138
Original Contributions
12-Lipoxygenase Products Modulate Calcium
Signals in Vascular Smooth Muscle Cells
Fumio Saito, Mark T. Hori, Yasufumi Ideguchi, Morris Berger, Michael Golub,
Naftali Stern, and Michael L. Tuck
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Previous studies have shown that inhibition of the lipoxygenase pathway of arachidonic acid metabolism
can prevent the development of elevated blood pressure in renin-dependent models of hypertension.
Agents that inhibit the lipoxygenase pathway such as phenidone and the flavonoid baicalein can selectively
attenuate contractile responses to angiotensin II in vivo as well as in isolated vascular tissue. In the
present study, the effects of lipoxygenase inhibitors on pressor-induced changes in cytosolic calcium were
examined in cultured rat vascular smooth muscle cells using the fluorescent dye fura-2. Two structurally
unrelated lipoxygenase inhibitors, baicalein and 5,8,11-eicosatriynoic acid, attenuated angiotensin
Il-stimulated increases in cytosolic calcium in both normal and calcium-poor buffer. The addition of 5-,
12-, or lSfSJ-hydroxyeicosatetraenoic acid alone to the cells had no acute effect on intracellular calcium
concentration. However, the addition of 12f£)-hydroxyeicosatetraenoic acid but not 5- or IS(S)hydroxyeicosatetraenoic acid restored the initial calcium response to angiotensin II in vascular smooth
muscle cells pretreated with both inhibitors; 5,8,11-eicosatriynoic acid also reduced [Arg*]-vasopressin
and endothelin-stimulated increases in intracellular calcium. The attenuation of vasopressor-induced
calcium transients by agents that inhibit lipoxygenase may explain their observed hypotensive effects in
vivo. Moreover, Hpoxygenase products, in particular 12(5J-hydroxyeicosatetraenolc acid, may act as
mediators for the intracellular actions of angiotensin II and possibly other pressor hormones in vascular
tissue by regulation of intracellular calcium metabolism. (Hypertension 1992^0:138-143)
KEY WORDS • lipoxygenase • angiotensin II • endothelin • vasopressins • calcium • muscle,
smooth, vascular
I
t is well recognized that many hormone-coupled
events are mediated by changes in intracellular
calcium concentration ([Ca2+],), either by release
from intracellular stores or as a result of influx from the
extracellular space.1'2 In vascular smooth muscle, an
important pathway that follows pressor hormone-receptor coupling is activation of phospholipase C. This
results in the hydrolysis of phosphatidylinositol diphosphate and the production of inositol trisphosphate (IP3)
and diacylglycerol (DG). 3 IP3 affects intracellular calcium release from the sarcoplasmic reticulum. DG
couples membrane-associated protein kinase C and
serves as a sustained source of arachidonic acid,4 presumably by activation of DG lipase.5 Arachidonic acid
can be metabolized via several enzyme systems to
biologically important compounds.
One of these enzyme complexes, the lipoxygenase
(LO) pathway, may contribute to the effector mechanisms of angiotensin II (Ang II). We have shown that
inhibition of the IX) pathway can reduce the in vivo and
From the Veterans Administration Medical Center, Sepulveda,
and UCLA School of Medicine, Los Angeles, Calif. (F.S., M.T.H.,
Y.I., M.B., M.G., M.L.T.) and Souraskv-Tel Aviv Medical Center
(N.S.), Tel Aviv University, Tel Aviv, Israel.
Supported by a Veterans Administration Merit Review and by
grant RO1-HL-41295 from the National Institutes of Health.
Address for correspondence: Michael L. Tuck, MD, Division of
Endocrinology (11 IE), Veterans Administration Medical Center,
16111 Plummer Street, Sepulveda, CA 91343.
Received July 8, 1991; accepted in revised form April 3, 1992.
in vitro vascular responses to Ang II, but not norepinephrine and potassium chloride.6 In bovine and human
endothelial cells, Ang II-induced chemoattractant generation is blocked by LO pathway inhibition but not by
cyclooxygenase (CO) inhibition.7 Ang II-dependent
inhibition of renin secretion can be reversed by LO but
not CO inhibition.8 Additionally, 12- and lSfSJ-hydroxyeicosatetraenoic acids (HETE), which are products of
LO pathway metabolism, can inhibit renal renin secretion, emulating the action of Ang II.9 Previous observations have also implicated LO products as mediators of
Ang II effects on the adrenal cortex,10-11 since various
LO blockers selectively inhibit aldosterone responses to
Ang II but not to adrenocorticotropic hormone or
potassium.
It is not known, however, what mechanisms in hormone-response coupling might mediate the attenuation
of vascular responses to Ang II during LO pathway
inhibition. Changes in [Ca2+]j affect multiple intracellular steps in Ang II action, and [Ca2+]j can be altered by
LO products. The LO inhibitor baicalein attenuates
degranulation and Ca2+ mobilization from intracellular
stores in human polymorphonuclear leukocytes.12
Moreover, 12-HETE enhances 45Ca uptake in rabbit
neutrophils13 and mobilizes calcium from liver mitochondria.14 LO but not CO products have been implicated as the major intermediates of arachidonic acidinduced contraction of canine cerebral arteries.15 Since
changes in [Ca2+]; are essential for Ang H-induced
Saito et al LJpoxygenase and Calcium
contractile responses in vascular smooth muscle cells
(VSMC), we examined the effect of LO pathway inhibition on Ang II- and other agonist-induced calcium
transients in cultured VSMC.
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
Methods
Culture of Vascular Smooth Muscle Cells
VSMC were isolated from rat thoracic aorta (250300 g, male Sprague-Dawley rats, Bantin and Kingman,
Freemont, Calif.) by enzymatic dispersion as previously
described.16 The harvested cells were grown in Dulbecco's modified Eagle's medium (Sigma Chemical Company, St. Louis, Mo.) supplemented with 10% fetal calf
serum (Hyclone Laboratory, Logan, Utah), 50 units/ml
penicillin, and 50 jig/ml streptomycin (Sigma). Cells,
from passage 5 to 20, were seeded onto 25-mm round
glass coverslips and grown to confluence in 4-6 days.
Before experimentation, cell lines were randomly
screened for smooth muscle actin expression as assayed
by immunofluorescent staining with anti-rat smooth
muscle actin (Enzo Diagnostics Inc., New York), visualized with fluorescein conjugated rabbit anti-mouse
immunoglobulin G (IgG) (Cappel Laboratories, Organon Teknica Corp., Westchester, Pa.).
Measurement of [Ca2+]t
Confluent cells attached to coverslips were deprived
of serum for 24 hours before the experiments and then
incubated with 4 /iM fura 2-acetoxymethylester (fura
2/AM) (Molecular Probes, Eugene, Ore.) for 40 minutes at 37°C in balanced salt solution (BSS) (mM: NaCl
145, KC1 5, CaCl2 2, MgSO4 • 7H2O 1, Na • PO4 • 7H2O
0.5, glucose 6, and HEPES 10, pH 7.4) without added
surfactants. Loaded cells were washed three times for
2-3 minutes and then incubated with fresh BSS for 15
minutes at 37°C to allow hydrolysis of the entrapped
ester. Coverslips were rinsed in fresh BSS, fixed to a
specially designed incubation well, and placed in a
thermoregulated holder (maintained at 37°C) on the
microscopic stage. Photon emission was monitored with
a Deltascan spectrofluorometer (Photon Technology
International, South Brunswick, N.J.) at 510 nm with
excitation wavelength alternating between 350 (F350)
and 380 (F380) nm. Changes in fura 2/AM lot number
and differences in the cell line and their field density
resulted in variability in the total loading and the
absolute magnitude of the total emission signal from a
high of approximately 1.5 x 106 to a low of 3 x 105 counts
per second. However, preliminary studies performed
under these conditions indicated that increasing the
post-loading time from 15 to 60 minutes had no further
effect on the stability of the baseline or on the final
calibrated data analysis. At the end of each experiment,
the minimum ratio (Rmin) of F350 and F380 (R350/
380) was determined in buffer containing no added
Ca 2+ , 4 mM ethylene glycol-bisO-aminoethyl ether)
N,N,N',N'-tetraacetic acid (EGTA), and 1 /xM ionomycin. The maximum ratio (Rmax) was determined in BSS
containing 10 mM Ca2+ and 1 /iM ionomycin. The
[Ca2+], was calculated as described by Grynkiewicz et
al,17 according to the formula:
139
where Xj represents the dissociation constant of fura-2
for calcium (224 nM), R represents R350/380 collected
in real time at 0.5-second intervals, and b represents the
ratio of F380 measured in EGTA plus ionomycin to that
of F380 in buffer with excess Ca2+ plus ionomycin. All
data were individually corrected for autofluorescence by
Mn2+ quenching,18 and each coverslip was calibrated
separately. Autofluorescence was typically 3 to 4x10*
counts per second and did not vary significantly during
the experimental time period. This methodology does
not totally exclude errors due to the development of
bleached intermediates or other non-calcium-sensitive
fluorescent products.19-20 Intracellular calcium values,
therefore, must be interpreted as approximations. However, the control and experimental cells should have
been similarly influenced. Thus, the relative changes
reported would not be affected by these potential
artifacts.
Experimental Protocol
The agonist-induced [Ca2+]| transients were measured after preincubation with either 0.0623 fil/ml
DMSO (vehicle), 1.0 /xM baicalein (a bioflavonoid
inhibitor of 15- and 12-LO),21 or 2.5 jtM 5,8,11-eicosatriynoic acid (ETI) (a substrate analogue for 5- and
12-LO)22^3 for 20 minutes. To evaluate if HETEs could
restore [Ca 2+ ] ; responses, 400 nM 12(5)-HETE, 5(5)HETE, or 15(5)-HETE (Biomol Research Laboratories, Plymouth Meeting, Pa.) or 0.25% ethanol (vehicle)
was added for 1 minute before the addition of Ang II
(10~8 M), arginine vasopressin (AVP) (10~7 M), or
endothehn (2xlO~7 M). In all cases, the observed rise in
calcium was immediate and rapidly reached peak levels
within 20 seconds of agonist challenge (see Figure 1 for
illustration). 5(5)-HETE and 15(5)-HETE, which are
naturally occurring products of alternate LO pathways
in some tissues, were used as controls for 12-HETE
add-back experiments for ETI and baicalein, respectively, because of the reported effects of these inhibitors
on these pathways, as a test of specificity.
Statistics
Results are expressed as mean±SEM. Data were
evaluated by analysis of variance with subset analysis by
Tukey's group-to-group comparison.
Results
Effect of Baicalein and ETI on Angiotensin IIInduced [Ca2+]t Transients
Basal [Ca2+]j levels were not significantly different
among the VSMC of the four experimental protocols
(Table 1). The addition of baicalein inhibited (p<0.05)
100 nM Ang II-induced increments in [Ca2+]j. However,
the initial phase of the calcium response to Ang II could
be restored by addition of 12(5)-HETE to VSMC
pretreated with baicalein. Restoration of [Ca 2+ ]| responses was not observed with addition of 15(5)-HETE
to baicalein-blocked cells. The order of additions appeared important for the restoration experiments. The
calcium signals could be consistently restored in treated
cells when 12(5)-HETE was added 1 minute before the
addition of Ang II. However, when Ang II and 12(5)HETE were added simultaneously or when 12(5)HETE was added after the application of Ang II,
140
Hypertension
Vol 20, No 2 August 1992
TABLE 1. Effect of Baicalein on AngioUnsin II-Indnced Calcium Transient
[Ca2+]i (nM)
Group
Peak
Basal
Change
Change (%)
147.0±17.1
146.6±11.7
2503±17.2
103.8 ±9.9
Control (n = 14)
77.9±5.0*
64.6±2.7*
Baicalein (1x10"* M) (n = 14)
199.5 ±13.6*
121.6±9.1
Baicalein+12W-HETE
(4xlO- 7 M)(/j = 13)
\2S5±\2.2
256.6±22.9t
131.1±16.Ot
107.8±13.&t§
Baicalein+15(S,)-HETE
86.6 ±27.6
145.6±2.6*
58.9±30.2*
88.1±62.9*
(4xlO" 7 M)(n=3)
Results (mean±SEM) showing the effect of 20-minute treatment with 1 fiM baicalein on 100 nM angiotensin
II-raediated intracellular calcium mobilization. [Ca2+]i, intracellular calcium concentration; HETE, hydroxyeicosatetraenoic acid.
*p<0.01 compared with control.
tp<0.01 compared with baicalein and baicalein plus 15(S)-HETE.
tp<0.05 compared with control.
§p<0.05 compared with baicalein.
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
calcium responses to Ang II could not be restored.
These results suggest that LO products must be available in the cell in sufficient concentration to transmit
the Ang II-induced calcium signals. ETI also inhibited
the peak Ang H-induced [Ca2+]i responses in VSMC as
shown in Table 2. This inhibition was likewise restored
by the addition of 12(SJ-HETE but not by 5 ^ - H E T E .
The averaged traces from 1 day of experiments showing
ETI inhibition of calcium transients and restoration by
12(SJ-HETE are shown in Figure 1. In all cases, 0.25%
ethanol vehicle with or without 5-, 12-, or 15(5>HETE
caused a small (10-15 nM), transient increase in [Ca2+]|.
These increments rapidly returned to baseline and
stabilized in approximately 30 seconds. There were no
significant differences in this effect between treatments.
To evaluate the contribution of extracellular calcium,
4 mM EGTA was added to the medium 19 minutes after
the addition of ETI and 1 minute before the addition of
Ang II. In this set of experiments addition of EGTA
alone decreased basal [Ca2+], by 12%. Subsequent Ang
H-stimulated transients were preserved, although they
were slightly (—5%) smaller than in the 2 mM calcium
buffer. In this calcium-poor buffer, there was no significant difference in basal [Ca2+]j between control and
ETI-treated cells. However, the Ang II-stimulated increase in [Ca2+]j was smaller in the ETI-treated cells
than in the control cells (Table 3).
ETI also inhibited AVP and endothelin-stimulated increase in [Ca2+]j in the buffer with 2 mM Ca2+. The
incremental changes in [Ca2+]i were: for 100 nM AVP,
1433±19.0 versus 56.0±16.0 nM, in the absence or presence of ETI, respectively, n=4 for each protocol,p<0.01;
and for 200 nM endothelin, 393.9±97.7 versus 123.5±37.0
nM, in the absence and presence of ETI, respectively, n=8
for each treatment, p<0.05 (Figures 2 and 3). The same
effects were observed in the calcium-poor medium
(changes in [Ca2+]j: 100 nM AVP, 3433±86.8 versus
95.9±44.9 nM, in the absence or presence of ETI, n=6 for
each treatment, p<0.05; 200 nM endothelin, 158.5±6.7
versus 75.8±18.8 nM, in the absence or presence of ETI,
n=4 for each treatment,p<0.01) (Figure 4).
Discussion
We reported that the nonspecific LO/CO pathway
blocker phenidone attenuated the development of hypertension in the Goldblatt two-kidney, one clip renovascular hypertensive rat model24-23 but not in the
deoxycorticosterone acetate-salt model of hypertension.25 In addition, intraperitoneal pretreatment with
LO pathway blockers such as phenidone, baicalein, and
esculetin can attenuate pressor responses to Ang II
infusion but not to norepinephrine in Sprague-Dawley
rats.6 Phenidone and baicalein also inhibited contractile
responses to Ang II but not to norepinephrine or
potassium chloride in isolated rat femoral artery segments.6 These studies offered preliminary support for a
selective inhibitory effect of LO pathway blocking
agents on the vascular actions of Ang II and provide
strong evidence that LO pathway products are involved
in the mediation of the actions of Ang II in vascular
tissue. Since changes in intracellular calcium represent
a major step in the cellular actions of Ang II in vascular
tissue, it is possible that LO pathway products alter this
function in VSMC.
TABLE 2. Effect of 5,8,11-Eicosatriyiioic Add on Angiotensin Il-Induced Caldum Traiuients
Group
Control (n = ll)
ETI(2.5xKr 6 M)(n-12)
En+X2(S)-HETE (4xlO"7 M) (n=9)
ETI+5W-HETE (4xlO" 7 M) (n=5)
Basal
104J±6.7
98.2±10J
93.8±13.9
112.2±19.7
[Ca2+), (nM)
Change
Peak
330.9±71.7
435.2±69.7
233.2±38.4*
124.6±36.0*
293.9 ±60.8t
387.7±62.1t
175-5±46.6*
63J±53.9*
Change (%)
346.1 ±79.9
152.8±37.7*
365.0±88.2t
85J±75.0'
Results (mean±SEM) showing the effect of 20-minute treatment with US jiM 5,8,11-eicosatriynoic acid, a suicide
substrate analogue for 5- and 12-lipoxygenase, on 100 nM angiotensin Il-mediated intracellular calcium mobilization.
ETI, 5,8,11-eicosatriynoic acid; [Ca2+]i, intracellular calcium concentration; HETE, hydroxyeicosatetraenoic acid.
*p<0.01 compared with control.
tp<0.01 compared with ETI and ETI plus 5(S>HETE.
Saito et al Iipoxygenase and Calcium
141
* fxO.05
Control
** (XO.01
I
2.S uU ET1
P ioo-
ET1 + 12(S)HETE
i
I
Ul
30
60
120
90
150
5 0
180
AVP
(n-4)
TIME (sac)
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FIGURE 1. Effect of the lipoxygenase blacker 5,8,11-eicosatriynoic acid (ETI) on 100 nM angiotensin II (Ang II)induced calcium mobilization in vascular smooth muscle
cells. Representative cytosolic calcium
concentration
([Ca 2+]j) tracings of the three different treatments for a single
day of experiments. Each line was generated by the graphic
averaging of the final data for three experimental runs. DMSO
vehicle or 2.5 pM ETI in vehicle was added 19 minutes before
tracing. For add-back experiments, either 0.25% ethanol
acid
vehicle, or 400 nM 12(S)-hydroxyeicosatetraenoic
(HETE) was added at time 0. Ang II (100 nM) was added at
60 seconds in all three traces. Note attenuation of initial
calcium response with ETI and restoration by 12(S)-HETE.
We now report that two structurally unrelated inhibitors of the LO pathway, baicalein and ETI, attenuate
Ang II-induced calcium transients as studied in cultured rat VSMC. Moreover, the finding that addition of
the LO pathway product 12-HETE can restore the
initial Ang II-induced [Ca2+]; responses in LO-blocked
VSMC provides evidence that this LO product may
participate in the modulation of Ang II-dependent
calcium transients in VSMC. In contrast, the absence of
an effect with 5-HETE and 15-HETE on LO-blocked
VSMC demonstrates the specificity of 12-HETE in the
vascular actions of Ang II. The fact that direct addition
of 12-HETE to cultured VSMC did not affect [Ca2+]j
suggests that this LO product may act more as a
cofactor in the action of Ang II in vascular tissue.
However, the current studies do not resolve this issue.
An important assumption in the proposed series of
events for LO pathway involvement in Ang II action in
the vasculature is that the LO pathway products are
present in sufficient amounts in vascular cells and can
respond to Ang II. There is some controversy regarding
the levels and biosynthetic origin of HETE production
TABLE 3. Effect of Caldum-Defideiit Buffer on 5,8,11-Ekusatrrynolc Add Attenuation of Angiotensin n-Induced Calcium Transients
[Ca2+], ( nM)
Group
Control (n=6)
ETI (2.5 MM) ("=6)
Basal
70.2±13.9
63.8±11.6
Change
113.3±13.9
22.4 ±3.6'
Results (mean±SEM) of the effect of 2.5 jtM ETI on 100 nM
angiotensin II induced calcium transients in calcium-deficient
buffer. ETI, 5,8,11-eicosatriynoic acid; [Ca2+]i, intracellular calcium concentration.
V<0.05 vs. control.
AVP+ET1
(N-5)
BIDT
(ifrt)
BflTT+ED
(n-8)
FIGURE 2. Bar graph shows effect of 5,8,11-eicosatriynoic
acid (ETI) on arginine vasopressin (AVP)- and endothelin
(ENDT)-stimulated increases in intracellular calcium concentration ([Ca 2+]i) in a buffer containing 2 mM calcium. Peak
calcium responses to 100 nMAVP and 200 nMENDT-1 with
and without 2.5 fiM ETI are shown. Due to variability in
calcium responses from cell line to cell line, changes are
expressed as percentage of basal [Ca 1+], levels for comparison. ETI-treated cells have mean maximal [Ca2+]t that is
reduced by 60.9% for AVP (**p<0.01 vs. AVP alone) and
68.6% for ENDT (*p<0.05 vs. ENDT alone).
in vascular tissue. Takayama et al26 reported finding
only minute amounts of LO pathway products in vascular tissue. Bailey et al27 provided evidence for 11- and
15-HETE synthesis in cultured smooth muscle cells but
attributed their production to the CO rather than LO
pathway. In contrast, Greenwald et al28 showed signifi600-,
400-
200-
60
120
180
B
300 _
200-
f
100-
60
120
180
TIME (tac)
FIGURE 3. Effect of 5,8,11-eicosatriynoic acid (ETI) on
arginine vasopressin (AVP) and endothelin 1-stimulated cytosolic calcium concentration ([Ca2*]i). Representative real
time tracings of endothelin (panel A) and AVP (panel B)
stimulated calcium transients showing the effect of20-minute
pretreatment with 2.5 u-M ETI. Addition of the agonist
occurred at 60 seconds as indicated by the arrows.
142
Hypertension
Vol 20, No 2 August 1992
* pxO.05
** p<aoi
T
1_
AVP
(n-6)
AVP+ETI
(N-6)
ENBl
(n_4)
BtDT+ETl
(jtM)
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FIGURE 4. Bar graph shows effect of 5,8,11-eicosatriynoic
acid (ETI) on arginine vasopressin (AVP)- and endothelin
(ENDT)-stimulated increase in cytosolic calcium concentration ([Ca2+]i) in calcium-chelated buffer. In these experiments, 4 mM EGTA was added to the incubation wells 1
minute before agonist. Mean reductions in [Ca2+], levels after
ETI treatment were 72% for AVP (*p<0.05 vs. AVP alone)
and 52.2% for ENDT ("p<0.01 vs. ENDT alone).
cant LO activity in freshly prepared rabbit aortic rings
and found that HETE production could be reduced by
LO pathway inhibitors. Larrue et al29 identified considerable basal activity of the 5-, 12-, and 15-LO pathways
in cultured rabbit aortic smooth muscle cells and reported that activated cells produced predominantly
12-HETE. We have also observed significant 12-HETE
production in de-endothelialized aortic tissue that was
reduced by LO pathway inhibitors.6 Significant amounts
of 5-, 12-, and 15-HETE products have also been noted
in cultured endothelial cells taken from bovine coronary
arteries.30 More recently, Moore and coworkers31 described LO activity in brain microvessels that produced
12fS)-HETE when stimulated by addition of exogenous
arachidonic acid or the calcium ionophore A23187.
The correlation between vascular contraction and a
rise in [Ca 2+ ] ; is well documented.3133 However, the
mechanism of calcium mobilization is influenced by
multiple factors and varies with different agonists. Norepinephrine and potassium chloride addition produces
a sustained contraction that is dependent on extracellular calcium.34-36 When added to vascular tissue, Ang
II causes a transient increase in [Ca2+], and a corresponding brief contractile response.34 Ang H-induced
calcium transients are maintained in a calcium-free
medium,34 suggesting that mobilization of intracellular
calcium stores is primarily responsible for the observed
changes in [Ca2+]i. The selective effects of LO inhibitors
on Ang II responses compared with other vascular
agonists in vivo and in vitro may be due to differences in
the cellular mechanism leading to the [Ca2+]i response.
This assumption is supported by our results in the
calcium-poor buffer, in which ETI also blocked Ang
H-stimulated increases in [Ca2+]i. In cultured VSMC,
both AVP- and endothelin-stimulated [Ca 2+ ]| transients
are reported to be preserved in the absence of extracellular calcium. In our experiments, AVP- and endothelin-induced [Ca2+], transients were inhibited by ETI in
both 2-mM and calcium-poor buffer. The fact that the
inhibitory effect of ETI on Ang II is also observed with
AVP- and endothelin-mediated [Ca2+]i increases may
indicate that LO inhibition alters the mobilization of
[Ca2+]i from intracellular sites via the same mechanism.
Considerable evidence indicates that IP3-mediated
[Ca2+]i release from sarcoplasmic reticulum is responsible for transient increase in [Ca 2+ ],. 37J8 IP3 and DG are
derived by hydrolysis of phosphatidylinositol 4,5-diphosphate by a specific receptor-linked phospholipase.
Further studies into interactions between LO products
and IP3-mediated calcium release are needed to elucidate possible interactions in this pathway.
In summary, we have shown that LO pathway inhibition in VSMC also leads to a reduction in Ang IIstimulated [Ca2+],. When 5-, 12-, and 15-HETE are
added alone to cultured VSMC, they have little effect
on [Ca2+],. However, under the experimental conditions
where VSMC are pretreated with LO inhibitors, the
inhibitory effect on Ang II calcium transients can be
reversed by addition of 12-HETE, but not by 5-HETE
or 15-HETE. Thus, the LO pathway of arachidonic acid
metabolism, in particular 12-HETE, may participate in
the contractile actions of Ang II through modulation of
the intracellular calcium responses to Ang II in VSMC.
The observation that LO inhibitors also block AVP- and
endothelin-stimulated calcium signals suggests that LO
products might be involved in effector mechanisms of
other vasoactive peptide agonists that use the calcium-DG messenger system.
Acknowledgments
We thank Andrew Tuck for the expert word processing
assistance in preparation of this manuscript.
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Hypertension. 1992;20:138-143
doi: 10.1161/01.HYP.20.2.138
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