Various imidazolines have cholinomimetic,
sympathomimetic, histaminelike, antihistamine and adrenergic properties. Second
generation central antihypertensives, such as moxonidine and rilmenidine, have
attenuated sedative (
2
adrenoceptor-mediated) side effects at equihypotensive doses compared with the
drugs of first generation (1). While recognized as potent agonists at peripheral
2 adrenergic receptors, several studies suggested that the drugs also had another
mode of action. Clonidine-like drugs, such as moxonidine, rilmenidine and dexmedetomidine,
reduce blood pressure by acting centrally at both
2 adrenergic and imidazoline
receptors. Imidazoline-binding sites (IBS) were discovered in central nervous
system (2) as well as in peripheral tissues from various species (3).
Imidazoline receptors engaged in circulatory system are classified in two groups:
the I
1 type, sensitive to clonidine and idazoxan,
an antagonist with an imidazoline structure, and the I
2 type, displaying a high
affinity for idazoxan (4), guanabenz (5, 6) cirazoline (7), and a medium-to-low
affinity for clonidine (4).
Imidazoline I
1 receptors are reported to play a role in the central regulation of blood pressure (8). The selective activation of central I
1imidazoline receptors results in an inhibition of peripheral sympathetic activity and produces arterial vasodilatation (9, 10). Imidazoline agents evoke diverse pharmacological responses in both peripheral tissues and the central nervous system, so that are difficult to attribute to known receptor signaling system (11).
The presence of presynaptic imidazoline receptors has been suggested in the human and rat heart but their functional role is unknown (12). Imidazoline I
1 receptors over
2 adrenoceptors in the heart atria and ventricles have been identified with the affinity to imidazolines at nanomolar range (13). An 85 kD protein may correspond to the functional I
1 receptor in atria. It has been shown that atrial I
1 receptors are up-regulated in spontaneously hypertensive rats (SHR) (14, 15). El-Ayoubi
et al. (14) observed that I
1 receptors are increased in hypertensive rats or humans. Therefore, there are remisses to suppose the engagement of imidazoline receptors in ino- and chronotropism of isolated heart atria (16).
Functional cardiac imidazoline I
1 receptors are tissue-specific, being differentially regulated in atria and ventricles in hypertension by chronic exposure to agonists (17).
In contrast to the I
1 imidazoline receptors, physiological role for I
2 sites has not yet been determined but it has been proposed that they play an active role in various physiological processes (18). For example, antiproliferative action of the imidazolines correlated with their affinity to the I
2 imidazoline binding sites in blood vessels (2, 19). The imidazoline I
2 binding sites (IBS) were described as imidazoline-guanidinium receptive site (IGRS) with idazoxan binding selectivity (20, 21). IBS appear to be heterogeneous in nature (22). Up today, their molecular structure, functional significance and their second-messenger system are unknown. I
2 receptor ligands interact with a domain on MAO, a catecholamine metabolizing enzyme, but this mechanism is not equally accessible in all tissues (11, 23, 24).
Clonidine is twice as potent as moxonidine at the I
1
receptor but has similar affinity for
2
and
1
adreceptors (25). In binding assay on cow brain the K
i
values for clonidine at
2A
and I
1 receptors were 3.8 and 1.0 nM, respectively
(26). Hypotensive effect of clonidine is mainly through the
2
adrenergic over I
1 imidazoline receptors (27).
Significant bradycardia of isolated rat heart was observed with clonidine, and
less with moxonidine, at 10
-6 M concentration.
It suggests that postsynaptic cardiac imidazoline I
1
receptors may be involved in these effects (28).
Moxonidine is pharmacologically similar to clonidine, but its affinity to imidazoline I
1 receptors over
2 adrenoceptors is 100-fold higher (29). The hypotensive mechanism of moxonidine originally suggested was through activation of central
2 adrenoceptors, but it appeared that primary action in hypertension is due to binding of moxonidine at imidazoline I
1 receptors in the rostroventrolateral part of the brainstem (RVLM) (30). Moxonidine is three times more selective for the I
1 receptor in RVLM than rilmenidine and has 40-70 times greater affinity for I
1 receptors than for
2 adrenoceptors (25). Moxonidine and rilmenidine injected intravenously lowered blood pressure, decreased plasma norepinephrine concentrations and inhibited stimulation-evoked cardioacceleration in pithed rabbits
via 2 adrenoceptor mechanism (31). Inhibition of norepinephrine release by moxonidine in pithed SHR was demonstrated by Raasch
et al. (32): the authors explain this effect by interaction with imidazoline I
1 receptor. Intravenous moxonidine may activate imidazoline I
1 receptors and
2 adrenoceptors present in the rat heart. Compared with clonidine selectivity, moxonidine and rilmenidine has approximately 3 to 10 times greater affinity for imidazoline receptors (14, 17).
Rilmenidine exhibited antiarrhythmic effects in different animal models of arrhythmia (33). This effect is likely to originate from effects on the central nervous system as well as from an action at peripheral sites (34).
Rilmenidine is neutral regarding metabolic parameters, but it influences left ventricular hypertrophy, microalbuminuria and insulin resistance positively in hypertensives at risk (35). Widimsky and Sirotiakova (36) observed decrease of arterial pressure and reduction in heart rate in hypertensive patients treated with 1mg rilmenidine daily. That may be a clinically relevant benefit in patients with an increased cardiovascular risk and metabolic disorders.
Agmatine (decarboxylated arginine) is widely distributed through the body, attaining
high levels in the rat aorta (57.41 ng/g) and in the rat heart (6.03 ng/g),
with the concentration in brain being relatively small (2.4 ng/g) (37). Molecular
mass of agmatine is 130 Da (24). The agent was found to be regionally distributed
in the rat brain (38). Agmatine is relased from the neurons and interacts with
various pre- and postsynaptic receptors including the I
1
imidazoline receptor,
2
adrenoceptor, NMDA receptor, nicotinic cholinergic receptor and 5-HT
3.
receptor which might be of physiological importance (39, 40). All that suggests
that agmatine may be a neurotransmitter (41). Their affinity (K
i)
in human brain for
2A
and I
1 imidazoline receptors is 46 980 and 33.4
nM, respectively (26), although the interaction of agmatine with
2
adrenoceptors is unclear (42, 43). It has been postulated that agmatine may
change uptake of norepinephrine, like clonidine does, thus reducing sympathetic
tone through imidazoline receptors (42).
Agmatine concentration-dependently releases adrenaline and noradrenaline from
chromaffin cells. This effect can be blocked by antagonists of the I
1
receptor. In chromaffin cells agmatine has a high affinity to
2
adrenoceptors and I
1 and I
2
binding sites, namely 4.0, 0.7 and 1.0 µM, respectively. It has a low affinity
to the
1
and ß adrenoceptors as well as to the 5-HT
3
serotonin and D
2 dopamine binding sites. Agmatine-uptake
into synaptosomes is blocked by the imidazoline derivative idazoxan and by phentolamine,
but not by clonidine, moxonidine and rilmenidine (41).
Agmatine has no effect on vascular contraction and blood pressure, contrary to moxonidine, which increases the vascular contraction and decreases blood pressure (39). However, Herman (44) suggested that it can regulate cardiovascular functions and modulate some processes in the peripheral and central nervous system, whereas it has only a weak blood pressure effect when applied within the RVLM area. The affinity of agmatine for IBS is rather low, while it also binds
2 adrenoceptors (41). In the experiments on rat hearts, agmatine increased norepinephrine level indicating a synergistic inhibitory action at I
1 imidazoline receptors under conditions of stimulated
2 adrenergic autoinhibition (15).
Research on newly synthesized imidazoline compounds with a high affinity to imidazoline receptors is still carried on. It is believed that the binding sites specifically recognizing the imidazoline structure or similar chemical structures, both in the brain and in certain peripheral tissues, including the heart, participate in the control of blood pressure (14).
Imidazoline receptors bind some agents that are not imidazolines, such as guanabenz (45), guanidinium, rilmenidine (46) and oxazole. Newly synthesized analogues of imidazolines and reference oxazolines elicit an appreciably increased selectivity for I
1 and I
2 receptors. A few of these compounds, namely "AGN" and "BU families", show prevailing affinities for I
1 and I
2 receptors. These compounds are the first imidazoline analogues described that are devoid of any significant affinity to
2 adrenergic receptors.
The isofuran derivative of imidazoline, AGN 192403, is about 10,000 time more selective for imidazoline receptors than for
2 adrenoceptors (47). It had no effect on blood pressure when injected intravenously in monkeys and rabbits. AGN was found to be an antagonists of imidazoline receptor in a number of studies (15, 48).
The benzofuran derivative, 2-BFI, appeared 1,000 to 10,000 times more selective
for [3H]idazoxan imidazoline specific binding sites than for
2
adrenoceptors. Selective for the I
2 imidazoline
binding sites agent 2-BFI has K
i of about 1
nM and a low activity for
2
adrenoceptors. It can elevate extracellular levels of norepinephrine in the
frontal cortex and hippocampus in rats (49).
BU239 was described as a ligand with a high selectivity to I
2
receptors in rabbit brain. In competitive binding assays on rat kidney membranes
K
i for BU239 was 4.3 nM (16, 50).
Distinction between the imidazoline receptor and the
2 adrenoceptor-mediated mechanism for imidazoline compounds is difficult. A combination of agonists and antagonists differing in affinities for each receptor is required for that (26). The most frequently used imidazoline receptor antagonist, idazoxan, is also a potent
2 adrenoceptor antagonist. It seems that important for affinity of agents towards I
1 receptors is their hydrophobicity, as expressed by log P ranging from 1 to 2 (51). Therefore, structural alterations of idazoxan can result in molecules with a marked selectivity for either
2 adrenoceptors or imidazoline receptors.
Beside phentolamine, potent nonselective
1 /
2 adrenoceptor antagonists with a low affinity at histaminergic receptors and less potent at imidazoline receptors are used. For now, there are no endogenous agonists selective for imidazoline receptors. All drugs binding to imidazoline receptors bind to
2 adrenergic receptors as well. It has not been possible to determine unambiguously whether drugs binding to I receptors act as agonists or antagonists and have actions comparable to their actions at the
2 adrenergic receptors.
Imidazoline derivatives have been demonstrated to interact with the sympathetic neurotransmission
via nonadrenergic presynaptic receptors in different experimental models. However, no systematic comparative studies were reported regarding the ino- and chronotropic activity of a representative set of imidazoline drugs towards imidazoline receptors in the heart preparations. On the other hand, single individual imidazolines have been reported to elicit pronounced pharmacological effects mediated through cardiac receptors. The direct effect of an imidazoline receptor ligand on cardiac receptors has not been established. Still, new imidazoline derivatives are potential drugs for antihypertensive therapy.
The aim of this study was to determine
in vitro the inotropic and chronotropic
effect of newly synthesized imidazoline receptor ligands: 2-BFI, BU239 and AGN192403
as well as the known imidazoline drugs, like clonidine, rilmenidine, moxonidine
and agmatine on isolated rat atria. The agents studied were selected from the
point of view of their hypothetical interaction with the adrenergic/imidazoline
receptors in cardiac cells. The main task of this project was to help to direct
rationally the further search for original circulatory and antihypertensive
imidazoline agents based on their cardiotropic properties.
MATERIALS AND METHODS
The procedure applied was designed in accordance with the respective Polish and European regulations and the guidelines established by the Ethics Committee for Animal Experiments of the Medical University of Gdansk, Poland.
Materials
Male Wistar rats (200-350 g) were used for
in vitro studies. The animals
were housed and fed in a laboratory kept at constant temperature of 22°C under
the standard conditions (12:12 h L:D cycle, standard pellet diet, tap water).
Methods
The animals were anesthetized with urethane (1.5 g/kg i.p.) and the heart was
rapidly excised. After cervical dislocation, thorax was quickly opened, the
still beating heart removed and placed in the preparation dish with a modified
Krebs-Henseleit solution. The ventricular tissue was cut away as far as possible.
The atria, left and right separately, were placed in the solution, gassed with
95 % O
2 and 5 % CO
2,
giving pH of 7.3-7.4, and kept at 35.5-37°C. The incubation medium contained
NaCl 118 mmol, KCl 4.7 mmol, CaCl
2 6 mmol, NaH
2PO
4
1 mmol, MgCl
2 1.2 mmol, NaHCO
3
25 mmol, glucose 11.1 mmol, EDTA 0.04 mmol, and ascorbic acid 0.1 mmol.
The muscle was tied at either end to stainless hooks under a tension of 0.5-1.0
g in an organ bath and was allowed to stabilize for 45-60 min. The left atrium
was electrically stimulated with two platinum electrodes by square-wave electrical
pulses (2.5 Hz, 4 ms) and voltage 10 V. Amplitude of contractile tension (mm)
of the left atrium and the rate of contractile action (min
-1)
of the spontaneously beating right atrium were recorded by means of an isometric
force transducer (Bio-Sys-Tech, Bialystok, Poland).
Experimental protocol
After equilibration period, the cumulative concentration-response curves were
constructed for increasing concentrations of the imidazolines studied ranging
from 10
-11 to 10
-3
M. Structure of compounds studied shows
Fig. 1.
|
Fig. 1. Chemical structure of compounds studied. |
In the next stage of the experiment, the inotropic and chronotropic responses
to imidazolines studied were 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 with variable slope were constructed
and analysed by means of GraphPad Prism4 software (GraphPad Software Inc., San
Diego, Ca). Each point of the curves was a mean of at least 6 experiments. The
changes of responses to each concentration was expressed as percent of the control
value (100 %) of atrial rate or amplitude preceding the administration of cumulative
concentrations of an agent with or without idazoxan or phentolamine. Data are
reported as mean SEM (standard error of mean). Based on the profile of concentration-response
behavior of isolated organs for the compounds studied, the -log EC
50
parameters were calculated (the concentration of the ligand producing the half
of the maximal effect observed). A significance level was taken as p
0.05 or p
0.01 in
comparison of the compound studied alone and the same compound pretreated with
idazoxan or phentolamine. Nonparametric analysis was done by U'Mann-Whitney
(unpaired) test using Statistica 7.1 software (StatSoft Inc., Tulsa, OK, USA)
Data obtained are shown in
Figs. 2-6 and in
Tables 1-3.
Drugs
Pure chemical substances were used for the preparation of bath solutions of the drugs studied.
Phentolamine and idazoxan were obtained from Sigma (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 from Tocris (Bristol, UK); 2-BFI (2-(2-benzofuranyl)-2-imidazoline) was
from Tocris (London, UK); rilmenidine was a gift from Servier (Paris, France)
and moxonidine was a gift from Dr. B. I. Armah, (BDF Research Laboratories,
Hamburg, Germany). Stock solutions 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. Stock solutions were
diluted with water ex tempore before individual experiments.
RESULTS
Inotropic activity
It has been demonstrated that clonidine, moxonidine and rilmenidine elicit the
positive inotropic activity on electrically stimulated left atria with maximum
effects of 132.1, 116.2 and 118.3 per cent, respectively (
Fig. 2A,
Table
1). The -log EC
50 values observed for clonidine,
moxonidine and rilmenidine were 5.2, 6.2 and 5.1, respectively (
Table 2).
|
Fig. 2. Effect of cumulative concentrations of the imidazoline compounds studied on contractility of the left rat heart atria (A) and on beating rate of the right rat heart atria (B).
The data are shown as means of at least 6 experiments SEM |
Table 1. Inotropic
and chronotropic effects of the cumulative concentrations of the compounds
studied. Maximum effect observed are expressed as % of control (at indicated
molar concentration). |
|
The presence of idazoxan 10
-5 M and 10
-3
M diminished positive inotropic effect of clonidine and rilmenidine (
Figs.
3A,
3C). The -log EC
50 values for
clonidine and rilmenidine increased after pretreatment with idazoxan 10
-3
and 10
-5 M (
Table 2). Surprisingly, moxonidine
produced positive inotropic effect at the presence of idazoxan 10
-5
M (
Fig. 3B). The antagonism at the presence of idazoxan 10
-3
M manifested itself at the high concentrations (10
-5
- 10
-3 M) of moxonidine. The -log EC
50
for moxonidine alone was 6.2 and it increased to 7.2 after pretreatment with
the 10
-5 M idazoxan (
Table 2).
|
Fig. 3. Effect of cumulative concentrations of clonidine (Clo) (A), moxonidine (Mox) (B) and rilmenidine (Ril) (C) alone and in the presence of fixed concentrations of idazoxan (Ida) on contractility of the left rat heart atria.
The data are shown as means of 6 experiments SEM; p 0.05 denotes significance level of differences between results obtained for the compounds alone and after pretreatment with idazoxan (Ida) as compared with the U'Mann-Whitney, unpaired test. |
Table 2. The -log
EC50 value for compounds studied alone
and at the presence of idazoxan (Ida) or phentolamine (Phen). |
|
AGN192403 in cumulative concentrations from 10
-11
to 10
-3 M does not act on amplitude of beating
of left atria. A pretreatment with idazoxan 10
-9
and 10
-6 M or phentolamine 10
-9
M increases inotropic activity of AGN 192403 but these effects are of no statistical
significance.
Positive inotropic effect of compound 2-BFI appeared only at very low agent's
concentrations (10
-11-10
-8
M). In higher concentrations, 2-BFI decreased the amplitude of contraction of
left atria up to 76.1 %, in comparison to the 100% of control. The presence
of idazoxan 10
-3 M partially diminished inotropic
effect of 2-BFI but concentration 10
-5 M of idazoxan
remained without effect on 2-BFI (
Fig. 4A). The -log EC
50
values of 2-BFI alone and pretreated with idazoxan 10
-5
M were equally 7.0, and for 2-BFI pretreated with idazoxan 10
-3
M both values were 9.5 (
Table 3).
|
Fig. 4. Effect of cumulative concentrations of 2-BFI (A), BU239 (B) and Agmatine (Agm) (C) alone and in the presence of fixed concentrations of idazoxan (Ida) on contractility of the left rat heart atria.
The data are shown as means of 6 experiments SEM; p 0.05 denotes significance level of differences between results obtained for the compounds alone and after pretreatment with idazoxan (Ida) as compared with the U'Mann-Whitney, unpaired test. |
Table 3. The -log
EC50 value for compounds studied alone
and at the presence of idazoxan (Ida) or phentolamine (Phen). |
|
Compound BU239, which is structurally related to 2-BFI and has been reported
to label I
2 receptors, produced positive inotropic
activity with the maximum effect observed of 117.4 % (
Fig. 4B). Idazoxan
at concentration 10
-7 M slightly diminished the
left atria amplitude of contraction evoked by BU239 whereas idazoxan 10
-9
M increased the activity of BU239. The presence of phentolamine 10
-9
M had no effect on inotropy of BU239. The -log EC
50
values were also diminished from 8.8 for BU239 alone to 8.3 for BU239 with idazoxan
10
-5 M, to 6.6 for BU239 with idazoxan 10
-7
M and to 7.2 for BU239 with phentolamine 10
-5
M (
Table 3).
The most marked positive inotropy was observed for agmatine with maximal effect
observed of 142.0 % and the -log EC
50 of 8.2
(
Fig. 4C, Table 3). Pretreatement with idazoxan 10
-7
M or 10
-3 M agmatine decreased the amplitude of
left atria. Phentolamine 10
-9 M antagonized the
inotropism of agmatine also. The -log EC
50 values
of agmatine with antagonists were diminished as has been presented in
Table
3.
Idazoxan in cumulative concentrations did not affect the amplitude of beating of left atria: the maximum effect was 105.7 %. Also phentolamine remained without effect on the inotropic effect of left atria.
Chronotropic activity
Clonidine produced a weak positive chronotropic effect on the right atria up
to maximum of 110.2 % (
Figs. 2B and
5A). Idazoxan at concentrations
10
-5 and 10
-3 M
markedly antagonized positive chronotropic activity of clonidine and decreased
the -log EC
50 value about 100-fold (
Table
2).
Rilmenidine and moxonidine had no effect, either alone or pretreated with idazoxan,
on right atria (
Figs. 5B, 5C).
|
Fig. 5. Effect of cumulative concentrations of clonidine (Clo) (A), moxonidine (Mox) (B), rilmenidine (Ril) (C) and AGN192403 (D) alone and in the presence of fixed concentrations of idazoxan (Ida) on beating rate of the right rat heart atria.
The data are shown as means of 6 experiments SEM; p 0.05, p 0.01 denotes significance level of differences between results obtained for the compounds alone and after pretreatment with idazoxan (Ida) as compared with the U'Mann-Whitney, unpaired test. |
AGN192403 increased weakly the rate of beating of the right atria (maximum effect
was 110.4 %). In experiments with preexposure to idazoxan 10
-9
M or 10
-6 M, the antagonism with regards to chronotropic
action occured with the statistical significance of p< 0.05. In the case of
preexposure with phentolamine 10
-9 M, there was
no change of this effects (
Fig. 5D). The -log EC
50
values were changed for AGN192403 when pretreated with idazoxan 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 the right atria (from maximum
effect of 106.5 to minimum of 93.0 %) (
Fig. 6A). In the presence of various
concentrations of idazoxan, the chronotropic effect of 2-BFI was not changed
(
Fig. 6A). The -log EC
50 values from
experiments with 2-BFI untreated and treated with either idazoxan or phentolamine
were ever in the range from 6.9 to 8.1 (
Table 3).
|
Fig. 6. Effect of cumulative concentrations of 2-BFI (A), BU239 (B) and Agmatine (Agm) (C) alone and in the presence of fixed concentrations of idazoxan (Ida) on beating rate of the right rat heart atria.
The data are shown as means of 6 experiments SEM; p 0.05 denotes significance level of differences between results obtained for the compounds alone and after pretreatment with idazoxan (Ida) as compared with the U'Mann-Whitney, unpaired test. |
BU239 in cumulated concentrations significantly increased the rate of beating
of the right atria and the maximum effect observed was 131.0 %. However, in
the presence of idazoxan 10
-7 and 10
-9
M the chronotropic effect of BU239 was reduced (
Fig. 6B). Phentolamine
10
-9 M, added to BU239 preincubated with idazoxan
10
-9, 10
-7 or 10
-5
M, had no effect. All the values of -log EC
50
from experiments involving BU239 were similar, ranging only from 7.3 to 8.3
(
Table 3).
Agmatine in cumulative concentrations accelerated the rate of the spontaneously
beating right heart atria to a maximum of 125.7 % (
Fig. 6C). Idazoxan
in concentrations 10
-7, 10
-5
or 10
-3 M attenuated agmatine's positive chronotropic
effect. Phentolamine 10
-9 M, added to agmatine
pretreated with idazoxan, significantly decreased the rate of beating of the
atria. The -log EC
50 value decreased about 100-fold
(
Table 3).
Neither idazoxan nor phentolamine in cumulative concentrations had any marked effect on chronotropic activity of right atria.
DISCUSSION
Inotropy
In this work manifestations of positive inotropic activity were shown in case
of agmatine, clonidine, rilmenidine and moxonidine. The narrow range of -log
EC
50 values for clonidine, rilmenidine and moxonidine
from 5.1 to 6.2 indicates a similar receptor-mediated effect of those drugs.
Idazoxan diminished significantly statistically the positive inotropic effect
of clonidine and rilmenidine, but not that of moxonidine. The positive inotropic
effect of rilmenidine was diminished by idazoxan, unless idazoxan was applied
at a high concentration 10
-3M, suggesting that
rilmenidine exhibits some selectivity for imidazoline receptors. Rilmenidine
is usually considered to be an I
1 receptor ligand.
However, recent evidences show that it may also label an I
2-like
site (52). Moreover, it was reported that the hypotensive effect of rilmenidine
in humans was potently antagonized by idazoxan, whereas it was weakly or not
at all antagonized by yohimbine. At the oral dose of 2 mg, rilmenidine has no
effect on beating rate of the heart (53, 54).
Molderings
et al. (52) observed that rilmenidine and oxymetazoline are
potent full agonists to
2
adrenoceptors in rabbit hearts, whereas in the human atrial appendages both
agents are antagonists at the
2
autoreceptors, like rauwolscine and idazoxan are. Prazosin is ineffective in
that preparation. The antagonistic activity of rilmenidine towards human
2A
adrenoceptors indicates that, in contrast to the suggestion based on rabbit
data, the hypotensive effect on humans is not due to activation of
2A
adrenoceptors but other, presumably I
1 imidazoline,
receptors are involved (52). In our work, the increasing -log EC
50
values for clonidine and rilmenidine at the presence of idazoxan 10
-3
M, suggest dual interaction of the imidazolines with the
2
adenergic and the imidazoline I
1 receptors.
In the case of clonidine it would confirm the hypothesis that its positive inotropic
effect at low doses is mainly due to a stimulation of postsynaptic a adrenoceptors,
whereas additional stimulation of the imidazoline receptors occurs at higher
drug doses. Certainly our results do not exclude the possibility that rilmenidine
and clonidine elicit their effects through putative presynaptic imidazoline
receptors, at least as regards the inotropic activity.
In our study moxonidine elicited a weak positive inotropic effect on left atrium.
This finding is in accordance with the results obtained by Raasch
et al.
(55). Evidently the presence of idazoxan potentiates the inotropic effect of
moxonidine. It suggests that moxonidine acts as an agonist-antagonist on both
adrenoceptors and imidazoline receptors. The -log EC
50
value increases in the presence of idazoxan at concentration 10
-5
M, but it decreases when 10
-3 M idazoxan is added
to the incubation medium. On the other hand, it has been reported, that moxonidine
reduced norepinephrine release independently of I
1
receptor, thus suggesting the prominent effect of
2
adrenoceptors in cardiac tissue (52). Moxonidine binds with different affinities
to cardiac imidazoline I
1 receptors,
2
adrenoceptors (56), and, at some conditions, to
1
adrenoceptors. Raasch
et al. (55) explain the increase of contractility
of left rat atria by stimulation of postsynaptic
1
adrenoceptors rather than the imidazoline I
1
receptors. Hovewer, in experiments consisting in chronic moxonidine treatment
of the spontaneously hypertensive rats Mukaddam-Daher and Gutkowska (16) and
El-Ayoubi
et al. (17) observed the specific binding with moxonidine at
the atrial I
1 subtype receptors.
A structurally related to moxonidine compound AGN192403 did not change the amplitude
of beating of the left atria. However, in the presence of various concentrations
of idazoxan, AGN192403 showed a weak nonsignificant positive inotropic effect,
potentiated with 10
-9 M of either phentolamine
or idazoxan, similarly as in experiments with moxonidine. Most authors suggest
that AGN192403 has no effect on circulatory system. However, its general behaviour
may suggest it to be a selective ligand of I
1
receptor (15, 57, 58) in the experiments on rat hearts, demonstrated that AGN192403
had no influence on norepinephrine level. According to these authors AGN192403
seems to be an antagonist to the imidazoline I
1
receptor and the potentiation of inotropic activity by idazoxan seems to result
from synergistic interactions.
In the case of agmatine most important are increases of the amplitude of the
left atria contraction. Idazoxan at concentration of 10
-7,
10
-5 and 10
-3 M,
and phentolamine at concentration of 10
-9 M, diminished
inotropic activity in a dose dependent manner. Except of imidazoline receptors,
agmatine has affinity to both the
/ß
adrenergic and dopaminergic receptors. Its effect is mediated probably by all
those receptor sites in cardiac tissue.
Compound 2-BFI and a more potent ligand at I
2
imidazoline receptor, BU239, evoked a very weak positive inotropic activity.
The presence of idazoxan diminished inotropic effect of 2-BFI and BU239 without
statistical significance. The -log EC
50 values
for BU239, 2-BFI and idazoxan were 8.8, 7.0 and 7.0, respectively. The reported
binding affinities, K
i for I
2
receptors in rat brain membrane are 4.2 and 7 nM for BU239 and 2-BFI, respectively
(19). Pharmacometric analysis of the data obtained involving 2-BFI and BU239
lead to conclusion that these ligands cannot be clearly identified as either
agonists or antagonists of the I
2 receptor.
This hypothesis is in accordance with the conclusion from the study on the relaxation
of rat jejnum evoked by 2-BFI and idazoxan (59).
In our conclusion, the positive inotropic action on isolated rat heart left
atria is with the following rank order for the agents studied: agmatine >> clonidine
> BU239
rilmenidine
moxonidine. Rilmenidine
and moxonidine act as partial agonists of the imidazoline I
1
receptors. In inotropic effects of these imidazolines both the I
1
and
2
receptors are engaged. Inotropic effect of clonidine and agmatine is mostly
due to the
adrenoceptors
activation. The role of I
2 imidazoline receptors
is not to convince.
Chronotropy
Agmatine and clonidine were found to elicit positive chronotropic effect on the right rat heart atria. Idazoxan markedly antagonized activity of clonidine, but independly of the dose used. It is well known that clonidine is an agonist not only of the
2 but also of the
1 adrenoceptors, present in the right atria.
Phentolamine 10
-9 M, added to agmatine, significantly
decreased positive chronotropic effect of agmatine. In opposite, in the experiments
of some authors (41, 60) agmatine did not influence contractions of isolated
rat heart atria. Agmatine has affinity to both the
adrenergic and the imidazoline receptors. Some investigators demonstrated that
agmatine recognizes
2
adrenoceptors but is without effect on these receptors (61). To date, there
are no proofs of action of agmatine attributable to agonism or antagonism at
the site
in vitro.
In the present study, rilmenidine and moxonidine had almost no effect on the spontaneously beating right atria. Pretreatment with idazoxan attenuated the chronotropic effect of the drugs, but this antagonism against the chronotropic effect of rilmenidine and moxonidine never reached statistical significance, presumably because the induced effects were very small. Moxonidine and rilmenidine are the most selective agonists for I
1 receptors among imidazoline agents. Nevertheless, some authors classify moxonidine and rilmenidine among selective
2 adrenoceptor agonists clainning that this receptor may be predominant in the chronotropic activity of the assumed I
1 imidazoline receptor agonists.
Compound AGN192403 possesses a small positive chronotropic activity on right
atrium and this effect is blocked by idazoxan 10
-6
M and phentolamine 10
-9 M as proved by the decreased
-log EC
50 values.
An I
2 imidazoline receptor ligand, 2-BFI, had a very weak negative chronotropic activity, probably due to its antagonistic activity towards the I
2 receptor subtype (19). After pretreatement with idazoxan the effect of 2-BFI remains unchanged.
Another I
2 receptor ligand, BU239, is the most
potent of all the agents studied in increasing the beating rate of the right
atria. An antagonist, idazoxan, applied at various concentrations diminished
this activity in an irregular manner. The -log EC
50
values determined after the preincubation with idazoxan in different concentrations,
were closely similar. It may suggest that the chronotropic mechanism of BU239
involves the imidazoline I
2 receptors.
In conclusion, regarding to the maximal efffect observed, the positive chronotropic action on isolated rat heart right atria were with the rank order for the agents studied: BU239 ³ agmatine >> clonidine > AGN192403.
In view of our research, the engagement of imidazoline receptors in the chronotropic response of rat heart atria to imidazoline drugs still remains disputable. Certainly, regarding to chronotropic effect of agmatine and clonidine we feel obliged to acknowledge an involvement of the
2/
1 adrenergic receptors. However, as concerns BU239, the results obtained by us demonstrate that its activity on the rat right heart atrial beating rate is exerted
via the imidazoline I
2 receptors.
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