Effect of Docosahexaenoic Acid on Voltage-Independent Ca²⁺ Entry Pathways in Cultured Vascular Smooth Muscle Cells Stimulated with 5-Hydroxytryptamine

Abstract

We previously reported that docosahexaenoic acid (DHA) inhibits an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured rat vascular smooth muscle cells (VSMCs) through a mechanism involving mainly voltage-dependent Ca²⁺ channels; however, the effect of DHA on voltage-independent pathways, such as store-operated and receptor-operated Ca²⁺ entry, and Ca²⁺ entry through Na⁺/Ca²⁺ exchanger (NCX), has not been clarified. In the present study, we investigated the effect of DHA treatment on the expression of transient receptor potential canonical (TRPC) channels, capacitative Ca²⁺ entry, and Ca²⁺ entry through NCX in rat cultured VSMCs stimulated with 5-hydroxytryptamine (5-HT). RT-PCR analysis detected TRPC1, TRPC4, and TRPC6 mRNA in cultured VSMCs. DHA treatment for 2 d slightly but significantly decreased TRPC1, but not TRPC4 and TRPC6, mRNA expression. Sarpogrelate, a selective serotonin 5-HT_2A receptor inhibitor, completely inhibited the 5-HT-induced increase in [Ca²⁺]_i in cultured VSMCs. Ca²⁺ influx by adding extracellular Ca²⁺ (1.3 mM) to the Ca²⁺-free condition in the presence of 5-HT was partially but significantly inhibited by sarpogrelate. DHA treatment for 2 d had no effect on Ca²⁺ influx when extracellular Ca²⁺ was added to the Ca²⁺-free condition in the presence of either 5-HT alone or 5-HT with sarpogrelate. KB-R7943, a selective inhibitor of reverse mode NCX, significantly suppressed the 5-HT-induced increase of [Ca²⁺]_i. Furthermore, DHA treatment for 2 d significantly decreased NCX1 mRNA expression. These results suggest that DHA seems to have little effect on capacitative Ca²⁺ entry. Through decreasing NCX1 expression, DHA may suppress the 5-HT-induced increase in [Ca²⁺]_i.

A large number of studies have shown that n-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid, protect against several types of cardiovascular diseases, such as myocardial infarction, arrhythmia, atherosclerosis, and hypertension.^1,2) Although the precise mechanisms are unclear, the protective effects of DHA and eicosapentaenoic acid are attributable to their direct effects on the function of vascular endothelial and smooth muscle cells (VSMCs). Furthermore, it is believed that DHA has more potent and beneficial effects on cardiovascular diseases than eicosapentaenoic acid. In fact, DHA activates large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), and this effect is suggested to reduce blood pressure.^3,4)

Our laboratory previously reported that treatment of cultured VSMCs with DHA significantly suppresses the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by 5-hydroxytryptamine (5-HT) and angiotensin II.^5,6) 5-HT and angiotensin II stimulate voltage-dependent L-type Ca²⁺ channels in VSMCs.^7,8) Furthermore, DHA treatment of VSMCs inhibits KCl (80 mM)-induced intracellular Ca²⁺ mobilization and Mn²⁺ influx.⁶⁾ Thus, it is likely that DHA suppresses receptor-mediated Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, the effect of DHA on voltage-independent Ca²⁺ entry is still largely unknown.

Transient receptor potential canonical (TRPC) proteins have been shown to be Ca²⁺ channels activated by store depletion and/or receptor stimulation.⁹⁾ TRPC1 and TRPC6 have been demonstrated to be expressed abundantly in primary culture of VSMCs isolated from rats.¹⁰⁾ Although the function and mechanism of Ca²⁺ entry via TRPC proteins are not fully defined, TRPC1 is recognized to have an important role in store-operated Ca²⁺-entry (SOCE) rather than receptor-operated Ca²⁺ entry (ROCE). Conversely, TRPC6 is predicted to function in ROCE.

In the present study, we investigated the effect of DHA on SOCE and ROCE, including TRPC expression, to understand further the detailed mechanisms of DHA action on intracellular Ca²⁺ mobilization in VSMCs.

MATERIALS AND METHODS

Materials

Fetal calf serum (FCS), penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Life Technologies (Grand Island, NY, U.S.A.). Fura-2 acetoxymethyl ester (fura-2/AM) and ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) were from Dojindo (Kumamoto, Japan). Sarpogrelate was a kind gift from Mitsubishi Tanabe Pharma (Osaka, Japan). DHA was from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other reagents were purchased from standard suppliers.

Cell Culture

VSMCs were enzymatically isolated from the aortic media of 6–7-week-old Wistar rats using collagenase and elastase and cultured in DMEM containing 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin, as described previously.¹¹⁾ To measure [Ca²⁺]_i, the cells were seeded and grown on coverglasses (8×16 mm; Matsunami Glass, Osaka, Japan). The cells were then cultured in DMEM containing 2% FCS in the presence or absence of 30 µM DHA. Primary VSMCs were used throughout the experiments. VSMCs for the experimental groups (shown as patterned columns in all figures) and control groups (shown as open columns in all figures) were obtained from the same animals so all experiments could be performed in a paired fashion. This study was conducted in accordance with the Care and Use of Laboratory Animals of the Animal Research Committee of Health Sciences University of Hokkaido.

RNA Extraction and RT-PCR

TRPC channels and Na⁺/Ca²⁺ exchanger (NCX), which are expressed in cultured VSMCs, were identified by qualitative RT-PCR. Total RNA in vascular media and cultured VSMCs was extracted using TRI Reagent^® (Sigma-Aldrich) according to the manufacturer’s instructions. One-step RT-PCR were carried out using a QIAGEN OneStep RT-PCR kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The thermocycling program consisted of 30 min at 50°C, 15 min at 95°C, and 43 cycles of 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C, and a final extension for 10 min at 72°C in a thermal cycler (TaKaRa PCR thermal cycler Dice; TaKaRa Bio, Shiga, Japan). The RT-PCR products were separated and visualized on an ethidium bromide-stained 1.0% agarose gel.

mRNA expression in VSMCs was also quantified by real-time RT-PCR using a 7500 Fast Real-Time PCR system (Life Technologies, Applied Biosystems, Foster City, CA, U.S.A.) and SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan).

The primers for TRPCs and NCXs are shown in Table 1. The primer for β-actin was described previously.¹²⁾ The expression of TRPC and NCX products was calculated relative to β-actin.

Table 1. Oligonucleotide Sequences of Primers Used for RT-PCR

	Predicted size (bp)	Sense/Antisence
For RT-PCR
TRPC1	402	5′-CTGCCACAGATGTTACAAGATTTTGGG-3′/5′-GGCGAACTTCCACTCTTTATCCTCATG-3′
TRPC2	487	5′-CAGTTTCACCCGATTGGCGTAT-3′/5′-CTTTGGGGATGGCAGGATGTTA-3′
TRPC3	529	5′-CCTGAGCGAAGTCACACTCCCAC-3′/5′-CCACTCTACATCACTGTCATCC-3′
TRPC4	492	5′-GCCTACACCTTTCAATGTCATCCC-3′/5′-CTTAGGTTATGTCTCTCGGAGGC-3′
TRPC5	220	5′-CTATCAGACCAGAGCTATTGATG-3′/5′-CTACCAGGGAGATGACGTTGTATG-3′
TRPC6	315	5′-GTGCCAAGTCCAAAGTCCCTGC-3′/5′-CTGGGCCTGCAGTACGTATC-3′
TRPC7	400	5′-ACCTTCACAGACTACCCCAAAC-3′/5′-AGAAGCTGAGGACAACCGCAAT-3′
NCX1	420	5′-TAAAACCATTGAAGGCACAGC-3′/5′-ACTTCCAGCTTGGTGTGTTC-3′
NCX2	351	5′-TCCTTCCAGGACCGCCTGC-3′/5′-GGCCTCCTCCTCCTCTGC-3′
NCX3	353	5′-ACTTTTGAATGTGATACCATTCAT-3′/5′-TTGGCCTCCTCTTCCTCCAT-3′
For real-time RT-PCR
TRPC1	96	5′-TTCTGTGAACAGCAAAGCAA-3′/5′-CATGCGCTAAGGAGAAGATG-3′
TRPC4	118	5′-TGACGGAGGAGAATGTTAAGG-3′/5′-CGCGTTGGCTGACTGTATT-3′
TRPC6	126	5′-ACTGGTGTGCTCCTTGCAG-3′/5′-TCAGCTGCATTCATGACGA-3′
NCX1	102	5′-AGCAAGGCGGCTTCTCTTTT-3′/5′-GCTGGTCTGTCTCCTTCATGT-3′
β-Actin	151	5′-CTGGCCGGGACCTGACAGA-3′/5′-GCGGCAGTGGCCATCTCAT-3′

Measurement of [Ca²⁺]_i

[Ca²⁺]_i was measured as described previously.^6,13) Cells seeded and grown on coverglasses were loaded with 5 µM fura-2/AM for 45 min at 37°C in Hank’s balanced salt solution containing 0.1% bovine serum albumin and 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (HBSS; pH 7.4). Fluorescence at 505 nm emission wavelength alternately excited at 340 and 380 nm was measured using fluorescence spectrophotometers (Hitachi F-4000 and F-2500, Japan). The cells were perfused continuously at 1 mL/min with HBSS, to which test drugs were added. R_max, the maximal fluorescence ratio, was measured by exposing the cells to 10 µM ionomycin in the presence of 5 mM Ca²⁺, followed by perfusion with Ca²⁺-free HBSS containing 1 mM EGTA to obtain R_min, the minimum ratio. The cells were finally exposed to 0.05% Triton X-100 to obtain autofluorescence. After the subtraction of autofluorescence, [Ca²⁺]_i was calibrated according to the equation of Grynkiewicz et al.,¹⁴⁾ assuming the K_d of the Ca²⁺-fura-2 interaction to be 225 nM in the cytosolic environment.

Statistical Analysis

Statistical analysis of the results was performed using Student’s unpaired t-test for unpaired data and ANOVA followed by Dunnett’s test for multiple comparisons. p-Values less than 0.05 were considered significant.

RESULTS

Effect of DHA on TRPC Expression

The expression profiles of TRPC mRNA in cultured VSMCs were examined by RT-PCR. PCR amplification was performed for 43 cycles in order to reach saturation. mRNAs of TRPC1, TRPC4, and TRPC6 were clearly detected in cultured VSMCs, whereas mRNAs of TRPC2, TRPC3, TPRC5 and TPRC7 were barely detected (Fig. 1A).

Fig. 1. TRPC mRNA Expression in Cultured VSMCs Isolated from Aorta

A: Representative fluorescence images of TRPC expression in cultured VSMCs. TRPC mRNA expression was determined by RT-PCR. B, C, and D: Effect of DHA on TRPC1 (B), TRPC4 (C), and TRPC6 (D) mRNA expression. Cells were treated without (Control) or with DHA (30 µM) for 2 d. TRPC mRNA expression was determined by real-time RT-PCR. Each column represents the mean±S.E.M. (n=3 for C, n=4 for D and E). * p<0.05 versus control.

The effect of DHA on the mRNA expression levels of TRPC1, TRPC4, and TRPC6 in cultured VSMCs was then investigated by real-time RT-PCR. As shown in Fig. 1B, TRPC1 mRNA expression was slightly but significantly decreased by DHA (30 µM) treatment for 2 d. DHA had no effect on TRPC4 and TRPC6 mRNA expression (Figs. 1C, D).

Role of 5-HT in SOCE and ROCE

As shown in Figs. 2A and B, 5-HT at 10 µM induced a rapid and transient increase in [Ca²⁺]_i in cultured VSMCs. Peak [Ca²⁺]_i and [Ca²⁺]_i at 5 min after stimulation by 5-HT were 230.29±47.20  and 117.71±14.5 nM, respectively, (mean±standard error of the mean (S.E.M.)) These increases were completely blocked by sarpogrelate (1 µM), a selective serotonin 5-HT_2A receptor antagonist (Figs. 2A, B).

Fig. 2. Effect of Sarpogrelate, a Selective Serotonin 5-HT_2A Receptor Antagonist, on the 5-HT-Induced Increase in [Ca²⁺]_i

A and B: Cells were incubated without or with sarpogrelate (1 µM) for 3 min before and during stimulation with 5-HT (10 µM). A: Representative results. B: Summary of the effect of sarpogrelate on the 5-HT-induced increase of [Ca²⁺]_i. Each column represents the mean±S.E.M. of (n=3 for control and 4 for +sarpogrelate). Basal: basal [Ca²⁺]_i before stimulation; Peak: peak [Ca²⁺]_i after stimulation; At 5 min: [Ca²⁺]_i at 5 min after stimulation. C and D: Cells were treated with Ca²⁺-free buffer (+1 mM EGTA), buffer in the presence of 5-HT (10 µM), buffer with or without sarpogrelate (1 µM), or buffer containing 1.3 mM Ca²⁺ in the presence of 5-HT with or without sarpogrelate. C: Representative results. D: Summary of the effect of the Ca²⁺-reload-induced increase of [Ca²⁺]_i in 5-HT-stimulated VSMCs. Basal: basal [Ca²⁺]_i before stimulation with extracellular Ca²⁺; Peak: peak [Ca²⁺]_i after stimulation with extracellular Ca²⁺. Each column represents the mean±S.E.M. (n=7). *** p<0.001, ** p<0.01 versus control.

When extracellular Ca²⁺ (1.3 mM) was removed, the basal level of [Ca²⁺]_i decreased, and a small transient increase by 5-HT stimulation was observed (Fig. 2C). Figure 2D shows the mean±S.E.M. values of peak [Ca²⁺]_i after the addition of extracellular Ca²⁺ to the Ca²⁺-free condition in the presence of 5-HT. This increase in [Ca²⁺]_i was partially but significantly inhibited by sarpogrelate.

Effect of DHA on 5-HT-Induced SOCE and ROCE

The effect of DHA on 5-HT-induced SOCE and ROCE was then investigated. As shown in Figs. 3A and C, DHA treatment for 2 d had no effect on the 5-HT-induced increase in [Ca²⁺]_i under the Ca²⁺-free condition, as described previously.⁶⁾ DHA treatment also had no effect on the [Ca²⁺]_i change induced by the addition of extracellular Ca²⁺ to the Ca²⁺-free condition in the presence of 5-HT without (Figs. 3A, B) or with (Figs. 3C, D) sarpogrelate.

Fig. 3. Effect of DHA on 5-HT-Induced SOCE and ROCE

Cells were treated without or with DHA (30 µM) for 2 d. A and B: Cells were treated with Ca²⁺-free buffer (+1 mM EGTA), buffer in the presence of 5-HT (10 µM), or buffer containing 1.3 mM Ca²⁺ in the presence of 5-HT. C and D: Cells were treated with Ca²⁺-free buffer (+1 mM EGTA), buffer in the presence of 5-HT (10 µM), buffer containing 5-HT with sarpogrelate (1 µM), or buffer containing 1.3 mM Ca²⁺ in the presence of 5-HT with sarpogrelate. A and C: Representative results. B and D: Summary of the effect of the Ca²⁺-reload-induced increase of [Ca²⁺]_i in 5-HT-stimulated VSMCs in the absence (B) or presence (D) of sarpogrelate. Each column represents the mean±S.E.M. (n=13 for control of B and 14 for DHA-treated of B and n=4 for D). Basal: basal [Ca²⁺]_i before stimulation with extracellular Ca²⁺; Peak: peak [Ca²⁺]_i after stimulation with extracellular Ca²⁺.

Role of NCX on the 5-HT-Induced [Ca²⁺]_i Change and the Effect of DHA on NCX mRNA Expression

As shown in Figs. 4A and B, KB-R7943 (10 µM), a specific inhibitor of reverse mode NCX, significantly inhibited both peak [Ca²⁺]_i and [Ca²⁺]_i at 5 min after stimulation with 5-HT. The reduction rate at peak [Ca²⁺]_i by KB-R7943 was almost the same as that after treatment with DHA for 2 d, and KB-R7943 had no effect on DHA-treated cells (Fig. 4C).

Fig. 4. Effect of KB-R7943, a Specific Inhibitor of Reverse Mode NCX, on the 5-HT-Induced Increase in [Ca²⁺]_i

A and B: Cells were incubated without or with KB-R7943 (10 µM) for 3 min before and during stimulation with 5-HT (10 µM). A: Representative results. B: Summary of the effect of KB-R7943 on the 5-HT-induced increase of [Ca²⁺]_i. Basal: basal [Ca²⁺]_i before stimulation; Peak: peak [Ca²⁺]_i after stimulation; At 5 min: [Ca²⁺]_i at 5 min after stimulation. Each column represents the mean±S.E.M. (n=8 for control of B; n=7 for +KB-R7943). C: Effect of KB-R7943 on the 5-HT-induced increase of peak [Ca²⁺]_i in DHA-treated cells. Cells were treated without or with DHA (30 µM) for 2 d. Cells were treated without or with KB-R7943 (10 µM) for 3 min before and during stimulation with 5-HT (10 µM). Each column represents the mean±S.E.M. (n=4) of the ratio expressed by taking the peak [Ca²⁺]_i value after 5-HT stimulation in untreated control cells as 1. ** p<0.01, * p<0.05 versus control.

The effect of DHA on NCX mRNA expression was then investigated. In rat cultured VSMCs, NCX1 mRNA, but not NCX2 and NCX3, was expressed abundantly (Fig. 5A). As shown in Fig. 5B, NCX1 mRNA expression was slightly but significantly decreased by DHA (30 µM) treatment for 2 d.

Fig. 5. NCX mRNA Expression in Cultured VSMCs Isolated from Aorta

A: Representative fluorescence images of NCX expression in cultured VSMCs. NCX mRNA expression was determined by RT-PCR. B: Effect of DHA on NCX1 mRNA expression. Cells were treated without (control) or with DHA (30 µM) for 2 d. NCX mRNA expression was determined by real-time RT-PCR. Each column represents the mean±S.E.M. (n=4). * p<0.05 versus control.

DISCUSSION

In the present study, we investigated the effect of DHA on SOCE and ROCE in cultured rat VSMCs. We first showed that cultured VSMCs express TRPC1, 4, and 6 mRNA, which is consistent with a previous report by Poburko et al.¹⁵⁾ In intact vascular media before enzymatic digestion, abundant TRPC3 mRNA expression in addition to TRPC1 and TRPC6 mRNA expression was detected, whereas TRPC4 mRNA expression was barely detected (data not shown). Therefore, the process of cell culture may affect the expression pattern of TRPCs in vascular media cells. DHA treatment for 2 d significantly inhibited the expression of TRPC1 mRNA, but not TRPC4 and TRPC6 mRNA. This condition of DHA treatment was the same as in our previous reports showing that DHA significantly inhibits the 5-HT- and angiotensin II-induced increase in [Ca²⁺]_i in the presence of extracellular Ca²⁺.^5,6,8)

Since TRPC1 has an important role in not only SOCE but also ROCE in VSMCs,¹⁰⁾ we then investigated the effect of 5-HT on SOCE and ROCE. The 5-HT-induced rapid and transient elevation of [Ca²⁺]_i was completely inhibited by sarpogrelate, suggesting that serotonin 5-HT_2A receptor activation mediates Ca²⁺ mobilization. Sarpogrelate also partially but significantly inhibited Ca²⁺ entry when extracellular Ca²⁺ was added to the Ca²⁺-free condition in the presence of 5-HT, suggesting that this capacitative Ca²⁺ entry is also partially dependent on the stimulation of serotonin 5-HT_2A receptors. Therefore, our result indicates that Ca²⁺ entry when extracellular Ca²⁺ is added to the Ca²⁺-free condition in the presence of 5-HT consists of both SOCE (i.e., which was not blocked by sarpogrelate) and ROCE (i.e., which was blocked by sarpogrelate). In contrast to 5-HT-induced capacitative Ca²⁺ entry, olmesartan (100 nM), a selective angiotensin II AT₁ receptor antagonist, has no effect on Ca²⁺ entry when extracellular Ca²⁺ is added to the Ca²⁺-free condition in the presence of 100 nM angiotensin II, although olmesartan completely inhibits the angiotensin II-induced increase in [Ca²⁺]_i in the presence of extracellular Ca²⁺ (unpublished data).

Although DHA treatment for 2 d significantly inhibited TRPC1 mRNA expression, it had no impact on 5-HT-induced SOCE and ROCE. We also confirmed that DHA had no impact on angiotensin II-induced SOCE (unpublished data). In astrocytes, it has been reported that acute DHA treatment inhibits SOCE.¹⁶⁾ In VSMCs, the addition of DHA at the time of agonist stimulation has no effect on intracellular Ca²⁺ mobilization, even in the presence of extracellular Ca²⁺.⁵⁾ Therefore, the inhibitory effect of DHA on SOCE may depend on cell type, being different between vascular and other cell types. Ye et al.¹⁷⁾ showed that DHA treatment for 3 d suppresses the TRPC1-mediated Ca²⁺ signaling pathway by partially displacing TRPC1 from membrane caveolar lipid rafts in vascular endothelial cells. Therefore, it is possible that DHA may affect not only TRPC1 mRNA expression but also TRPC1 distribution in the membrane of VSMCs. However, even if DHA modulates TRPC1 function in VSMCs, it seems to have little effect on SOCE and ROCE, as indicated in our present study. Yet, there is increasing evidence of a role for TRPC1 in VSMCs. Kwan et al.¹⁸⁾ reported that TRPC1 and BK_Ca co-localize in VSMCs, and Ca²⁺ influx through TRPC1 activates BK_Ca to induce membrane hyperpolarization. Ávila-Medina et al.¹⁹⁾ reported that TRPC1 and Orai1 co-localize with voltage-dependent Ca_V1.2 L-type Ca²⁺ channels, and 5-HT-induced TRPC1-dependent SOCE can trigger the activation of voltage-dependent Ca_V1.2 L-type Ca²⁺ channels. Therefore, it might still be worthwhile continuing to determine the effect of DHA on TRPC1 function in VSMCs.

The accumulation of intracellular Na⁺ as a result of Na⁺ entry forces NCX to the reverse mode, leading to Ca²⁺ entry from the extracellular space in VSMCs.^20,21) In the present study, we also showed that KB-R7943 significantly suppressed the 5-HT-induced increase in [Ca²⁺]_i, suggesting the involvement of reverse mode NCX in Ca²⁺ influx by 5-HT stimulation. Furthermore, KB-R7943 had no effect in DHA-treated cells. Therefore, it is possible that DHA suppresses the reverse mode function of NCX. There are three NCX isoforms (NCX1, NCX2, and NCX3); NCX1 is widely expressed in the heart, kidney, brain, arteries, and other organs, whereas NCX2 and NCX3 expression is limited mainly to the brain and skeletal muscle.²²⁾ In fact, we confirmed the abundance of NCX1, but not NCX2 and NCX3, expression in rat VSMCs (Fig. 5A). Although the precise mechanism by which the reverse mode of NCX is activated by 5-HT is still unclear, it was reported recently that the reverse mode of NCX1 contributes to angiotensin II-induced Ca²⁺ influx in VSMCs.²³⁾ The authors made the point that angiotensin II-induced SOCE may increase the concentration of intracellular Na⁺ through mechanisms involving the TRPC family, and this intracellular Na⁺ may lead to the activation of the reverse mode of NCX. Therefore, the suppression of NCX1 expression by DHA treatment may contribute to the inhibition of Ca²⁺ influx in VSMCs. Thus, it is worthwhile continuing to investigate the effect of DHA on the reduction of [Ca²⁺]_i via NCX in our future studies.

In conclusion, DHA may suppress the 5-HT-induced increase in [Ca²⁺]_i through decreasing NCX1 expression, in addition to suppressing receptor-mediated Ca²⁺ influx through voltage-dependent Ca²⁺ channels. Inhibition of SOCE and ROCE could be excluded as the inhibitory mechanism of receptor-mediated Ca²⁺ influx by DHA treatment in VSMCs.

Acknowledgments

This study was supported in part by a Grant-in-Aid for the 2009–2010 Research Project of the Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido. The authors would like to thank Dr. Akihiro Nezu and Dr. Akihiko Tanimura for their valuable help.

Conflict of Interest

The authors declare no conflict of interest.

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