Mean arterial pressure (MAP) and heart rate (HR) were recorded before (baseline) and after intravenous administration of CH₂Cl₂/MeOH extract from Dictyota pulchella (CME, 5, 10, 20 and 40 mg/kg body weight, randomly) in conscious normotensive rats. CME elicited a dose-dependent hypotension (−4 ± 1; −8 ± 2; −53 ± 8 and −63 ± 3 mmHg) and bradycardia (−8 ± 6; −17 ± 11; −257 ± 36 and −285 ± 27 b.p.m.) as illustrated in Figure 1A,B.

It is well-known that anesthesia modifies the levels of blood pressure and heart rate interfering with central regulatory mechanisms involved in BP regulation, such as the baroreflex, by producing depression of the central nervous system synapses, altering the autonomic responses [25]. In order to avoid the possible influence of anesthesia and surgical stress on cardiovascular parameters [26], our studies were carried out in conscious freely moving rats.

In the present study, the acute administration of CME induced marked hypotension and bradycardia in conscious rats (Figure 1B). It is important to note that reduction in blood pressure due to vasodilation is usually followed by reflex tachycardia. However, under our experimental conditions, in addition to hypotension, CME induced bradycardia, which could be explained, at least in part, by a possible direct effect of the CME on the heart. Although this is an interesting possibility, this hypothesis awaits further investigation.

Although we demonstrated that CME can produce hypotension in normotensive rats, the beneficial effects of this marine algae on experimental hypertension and its clinical relevance still awaits further investigation.

Based on the reports highlighting that the vascular smooth muscle tone plays an important role in maintaining blood pressure [27], we investigated the possible vasorelaxant effect elicited by Dictyota pulchella in isolated superior mesenteric arteries. An important layer in the regulation of vascular tone is the endothelium. For many years, this layer was considered to be an inert barrier that separated flowing blood from underlying tissue. Over time, it has been established that the endothelium plays an active role in regulating hemostasis, cellular and nutrient trafficking, and vasomotor tone [28]. Endothelial cells are able to produce both vasoconstrictive and vasodilating substances. The main endothelium-derived relaxing factors (EDRF) are nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Among the contracting factors are endothelin-1, thromboxane A₂ and reactive oxygen species [29]. To evaluate the possible role played by the endothelium in the hypotension elicited by Dictyota pulchella observed in vivo, mesenteric artery rings were pre-contracted with phenylephrine (1 μM), a α₁-adrenoceptors agonist. In the presence of this contracting agent, CME (0.01–500 μg/mL) induced a concentration-dependent relaxation (Maximum Response = 101.4 ± 4.5%; EC₅₀ = 22.35 ± 5.09 μg/mL, n = 6) and this effect was not modified by vascular endothelium removal (Maximum Response = 103.3 ± 8.3%; EC₅₀ = 21.43 ± 8.98 μg/mL, n = 6) (Figure 2A). Similar results were found in the presence of Hexane/EtOAc phase from Dictyota pulchella (HEP) (0.01–500 μg/mL), which induced a concentration-dependent vasodilatation in both intact (Maximum Response = 80.6 ± 5.8%; EC₅₀ = 24.1 ± 8.95 μg/mL, n = 6) or denuded-endothelium (Maximum Response = 95.6 ± 7.5%; EC₅₀ = 23.7 ± 5.65 μg/mL, n = 6) (Figure 2B).

Potassium channels (K⁺ channels) appear to play a crucial role in controlling the cellular membrane potential and in the vascular tone. Potassium channel openers exert their biological effects by increasing the probability of opening K⁺ channels and a number of substances that act on K⁺ channels have been shown to dilate arteries by causing hyperpolarization in vascular smooth muscle cells [30]. In addition several natural products have been shown to induce vasorelaxant effects through the activation of K⁺ channels [31–33]. Aiming to investigate involvement of potassium channels (K⁺) in the vasorelaxant activity elicited by CME, the preparations were pre-incubated with Tyrode’s modified solution containing KCl (20 mM) or with tetraethylammonium (TEA, 3 mM), a non-selective K⁺ channel blocker. In both preparations, the vasorelaxant activity was not changed (Maximum Response = 102.3 ± 4.8%; EC₅₀ = 25.40 ± 6.05 μg/mL; and Maximum Response = 111.2 ± 5.3%; CE₅₀ = 16.70 ± 3.61 μg/mL; n = 7, respectively) as shown in Figure 3A. These responses were no different from the control curve, suggesting that K⁺ channels are not involved in the vasorelaxant effect elicited by CME.

To test the hypothesis that CME produces vasorelaxant effects independent of the vasoconstrictor agent used in the preparation, mesenteric artery rings were incubated with tromboxane A₂ agonist U-46619 (100 nM). In the presence of U-46619, CME induced concentration-dependent vasodilatation (Maximum Response = 90.3 ± 7.8%; EC₅₀ = 24.63 ± 4.04 μg/mL, n = 6) similar to the response found by Phe-contracted mesenteric artery rings (Figure 3B).

In addition, CME produced relaxation in isolated arteries pre-contracted with KCl (60 mM). KCl depolarization elicits contraction by allowing the extracellular Ca²⁺ influx through voltage-dependent (L- and T-type) Ca²⁺ channels and subsequent calcium release from the sarcoplasmic reticulum. Due to the fact that the membrane potential is essentially clamped by the high K⁺ solution, the mechanism by which relaxation can be produced is through blockade of voltage-dependent Ca²⁺ channels, but not by other mediators that cause hyperpolarization [34]. After exposure to high concentrations of extracellular K⁺ (KCl, 60 mM), CME induced concentration-dependent vasodilatation (Maximum Response = 97.7 ± 4.0%; EC₅₀ = 34.57 ± 5.11 mg/mL; n = 6). Under the same experimental condition, HEP induced concentration-dependent vasodilatation (Maximum Response = 113.5 ± 6.1%; EC₅₀ = 10.92 ± 2.81 μg/mL; n = 6) (Figure 3C). Furthermore, both CME and HEP relaxed arterial segments pre-contracted with KCl (60 mM) suggesting that CME and HEP block Ca²⁺ entry through voltage-dependent Ca²⁺ channels (Ca_v).

In order to further investigate the hypothesis that CME and HEP act through the blockade of calcium influx, CME and HEP were tested in the presence of CaCl₂-induced contractions in a depolarizing medium without calcium. This protocol was based on the fact that CaCl₂-induced contractions are elicited, almost exclusively, through Ca²⁺ influx, since the depolarization promoted by high concentrations of extracellular K⁺ induces the opening of voltage-dependent Ca²⁺ channels [35]. Under this experimental condition, CME and HEP (0.03; 0.3; 10; 30 and 100 μg/mL) produced a non-parallel and concentration-dependent rightward shift of the CaCl₂ concentration–response curve significantly reducing the maximal response (Maximum Response = 116.1; 122.7; 84; 40.7; 7.6% and 130; 126.2; 46.8; 22.8; 7.3%, respectively) as illustrated in Figure 4A,B.

Voltage-dependent calcium channels (Ca_v) are transmembrane proteins that provide influx of calcium for a variety of intracellular activities in excitable cells. In blood vessels, this calcium entry produces vasoconstriction and blockers of Ca_v have been used to treat cardiovascular disorders. Voltage-dependent calcium channels consist of different subunits: the α₁-subunit, which contains four homologous transmembrane domains encompassing the pore, the voltage sensor and the selectivity filter; and the β, α2δ and γ auxiliary subunits. The α₁-subunits have been classified as Ca_v1.1, Ca_v1.2, Ca_v1.3, Ca_v1.4 (L-type Ca_v), Ca_v2.1 (P/Q-type Ca_v), Ca_v2.2 (N-type Ca_v), Ca_v2.3 (R-type Ca_v), Ca_v3.1, Ca_v3.2 and Ca_v3.3 (T-type Ca_v). In the vascular smooth muscle cells two types are expressed: L-type and T-type. The first ones are more expressed in these cells and exert an important role in the regulation of the vascular tone [36,37].

In order to examine which subtype of Ca_v was involved in the vasorelaxant effect elicited by CME, a contraction with Bay K 8644 (200 nM), a L-type Ca²⁺ channel agonist, was evoked. CME induced concentration-dependent vasodilatation (Maximum Response = 113.3 ± 6.7%; EC₅₀ = 19.45 ± 6.66 μg/mL, n = 7) and it was similar to the response found under Phe-induced contractions (Figure 5). These data indicate that L-type Ca_v channels could be involved in the vasorelaxant effect induced by CME.

In order to investigate a role for dihydropyridine calcium channels, rings were incubated with nifedipine. In the presence of nifedipine, CME (0.01–500 μg/mL) induced a concentration-dependent relaxation (Maximum Response = 63.6 ± 2.9%; EC₅₀ = 25.4 ± 11.0 μg/mL, n = 6). The maximum response was different to the response found by Phe-contracted mesenteric artery rings (Figure 6A). In addition, in the presence of nifedipine, HEP (0.01–500 μg/mL) induced a concentration-dependent relaxation (Maximum Response = 69.8 ± 1.7%; EC₅₀ = 24.4 ± 10.3 μg/mL, n = 6). The maximum response was different to the response found by Phe-contracted mesenteric artery rings (Figure 6B). Based on these findings, it can be suggested that the endothelium-independent vasodilatation induced by both CME and HEP involves, at least in part, the inhibition of the Ca²⁺ influx through blockade of calcium channels.

The brown alga Dictyota pulchella Hörnig and Schnetter (Dictyotaceae, Phaeophyceae) was collected at Bessa Beach, João Pessoa, State of Paraíba, Brazil, at coordinates 07°04′01″S and 34°49′35″W in February 2009 at a depth of 1 m and it was identified by George Emmanuel C. de Miranda. A voucher specimen (JPB 41771) is deposited at Lauro Pires Xavier Herbarium of the Federal University of Paraíba.

The freeze-dried material (240 g) was extracted with CH₂Cl₂/MeOH (2:1) at room temperature. The concentrated extract (10 g) was partitioned by vacuum filtration with silica gel that uses the solvents Hexano/EtOAc (9:1) to give the fraction Hexano/EtOAc (9:1) (850 mg).

The CH₂Cl₂/MeOH extract (CME) and Hexane/EtOAc phase (HEP) were prepared in a mixture of distilled water (in vitro experiments) or NaCl 0.9 % solution (in vivo experiments) and cremophor (0.013% v/v) and kept at −4° C. The stock solution was diluted to the desired concentrations with distilled water or isotonic saline just before use. The final concentration of cremophor in the bath had no effect when tested in control preparations (data not shown).

The following drugs were used: Cremophor EL, l-phenylephrine chloride (Phe), acetylcholine chloride (Ach), tetraethylammonium (TEA), S(−)-Bay K 8644, sodium nitroprusside, Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and 9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F_2α (U 46619), were purchased from Sigma Chemical (Sigma Chemical Co., St. Louis, MO, USA). Heparin sodium salt (Roche Brazil, São Paulo, Brazil), sodium thiopental (Cristália, São Paulo, SP, Brazil). The substances were dissolved in distilled water (in vitro experiments) or in NaCl 0.9% solution (in vivo experiments).

The composition of the Tyrode’s solution used to bath isolated rings was (mM): NaCl, 158.3; KCl, 4.0; CaCl₂, 2.0; MgCl₂, 1.05; NaH₂PO₄, 0.42; NaHCO₃, 10.0 and glucose, 5.6.

Male Wistar rats (250–300 g) were used in all experiments. They were housed in conditions of controlled temperature (21 ± 1 °C) and exposed to a 12 h light-dark cycle with free access to food (Labina^®, PURINA, Brazil) and tap water. All procedures described in the present study are in agreement with Institutional Animal Care and Use Committee of the Federal University of Paraiba (CEPA/LTF protocol No. 0208/10).

Intra-aortic blood pressure was recorded using a technique described by Braga [37] under sodium thiopental anesthesia (45 mg/kg, i.v.), the lower abdominal aorta and inferior vena cava were cannulated via left femoral artery and vein using polyethylene catheters. Thereafter, catheters were filled with heparinized saline solution and tunneled under the skin to emerge between the scapulae. Arterial pressure was measured 24 h after surgery by connecting the arterial catheter to a pre-calibrated pressure transducer (MLT0380/D, ADInstruments, Australia) and connected to a computer (Mikro-tip Blood pressure system, ADInstruments, Australia) running the LabChart software (ADInstruments, Australia). The data were sampled at 2000 Hz. For each pulse pressure, the computer calculated mean arterial pressure (MAP) and heart rate (HR). The venous catheter was used for drug administration.

Data are expressed as the mean ± SEM. When appropriate, the significance of differences was determined using one-way ANOVA following Bonferroni’s post test with GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA). Throughout the results, the “maximal relaxation” corresponds to the maximum response of pre-contracted tissues to the highest concentration of drug tested. P < 0.05 was considered significant.