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. 2012 Nov 24;94(1):17–24. doi: 10.1111/iep.12000

Changes of imidazoline receptors in spontaneously hypertensive rats

Guang-Yuan Mar *, Ming-Ting Chou †, Hsien-Hui Chung ‡, Nien-Hua Chiu §, Mei-Fen Chen , Juei-Tang Cheng †,‡
PMCID: PMC3575869  PMID: 23176371

Abstract

The role of imidazoline receptors in the regulation of vascular function remains unclear. In this study, we evaluated the effect of agmatine, an imidazoline receptor agonist, on systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) and investigated the expressions of imidazoline receptors by Western blot. The isometric tension of aortic rings isolated from male SHRs was also estimated. Agmatine decreased SBP in a dose-dependent manner in SHRs but not in the normal group [Wistar–Kyoto (WKY) rats]. This reduction in SBP in SHRs was abolished by BU224, a selective antagonist of imidazoline I2-receptors. Higher expression of imidazoline receptors in SHR was observed. Moreover, agmatine-induced relaxation in isolated aortic rings precontracted with phenylephrine or KCl. This relaxation was also abolished by BU224 but was not modified by efaroxan, an imidazoline I1-receptor antagonist. Agmatine-induced relaxation was also attenuated by PNU 37883, a selective blocker of vascular ATP-sensitive potassium (KATP) channels. Additionally, vasodilatation by agmatine was reduced by an inhibitor of protein kinase A (PKA). We suggest that agmatine can lower blood pressure in SHRs through activation of the peripheral imidazoline I2-receptor, which is expressed more highly in SHRs.

Keywords: agmatine, ATP-sensitive K+ channels, forskolin, imidazoline receptor, spontaneously hypertensive rats, vasodilatation

Hypertension is one of the major risk factors for cardiovascular diseases, contributing to the pathogenesis of atherosclerosis, myocardial infarction and stroke. Despite the fact that hypertension is thus one of the most important cardiovascular disorders, and that searching for better agent(s) to handle hypertension is an urgent question (Cohen & Townsend 2011) few such compounds are currently being explored.

Imidazoline receptors have been thought to play a role in the cardiovascular system (Regunathan et al. 1996; Yang et al. 2005). Previous studies indicate that the antihypertensive agent rilmenidine lowered blood pressure via activation of central imidazoline I1-receptors leading to peripheral sympathoinhibition (Esler 1998; Van Zwieten & Peters 1999). However, treatment of hypertension with rilmenidine often leads to adverse side effects including mental depression, insomnia and drowsiness. Therefore, the development of improved therapies acting via this pathway for the management of hypertension is necessary. Moreover, the way that these peripheral imidazoline receptors affects cardiovascular function remains unclear.

Additionally, membrane potential is a major determinant of vascular tone. Changes in potassium (K+) channel activity may lead to altered Ca+ channel activity and may result in vasodilatation (Nelson & Quayle 1995; Ko et al. 2008). The opening of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels can lower intracellular Ca+ concentration (Quayle & Standen 1994; Mishra & Aaronson 1999), whereas the attenuation of KATP channel activity may result in vasoconstriction and the depolarization of vascular smooth muscle (Davis et al. 1991). Thus, this process plays an important role in the regulation of vascular tone. Although the relationship between imidazoline receptors and KATP channels has been mentioned in earlier studies (Chan et al. 1991; Dunne 1991; Plant et al. 1991; Schwietert et al. 1992; Rustenbeck et al. 1995) the detailed mechanism remains unclear.

It has been documented that agmatine (an endogenous agonist of imidazoline receptors) can lower blood pressure in anaesthetized hypertensive rats (Raasch et al. 2002). In this study, we checked the effects of agmatine on systolic blood pressure (SBP) in conscious spontaneously hypertensive rats (SHRs). Also, we indentified the expression of imidazoline receptors in SHRs. In an attempt to identify the role of peripheral imidazoline receptors in the regulation of vascular tone, changes by agmatine in isometric tension of aortic rings isolated from SHRs were also examined. Additionally, specific antagonists or blockers were applied to elucidate the potential action mechanism(s) of agmatine. The results obtained highlight the potential of peripheral imidazoline receptors as novel targets in the development of therapeutic agents to treat hypertension.

Materials and methods

Experimental animals

We obtained 12-week-old male rats with spontaneous hypertension (SHR) and age-matched male Wistar–Kyoto rats (WKY) from the National Animal Center (Taipei, Taiwan) and housed them in our animal facility. The rats were maintained in a temperature-controlled room (25 ± 1 °C) under a 12-h light–dark cycle (lights on at 06:00). All rats were provided water and standard chow (Purina Mills, LLC, St Louis, MO, USA) ad libitum. All animal-handling procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (USA) as well as the guidelines of the Animal Welfare Act.

Ethical approval

Ethical approval from the Chi-mei Medical Centre was obtained (code numbe 100110403).

Measurement of blood pressure

Stock solutions of agmatine (Sigma-Aldrich, St Louis, MO, USA) or 2-BFI, a selective imidazoline I2-receptor agonist (Tocris Cookson, Bristol, UK), were prepared by dissolving 1, 3 and 5 mg of agent in 1 ml saline respectively. Agmatine or 2-BFI was administered via intravenous injection at a volume under 1 ml/kg. BU224, a selective imidazoline I2-receptor antagonist (Tocris Cookson) was injected into rats intravenously 30 min before treatment with agmatine. Control rats were injected with an equal volume of vehicle (saline). Thirty minutes following agmatine or vehicle injection, SBP and heart rate (HR) were determined using a non-invasive tail-cuff monitor (MK2000; Muromachi Kikai, Tokyo, Japan). Values are presented as the mean ± SEM and values for each animal were determined in triplicate.

Preparation of isolated aortic rings

In all vascular experiments, we used aortas from SHRs. Each rat was sacrificed by decapitation under anaesthesia with pentobarbital (50 mg/kg). Thoracic aortas were rapidly removed and placed in oxygenated Krebs' buffer (95% O2, 5% CO2). Aortas were then excised from fat and connective tissue and cut into ring segments approximately 3 mm long. The rings were then mounted in organ baths filled with 10 ml oxygenated Krebs' buffer (95% O2, 5% CO2) at 37 °C. Krebs' buffer consists of the following (in mmol/l): NaCl 135; KCl 5; CaCl2 2.5; MgSO4 1.3; KH2PO4 1.2; NaHCO3 20; and d-glucose 10 (pH 7.4).

Each preparation was connected to strain gauges (FT03; Grass Instrument, Quincy, MA, USA) and isometric tension was recorded using chart software (MLS023, powerlab; AD Instruments, Bella Vista, NSW, Australia). Rings were mounted and allowed to stabilize for 2 h. Each preparation was then gradually stretched to achieve an optimal resting tension of 1 g.

Vasodilatation caused by agmatine

After the resting tension stabilized, a solution of either phenylephrine (Sigma-Aldrich) or KCl prepared in distilled water was added to the bathing buffer to induce a rapid increase in vascular tone followed by stable vasoconstriction (tonic contraction). The final concentration in the organ bath of both phenylephrine and KCl was 1 and 50 mmol/l respectively. Rings in the treatment group were exposed to agmatine (1–100 μmol/l), and alterations in tonic contraction (vasodilatation) were recorded. In addition, forskolin (Sigma-Aldrich), a direct activator of adenylyl cyclase was used as a positive control to investigate aortic relaxation. Relaxation is expressed as the per cent decrease in maximal tonic contraction. Concentration–relaxation curves were generated by a cumulative addition of agonist.

Removal of endothelium

To preclude a possible role of the endothelium in agmatine-induced vasodilatation, tests were conducted in endothelium-denuded preparations. The endothelium was removed by gently rubbing it against the teeth of a pair of forceps. The successful removal of endothelium was confirmed by demonstrating the failure of 1 μmol/l acetylcholine to relax rings that had been precontracted with phenylephrine as described previously (Tsai et al. 2002).

Effects of blockers on agmatine-induced vasodilatation

Aortic rings were exposed to glibenclamide, efaroxan (a selective imidazoline I1-receptor antagonist; Research Biochemical, Wayland, MA, USA) or BU224, for 15 min prior to the addition of agmatine into the organ bath. PNU 37883 (Tocris Cookson), an antagonist selective for the vascular form of KATP channels, and H-89 (Sigma-Aldrich), a protein kinase A (PKA) inhibitor, were administered in the same manner. Agmatine-induced vasodilatation was examined after treatment with each of these inhibitors and compared with vehicle (distilled water)-treated controls.

Western blotting analysis

The aortic tissues were put in ice-cold homogenized buffer containing 10 mmol/l Tris–HCl (pH 7.4), 20 mmol/l EDTA, 10 mmol/l EGTA, 20 mmol/l β-glycerolphosphate, 50 mmol/l NaF, 50 mmol/l sodium pyrophosphate, 1 mmol/l phenylmethylsulfonyl fluoride and the protease inhibitors 25 μg/ml leupeptin and 25 μg/ml aprotinin. The mixture was centrifuged at 1000 g for 10 min at 4 °C. The supernatant containing the membrane fraction was centrifuged at 48,000 g for 30 min at 4 °C. The supernatant was removed, and the pellet was resuspended in Triton X-100 lysis buffer on ice for 30 min, homogenized and then centrifuged at 14,010 g for 20 min at 4 °C. Finally, the supernatant was transferred to a new Eppendorf tube and stored at −80 °C. The membrane extracts (20–80 μg) were separated by performing SDS–polyacrylamide gel electrophoresis, and the proteins were transferred onto a BioTraceTM polyvinylidene fluoride (PVDF) membrane (Pall Corporation, Pensacola, FL, USA). Following blocking, the blots were developed using antibodies for imidazoline receptors (IR; Abcam, Cambridge, UK). The blots were subsequently hybridized using horseradish peroxidase-conjugated goat anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and developed using the Western Lightning Chemiluminescence Reagent PLUS (PerkinElmer Life Sciences Inc., Boston, MA, USA). Densities of the obtained immunoblots at 37 KDa for imidazoline receptors (IR) and 43 KDa for actin were quantified using gel-pro analyser software 4.0 (Media Cybernetics, Silver Spring, MD, USA).

Statistical analysis

All values are presented as the mean ± SEM from one group of animals or samples. Analysis of variance and Dunnett's post hoc test were used to evaluate any significant differences between groups. P < 0.05 was taken to indicate a significant difference.

Results

Effect of imidazoline I2-receptor antagonist on agmatine-induced reduction of blood pressure

Tail-cuff measurements of SBP revealed markedly higher value in 12-week-old SHRs than in age-matched controls (WKY). Intravenous injections of agmatine (1, 3 or 5 mg/kg) decreased SBP markedly in conscious SHRs 30 min postinjection in a dose-dependent manner (Figure 1). However, agmatine did not modify SBP in WKY rats (113 ± 1.98 mmHg in controls vs. 107 ± 1.78 mmHg after injection of 5 mg/kg agmatine; N = 8, P > 0.05). Additionally, 2-BFI, an imidazoline I2-receptor agonist (1, 3, or 5 mg/kg, i.v.) also exhibited decreases in SBP similar to agmatine in conscious SHRs. (Figure 1) but 2-BFI did not modify SBP in WKY rats (110 ± 0.91 mmHg in controls vs. 112 ± 1.45 mmHg after injection of 5 mg/kg 2-BFI; N = 8, P > 0.05). Administration of BU224 (0.5, 1.0 or 1.5 mg/kg, i.v.) blocked the agmatine-dependent decrease in SBP in conscious SHRs in a dose-dependent manner, and similar inhibitory results were observed in that there was a 2-BFI-dependent decrease of SBP in conscious SHRs (Table 1). However, BU224 alone did not alter blood pressure in SHRs. Also, the heart rate in SHRs was not modified by treatment with either agmatine or 2-BFI (data not shown).

Figure 1.

Figure 1

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The mediation of imidazoline I2-receptors in agmatine-induced changes of systolic blood pressure (SBP) in conscious spontaneously hypertensive rats (SHRs). A concentration-dependent decrease in SBP of SHRs upon treatment with agmatine (•) or 2-BFI (○). Data represent the mean ± SEM of eight animals. *P<0.05, **P<0.01 and ***P<0.001 compared with vehicle-treated SHRs.

Table 1.

A concentration-dependent inhibitory effect of BU224 on the agmatine-induced or 2-BFI-induced reduction of systolic blood pressure (SBP) in conscious spontaneously hypertensive rats (SHRs)

SBP (mmHg)
Agmatine (5 mg/kg)
 +Vehicle 145.50 ± 1.15
 +BU224
 0.5 mg/kg 158.75 ± 4.02*
 1.0 mg/kg 166.25 ± 4.36**
 1.5 mg/kg 182.75 ± 0.41***
2-BFI (5 mg/kg)
 +Vehicle 144.00 ± 1.46
 +BU224
 0.5 mg/kg 155.75 ± 2.07**
 1.0 mg/kg 169.00 ± 1.46***
 1.5 mg/kg 180.50 ± 0.56***
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Data represent the mean ± SEM of eight animals. *P<0.05, **P<0.01 and ***P<0.001 compared with vehicle-treated SHRs.

Effects of imidazoline receptor blockade on agmatine-induced vasodilatation

Aortic ring strips are contract after application of phenylephrine (1 μmol/l) or KCl (50 mmol/l; Wynne et al. 2004). Similar to the previous report (Raasch et al. 2002), agmatine did not modify the vascular tone of aortic rings. At the maximum concentration tested (100 μmol/l), agmatine significantly attenuated the tonic contraction of SHR aortic rings induced by phenylephrine to 68.13 ± 4.02% of the maximum contraction. Similarly, 100 μmol/l agmatine also decreased KCl-induced tonic vasoconstriction to 62.07 ± 3.00% of the maximum contraction. However, agmatine at the same concentration attenuated the tonic contraction of aortic rings in the control group induced by phenylephrine to 84.58 ± 1.41% of the maximum contraction, and KCl- induced tonic vasoconstriction to 73.69 ± 2.05% of the maximum contraction respectively. BU224 (0.1–1 μmol/l) produced a significant concentration-dependent attenuation of agmatine-induced relaxation of phenylephrine- and KCl-precontracted aortic rings (Table 2). However, BU224 alone did not alter the vascular tone of aortic rings. In contrast, efaroxan (0.1–1 μmol/l) failed to abolish the relaxant effect of agmatine on tonic contraction in both phenylephrine- and KCl-pretreated aortic rings (Table 2).

Table 2.

Inhibitory effects of efaroxan or BU224 on the vasorelaxation of agmatine (100 μmol/l) induced in isolated SHR aortic rings precontracted with 1 μmol/l phenylephrine (PE) or 50 mmol/l KCl

Contraction force (%) PE (%) KCl (%)
Baseline (precontracted state) 100 100
Agmatine (100 μmol/l)
 +Vehicle 68.13 ± 4.02 62.07 ± 3.00
 +Efaroxan
 0.1 μmol/l 65.20 ± 1.02 59.17 ± 5.03
 1.0 μmol/l 68.78 ± 2.71 60.77 ± 4.70
 +BU224
 0.1 μmol/l 82.94 ± 0.92* 74.85 ± 2.52*
 1.0 μmol/l 88.38 ± 0.62** 84.87 ± 1.61***
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Data represent the mean ± SEM of eight animals. *P<0.05, **P<0.01 and ***P<0.001 compared with vehicle-treated control.

The expression of imidazoline receptors in aorta between WKY and SHR

As shown in Figure 2, the blot densities for imidazoline receptor bands were corrected to those for actin, while the Nischarin-antibody recognized imidazoline receptors. The expression of imidazoline receptors in aortas of SHR rats was significantly higher than in aortas from WKY (Figure 2). Differences between the two groups were confirmed by quantitation of protein levels (Moreover, as shown in Figure 3, vasodilatation induced by agmatine in aortic rings of SHRs was abolished by the Nischarin-antibody used to characterize the expression of imidazoline receptors.

Figure 2.

Figure 2

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The expression of imidazoline receptors in aorta obtained from WKY rats or SHRs. Data represent mean ± SEM of eight animals. *P<0.05 compared with WKY group.

Figure 3.

Figure 3

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Concentration-dependent inhibition of agmatine-induced relaxation in phenylephrine (▪) or KCl (□) precontracted aortic rings isolated from SHRs by pretreatment with imidazoline receptors (IR) antibody. Data represent the mean ± SEM of eight animals. *P<0.05, **P<0.01 and ***P<0.001 compared with agmatine-treated group.

Role of the endothelium in agmatine-induced vasodilatation

Preliminary experiments indicated that 1 μmol/l acetylcholine induced relaxation to 34.61 ± 1.64% (N = 8) of the maximal contraction in aortic rings precontracted with 1 μmol/l phenylephrine. The relaxation effect of acetylcholine was greatly reduced in endothelium-removed aortic rings to 95.60 ± 0.62% (N = 8) of maximal contraction. The successful removal of the endothelium was also confirmed by histology (data not shown). However, the relaxation effect of agmatine at 100 μmol/l on phenylephrine- or KCl-induced tonic vasoconstriction was 70.04 ± 3.84% or 67.94 ± 3.27% which was not significantly different from 74.76 ± 2.75% or 73.77 ± 1.97% in SHR aortic rings without an intact of endothelium (N = 8, P > 0.05).

The role of vascular ATP-sensitive K+ (KATP) channels in agmatine-induced vasodilatation

PNU 37883 (0.1–10 nmol/l) produced a concentration-dependent attenuation of the relaxation of agmatine (100 μmol/l) in phenylephrine- or KCl- precontracted aortic rings (Figure 4). However, treatment with PNU 37883 (10 nmol/l) alone did not modify the vascular tone.

Figure 4.

Figure 4

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PNU 37883 produces a concentration-dependent inhibition of agmatine (100 μmol/l) induced relaxation in isolated aortic rings precontracted with 1 μmol/l phenylephrine (▪) or 50 mmol/l KCl ((□). Data represent the mean ± SEM of eight animals in each column. *P<0.05, **P<0.01 and ***P<0.001 compared with the agmatine-treated group.

The role of cAMP and PKA in agmatine-induced vasodilatation

We used forskolin (10 μmol/l), a direct activator of adenylate cyclase, as a positive control for the induction of cyclic adenosine monophosphate (cAMP), as described previously (Zhang et al. 1994). In aortic rings precontracted with phenylephrine (1 μmol/l) or KCl (50 mmol/l), forskolin-induced vasodilatation was abolished by pretreatment with glibenclamide (1 μmol/l). Forskolin-induced vasodilatation was decreased by H-89 at a concentration (1 μmol/l) known to inhibit PKA (Wellman et al. 1998). The vasodilatation induced by agmatine was also attenuated by H-89 or glibenclamide (Table 3) in a manner similar to forskolin-induced vasodilatation. However, treatment with H-89 or glibenclamide alone did not alter the vascular tone.

Table 3.

The effect of protein kinase A (PKA) or KATP channel inhibitor on the vasorelaxation induced by agmatine (100 μmol/l) or forskolin (10 μmol/l) in isolated SHR aortic rings precontracted with 1 μmol/l phenylephrine (PE) or 50 mmol/l KCl

Contraction force PE (%) KCl (%)
Baseline (precontracted state) 100 100
Agmatine (100 μmol/l)
 +Vehicle 69.67 ± 3.33 60.35 ± 2.95
 +Glibenclamide (1 μmol/l) 88.77 ± 1.05** 87.06 ± 0.74***
 +H-89 (1 μmol/l) 88.57 ± 1.77** 78.93 ± 1.68***
Forskolin (10 μmol/l)
 +Vehicle 34.65 ± 2.34 29.24 ± 2.39
 +Glibenclamide (1 μmol/l) 98.34 ± 6.06*** 100.21 ± 6.30***
 + H-89 (1 μmol/l) 101.30 ± 4.21*** 98.80 ± 9.36***
Glibenclamide (1 μmol/l) 98.13 ± 2.47*** 97.46 ± 3.16***
H-89 (1 μmol/l) 100.23 ± 1.48*** 99.44 ± 2.38***
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Data represent mean ± SEM of eight animals. *P<0.05, **P<0.01 and ***P<0.001 as compared with vehicle-treated control respectively.

Discussion

In the present study, we found that agmatine induced a dose-dependent reduction in SBP in SHRs but not in WKY rats. This is consistent with a previous report (Raasch et al. 2002). The effect of agmatine on SBP occurs mainly through the activation of imidazoline receptors in peripheral tissues as the dose of agmatine used in the present study is below the dose needed to cross the blood–brain barrier (Aricioglu & Altunbas 2003; Lavinsky et al. 2003; Piletz et al. 2003). Also, direct injection of agmatine into brain induced an increase in blood pressure (Raasch et al. 2002).

Activation of imidazoline receptors has been suggested to regulate cardiovascular functions (Messerli 2000; Monroy-Ordonez et al. 2008). Vascular tone is an important factor in the regulation of blood pressure (Khalil 2011). Although blood pressure is regulated by multiple factors, there is general agreement that the level of blood pressure per se, and particularly in hypertension, is determined in large part by total peripheral resistance which is primarily a function of the resistance of terminal arterioles (Guinea et al. 2010). Thus, we investigated the aortic response to elucidate blood pressure regulation in hypertension. In the present study, isolated aortic rings were precontracted with phenylephrine (1 μmol/l) or KCl (50 mmol/l) as described previously (Wynne et al. 2004). We used two different preconstrictors in this investigation mainly to establish that the direct relaxation of the aorta by agmatine occurs regardless of whether the contraction is induced by adrenergic α-receptors or by ion channels. Actually, agmatine induced a concentration-dependent relaxation of aortic rings that were precontracted with either phenylephrine or KCl. This is consistent with a previous report that agmatine induces relaxation in phenylephrine precontracted rat thoracic aorta (Gerova & Torok 2004). To clarify which imidazoline receptor subtype could be activated by agmatine, we used imidazoline I1-receptor and I2-receptor antagonists to investigate this relaxation. Actually, the relaxation of agmatine was attenuated by pretreatment with BU224 at a concentration sufficient to block imidazoline I2-receptors but not by efaroxan at effective concentration to block imidazoline I1-receptors. Thus, a direct effect of agmatine on imidazoline I2-receptors can be identified. We used agmatine in the assay of vascular tone in isolated aorta because it is a well-known agonist of imidazoline receptor. Moreover, we also tested the vasomotor effects of agmatine in aorta from WKY rats. However, aortic relaxation by agmatine was not significant in WKY rats, except at the highest concentration of agmatine which may cause relaxation. Actually, as shown in Figure 2, expression of imidazoline receptors is higher in SHRs that in WKY rats. The Nischarin-antibody used in the present study seems to be linked to the imidazoline I1-receptor because it might be an anchor protein. Therefore, it has been used as a surrogate parameter in investigations on imidazoline I1-receptors. Moreover, this Nischarin-antibody blocked the relaxation by agmatine in aortic rings. It is possible that this antibody can bind the same sites as agmatine in aorta. Thus one could speculate that there is higher expression of imidazoline receptors in the aorta of SHRs. However, the subtype of imidazoline receptor in the aorta needs to be characterized in the future. In the absence of specific antibody for imidazoline receptors, we only can assume that peripheral imidazoline receptors are changed in hypertension. Notably this is consistent with the similar action of agmatine observed in another hypertensive model (Li & He 2001). Agmatine is known to be an agonist of imidazoline receptors (Gao et al. 1995; Wu et al. 2008), which are classified into at least 3 subtypes – I1-, I2- and I3-imidazoline receptors (Morgan & Chan 2001; Kaliszan et al. 2006). In recent studies agmatine has been used as both agonist and antagonist at the imidazoline receptors, but can bind to imidazoline receptors/recognition sites without inducing an effect (Molderings & Haenisch 2012). The antihypertensive effects of moxonidine are also thought to occur through activation of I1-imidazoline receptors, which have been reported to lower sympathetic nervous tone (Benedict 1999). The previous studies showed that intravenous application of agmatine decreased arterial pressure and sympathetic nerve activity due to blockade of transmission through sympathetic ganglia (Sun et al. 1995) and inhibition of carotid baroreflex in anaesthetized rats (Qin & He 2001). However, these reports did not explore the vasodilatation of agmatine as shown in the present study. The heart rate in SHR rats did not increase despite the vasodilatation induced by agmatine in the same manner as was shown in a previous report (Raasch et al. 2002) - this contradictory result may be due to multiple mechanisms and requires additional studies. In particular, additional experiments to understand the mechanisms of autoregulation in circulation homoeostasis are required. We found that 2-BFI shows an effect similar to agmatine in SHRs (Figure 1). It is well known that 2-BFI is a specific agonist of peripheral imidazoline I2-receptors (Sanchez-Blazquez et al. 2000). Thus, the activation of peripheral imidazoline I2-receptors is involved in the decrease in blood pressure in SHRs. In a previous study (Gerova & Torok 2004), agmatine produced a marked hypotensive action in anaesthetized normal rats. However, blood pressure in WKY rats was not altered by agmatine in this study. The difference may be due to the fact that we measured the blood pressure in conscious rats as opposed to anaesthetized rats (Gerova & Torok 2004).

The endothelium plays a key role in the regulation of vascular tone while a dysfunctional endothelium and/or reduced nitric oxide (NO) has been shown to be important in the aberrant contractility associated with vascular disease (Demougeot et al. 2005; Bagnost et al. 2008). According to the previous study (Musgrave et al. 2003), the endogenous imidazoline ligands are capable of stimulating NO synthetase to relax rat aorta by an endothelium-dependent mechanism. However, in the present study, relaxation induced by agmatine was not influenced by the removal of the endothelium. This difference may be due to the variation of incubation in organ bath and/or other factors. Thus, the role of endothelium in the relaxation of the aorta induced by agmatine seems negligible. This is consistent with a previous report demonstrating the presence of I2-imidazoline receptors in vascular smooth muscle (Regunathan et al. 1996). Based upon this we suggest that activation of I2-imidazoline receptors seems to be responsible for the vasodilatation effect of agmatine. In addition we focused on the cellular signals downstream of imidazoline I2-receptors.

Potassium channels play an important role in the regulation of vascular relaxation (Ko et al. 2008). ATP-sensitive potassium (KATP) channels are composed of four inwardly rectifying K+ channel subunits and four regulatory sulphonylurea receptors (Brayden 2002). Part of the mechanism for contraction of endogenous vasoconstrictors is due to inhibition of KATP channels (Nakhostine & Lamontagne 1993; Brayden 2002). The activation of KATP channels induces hyperpolarization and consequently relaxes vascular smooth muscle cells. KATP channels dysfunction in aortic cells leads to the impaired vasodilatation and hypertension in deoxycorticosterone acetate (DOCA)-salt hypertensive rats (Ghosh et al. 2004). In the present study, the relaxation induced by agmatine in rat aortic rings was abolished by pretreatment with glibenclamide at a concentration sufficient to block KATP channels, as described previously (Tsai et al. 2002; Wong et al. 2004). Thus, it is possible that KATP channels are involved in the aortic relaxation induced by agmatine. Actually, aortic relaxation by agmatine was dose-dependently inhibited by PNU 37883 that is known as specific blocker of vascular KATP channels (Cui et al. 2003; Teramoto 2006). Forskolin, a direct activator of adenylate cyclase, increases intracellular levels of cAMP and activates PKA which can then induce the opening of KATP channels (Wellman et al. 1998). In the present study, we demonstrate that forskolin-induced vasodilatation may also be blocked by glibenclamide. Forskolin-induced vasodilatation was abolished by H-89, a PKA inhibitor (Wellman et al. 1998). At doses greater than those used in the present study, H-89 has been shown to inhibit other kinases (Cho et al. 2009). We do not believe that the off-target effect of H-89 is responsible for the effects observed in the present study as we used the inhibitor at concentration lower than that required to induce such off-target effects. The effect of H-89 on agmatine-treated aortic rings was similar to that observed in forskolin-treated aortic rings. These data suggest that the mechanism underlying agmatine-induced vasodilatation may involve the cAMP-PKA-mediated opening of KATP channels. According to previous reports, I2-imidazoline receptors are related to the monoamine oxidase pathway in the central nervous system (Sastre & Garcia-Sevilla 1993; Sastre-Coll et al. 2001). However, the expression of imidazoline I2-receptors in rat aortas has been previously identified (Regunathan et al. 1996) and the present study emphasized the role of peripheral imidazoline receptors in hypertension. Thus, the present study suggests that an effect via central nervous system on monoamine oxidase can beexcluded. Furthermore the subtypes of imidazoline I2-receptors which have been classified as I2a and I2b (Diamant et al. 1992) may indicate that there are tissue differences in expression and responsiveness. Taken together, our results provide novel insight into the mechanisms for agmatine-induced aortic relaxation. We believe that agmatine-induced aortic relaxation is mediated through multiple mechanisms involving cAMP, PKA and/or KATP pathways in either PE or KCl-precontraction.

In conclusion, we found that imidazoline receptor is more highly expressed in the aorta of SHRs and this provides an explanation for the agmatine-induced decrease in blood pressure in SHRs but not in WKY rats. Hence peripheral imidazoline receptors represent an attractive new target in the development of therapeutic agents for the treatment of hypertension.

Acknowledgments

We thank Mr. K.F. Liu for technical assistance. The present study was supported in part by a grant from Chie-Mei Medical Center (CMF-HT-9801).

Funding source

None.

Conflicts of Interest

None.

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