Vol.

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1990

941-947

Plasma concentrations of endothelin-1 in spontaneously hypertensive rats and DOCA-salt hypertensive rats Nobuhiro Suzuki#, Takashi Miyauchi *, Yoko Tomobe*, Hirokazu Matsumoto, Katsutoshi Goto*, Tomoh Masaki*, and Masahiko Fujino Tsuku‘ba Research Laboratories, Takeda Chemical Industries, Ltd., 7 Wadai, Tsukuba, Ibaraki 300-42, Japan *Department of Pharmacology, Institute University Received

February

13,

of Basic Medical Sciences,

of Tsukuba, Tsukuba, Ibaraki 305, Japan 1990

To investigate the possible involvement of endothelin-1 (ET-l), an endothelium-derived potent vasoconstrictor peptide, in the pathophysiology of hypertension, plasma ET-l levels in 15week-old spontaneously hypertensive rats (SHR) and DOCA-salt hypertensive rats were measured with a sandwich-type enzyme immunoassay. The vasocontractile effect of ET-l in aortic helical preparations was significantly more sensitive in DOCA-salt hypertensive rats than in control sham-operated rats, but plasma levels of ET-l did not differ between them. Plasma ET-l levels in genetically hypertensive rats (SHR and stroke-prone SHR) were significantly lower than those in age-matched normotensive Wistar-Kyoto (WKY) rats. The plasma concentrations of big ET-l, a precursor of ET-l, in both SHR and SHR-SP were significantly lower than those of WKY, suggesting that the production of ET-l is decreased in rats of genetic hypertension. Although the vascular reactivity to ET-l increased in both DOCAsalt hypertensive and genetically hypertensive rats, present findings of the plasma ET-l levels suggest that the role of ET-l in the vascular control system may be different in DOCA-salt hypertensive rats and genetically hypertensive rats. 0 1990Academic PfeSS,Inc. Endothelin-1 endothelial

(ET-l) is a potent vasoconstrictor peptide produced by vascular

cells (1).

ET-l

consists of 21 amino acids and is thought

produced from a less active intermediate,

big ET-l (1,2).

to be

Since ET-l produces

potent v,asoconstrictions and extremely long-lasting pressor responses, ET-l may be considered to be important for the control of blood pressure and/or local blood flow (1,2;,3). We have previously reported that the vasocontractile effects of ET-l were more sensitive

in spontaneously

normotensive Wistar-Kyoto

(WKY)

hypertensive

rats (4,5), indicating

rats (SHR) that ET-l

than

in

may be

“To whom correspondence should be addressed. Abbreviations: ET-l, endothelin-1; SHR, spontaneously hypertensive rat; SHR-SP, stroke-prone spontaneously hypertensive rat; WKY rat, Wistar-Kyoto rat; DOCA, deoxycorticosterone acetate; EIA, enzyme immunoassay; HPLC, high performance liquid chromatography; TFA, trifluorocaetic acid. 0006-291X/90

941

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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in the pathophysiology

investigate

of SHR.

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RESEARCH

Therefore,

human

sandwich-type the existence

blood (6,7). In the present

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it is of particular

how the plasma level of ET-l is regulated

Recently, we developed sensitive for ET-l and big ET-l and reported in normal

AND

in hypertensive

interest

to

state.

enzyme immunoassays (EIAs) of circulating ET-1 and big ET-l

study,

the plasma

concentrations

of

both ET-l and big ET-l were measured in SHR, stroke-prone SHR (SHR-SP) and age-matched WKY rats. Furthermore, since the vasocontractile effects of ET-l in DOCA-salt concentrations hypertensive

hypertensive of ET-l

rats

were

not known,

and the vasocontractile

rats and control sham-operated

Materials

we studied effects

both the plasma

of ET-l

in DOCA-salt

rats.

and Methods

Preparation

of animals and measurement of blood pressure: Rats were purchased from CLEA Japan Inc. (Japan). Male Wistar rats (gweek-old) were separated into two groups. One group was unilaterally nephrectomized under The other group was sham-operated. Na-pentobarbital (50 mg/kg) anesthesia. Two weeks after the nephrectomy or the sham-operation, DOCA (3 mg/lOOg body weight) in olive oil and the vehicle (i.e. olive oil 0.1 ml/lOOg body weight) were injected subcutaneously twice a week into the former and the latter groups, respectively. The DOCA-treated group and the vehicle-treated group were allowed free access to 1 % NaCl drinking solution and tap water for 4 weeks, respectively. All the rats used received a standard laboratory diet. The systolic blood pressure of conscious SHR, SHR-SP, WKY rats (each strain, n = 20), DOCA-salt hypertensive (n = 11) and control rats (n = 10) was measured by a tail-cuff sphygmomanometer (Riken Kaihatsu PS-100, Kanagawa, Japan). Collection of plasma samples: Each rat was bled from the abdominal aorta The blood sample was put in chilled tubes containing under anesthesia. aprotinin (300 KIU/ml) and EDTA (2 mg/ml), and centrifuged at 2000 x g for 15 min at 4 “C. The plasma was stored at -30 “C until used. Preparation

of the rat

aorta

strip

and

measurement

of tension:

Descending thoracic aorta from both DOCA-salt hypertensive and sham-operated rats were immediately excised and placed in a Krebs-Ringer solution of the following composition (mM) : NaCl, 113; KCl, 4.8; CaC12, 2.2; KHzPO+, 1.2; MgS04,1.2; NaHC03,25; and glucose, 5.5. The arterial segments were cut mto 2 x 1.5 mm helical strips. They were gently treated so as not to injure the intimal surface and the presence of endothelium was comfirmed by the dilator response to acetylcholine (10-s M). The helical strips were suspended in siliconized glass organ baths and the isometric contraction was measured as described previously (5). The dose-response relationship for ET-l was determined by means of a cumulative application. For normalization, the response to ET-l was expressed by the percent of the maximum response to K+ (100 mM). Sandwich-EIA: Sandwich-EIA for ET-l was carried out as previously described using immobilized mouse monoclonal antibody AwETN40, which recognizes the N-terminal portion of ET-l? and peroxidase-labeled rabbit anti-ET-l C-terminal peptide(l5-21)Fab’ (6). Similarly, in the sandwich-EIA for big-ET-I, immobilized AwETN40 and peroxidase-labeled rabbit anti-human big-ET-1 C-terminal peptide(22-38)Fab’ (7,8) was used. The detection limits of these EIAs were 0.2 pg/well for both ET-l and human big-ET-l. Plasma, 1 ml, was acidified with 3 ml of 4% acetic acid and immunoreactive ET-l and big-ET-l were extracted with a Sep-pak C-18 cartridge (Waters Associates, Milford MA) as previously described (6). The eluates were reconstituted with 0.25 ml of assay buffer and subjected to the EIAs. Recoveries with this method were 80.5 + 1.7% (n = 12) and 69.1 + 1.3% (n= 12) when 50 pg of ET-1 and big ET-l were added to 1 ml plasma, respecvtively. Recovery did not

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differ significantly between various strains. Immunoreactive ET and big-ET levels were corrected for these recovery values. Reverse-phase HPLC: Plasma samples from l&week-old WKY rats (20 ml) and SHR-SP (50 ml) were diluted 3 times with acetic acid/ethanol/H20 =4.5/15/80.5 and applied to a YMC-GEL, ODS-AM 120-S50 column (12 x 70 mm, Yamamura Chemical Labs., Ltd., Japan). The adsorbed materials were eluted with acetic acid/ethanol/Hz0 = 4/86/10 and lyophilized. They were separated on a TSK ODS-80 column (4.6 x 250 mm, TOSOH, Co., Ltd., Japan) with increased concentration of CH3CN at a flow rate of 1 ml/min (3). Each fraction (0.5 ml) was lyophilized, reconstituted with the EIA buffer, and subjected to the EIAs for ET-l and big-ET. Statistics : Values are expressed as mean I!I S.E.M. Statistical analysis was carried out by the Student’s t-test for unpaired values.

Results Blood

pressure

The systolic and

of rats blood pressures

246 f 3.3 mmHg,

of 15-week-old

respectively

SHR and SHR-SP

and were

significantly

were

higher

223 f 3.7 than that of

age-matched WKY rats (156 f 2.7 mmHg, p < 0.01). Furthermore, SHR-SP exhibited significantly higher systolic blood pressure than SHR (p < 0.01). The systolic

blood pressure

experiment)

was

136 f

of 12-week-old 4.3 mmHg,

DOCA-salt

rats

(on the day

and was significantly

higher

control sham-operated rats (130 I!I 3.9 mmHg, p < 0.01). Contractile effects of ET-1 on aortic strips from DOCA-salt ET-l DOCA-salt

caused

a dose-dependent

and control rats (Figure

vasoconstriction

in aortic

1A). In the aortic strips

of the

than that of

rats strips

from

both

from these rats, there

B

A -c-

Endothelin-1

Doca-salt be/ml)

Endothelin - 1

NS

-logM

Fig. Y A.Vasocontractile effects of ET-l on the aortic strips from DOCA-salt rats (n = 11) ( O) and control sham-o erated rats (n = ll)( 0 1. Contractile responses were expressed as percentages o f maximum tension induced by 100 mM K+. B. Plasma concentrations of immunoreactive ET-l in DOCA-salt rats (n = 11) and control sham-operated rats (n = 10). N.S. denotes not significant.

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was no significant difference in the maximum responses to ET-l. EDso value in DOCA-salt rats (8.22 x lo-11 M) was significantly that in control

However, the smaller

than

rats (2.33 x lo-10 M, p C O.Ol), indicating that the sensitivity

to

ET-l was greater in DOCA-salt rats than in control rats. Plasma ET-l level in DOCA-salt rats Plasma levels of ET-l in DOCA-salt

hypertensive

rats were not significantly

different from those of control sham-operated rats (Figure 1B).

Plasma

ET-l

and big ET-1 levels in SHR, SHR-SP,

and WKY rats

Plasma levels of ET-l in both 15-week-old SHR and SHR-SP were significantly lower than those in age-matched WKY rats (Figure 2A).

This prompted us to

measure the plasma level of the precursor peptide, big ET-l,

in genetically

hypertensive rats. Plasma levels of big ET-l in both SHR and SHR-SP were also significantly lower than those in WKY rats (Figure 2B). In hypertensive rats. SHR-SP were found to have significantly lower ET-l and big ET-l levels in plasma than SHR (Figure 2A, 2B).

Reverse-phase

HPLC

of WKY plasma

and SHR plasma

extracts

Immunoreactive ET-l and big ET-l in rat plasma detected by sandwich-EIAs for ET-l and big ET-l were analyzed by reverse-phase HPLC. Immunoreactive

ET-l

extracted from WKY rat and SHR-SP plasma appeared as a single peak at

the elution position of synthetic ET-l. immunoreactive big ET-l extracted

Similarly, more than 94% and 87% of the from WKY rat and SHR-SP plasma,

respectively, were eluted at the position of synthetic human big ET-l (Figure 3).

A

Endothelin-1

B

Big

Endothelin-1

A 2%

2-l

(m/ml)

Fi . 2 Plasma concentrations of immunoreactive (A) ET-l (each strain, n = 20) WKY rats, SHR and SHRan (B) big ET-l (each strain, n = 10) in 15-week-old different between the two groups. SP. *P < 0.05, **P < 0.01: significantly

-?I-

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-0-I.1 0 2

1,

0

I

10

30

20 Retention

Time

40

(min)

Fi . 3 Reverse-phase HPLC of extracts of ( A ) WKY rat plasma (20 ml) and ( B ) !&kSP plasma (50 ml). Immunoreactive ET-l and big ET-l extracted from plasma with a YMC-GEL ODS-AM120-S50 column (12 x 70 mm) were applied to a TSK ODS-80TM column (4.6 x 250 mm) and eluted with increased concentrations of CH$N(----) at a flow rate of 1 mbmin. Each fraction (0.5 ml) was lyophilized, reconstituted with EIA buffer, and subjected to EIAs for ET-l (open bars) and big ET-l (solid bars). Arrows indicate the eluted positions of synthetic endothelins. h big ET-l, human big ET-l.

SHR plasma also showed a similar elution pattern (data not shown). These results indicate that rat ET-l and big ET-l were major immunoreactive substances in plasma detected by the sandwich-EL&. Furthermore, there was no qualitative difference in plasma ET-l and big ET-l between normotensive and 945

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hypertensive

rats.

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Therefore,

AND

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it was suggested

big ET-l levels in genetically hypertensive differences in plasma endothelins.

RESEARCH

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that the reduced plasma ET-l rats

were

and

not due the qualitative

Discussion We have previously shown that the vasocontractile effect of ET-l was greater in lZweek-old (hypertensive stage) SHR than in WKY rats, despite no differences between

B-week-old

been shown arteries

(prehypertensive

that the vascular

from

SHR-SP

(9).

stage) SHR and WKY

reactivity Thus,

to ET-l

rats (5). It has also

is increased

hyperresponsiveness

in the femoral

to ET-l

in genetic

hypertension was indicated. In the present study, increased vascular to ET-l was also observed in DOCA-salt hypertensive rats, a model

reactivity animal of

hypertension

a possible

caused by non-genetic

etiologies.

These findings

role of ET-l in the development and/or maintenance In the present study, plasma levels of ET-l significantly ET-l

lower

than in WKY

has not yet been reported,

is the same as or quite similar

rats.

of hypertension in rats. in SHR and SHR-SP were

Although

reverse-phase to human

suggest

the precise sequence of rat big

HPLC

big ET-l.

analysis Plasma

has revealed

that it

levels of big ET-l

in

SHR and SHR-SP were also lower than in WKY rats; an important finding indicating that the production of ET-l might be decreased in genetically hypertensive ET-l

rats.

in SHR-SP

Furthermore, were

the plasma concentrations

significantly

lower

correlated with higher blood pressure for the reduced production of ET-l

than

those in SHR,

was significantly

lower

In contrast to genetically DOCA-salt hypertensive normotensive DOCA-salt

rats.

which

and big may be

in SHR-SP than SHR. Although the reason in genetically hypertensive rats is still

unclear, the result of the present study is in agreement that the concentration of immunoreactive endothelin kidney

of both ET-l

in SHR compared

with the previous report in the inner medulla of

with WKY

rats (10).

hypertensive rats, the plasma concentration of ET-l in rats did not differ significantly from that in control

Thus, the reduction

rats, irrepective

of a marked

in the production elevation

of ET-l

did not occur in

of blood pressure.

Therefore,

the role of ET-l in the control of blood pressure or peripheral vascular resistance may differ between DOCA-salt hypertensive rats and SHR. If the production of ET-l from the vascular endothelial cells in DOCA-salt rats is similar to that in the control rats, the increased vascular reactivity to ET-l be assumed to be one of the causal factors for hypertension

in DOCA-salt of this type.

rats can

We have previously reported that the venous plasma concentration of ET-l in patients with untreated essential hypertension did not differ from that in agematched normal humans (11). Since ET-l produces potent vasoconstrictions in human

resistant

vessels

(12,13,14),

an intriguing 946

question

remains

to be solved

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as to whether such phenomenon in blood vessels applicable to the tissues of hypertensive humans. In summary, hypertensive plasma animals

although rats

vascular

and genetically

E:T-1 level displays of distinct

reactivity

different

etiology.

in the vascular

control

system

and genetically

hypertensive

tendencies

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from hypertensive

to ET-l

hypertensive

RESEARCH

increased rats

(SHR

between

rats can be

in both DOCA-salt and SHR-SP),

the

the two hypertensive

It can be suggested,

therefore,

may be different

in DOCA-salt

that the role of ET-1 hypertensive

rats

rats (SHR and SHR-SP).

Acknowledgments We thank Dr. Tadashi Yasuhara, useful advice regarding our study. excellent technical assistance.

Dr. Masao Tsuda and Dr. Akira Saito for their We also thank Miss Noriko Narushima for her

References M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., 1. Yanagisawa, Mitsui, Y., Yazaki, Y., Goto, K. and Masaki T. (1988) Nature 332,411-415 Yanagisawa, M. and Masaki, T. (1989) Trends. Pharmacol. Sci. 10,374-378 M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. i: Inoue, A., Yanagisawa, and Masaki, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86,2863-2867 M., Kimura, S., Goto, K. 4. Tomobe, Y., Miyauchi, T., Saito, A., Yanagisawa, and Masaki, T. (1988) Eur. J. Pharmacol. 152,373-374 5. Miyauchi, T., Ishikawa, T., Tomobe, Y., Yanagisawa, M., Kimura, S., Sugishita, Y., Ito, I., Goto, K. and Masaki, T. (1989) Hypertension 14,427-434 6. Suzuki, N., Matsumoto, H., Kitada, C., Masaki, T. and Fujino, M. (1989) J. Immunol. Methods 118,245-250 7. Suzuki, N., Matsumoto, H., Kitada, C., Kimura, S., Miyauchi, T. and Fujino, M. (1990) J. Immunol. Methods in press 8. Suzuki, N,,Matsumoto, H., Kitada, C., Kimura, S., and Fujino, M. (1989) J. Biochem. (Tokyo) 1(X,736-741 14,335 (abstract) 9. Joshua, I.G. and Bohr, D.F. (1989) Hypertension 10. Kitamura, K., Tanaka, T., Kato, J., Eto, T., and Tanaka, K. (1989) Biochem. Biophys. Res. Commun. 162,38-44 11. Miyauchi, T., Yanagisawa, M:, Suzuki, N., Iida, K., Sugishita, Y., Fujino, M., Saito, T., Goto, K. and Masaki, T. (1989) Circulation 80, H-573 (abstract) 12. Brain, S.D., Crossman, D.C., Buckley, T.L. and Williams, T.G. (1989) J. Cardiovas. Pharmacol. 13(Suppl. 5), S147-S149 13. Miyauchi, T., Tomobe, Y., Yanagisawa, M., Su ‘shita, Y., Iwao, I., Kimura, S., Goto, K. and Masaki, T. (1989) J. Am. Coll. F ardiol. 13,85A (abstract) 14. Chester,A.H., Dashwood, M.R., Clarke, J.G., Larkin, S.W., Davies, G.J., Tadjkanmi, S., Maseri, A. and Yacoub, M.H. (1989) Am. J. Cardlol. 63,13951398