Hugues Greney

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.

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 μm 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 α2‐adrenoceptors. The low affinity binding of [3H]‐clonidine was insensitive to catecholamines, as well as to other α2‐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 α2‐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.

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

Abstract: 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 brain‐stem 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.

Respective contributions of α-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 α2‐adrenoceptors (α2ARs). 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 α2‐adrenergic agonist activity may alter blood pressure (BP) and (ii) to seek a possible interaction between such a drug and an α2ARs agonist α‐methylnoradrenaline (α‐MNA). We selected S23515 and S23757, two imidazoline‐like drugs with negligible affinities and activities at α2ARs 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 μg kg−1 i.c.) was prevented by S23757 (1 mg kg−1 i.c.) and efaroxan (10 μg kg−1 i.c.), while these compounds, devoid of haemodynamic action by themselves, did not alter the hypotensive effect of α‐MNA (3 and 30 μg kg−1 i.c.). Moreover, the α2ARs antagonist rauwolscine (3 μg kg−1 i.c.) did not prevent the effect of S23515. Finally, whilst 3 μg kg−1 of S23515 or 0.5 μg kg−1 of α‐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 α2‐adrenergic properties lowers BP and interacts synergistically with an α2ARs agonist.

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.

IRAS is an anti-apoptotic protein.

Abstract: Active cell death, also known as apoptosis, has been implicated in the pathophysiology of diseases such as cancer, heart failure and neurodegenerative disorders. We report the anti‐apoptotic function of IRAS, which was previously shown to bind imidazoline ligands. The amino acid sequence of human IRAS (hIRAS) is unrelated to known proteins, except for rat IRAS and a mouse homologue named nischarin, which binds the alpha5 integrin subunit of the fibronectin receptor. When stably transfected into PC12 cells, hIRAS localizes to the cytosol as a 167 kDa immunoreactive protein. Clonal PC12 cell lines expressing hIRAS displayed normal serum growth responses. However, hIRAS expression led to prolonged cell survival against known apoptotic stimuli: serum starvation or thapsigargin or staurosporine treatments. The apoptotic population of hIRAS‐expressing cells was significantly reduced, and this protection was achieved by a decrease in caspase‐3 activity, phosphatidylserine translocation, and nuclear fragmentation. Similar protective effect was obtained in COS7 cells transiently transfected with hIRAS. A partial activation of the PI3 kinase pathway is possibly implicated in the anti‐apoptotic effect of IRAS. Thus, IRAS appears to represent a previously unknown anti‐apoptotic protein involved in the regulation of cell survival.

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.

σ2-Receptor Ligand-Mediated Inhibition of Inwardly Rectifying K+ Channels in the Heart

The σ2-receptor agonist, ifenprodil, was suggested as an inhibitor of G protein-coupled inwardly rectifying potassium channels. Nevertheless, an analysis of the role of σ2 receptors in cardiac electrophysiology has never been done. This work aims i) to identify the roles of cardiac σ2 receptors in the regulation of cardiac K+ channel conductances and ii) to check whether σ2-receptor agonists exhibit class III antiarrhythmic properties. The σ2-receptor agonists ifenprodil, threo-ifenprodil, LNP250A [threo-8-[1-(4-hydroxyphenyl)-1-hydroxy-propan-2-yl]-1-phenyl-1,3,8-triazaspiro[4,5]decane-4-one] (a derivative of ifenprodil devoid of α1-adrenergic and N-methyl-d-aspartate glutamate receptor-blocking properties), and 1,3-di(2-tolyl)guanidine were used to discriminate the effects linked to σ2 receptors from those of the σ1 subtype, induced by (±)-N-allylnormetazocine (SKF-10,047). The σ2-receptor antagonist 3-α-tropanyl-2(pCl-phenoxy)butyrate (SM-21) was employed to characterize σ2-mediated effects in patch-clamp experiments. In rabbits, all σ2-receptor agonists reduced phenylephrine-induced cardiac arrhythmias. They prolonged action potential duration in rabbit Purkinje fibers and reduced human ether-a-go-go-related gene (HERG) K+ currents. (+)-SKF-10,047 was completely inactive in the last two tests. The effects of threo-ifenprodil were not antagonized by SM-21. In HERG-transfected COS-7 cells, SM-21 potentiated the ifenprodil-induced blockade of the HERG current. These data suggest that σ2-receptor ligands block IKr and that this effect could explain part of the antiarrhythmic properties of this ligands family. Nevertheless, an interaction with HERG channels not involving σ2 receptors seems to share this pharmacological property. This work shows for the first time that particular caution has to be taken toward ligands with affinity for σ2 receptors. The repolarization prolongation and the early-afterdepolarization can be responsible for “torsades de pointe” and sudden cardiac death.

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.

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.