326

Correlation With Blood Pressure of the

Acetylcholine-Induced Endothelium-Derived
Contracting Factor in the Rat Aorta
Yoshio Iwama, Toshio Kato, Masahito Muramatsu, Hiroshi Asano, Kiyokazu Shimizu,
Yukio Toki, Yutaka Miyazaki, Kenji Okumura, Hidekazu Hashimoto,
Takayuki Ito, and Tatsuo Satake

To examine a relation between the production of acetylcholine-induced endothelium-derived contracting

factor and an increase in blood pressure, endothelium-dependent contraction and relaxation were
evaluated by measuring the isometric tension of aortic rings from spontaneously hypertensive rats and
Wlstar-Kyoto rats at 5,10, 20, and 30 weeks of age. In norepinephrine-precontracted rings, acetylcholine
(10"' to 10"' M)-induced relaxations diminished at the doses of 10"' to 10"5 M in both strains except at
5 weeks of age. Treatment with a thromboxane A2/prostaglandin H2 antagonist (ONO-3708) prevented this
reduction in acetylcholine-induced relaxations in both strains and induced dose-dependent relaxations,
which were completely inhibited by treatment with a nitric oxide inhibitor, ArG-nitro-L-arginine methyl
ester. In aorta treated with /VG-nitro-L-arginine methyl ester without precontraction, acetylcholine induced
dose-dependent contractions, which were greater in spontaneously hypertensive rats than in Wistar-Kyoto
rats. These acetylcholine-induced contractions, which were observed only in rings with endothelium, were
completely inhibited by treatment with ONO-3708 but not with a thromboxane A2 synthetase inhibitor
(OKY-046). There was a statistically significant correlation between the acetylcholine-induced contrac-
tions and blood pressure. Release of 6-ketoprostaglandln F,o by acetylcholine from the aorta was greater
in spontaneously hypertensive rats. In vivo administration of another thromboxane A2/prostaglandin H2
antagonist (ONO-8809) (10 or 30 /ig per body per day) for 3 weeks (5-8 weeks of age) did not affect blood
Downloaded from http://ahajournals.org by on November 19, 2019

pressure in either rat strain. These results suggest that the production of acetylcholine-induced
endothelium-derived contracting factor, which is most likely prostaglandin H2, is closely associated with
the increase in blood pressure that occurs in young to adult rats. (Hypertension 1992;19-J26-332)
KEY WORDS • endothelium • endothelium-derived contracting factor • endothelium-derived
relaxing factor • prostaglandin H2 • spontaneously hypertensive rats

T he endothelium produces various vasoactive sub-
stances and controls vascular smooth muscle
tone. Since Furchgott and Zawadzki1 first re-
ported the presence of endothelium-derived relaxing
factor (EDRF) in 1980, many vessels were found to
produce EDRF in response to a variety of stimuli.2-3
The primary component of EDRF was thereafter shown
to be nitric oxide.4'5 It was also clarified that endotheli-
um-derived contracting factor (EDCF) is also produced
by stimulated vessels,6 and increased production of
EDCF has been reported in anoxia,7 hypertension,8 and
aging.9 The nature of EDCF varies with the species and
the anatomical site of its production, but thromboxane
A2 (TXA2),10 superoxide anion, 1112 endothelin,13 and
prostaglandin H2 (PGH2)14 have been suggested as
EDCF. The EDCF released from the rat thoracic aorta
by acetylcholine stimulation is likely to be PGH2, as we
have previously suggested.14
From the Second Department of Internal Medicine, Nagoya
University School of Medicine, Nagoya, Japan.
Address for correspondence: Takayuki Ito, MD, The 2nd De-
partment of Internal Medicine, Nagoya University School of
Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan.
Received October 2, 1990; accepted in revised form December
16, 1991.
The present study was designed to confirm the hy-
pothesis that increased production of the EDCF in-
duced by acetylcholine in rat aorta coincides with the
increase in blood pressure. For this purpose, we sepa-
rately evaluated EDCF-induced contractile responses
and EDRF-induced relaxations in the rat thoracic aorta
under acetylcholine stimulation by inhibiting either
EDRF or EDCF with a nitric oxide inhibitor or a
TXA 2 /PGH 2 receptor antagonist, respectively, and also
by examining changes in the production of these factors
in rats of various age groups. Furthermore, we examined
whether blood pressure in rats in vivo was reduced by
inhibition of the EDCF production.
Methods
Male spontaneously hypertensive rats (SHR) and
Wistar-Kyoto (WKY) rats (Charles River Japan Inc.,
Atsugi, Japan) in 5-, 10-, 20-, and 30-week-old groups
were used. Each group consisted of rats aged 5-6,
10-11, 20-22, and 30-32 weeks. Blood pressures were
measured in conscious rats using the tail-cuff method.
The rats were decapitated, the thoracic aorta was
excised, and the connective tissue was removed in
cooled Krebs-Henseleit solution with the following
 Iwama et al Endothelium-Derived Contracting Factor
composition (mM): NaCl 118, KC1 4.7, CaCl2 2.55,
MgSO41.18, KH2PO41.18, NaHCOj 24.88, glucose 11.1,
EDTA Ca-2Na 0.026, and then cut into rings 5 mm in
length. Two stainless steel wires were inserted into the
vascular lumen, and one was connected to a force
transducer (model TB-612T, Nihonkohden, Tokyo).
The rings were suspended in a chamber containing 30
ml Krebs-Henseleit solution that was bubbled with a
95% O2-5% CO2 mixture and maintained at 37°C. The
rings were incubated for 90 minutes for equilibration at
a resting tension of 2 g with buffer exchanges every 30
minutes during this period. The rings were manipulated
carefully so as not to produce unnecessary tension or to
damage the endothelium. The endothelium was re-
moved by rubbing the vascular lumen with a thin swab.
Acetylcholine (lfr7 M)-induced relaxation less than 5%
of precontraction with norepinephrine (10~7 M) was
considered to indicate the absence of endothelium.
Precontraction of the Arteries
Contractions in response to 10"7 M norepinephrine in
aortic rings with and without endothelium were esti-
mated by measuring the changes in tension of rings in
each group of WKY rats and SHR.
Contractions to norepinephrine (10~8 to 10"5 M) in
the rings without endothelium reached a plateau at the
concentration of 10"5 M. Ratios of the contraction
induced by 10~7 M norepinephrine to that induced by
10"5 M norepinephrine were as follows: 92.7 ±1.8% (5
weeks), 86.0±2.9% (10 weeks), 91.6±0.7% (20 weeks),
and 91.7±1.1% (30 weeks) in WKY rats and 94.3±1.5%
Downloaded from http://ahajournals.org by on November 19, 2019
(5 weeks), 86.8±2.8% (10 weeks), 90.1 ±1.6% (20
weeks), and 89.5±3.1% (30 weeks) in SHR. Since these
ratios were similar, the concentration of 10"7 M norepi-
nephrine was considered an effective dose for all tissues.
To determine the optimal resting tension, contrac-
tions to 10"7 M norepinephrine in rings without endo-
thelium were estimated under resting tension of 1 g, 2 g,
and 3 g. Because contractions developed under these
resting tensions were similar in each age group (data not
shown), resting tension of 2 g was applied to all rings.
Responses to Acetylcholine and Effects of
Thromboxane A2IProstaglandin H2 Receptor
Antagonists and a Nitric Oxide Inhibitor
The aortic rings with or without endothelium from
SHR and WKY rats in 5-, 10-, 20-, and 30-week-old
groups were contracted with norepinephrine (10~7 M).
When a plateau was reached, the rings were relaxed by
cumulative addition of 10~8 to 10~5 M acetylcholine.
The rings were treated with a TXA 2 /PGH 2 receptor
antagonist ONO-370815 (10~6 M) or a novel TXA 2 /
PGH2 receptor antagonist ONO-NT-12616 (3xlO~6 M)
15 minutes before precontraction with norepinephrine.
When the rings were treated with both a nitric oxide
inhibitor, N -nitro-L-arginine methyl ester 17 (L-
NAME) (10' 3 M), and ONO-3708, L-NAME was added
30 minutes before precontraction.
In other experiments, 10"* to 10"5 M acetylcholine
was applied to the rings without precontraction. The
rings were treated with L-NAME or ONO-3708 15
minutes before acetylcholine application. When the
rings were treated with both L-NAME and ONO-3708,
327
L-NAME was added 30 minutes before acetylcholine
application.
When the rings were precontracted, the relaxations
were expressed as percentages of the contraction in-
duced by 10"7 M norepinephrine, and when the rings
were not precontracted, they were expressed as percent-
ages of the contraction induced by 10"7 M norepineph-
rine in another adjacent ring from the same rat.
Effects of a Thromboxane A2 Synthetase Inhibitor
Under resting tension, 10~8 to 10~5 M acetylcholine
was added cumulatively 15 minutes after the treatment
with both 10"3 M L-NAME and 10"5 M OKY-046,18 a
TXA 2 synthetase inhibitor, and vascular responses
were examined.
Concentrations of 6-Ketoprostaglandin Fla
Only a resting tension was applied to the aortic rings
from SHR and WKY rats in the 30-week-old group, and
10"5 M acetylcholine was added 15 minutes after treat-
ment with 10~3 M L-NAME. Before the treatment with
L-NAME, immediately before the addition of acetyl-
choline and at the peak response to 10~5 M acetylcho-
line, 0.5 ml of the solution in the chamber was collected
and the 6-ketoprostaglandin F l a (6-keto-PGFla) con-
centration was determined by the method of Jaffe et al19
and Powell20 using a radioimmunoassay kit prepared by
New England Nuclear, Boston, Mass.
Administration of a Thromboxane A2IProstaglandin H2
Receptor Antagonist In Vivo
ONO-3708 is a short-acting agent in vivo,15 and
ONO-8809 is a long-acting prodrug, which is trans-
formed to ONO-NT-126 in vivo (unpublished observa-
tion). To evaluate the effect of inhibition of EDCF
production on blood pressure, ONO-8809 (10 or 30 fig
per body per day) was administered to 5-week-old WKY
rats and SHR for 3 weeks. The systolic blood pressures
of WKY rats and SHR were measured at 5 and 8 weeks
of age by the tail-cuff method in the unanesthetized
state.
Drugs
The following drugs from Sigma Chemical Co., St.
Louis, Mo. were used: /-norepinephrine bitartrate, ace-
tylcholine chloride, and N°-nitro-L-arginine methyl es-
ter. Ono Pharmaceutical Company, Osaka, Japan, pro-
vided (£)-3[4-(l-imidazorylmethyl)phenyl]-2-propenoic
acid hydrochloride monohydrate (OKY-046),
(9,ll)(ll,12)-dideoxa-9a, lla-dimethylmethano-11,12-
methano-13,14-dihydro-13-aza-14-oxo-15-cyclopentyl-
16,17,18,19,20-pentanor-15-epi-TXA2 (ONO-3708),
9,ll-epithio-ll,12-methano-TXA2 (STA2), n-decyi (Z)-
6-[(lS,2S,3i?,4/?)-3-(4-bromobenzenesulfonylaminometh-
yl)bicyclo [2.2.1] hept-2-yl]-5-hexenoate (ONO-8809),
and 5(Z)-6-[(LR,2^,3^45)-3-(A^-4-bromobenzenesuhco-
nylaminomethyl) bicyclo[2.2.1]heptane-2-yl] hex-5-enoic
acid (ONO-NT-126). STA2, ONO-8809, and ONO-NT-
126 were dissolved in ethanol. The final ethanol concen-
tration in the bath solution was below 0.1%. Vehicle did
not affect the acetylcholine-induced responses or the
resting tension. The other drugs were dissolved in dis-
tilled water.
 328 Hypertension Vol 19, No 4 April 1992

TABLE 1. Changes in Systolic Blood Pressure in Each Age Group TABLE 2. Contractions to 10 7 M Norepinephrine In Rat Aortic
Rings With and Without Endothelium
Group 5 weeks 10 weeks 20 weeks 30 weeks
WKY 97±3 109±4 134±3 141±2 Group 5 weeks 10 weeks 20 weeks 30 weeks
SHR 108+3 162±3 184±4 192±3 Endothelium(+)
WKY 0.35±0.02 0.40±0.03 0.36±0.01 0.41 ±0.04
Results (mm Hg) are expressed as mean±SEM. n=6-9. WKY,
Wistar-Kyoto rats; SHR, spontaneously hypertensive rats. SHR 0.33±0.02 037±0.02 0.32±0.01 0.34±0.01
Endothelium(-)
WKY 0.63±0.04 0.69±0.05 0.64±0.04 0.62±0.02
Statistical Analysis
SHR 0J7±0.03 0.62±0.03 0.64±0.05 0.60±0.03
Results were expressed as mean±SEM. For statistical
Results (g) are expressed as mean±SEM. n=5-6. There is no
analysis, Student's t test for paired or unpaired observa- significant difference between Wistar-Kyoto (WKY) rats and
tions and the Wilcoxon test were used. The regression spontaneously hypertensive rats (SHR) with or without endothe-
line was determined by the least-squares method. Values lium in each age. There is no age-dependent change in each strain.
of /?<0.05 were considered to be significant.
Results
the presence of ONO-3708 and OKY-046, but in rings
Blood Pressure with endothelium, the contractions increased in the
The changes in systolic blood pressure of SHR and presence of L-NAME.
WKY rats in each age group are shown in Table 1.
Responses to Acetylcholine
Contractions to Norepinephrine Figure 1A shows typical responses. When acetylcho-
Contractions to 10"7 M norepinephrine in aortic rings line was added cumulatively after contraction induced
with and without endothelium are shown in Table 2. by norepinephrine, aortic rings of 30-week-old SHR
There were no age-dependent or hypertension-depen- were relaxed at acetylcholine concentrations of 10~8 to
dent changes in contractions to 10 M norepinephrine 10~7 M, but relaxations were weaker at acetylcholine
in either strain. These contractions were not affected by concentrations of 10~6 to 10~5 M. In the rings without
eiidulhelium, nu relaxations to acetylcholine were ob-
served in either SHR or WKY rats at all ages (data not
HE 10 ACh -logM shown).
In the 5-week-old group, both SHR and WKY rats
showed dose-dependent relaxations to acetylcholine,
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but the relaxations decreased in the 10-week-old and

older groups at acetylcholine concentrations of 10"6 to
10"5 M in both strains (Figure 2). The degree of these
decreases was greater in SHR than in WKY rats at all
ages between 10 and 30 weeks (Figure 2).

0N0-3708 HE 10
10"'M

8 76 5

0H0-370B 10-6H
1
T t 10 mln
J 7
NNM 10" M NE 10~ H
FIGURE 1. Typical records of acetylcholine (ACh)-induced
responses in 30-week-old spontaneously hypertensive rat aor-
tic rings. Panel A: Rings were contracted with 10~7 M
norepinephrine (NE), and ACh was cumulatively added.
Panel B: W6 M ONO-3708 was added 15 minutes before
contraction by NE. Panel C: 10'3 M NG-nitro~L-arginine
methyl ester (NNM, L-NAME in text) and 10~* M ONO-3708
were added 30 and 15 minutes, respectively, before contraction
byNE.
IDOL
I 5 7 6 5
FIGURE 2. Line graphs show acetylcholine (ACh)-induced
endothelium-dependent responses and the effect of ONO-3708
(10~6 M) in spontaneously hypertensive rat (SHR) and
Wistar-Kyoto (WKY) rat aortic rings (n=6 rats). ACh was
added cumulatively after contraction induced by 10'7 M
norepinephrine. Relaxation was expressed as percent contrac-
tion induced by 10~7 M norepinephrine. Results are shown as
mean±SEM. **p<0.01 between presence and absence of
ONO-3708. t p < 0 0 5 , tfp<0.01 between SHR and WKY
rats.
 Iwama et al Endothelium-Derived Contracting Factor 329

o—o i n ui-i7ii
• — • i n en-i7ii
FIGURE 3. Line graphs show effects of
A—am u t - m i + i n
A—*HT MB-I7II+III
ONO-3708 and tF-nitro-L-arginine methyl
•lUil.E ester (NNM, L-NAME in text) on acetylcho-
10W 201 301 line (ACh)-induced cndothelium-dependent
relaxation in spontaneously hypertensive rat
(SHR) and Wistar-Kyoto (WKY) rat aortic
rings (n=6). ACh was added cumulatively
after contraction induced by 10'7 M norepi-
nephrine. Relaxation was expressed as per-
cent response to contraction induced by 10~7
8 7 6 5 7 6 5 7 85 8 7 8 5 M norepincphrine. Results are shown as
ACb-logU mean±SEM.

Effects of Thromboxane A2IProstaglandin H2 acetylcholine dose-dependent relaxations were ob-

Receptor Antagonists
Figure IB shows typical responses of 30-week-old
SHR aortic rings treated with ONO-3708. The decrease
in the relaxation, which was observed at acetylcholine
concentrations of 10"6 to 10~5 M without ONO-3708,
disappeared in both SHR and WKY rats, and similar
served at all ages (Figures 2 and 3). The aorticringsof
30-week-old SHR contracted with 10~7 M norepineph-
rine showed acetylcholine (10~8 to 10"5 M) dose-depen-
dent relaxations in the presence of ONO-NT-126
(3xlO" 6 M) as well as in the presence of ONO-3708
(data not shown).

Effects of a Nitric Oxide Inhibitor and a

Thromboxane A2 Synthetase Inhibitor
* ACh - l o g M
Figure 4A shows the typical acetylcholine dose-
dependent contractile responses of 20-week-old SHR
aortic rings after treatment with L-NAME under rest-
t ing tension. These contractile responses increased with
NNM age and were always greater in SHR than in WKY rats
(Figure 5).
0.2g In the rings without endothelium, no contractile
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responses were observed in either SHR or WKY rats at

10 min all ages (data not shown).
OKY-046 did not affect these contractile responses
(data not shown).
0N0 Effects of a Thromboxane A2IProstaglandin H2
8 7 6 5 ACh - l o g M Receptor Antagonist and a Nitric Oxide Inhibitor
1
Figure 1C shows the typical acetylcholine dose-de-
t pendent responses when 30-week-old SHR aortic rings
NNM were treated with both L-NAME and ONO-3708 before
FIGURE 4. Typical records of acetylcholine (ACh)-induced contraction induced by norepinephnne. The relaxations
responses in 20-week-old spontaneously hypertensive rat aor- in response to the cumulative addition of acetylcholine
tic rings under resting tension. ACh (10'' to 10'' M) was disappeared completely in both SHR and WKY rats at
added cumulatively 15 minutes after treatment with 10' M 3 all ages (Figure 3). Figure 4B shows the typical acetyl-
FF-nitro-L-arginine methyl ester (NNM, L-NAME in text) choline dose-dependent responses when 20-week-old
(panel A) or both 10r3 M NNM and W6 M ONO-3708 SHR aortic rings were treated with both L-NAME and
ONO-3708 under resting tension. No contractile re-
(ONO) (panel B).
O—Olll UH FlGURE 5. Line graphs show acetylcholine
• — * I I T im
A—"Mil • • • + 111-1711 (ACh)-induced cndothehum-dependent con-
*—*!IT n i + tU-3701
niu±S.E traction in spontaneously hypertensive rat
51 301 (SHR) and Wistar-Kyoto (WKY) rat aortic
rings under resting tension (n=6). ACh (1O'S
to 10'5 M) was added cumulatively 15 min-
utes after treatment with 10'3 M t^-nitro-L-
arginine methyl ester (NNM, L-NAME in
text) or both l(T3 M NNM and lOr6 M
ONO-3708. Contraction was expressed as
percent contraction induced by IO~7 M nor-
epinephrine. Results are shown as
8 5 7 6 5 5 mean±SEM. *p<0.05, **p<0.01 between
ACb-lo(H SHR and WKY rats.
 330 Hypertension Vol 19, No 4 April 1992

IZZISHR
60
Pi/ml
500 50- y . 0.694 x - 60.5 r -0.976
pxO.05
400 40

300 30 y - 0.557 x - 49.6 ' -0.961

p<0.05
200 N.S. 20
mean ± S-E
100 10 O WKY
*p<0.05
• SHR
0
before lOmin
100 120 140 160 180 200
FIGURE 6. Bar graph shows changes in concentrations of Blood Pressure (mtnHg)
6-ketoprostaglandin Fla in the solution in 30-week-old spon-
taneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) FIGURE 7. Scatter plot shows relation between 10'5 M
rat aortic rings (n=6). Under resting tension, 10~5 M acetyl- acetylcholine-induced endothelium-dependent contraction
choline was added 15 minutes after treatment with 10'3 M and blood pressure in Wistar-Kyoto (WKY) rats and sponta-
NG-nitro-L-arginine methyl ester. Left, before acetylcholine neously hypertensive rats (SHR). Ordinate shows acetylcho-
addition. Right, 10 minutes after 10~5 M acetylcholine addi- line (10~s M)-induced endothelium-dependent contraction of
tion. Results are expressed as mean±SEM. *p<0.05 between the rings with endothelium under resting tension as shown in
SHR and WKY rats. Figure 5. Endothelium-dependent contraction was expressed
as percent of contraction induced by 10~7 M norepinephrine.
Abscissa shows systolic blood pressure. Each point represents
mean of six rats in 5-, 10-, 20-, and 30-week-old rat groups.
sponses to acetylcholine were observed in either SHR
or WKY rats at all ages (Figure 5),
Changes in Blood Pressure by Administration of a
Concentrations of 6-Ketoprostaglandin F,a Thromboxane A2/Prostaglandin H2
The 6-keto-PG F l a concentration in the solution Receptor Antagonist
before addition of L-NAME and acetylcholine was After oral administration of ONO-8809, the systolic
similar at 128±12 pg/ml and 97±19 pg/ml for SHR and
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blood pressures in both strains were not reduced com-

WKY rats, respectively. It was not affected by addition
of L-NAME (data not shown). It was, however, 432±30
pg/ml in SHR and 313 ±29 pg/ml in WKY rats 10
minutes after addition of 10 M acetylcholine, being
significantly higher in SHR (Figure 6).
Contractions to a Thromboxane A2 Analogue
Contractions of aortic rings without endothelium to a
thromboxane A2 analogue, STA2 (10~7 M), were deter-
mined in WKY rats and SHR (Table 3). There was no
age-dependent change in either strain, and there was no
difference between WKY rats and SHR in sensitivity to
STA2 except in the 10-week-old group.
Relation Between Endothelium-Dependent
Contraction and Blood Pressure
Relation between 10"5 M acetylcholine-induced endo-
thelium-dependent contraction and the systolic blood
pressure is shown in Figure 7. There is a statistically
significant correlation between acetylcholine-induced en-
dothelium-dependent contraction and blood pressure.
pared with control as shown in Table 4.
Discussion
The present study evaluated vascular responses to
EDCF and EDRF in aortic rings of SHR and WKY rats
after cumulative addition of acetylcholine.
When the aortic rings of SHR and WKY rats were
treated with ONO-3708 and then contracted with nor-
epinephrine, rings from both strains showed similar
acetylcholine dose-dependent relaxations in all age
groups. Since ONO-3708 inhibits the action of the
acetylcholine-stimulated EDCF, which is most likely
PGH2,14 and the relaxations due to inhibition of EDCF
were completely suppressed by a nitric oxide inhibitor,
L-NAME, the relaxations observed after treatment
with ONO-3708 are considered to have been caused by
acetylcholine-induced EDRF (nitric oxide). The degree
of relaxation was similar in SHR and WKY rats from 5
to 30 weeks of age. The present findings indicate that
EDRF production is maintained in rats from age 5 to 30
weeks, as has been noted in previous studies.8-9

TABLE 3. Contractions to 10"' M Thrombozant A] Analogue in Rat Aortic Rings Without Endothetinm in Each Age Group
Group 5 weeks 10 weeks 20 weeks 30 weeks
WKY 0.78±0.04 -i 0.90±0.04 -i 0.80±0.04 -] 0.80±0.01 -1
NS • NS NS
SHR 0.72±0.03 -1 0.72±0.03 - 1 0.73 ± 0 . 0 3 - ' 0.78±0.07 -•
Results (g) are expressed as mean±SEM. n=5. There is no age-dependent change in each strain. NS, statistically
nonsignificant.
•p<0.05 between Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at 10 weeks.
 Iwama et al Endothelium-Derived Contracting Factor
TABLE 4. Changes in Systolic Blood Pressure of Rats After Oral
Administration of ONO-8809
Dose of agent n 5 weeks 8 weeks
SHR
ONO-8809
(10 /tg/body/day) 7 110±13 149±3.7
ONO-8809
(30 /tg/body/day) 5 107±3.3 143 ±3.8
Control 6 107±2.5 144±2.8
WKY
ONO-8809
(10 /tg/body/day) 6 97±1.6 109±2.2
ONO-8809
(30 /ig/body/day) 6 98+4.0 118±8.0
Control 6 98±2.6 106±4.2
Five-week-old spontaneously hypertensive rats (SHR) and
Wistar-Kyoto (WKY) rats were orally administered ONO-8809
(once a day) for 3 weeks. Results (mm Hg) are expressed as
mean±SEM. n, Number of rats.
When EDRF was inhibited by treatment with
L-NAME, endothelium-dependent contractile re-
sponses were observed in 10-week-old or older rats.
Since these contractile responses were completely sup-
pressed by ONO-3708 but were not affected by a TXA2
synthetase inhibitor, OKY-046, they are most likely
mediated by endothelium-derived PGH 2 . U These con-
tractile responses increased markedly from 5 to 10
weeks of age but more gradually thereafter in SHR. In
WKY rats, however, they increased gradually from 5 to
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20 weeks of age and were consistently smaller than in
SHR. The increase in the contractile responses in SHR
and WKY rats paralleled the increase in the blood
pressure, suggesting a relation between the increased
production of EDCF and the increase in blood pressure.
The greater contractile responses to EDCF in SHR
than in WKY aortic rings may be due to higher PGH2
production or more marked enhancement of the re-
sponsiveness of vascular smooth muscles to PGH2 in
SHR. We found that there was no age-dependent change
in contractions of rings without endothelium to STA2.
But in the 10-week-old group, contraction to STA2 was
greater in WKY rats than in SHR, indicating that pro-
duction of the EDCF in 10-week-old WKY rats might be
overestimated. We also found that the 6-keto-PG F l a
production, which is considered to be correlated with
PGH2 production,14-21 was significantly greater in SHR
than in WKY rats. It has been reported that PGI2
synthetase activity was the same in vessels from SHR and
WKY rats.22 Therefore, the EDCF (PGH2) production
may also be higher in SHR than in WKY rats.
Whether the increase in EDCF is a cause or a result of
hypertension is of interest, but this cannot be determined
from our present finding alone. In hypertension, endo-
thelial function is reported to be altered and endotheli-
um-dependent relaxations to be decreased.23"28 In addi-
tion, the endothelium-dependent relaxations are
reported to be normalized by antihypertensive treat-
ments.29-30 Moreover, the blood pressure of SHR was not
reduced by oral administration of ONO-8809, a novel
TXA 2 /PGH 2 receptor antagonist, which inhibits the con-
traction to the EDCF (PGH2) as well as ONO-3708.
These observations suggest that the increase in EDCF in
331
SHR is more likely to be a result than a cause of
hypertension. In considering the close relation between
the endothelium-dependent contraction and blood pres-
sure, it is speculated that some genetic factor may
promote the increase of both EDCF and blood pressure
simultaneously. In any case, increased production of
EDCF may be a deteriorating factor in increasing blood
pressure.
Recently, endothelium-dependent relaxations to ace-
tylcholine were reported to be reduced in the arteries of
patients with essential hypertension.31-32 Also, the reduc-
tion in the endothelium-dependent relaxations in resist-
ant arteries was suggested to act as a deteriorating factor
in hypertension by producing injuries in various organs.
The possible involvement of increased EDCF in condi-
tions characterized by a reduction of such endothelium-
dependent relaxations needs further clarification.
In conclusion, the degree of acetylcholine-induced en-
dothelium-dependent relaxations in aortic rings was simi-
lar in SHR and WKY rats at 5-30 weeks of age, but the
production of acetylcholine-induced EDCF, which is most
likely PGH2, was consistently more enhanced at younger
ages in SHR than in WKY rats. These findings suggest a
close relation between increased EDCF production and
the increase in blood pressure in young to adult rats.
Acknowledgment
We thank Keiko Sakai for her secretarial assistance.
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