INTRODUCTIONVarious imidazolines have cholinomimetic,

sympathomimetic, histaminelike, antihistamine andadrenergic properties. Second generation centralantihypertensives, such as moxonidine and

rilmenidine, have attenuated sedative (α2adrenoceptor-mediated) side effects atequihypotensive doses compared with the drugs offirst generation (1). While recognized as potentagonists at peripheral α2 adrenergic receptors,several studies suggested that the drugs also had

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, 1, 131–142www.jpp.krakow.pl

A. RADWANSKA1, J. DLUGOKECKA1, R. WASILEWSKI2, R. KALISZAN1

TESTING CONCEPTION OF ENGAGEMENT OF IMIDAZOLINE RECEPTORS IN IMIDAZOLINE DRUGS EFFECTS ON ISOLATED RAT HEART ATRIA

1Department of Biopharmaceutics and Phamacodynamics, Medical University of Gdansk, Gdansk, Poland;2Department of Pharmacology, Medical University of Gdansk, Gdansk, Poland

Recently, attention has been payed to the role of imidazolines in physiology of the heart. However,no systematic comparative studies were reported regarding the activity of a representative set ofspecific ligands towards imidazoline receptors in the heart preparations. The aim of this project wasto test effects of a set of ligands on the pharmacological function of putative imidazoline receptorsin isolated rat heart atria. Known imidazoline drugs with a postulated high affinity to imidazolineI1 receptor: AGN192403, rilmenidine, moxonidine and clonidine were used. The specific ligands ofimidazoline I2 receptor: 2-BFI, BU239 and putative natural ligand for imidazoline I1, I2 and I3receptors, agmatine, were tested also. The spontaneously beating right and left atria, drivenelectrically, were studied. Dose-response curves for amplitude and rate of the contractions of theatria were produced by administration of increasing doses of the agents. Phentolamine as α1/α2adrenergic receptors blocker and idazoxan as I2/I1/α2 receptors blocker were added in order toinhibit ino- and chronotropic effects of the compounds studied. The -log EC50 parameters werecalculated. The positive inotropic effect on left atria were evoked with the rank order of potency:agmatine >> clonidine > BU239 > rilmenidine ≥ moxonidine and these effects were generallydiminished by idazoxan. Moxonidine produced a weak positive inotropic effect potentiated byidazoxan. Rilmenidine and moxonidine were assumed to act as partial agonists of imidazoline I1receptor. AGN192403 did not change the amplitude of beating of left atria. The positivechronotropic effects on spontaneously beating right heart atria were with in the following order ofpotency: BU239 ≥ agmatine >>> clonidine > AGN192403. Idazoxan markedly antagonizedchronotropic effect of both BU239 and agmatine. 2-BFI weakly diminished the rate of beating ofatria; moxonidine and rilmenidine had no effect. In conclusion, imidazoline receptors of the I1subtype may be involved in inotropic reaction of the agents studied, but this effect depends mainlyon the α2/α1 adrenergic receptors. Engagement of I2 imidazoline receptors, along with the α2adrenergic ones, in chronotropic activity of isolated right atria of rat has been demonstrated.

K e y w o r d s : imidazoline receptors, rat heart atria, chronotropic and inotropic effect

another mode of action. Clonidine-like drugs, suchas moxonidine, rilmenidine and dexmedetomidine,reduce blood pressure by acting centrally at both α2adrenergic and imidazoline receptors. Imidazoline-binding sites (IBS) were discovered in centralnervous system (2) as well as in peripheral tissuesfrom various species (3).

Imidazoline receptors engaged in circulatorysystem are classified in two groups: the I1 type,sensitive to clonidine and idazoxan, an antagonistwith an imidazoline structure, and the I2 type,displaying a high affinity for idazoxan (4),guanabenz (5, 6) cirazoline (7), and a medium-to-low affinity for clonidine (4).

Imidazoline I1 receptors are reported to play arole in the central regulation of blood pressure (8).The selective activation of central I1imidazolinereceptors results in an inhibition of peripheralsympathetic activity and produces arterialvasodilatation (9, 10). Imidazoline agents evokediverse pharmacological responses in bothperipheral tissues and the central nervous system, sothat are difficult to attribute to known receptorsignaling system (11).

The presence of presynaptic imidazoline receptorshas been suggested in the human and rat heart buttheir functional role is unknown (12). Imidazoline I1receptors over α2 adrenoceptors in the heart atria andventricles have been identified with the affinity toimidazolines at nanomolar range (13). An 85 kDprotein may correspond to the functional I1 receptorin atria. It has been shown that atrial I1 receptors areup-regulated in spontaneously hypertensive rats(SHR) (14, 15). El-Ayoubi et al. (14) observed that I1receptors are increased in hypertensive rats orhumans. Therefore, there are remisses to suppose theengagement of imidazoline receptors in ino- andchronotropism of isolated heart atria (16).

Functional cardiac imidazoline I1 receptors aretissue-specific, being differentially regulated in atriaand ventricles in hypertension by chronic exposureto agonists (17).

In contrast to the I1 imidazoline receptors,physiological role for I2 sites has not yet beendetermined but it has been proposed that they play anactive role in various physiological processes (18).For example, antiproliferative action of theimidazolines correlated with their affinity to the I2imidazoline binding sites in blood vessels (2, 19).The imidazoline I2 binding sites (IBS) weredescribed as imidazoline-guanidinium receptive site(IGRS) with idazoxan binding selectivity (20, 21).IBS appear to be heterogeneous in nature (22). Uptoday, their molecular structure, functionalsignificance and their second-messenger system areunknown. I2 receptor ligands interact with a domain

on MAO, a catecholamine metabolizing enzyme, butthis mechanism is not equally accessible in alltissues (11, 23, 24).

Clonidine is twice as potent as moxonidine at theI1 receptor but has similar affinity for α2 and α1adreceptors (25). In binding assay on cow brain theKi values for clonidine at α2A and I1 receptors were3.8 and 1.0 nM, respectively (26). Hypotensiveeffect of clonidine is mainly through the α2adrenergic over I1 imidazoline receptors (27).Significant bradycardia of isolated rat heart wasobserved with clonidine, and less with moxonidine,at 10-6 M concentration. It suggests that postsynapticcardiac imidazoline I1 receptors may be involved inthese effects (28).

Moxonidine is pharmacologically similar toclonidine, but its affinity to imidazoline I1 receptorsover α2 adrenoceptors is 100-fold higher (29). Thehypotensive mechanism of moxonidine originallysuggested was through activation of central α2adrenoceptors, but it appeared that primary action inhypertension is due to binding of moxonidine atimidazoline I1 receptors in the rostroventrolateralpart of the brainstem (RVLM) (30). Moxonidine isthree times more selective for the I1 receptor inRVLM than rilmenidine and has 40-70 times greateraffinity for I1 receptors than for α2 adrenoceptors(25). Moxonidine and rilmenidine injectedintravenously lowered blood pressure, decreasedplasma norepinephrine concentrations and inhibitedstimulation-evoked cardioacceleration in pithedrabbits via α2 adrenoceptor mechanism (31).Inhibition of norepinephrine release by moxonidinein pithed SHR was demonstrated by Raasch et al.(32): the authors explain this effect by interactionwith imidazoline I1 receptor. Intravenousmoxonidine may activate imidazoline I1 receptorsand α2 adrenoceptors present in the rat heart.Compared with clonidine selectivity, moxonidineand rilmenidine has approximately 3 to 10 timesgreater affinity for imidazoline receptors (14, 17).

Rilmenidine exhibited antiarrhythmic effects indifferent animal models of arrhythmia (33). Thiseffect is likely to originate from effects on the centralnervous system as well as from an action atperipheral sites (34).

Rilmenidine is neutral regarding metabolicparameters, but it influences left ventricularhypertrophy, microalbuminuria and insulinresistance positively in hypertensives at risk (35).Widimsky and Sirotiakova (36) observed decrease ofarterial pressure and reduction in heart rate inhypertensive patients treated with 1mg rilmenidinedaily. That may be a clinically relevant benefit inpatients with an increased cardiovascular risk andmetabolic disorders.

132

Agmatine (decarboxylated arginine) is widelydistributed through the body, attaining high levels inthe rat aorta (57.41 ng/g) and in the rat heart (6.03ng/g), with the concentration in brain beingrelatively small (2.4 ng/g) (37). Molecular mass ofagmatine is 130 Da (24). The agent was found to beregionally distributed in the rat brain (38). Agmatineis relased from the neurons and interacts withvarious pre- and postsynaptic receptors including theI1 imidazoline receptor, α2 adrenoceptor, NMDAreceptor, nicotinic cholinergic receptor and 5-HT3.receptor which might be of physiological importance(39, 40). All that suggests that agmatine may be aneurotransmitter (41). Their affinity (Ki) in humanbrain for α2A and I1 imidazoline receptors is 46 980and 33.4 nM, respectively (26), although theinteraction of agmatine with α2 adrenoceptors isunclear (42, 43). It has been postulated that agmatinemay change uptake of norepinephrine, like clonidinedoes, thus reducing sympathetic tone throughimidazoline receptors (42).

Agmatine concentration-dependently releasesadrenaline and noradrenaline from chromaffin cells.This effect can be blocked by antagonists of the I1receptor. In chromaffin cells agmatine has a highaffinity to α2 adrenoceptors and I1 and I2 bindingsites, namely 4.0, 0.7 and 1.0 µM, respectively. It hasa low affinity to the α1 and β adrenoceptors as wellas to the 5-HT3 serotonin and D2 dopamine bindingsites. Agmatine-uptake into synaptosomes is blockedby the imidazoline derivative idazoxan and byphentolamine, but not by clonidine, moxonidine andrilmenidine (41).

Agmatine has no effect on vascular contractionand blood pressure, contrary to moxonidine, whichincreases the vascular contraction and decreasesblood pressure (39). However, Herman (44)suggested that it can regulate cardiovascularfunctions and modulate some processes in theperipheral and central nervous system, whereas it hasonly a weak blood pressure effect when appliedwithin the RVLM area. The affinity of agmatine forIBS is rather low, while it also binds α2adrenoceptors (41). In the experiments on rat hearts,agmatine increased norepinephrine level indicating asynergistic inhibitory action at I1 imidazolinereceptors under conditions of stimulated α2adrenergic autoinhibition (15).

Research on newly synthesized imidazolinecompounds with a high affinity to imidazolinereceptors is still carried on. It is believed that thebinding sites specifically recognizing theimidazoline structure or similar chemical structures,both in the brain and in certain peripheral tissues,including the heart, participate in the control ofblood pressure (14).

Imidazoline receptors bind some agents that are notimidazolines, such as guanabenz (45), guanidinium,rilmenidine (46) and oxazole. Newly synthesizedanalogues of imidazolines and reference oxazolineselicit an appreciably increased selectivity for I1 and I2receptors. A few of these compounds, namely "AGN"and "BU families", show prevailing affinities for I1 andI2 receptors. These compounds are the first imidazolineanalogues described that are devoid of any significantaffinity to α2 adrenergic receptors.

The isofuran derivative of imidazoline, AGN192403, is about 10,000 time more selective forimidazoline receptors than for α2 adrenoceptors (47).It had no effect on blood pressure when injectedintravenously in monkeys and rabbits. AGN wasfound to be an antagonists of imidazoline receptor ina number of studies (15, 48).

The benzofuran derivative, 2-BFI, appeared 1,000to 10,000 times more selective for [3H]idazoxanimidazoline specific binding sites than for α2adrenoceptors. Selective for the I2 imidazolinebinding sites agent 2-BFI has Ki of about 1 nM and alow activity for α2 adrenoceptors. It can elevateextracellular levels of norepinephrine in the frontalcortex and hippocampus in rats (49).

BU239 was described as a ligand with a highselectivity to I2 receptors in rabbit brain. Incompetitive binding assays on rat kidney membranesKi for BU239 was 4.3 nM (16, 50).

Distinction between the imidazoline receptor andthe α2 adrenoceptor-mediated mechanism forimidazoline compounds is difficult. A combinationof agonists and antagonists differing in affinities foreach receptor is required for that (26). The mostfrequently used imidazoline receptor antagonist,idazoxan, is also a potent α2 adrenoceptorantagonist. It seems that important for affinity ofagents towards I1 receptors is their hydrophobicity,as expressed by log P ranging from 1 to 2 (51).Therefore, structural alterations of idazoxan canresult in molecules with a marked selectivity foreither α2 adrenoceptors or imidazoline receptors.

Beside phentolamine, potent nonselective α1 /α2adrenoceptor antagonists with a low affinity athistaminergic receptors and less potent atimidazoline receptors are used. For now, there are noendogenous agonists selective for imidazolinereceptors. All drugs binding to imidazoline receptorsbind to α2 adrenergic receptors as well. It has notbeen possible to determine unambiguously whetherdrugs binding to I receptors act as agonists orantagonists and have actions comparable to theiractions at the α2 adrenergic receptors.

Imidazoline derivatives have been demonstrated tointeract with the sympathetic neurotransmission vianonadrenergic presynaptic receptors in different

133

experimental models. However, no systematiccomparative studies were reported regarding the ino-and chronotropic activity of a representative set ofimidazoline drugs towards imidazoline receptors in theheart preparations. On the other hand, single individualimidazolines have been reported to elicit pronouncedpharmacological effects mediated through cardiacreceptors. The direct effect of an imidazoline receptorligand on cardiac receptors has not been established.Still, new imidazoline derivatives are potential drugsfor antihypertensive therapy.

The aim of this study was to determine in vitro theinotropic and chronotropic effect of newly synthesizedimidazoline receptor ligands: 2-BFI, BU239 andAGN192403 as well as the known imidazoline drugs,like clonidine, rilmenidine, moxonidine and agmatineon isolated rat atria. The agents studied were selectedfrom the point of view of their hypothetical interactionwith the adrenergic/imidazoline receptors in cardiaccells. The main task of this project was to help to directrationally the further search for original circulatory andantihypertensive imidazoline agents based on theircardiotropic properties.

MATERIALS AND METHODSThe procedure applied was designed in

accordance with the respective Polish and Europeanregulations and the guidelines established by theEthics Committee for Animal Experiments of theMedical University of Gdansk, Poland.Materials

Male Wistar rats (200-350 g) were used for invitro studies. The animals were housed and fed in alaboratory kept at constant temperature of 22°Cunder the standard conditions (12:12 h L:D cycle,standard pellet diet, tap water).Methods

The animals were anesthetized with urethane (1.5g/kg i.p.) and the heart was rapidly excised. Aftercervical dislocation, thorax was quickly opened, thestill beating heart removed and placed in thepreparation dish with a modified Krebs-Henseleitsolution. The ventricular tissue was cut away as far aspossible. The atria, left and right separately, wereplaced in the solution, gassed with 95 % O2 and 5 %CO2, giving pH of 7.3-7.4, and kept at 35.5-37°C. Theincubation medium contained NaCl 118 mmol, KCl4.7 mmol, CaCl2 6 mmol, NaH2PO4 1 mmol, MgCl21.2 mmol, NaHCO3 25 mmol, glucose 11.1 mmol,EDTA 0.04 mmol, and ascorbic acid 0.1 mmol.

The muscle was tied at either end to stainlesshooks under a tension of 0.5-1.0 g in an organ bathand was allowed to stabilize for 45-60 min. The leftatrium was electrically stimulated with twoplatinum electrodes by square-wave electricalpulses (2.5 Hz, 4 ms) and voltage 10 V. Amplitudeof contractile tension (mm) of the left atrium and therate of contractile action (min-1) of thespontaneously beating right atrium were recordedby means of an isometric force transducer (Bio-Sys-Tech, Bialystok, Poland).Experimental protocol

After equilibration period, the cumulativeconcentration-response curves were constructed forincreasing concentrations of the imidazolines studiedranging from 10-11 to 10-3 M. Structure of compoundsstudied shows Fig. 1.

In the next stage of the experiment, the inotropicand chronotropic responses to imidazolines studiedwere measured at the presence of fixed concentrations(from 10-9 to 10-3 M) of the imidazoline blockers(idazoxan or/and phentolamine).Data presentation and statistical evaluation

Cumulative concentration-response curves withvariable slope were constructed and analysed by

134

NH

NNH

Cl

Cl x HCl

Clonidine

NH

NN

N

NH

Me

OMe

Moxonidine

O

NNH

+

Rilmenidine

x H2SO4-

NH2

MeMe

AGN192403

x HCl

NH

N

O x HCl

2-BFI

NH

N

N

N

BU239

NH2 NH

NH2

NH

x H2SO4

Agmatine

NH

N

O

O

x HCl

Idazoxan

Fig.1. Chemical structure of compounds studied.

x HCl

means of GraphPad Prism4 software (GraphPadSoftware Inc., San Diego, Ca). Each point of thecurves was a mean of at least 6 experiments. Thechanges of responses to each concentration wasexpressed as percent of the control value (100 %) ofatrial rate or amplitude preceding the administrationof cumulative concentrations of an agent with orwithout idazoxan or phentolamine. Data are reportedas mean SEM (standard error of mean). Based on theprofile of concentration-response behavior of isolatedorgans for the compounds studied, the -log EC50parameters were calculated (the concentration of theligand producing the half of the maximal effectobserved). A significance level was taken as p ≤ 0.05or p ≤ 0.01 in comparison of the compound studiedalone and the same compound pretreated withidazoxan or phentolamine. Nonparametric analysiswas done by U'Mann-Whitney (unpaired) test usingStatistica 7.1 software (StatSoft Inc., Tulsa, OK, USA)

Data obtained are shown in Figs. 2-6 and inTables 1-3.Drugs

Pure chemical substances were used for thepreparation of bath solutions of the drugs studied.

Phentolamine and idazoxan were obtained fromSigma (Steinheim, Germany); clonidine from

Boehringer (Ingelheim, Germany); agmatine [(4-aminobutyl)guanidine], AGN192403 (2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane) and BU239 (2-(4,5-dihydroimidazol-2-yl)quinoxaline) were fromTocris (Bristol, UK); 2-BFI (2-(2-benzofuranyl)-2-imidazoline) was from Tocris (London, UK);rilmenidine was a gift from Servier (Paris, France) andmoxonidine was a gift from Dr. B. I. Armah, (BDFResearch Laboratories, Hamburg, Germany). Stocksolutions of each agent for in vitro studies were 10-3 M.The concentrations of idazoxan were 10-3, 10-5, 10-6,10-7 and 10-9 M and phentolamine 10-9 M. Stocksolutions were diluted with water ex tempore beforeindividual experiments.

RESULTSInotropic activity

It has been demonstrated that clonidine,moxonidine and rilmenidine elicit the positiveinotropic activity on electrically stimulated leftatria with maximum effects of 132.1, 116.2 and118.3 per cent, respectively (Fig. 2A, Table 1). The-log EC50 values observed for clonidine,moxonidine and rilmenidine were 5.2, 6.2 and 5.1,respectively (Table 2).

135

Fig.2. Effect of cumulativeconcentrations of the imidazolinecompounds studied oncontractility of the left rat heartatria (A) and on beating rate ofthe right rat heart atria (B).The data are shown as means ofat least 6 experiments SEM

Compound studied Amplitude of contractility Rate of contraction Clonidine 132.1 19.8 (10-3) 110.2 5.9 (10-7)

Moxonidine 116.2 3.1 (10-5) 99.1 0.9 (10-11) Rilmenidine 118.3 4.7 (10-3 ) 100.9 1.2 (10-4) AGN192403 100.4 0.2 (10-10) 110.4 1.5 (10-4)

2-BFI 76.1 8.8 (10-5) 93.5 4.6 (10-3) BU239 117.4 6.1 (10-5) 131.0 12.5 (10-3)

Agmatine 142.0 9.3 (10-3) 125.7 2.4 (10-3)

Table 1. Inotropic and chronotropic effects of the cumulative concentrations of the compounds studied. Maximum effect observed areexpressed as % of control (at indicated molar concentration).

The presence of idazoxan 10-5 M and 10-3 Mdiminished positive inotropic effect of clonidine andrilmenidine (Figs. 3A, 3C). The -log EC50 values forclonidine and rilmenidine increased afterpretreatment with idazoxan 10-3 and 10-5 M (Table2). Surprisingly, moxonidine produced positiveinotropic effect at the presence of idazoxan 10-5 M(Fig. 3B). The antagonism at the presence ofidazoxan 10-3 M manifested itself at the highconcentrations (10-5 - 10-3 M) of moxonidine. The -log EC50 for moxonidine alone was 6.2 and itincreased to 7.2 after pretreatment with the 10-5 Midazoxan (Table 2).

AGN192403 in cumulative concentrations from10-11 to 10-3 M does not act on amplitude of beatingof left atria. A pretreatment with idazoxan 10-9 and10-6 M or phentolamine 10-9 M increases inotropicactivity of AGN 192403 but these effects are of nostatistical significance.

Positive inotropic effect of compound 2-BFIappeared only at very low agent's concentrations(10-11-10-8 M). In higher concentrations, 2-BFIdecreased the amplitude of contraction of left atriaup to 76.1 %, in comparison to the 100% of control.The presence of idazoxan 10-3 M partiallydiminished inotropic effect of 2-BFI but

136

Fig.3. Effect of cumulativeconcentrations of clonidine(Clo) (A), moxonidine (Mox)(B) and rilmenidine (Ril) (C)alone and in the presence offixed concentrations ofidazoxan (Ida) on contractilityof the left rat heart atria.The data are shown as means of6 experiments SEM; p 0.05denotes significance level ofdifferences between resultsobtained for the compoundsalone and after pretreatmentwith idazoxan (Ida) ascompared with the U'Mann-Whitney, unpaired test.

Compound studied Amplitude of contractility -log EC50

Rate of contraction -log EC50

Clonidine (Clo) 5.2 10.6 Clo +Ida 10-5 5.7 8.9 Clo +Ida 10-3 9.8 8.9

Moxonidine (Mox) 6.2 n.a Mox +Ida 10-5 7.2 n.a. Mox +Ida 10-3 5.3 n.a.

Rilmenidine (Ril) 5.1 n.a Ril +Ida 10-5 7.2 n.a Ril +Ida 10-3 9.9 n.a.

AGN192403 (AGN) 7.5 7.0 AGN +Ida 10-9 10.6 7.5 AGN +Ida 10-6 n.a. 3.9

AGN + Phen 10-9 n.a. 4.0

Table 2. The -log EC50 value for compounds studied alone and at the presence of idazoxan (Ida) or phentolamine (Phen).

concentration 10-5 M of idazoxan remained withouteffect on 2-BFI (Fig. 4A). The -log EC50 values of 2-BFI alone and pretreated with idazoxan 10-5 M wereequally 7.0, and for 2-BFI pretreated with idazoxan10-3 M both values were 9.5 (Table 3).

Compound BU239, which is structurally related to2-BFI and has been reported to label I2 receptors,produced positive inotropic activity with themaximum effect observed of 117.4 % (Fig. 4B).Idazoxan at concentration 10-7 M slightly diminishedthe left atria amplitude of contraction evoked byBU239 whereas idazoxan 10-9 M increased the

activity of BU239. The presence of phentolamine 10-9M had no effect on inotropy of BU239. The -log EC50values were also diminished from 8.8 for BU239alone to 8.3 for BU239 with idazoxan 10-5 M, to 6.6for BU239 with idazoxan 10-7 M and to 7.2 for BU239with phentolamine 10-5 M (Table 3).

The most marked positive inotropy was observedfor agmatine with maximal effect observed of 142.0% and the -log EC50 of 8.2 (Fig. 4C, Table 3).Pretreatement with idazoxan 10-7 M or 10-3 Magmatine decreased the amplitude of left atria.Phentolamine 10-9 M antagonized the inotropism of

137

Fig.4. Effect of cumulativeconcentrations of 2-BFI (A),BU239 (B) and Agmatine (Agm)(C) alone and in the presence offixed concentrations of idazoxan(Ida) on contractility of the leftrat heart atria.The data are shown as means of6 experiments SEM; p 0.05denotes significance level ofdifferences between resultsobtained for the compoundsalone and after pretreatmentwith idazoxan (Ida) as comparedwith the U'Mann-Whitney,unpaired test.

Compound studied Amplitude of contractility -log EC50

Rate of contraction -log EC50

2-BFI 7.0 6.9 2-BFI +Ida 10-5 7.0 8.1 2-BFI +Ida 10-3 9.5 7.1

BU239 (BU) 8.8 7.3 BU +Ida 10-9 8.3 7.0 BU +Ida 10-7 6.6 8.3 BU +Ida 10-5 n.a. 7.7

BU + Phen 10-9 7.2 7.3 Agmatine (Agm) 8.2 8.2 Agm +Ida 10-9 7.7 7.6 Agm +Ida 10-7 7.6 7.2 Agm +Ida 10-5 n.a. 5.4 Agm +Ida 10-3 7.8 5.8

Agm + Phen 10-9 6.7 6.3

Table 3. The -log EC50 value for compounds studied alone and at the presence of idazoxan (Ida) or phentolamine (Phen).

agmatine also. The -log EC50 values of agmatinewith antagonists were diminished as has beenpresented in Table 3.

Idazoxan in cumulative concentrations did notaffect the amplitude of beating of left atria: themaximum effect was 105.7 %. Also phentolamineremained without effect on the inotropic effect ofleft atria.

Chronotropic activityClonidine produced a weak positive chronotropic

effect on the right atria up to maximum of 110.2 %(Figs. 2B and 5A). Idazoxan at concentrations 10-5and 10-3 M markedly antagonized positivechronotropic activity of clonidine and decreased the-log EC50 value about 100-fold (Table 2).

138

Fig.5. Effect of cumulativeconcentrations of clonidine (Clo)(A), moxonidine (Mox) (B),rilmenidine (Ril) (C) andAGN192403 (D) alone and in thepresence of fixed concentrationsof idazoxan (Ida) on beating rateof the right rat heart atria.The data are shown as means of6 experiments SEM; p 0.05, p0.01 denotes significance levelof differences between resultsobtained for the compoundsalone and after pretreatmentwith idazoxan (Ida) as comparedwith the U'Mann-Whitney,unpaired test.

Fig.6. Effect of cumulativeconcentrations of 2-BFI (A),BU239 (B) and Agmatine (Agm)(C) alone and in the presence offixed concentrations of idazoxan(Ida) on beating rate of the rightrat heart atria.The data are shown as means of6 experiments SEM; p 0.05denotes significance level ofdifferences between resultsobtained for the compoundsalone and after pretreatmentwith idazoxan (Ida) as comparedwith the U'Mann-Whitney,unpaired test.

Rilmenidine and moxonidine had no effect, eitheralone or pretreated with idazoxan, on right atria(Figs. 5B, 5C).

AGN192403 increased weakly the rate ofbeating of the right atria (maximum effect was 110.4%). In experiments with preexposure to idazoxan10-9 M or 10-6 M, the antagonism with regards tochronotropic action occured with the statisticalsignificance of p< 0.05. In the case of preexposurewith phentolamine 10-9 M, there was no change ofthis effects (Fig. 5D). The -log EC50 values werechanged for AGN192403 when pretreated withidazoxan 10-6 M and with phentolamine 10-9 M,from 7.0 to 3.9, and 4.0, respectively.

2-BFI decreased weakly the rate of beating of theright atria (from maximum effect of 106.5 tominimum of 93.0 %) (Fig. 6A). In the presence ofvarious concentrations of idazoxan, the chronotropiceffect of 2-BFI was not changed (Fig. 6A). The -logEC50 values from experiments with 2-BFI untreatedand treated with either idazoxan or phentolaminewere ever in the range from 6.9 to 8.1 (Table 3).

BU239 in cumulated concentrations significantlyincreased the rate of beating of the right atria and themaximum effect observed was 131.0 %. However,in the presence of idazoxan 10-7 and 10-9 M thechronotropic effect of BU239 was reduced (Fig.6B). Phentolamine 10-9 M, added to BU239preincubated with idazoxan 10-9, 10-7 or 10-5 M, hadno effect. All the values of -log EC50 fromexperiments involving BU239 were similar, rangingonly from 7.3 to 8.3 (Table 3).

Agmatine in cumulative concentrationsaccelerated the rate of the spontaneously beatingright heart atria to a maximum of 125.7 % (Fig. 6C).Idazoxan in concentrations 10-7, 10-5 or 10-3 Mattenuated agmatine's positive chronotropic effect.Phentolamine 10-9 M, added to agmatine pretreatedwith idazoxan, significantly decreased the rate ofbeating of the atria. The -log EC50 value decreasedabout 100-fold (Table 3).

Neither idazoxan nor phentolamine in cumulativeconcentrations had any marked effect onchronotropic activity of right atria.

DISCUSSIONInotropy

In this work manifestations of positive inotropicactivity were shown in case of agmatine, clonidine,rilmenidine and moxonidine. The narrow range of -log EC50 values for clonidine, rilmenidine andmoxonidine from 5.1 to 6.2 indicates a similarreceptor-mediated effect of those drugs. Idazoxandiminished significantly statistically the positive

inotropic effect of clonidine and rilmenidine, but notthat of moxonidine. The positive inotropic effect ofrilmenidine was diminished by idazoxan, unlessidazoxan was applied at a high concentration 10-3M,suggesting that rilmenidine exhibits some selectivityfor imidazoline receptors. Rilmenidine is usuallyconsidered to be an I1 receptor ligand. However,recent evidences show that it may also label an I2-like site (52). Moreover, it was reported that thehypotensive effect of rilmenidine in humans waspotently antagonized by idazoxan, whereas it wasweakly or not at all antagonized by yohimbine. Atthe oral dose of 2 mg, rilmenidine has no effect onbeating rate of the heart (53, 54).

Molderings et al. (52) observed that rilmenidineand oxymetazoline are potent full agonists to α2adrenoceptors in rabbit hearts, whereas in the humanatrial appendages both agents are antagonists at the α2autoreceptors, like rauwolscine and idazoxan are.Prazosin is ineffective in that preparation. Theantagonistic activity of rilmenidine towards human α2Aadrenoceptors indicates that, in contrast to thesuggestion based on rabbit data, the hypotensive effecton humans is not due to activation of α2Aadrenoceptors but other, presumably I1 imidazoline,receptors are involved (52). In our work, the increasing-log EC50 values for clonidine and rilmenidine at thepresence of idazoxan 10-3 M, suggest dual interactionof the imidazolines with the α2 adenergic and theimidazoline I1 receptors. In the case of clonidine itwould confirm the hypothesis that its positive inotropiceffect at low doses is mainly due to a stimulation ofpostsynaptic α adrenoceptors, whereas additionalstimulation of the imidazoline receptors occurs athigher drug doses. Certainly our results do not excludethe possibility that rilmenidine and clonidine elicittheir effects through putative presynaptic imidazolinereceptors, at least as regards the inotropic activity.

In our study moxonidine elicited a weak positiveinotropic effect on left atrium. This finding is inaccordance with the results obtained by Raasch et al.(55). Evidently the presence of idazoxan potentiatesthe inotropic effect of moxonidine. It suggests thatmoxonidine acts as an agonist-antagonist on bothadrenoceptors and imidazoline receptors. The -log EC50 value increases in the presence of idazoxanat concentration 10-5 M, but it decreases when 10-3 Midazoxan is added to the incubation medium. On theother hand, it has been reported, that moxonidinereduced norepinephrine release independently of I1receptor, thus suggesting the prominent effect of α2adrenoceptors in cardiac tissue (52). Moxonidine bindswith different affinities to cardiac imidazoline I1receptors, α2 adrenoceptors (56), and, at someconditions, to α1 adrenoceptors. Raasch et al. (55)explain the increase of contractility of left rat atria by

139

stimulation of postsynaptic α1 adrenoceptors ratherthan the imidazoline I1 receptors. Hovewer, inexperiments consisting in chronic moxonidinetreatment of the spontaneously hypertensive ratsMukaddam-Daher and Gutkowska (16) and El-Ayoubiet al. (17) observed the specific binding withmoxonidine at the atrial I1 subtype receptors.

A structurally related to moxonidine compoundAGN192403 did not change the amplitude of beating ofthe left atria. However, in the presence of variousconcentrations of idazoxan, AGN192403 showed aweak nonsignificant positive inotropic effect,potentiated with 10-9 M of either phentolamine oridazoxan, similarly as in experiments with moxonidine.Most authors suggest that AGN192403 has no effect oncirculatory system. However, its general behaviour maysuggest it to be a selective ligand of I1 receptor (15, 57,58) in the experiments on rat hearts, demonstrated thatAGN192403 had no influence on norepinephrine level.According to these authors AGN192403 seems to be anantagonist to the imidazoline I1 receptor and thepotentiation of inotropic activity by idazoxan seems toresult from synergistic interactions.

In the case of agmatine most important areincreases of the amplitude of the left atriacontraction. Idazoxan at concentration of 10-7, 10-5and 10-3 M, and phentolamine at concentration of 10-9 M, diminished inotropic activity in a dosedependent manner. Except of imidazoline receptors,agmatine has affinity to both the α/β adrenergic anddopaminergic receptors. Its effect is mediatedprobably by all those receptor sites in cardiac tissue.

Compound 2-BFI and a more potent ligand at I2imidazoline receptor, BU239, evoked a very weakpositive inotropic activity. The presence of idazoxandiminished inotropic effect of 2-BFI and BU239without statistical significance. The -log EC50 valuesfor BU239, 2-BFI and idazoxan were 8.8, 7.0 and7.0, respectively. The reported binding affinities, Kifor I2 receptors in rat brain membrane are 4.2 and 7 nM for BU239 and 2-BFI, respectively (19).Pharmacometric analysis of the data obtainedinvolving 2-BFI and BU239 lead to conclusion thatthese ligands cannot be clearly identified as eitheragonists or antagonists of the I2 receptor. Thishypothesis is in accordance with the conclusion fromthe study on the relaxation of rat jejnum evoked by2-BFI and idazoxan (59).

In our conclusion, the positive inotropic actionon isolated rat heart left atria is with the followingrank order for the agents studied: agmatine >>clonidine > BU239 ≥ rilmenidine ≥ moxonidine.Rilmenidine and moxonidine act as partial agonistsof the imidazoline I1 receptors. In inotropic effectsof these imidazolines both the I1 and α2 receptorsare engaged. Inotropic effect of clonidine and

agmatine is mostly due to the α adrenoceptorsactivation. The role of I2 imidazoline receptors isnot to convince.Chronotropy

Agmatine and clonidine were found to elicitpositive chronotropic effect on the right rat heart atria.Idazoxan markedly antagonized activity of clonidine,but independly of the dose used. It is well known thatclonidine is an agonist not only of the α2 but also ofthe α1 adrenoceptors, present in the right atria.

Phentolamine 10-9 M, added to agmatine,significantly decreased positive chronotropic effectof agmatine. In opposite, in the experiments of someauthors (41, 60) agmatine did not influencecontractions of isolated rat heart atria. Agmatine hasaffinity to both the α adrenergic and the imidazolinereceptors. Some investigators demonstrated thatagmatine recognizes α2 adrenoceptors but is withouteffect on these receptors (61). To date, there are noproofs of action of agmatine attributable to agonismor antagonism at the site in vitro.

In the present study, rilmenidine and moxonidinehad almost no effect on the spontaneously beatingright atria. Pretreatment with idazoxan attenuated thechronotropic effect of the drugs, but this antagonismagainst the chronotropic effect of rilmenidine andmoxonidine never reached statistical significance,presumably because the induced effects were verysmall. Moxonidine and rilmenidine are the mostselective agonists for I1 receptors among imidazolineagents. Nevertheless, some authors classifymoxonidine and rilmenidine among selective α2adrenoceptor agonists clainning that this receptormay be predominant in the chronotropic activity ofthe assumed I1 imidazoline receptor agonists.

Compound AGN192403 possesses a smallpositive chronotropic activity on right atrium andthis effect is blocked by idazoxan 10-6 M andphentolamine 10-9 M as proved by the decreased -log EC50 values.

An I2 imidazoline receptor ligand, 2-BFI, had avery weak negative chronotropic activity, probablydue to its antagonistic activity towards the I2 receptorsubtype (19). After pretreatement with idazoxan theeffect of 2-BFI remains unchanged.

Another I2 receptor ligand, BU239, is the mostpotent of all the agents studied in increasing thebeating rate of the right atria. An antagonist, idazoxan,applied at various concentrations diminished thisactivity in an irregular manner. The -log EC50 valuesdetermined after the preincubation with idazoxan indifferent concentrations, were closely similar. It maysuggest that the chronotropic mechanism of BU239involves the imidazoline I2 receptors.

140

In conclusion, regarding to the maximal efffectobserved, the positive chronotropic action onisolated rat heart right atria were with the rank orderfor the agents studied: BU239 ≥ agmatine >>clonidine > AGN192403.

In view of our research, the engagement ofimidazoline receptors in the chronotropic responseof rat heart atria to imidazoline drugs still remainsdisputable. Certainly, regarding to chronotropiceffect of agmatine and clonidine we feel obliged toacknowledge an involvement of the α2/α1 adrenergicreceptors. However, as concerns BU239, the resultsobtained by us demonstrate that its activity on the ratright heart atrial beating rate is exerted via theimidazoline I2 receptors.

REFERENCES1. Molderings GJ. Imidazoline receptors: basic knowledge,

recent advances and future prospects for therapy anddiagnosis. Drugs Future 1997; 22: 757-772.

2. De Vos H, Bricca G, De Keyser J, De Backer J-P, BousquetP, Vauquelin G. Imidazoline receptors, non-adrenergicidazoxan binding sites and α2 adrenoceptors in the humancentral nervous system. Neuroscience 1994; 59: 589-598.

3. Bousquet P, Greney H, Bennai F et al. Imidazoline receptorsand cardiovascular regulations. A statement. Ann NY AcadSci 1995; 763: 526-530.

4. Ernsberger P. Heterogeneity of imidazoline binding sites:proposed I1 and I2 subtypes. Fund Clin Pharmacol 1992;6(Suppl.1): 55.

5. Miralles A, Olmos G, Sastre M, Barturen F, Martin J, Garcia-Sevilla JA. Discrimination and pharmacologicalcharacterization of I2-imidazoline sites with [3H]idazoxanand α2-adrenoceptor with [3H]RX821002 (2-methoxyidazoxan) in the human and rat brains. J PharmacolExp Ther 1993; 264: 1187-1197.

6. Wikberg JES, Uhlen S, Chjalari V. Evidence that drugbinding to non-adrenergic [3H]idazoxan binding sites (I-receptors) occurs to interacting or interconvertible affinityforms of the receptor. Pharmacol Toxicol 1992; 70: 208-219.

7. Greney H, Molines D, Bousquet P, Dontenwill M.Heterogeneity of imidazoline binding sites revealed by acirazoline derivative. Eur J Pharmacol 1994; 271: 533-536.

8. Morgan NG, Chan SLF. Imidazoline receptors and theirligands as potentiators of nutrient-induced insulin secretion.Drug Design Reviews 2004; 1: 185-193.

9. Scheen AJ. Pharma Clinics. Medication of the month.Moxonidine (Moxon). Rev Med Liege 2000; 55: 61-63.

10. Camilleri G, Portal B, Quiniou G, Clerson P. Comparison ofthe efficacy and the safety of two imidazoline receptorsagonists: rilmenidine and moxonidine. Ann Cardiol Angiol2001; 50: 169-174.

11. Raddatz R, Savic SL, Laniers SM. Imidazoline bindingdomains on MAO-B. Localization and accessibility. Ann NYAcad Sci 1999; 881: 26-31

12. Molderings GJ, Göthert M. Imidazoline binding sites andreceptors in cardiovascular tissue. Gen Pharmacol 1999;32: 17-22.

13. Trendelenburg A-U, Sutej I, Wahl CA, Molderings GJ,Rump LC, Starke K. A re-investigation of questionablesubclassifications of presynaptic α2-autoreceptors: rat venacava, rat atria, human kindey and guinea-pig urethra.Naunyn-Schmiedeberg's Arch Pharmacol 1997; 356: 721-737.

14. El-Ayoubi R, Gutkowska J, Regunathan S, Mukaddam-Daher S. Imidazoline receptors in the heart: characterization,distribution and regulation. J Cardiovasc Pharmacol 2002;39: 875-883.

15. Schafer U, Burgdorf C, Engelhardt A, Raasch W, Kurz T,Richardt G. Moxonidine displays a presynaptic alpha-2-adrenoceptor-dependent synergistic sympathoinhibitoryaction at imidazoline-I1 receptors. Ann NY Acad Sci 2003;1009: 265-269.

16. Mukaddam-Daher S, Gutkowska J. Imidazoline receptors inthe heart: a novel target and a novel mechanism of actionthat involves atrial natriuretic peptides. Braz J Med Biol Res2004; 37: 1239-1245.

17. El-Ayoubi R, Menaouar A, Gutkowska J, Mukaddam-DaherS. Imidazoline receptors, but not alpha2-adrenoceptors areregulated in SHR heart by chronic moxonidine treatment. JPharmacol Exp Ther 2004; 310: 446-451.

18. Bousquet P, Bruban V, Schann S, Feldman J. Imidazolinereceptors: a challenge. Pharm Acta Helv 2000; 74: 205-209.

19. Regunathan S, Youngson C, Raasch W, Wang H, Reis DJ.Imidazoline receptors and agmatine in blood vessels: a novelsystem inhibiting vascular smooth muscle proliferation. JPharmacol Exp Ther 1996; 276: 1272-1282.

20. Coupry I, Limon I, Tesson F, Lachaud V, Gargalidis-Moudanos C, Parini A. Imidazoline-guanidine site: a subtypeof imidazoline receptors. Therapie 1992: 47: 519-524.

21. Holt A, Wieland B, Baker GB. Allosteric modulation ofsemicarbazide-sensitive amine oxidase activities in vitro byimidazoline receptor ligands. Br J Pharmacol 2004; 143:495-507.

22. MacInnes N, Handley S. Autoradiographic localization of[3H]2-BFI imidazoline I2 binding sites in mouse brain. EurJ Pharmacol 2005; 516: 139-44.

23. Romer L, Wurster S, Savola J-M, Raasmaja A.Iidentification and characterization of the imidazoline I2-binding sites in the hamster brown adipose tissue as a studymodel for imidazoline receptors. Arch Physiol Biochem2003; 111: 159-166.

24. Raasch W, List B, Hauser W, Schafer U, Dominiak P.Presynaptic release of noradrenaline is mediated not onlythrough α2-adrenoceptors but also through imidazolinebinding sites. Dtsch Med Wschr 1999; 124: S114.

25. Khan ZP, Ferguson C, Jones RM. Alpha-2 and imidazolinereceptor agonists. Their pharmacology and therapeutic role.Anaesthesia 1999; 54: 146-165.

26. Head GA, Mayorov DN. Imidazoline receptors, novel agentsand therapeutic potential. Cardiovasc Hematol Agents MedChem 2006; 4: 17-32.

27. Chan CKS, Sannajust F, Head GA. Role of imidazolinereceptors in the cardiovascular actions of moxonidine,rilmenidine and clonidine in conscious rabbits. J PharmacolExp Ther 1996; 276: 411-420.

28. Mukaddam-Daher S, Menaouar A, Gutkowska J. Receptorsin moxonidine stimulated atrial natriuretic peptide releasefrom isolated normotensive rat hearts. Eur J Pharmacol2006; 541: 73-79.

141

29. Mukaddam-Daher S, Gutkowska J. Atrial natriuretic peptideis involved in renal actions of moxonidine. Hypertension2000; 35: 1215-1220.

30. Urban R, Szabo B, Starke K. Involvement of peripheralpresynaptic inhibition in the reduction of symphathetic toneby moxonidine, rilmenidine and UK14.304. Eur JPharmacol 1995; 282: 29-37.

31. Szabo B, Bock C, Nordheim U, Niedewrhoffer N.Mechanism of the sympatho-inhibition produced by theclonidine-like drugs rilmenidine and moxonidine. Ann NYAcad Sci 1999; 881: 264.

32. Raasch W, Jungbluth B., Schäfer U, Hauser W, Dominiak P.Modification of noradrenaline release in pithedspontaneously hypertensiverats by I1-binding sites inaddition to alpha2-adrenoceptors. J Pharmacol Exp Ther2003; 304: 1063-1071.

33. Bousquet P. I1 receptors, cardiovascular function andmetabolism. Am J Hypertens 2001; 14: 317-321.

34. Alemany R, Olmos G, Escriba PV, Menargues A, Obach R,Garcia-Sevilla JA. LNS, a selective ligand for imidazoline I2receptors, on glial fibrillary acidic protein concentration. EurJ Pharmacol 1995; 280: 205-210.

35. Kourilsky O. ITERIUM - Clinical benefits from aninnovative antihypertensive treatment. J Cardiol 2003;10(Suppl. D): 8-13.

36. Widimsky J, Sirotiakova J. Efficacy and tolerability ofrilmenidine compared with israpidine in hypertensivepatients with features of metabolic syndrom. Curr Med ResOpin 2006; 22: 1287-1294.

37. Raasch W, Regunathan S, Li G, Reis DJ. Agmatine, thebacterial amine, is widely distributed in mammalian tissues.Life Sci 1995; 56: 2319-2330.

38. Otake K, Ruggiero DA, Regunathan S, Wang H, Milner TA,Reis DJ. Regional localization of agmatine in the rat brain:an immunocytochemical study. Brain Res 1998; 787: 1-14.

39. Raasch W, Schafer U, Chun J, Dominiak P. Biologicalsignificance of agmatine, an endogenous ligand atimidazoline binding sites. Brit J Pharmacol 2001; 133:755-780.

40. Reis DJ, Regunathan S. Agmatine: an endogenous ligand atimidazoline receptors may be a novel neurotransmitter inbrain. J Auton Nerv Syst 1998; 72: 80-85.

41. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, ReisDJ. Agmatine: an endogenous clonidine displacingsubstance in brain. Science 1994; 263: 966-969.

42. Pinthong D, Wright IK, Hannar C et al. Agmatine recognizesalpha-2 adrenoceptor binding sites but neither activates norinhibits alpha-2 adrenoceptors. Naunyn Schmiedebergs ArchPharmacol 1995; 351: 10-16.

43. Pineda J, Ruiz-Ortega JA, Martin-Ruiz, Ugedo L. Agmatinedoes not have activity at alpha 2-adrenoceptors whichmodulate the firning rate of locus coreuleus neurones: anelectrophysiological study on rats. Neurosci Lett 1996; 219:103-106.

44. Herman ZS. Agmatine - a novel endogenous ligand ofimidazoline receptors. Pol J Pharmacol 1997; 49: 85-88.

45. Bricca G, Greney H, Dontenwill-Kieffer M, Zhang J,Belcourt A, Bousquet P. Heterogenity of the specificimidazoline binding of [3H]-idazoxan in the human cerebralcortex. Neurochem Int 1993; 22: 153-163.

46. Reis DJ, Regunathan S, Meeley MP. Imidazole receptors andclonidine-displacing substances in relationship to controlblood pressure, neuroprotection and adrenomedullarysecretion. Am J Hypertens 1992; 5: S51-S57.

47. Munk SA, Lai RK, Burke JE et al. Synthesis andpharmacologic evaluation of 2-endo-amino-3-exo-isopropylbicyclo[2,2,1]heptane: a potent imidazoline1receptor specific agent. J Med Chem 1996; 39: 193-195.

48. Velliquette RA, Ernsberger PJ. The role of I1-imidazolineand α2-adrenergic receptors in the modulation of glucosemetabolism in the spontaneously hypertensive obese ratmodel of metabolic syndrome X. J Pharmacol Exp Ther2003; 306: 646-657.

49. Angel I, Le Rouzic M, Pimoule C, Graham D, Arbilla S.[3H]-Cirazoline as a tool for the characterization ofimidazoline sites. Ann NY Acad Sci 1995; 763: 112-124.

50. Hudson AL, Gough R, Tyacke RJ et al. Novel selectivecompounds for the investigation of imidazoline receptors.Ann NY Acad Sci 1999; 881: 81-91.

51. Garaj V, Remko M. Conformational study of drugs witheffect on I1-imidazoline receptors. Ceska Slov Farm 2002;51: 196-199.

52. Molderings GJ, Menzel S, Kathmann M, Schlicker E,Gothert M. Dual interaction of agmatine with the rat alpha(2D)-adrenoceptor: competitive antagonism and allostericactivation. Br J Pharmacol 2000; 130: 1706-1712.

53. Head GA, Godwin SJ, Sannajust F. Differential receptorsinvolved in the cardiovascular effects of clonidine andrilmenidine in conscious rabbits. J Hypertens Suppl 1993;11(5): S322-S325.

54. Feldman J, Tibirica E, Bricca G, Dontenwill M, Belcourt A,Bousquet P. Evidence for the involvement of imidazolinereceptors in the central hypotensive effect of rilmenidine inthe rabbit. Br J Pharmacol 1990; 100: 600-604.

55. Raasch W, Chun KRJ, Dendorfer A, Dominiak P. Positiveinotropic effects of imidazoline derivatives are not viaimidazoline binding sites, but alpha1-adrenergic receptors.Jpn J Pharmacol 2000; 84: 1-6.

56. Haxiu MA, Dreshaj I, Schafer SG, Ernsberger P. Selectiveantihyprtensive action of moxonidine is mediated by I1-imidazoline receptors in the rostral ventrolateral medulla. JCardiovasc Pharmacol 1994; 24(Suppl. 1): S1-S8.

57. Brede M, Philipp M, Kaus A, Muthing V, Hein L. α2-Adrenergic receptor subtypes - novel functions uncovered ingene-targeted mouse models. Biol Cell 2004; 96: 343-348.

58. Dardonville C, Rozas I. Imidazoline binding sites and theirligands: an overview of the different chemical structures.Med Res Rev 2004; 24: 639-661.

59. Kaliszan W, Petrusewicz J, Kaliszan R. Imidazoline rceptorsin relaxation of acetylocholine-constricted isolated ratjejnum. Pharmacol Reports 2006; 58: 700-710.

60. Raasch W, Schäfer U, Qadri F, Dominiak P. Agmatine, anendogenous ligand of imidazoline binding sites, does notantagonize the clonidine mediated blood pressure reaction.Br J Pharmacol 2002; 135: 663-672.

61. Pintong D, Wright LK, Hammer C et al. Agmatinerecognizes alpha-2 adrenoceptor binding sites but neitheractivates nor inhibits alpha-2-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 1995; 351: 10-16.

R e c e i v e d : July 27, 2008,A c c e p t e d : February 20, 2009.

Author’s address: Dr Aleksandra Radwanska, Department ofBiopharmaceutics and Phamacodynamics, Medical Universityof Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland. Phone:+48 58 349 32 60; Fax: +48 58 349 32 62; e-mail: alex@amg.gda.pl

142