Fundam Clin Pharmacol(l992)6 , Suppl I , 7s-13s 0 Elsevier. Paris
L 1 - IMIDAZOLINE RECEPTORS: HISTORICAL PERSPECTIVE J. Paul Hieble and Robert R Ruffolo, Jr. Department of Pharmacology, Smith Kline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, U.S.A.
I. INTRODUCTION Although both phenethylamines and imidazolines activate al-adrenoceptor a n d a2-adrenoceptors, t h e two structural classes do not interact with a-adrenoceptors in a n identical manner. Thus, desensitization of t h e rat vas deferens to t h e contractile action of oxymetazoline, a n imidazoline, produced nearly complete blockade of contraction induced by tetrahydrozoline, another imidazoline, but had no effect on t h e contractile response to t h e phenethylamine agonists, such as noradrenaline, methoxamine or phenylephnne (Ruffolo et al. 1977). Additional evidence for a different mode of interaction of imidazolines and phenethylamines at aadrenoceptors was provided by t h e observation t h a t optically active phenethylamines conform to t h e EassonStedman hypothesis, a n d appear to attach to t h e a-adrenoceptor via a three-point attachment, whereas imidazolines do not conform to t h e Easson-Stedman Hypothesis and interact with t h e receptor by a two-point attachment (Ruffolo et a1 .1983). Furthermore, structure activity relationships for agonist affinity and efficacy at both a l - and a2-adrenoceptors differ substantially between the imidazolines a n d phenethylamines (Ruffolo, 1991). Although subsequent evidence will show t h a t certain imidazolines can interact with non-adrenergic receptors, i t should be stressed t h a t these a n d other imidazolines function as classical agonists and antagonists at the al-and a 2 - adrenoceptor. The contractile or neuroinhibitory actions of imidazdine agonists such as clonidine, cirazoline, oxymetazoline a n d U K 14,304 a r e sensitive to blockade by a n imidazoline antagonists, such as prazosin or rauwolscine (Ruffolo et al. 1982; Ruffolo and Zeid, 1985; Oriowo et al. 1991). Imidazoline agonists can be relatively nonselective for a1 vs a2-adrenoceptors (e.g. oxymetazoline), selective for al-adrenoceptors (St587, cirazoline) or a2-adrenoceptors (clonidine, U K 14,304, DPI). In general, t h e receptor dissociation constants in a given tissue preparation for antagonism against t h e action of imidazoline and phenthylamine agonists are equivalent. Imidazolines, such as clonidine. para-aminoclonidine a n d oxymetazoline, a r e potent inhibitors of the binding of [3Hl epinephrine or [3Hl noradrenaline to brain homogenates (U'Prichard a n d Snyder, 1978) and to a cell line expressing the ag-adrenoceptor (Kahn et al.. 1982). Conversely, epinephrine, noradrenaline and phenylephrine produce potent, stereoselective inhibition of i3Hl clonidine binding in a variety of tissue preparations. Some early radioligand binding experiments did suggest differences i n t h e interaction of phenethylamines a n d imidazolines with a-adrenoceptors- In t h e rat v a s deferens, imidazoline agonists (oxymetazoline, naphazoline) were significantly more p o t e n t t h a n phenethylamines (epinephrine, noradrenaline, phenylephrine) a g a i n s t L3H1 dihydroazapetine binding, which presumably labeled a l adrenoceptors in t h i s tissue (Ruffolo et al.. 1976), b u t clonidirie, epinephrine a n d noradrenaline were essentially equipotent against L3Hl WB4101, another al-adrenoceptor radioligand (U' Prichard a n d Snyder, 1979). Clonidine was also nearly equipotent with t h e phenethylamine agonists against [3Hl WB-4101 in membranes from rat heart (Yamada et al. 1980) and rat brain (Greenberg a n d Snyder, 19771, and clonidine, noradrenaline a n d epinephrine all produced potent displacement of t h e nonselective a - a d r e n o c e p t o r antagonist, dihydro-ergokryptine, to rat brain (Greenberg and Snyder, 1977). 1I.INVOLVEMENT O F IMIDAZOLINE RECEPTORS IN T H E ANTIHYPERTENSIVE ACTION OF CLONIDINE I t is well documented t h a t clonidine lowers blood pressure via a n action within t h e central nervous system to decrease sympathetic outflow to t h e periphery (see review by Kobinger, 1978). A prejunctional receptor on a noradrenergic neuron does not appear to be involved, since neither neurotransmitter depletion with reserpine (Haeusler, 1974; Kobinger a n d Pichler, 1976) or destruction of t e r m i n a l s with 6hydroxydopamine (Dollery a n d Reid, 1973; Finch, 1975) substantially attenuates t h e ability of clonidine to decrease blood pressure.
J P Hieble and R R Ruffolo, J r
The selective a2-adrenoceptor antagonists, yohimbine and rauwolscine, will produce dose related blockade of the hypotensive effect produced by intravertebral administration of clonidine to the anesthetized cat (Timmermans et al. 1981). Experiments in anesthetized dogs (Schmitt et al. 1973) also show blockade by yohimbine of the action of intravertebral clonidine In the anesthetized cat (Hamilton et al. 1980) or r a t (Borkowski and Finch, 19791, centrally administered yohimbine can block the hypotensive response to intracerebroventricular clonidine. This evidence striongly supports the premise t h a t clonidine lowers blood pressure via an action at central ap-adrenoceptors. a-Methyldopa also acts within the central nervous system to lower blood presssure, via its metabolite, a-methy lnoradrenaline, a potent and selective ap-adrenoceptor agonist. Although the therapeutic and sideeffect profiles of systemically administered clonidine and a-methylnoradrenaline a r e quite similar, it appears that their sites of action within the brain are different. Both phenethylamine and imidazoline agonists are known t o produce hypotensive effects when administered to several diverse sites within the central nervous system (see review by Brody et al., 1984). Local administration of a-methylnoradrenaline t o the nucleus tractus solitarius produces a dramatic hypotensive response in the anesthetized rat, whereas clonidne is active via this route only at very high doses (Kubo and Misu, 1981). In contrast, very low doses of clonidine are hypotensive in the anesthetized cat when administered to the nucleus reticularis lateralis; amethylnoradrenaline is inactive at this site (Bousquet et d., 1984). Earlier studies had shown t h a t lesions on the ventral surface of the brain stem, in the general area of the nucleus reticularis lateralis, would abolish the hypotensive action of intravenous clonidine in the cat (Bousquet et al. 1975). Differences in the central action of clonidine and a-methylnoradrenaline could be attributed to differences in intrinsic activity between the two agonists, or the potent R-adrenoceptor agonist activity of a-methylnoradrenaline, since R-adrenoceptor stimulation a t several central sites is known t o elevate blood pressure (see Brody et al., 1984). However, differences exist even between two selective ap-adrenoceptor agonists, clonidine and guanfacine, since only clonidine is effective in reducing bood pressure in cats upon topical administration to the ventral surface of the medulla (Scholtysik et al., 1975). Clonidine and guanfacine are essentially equipotent when administered into the left lateral ventricle of anesthetized cats (compare data of Scholtysik et al. 1975 and Hamilton et al. 1980). The differences in hypotensive action of structurally dissimilar ap-adrenoceptor agonist when administered to the nucleus reticularis lateralis (NRL)suggested the presence in this region of a receptor sensitive to the imidazoline moiety present in clonidine (Bousquet et al. 1984). This premise is supported by the hypotensive action of imidazolines having no a2-adrenoceptor agonist activity, such as cirazoline and ST587. These al-adrenoceptor agonists lower blood pressure in the anesthetized cat when directly administered to the NRL, but have no hypotensive effect when administered intravenously or into the vertebral artery (DeJonge et al. 1981). Experimental support for imidazoline receptors in the NRL h a s been provided by studies in anesthetized rat showing that a variety of imidazoline containing agents can lower blood pressure and heart rate when injected into this region (Ernsberger et al.. 1990). The hypotensive action of locally administered clonidine could be antagonized by local administration of idazoxan, a n ap-adrenoceptor antagonist containing an imidazoline ring, but not by the non-imidazoline, SK&F 86466, which h a s a similiar a p adrenoceptor affinity to idazoxan. A similiar profile was a served for rilmenidine (Gomez et al. 19911, a novel oxazoline derivative having antihypertensive activity in both experimental animals and in human subjects (Safar, 1989). In anesthetized rabbits, intracisternal administration of idazoxan was more effective than an equal dose of rauwolscine against the hypotensive effect of intracisternal rilmenedine (Bricca et al.. 1990). Intracisternal administration should allow access to many of the putative sites of action for a centrally acting sympatholytic drug, and selective blockade by idazoxan of the action of rilmenidine administered uia this route strengthens the case for a functional role for the imidazoline receptor in the NRL. Studies in anesthetized rats show t h a t intracisternal administration of idazoxan, but not yohimbine, can block the hypotensive action of intravenous clonidine (Tibirica et al. 1991); the dose of yohimbine used was sufficient t o block the inhibitory effect of clonidine on noradrenaline turnover in the locus coeruleus. Thus it appears that under these conditions, the cardiovascular action of systemic clonidine was primarily a result of imidazoline receptor activation. Hence there is now convincing evidence for a non-adrenergic receptor in the brainstem of both cats and rats which can be activated by imidazoline containing compounds to inhibit sympathetic outflow t o the periphery. This finding provided an impetus for further characterization of this receptor via radioligand binding techniques.
111. IDENTIFICATION OF NON-ADRENERGIC IMIDAZOLINE BINDING SITES. A. Studies With [3Hl Clondine and [3Hl para-Aminoclonidine I t was noted earlier that in most tissue preparations the binding of r3H] clonidine can be completely displaced by phenethylamine a-adrenoceptor agonists. However, in membrane homogenates from bovine ventrolateral medulla, only 70% of the sites labeled by I3H1 p-aminoclonidine could be displaced by the phenethylamines. The remaining, phenethylamine insensitive, sites were sensitive to imidazoline aadrenoceptor agonists a s well a s certain molecules bearing an imidazole moitey, structurally similar to the imidazoline ring, such a s histamine and cimetidine (Ernsberger et al.. 1987). These non-adrenergic imidazoline binding sites appeared t o the concentrated in the ventrolateral medulla, a s compared t o the frontal cortex, a finding consistent with an action of clonidine on medullary imi dazoline receptors to lower blood pressure. Using [3H] donidine, Bricca et a1 (1989) confirmed that about 25% of the binding sites in bovine ventral medulla were insensitive t o displacement by noradrenaline. In whole r a t brainstem, no noradrenaline insensitive binding could be detected, while in the area in human brain corresponding t o the NRL, noradrenaline, epinephrine or a-methylnoradrenaline had virtually no effect on i3H1 clonidine binding, but imidazoline agonists, such as cirazoline, and imidazoline antagonists, such a s idazoxan, produced potent displacement. Recent experiments have shown that another imidazoline agonist radioligand [3Hlmoxonidine, can label the sites identified by [3H] clonidine or [3H] p-aminoclonidine, and that, in addition to the central nervous system, these sites can also be detected in the renal medulla and adrenal chromafin tissue (Ernsberger et al., 1992). The ability of a series of a2-adrenoceptor agonists, including both imidazolines and phenethylamines, to lower blood pressure or heart rate upon direct injection to the NRL in anesthetized rats correlated well with their ability t o inhibit the non-adrenergic component of [3Hl p-aminoclonidine binding in bovine lateral medulla, but not with their ability to inhibit binding to central ap-adrenoceptors in bovine brain (Ernsberger et al. 1990). This suggests a functional role for the l3H1 p-aminoclonidine binding site at this location. R. Studies with p H ] ldazoxan In addition to the imidazoline sites labeled by clonidine and p-aminoclonidine described above, many investigators have studied the high affinity non-adrenergic site which can be labeled by [3Hl idazoxan. Idazoxan behaves functionally a s a potent and selective antagonist at the a2-adrenoceptor (Doxey et al. 1984). Although [3H1 idazoxan was initially found t o be a useful radioligand for labelling the a2-adrenoceptor (Pimoule et al.. 1983; Doxey et al. 1984), autoradiographic studies in rat brain (Boyajian et al. 1987) showed that L3H1 idazoxan could label additional sites that were not labelled by [3Hl rauwolscine. Although initially thought to be an additional population of ag-adrenoceptors, subsequent studies in a variety of tissues, including rabbit cortex (Hamilton et al. 1988; Covents et al. 1989), guinea pig cortex (Wikberg, 19891, rabbit adipocyte (Langin and Lafontan, 1989; Portillo et al. 1991), rabbit colon (Portillo et al. 1991), rabbit urethra (Yablonsky et al. 1988), rabbit kidney (Coupry et al. 1987, 1989; Parini et al. 19891, rat and human kidney (Michel et al. 1989), human platelet (Michel et al. 1990), human adipocytes (Langin et al.. 1990) and human brain (Covents et al. 1989; De Vos et al., 1991) showed this binding side to be non-adrenergic. Idazoxan binding sites have also been identified in an insulin secreting tissue culture line derived from a rat islet cell tumor (Remaury and Pans, 1992). The idazoxan sites in this diverse group of tissues share several characteristics: 1) a low affinity for the endogenous catecholamines, noradrenaline and epinephrine (Ki> 10pM); 2) a low affinity for non-imidazoline a2-adrenoceptor antagonists, such a s yohimbine and rauwolscine (Kj > 10pM); and 3) a high affinity for cirazoline (Ki = 1-50 nM), an imidazoline having low affinity for the a2-adrenoceptor. Although this pharmacologic profile is similar t o t h a t reported for the clonidine or p-aminoclonidine binding site, clonidine is either weak (Kj= 200-1000 nM) or inactive (Kj > [email protected]
) as an inhibitor of [3H1 idazoxan binding (Michel and Insel, 1989, see below), and guanabenz, which is totally inactive a s an inhibitor of [3Hl p-aminoclonidine binding (Kj > 100 pM; Emsberger 1990), is a potent inhibitor of L3HI idazoxan binding (Ki= 2-100 nM). Furthermore, imidazole derivatives, such as histamine, cimetidine and imidazole acetic acid, have affinisty for p-amninoclonidine, but not idazoxan, binding sites (Wikberg and Uhlen, 1990; Langin and Lafontan, 1989; Wikberg et al. 1991). Interestingly, the 2-methoxy derivative of idazoxan, RX821002, which h a s similiar a2-adrenoceptor antagonist activity and selectivity to idazoxan in functional assays (Stillings et al. 1985) does not inhibit the
JP Hieble and RR Ruffolo, Jr
binding of I3H1 idazoxan to its non-adrenergic sites (Kj > lOpM), and, when radiolabeled, binds only to a2adrenoceptors (Langin et al. 1990). The idazoxan binding site is not co-expressed with the a2-adrenoceptor when cells are transfected with genes for two ag-adrenoceptor subtypes (Michel et al. 1990). Furthermore, the idazoxan binding site is regulated independently from the a2-adrenoceptor (Michel et al.. 1990; Portillo et al. 1991) and be separated from the a2-adrenoceptor by several protein purification procedures (Parini et al. 1989; Michel et al. 1990)The idazoxan site can be solubilized from guinea pig cortex, and retains its binding characteristics in the purified state (Wikberg and Uhlen. 1990). At the present time, no functional effect can be definitely attributed to the idazoxan binding site. Since the in vivo effects of idazoxan and RX821002 on adipocyte a2-adrenoceptor number, and on the levels of catecholamines, glucose or free fatty acids in the plasma are similar, a contribution of the idazoxan binding site to these metabolic functions is unlikely (Portillo et al. 1991). It has been suggested that the ability of idazoxan to inhibit prolactin secretion in the rat (Kurlich et al. 1989) may be attributed to a n action on the idazoxan binding site in the pituitary gland (Wikberg and Uhlen, 1990). The ability of phentolamine and other imidazolines to stimulate insulin secretion in isolated mouse pancreatic islets (Schultz and Hasselblatt, 1989) does not appear to be dependent on aq-adrenoceptor blockade, and has been postulated to result from an interaction with imidazoline receptors (Molderings et al. 1991). However a correlation between the ability t o induce this functional effect and affinity for any of the imidazoline binding sites has not been attempted. IV. HETEROGENEITY OF IMIDAZOLINES SITES As noted above. two distinct imidazoline binding sites have been identified, using [3Hl clonidine or [3Hl para-aminoclonidine and c3H1 idazoxan. However, i t appears likely that there are two distinct idazoxan binding sites (Michel and Insel, 1989). Comparing the affinities for clonidine as an inhibitor of [3H] idazoxan binding, it is clear t h a t the tissues examined fall into two groups, one in which clonidine h a s moderate affinity for the idazoxan site, and another where it is virtually inactive. While this may result in part from species differences, since many of the tissue in the first group are from the rabbit, and human tissue sources show a uniform lack of clonidine affinity, recent evidence from the guinea pig shows that two tissues (cortex and ileum) from a single species can show dissimilar affinities for clonidine (Wikberg et al.. 1991). The fact that a structurally dissimilar agonist, (-1 medetomidine, shows a stereospecific ability to differentiate the idazoxan binding sites in these guinea pig tissues, and that (-1 medetomidine h a s an opposite affinity profile compared t o clonidine, supports the presence t o two distinct sites for idazoxan in the guinea pig (Wikberg et al.1991). Recent functional experiments measuring the ability of imidazolines t o inhibit noradrenaline release in isolated rabbit pulmonary artery and aorta suggest the possibility of a prejunctional imidazoline receptor, since following a l k y l a t i o n / b l o c k a d e of t h e p r e j u n c t i o n a l a 2 . a d r e n o c e p t o r with phenoxybenzarnindrauwolscine, several imidazolines could inhibit transmitter overflow to a greater degree than noradrenaline (Gothert and Molderings, 1991). In rabbit pulmonary arteries with intact a 2 adrenoceptors the potency of rauwolscine against prejunctional receptor stimulation depended on the agonist employed and was generally less than that predicted for a2- adrenoceptor blockade (Molderings et al. 1991). However, this putative "imidazoline receptor" has characteristics markedly different from the binding sites identified for 13Hl clonidinelp-aminoclonidine or r3H1 idazoxan, since the receptor dissociation constant of rauwolscine against the "imidazoline" agonists was in the 150-500 nM range, and the receptor could be activated by noradrenaline. Hence a perhaps more likely explanation is t h e presence of multiple prejunctional a-adrenoceptors in this tissue, with one having novel characteristics. This hypothesis is made more plausible by recent observations suggesting prejunctional 1x2-adrenoceptor heterogeneity in the rat vas deferens (Oriowo et al.. 1991). V. CONCLUSIONS I t is now established that there are two distinct non-adrenergic sites recognizing some feature of imidazolines and related compounds; one of these is identified by L3H1 clonidine or [3Hl p-aminoclonidine, the other by L3H1 idazoxan. Furthermore, recent data supports the premise of Michel and Insel (1989) that the 13Hl idazoxan site can be further subdivided into two groups. The [3Hl clonidine site has been well characterized only in a discrete area within the medulla of several species, including humans, where i t appears to mediate a sympathoinhibtory action. There is strong
experimental evidence that this binding site is associated, at least in part, with the blood pressure lowering action of clonidine and other imidazolines when administered by local injection to this portion of the brain. The relative contribution of imidazoline and a2-adrenoceptors t o the cardiovascular actions of centrally acting antihypertensive drugs when administered systemically t o experimental animals or humans has yet to be established, but agonists acting selectively at imidazoline site could offer superiority to agents activating both imidazoline and a2-adrenoceptors. The binding site for [3H] idazoxan has been found in a variety of tissue sites, including platelet, adipocyte and renal tubular epithelial cell, in addition to the central nervous system. No functional effect has yet been attributed to activation of this site. In view of the increasing interest in “imidazoline receptors it is likely that additional information on their molecular structure, pharmacological properties and functional role within the body will soon become available. This information will help t o answer the question of whether the various imidazoline binding sites represent useful targets for novel therapeutic agents. REFERENCES Borkowsli, K. & Finch, L. (1979) A comparison of the cardiovascular effects of centrally administered clonidine and adrenaline in the anesthetized rat. J. Pharm. Pharmacol. 31,16-19. Bousquet, P., Feldman, J. & Schwartz, J. (1984) Central cardiovascular effects of alpha adrenergic drugs: Differences between catecholamines and imidazolines. J. Pharmacol. Exp. Ther. 230, 232-236. Bousquet, P., Feldman, J., Velly, J & Bloch, R. (1975) Role of the ventral surface of the brain stem in the hypotensive action of clonidine. Europ. J. Pharmacol. 34,151-156. Boyajian, C.L., Loughlin, S.E. & Leslie, F.M. (1987) Anatomical evidence for alpha2.adrenoceptor heterogeneity: Differential autoradiographic distributions of I3HI rauwolscine and 13Hl idazoxan in rat brain J. Pharmacol. Exp. Ther. 241, 1079-1091. Bricca, G., Dontenwill, M., Molines, A., Feldman, J., Belcourt, A. & Bousquet, P. (1989) The imidazoline preferring receptor: Binding studies in bovine, rat and human brainstem. Europ. J. Pharmacol. 162, 1-9. Brody, M.J., O’Neill, T.P. & Porter, J.P. (1984) Role of central catecholaminergic systems in pathogenesis and treatment of hypertension. J . Cardiovasc. Pharmacol. 6 (Suppl 5), S7274741. Covents, A., Covents, D., DeBacker, J-P., DeKeyser, J. & Vauquelin, G. (1989) High affinity binding of I3H1 rauwolscine and [3H1 RX781094 to ap-adrenergic receptors and non-stereoselective sites in human and rabbit brain cortex membranes. Biochem. Pharmacol. 38, 455-463. Coupry, I., Lachaud, V., Podevin, R.A., Koenig E. & Parini A. (1989). Different affinities of a2-agonists for imidazoline and ag-adrenergic receptors. Am. J. Hyperten. 2,468-470. Coupry, I., Podevin, R.A., Dausse, J-P. & Parini, A. (1987) Evidence for imidazoline binding sites in basolateral membranes from rabbit kidney. Biochem. Biophys. Res. Comm. 147, 1055-1060. DeJonge, A., van Meel, J.C.A., Timmermans, P.B.M.W.M. & van Zwieten, P.A. (1976) A lipophilic selective al-adrenoceptor agonist: 2(2-chloro-5-trifluoromethylphenyliminoimidazoli~ine)(ST 587). Life Sci. 28,2009-201 De Vos, H., Covents, A., De Keyser, J., De Backer, J-P., Van Megen, I.J.B., Ebinger, G. & Vauquelin, G. (199 1) Autoradiographic distribution of a2-adrenoceptors, NAIBS, and 5 - H n receptors ~ in human brain using L3HI idazoxan and [3H1 rauwolscine. Brain Res. 566, 13-20. Dollery, C.T. & Reid, J.L. (1973) Central noradrenergic neurons and the cardiovascular action of clonidine in the rabbit. Br. J. Phannacol. 47,206-216. Doxey, J.C., Lane, A.C., Roach, A.G. & Virdee, N.K. (1984) Comparison of the a-adrenoceptor antagonist profiles of idazoxan (RX781094), yohimbine, rauwolscine and corynanthine. NaunynSchmiedeberg’s Arch. Pharmacol 325, 136-144. Ernsberger, P., Giuliano, R., WIllette, R.N. &, Reis, D.J. (1990). Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostra1 ventrolateral medulla. J. Pharmacol. Exp. Ther. 253,408418. Ernsberger, P., Haxhiu, M.A., Damon, T.H. & Wendorff, L.A. (1992). Imidazoline I1 receptors and brainstem autonomic control, membrane binding, autoradiographic and functional studies. FASEB J. 6, A1874.
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