976
Relaxation of Porcine Coronary
Artery to Bradykinin
Role of Arachidonic Acid
Neal L. Weintraub, Shobha N. Joshi, Carrie A. Branch, Alan H. Stephenson,
Randy S. Sprague, Andrew J. Lonigro
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract Bradykinin-induced relaxation of precontracted,
porcine coronary artery (PCA) rings is mediated by distinctly
different endothelium-derived relaxing factors depending on
the contractile agent used. Thus when contracted with KC1,
bradykinin-induced relaxation of PCA rings is mediated solely
by nitric oxide (NO), whereas when contracted with the
thromboxane mimetic U46619, a small component of the
relaxation is attributable to NO and a large component is
attributable to a non-NO mechanism that is independent of
cyclooxygenase activity. We hypothesized that the non-NO
component was mediated by arachidonic acid (AA) or by a
non-cyclooxygenase product of AA metabolism. Bradykinininduced relaxations of PCA rings precontracted with U46619
in the presence of indomethacin (10 /imol/L) were moderately
attenuated by the NO synthase inhibitor W-nitro-L-arginine
methyl ester (L-NAME, 100 fxmolfL), whereas when precontracted with KC1, L-NAME abolished the relaxations. AA
produced endothelium-dependent relaxations of rings precon-
traded with U46619 that were unaffected by L-NAME,
whereas AA did not relax rings precontracted with KC1. In
rings precontracted with U46619, in the presence of L-NAME
and indomethacin the phospholipase inhibitors quinacrine (50
fimolfL) and 4-bromophenacyl bromide (10 jtmol/L) attenuated bradykinin- but not AA-induced relaxations. Inhibitors of
both lipoxygenase (BW 755c [100 /imol/L] and nafazatrom [20
/xmol/L]) and cytochrome P-450 (proadifen [10 iimol/L] and
clotrimazole [10 /xmol/L]) pathways did not eliminate bradykinin- or AA-induced relaxations, although clotrimazole partially attenuated AA-induced relaxations. These findings suggest that bradykinin-induced relaxation of PCA rings is
mediated by AA through a mechanism that is not dependent
on cyclooxygenase, lipoxygenase, or cytochrome P-450 pathways. (Hypertension. 1994;23[part 2]:976-981.)
T
acid may in turn be reacylated21 or metabolized further
through three known pathways, ie, the cyclooxygenase
pathway* to prostaglandins and thromboxanes, the lipoxygenase pathway22 to leukotrienes and oxygenated
fatty acids, and the cytochrome P-450 monooxygenase
pathway23 to epoxides and oxygenated fatty acids. Several of the non-cyclooxygenase-mediated products of
arachidonic acid metabolism are capable of relaxing
blood vessels.2426
In view of the finding that bradykinin produces relaxation of PCA rings through a mechanism that is in large
part independent of NO synthase and cyclooxygenase,
we hypothesized that bradykinin-induced relaxation of
PCA rings is mediated by arachidonic acid itself or
through a product of arachidonic acid metabolism that
is independent of cyclooxygenase activity.
he concept of the vascular endothelium as a
fundamental participant in circulatory control
is rapidly evolving.1-2 In addition to several
vasoconstricting agents,3-5 at least two vasorelaxing substances of vascular endothelial origin have been identified: prostaglandin I2 (PGI 2 ), 6 a product of cyclooxygenase-mediated arachidonic acid metabolism, and nitric
oxide (NO), 78 or a closely related compound,9 which is
produced by the action of NO synthase on L-arginine.
More recently, endothelium-dependent relaxations of
several vascular tissues have been reported to occur
through a mechanism(s) distinct from one involving
either cyclooxygenase or NO synthase. 1013 Thus relaxation of isolated porcine coronary arterial (PCA) rings
to bradykinin, for example, persisted despite inhibition
of both cyclooxygenase and NO synthase.1315 It was
proposed that this relaxation was mediated by an endothelium-derived hyperpolarizing factor.16
Bradykinin activates phospholipase A 2 , 1718 purportedly through receptor-mediated increases in intracellular calcium.19 The activation of phospholipase A2 results
in hydrolysis of tissue phospholipids, in turn releasing
fatty acids, including arachidonic acid.19-20 Arachidonic
From the Departments of Internal Medicine (N.L.W., S.NJ.,
C.A.B., R.S.S., A.J.L.) and Pharmacological and Physiological
Science (A.H.S., A.J.L.), Saint Louis (Mo) University School of
Medicine.
Correspondence to Neal L. Weintraub, MD, Saint Louis University, School of Medicine, Division of Clinical Pharmacology,
Rm M205, 1402 S Grand Blvd, St Louis, MO 63104.
Key Words • endothelium-derived relaxing factor
coronary artery • arachidonic acid • bradykinin
•
Methods
Pig hearts were obtained from a local slaughterhouse;
placed immediately into ice-cold modified Krebs-Ringer bicarbonate (KRB) solution containing (in mmol/L) NaCI 118.3,
KC1 4.7, CaCl2 2.5, MgSO, 1.2, KH 2 PO, 1.2, NaHCO3 25.0,
Na-EDTA 0.026, and glucose 11.1; and transported to the
laboratory. The right coronary artery was dissected, cleansed
of all adherent fat and connective tissue, and cut into rings 3 to
5 mm wide. Each ring was mounted on two stainless-steel
posts, one fixed and the other movable. The latter post was
attached to an isometric force transducer (model FT-03, Grass
Instrument Co) coupled to a polygraph (model 7, Grass) for
continuous recording of ring tension. The rings were suspended in water-jacketed (37°C) organ baths (Radnoti Glass
Weiniraub et al
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Endothelium-Arachidonic Acid Interactions
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Arichidonlc Acid (/JJ)
Technology, Inc) containing 10 or 20 mL KRB solution and
continuously aerated with 95% C>2-5% CO2. Basal ring tension
was increased incrementally. With each increment, the ring
was contracted with KCI (60 mmol/L) until a maximal contraction was identified. The rings were maintained at the basal
tension that produced a maximal contraction (range, 9 to 12 g;
mean, 10.0±0.1 g) for the remainder of the experiment.
Indomethacin (10 jimol/L) was in the KRB solution throughout all experiments. When used, JV"-nitro-L-arginine methyl
ester (L-NAME) was introduced into the bath 45 minutes
before the initial contraction to achieve a final concentration
of 100 jtmol/L and thereafter was included throughout the
experiment.
Rings were contracted with either the thromboxane mimetic
U46619 (2.5 to 30 nmol/L) or KCI (20 to 30 mmol/L) to
achieve 50% to 80% of the maximal contraction obtained with
KCI (60 mmol/L). Relaxation in response to vasoactive agents
was expressed as the percent decrease from the U46619- or
KCl-induced tension. After contractions had stabilized, the
rings were relaxed with bradykinin (0.3 to 100 nmol/L) to
evaluate the functional integrity of the endothelium. The
endothelium was considered to be intact when the maximal
relaxation to bradykinin was £40% in rings contracted with
U46619; only vessels with intact endothelium were studied
further. The baths were rinsed, and after a stabilization period
(minimum of 30 minutes), the rings were contracted, and
relaxation responses to bradykinin, arachidonic acid, or nitroglycerin were obtained. In preliminary experiments, repeated
application of arachidonic acid in rings contracted with
U46619 revealed potentiation of relaxation with the second
concentration-response determination but no further potentiation with subsequent determinations. This potentiation was
obviated by incubating the rings with arachidonic acid (100
jtmol/L) for 45 minutes before the initial exposure to bradykinin. In some experiments the phospholipase inhibitors
quinacrine and 4-bromophenacyl bromide (BPB) were administered 30 minutes before the rings were contracted. The
inhibitors of lipoxygenase (BW 755c, nafazatrom) and cytochrome P-450 monooxygenase (proadifen, clotrimazole)
were administered 60 minutes before the rings were contracted. Mechanical disruption of the endothelium was
achieved by rubbing rings with a wooden stick and a roughened
10
100
1000
977
FIG 1. Plots of relaxation responses to
bradykinin (BK), arachidonic acid (AA),
and nitroglycerin (GTN) in porcine coronary artery rings. A, BK relaxations were
determined in 1146619-precontracted
rings that were untreated, pretreated
with W-nitro-L-arginine methyl ester (LNAME) (100 ^mol/l.), or mechanically
denuded of endothelium (n=9). B, BK
relaxations of KCI-precontracted rings,
untreated or pretreated with L-NAME
(n=7). C, AA relaxations of U46619-precontracted, endothelium-intact (E+)
rings in the presence of L-NAME (n=7);
1146619-precontracted, endothelium-denuded (E-) rings (n=6); and KCI-precontracted, endothelium-intact (E+)
rings (n=5). D, GTN-induced relaxations
of endothelium-denuded rings precontracted with U46619 or KCI (n=5). Indcmethacin (10 ^mol/L) was included in all
experiments. Results are expressed as
mean±SEM. *P<.05, untreated vs
L-NAME-pretreated or endothelium-denuded (BK data) rings; U46619-precorv
tracted, endothelium-intact rings vs
1146619-precontracted, denuded rings
or KCI-precontracted, intact rings (AA
data).
NKroglyctrin (nM)
23-gauge needle. The ring was considered denuded of endothelium when maximal relaxation to bradykinin (100 nmol/L)
was £10%. All denuded rings were relaxed with nitroglycerin
(0.3 /imol/L), and only the rings that relaxed at least 50% were
studied further.
Chemicals
Bradykinin (acetate salt), arachidonic acid (sodium salt),
U46619, indomethacin, L-NAME, quinacrine, BPB, proadifen, clotrimazole, and cromokalim were purchased from
Sigma Chemical Co. Nitroglycerin was purchased from American Regent Laboratories, Inc. BW 755c and nafazatrom were
the gifts of The Wellcome Research Laboratories and Miles
Laboratories, Inc, respectively. Indomethacin, BPB, clotrimazole, and cromokalim were dissolved in ethanol; U46619 was
dissolved in methanol; and nafazatrom was dissolved in dimethyl sulfoxide (DMSO). Final bath concentrations of ethanol and DMSO did not exceed 0.1%. All other chemicals were
dissolved in distilled water.
Statistical Analysis
All values are given as mean±SEM. Differences between
values were analyzed by Student's t tests for paired and
unpaired data, as appropriate. Differences between mean
values of multiple groups were analyzed by analysis of variance, with a least significant difference test applied if the F
ratio was significant. Values of P<.05 were considered statistically significant.
Results
Responses to Bradykinin
Bradykinin (0.3 to 100 nmol/L) produced concentration-dependent relaxations of PCA rings precontracted
with U46619 (Fig 1A). In rings pretreated with
L-NAME (100 /imol/L), responses to bradykinin were
reduced (Fig 1A); however, significant relaxation persisted, suggesting the presence of a non-NO-mediated
mechanism of vasorelaxation. Endothelium dependency
of the response was demonstrated by the observation
that mechanical disruption of the endothelium abol-
978
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Hypertension
Vol 23, No 6, Part 2 June 1994
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100
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Araotildonic Add (iM)
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Bradyfcinln (nM)
1000
Nilroglyoarin (nU)
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100
10
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100
Ariotiidonic Aaid (jM)
10
100
1000
mtroglyoarin (nU)
FK3 2. Plots of effects of the phospholipase inhibitors quinacrine and 4-bromophenacyl bromide (BPB) on bradykinin (BK), arachidonic
acid (AA), and nitroglycerin (GTN) relaxations of porcine coronary artery rings precontracted with U46619 in the presence of
/V"-nitrc-L-arginine methyl ester and indomethacin. A, BK relaxations were determined before (control) and after treatment with
quinacrine (50 jtmol/L) (n=6). B, AA relaxations before (control) and after quinacrine (n=5). C, QTN relaxations before (control) and after
quinacrine (n=5). D, BK relaxations were determined before (control) and after treatment with BPB (10 jimol/L) (n=6). E, AA relaxations
before (control) and after BPB (n=6). F, GTN relaxations before (control) and after BPB (n=6). Results are expressed as mean±SEM.
*P<.05, control vs treatment with quinacrine or BPB.
ished bradykinin-induced relaxation (Fig 1A). In contrast, when rings were contracted with KC1 (20 to 30
mmoI/L) to oppose hyperpolarization, pretreatment
with L-NAME (100 /xmol/L) abolished relaxation responses to bradykinin, suggesting that the relaxation to
bradykinin in rings precontracted with KC1 is dependent
on NO (Fig IB).
Responses to Arachidonic Acid
Arachidonic acid (0.3 to 100 /xmol/L) produced concentration-dependent relaxations of PCA rings precontracted with U46619 (Fig 1C). Arachidonic acid-induced relaxations of U46619-contracted rings were not
affected by pretreatment with L-NAME (100 /xmol/L)
(data not shown) but were markedly attenuated by
mechanical disruption of the endothelium (Fig 1C).
Precontraction of rings with KC1 (20 to 30 mmol/L)
abolished the relaxations produced by arachidonic acid
(Fig 1C). In contrast, precontraction of rings with KC1
(20 to 30 mmol/L) did not attenuate nitroglycerininduced relaxations (Fig ID).
Effects of Phospholipase Inhibitors on
Responses to Vasorelaxants
In rings precontracted with U46619 in the presence of
L-NAME (100 jimol/L), quinacrine (50 /xmol/L) attenuated the relaxation responses to bradykinin (Fig 2A)
but not to arachidonic acid (Fig 2B) or to nitroglycerin
(Fig 2C). BPB (10 /xmol/L) abolished bradykinin-induced relaxation (Fig 2D). In contrast, relaxations in
response to arachidonic acid (Fig 2E), nitroglycerin (Fig
2F), and cromokalim (data not shown) were not inhib-
ited by treatment with BPB. These results suggest that
under these experimental conditions, bradykinin requires phospholipase activity to manifest its vasorelaxant properties, whereas arachidonic acid does not.
Effects of Inhibition of the Cytochrome P-450 and
Lipoxygenase Pathways of Arachidonic Acid on
Relaxation Responses
Concentrations of proadifen or clotrimazole >10
jtmol/L were not used because of persistent contraction
of the PCA ring and precipitation of the compound in
the bath, respectively. Neither proadifen (10 /imol/L)
nor clotrima2ole (10 ^mol/L) inhibited relaxation responses to bradykinin (Table). Proadifen had little
effect on arachidonic acid-induced relaxation, but clotrimazole partially attenuated relaxation produced by
arachidonic acid (Table).
Not only did BW 755c fail to inhibit bradykinininduced relaxations in PCA rings contracted with
U46619, it potentiated the relaxations (Table). This
potentiation was not evident after removal of BW 755c
(data not shown). BW 755c had little effect on arachidonic acid-induced relaxation (Table). A second lipoxygenase inhibitor, nafazatrom (20 ^tmol/L), did not inhibit relaxation responses to bradykinin or arachidonic
acid (Table).
Discussion
The findings of the present study are consistent with
previous studies 1416 in which endothelium-dependent
relaxations of the PCA produced by bradykinin were
reported to be resistant to the application of either
Weintraub et al Endothelium-Arachidonic Acid Interactions
979
Effects of Inhibitors of LJpoxygenase and Cytochrome P-450 Enzymes on Bradyklnin- and Arachldonic Acid-Induced
Relaxations of Porcine Coronary Artery Rings
Arachldonic Add, /unol/L
Bradykinin, nmol/L
Treatment
Control
100
1
10
100
0.0±0.0
1
30.8±7.7
68.8±8.4
1.3±1.2
14.6±5.9
62.6±8.8
10
0.0±0.0
35.5±14.3
80.2±7.5
0.0±0.0
10.1+3.7
54.0+8.8
11.8±5.6
72.6±7.8
88.8±2.7
0.9+0.5
22.7±7.4
68.3±9.6
Clotrimazole, 10 jtmol/L
3.4±1.8
69.8±8.3
86.8±3.8
1.1±0.7
9.7±4.2*
Control
0.0±0.0
41.4±11.8
69.6±10.9
4.0±2.0
45.6+10.3
92.6±3.7
BW755C, lOO^mol/L
0.0±0.0
71.4+9.1*
88.0±7.2*
4.6+1.5
66.0±7.2*
95.6±1.7
Control
0.0±0.0
43.2±8.3
73.6±8.6
0.0+0.0
23.8±9.4
72.6±12.0
Nafazatrom, 20 ^mol/L
0.0+0.0
39.8±9.9
80.0±5.3
1.2±1.1
25.8±10.6
73.0±13.6
Proadifen, 10 ^mol/L
Control
52.1 ±8.2*
Rings were contracted with U46619 in the presence of /V-nitro-L-arginine methyl ester (100 /xmol/L) and indomethadn (10 ^mol/L),
n=5 to 7.
*P<.05, treatment value vs control value.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
methylene blue or hemoglobin (both of which inhibit
NO-mediated vasorelaxation1-2) and to the administration of either NO synthase or cyclooxygenase inhibitors.
Furthermore Cowan and Cohen15 demonstrated that
increased cyclic GMP concentrations in PCA rings after
exposure to bradykinin were prevented by W-monomethyl-L-arginine or methylene blue and that cyclic
AMP (which increases in response to PGI2) was not
stimulated by bradykinin. Therefore endothelium-dependent relaxation of the PCA occurs through a process
that is distinct from either of the two previously identified endothelium-derived factors, NO and PGI2.
Although L-NAME had only a minor effect on bradykinin-induced relaxation of PCA rings contracted with
U46619, it abolished bradykinin relaxation of rings
contracted with KC1. These findings are in agreement
with those of Cowan and Cohen15 and Nagao and
Vanhoutte.16 One interpretation of these results is that
the major component of bradykinin-induced relaxation
of PCA rings contracted with U46619 occurs through a
mechanism that is not affected by inhibition of NO
synthase but is blocked by KC1, which opposes hyperpolarization. This relaxing mechanism might then result
from membrane hyperpolarization and its attendant
effects on cytosolic free calcium. Agents such as bradykinin, which produce endothelium-dependent relaxation of the PCA, have also been reported to hyperpolarize PCA endothelial and smooth muscle cells.1327
Hyperpolarization is believed to occur secondary to an
enhanced efflux of potassium ions from vascular smooth
muscle cells after the activation of potassium channels.
The resultant hyperpolarization then is believed to close
voltage-dependent calcium ion (Ca 2+ ) channels, thereby
reducing Ca2+ entry into the smooth muscle cells and
resulting in vasorelaxation.28 Hyperpolarization of PCA
smooth muscle cells in response to bradykinin was
reported to be dependent on the presence of the
endothelium.16 Thus the possibility that the relaxant
effect of bradykinin on PCA rings is mediated by the
release of a hyperpolarizing factor is a reasonable
hypothesis.
In the present study we hypothesized that the nonNO-, cyclooxygenase-independent relaxation of PCA
rings produced by bradykinin was mediated by arachi-
donic acid or through products of a non-cyclooxygenase
pathway of arachidonic acid metabolism. This hypothesis was formulated in view of the findings that bradykinin receptor activation activates phospholipases in a
variety of tissues, resulting in the release of arachidonic
acid17; arachidonic acid contributes to bradykinin-induced relaxation of isolated canine coronary artery
rings through its conversion to cytochrome P-450 metabolites24; arachidonic acid29 as well as several metabolites of arachidonic acid can activate potassium channels on smooth muscle cells3032; and potassium channel
activation was reported to be a mechanism by which
11,12-epoxyeicosatrienoic acid, a product of epoxygenase-mediated arachidonic acid metabolism, produced
relaxation of isolated cat cerebral arteries.31 As a first
step in addressing this hypothesis, we characterized
responses of PCA rings to the administration of arachidonic acid. In the presence of indomethacin, arachidonic acid produced concentration-dependent relaxations of rings contracted with U46619, which were not
affected by pretreatment with L-NAME but were markedly attenuated by removal of the endothelium. In
contrast, when the rings were contracted with KC1,
arachidonic acid did not produce relaxation. These data
suggest that arachidonic acid primarily relaxes PCA
rings through an endothelium-dependent mechanism
that does not require NO but is inhibited by excess K + ,
suggesting that the relaxation may result from potassium channel activation. This finding is consistent with
the hypothesis that arachidonic acid (or one of its
metabolites) effects the relaxation of PCA rings in
response to bradykinin. The endothelium-dependent
relaxation produced by bradykinin does not appear to
be mediated by free arachidonic acid acting directly on
vascular smooth muscle cells, however, because arachidonic acid-induced relaxation was also found to be
endothelium dependent.
If arachidonic acid does mediate bradykinin-induced
relaxation of the PCA, then agents that inhibit bradykinin-induced arachidonic acid release from tissue
phospholipids would also be expected to attenuate
bradykinin relaxation responses. Arachidonic acid can
be liberated from cellular membranes through hydrolysis by phospholipase A2 or by the sequential actions of
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Vol 23, No 6, Part 2 June 1994
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phospholipase C and diglyceride lipase.21 Quinacrine,
which inhibits phospholipases by interfering with the
substrate-enzyme interface, and BPB, which inhibits
phospholipases by alkylating a histidine moiety near the
active site of the enzyme,19 are nonselective agents that
have been reported to inhibit phospholipase C as well as
phospholipase A2.19-33 These agents would be expected
to inhibit bradykinin-induced relaxation if this response
is dependent on the release of arachidonic acid from
tissue stores. Indeed, bradykinin relaxation was inhibited
by both quinacrine and BPB, whereas relaxations to
nitroglycerin and arachidonic acid were not attenuated
by either compound. Furthermore BPB did not attenuate
relaxation produced by the ATP-dependent potassium
channel activator cromokalim. These observations suggest that quinacrine and BPB may have inhibited bradykinin-induced relaxation by interfering with a proximal
step in the signal transduction process, ie, arachidonic
acid release, as opposed to a distal one, ie, arachidonic
acid metabolism. These findings are consistent with our
hypothesis that arachidonic acid, or one of its metabolites, mediates bradykinin-induced relaxation of the
PCA. To evaluate the possibility that a lipoxygenase or
cytochrome P-450 product of arachidonic acid metabolism mediates the bradykinin-induced endothelium-dependent relaxation of PCA rings, responses to bradykinin
and arachidonic acid were studied in the presence and
absence of inhibitors of both enzymatic pathways. The
lipoxygenase inhibitor nafazatrom did not affect either
bradykinin- or arachidonic acid-induced relaxation,
whereas the combined cyclooxygenase/lipoxygenase inhibitor BW 755c potentiated relaxation to bradykinin and
had little effect on relaxation in response to arachidonic
acid. It is therefore unlikely that lipoxygenase products
of arachidonic acid metabolism mediate either bradykinin- or arachidonic acid-induced relaxation of the PCA.
The mechanism by which BW 755c potentiated relaxation was not addressed in the present study. The
cytochrome P-450 inhibitor clotrimazole partially attenuated arachidonic acid-induced relaxation of PCA rings.
It is therefore possible that the relaxation produced by
arachidonic acid administration is in part mediated by
epoxygenase metabolites, although this question remains
to be resolved, because a second inhibitor of cytochrome
P-450, proadifen, had little effect on arachidonic acidinduced relaxations. In contrast, bradykinin-induced relaxations were unaffected by either of the cytochrome
P-450 inhibitors; therefore, epoxygenase products of
arachidonic acid metabolism most likely do not mediate
bradykinin-induced relaxation of the porcine coronary
artery.
The finding that relaxations in response to arachidonic acid were inhibited by clotrimazole, whereas
relaxations in response to bradykinin were not, does not
preclude a role for arachidonic acid in mediating bradykinin relaxation. It has been reported that tissues may
produce different proportions of eicosanoids depending
on the particular agonist used, possibly related to release of arachidonic acid into distinct subcellular sites
where it may be metabolized by different enzyme pathways.21 Thus it is possible that arachidonic acid may be
producing relaxation in response to bradykinin through
a metabolic pathway that is insensitive to the inhibitors
used in the present study. Alternatively, arachidonic
acid has been proposed to participate in the cellular
signaling process through complex effects such as increasing [Ca2*],.34-35
In summary, we have demonstrated that arachidonic
acid, similar to bradykinin, produces endothelium-dependent relaxation of PCA rings through a mechanism
that is sensitive to excess potassium. A role for arachidonic acid in mediating bradykinin relaxation is suggested by the observation that the phospholipase inhibitors quinacrine and BPB attenuated bradykinin- but
not arachidonic acid-induced relaxation of PCA rings.
In view of the observation that inhibitors of cyclooxygenase-, lipoxygenase-, and cytochrome P-450-mediated metabolism of arachidonic acid did not attenuate
bradykinin relaxation, arachidonic acid may produce
relaxation through enzymatic conversion that is insensitive to the inhibitors tested or by participating in the
cellular signaling process as a second messenger.
Ack nowledgmen t s
This work was supported in part by National Institutes of
Health grant HL-30572. N.L.W. and C.A.B. were supported by
National Research Service Award Post-Doctoral Research
Training Grant 2 T 32 HL-07050-18.
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N L Weintraub, S N Joshi, C A Branch, A H Stephenson, R S Sprague and A J Lonigro
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Hypertension. 1994;23:976-981
doi: 10.1161/01.HYP.23.6.976
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