European Journal of Pharmacology, 215 (1992) 325-328

325

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 21053

Short communication

Effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone M a r y B. E n g l e r Laboratory of CardioL'ascular Physiology, Department of Physiological Nursing, University of California at San Francisco, N611Y-0610, San Francisco, CA 94143-0610, U.S.A. Received 28 January 1992, accepted 10 March 1992

The comparative effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone were investigated. Docosahexaenoic acid (1-255 /zM) and eicosapentaenoic acid (31-255 /~M) inhibited phenylephrine-induced contractions, (8-63%) and (20-65%), respectively, which were not altered by indomethacin, NDGA, or by removal of the endothelium. Linoleic acid (18 : 2n6) and arachidonic acid (20 : 4n6) also induced significant relaxation. Therefore, fatty acid-induced relaxation of the rat aorta is specific to polyunsaturated fatty acids, 20 : 5n3, 22 : 6n3, 18 : 2n6 and 20 : 4n6. Docosahexaenoic acid; Eicosapentaenoic acid; Fatty acids (omega-3, omega-6, omega-9); Fish oil

1. Introduction

Epidemiologic studies suggest that a diet high in omega-3 polyunsaturated fatty acids may be protective against the occurrence of cardiovascular disease. Diets containing fish oil rich in omega-3 fatty acids, docosahexaenoic (22:6n3) and eicosapentaenoic (20:5n3), have been shown to produce hypotensive effects in both humans and animals (Bonaa et al., 1990; Schoene and Fiore, 1981). The relaxant effects of omega-3 fatty acids (Engler et al., 1990, 1991) may contribute to such hypotensive effects by modulating vascular tone. There is no known comparative data in which the effects of omega-3, omega-6 and omega-9 fatty acids are investigated. To gain an understanding of the omega-3 fatty acids and their vascular relaxant properties, the current investigation was performed to examine the vascular effects of a homologous series n-3, n-6 and n-9 fatty acids which differ in carbon chain length and the number and position of double bonds. The mediation of the relaxant responses by endogenous eicosanoids and the endothelium was also examined.

Correspondence to: M.B. Engler, Laboratory of Cardiovascular Physiology, Department of Physiological Nursing, University of California at San Francisco, N611Y-0610, San Francisco, CA 94143-0610, U.S.A. Tel. 1.415.476 0984, fax 1.415.476 8899.

2. Materials and methods

Aortic rings from male Sprague-Dawley rats (300400 g, Taconic Farms, N.Y.) were isolated and fixed in 40 ml smooth muscle baths to isometric force displacement transducers. The rings (3 mm segments) were equilibrated in Krebs-Ringer bicarbonate buffer (pH 7.4) containing (mM): NaC1 118.3 mM, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgSO 4 1.2, N a H C O 3 25 and glucose 11.1 at a preload tension of 1.5 g for 60-90 min. The rings were bubbled continuously with 95% O 2, 5% CO 2 at 37 _+ 0.5°C. Isometric force was measured with a force-displacement transducer (Grass FTO3, Grass Instruments Co., Quincy, MA) connected to universal amplifiers (Model 13-4615-58, Gould, Inc., Cleveland, OH) and a recorder (Model 28005, Gould, Inc., Cleveland, OH).

2.1. Experimental protocols Cumulative concentration-response curves (1-255 /zM) were generated for n-3, n-6 and n-9 fatty acids (table 1) in vessels precontracted with phenylephrine (10 -7 M). To delineate the possible mechanisms of those fatty acids (18 : 2n6, 20 : 4n6, 20 : 5n3, 22 : 6n3) which evoked significant relaxant responses, the effects of indomethacin (INDO, 14/zM, 30 min), a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA, 25 ~M, 15 min), an antioxidant, lipoxygenase inhibitor, and an inhibitor of endothelium-derived relaxing factor

326 TABLE

1

linolenate, arachidonate, and erucate from Nu Chek Prep at > 99% purity. Phenylephrine HCl (WinthropBreon Laboratories, New York, NY) and acetylcholine (Sigma) were dissolved in Krebs buffer. Indomethacin (Sigma) and NDGA (Biomol Research Laboratory, Philadelphia, PA) were dissolved in ethanol and diluted in 0.9% NaC1 so that the final concentrations of ethanol did not exceed 0.01% in the tissue baths. The fatty acids were dissolved in methanol, gassed and capped with nitrogen, and stored at -70°C. The samples were prepared immediately prior to experimentation by completely evaporating the methanolic solution of the fatty acid to dryness under a stream of nitrogen and reconstituting it by vortexing in nitrogen-saturated 0.9% NaC1.

Omega-3, omega-6 and omega-9 fatty acids. Fatty acids n-3 18:3

c~-Linolenic

Eicosapentaenoic Docosahexaenoic

20 : 5 22 : 6

n-6 18:2 18:3 20: 3

Linoleic

y-Linolenic Dihomo-y-linolenic Arachidonic

20 : 4

rt-9 18:1 20 : 1

Oleic

22 : 1

Erucic

11-Eicosenoic

2.3 Statistical et'aluation

(EDRF), were evaluated after the subsequent addition of the fatty acid. In experiments designed to assess the role of the endothelium in fatty acid-induced relaxant responses, fatty acids were administered in the presence and absence of an intact endothelium. An intact endothelium was verified in precontracted (phenylephrine, 2 × 10 -8 M, EC50) vessels which relaxed to acetlycholine (1 /xM).

Data were evaluated for statistical significance by applying the Student's t-test for unpaired data. Analysis of Variance for multiple comparisons was performed followed by the least significant difference (LSD) procedure for significant F values. A P value of less than 0.05 was considered statistically significant. All values are reported as mean _+ S.E. (%).

2.2. Drugs 3. Results

The following chemicals were used: sodium docosahexaenoate ( > 95% purity) from Nu Check Prep (Elysian, MN), 5,8,11,14,17-eicosapentaenoic acid (sodium salt > 90% purity) from Sigma Chemical Co. (St. L~uis, MO), y-linolenic acid, sodium oleate, sodium linoleate, sodium linolenate, l l-eicosenoate, sodium dihomo-y-

TABLE

3.1. Effect of n-3, n-6, n-9 fatty acids on aortic tone The fish oil n-3 fatty acids, 20:5 and 22"6, inhibited phenylephrine-induced contractions in a concentra-

2

Effect of n-3, 6 and 9 fatty acids on precontracted aortic smooth muscle tone. Data (percent change in isometric tension) are expressed as mean + S.E. ( % ) . Fatty

n

acid

% Relaxation l ~M

n-3 18:3 20:5 22:6

6 20 20

n-6 18:2

8

18:3 20:3 20:4

6 20 8

3/xM

. . - - 8 _ + 2 % ~,h - 11 + 2 %

.

.

. -- 1 3 + 2 %

~,i

. 1 2 _ + 3 % b.i

7,aM

. . -- 1 9 _ + 3 % d.i

. ~.h

- 1 7 _ + 3 % ~.i .

-22_+4%

. - -

17+3%

~.i

. ~.i

15 # M

31/.tM

255 , a M

- - 2 3 _ + 3 % c,i

2 0 + 1 % e,h - - 2 7 + 3 % c,i

-40+4% -65+4% -63_+3%

i i i

-23+4%

c,f.i

-25_+4%

c,i

-22+4%

c.i

-24+3%

d.i

-50+_4% 36+4% -55_+6% - 3 9 _ + 1%

i i i i

.

- 19_+3% ¢,f.i

n-9 18:1 20:1 22:1

19 4 4

.

.

.

.

. .

.

. .

. .

. .

~ Significantly different than n-9 fatty acids ( 1 8 : 1, 20: 1, 2 2 : 1), 1 8 : 3 n 6 , 2 0 : 3 n 6 , 1 8 : 3 n 3 , 2 0 : 5 n 3 ; b t h a n n-9 fatty acids, 1 8 : 3 n 6 , 2 0 : 3 n 6 , 1 8 : 3 n 3 ; c t h a n n - 9 fatty acids, 2 0 : 3 n 6 , 1 8 : 3 n 3 ; J t h a n n-9 fatty acids, 2 0 : 3 n 6 ; c t h a n 2 0 : 3 n 6 ; f than 20:5n3; and g than n-9 fatty acids, P < 0 . 0 5 . Significance at h p < 0.05, i p < 0.01 as compared to control responses. ( - ) represents data which were not significantly different from control

responses.

327

tion-dependent manner (table 2). These responses were not altered by removal of the endothelium, a-Linolenic acid (18:3n3) induced a significant response only at the highest concentration (255 /zM), as did the n-6 fatty acid, dihomo-y-linolenic acid (20:3n6). A significant relaxant effect was observed for arachidonic (20:4n6) and linoleic (18:2n6) acids for the full concentration response curve (1-255 /xM) (table 2). In contrast, -y-linolenic acid (18:3n6) evoked relaxations beginning at a concentration of 63 /xM (23 + 4%, P < 0.01) and above. The fatty acids of the n-9 family, ll-eicosenoic (20: 1), erucic (22: 1), and oleic (18: 1) acids elicited no significant change in smooth muscle tone. The rank order of fatty acid efficacy in producing relaxation at various concentrations are as follows: 1 /~M-20:4n6> 18:2n6>22:6n3; 15 / x M - 1 8 : 2 n 6 > 22 : 6n3 > 20 : 4n6; 31 /zM-22 : 6n3 > 18 : 2n6 > 20 : 4n6 > 20:5n3; 127 / z M - 2 0 : 5 n 3 > 2 2 : 6 n 3 > 1 8 : 2 n 6 > 20 : 4n6 > 18 : 3n6.

3.2. Effect of cyclooxygenase and lipoxygenase inhibitors on n-3 and n-6 fatty acid-induced relaxations No significant differences in relaxation responses were apparent in precontracted rings with 18:2n6, 20:5n3 or 22:6n3 in the presence of both I N D O and N D G A relative to the fatty acids alone. However, a significant inhibition (11%, P