Norepinephrine stimulates arachidonic acid release from
vascular smooth muscle via activation of
cPLA2
Edward F.
LaBelle and
Erzsebet
Polyak
Department of Physiology, Allegheny University of the Health
Sciences, Allegheny University Hospital: Graduate, Philadelphia,
Pennsylvania 19146
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ABSTRACT |
The mechanism of agonist-activated arachidonate release was
studied in segments of rat tail artery. Tail artery segments were prelabeled with
[3H]arachidonate and
then stimulated with norepinephrine (NE), and the radioactivity of the
extracellular medium was determined. NE stimulated arachidonate release
from the tissue without increasing arachidonic acid levels within
cellular cytosol or crude membranes. About 90% of the extracellular
radioactivity was shown to be unmetabolized arachidonate by TLC.
Arachidonic acid release was not inhibited by the removal of the
endothelium from the artery. NE exerted a half-maximal effect at
a concentration of 0.2 µM. NE-stimulated arachidonate release was not
inhibited by blockers of phospholipase C (U-73122), diacylglycerol
lipase (RHC-80267), secretory phospholipase A2 (manoalide),
calcium-insensitive phospholipase A2 (HELSS), or
-adrenergic receptors (propranolol). NE-stimulated arachidonic acid
release was inhibited by blockers of cytosolic phospholipase A2
(cPLA2)
(AACOCF3),
1-adrenergic receptors
(prazosin), and specific G proteins (pertussis toxin). This indicated
that NE stimulated arachidonate release from vascular smooth muscle via activation of
-adrenergic receptors, either
Gi or
Go, and
cPLA2. NE-activated arachidonic
acid release from vascular smooth muscle may play a role in force
generation by the tissue. Perhaps arachidonic acid extends the effect
of NE on one specific smooth muscle cell to its nearby neighbor cells.
rat tail artery; calcium; force regulation; phospholipase C; cytosolic phospholipase A2
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INTRODUCTION |
IT HAS BEEN DEMONSTRATED that agonists that induce
force in vascular smooth muscle, such as norepinephrine (NE), also
activate specific phospholipases in this tissue, such as phospholipase C (PLC) and phospholipase D (PLD) (15, 16, 18, 27). Phosphoinositidase C activation results in the release of inositol 1,4,5-trisphosphate (IP3) within the cytosol of the
smooth muscle cell, which can stimulate calcium release from
subcellular stores, which activates smooth muscle contraction (45, 49).
Diacylglycerol released from phosphatidylinositol 4,5-bisphosphate by
this same enzyme can also activate protein kinase C (PKC), which can
also influence smooth muscle force (22, 35). Still other phospholipases
have been demonstrated in vascular smooth muscle, such as
phosphatidylcholine (PC)-specific PLC and PLD, which
produce either diacylglycerol and choline phosphate or phosphatidic
acid and choline in this tissue (16, 38). Although diacylglycerol
released in this process can activate PKC, there is much uncertainty
about the function of the phosphatidate and the hydrophilic choline
derivatives released.
Many investigators have found that yet another phospholipase, known as
phospholipase A2
(PLA2), exists in many mammalian
tissues and that this phospholipase can be activated by agonists to
release arachidonic acid from the tissues (1, 9). Arachidonic acid itself is the precursor of a series of eicosanoids that appear to play
a role in many important processes (34).
PLA2 can also be activated by
contractile agonists in smooth muscle (14, 29). Muthalif et al. (33)
have demonstrated that NE could stimulate arachidonic acid release from
cultured rabbit aortic smooth muscle cells via the activation of a
specific PLA2 known as cytosolic PLA2
(cPLA2). Many different isoforms
of PLA2 have been detected in
mammalian tissues, such as cPLA2,
secretory PLA2
(sPLA2), and calcium-insensitive
PLA2
(iPLA2) (8, 32). Some studies
have shown that cPLA2 appears most
important in agonist-activated tissue (30, 39). These studies have
suggested that cPLA2 could be activated by pertussis toxin-sensitive G proteins (2, 36), by tyrosine
phosphorylation (12), by calcium activation (12, 23), and by
calcium-dependent translocation to the membrane (7). The
low-molecular-weight sPLA2 has
been detected in many cell types, but since it is only active at
millimolar levels of calcium, it is unlikely to mediate agonist effects
within cells (6). iPLA2 has been
detected in vasopressin-activated cultured smooth muscle cells (29).
There is also the possibility that arachidonic acid might be released
via the activation of PLC and diacylglycerol lipase (17, 21).
The function of the released arachidonate and associated eicosanoids in
smooth muscle is unclear. There is evidence that arachidonic acid can
influence membrane ion channel activity (37, 48) and activate PKC (41),
whereas certain eicosanoids, such as leukotrienes and thromboxanes, can
activate surface receptors that release
IP3 and activate intracellular
calcium release from subcellular stores (42, 43). Gong et al. (13) have
shown that arachidonic acid can inhibit myosin light chain phosphatase, and they have suggested that this effect of arachidonic acid might be
responsible for the GTP-dependent calcium sensitization of vascular
smooth muscle (14).
The purpose of the current study was to determine whether the
contractile agonist NE could stimulate the release of arachidonic acid
from smooth muscle of rat tail artery and to determine the mechanism of
this process. We find that NE can stimulate arachidonate release from
this tissue and that the process appears to require activation of
cPLA2. This system also appears to
be sensitive to pertussis toxin, which indicates the involvement of a G
protein that is different from the G protein earlier shown to be
required for the activation of PLC and PLD in this tissue (25, 28). There does not appear to be an agonist-dependent increase in free arachidonic acid levels within the smooth muscle cells.
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MATERIALS AND METHODS |
Male Sprague-Dawley rats (300-500 g) were purchased from Taconic
Farms (Germantown, NY). The
[3H]arachidonic acid,
together with a kit used to measure levels of
6-ketoprostaglandin
F1
(6-keto-PGF1
), was purchased from New England Nuclear (Boston, MA). BSA, NE, prazosin,
isoproterenol, phenylephrine, propranolol, desipramine,
and indomethacin were purchased from Sigma Chemical (St. Louis, MO).
Manoalide,
1-(6-{[17
-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), arachidonyl trifluoromethyl ketone
(AACOCF3),
E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), tricyclodecan-9-yl xanthogenate · K (D609),
and pertussis toxin were purchased from Biomol (Plymouth Meeting, PA).
A monoclonal antibody directed against
cPLA2 was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA).
Arachidonate release measurements.
Rats were killed by excess CO2
exposure, and the tail arteries were quickly removed. The arteries were
rinsed with physiological saline solution (PSS) containing (in mM)
116.7 NaCl, 1.2 NaH2PO4,
4.5 KCl, 2.5 CaCl2, 1.2 MgSO4, 2.4 Na2SO4,
22.5 NaHCO3, 5 HEPES, and 5 D-glucose. The PSS solution was
gassed with 95% O2-5%
CO2 until the pH was 7.4. The
arteries were dissected to remove extraneous fat and connective tissue,
and then they were cut with a scalpel into segments of identical length
(5 mm). The segments were incubated for 1 h at 37°C in 1 ml PSS
solution that had been equilibrated with
O2/CO2
gas. They were then incubated for 3 h at 37°C in 1 ml of solution
containing PSS, BSA (10 µg/ml), and
[3H]arachidonic acid
(5 µCi), after equilibration with
O2/CO2
gas. The 18 segments obtained from one rat tail artery were divided into 6 groups of 3 segments each, and each group was treated in a
single test tube. The segments were divided into these
groups to correct for any slight variability in the size of an
individual segment. Each group of three segments was rinsed three times
for 10 min with 2 ml of nonradioactive solution containing PSS/BSA (10 mg/ml) plus either DMSO (1%) or any of a variety of inhibitors dissolved in DMSO. The groups were then treated for 20 min with 1 ml
PSS/BSA either with or without inhibitor or with or without NE, and
aliquots of the extracellular medium were removed after 5, 10, 15, or
20 min of incubation. Each tube containing a group of artery segments
was continuously gassed with
O2/CO2.
The radioactivity of each aliquot was determined by liquid
scintillation counting. In some experiments the segments were
homogenized in liquid-N2-cooled mortars at the end of the NE treatment, and the lipids were extracted with chloroform-methanol as described in Ref. 27. The
radioactivity of each lipid extract was determined.
Inositol phosphate measurements. The
production of inositol phosphates in rat tail artery was determined by
the procedure of Gu et al. (15).
Endothelium removal. In some
experiments the rat tail artery was perfused with
N2 gas for 5 min to destroy
endothelial cells (26). In our earlier study, it was proven that this
procedure removed the arterial endothelium when ACh was shown to have
no effect on force generated by denuded tail artery segments (26).
Subcellular fractionation and lipid
identification. To determine intracellular levels of
arachidonic acid, rat tail artery segments labeled with
[3H]arachidonate as
described above were homogenized in
liquid-N2-cooled mortars together
with frozen sucrose (0.25 M). The homogenates were then thawed at
4°C, homogenized further in Ten-brock glass-glass homogenizers, and centrifuged at 27,200 g for 10 min to separate supernatant
cytosol from crude membranes in the pellet. Lipids were extracted from
the cytosol and membrane fractions with chloroform-methanol as
described in Ref. 27. Lipids were also extracted from the extracellular
medium to prove that extracellular radioactivity represented mostly
arachidonic acid. Lipid extracts were separated by TLC using silica gel
LK5 plates eluted with solvent mixtures containing ethyl
acetate-isooctane-acetic acid-water (55:25:10:50; upper phase only).
After the separation the thin-layer plates were sprayed with
scintillation fluid and exposed to X-ray film, and sections of the
plates that produced dark spots on the X-ray film were scraped into
scintillation vials, mixed with water (1 ml) and Ecolume scintillation
fluid, and the radioactivity determined. The
Rf for standard arachidonate in
this thin-layer system was found to be 0.88, whereas the
Rf for standard
6-keto-PGF1
was found to be
0.24.
RIA for 6-keto-PGF1
.
The amount of the prostacyclin derivative
6-keto-PGF1
released from rat
tail artery was measured with the assistance of a RIA kit. Segments of
rat tail artery were incubated in the presence and absence of NE (10 µM) for 10 min at 37°C. Aliquots of the incubation medium were
incubated for 16 h at 4°C with 0.01 µCi standard
[3H]6-keto-PGF1
together with antibody directed against
6-keto-PGF1
. The mixtures were
treated with charcoal for 15 min and then were centrifuged at 1,000 g for 10 min. The radioactivity in the
supernatant was measured and compared with radioactivity determined
when [3H]prostaglandin
was mixed with antibody and known amounts of nonlabeled prostaglandin.
Immunoblotting. The presence of
cPLA2 in rat tail artery was
determined by immunoblotting using the procedure of LaBelle et al.
(25).
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RESULTS |
NE stimulated arachidonic acid release from segments of rat tail
artery. Arachidonic acid release was also observed in the absence of
NE.
The rate of efflux of arachidonic acid from the tissue was constant for
~12 min, in both the presence and absence of NE (Fig. 1A).
NE significantly stimulated arachidonic acid release from rat tail
artery after 30 s of treatment (Fig.
1B). When the extracellular medium
obtained in the experiment shown in Fig.
1A was extracted with
chloroform-methanol and the extract was separated by TLC, ~95% of
the extracellular radioactivity was shown to migrate with standard
arachidonic acid (Fig. 2). When the
production of a key cyclooxygenase metabolite of arachidonic acid
(6-keto-PGF1
) was measured in
segments of rat tail artery, we found that 0.03 ± 0.006 pmol/mg wet
wt of this prostacyclin derivative was released from the tissue in the
absence of agonist, whereas 0.34 ± 0.014 pmol/mg wet wt of
6-keto-PGF1
was released in the
presence of NE (n = 4, P < 0.01). The cyclooxygenase
inhibitor indomethacin (10 µM) failed to inhibit arachidonate release
from rat tail artery, either in the presence or absence of NE (data not
shown). This provided evidence that the radioactive material expelled
from the tissue was only arachidonic acid and not a mixture of
arachidonic acid plus a substantial amount of cyclooxygenase product.
When rat tail artery was treated to remove endothelial cells, NE could still stimulate arachidonic acid release from the tissue, which proved
that arachidonic acid was being released from the smooth muscle cells
of the artery and not the endothelium (Fig.
3). NE stimulated arachidonic acid release
from rat tail artery half-maximally at a concentration of ~200 nM
(Fig. 4). When the concentration of NE
required to stimulate arachidonic acid release half-maximally was
determined in the presence of desipramine (0.1 µM) to inhibit neuronal NE reuptake, no change in the sensitivity of the tissue to NE
was observed (data not shown).

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Fig. 1.
Effect of norepinephrine (NE) on arachidonic acid (AA) release from rat
tail artery. A: segments of rat tail
artery (0.5 cm) were incubated for 3 h with
[3H]AA (5 µCi),
rinsed free of extracellular radioactive material, and then treated
with ( ) or without ( ) NE (10 µM) for times indicated, and
aliquots of incubation medium were removed for determination of
radioactivity. Values represent triplicate determinations (±SE) of
extracellular radioactivity. These results were repeated with nearly
identical results 10 times using tissue from 10 rats. Counts per minute
(cpm) determined in presence of NE were significantly higher than
control cpm (P < 0.05).
B: experiment identical to
experiment shown in A, except that
incubation times were shorter. These results represent combined data
from 2 separate experiments performed with tissue from 2 rats and
repeated with essentially identical results using 2 additional rats.
The cpm determined in the presence of NE were significantly higher than
control (P < 0.05).
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Fig. 2.
TLC of extracellular radioactivity proves identity of AA. Extracellular
medium from rat tail artery rings that had been labeled with
[3H]AA and treated
with NE for 20 min was extracted with chloroform-methanol and the lipid
extract separated by TLC. Single-centimeter zones were scraped from the
plate and the radioactivity in each zone determined. Arrow, location of
standard AA. These results were repeated 4 times using tissue from 4 rats.
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Fig. 3.
Effect of endothelial (Endo) denudation on AA release from rat tail
artery. Segments of rat tail artery were either treated to remove
endothelial cells as described in MATERIALS AND
METHODS or left untreated, and then AA release was
measured as described in legend to Fig. 1. NE-stimulated AA release was
determined for 5-20 min. Values represent cpm AA/min (means ± SE; n = 4). These results were
repeated 3 times using tissue from 3 rats with essentially identical
results. Con, control.
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Fig. 4.
Effect of NE concentration on AA release from rat tail artery. Segments
of rat tail artery were labeled with
[3H]AA, treated with
indicated concentrations of NE for 5-20 min, and radioactivity of
the medium determined as described in legend to Fig. 1. Values are
means ± SD (n = 4). These results
were repeated 4 times using tissue from 4 rats with essentially
identical results. All values obtained with NE were significantly
higher than the control (P < 0.01).
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When rat tail artery segments were treated with buffer containing 80 mM
KCl, the cell membranes were depolarized and calcium was induced to
enter the cells via potential-sensitive channels (25). This resulted in
the development of force by the tissue (25). The treatment of tail
artery segments with 80 mM KCl-containing buffer did not influence
arachidonic acid release from the tissue (Fig.
5)

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Fig. 5.
Effect of NE and potassium on AA release from rat tail artery. AA
release from segments of rat tail artery was measured as described in
legend to Fig. 1. Segments were either unstimulated, as indicated, or
treated with NE (10 µM) for 5-20 min or with physiological
saline solution (PSS) containing 80 mM KCl together with diminished
NaCl (41.2 mM). Values are means ± SD
(n = 4). These results were repeated 3 times using tissue from 3 rats with essentially identical results.
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Some investigators have suggested that agonists could stimulate
arachidonic acid release from tissue via the stimulation of PLC
followed by diacylglycerol lipase (11, 17). However, neither the
phosphoinositidase inhibitor U-73122 nor the diacylglycerol lipase
inhibitor RHC-80267 could prevent NE from stimulating arachidonic acid
release from rat tail artery (Fig. 6). This
suggested that NE did not stimulate arachidonic acid release via either
phosphoinositidase or diacylglycerol lipase. When we measured the
effects of U-73122 on NE-stimulated production of inositol phosphates
in rat tail artery, this compound was shown to inhibit NE-activated
increases in inositol monophosphate, inositol diphosphate,
and IP3 (data not shown).
NE-stimulated arachidonic acid release from rat tail artery was also
insensitive to inhibition by D609 (data not shown), which has been
shown to inhibit PC-specific PLC (40). This indicated that
agonist-stimulated arachidonic acid release did not occur in response
to stimulation of PC-specific PLC. We have shown that NE could
stimulate phosphoinositidase in this tissue by the determination of
IP3 production (15, 27), and we
demonstrated PC-specific PLC activation by the determination of choline
phosphate release (16).

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Fig. 6.
Effect of phospholipase C and diacylglycerol lipase inhibitors on AA
release from rat tail artery. A: AA
release from segments of rat tail artery was measured as described in
legend to Fig. 1. U-73122 (10 µM) was included during both the rinse
step and the subsequent agonist incubation as described. AA release was
measured for 5-20 min. Values are means ± SE
(n = 4). These results were repeated 3 times using tissue from 3 rats with essentially identical results.
B: AA release from segments of rat
tail artery was measured as described in legend to Fig. 1. RHC-80267
(20 µM) was included during both the rinse step and the subsequent
agonist incubation as described. Values are means ± SE
(n = 4). These results were repeated 3 times using tissue from 3 rats with essentially identical results.
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The compound AACOCF3 has been
shown to be a selective inhibitor of
cPLA2 (30, 47).
AACOCF3 blocks the effects of NE
on arachidonic acid release in rat tail artery (Fig.
7). The concentration of
AACOCF3 that half-maximally
blocked the effects of NE on arachidonic acid release was ~10 µM.
The presence of cPLA2 in rat tail
artery was proven by means of immunoblotting with an antibody to
cPLA2 (Fig.
8). Neither the inhibitor of
iPLA2 (HELSS) nor the inhibitor of
sPLA2 (manoalide) could block
NE-stimulated arachidonic acid release from rat tail artery (Fig.
9). This indicated that the stimulation of
arachidonic acid release in this tissue by NE was most likely not
mediated by either iPLA2 or
sPLA2.

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Fig. 7.
Effect of cytosolic PLA2
(cPLA2) inhibitor arachidonyl
trifluoromethyl ketone (AACOCF3)
on AA release from rat tail artery. AA release from segments of rat
tail artery was measured as described in legend to Fig. 1.
AACOCF3 was included at indicated
concentrations during the rinse step and during the incubation with NE
(5-20 min) as described. Values are means ± SE
(n = 4). These results were obtained
by combining data from 8 experiments using 8 different rats. ,
Control; , NE.
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Fig. 8.
Identification of cPLA2 in rat
tail artery. Presence of cPLA2 in
rat tail artery was determined by immunoblotting using an antibody
directed against cPLA2. These
results were repeated twice with essentially identical results. Numbers
on right are molecular
weight.
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Fig. 9.
Effect of inhibitors of calcium-insensitive
PLA2 and secretory
PLA2, HELSS and manoalide,
respectively, on AA release from rat tail artery. AA release from
segments of rat tail artery was measured as described in legend to Fig.
1. Either HELSS (10 µM; A) or
manoalide (10 µM; B) was included
during the rinse step and the subsequent NE incubation (5-20 min)
as described. Values are means ± SE
(n = 4). These results were repeated 6 times for HELSS and 3 times for manoalide, with essentially identical
results.
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Pertussis toxin was shown to totally block NE-stimulated arachidonic
acid release from rat tail artery (Fig.
10). This indicated that NE exerted its
effects on arachidonic acid release via a pertussis toxin-sensitive G
protein, most likely either Gi or Go. NE was shown to exert its
effects on arachidonic acid release via the
-adrenergic receptor
when the
1-adrenergic receptor inhibitor prazosin was shown to totally block NE-stimulated arachidonic acid release (Fig. 11). The
-adrenergic receptor propranolol failed to inhibit
NE-stimulated arachidonic acid release, thereby proving that
-adrenergic receptors were not involved in this process (data not
shown). Likewise phenylephrine, an
-adrenergic receptor agonist,
stimulated arachidonic acid release from rat tail artery, whereas the
-receptor agonist isoproterenol failed to stimulate arachidonic acid
release from this tissue (data not shown).

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Fig. 10.
Effect of pertussis toxin (PT) on AA release from rat tail artery.
Segments of rat tail artery were incubated for 90 min with
[3H]AA, treated for 90 min longer with or without pertussis toxin (5 µg/ml), rinsed 3 times
for 10 min with PSS/BSA solution, and then treated with or without NE
for 5-20 min. Aliquots of the incubation medium were removed for
radioactivity determination. Values represent cpm AA/min (means ± SD; n = 4). These results were
repeated 6 times using tissue from 6 rats with essentially identical
results.
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Fig. 11.
Effect of prazosin on AA release from rat tail artery. AA release from
segments of rat tail artery was measured as described in legend to Fig.
1. Prazosin (10 µM) was included during the rinse step and the
subsequent NE incubation (5-20 min) as described. Values are means ± SE (n = 4). These results were
repeated 3 times using tissue from 3 rats with essentially identical
results.
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When rat tail artery segments prelabeled with
[3H]arachidonic acid
were stimulated with NE, homogenized, and separated into cytosol and
crude membranes by centrifugation, NE was shown to have no stimulatory
effects on the levels of arachidonic acid within the tissue (Table
1).
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DISCUSSION |
Many agonists have been shown to stimulate arachidonic acid release
from different mammalian tissues (1, 9, 34). Arachidonic acid itself
has been shown to serve as the precursor of a family of compounds known
as eicosanoids (34). Both arachidonic acid and the eicosanoids have
been shown to have effects on potassium channels (37), PKC (41), and
receptor-mediated IP3 release (42,
43). It has also been suggested that arachidonic acid could inhibit
myosin light chain phosphatase in smooth muscle and thereby enhance
smooth muscle force (13, 14). Many investigators have found evidence
for the direct activation of plasma membrane calcium channels by
arachidonic acid in oligodendrocytes (44) as well as in smooth muscle
cells (5, 48, 50).
There have been numerous reports that arachidonate release could be
stimulated by the combined activities of the enzymes PLC and
diacylglycerol lipase (11, 17). Other studies have suggested that
arachidonate release was directly stimulated by
PLA2 (4, 46). Several different
PLA2 enzymes that might be
important, such as sPLA2,
cPLA2, and
iPLA2, were found in mammalian
tissues (1, 8, 9, 32, 34). The activation of
cPLA2 in many tissues has been
shown to be dependent on G protein activation (2, 36), tyrosine
phosphorylation (12), calcium activation (12, 23), and membrane
translocation (7). Mitogen-activated protein kinase has also been shown
to activate cPLA2 in certain tissues (31) but not in others (3). Some very recent studies have
indicated that cPLA2 activation
might require PLD activation (21). It remains unclear just how
arachidonic acid is produced in vascular smooth muscle during agonist
activation.
Our data indicate that NE rapidly stimulates arachidonic acid release
from the smooth muscle of rat tail artery. This process is activated at
a fairly low concentration of NE (0.2 µM), which is much lower than
the NE concentration shown to activate either PLC or PLD in our earlier
studies (1-10 µM) (16, 27). Because 80 mM KCl failed to
stimulate arachidonic acid release from rat tail artery, it could be
concluded that arachidonic acid release might be involved in the
mechanism of NE-induced force rather than a mere secondary consequence
of force development in smooth muscle. Likewise, if KCl fails to
stimulate arachidonic acid release, one can conclude that arachidonic
acid release is not a simple consequence of increased levels of
cytosolic calcium (25).
Our data also indicate that arachidonic acid release from vascular
smooth muscle most likely does not require the activation of either
phosphoinositidase C or PC-specific PLC combined with diacylglycerol
lipase, since the inhibitors of these enzymes fail to block
NE-activated arachidonic acid release (Fig. 6). The relative insensitivity of PLC and PLD to NE demonstrated in our earlier studies
also suggests that arachidonic acid release is not secondary to either
PLC or PLD in our tissue. Finally, pertussis toxin has never inhibited
PLC or PLD in rat tail artery (25, 28), whereas it does block
arachidonic acid release (Fig. 10).
These experiments suggest that arachidonic acid release in rat tail
artery does not rely on either PLC or PLD activation and might rely on
PLA2 activation. Evidence in
support of cPLA2 activation is
shown in Fig. 7, wherein the selective inhibitor
AACOCF3 (30, 47) blocks
arachidonate release in rat tail artery. The insensitivity of this
process to manoalide and HELSS suggests that neither
sPLA2 nor
iPLA2 are required during
NE-activated arachidonic acid release in rat tail artery (19, 20).
Manoalide has been shown to be selective for
sPLA2 (20) and HELSS selective for
iPLA2 (12). All of these
inhibitors can easily penetrate cells since they are extremely
hydrophobic (DMSO soluble). Lehman et al. (29) have shown that
iPLA2 appeared to be activated by
vasopressin in cultured smooth muscle cells (A10 cells). This may
reflect a difference between agonists (NE vs. vasopressin) or a
difference between cultured smooth muscle cells and intact smooth
muscle. Muthalif et al. (33) introduced oligonucleotides complementary to mRNA specific for cPLA2 into
cultured rabbit aortic smooth muscle cells, and these oligonucleotides
were able to block the effects of NE on arachidonic acid release. This
provided more evidence for a role of
cPLA2 in agonist-activated smooth
muscle cells. Wright and Malik (52) demonstrated that NE could
stimulate prostacyclin release from intact rat aorta and that this
process was sensitive to an inhibitor of
PLA2
[7,7-dimethyl-(5Z,8Z)-eicosadienoic acid]. These results were consistent with our
evidence that NE stimulates arachidonic acid release via
PLA2 activation in intact rat tail
artery. The direct demonstration of
cPLA2 in rat tail artery by
immunoblotting is consistent with its function in this tissue.
No increase in the concentration of arachidonic acid was observed
within the cells of the rat tail artery after agonist activation. This
would suggest that arachidonate is unlikely to ever reach levels high
enough to inhibit myosin light chain phosphatase in smooth muscle
during agonist activation. Gong et al. (13) have shown that 300 µM
arachidonic acid could inhibit myosin light chain phosphatase in
permeable vascular smooth muscle. Khan et al. (24) have indicated that
free arachidonate levels in tissues never get beyond low nanomolar
levels, although a certain amount of this fatty acid might exist in the
cytosol attached to fatty acid binding proteins (24). If NE can
stimulate arachidonic acid release from rat tail artery, it must
increase arachidonic acid levels transiently in certain membrane
fractions, which was not detected in our study of crude membranes shown
in Table 1. Further work is necessary to determine the arachidonic acid
levels in more purified membranes from smooth muscle.
PLA2 activation has been detected
on the nuclear membrane (33), and Wolf et al. (51) have shown that
iPLA2 activation on the membrane
of subcellular stores has occurred in response to decreases in the calcium content of the stores. This indicates that
PLA2 activation may not be limited
to plasma membrane.
Some 6-keto-PGF1
was released
from tail artery in this study. However, the amount of arachidonate
released was presumably much greater than the amount of
6-keto-PGF1
, since no
radioactivity was detected on the portion of the thin-layer plate where
6-keto-PGF1
would be expected
to migrate (Fig. 2), and indomethacin failed to inhibit the release of
radioactive material from the tissue. Other eicosanoids were no doubt
released in very low amounts, but nearly all of the arachidonate
released from the cells remained as free fatty acid, which suggests
that arachidonate itself plays a role in this tissue. This does not
rule out a role for the eicosanoids that might be released during our
study.
Although this study does not elucidate the function of arachidonic acid
in vascular smooth muscle, the possibility exists that it might be
released from the cells to extend the function of NE beyond the
synaptic cleft. Perhaps NE is itself metabolized very soon after being
released from the neurons that impinge on smooth muscle, but
arachidonic acid can diffuse quite a distance from the cell membrane
directly under the synapse and exert effects on more distant smooth
muscle cells.
In summary, we have demonstrated that NE stimulates arachidonic acid
release from the rat tail artery. This process is sensitive to an
inhibitor of cPLA2 and most likely
results from cPLA2 activation, perhaps via pertussis toxin-sensitive G protein activation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Ronald Coburn for discussions and advice concerning
this subject, Dr. Haiwei Wang and David Nicholaisen for technical assistance, and Dr. Carl Baron for assistance with lipid
separations.
 |
FOOTNOTES |
This study was supported by National Heart, Lung, and Blood Institute
Grant HL-37413 (to E. F. LaBelle).
Present address of E. Polyak: Univ. of Pennsylvania, Dept. of Cell and
Developmental Biology, Philadelphia, PA 19104.
Address for reprint requests: E. F. LaBelle, Dept. of Physiology,
Allegheny Univ. of the Health Sciences, 2900 Queen La., Philadelphia,
PA 19129.
Received 10 July 1997; accepted in final form 18 December 1997.
 |
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