Role of imidazoline receptors in cardiovascular regulation.

The involvement of nonadrenergic imidazoline specific receptors in the central control of the vasomotor tone and in the mechanism of action of drugs bearing an imidazoline structure, or analogs, is now well documented. Imidazoline-specific binding sites were found in many tissues and species. Moreover, until now, it is only in the brainstem that such binding sites are associated with a function: the hypotensive effect of imidazoline-like drugs. Rilmenidine, which is an oxazoline structurally related to the reference imidazolines, exerts a central hypotensive effect of central origin involving imidazoline receptors. The selectivity of rilmenidine for the imidazoline receptors compared to alpha 2-adrenergic receptors could explain the low incidence of sedative side effects observed with this antihypertensive drug. A specific anti-imidazoline radioimmunoassay allowed us to detect the presence of an immunoreactive imidazoline-like substance in human sera. High levels of this immunoreactive substance are associated with high blood pressure in 20-30% of the hypertensive patients. This observation indicates that high levels of this immunoreactive substance in the serum can be associated with some kinds of primary hypertension. The cause-and-effect relation between these 2 phenomena has not yet been determined. This substance is in process of purification; it could be a candidate to be an endogenous ligand of the imidazoline receptors.

Relevance of the use of [3H]-clonidine to identify imidazoline receptors in the rabbit brainstem.

1. [3H]-clonidine binding was investigated in membranes isolated from the ventral medulla oblongata of the rabbit, where clonidine produced a hypotensive effect which was not mediated by adrenoceptors. [3H]-clonidine specific binding, as defined by the difference between the binding of [3H]-clonidine in the presence and in the absence of 10 microM cirazoline, occurred at two sites: a high affinity site with a KD = 2.9 +/- 0.7 nM and a Bmax of 40 +/- 8 fmol mg-1 protein and a low affinity site with a KD = 18.2 +/- 0.4 nM and a Bmax of 66 +/- 14 fmol mg-1 protein. 2. The high affinity sites being catecholamine-sensitive were identified as alpha 2-adrenoceptors. The low affinity binding of [3H]-clonidine was insensitive to catecholamines, as well as to other alpha 2-adrenoceptor specific probes, and could be inhibited with high affinity only by compounds which lowered blood pressure when directly injected in the nucleus reticularis lateralis of the ventral brainstem, or by antagonists. 3. It was concluded that in the ventral medulla of the rabbit, [3H]-clonidine labelled alpha 2-adrenoceptors and imidazoline receptors (IRs). Only the latter were related to the hypotensive effects of clonidine and rilmenidine directly injected into the rostroventrolateral medulla oblongata (RVLM) of the rabbit. The methodological problems regarding the study of IRs with [3H]-clonidine are discussed.

Respective contributions of alpha-adrenergic and non-adrenergic mechanisms in the hypotensive effect of imidazoline-like drugs.

The hypotensive effect of imidazoline-like drugs, such as clonidine, was first attributed to the exclusive stimulation of central alpha2-adrenoceptors (alpha2ARs). However, a body of evidence suggests that non-adrenergic mechanisms may also account for this hypotension. This work aims (i) to check whether imidazoline-like drugs with no alpha2-adrenergic agonist activity may alter blood pressure (BP) and (ii) to seek a possible interaction between such a drug and an alpha2ARs agonist alpha-methylnoradrenaline (alpha-MNA). We selected S23515 and S23757, two imidazoline-like drugs with negligible affinities and activities at alpha2ARs but with high affinities for non-adrenergic imidazoline binding sites (IBS). S23515 decreased BP dose-dependently (-27+/-5% maximal effect) when administered intracisternally (i.c.) to anaesthetized rabbits. The hypotension induced by S23515 (100 microg kg(-1) i.c.) was prevented by S23757 (1 mg kg(-1) i.c.) and efaroxan (10 microg kg(-1) i.c.), while these compounds, devoid of haemodynamic action by themselves, did not alter the hypotensive effect of alpha-MNA (3 and 30 microg kg(-1) i.c.). Moreover, the alpha2ARs antagonist rauwolscine (3 microg kg(-1) i.c.) did not prevent the effect of S23515. Finally, whilst 3 microg kg(-1) of S23515 or 0.5 microg kg(-1) of alpha-MNA had weak hypotensive effects, the sequential i.c. administration of these two drugs induced a marked hypotension (-23+/-2%). These results indicate that an imidazoline-like drug with no alpha2-adrenergic properties lowers BP and interacts synergistically with an alpha(ARs agonist.

Imidazoline receptors. A new concept in central regulation of the arterial blood pressure.

Clonidine-like antihypertensive substances bind to nonadrenergic imidazoline specific sites within the nucleus reticularis lateralis (NRL), their medullary privileged site of action. Rilmenidine, a new central antihypertensive agent, was tested in the anesthetized rabbit. For the same hypotensive effect, cumulative doses given intracisternally proved 50 times more active than of those given systemically. Idazoxan, an alpha 2-adrenergic antagonist structurally related to the imidazolines, given centrally as pretreatment proved more potent in preventing the hypotensive effects than the same molar dose of yohimbine. When injected within the NRL area of the anesthetized rabbit, rilmenidine, like clonidine, always exhibited a hypotensive effect. The influence of clonidine and rilmenidine upon the NRL noradrenergic neurons involved in the blood pressure regulation, and upon those of the locus coeruleus (LC) involved in the sedative effect was studied by differential voltammetry. It was observed that rilmenidine was two times more selective than clonidine in inhibiting the NRL, as opposed to LC, neuronal activity. In addition, binding experiments of tritiated clonidine to human cortical and NRL membrane preparations showed that rilmenidine, as compared to clonidine, has a two to three times higher selectivity for the imidazoline receptors. In conclusion, we are now able to discriminate between the mechanism of the hypotensive effect of imidazoline-like drugs and that of their sedative action. Rilmenidine is the first example of an hypotensive drug more selective for imidazolin preferring receptors than for classical alpha 2-adrenoceptors.

Characterization of imidazoline binding protein(s) solubilized from human brainstem: studies with [3H]idazoxan and [3H]clonidine.

Imidazoline binding sites from the human brainstem were solubilized with 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane-sulfonate (CHAPS). [3H]idazoxan and [3H]clonidine were used as ligands to characterize the solubilized binding sites. In both the soluble and membrane fractions, [3H]idazoxan binding was saturable, stereoselective, sensitive to imidazolines and insensitive to (-)norepinephrine and to amiloride. The affinities of [3H]idazoxan for the soluble and membrane sites were similar (KD = 25 +/- 11 nM and 20 +/- 3 nM). In both soluble and membrane fractions, the alpha 2-adrenoceptor binding being masked with (-)norepinephrine, [3H]clonidine bound to a low affinity site which was insensitive to (-)norepinephrine and which exhibited the same selectivity for various drugs as the [3H]idazoxan binding site. alpha 2-adrenoceptor binding was present in the membrane and the soluble fractions although it was difficult to detect in the soluble fraction because of inhibition of [3H]rauwolscine binding by the CHAPS detergent.

Binding of new cirazoline derivative to imidazoline receptors from human brain.

Imidazoline compounds are known to interact with alpha 2-adrenoceptors as well as with specific non-adrenergic binding sites. Such binding sites are present in the brain and in peripheral tissues. Hypotensive effects of imidazolines were shown to be related to specific interaction with imidazoline binding sites within the brainstem. Heterogeneity of these sites based on differences in selectivities was reported. In order to facilitate the characterization of human brain imidazoline receptors, we synthetized new ligands by substitutions on the cirazoline phenyl ring. Affinities of these cirazoline derivatives were determined in two imidazoline binding site models, namely the human brain and the rabbit kidney. Interaction of these compounds with imidazoline binding sites from the human brain appeared more sensitive to structural variations of the imidazoline than those with rabbit kidney sites. Moreover, no correlation was found between affinities for imidazoline binding sites and those for alpha 2-adrenoceptors of the rat brain. Arylazide derivative of 2-(5-amino-2-methyl-phenoxymethyl)-imidazoline exhibited a higher affinity for human brain imidazoline binding sites than for human brain alpha 2-adrenoceptors. Photoincorporation of this azido-compound in human brain imidazoline binding sites was achieved and blockade of [3H]idazoxan imidazoline specific binding observed. These new tools may allow fine characterization of the different subtypes of imidazoline binding proteins.

Heterogeneity of the specific imidazoline binding of [3H]idazoxan in the human cerebral cortex.

The aim of the present study was to verify whether [3H]idazoxan can be considered as a highly selective ligand for imidazoline preferring receptors (IPR). In human frontal cortex membrane preparations [3H]idazoxan at a low concentration (2 nM) only labelled imidazoline sensitive, catecholamine insensitive sites. Binding was of high affinity, saturable and stereospecific. The rank order of potency of different compounds able to inhibit this binding was cirazoline > (+/-)-idazoxan > guanoxan > (-)-idazoxan > tolazoline > UK-14304 > clonidine. Amiloride, imidazol-4-acetic acid and histamine had no significant affinity for IPR labelled by [3H]idazoxan. [3H]idazoxan bound to 2 different sites (KD1 = 1 nM and KD2 = 16.4 nM). Clonidine behaved as a non competitive, non allosteric inhibitor of [3H]idazoxan binding. Both [3H]idazoxan binding sites were equally affected by clonidine. In membrane preparations obtained from the Nucleus Reticularis Lateralis region (NRL) of the brainstem, [3H]idazoxan binding was similar to that in cortical membranes, particularly with regard to specificity and kinetics. However, in the NRL region binding sites were 4-5 times more numerous than in the frontal cortex. Non linear analyses of saturation data obtained with NRL membrane preparations were compatible with both a one site and a two sites model. No significant effects of 1 mM MgCl2 alone or with 100 microM Gpp(NH)p were observed on either [3H]idazoxan binding or the competition with clonidine or rilmenidine. As in the cortical membrane, clonidine was a non competitive inhibitor of [3H]idazoxan binding to membranes from the NRL region. In conclusion, we show that when a low concentration is used, [3H]idazoxan binding to human brain involves sites almost completely insensitive to catecholamines and specific for imidazolines or related compounds. This binding involves two distinct sites. We also report that [3H]idazoxan imidazoline binding sites are not coupled with a G protein. Because of the non competitive interaction between clonidine and [3H]idazoxan for the binding sites of the latter, we are unable to conclude that the binding sites of the two drugs are identical. However, the non competitive, non allosteric interaction suggests a complex model of multiple binding sites.

I(1) imidazoline receptors involved in cardiovascular regulation: where are we and where are we going?

Clonidine-like drugs (hybrid drugs) reduce blood pressure by acting centrally at both alpha(2)-adrenergic receptors (alpha(2)AR) and I(1) receptors (I(1)R). Some attempts at cloning I(1)R have failed, probably because of the lack of selectivity of the ligands. Recently, compounds acting exclusively at I(1)R were synthesized: LNP 911, LNP509, and S23515. For example, LNP911 has a K(d) value of 1.7 nmol/L at I(1)R. LNP509 and S23515 reduce blood pressure when injected centrally in anesthetized animals, whereas S23757 behaves as an antagonist of hypotensive imidazolines. LNP509 reduces blood pressure even in genetically engineered mice lacking functional alpha(2)AR. An exclusive action at central I(1)R is therefore sufficient to modify blood pressure. With the help of drugs selective for I(1)R and alpha-methylnoradrenaline, selective for alpha(2)AR, we showed that imidazoline and alpha(2)-adrenergic mechanisms interact synergistically in controlling the blood pressure. Such a synergism may explain the very powerful hypotensive effects of hybrid drugs. The new ligands selective for I(1)R will be very helpful to investigate the molecular features and the signaling system of I(1)R.

Identification of human I1 receptors and their relationship to alpha 2-adrenoceptors.

I1 imidazoline receptors (I1R) were defined as receptors insensitive to catecholamines and highly sensitive to [3H]clonidine and analogs. By contrast, the I2R subtype is more sensitive to [3H]idazoxan. [3H]clonidine and [3H]idazoxan imidazoline specific binding sites (IBS) have been detected in crude human membranes. Pharmacologic characterization by binding assays clearly differentiates IBS from alpha 2-adrenoceptors, whereas differences between [3H]clonidine and [3H]idazoxan IBS are less clear in crude preparations. In fact, only moderate affinity for [3H]clonidine was detectable in such preparations. However, purification procedures allowed detection of high affinity [3H]clonidine IBS in the human brain, corresponding to the I1R. Difficulties in the characterization of the I1R in crude membranes are due to multiple factors including heterogeneity of IBS, their low Bmax value, the existence of allosteric modulation, and possibly the presence of natural binding inhibitors. Immunologic studies with specific anti-idiotypic antibodies revealed a 43-kD protein as the best candidate for I1R as binding activity coincides with immunodetection. No cross-reaction was found with anti-monoamine oxidase (MAO) A/B antibodies and the 43-kD protein, ruling out the possibility of this protein being an MAO-associated I2R. Neither anti-alpha 2A- nor anti-alpha 2B-specific antibodies were able to immunodetect the 43-kD protein in crude membrane preparations or in purified fractions. These results and further biochemical characterization (pHi, N-glycosylation) of the 43-kD protein definitely assessed that human brain I1R and alpha 2-adrenoceptors clearly differ physically. However, coexpression of I1R and alpha 2-adrenoceptors in synaptic plasma membranes of the bovine brainstem reinforce the possibility of a functional relationship between the two types of receptor.

Participation of imidazoline receptors and alpha(2-)-adrenoceptors in the central hypotensive effects of imidazoline-like drugs.

The central hypotensive effect of imidazoline-like drugs (IMs) involves non-adrenergic imidazoline receptors (IRs). IMs cause hypotension irrespective of their affinity and selectivity for one or the other alpha-adrenoceptor subtypes. LNP 509, which binds to I1Rs (Ki = 5.10(-7) M) but roughly not to alpha 2-adrenoceptors (A2Rs) (Ki > 10(-5) M), causes hypotension when injected alone into the brainstem. As far as hybrid drugs, that is, those with mixed binding profiles (I1/alpha 2), are concerned, a significant correlation was reported between their central hypotensive effect and their affinity for IRs. Imidazoline antagonists such as idazoxan competitively antagonized the centrally induced hypotensive effect of IMs. Yohimbine, an A2Rs antagonist, blocks the hypotensive effect of hybrids but usually in a noncompetitive manner. Mutation of A2Rs prevented the hypotensive effects of drugs highly selective for A2Rs, but also that of hybrids such as clonidine. These data indicate that triggering of the hypotensive effects of IMs (1) needs implication of IRs; (2) appears to be facilitated by additional activation of A2Rs; and (3) requires integrity of A2Rs along the sympathetic pathways.

Heterogeneity of imidazoline binding sites revealed by a cirazoline derivative.

The affinity of AMPI (2-[3-aminophenoxy]methyl imidazoline) for [3H]clonidine and [3H]idazoxan imidazoline binding sites was determined in various rabbit and human tissues. Although cirazoline showed a high affinity (nM range) in all the tested tissues, its derivative, AMPI, had a high affinity (nM range) in rabbit brain and kidney but a low affinity (microM range) in the human brain. These differences in affinities were very similar to those obtained with amiloride. The same results were obtained when considering [3H]clonidine or [3H]idazoxan specific imidazoline binding sites.

Polyclonal anti-idiotypic antibodies to idazoxan and their interaction with human brain imidazoline binding sites.

Polyclonal antibodies were raised in rabbits against purified polyclonal anti-idazoxan antibodies. The anti-idiotypic antibodies thus obtained, proved able to inhibit [3H]idazoxan specific binding to anti-idazoxan antibodies. Applied to human nucleus reticularis lateralis membrane preparations, these antibodies (20 micrograms) inhibited about 50 and 70% of the imidazoline specific binding of [3H]idazoxan and [3H]clonidine, respectively. Furthermore, they specifically immunoprecipitated 50% of [3H]idazoxan binding activity of imidazoline binding sites solubilized from the same tissue. [3H]Rauwolscine binding to alpha 2-adrenoceptors in rat cortex was not significantly affected by these antibodies. The antibodies labeled a 43 kDa protein in Western blots of partially purified imidazoline binding sites from human brain. In conclusion, these anti-idiotypic antibodies recognize imidazoline binding sites from human brain and allow the detection of a 43 kDa binding protein associated with or representing the imidazoline receptor expressed in human brain.

Isolation of a human cerebral imidazoline-specific binding protein.

The first isolation of a human brain specific imidazoline binding protein is described. This protein was obtained using affinity chromatography and was revealed with the aid of an anti-idiotypic antibody specific for imidazoline binding sites. The protein (43 kDa) differs from other imidazoline binding proteins previously isolated from peripheral tissues, in particular by being also sensitive to clonidine.

Human brain imidazoline receptors: further characterization with [3H]clonidine.

The aim of the present study was to further characterize [3H]clonidine binding in the ventrolateral medulla of the human brainstem, the region involved in the vasodepressor effect of imidazoline drugs of the clonidine type. Under basal conditions, [3H]clonidine can bind both to the imidazoline receptors and to the alpha-adrenoceptors. The latter represent only a small part of the total [3H]clonidine binding with a Bmax of 61 +/- 13 fmol/mg proteins and a KD of 4.9 +/- 2.2 nM. Most of the binding was associated with imidazoline receptors with a KD of 67 +/- 13 nM and a Bmax of 677 +/- 136 fmol/mg protein. alpha-Adrenoceptor binding of [3H]clonidine could be completely prevented when membranes were either treated during preparation with the aIkylating agent phenoxybenzamine or incubated in the presence of 30 microM (-)-noradrenaline or in the presence of the non-hydrolysable analogue of GTP, guanylyl imidodiphosphate (Gpp(NH)p). When the alpha-adrenoceptors binding was prevented, we demonstrated the insensitivity of [3H]clonidine binding to Gpp(NH)p and showed that the competition between clonidine and idazoxan for imidazoline receptors was insensitive to Gpp(NH)p suggesting that imidazoline receptors are not G protein coupled receptors. The specificity of [3H]cloniding binding to imidazoline receptors in the human ventrolateral medulla indicates that these receptors are different from imidazole receptors as defined with p-aminoclonidine in the bovine brainstem.

Differential sensitivity to inverse agonists of GABA(A)/benzodiazepine receptors in rats with genetic absence-epilepsy.

A strain of Wistar rats, genetic absence epilepsy rats from Strasbourg (GAERS), was selected and inbred over 40 generations for occurrence of spontaneous spike-wave discharges characteristic of absence seizures, simultaneously with a strain of non-epileptic rats (NER). GAERS demonstrate an excessive sensitivity to antagonists of the GABA(A) receptor. The sensitivity to convulsions induced by various inverse agonists of the GABA(A)/benzodiazepine receptor was compared in GAERS and NERs. The beta-carbolines FG 7142 and DMCM, and the imidazobenzodiazepines RO 19-4603 and the alpha 5-selective RY 024 were several times more convulsant in GAERS than in NERs. The largest differences were found with the non-selective RO 19-4603- and FG 7142. The proconvulsant imidazobenzodiazepine RO 15-4513, binding also to diazepam-insensitive receptors, had low efficacy. The high affinity binding of GABA(A)/BZD receptors with (3H) RO 15-1788 in the brain of naive rats and after administration of FG 7142 did not differ in GAERS and NERs. The data indicate that the hypersensitivity of GAERS to various inverse agonists of the GABA(A)/benzodiazepine receptor involves cortical GABA(A) receptors and is not related to differential activity of a subunit-selective receptor.

Further biochemical characterization of imidazoline binding sites from the human brainstem.

Biochemical characteristics of imidazoline specific binding sites from the human brainstem were further investigated using [3H]idazoxan as radiolabeled ligand. The study of the interaction of [3H]idazoxan binding sites with heparin and lectins (soybean and lentil lectin) confirm the heterogeneity of these sites in the human brain. In fact, about 10-15% of [3H]idazoxan binding sites were retained by each of the three supports used, leading to the hypothesis that two populations of sites, with different biochemical characteristics, coexist in this tissue. A small proportion of [3H]idazoxan binding sites was retained on an affinity chromatography support consisting of a clonidine-derived Pharmalink column. The binding activity of these clonidine-eluted sites was markedly and dose-dependently improved by the addition of 'treated fall-through' fraction from the same column. On the other hand, this 'treated fall-through' fraction inhibited the binding activity detected in the solubilized human brainstem membranes. These results also suggest the existence of heterogeneous imidazoline specific binding sites in the human brainstem and the existence of endogenous factors able to discriminate between them.

I1-imidazoline receptors: an update.

BACKGROUND: The site of the hypotensive action of imidazoline compounds, such as clonidine, was first identified within the nucleus reticularis lateralis of the rostroventrolateral part of the medulla (NRL/RVLM). It was shown that imidazolines and related substances reduced blood pressure when applied in this area whereas no catecholamine was capable of such an effect. IMIDAZOLINE-SPECIFIC BINDING: We previously suggested the existence of receptors specific for imidazoline-like compounds that differed from the alpha-adrenergic receptors. Imidazoline-binding sites were subsequently reported in the brain and in a variety of peripheral tissues, including the human kidney, and as expected these specific binding sites do not bind the catecholamines. The imidazoline-binding sites are classified into two subgroups: the I1-type, which is sensitive to clonidine and idazoxan, and the I2-type, sensitive to idazoxan and largely insensitive to clonidine. Numerous studies have confirmed the involvement of these receptors in various regulations and pathological processes, hypertension being the most notable. DRUGS: Functional studies have confirmed that the hypotensive effects of clonidine-like drugs involve I1-imidazoline receptors while their most frequent side effects only involve alpha2-adrenergic receptors. Recent studies have shown that a contribution of both receptor types might be necessary to trigger the hypotensive effect of central origin. Rilmenidine, an oxazoline analogue to the imidazolines, has been proposed as the prototype of a new class of antihypertensive drugs selective for I1-imidazoline receptors. At hypotensive doses, this drug is devoid of any significant sedative effect. As with clonidine, it evokes hypotension when injected into the NRL region and it completely displaces the [3H]clonidine bound to specific imidazoline-binding sites in human medullary membrane preparations, but it has proved more selective for cerebral imidazoline receptors than clonidine. This selectivity might explain the low incidence of side effects evoked by rilmenidine. CONCLUSION: Rilmenidine is the first example of a drug exhibiting a favourable selectivity between I1-imidazoline receptors and alpha2-adrenergic receptors, for example reducing blood pressure but avoiding sedation and mouth dryness.