G. Bricca

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.

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.

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.

Production and characterization of anti-clonidine antibodies not cross-reacting with catecholamines

Polyclonal antibodies against clonidine were developed, with para-aminoclonidine coupled to bovine serumalbumin or hemocyanine with glutaraldehyde used as antigens. The selected antibody (from rabbits) cross-reacted with high specificity with clonidine and its structurally closely related analogues but it recognized neither catecholamines nor various endogenous imidazole molecules such as histamine, purine, adenine, and adenosine, thus appearing to be specific for the aminoimidazoline structure. An interesting cross-reactivity was observed with the bovine clonidine displacing substance, the probable endogenous ligand for receptors involved in the hypotensive effect of clonidine-type substances. This suggested that this molecule should contain an aminoimidazoline or guanidine moiety.

Production and characterization of an iminoimidazolidine specific monoclonal antibody using para-aminoclonidine as antigen

Para-aminoclonidine coupled to hemocyanin was used to produce mouse monoclonal antibodies directed against clonidine. The properties of one of these, called mFE7, secreted by a clone of hybrid myeloma, are described. This antibody displayed total crossreactivity with imidazolidines and no crossreactivity at all with catecholamines or other known naturally occurring substances tested. A liquid phase radioimmunoassay permitted the detection of immunoreactivity in human brain extracts. The mFE7 antibody could be useful for immunopurifying the endogenous ligand of Imidazolines Preferring Receptors (IPR) which are catecholamines insensitive.

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.