Calcium-mediated mechanisms of eicosapentaenoic acid-induced relaxation in hypertensive rat aorta

AJH

1999;12:1225–1235

Calcium-Mediated Mechanisms of Eicosapentaenoic Acid-Induced Relaxation in Hypertensive Rat Aorta Mary B. Engler, Yunn-Hwa Ma, and Marguerite M. Engler

We have previously demonstrated the vasorelaxant properties of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in normotensive and spontaneously hypertensive rat (SHR) aorta, although the mechanism(s) of action are not fully understood. Because endothelial dysfunction and increased intracellular free calcium concentration ([Ca2ⴙ]i) are seen in hypertensive rat aorta, we investigated the potential role of Ca2ⴙ signaling, endothelium and derived factors, and the opening of potassium (Kⴙ) channels in EPA-induced relaxation. In the presence of extracellular Ca2ⴙ, EPA induced significant relaxations at > 10 ␮mol/L (P < .01) in norepinephrine (NE) (10ⴚ6 mol/L)contracted aortic rings and at 30 ␮mol/L (P <. 001) in high Kⴙ (80 mmol/L)-contracted aortic rings. In the absence of extracellular Ca2ⴙ, EPA (10 to 30 ␮mol/L) inhibits the tonic component of NEinduced contraction (P < .0001). The relaxant properties of EPA in SHR aorta appear specific to Ca2ⴙ release from an internal storage site associated with NE-induced tonic contraction. Further studies with the use of fura-2 to measure [Ca2ⴙ]i in cultured vascular smooth muscle (VSM) cells from SHR aorta indicated that EPA (30 ␮mol/ L)-pretreatment attenuated angiotensin II (50 nmol/ L)-induced Ca2ⴙ transient by 95%, suggesting that an inhibitory effect on the Ca2ⴙ signaling may

underlie EPA-induced relaxation of the vessel preparation. In addition, EPA per se induced an increase in [Ca2ⴙ]i with a duration of approximately 20 min in VSM cells, and the effect was not altered by removal of extracellular Ca2ⴙ. There was no increase in the level of inositol-1,4,5trisphosphate in response to EPA (30 ␮mol/L). The actions of EPA are independent of endotheliumderived factors, cyclooxygenase metabolites, and activation of Kⴙ channels since endothelium removal, N␻-nitro-L-arginine methyl ester hydrochloride, (L-NAME, 100 ␮mol/L), indomethacin (10 ␮mol/L), tetraethylammonium (1 mmol/L), and glibenclamide (10 ␮mol/L) did not affect EPA-induced vasodilation in NEprecontracted aortic rings. These results suggest that EPA directly modulates intracellular Ca2ⴙ signaling in VSM cells, and that this may contribute to the vasorelaxant effect and, at least in part, the blood pressure-lowering effect of fish oil. Am J Hypertens 1999;12:1225–1235 © 1999 American Journal of Hypertension, Ltd.

Received September 19, 1997. Accepted April 26, 1999. From the Laboratory of Cardiovascular Physiology, Department of Physiological Nursing, University of California, San Francisco, California (MBE, MME); and Chang Gung University, Department of Physiology and Pharmacology, Taiwan, Republic of China (Y-HM). This work was supported by grant NR02407 from the National

Institutes of Health, Bethesda, MD, USA, and grant NSC 88-2314B-182A-110 from the National Science Council, Taiwan. Address correspondence and reprint requests to Mary B. Engler, PhD, University of California, San Francisco N611Y, Box 0610, San Francisco, CA 94143-0610; e-mail: [email protected]

© 1999 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.

KEY WORDS:

Omega-3 fatty acid, fish oil, spontaneously hypertensive rat, endothelium, potassium channels, cyclooxygenase, calcium, vascular smooth muscle.

0895-7061/99/$20.00 PII S0895-7061(99)00060-6

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t has been established that the omega-3 fatty acids, docosahexaenoic (DHA) and eicosapentaenoic acid (EPA), found in fish oil have a blood pressure-lowering effect.1 These fatty acids have proved to be effective in the treatment of mild hypertension and as an adjunct therapy to antihypertensive medications.2 It is acknowledged that further study of these nutrients related to the efficacy of individual omega-3 fatty acids, additional cardiovascular benefits, and possible disadvantages with increased dietary intake are needed. In hypertensive rat models, several dietary fish-oil investigations have demonstrated a reduction in blood pressure as well as prevention of hypertension development.3–7 These effects are largely attributed to changes in eicosanoid metabolism.3,4,6 Replacement of omega-6 fatty acids (such as arachidonic acid) with omega-3 fatty acids in vascular tissue and platelets leads to favorable changes in the eicosanoid profile. The overall shift in eicosanoids is due to increased production of prostacyclin (PGI2/3) and a reduction in thromboxane A2 or prostaglandin H2 (PGH2) synthesis and release.3,4,6,8 Platelet aggregation is thus prevented and vasodilation is promoted.8 The omega-3 fatty acids may also alter vascular smooth muscle cell (VSMC) membrane composition and, in turn, important receptor and enzyme activities, as well as functioning of ion channels.9,10 Our previous findings with normotensive Sprague-Dawley rat aorta suggest that the relaxant properties of EPA may be related to intracellular calcium mechanisms.11 This has implications for intracellular calcium regulation and contractile properties of VSMCs, especially since an increase in basal intracellular free calcium concentration ([Ca2⫹]i) has been observed in hypertensive rat aorta.12–14 Endothelial dysfunction has also been demonstrated in aortic tissue from hypertensive rats.15,16 Normally, endothelium-derived relaxing factors such as nitric oxide (NO) and PGI2/3 cause relaxation. Endothelium-derived hyperpolarizing factor (EDHF) has also been found to cause hyperpolarization of VSM and relaxation.17 Although the identity of EDHF is speculative, epoxyeicosatrienoic acids (EETs) metabolized from arachidonic acid by cytochrome P-450 appear to closely correlate to EDHF.18 EDHF reportedly activates Ca2⫹-activated rather than ATP-sensitive K⫹-channels.18 Vascular tone and membrane potential are regulated by these K⫹ channels as well as voltagedependent Ca2⫹-channels.19 The role of K⫹-channels in EPA-induced vasorelaxation and possible changes in [Ca2⫹]i has yet to be defined. The endothelium also produces several cyclooxygenase-dependent contracting factors (ie, PGH2, thromboxane A2, superoxide anions) and endothelin-1, which may, in part, mediate the endothelial dysfunc-

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tion seen in hypertensive aorta.20 We have previously demonstrated an endothelium-dependent vasorelaxant effect of EPA at low concentration (⬍ 10 ␮mol/L) in spontaneously hypertensive rat (SHR) aorta.21 Vessels from animals supplemented with fish oil in the diet have been shown to exhibit facilitated endothelium-dependent relaxations22,23 as well as an inhibition of endothelium-dependent contractions.23,24 Since VSM contraction is associated with a transiently increased [Ca2⫹]i or enhanced Ca2⫹ sensitivity (or both) of the contractile elements,25 modulation of cytosolic Ca2⫹ level may thus modulate vascular tone. Receptor activation-induced Ca2⫹ transients can result from both a release of intracellular Ca2⫹ stores (ie, sarcoplasmic reticulum [SR] and an influx of extracellular Ca2⫹).25 After contraction, muscle relaxation is associated with a reduction in [Ca2⫹]i due to sequestration of [Ca2⫹]i by Ca2⫹-ATPase of SR and Ca2⫹ efflux across the cellular membrane by the Ca2⫹ATPase and Na⫹/Ca2⫹ exchanger.25 The purpose of this study was to further investigate the relaxant mechanism(s) of action of EPA that may involve Ca2⫹ signaling in SHR aorta. The effects of EPA on NE- and high K⫹-induced contractions were examined under normal Ca2⫹ conditions. Since VSM contraction in the absence of extracellular Ca2⫹ is a useful technique to examine intracellular Ca2⫹ mobilization, the effects of EPA on the biphasic contractile responses of NE were investigated. To further delineate the mechanism of EPA, intracellular Ca2⫹, and inositol-1,4,5-trisphosphate, Ins(1,4,5)P3, were measured after EPA administration in cultured VSM cells from SHR. The potential role of the endothelium and derived factors (NO, EDHF, prostanoids or cyclooxygenated products) and opening of K⫹-channels in EPA-induced vasorelaxation were also examined in the isolated vessel preparation. A preliminary report was presented in November 1998 at the 71st Scientific Sessions of the American Heart Association in Dallas, TX.26 METHODS Animals Male SHR (n ⫽ 75) (Harlan Sprague Dawley, Inc., Indianapolis, IN; Taconic, Germantown, NY) aged 16 to 17 weeks and mean weight 316.4 ⫾ 2.0 g were used in the study. Animal care and use were approved by the Committee on Animal Research, University of California, San Francisco, CA. Blood Pressure Before anesthesia, systolic blood pressure was measured with the indirect tail cuff plethysmography method at room temperature. The device was a photoelectric tail cuff-system (model 179, IITC Inc., Woodland Hills, CA). The average of three successive measurements was taken as the mean systolic pressure value. The mean systolic blood pressure was 186.2 ⫾ 1.6 mm Hg (n ⫽ 75).

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Chemicals Acetylcholine (ACH) chloride, tetraethylammonium (TEA), N␻-nitro-l-arginine methyl ester hydrochloride (L-NAME), ethylene glycol-bis (␤-aminoethylether)-N,N⬘-tetra-acetic acid (EGTA), glibenclamide (Glb), phentolamine, indomethacin (INDO), EPA (sodium salt, ⬎ 99% purity), norepinephrine (NE, (-)-arterenol bitartrate salt), angiotensin II (AII, acetate salt, synthetic, human plasma), saponin, thapsigargin, elastase (EC 3.4.21.36, type III, porcine pancreas), and fluorescein isothiocyanate (FITC) conjugated anti-␣smooth muscle actin antibody were obtained from Sigma Chemical Co., St. Louis, MO. EPA was dissolved in nitrogen-saturated methanol and stored at ⫺70°C under nitrogen gas. Just before experimentation, EPA in methanol was evaporated under nitrogen and reconstituted in nitrogen-saturated NaCl (0.9%). The fatty acid vehicle (0.9% NaCl) was also saturated with nitrogen before addition to control vessels. Glb was dissolved in dimethyl sulfoxide to a final DMSO concentration less than 0.1%. INDO was dissolved in sodium carbonate solution (0.1 mol/L). Collagenase (CLSIII) and soybean trypsin inhibitor were purchased from Worthington Biochemical (Freehold, NJ). Fura-2 acetoxymethyl ester (fura-2 AM) and Pluronic F127 from Molecular Probes (Eugene, OR) were dissolved in concentrated form and stored in DMSO before experimentation. Minimum essential medium (with Earl’s salt and glutamine) was from Mediatech (Herndon, VA). Fetal bovine serum (AFA4752) was obtained from Hyclone (Logan, UT). Tryptose phosphate broth, glutamine, penicillin, streptomycin, and Waymouth medium were obtained from the tissue culture facility, University of California, San Francisco, CA. Vessel Preparation The animals were anesthetized with a mixture of 70% oxygen, 30% nitrous oxide, and 5% halothane. The descending thoracic aorta was excised after thoracotomy. The aorta was transferred immediately to ice-cold Krebs-Ringer bicarbonate buffer solution (composition in mmol/L): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3, and 11.1 glucose), trimmed of all connective and fat tissue and cut into ring segments (3.0 mm length). In some rings, the endothelium was removed by gently rubbing the intimal surface with the tip of a small curved forceps. Each ring was placed into a tissue bath (Radnoti Glass Technology, Inc., Monrovia, CA) filled with 15 mL Krebs bicarbonate buffer (37°C) and bubbled with 95% O2/5% CO2. The rings were attached to force-displacement transducers (model FT03, Grass Instrument Co., Quincy, MA) coupled to an eight-channel chartwriter (model WR3701, Western Graphtec, Inc., Irvine, CA) for isometric tension measurement. Tension adjustments and bath washes were automated by using a computerized sys-

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tem (STC400, Buxco Electronics, Inc., Troy, NY). A PO-NE-MAH data acquisition system (Gould, Inc., Cleveland, OH) was used for acquiring the data. Aortic rings were equilibrated for 60 to 90 min at an optimal resting tension of 2 g. Tissue viability was determined with contraction to KCl (30 mmol/L) and the integrity of the endothelium was assessed by relaxation to ACH (1 ␮mol/L) in KCl-contracted rings. The absence of functional endothelium (deendothelialization) was confirmed by the complete lack of relaxation in response to ACH in rubbed preparations. Experimental Procedures of the Vessel Preparation After maximal contraction to NE (10⫺6 mol/L) or high (K⫹) solution (80 mmol/L), the relaxant effects of EPA (1 to 30 ␮mol/L) in SHR aorta were evaluated. (This concentration of EPA was used since it represents the physiological level found in normal human plasma.27) In some rings, the endothelium was removed before NE-induced contraction and administration of EPA. Phentolamine (1 ␮mol/L) was used in high K⫹-contracted rings to prevent release of endogenous catecholamines. In some experiments, NE-contracted rings were pretreated (20 min) with the cyclooxygenase inhibitor, INDO (10 ␮mol/L) or the NO synthesis inhibitor, L-NAME (100 ␮mol/L), (or both). INDO and L-NAME were used to determine the possible involvement of prostanoids or cyclooxygenase products and endothelial NO in EPA-induced relaxations. To rule out the possible involvement of K⫹ channels in EPA-induced relaxations, TEA (1 mmol/L) and Glb (10 ␮mol/L), the Ca2⫹-activated and ATP-sensitive K⫹-channel blockers, respectively, were incubated 20 min before EPA (1 to 30 ␮mol/L)-induced relaxation in NE-contracted rings. In a separate set of experiments to determine the inhibitory effect of EPA on the sustained or tonic component of NE (10⫺6 mol/L)-induced contraction, Ca2⫹-free solution containing EGTA (2 mmol/L) was used. Aortic rings were equilibrated initially in Krebs solution with CaCl2 (2.5 mmol/L) and were then washed four times at 4-min intervals for a total of 20 min in Ca2⫹-free EGTA containing solution. Under these conditions, NE produced a biphasic contractile response with an initial phasic component followed by a tonic or second component. Cumulative concentration-response curves to EPA (1 to 30 ␮mol/L) were generated at the plateau of the tonic NE-contractile component. The last set of experiments also included Ca2⫹-free solution with and without EGTA (2 mmol/L); however, EPA (5, 10, 30 ␮mol/L) was incubated (20 min) in the tissue bath before NE (10⫺6 mol/L) administration. These experiments were performed to investigate the possible inhibition of EPA on the phasic compo-

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nents of NE-induced contraction in Ca2⫹-free solution with and without EGTA. Cell Culture From abdominal aorta of male SHR (Taconic), primary cultures of VSM cells were obtained with enzyme digestion. Briefly, the adventitia of each vessel was removed using forceps, the vessel was cut longitudinally, and the endothelium was mechanically removed. The artery from each rat was cut into small pieces (1 mm ⫻ 1 mm), which were incubated with collagenase (CLSIII 402 U/mL), elastase (type 3, 1.3 U/mL) and soybean trypsin inhibitor (1 mg/mL) for three 30-min intervals. This enzyme mixture was prepared fresh for each experiment. Cell suspensions were pooled and evenly divided into six aliquots and transferred into a 6-well plate with glass cover slips containing minimum essential medium with 10% fetal bovine serum, 2% tryptose phosphate broth, glutamine (20 mmol/L), penicillin (50 U/mL), and streptomycin (growth medium, 50 U/mL) in a humidified atmosphere of 5% CO2/ 95% air at 37°C. Culture medium was changed every 2 to 3 days for 1 week and cells on the coverslips were used for experiments. The remaining cells in the six-well plates were allowed to grow and were subcultured with trypsin– versene and 0.2% pancreatin. VSM cells thus obtained were used for experiments within 3 to 5 passages. Smooth muscle cell identification was made by immunofluorescence with an fluorescein isothiocyanate conjugated anti-␣-smooth muscle actin antibody. In all cell preparations tested before experimentation, greater than 95% of cells stained positive for this antigen. Intracellular [Ca2ⴙ] Measurement The [Ca2⫹]i in a small group (5 to 10) cells was determined by using fura-2 AM and a Nikon inverted epifluorescence microscope with the UMANS analytic software (Bio-Rad, Bio-Rad Laboratories, Hercules, CA). Cells plated on a cover slip (0.07 mm) were incubated with a mixture containing fura-2 AM (6.3 ␮mol/L), pluronic F-127 (0.03%), and bovine serum albumin (0.4%) in salt solutions at room temperature for 1 h. The salt solutions (pH 7.2) used in the experiments contained bovine serum albumin (0.05%) and the following (mmol/L): NaCl (140), KCl (5), MgSO4 (1), Na2HPO4 (1), CaCl2 (0 or 2), glucose (25), HEPES (25). Fluorescent measurements were conducted at 32°C. The ratio of fluorescence intensity at excitation wavelengths 340 and 380 nm was used to determine [Ca2⫹]i. For calibration, saponin (50 ␮g/mL) and EGTA (5 mmol/L) were used to determine maximum and minimum fluorescence ratios. [Ca2⫹]i values were calculated as previously described,28 by using a dissociation constant of 224 nmol/L.

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Ins(1,4,5)P3 Measurement Cells in six-well plates were treated with effectors in the presence of lithium acetate (10 mmol/L) for 10 min before harvest. The response was terminated by addition of 50% ice-cold perchloric acid (0.2 of volume) at the indicated time. After 20 min incubation on ice, the cells and medium were collected with a cell lifter and the mixture was centrifuged at 2000 g for 15 min at 4°C. Supernatants were titrated to pH 7 to 8.5 with 10N KOH and kept on ice. After removal of insoluble potassium perchlorate by centrifugation, total mass of Ins(1,4,5)P3 was assayed with a kit from Amersham (TRK 1000, Pharmacia Biotech, Inc., Piscataway, NJ). Ins(1,4,5)P3 standards were prepared in the same solutions used for experimentation. The levels of Ins(1,4,5)P3 with both samples and standards were determined in duplicate. Calculations and Statistical Analysis Relaxations were expressed as a percentage of the maximum tension produced by the contractile agonist. Area under the curve of Ca2⫹ responses was calculated from 0 to 15 min after addition of EPA with Prism (GraphPad Software, Inc., San Diego, CA). All results are shown as mean ⫾ SEM. Statistical significance was determined by applying the Student t test for unpaired observations. One-way analysis of variance (ANOVA) was performed for multiple comparisons followed by the Scheffe procedure for statistically significant F values. The Kruskal-Wallis test for group comparisons was used when the assumption of equal variances required for the ANOVA was not satisfied. Concentration–response curve comparisons were analyzed with repeated measures ANOVA. The GreenhouseGeisser adjustment for multisample asphericity was used to avoid excessive Type I error.29 Significance was set at P ⫽ .05, and when multiple comparisons were performed at each concentration, the test of simple main effects with Bonferroni correction was used (eg, 0.05/5 ⫽ 0.01). RESULTS Relaxing Effect of EPA in Contracted SHR Aortic Rings As illustrated in Figure 1, EPA induced similar relaxation responses (difference not significant, [ns]) after contraction to the agonists NE (10⫺6 mol/L) and high K⫹ (80 mmol/L) solution (EPA and KClEPA). Although compared to control responses, EPA induced relaxations that were significantly greater in NE-contracted rings at ⬎ 10 ␮mol/L (P ⬍ .01) and in high K⫹-contracted rings at 30 ␮mol/L (P ⬍ .001). Similar maximal contractile responses were noted in SHR aortic rings contracted with NE (10⫺6 mol/L) or high K⫹ (80 mmol/L) solution. These maximal responses were 1531.4 ⫾ 65.7 mg, n ⫽ 9 and 1385 ⫾ 94.7 mg, n ⫽ 6; (ns), respectively. Overall, with repeated measures ANOVA, removal of the endothelium (EPA

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FIGURE 1. Comparison of relaxation induced by EPA in SHR aortic rings (intact and de-endothelialized [E ⫺]) contracted with NE, 10⫺6 mol/L) or high K⫹ solution (80 mmol/L) (KCl-EPA). The (ⴙ) indicates pretreatment (20 min) with INDO (10 ␮mol/L) or L-NAME (100 ␮mol/L) or both in NE-contracted rings (n ⫽ 3 to 16 animals per group). **Significant at P ⬍ .01, *** P ⬍ .001, **** P ⬍ .0001 as compared to EPA group.

E ⫺) or addition of L-NAME (EPA ⫹ L-NAME) did not significantly alter EPA-induced relaxations when compared to intact vessel responses (EPA), Figure 1. Maximal contractile responses to NE in both groups were 2095 ⫾ 175.7 mg for the EPA ⫹ L-NAME group (n ⫽ 3) and 1718.7 ⫾ 125.5 mg for the EPA E⫺ group (n ⫽ 4). No significant difference in the NE responses was noted between the EPA E⫺ group and the intact EPA group (1531.4 ⫾ 65.7 mg, ns); however, responses in the EPA ⫹ L-NAME group were much greater as compared to the EPA group (P ⬍ .01). The maximal contractile response to NE after INDO treatment in the EPA ⫹ INDO group (n ⫽ 3) was diminished (800.3 ⫾ 75.1 mg, P ⬍ .001) as compared to the EPA group. Addition of INDO (EPA ⫹ INDO) or combination INDO and L-NAME (EPA ⫹ INDO/L-NAME, n ⫽ 16) caused enhanced relaxations at EPA concentrations 3 to 30 ␮mol/L (P ⬍ .01) and 1 to 30 ␮mol/L (P ⬍ .001), respectively (Figure 1). Further statistical analysis of the differences between the EPA ⫹ L-NAME and the EPA ⫹ INDO group responses revealed greater relaxations to EPA (⬎ 10 ␮mol/L, P ⬍ .01) in the presence of INDO. The maximal contractile response to NE was 1717.6 ⫾ 81.6 mg (n ⫽ 16) in the presence of both inhibitors (INDO, L-NAME). Parallel control responses in NE-contracted and high K⫹-contracted rings without inhibitors ranged from 0% to 8.8% and ⫺6% to ⫺13% in the presence of both inhibitors (INDO, L-NAME). In preliminary experiments, ACH (10⫺9 to 10⫺4 mol/L) induced endothelium-dependent relaxations (⫺2.4 ⫾ 0.6% to ⫺51.3 ⫾ 4.8%, n ⫽ 6) in KCl (30 mmol/L)-contracted intact rings. INDO (10 ␮mol/L) did not alter ACH-induced relaxations (⫺4.2 ⫾ 1.2%

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FIGURE 2. Comparison of relaxation induced by EPA in SHR aortic rings (intact) contracted with NE (10⫺6 mol/L) in the presence (20 min) and absence of TEA (1 mmol/L) and Glb (10 ␮mol/L) (n ⫽ 4 to 9 animals per group).

to ⫺69.1 ⫾ 7.7%, n ⫽ 6, ns); however, L-NAME (100 ␮mol/L) pretreatment significantly inhibited ACH (10⫺7 to 10⫺4 mol/L)-induced relaxations (1.2% ⫾ 1.8% to 9.4% ⫾ 2.5%, n ⫽ 6, P ⬍ .0001). Effect of Kⴙ-Channel Blockers on EPA-Induced Relaxations Because K⫹-channel activation induces relaxation of arteries,25 the role of K⫹-channel opening in EPA-induced relaxation was examined with K⫹channel blockers, Glb and TEA. As illustrated in Figure 2, no significant inhibition of EPA (1 to 30 ␮mol/ L)-induced relaxation in NE-contracted rings was observed by the treatment of Glb or TEA. NE Contractions After Pre-Incubation with EPA in Ca2ⴙ-Free Solutions It has been established that extracellular Ca2⫹ is required for most vasoconstrictor-induced contractions.25 In our aortic preparations, removal of extracellular Ca2⫹ with Ca2⫹-free solution containing EGTA (2 mmol/L) caused a reduction of NE (10⫺6 mol/L)-induced contraction, ie, 43.7% ⫾ 2.2% of that obtained in Krebs (Ca2⫹-containing) solution (n ⫽ 10). When EPA (5, 10 ␮mol/L) was preincubated for 20 min, NE (10⫺6 mol/L)-induced initial phasic contractions in Ca2⫹-free solution with 2 mmol/L of EGTA was not altered. These responses were 44.8% ⫾ 3.0% (EPA at 5 ␮mol/L, n ⫽ 10) and 49.2% ⫾ 6.7% (EPA at 10 ␮mol/L, n ⫽ 10) of the effects of NE in Ca2⫹-containing solutions, respectively. In additional experiments with Ca2⫹-free solution without EGTA, EPA (10, 30 ␮mol/L) also exhibited no significant inhibitory effect on NE (10⫺6 mol/L)induced phasic contractions. Under these conditions, the responses to NE were 49.2% ⫾ 1.6% (control, n ⫽ 4), 52.1% ⫾ 4.1% (EPA at 10 ␮mol/L, n ⫽ 4), 49.2% ⫾ 1.8% (EPA at 30 ␮mol/L, n ⫽ 4) of the effects of NE in Ca2⫹-containing solutions, respectively.

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FIGURE 3. Relaxant effect of EPA on the sustained component of NE (10⫺6 mol/L)-induced contraction in Ca2⫹-free, EGTA (2 mmol/L) solution (n ⫽ 10 animals per group). *** P ⬍ .001, ****P ⬍ .0001 indicates statistical difference compared to parallel control data.

In separate experiments in SHR aorta, we found that EPA pretreatment for 20 min did not affect NE-induced contraction in Ca2⫹-containing solution. Control responses (n ⫽ 10) under these conditions to NE (10⫺9 to 5 ⫻ 10⫺5 mol/L) were 423 ⫾ 52 mg to 1857 ⫾ 105 mg, which were not significantly different from those of the EPA (5, 10 mol/L)-treated groups (n ⫽ 10). Relaxing Effect of EPA on NE-induced Sustained Contraction in Ca2ⴙ-Free Solutions To determine if EPA induces relaxation of precontracted aorta in the absence of extracellular Ca2⫹, vessels were exposed to Ca2⫹-free solution containing EGTA (2 mmol/L) for 20 min before addition of NE (see METHODS section). In Figure 3, a significant relaxant response was seen with EPA (ⱖ 10 ␮mol/L, P ⬍ .001) when administered at the plateau of the tonic component of NE (10⫺6 mol/L)-induced contraction in Ca2⫹-free solution containing EGTA (2 mmol/L). EPA-induced relaxations were expressed as a percentage of maximal NE-induced contraction in Krebs (Ca2⫹-containing) solution. The actual plateau contractile responses to NE in Krebs (Ca2⫹-containing) solution were 1434.9 ⫾ 61 mg for the control group (n ⫽ 10) and 1473.3 ⫾ 104.9 mg for the EPA-treated group (n ⫽ 10) (ns). Before administration of the vehicle (control) or EPA, the tonic components of NE-induced contractions in Ca2⫹-free solution with EGTA were 30.7 ⫾ 12.2 mg (control, n ⫽ 10) and 34.1 ⫾ 12.3 mg (EPA, n ⫽ 10) (ns). Effect of EPA on Intracellular [Ca2ⴙ] To determine the effect of EPA on vasoconstrictor-induced transient increase in the intracellular calcium, [Ca2⫹]i was measured by using fura-2 in VSM cells from aorta of SHR.

FIGURE 4. EPA inhibited AII-induced Ca2⫹ transients in VSMC of SHR. Vehicle (A), EPA (31 ␮mol/L) (B), and AII (50 nmol/L) (A, B) were administered as indicated by arrowheads. AII was added in the absence of EPA or vehicle. Tracings are representative of three similar experiments.

Figure 4 shows that both EPA (30 ␮mol/L) and AII (50 nmol/L) induced Ca2⫹ transients with a duration of approximately 30 min v 2 min, respectively. Thirty minutes after EPA pretreatment, the magnitude of AII-induced Ca2⫹ transient was only 5% ⫾ 2% (n ⫽ 3) of that observed without EPA pretreatment. To examine the mechanism(s) by which EPA inhibits the Ca2⫹ transient elicited by subsequent addition of vasoconstrictors, the source of Ca2⫹ in EPA-induced Ca2⫹ transients was determined in VSM cells. When the effect of EPA in the absence of extracellular Ca2⫹ was measured, Ca2⫹ was removed from the physiological salt solution 2 min before addition of EPA to prevent depletion of intracellular Ca2⫹ stores. Figure 5 illustrates that both the EPA-induced peak increase of [Ca2⫹]i, or total increase of cellular Ca2⫹, were not significantly different in the presence (⫹ Ca2⫹) and absence (⫺ Ca2⫹) of extracellular Ca2⫹. These results suggest that EPA-induced Ca2⫹ transient is primarily caused by Ca2⫹ release from the intracellular stores. Since receptor activation-mediated phospholipase C-activation and subsequent Ins(1,4,5)P3 production is a common pathway to stimulate Ca2⫹ release, we determined whether EPA-induced Ca2⫹ transient is mediated by Ins(1,4,5)P3 in VSM cells of SHR. Figure 6 shows that EPA, to a small (but not significant) degree, altered the total cellular Ins(1,4,5)P3 level within 5 min

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FIGURE 6. Effect of EPA on total cellular mass of Ins(1,4,5)P3 in VSMCs of SHR. Total cellular mass was measured at various time points after addition of EPA (30 ␮mol/L, n ⫽ 3) and at one min after addition of thrombin (1 U/mL, n ⫽ 3) (see METHODS section). *P ⬍ .05 as compared to Ins(1,4,5)P3 level at time zero.

FIGURE 5. EPA induced similar peak increase (A) and total increase (B) of intracellular Ca2⫹ concentration in the presence (⫹ Ca2⫹, n ⫽ 7) and absence (⫺ Ca2⫹, n ⫽ 6) of extracellular Ca2⫹ in VSMCs of SHR. The total increase of [Ca2⫹]i was determined by analysis of the area under the curve as described in the METHODS section. Ca2⫹-containing physiological salt solution was replaced with Ca2⫹-free solution 2 min before addition of EPA (31␮mol/L), and the basal [Ca2⫹]i were 25 ⫾ 12 and 7 ⫾ 2 nmol/L with and without extracellular Ca2⫹, respectively.

during administration of EPA (n ⫽ 3), suggesting that EPA-induced Ca2⫹ release may not be mediated by Ins(1,4,5)P3. With the same batch of VSM cells from SHR, thrombin (1 U/mL) induced a 2.7-fold increase in total Ins(1,4,5)P3 production over the basal level after 1 min of administration (n ⫽ 3, P ⬍ .01), suggesting that the cells have the capability to respond. DISCUSSION These results demonstrate the relaxant properties of physiological concentrations27 of EPA in SHR aorta contracted by different stimuli, ie, ␣-adrenergic receptor activation by NE and depolarization by high K⫹ solution. Both NE and high K⫹ solution increase [Ca2⫹]i. However, ␣-adrenoceptor activation stimulates phosphatidylinositol turnover, releases Ca2⫹

from internal stores, and induces Ca2⫹ influx through Ca2⫹ channels and nonselective cation channels to increase [Ca2⫹]i.25,30,31 Unlike NE, high K⫹ increases [Ca2⫹]i by depolarization, which induces Ca2⫹ influx through voltage-gated or L-type Ca2⫹ channels without inducing phosphatidylinositol turnover or altering Ca2⫹ sensitivity.25,32–34 Since EPA induced relaxation in vessels precontracted with NE or high K⫹, it is unlikely that it primarily acts at earlier signaling steps, like a receptor blocking agent. Instead, EPA might act on a Ca2⫹ signaling mechanism common to both vasoactive agents, such as Ca2⫹ channel activation or Ca2⫹ sequestration system. Our results demonstrated that pretreatment of EPA dramatically attenuated AIIinduced Ca2⫹ transients in VSM cells (Figure 4). This mechanism might explain the effect of EPA in causing relaxation after vasoconstrictor-induced contraction. In addition, Locher et al have shown that pretreatment of VSM cells with EPA attenuated AII-, low-densitylipoprotein- and platelet-derived growth factor-induced Ca2⫹ transients by half, 35 indicating that EPA may attenuate Ca2⫹ transients elicited by vasoconstrictors in general. Incorporation of EPA into the VSM cell membrane can alter the physical state of the cell membrane (ie, increase fluidity) and, in turn, influence ion transport, enzyme, and receptor activities.9,10 In contrast, a decrease in VSM membrane fluidity has been reported in hypertensive rats.36 We recently found that EPA levels are increased in aortic rings after administration of EPA into tissue baths (unpublished observations). This may normalize the decreased membrane fluidity seen in VSM cells from hypertensive rats. The resulting membrane changes induced by EPA may also

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contribute to the modulating role of the omega-3 fatty acid on VSM Ca2⫹ mechanisms. It has been shown that EPA suppresses voltage-gated L-type Ca2⫹ currents in neonatal and adult rat cardiac myocytes.37–39 In our study, EPA might attenuate vasoconstrictorinduced Ca2⫹ entry by inhibition of the L-type Ca2⫹ channel in VSM cells. However, after removal of extracellular Ca2⫹, EPA preserved its capability to induce relaxation of arteries, suggesting mechanisms other than L-type Ca2⫹ channel inhibition might be involved in its action as a vasodilator. In this study with SHR aorta, EPA (ⱖ 10 ␮mol/L) relaxed the tonic contractions to NE in Ca2⫹-free solution containing EGTA. These results are similar to our previous findings with normotensive SpragueDawley rat aorta.11 In rat aorta, there is a biphasic response to NE in Ca2⫹-free solution40 which might be related to release of Ca2⫹ from heterogeneous Ca2⫹ pools in the cytoplasm.41 The phasic component results from release of Ca2⫹ stored in the SR, and the second NE component (tonic) is believed to represent release of Ca2⫹ from an internal storage compartment that is close to or directly connected to the plasma membrane.42 The omega-3 fatty acid may be acting by specifically interfering with Ca2⫹ release from the second internal storage component of NE contractions in Ca2⫹-free solution. However, we cannot rule out the possibility that EPA might also interfere with the downstream biochemical processes, such as phophorylation and dephosphorylation, to induce relaxation. Interestingly, on incubation with EPA under the same Ca2⫹-free conditions, NE-induced phasic contractions were not inhibited. It is clear that the vasorelaxant responses to EPA in this study are independent of the endothelium and derived factors, (ie, NO, EDHF). This is based on similar EPA responses in intact and deendothelialized rings. Additionally, our results with L-NAME, which blocked ACH-induced relaxations, confirm that the relaxing factor released by ACH in SHR aorta is NO. It is evident that EPA relaxations are not mediated by NO because L-NAME had no effect on relaxation. We previously reported on the endothelium-dependent relaxations to EPA at low concentrations in the SHR model.21 In the current study, using a new statistical method of analysis29 that addresses the overall effect of all concentrations in a response curve, we found no significant differences between EPA responses in intact and deendothelialized SHR aorta. In our previous study,21 data analysis between the groups was performed with use of vertical contrasts at each concentration level of EPA. This approach increased the risk of type I error that results from multiple comparisons.29 Although vertical contrasts of the present data demonstrate results similar to those of our previous study,21 repeated measures ANOVA with the Green-

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house-Geisser adjustment were used with correction for multisample asphericity to avoid the risk of type I error. Because EPA (10 to 30 ␮mol/L)-induced relaxations were enhanced in the presence of INDO, it is possible that blockade of the cyclooxygenase enzyme suppressed normally elevated basal levels of cyclooxygenase-dependent endothelium-derived contracting factors (thromboxane A2, PGH2, and superoxide anions) seen in hypertension. Thus, vascular tone would be expected to be decreased and EPA-induced relaxations would be greater in the presence of cyclooxygenase enzyme inhibition. We did find a reduction in the maximal contractile response to NE as well as greater relaxation to EPA in the presence of INDO. A similar enhancement of EPA-induced relaxations was also seen with the combination inhibition of cyclooxygenase and NO synthesis with INDO and L-NAME in NE-contracted rings. This occurred, although the maximal contractile response to NE was not diminished. It is known that endothelium-dependent relaxation under conditions of combined inhibition of prostaglandins and NO is associated with membrane hyperpolarization.43 It is unlikely that endotheliumdependent hyperpolarization is involved in EPA-induced relaxation, because removal of the endothelium and use of the K⫹ channel blockers, TEA, and Glb, had no effect on EPA responses. Therefore, opening of either Ca2⫹-activated or ATP-sensitive K⫹-channels and subsequent hyperpolarization are not likely to mediate EPA-induced relaxation in SHR aorta. Endothelin-mediated responses are not affected by inhibitors of cyclooxygenase or NO,44 thereby ruling out inhibition of endothelin as a factor in the enhanced EPA relaxations with cyclooxygenase and NO inhibitors present. The reduced EPA relaxations in the absence of inhibitors may be due to an increase in resting SHR vascular tone. This may be related to a reduction in aortic NO production and synthesis, or an imbalance in vasoconstriction caused by endothelium-derived contracting factors in this SHR model. Fatty acids may directly or indirectly affect ion channels.45 Indirect effects occur with metabolic conversion of the fatty acid to an active oxygenated metabolite which, in turn, interacts with the ion channel. Direct effects result from actions by the fatty acid on the ion channel or an associated site within the cell membrane. Although a recent study by Asano et al showed that EPA (30 ␮mol/L) induces K⫹ currents in rat A7r5 smooth muscle cells,46 we were unable to demonstrate that the relaxant properties of the omega-3 fatty acid in SHR might be related to membrane hyperpolarization through activation of K⫹-channels. This may be due to the differences in aortic tissue preparations as well as rat strains. However, this study46 reported an additional inhibition by EPA (⬎3

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␮mol/L) on receptor (vasopressin, endothelin-1)-mediated Ca2⫹-permeable nonselective cation current. A similar action may be reflected in our findings with EPA-induced relaxation in NE-adrenergic receptormediated contraction. Because NE-induced contraction can open the nonselective cation channel,25 EPA may prevent the depolarization of the membrane associated with such channel opening and resultant activation of L-type Ca2⫹-channel, which further increases Ca2⫹-influx. Although EPA relaxes preconstricted vessels, we found that EPA itself induced an increase in [Ca2⫹]i in VSM cells (Figure 4). Previous studies with T cells47 and endothelial cells48 also demonstrated that EPA stimulates an increase in [Ca2⫹]i. Compared to a physiological concentration of AII-induced Ca2⫹ transient, the amplitude of EPA (30 ␮mol/L)-induced Ca2⫹ transient is smaller with a longer duration in our studies with VSM cells. Because no contraction was observed after EPA treatment in the vessel preparation, the slightly increased [Ca2⫹]i may be important for the subsequent signaling without triggering contraction of VSM. EPA-induced increase in [Ca2⫹]i may be due to release of intracellular stores or entry from the extracellular space. In the presence and absence of extracellular Ca2⫹, EPA induced a similar amount of increase in cytosolic Ca2⫹ in VSM cell preparations (Figure 5b). The independence of extracellular Ca2⫹ suggests that the increased cytosolic Ca2⫹ was of intracellular origin. According to the “capacitative hypothesis” proposed by Takemura and colleagues,49 the emptiness of Ca2⫹ store per se can be a signal to trigger Ca2⫹ entry; therefore, depletion of Ca2⫹ stores by thapsigargin, an SR-Ca2⫹-ATPase inhibitor, induces Ca2⫹ influx. However, EPA-induced intracellular Ca2⫹ release did not stimulate Ca2⫹ influx, as extracellular Ca2⫹ removal did not affect EPA-induced Ca2⫹ transients. This is consistent with previous findings in T cells that depletion of the Ins(1,4,5)P3-sensitive pool by EPA did not induce any influx of Ca2⫹ measured by Mn2⫹-quenching technique.47 It is possible that Ca2⫹ entry after EPA-induced release was blocked by additional effects of EPA on the Ca2⫹ entry mechanism, such as Ca2⫹ channels. Whether the absence of Ca2⫹ entry after EPA-induced Ca2⫹ release in VSM cells is due to an inhibitory effect of EPA on the Ca2⫹ entry mechanism requires additional study. Nevertheless, it is likely that EPA acts on the Ca2⫹ sequestration system to cause release of intracellular Ca2⫹ stores, which further prevents subsequent Ca2⫹ transient induced by vasoconstrictors. Although EPA incorporation into plasma membrane might affect protein configuration and enzyme activities, administration of EPA did not induce accu-

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mulation of Ins(1,4,5)P3 (Figure 6), suggesting that EPA-induced Ca2⫹ release is not mediated by production of Ins(1,4,5)P3. Previous studies50 suggest that AII-stimulated Ins(1,4,5)P3 production was inhibited by EPA at a concentration of 100 ␮mol/L in VSM cells. However, 30 ␮mol/L of EPA, as used in the current study, did not affect AII-induced InsP3 production. Therefore, the effect of EPA on Ca2⫹ signaling or vascular tone may not be explained by Ins(1,4,5)P3 production. In conclusion, EPA inhibits vascular contraction to ␣-adrenergic stimulation and high K⫹ depolarization in SHR aorta. The actions of EPA in the vessels appear to be independent of endothelium, cyclooxygenase metabolism, and activation of K⫹-channels, but related to modulation of Ca2⫹ signaling. In cultured VSM cells, EPA released intracellular Ca2⫹ and attenuated vasoconstrictor-induced Ca2⫹ transients, which may underlie the vasorelaxant effects of EPA and contribute, in part, to the blood pressure-lowering properties of fish oil. ACKNOWLEDGMENT We thank Diane Heininger for typing the manuscript and providing expert technical graphics support.

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