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.

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.

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.

Coupling of I(1) imidazoline receptors to the cAMP pathway: studies with a highly selective ligand, benazoline.

Clonidine and benazoline are two structurally related imidazolines. Whereas clonidine binds both to alpha(2)-adrenoceptors (alpha(2)R) and to I(1) imidazoline receptors (I(1)R), benazoline showed a high selectivity for imidazoline receptors. Although the alpha(2)R are negatively coupled to adenylate cyclase, no effect on cAMP level by activation of I(1)R has been reported so far. We therefore aimed to compare the effects of clonidine and benazoline on forskolin-stimulated cAMP levels in cell lines expressing either I(1)R only (PC12 cells), alpha(2)R only (HT29 cells), or I(1)R and alpha(2)R together (NG10815 cells). Clonidine proved able to decrease the forskolin-stimulated cAMP level in the cells expressing alpha(2)R and this effect could be blocked by rauwolscine. In contrast, in cells lacking these adrenoceptors, clonidine had no effect. On the other hand, benazoline and other I(1) receptor-selective imidazolines decreased forskolin-stimulated cAMP level in the cells expressing I(1)R, in a rauwolscine- and pertussis toxin-insensitive manner. These effects were antagonized by clonidine. According to these results, we demonstrated that 1) alpha(2)R and I(1)R are definitely different entities because they are expressed independently in different cell lines; 2) alpha(2)R and I(1)R are both implicated in the cAMP pathway in cells (one is sensitive to pertussis toxin and the other is not); and 3) I(1)R might be coupled to more then one transduction pathway. These new data will be essential to further understand the physiological implications of the I(1)R and the functional interactions between I(1) receptors and alpha(2)-adrenoceptors.

Imidazoline receptors in cardiovascular and metabolic diseases.

The site of the hypotensive action of imidazoline compounds, such as clonidine, was first identified within the rostroventrolateral part of the brainstem. Afterwards, it was shown that imidazolines reduced blood pressure when applied in this area, whereas no catecholamine was capable of such an effect. These data led us to suggest the existence of receptors specific for imidazolines different from the alpha-adrenergic receptors. Soon after, the existence of imidazoline binding sites (IBS) was reported in the brain and in a variety of peripheral tissues including pancreatic gland and kidney. As expected, these specific binding sites do not bind the catecholamines. The IBS are classified in two groups: the I1 type, sensitive to clonidine and idazoxan; and the I2 type, sensitive to idazoxan and largely insensitive to clonidine. Imidazoline receptors were shown to be involved in several physiological regulations and pathological processes such as hypertension, diabetes mellitus and some mood disorders. Evidence for their implication in the nervous regulation of blood pressure and in the insulin secretion control will be presented. The hypotensive effects of clonidine-like drugs involve imidazoline receptors (I1Rs), while their most frequent side-effects only involve alpha2-adrenergic receptors. A new class of centrally acting antihypertensive drugs selective for I1Rs is now available. At hypotensive doses, these drugs are devoid of significant side effects. It was shown that the good acceptability of these drugs is likely due to their selectivity for I1Rs.

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.

Does a second generation of centrally acting antihypertensive drugs really exist?

The site of the hypotensive action of imidazoline compounds, such as clonidine, was first identified within the rostroventrolateral part of the brainstem: the nucleus reticularis lateralis. After that, it was shown that imidazolines and related substances reduced blood pressure when applied in this area whereas catecholamines were not capable of producing such an effect. These data led us to suggest the existence of receptors specific for imidazoline-like compounds different from the alpha2-adrenoceptors. Soon after, the existence of imidazoline binding sites was reported in the brain and in a variety of peripheral tissues including the human kidney. As expected, these specific binding sites do not bind the catecholamines. The imidazoline binding sites are already subclassified in two groups: the I1-subtype sensitive to clonidine and idazoxan, and the I2-subtype, sensitive to idazoxan and nearly insensitive to clonidine. Functional studies confirmed that the hypotensive effects of clonidine-like drugs involved imidazoline receptors while their most frequent side effects only involved alpha2-adrenoceptors. However, recent functional evidence suggests that a cross talk between imidazoline receptors and alpha2-adrenoceptors is necessary to trigger a hypotensive effect within the ventral brainstem. Rilmenidine and Moxonidine are the leader compounds of a new class of antihypertensive drugs selective for imidazoline receptors. At hypotensive doses, these drugs are devoid of significant sedative effect. Rilmenidine evoked hypotension when injected within the nucleus reticularis lateralis region; it competed for [3H]-clonidine bound to specific imidazoline binding sites in human medullary membrane preparations but proved more selective for cerebral imidazoline receptors than clonidine. It is suggested that this selectivity might explain the low incidence of their side effects. Additional potentially beneficial actions on cardiac arrhythmias or congestive heart failure enlarge the therapeutic interest of imidazoline-related drugs. Recent binding and functional data throw a new light on the optimal pharmacological profile of this second generation of centrally acting antihypertensive drugs.

Evidence for the existence of imidazoline-specific binding sites in synaptosomal plasma membranes of the bovine brainstem.

Nonadrenergic imidazoline-specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brainstem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3-8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline-specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p-iodoclonidine (subnanomolar), clonidine (0.2 nM), and efaroxan (11 nM), but idazoxan did not compete significantly for the p-[125I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I1 imidazoline receptors. In summary, we describe for the first time that high-affinity I1 receptors of the bovine brainstem are located on (pre)synaptic membranes.

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.

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.

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.

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.