechanism operates in parallel to, or in addition to inositol 1,4,5-trisphosphate-gated Ca release and cADP-ribose gated Ca release(1) . Platelet-derived growth factor (18) stimulates the production of both sphingosine and inositol trisphosphate. The contribution in vivo of these pathways to intracellular Ca release is likely to differ among cell and tissue types. We have observed tissue specific differences in Ca release in response to SPC, suggesting that sphingolipid-mediated Ca release could play a role in the tissue-specific resonse to cell signaling. (^2)

Because unpermeabilized cells also release Ca in response to SPC, a second mechanism mediating the cellular response to sphingolipids must also be present. This pathway is likely to be mediated by the activation of the phosphoinositide cascade through binding of SPC at specific plasma membrane receptors. Release of Ca from intact cells may represent activation of the lysophosphatidic acid receptor(19) .

The mechanism of Ca release in response to sphingolipids has not been completely characterized. In vivo, sphingosine accumulates in concentrations that are sufficient to cause Ca release(18, 20) . However, to cause Ca release, sphingosine must either be metabolized to another derivative or must stimulate the elaboration of another messenger(5) . Sphingosine 1-phosphate has been suggested to be the biologically active ligand directly mediating Ca release(6, 21) . However, SPC is a stable, soluble direct acting agonist of Ca release. Because of the highly lipophilic and labile nature of sphingosine-1-phosphate, (^3)SPC has been studied as a surrogate agonist for Ca release.

In cardiac muscle, the potential role for sphingolipids as regulators of Ca homeostasis appears more complex. Sabbadini and co-workers (22, 23, 24) have established that sphingosine inhibits Ca efflux through the ryanodine receptor and L-type Ca channels(22, 23) . SPC, on the other hand, causes release of Ca from sarcoplasmic reticulum(24) .

The molecular properties of the sphingolipid-gated Ca-permeable channel in endothelial cells suggest it to be a channel with subunits of intermediate molecular mass, of some 40-70 kDa. Based on its size and on its electrophysiologic properties, the channel may resemble the ionotropic glutamate receptors (cf. (25) ) or the purinergic P2x receptor(26, 27) . ATP-gated cation channels are only partially blocked by divalent and trivalent cations(28) . The findings reported here support the hypothesis that the channel represents a new member of the intracellular ligand-gated Ca channel family.

In this report, we describe a novel system with which to characterize the molecular properties of other intracellular ion channels. This system is also adaptable for the investigation of surface membrane receptors. We have also observed Ca efflux from nonpermeabilized oocytes previously injected with the neuromedin B receptor (NMB-R/pcDNAI) (29) or the platelet-derived growth factor receptor (PR-FR(30) ) (not shown). Although we describe the use of a fluorescent Ca indicator, this system can easily be adapted for the use of other types of fluorescent indicators, as well as for the use of radionuclides.


FOOTNOTES

*
This work was supported by Grant HL02361 from the NHLBI, National Institutes of Health. 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.

§
To whom correspondence should be addressed: Box 3547, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-4151; Fax: 919-681-8978; lak{at}hodgkin.mc.duke.edu.

(^1)
The abbreviations used are: SPC, sphingosyl phosphorylcholine; kb, kilobase(s).

(^2)
S. Kim, V. Lakhani, D. J. Costa, and L. A. Kindman, unpublished observations.

(^3)
S. Spiegel and D. Gill, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. C. Edgell (Dept. of Pathology, University of North Carolina-Chapel Hill) for providing us with EA.hy926 cells, J. F. Battey (National Institutes of Health) for the neuromedin B receptor construct, and Dr. L. T. Williams (University of California at San Francisco) for the platelet-derived growth factor and fibroblast growth factor receptor chimeras PR-FR-wt and PR-FR Y/F766. We thank Dr. J. Almenoff for helpful comments.


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