Citrus bergamia Risso Elevates Intracellular Ca2+ in Human Vascular Endothelial Cells due to Release of Ca2+ from Primary Intracellular Stores

The purpose of the present study is to examine the effects of essential oil of Citrus bergamia Risso (bergamot, BEO) on intracellular Ca2+ in human umbilical vein endothelial cells. Fura-2 fluorescence was used to examine changes in intracellular Ca2+ concentration [Ca2+]i . In the presence of extracellular Ca2+, BEO increased [Ca2+]i , which was partially inhibited by a nonselective Ca2+ channel blocker La3+. In Ca2+-free extracellular solutions, BEO increased [Ca2+]i in a concentration-dependent manner, suggesting that BEO mobilizes intracellular Ca2+. BEO-induced [Ca2+]i increase was partially inhibited by a Ca2+-induced Ca2+ release inhibitor dantrolene, a phospholipase C inhibitor U73122, and an inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker, 2-aminoethoxydiphenyl borane (2-APB). BEO also increased [Ca2+]i in the presence of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca2+ uptake. In addition, store-operated Ca2+ entry (SOC) was potentiated by BEO. These results suggest that BEO mobilizes Ca2+ from primary intracellular stores via Ca2+-induced and IP3-mediated Ca2+ release and affect promotion of Ca2+ influx, likely via an SOC mechanism.


Introduction
Bergamot essential oil (BEO) is obtained from bergamot (Citrus bergamia Risso), a roughly pear-shaped citrus fruit. BEO is widely used in aromatherapy to alleviate symptoms of stress-induced anxiety, mild mood disorders, and cancer pain [1] and it has anxiolytic [2] and analgesic [1,3,4] effects in rodents.
Besides these effects of BEO, bergamottin, isolated from nonvolatile fraction of BEO, significantly decreased the typical electrocardiographic signs of coronary arterial spasm, the force of the contraction and the incidence of cardiac arrhythmias induced by vasopressin in the guinea pig [5]. Given the potential roles of bergamottine in cardiovascular function, it is of interest to know the roles of BEO in Ca 2+ mobilization in endothelial cells. Vasorelaxant effect of BEO may involve Ca 2+ mobilization from intracellular stores and/or from the extracellular pool in endothelial cells. Several studies demonstrating the relationship between endothelial cells and smooth muscle relaxation have been reported. Our previous study implicated that a change in cytosolic Ca 2+ levels during stimulation of endothelial cells was a basic mechanism by which endothelial cells modulate vasomotor activity [6]. Another report indirectly supports this view, demonstrating that the vasorelaxant effect of the essential oil of Ocimum gratissimum is partly dependent on the integrity of the vascular endothelium [7].

Human Vascular Endothelial Cell Culture.
A human endothelium-derived cell line EA.hy926 was purchased from the American Type Culture Collection (Manassas, VA, USA) [8]. EA cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 20% fetal bovine serum (FBS) and 10% hypoxanthine-aminopterin-thymidine 50 supplement (Life Technologies). Cell cultures were maintained at 37 ∘ C in a fully humidified 95% air 5% CO 2 atmosphere. The media were removed and replaced with fresh medium three times in a week. The cells were detached by exposure to trypsin, reseeded on gelatin-coated coverslips, and maintained in culture for 2 to 4 days before use. Measurements were performed on subconfluent cells.

Cell Viability Assays.
The thiazolyl blue tetrazolium bromide (MTT) assay was used to determine the effects of BEO on cell viability. EA cells were cultured using DMEM with 100% (v/v) FBS (Gibco Invitrogen), 100 units/mL penicillin, 100 g/mL streptomycin (Gibco Invitrogen), and MEM nonessential amino acids (Invitrogen) in 96-well plates (Nunclon, Denmark) for 48 h until 80-90% confluence has been reached. To evaluate the effect of BEO, cells were for 15 min with varying concentrations (0.001, 0.005, 0.01, 0.05, or 0.1% [v/v in DMSO]) of the BEO and 0.25% DMSO. Then cells were washed with fresh PBS and replaced with serumfree media. In addition, cells were loaded with 10 L MTT (5 mg/mL) and incubated at 37 ∘ C for 3 h. MTT solution was removed and replaced with 100 L DMSO in a dark place for 2 h. The change in color was read at 540 nm using a plate reader.

Ca 2+
Measurements. Cells were loaded with fura-2 AM, and [Ca 2+ ] i was measured using a microfluorometer system consisting of an inverted microscope (IX71, Olympus, Japan) and a PTI Filter Scan power illuminator system (Photon Technology International). Fura-2 AM (2 M) was added to the bath and the cells were incubated for 25 min at 37 ∘ C. The cells were illuminated alternatively at wavelengths of 340 and 380 nm through a chopper wheel (frequency = 50 Hz). Fluorescence was measured at 510 nm, and autofluorescence was subtracted from the signals obtained. The Ca 2+ -free concentration was calculated from the ratio of the fluorescence signals emitted at each excitation wavelength. The calibration procedure was identical to that described previously [9].

Statistical Analysis.
Pooled data are presented as means ± SEMs and significant differences were determined using paired t-test or ANOVA followed by Scheffe's post-hoc analysis. A value of < .05 was considered significant.  (Figure 1). Differences between groups were analyzed using the ANOVA followed by Scheffe's post-hoc analysis. There was no significant effect to the percentage of viable cells at all concentrations of BEO in EA cells (F (6.27) = 1.541, = .214).

Elevation of [ 2+ ] by BEO in Human Vascular
Endothelial Cells. BEO increased [Ca 2+ ] i in a concentrationdependent manner in EA cells (Figure 2(a)). The Evidence-Based Complementary and Alternative Medicine concentration-response relationship for mobilization of Ca 2+ from intracellular stores by BEO is summarized in Figure 2(b).  [10]. BEO-induced intracellular Ca 2+ increase was significantly and reversibly inhibited by the CICR inhibitor, dantrolene ( < .001, = 10, Figure 3(a)).
These data indicate that BEO elevates [Ca 2+ ] i in part by the release of Ca 2+ from intracellular stores via a CICR mechanism. To determine whether BEO releases Ca 2+ from intracellular Ca 2+ stores via IICR, we tested the effects of BEO in the presence of U73122, the specific inhibitor of phospholipase C (PLC) [11], to inhibit IP 3 synthesis, or 2-APB, a membrane-permeable inhibitor of IP 3 -gated ER Ca 2+ channels [12]. BEO-induced intracellular Ca 2+ increase was significantly inhibited by both U73122 ( < .001, = 10, Figure 3(b)) and 2-APB ( < .001, = 15, Figure 3(c)). These data indicate that PLC-mediated synthesis of IP 3 and IP 3 binding to IP 3 -gated Ca 2+ channels in the ER contribute to BEO-induced Ca 2+ release from intracellular stores.
A portion of Ca 2+ released from the ER is taken up by proximate mitochondria, which can also release Ca 2+ and thereby regulate [Ca 2+ ] i . To determine whether mitochondria participate in the reuptake of BEO-induced Ca 2+ release, we examined the effects of BEO on [Ca 2+ ] i in a Ca 2+free solution in the presence of BHQ (an SR/ER Ca 2+ -ATPase inhibitor) and CCCP (a mitochondrial Ca 2+ uptake inhibitor). In cells treated with BHQ and/or CCCP, [Ca 2+ ] i transiently increased and then decreased slowly to a steady state, suggesting that an SR/ER Ca 2+ -ATPase and mitochondrial Ca 2+ uptake participate in the regulation of [Ca 2+ ] i under basal conditions (Figure 4(a) EA cells by subsequent application of BEO in the presence of BHQ and CCCP was higher than that in the presence of BHQ only. An area under the curve in each condition was 72.97 ± 8.81 and 31.62 ± 2.75 arbitrary unit, respectively. The difference between two conditions was 41.35 ± 8.12 arbitrary unit, suggesting that mitochondrial Ca 2+ stores may contribute to the regulation of the BEO-induced increase in Considering the inhibitory effect of La 3+ , noted above, it is suggested that Ca 2+ -entry pathway(s) is (are) activated by BEO. Thus, we next examined whether BEO modulated Ca 2+ entry via an SOC mechanism. Exposure of EA cells to the BHQ in a Ca 2+ -free solution induced a transient increase in [Ca 2+ ] i , which then decreased slowly to a steady state (Figure 4(a)). Reapplication of extracellular Ca 2+ following emptying of intracellular Ca 2+ stores with BHQ caused an increase in [Ca 2+ ] i , indicating activation of an SOC mechanism. When BEO was applied after SOC was evoked, SOC was further enhanced, suggesting that BEO also activates Ca 2+ influx through an SOC pathway (Figure 4(b)). However, using this protocol alone in this experiment does not rule out that other calcium entry pathways may be activated by BEO.

Discussion
In the present study, we firstly demonstrate that BEO mobilized Ca 2+ from extracellular and intracellular sources in endothelial cells. Our present results also suggest that BEO increased intracellular Ca 2+ level through both mobilization of intracellular Ca 2+ stores, ER and mitochondria, and promotion of Ca 2+ influx, via an SOC mechanism. These findings will provide insight into the physiological mechanisms involved in Ca 2+ regulation in endothelial cells following exposure to BEO. Some essential oils contain photoactive molecules like furocoumarins. For instance, essential oil of Citrus bergamia contains psoralens which bind to DNA under ultraviolet A light exposure producing mono-and biadducts that are cytotoxic and highly mutagenic [13]. Therefore, the results observed in the present study may be results from the effects of photoirritation of BEO. Since, however, intracellular Ca 2+ level rapidly returned to a baseline level after washing out of BEO and cell viability was normal in doses tested, we think that phototoxic or cytotoxic effect is little on intracellular Ca 2+ level by BEO. In addition, in vitro studies have shown that BEO reduces glutamate receptor-mediated cell death induced by N-methyl-D-aspartate [14]. Nevertheless, further researches are necessary to evaluate phototoxic potential of BEO in endothelial cells.
BEO has been reported to decrease the blood pressure in healthy human and have dilating effect on mouse artery [15,16]. In the recent study, limonene, one of the major components of BEO (37.26%), increased cytosolic Ca 2+ concentration by the direct activation of adenosine A 2A receptors [17]. In endothelium, an adenosine A 2A receptor has an important role in NO release. Adenosine A 2A receptor induced NO-dependent vasodilation by intracellular Ca 2+ increase [18]. Thus, we suggest that the effect of BEO may be attributable to limonene by activation of adenosine A 2A receptors.
Contraction and relaxation of smooth muscle are regulated not only by changes in cytoplasmic calcium concentration but also by other important signaling mechanisms, that is, independent of the changes in [Ca 2+ ] i , known as Ca 2+ sensitization [19]. Although the increase in [Ca 2+ ] i initiates smooth muscle contraction via activating myosin light chain kinase, Ca 2+ sensitization mediates smooth muscle contraction by modulating myosin light chain phosphatase [20]. The increase in [Ca 2+ ] i in endothelial cell plays a role in synthesis and the release of vasoactive compounds such as nitric oxide or prostaglandins [21], thereby altering Ca 2+ sensitization in smooth muscle cells. It has been reported that linalyl acetate, one of the main components of BEO, induces relaxation of the smooth muscle via partially endothelium-dependent pathway [22]. Further research will be needed to reveal the mechanism by which the calcium releasing actions by BEO in endothelial cells regulate to the vasodilator action.
In conclusion, before BEO can be considered for use in treating vascular-related diseases, further studies are necessary to define the Ca 2+ -elevating pathways enlisted by BEO under pathological conditions.