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INTRODUCTION |
One of the biochemical pathways for metabolism of arachidonic acid
is the cytochrome P450 monooxygenase pathway, which results in
formation of 4 regio- and stereoisomeric products; cis-5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids
(EETs).1 Compared with our
knowledge of the lipoxygenase and cyclooxygenase pathways for
arachidonic acid metabolism, relatively little is understood about the
epoxygenase pathway. Numerous physiological roles have been suggested
for the EETs and collectively, the EETs appear to have potent effects
on ion channels (1-3).
One of the mechanisms for regulation of intracellular calcium dynamics
in response to hormones and other agonists is through the
"capacitative pathway" as originally described by Putney (4, 5).
Activation of this pathway occurs through G protein receptor-mediated activation of phospholipase C, which catalyzes the hydrolysis of
phosphatidylinositol bisphosphate yielding inositol trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Ins(1,4,5)P3
binds to its receptor on the intracellular calcium stores, initiating
release of stored calcium (6). As the stores are depleted of calcium, a
second messenger termed "calcium influx factor" (CIF) is released
(7). CIF induces influx of extracellular calcium through second
messenger operated channels (SMOC) in the plasma membrane, thereby
coupling calcium entry to depletion of internal stores. To date, the
identity of CIF remains unknown.
In previous work from our laboratory (8), we reported that capacitative
calcium influx was linked to arachidonic acid release by activation of
the 85-kDa cytosolic phospholipase A2 (cPLA2) in human U937 lymphoma cells and in rat cortical astrocytes (9), suggesting that arachidonic acid, or a metabolite thereof, was a
component of CIF. Recent work from several other groups has shown that
the actions of cytochrome P450 monooxygenase may also be coupled to
capacitative calcium influx (10, 11). Hoebel et al. (11)
recently reported that functional P450 activity was critical to
regulation of store-operated calcium influx and proposed that an EET
may constitute the driving force for capacitative calcium entry in
endothelial cells. Graier et al. (10) have presented
evidence suggesting that 5,6-EET stimulates capacitative calcium influx
in endothelial cells, consistent with CIF. However, if 5,6-EET is CIF
or a component thereof, then it should have similar effects in all
cells lines which signal through Ins(1,4,5)P3.
Although CIF is hypothesized to have its primary actions at an
intracellular level, it may also be released into the extracellular environment, acting as a paracrine signal for SMOC calcium influx independent of release of calcium from intracellular stores. In Jurkat
cells, CIF was released to the extracellular medium upon stimulation
with phytohemmaglutinin (7). Endothelium-derived hyperpolarizing
factor, which induces NO- and prostaglandin I2-independent relaxation of vascular smooth muscle, is released in response to
agonists operating through
Ins(1,4,5)P3-dependent signaling (12-14).
Harder et al. (15) and Gebremedhin et al. (16)
reported that cat brain converted arachidonic acid to EETs, which
dilated cerebral arteries, implying that endothelium-derived
hyperpolarizing factor may be an EET. Hecker et al. (17)
reported an NO- and cyclooxygenase-independent relaxation of porcine
aortic rings which was mediated by calcium-activated K+
channels (KCa) and required the combined actions
of PLA2 and P450, precisely those systems which may mediate
CIF. Additional reports indicate that KCa are
closely coupled to capacitative calcium influx (18, 19). Taken
together, these reports lead to the speculation that
endothelium-derived hyperpolarizing factor may represent a paracrine
function of CIF. In the present report we present evidence suggesting
that 5,6-EET is a CIF in astrocytes and when released into the
extracellular environment may participate in regulation of local
cerebrovascular tone.
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EXPERIMENTAL PROCEDURES |
Materials
Young adult rats and rat pups (1-2 days old) were purchased
from Hilltop Lab Animals (Scottdale, PA). Dulbecco's modified essential medium and Dulbecco's phosphate-buffered saline (DPBS) with
or without calcium were purchased from Life Technologies, Inc. (Grand
Island, NY). Fetal calf serum was from Hyclone (Logan, UT). Arachidonic
acid and EETs (free acids) were purchased from Cayman Chemical (Ann
Arbor, MI). Fura-2 AM and calcium calibration buffers were obtained
from Molecular Probes (Eugene, OR). SKF96365, arachidonyltrifluoromethyl ketone (AAOCF3), econazole,
SKF525A, and thapsigargin were purchased from Calbiochem (San Diego,
CA). Indomethacin was obtained from Sigma.
N-Methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) was synthesized by Dr. J. R. Falck (University of Texas Southwestern Medical Center, Dallas, TX).
Methods
Cell Culture--
Astrocytes were prepared from 1-2-day-old rat
pups as described previously (20). In brief, cortices were isolated,
cleaned of white matter and meninges, minced, and trypsin-digested for 10 min. The dissociated tissue was diluted into Dulbecco's modified essential medium (supplemented with 10% fetal calf serum and 2 mM glutamine) and seeded into 75-cm2 flasks at
an initial density of 1-2 × 106 cells per flask.
Flasks were cultured to confluency (10-14 days) in 5%
CO2/air at 37 °C. Medium was changed every 2-3 days.
During medium changes, flasks were forcefully shaken to remove
microglia, which were decanted with the medium. On reaching confluency,
flasks were trypsinized (0.25% trypsin, 0.02% EDTA in saline) for 1 min. The trypsin/EDTA solution was aspirated and the cell monolayer (still adherent to the flask) was covered with 10 ml of Dulbecco's modified essential medium and incubated at 37 °C for 10 min,
sufficient time to lift the cells from the flask bottom. Lifted cells
were washed and plated onto collagen-coated 35 × 100-mm tissue
culture dishes at a density of 3.2 × 105 cells per
dish. For arachidonic acid release assays, 0.5 × 105
cells were plated into 25-mm diameter tissue culture wells (6 wells/plate). Cells were then cultured to confluency (2 weeks). Astrocyte cultures were characterized for purity as described previously (20) and found to be >98% pure as assessed by the presence
of glial fibrillary acidic protein. Cells were used for experiments at
a total of 4 weeks after removal from the rat.
Fura-2 AM Loading--
[Ca2+]i was
measured with the ratiometric dye, Fura-2, as described previously
(21). Astrocytes were washed 3 times with DPBS supplemented with 2%
fatty acid-free bovine serum albumin and 1 mM glucose and
placed in 1.5 ml of this media for loading. Cells were loaded with 5 µM Fura-2 AM for 50 min. at room temperature. This
loading procedure resulted in complete dye hydrolysis as determined by
scanning the excitation spectra of loaded cells, with negligible
sequestration of dye in subcellular organelles (22). The intracellular
concentration of Fura-2 was monitored as described previously by Poenie
et al. (23).
Measurement of
[Ca2+]i--
[Ca2+]i was
measured at room temperature using a Ratiomaster (Photon Technologies
Int., South Brunswick, NJ) microspectrophotometry system as described
previously (24). Excitation light was provided by a xenon arc lamp
coupled to a scanning monochrometer which alternated excitation light
between 350 and 380 nm. Bandpass was set at ±2 nm. Excitation light
was delivered to the cells via fiber optics through the epifluorescence
port of a Zeiss Standard 16 microscope coupled to a Zeiss Achroplan
20× water immersion lens. Use of the water immersion lens allowed
in situ measurements of the cells while in the culture dish
and easy manipulation. However, the optical properties of the lens
necessitated ratioing of Fura-2 at 350 and 380 nm. Emission was
measured at 510 nm, via a microphotometer. The entire system for data
collection and analysis was computer driven.
After loading, astrocytes were washed twice and placed in 2 ml of DPBS
supplemented with 1 mM glucose and 1% fatty acid-free bovine serum albumin. For measurement of [Ca2+]i,
a field of 1-2 astrocytes was selected using slit width adjustments on
the microphotometer. Basal [Ca2+]i was recorded
for several seconds, at a sampling rate of 1 ratio every 0.5 s.
For experiments using agonists (arachidonic acid, 5,6-EET, and
thapsigargin) or inhibitors (econazole, SKF96365, SKF525A,
AAOCF3, and MS-PPOH), these agents were added to the tissue
culture dish in a volume of 200 µl. This volume assured almost
immediate mixing with the buffer, as determined by dye diffusion. For
experiments using indomethacin, Fura-2-loaded astrocytes were incubated
with 10 µM indomethacin for 15 min prior to measurement of [Ca2+]i. Each experiment was performed on a
separate culture of astrocytes.
[Ca2+]i was calculated as described previously
(25) using a correction for intracellular viscosity (23).
Autofluorescence at 350 and 380 was recorded from wells of astrocytes
not loaded with Fura-2 and was subtracted from all measurements.
Agonist-stimulated [Ca2+]i concentrations were
normalized to the basal [Ca2+]i level in each
experiment and are expressed as a percent of basal. Leakage of Fura-2
into the medium was monitored by measurement of intracellular Fura-2
concentration at the isobestic wavelength, 362 nm (25).
For calcium-free experiments, cells were placed in calcium-free DPBS
containing 1 mM glucose and 1% fatty acid-free bovine serum albumin immediately prior to the experiment. EGTA (0.5 mM) was added to chelate any residual calcium. Astrocytes
were not exposed to calcium-free conditions for longer than 15 min. As described previously, this treatment did not result in lifting of cells
from the monolayer.
Arachidonic Acid Release--
Arachidonic acid release was
measured as described previously (8, 26). Briefly, 1 × 106 cells grown in a 25-mm diameter well were radiolabeled
by addition of 0.5 µCi of [3H]arachidonic acid to the
growth medium for 24 h prior to the assay. Incorporation of
radiolabel was 95%. After labeling, the cells were washed three times
and placed in DPBS supplemented with 1 mM glucose and 1%
fatty acid-free bovine serum albumin. Next, cells were preincubated
with 10 µM econazole, 5 µM
AAOCF3, 1 µM MS-PPOH, or 10 µM
SKF525A for 2 min in a final volume of 1.0 ml. Following preincubation,
cells were stimulated with 1 µM thapsigargin for 2 min.
Radioactivity released into the medium after thapsigargin stimulation
was determined by scintillation counting. Controls consisted of
unstimulated cells, or cells treated with inhibitors alone. Econazole,
AAOCF3, MS-PPOH, and SKF525A did not significantly alter
basal arachidonic acid release as compared with untreated controls. For
experiments conducted under calcium-free conditions, calcium-free DPBS
supplemented with 1 mM glucose, 1% fatty acid-free bovine
serum albumin, and 0.5 mM EGTA was utilized for all
incubations. Results are expressed as percent of control (unstimulated) cells.
Preparation and Handling of EETs--
EETs are highly labile in
the aqueous environment. Concentrated EET stock solutions were stored
in aliquots at
70 °C in acetonitrile. A fresh aliquot was used for
each experiment. Just prior to use, the acetonitrile solution was dried
under N2 and EETs were resuspended in ethanol and kept on
ice. Aliquots were removed and resuspended in DPBS as required for
agonist stimulation. The final concentration of ethanol in all
experiments did not exceed 0.1%.
Cranial Window--
The acute cranial window technique and
in vivo microscopy were utilized to examine the effect of
5,6-EET on rat cerebral arteriolar diameter, as described previously
(27). Briefly, young adult male Sprague-Dawley rats were anesthetized
with thiopental (75 mg/kg) and supplemented with pentobarbital. After
completion of a tracheotomy, each rat was ventilated with room air. The
end-expiratory CO2 of each rat was continuously monitored
with a capnometer (Transverse Medical Monitors, model 2200) and was
maintained at approximately 30 mm Hg by adjusting the respirator rate
and volume. Arterial blood pressure was measured via a cannula inserted
into the right femoral artery. Arterial samples were periodically
analyzed with a Corning Blood Gas Analyzer to ensure normal
PaO2, PaCO2, and blood
pH. A cannula was also inserted into the right femoral vein for
systemic administration of supplemental anesthetic.
Pial arteries were visualized using a cranial window implanted into the
scalp through a midline incision. The skin and fascia were retracted
and a 3-mm diameter craniotomy was made over the left parietal cortex
using a trephine. With the aid of a surgical microscope, microscissors
were used to remove the dura and expose the pial surface of the brain.
A 12-mm diameter cranial window frame with a 6.5-mm diameter glass
window was implanted over the craniotomy. The cranial window was
equipped with three openings. Two openings were used as an inlet and
outlet for filling the space under the cranial window with test
solutions. The inlet and outlet valves were positioned such that the
test solutions flowed over the cortical surface as viewed through the
cranial window. The third opening of the cranial window was connected to a Statham pressure transducer for continuous measurement of intracranial pressure. The outlet of the window was connected to
plastic tubing whose open end was placed at a fixed level to give a
constant intracranial pressure of 5 mm Hg throughout the experiment.
The space under the window and the plastic tubing were filled with
artificial CSF. This fluid was equilibrated with gas containing 5.9%
CO2, 6.6% O2, and 87.5% N2, which
produces pH and gas tensions in a normal range for CSF. The vehicle for all agents applied under the cranial window was artificial CSF. The
diameter responses of three to five arterioles were studied in each rat
using a Vickers image-splitting device as described previously (28).
The responses of the arterioles in a given rat were averaged, and this
single number was used to compute the average for a group of rats.
After implantation of the cranial window, baseline arteriolar diameter
was established by washing the window with 1 ml of artificial CSF at
5-min intervals. At the end of each 5-min interval, baseline
measurements of pial arteriolar diameter were recorded. Next, 5,6-EET
was infused under the window in increasing concentrations (10
9-10
5 M) in a total volume
of 1 ml of artificial CSF at 5-min intervals. Pial arteriolar diameters
were measured at 2 and 5 min after the infusion of each test solution.
Statistics--
Both one-way and repeated measures ANOVA were
performed and were followed by Tukey-Kramer comparisons to determine
differences between the groups using Super Anova statistical software
for Macintosh. A value of p < 0.05 was considered significant.
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RESULTS |
Exogenous Arachidonic Acid Stimulates Calcium Influx in
Astrocytes--
Basal [Ca2+]i in astrocytes was
88 ± 5 nM, consistent with published reports (21).
Exogenous arachidonic acid (1-100 µM) stimulated a
dose-dependent elevation in [Ca2+]i
in astrocytes (data not shown). A representative tracing of the
[Ca2+]i elevation stimulated by arachidonic acid
is shown in Fig. 1A.
Arachidonic acid (10 µM) stimulated an elevation in [Ca2+]i in rat cortical astrocytes to 265 ± 14% of basal (n = 6). The elevation in
[Ca2+]i was not immediate, but appeared to
require approximately 175 s for initiation. Although lag time
after application of an agonist is often difficult to compare
accurately, the lag time for the arachidonic acid response greatly
exceeded the usual lag time for other agonists and was consistently
observed in all experiments. This delay suggests that metabolism of
arachidonic acid may be necessary to produce an elevation in
[Ca2+]i. Pretreatment with the cyclooxygenase
inhibitor indomethacin (10 µM, 15 min) had no effect on
either basal or arachidonic acid-induced elevation of
[Ca2+]i (Fig. 1A, broken line),
suggesting that a cyclooxygenase metabolite was not involved in
arachidonic acid-induced elevation of
[Ca2+]i.

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Fig. 1.
Arachidonic acid (AA)-induced elevation of
[Ca2+]i in astrocytes is inhibited by
econazole. Astrocytes were loaded with Fura-2 and
[Ca2+]i was measured as described under
"Methods." Basal [Ca2+]i in astrocytes was
88 ± 5 nM. Results were normalized and expressed as a
percent of basal [Ca2+]i. The solid
line of A, 10 µM arachidonic acid was
added at the indicated time point. The broken line, cells
were pretreated with 10 µM indomethacin for 15 min,
followed by stimulation with 10 µM AA. Indomethacin had
no effect on AA-induced elevation of [Ca2+]i. In
B (solid line), separate astrocyte cultures were
treated with 10 µM econazole, followed by 10 µM arachidonic acid. The broken line,
astrocytes were stimulated with 10 µM AA in
Ca2+-free DPBS. Tracings are representative of six separate
experiments performed on different days.
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Removal of extracellular [Ca2+]i (Fig.
1B, broken line) completely
blocked AA-induced elevation of [Ca2+]i,
suggesting that AA-induced [Ca2+]i elevation in
astrocytes is due to influx of extracellular Ca2+. Addition
of the cytochrome P450 inhibitor econazole (10 µM) had no
effect on basal [Ca2+]i (Fig. 1B).
However, a 2-min econazole pretreatment dramatically attenuated the
arachidonic acid-induced elevation of [Ca2+]i to
130 ± 9% of basal (n = 6). Similar results were observed with a second inhibitor of P450, SKF525A (data not shown). These results suggest that the arachidonic acid-induced elevation in
astrocyte [Ca2+]i may require cytochrome P450
metabolism of arachidonic acid.

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Fig. 2.
Thapsigargin stimulated elevation of
[Ca2+]i in the astrocyte is blocked by inhibition
of cytochrome P450 and cPLA2. A, for
measurement of [Ca2+]i (open bars)
astrocytes were loaded with Fura-2 and the maximum
[Ca2+]i elevation achieved after thapsigargin
stimulation was measured as described in the legend to Fig. 1.
Arachidonic acid release (closed bars) was measured in
[3H]arachidonate-labeled astrocytes as described under
"Methods." For all experiments, astrocytes were placed in DPBS with
or without extracellular calcium as indicated. Inhibitors were added
for 2 min prior to thapsigargin stimulation. Results represent
mean ± S.E. for four to 10 separate experiments
([Ca2+]i) and for three separate experiments
(arachidonic acid release); *, p < 0.01 versus 1 mM Ca2+. B,
representative tracings of thapsigargin-stimulated
[Ca2+]i elevation are shown for all conditions in
A. Arrow indicates addition of 1 µM
thapsigargin.
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Arachidonic Acid Release Is Coupled to Thapsigargin-stimulated
[Ca2+]i Elevation in Astrocytes--
If a
metabolite of arachidonic acid is a component of CIF, then arachidonic
acid should be released in response to depletion of intracellular
calcium stores. Using [3H]arachidonic acid-labeled
astrocytes, we investigated the total release of arachidonic acid and
metabolites in response to a 2-min stimulation with thapsigargin.
Activation of the capacitative pathway with thapsigargin induced
release of arachidonic acid (and/or metabolites) of 399 ± 43% of
basal (Fig. 2A, filled bars). In calcium-free medium
supplemented with EGTA, thapsigargin-stimulated release of arachidonic
acid (and/or metabolites) was reduced to 233 ± 13% of basal.
Similar to our observations in U937 (8), these results suggest that a
portion of the thapsigargin-stimulated arachidonic acid release is
coupled to influx of extracellular calcium, while a portion is also
coupled to depletion of intracellular calcium stores. Thus,
PLA2 activity may be associated with release of calcium
from intracellular stores. Inhibition of cytochrome P450 with
econazole, SKF525A, or MS-PPOH did not inhibit thapsigargin-induced arachidonic acid release. Inhibition of cPLA2 with
AAOCF3 inhibited thapsigargin-stimulated arachidonic acid
release to control levels.
Thapsigargin-induced Elevation of [Ca2+]i Is
Blocked by Inhibitors of Cytochrome P450 and cPLA2--
We
have previously shown that capacitative calcium influx requires the
action of cPLA2 and release of arachidonic acid in U937
cells (8). The results above suggest that arachidonic acid elevates
[Ca2+]i in the astrocyte, possibly through a P450
metabolite. Therefore, we investigated whether cPLA2 and
P450 were also linked to capacitative calcium influx in the astrocyte.
Thapsigargin is a pharmacological agent that inhibits the
Ca2+-ATPase of the intracellular calcium store and
activates capacitative calcium influx independent of phospholipase C
(29). The thapsigargin-stimulated elevation of
[Ca2+]i is dependent on 2 sources of calcium,
release from intracellular stores followed by capacitative influx of
extracellular calcium. If capacitative calcium influx is mediated by
release of arachidonic acid and subsequent metabolism via P450, then
capacitative calcium influx should be inhibited by blockade of either
cPLA2 or P450. To test this we utilized econazole, SKF525A
(30), and MS-PPOH (31), inhibitors of cytochrome P450 and
AAOCF3, a selective inhibitor of cPLA2 (32). A
summary of these experiments appears in Fig. 2A and
representative tracings in Fig. 2B.
In the astrocytes, thapsigargin elevated [Ca2+]i
to 432 ± 62% of basal (Fig. 2, A and B, trace
1). To further dissect this response, the relative size of
thapsigargin-releasable intracellular calcium stores in astrocytes was
determined by stimulation with thapsigargin in calcium-free DPBS
supplemented with 0.5 mM EGTA. Under these conditions,
thapsigargin elevated [Ca2+]i by only 196 ± 23% of basal (Fig. 2, A and B, trace 2),
approximately half that observed in experiments where extracellular calcium was 1 mM.
To assess the effect of cPLA2 or P450 inhibitors on
capacitative calcium influx, astrocytes were pretreated with 5 µM AAOCF3, 10 µM econazole, 10 µM SKF525A, or 1 µM MS-PPOH for 2 min.
Inhibition of cPLA2 with AAOCF3 or inhibition
of cytochrome P450 with econazole or SKF525A had no effect on
thapsigargin-stimulated elevation of [Ca2+]i in
calcium-free medium, indicating that these compounds had no effect on
release of calcium from intracellular stores (data not shown). In DPBS
containing 1 mM extracellular calcium (Figs. 2,
A and B, trace 5) inhibition of cPLA2
with AAOCF3 blocked capacitative calcium influx and
produced [Ca2+]i levels consistent with release
of calcium from intracellular stores alone. In calcium replete medium,
inhibition of cytochrome P450 with 10 µM econazole or 10 µM SKF525A also inhibited capacitative calcium influx
(Fig. 2, A and B, traces 3 and 4).
These two P450 inhibitors blocked the maximum thapsigargin-stimulated
[Ca2+]i elevation to the same extent as
calcium-free medium, suggesting that capacitative Ca2+
influx was inhibited. However, the sustained phase of
[Ca2+]i elevation was only partially inhibited.
The cytochrome P450 enzymes comprise a large family of isoforms (33) of
which econazole and SKF5125A are generalized inhibitors which block many isoforms. We therefore used an additional inhibitor of cytochrome P450, MS-PPOH (31). MS-PPOH is a "suicide substrate" inhibitor of
P450 arachidonic acid epoxygenase, designed to resemble the substrate
arachidonic acid and inactivate the enzyme. In rat renal microsomes,
MS-PPOH was a potent and selective inhibitor of arachidonic acid
epoxygenase activity (31). In the astrocyte, 1 µM MS-PPOH inhibited thapsigargin-stimulated capacitative calcium influx to a
level consistent with depletion of intracellular calcium stores only
(Fig. 2, A and B, trace 6). Taken together, these results suggest that formation of CIF requires the combined actions of
cPLA2 and cytochrome P450 and may involve an epoxide of
arachidonic acid.
5,6-EET Elevates [Ca2+]i in
Astrocytes--
If an EET is a component of CIF, then application of
exogenous EET should stimulate capacitative calcium influx directly
through SMOC, without affecting intracellular calcium stores.
Therefore, we examined the effect of all 4 EETs on
[Ca2+]i in astrocytes. At a concentration of
10
6 M, 8,9- (n = 3), 11,12- (n = 3), and 14,15-EET (n = 6) had no effect on [Ca2+]i (data not shown). As shown in
Fig. 3, 5,6-EET induced a
dose-dependent increase in [Ca2+]i in
astrocytes, which was elevated to 150% of basal by 10
10
M 5,6-EET and to a maximum of 310% of basal by
10
7 M. The nanomolar to picomolar activity of
5,6-EET suggests that this response was not a result of lipid-induced
alterations in membrane fluidity. However, the dose-response curve for
5,6-EET was bell-shaped, and at higher concentration (10
6
M) 5,6-EET consistently produced a submaximal response in
all experiments. However, this decreased response was not statistically significant. Consistent with reports by Graier et al. (10), we have observed similar 5,6-EET-induced increases in
[Ca2+]i in cultured endothelial cells (data not
shown). The hydration products of 14,15- and 5,6-EET, 14,15- and
5,6-dihydroxyeicosatrienoic acid, had no effect on
[Ca2+]i in the astrocyte.

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Fig. 3.
5,6-EET stimulates elevation of
[Ca2+]i in astrocytes. Astrocytes were
loaded with Fura-2 as described in the legend to Fig. 1. 5,6-EET was
added at the indicated concentrations and the maximum elevation in
[Ca2+]i was measured as described under
"Methods." Results represent mean ± S.E. for the maximum
[Ca2+]i achieved upon addition of 5,6-EET in four
to eight separate experiments. Please note that the S.E. for
10 10 M is too small to show graphically;
a, p < 0.01 versus all other
concentrations; b, p < 0.01 versus 10 10, 10 8, and
10 7 M; c, p < 0.01 versus 10 9 and 10 10
M.
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5,6-EET Induces Influx of Extracellular Calcium, and Not Depletion
of Intracellular Calcium Stores--
If 5,6-EET is CIF, then it should
activate influx of extracellular calcium through SMOC, and should not
induce release of calcium from intracellular stores. When astrocytes
were placed in calcium-free DPBS supplemented with 0.5 mM
EGTA to chelate residual calcium, 5,6-EET did not elevate
[Ca2+]i, suggesting that 5,6-EET-induced influx
of extracellular calcium without effect on intracellular calcium stores
(Fig. 4A, broken line).

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Fig. 4.
5,6-EET-stimulated
[Ca2+]i elevation is blocked by SKF96365.
A, Fura-2 loaded astrocytes were stimulated with 156 nM 5,6-EET at the indicated time point in DPBS with ( ) or
without (- - -) 1 mM Ca2+. B,
separate cultures of Fura-2 loaded astrocytes in DPBS containing 1 mM Ca2+ were pretreated with SKF96365 followed
by 5,6-EET as shown. Tracings are representative of four separate
experiments.
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In addition to SMOC calcium influx channels, many cells also have
receptor-operated channels (34). Receptor-operated channels are
activated by a ligand binding to its receptor, which directly controls
the opening of a calcium channel. In contrast, SMOC are controlled by a
second messenger (CIF). To discriminate between receptor operated
channels and SMOC we first utilized the inhibitor of SMOC, SKF96365
(35). Fig. 4A shows a typical elevation in [Ca2+]i observed in response to 156 nM 5,6-EET in astrocytes. The 5,6-EET-induced elevation in
[Ca2+]i was completely inhibited by pretreatment
with 1 µM SKF96365 (Fig. 4B). Nimodipine (50 µM) had no effect on 5,6-EET-stimulated elevation of
[Ca2+]i, indicating 5,6-EET did not activate
voltage-gated calcium channels. Additionally, pretreatment with 100 µM neomycin for 15 min also had no effect on
5,6-EET-stimulated elevation of [Ca2+]i,
suggesting that 5,6-EET did not require phospholipase C activation to
elevate [Ca2+]i (data not shown).
Further support for 5,6-EET acting directly on SMOC is provided in Fig.
5A. In these experiments,
astrocytes were treated with 1 µM thapsigargin in 1 mM calcium-containing DPBS. After the maximum elevation in
[Ca2+]i was attained in response to thapsigargin,
156 nM 5,6-EET was added. If 5,6-EET activated
receptor-operated channels, it is likely that an additional increase in
[Ca2+]i would be produced (4). However, there was
no further elevation in [Ca2+]i, providing
further evidence that 5,6-EET acts directly on SMOC.

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Fig. 5.
A, 5,6-EET-stimulated
[Ca2+]i elevation is not additive to that of
thapsigargin. Fura-2 loaded astrocytes were stimulated with 1 µM thapsigargin as indicated. After the maximal response
to thapsigargin had been attained, 156 nM 5,6-EET was
added. No further elevation in [Ca2+]i was
observed. Results shown are representative tracings from three separate
experiments performed on different days. B, 5,6-EET
overcomes econazole-inhibition of thapsigargin-stimulated
[Ca2+]i elevation. Fura-2 loaded astrocytes were
pretreated with 10 µM econazole, followed by 1 µM thapsigargin. Note that the thapsigargin response is
reduced to the same degree as shown in Fig. 2. After the maximal
response to thapsigargin was attained, 156 nM 5,6-EET was
added. [Ca2+]i was measured as described in the
legend to Fig. 1. The tracing is representative of three separate
experiments performed on different days.
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Inhibition of Thapsigargin-stimulated [Ca2+]i
Elevation by Econazole Is Overcome by Addition of 5,6-EET--
If
5,6-EET is a CIF, then inhibition of calcium influx with the P450
inhibitor econazole should be reversed by addition of exogenous
5,6-EET. To test this, Fura-2 loaded astrocytes were pretreated with 10 µM econazole for 2 min as shown in Fig. 5B. Next, cells were stimulated with 1 µM thapsigargin. As
described previously, in the presence of econazole the
thapsigargin-stimulated elevation in [Ca2+]i was
reduced to a level consistent with depletion of intracellular calcium
stores alone. Upon attaining the maximum sustained
[Ca2+]i in response to thapsigargin, 156 nM 5,6-EET was added. As can be seen in Fig. 5B,
5,6-EET rapidly increased [Ca2+]i in the presence
of econazole.
14,15-EET Prevents 5,6-EET-stimulated Elevation of
[Ca2+]i in Astrocytes--
Our previous work
(20, 36) and that of others (37, 38) indicates that the primary EET
metabolites in astrocytes are 5,6- and 14,15-EET. As described above,
14,15-EET had no effect on [Ca2+]i in astrocytes.
However, in six separate experiments, we found that pretreatment of
astrocytes with 156 nM 14,15-EET blocked 5,6-EET-induced
elevation of [Ca2+]i. A representative tracing of
these results is shown in Fig. 6.
Subsequent addition of a second dose of 156 nM 5,6-EET counteracted the inhibitory effect of 14,15-EET.

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Fig. 6.
14,15-EET inhibits 5,6-EET-stimulated
elevation of [Ca2+]i. Fura-2 loaded
astrocytes were stimulated with 156 nM 14,15 EET followed
by 156 nM 5,6-EET as indicated.
[Ca2+]i was measured as described in the legend
to Fig. 1. Results are representative of six separate experiments
performed on different days.
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5,6-EET May Also Act as a Paracrine Signal--
Our present
results in astrocytes suggest that 5,6-EET may be the elusive CIF of
the capacitative calcium influx pathway. We have previously shown that
5,6-EET causes dilation of cerebral arterioles when applied topically
under an in vivo cranial window in rabbits (36). Leffler
et al. (39) have recently shown that 5,6-EET is also a
potent dilator of piglet cerebral arterioles. However, the effects of
EETs may differ with species. Since the astrocytes used in the above
tissue culture experiments were of rat cortical origin, we tested the
activity of 5,6-EET on vascular diameter in rat brain using the cranial
window technique (27). As shown in Fig.
7, 5,6-EET caused dilation of cerebral
arterioles when topically applied to the brain surface. Curiously, the
dose-response of 5,6-EET on pial arteriolar diameter, like
5,6-EET-induced [Ca2+]i elevation in astrocytes
(Fig. 3), was bell-shaped. 5,6-EET produced a linear increase in
arteriolar diameter over the concentration range
10
9-10
6 M. However, a higher
dose of 10
5 M produced a very significantly
reduced dilator response. A similar trend at the higher concentration
of 5,6-EET has been reported by Leffler et al. (39) in the
piglet cerebral microcirculation. This reduced dilator response at
10
5 M suggests inhibition of the dilator
stimulus or activation of an opposing constrictor response.

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Fig. 7.
The effect of 5,6-EET on in vivo
rat cerebral arteriolar diameter. 5,6-EET was diluted with
artificial CSF and cumulative doses were applied under the rat cranial
window chamber. Diameter responses were recorded at 3 and 5 min after
application. The maximal response was at 3 min and is shown in the
figure. Values are the average ± S.E. for responses in 4 rats.
a, p < 0.01 versus control for
10 9-10 6 M; b,
p < 0.01 versus 10 7,
10 6 M. Please note that S.E. for
10 7 is too small to show graphically.
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DISCUSSION |
Since the first reports identifying store-controlled or
capacitative calcium influx, the link between intracellular calcium stores and influx of calcium through the plasma membrane has remained obscure. Randriamampita and Tsien (7) isolated a CIF-like substance from stimulated Jurkat cells, which was of molecular weight less than
500 and moderately hydrophobic. Parekh et al. (40) reported that CIF formation involved a phosphatase and a diffusible second messenger. Fasolato et al. (41) have presented evidence
suggesting that formation of CIF involved hydrolysis of GTP and
possibly a low molecular weight G protein. It has been hypothesized
that CIF represents a complex of several different biochemical
components (4, 42). Our previous results suggest that the link to CIF requires the activation of cPLA2 and release of arachidonic
acid from cellular phospholipids in U937 cells (8). Other groups have
reported similar correlations between arachidonic acid release and
calcium influx (43, 44). Cytochrome P450 activity has also been coupled
to the formation of CIF (45, 46) and is one of the major pathways
through which arachidonic acid is metabolized. In the present report,
we present evidence suggesting that the link to CIF involves 5,6-EET, a
cytochrome P450 metabolite of arachidonic acid.
Several lines of evidence support a role for 5,6-EET as a component of
CIF. First, CIF appears to be ephemeral in nature, having a short
half-life. This property has made its identification difficult (4, 7).
5,6-EET is a relatively short-lived metabolite of arachidonic acid,
which is rapidly degraded in the aqueous environment, consistent with
the properties of CIF. Second, in order to function as CIF, arachidonic
acid must first be released from its intracellular phospholipid storage
pool through the action of PLA2, followed by metabolism via
cytochrome P450. We have previously reported a coupling between
cPLA2, arachidonic acid release, and depletion of calcium
from intracellular stores (8). In the present report, we demonstrate
that thapsigargin-stimulated arachidonic acid release is coupled to
release of calcium from intracellular stores in the astrocytes.
Consistent with our data from U937 cells, inhibition of
cPLA2 activity in the astrocyte effectively blocked capacitative calcium influx, but not release of intracellular calcium
stores. Similar to reports from other laboratories (10, 11, 45),
inhibition of cytochrome P450 with econazole or SKF525A also inhibited
capacitative calcium influx. Furthermore, MS-PPOH, a specific inhibitor
of the P450 system that metabolizes arachidonic acid to 5,6-EET, also
inhibited capacitative calcium influx, to a level consistent with
release of calcium from intracellular stores alone. Thus, the two
enzymatic systems which produce 5,6-EET are both coupled to
capacitative calcium influx.
We have also shown that 5,6-EET dose dependently activates calcium
influx in the astrocyte at nano- to picomolar concentrations. The
influx of calcium initiated by 5,6-EET is consistent with CIF for
several reasons. First, 5,6-EET did not induce calcium influx in
calcium-free medium, suggesting that it has no effect on intracellular
calcium stores. Second, 5,6-EET-stimulated calcium influx was blocked
by inhibition of SMOC with SKF96365. Third, the elevation in
[Ca2+]i produced by 5,6-EET was not additive to
that produced by thapsigargin. Fourth, exogenous 5,6-EET could rapidly
overcome econazole inhibition of thapsigargin-stimulated
[Ca2+]i elevation. Finally, the
5,6-EET-stimulated [Ca2+]i elevation was
unaffected by blockade of voltage-gated channels with nimodipine or
inhibition of phospholipase C with neomycin.
Graier et al. (10) have elaborately demonstrated that
5,6-EET is consistent with CIF in endothelial cells. This group has shown that inhibition of P450 blocked capacitative calcium influx, while induction of P450 with dexamethasone/clofibrate enhanced thapsigargin-stimulated [Ca2+]i elevation. Thus,
reports from endothelial cells (10), U937 cells (8), and our present
report in astrocytes all support the hypothesis that 5,6-EET is coupled
to CIF.
In addition to the effects of 5,6-EET on elevation of
[Ca2+]i, we have also found that 14,15-EET may
also participate in 5,6-EET-mediated [Ca2+]i
regulation. 14,15-EET effectively blocked 5,6-EET-induced elevation of
[Ca2+]i. This effect could be overcome with
additional doses of 5,6-EET. However, loss of 14,15-EET's ability to
prevent 5,6-EET-induced calcium influx could alternatively be explained
by a time-dependent inactivation of 14,15-EET due to its
metabolism to the vicinal diol by epoxide hydrolase (20). Malcolm
et al. (47) have previously reported that 14,15-EET
inhibited capacitative calcium entry in platelets. We (20) and others
(15, 48) have previously reported that 14,15-EET is synthesized and
released by astrocytes. Our laboratory has also reported that 14,15-EET
can be taken up and incorporated into cellular phospholipids and may
act as a regulator of several isoforms of protein kinase C (49). It is
tempting to speculate that 14,15- and 5,6-EET exert opposing actions on capacitative calcium influx, thereby acting as regulatory elements for
[Ca2+]i homeostasis.
Although 5,6-EET is rapidly degraded in vitro, its life span
in a lipid environment is unknown. Studies by Randriamampita and Tsein
(7) suggest that CIF can be released from cells and initiate
capacitative calcium influx in unstimulated cell cultures. In
vivo, release of 5,6-EET may act as a paracrine signal for other
nearby cells. Alkayed et al. (48) have shown that EETs are
released from astrocytes stimulated with glutamate and that expression
of P450 2C11 protein is markedly elevated by glutamate exposure. We and
others have shown that in the astrocyte, glutamate stimulation is
closely coupled to activation of phospholipase C and capacitative
calcium influx (21, 50). According to our present results, activation
of this pathway with glutamate would result in formation of 5,6-EET as
CIF. Since astrocytes are known to respond to neuronal activity, we
hypothesize that 5,6-EET may be released from astrocytes and act on the
brain microvasculature to induce vasodilation in response to changes in
neuronal activity. In support of this hypothesis, we have demonstrated
that application of exogenous 5,6-EET causes dilation of cerebral
vessels in the rat. We have reported similar results in the rabbit
using 5,6-EET synthesized by rat cortical astrocytes (36). In the
rabbit, 5,6-EET-induced vasodilation was blocked by indomethacin.
Leffler (39) have recently reported that 5,6-EET induced vasodilation in newborn pig pial arterioles. They also found that 5,6-EET-induced dilation was blocked with indomethacin and restored by a low dose (10
12 M) of iloprost, a prostaglandin
I2 mimetic. In the study by Leffler (39), 5,6-EET did not
elevate 6-keto-prostaglandin F1
levels in the CSF,
suggesting that 5,6-EET did not directly activate prostaglandin
I2 synthesis. Leffler suggests that 5,6-EET induced vasodilation requires a "permissive" concentration of
prostaglandin. Prostaglandins, as well as other agonists, often act
through G-proteins coupled to regulation of cyclic AMP. It has been
reported that alterations in cAMP levels may act to modulate
Ins(1,4,5)P3-mediated signaling and capacitative calcium
influx (51, 52). Thus, we speculate that the permissive effect of
prostaglandins on 5,6-EET-induced vasodilation are not directly due to
a cyclooxygenase metabolite of 5,6-EET, but rather that a prostanoid
may modulate a 5,6-EET-induced signaling component of capacitative
calcium influx.
Interestingly, we found that the [Ca2+]i
elevation and arteriolar dilation induced by 5,6-EET were diminished at
the highest concentrations of 5,6-EET, resulting in a bell-shaped dose
response (Figs. 3 and 7). Similar results were observed by Leffler (39)
for 5,6-EET-induced arteriolar dilation in the piglet. The exact reason
for these bell-shaped dose-response curves is uncertain. At the
cellular level we can speculate that as 5,6-EET activates SMOC calcium
influx and [Ca2+]i rises above a certain level,
additional opposing intracellular mechanisms may be activated, such as
reduced Ins(1,4,5)P3-receptor sensitivity (53-55). At the
in vivo level we speculate that as intracellular calcium is
initially elevated in vascular smooth muscle by 5,6-EET,
calcium-activated K+ channels induce hyperpolarization and
smooth muscle relaxation. However, as [Ca2+]i
rises above a certain point, calcium-mediated contractile mechanisms
may be activated, which counteract the initial dilation.
In summary, we report that 5,6-EET may be a component of CIF in the
astrocyte. Furthermore, our results suggest that 5,6-EET released by
astrocytes may regulate pial arteriole diameter. Thus, Ins(1,4,5)P3-mediated signaling in astrocytes may be
coupled to regulation of cerebral blood flow.