2-Ethylpyrazine Induces Vasodilatation by Releasing Nitric Oxide in the Endothelium

Abstract

Oxygen transportation and regulation of some physiological processes are facilitated by blood flow. Furthermore, blood flow is regulated by various factors such as nitric oxide (NO) and the autonomic nerve system. In modern life, many people suffer from chilliness (hiesho) because of mental stress and an excessive use air-conditioning systems, which induces vasoconstriction in the peripheral skin. In this study, we focused on pyrazine derivatives, particularly compounds that are used as food flavoring materials, and investigated their effects on vascular function and blood flow. We examined the vasodilatory effect of pyrazine derivatives in the rat thoracic aorta and found 2-ethylpyrazine (2-EP) to be the most active pyrazine compound. Additionally, we found that 2-EP induces vasodilatation through the activities of endothelium-derived relaxing factors. 2-EP activates NO synthesis through the effect of endothelial NO synthase in the endothelium. As a result, cyclic GMP levels rise in smooth muscle cells and vasodilatation is induced. We also confirmed that 2-EP increases peripheral blood flow in rats. From these results, we concluded that 2-EP induces vasodilatation by inducing the release of NO and increasing peripheral blood flow.

Blood flow has important roles such as delivering important compounds, including oxygen,¹⁾ carbon dioxide, and various hormones, in the body and maintaining homeostasis by regulating osmotic pressure²⁾ and body temperature.³⁾ Furthermore, blood flow is regulated by various factors such as nitric oxide (NO)⁴⁾ and prostanoids,⁵⁾ and the autonomic nerve system.⁶⁾ NO plays important roles including causing vasodilatation in the endothelium and controlling physiological conditions. It also activates guanylate cyclase, which catalyzes cyclic GMP (cGMP) production from guanosine triphosphate, in smooth muscle cells.^7,8) cGMP induces Ca²⁺ ion transport out of cells. This causes hyperpolarization, which leads to vessel relaxation.^7,8) As a result, NO induces vasodilatation and increases peripheral blood flow.

Due to modern lifestyle, many people suffer from “hiesho”^9,10) or chilliness because of mental stress¹¹⁾ and excessive use of air-conditioning systems, which induces vasoconstriction in the peripheral skin.¹²⁾ Hiesho decreases QOL; therefore, it is very important to control peripheral blood flow and improve the symptoms of “hiesho.”

Pyrazine derivatives are well-known chemicals used for flavoring foods such as peanuts¹³⁾ and barley tea,¹⁴⁾ which are prepared by roasting (Maillard reaction).¹⁵⁾ Pyrazine derivatives are heterocyclic compounds (Fig. 1). 2,3,5,6-Tetramethylpyrazine (TMP) is one of the most commonly used pyrazine derivatives. The Chinese medicine “Senkyu” contains various pyrazine derivatives; however, TMP has been reported as the key chemical in “Senkyu.”¹⁶⁾ “Senkyu” is used for the treatment of blood flow failure.¹⁷⁾

Fig. 1. Chemical Structures of Pyrazine and 2-Ethylpyrazine

a) Pyrazine, b) 2-ethylpyrazine.

It has been reported that some pyrazines have antiplatelet activity.¹⁸⁾ In addition, it has been noted that some pyrazine derivatives have effects on vascular muscle cells¹⁹⁾; however, there is no report of the effects of pyrazine derivatives on vascular endothelial cells and blood vessels.

In this study, we investigated the effects of pyrazine derivatives on vasodilatation and peripheral blood flow in rats.

MATERIALS AND METHODS

Chemicals

All the pyrazine derivatives used in this study were purchased from Tokyo Chemical Industry (Tokyo, Japan). Norepinephrine (NE), acetylcholine, indomethacin, and chlorpheniramine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). N^G-Nitro-L-arginine methyl ester (L-NAME) was purchased from Dojindo Laboratories (Kumamoto, Japan). 1H-[1,2,4]Oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Animals

Specific pathogen-free male Wistar rats purchased from Nihon SLC (Yokohama, Japan) were used in this study. The rats were housed under a 12 : 12-h light/dark cycle with lights in a temperature-controlled room maintained at 24±2.0°C. Humidity was maintained at 55±10%, and food (CE-2; Clea, Tokyo, Japan) and water were provided. Blood flow measurement was performed at the age of 9 to 10 weeks. The rats were treated in accordance with the ethical guidelines for animal care, handling, and termination established by Kirin Co., Ltd.

Assessment of Vasodilatation Using a Rat Aorta Ring

The rats were adapted to the environment for at least one week prior to the experiment. Anesthesia was induced with diethyl ether by inhalation, followed by exsanguination by abdominal aorta dissection. The thoracic aorta was removed and cut into rings (2 mm in length). The rings were placed in a 5-mL organ bath filled with Krebs buffer solution (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 25.0 mM NaHCO₃, and 10 mM glucose; pH 7.35) that was continuously bubbled with 95% O₂/5% CO₂. Tension was measured using an isometric force transducer (micro easy Magnus UC-2TD; Iwashiya Kishimoto Ika Sangyo, Kyoto, Japan). Resting tension was 2 mN. The rings were equilibrated for a period of 10 min before the experiment was initiated. The vessels in the rings were contracted by adding 0.1 µM NE in the organ bath.^19,20) Relaxing ration is expressed as a percentage of the steady-state obtained with 1 µM acetylcoline addition (maximal relaxation).

The endothelium was removed from each rat aorta sample to investigate the vascular endothelium.²¹⁾ The mechanism of vasodilatation was studied using the following: 100 µM L-NAME as an inhibitor of endothelial NO synthase (eNOS),²²⁾ 100 µM indomethacin as a cyclooxygenase (COX) inhibitor,²³⁾ and 50 µM ODQ as a guanylate cyclase inhibitor.²⁴⁾

Measurement of cGMP Concentration in Rat Aorta

The rats were anesthetized with diethyl ether by inhalation and then exsanguinated by abdominal aorta dissection. The thoracic aorta was removed, cut into rings (2 mm in length), and incubated in the presence of 100 µM 2-EP, 1 mM 2-EP, or 100 µM acetylcholine for 15 min. After incubation, the rings were homogenized in 0.1 mM HCl using a multi-bead shocker (Yasui Kikai Corporation, Osaka, Japan). The homogenates were centrifuged at 15000 rpm for 10 min, after which the amount of cGMP was measured using Direct cGMP ELISA kit (Enzo Life Science, Inc., Farmingdale, NY, U.S.A.). Protein concentrations were measured using a protein assay kit (Bio-Rad, Hercules, CA, U.S.A.). Bovine serum albumin was used as the standard in the analysis. cGMP amount was expressed as pmol of cGMP/mg protein from an aorta ring.²⁵⁾

Blood Flow Measurement

On the day of this experiment, the animals were not allowed access to food for 16 h prior to being anesthetized. The rats were anesthetized with 1 g/kg urethane by intraperitoneal injection. Next, the rats were cannulated intratracheally and their tails were connected to a laser Doppler blood flow meter (ALF-21; Advance Co., Tokyo, Japan). Analog signals were converted to digital signals using an A/D converter (Power-Lab; AD Instruments, Dunedin, New Zealand). Before the rats were administered the test sample, they were stabilized for 15 min because the recording signal was fluctuated. After stabilization, the samples were injected into the rats and blood flow measurement was started.²⁶⁾ The data obtained have been expressed as mean±standard error of the mean (S.E.M.).

Statistical Analysis

Data are expressed as mean±standard error. The data obtained from all the experiments were analyzed using the Dunnett test. p values less than 0.05 were considered statistically significant.

RESULTS

Comparing the Vasodilatory Effects of Nine Pyrazine Derivatives on Rat Aorta Rings

We found that nine pyrazine derivatives induced vasodilatation in the rat aorta rings that were contracted by phenylephrine (Fig. 2). 2-EP induced vasodilatation more strongly than the other derivatives did (Fig. 2).

Fig. 2. Screening the Vasodilation Effect of Pyrazine Derivatives with Rat Aorta on 1 µM Phenylephrine-Contracted

Each compound was added 1 mM in organ bath. The data for the figure are expressed as means±S.E. (n=4).

Vasodilatory Effect of 2-EP in the Presence of L-NAME, ODQ, or Indomethacin

The vasodilatory effect of 2-EP was not observed when the vascular endothelium was removed from the rings (Fig. 3). Furthermore, it was observed that 2-EP-induced vasodilatation was reduced by L-NAME or ODQ but unaffected by indomethacin (Fig. 3).

Fig. 3. Vasodilatation Effects of 1 mM 2-Ethylpyrazine (2-EP) on 1 µM Phenylephrine-Contracted Rat Aorta Rings in the Presence of Inhibitor

The differences between intakt and denuded, L-NAME, ODQ were statistically significant by Dunnet test (p<0.05, n=4). The data for the figure are expressed as means±S.E.

cGMP Production in the Rat Aorta Rings

It was observed that cGMP levels in the rat aorta rings were increased by acetylcholine (positive control) and also by 2-EP; however, the effect of 2-EP was concentration-dependent (Fig. 4).

Fig. 4. cGMP Releasing from Rat Aorta with 100 µM or 1 mM 2-Ethylpyrazine (2-EP) or 100 µM Acetylcoline (Ach)

The differences between control and 2-EP or Ach were statistically significant by Dunnet test (p<0.05, n=4).

Effect of 2-EP on Blood Flow

Compared to the control treatment, 2-EP increased blood flow in a concentration-dependent manner (Fig. 5).

Fig. 5. Rat Tail’s Blood Flow Change by Intaking 2-Ethylpyrazine

Control (●), 10 µg/kg (▲), 100 µg/kg (■). The differences between control and 2-EP administration were statistically significant by Dunnet test (p<0.05, n=10). The data for the figure are expressed as means±S.E. of percentages of 0 min values.

DISCUSSION

In this study, we compared the vasodilatory effects of nine pyrazine derivatives on rat aorta rings. Our findings revealed that 2-EP had the strongest vasodilatory effect on the rat aorta rings (Fig. 2). The side-chain structure of pyrazine derivatives is very important in vasodilatation. For instance, an ethyl group in the side chain can result in a strong vasodilatory effect (Fig. 2).

It was observed that 2-EP-induced vasodilatation was diminished in denuded aorta rings, which indicates that 2-EP-induced vasodilatation is dependent on the vascular endothelium (Fig. 3). Vasodilatation in the endothelium may occur via the NO–cGMP pathway^7,8) or the prostanoid–cAMP pathway.²⁷⁾ In the experiment involving inhibitors, it was revealed that 2-EP-induced vasodilatation occurs via the NO–cGMP pathway. This is because L-NAME and ODQ inhibited 2-EP-induced vasodilatation (Fig. 3). On the other hand, indomethacin, which is an inhibitor of prostanoid synthesis, did not inhibit 2-EP-induced vasodilatation. NO activates guanylate cyclase in vascular smooth muscle cells. As a result, cGMP levels increase and Ca²⁺ ions are released from the vascular smooth muscle cells, which result in vasodilatation.^7,8) Our results showed that there was an increase in cGMP levels in the rat aorta (Fig. 4). Moreover, it was shown that 2-EP-induced vasodilatation occurred through NO production via eNOS in the endothelium. Generally, eNOS is activated by Ca-calmodulin. Thus, 2-EP might activate Ca-calmodulin.

Furthermore, our results confirmed that oral 2-EP improves peripheral blood flow in a concentration-dependent manner (Fig. 5). It was observed that peripheral blood flow in the control group was slightly lower than it was before the treatment was administered. In the present study, anesthesia was induced with urethane, which activates sympathetic nerves by releasing adrenalin.²⁸⁾ Vasoconstriction and a decline in peripheral blood flow were noted after urethane was administered to the animals.²⁶⁾ Blood flow in the 2-EP-treated groups was higher than that in the control group. Thus, 2-EP has a vasodilatory effect and increases peripheral blood flow in rats.

Hesperidin has been reported to have an effect on NO synthesis in the endothelium.²⁹⁾ It also increased peripheral blood flow and improved “hiesho” symptoms in a clinical study.³⁰⁾ From our results, 2-EP and hesperidin cause vasodilatation via the same mechanism. It can therefore be assumed that 2-EP has the potential to improve microcirculation and the symptoms of “hiesho” in humans.

On the other hand, we could not determine the receptors involved in the vasodilatation induced by 2-EP. There are various receptors involved in NO synthesis. Therefore, we surveyed the relationship between 2-EP-induced vasodilatation and acetylcholine or histamine receptors. However, the results showed that the two receptors are not involved in 2-EP-induced vasodilatation (data not shown).

TMP, which is the key chemical in the Chinese medicine “Senkyu,” activates adrenergic and serotonin receptors.³¹⁾ In addition, it has an effect on platelet aggregation¹⁸⁾ and blood viscosity.¹⁸⁾ Furthermore, 2,5-dimethylpyrazine has effects on β-adrenergic receptors and uterine contraction.³²⁾ Therefore, since 2-EP and 2,5-dimethylpyrazine have similar chemical structures, it could be assumed that the two compounds have similar effects.

Blood flow is controlled by the autonomic nerve system.⁶⁾ In the present study, we focused on investigating vasodilatation through the endothelium and whether 2-EP has an effect on the autonomic nerve system.

In general, NO has effects on hypertension,³³⁾ arteriosclerosis,³⁴⁾ and neuroprotection³⁵⁾; therefore, we expect that 2-EP might be effective in improving these conditions. A limitation of the present study is that the vasodilatory effect of 2-EP was investigated in an ex vivo experiment using rats. Therefore, the vasodilatory effect of 2-EP should be confirmed in a clinical study.

In this study, we revealed that pyrazine derivatives activate the release of NO in the endothelium. The results indicate that 2-EP causes vasodilatation and improves blood flow in rats.

Acknowledgments

We thank Kyoko Kato, Eri Sano, and Guanying Wang for their helpful discussion and technical support.

Conflict of Interest

All the authors are employees of Kirin Co., Ltd., the study sponsor.

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