M. Dontenwill

The imidazoline preferring receptor: binding studies in bovine, rat and human brainstem

The binding of [3H]clonidine to brainstem membrane preparations was studied in an attempt to characterize imidazoline-sensitive, catecholamine-insensitive receptors. Human samples and samples from two animal species were used. [3H]Clonidine binding was always saturable, reversible and specific with a KD value of 6-7 nM. The Bmax values were 45.5 +/- 5.5, 145 +/- 34 and 65 +/- 33 fmol/mg protein in the whole rat medulla oblongata, the nucleus reticularis lateralis region of bovine and that of human, respectively. In the whole rat brainstem we could not demonstrate the presence of [3H]clonidine binding sites that were insensitive to catecholamines. In bovine and human nucleus reticularis lateralis (NRL) preparations, the amount of specifically bound labelled clonidine that was not displaced by an excess of (-)-norepinephrine was 25 and 100%, respectively. Substances that had a structure similar to that of clonidine were able to compete with [3H]clonidine binding within the human NRL. Cirazoline was the most potent to inhibit [3H]clonidine binding although yohimbine was also able to displace binding in the human NRL but with lower apparent affinity. Competition assays with idazoxan stereoisomers clearly showed that this binding was stereospecific. Therefore the human NRL region provides the first model of an homogenous population of imidazoline-preferring, non-alpha-adrenergic membrane receptors.

Evidence for the involvement of imidazoline receptors in the central hypotensive effect of rilmenidine in the rabbit.

1 Rilmenidine has recently been introduced as a new centrally‐acting antihypertensive agent. We examined its cardiovascular effects after intracerebral injection to anaesthetized rabbits. Cumulative doses of rilmenidine injected intracisternally (1 to 300 μg kg−1) led to dose‐dependent decreases in arterial blood pressure and heart rate. The effective doses of rilmenidine were lower when injected centrally than when injected intravenously. 2 Pretreatment with the same dose of yohimbine or idazoxan shifted the rilmenidine dose‐response curves for its hypotensive and bradycardic effects to the right. Idazoxan, which has an imidazoline structure, proved to be a more active antagonist than yohimbine of rilmenidine centrally‐mediated cardiovascular effects. 3 The dose‐response curve for the central hypotensive effect of rilmenidine was also shifted to the right after pretreatment with a bovine brain extract. This extract contains the endogenous ligand of the imidazoline‐preferring receptors which is not a catecholamine. 4 Rilmenidine, like clonidine, proved to be active when micro‐injected into the rabbit nucleus reticularis lateralis region. 5 In conclusion, rilmenidine exhibited in the rabbit a central hypotensive effect which originated in the same area as where clonidine acts. Specific imidazoline‐preferring receptors appear to be involved in this hypotensive effect.

Rilmenidine selectivity for imidazoline receptors in human brain

The selectivity of three centrally acting antihypertensive agents for the medullary imidazoline-preferring receptors (IPR) versus cortical alpha-adrenoceptors was investigated in human brain. [3H]Clonidine binding was studied in various membrane preparations. Competition experiments were performed. Cortical membrane preparations were used as they mainly contained classical alpha-adrenoceptors whereas medullary membrane preparations from the nucleus reticularis lateralis contained only IPR insensitive to catecholamines. Rilmenidine, a new antihypertensive agent, appeared 2.5 and 3.5 times more selective than clonidine and guanfacine, respectively, for medullary IPR sites than for cortical alpha-adrenoceptors, thus providing a possible explanation for the low sedative effects of this new molecule.

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.

Imidazoline receptors: Qualitative structure-activity relationships and discovery of tracizoline and benazoline. Two ligands with high affinity and unprecedented selectivity☆

The observation that all the attempts to characterize imidazoline (I) receptors have been carried out with non-selective or poorly selective ligands prompted us to undertaken research aimed at developing selective ligand(s). In previous work using, as a starting point, cirazoline I, a potent alpha 1-adrenergic receptor agonist that also binds to I receptors, we showed that removal of the cyclopropyl ring (2) retains high affinity for I2 receptors while reducing alpha 1-adrenergic agonist activity. However, it was felt that this residual, albeit modest, alpha 1-adrenergic agonist activity might diminish the usefulness of compound 2, and we now report on our continuing efforts in this field. Starting from compound 2, we first eliminated the alpha 1-agonist component by isosteric replacement and then, by means of conformational restrictions on compound 7, succeeded in discovering tracizoline (9) and benazoline (12). These two new ligands with high affinity (pKi value 8.74 and 9.07, respectively) and unprecedented selectivity with respect to both alpha 2- (I2/alpha 2 7,762 and 18,621) and alpha 1- (I2/alpha 1 2,344 and 2,691) adrenergic receptors, are valuable tools in the study of I receptor structure and function. In addition, the large number of derivatives studied has allowed us to establish congruent qualitative structure-activity relationships and identify some structural elements governing affinity and selectivity.

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.

New concepts on the central regulation of blood pressure: Alpha2-adrenoceptors and “Imidazoline receptors”

The most usual hypothesis to explain the central hypotensive effect of clonidine-like substances was to admit that these drugs stimulated alpha 2-adrenoceptors within the brainstem. Now it has been demonstrated that neither the endogenous ligand to the alpha-adrenoceptors, noradrenaline, nor any other catecholamine or phenylethylamine was hypotensive in the medullary nucleus reticularis lateralis, where all imidazolines proved to be such. Recently, a membrane receptor population sensitive to clonidine and insensitive to catecholamines was described within the nucleus reticularis lateralis; this subgroup of receptors represented 20 to 30 percent of the [3H]clonidine binding sites in the bovine nucleus reticularis lateralis and 100 percent within the human nucleus reticularis lateralis region. Thus, the existence of such imidazoline specific receptors was clearly established and the endogenous ligand for those receptors, which is neither a catecholamine nor likely a peptide, is under processing for purification. Therefore, it appeared that the hypotensive effect of substances with an imidazoline or imidazoline-like structure might be due to their action within medullary receptors specific for this endogenous ligand temporarily named clonidine displacing substance. Rilmenidine, structurally close to imidazolines, also interfered with these receptors. The central component of its hypotensive effect was recently confirmed in rabbits, where its central cardiovascular effects were antagonized by the clonidine displacing substance. Although exhibiting a lower affinity than the reference substance for these receptors, rilmenidine might have a higher selectivity, thus explaining its restricted side effects. A structure-activity study with this molecule would bring a confirmation to these first observations.

Characterization of a partial cDNA clone detected by imidazoline receptor-selective antisera

A cDNA clone has been isolated from a human hippocampal cDNA expression library by relying on the selectivity of two antisera that are specific for imidazoline binding proteins. A 1789 bp cDNA clone was sequenced and shown to contain a single open-reading frame that predicts a 66 kDa polypeptide, but it is truncated based on its lack of a stop codon and poly-A+ tail. Two regions of homology exist for the predicted amino acid sequence in common with chromogranin-A and B proteins, a zinc finger protein, and the ryanodine receptor. Northern blot analyses of poly-A+ mRNA from 36 human tissues indicated two differentially expressed transcripts of 6.0 and 9.5 kb. The 6.0 kb mRNA form was enriched in brain and endocrine tissues as compared to other tissues, but not in strict concordance with I1-imidazoline binding sites. The highest overall amounts of the combined transcripts were found in pituitary. In situ hybridization histochemistry revealed an enrichment of the message in neuronal cell bodies of the rat hippocampus and cerebellar cortex. This clone has some of the properties expected of an imidazoline receptor.

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.

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.