Effect of omega3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine-induced contractions MARYB. ENGLER
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Labomtory of Cardiovascular Physiology, Department sf PhysioBsgical Nursing, University of CaBi$ornia at San Francisco, San Francisco, CA 94643-0640, U.S.A. Received August 2, 1991 ENGLER,M. B. 199%.Effect of omega-3 fatty acids, dscosahexaensic and eicosapentaenoic, on norepinephrine-induced contractions. Can. J. Physiol. Pharmacol. 70: 675 -679. The relaxant responses of the rat thoracic aorta to omega-3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine- and potassium-induced contractions were investigated. Relaxation was enhanced in vessels contracted with norepinephrine. Docosahexaenoic acid at concentrations as low as 1, 3, and 10 pM evoked significant relaxant responses (15,23, 30%) in norepinephrine-contracted vessels as compared with responses (5,9, 12%) in potassium-contracted vessels. Results for eicosapentaenoic acid under similar conditions were 3, 8, and 19% in norepinephrine-contracted vessels and 3, 3, and 8% in potassium-contracted vessels. Pretreatment with eicosapentaenoic (18 pM) or docosahexaenoic acids (1 10 pM) decreased the contractile response to physiologic concentrations of norepinephrine. In the presence of calcium-free medium, the omega-3 fatty acids (1 - 30 pM) significantly abolished sustained norepinephrine contractions but did not reduce the phasic contractions when incubated prior to norepinephrine contraction. Comparatively, the effects of docosahexaenoic acid were greater than eicosapentaenoic acid. These findings suggest that the relaxant effects of the omega-3 fatty acids are specific to the mode of contraction, i.e., a-adrenoceptor stimuli. This effect may be related to intracellular calcium mechanisms, since both fatty acids reversed norepinephrine-induced sustained contractions in the absence of extracellular calcium. Keg?words: omega-3 polyunsaturated fatty acids, eicosapentaenoic acid, docosahexaenoic acid, vascukr responses, fish oils. ENGLEW, M. B. 1982. Effect of omega-3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine-induced contractions. Can. J. Physiol. P h a m c o l . 70 : 675 -679. On a examint les rkponses relaxantes de 17aoftethoracique du rat aux acides gras omega-3, docosahexaenoique et eicosapentaenoique, sur Ses contractions induites par le potassium et la norkpinkphrine. La relaxation a CtC augmentke dans les des i concentrations aussi faibles que 1, 3 et 16 pM, vaisseaux contractCs avec la noripinephrine. L'acide dascosahexaenoique, ? a provoque des rkponses relaxantes significatives (15, 23, 30%) dans les vaisseaux contractks par la norkpinkphrine, comparativement aux rkponses (5, 9, 12%) obsehvCes dans les vaisseaux contract& par le potassium. Dans des conditions simihires, les rCponses de l'acide Cicosapentaenoique ont kt6 de 3, 8 et 19% dans les vaisseaux contract& par la norkpinehrine et de 3, 3 et $% dans les vaisseaux contract& par le potassium. Un prktraitement avec les acides docosahexaenoique (1 - 10 pM) ou Cicosapentaenasique (10 pM) a diminuC la rkponse contractile aux concentrations physiologiques de nort5pinCphrine. Dans un milieu sans calcium, Ies acides gras omCga-3 (1 -36 pM) ont CliminC de manikre significative les contractions soutenues induites par la norkpinCphrine, mais ils n'ont pas rkduit les contractions phasiques %orsqu'incubCs avant I'iduction des contractions. En comparaison, les effets de l'acide docosahexaenoique ont kt6 supkrieurs h ceux de l'acide Cicosapentaenoique. Ces rksultats sugg5rent que les effets relaxants des acides gras omkga-3 sont spkcifiques au m d e de contraction, c.-h-d. a w stimuli des rkcepteurs a-adrenergiques. Cet effet pourrait etre relit5 aux mkcanisrnes du calcium intracelldaire, Ctant donne que les deux acides gras ont neutralis6 les contractions soutenues, induites par la norkpinkphrine, en l'absence de calcium extracellulaire. Mots elks : acides gras polyinsaturCs smCga-3, acide Cicosapentaenoique, acide docosahexaenoique, rCponse vasculaire, huiles de poisson. [Traduit par la redaction]
Several studies have provided evidence indicating the antihypertensive effects of omega-3 polyunsaturated fatty acids (Bsnaa et al. 1990; Knapp and Fitzgerdd 1989; Bonaa 1989). Docosahexaensic acid (DHA) and eicosapentaenoic acid (EPA) are the omega-3 fatty acids found in fish oil that are thought to be the active biological components. One major effect of physiological significance exerted by these fatty acids is their relaxing effect on vascular smooth muscle (Engler 1989; Engler et al. 1990; Yanagisawa and Lefer 1987). The precise rnechanism(s) of action still remain to be established. Little information is currently available regarding the comparative effects of DHA and EPA or on various calcium flux mechanisms that may contribute to their vasorelaxant effects. Previous investigations have demonstrated that DHA reversed contractions of rat aortic rings induced by phenylephrine, an a-adrenergic agonist, and by U44-069, a stable PGH2 analogue (Engler et al . 1990). Both phenylepkrine and UUQ69 mobilize intracellular calcium during the contractile Printed in Canada / Imprime au Canada
response. Moreover, the pretreatment of verapamil, a voltagesensitive calcium channel blocker, had no significant effect on the relaxant effect induced by DHA (Engler 1990). A calcium-mediated mechanism of omega-3 fatty acid induced relaxation is likely since the vasorelaxant effects of both EBA and DHA in the rat aorta have been reported to be independent of the endothelium and not affected by the use of lipoxygenase and cyclooxygenase inhibitors (Engler 1989; Engler et al. 1990). The attenuation of noradrenaline and angiotensin II contractions by EBA in the rabbit aorta and isolated perfused rabbit ear is also not dependent on the endothelium or eicosanoid production (Juan et al. 1987; Juan and Sametz 1986). Furthermore, the vasoactive effects of EPA are not attributed to elevated levels of cyclic nucleotides (CAMP, cGMP) (Juan et al. 1987). The above findings exclude the role of the endothelium and endothelium-derived relaxing factor (EDWF), cyclic nucleotides, and prostanoids/eicosanoids as possible mediators in the vasoactive effects of DHA and EPA. It is interesting that a
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CAN. J. PHYSIOL. PHAWMACBL. VOL. 70, 1992
reduction of vasoconstrictor responses and augmentation s f vasodilatory responses by EPA has d s s been reported in ves1989; sels from animals fed EPA-enriched diets (Pfister et Yoshimura et al. 1986). The results of these studies suggest similar modes of action by EPA in both dietary EPA feeding and the direct administration of the fatty acid in isolated vascular preparations. The present study was designed to characterize the effects of DHA and EPA and to provide evidence for their possible effect on calcium permeability in vascular smooth muscle. The evidence presented suggests that the omega-3 fatty acids may act at the intracellular level of calcium mechanisms.
Methods Tissue prepamtion Adult male nomotensive Sprague -Dawley rats (3648- 350 g) were anesthetized with a mixture of 70% oxygen, 38% nitrous oxide, and 5 % halothane. Thoracic aortas were dissected free from thoracotomized animals and immersed in Krebs -Ringer bicarbonate buffer (pH 7.4) that contained the following (mM): NaCl, 118.3; KC%,4.7; CaCl,, 2.5; W 2 P 0 4 , 1.2: MgSO,, 1.2; NaHCO,, 25.0; and glucose, 11.1 at 4OC. The aortas were cleaned of adherent connective tissue and superficial fat and cut into ring segments (3 m).Dismption of the endothelium and unnecessary stretching was avoided during vessel preparation. Each aortic ring with intact endothelium was mounted in a tissue bath containing 10 mL of Krebs -Ringer solution at 37 0.5 "C and bubbled continuously with 95 % 0,and 5 % C0,. Isometric tension was recorded on a Grass model 7B polygraph with Grass FT03 force-displacement transducers (Grass Instrument Co., Quincy, Ma.). The vascular rings were equilibrated for 60 -90 min under 2 g resting tension. Control aortic rings were treated with 0.9% NaCl. At the start of each experiment, the rings were challenged w i h 30 mhf KC1 to establish tissue viability.
+
Relaxant responses to omega-3 fatty acids Aortic rings were precontracted with either maximal concentrations of M norepinephrine (NE) or 30 mM KC1 to detemine the specificity of antagonism by DHA or EPA. The concentrations of NE and KC1 were selected due to similar induced magnitudes of maximal contraction in control vessels. At the contraction plateau, cumulative concentration -response curves for DHA or EPA (1 - 108 yM)were generated. The relaxant responses were then determined and expressed as the percentage of decrease in the contractile response to NE or KCl. Inhibitory eflect of omega-3 fatty acids on ME contmcfions Experiments were conducted to study the inhibitory effect of BHA and EPA on NE contractions. Tissues were pretreated with DHA or EPA at three different concentrations (1, 3, and 10 pM)and exposed for 5 min before the NE contractions were obtained. Full concentration-~sponse curves to 1 x M - 1 X los5 M NE were generated after 5 ranin incubation to either BHA or EPA at the three different concentrations. Control tissues pretreated with 8.9 % NaCl vehicle were used in parallel. Relaant responses to omega-3 fatty acids in calcium--ee medium To detemine the effects of omega-3 fatty acids on intracellular contractile mechanisms, aortic rings were initially equilibrated in buffer solution containing 2.5 mM calcium and them were rinsed four times at 4-min intends for a totd of 28 min in calcium-free buffer containimg I .O mh4 ehyleneglycol-bis(0-aminoehy1eher)-M'-tetraacetic acid (EGTA), a calcium chelating agent. After equilibration, the tissues were e x p o d to M NE in the calcium-free medium. At the sustained plateau contraction after the initial phasic NE-induced contraction, cumulative concentration - response curves to DHA or EPA (1 -30 pM) were generated. The phasic and sustained contractions induced by NE in calcium-free medium are expressed as the actual developed tension in milligrams. DHA- and EPA-induced
relaxations are expressed as the change in tension (mg) from the NE-sustained plateau contraction.
Inhkbttoqg, eflect of omega-3 f a t ~acids on NE confaactions in calctulcra2-4pes medium Using the same calcium-free medium and 1 EM EGTA protocol, the effect of omega-3 fatty acids on intracellular calcium mobilization was investigated by incubating aortic rings in calcium-free medium and 1.0 mM EGTA with BHA or EPA (3 and 10 pM) for 20 min. A f e r incubation with DHA or EPA at the two different concentrations, vessels were contracted with 10-% ME. The phasic and sustained contractions induced by NE in calcium-free medium aker omega-3 fatty acid incubation were expressed as the acmal developed tension in milligrams.
Drugs and chemicals Norepinephrine ( -)-arterenol bitartrate), EGTA, and EPA sodium salt were purchased from Sigma Chemical Co. (St. Louis, Ms.). BHA sodium s d t was obtained from Nu Chek Prep (Elysian, Minn.). The omega-3 fatty acids EPA and %$HAwere dissolved in nitrogensaturated methanol and stored at -70°C under nitrogen. The samples were prepared immediately prior to experimentation by evaporating the fatty acids in methanol under nitrogen and then were reconstituted in nitrogen-saturated 0.9 % NaC1. Analysis of data Data were evaluated for statistical significance by applying the Student's t-test for unpaired observations and analysis of variance for multiple comparisons. Statistically significant E values for multiple comparisons were tested using the Scheffe method. A p value of less than 0.05 was considered statistically significant. All values are reported as the mean f SE.
Relaxant responses to DHA and EPA The relaxant effects of DHA and EPA (1 - 100 $4) on isoNNE or lated aortic rings precontracted with 1 x 10-% 30 mM KC1 are illustrated in Fig. I. DHA at concentrations as low as I pM significantly reversed the NE-induced contractile response (Fig. 1A). This effect was noted at DHA csncentrations up to 100 pM. In contrast, EPA-induced relaxations were demonstrated at concentrations I0 - I00 pM (Fig. 1A). When comparing relaxant responses between the two ornega-3 fatty acids, there was no significa~~t difference at concentrations ranging from 18 to 100 pM. However, the relaxant effects sf DHA were greater than EPA at physiologic concentrations of 1 pM ( p < 0.081) and 3 pM ( p < 0.0 1). In vessels contracted with 38 mM KC1, no significant reversal of the contractile response was noted for EPA except at the highest concentration (100 pM) used (Fig. 1B). Modest relaxations (14 -2 1%) were seen for DHA at concentrations greater than 30 pM (Fig. 1B). There was no significant difference in responses between DHA and EPA in KCl-induced csntractions. Inhibitions of NE eontractisns by DHA and EPA In association with the study s f DHA- and EPA-induced relaxation, experiments were dso done to investigate the inhibitory effect of omega-3 fatty acids on NE-induced contractions. Pretreatment of vessels with DHA or EPA (5 min) prior to NE exposure resulted in a significant decrease in NE contractisns (18-"10-8 M) (Fig. 2). A rightward shift in the NE contractile response curve at concentrations (10-9-M) was observed in aortic rings treated with DHA (I - 10 pM) (Fig. 2A) and EPA (18 pM) (Fig. 2B). However, the maximal force development was not different from the
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t
I
I
1
10
1 00
-9
CONCENTRATION (pM)
-8
-7
-5
-6
NOREPINEPHRINE (Bog M) W
CONCENTRATION (pM) PIG. 1. Concentration -response curves to docssabexaenoic acid (BHA) and eicosapentaenoic acid (EPA). (A) Vehicle control ( O ) , DHA (A), and EPA (a) in vessels contracted with lo-' Mi norepinephrine (NEI0 (B) Vehicle control (e), DWA (A), and EPA (o) in vessels contracted with 30 rdvf KCl. Each point represents the mean of results from 5 to 16 aortic rings (three to seven animals) with the mean f SE indicated by a vertical line. *p < 0.05, **p < 0.01, and ***p < O.WB (statistical difference compared with the parallel vehicle data).
vehicle control data. The EC50 values (the concentration eliciting half-maximal response) for each ring were determined from the concentration-response curves of these experiments. Table 1 summarizes the mean ECS0 and force data for omega-3 fatty acid treated and control arterial segments. A significant decrease in sensitivity to NE was noted in vessels treated with DBH (3 and 18 pM).
Efects of DHA and EM on NE-induced contraction in calcium-flee bufler To determine if the reversal of NE-induced contractions by the omega-3 fatty acids is related to inhibition of intracellular calcium mobilization, the effect of DHA m d EPA on NE-induced contmctisns in calcium-free buffer containing 1.0 m M EGTA was determined (Fig. 3). A significant relaxant effect was noted with both D M and EPA (1 -30 pM$ in NE contracted vessels. The fatty acids were administered during the sustained phase of norepinephrine contraction in calciumfree, EGTA buffer. Further analysis demonstrated no significant difference in relaxation effects between DHA and EPA. As shown in Figs. BA and 3, BHA at 1 -338 pM concentrations depressed NE-induced contractions in normd cdcium buffer as well as in calcium-free EGTA buffer. Similar comparisons with EPA (Figs. 1A and 3) reveal a greater relaxant effect in cdcium-free buffer versus normal cdcium buffer.
NOREPINEPHRINE (log M) FIG. 2. Concentration -response curves for the contractile response to norepinephrine (NE). (A) Vehicle control (Q), DHA pretreatment (I pM, A ; 3 pM,m; 10 pM, e) in vessels contracted with lo-" I W 5 M NE. (B) Vehicle control (a)and EPA pretreatment (1 pM, A ; 3 pM, =; 10 pM, 0 ) in NE-contracted vessels. Each point represents the mean s f results from 7 to 12 aortic rings (five to six animals) with the m a n f SE indicated by a vertical line. *p < 0.05, **p < 0.01, and ***p < 0.001 (statistical difference compared with the parallel vehicle data).
TABLEI. Sensitivity and maximum values in control and omega-3 fatly acid treated aortic rings ~ a t t yacid
Maximum developed tension (g)
EGO
(M)
control DHA 1 beM 3 PM 28 pM EPA
NOTE:Each value represents the mean f SE. **p < 0.01 (statistical difference compared with the parallel vehicle data). ***p < 0.061 (statistical difference compared with the parallel vehicle data).
Eflects of DHA- and EPA-pretreated aortic rings on NEinduced contractiores in calcium-flee bufler A series of experiments were d s o done to examine the possible imhibitory effect of DHA and EPA on RE-induced csntractions in calcium-free, EGTA buffer. Tissues pretreated
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CAN. J . PHYSIOL. PHARMACOL. VOL. 70, 1992
TABLE 2. Effects of BWA and EPA pretreatment on NE-induced contraction in calcium-free buffer
Contraction
rCONTROL
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DHA Ex4 EPA
CONCENTRATION (pM)
Fro. 3. Effects of docssahexaensic acid (DHA) and eicssapentaensic acid (EPA) on NE-induced contraction in calcium-free buffer. Values represent change in tension from NE sustained contraction: control, +230 f 4.5 mg; DHA, +232 f 26.6 mg; EBA, +203 f 35.7 mg. Each bar depicts the mean of results from five to six aortic rings (two to three animals) with the mean f SE indicated by a vertical line. *p < 0.05 and **p < 0.01 (statistical difference compared with the parallel vehicle data).
with DHA or EPA (3 and 10 pM) prior to NE administration displayed no significant reduction in the NE-induced phasic or sustained contractions (Table 2).
Although antihypertensive properties have been ascribed to the omega-3 fatty acids, few reports are available on the direct effects of these fatty acids on the vasculature. In the present study, the vascular responses of the rat aorta to the omega-3 fatty acids DHA and EPA were investigated. This is the first such report to fully describe the comparative vascular effects of both DHA and EPA and to examine various calcium flux mechanisms that may contribute to this effect. Cumulative addition of DHA or EPA produced a concentration-dependent relaxant effect in vessels contracted with NE, an a-adrenoceptor agonist, without substantially affecting KCl-induced contractions. The relaxant effect of DHA was more pronounced compared with EPA at physiologic concentrations 41- 18 pM) . Furthermore, the inhibitory effects of DHA were greater than EPA at low concentrations of NE-induced contractions. The relaxing effect of the omega-3 fatty acids in aortic rings contracted with NE may be primarily mediated through an intracellular mechanism of action, e.g., by inhibition of intracellular calcium release or interference with contractile protein function. This study provides evidence for the selective action of omega-3 fatty acids on norepinephrine agonistinduced contractions. The lack of substantid relaxation induced by low concentrations of the omega-3 fatty acids on KCI-induced contraction suggests that the voltage-operated channel of calcium influx is not a primary site of inhibition or antagonism. It is possible that the omega-3 fatty acids at high concentrations 4 > BOQ pM) may possess additional properties that regulate voltage-sensitive calcium channels. Conversely, the more pronounced relaxant effects of DHA and EPA in NE-contracted vessels and the inhibition sf (10-9-10-8 M) NE contractions (rightward shift) may be related to a preferential inhibition of edcium influx associated
Fatty acid
n
NE phasic
NE sustained
Vehicle control DHA 3 bkM 10 yM EPA
4
+279&17.8
+230&6.5
4 3
+328&47.4 +340+20
+307&32.9 +260f40.4
NOTE:Each value represents the mean
* SE in milligrams tension.
with receptor-operated channels and (or) a decreased calcium sensitivity of the myofilaments. Isolated aortic strips from rats fed high dietary eicosapentaenoic acid for 3 weeks show a similar shift to the right of the concentration-response curve to NE, with no effect on contractions induced by KC1 (Lockette et al. 1982). These effects were thought to be attributed to direct changes in prostaglandin metabolism. Fish oil administration has also been demonstrated to improve both EDRF release and relaxation responses of smooth muscle to EDRF (Shimokawa and Vanhoutte 1988, 1989). Calcium transport in isolated cardiac myocytes from fish oil fed rats and mice is altered by DHA and EPA (Swanson et al. 1989; Karmazyn et d. 1987). Specifically, elevated levels of DHA in sarcoplasmic reticulum (SR) phospholipids are associated with a lower relative activity of Ca2+-Mg2+ ATPase, a reduced initial rate of calcium transport, and a reduced maximal uptake of calcium in cardiac SR (Swanson et al. 1989). There are no h o w n vascular studies of DHA and EPA that document such calcium-mediated effects. However, the findings of the present study demonstrating the ability of DHA and EPA to reverse the sustained component of the NE contractile response in calcium-free, EGTA-containing buffer suggests an important role for these omega-3 fatty acids in intracellular calcium regulation. In the present study, DHA and EPA did not affect the initial phase of NE contraction in calcium-free buffer. The initial phase of NE contraction is thought to be mediated by inositol 1,4,5-trisphosphate (IP3) formation, which causes the release of intracellular calcium (Manolopoulos et al. 1991). This suggests that the vasoactive effects of the omega-3 fatty acids are probably not attributed to an inhibition of IB3 formation. It is h o w n that in the absence of extracellular cdcium, two distinct intracellular calcium pools mediate the phasic and sustained components of NE-induced contractions in the rat aorta (Heaslip and Wahwan 1982). Therefore, the failure of omega-3 fatty acids to inhibit the phasic NE contraction in calcium-free medium may be due to their primary effect on a different intracellular calcium pool mediated by the sustained component of NE-induced contraction. In summary, the present investigation demonstrates the differential vasorelaxant effects of the omega-3 fatty acids DHA and EPA. The effects s f DHA are more pronounced compared with those of EPA. Regulation of intracellular calcium may be the primary mechanism of the antihypertensive effects of the omega3 fatty acids. The precise mechanism(s) of action remains to be elucidated.
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BNGLER
Acknowledgements This work was supported in part by the Academic Senate Committee on Research, University sf California, San Francisco, Calif.; the American Heart Association, San Francisco Chapter; and the National Center for Resources, National Institutes of Health, Bethesda, Md. (Biomedical Research Support Grant S07WR056M). The author expresses appreciation to Lori Hudson for her excellent secretarial assistance in the preparation of this manuscript. Bonaa, K. 1989. Epidemiological and intervention studies on the effect of marine polyunsaturated fatty acids on blood pressure. J. Intern. Med. 225(Suppl I): 105- 110. Bonaa, K. H., Bjerve, K. S., Straume, B., et a&. 1990. Effect of eicosapentaenoic and docosahexaenoic acids on blood pressure in hypertension. N. Engl. J. Med. 322: 795 -801. Engler, M. B. 1989. The vascular effects of omega-3 polyunsaturated fatty acids. Physiologist, 32: 170. Engler, M. B. 1990. Vascular responses to omega-3 fatty acids: Role of calcium. Circulation, 82: 111-607. Engler, M. B., Karanian, J. W., and Salem, N., Jr. 1990. Bocosahexaenoic acid (22:6n3)-induced relaxation of the rat aorta. Eur. J. Phamacol. 185: 223-226. Heaslip, R. J., and Rahwan, R. G. 1982. Evidence for the existence of two distinct pools of intracellular calcium in the rat aorta accessible to mobiIization by norepinephrine. J. Phamacol. Exp. Ther . 221: 9-13. Juan, H., and Sametz, W. 1986. Vasoconstriction induced by noradrenaline and angiotensin II is antagonized by eicosapentaenoic acid independent of formation of trienoic eicosanoids. Naunyn-Schmiedeberg's Arch. Phamacol. 332: 288 - 292. Juan, H.,Sutter, B., and Sametz, W. 1987. Influence of eicosapentaenoic acid on noradrenaline and angiotensin-inducedcontractions of the rabbit aorta: mode of action. In Prostaglandins in clinical research. Edited by H. Siminger and K. Schror. Alan R. Liss, Inc., New York. pp. 57-62.
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Kslrmazyn, M., Horackova, M., and Murphy, M. 6. 1987. Effects of dietary cod liver oil on fatty-acid composition and calcium transport in isolated adult rat ventricular myocytes and on the response of isolated hearts to ischemia and reperfusion. Can. J. Bhysiol. Phamacol. 65: 201 -209. Knapp, PI. R., and Fitzgedd, G . A. 1989. The antihypertensive effects of fish oil. N. Engl. J. Med. 328: 1037- 1043. Lockette, W. E., Webb, R. C., Culp, B. R., and Pitt, B. 1982. Vascular reactivity and high dietary eicosapentaenoic acid. Prostaglandins, 24: 631 -639. Manolopulos, V. G . , Pipili-Synetos, E., Hertog, A. D., and Nelemans, A. 1991. Inositol phosphates formed in rat aorta after alpha,-adrenoceptor stimulation are inhibited by forskolin. Eur. J. Pharmcsl. 207: 29 - 36. Pfister, S. Lo,Rosolswsky, M., Schranitz, J. M., et a&. 1989. Eicosapentaenoic acid alters vascular reactivity and platelet adhesion in Watanabe heritable hyperlipidemic rabblts. Eur. J. Pharmcol. 161: 85-$9. Shirnokawa, H. , and Vanhoutte, P. M. 1988. Dietary cod-liver oil improves endothelium-dependent responses in hypercholesterolemic and atherosclerotic porcine coronary arteries. Circulation, 78: 1421- 1430. Shhokawa, H., and Vanhoutte, P. M. 1989. Dietary 03 fatty acids and endothelium-dependent relaxations in porcine c6ronary Aeries. Am. J. Physiol. 256: H968 -H973. Swanson, J. E., Lokesh, B. W., and Kinsellla, J. E. 1989. Ca2+-Mg2+ AThse of mouse cardiac sarcsplasmic reticulum is affected by membrane n -6 and n -3 polyunsaturated fatty acid content. J. Nutr. 119: 364-392. Ymagisawa, A., and kefer, A. M. 1987. Vasoactive effects of eicosapentaenoic acid on isolated vascular smooth muscle. Basic Res. Cardiol. 82: 186-196. Yoshimura, T.,Hto, M., Matsui, KO,and Fujisaki, S. 1986. Effects of highly purified eicosapentaenoic acid on vascular reactivity to angiotensin II and norepinephrine in the rabbit. Prostaglandins, 32: 179- 188.
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Labomtory of Cardiovascular Physiology, Department sf PhysioBsgical Nursing, University of CaBi$ornia at San Francisco, San Francisco, CA 94643-0640, U.S.A. Received August 2, 1991 ENGLER,M. B. 199%.Effect of omega-3 fatty acids, dscosahexaensic and eicosapentaenoic, on norepinephrine-induced contractions. Can. J. Physiol. Pharmacol. 70: 675 -679. The relaxant responses of the rat thoracic aorta to omega-3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine- and potassium-induced contractions were investigated. Relaxation was enhanced in vessels contracted with norepinephrine. Docosahexaenoic acid at concentrations as low as 1, 3, and 10 pM evoked significant relaxant responses (15,23, 30%) in norepinephrine-contracted vessels as compared with responses (5,9, 12%) in potassium-contracted vessels. Results for eicosapentaenoic acid under similar conditions were 3, 8, and 19% in norepinephrine-contracted vessels and 3, 3, and 8% in potassium-contracted vessels. Pretreatment with eicosapentaenoic (18 pM) or docosahexaenoic acids (1 10 pM) decreased the contractile response to physiologic concentrations of norepinephrine. In the presence of calcium-free medium, the omega-3 fatty acids (1 - 30 pM) significantly abolished sustained norepinephrine contractions but did not reduce the phasic contractions when incubated prior to norepinephrine contraction. Comparatively, the effects of docosahexaenoic acid were greater than eicosapentaenoic acid. These findings suggest that the relaxant effects of the omega-3 fatty acids are specific to the mode of contraction, i.e., a-adrenoceptor stimuli. This effect may be related to intracellular calcium mechanisms, since both fatty acids reversed norepinephrine-induced sustained contractions in the absence of extracellular calcium. Keg?words: omega-3 polyunsaturated fatty acids, eicosapentaenoic acid, docosahexaenoic acid, vascukr responses, fish oils. ENGLEW, M. B. 1982. Effect of omega-3 fatty acids, docosahexaenoic and eicosapentaenoic, on norepinephrine-induced contractions. Can. J. Physiol. P h a m c o l . 70 : 675 -679. On a examint les rkponses relaxantes de 17aoftethoracique du rat aux acides gras omega-3, docosahexaenoique et eicosapentaenoique, sur Ses contractions induites par le potassium et la norkpinkphrine. La relaxation a CtC augmentke dans les des i concentrations aussi faibles que 1, 3 et 16 pM, vaisseaux contractCs avec la noripinephrine. L'acide dascosahexaenoique, ? a provoque des rkponses relaxantes significatives (15, 23, 30%) dans les vaisseaux contractks par la norkpinkphrine, comparativement aux rkponses (5, 9, 12%) obsehvCes dans les vaisseaux contract& par le potassium. Dans des conditions simihires, les rCponses de l'acide Cicosapentaenoique ont kt6 de 3, 8 et 19% dans les vaisseaux contract& par la norkpinehrine et de 3, 3 et $% dans les vaisseaux contract& par le potassium. Un prktraitement avec les acides docosahexaenoique (1 - 10 pM) ou Cicosapentaenasique (10 pM) a diminuC la rkponse contractile aux concentrations physiologiques de nort5pinCphrine. Dans un milieu sans calcium, Ies acides gras omCga-3 (1 -36 pM) ont CliminC de manikre significative les contractions soutenues induites par la norkpinCphrine, mais ils n'ont pas rkduit les contractions phasiques %orsqu'incubCs avant I'iduction des contractions. En comparaison, les effets de l'acide docosahexaenoique ont kt6 supkrieurs h ceux de l'acide Cicosapentaenoique. Ces rksultats sugg5rent que les effets relaxants des acides gras omkga-3 sont spkcifiques au m d e de contraction, c.-h-d. a w stimuli des rkcepteurs a-adrenergiques. Cet effet pourrait etre relit5 aux mkcanisrnes du calcium intracelldaire, Ctant donne que les deux acides gras ont neutralis6 les contractions soutenues, induites par la norkpinkphrine, en l'absence de calcium extracellulaire. Mots elks : acides gras polyinsaturCs smCga-3, acide Cicosapentaenoique, acide docosahexaenoique, rCponse vasculaire, huiles de poisson. [Traduit par la redaction]
Several studies have provided evidence indicating the antihypertensive effects of omega-3 polyunsaturated fatty acids (Bsnaa et al. 1990; Knapp and Fitzgerdd 1989; Bonaa 1989). Docosahexaensic acid (DHA) and eicosapentaenoic acid (EPA) are the omega-3 fatty acids found in fish oil that are thought to be the active biological components. One major effect of physiological significance exerted by these fatty acids is their relaxing effect on vascular smooth muscle (Engler 1989; Engler et al. 1990; Yanagisawa and Lefer 1987). The precise rnechanism(s) of action still remain to be established. Little information is currently available regarding the comparative effects of DHA and EPA or on various calcium flux mechanisms that may contribute to their vasorelaxant effects. Previous investigations have demonstrated that DHA reversed contractions of rat aortic rings induced by phenylephrine, an a-adrenergic agonist, and by U44-069, a stable PGH2 analogue (Engler et al . 1990). Both phenylepkrine and UUQ69 mobilize intracellular calcium during the contractile Printed in Canada / Imprime au Canada
response. Moreover, the pretreatment of verapamil, a voltagesensitive calcium channel blocker, had no significant effect on the relaxant effect induced by DHA (Engler 1990). A calcium-mediated mechanism of omega-3 fatty acid induced relaxation is likely since the vasorelaxant effects of both EBA and DHA in the rat aorta have been reported to be independent of the endothelium and not affected by the use of lipoxygenase and cyclooxygenase inhibitors (Engler 1989; Engler et al. 1990). The attenuation of noradrenaline and angiotensin II contractions by EBA in the rabbit aorta and isolated perfused rabbit ear is also not dependent on the endothelium or eicosanoid production (Juan et al. 1987; Juan and Sametz 1986). Furthermore, the vasoactive effects of EPA are not attributed to elevated levels of cyclic nucleotides (CAMP, cGMP) (Juan et al. 1987). The above findings exclude the role of the endothelium and endothelium-derived relaxing factor (EDWF), cyclic nucleotides, and prostanoids/eicosanoids as possible mediators in the vasoactive effects of DHA and EPA. It is interesting that a
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CAN. J. PHYSIOL. PHAWMACBL. VOL. 70, 1992
reduction of vasoconstrictor responses and augmentation s f vasodilatory responses by EPA has d s s been reported in ves1989; sels from animals fed EPA-enriched diets (Pfister et Yoshimura et al. 1986). The results of these studies suggest similar modes of action by EPA in both dietary EPA feeding and the direct administration of the fatty acid in isolated vascular preparations. The present study was designed to characterize the effects of DHA and EPA and to provide evidence for their possible effect on calcium permeability in vascular smooth muscle. The evidence presented suggests that the omega-3 fatty acids may act at the intracellular level of calcium mechanisms.
Methods Tissue prepamtion Adult male nomotensive Sprague -Dawley rats (3648- 350 g) were anesthetized with a mixture of 70% oxygen, 38% nitrous oxide, and 5 % halothane. Thoracic aortas were dissected free from thoracotomized animals and immersed in Krebs -Ringer bicarbonate buffer (pH 7.4) that contained the following (mM): NaCl, 118.3; KC%,4.7; CaCl,, 2.5; W 2 P 0 4 , 1.2: MgSO,, 1.2; NaHCO,, 25.0; and glucose, 11.1 at 4OC. The aortas were cleaned of adherent connective tissue and superficial fat and cut into ring segments (3 m).Dismption of the endothelium and unnecessary stretching was avoided during vessel preparation. Each aortic ring with intact endothelium was mounted in a tissue bath containing 10 mL of Krebs -Ringer solution at 37 0.5 "C and bubbled continuously with 95 % 0,and 5 % C0,. Isometric tension was recorded on a Grass model 7B polygraph with Grass FT03 force-displacement transducers (Grass Instrument Co., Quincy, Ma.). The vascular rings were equilibrated for 60 -90 min under 2 g resting tension. Control aortic rings were treated with 0.9% NaCl. At the start of each experiment, the rings were challenged w i h 30 mhf KC1 to establish tissue viability.
+
Relaxant responses to omega-3 fatty acids Aortic rings were precontracted with either maximal concentrations of M norepinephrine (NE) or 30 mM KC1 to detemine the specificity of antagonism by DHA or EPA. The concentrations of NE and KC1 were selected due to similar induced magnitudes of maximal contraction in control vessels. At the contraction plateau, cumulative concentration -response curves for DHA or EPA (1 - 108 yM)were generated. The relaxant responses were then determined and expressed as the percentage of decrease in the contractile response to NE or KCl. Inhibitory eflect of omega-3 fatty acids on ME contmcfions Experiments were conducted to study the inhibitory effect of BHA and EPA on NE contractions. Tissues were pretreated with DHA or EPA at three different concentrations (1, 3, and 10 pM)and exposed for 5 min before the NE contractions were obtained. Full concentration-~sponse curves to 1 x M - 1 X los5 M NE were generated after 5 ranin incubation to either BHA or EPA at the three different concentrations. Control tissues pretreated with 8.9 % NaCl vehicle were used in parallel. Relaant responses to omega-3 fatty acids in calcium--ee medium To detemine the effects of omega-3 fatty acids on intracellular contractile mechanisms, aortic rings were initially equilibrated in buffer solution containing 2.5 mM calcium and them were rinsed four times at 4-min intends for a totd of 28 min in calcium-free buffer containimg I .O mh4 ehyleneglycol-bis(0-aminoehy1eher)-M'-tetraacetic acid (EGTA), a calcium chelating agent. After equilibration, the tissues were e x p o d to M NE in the calcium-free medium. At the sustained plateau contraction after the initial phasic NE-induced contraction, cumulative concentration - response curves to DHA or EPA (1 -30 pM) were generated. The phasic and sustained contractions induced by NE in calcium-free medium are expressed as the actual developed tension in milligrams. DHA- and EPA-induced
relaxations are expressed as the change in tension (mg) from the NE-sustained plateau contraction.
Inhkbttoqg, eflect of omega-3 f a t ~acids on NE confaactions in calctulcra2-4pes medium Using the same calcium-free medium and 1 EM EGTA protocol, the effect of omega-3 fatty acids on intracellular calcium mobilization was investigated by incubating aortic rings in calcium-free medium and 1.0 mM EGTA with BHA or EPA (3 and 10 pM) for 20 min. A f e r incubation with DHA or EPA at the two different concentrations, vessels were contracted with 10-% ME. The phasic and sustained contractions induced by NE in calcium-free medium aker omega-3 fatty acid incubation were expressed as the acmal developed tension in milligrams.
Drugs and chemicals Norepinephrine ( -)-arterenol bitartrate), EGTA, and EPA sodium salt were purchased from Sigma Chemical Co. (St. Louis, Ms.). BHA sodium s d t was obtained from Nu Chek Prep (Elysian, Minn.). The omega-3 fatty acids EPA and %$HAwere dissolved in nitrogensaturated methanol and stored at -70°C under nitrogen. The samples were prepared immediately prior to experimentation by evaporating the fatty acids in methanol under nitrogen and then were reconstituted in nitrogen-saturated 0.9 % NaC1. Analysis of data Data were evaluated for statistical significance by applying the Student's t-test for unpaired observations and analysis of variance for multiple comparisons. Statistically significant E values for multiple comparisons were tested using the Scheffe method. A p value of less than 0.05 was considered statistically significant. All values are reported as the mean f SE.
Relaxant responses to DHA and EPA The relaxant effects of DHA and EPA (1 - 100 $4) on isoNNE or lated aortic rings precontracted with 1 x 10-% 30 mM KC1 are illustrated in Fig. I. DHA at concentrations as low as I pM significantly reversed the NE-induced contractile response (Fig. 1A). This effect was noted at DHA csncentrations up to 100 pM. In contrast, EPA-induced relaxations were demonstrated at concentrations I0 - I00 pM (Fig. 1A). When comparing relaxant responses between the two ornega-3 fatty acids, there was no significa~~t difference at concentrations ranging from 18 to 100 pM. However, the relaxant effects sf DHA were greater than EPA at physiologic concentrations of 1 pM ( p < 0.081) and 3 pM ( p < 0.0 1). In vessels contracted with 38 mM KC1, no significant reversal of the contractile response was noted for EPA except at the highest concentration (100 pM) used (Fig. 1B). Modest relaxations (14 -2 1%) were seen for DHA at concentrations greater than 30 pM (Fig. 1B). There was no significant difference in responses between DHA and EPA in KCl-induced csntractions. Inhibitions of NE eontractisns by DHA and EPA In association with the study s f DHA- and EPA-induced relaxation, experiments were dso done to investigate the inhibitory effect of omega-3 fatty acids on NE-induced contractions. Pretreatment of vessels with DHA or EPA (5 min) prior to NE exposure resulted in a significant decrease in NE contractisns (18-"10-8 M) (Fig. 2). A rightward shift in the NE contractile response curve at concentrations (10-9-M) was observed in aortic rings treated with DHA (I - 10 pM) (Fig. 2A) and EPA (18 pM) (Fig. 2B). However, the maximal force development was not different from the
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t
I
I
1
10
1 00
-9
CONCENTRATION (pM)
-8
-7
-5
-6
NOREPINEPHRINE (Bog M) W
CONCENTRATION (pM) PIG. 1. Concentration -response curves to docssabexaenoic acid (BHA) and eicosapentaenoic acid (EPA). (A) Vehicle control ( O ) , DHA (A), and EPA (a) in vessels contracted with lo-' Mi norepinephrine (NEI0 (B) Vehicle control (e), DWA (A), and EPA (o) in vessels contracted with 30 rdvf KCl. Each point represents the mean of results from 5 to 16 aortic rings (three to seven animals) with the mean f SE indicated by a vertical line. *p < 0.05, **p < 0.01, and ***p < O.WB (statistical difference compared with the parallel vehicle data).
vehicle control data. The EC50 values (the concentration eliciting half-maximal response) for each ring were determined from the concentration-response curves of these experiments. Table 1 summarizes the mean ECS0 and force data for omega-3 fatty acid treated and control arterial segments. A significant decrease in sensitivity to NE was noted in vessels treated with DBH (3 and 18 pM).
Efects of DHA and EM on NE-induced contraction in calcium-flee bufler To determine if the reversal of NE-induced contractions by the omega-3 fatty acids is related to inhibition of intracellular calcium mobilization, the effect of DHA m d EPA on NE-induced contmctisns in calcium-free buffer containing 1.0 m M EGTA was determined (Fig. 3). A significant relaxant effect was noted with both D M and EPA (1 -30 pM$ in NE contracted vessels. The fatty acids were administered during the sustained phase of norepinephrine contraction in calciumfree, EGTA buffer. Further analysis demonstrated no significant difference in relaxation effects between DHA and EPA. As shown in Figs. BA and 3, BHA at 1 -338 pM concentrations depressed NE-induced contractions in normd cdcium buffer as well as in calcium-free EGTA buffer. Similar comparisons with EPA (Figs. 1A and 3) reveal a greater relaxant effect in cdcium-free buffer versus normal cdcium buffer.
NOREPINEPHRINE (log M) FIG. 2. Concentration -response curves for the contractile response to norepinephrine (NE). (A) Vehicle control (Q), DHA pretreatment (I pM, A ; 3 pM,m; 10 pM, e) in vessels contracted with lo-" I W 5 M NE. (B) Vehicle control (a)and EPA pretreatment (1 pM, A ; 3 pM, =; 10 pM, 0 ) in NE-contracted vessels. Each point represents the mean s f results from 7 to 12 aortic rings (five to six animals) with the m a n f SE indicated by a vertical line. *p < 0.05, **p < 0.01, and ***p < 0.001 (statistical difference compared with the parallel vehicle data).
TABLEI. Sensitivity and maximum values in control and omega-3 fatly acid treated aortic rings ~ a t t yacid
Maximum developed tension (g)
EGO
(M)
control DHA 1 beM 3 PM 28 pM EPA
NOTE:Each value represents the mean f SE. **p < 0.01 (statistical difference compared with the parallel vehicle data). ***p < 0.061 (statistical difference compared with the parallel vehicle data).
Eflects of DHA- and EPA-pretreated aortic rings on NEinduced contractiores in calcium-flee bufler A series of experiments were d s o done to examine the possible imhibitory effect of DHA and EPA on RE-induced csntractions in calcium-free, EGTA buffer. Tissues pretreated
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CAN. J . PHYSIOL. PHARMACOL. VOL. 70, 1992
TABLE 2. Effects of BWA and EPA pretreatment on NE-induced contraction in calcium-free buffer
Contraction
rCONTROL
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DHA Ex4 EPA
CONCENTRATION (pM)
Fro. 3. Effects of docssahexaensic acid (DHA) and eicssapentaensic acid (EPA) on NE-induced contraction in calcium-free buffer. Values represent change in tension from NE sustained contraction: control, +230 f 4.5 mg; DHA, +232 f 26.6 mg; EBA, +203 f 35.7 mg. Each bar depicts the mean of results from five to six aortic rings (two to three animals) with the mean f SE indicated by a vertical line. *p < 0.05 and **p < 0.01 (statistical difference compared with the parallel vehicle data).
with DHA or EPA (3 and 10 pM) prior to NE administration displayed no significant reduction in the NE-induced phasic or sustained contractions (Table 2).
Although antihypertensive properties have been ascribed to the omega-3 fatty acids, few reports are available on the direct effects of these fatty acids on the vasculature. In the present study, the vascular responses of the rat aorta to the omega-3 fatty acids DHA and EPA were investigated. This is the first such report to fully describe the comparative vascular effects of both DHA and EPA and to examine various calcium flux mechanisms that may contribute to this effect. Cumulative addition of DHA or EPA produced a concentration-dependent relaxant effect in vessels contracted with NE, an a-adrenoceptor agonist, without substantially affecting KCl-induced contractions. The relaxant effect of DHA was more pronounced compared with EPA at physiologic concentrations 41- 18 pM) . Furthermore, the inhibitory effects of DHA were greater than EPA at low concentrations of NE-induced contractions. The relaxing effect of the omega-3 fatty acids in aortic rings contracted with NE may be primarily mediated through an intracellular mechanism of action, e.g., by inhibition of intracellular calcium release or interference with contractile protein function. This study provides evidence for the selective action of omega-3 fatty acids on norepinephrine agonistinduced contractions. The lack of substantid relaxation induced by low concentrations of the omega-3 fatty acids on KCI-induced contraction suggests that the voltage-operated channel of calcium influx is not a primary site of inhibition or antagonism. It is possible that the omega-3 fatty acids at high concentrations 4 > BOQ pM) may possess additional properties that regulate voltage-sensitive calcium channels. Conversely, the more pronounced relaxant effects of DHA and EPA in NE-contracted vessels and the inhibition sf (10-9-10-8 M) NE contractions (rightward shift) may be related to a preferential inhibition of edcium influx associated
Fatty acid
n
NE phasic
NE sustained
Vehicle control DHA 3 bkM 10 yM EPA
4
+279&17.8
+230&6.5
4 3
+328&47.4 +340+20
+307&32.9 +260f40.4
NOTE:Each value represents the mean
* SE in milligrams tension.
with receptor-operated channels and (or) a decreased calcium sensitivity of the myofilaments. Isolated aortic strips from rats fed high dietary eicosapentaenoic acid for 3 weeks show a similar shift to the right of the concentration-response curve to NE, with no effect on contractions induced by KC1 (Lockette et al. 1982). These effects were thought to be attributed to direct changes in prostaglandin metabolism. Fish oil administration has also been demonstrated to improve both EDRF release and relaxation responses of smooth muscle to EDRF (Shimokawa and Vanhoutte 1988, 1989). Calcium transport in isolated cardiac myocytes from fish oil fed rats and mice is altered by DHA and EPA (Swanson et al. 1989; Karmazyn et d. 1987). Specifically, elevated levels of DHA in sarcoplasmic reticulum (SR) phospholipids are associated with a lower relative activity of Ca2+-Mg2+ ATPase, a reduced initial rate of calcium transport, and a reduced maximal uptake of calcium in cardiac SR (Swanson et al. 1989). There are no h o w n vascular studies of DHA and EPA that document such calcium-mediated effects. However, the findings of the present study demonstrating the ability of DHA and EPA to reverse the sustained component of the NE contractile response in calcium-free, EGTA-containing buffer suggests an important role for these omega-3 fatty acids in intracellular calcium regulation. In the present study, DHA and EPA did not affect the initial phase of NE contraction in calcium-free buffer. The initial phase of NE contraction is thought to be mediated by inositol 1,4,5-trisphosphate (IP3) formation, which causes the release of intracellular calcium (Manolopoulos et al. 1991). This suggests that the vasoactive effects of the omega-3 fatty acids are probably not attributed to an inhibition of IB3 formation. It is h o w n that in the absence of extracellular cdcium, two distinct intracellular calcium pools mediate the phasic and sustained components of NE-induced contractions in the rat aorta (Heaslip and Wahwan 1982). Therefore, the failure of omega-3 fatty acids to inhibit the phasic NE contraction in calcium-free medium may be due to their primary effect on a different intracellular calcium pool mediated by the sustained component of NE-induced contraction. In summary, the present investigation demonstrates the differential vasorelaxant effects of the omega-3 fatty acids DHA and EPA. The effects s f DHA are more pronounced compared with those of EPA. Regulation of intracellular calcium may be the primary mechanism of the antihypertensive effects of the omega3 fatty acids. The precise mechanism(s) of action remains to be elucidated.
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BNGLER
Acknowledgements This work was supported in part by the Academic Senate Committee on Research, University sf California, San Francisco, Calif.; the American Heart Association, San Francisco Chapter; and the National Center for Resources, National Institutes of Health, Bethesda, Md. (Biomedical Research Support Grant S07WR056M). The author expresses appreciation to Lori Hudson for her excellent secretarial assistance in the preparation of this manuscript. Bonaa, K. 1989. Epidemiological and intervention studies on the effect of marine polyunsaturated fatty acids on blood pressure. J. Intern. Med. 225(Suppl I): 105- 110. Bonaa, K. H., Bjerve, K. S., Straume, B., et a&. 1990. Effect of eicosapentaenoic and docosahexaenoic acids on blood pressure in hypertension. N. Engl. J. Med. 322: 795 -801. Engler, M. B. 1989. The vascular effects of omega-3 polyunsaturated fatty acids. Physiologist, 32: 170. Engler, M. B. 1990. Vascular responses to omega-3 fatty acids: Role of calcium. Circulation, 82: 111-607. Engler, M. B., Karanian, J. W., and Salem, N., Jr. 1990. Bocosahexaenoic acid (22:6n3)-induced relaxation of the rat aorta. Eur. J. Phamacol. 185: 223-226. Heaslip, R. J., and Rahwan, R. G. 1982. Evidence for the existence of two distinct pools of intracellular calcium in the rat aorta accessible to mobiIization by norepinephrine. J. Phamacol. Exp. Ther . 221: 9-13. Juan, H., and Sametz, W. 1986. Vasoconstriction induced by noradrenaline and angiotensin II is antagonized by eicosapentaenoic acid independent of formation of trienoic eicosanoids. Naunyn-Schmiedeberg's Arch. Phamacol. 332: 288 - 292. Juan, H.,Sutter, B., and Sametz, W. 1987. Influence of eicosapentaenoic acid on noradrenaline and angiotensin-inducedcontractions of the rabbit aorta: mode of action. In Prostaglandins in clinical research. Edited by H. Siminger and K. Schror. Alan R. Liss, Inc., New York. pp. 57-62.
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Kslrmazyn, M., Horackova, M., and Murphy, M. 6. 1987. Effects of dietary cod liver oil on fatty-acid composition and calcium transport in isolated adult rat ventricular myocytes and on the response of isolated hearts to ischemia and reperfusion. Can. J. Bhysiol. Phamacol. 65: 201 -209. Knapp, PI. R., and Fitzgedd, G . A. 1989. The antihypertensive effects of fish oil. N. Engl. J. Med. 328: 1037- 1043. Lockette, W. E., Webb, R. C., Culp, B. R., and Pitt, B. 1982. Vascular reactivity and high dietary eicosapentaenoic acid. Prostaglandins, 24: 631 -639. Manolopulos, V. G . , Pipili-Synetos, E., Hertog, A. D., and Nelemans, A. 1991. Inositol phosphates formed in rat aorta after alpha,-adrenoceptor stimulation are inhibited by forskolin. Eur. J. Pharmcsl. 207: 29 - 36. Pfister, S. Lo,Rosolswsky, M., Schranitz, J. M., et a&. 1989. Eicosapentaenoic acid alters vascular reactivity and platelet adhesion in Watanabe heritable hyperlipidemic rabblts. Eur. J. Pharmcol. 161: 85-$9. Shirnokawa, H. , and Vanhoutte, P. M. 1988. Dietary cod-liver oil improves endothelium-dependent responses in hypercholesterolemic and atherosclerotic porcine coronary arteries. Circulation, 78: 1421- 1430. Shhokawa, H., and Vanhoutte, P. M. 1989. Dietary 03 fatty acids and endothelium-dependent relaxations in porcine c6ronary Aeries. Am. J. Physiol. 256: H968 -H973. Swanson, J. E., Lokesh, B. W., and Kinsellla, J. E. 1989. Ca2+-Mg2+ AThse of mouse cardiac sarcsplasmic reticulum is affected by membrane n -6 and n -3 polyunsaturated fatty acid content. J. Nutr. 119: 364-392. Ymagisawa, A., and kefer, A. M. 1987. Vasoactive effects of eicosapentaenoic acid on isolated vascular smooth muscle. Basic Res. Cardiol. 82: 186-196. Yoshimura, T.,Hto, M., Matsui, KO,and Fujisaki, S. 1986. Effects of highly purified eicosapentaenoic acid on vascular reactivity to angiotensin II and norepinephrine in the rabbit. Prostaglandins, 32: 179- 188.
