(Received for publication, December 12, 1994; and in revised form, January 6, 1995)
From the
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.
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). (
)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.
Figure 1: Schematic view of oocyte holder. A, without cuvette; B, in a standard optical cuvette accompanied by a Cell Stirrer stir bar.
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 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).
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.
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. (
)
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, (
)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.