Blood pressure reduction by fish oil in adult rats with established hypertension — Dependence on sodium intake

Prostaglandins Leukotrienes and Essential Fatty Acids (1991) 44, 113-117 0 Longmnn Group UK Ltd WI

Blood Pressure Reduction by Fish Oil in Adult Rats with Established Hypertension - Dependence on Sodium Intake P. R. C. Howe, P. F. Rogers and Y. Lungershausen Hypertension Research Unit, CSIRO, Division of Human Nutrition, Adelaide,

SA 5000, Australia

P.O. Box 10041, Gouger Street,

(Reprint requests to PRCH)

The effects of fish oil combined with dietary sodium restriction on blood pressure and mesenteric vascular resistance were examined in a series of experiments with adult normotensive (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Rats were fed normal or low sodium diets containing fish oil, olive oil or &Bower oil. Small hut significant reductions of blood pressure (measured directly in conscious rats) were seen in SHRSP hut not in WKY after 8 weeks on a fish oil/low sodium diet, compared with rats fed olive or safflower oil diets with normal sodium content. This antihypertensive effect was not dependent on neurally mediated vasoconstriction hut was associated with a reduction of basal resistance in the blood-perfused mesenteric artery. Subcutaneous injection of lish oil reduced blood pressure in adult SHRSP on a normal sodium diet. However, there was a further fall in blood pressure when sodium intake was reduced. The results indicate the antihypertensive effect of fish oil can he enhanced by restricting sodium intake.

ABSTRACT.

rats with established hypertension, in which the fish oil was administered either by diet or by subcutaneous injection.

INTRODUCTION Several dietary intervention trials have now shown that supplementing the diet with fish oil causes a modest reduction of blood pressure in hypertensive subjects (l-3). The nature of this effect has been investigated in spontaneously hypertensive rats (SHR) (4-9). Feeding a diet containing eicosapentaenoic acid (EPA)-rich fish oil can attenuate the development of hypertension in young SHR (4-8). However, there has been only one report of an antihypertensive effect of fish oil in adult SHR with established hypertension (9). The mechanism mediating this antihypertensive effect is unknown, but it may involve an effect of EPA on prostanoid synthesis, resulting in suppression of the vasoconstrictor prostaglandin, thromboxane-A2 (9). We have recently shown that feeding a high sodium diet can counteract the antihypertensive effect of fish oil in young rats, suggesting that it may be possible to obtain greater reductions of blood pressure with fish oil by restricting dietary sodium intake (8). This possibility is addressed in the present report on a series of experiments which we have undertaken in normotensive adult rats and in

METHODS Dietary administration

Synthetic isocaloric diets were prepared as previously described (8) with the total lipid content (5% of dry weight) comprising fish oil, olive oil or safflower oil. Two types of fish oil were used: HIMEGA (Roussel Pty Ltd, Australia), containing 31% EPA and 21% docosahexaneoic acid (DHA), or NIH n-3 ester concentrate (National Institutes of Health, USA), containing 38% EPA and 29% DHA. The sodium content of the fish oil diet was either 0.23% (normal) or 0.04% of dry weight (low). Blood pressure measurement Adult (4-6-month-old) male stroke-prone SHR (SHRSP) or normotensive (WKY) rats obtained from the CSIRO breeding colony were fed the above diets for 8 weeks, after which catheters were implanted in the abdominal aorta for direct recording of blood pressure in the conscious state (10). After allowing 2 days for recovery and re-

Date received 19 March 1991 Date accepted 25 March 1991 113

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of oils

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Prostaglandins Leukotrienes

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establishment of normal food intakes, the catheters were connected to a Statham P23ID pressure transducer and Neotrace polygraph, enabling pulsatile blood pressure, mean arterial pressure (MAP) and heart rate to be recorded daily while the rats remained unrestrained in their individual cages. The resting levels of blood pressure, taken when heart rate reached a steady minimum, were averaged over 4 consecutive days. In one experiment, after the final recording of resting blood pressure, a vasopressin antagonist (Pmp-0-Me-Tyr-[Arg]-Vasopressin, Peninsula Laboratories, USA, 60 pg/kg) then a ganglion-blocking drug (Pentolinium tartrate, Sigma, USA, 2 mg/kg) were administered via the arterial catheter and acute changes in blood pressure were recorded. Mesenteric artery perfusion Rats were finally anaesthetised with Nembutal (40 mg/kg, i.p.) and prepared for in situ perfusion of the mesenteric vascular bed with the rat’s own blood (8, 11). Following tracheotomy, the carotid artery and superior mesenteric artery were cannulated and interconnected via an extracorporeal circuit. 10 min later, 1000 units of heparin was given and blood was pumped into the mesenteric artery at a constant rate of 0.5 ml/min for a further 10 min using a Gilson Minipuls perfusion pump. The flow rate was then varied in the range of 0.25-3.0 ml/min and changes of mesenteric perfusion pressure were recorded. Pressure/flow gradients were derived by linear regression of all the data on change in perfusion pressure versus flow rate obtained for each treatment group. Subcutaneous injection of oils Fish oil and olive oil were also administered by subcutaneous injection. Male SHRSP aged 3-3.5 months received bi-weekly injections of 0.5 ml of olive oil or HIMEGA fish oil for 7 weeks. The injection was given alternately into the inside flank of the right or left leg. After 5 weeks, catheters were implanted for direct measurement of blood pressure. Resting blood pressure was recorded on 5 consecutive days. All rats were then transferred from a commercial colony diet (0.2% sodium by dry weight) to our synthetic low sodium (0.04%) diet. Further daily recordings of blood pressure were taken 3-7 days later. Statistical analysis Results are expressed as the mean f SEM of individual values in each treatment group. Differences in repeated measurements of blood pressure were compared by split-plot analysis of variance

(ANOVA). Differences in mesenteric pressure/flow gradients were compared with an unpaired t-test. A confidence level of p < 0.05 was considered significant.

RESULTS Effects of dietary fish oil In an initial experiment, 4-month-old WKY and SHRSP were fed diets containing either HIMEGA fish oil with low sodium or olive oil with normal sodium. Figure 1 shows measurements of resting arterial blood pressure taken after 8 weeks. The elevation of blood pressure in adult SHRSP compared with age-matched WKY is clearly evident. Blood pressure readings averaged over 4 consecutive days were lower in SHRSP fed the fish oil/low sodium diet than in SHRSP fed the control diet with olive oil and normal sodium, although the decrease was significant for systolic pressure only. In the WKY, however, differences in blood pressure between diets were even smaller and were not significant. In a second experiment, 4-6-month-old SHRSP were fed diets containing either NIH fish oil with low or normal sodium or safflower oil with normal sodium. Compared with the safflower oil diet, both fish oil diets significantly reduced MAP; however, both fish oil diets failed to lower systolic pressure and diastolic pressure was reduced significantly by the fish oil/low sodium diet only (Fig. 2). Intraarterial administration of a vasopressin antagonist did not significantly affect resting blood pressure; MAP fell by less than 10 mm Hg. Average values for each treatment group were: 152.9 + 3.9, 161.1 + 5.6 and 169.4 + 2.8 mm Hg for fish oil/low mm Hg

225 -

WKY

SHRSP

200 L

175-

OOlive oil (normal Na) 6ElHlMEGA fish oil (low No)

150125loo75-

DBP

SBP

DBP

MAP

Fig. 1 Direct measurements (mean + SEM) of systolic (SBP). diastolic (DBP) and mean (MAP) arterial blood press&es in conscious’adult SHRSP and’WKY rats fed diets containing either olive oil and normal (0.23%) sodium (n = 6, 7) or fish oil and low (0.04%) sodium (n = 8, 7) for 8 weeks. Asterisk indicates significantly lower SBP after fish oil than after olive oil (determined by split-plot ANOVA of repeated blood pressure measurements taken daily for 4 days; p < 0.05).

Sodium

225 1

125

SBP

DBP

MAP

Fig. 2 Direct measurements (mean f SEM) of systolic (SBP), diastolic (DBP) and mean (MAP) arterial blood pressures in conscious adult SHRSP fed diets containing either safflower oil and normal (0.23%) sodium or fish oil and low (0.04%) or normal sodium for 8 weeks (n = 7 in each case). Asterisks denote values that differ significantly from safflower oil/normal sodium diet (determined by split-plot ANOVA of 3 consecutive.daily blood pressure measurements; * p < 0.05; ** p < 0.01).

Vascular

resistance

in the in situ

blood-perfused

Repeated subcutaneous injection of fish oil resulted in a significant reduction of blood pressure in adult SHRSP (Fig. 3). The mean value for the repeated daily measurement of MAP in these rats was 173.3 + 5.3 mm Hg (n = 8) compared with 189.6 + 4.8 mm Hg (n = 7) in SHRSP injected with olive oil. Shortly after lowering the sodium content of the diet, there was a further reduction of MAP, so that the combination of fish oil injections with the low sodium diet gave an overall reduction of 34 mm Hg.

rate

(mi/min)

DISCUSSION Blood pressure reduction In a recent study in our laboratory using young SHRSP (8), we were able to confirm earlier reports that the development of genetic hypertension could be attenuated by chronic dietary administration of

mesentry

Increase Flow

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Hypertension

Effects of subcutaneous fish oil

sodium, fish oil/normal sodium and safflower oil/normal sodium diets respectively. Subsequent administration of a ganglion blocker caused blood pressure to fall immediately to basal levels. After this treatment, diet induced differences in MAP were still evident but the antihypertensive effect of the fish oil/normal sodium diet was now equivalent to that of the fish oil/low sodium diet and both were significant (basal MAP = 75.9 + 1.9, 74.1 + 1.1 and 86.3 + 2.6 mm Hg for the fish oil/low sodium, fish oil/normal sodium and safflower oil/normal sodium diets respectively; n = 7 in each case). Effects of the diets on vascular resistance were Table

and

assessed from the relationship between flow rate and perfusion pressure in the in situ blood-perfused mesenteric artery. Data from both experiments are shown in the Table. A perfusion/flow gradient was derived from the incremental rise of perfusion pressure in response to increases in the rate of blood flow. The pressure/flow gradient in SHRSP fed diets containing fish oil with either low or normal sodium was lower than in SHRSP fed either the olive oil or safflower oil control diet. The mesenteric pressure/ flow gradient was also lower in WKY than in SHRSP but was unaffected by diet.

USafflower oil, (normal No) tS2NIH Fish oil. normal No) mNIH fish oil, low Na)

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Intake

in perfusion

pressure

(mm

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sodium fish

sodium

Safflower

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Olive

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oil

sodium

are means fSEM of values versus flow rates. Asterisks or olive oil/normal sodium

from individual rats. Gradients were indicate significantly different (p < diets (see text for details of diets).

derived by linear 0.05) from mean

rcgrcssion gradient

for

of incrc;rscs in perfusion rats (‘cd s;rftt,,wer ~ut/m~rm;rt

116

Prostaglandins Leukotrienes

and Essential Fatty Acids

OOlive

zoo-

M

MAP (mm Hd

Antihypertensive

oil

HIMEGA fish

oil

175-

150-

125 1 Normal

Sodium

Low Sodium

Fig. 3 Direct measurements (mean f SEM) of mean arterial pressure (MAP) in conscious adult SHRSP given bi-weekly subcutaneous injections (0.5 ml) of olive oil or fish oil for 6 weeks and again, 1 week later, after dietary sodium was reduced from normal (0.3%) to low (0.04%). Asterisks indicate significantly different from (*) olive oil/normal sodium, (**) all other groups (Student’s t-test).

fish oil. Furthermore, we found that we could counteract this antihypertensive effect by raising the sodium content of the diet. This prompted us to further examine the effects of fish oil and its interaction with dietary sodium on blood pressure in adult rats with established hypertension. Our results confirm that the antihypertensive effect of fish oil, when sodium intake is restricted, is not confined to an early stage in the pathogenesis of hypertension but can also occur in adult SHRSP after blood pressure has reached hypertensive levels. However, it is not clear from our experiments or those of Yin et al (9) whether the small effect of dietary fish oil on blood pressure in adult rats represents an actual reduction or merely a prevention of the continuing rise of blood pressure with age. Nevertheless, the large difference in blood pressure which was seen when the fish oil was given subcutaneously suggests that fish oil does lower blood pressure in adult hypertensive rats as indeed it does in hypertensive humans (l-3). It is difficult to characterise the antihypertensive effect of fish oil because the blood pressure responses to the dietary intervention are so small. Small reductions of blood pressure have also been attributed to dietary n-6 fatty acids (12, 13). We have found, however, that SHRSP reared on diets containing safflower oil, sunflower oil, olive oil, beef fat or commercial rat chow have similar blood pressures [unpublished data]; only the fish oil diets have consistently lowered blood pressure. Taken overall, our observations from the present study along with those in young SHRSP (8) and from unpublished experiments indicate that fish oil can lower blood pressure in rats with established hyperthan during the tension, but less effectively developing phase of hypertension, and that the antihypertensive effect is probably dose-dependent and mediated by n-3 fatty acids.

mechanism

As in young SHRSP (S), the reduction of resting blood pressure by fish oil in adult SHRSP cannot be attributed to attenuation of sympathetic vasoconstriction, since the difference in blood pressure persisted after ganglion blockade. Pretreatment of conscious SHRSP with an antagonist of the pressor effect of vasopressin results in a far more precipitous drop in pressure after ganglion blockade, even though administration of the antagonist by itself has little effect on resting blood pressure (Jablonskis et al, in preparation). This suggests that the acute loss of sympathetic vasoconstrictor tone can be partially compensated by an increase in vasopressin-mediated pressor activity. Hence, we assessed the contribution of sympathetic nerve activity to the resting level of blood pressure by administering the ganglion blocker in the presence of the vasopressin antagonist. Moreover, the lack of effect of the initial administration of the vasopressin antagonist on blood pressure precludes the possibility that the antihypertensive effect of fish oil is due to a decreased pressor responsiveness to vasopressin. In young SHRSP fed a fish oil diet, we observed that basal resistance in the blood-perfused mesenteric artery was reduced while the pressor responsiveness to phenylephrine remained normal. We concluded that these observations were consistent with the suppression of a circulating pressor substance, such as thromboxane AZ (TXAz), by fish oil. There is evidence that EPA can inhibit the synthesis of TXA2 (14). Moreover, reductions of serum TXB2, the stable metabolite of TXA*, have been found to accompany blood pressure reductions by dietary fish oil in man (1) and in SHR (9). SHR have raised levels of serum, renal and vascular TXA2 (15-17) and the latter may contribute to the vascular hypertrophy in this strain (17). Chronic administration of a thromboxane synthetase inhibitor has been shown to lower blood pressure in adult SHR (18, 19) and to attenuate the development of hypertension in SHR without affecting blood pressure in WKY (20). Thus the hypothesis that dietary fish oil lowers blood pressure by inhibiting a vasoconstrictor effect of TXA2 could explain why, in the present study with adult rats, the fish oil diets lowered blood pressure and mesenteric vascular resistance in SHRSP but not in WKY.

Dependence on sodium intake It is unlikely that the diet-induced reductions of blood pressure seen in this study are attributable solely to the reduction of sodium intake, as the low sodium diet did not significantly affect blood pressure in those rats given subcutaneous injections of olive oil (Fig. 3). Moreover, in young SHRSP (8), changes of sodium intake affected blood pressure in

Sodium Intake and Hypertension

those fed fish oil but not in those fed olive oil. Finally, both the difference in basal blood pressures after ganglion blockade and the difference in mesenteric pressure/flow gradients between rats fed fish oil or safflower oil were independent of the sodium content of their diets. As sodium restriction influenced the ability of the fish oil diet to lower resting blood pressure but did not affect its ability to reduce the basal blood pressure after ganglion blockade, it seems that these two dietary strategies affect blood pressure through different mechanisms. While fish oil appears to affect vasoconstriction through a non-neural mechanism, the chronic pressor effect of sodium in SHR is thought to be mediated by increased sympathetic vasoconstriction (21), which would account for the lack of effect of dietary sodium content on the basal levels of blood pressure after ganglion blockade. However, by inhibiting synthesis of renal prostaglandins and compromising their role in sodium excretion (15), fish oil may be able to indirectly augment the pressor effect of dietary sodium. Such a mechanism would explain why it was necessary to restrict dietary sodium intake in order to observe the maximum antihypertensive effect of the fish oil. References I. Knapp H R, Fitzgerald G A, The antihypertensive effects of fish oil: a controlled study of polyunsaturated fatty acid supplements in essential hypertension. The New England Journal of Medicine 320: 1037-1043, 1989 2. Norris P G. Jones C J H, Weston M J, Effect of dietary supplementation with fish oil on systolic blood pressure in mild essential hypertension. British’Medical Journal 293: 104ii)5, 1986 3. Bonaa K H. Bierve K S. Straume B. Gram I T. Thelle D, Effect of eicosapentaenoic and docosahexanoic acids on blood pressure in hypertension. New England Journal of Medicine 322: 795-Sol,1990 4. Schoene N W, Fiore D, Effect of a diet containing fish oil on blood pressure in spontaneously hypertensive rats. Progress in Lipid Research 20: 569-570,1981 5. Singer P, Berger I, Moritz V, Forster D, Taube C, N-6 and n-3 PUFA in liver lipids, thromboxane formation and blood pressure from SHR during diets supplemented with evening primrose, sunflowerseed or fish oil. Prostaglandins Leukotrienes and Essential Fatty Acids 39: 207-211,199O 6. Mtabaji J P, Manku M S, Horrobin D F, Release of fatty acids by perfused vascular tissue in normotensive and hypertensive rats. Hypertension 12: 39-45. 1988 7. Head R J, Mano M T, Bexis S, Howe P R C, Smith R M. Dietary fish oil administration retards the development of hypertension and influences vascular neuroeffector function in the stroke prone spontaneously hypertensive rat (SHRSP). Prostaglandins Leukotrienes and Essential Fatty Acids (this Proceedings, in the press)

8. Howe P R C, Lungershausen Y, Rogers P F, Gerkens J F, Head R J, Smith R M, Effects of dietary sodium and fish oil on blood pressure development in stroke-prone spontaneously hypertensive rats. Journal of Hypertension (in the press) 9. Yin K, Chu Z M, Beilin L J, Effect of fish oil feeding on blood pressure and vascular reactivity in spontaneously hypertensive rats. Clinical and Experimental Pharmacology and Physiology 17: 235-239.1990 IO. Howe P R C. Rogers P F, Morris M, Chalmers J P, Smith R M, Plasma catecholamines and neuropeptide-Y as indices of sympathetic nerve activity in normotensive and stroke-prone spontaneously hypertensive rats. Journal of Cardiovascular Pharmacology 8: I1 13-1121, 1986 Il. Armsworth S J, Gerkens J F, Smith A J, Frusemide inhibition of sympathetic vasoconstriction in the rat in situ blood perfused mesentery. Clinical and Experimental Pharmacology and Physiology 13: 495-503. 1986 12. Cachofeiro V. Francisco D, Lahera V, Canizo F J, Rodriguez F J. Tresguerres J A, The hypotensive effect of high linoleic acid intake is independent of the origin of hypertension. Journal of Hypertension 5: s555-S557.1987 13. Hoffmann P, Taube C H. Heinroth-Hoffmann I, Fahr A, Beitz J, et al, Antihypertensive action of dietary polyunsaturated fatty acids in spontaneously hypertensive rats. Archives Internationales Pharmacodvnamie 276: 222-235. 1985 14. Gibson R A, The effect of diets containing fish and fish oils on disease risk factors in humans. Australian and New Zealand Journal of Medicine 18: 713-722.1988 15. Codde J P, Beilin L J, Croft K D, Vandongen R. The effect of dietary fish oil and salt on blood pressure and eicosanoid metabolism of spontaneously hypertensive rats. Journal of Hypertension 5: 137-142, 1987 16 Shibouta Y. Terashita Z, Inada Y. Nishikawa K, Kikuchi S, Enhanced thromboxane A2 biosynthesis in the kidney of spontaneously hypertensive rats during development of hypertension. European Journal of Pharmacology 70: 247-256, 1981 17 Ishimitsu T. Uehara Y, Ishii M, Sugimoto T. Enhanced generation of vascular thromboxane A in spontaneously hypertensive rats and its role in the rapid proliferation of vascular smooth muscle cells. American Journal of Hypertension 1: 38S-40s. 1988 IS. Gomi T, Ikeda T, Ishimitsu T, Uehara Y. Effects of OKY-046. a selective thromboxane synthetase inhibitor, on blood pressure and thromboxane synthesis in spontaneously hypertensive rats. Prostaglandins Leukotrienes and Essential Fatty Acids 37: 139-144, 1989 19. Uderman H D. Jackson E K, Puett D, Workman R J, Thromboxane synthetase inhibitor UK38, 485 lowers blood pressure in the adult spontaneously hypertensive rat. Journal of Cardiovascular Pharmacology 6: 969-972. 1984 20. Stier C T. Itskovitz H. Thromboxane A and the development of hypertension in spontaneously rats. European Journal of Pharmacology 146: 129-135. 1988 21. Dietz R, Schomig A, Rascher W, Strasser R, Luth J, Ganten U, Kubler W, Contribution of the sympathetic nervous system to the hypertensive effect of a high sodium diet in stroke-prone spontaneously hypertensive rats. Hypertension 4: 773-781. 1982

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