Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and 0.25% trypsin-EGTA (ethylene glycol tetraacetic acid) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fura-2 acetoxymethyl ester (fura-2/AM) was purchased from Dojindo Laboratories (Kumamoto, Japan). Acetylcholine chloride (ACh) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fucoidan (MW=c.a. 20 kDa), adenosine-5′-triphosphate (ATP), compound 48/80, and histamine were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Heparin was obtained from Ajinomoto Co., Inc. (Tokyo, Japan).

HeLa cells and astrocytes (a gift from Dr R Susuki, Photon Medical Research Center, Hamamatsu University School of Medicine) were cultured at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide (CO₂) in DMEM supplemented with 10% FBS and 100 units/ml penicillin-streptomycin. These cells were maintained in 60-mm dishes (Corning Incorporated, Corning, NY, USA). For studies of Ca²⁺ response, the cells were subcultured in 35-mm glass-bottom dishes (Iwaki Glass, Chiba, Japan) at approximately 0.5×10⁵ cells per dish.

Human umbilical vein endothelial cells (HUVECs), purchased from Health Science Research Resources Bank (HSRRB; Osaka, Japan), were maintained in E-STIM Endothelial Cell Culture Medium (BD Biosciences, Franklin Lakes, NJ, USA) with 10% FBS, 10 ng/ml EGF, and 200 µg/ml endothelial cell growth supplements (ECGS) on 75-mm flasks. They were cultured in a humidified incubator containing 5% CO₂ and 95% air at 37°C. The HUVECs were removed from the substrate with 0.25% trypsin and 0.02% EDTA (BD Biosciences) and passaged on coverslips in 35-mm-diameter culture dishes at a density of 1×10⁴ cells/ml. The subcultures were used for [Ca²⁺]_i measurements on day 2 after passage.

The [Ca²⁺]_i was measured using the fluorescent Ca²⁺ indicator dye fura-2. For staining, HeLa cells, HUVECs, and astrocytes were incubated in a medium containing fura-2/AM (2.5 µM) for 20 min at 37°C. The cells were then rinsed twice with the medium prepared for calcium imaging. The medium, here referred to as recording medium, contained (in mM): NaCl, 140; KCl, 5; CaCl₂, 2; MgCl₂, 1.2; glucose, 2; and HEPES (pH 7.4), 10. A calcium-free recording medium was also used in which CaCl₂ was removed, MgCl₂ was increased to 3.2 mM, and 1 mM EGTA was added (pH 7.4).

Fluorescence imaging for [Ca²⁺]_i was performed 24 h after subculture for HeLa cells and astrocytes, and 48 h after subculture for HUVECs. The cells in each culture dish loaded with fura-2 AM were placed on the stage of an inverted microscope (IX 70; Olympus, Tokyo, Japan). They were superfused continuously using silicone tubing warmed to 37°C and connected to a reservoir syringe held at a height of 70 cm. The cells were illuminated at wavelengths of 340 and 380 nm, alternating every 3 sec. Time-lapse images of the fluorescence emitted were obtained at 510 nm with a 40× objective lens (UApo 40x/340, NA 0.9; Olympus). An intensified charge-coupled device (CCD) camera (C4742-95; Hamamatsu Photonics K.K., Hamamatsu, Japan) was used to capture the images. Approximately 10–20 cells were observed in a microscopic field, and the changes in [Ca²⁺]_i in individual cells were determined using the fluorescence ratio of 340/380 nm and an image-analysis software (Aquacosmos, Hamamatsu Photonics). The changes in [Ca²⁺]_i, here referred to as Ca²⁺ responses, were quantified by calculating the areas under the response curve recorded in each cell image, which are expressed as the integrated amplitude of Ca²⁺ responses.

In order to examine the effects of fucoidan on expressions of specific G-protein coupled receptors, RT-PCR was used to identify the mRNA expressions of H1R and P2YRs. HeLa cells were serum-starved for 24 h and treated with 0.5 and 1.0 mg/ml fucoidan respectively, for 3 h. The cells were harvested and extracted with RNA. Total RNA pellet was resolved in 10 µl of diethylpyrocarbonate-treated water, and 1 µg of each RNA sample was used for the RT reaction. RNA samples were reverse-transcribed to cDNA by RT EasyTM II kit (First-strand cDNA for Real-Time PCR) (Foregene Co., Ltd., Chengdu, China). RT-qPCR was conducted by Real Time PCR EasyTM-SYBR-Green I kit (Foregene Co., Ltd.). TaqMan primers and the probe for H1R were listed in Table I. Amplicon size and reaction specificity were confirmed by agarose gel electrophoresis. Identities of the PCR products were verified by sequencing using a genetic analysis system. Semi-quantitive RT-PCR was used to test the P2YRs expressions. cDNA was amplified by PCR using 2X Taq PCR StarMix (GenStar Biosolutions Co., Ltd., Beijing, China) according to the manufacturer's instructions. The conditions consisted of an initial denaturation step at 95°C for 2 min, followed by 40 cycles of 30 sec denaturation step at 95°C, 30 sec annealing step at 53.5°C to 56.5°C, and 1 min extension step at 72°C. A final extension step of 5 min at 72°C was also performed. PCR products were separated by electrophoresis on a 1% agarose gel. All PCR results were derived with cycle number producing a signal in the linear portion of the amplification curve. The primers were designed as presented in Table II. The GAPDH gene was used as the internal standard, and data were expressed as the ratio of H1R mRNA to GAPDH mRNA or P2YRs mRNA to GADPH mRNA, respectively.

For the quantification of Ca²⁺ responses, 10–20 cells were analyzed in each observation. All observations were repeated using 3–5 culture dishes. The integrated amplitude of the Ca²⁺ response was calculated using an extended baseline for subtraction. Statistical significance was determined by one-way ANOVA followed by Student's t-test, and differences were considered significant at P<0.05.

Histamine (2.5 µM) was applied three times to induce Ca²⁺ responses. The first challenge induced a rapid rise in [Ca²⁺]_i followed by a sustained plateau in HeLa cells in the presence of extracellular Ca²⁺ (Fig. 1A). When the cells were superfused with the recording medium containing fucoidan (0.5 mg/ml), the second histamine challenge (in the same medium) was followed, and only a small response was induced, i.e., a small peak with a small or no plateau preceded by a long delay time (Fig. 1A). The third challenge, which took place after the removal of fucoidan, induced a quite large response (Fig. 1A), suggesting that the suppressive effect of fucoidan was reversible. The recovery of the responses was observed within 2 min after removal of fucoidan.

A dose-response curve for the suppression of histamine-induced responses was obtained by using different concentrations of fucoidan (Fig. 1B). In the presence of extracellular Ca²⁺ (filled circles), the Ca²⁺ responses were gradually suppressed by fucoidan in a dose-dependent manner. The response was completely suppressed at a concentration of 1.5 mg/ml. Similarly, in the absence of extracellular Ca²⁺ (EGTA 1 mM present) (filled squares), fucoidan suppressed Ca²⁺ responses gradually as its concentration increased. In both the presence and absence of extracellular Ca²⁺, 1.5 mg/ml of fucoidan was sufficient to completely abolish histamine-induced Ca²⁺ responses.

In the presence of extracellular Ca²⁺, challenges with histamine induced Ca²⁺ responses of a similar amplitude each time. However, in the absence of extracellular Ca²⁺, stimulation with an agonist, such as histamine, depleted intracellular Ca²⁺ pool, and thus the second or the third challenge in the same cell always induced a very small or no response at all. Therefore, we compared these responses statistically in the absence or presence of extracellular Ca²⁺ by integrating the amplitudes of the first responses induced by the first 3-min challenge only. For the control, we used 3-min applications of agonists after recording the baseline for 3 min. For the test, fucoidan was added to the recording medium during the same baseline period (3 min). Then, without removal of fucoidan, the subsequent doses of agonists were further added to the medium (Fig. 2A and C). In both presence and absence of extracellular Ca²⁺, fucoidan at a concentration of 0.5 mg/ml suppressed the Ca²⁺ responses induced by histamine very effectively (Fig. 2B and D) as compared to the control.

Application of 5 µM ATP to HeLa cells rapidly induced oscillating Ca²⁺ responses, as visualized by fura-2 imaging in the presence and absence of extracellular Ca²⁺. The amplitudes of such responses were as large as those induced by histamine. We measured Ca²⁺ responses by varying the concentration of fucoidan in the presence and absence of extracellular Ca²⁺. In the absence of calcium ions (EGTA, 1 mM) (filled squares, Fig. 1C), Ca²⁺ responses were gradually suppressed as fucoidan concentration increased from 0.005 to 5.0 mg/ml. Responses were almost completely suppressed at a fucoidan concentration of 5.0 mg/ml. The suppression of ATP-induced Ca²⁺ responses by fucoidan appeared to be dose-dependent. Although the dose-dependent suppression curve observed in the presence of external Ca²⁺ (filled circles) was roughly parallel to that observed in the absence of extracellular Ca²⁺ (plus EGTA), the curve obtained with 2 mM extracellular Ca²⁺ did not reach zero. Even at a fucoidan concentration of 5.0 mg/ml, responses could not be suppressed completely in the presence of extracellular Ca²⁺.

Compound 48/80 is an oligomeric mixture of the condensation products of p-methoxyphenethylamine and formaldehyde. It activates G proteins by a mechanism analogous to that of mastoparan, which interacts directly with G proteins to mimic the role of the intracellular loop of G-protein-coupled receptors (24,25). We measured the Ca²⁺ responses induced by compound 48/80 in the absence of extracellular Ca²⁺. Compound 48/80 induced rapid Ca²⁺ responses in HeLa cells (Fig. 3A), and it was suppressed significantly by fucoidan at a concentration of 0.5 mg/ml (Fig. 3A). The responses induced by compound 48/80 were very sensitive to fucoidan.

Acetylcholine (ACh) has been shown to act through two major types of receptors, nicotinic and muscarinic receptors. Nicotinic receptors are ligand-gated ion channels (nAChR, also known as ionotropic cholinergic receptors), while muscarinic receptors belong to the G-protein coupled receptor superfamily of seven transmembrane domain proteins (mAChR, also known as metabotropic cholinergic receptors). Our fura-2 Ca²⁺ imaging showed that nAChR and mAChR agonists ACh (10 µM) and carbachol (CCh, 10 µM) increased the [Ca²⁺]_i in HeLa cells. Both of these Ca²⁺ transients were completely blocked by addition of atropine (10 µM) (Fig. 3B), a muscarinic antagonist. When fucoidan was applied at 0.5 mg/ml, the ACh- or CCh-induced Ca²⁺ responses were suppressed to an extent similar to that of inhibition by atropine (10 µM) (Fig. 3B). This suppressive effect was reversed immediately after fucoidan was removed from the medium.

In order to examine the cell specificity of the effects of fucoidan on Ca²⁺ responses, we verified the sensitivity of Ca²⁺ responses induced by an agonist common to different cell types, specifically HUVECs and astrocytes. Histamine induced Ca²⁺ response in HUVECs and astrocytes similar to those induced in HeLa cells. In HUVECs, the Ca²⁺ responses induced by histamine (5 µM) were suppressed to approximately 30% the magnitude of the first uninhibited application by the addition of fucoidan at 0.5 mg/ml and to approximately 10% by 1.5 mg/ml (Fig. 3C). In astrocytes, Ca²⁺ responses induced by histamine (10 µM) were reduced to approximately 36 and 6% the magnitude of the first uninhibited application when the cells were given fucoidan at 0.5 and 1.5 mg/ml, respectively (Fig. 3D), suggesting that histamine receptors expressed both in HUVECs and astrocytes were sensitive to fucoidan in a manner similar to that of HeLa cells.

Heparin is also a sulfated polysaccharide with a structure similar to that of fucoidan. It has been shown that heparin via micropipette-injection into cells inhibited Ca²⁺ responses by inhibiting the inositol 1,4,5-trisphosphate (InsP₃) receptor (26,27). In our study, heparin was applied extracellularly. Fig. 4 shows a representative single cell trace of [Ca²⁺]_i during a heparin test. In a series of trials, HeLa cells were stimulated first with histamine (2.5 µM) and then superfused with a recording medium containing 40 unit/ml of heparin. Without removal of heparin, the same concentration of histamine (2.5 µM) was re-administered to evaluate the effects of heparin on the Ca²⁺ response. By comparing the peak Ca²⁺ response induced by histamine in the presence of extracellular Ca²⁺ (Fig. 4A and B) with that in the absence (Fig. 4C and D), heparin was found to neither facilitate nor suppress the generation of Ca²⁺ responses. The removal of heparin did not increase the peak of Ca²⁺ responses induced by the next histamine stimulation. The Ca²⁺ responses induced by ATP (5 µM) were also insensitive to heparin (data not shown).

As described above, fucoidan showed inhibitory effects on Ca²⁺ responses linked to a wide range of receptors. We suspected that the inhibition of Ca²⁺ responses by fucoidan would be attributable to one of its physical properties related to interaction with calcium ions. Fucoidan is a sulfated polysaccharide and has many negatively charged sulfate groups. These may chelate positively charged Ca²⁺ in the extracellular medium. Assuming that this is the case, the shortage of external Ca²⁺ would lead to a reduction or complete loss of Ca²⁺ response. Our data, obtained using a Ca²⁺−sensitive electrode (Fig. 5), show that fucoidan has only a very small effect on the effective concentration of calcium ions in solution. This excluded the possibility that the suppression of Ca²⁺ response by fucoidan was due to its effect on Ca²⁺ chelating. The biological effects of fucoidan can then be solely attributed to its interactions with membrane proteins.

To identify the histamine receptors and P2YRs expressed in HeLa cells and the effect of fucoidan on expressions of H1R and P2YRs, RT-PCR of H1R, P2YR1, P2YR2, and P2YR11 were performed. H1R expression in HeLa cells treated by 0.5 or 1.0 mg/ml fucoidan for 3 h was significantly lower than that of untreated cells (Fig. 6A). Consistently, the expressions of P2YR1 and P2YR11 in HeLa cells induced by either 0.5 or 1.0 mg/ml for 3 h were decreased significantly compared with untreated cells. And the expression of P2YR2 was also inhibited by 1.0 mg/ml fucoidan treatment for 3 h compared with that of the untreated cells (Fig. 6B, C and D). These results suggested that fucoidan suppressed the histamine-induced Ca²⁺ response by interacting with the H1 type metabotropic histamine receptors, and inhibited ATP-evoked [Ca²⁺]_i by inhibiting the purinergic G-protein-coupled P2Y receptors.