Pergamon Press
Life Sciences, Vol . 25, pp . 769-774 Printed in the U .S .A .
[ 3H]PARA-AMINO-CLONIDINE : A NOVEL LIGAND WHICH BINDS WITH HIGH AFFINITY TO a-ADRENERGIC RECEPTORS Bruno R. Rouot and Solomon H . Snyder Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences Johns Hopkins University School of Medicine Baltimore, Maryland 21205 (Received in final form July 23, 1979) Summary Pare-amino-clonidine (PAC) is an a-adrenergic agoaist with extraordinarily high potency is some peripheral tissues . We have demonstrated the labeling of a-adrenergic binding sites is central and peripheral tissues with [3H]PAC and compared properties of this binding to those of[3H]clonidiae . [3H]PAC binds saturably with a dissociation constant (ICD) of about 0 .9 nM to rat cerebral cortex membranes . It has about 2-3 times the affinity of [3H]clonidine for a-receptor binding sites . The greater affinity is attributable mainly to a slower dissociation of [3H]PAC than [3H]clonidine from binding sites . The relative and absolute potencies of various adrenergic agonists and antagonists in competing for [3H]PAC and [3H]clonidine binding are [3H]PAC can also be utilized to essentially the same . label a-adrenergic binding sites in the kidney and spleen where the relative potencies of PAC and clonidine are the same as in the brain. a-Adrenergic receptors in mammalian tissues can be labeled with a variety of [3H]ligands including both agonists and antagonists . The antagonist [3H]WB-4101 (2-([2',6'-dimethoxy]phenoxyethylamino methylbenzodioxane) labels post synaptic a-adrenergic receptors whose drug specificity is characteristic of alreceptors (1,2) . The agonists [3H]clonidiae, [3H]epinephrine and [3H]aorepinephrine also label poataynaptic a-adrenergic receptors, but the sites labeled by these [3H]agonists are physically distinct from those labeled by [3H]WB-4101 (1,3-5) . The drug specificity of the sites labeled by these [3H]agonists are similar to those of a-adrenergic autoreceptors localized to sympathetic nerve endings and referred to as a2-receptors (2,6,7) but are not presynaptic since destruction of noradrenergic innervation does not reduce binding (1,8) . Accordingly, the sites labeled by [3H]clonidine, [3H]epinephrine and [3H]aorepinephrine are designated poetaynaptic a2-receptors (8) . Some tissues, such as the vas deferens and heart possess poatsynaptic al-receptors but no postsynaptic a2-receptors, while other tissues such as the rabbit duodenum possess postsynaptic a2- but not al-receptors . Pharmacological potencies of agents in peripheral tissues containing predominantly either al- or ay-postsynaptic receptors correlate cloael with affinities of drugs for the respective types of binding sites (8) . [3H~ Dihydroergocryptine([3H]DHE) labels both types of a-receptors (9-11) . 0024-3205/79/090769-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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Thus, There are limitations is the use of any one of these [31i ligands . [~]Epinephrine [ 3H]WB-4101 cannot be employed for stades of a2-receptors . and [ 3H]norepinephrine may label ß- as well as a-receptors (12) . [ 3H]DHE can [3H]clonidine of bind to dopamine as well se to a-adrenergic receptors (13) . high specific radioactivity has not been readily available from commercial sources . Recently p-amino-clonidine (PAC ; 2-([2,6-dichloro 4-amino]phenylimino~ imidazolidine) has been reported to be a uniquely potent a-adrenergic agonist (14-15) . In the rat aorta p-amino-clonidine elicits half maximal contractile effects at 0 .01 aM, three thousand times more potent than clonidine and 150 times more potent than norepinephrine . In pithed rate, after intravenous injection, PAC is respectively 5 times and 2 times more potent than clonidine and norepinephrine in producing a transient raise in blood pressure . Because of its polar properties, PAC, unlike clonidine, does not penetrate easily the blood brain barrier but intraventricular injection of PAC produces a decrease In the present of blood pressure similar to those induced by clonidine. study we describe the labeling of a-adrenergic receptor sites is rat central and peripheral tissue membranes and compare properties of this binding with those of [3H]clonidine . Methods Tissue preparation : Methode were generally similar to previous a-receptor Fresh cortex, kidney and spleen from adult male binding studies (1,3-6) . Sprague-Dawley rats (175-200 g) and frozen calf cerebral cortices from a local slaughterhouse were homogenized in about 20 volumes of ice-cold 50 mM Tris-HC1 buffer, pH 7.7 at 25° C, with a Brinkmann Polytron PT 10 . After two ceatrithe fugatioas at 50,000 x g for 10 min with intermediate rehomogenizatioa, final pellets were resuspeaded in 49 volumes (cortex, kidney) or 99 volumes Protein concentrations (spleen) of 50 mM Tris-HC1 buffer, pH 7 .7 at 25 ° C. were determined by the method of Lowry et al . (16) . Ligands and drugs: Unlabeled p-amino-clonidine was prepared by reduction of the corresponding p-vitro derivative using iron in hydrochloric acid medium . [3H]PAC was synthesized according to the method described elsewhere (17) . Briefly, addition of an excess of iodine monochloride is acetic acid allowed Reduction was carried the introduction of two iodines in the benzene ring . out in basic medium with palladium on alumina as a catalyst permitting the selective removal of iodine atoms without affecting the chlorine-carbon bond . Tritiation performed by New England Nuclear Corporation on the düodo derivative of PAC yielded [3H]PAC with a specific activity of 46 Ci/mmole . clonidine and [ 3fi]clonidine (26 .7 Ci/mmole) were generously donated by Boehringer Lngelheim. WB-4101 was a gift of Ward-Blenkinsop, Cimetidine and Other drugs were obtained from metiamide were from Smith Rline and French . the pharmaceutical companies of origin or commercial sources . To triplicate incubation tubes were added various Bindin sass s : concentrations of H]PAC or [3H]clonidine (generally 30 ul), 20 yl of O.1X The volume ascorbic acid with or without (-)-norepinephrine or other drugs . was made up to 1 ml with Tris HC1, pH 7.7 at 25° C, and incubation was initiated by addition of the membrane suspension containing 20 mg or 10 mg Incubations were run for 30 min at 25 ° C and (spleen) of wet weight tissue . terminated by filtration through GF/B Whatman glass fiber filters followed by Filters were subsequently counted by liquid 3 x 5 ml ice cold buffer washes . scintillation spectrometry in 9 ml Formula 947 (New England Nuclear) or Specific Econofluor (New England Nuclear) + 3X protosol at 44X efficiency . binding was measured as the excess over blanks containing 10 uM (rat and calf In routine cortex) or 100 uM (peripheral tissues) of (-)-norepinephrine .
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experiments utilizing 0 .13 nM [3H]PAC, total binding was about 1800 cpm while nonspecific binding was about 300 cpm. Thus, specific binding represented about 85Z of total binding . Results and Discussion [3H]PAC binds saturabl 3to rat cerebral cortex membranes with a saturation pattern similar to that of y [ H]clonidine (Table I) . The dissociation constant determin d by Scatchard analysis is about 0.9 nM, a little leas than half the Kn for [~]clonidine binding . The maximal number of binding sites (Borax) is the same for [3H]PAC and [3H]clonidine, about 13 pmoles/g wet weight of tissue . TABLE I Equilibrium and Kinetic Constants for [3H]p-Amino Clonidine and [3H]Clonidine Binding Sites in Rat Cerebral Cortex Membranes
[ 3H]PAC
[3A]Clonidine*
E~ilibrium (nM) KD B~ (pmole/g tissue) (fmole/mg protein)
0 .87 13 .0 266
+ + +
0 .25 1 .7 37
2 13
_+ 0 .3 -
Kinetic Dissociation : k l slow component fast component
(min 1)
0 .333 + 0 .304 +
0 .004 0 .088
0 .063 0 .68
+ 0 .008 + 0 .15
Association : maximal at (min) half maximal (min) kl (nM-1 min-1)
10 1 -
30 3 0 .16
+ 0 .045
Saturation experiments for [3H ]PAC were performed with seven concentrations and values shown are means _+ standard error of six experiments carried out in triplicate . Dissociation and association experiments were performed as described in Methods and are means of four or five experiments performed in triplicate . *Equilibrium and kinetic constants of [3H]clonidine are from reference 5, representing studies conducted in our laboratory under conditions identical t~ those employed for [3H]PAC . In limited studies values for [ H]clonidine and [3H]PAC have been replicated in side-by-side experiments . In order to determine the reason for the greater affinity of PAC than clonidine for a-receptor binding sites, we evaluated the kinetics of binding . Dissociation was determined by incubating the rat cortex membranes to equili brium for 30 min at 25°C whereupon 10 tiùf (-)-norepinephrine was added and residual binding determined at various intevals . Ae reported previously (5) [3H]clonidine dissociation under these conditions is biphasic . A similar biphasic dissociation of [3H]PAC is apparent . The rate constants for disaoc ation of both rapid and slow components are about half as great for [3H]PAC ae [~]clonidine. The half life for the rapid and slow pha es of dissociation for [3H]PAC are 2 .4 min and 21 min, respectively . For [~]clonidine the rapid and slow rates of dissociation display respective half life of 1 min and 13 min.
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As compared to [3R]clonidine, the association of [3H,]PAC is more rapid, attaining maximal values after 10-12 min incubation with half maximal binding apparent at about 1 min . Because of this rapid association at 25 ° C and because of the two dissociation components, it is not possible to determine an association rate constant with accuracy . Nevertheless, it is likely that the slower dissociation rates are largely responsible for the greater affinity of [3H]PAC than [3R]clonidine for a-receptor binding sites, since quantitatively they readily account for the difference is affinity o the two [3I{]ligands. It should be noted that whereas both [ 3 H]PAC and [clonidine display a biphasic dissociation, only [ 3H]clonidine shows a biphasic Scatchard plot in moat saturation experiments (5) . In the six saturation experiments performed [3H]PAC saturation is always monophasic . To determine whether the specificity of binding of [ 3H]PAC differs from that of 3H clonidine, we evaluated the influence of a variety of agonists and antagonists on binding of the two [ 3R]ligands ~Table II The potencies of all drugs examined are essentially the same at I 3IiJPAC and ~3H]clonidine binding sites . In both cases binding displays the characteristics expected of aadrenergic receptors . Binding is etereospecific for the isomers of norepinephrine . The imidazoline agoniats, clonidine, oxymetazoline and PAC itself display several times greater affinity than norepinephrine . The Ki for PAC is about a third that of clonidine. Among antagonists examined phentolamine is the moat potent with a K1 value of 3 nM . The specificity of the [3R]ligands for a2-receptors is indicated by the relatively limited potency of WB-4101 whose Ki for [ 3H]PAC and [3H clonidine sites is about 130 nM, in contrast to its Ki for sites labeled by ~31;]WB-4101 itself, which is about 0 .4 nM (8) . There have been reports that clonidine is active at histamine H2-receptors (18-20) . Accordingly, we evaluated the H2-antagonist metiamide and cimetidine, which appear fairly weak in competing for [3H]PAC or [3ü]clonidine binding . Recently clonidine was reported to be an adenosine antagonist (21) . However, in the rat cortex we fail to detect any significant influence of adenosine on [3H]PAC or [3H]clonidine binding at a concentration of 10 -4 M. Similar findings have also been reported on the binding of [3H]epinephrine, [3H}norepinephrine and [3fi]WB-4101 suggesting that adenosine does not act on tie initial receptor-transmitter interaction (22) . To ascertain whether [ 3H]PAC can be utilized to label peripheral areceptors, we examined the kidney and spleen . Substantial levels of [3H]PAC binding are detectable in both tissues with affinity and drug specificity characteristic of poatsynaptic a2-receptors, resembling results obtained earlier with ( 3H]clonidine itself in these tissues (8) . In competing for [3H]PAC binding, PAC is 2-3 times more potent than clonidine in the kidney and spleen, similar to results obtained in brain membranea,(Table III) . One rationale for studying PAC was its extremely high potency as a poetsynaptic a-agonist to contract rat aortic stripe . In other peripheral tissues such as the rat vas deferens PAC is about 5-8 times more potent than clonidine in inhibiting contraction induced by phenylephrine (N . Bieth J . 3chwartz, unpublished) . We have attempted to demonstrate binding of [3RjPAC to aortic strips of rat, rabbit and dog aorta with no success. It is not clear whether the uniquely high potency of PAC in aortic strips indicates the presence of a different type of a-receptor in this tissue or whether tissue penetration factors determine the relative potencies of PAC and clonidine in the aorta. In~aummary, [3A]PAC provides a potent ligand for binding studies labeling central or peripheral a-adrenergic receptors . The specificity of binding ie similar to that obtained with 3H clonidine suggesting that the two ligands
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TABLE II Drug Effects on [31i]p-Amino-Clonidine and [3H]Clonidine Binding
[3H]PAC
[3A]Clonidine Ri
onist (-)-Norepinephrine (+)-Norepiaephrine (-)-Epinephrine Oxymetazoline Clonidine p-amino-Clonidine (+)-Isoproterenol Antagonists Pheatolamine Phenozybeazamine Yohimbiae WB-4101 Miscellaneous Drugs (+)-Propranolol Metismide Cimetidiae
10 .5 363 2 .9 1 .7 2 .8 0 .91 2920 3.1 17 .2 57 138 8500 14600 15400
(nM)
+ 3 .2(3) + 106 (3) + 0 .2(3) + 0 .3(3) + 0 .4(3) + 0 .04(3) + 660
6 .1 170 2 .1 1.2 2 .0 0 .63
+ + + + + + -
1 .2(5)* 8 (2)* 0 .3(3)* 0 .2(2)* 0 .2(4)* (1)*
+ + + _+
2 .0 20 47 108
+ + + _+
0 .2(2)* 4 .2(2)* 2 .5(3)* 12 .5(3)*
0 .1(3) 0.2(3) 2 .4(3) 20 (3)
+ 1400 + 700 + 1700
(3) (3) (3)
8500 15000
+ 1200 + 4000
(3) (3)
Drug inhibition of the binding of 0 .1-0 .15 nM [3H]PAC or 0 .4 nM [3H]Clonidine using 5-6 concentrations of each unlabeled drug, was determined as described is Methods . IC50 values were determined graphically by log probat analysis and apparent K.~ values calculated from the equation Ri = ICgO/ (1 + [3H]PAC ]RD) . Values are means _+ standard errors of experiments each performed in triplicate . Numbers in parentheses are the number of determinations for each compound . *Ri values of a-agonists and a-antagonists with [3H]Clonidine were calculated by U'Prichard from ref . 5 for experiments conduct ed in the same laboratory under conditions identical to those employed for [3H]PAC . In limited studies, _Ri values for drugs were determined for [3H]PAC and [3H]Clonidine in simultaneous experiments . TABLE III Clonidine and p-Amino-Clonidine Influences on oa [3H]p-Amino-Clonidine Binding in Three Tissues
Tissue
Calf Cortex
Rat Kidney
Rat Spleen
IC 50 (nM) Clonidine p-Amino-Clonidine
1.8 + 0 .1 0.9 + 0 .1
8 .5 + 2 .5 4 .3 + 1 .5
10 .7 + 5.6 4.2 + 0 .7
Experiments were performed using a concentration of [3H]PAC of 0 .2 nM for the calf cortex and 1 nM for the kidney and the spleen . Values are mean _+ standard errors of 3 experiments each performed in triplicate .
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label the same sites . Since the binding of [3ü]clonidine in brain and peripheral tissues is not affected by catecholamine depletion utilizing 6hydroxydopamine, it is likely that both [3H]PAC and [3H]clonidine bind to postsynaptic a-receptors. The drug specificity for binding of both liganda indicatesthat they label poatsynaptic a2-receptors (8) . Acknowledgements This work was supported by USPHS grants MH-18501 and DA-00266 as well as the John Hartford and McRnight Foundatons . We thank Guillermo Suarez for his helpful assistance and Pam Morgaa and Billie Little for manuscript preparation . B .R . acknowledges the support of the Délégation Général ~ la Recherche Scientifique et Technique . References 1. 2. 3. 4. 5. 6, 7. 8. 9. 10 . 11 . 12 . 13, 14 . 15 . 16, 17 . 18 . 19 . 20 . 21 . 22 .
D .C . U'PRICHARD, D.A . GREENBERG and S .H . SNYDER, Mol. Pharmacol . _13 454-473 (1977) . S . BERTHELSEN and W.A . PETTINGER, Life Sciences 21 595-606 (1977) . D,C. U'PRICHARD and S .H, SNYDER, J. Biol . Chem . 252 6450-6463 (1977) . D .C . U'PRICHARD and S .H . SNYDER, Life Sciences _20 527-534 (1977) . D .C . U'PRICHARD, W.D . BECHTEL, B . ROUOT and S .H . SNYDER, Mol . Pharmacol, in press (1979) . K. STARRE, T. ENDO and H.D . TRAUBE, Naunyn-Schmied . Arch . Pharmacol . _291 55-78 (1975) S.Z. LANGER, Biochem. Pharmacol, _23 1793-1800 (1974) . D .C . U'PRICHARD and S .H . SNYDER, Life Sciences _24 79-88 (1979) . S .J . PEROUTRA, D .A . GREENBERG, D .C . U'PRICHARD and S.H . SNYDER, Mol . Pharmacol. _14 403-412 (1978) . L,T. WILLIAMS, D. MULLIRIN and R.J . LEFKOWITZ, J . Biol . Chem . _251 69156923 (1976) . P . GUICHSNEY, R.P . GARAY, C, LEVY-MARCHAL and P, MEYER, Proc . Natl . Acad . Sci ., USA _75 6285-6289 (1978) . D .C . U'PRICHARD, D.B . BYLUND and S .H . SNYDER, J, Biol . Chem . _253 50905102 (1978) . M. TITTLER, P, WEINREICH and P, SEEMAN, Proc . Natl . Acad . Sci ., USA _74 3750-3753 (1977), B. ROUOT, G. LECLERC, N. BIETH, C.G . WERMUTH and J . SCHWARTZ, C,R, Acad . Sci . (Paris) _286 909-912 (1978) . G, LECLERC, B . ROUOT, J . SCHWARTZ, J. VELLY and C,G . WERMDTH, Brit, J . Pharmacol, in press (1979) . 0. LOWRY, N. ROSEBROUGH, A. FARR and J . RANDALL, J . Biol, Chem . _193 265275 (1951) . B. ROUOT, J.D . EHRHARDT, G, LECLERC and J . SCHWARTZ,J . Label Compounds Radiopharm., in press (1979) . L. FINCH, C.A . HARVEY, P.E, HICRS and D,A.A . OWEN, Neuropharmacol, _17 307-313 (1978) . H. KARPPANEN, I, PAARRARI, P . PAAKKARI, R. HUOTARI and A-L . ORMA, Nature _259 587-588 (1976) . Y, AUDIGIER, A. VIRION and J .C . SCHWARTZ, Nature 262 307-308 (1976) . T.W . STONE and D,A. TAYLOR, J. Pharm. Pharmacol . 30792-793 (1978) . D,C . U'PRICHARD and S .H . SNYDER, J. Biol . Chem . 253 3444-3452 (1978) .
Life Sciences, Vol . 25, pp . 769-774 Printed in the U .S .A .
[ 3H]PARA-AMINO-CLONIDINE : A NOVEL LIGAND WHICH BINDS WITH HIGH AFFINITY TO a-ADRENERGIC RECEPTORS Bruno R. Rouot and Solomon H . Snyder Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences Johns Hopkins University School of Medicine Baltimore, Maryland 21205 (Received in final form July 23, 1979) Summary Pare-amino-clonidine (PAC) is an a-adrenergic agoaist with extraordinarily high potency is some peripheral tissues . We have demonstrated the labeling of a-adrenergic binding sites is central and peripheral tissues with [3H]PAC and compared properties of this binding to those of[3H]clonidiae . [3H]PAC binds saturably with a dissociation constant (ICD) of about 0 .9 nM to rat cerebral cortex membranes . It has about 2-3 times the affinity of [3H]clonidine for a-receptor binding sites . The greater affinity is attributable mainly to a slower dissociation of [3H]PAC than [3H]clonidine from binding sites . The relative and absolute potencies of various adrenergic agonists and antagonists in competing for [3H]PAC and [3H]clonidine binding are [3H]PAC can also be utilized to essentially the same . label a-adrenergic binding sites in the kidney and spleen where the relative potencies of PAC and clonidine are the same as in the brain. a-Adrenergic receptors in mammalian tissues can be labeled with a variety of [3H]ligands including both agonists and antagonists . The antagonist [3H]WB-4101 (2-([2',6'-dimethoxy]phenoxyethylamino methylbenzodioxane) labels post synaptic a-adrenergic receptors whose drug specificity is characteristic of alreceptors (1,2) . The agonists [3H]clonidiae, [3H]epinephrine and [3H]aorepinephrine also label poataynaptic a-adrenergic receptors, but the sites labeled by these [3H]agonists are physically distinct from those labeled by [3H]WB-4101 (1,3-5) . The drug specificity of the sites labeled by these [3H]agonists are similar to those of a-adrenergic autoreceptors localized to sympathetic nerve endings and referred to as a2-receptors (2,6,7) but are not presynaptic since destruction of noradrenergic innervation does not reduce binding (1,8) . Accordingly, the sites labeled by [3H]clonidine, [3H]epinephrine and [3H]aorepinephrine are designated poetaynaptic a2-receptors (8) . Some tissues, such as the vas deferens and heart possess poatsynaptic al-receptors but no postsynaptic a2-receptors, while other tissues such as the rabbit duodenum possess postsynaptic a2- but not al-receptors . Pharmacological potencies of agents in peripheral tissues containing predominantly either al- or ay-postsynaptic receptors correlate cloael with affinities of drugs for the respective types of binding sites (8) . [3H~ Dihydroergocryptine([3H]DHE) labels both types of a-receptors (9-11) . 0024-3205/79/090769-06$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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Thus, There are limitations is the use of any one of these [31i ligands . [~]Epinephrine [ 3H]WB-4101 cannot be employed for stades of a2-receptors . and [ 3H]norepinephrine may label ß- as well as a-receptors (12) . [ 3H]DHE can [3H]clonidine of bind to dopamine as well se to a-adrenergic receptors (13) . high specific radioactivity has not been readily available from commercial sources . Recently p-amino-clonidine (PAC ; 2-([2,6-dichloro 4-amino]phenylimino~ imidazolidine) has been reported to be a uniquely potent a-adrenergic agonist (14-15) . In the rat aorta p-amino-clonidine elicits half maximal contractile effects at 0 .01 aM, three thousand times more potent than clonidine and 150 times more potent than norepinephrine . In pithed rate, after intravenous injection, PAC is respectively 5 times and 2 times more potent than clonidine and norepinephrine in producing a transient raise in blood pressure . Because of its polar properties, PAC, unlike clonidine, does not penetrate easily the blood brain barrier but intraventricular injection of PAC produces a decrease In the present of blood pressure similar to those induced by clonidine. study we describe the labeling of a-adrenergic receptor sites is rat central and peripheral tissue membranes and compare properties of this binding with those of [3H]clonidine . Methods Tissue preparation : Methode were generally similar to previous a-receptor Fresh cortex, kidney and spleen from adult male binding studies (1,3-6) . Sprague-Dawley rats (175-200 g) and frozen calf cerebral cortices from a local slaughterhouse were homogenized in about 20 volumes of ice-cold 50 mM Tris-HC1 buffer, pH 7.7 at 25° C, with a Brinkmann Polytron PT 10 . After two ceatrithe fugatioas at 50,000 x g for 10 min with intermediate rehomogenizatioa, final pellets were resuspeaded in 49 volumes (cortex, kidney) or 99 volumes Protein concentrations (spleen) of 50 mM Tris-HC1 buffer, pH 7 .7 at 25 ° C. were determined by the method of Lowry et al . (16) . Ligands and drugs: Unlabeled p-amino-clonidine was prepared by reduction of the corresponding p-vitro derivative using iron in hydrochloric acid medium . [3H]PAC was synthesized according to the method described elsewhere (17) . Briefly, addition of an excess of iodine monochloride is acetic acid allowed Reduction was carried the introduction of two iodines in the benzene ring . out in basic medium with palladium on alumina as a catalyst permitting the selective removal of iodine atoms without affecting the chlorine-carbon bond . Tritiation performed by New England Nuclear Corporation on the düodo derivative of PAC yielded [3H]PAC with a specific activity of 46 Ci/mmole . clonidine and [ 3fi]clonidine (26 .7 Ci/mmole) were generously donated by Boehringer Lngelheim. WB-4101 was a gift of Ward-Blenkinsop, Cimetidine and Other drugs were obtained from metiamide were from Smith Rline and French . the pharmaceutical companies of origin or commercial sources . To triplicate incubation tubes were added various Bindin sass s : concentrations of H]PAC or [3H]clonidine (generally 30 ul), 20 yl of O.1X The volume ascorbic acid with or without (-)-norepinephrine or other drugs . was made up to 1 ml with Tris HC1, pH 7.7 at 25° C, and incubation was initiated by addition of the membrane suspension containing 20 mg or 10 mg Incubations were run for 30 min at 25 ° C and (spleen) of wet weight tissue . terminated by filtration through GF/B Whatman glass fiber filters followed by Filters were subsequently counted by liquid 3 x 5 ml ice cold buffer washes . scintillation spectrometry in 9 ml Formula 947 (New England Nuclear) or Specific Econofluor (New England Nuclear) + 3X protosol at 44X efficiency . binding was measured as the excess over blanks containing 10 uM (rat and calf In routine cortex) or 100 uM (peripheral tissues) of (-)-norepinephrine .
Vol . 25, No . 9,
[ 3H]PAC Binding to a-Adrenergic Receptors
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experiments utilizing 0 .13 nM [3H]PAC, total binding was about 1800 cpm while nonspecific binding was about 300 cpm. Thus, specific binding represented about 85Z of total binding . Results and Discussion [3H]PAC binds saturabl 3to rat cerebral cortex membranes with a saturation pattern similar to that of y [ H]clonidine (Table I) . The dissociation constant determin d by Scatchard analysis is about 0.9 nM, a little leas than half the Kn for [~]clonidine binding . The maximal number of binding sites (Borax) is the same for [3H]PAC and [3H]clonidine, about 13 pmoles/g wet weight of tissue . TABLE I Equilibrium and Kinetic Constants for [3H]p-Amino Clonidine and [3H]Clonidine Binding Sites in Rat Cerebral Cortex Membranes
[ 3H]PAC
[3A]Clonidine*
E~ilibrium (nM) KD B~ (pmole/g tissue) (fmole/mg protein)
0 .87 13 .0 266
+ + +
0 .25 1 .7 37
2 13
_+ 0 .3 -
Kinetic Dissociation : k l slow component fast component
(min 1)
0 .333 + 0 .304 +
0 .004 0 .088
0 .063 0 .68
+ 0 .008 + 0 .15
Association : maximal at (min) half maximal (min) kl (nM-1 min-1)
10 1 -
30 3 0 .16
+ 0 .045
Saturation experiments for [3H ]PAC were performed with seven concentrations and values shown are means _+ standard error of six experiments carried out in triplicate . Dissociation and association experiments were performed as described in Methods and are means of four or five experiments performed in triplicate . *Equilibrium and kinetic constants of [3H]clonidine are from reference 5, representing studies conducted in our laboratory under conditions identical t~ those employed for [3H]PAC . In limited studies values for [ H]clonidine and [3H]PAC have been replicated in side-by-side experiments . In order to determine the reason for the greater affinity of PAC than clonidine for a-receptor binding sites, we evaluated the kinetics of binding . Dissociation was determined by incubating the rat cortex membranes to equili brium for 30 min at 25°C whereupon 10 tiùf (-)-norepinephrine was added and residual binding determined at various intevals . Ae reported previously (5) [3H]clonidine dissociation under these conditions is biphasic . A similar biphasic dissociation of [3H]PAC is apparent . The rate constants for disaoc ation of both rapid and slow components are about half as great for [3H]PAC ae [~]clonidine. The half life for the rapid and slow pha es of dissociation for [3H]PAC are 2 .4 min and 21 min, respectively . For [~]clonidine the rapid and slow rates of dissociation display respective half life of 1 min and 13 min.
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[ 3H]PAC Binding to a-Adrenergic Receptors
Vol . 25, No . 9, 1979
As compared to [3R]clonidine, the association of [3H,]PAC is more rapid, attaining maximal values after 10-12 min incubation with half maximal binding apparent at about 1 min . Because of this rapid association at 25 ° C and because of the two dissociation components, it is not possible to determine an association rate constant with accuracy . Nevertheless, it is likely that the slower dissociation rates are largely responsible for the greater affinity of [3H]PAC than [3R]clonidine for a-receptor binding sites, since quantitatively they readily account for the difference is affinity o the two [3I{]ligands. It should be noted that whereas both [ 3 H]PAC and [clonidine display a biphasic dissociation, only [ 3H]clonidine shows a biphasic Scatchard plot in moat saturation experiments (5) . In the six saturation experiments performed [3H]PAC saturation is always monophasic . To determine whether the specificity of binding of [ 3H]PAC differs from that of 3H clonidine, we evaluated the influence of a variety of agonists and antagonists on binding of the two [ 3R]ligands ~Table II The potencies of all drugs examined are essentially the same at I 3IiJPAC and ~3H]clonidine binding sites . In both cases binding displays the characteristics expected of aadrenergic receptors . Binding is etereospecific for the isomers of norepinephrine . The imidazoline agoniats, clonidine, oxymetazoline and PAC itself display several times greater affinity than norepinephrine . The Ki for PAC is about a third that of clonidine. Among antagonists examined phentolamine is the moat potent with a K1 value of 3 nM . The specificity of the [3R]ligands for a2-receptors is indicated by the relatively limited potency of WB-4101 whose Ki for [ 3H]PAC and [3H clonidine sites is about 130 nM, in contrast to its Ki for sites labeled by ~31;]WB-4101 itself, which is about 0 .4 nM (8) . There have been reports that clonidine is active at histamine H2-receptors (18-20) . Accordingly, we evaluated the H2-antagonist metiamide and cimetidine, which appear fairly weak in competing for [3H]PAC or [3ü]clonidine binding . Recently clonidine was reported to be an adenosine antagonist (21) . However, in the rat cortex we fail to detect any significant influence of adenosine on [3H]PAC or [3H]clonidine binding at a concentration of 10 -4 M. Similar findings have also been reported on the binding of [3H]epinephrine, [3H}norepinephrine and [3fi]WB-4101 suggesting that adenosine does not act on tie initial receptor-transmitter interaction (22) . To ascertain whether [ 3H]PAC can be utilized to label peripheral areceptors, we examined the kidney and spleen . Substantial levels of [3H]PAC binding are detectable in both tissues with affinity and drug specificity characteristic of poatsynaptic a2-receptors, resembling results obtained earlier with ( 3H]clonidine itself in these tissues (8) . In competing for [3H]PAC binding, PAC is 2-3 times more potent than clonidine in the kidney and spleen, similar to results obtained in brain membranea,(Table III) . One rationale for studying PAC was its extremely high potency as a poetsynaptic a-agonist to contract rat aortic stripe . In other peripheral tissues such as the rat vas deferens PAC is about 5-8 times more potent than clonidine in inhibiting contraction induced by phenylephrine (N . Bieth J . 3chwartz, unpublished) . We have attempted to demonstrate binding of [3RjPAC to aortic strips of rat, rabbit and dog aorta with no success. It is not clear whether the uniquely high potency of PAC in aortic strips indicates the presence of a different type of a-receptor in this tissue or whether tissue penetration factors determine the relative potencies of PAC and clonidine in the aorta. In~aummary, [3A]PAC provides a potent ligand for binding studies labeling central or peripheral a-adrenergic receptors . The specificity of binding ie similar to that obtained with 3H clonidine suggesting that the two ligands
Vol . 25, No . 9,
[3H]PAC Binding to a-Adrenergic Receptors
1979
773
TABLE II Drug Effects on [31i]p-Amino-Clonidine and [3H]Clonidine Binding
[3H]PAC
[3A]Clonidine Ri
onist (-)-Norepinephrine (+)-Norepiaephrine (-)-Epinephrine Oxymetazoline Clonidine p-amino-Clonidine (+)-Isoproterenol Antagonists Pheatolamine Phenozybeazamine Yohimbiae WB-4101 Miscellaneous Drugs (+)-Propranolol Metismide Cimetidiae
10 .5 363 2 .9 1 .7 2 .8 0 .91 2920 3.1 17 .2 57 138 8500 14600 15400
(nM)
+ 3 .2(3) + 106 (3) + 0 .2(3) + 0 .3(3) + 0 .4(3) + 0 .04(3) + 660
6 .1 170 2 .1 1.2 2 .0 0 .63
+ + + + + + -
1 .2(5)* 8 (2)* 0 .3(3)* 0 .2(2)* 0 .2(4)* (1)*
+ + + _+
2 .0 20 47 108
+ + + _+
0 .2(2)* 4 .2(2)* 2 .5(3)* 12 .5(3)*
0 .1(3) 0.2(3) 2 .4(3) 20 (3)
+ 1400 + 700 + 1700
(3) (3) (3)
8500 15000
+ 1200 + 4000
(3) (3)
Drug inhibition of the binding of 0 .1-0 .15 nM [3H]PAC or 0 .4 nM [3H]Clonidine using 5-6 concentrations of each unlabeled drug, was determined as described is Methods . IC50 values were determined graphically by log probat analysis and apparent K.~ values calculated from the equation Ri = ICgO/ (1 + [3H]PAC ]RD) . Values are means _+ standard errors of experiments each performed in triplicate . Numbers in parentheses are the number of determinations for each compound . *Ri values of a-agonists and a-antagonists with [3H]Clonidine were calculated by U'Prichard from ref . 5 for experiments conduct ed in the same laboratory under conditions identical to those employed for [3H]PAC . In limited studies, _Ri values for drugs were determined for [3H]PAC and [3H]Clonidine in simultaneous experiments . TABLE III Clonidine and p-Amino-Clonidine Influences on oa [3H]p-Amino-Clonidine Binding in Three Tissues
Tissue
Calf Cortex
Rat Kidney
Rat Spleen
IC 50 (nM) Clonidine p-Amino-Clonidine
1.8 + 0 .1 0.9 + 0 .1
8 .5 + 2 .5 4 .3 + 1 .5
10 .7 + 5.6 4.2 + 0 .7
Experiments were performed using a concentration of [3H]PAC of 0 .2 nM for the calf cortex and 1 nM for the kidney and the spleen . Values are mean _+ standard errors of 3 experiments each performed in triplicate .
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[ 3H]PAC Binding to a-Adrenergic Receptors
Vol . 25, No . 9,
1979
label the same sites . Since the binding of [3ü]clonidine in brain and peripheral tissues is not affected by catecholamine depletion utilizing 6hydroxydopamine, it is likely that both [3H]PAC and [3H]clonidine bind to postsynaptic a-receptors. The drug specificity for binding of both liganda indicatesthat they label poatsynaptic a2-receptors (8) . Acknowledgements This work was supported by USPHS grants MH-18501 and DA-00266 as well as the John Hartford and McRnight Foundatons . We thank Guillermo Suarez for his helpful assistance and Pam Morgaa and Billie Little for manuscript preparation . B .R . acknowledges the support of the Délégation Général ~ la Recherche Scientifique et Technique . References 1. 2. 3. 4. 5. 6, 7. 8. 9. 10 . 11 . 12 . 13, 14 . 15 . 16, 17 . 18 . 19 . 20 . 21 . 22 .
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