The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 70-76

The Hypotensive Effect of Docosahexaenoic Acid Is Associated with the Enhanced Release of ATP from the Caudal Artery of Aged Rats^1,2

Michio Hashimoto, Kazumasa Shinozuka^*, Shuji Gamoh, Yoko Tanabe, Md Shahdat Hossain, Young Mi Kwon^*, Noriaki Hata, Yoshihisa Misawa, Masaru Kunitomo^*, and Sumio Masumura³

Department of Physiology, Shimane Medical University, Izumo 693-8501, Japan; ^* Department of Pharmacology, Faculty of Pharmaceutical Science, Mukogawa Women's University, Koshien Kyuban-cho, Nishinomiya 663-8179, Japan; and  Applied Research Department, Harima Chemicals Incorporated, Tsukuba 300-2635, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Fish oils have been shown to lower blood pressure in hypertensive subjects. To determine the mechanism of this hypotensive effect, we examined the effects of docosahexaenoic acid (DHA), one of the (n-3) polyunsaturated fatty acids in fish oil, on blood pressure and on the release of adenyl purines, such as ATP, ADP, AMP and adenosine, from the caudal arteries of aged rats. Aged female Wistar rats (100 wk) were fed a high cholesterol diet and were administered intragastrically ethyl all-cis-4,7,10,13,16,19-docosahexaenoate [300 mg/(kg·d)] for 12 wk (DHA group) or vehicle alone (control group). Compared with the controls, rats supplemented with DHA had significantly greater (10.1%) DHA concentrations in the caudal arteries. This was associated with more total (n-3) arterial fatty acids, a greater unsaturation index of arterial fatty acids, 43.9% lower plasma noradrenaline levels and the repression of the elevation in blood pressure observed with advancing age. The amount of purines released, both spontaneously and in response to noradrenaline, from arterial segments of DHA-supplemented rats was significantly higher than that released from tissues of control rats. Regression analysis revealed significant negative relationships between the total amount of purines released from the artery and the systolic (SBP) and diastolic (DBP) blood pressures. These results suggest that in aged rats, supplementation with DHA alters the membrane fatty acid composition as well as the amount of ATP released from vascular endothelial cells and decreases plasma noradrenaline, and that these factors may ameliorate the rise in blood pressure normally associated with advancing age.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The (n-3) polyunsaturated fatty acids are involved in the regulation of various biological functions. Long-term supplementation with fish oil, which has a high content of (n-3) polyunsaturated fatty acids, has beneficial effects on the cardiovascular system, such as a moderate reduction in arterial blood pressure in normotensive and hypertensive subjects (Knapp and Fitzgerard 1989, Singer et al. 1986). We have reported that aged rats fed a standard diet or a high cholesterol diet supplemented with eicosapentaenoic acid [one of the (n-3) polyunsaturated fatty acids in fish oil] for a short time (5 wk) have lower blood pressure, associated with an increase in the release of ATP from the caudal artery (Hashimoto et al. 1998). Docosahexaenoic acid (DHA),⁴ an (n-3) polyunsaturated fatty acid, is also one of the major active components in fish oil. Little is known, however, about the antihypertensive effects of DHA alone on blood pressure, other than some results from in vitro studies.

Vascular tone is regulated by the responsiveness of the vascular smooth muscle to a variety of vasoactive substances, such as the endothelium-derived relaxing factor (EDRF) and endothelin. In addition, ATP is being recognized for its extracellular action in various tissues, including blood vessels (Burnstock 1991, Daiziel and Westfall 1994, Gordon 1986); ATP can cause vasodilatation by stimulating the release of EDRF from endothelial cells (De Mey and Vanhoutte 1981), by a direct action on vascular smooth muscle cells (Burnstock 1987) and by hyperpolarizing smooth muscle cells (Nishiye et al. 1990). Adenosine enhances vasodilatation by direct action on vascular endothelial cells (Li et al. 1995) and on smooth muscle cells (Burnstock 1987). In addition both ATP and adenosine can reduce the release of noradrenaline from vascular sympathetic nerves (Shinozuka et al. 1988). In vitro, studies using cultured endothelial and smooth muscle cells isolated from the caudal artery of rats have indicated that the main source of the released adenyl purines, such as ATP and its metabolites as a consequence of ₁-adrenoceptor stimulation, is the endothelium and, to a lesser extent, the smooth muscle (Shinozuka et al. 1994). Because ATP is degraded sequentially into ADP, AMP and eventually adenosine in vascular tissues by ectonucleotidases (Sedaa et al. 1990, Slakey and Gordon 1990), the source of released purine is considered to be ATP. Indeed, Shinozuka et al. (1994) have shown that the amount of ATP released in response to noradrenaline from the caudal arteries of rats is increased by the 5'-nucleotidase inhibitor, ,-methylene ADP.

Using endothelial cells isolated from rat caudal arteries, we have shown that the amount of ATP released in response to ₁-adrenoceptor stimulation decreases with advancing age, and that this extracellular ATP and its metabolites play a role in blood pressure changes associated with aging (Hashimoto et al. 1995). These findings suggest that the hypotensive effect of (n-3) polyunsaturated fatty acids on blood pressure may be associated with augmentation of ATP release from vascular beds. Moreover, meta-analyses of controlled trials in humans have demonstrated that a dose-response hypotensive effect of fish oil occurs in hypertensive patients, but little or no effect is observed in healthy normotensive subjects, and that the most consistent hypotensive effect of the oil occurs among hypercholesterolemic subjects (Morris et al. 1993). We therefore examined the effects of long-term DHA supplementation on blood pressure, on levels of plasma vasoactive substances and on the release of adenine nucleotides and adenosine from caudal arteries of aged hypercholesterolemic rats (100 wk), in which blood pressure was higher than that of young rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  Rats were handled and killed in accordance with the Guidelines for Animal Experimentation of Shimane Medical University (see below), compiled from the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science. Rats were kept in an environmentally controlled room at 23 ± 2°C and relative humidity of 50 ± 10%, with automatic lighting from 0800 to 2000 h. Female Wistar rats, 100-105 wk, were randomly divided into two groups as follows: the control group, fed a high cholesterol-containing diet (HC diet, the standard F1 diet containing no fish products contains 1% cholesterol and 1% cholic acid, Funabashi Farm, Chiba, Japan) (Table 1) and the DHA group intragastrically administered DHA-95E [300 mg/(kg·d), an ethyl all-cis-4,7,10,13,16,19-docosahexaenoate, an ethyl-ester form with a purity of almost 95%, Harima Chemicals, Tokyo, Japan] with the HC diet. DHA-95E was gently emulsified in 50 g/L gum arabic solution (Wako, Osaka, Japan) in ice-cold water with an ultrasonic cell homogenizer (Taitec VP-5, Taitec, Tokyo, Japan) before intragastric administration. The 5 g/L gum arabic solution without DHA-95E was administered to the control group. The rats were housed for 12 wk.

Body weight and blood pressure were determined by the tail-cuff plethysmographic method (Ueda, UR-1000, Tokyo, Japan) and were measured as previously described (Hashimoto et al. 1995) before and after the experiment. After measurement of systolic and mean blood pressure (P_S and P_M) by a plethysmography, the diastolic blood pressure (P_D) was calculated by the following equation: where P_S and P_M were measured by a plethysmography.

After the second measurement of systolic blood pressure, rats were anesthetized intraperitoneally with sodium pentobarbital (65 mg/kg). Blood was collected from the inferior vena cava into heparinized syringes, placed into polyethylene tubes containing EDTA (1 mmol/L) to prevent clotting and centrifuged at 1000 × g for 20 min at 4°C. After being checked for platelet contamination with an automated hematology analyzer (K-2000, Toa Medical Electronics, Kobe, Japan) (<10⁹/L), plasma levels of ATP, ADP, AMP and adenosine were measured by HPLC-fluorescence, and the residual plasma was used to measure plasma levels of total cholesterol, triglyceride and fatty acids.

Tissue preparation and purine release.  Tissue preparation and purine release assays were done as described (Hashimoto et al. 1995). After blood collection, a segment of the caudal artery (~11 cm in length and 10 mg wet weight) was removed and suspended in a water-jacketed organ chamber containing 2.0 mL modified Krebs solution continuously bubbled with 95% O₂ and 5% CO₂ at 37°C. The composition of the solution was as follows (mmol/L): NaCl, 110; KCl, 4.6; CaCl₂, 2.5; NaHCO₃, 24.8; KH₂PO₄, 1.2; MgSO₄, 1.2; and glucose, 5.6. The artery was allowed to equilibrate for 60 min, and the medium was replaced every 3 min during the latter half of the equilibration period.

To determine spontaneous purine release, the bathing solution was incubated for 3 min before collection. After the first sampling (to determine spontaneous release), the tissue was stimulated with noradrenaline (1 µmol/L) for 3 min, and the bathing solution (stimulation sample) was also collected. ATP, ADP, AMP and adenosine were measured (pmol/mg wet tissue) by HPLC-fluorescence. After the release experiments, the arteries were stored at 80°C.

Vascular reactivity in aortic rings.  A ring segment (3 mm) of thoracic aorta 5 mm from the aortic arch was excised from each aorta and immersed in physiological saline solution (PSS) containing (mmol/L) NaCl, 110; KCl, 4.6; CaCl₂, 2.5; NaHCO₃, 24.8; KH₂PO₄, 1.2; MgSO₄, 1.2; and glucose, 5.6. The vascular reactivity of the aortic segment was measured as described previously (Hashimoto 1990). The segment was suspended in an organ bath containing 20 mL PSS maintained at 37°C and continuously bubbled with 95% O₂/5% CO₂. The tension response of the segment was isometrically recorded with a force-displacement transducer (Orientec T7-30, Orientec, Tokyo, Japan), and the basal tension was adjusted to 1 g during equilibration for 60 min. A curve of the cumulative concentration response to prostaglandin F₂ (PGF₂) was generated to find a concentration producing 50-70% of the maximal contractile response (EC_50-70). Thus, after the equilibration, the endothelium-dependent relaxation response to acetylcholine (ACh) and the endothelium-independent relaxation response to sodium nitroprusside (SNP) were estimated. The ring segment was then rinsed with normal PSS for 60 min, precontracted by PGF₂ (1-7 µmol/L) under the previously determined EC_50-70 and relaxed by the cumulative addition of ACh and SNP.

Fatty acid composition of plasma and tissue.  Concentrations of total cholesterol and triglyceride in plasma were each determined by analytical kits (the Total Cholesterol E-test and the Triglyceride E-test; Wako). Fatty acid levels in plasma were determined by the one-step reaction (Lepage and Roy 1986) with some modification. The mixture of 100 µL plasma, augmented with 2 mL methanol/toluene (4:1, v/v) containing 10 µg tricosanoic acid as an internal standard, and 200 µL acetyl chloride, was incubated at 100°C for 60 min; then 60 g/L aqueous potassium carbonate containing 100 g/L sodium chloride was added. The mixture was shaken for 10 min at room temperature and centrifuged at 1800× g for 5 min. The toluene phase, containing the fatty acid methyl esters, was subjected directly to gas chromatography (GC) on a Model 5890 II gas chromatograph (Hewlett-Packard, Avondale, PA) equipped with a flame ionization detector and an automatic sampler (Model 7673) utilizing a 25 m × 0.25-mm i.d. fused-silica column (DB-WAX P/N 122-7032, J & W Scientific, Folsom, CA) programmed from 100 to 180°C at 20°C/min, 180 to 240°C at 2°C/min, 240 to 260°C at 4°C/min and at 260°C for 5 min. The identities of the peaks were established by comparison with those of reference compounds and, in part, by JMS-D 300 gas chromatography-mass spectrometry (JEOL, Tokyo, Japan).

Fatty acid levels in caudal arteries were measured by a similar procedure. The stored caudal arteries (10-20 mg) were transferred to a capsule precooled in liquid N₂, crushed with an amalgam mixer (UT-1600, Sharp, Osaka, Japan) and suspended in 200 µL of Dulbecco's PBS() containing 0.005% BHT. The fatty acid content of 100 µL of this suspension was analyzed by GC as described above.

Fatty acid concentrations in plasma and in the caudal artery were expressed as mmol/L plasma and as µmol/g wet tissue, respectively. The average degree of fatty acid unsaturation, the unsaturation index (UI), was calculated as a function of the sum of the mole percentages of the unsaturated fatty acids times the number of olefinic double bonds (Stubbs and Smith 1984).

Plasma noradrenaline.  Plasma noradrenaline concentrations were measured by the method described previously (Shinozuka et al. 1997). Briefly, plasma was immediately acidified to pH 2 with 0.4 mol/L perchloric acid containing 1.3 mmol/L EDTA-2Na and 5.3 mmol/L Na₂S₂O₅. The samples were isolated by batch alumina chromatography and analyzed by HPLC-electrochemical detection. Alumina was added to each acidified sample and pH was then adjusted to 8.6 with 1.5 mol/L Tris-HCl (pH 9) containing 50 mmol/L EDTA-2Na. Samples were vortexed for 30 s and placed on a rotator in a cold room at 4°C for 15 min. The supernatant was discarded, the alumina washed twice with distilled water and the noradrenaline eluted with 0.1 mol/L perchloric acid.

The HPLC-electrochemical detection system comprised a solvent delivery system (LC-9A pump, Shimazu, Kyoto, Japan), a sample processor (AS-8020, Tosoh, Tokyo), an ODS column (Eicompak CA-50 ODS, Eicom, Kyoto, Japan), and an electrochemical detector (ECD-300, Eicom) equipped with a glass carbon electrode. The solvent system, pumping at the rate of 1 mL/min, contained 80 mmol/L Na₂HPO₄, 0.5 mmol/L EDTA-2Na, 0.1 mmol/L sodium octyl sulfate and 50 g/L methanol (pH 2.7). The electrochemical detector was set at 0.7 V, which provided a limit of detection of ~10 fmol of noradrenaline.

Plasma nitrite and nitrate.  Plasma nitrite and nitrate levels were measured by the method adapted from Misko et al. (1993). Nitrate was converted to nitrite by the action of nitrate reductase from Aspergillus niger (Sigma Chemical, St. Louis, MO).

Statistical analysis.  Results are expressed as means ± SEM. All statistical analyses were conducted using the SAS statistical package (version 6.12, SAS Institute, Cary, NC). The effects of DHA on blood pressures (Fig. 1) and on total adenyl purines released from rat caudal artery (Fig. 2) were determined by analysis of covariance, using blood pressure before DHA administration (0 wk) as the covariable (Fig. 1) and using the effects of stimulation of noradrenaline as the covariable (Fig. 2). A t test for simple comparison was done to identify significant differences between the control group and the DHA group (Tables 2 and 3 and Fig. 3). P < 0.05 was considered significant. Pearson's correlation coefficients were used to evaluate linear relationships between variables.

View larger version (20K): [in this window] [in a new window]   Fig 1. Effect of docosahexaenoic acid (DHA) supplementation on systolic and diastolic blood pressures of aged rats. Systolic and diastolic blood pressures of DHA-treated (n = 11) and control (n = 17) rats were measured 0, 5 and 12 wk after supplementation. Values are means ± SEM. *P < 0.05, control group vs. DHA group.

View larger version (27K): [in this window] [in a new window]   Fig 2. Comparison of the release of adenyl purines from the caudal arteries of docosahexaenoic acid (DHA) treated and control rats. The ordinate indicates spontaneous and 1.0 µmol/L noradrenaline (NA)-evoked release of purines for 3 min. All values are expressed as means (control, n = 17; DHA, n = 11); the vertical bars on each column indicate the SEM for total purine release. Bars with different letters are significantly different, P < 0.05.

View larger version (33K): [in this window] [in a new window]   Fig 3. Plasma concentrations of adenyl purines of docosahexaenoic acid (DHA) treated and control rats. All values are expressed as means (control, n = 17; DHA, n = 11); the vertical bars on each column indicate the SEM for total plasma adenyl purines.

	RESULTS

Abstract Introduction Methods Results Discussion References

Blood pressure, body weight and food intake.  DHA supplementation for 5 wk did not affect blood pressure (Fig. 1). After 12 wk, the systolic blood pressure (SBP) of DHA-supplemented rats was significantly lower than that in controls (P < 0.05), and the diastolic blood pressure (DBP) tended to be lower (P = 0.0875) (Fig. 1).

No significant differences were observed in body weight or food intake, measured after 12 wk, between control and DHA groups [370 ± 11 vs. 353 ± 12 g for body weight, 15.1 ± 0.7 vs. 14.6 ± 0.6 g/(rat·d) for food intake]. During these experiments, no deaths occurred in either group.

Plasma total cholesterol, triglyceride, noradrenaline and nitrite + nitrate.  The plasma total cholesterol, triglyceride and noradrenaline concentrations were 47.3, 30.4 and 43.9% lower, respectively, in the DHA group than in the control group (P < 0.05, Table 2). Plasma concentration of nitrite + nitrate in hypercholesterolemic aged rats was not affected by DHA supplementation (Table 2).

Vascular reactivity of aortic rings.  Cumulative addition of ACh (10⁹-10⁵ mol/L) to a ring segment with the endothelium intact produced concentration-dependent relaxation. When the endothelium was removed by gentle rubbing with a cotton probe, the ACh did not elicit relaxation. This result is consistent with the fact that ACh elicits endothelium-dependent relaxation of rat aorta. There were no significant differences in the maximal relaxation induced by ACh or the EC₅₀ value for ACh between control (n = 17) and DHA (n = 11) groups (91.6 ± 1.6 vs. 90.8 ± 1.6% for maximal relaxation and 4.61 ± 1.2 vs. 3.84 ± 0.6 × 10⁸mol/L for the EC₅₀ value, respectively). SNP, a nonspecific vasodilator, elicited endothelium-independent relaxation of rat aorta. There were also no significant differences in the maximal relaxation induced by SNP or the EC₅₀ value between control (n = 17) and DHA (n = 11) groups (103 ± 1.06 vs. 101 ± 3.04% for maximal relaxation and 2.79 ± 0.4 vs. 3.57 ± 1.2 × 10⁹mol/L for the EC₅₀ value, respectively).

Fatty acid profiles of plasma and arterial lipids.  Plasma concentrations of palmitic, oleic, linoleic, linolenic and arachidonic acids in DHA-supplemented rats were significantly lower than those in controls (Table 3). In contrast, supplementation markedly increased plasma concentrations of eicosapentaenoic acid and DHA (P < 0.05). Thus, plasma total (n-3) fatty acids and the unsaturation index (UI) of the fatty acids were significantly higher in DHA-supplemented rats than in controls.

The arterial concentrations of palmitic and oleic acid were lower and the DHA concentration was higher (P < 0.05) in DHA-supplemented rats than in controls, leading to significantly more total (n-3) fatty acids and a higher UI of the arterial fatty acids (Table 3).

Examination of plasma and caudal arterial fatty acids in rats of both groups by regression analysis revealed a significant positive relationship between plasma and the arterial arachidonic acid levels (r = 0.404, P < 0.05), as well as the DHA levels (r = 0.460, P < 0.05).

Plasma adenine nucleotides and adenosine.  Plasma concentrations of total adenine nucleotides plus adenosine tended to be greater in DHA-supplemented rats than in controls (P = 0.0944; Fig. 3).

Release of adenyl purines from caudal arteries.  Significantly more adenine nucleotides and nucleosides were spontaneously released from the caudal arteries over a 3-min period from the arteries of DHA-supplemented rats than from controls (Fig. 2). Treatment of these tissue samples with noradrenaline (1.0 µmol/L for 3 min) significantly increased the release of total adenyl purines, but the difference between groups was maintained (Fig. 2).

Regression analysis indicated a significant positive relationship between the adenyl purine release in vitro and arterial fatty acids [spontaneous and noradrenaline-evoked release of total purines vs. the total (n-3) arterial fatty acids: r = 0.525 and r = 0.439, respectively, P < 0.05] and also a significant positive relationship between the UI of the arterial fatty acids and the spontaneous (r = 0.407, P = 0.0317) and noradrenaline-evoked (r = 0.417, P = 0.0274) release of total purines from the caudal arteries. These results indicate that the DHA supplementation-induced increase in the average number of double bonds of the arterial fatty acids may accelerate both the spontaneous and noradrenaline-evoked release of adenyl purines from the arterial bed of aged rats.

Systolic blood pressure and adenyl purines.  Regression analysis of the relationship between blood pressure and the adenyl purines released in vitro spontaneously or evoked by noradrenaline revealed significant negative relationships (vs. spontaneous, r = 0.529, P = 0.0038; vs. noradrenaline-evoked, r = 0.532, P = 0.0036). Similarly, significant inverse relationships were observed between spontaneous (r = 0.468, P = 0.0162) and noradrenaline-evoked (r = 0.484, P = 0.0090) release of total purines and the DBP, indicating that systolic and diastolic blood pressures were lower in rats with caudal arteries that in vitro released more purines.

Regression analysis revealed significant positive relationships between plasma noradrenaline and the SBP (r = 0.433, P = 0.0189) or the DBP (r = 0.412, P = 0.0262), indicating that the blood pressure was lower in rats in which the plasma noradrenaline level was lower.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The results of this study indicate a possible beneficial effect of DHA supplementation on blood pressure. The blood pressure of aged hypercholesterolemic rats was inversely related to the DHA diet-induced change in the unsaturation index of arterial fatty acids. This observation is in agreement with the results of population-based intervention studies, showing that polyunsaturated fatty acids in fish oil lower blood pressure in subjects with hypertension (Bonaa et al. 1990). DHA supplementation to aged rats led to accumulation of (n-3) fatty acid in the endothelial lining of the caudal arteries, which may have repressed the blood pressure elevation usually seen with advancing age. Dietary (n-3) polyunsaturated fatty acids augmented endothelium-dependent relaxation of the porcine coronary artery due to augmented release of EDRF/nitric oxide (Shimokawa and Vanhoutte 1989). Further, Lawson et al. (1991) have suggested that treatment of isolated rat aortic rings with DHA increases endothelium-dependent vasodilatation. In this study, however, DHA administration did not affect ACh-induced endothelium-dependent relaxation of thoracic aortic rings or plasma nitrite + nitrate concentrations, an index of nitric oxide production, in aged hypercholesterolemic rats. Although this discrepancy may be related to the strain, sex and/or age of the rats, and/or to the relaxation response to ACh in the vascular beds, we speculate that nitric oxide is not involved in the blood pressure-lowering effect induced by DHA supplementation.

The increased DHA levels in both plasma and the caudal arteries of DHA-supplemented rats were closely associated with an increase in the arterial release of adenyl purine, ATP and its metabolites, and inversely associated with the increase in blood pressure seen with aging in rats. Dietary fish oils have plasma cholesterol- (Harris et al. 1988) and triglyceride- (Sanders et al. 1985) lowering effects. In this study, plasma cholesterol of aged hypercholesterolemic rats was markedly decreased by dietary supplementation with DHA. One possible explanation for this is that the DHA supplementation-induced increase in the adenyl purine release from the aged rat arteries observed was caused by an antihypercholesterolemic effect of DHA. In a previous study, however, despite a marked decrease in plasma cholesterol level (from 7.43 ± 1.46 to 4.94 ± 0.56 mmol/L), DHA supplementation for 5 wk to aged hypercholesterolemic rats did not have a blood pressure-lowering effect and did not increase ATP release from the caudal artery (unpublished data). Therefore, we conclude that a DHA-induced increase in purine release is not related to any cholesterol-lowering effect of DHA.

How spontaneous release or ₁-adrenoceptor-stimulated release of adenine nucleotides and adenosine is increased by dietary DHA supplementation has not yet been elucidated. One possibility is that selective changes in the number or affinity, or both, of ₁-adrenoceptors in the endothelial cells of rat caudal arteries play an important role in altering the release of ATP from vessels. Changes in the content of unsaturated fatty acids in the membrane correlated with changes in membrane fluidity that ultimately affect cell function. For example, (n-3) polyunsaturated fatty acids modify the fluid mobility gradient of the phospholipid bilayer (Furchgott et al. 1984, Malasanos and Stacpoole 1991). An increase in the activity of membrane enzymes, such as 5'-nucleotidase and adenylate cyclase upon DHA enrichment have been reported in human skin fibroblasts (Brown and Subbaiah 1994).

ATP can produce endothelium-dependent vasodilatation via P_2y-purinoceptors in the precontracted arteries, and the ATP metabolite adenosine also produces direct vasodilatation via cAMP accumulation in arterial smooth muscle cells by A₂-purinoceptor stimulation (Burnstock and Ralevic 1994). Furthermore, both ATP and adenosine inhibit noradrenaline release from the sympathetic nerves via presynaptic P₃-purinoceptors (Shinozuka et al. 1988). Therefore, endogenous purines released via noradrenaline stimulation from arteries could produce vasodilatation via P_2y-, A₂- and P₃-purinoceptor stimulation and thus decrease blood pressure. There is an association between the age-related decrease in ATP release from rat caudal arteries and the increase in blood pressure (Hashimoto et al. 1995). All of the foregoing evidence suggests that the increase in the release of ATP and its metabolites by DHA supplementation underlies the antihypertensive actions of DHA.

Plasma noradrenaline is derived in part from perivascular sympathetic nerve endings, and the released amine acts as a vasoconstrictor in peripheral vascular beds, leading to increased blood pressure (Singer et al. 1990). In this study, DHA supplementation decreased plasma levels of noradrenaline in aged hypercholesterolemic rats. This decrease is consistent with the report by Singer et al. (1983) that human plasma levels of noradrenaline decrease by 34.4% after consumption of a mackerel diet that contains DHA-rich fish oil and are associated with significant decreases in systolic and diastolic blood pressure and plasma cholesterol and triglyceride. Thus, it may be postulated that the decrease in plasma noradrenaline seen in DHA-supplemented rats plays some part in the blood pressure-lowering effect induced by DHA supplementation. Further, the decrease in the level of noradrenaline may be due to presynaptic inhibition by plasma adenyl purines, as shown Shinozuka et al. (1991) in that endogenous purines from blood vessels inhibit the release of noradrenaline from the vascular adrenergic nerves.

DHA is the most unsaturated membrane fatty acid in mammalian biological systems. It accounts for a small percentage of the fatty acid content of many tissues but comprises 30-40% of phospholipids of the cerebral cortex and retina (Treen et al. 1992). Ingestion of fish oils generally augments the susceptibility of cellular membranes to lipid peroxidation (Hu et al. 1989, Kaasgaard et al. 1992) and increases the requirement for vitamin E (Hu et al. 1989, Kaasgaard et al. 1992, Meydani et al. 1991); thus DHA supplementation may induce a high need for vitamin E, a lipophilic membrane antioxidant. Moreover, a deficiency in brain DHA has been detected in Alzheimer's disease (Soderberg et al. 1991) and has also been shown to reduce learning ability for discrimination (Fujimoto et al. 1989). Based on these results and our present studies, long-term supplementation with DHA may be considered useful in the prevention of hypercholesterolemia, hypertension, the subsequent development of cardiovascular diseases and changes in the central nervous system with advancing age.

In conclusion, there was a significant positive correlation between the unsaturation index of arterial fatty acids and the amount of ATP released from the artery, and a significant inverse correlation between the amount of ATP released and blood pressure. Because ATP and its metabolites can produce vasodilatation, we postulate that DHA accumulation in the vascular beds may accelerate ATP release from vascular endothelial cells and repress the blood pressure elevation seen with advancing age.

	FOOTNOTES

2

2

Manuscript received 15 July 1998. Initial reviews completed 13 August 1998. Revision accepted 19 October 1998.

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