Fundam Clin Pharmacol (1992) 6, Suppl I , 15s-21s
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PBousquet, GBricca, M.DontenwiU, J.Feldman, H.Greney, kBelcourt,J. Stutzmann,E.Tibiriw+ Laboratoire de Pharmacologie Cardiovasculaire e t RBnale, CNRS URA 589, Facultk d e MBdecine, Universitd Louis Pasteur, STRASBOURG - FRANCE * Instituto Oswaldo Cruz,departamento d e Fisiologia e Farmacodinamica, av; Brasil, 4365 Manguinhos C.P. 926 20040 RIO DE JANEIRO - BRAZIL The involvement of the brain in t h e cardiovascular regulation and in dysfunctioning associated with some forms of the hypertensive diseases presents this organ consequently as a target for cardiovascular drugs and causes presently a new wave of interest for the research in this field. Once again, t h e precise study of the mechanism of action of these drugs allowed t h a t revival of interest. attempting to explain the If the 70's and the first half of the 80's were the period of t h e a-adrenergic theory (1-4) cardiovascular effects of drugs widely used as antihypertensive agents such as clonidine, a new hypothesis recently arose: t h e involvement of receptors, unknown until now, specifically sensitive to imidazolines and catecholamines insensitive (5). Indeed, as soon as 1975, we observed the incapability of noradrenaline to reproduce the cardiovascular effects, especially t h e hypotensive one, of clonidine and/or its analogues, as soon as i t was injected directly within the Nucleus Reticularis Lateralis (NRL) of the medulla oblongata, t h e privileged site of action of these substances (6, 7). This first observation introduced a doubt as to t h e validity of the theory involving cerebral aadrenoceptors to explain these effects. Furthermore, no direct correlation could b e established between t h e affinities of clonidine-like compounds for a-adrenoceptors and their efficiencies upon these receptors(2, 8).
These observations corroborated those of Ruffolo's group indicating that, in a variety of peripheral tissues also, phenylethylamines a n d imidazoles had dissimilar mechanisms of action (see Hieble a n d Ruffolo's review in this issue). We then performed a wide structure/activity relationship study during which we observed t h a t aadrenergic substances bearing a catecholamine or phenylethylamine structure proved never capable to reproduce t h e hypotensive effect of clonidine when all t h e imidazolines tested induced dose-dependent effects, whatever their selectivity for one or the other sub-type of a-adrenoceptors was. This study was performed by injecting the drugs directly within t h e anaesthetized cat's NRL (5).These d a t a led us to suggest t h e existence of receptors specifically sensitive to t h e imidazoline structure which initiated t h e hypotensive effects of clonidine-type substance and did not belong to t h e classical a-adrenoceptor family. These receptors do not belong to t h e histamine receptors family either, since at least in our hands, antihistamines bearing an imidazole structure such as metiamide or cimetidine could not prevent t h e hypotensive effect of clonidine and in binding assays [3Hlclonidine binding in t h e NRL region was insensitive to histamine itself. By analogy with t h e terminology used for benzodiazepine receptors or opiate receptors, we provisionally named these receptors, imidazoline receptors or imidazoline preferring receptors. Since, a rather large amount of biochemical and functional experimental d a t a confirmed t h a t hypothesis (8,9). Very recently, a therapeutical extension strenghthened this new concept. Indeed, if all these hypothesis were to b e confirmed, i t would become
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possible t o discriminate the mechanism associated with the hypotensive effect of these clonidine-like substances from the one presiding t o their most frequent side-effect, sedation, which is clearly related by to the involvement of a-adrenoceptors as shown by a series of experimental arguments (10).
Biochemical data The first confirmation of the existence of specific imidazoline sensitive binding sites insensitive to catecholamines was brought the first time, by the study of the tritiated para-aminoclonidine (PAC)binding to membranes prepared from the ventrolateral area, including the NRL, of the bovine medulla (8). In these preparations, it was demonstrated that 30% of labelled PAC remained undisplaced by noradrenaline in excess (11).Since, this observation has been confirmed in other species, especially the rabbit which proved highly
sensitive to the central cardiovascular effects of substances with an imidazoline structure (12).The proportion of sites specifically sensitive t o imidazolines was even higher in human membranes prepared from the same area of the brain stem (13, 14). Several tritium labelled drugs have been used t o study the characteristics of t h a t specific binding: idazoxan, clonidine and PAC. With each of these substances, the binding was saturable, reversible, sensitive
to different chemical families but always insensitive t o catecholamines. Some correlations between the affinity of these imidazoline compounds for the NRL imidazoline specific binding sites and their hypotensive efficiency have been reported (8, 15). The difficulty with such correlations probably resided in the fact that the interferences between some of clonidine-related substances were not strictly competitive (Bricca e t al., in press). However, the interaction of these drugs in that area was, a t least qualitatively, associated with an influence upon the arterial blood pressure or with an antagonistic activity toward substances capable to modify the vasomotor tone. Further arguments to this point will be presented later in this review. The association of a specific binding site with a function definitely defines a true receptor (16). The choice of the ligand t o perform the fine study of this binding is not innocent. As a matter of fact, clonidine exhibited finally a rather mediocre affinity for the "imidazoline" site labelled by tritiated clonidine insensitive t o noradrenaline and in any case lower than its affinity for the classical a2-adrenoceptors labelled by tritiated rauwolscine o r clonidine (14). In return, idazoxan exhibits a high affinity for an "imidazoline" type binding site which seems t o be different from the one labelled by clonidine as these two compounds did not interfere competitively with the "idazoxan" site (17). In fact, clonidine not only poorly displaced tritiated idazoxan from its binding sites but in addition clonidine's behaviour is different from a competitive interaction for there are no modifications of the affinity constant together with a decrease of the number of the binding sites number labelled by [3Hlidazoxan in the presence of clonidine (Bricca e t al. in press). Thus, imidazoline binding sites different from the classical ap-adrenoceptors exist in the brain. Some of them are involved in the central regulation of the cardiovascular function. However, at the time, all the available ligands proved capable of binding, a t various extent, t o a2-adrenoceptors and "imidazoline" receptors a s well. This represents an hindrance to the advances in the biochemical research which would not
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be released until t h e discovery of a chemical tool devoid of a n y affinity for t h e a - a d r e n o c e p t o r s a n d specifically selective of t h e "imidazoline" receptors. I t should be pointed out that t h e limit evoked before was not really surprizing as t h e imidazoline structure had been developed in that series of drugs to stimulate adrenoceptors. I t i s perhaps useful to call everyone's recollection about clonidine designed as a n a-adrenergic peripheral vasoconstrictive substance. Paradoxically, i t will probably be necessary to q u i t the domain of a strict imidazoline structure to conceive ligands selective of the imidazoline sensitive receptors. However, t h a t paradox is only apparent and these receptors would have probably soon or later to be redifined and renamed. The existence of specific imidazoline binding sites insensitive to catecholamines was confirmed by various techniques including autoradiography within various parts of t h e brain (8, 11-15,17, 18).Such binding sites were also found in peripheral tissues such as t h e liver (19,201, the adipose tissue (21, 22) a n d the kidney (23).The functions of these sites within the various organs a n d tissues have not yet been clearly established;
some might be associated with a Na+/H+ exchanger in t h e kidney for instance (24). Recently subcellular fractionation allowed to locate such sites in liver and brain mitochondria1 preparations (20, 25). In most cases, the biochemical studies of these specific binding sites in t h e peripheral tissues were performed with tritiated idazoxan as ligand a n d once again clonidine proved generally to be a weak competitor vs t h a t idazoxan binding. Studies a r e in progress to try to elucidate t h e complex interactions between clonidine a n d idazoxan (see A. Parini's review in this issue). Most of t h e time, cirazoline, a st7uctural analogue closely related to clonidine, appeared to exhibit t h e greater regularity in its binding to these imidazoline specific sites and proved to be not only a potent competitor of idazoxan and clonidine as well (14,17) b u t also t h e most potent competitor in all t h e models of imidazoline specific binding. To discriminate between the imidazoline specific binding sites and the a2-adrenoceptors, biochemistry has already provided with several evidences such as the insensitivity to GTP or Gpp(NH)p of t h e imidazoline binding when t h e binding of the same ligands to a2-adrenoceptors i s sensitive (17, Bricca et al. in press). Furthermore, the partial purification of the kidney imidazoline binding site allowed to physically separate the idazoxan site insensitive to catecholamines from t h e a2-adrenoceptors (26). T h e transduction mechanism of the signal coupled with t h e "imidazoline" receptors is not yet known but i t seems not to be mediated through a G-protein. A coupling with a n ionic channel, in particular with a potassium channel because of t h e high sensitivity of t h e idazoxan binding to potassium ions, was suggested (27) b u t further investigations a r e necessary to ascertain this hypothesis.
Functional data The association of some "imidazoline" binding sites with t h e modulation of t h e vasomotor tone was strongly suggested by t h e initial structure/activity relationship study evoked above (5). However, a n experimental procedure allowing to confirm t h i s point has h a d to be developed especially to discriminate between the mode of action leading to t h e sedative effects of clonidine-type substances a n d t h e mechanism involved in t h e hypotensive effect. Thus, we have recently developed a technology to analyse in uiuo the metabolism of t h e various populations of catecholaminergic neurones. T h e metabolic activity is evaluated
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electrochemically by differential voltammetry which allows to m e a s u r e t h e variation i n the DOPAC concentration within t h e extracellular compartment. The activity t h u s measured i s regarded as a n acurate index of t h e global neuronal activity (28). The influence of central antihypertensive drugs of t h e clonidine family upon the locus coeruleus (LC), t h e alleged site of their sedative action
(lo), a n d upon the NRL neurones,
the privileged site of their hypotensive effect (7). has been analysed. In both structures, we dealt with populations of noradrenergic neurones. First we demonstrated t h a t rather low doses of clonidine given systemically were able to inhibit the NRL neuronal acitivity a n d produced a decrease in the arterial blood pressure when the LC activity remained unaffected by t h e same dose of t h e drug
(29). To observe the beginning of a decrease in t h e activity of the LC neurones, t h e dose h a d to be increased by a factor of 5 at least. This showed already a functional selectivity of clonidine for t h e NRL area containing imidazoline preferring receptors. A correlation h a s been established between t h e importance of t h e hypotensive effect provoked by
different doses of clonidine a n d t h e inhibitory effect of the neuronal activity within t h e NRL (29). Of course, such a correlation could not be shown with the effects observed when high doses of clonidine were injected in the LC. In order to be able to use selective antagonists under optimal conditions in these studies, we reassessed these conditions for t h e two classical antagonists of t h e clonidine's effects, idazoxan a n d yohimbine, and we observed that, to assure their selectivity for one or the other type of receptors, these substances are to be used at doses much lower than t h a t usually given (30).
So, idazoxan, which h a s a n imidazoline structure, i s selective for imidazoline receptors when used at t h e low dose of Snmoles/kg directly injected intracisternally when yohimbine, at the s a m e dose, was exclusively active upon t h e classical a2-adrenoceptors. Under these conditions, a pretreatment with idazoxan prevented very efficiently t h e hypotensive effect of clonidine as well as i t s inhibitory effect of t h e catecholaminergic neurones of t h e NRL. This pretreatment however proved completely unefficient on t h e inhibitory action of higher doses of clonidine on t h e metabolic acitivity of LC neurones. A t t h e opposite, yohimbine was active as antagonist in the LC at the dose which was uneficient to prevent the inhibitory effects of clonidine upon t h e NRL neurones (31). Thus, not only a topographical selectivity for t h e sensitivity of the catecholaminergic neurones to clonidine exists b u t i t also clearly appeared that t h e action of that substance fitted into the frame of a n imidazoline coherence in the NRL area as well as in t h e frame of at typically a2adrenergic coherence within the LC. These works represented t h e first success in t h e distinction between t h e mechanisms involved respectively in the sedative and the hypotensive effects of these substances. Thus, t h e avenue was open to the development of hypotensive substances selectively acting on NRL imidazoline preferring receptors t h u s being much less sedative than the reference substances. Rilmenidine represents the first example of a therapeutic agent which is hypotensive via a central mechanism very close to t h a t of clonidine b u t much less sedative, not to say not at all, at t h e doses for which it is hypotensive (32).This lack of sedative effects might be related with a peripheral hypotensive action of rilmenidine but, in all cases, represents t h e expression of a functional
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selectivity of rilmenidine for imidazoline receptors versus t h e ag-adrenoceptors when compared to t h e reference substance, clonidine. Indeed, the binding studies showed a preferential binding of rilmenidine t o the imidazoline receptors in t h e NRL. Such a preference, evaluated by comparing t h e affinities ratio of rilmenidine and of t h e reference substance for t h e imidazoline receptors and t h e a2-adrenoceptors (331, was also found in t h e voltammetry studies; indeed rilmenidine proved even twice more selective than clonidine
for t h e NRL a r e a (34). In t h e same way, we have observed t h a t t h e imidazoline-structured antagonist, idazoxan, proved more efficient to prevent the central hypotensive effects of rilmenidine than t h e classical a 2 blocking antagonist (34), yohimbine.
Immunological approach Using para-aminoclonidine coupled to hemocyanine by glutaraldehyde as immunogen, we developped an anti-clonidine antibody with a double aim in view. On t h e one hand, such antibodies a r e useful tools to purify t h e imidazoline preferring receptors endogenous ligand and t h e receptor itself by t h e use of antiidiotypic antibodies. On t h e other hand, to verify t h e hypothesis according which a dysfunctioning in the system involving t h e imidazoline preferring receptors might be related to a n d explain some forms of t h e hypertensive disease. First a polyclonal antibody was developed and its characteristics were highly interesting as i t crossreacted with a high affinity with substances structurally close to clonidine a n d did absolutely not recognize catecholamines or a n y other endogenous mediator bearing a n imidazole structure such as histamine for instance (36). Then, a monoclonal antibody was also developed with t h e same immunogen a n d again we obtained an antibody exhibiting a selectivity spectrum very close to t h a t of the imidazoline preferring receptors (37). Both antibodies were used in radioimmunoassays which showed t h a t a n immunoreactive substance
existed in the plasma. Preliminary tests revealed t h a t t h a t substance was present in rather low concentrations in normotensive subjects devoid of any cardiovascular disease when high amounts were measured in some hypertensive patients. However, t h e association of high concentrations of this immunoreactive substance with
a cardiovascular pathological process, in particular some forms of arterial hypertension, did not provide yet with any evidence as to t h e existence of a cause-effect relationship nor about its meaning. Further experiments are in progress to investigate t h a t point. The identity of t h e immunoreactive substance t h u s detected and the endogenous ligand of t h e imidazoline preferring receptors has not been established, t h e endogenous mediator having not yet been completely purified but its purification i s under process by other biochemical techniques (38).
References 1.
2. 3.
SCHMITT H. The pharmacology of clonidine and related products. In handbuch der Experimentellen Pharmakologie, ed. by F. Gross, pp. 299-396,Springer, Berlin, 1977. KOBINGER W. Central alpha-adrenergic systems as targets for hypotensive drugs. Rev Physiol Biochem F'harmacol 1978;81:39-100. TIMMERMANS PBMW, VAN ZWIETEN PA. Alphag-adrenoceptors classification, localization, mechanisms and targets for drugs. J Med Chem 1982;25: 1389-1401.
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P Bouquet el a/
4. 5. 6. 7. 8.
9.
10. 11.
12. 13.
14.
15.
16. 17.
18.
19. 20.
21.
22.
23. 24. 25. 26.
27.
BOUSQUET P, SCHWARTZ J. Alpha-adrenergic drugs: pharmacological tools for study of the central vasomotor control. Biochem Pharmacol 1983; 32: 1459-1465. BOUSQUET P, FELDMAN J, SCHWARTZ J, Central cardiovascular effects of alpha-adrenergic drugs: Difference between catecholamines and imid.azolines. J Pharm Exp Ther 1984; 230: 230-236. BOUSQUET P, FELDMAN J , VELLY J , BLOCH R. Role of the ventral surface of the brain stem in the hypotensive action of clonidne. Eur J Pharmacol 1975; 34: 151-156. BOUSQUET P, FELDMAN J , BLOCH R, SCHWARTZ J . The nucleus reticularis lateralis a region highly sensitive to clonidine. Eur J Pharmacol 1981; 69: 389-392. ERNSBERGER, P.R., GIULIANO, R., WILLETTE, R.N. & REIS D.J. : Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostral ventrolateral medulla. J Pharm Exp "her 1990; 253: 408-418. MEELEY MP, ERNSBERGER PR, GRANATA AR, REIS DJ. A endogenous clonidine-displacing substance from bovine brain: receptor binding and hypotensive actions in the ventrolateral medulla. Life Sci 1986; 38: 1119-1126. AGHAJANIAN GR, VAN DEN MAELEN CP. Alpha2-adrenoceptor mediated hyperpolarisation of locus coeruleus neurons: intracellular studies in vivo. Science 1982; 215: 1394-1396. ERNSBERGER PR, MEELEY MP, MA" J J , REIS DJ. Clonidine binds to imidazole binding sites as well as alphap-adrenoceptors in the ventrolateral medulla. Eur J Pharmacol 1987; 134: 1-13. HAMILTON CA, REID JD, YAKUBU MA. I3H]yohimbine and [3Hlidazoxan bind to different sites on rabbit forebrain and kidney membranes. Eur J Pharmacol 1988; 146: 345-348 BRICCA G, DONTENWILL M, MOLINES A, FELDMAN J , BELCOURT A, BOUSQUET P. Evidence for the existence of a homogenous population of imidazoline receptors in the human brainstem. Eur J Pharmacol 1988; 150: 401-402. BRICCA G, DONTENWILL M, MOLINES A, FELDMAN J, BELCOURT A, BOUSQUET P. The imidazoline preferring receptor: binding studies in bovine,rat and human brainstem. E u r J Pharmacol 1989;162:1-9 ERNSBERGER PR, GIULIANO R, WILLE'R'E RN, GRANATA AR, REIS DJ. Hypotensive action of clonidine analogs correlates with binding affinity a t imidazole and not alphag-adrenergic receptors in the rostral ventrolateral medulla. Hypertens. 1988; 6: suppl. 4, S5543557. LADURON PM. Stereospecificity in binding studies. A useful criterion though insufficient to prove the presence of receptors. Biochem Pharmacol 1988; 37: 37-40 WIKBERG JES, UHLEN S. Further characterization of the guinea pig cerebral cortex idazoxan receptor, solubilization distinction from the imidazoline site, and demonstration of cirazoline as an idazoxan receptor selective drug. J Neurnchem 1990; 55: 192-203. KAMISAKI Y, ISHIKAWA T, TAKA0 Y, OMODANI H, KUNO N, ITOH T. Binding of l3H]paminoclonidine to two sites, alphag-adrenoceptors and imidazoline binding s i t s : distribution of imidazoline binding sites in rat brain. Br Research 1990; 514: 15-21. ZONNENSCHEIN R, DIAMANT S, ATLAS D. Imidazoline receptors in rat liver cell: a novel receptor or a subtype of a2-adrenoceptors? Eur J Pharmacol 1990; 190: 203-215. TESSON F, PRIP-BUUS C, LEMOINE A, PEGORIER J P , PARINI A. Sub-cellular distribution of imidazoline guanidinium receptive sites in human and rabbit liver. Major localisation to the mitochondria1 outer membrane. J Biol Chem 1991; 266: 155-160. LANGIN D, LAFONTAN M. [3H1-idazoxan binding a t non-alpha2-adrenoceptorsin rabbit adipocyte membranes. Eur J Pharmacol 1989; 159: 199-203. GALITZKY J , LAFONTAN M, PARIS H, BERLAN M. : Human fat cell alpha2-adrenoceptors. I1 comparative study of partial and full agonist binding parameters using [3Hlclonidine and [3H]UK 14304. J Pharm Exp Ther 1989; 249: 592. COUPRY I, PODEVIN RA, DAUSSE JP, PARINI A. Evidence for imidazoline binding sites in basolateral membranes from rabbit kidney. Biochem Biophys Res Commun 1987; 147: 1055-1060. BIDET M, POUJEOL P, PARINI A. Effect of imidazolines on NA+ transport and intracellular pH in renal proximal tubule cells. Bioch Biophys Acta 1990; 1024: 173-178. TESSON F, PARINI A. Identification of a n imidazoline-guanidium receptive site in mitochondria1 from rabbit cerebral cortex. Eur J Pharmacol Molecular Pharmacology Section 1991; 208: 81-83. PARINI A.,COUPRY I.,GRAHAM RM,UZIELLI 1,ATLAS D, LANIER SM, Characterization of a n imidazolindguanidinium receptive site distinct from the alpha2 -adrenergic recept0r.J Biol Chem 1989;264: 11874-11878. COUPRY I, ATLAS D, PODEVIN RA, UZIELLI I, PARINI A. Imidazoline-guanidiniumreceptive site in renal proximal tubule: Asymetric distribution, regulation by cations and interaction with a n endogenous clonidine displacing substance. J Pharm Exp Ther 1989; 252: 293-299.
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29.
30.
31.
32.
33. 34.
35.
36.
37.
38.
BUDA M, DE SIMON1 G, GONON F, PUJOL J F . Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. I. Nature and origin of contributors to the oxidation current at +0.1 V. Brain Res 1983; 273: 197-206. TIBIRICA E, MERMET C, FELDMAN J , GONON F, BOUSQUET P. Correlation between the inhibitory effect on catecholaminergic ventrolateral medullary neurons and the hypotension evoked by clonidine : a voltammetric approach. J Pharm Exp Ther 1989 250: 642-647. TIBIRICA E, FELDMAN J , BOUSQUET P. Differences in the ability of yohimbine to antagonize the hypotensive effect of clonidine in the normotensive and spontaneously hypertensive anaesthetized rats. J Pharmacol Exp Ther 1988; 244: 1062-1066. TIBIRICA E, FELDMAN J , MERMET C, GONON F, BOUSQUET P. An imidazoline specific mechanism for the hypotensive effect of clonidine : A study with yohimbine and idazoxan. J Pharm Exp Ther 1991; 256: 606-613. FILLASTRE J.P., LETAC B., GALJNIER F., LE BIHAN G., SCKWARTZ J. A multicenter double blind study of rilmenidine and clonidine in 333 hypertensive patients. Am. J . Cardiol. 1988; 61:81D-85D BRICCA G,DONTENWILL M,MOLINES A,FELDMAN J , TIBIRICA E,BELCOURT A,BOUSQUET P. Rilmenidine selectivity for imidazoline receptors in the human brain. Eur J Pharmacoll989 163: 373-377 TIBIRICA E, FELDMAN J , MERMET C, MONASSIER L, GONON F, BOUSQUET P. Selectivity of rilmenidine for the nucleus reticularis lateralis, a ventrolateral medullary structure containing imidazolinepreferring receptors. Eur J Pharmacol 1991; 209: 213-221. FELDMAN J , TIBIRICA E, BRICCA G, DONTENWILL M, BELCOURT A, BOUSQUET P. Evidence for the involvement of imidazoline receptors in the central hypotensive effect of rilmenidine in the rabbit. Br J Pharmacol 1990; 100: 600-604. DONTENWILL M, BRICCA G, MOLINES A, BOUSQUET P, BELCOURT A. Production a n d characterization of anti-clonidine antibodies not cross-reacting with catecholamines. Eur J Pharmacol 1988; 149: 249-255. DONTENWILL M, MOLINES A, BRICCA G, STUTZMA”, KEMPF J , BELCOURT A, BOUSQUET P. Production and characterization of a n iminoimidazolidine specific monoclonal antibody using para-amino clonidine as antigen. Life Sciences 1992 (in press). BELCOURT A, GRENEY H, BRICCA G, DONTENWILL M, BOUSQUET P Partial purification of the human endazoline, the specific endogenous ligand for imidazoline receptors. Eur J Pharmaco11990;183:852853.
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PBousquet, GBricca, M.DontenwiU, J.Feldman, H.Greney, kBelcourt,J. Stutzmann,E.Tibiriw+ Laboratoire de Pharmacologie Cardiovasculaire e t RBnale, CNRS URA 589, Facultk d e MBdecine, Universitd Louis Pasteur, STRASBOURG - FRANCE * Instituto Oswaldo Cruz,departamento d e Fisiologia e Farmacodinamica, av; Brasil, 4365 Manguinhos C.P. 926 20040 RIO DE JANEIRO - BRAZIL The involvement of the brain in t h e cardiovascular regulation and in dysfunctioning associated with some forms of the hypertensive diseases presents this organ consequently as a target for cardiovascular drugs and causes presently a new wave of interest for the research in this field. Once again, t h e precise study of the mechanism of action of these drugs allowed t h a t revival of interest. attempting to explain the If the 70's and the first half of the 80's were the period of t h e a-adrenergic theory (1-4) cardiovascular effects of drugs widely used as antihypertensive agents such as clonidine, a new hypothesis recently arose: t h e involvement of receptors, unknown until now, specifically sensitive to imidazolines and catecholamines insensitive (5). Indeed, as soon as 1975, we observed the incapability of noradrenaline to reproduce the cardiovascular effects, especially t h e hypotensive one, of clonidine and/or its analogues, as soon as i t was injected directly within the Nucleus Reticularis Lateralis (NRL) of the medulla oblongata, t h e privileged site of action of these substances (6, 7). This first observation introduced a doubt as to t h e validity of the theory involving cerebral aadrenoceptors to explain these effects. Furthermore, no direct correlation could b e established between t h e affinities of clonidine-like compounds for a-adrenoceptors and their efficiencies upon these receptors(2, 8).
These observations corroborated those of Ruffolo's group indicating that, in a variety of peripheral tissues also, phenylethylamines a n d imidazoles had dissimilar mechanisms of action (see Hieble a n d Ruffolo's review in this issue). We then performed a wide structure/activity relationship study during which we observed t h a t aadrenergic substances bearing a catecholamine or phenylethylamine structure proved never capable to reproduce t h e hypotensive effect of clonidine when all t h e imidazolines tested induced dose-dependent effects, whatever their selectivity for one or the other sub-type of a-adrenoceptors was. This study was performed by injecting the drugs directly within t h e anaesthetized cat's NRL (5).These d a t a led us to suggest t h e existence of receptors specifically sensitive to t h e imidazoline structure which initiated t h e hypotensive effects of clonidine-type substance and did not belong to t h e classical a-adrenoceptor family. These receptors do not belong to t h e histamine receptors family either, since at least in our hands, antihistamines bearing an imidazole structure such as metiamide or cimetidine could not prevent t h e hypotensive effect of clonidine and in binding assays [3Hlclonidine binding in t h e NRL region was insensitive to histamine itself. By analogy with t h e terminology used for benzodiazepine receptors or opiate receptors, we provisionally named these receptors, imidazoline receptors or imidazoline preferring receptors. Since, a rather large amount of biochemical and functional experimental d a t a confirmed t h a t hypothesis (8,9). Very recently, a therapeutical extension strenghthened this new concept. Indeed, if all these hypothesis were to b e confirmed, i t would become
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possible t o discriminate the mechanism associated with the hypotensive effect of these clonidine-like substances from the one presiding t o their most frequent side-effect, sedation, which is clearly related by to the involvement of a-adrenoceptors as shown by a series of experimental arguments (10).
Biochemical data The first confirmation of the existence of specific imidazoline sensitive binding sites insensitive to catecholamines was brought the first time, by the study of the tritiated para-aminoclonidine (PAC)binding to membranes prepared from the ventrolateral area, including the NRL, of the bovine medulla (8). In these preparations, it was demonstrated that 30% of labelled PAC remained undisplaced by noradrenaline in excess (11).Since, this observation has been confirmed in other species, especially the rabbit which proved highly
sensitive to the central cardiovascular effects of substances with an imidazoline structure (12).The proportion of sites specifically sensitive t o imidazolines was even higher in human membranes prepared from the same area of the brain stem (13, 14). Several tritium labelled drugs have been used t o study the characteristics of t h a t specific binding: idazoxan, clonidine and PAC. With each of these substances, the binding was saturable, reversible, sensitive
to different chemical families but always insensitive t o catecholamines. Some correlations between the affinity of these imidazoline compounds for the NRL imidazoline specific binding sites and their hypotensive efficiency have been reported (8, 15). The difficulty with such correlations probably resided in the fact that the interferences between some of clonidine-related substances were not strictly competitive (Bricca e t al., in press). However, the interaction of these drugs in that area was, a t least qualitatively, associated with an influence upon the arterial blood pressure or with an antagonistic activity toward substances capable to modify the vasomotor tone. Further arguments to this point will be presented later in this review. The association of a specific binding site with a function definitely defines a true receptor (16). The choice of the ligand t o perform the fine study of this binding is not innocent. As a matter of fact, clonidine exhibited finally a rather mediocre affinity for the "imidazoline" site labelled by tritiated clonidine insensitive t o noradrenaline and in any case lower than its affinity for the classical a2-adrenoceptors labelled by tritiated rauwolscine o r clonidine (14). In return, idazoxan exhibits a high affinity for an "imidazoline" type binding site which seems t o be different from the one labelled by clonidine as these two compounds did not interfere competitively with the "idazoxan" site (17). In fact, clonidine not only poorly displaced tritiated idazoxan from its binding sites but in addition clonidine's behaviour is different from a competitive interaction for there are no modifications of the affinity constant together with a decrease of the number of the binding sites number labelled by [3Hlidazoxan in the presence of clonidine (Bricca e t al. in press). Thus, imidazoline binding sites different from the classical ap-adrenoceptors exist in the brain. Some of them are involved in the central regulation of the cardiovascular function. However, at the time, all the available ligands proved capable of binding, a t various extent, t o a2-adrenoceptors and "imidazoline" receptors a s well. This represents an hindrance to the advances in the biochemical research which would not
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be released until t h e discovery of a chemical tool devoid of a n y affinity for t h e a - a d r e n o c e p t o r s a n d specifically selective of t h e "imidazoline" receptors. I t should be pointed out that t h e limit evoked before was not really surprizing as t h e imidazoline structure had been developed in that series of drugs to stimulate adrenoceptors. I t i s perhaps useful to call everyone's recollection about clonidine designed as a n a-adrenergic peripheral vasoconstrictive substance. Paradoxically, i t will probably be necessary to q u i t the domain of a strict imidazoline structure to conceive ligands selective of the imidazoline sensitive receptors. However, t h a t paradox is only apparent and these receptors would have probably soon or later to be redifined and renamed. The existence of specific imidazoline binding sites insensitive to catecholamines was confirmed by various techniques including autoradiography within various parts of t h e brain (8, 11-15,17, 18).Such binding sites were also found in peripheral tissues such as t h e liver (19,201, the adipose tissue (21, 22) a n d the kidney (23).The functions of these sites within the various organs a n d tissues have not yet been clearly established;
some might be associated with a Na+/H+ exchanger in t h e kidney for instance (24). Recently subcellular fractionation allowed to locate such sites in liver and brain mitochondria1 preparations (20, 25). In most cases, the biochemical studies of these specific binding sites in t h e peripheral tissues were performed with tritiated idazoxan as ligand a n d once again clonidine proved generally to be a weak competitor vs t h a t idazoxan binding. Studies a r e in progress to try to elucidate t h e complex interactions between clonidine a n d idazoxan (see A. Parini's review in this issue). Most of t h e time, cirazoline, a st7uctural analogue closely related to clonidine, appeared to exhibit t h e greater regularity in its binding to these imidazoline specific sites and proved to be not only a potent competitor of idazoxan and clonidine as well (14,17) b u t also t h e most potent competitor in all t h e models of imidazoline specific binding. To discriminate between the imidazoline specific binding sites and the a2-adrenoceptors, biochemistry has already provided with several evidences such as the insensitivity to GTP or Gpp(NH)p of t h e imidazoline binding when t h e binding of the same ligands to a2-adrenoceptors i s sensitive (17, Bricca et al. in press). Furthermore, the partial purification of the kidney imidazoline binding site allowed to physically separate the idazoxan site insensitive to catecholamines from t h e a2-adrenoceptors (26). T h e transduction mechanism of the signal coupled with t h e "imidazoline" receptors is not yet known but i t seems not to be mediated through a G-protein. A coupling with a n ionic channel, in particular with a potassium channel because of t h e high sensitivity of t h e idazoxan binding to potassium ions, was suggested (27) b u t further investigations a r e necessary to ascertain this hypothesis.
Functional data The association of some "imidazoline" binding sites with t h e modulation of t h e vasomotor tone was strongly suggested by t h e initial structure/activity relationship study evoked above (5). However, a n experimental procedure allowing to confirm t h i s point has h a d to be developed especially to discriminate between the mode of action leading to t h e sedative effects of clonidine-type substances a n d t h e mechanism involved in t h e hypotensive effect. Thus, we have recently developed a technology to analyse in uiuo the metabolism of t h e various populations of catecholaminergic neurones. T h e metabolic activity is evaluated
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electrochemically by differential voltammetry which allows to m e a s u r e t h e variation i n the DOPAC concentration within t h e extracellular compartment. The activity t h u s measured i s regarded as a n acurate index of t h e global neuronal activity (28). The influence of central antihypertensive drugs of t h e clonidine family upon the locus coeruleus (LC), t h e alleged site of their sedative action
(lo), a n d upon the NRL neurones,
the privileged site of their hypotensive effect (7). has been analysed. In both structures, we dealt with populations of noradrenergic neurones. First we demonstrated t h a t rather low doses of clonidine given systemically were able to inhibit the NRL neuronal acitivity a n d produced a decrease in the arterial blood pressure when the LC activity remained unaffected by t h e same dose of t h e drug
(29). To observe the beginning of a decrease in t h e activity of the LC neurones, t h e dose h a d to be increased by a factor of 5 at least. This showed already a functional selectivity of clonidine for t h e NRL area containing imidazoline preferring receptors. A correlation h a s been established between t h e importance of t h e hypotensive effect provoked by
different doses of clonidine a n d t h e inhibitory effect of the neuronal activity within t h e NRL (29). Of course, such a correlation could not be shown with the effects observed when high doses of clonidine were injected in the LC. In order to be able to use selective antagonists under optimal conditions in these studies, we reassessed these conditions for t h e two classical antagonists of t h e clonidine's effects, idazoxan a n d yohimbine, and we observed that, to assure their selectivity for one or the other type of receptors, these substances are to be used at doses much lower than t h a t usually given (30).
So, idazoxan, which h a s a n imidazoline structure, i s selective for imidazoline receptors when used at t h e low dose of Snmoles/kg directly injected intracisternally when yohimbine, at the s a m e dose, was exclusively active upon t h e classical a2-adrenoceptors. Under these conditions, a pretreatment with idazoxan prevented very efficiently t h e hypotensive effect of clonidine as well as i t s inhibitory effect of t h e catecholaminergic neurones of t h e NRL. This pretreatment however proved completely unefficient on t h e inhibitory action of higher doses of clonidine on t h e metabolic acitivity of LC neurones. A t t h e opposite, yohimbine was active as antagonist in the LC at the dose which was uneficient to prevent the inhibitory effects of clonidine upon t h e NRL neurones (31). Thus, not only a topographical selectivity for t h e sensitivity of the catecholaminergic neurones to clonidine exists b u t i t also clearly appeared that t h e action of that substance fitted into the frame of a n imidazoline coherence in the NRL area as well as in t h e frame of at typically a2adrenergic coherence within the LC. These works represented t h e first success in t h e distinction between t h e mechanisms involved respectively in the sedative and the hypotensive effects of these substances. Thus, t h e avenue was open to the development of hypotensive substances selectively acting on NRL imidazoline preferring receptors t h u s being much less sedative than the reference substances. Rilmenidine represents the first example of a therapeutic agent which is hypotensive via a central mechanism very close to t h a t of clonidine b u t much less sedative, not to say not at all, at t h e doses for which it is hypotensive (32).This lack of sedative effects might be related with a peripheral hypotensive action of rilmenidine but, in all cases, represents t h e expression of a functional
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selectivity of rilmenidine for imidazoline receptors versus t h e ag-adrenoceptors when compared to t h e reference substance, clonidine. Indeed, the binding studies showed a preferential binding of rilmenidine t o the imidazoline receptors in t h e NRL. Such a preference, evaluated by comparing t h e affinities ratio of rilmenidine and of t h e reference substance for t h e imidazoline receptors and t h e a2-adrenoceptors (331, was also found in t h e voltammetry studies; indeed rilmenidine proved even twice more selective than clonidine
for t h e NRL a r e a (34). In t h e same way, we have observed t h a t t h e imidazoline-structured antagonist, idazoxan, proved more efficient to prevent the central hypotensive effects of rilmenidine than t h e classical a 2 blocking antagonist (34), yohimbine.
Immunological approach Using para-aminoclonidine coupled to hemocyanine by glutaraldehyde as immunogen, we developped an anti-clonidine antibody with a double aim in view. On t h e one hand, such antibodies a r e useful tools to purify t h e imidazoline preferring receptors endogenous ligand and t h e receptor itself by t h e use of antiidiotypic antibodies. On t h e other hand, to verify t h e hypothesis according which a dysfunctioning in the system involving t h e imidazoline preferring receptors might be related to a n d explain some forms of t h e hypertensive disease. First a polyclonal antibody was developed and its characteristics were highly interesting as i t crossreacted with a high affinity with substances structurally close to clonidine a n d did absolutely not recognize catecholamines or a n y other endogenous mediator bearing a n imidazole structure such as histamine for instance (36). Then, a monoclonal antibody was also developed with t h e same immunogen a n d again we obtained an antibody exhibiting a selectivity spectrum very close to t h a t of the imidazoline preferring receptors (37). Both antibodies were used in radioimmunoassays which showed t h a t a n immunoreactive substance
existed in the plasma. Preliminary tests revealed t h a t t h a t substance was present in rather low concentrations in normotensive subjects devoid of any cardiovascular disease when high amounts were measured in some hypertensive patients. However, t h e association of high concentrations of this immunoreactive substance with
a cardiovascular pathological process, in particular some forms of arterial hypertension, did not provide yet with any evidence as to t h e existence of a cause-effect relationship nor about its meaning. Further experiments are in progress to investigate t h a t point. The identity of t h e immunoreactive substance t h u s detected and the endogenous ligand of t h e imidazoline preferring receptors has not been established, t h e endogenous mediator having not yet been completely purified but its purification i s under process by other biochemical techniques (38).
References 1.
2. 3.
SCHMITT H. The pharmacology of clonidine and related products. In handbuch der Experimentellen Pharmakologie, ed. by F. Gross, pp. 299-396,Springer, Berlin, 1977. KOBINGER W. Central alpha-adrenergic systems as targets for hypotensive drugs. Rev Physiol Biochem F'harmacol 1978;81:39-100. TIMMERMANS PBMW, VAN ZWIETEN PA. Alphag-adrenoceptors classification, localization, mechanisms and targets for drugs. J Med Chem 1982;25: 1389-1401.
20s
P Bouquet el a/
4. 5. 6. 7. 8.
9.
10. 11.
12. 13.
14.
15.
16. 17.
18.
19. 20.
21.
22.
23. 24. 25. 26.
27.
BOUSQUET P, SCHWARTZ J. Alpha-adrenergic drugs: pharmacological tools for study of the central vasomotor control. Biochem Pharmacol 1983; 32: 1459-1465. BOUSQUET P, FELDMAN J, SCHWARTZ J, Central cardiovascular effects of alpha-adrenergic drugs: Difference between catecholamines and imid.azolines. J Pharm Exp Ther 1984; 230: 230-236. BOUSQUET P, FELDMAN J , VELLY J , BLOCH R. Role of the ventral surface of the brain stem in the hypotensive action of clonidne. Eur J Pharmacol 1975; 34: 151-156. BOUSQUET P, FELDMAN J , BLOCH R, SCHWARTZ J . The nucleus reticularis lateralis a region highly sensitive to clonidine. Eur J Pharmacol 1981; 69: 389-392. ERNSBERGER, P.R., GIULIANO, R., WILLETTE, R.N. & REIS D.J. : Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostral ventrolateral medulla. J Pharm Exp "her 1990; 253: 408-418. MEELEY MP, ERNSBERGER PR, GRANATA AR, REIS DJ. A endogenous clonidine-displacing substance from bovine brain: receptor binding and hypotensive actions in the ventrolateral medulla. Life Sci 1986; 38: 1119-1126. AGHAJANIAN GR, VAN DEN MAELEN CP. Alpha2-adrenoceptor mediated hyperpolarisation of locus coeruleus neurons: intracellular studies in vivo. Science 1982; 215: 1394-1396. ERNSBERGER PR, MEELEY MP, MA" J J , REIS DJ. Clonidine binds to imidazole binding sites as well as alphap-adrenoceptors in the ventrolateral medulla. Eur J Pharmacol 1987; 134: 1-13. HAMILTON CA, REID JD, YAKUBU MA. I3H]yohimbine and [3Hlidazoxan bind to different sites on rabbit forebrain and kidney membranes. Eur J Pharmacol 1988; 146: 345-348 BRICCA G, DONTENWILL M, MOLINES A, FELDMAN J , BELCOURT A, BOUSQUET P. Evidence for the existence of a homogenous population of imidazoline receptors in the human brainstem. Eur J Pharmacol 1988; 150: 401-402. BRICCA G, DONTENWILL M, MOLINES A, FELDMAN J, BELCOURT A, BOUSQUET P. The imidazoline preferring receptor: binding studies in bovine,rat and human brainstem. E u r J Pharmacol 1989;162:1-9 ERNSBERGER PR, GIULIANO R, WILLE'R'E RN, GRANATA AR, REIS DJ. Hypotensive action of clonidine analogs correlates with binding affinity a t imidazole and not alphag-adrenergic receptors in the rostral ventrolateral medulla. Hypertens. 1988; 6: suppl. 4, S5543557. LADURON PM. Stereospecificity in binding studies. A useful criterion though insufficient to prove the presence of receptors. Biochem Pharmacol 1988; 37: 37-40 WIKBERG JES, UHLEN S. Further characterization of the guinea pig cerebral cortex idazoxan receptor, solubilization distinction from the imidazoline site, and demonstration of cirazoline as an idazoxan receptor selective drug. J Neurnchem 1990; 55: 192-203. KAMISAKI Y, ISHIKAWA T, TAKA0 Y, OMODANI H, KUNO N, ITOH T. Binding of l3H]paminoclonidine to two sites, alphag-adrenoceptors and imidazoline binding s i t s : distribution of imidazoline binding sites in rat brain. Br Research 1990; 514: 15-21. ZONNENSCHEIN R, DIAMANT S, ATLAS D. Imidazoline receptors in rat liver cell: a novel receptor or a subtype of a2-adrenoceptors? Eur J Pharmacol 1990; 190: 203-215. TESSON F, PRIP-BUUS C, LEMOINE A, PEGORIER J P , PARINI A. Sub-cellular distribution of imidazoline guanidinium receptive sites in human and rabbit liver. Major localisation to the mitochondria1 outer membrane. J Biol Chem 1991; 266: 155-160. LANGIN D, LAFONTAN M. [3H1-idazoxan binding a t non-alpha2-adrenoceptorsin rabbit adipocyte membranes. Eur J Pharmacol 1989; 159: 199-203. GALITZKY J , LAFONTAN M, PARIS H, BERLAN M. : Human fat cell alpha2-adrenoceptors. I1 comparative study of partial and full agonist binding parameters using [3Hlclonidine and [3H]UK 14304. J Pharm Exp Ther 1989; 249: 592. COUPRY I, PODEVIN RA, DAUSSE JP, PARINI A. Evidence for imidazoline binding sites in basolateral membranes from rabbit kidney. Biochem Biophys Res Commun 1987; 147: 1055-1060. BIDET M, POUJEOL P, PARINI A. Effect of imidazolines on NA+ transport and intracellular pH in renal proximal tubule cells. Bioch Biophys Acta 1990; 1024: 173-178. TESSON F, PARINI A. Identification of a n imidazoline-guanidium receptive site in mitochondria1 from rabbit cerebral cortex. Eur J Pharmacol Molecular Pharmacology Section 1991; 208: 81-83. PARINI A.,COUPRY I.,GRAHAM RM,UZIELLI 1,ATLAS D, LANIER SM, Characterization of a n imidazolindguanidinium receptive site distinct from the alpha2 -adrenergic recept0r.J Biol Chem 1989;264: 11874-11878. COUPRY I, ATLAS D, PODEVIN RA, UZIELLI I, PARINI A. Imidazoline-guanidiniumreceptive site in renal proximal tubule: Asymetric distribution, regulation by cations and interaction with a n endogenous clonidine displacing substance. J Pharm Exp Ther 1989; 252: 293-299.
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28.
29.
30.
31.
32.
33. 34.
35.
36.
37.
38.
BUDA M, DE SIMON1 G, GONON F, PUJOL J F . Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. I. Nature and origin of contributors to the oxidation current at +0.1 V. Brain Res 1983; 273: 197-206. TIBIRICA E, MERMET C, FELDMAN J , GONON F, BOUSQUET P. Correlation between the inhibitory effect on catecholaminergic ventrolateral medullary neurons and the hypotension evoked by clonidine : a voltammetric approach. J Pharm Exp Ther 1989 250: 642-647. TIBIRICA E, FELDMAN J , BOUSQUET P. Differences in the ability of yohimbine to antagonize the hypotensive effect of clonidine in the normotensive and spontaneously hypertensive anaesthetized rats. J Pharmacol Exp Ther 1988; 244: 1062-1066. TIBIRICA E, FELDMAN J , MERMET C, GONON F, BOUSQUET P. An imidazoline specific mechanism for the hypotensive effect of clonidine : A study with yohimbine and idazoxan. J Pharm Exp Ther 1991; 256: 606-613. FILLASTRE J.P., LETAC B., GALJNIER F., LE BIHAN G., SCKWARTZ J. A multicenter double blind study of rilmenidine and clonidine in 333 hypertensive patients. Am. J . Cardiol. 1988; 61:81D-85D BRICCA G,DONTENWILL M,MOLINES A,FELDMAN J , TIBIRICA E,BELCOURT A,BOUSQUET P. Rilmenidine selectivity for imidazoline receptors in the human brain. Eur J Pharmacoll989 163: 373-377 TIBIRICA E, FELDMAN J , MERMET C, MONASSIER L, GONON F, BOUSQUET P. Selectivity of rilmenidine for the nucleus reticularis lateralis, a ventrolateral medullary structure containing imidazolinepreferring receptors. Eur J Pharmacol 1991; 209: 213-221. FELDMAN J , TIBIRICA E, BRICCA G, DONTENWILL M, BELCOURT A, BOUSQUET P. Evidence for the involvement of imidazoline receptors in the central hypotensive effect of rilmenidine in the rabbit. Br J Pharmacol 1990; 100: 600-604. DONTENWILL M, BRICCA G, MOLINES A, BOUSQUET P, BELCOURT A. Production a n d characterization of anti-clonidine antibodies not cross-reacting with catecholamines. Eur J Pharmacol 1988; 149: 249-255. DONTENWILL M, MOLINES A, BRICCA G, STUTZMA”, KEMPF J , BELCOURT A, BOUSQUET P. Production and characterization of a n iminoimidazolidine specific monoclonal antibody using para-amino clonidine as antigen. Life Sciences 1992 (in press). BELCOURT A, GRENEY H, BRICCA G, DONTENWILL M, BOUSQUET P Partial purification of the human endazoline, the specific endogenous ligand for imidazoline receptors. Eur J Pharmaco11990;183:852853.
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