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Chronic antidepressant treatment enhances agonist affinity of brain α1-adrenoceptors
European Journal of Pharmacology,
35
87 (1983) 35-41
Elsevier Biomedical Press
CHRONIC ANTIDEPRESSANT a,-ADRENOCEPTORS
TREATMENT
DAVID B. MENKES *, GEORGE K. AGHAJANIAN Departments of Pharmacology and Psychiatry, Street, New Haven, CT 06508, U.S.A.
ENHANCES
AGONIST AFFINITY OF BRAIN
and DOROTHY W. GALLAGER **
Yale University School of Medicine and the Connecticut Mental Health Center, 34 Park
Received 1 June 1982, revised MS received 20 August 1982, accepted 26 October 1982
D.B. MENKES,
G.K. AGHAJANIAN and D.W. GALLAGER, Chronic antidepressant treatment enhances agonist European J. Pharmacol. 87 (1983) 35-41. Binding of [3H]prazosin, a selective a,-adrenoceptor antagonist, to thalamic membranes was studied following chronic treatment of rats with tricyclic antidepressants. Rosenthal analysis showed no change in the number or affinity affinity
of brain a,-adrenoceptors,
of antagonist binding sites; however, the ability of the a,-agonist phenylephrine to compete for these sites was significantly enhanced after chronic tricyclic treatment. This result is consistent with previous studies showing physiological a,-supersensitivity in thalamus after chronic antidepressant administration. a,-Adrenoceptors
Antidepressants
Prazosin
Tricyclics
1. Introduction Recent studies on the mechanism of action of antidepressant drugs have focussed on alterations in amine receptor function as models of the delayed clinical effects of these agents (cf., Charney et al., 1981; Sulser et al., 1978). We recently reported that chronic treatment of rats with tricyclic antidepressants, but not certain other types of psychotropic drugs, enhances q-adrenoceptormediated activation of neurons in the lateral geniculate nucleus (Menkes and Aghajanian, 1981). The molecular mechanism of this supersensitivity has been obscure, however, since a,-adrenoceptor binding, measured with the antagonist ligand [‘H]WB-4101, is generally unaltered in brain after chronic tricyclics (Rosenblatt et al., 1979; Peroutka and Snyder, 1980; Tang et al., 198 1; see however Rehavi et al., ,198O).
* Present address: Otago Hospital Board, Dunedin, New Zealand. ** To whom all correspondence should be addressed: Dept. of Psychiatry, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, U.S.A. 0014-2999/83/0000-0000/$03.00
0 1983 Elsevier Biomedical Press
Chronic treatment
Evidence has been accumulating that changes in agonist, as compared to antagonist, binding may provide a more sensitive index of physiologically relevant receptor changes. However, the lack of specific high-affinity agonist radioligands for certain adrenergic receptors has prompted the use of competition experiments, in which the affinity of an agonist is measured by its ability to compete for 3H-antagonist sites. Thus, a-adrenergic denervation supersensitivity to noradrenaline in rat vas deferens (Hata et al., 1981) and mesenteric artery (Colucci et al., 1981) has been found to correlate with enhanced agonist affinity for [3H]WB-4101 sites, despite no change or a decrease in the density of the antagonist sites. Similarly, desensitization of P-adrenoceptor function in frog erythrocytes (Wessels et al., 1979), human astrocytoma cells (Su et al., 1980), and rat glioma cells (Fishman, 1981) correlates better with agonist competition for 3H-antagonist binding sites than with simple 3H-antagonist site density or affinity. To study brain a,-adrenoceptor changes after chronic antidepressant treatment, we used the specific antagonist ligand [ 3H]prazosin to measure the density and affinity of antagonist binding sites
36
(Greengrass and Bremner, 1979; Hornung et al., 1979; Miach et al., 1980). In order to assess the agonist affinity of the receptors, the ability of the a,-agonist phenylephrine to compete for [3H]prazosin sites was also assessed. Three dissimilar tricyclics (TCA) were used in this study (amitriptyline, desipramine, and iprindole), chosen on the basis of their differing acute effects on neuronal firing (Nyback et al., 1975), amine uptake (Sulser and Sanders-Bush, 1971; Ross et al., 1971) and receptor binding (UPrichard et al., 1978; Tang and Seeman, 1980).
2. Materials and methods 2.1. Preparation
of animals
Male albino rats (Charles River Co.), initially weighing 200-250 g, were used. Groups of animals were pretreated for 3 weeks with daily intraperitoneal injections of amitriptyline HCl (AMI, 10 mg/kg, Merck, Sharp & Dohme), desipramine HCl (DMI, 10 mg/kg, USV Pharmaceuticals), iprindole (IPR, 5 or 10 mg/kg, Wyeth) or the same vol of 0.9% saline (1 ml/kg). In chronic TCA experiments, animals were killed between 14:OO and 16:00, 24 h after the last injection. For acute treatments, animals (n = 6/treatment) were killed 1 h after injection with AM1 (10 mg/kg), DMI (10 mg/kg), IPR (5 mg/kg) or saline. Thalami (1 lo- 130 mg) were rapidly dissected on an icecooled glass plate according to the method of Glowinski and Iversen (1966). Briefly, the anterior border of the thalamic region was dissected by a transverse section through the optic chiasm and anterior commissure, posteriorly by a transection at the anterior border of the superior colliculus and ventrally to the posterior border of the mammillary bodies. The hypothalamic area was dissected away, using the anterior commissure as a horizontal reference. The remaining tissue (including thalamus and subthalamus) was frozen on dry ice and stored at -70°C until assay. In some experiments cerebral cortical regions from treated and control animals were also dissected and frozen for subsequent assay.
2.2. Radioligand
binding
Binding of [ ‘Hlprazosin to crude thalamic membranes was performed using a modification of a method described previously (Greengrass and Bremner, 1979). Individual thalami were homogenized in 200 vol of ice-cold 50 mM Tris HCl (pH 7.4) using a Brinkmann Polytron (15 s, setting 8). Following centrifugation at 39 000 X g, pellets were re-homogenized and centrifuged three times in the same buffer. Further washings were not performed because preliminary experiments showed that this resulted in an excessive loss of protein. Binding was studied in pooled thalamic membranes from 8-10 rats in each treatment group, resuspended 1 : 160 (initial wt/vol) in 50 mM Tris HCI (pH 8.0). Final protein concentrations, determined according to Lowry et al. (195 1), were in the range of 0.35-0.45 mg/ml. In thalamic membranes, binding of [ ‘Hlprasozin is linear with tissue concentration in the range of 0.25- 1.80 mg protein/ml. In experiments where cerebral cortical membranes were assayed, membranes were prepared similarly to thalamic membranes as described above except that pooled membranes were resuspended in 1 : 80 (initial wt/vol) 50 mM Tris HCl. [3H]Prazosin binding of these membranes is linear with tissue concentration within this protein range (0.29- 1.29 mg protein/ml). Incubation mixtures (final vol 1 ml) contained [ 3H]prazosin (20.2 Ci/mmol, Amersham), 0.05% ascorbic acid, with or without 150 mM NaCl. The binding reaction, initiated by the addition of the membrane suspension (0.8 ml), was allowed to continue for 30 min at 25°C and was terminated by rapid filtration through Whatman GF/B filters, with 3 x 6 ml rinses of ice cold 50 mM Tris HCl (pH 7.4). Consistent with previous results (Greengrass and Bremner, 1979), binding equilibrium was attained within 5- 10 min, and did not change over the next 30 min. Specific binding was defined as that displaceable by the a,-antagonist (WB-4101 10m6 M, Ward-Blenkinsop). At ligand concentrations in the range of 1 nM, specific binding constituted 75-80% of total binding. Samples were counted in Betafluor (National Diagnostics) using a Packard Tri-Carb liquid scintillation spectrometer.
31
To estimate the density and affinity of [3H]prazosin sites, the binding of various concentrations (0.03-1.50 nM) of [3H]prazosin to thalamic membranes was analyzed by the method of Rosenthal (1967). These experiments were based on quadruplicate determinations at each ligand concentration. Agonist affinity was estimated by studying the ability of various concentrations of phenylephrine (0.1 PM-1.0 mM, Sigma) to compete for [3H]prazosin sites in the presence of a fixed concentration of radioligand (1 .O nM). Phenylephrine was chosen based upon its selectivity for the a,-receptor and its resistance to oxidation in the mild basic conditions (pH 8) used in this binding assay. Phenylephrine/[ 3H]prazosin competition curves, normalized by the maximum specific binding in each group, were computer analyzed using the probit procedure (Statistical Analysis System, Inc.). IC,, values were then converted to Ki by the Cheng and Prusoff (1973) equation. Competition studies, performed in triplicate with and without 150 mM NaCl, were conducted on 3 separate groups of chronically treated animals, each with 8- 10 rats per treatment group. In cerebral cortical membranes, competition studies were performed in triplicate without addition of salt on 2 separate groups of animals, each with 8-10 rats per treatment group. A single group of animals with 6 rats per treatment group were used in the acute TCA treatment studies. Assays on thalamic membranes for this acute group were performed in quadruplicate without addition of salt.
l&l SPECIFIC
3H-PRAZOSIN
(f mole/mg
140 SOUND
protein)
Fig. 1. Rosenthal plot of [ 3H]prazosin binding to thalamic n,-adrenoceptors following chronic treatment with tricyclic antidepressants. Data from each drug group are based on pooled thalamic membranes from S-10 rats. Estimated B,,,,, values were 192.1, 184.2, 186.1 and 194.9 fmol/mg protein for saline, AMI, DMI and IPR, respectively. K, values, estimated from the slope of the least squares regression lines, were 0.34 nM for DMI and 0.31 nM for saline, AMI and IPR. 0 SAL, 0 AMI, A DMI, A IPR.
3. Results Fig. 1 shows a Rosenthal plot of the binding of [ 3H]prazosin to a,-adrenoceptors in thalamic membranes. Chronic antidepressant treatment is seen to affect neither B,,,,, nor K,, in accord with previous studies using [3H]WB-4101 in other brain areas (see Introduction). Competition studies showed that chronic antidepressant treatment significantly enhanced the ability of phenylephrine to inhibit the binding of [ 3H]prazosin to thalamic a,-adrenoceptors. This change was seen as a shift to the left in the
Fig. 2. Competition of phenylephrine for [ 3Hlprazosin binding sites in thalamic membranes from rats chronically treated with DMI (0) or vehicle (0 0.9% saline). Each point represents the mean (k S.E.M.) maximum specific binding in 3 different experiments, each using pooled membranes from S-10 animals per group. Curves were drawn according to predicted values from probit analysis (see Methods). Statistical analysis is presented in table 1.
38
TABLE
1
Effect of chronic antidepressant treatment on parameters of phenylephrine competition for [ ‘Hlprazosin binding sites in thalamic membranes. Values for Ki and slope represent mean+ S.E.M. from 3 separate experiments (each with triplicate replications). Overall analysis of variance showed a significant effect on K, of pretreatment drug (F3.63 14.51, P < 0.01) and in vitro NaCl (F,,,, 77.48, P < 0.01). as well as a drugXNaC1 interaction (F 3,63 9.50, P < 0.01). Separate analyses of variance for the with and without NaCl conditions explained this interaction, since the drug effect was evident without (F3,30 13.64, P < 0.01) but not with NaCl (F3.30 1.67, NS). Slope was not significantly affected by drug (F3,s2 1.98, NS) or NaCl (F ,,62 0.08, NS). Individual treatment group differences from control were assessed with Duncan’s multiple range statistic (Duncan, 1955). Slope (A probit/A
Ki (PM)
SAL AMI DMI IPR
log (PE))
- NaCl
+ NaCl
- NaCl
+ NaCl
5.42f 2.38 f 2.23 f 2.66 f
1.46 f 0.27 1.16*0.46 1.03fO.33 1.32*0.16
1.11*0.20 0.89+0.07 0.85 f 0.09 0.85 f 0.01
0.95 0.94 0.95 0.92
1.41 0.98 a 0.22 a 0.35 a.b
f f + f
0.05 0.01 0.08 0.05
’ P < 0.01. b Data from 2 different chronic doses (5 and 10 mg/kg) of IPR were combined since effects similar (Ki (5 mg/kg) 2.25 pM, n = 8; Ki (10 mg/kg) 3.36 pM, n = 10 and 2.38 PM, n = 10).
competition curve (fig. 2) or, equivalently, a decrease in Ki (fig. 3) after 3 weeks treatment with AMI, DMI, or IPR. The presence of 150 mM NaCl in the binding mixtures was found to significantly reduce Ki in all treatment groups; this effect was most striking in the control group and was found to largely eliminate the antidepressant effect (fig. 3, table 1). The slope of the competition curves, which pro-
on agonist
affinity
were
vides an estimate of the Hill coefficient (Hill, 1910), was also evaluated using probit analysis. The slight decrease in slope seen in membranes from antidepressant-treated animals (fig. 2, table 1) failed to reach statistical significance. Hill coefficients for phenylephrine/[3H]prazosin competition curves obtained in the present study are in agreement with those reported (Glossmann and Hornung, 1981). The increase in agonist affinity
TABLE
2
Effect of chronic antidepressant treatment on phenylephrine competition for [ 3H]prazosin binding sites in cerebral cortical membranes. Values for Ki and slope represent mean&S.E.M. for 2 separate experiments (with triplicate replications). Overall analysis of variance showed a significant effect of drug pretreatment on Ki (F3.,* 7.18 P < 0.05). However slope was not significantly altered by drug pretreatment (F3,,2 3.24, NS). Individual treatment group differences from control were assessed with Duncan’s multiple range statistic (Duncan, 1955).
Fig. 3. Effect of chronic antidepressant treatment on K, values for inhibition of [3H]prazosin binding by phenylephrine. Values indicate mean ( f S.E.M.), Ki from three separate experiments, each with S-10 animals per treatment group. Statistical analysis of these data is presented in table 1. 0 No addition, w 150 mM NaCl.
SAL AM1 DMI IPR
Ki (PM)
Slope (A probit/A
3.56 f 0.49 2.54+0.14 a 1.47 f 0.25 b 1.36 f 0.25 b
0.88 0.97 0.76 0.81
B P < 0.05. b P < 0.01.
f 0.05 f 0.07 f 0.03 kO.05
log (PE))
39 TABLE
3
Effect of acute antidepressant treatment on phenylephrine competition for [3H]prazosin binding site in thalamic membranes. Values for Ki and slope represented mean f S.E.M. for quadruplicate replications from thalamic tissue pooled from 6 rats per treatment group. Overall analysis of variance showed no significant differences for drug pretreatment on either Ki (F4,,6 0.75, NS) or slope (F4 ,6 3.54, NS). Data were analyzed by probit procedure as descibed in Methods.
SAL AMI DMI IPR
Ki (PM)
Slope (Aprobit/Alog
5.00*0.47 5.11 kO.89 3.94k0.48 4.84 f 0.67
0.84*0.05 0.74 f 0.05 0.87 f 0.03 0.95 f 0.05
(PE))
for [3H]prazosin was not restricted to the thalamic membranes in TCA treatments. Significant decreases in the Ki for phenylephrine were also seen in cerebral cortical membranes prepared from animals treated for 3 weeks with AMI, DMI, or IPR (table 2). As in the thalamic membranes from TCA treated animals, no significant changes in the slope of the phenylephrine competition curves were seen in cerebral cortical membranes from antidepressant-treated rats (table 2). In contrast, in thalamic membranes prepared from animals receiving a single (acute) injection of TCA 1 h prior to sacrifice, no statistically significant alteration in phenylephrine affinity was observed (table 3).
4. Discussion This study shows that chronic but not acute treatment of rats with tricyclic antidepressants enhances the ability of an agonist, phenylephrine, to compete for antagonist sites labelled with [3H]prazosin. This alteration, which was found to occur without any change in the density or antagonist affinity of [ 3H]prazosin sites, suggests that a-adrenoceptors become more ‘agonist preferring’ after antidepressant treatment. This interpretation would be consistent with previous physiological (Menkes et al., 1980; Menkes and Aghajanian, 1981) and behavioral (Davis et al., 1981; Maj et al., 1979, 1980) studies showing a-adrenergic supersensitivity after chronic antidepressants.
As in these latter studies, the present result was obtained with drugs of differing acute effects on central noradrenergic systems; in particular, AMI, DMI, and IPR have markedly different affinities for a,-adrenoceptors (UPrichard et al., 1978; Tang and Seeman, 1980). This consideration makes it unlikely that direct interaction of these drugs with the receptor can account for the comparable shift in agonist affinity seen following chronic treatment. Furthermore, the acute effects of the TCAs were tested in thalamic membranes from animals receiving a single injection of drug. In these studies, acute administration of TCAs failed to affect phenylephrine affinity in contrast to the enhanced affinity for phenylephrine observed after chronic treatment with the same drugs. Since brain levels of TCAs are higher 1 h after a single dose than 24 h following a chronic daily sequence (Vetulani et al., 1976), the presence of residual drug cannot explain the agonist affinity changes observed after chronic drug administration. The possibility that enhanced agonist affinity is related to the physiological supersensitivity observed previously is strengthened by several considerations. First, prazosin is a remarkably efficaselective antagonist of cu,-adrenoceptor cious, mediated activations of neurons in the dorsal lateral geniculate nucleus (Menkes et al., 1981; Rogawski and Aghajanian, 1982). Thus it is likely that thalamic [3H]prazosin binding is of physiological relevance. Second, recent autoradiographic studies (Unnerstall et al., 1982) have indicated that [3H]prazosin sites are concentrated in various regions of the thalamus, including the dorsal lateral geniculate nucleus, paralleling the density of noradrenergic input (Fuxe, 1965). Third, increased agonist affinity has also been observed in cerebral cortical membranes from chronic TCA treated animals, thus this effect may be relevant to chronic TCA effects in other brain areas. Finally, the present results are consistent with peripheral studies of a,-adrenoceptor regulation, in which denervation supersensitivity has been found to correlate with enhanced agonist affinity for [3H]WB4101 sites (Colucci et al., 1981; Hata et al., 1981). As has been reported previously, the presence of 150 mM NaCl in the binding assay increases the affinity of phenylephrine for [ ‘Hlprazosin sites
40
(Glossmann and Hornung, 1981). This in vitro modulation by sodium ions is retained in membranes from TCA treated rats. Interestingly, high salt largely eliminates Ki differences between antidepressant treated and control membranes for phenylephrine (fig. 3). However, the possibile physiological significance of these in vitro sodium ion effects is obscure. While the receptor site itself is presumably located on the external surface of the membrane, it is not clear whether the cation-sensitive site is associated with the extracellular surface or the cytosol, since homogenization would disrupt these distinctions. In addition, in contrast to phenylephrine, some non-selective (Yagonists appear to have reduced affinity for [ 3H]prazosin sites in the presence of 150 mM NaCl (Glossmann and Hornung, 1981). The results of this study suggest a mechanism whereby a,-adrenoceptors become functionally supersensitive with tricyclic treatment. This finding may have relevance for the noradrenergic hypothesis of depression (Schildkraut, 1965; Bunney and Davis, 1965) which posits a functional deficit of noradrenaline in the disease. On the other hand based on studies of noradrenaline-stimulated cyclic AMP accumulation and /3-adrenoceptor density (Banerjee et al., 1977), Sulser and co-workers (Sulser et al., 1978) have suggested the opposite: that antidepressant treatment decreases noradrenaline P-receptor sensitivity and thereby remedies a noradrenergic hyperactivity in depression. Taken together, these findings suggest that tricyclic antidepressants may induce reciprocal changes in (Yand fi-adrenergic sensitivity (cf. Charney et al., 198 1; Maggi et al., 1980). While clinical neuroendocrine studies have indicated a-adrenergic subsensitivity in certain depressives (Matussek et al., 1980; Checkley et al., 1981), it remains to be determined whether regulation of cr- or /3-adrenoceptors (or both) is related to the therapeutic action of antidepressants in man.
Acknowledgements We thank Edward A. Wakeman, Annette Zimnewicz and Nancy Margiotta for technical help, and Leslie Fields for typing the manuscript. Supported by USPHS Grants MH-
25642, MH-17871, MH-14459, a grant from the Esther A. and Joseph Klingenstein Fund to D.W.G.. and the State of Connecticut.
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Lowry, O.H., M.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Ma@, A., D.C. UPrichard and S.J. Enna, 1980, Beta-adrenergic regulation of a-adrenergic receptors in the central nervous system, Science 207, 645. Maj, J., E. Mogilnicka and V. Klimek, 1979, The effect of repeated administration of antidepressant drugs on responsiveness of rats to catecholamine agonists, J. Neural Transm. 44, 221. Maj, J., E. Mogilnicka and A. Kordecka-Magiera, 1980, Effects of chronic administration of antidepressant drugs on aggressive behavior induced by clonidine in mice, Pharmacol. B&hem. Behav. 13, 153. Matussek, N., M. Ackenheil, H. Hippius, F. Muller, H-Th. Schroeder, H. Schultes and B. Wasilewski, 1980, Effects of clonidine on growth hormone release in psychiatric patients and controls, Psychiat. Res. 2, 25. Menkes, D.B. and G.K. Aghajanian, 1981, Alpha-l-adrenoceptor-mediated responses in the lateral geniculate nucleus are enhanced by chronic antidepressant treatment, European J. Pharmacol. 74, 27. Menkes, D.B., G.K. Aghajanian and R.B. McCall, 1980, Chronic antidepressant treatment enhances ol-adrenergic and serotonergic responses in the facial nucleus, Life Sci. 27, 45. Menkes, D.B., J.M. Baraban and G.K. Aghajanian, 1981, Prazosin selectively antagonizes neuronal responses mediated by a,-adrenoceptors in brain, Naunyn-Schmiedeb. Arch. Pharmacol. 3 17, 273. Miach, P.J., J.P. Dausse, A. Cardot and P. Meyer, 1980, [ 3Hlprazosin binds specifically to n-adrenoceptors in rat brain, Naunyn-Schmiedeb. Arch. Pharmacol. 312, 23. Nyback, H.V., J.R. Walters, G.K. Aghajanian and R.H. Roth, 1975, Tricyclic antidepressants: effects on the firing rate of brain noradrenergic neurons, European J. Pharmacol. 32, 302. Peroutka, S.J. and S.H. Snyder, 1980, Chronic antidepressant treatment decreases spiroperidol-labelled serotonin receptor binding, Science 210, 88. Rehavi, M., 0. Ramot, B. Yavetz and M. Sokolovsky, 1980, Amitriptyline: long term treatment elevates a-adrenergic and muscarinic receptor binding in mouse brain, Brain Res. 194, 443. Rogawski, M.A. and G.K. Aghajanian, 1982, Activation of lateral geniculate neurons by locus coeruleus or dorsal
noradrenergic bundle stimulation: selective blockade by the n,-adrenoceptor antagonist prazosin, Brain Res. (in press). Rosenblatt, J.E., C.E. Pert, J.F. Tallman, A. Pert and W.E. Bunney, 1979, The effect of imipramine and lithium on aand P-receptor binding in rat brain, Brain Res. 160, 185. Rosenthal, H.E., 1967, Graphic method for the determination and presentation of binding parameters in a complex system, Anal. Biochem. 20, 525. Ross, S.B., A.L. Renyi and S.O. Ogren, 1971, A comparison of the inhibiting activities of iprindole and imipramine on the uptake of 5-hydroxytryptamine and noradrenaline in brain slices, Life Sci. 10, 1267. Schildkraut, J.J., 1965, The catecholamine hypothesis of affective disorders: a review of supporting evidence, Am. J. Psychiat. 122, 509. Su, Y-F., T.K. Harden and J.P. Perkins, 1980, Catecholaminespecific desensitization of adenylate cyclase. Evidence for a multistep process, J. Biol. Chem. 255, 7410. Sulser, F. and E. Sanders-Bush, 1971, Effect of drugs on amines in the CNS, Ann. Rev. Pharmacol. 11, 209. Sulser, F., J. Vetulani and P.L. Mobley, 1978, Mode of action of antidepresssant drugs, Biochem. Pharmacol. 27, 257. Tang, S.W. and P. Seeman, 1980, Effect of antidepressant drugs on serotonergic and adrenergic receptors, Naunyn-Schmiedeb. Arch. Pharmacol. 311, 255. Tang, SW., P. Seeman and S. Kwan, 1981, Differential effect of chronic desipramine and amitriptyline treatment on rat brain adrenergic and serotonergic receptors, Psychiat. Res. 4, 129. Unnerstall, J.R., L.M. Orensanz, 1. Fernandez and M.J. Kuhar, 1982, On the selectivity of ‘H WB-4101 as an n,-ligand: an autoradiographic study. Abstr. VI European Neurosci. Cong. (in press). U’Prichard, DC., D.A. Greenberg, P.P. Sheehan and S.H. Snyder, 1978, Tricyclic antidepressants: therapeutic properties and affinity for a-noradrenergic receptor binding sites in brain, Science 199, 197. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976, A possible common mechanism of action of antidepressant treatments, Naunyn-Schmiedeb. Arch. Pharmacol. 293, 109. Wessels, M.R., D. Mullikin and R.J. Lefkowitz, 1979, Selective alteration in high-affinity agonist binding: a mechanism of P-adrenergic desensitization, Mol. Pharmacol. 16, 10.
35
87 (1983) 35-41
Elsevier Biomedical Press
CHRONIC ANTIDEPRESSANT a,-ADRENOCEPTORS
TREATMENT
DAVID B. MENKES *, GEORGE K. AGHAJANIAN Departments of Pharmacology and Psychiatry, Street, New Haven, CT 06508, U.S.A.
ENHANCES
AGONIST AFFINITY OF BRAIN
and DOROTHY W. GALLAGER **
Yale University School of Medicine and the Connecticut Mental Health Center, 34 Park
Received 1 June 1982, revised MS received 20 August 1982, accepted 26 October 1982
D.B. MENKES,
G.K. AGHAJANIAN and D.W. GALLAGER, Chronic antidepressant treatment enhances agonist European J. Pharmacol. 87 (1983) 35-41. Binding of [3H]prazosin, a selective a,-adrenoceptor antagonist, to thalamic membranes was studied following chronic treatment of rats with tricyclic antidepressants. Rosenthal analysis showed no change in the number or affinity affinity
of brain a,-adrenoceptors,
of antagonist binding sites; however, the ability of the a,-agonist phenylephrine to compete for these sites was significantly enhanced after chronic tricyclic treatment. This result is consistent with previous studies showing physiological a,-supersensitivity in thalamus after chronic antidepressant administration. a,-Adrenoceptors
Antidepressants
Prazosin
Tricyclics
1. Introduction Recent studies on the mechanism of action of antidepressant drugs have focussed on alterations in amine receptor function as models of the delayed clinical effects of these agents (cf., Charney et al., 1981; Sulser et al., 1978). We recently reported that chronic treatment of rats with tricyclic antidepressants, but not certain other types of psychotropic drugs, enhances q-adrenoceptormediated activation of neurons in the lateral geniculate nucleus (Menkes and Aghajanian, 1981). The molecular mechanism of this supersensitivity has been obscure, however, since a,-adrenoceptor binding, measured with the antagonist ligand [‘H]WB-4101, is generally unaltered in brain after chronic tricyclics (Rosenblatt et al., 1979; Peroutka and Snyder, 1980; Tang et al., 198 1; see however Rehavi et al., ,198O).
* Present address: Otago Hospital Board, Dunedin, New Zealand. ** To whom all correspondence should be addressed: Dept. of Psychiatry, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508, U.S.A. 0014-2999/83/0000-0000/$03.00
0 1983 Elsevier Biomedical Press
Chronic treatment
Evidence has been accumulating that changes in agonist, as compared to antagonist, binding may provide a more sensitive index of physiologically relevant receptor changes. However, the lack of specific high-affinity agonist radioligands for certain adrenergic receptors has prompted the use of competition experiments, in which the affinity of an agonist is measured by its ability to compete for 3H-antagonist sites. Thus, a-adrenergic denervation supersensitivity to noradrenaline in rat vas deferens (Hata et al., 1981) and mesenteric artery (Colucci et al., 1981) has been found to correlate with enhanced agonist affinity for [3H]WB-4101 sites, despite no change or a decrease in the density of the antagonist sites. Similarly, desensitization of P-adrenoceptor function in frog erythrocytes (Wessels et al., 1979), human astrocytoma cells (Su et al., 1980), and rat glioma cells (Fishman, 1981) correlates better with agonist competition for 3H-antagonist binding sites than with simple 3H-antagonist site density or affinity. To study brain a,-adrenoceptor changes after chronic antidepressant treatment, we used the specific antagonist ligand [ 3H]prazosin to measure the density and affinity of antagonist binding sites
36
(Greengrass and Bremner, 1979; Hornung et al., 1979; Miach et al., 1980). In order to assess the agonist affinity of the receptors, the ability of the a,-agonist phenylephrine to compete for [3H]prazosin sites was also assessed. Three dissimilar tricyclics (TCA) were used in this study (amitriptyline, desipramine, and iprindole), chosen on the basis of their differing acute effects on neuronal firing (Nyback et al., 1975), amine uptake (Sulser and Sanders-Bush, 1971; Ross et al., 1971) and receptor binding (UPrichard et al., 1978; Tang and Seeman, 1980).
2. Materials and methods 2.1. Preparation
of animals
Male albino rats (Charles River Co.), initially weighing 200-250 g, were used. Groups of animals were pretreated for 3 weeks with daily intraperitoneal injections of amitriptyline HCl (AMI, 10 mg/kg, Merck, Sharp & Dohme), desipramine HCl (DMI, 10 mg/kg, USV Pharmaceuticals), iprindole (IPR, 5 or 10 mg/kg, Wyeth) or the same vol of 0.9% saline (1 ml/kg). In chronic TCA experiments, animals were killed between 14:OO and 16:00, 24 h after the last injection. For acute treatments, animals (n = 6/treatment) were killed 1 h after injection with AM1 (10 mg/kg), DMI (10 mg/kg), IPR (5 mg/kg) or saline. Thalami (1 lo- 130 mg) were rapidly dissected on an icecooled glass plate according to the method of Glowinski and Iversen (1966). Briefly, the anterior border of the thalamic region was dissected by a transverse section through the optic chiasm and anterior commissure, posteriorly by a transection at the anterior border of the superior colliculus and ventrally to the posterior border of the mammillary bodies. The hypothalamic area was dissected away, using the anterior commissure as a horizontal reference. The remaining tissue (including thalamus and subthalamus) was frozen on dry ice and stored at -70°C until assay. In some experiments cerebral cortical regions from treated and control animals were also dissected and frozen for subsequent assay.
2.2. Radioligand
binding
Binding of [ ‘Hlprazosin to crude thalamic membranes was performed using a modification of a method described previously (Greengrass and Bremner, 1979). Individual thalami were homogenized in 200 vol of ice-cold 50 mM Tris HCl (pH 7.4) using a Brinkmann Polytron (15 s, setting 8). Following centrifugation at 39 000 X g, pellets were re-homogenized and centrifuged three times in the same buffer. Further washings were not performed because preliminary experiments showed that this resulted in an excessive loss of protein. Binding was studied in pooled thalamic membranes from 8-10 rats in each treatment group, resuspended 1 : 160 (initial wt/vol) in 50 mM Tris HCI (pH 8.0). Final protein concentrations, determined according to Lowry et al. (195 1), were in the range of 0.35-0.45 mg/ml. In thalamic membranes, binding of [ ‘Hlprasozin is linear with tissue concentration in the range of 0.25- 1.80 mg protein/ml. In experiments where cerebral cortical membranes were assayed, membranes were prepared similarly to thalamic membranes as described above except that pooled membranes were resuspended in 1 : 80 (initial wt/vol) 50 mM Tris HCl. [3H]Prazosin binding of these membranes is linear with tissue concentration within this protein range (0.29- 1.29 mg protein/ml). Incubation mixtures (final vol 1 ml) contained [ 3H]prazosin (20.2 Ci/mmol, Amersham), 0.05% ascorbic acid, with or without 150 mM NaCl. The binding reaction, initiated by the addition of the membrane suspension (0.8 ml), was allowed to continue for 30 min at 25°C and was terminated by rapid filtration through Whatman GF/B filters, with 3 x 6 ml rinses of ice cold 50 mM Tris HCl (pH 7.4). Consistent with previous results (Greengrass and Bremner, 1979), binding equilibrium was attained within 5- 10 min, and did not change over the next 30 min. Specific binding was defined as that displaceable by the a,-antagonist (WB-4101 10m6 M, Ward-Blenkinsop). At ligand concentrations in the range of 1 nM, specific binding constituted 75-80% of total binding. Samples were counted in Betafluor (National Diagnostics) using a Packard Tri-Carb liquid scintillation spectrometer.
31
To estimate the density and affinity of [3H]prazosin sites, the binding of various concentrations (0.03-1.50 nM) of [3H]prazosin to thalamic membranes was analyzed by the method of Rosenthal (1967). These experiments were based on quadruplicate determinations at each ligand concentration. Agonist affinity was estimated by studying the ability of various concentrations of phenylephrine (0.1 PM-1.0 mM, Sigma) to compete for [3H]prazosin sites in the presence of a fixed concentration of radioligand (1 .O nM). Phenylephrine was chosen based upon its selectivity for the a,-receptor and its resistance to oxidation in the mild basic conditions (pH 8) used in this binding assay. Phenylephrine/[ 3H]prazosin competition curves, normalized by the maximum specific binding in each group, were computer analyzed using the probit procedure (Statistical Analysis System, Inc.). IC,, values were then converted to Ki by the Cheng and Prusoff (1973) equation. Competition studies, performed in triplicate with and without 150 mM NaCl, were conducted on 3 separate groups of chronically treated animals, each with 8- 10 rats per treatment group. In cerebral cortical membranes, competition studies were performed in triplicate without addition of salt on 2 separate groups of animals, each with 8-10 rats per treatment group. A single group of animals with 6 rats per treatment group were used in the acute TCA treatment studies. Assays on thalamic membranes for this acute group were performed in quadruplicate without addition of salt.
l&l SPECIFIC
3H-PRAZOSIN
(f mole/mg
140 SOUND
protein)
Fig. 1. Rosenthal plot of [ 3H]prazosin binding to thalamic n,-adrenoceptors following chronic treatment with tricyclic antidepressants. Data from each drug group are based on pooled thalamic membranes from S-10 rats. Estimated B,,,,, values were 192.1, 184.2, 186.1 and 194.9 fmol/mg protein for saline, AMI, DMI and IPR, respectively. K, values, estimated from the slope of the least squares regression lines, were 0.34 nM for DMI and 0.31 nM for saline, AMI and IPR. 0 SAL, 0 AMI, A DMI, A IPR.
3. Results Fig. 1 shows a Rosenthal plot of the binding of [ 3H]prazosin to a,-adrenoceptors in thalamic membranes. Chronic antidepressant treatment is seen to affect neither B,,,,, nor K,, in accord with previous studies using [3H]WB-4101 in other brain areas (see Introduction). Competition studies showed that chronic antidepressant treatment significantly enhanced the ability of phenylephrine to inhibit the binding of [ 3H]prazosin to thalamic a,-adrenoceptors. This change was seen as a shift to the left in the
Fig. 2. Competition of phenylephrine for [ 3Hlprazosin binding sites in thalamic membranes from rats chronically treated with DMI (0) or vehicle (0 0.9% saline). Each point represents the mean (k S.E.M.) maximum specific binding in 3 different experiments, each using pooled membranes from S-10 animals per group. Curves were drawn according to predicted values from probit analysis (see Methods). Statistical analysis is presented in table 1.
38
TABLE
1
Effect of chronic antidepressant treatment on parameters of phenylephrine competition for [ ‘Hlprazosin binding sites in thalamic membranes. Values for Ki and slope represent mean+ S.E.M. from 3 separate experiments (each with triplicate replications). Overall analysis of variance showed a significant effect on K, of pretreatment drug (F3.63 14.51, P < 0.01) and in vitro NaCl (F,,,, 77.48, P < 0.01). as well as a drugXNaC1 interaction (F 3,63 9.50, P < 0.01). Separate analyses of variance for the with and without NaCl conditions explained this interaction, since the drug effect was evident without (F3,30 13.64, P < 0.01) but not with NaCl (F3.30 1.67, NS). Slope was not significantly affected by drug (F3,s2 1.98, NS) or NaCl (F ,,62 0.08, NS). Individual treatment group differences from control were assessed with Duncan’s multiple range statistic (Duncan, 1955). Slope (A probit/A
Ki (PM)
SAL AMI DMI IPR
log (PE))
- NaCl
+ NaCl
- NaCl
+ NaCl
5.42f 2.38 f 2.23 f 2.66 f
1.46 f 0.27 1.16*0.46 1.03fO.33 1.32*0.16
1.11*0.20 0.89+0.07 0.85 f 0.09 0.85 f 0.01
0.95 0.94 0.95 0.92
1.41 0.98 a 0.22 a 0.35 a.b
f f + f
0.05 0.01 0.08 0.05
’ P < 0.01. b Data from 2 different chronic doses (5 and 10 mg/kg) of IPR were combined since effects similar (Ki (5 mg/kg) 2.25 pM, n = 8; Ki (10 mg/kg) 3.36 pM, n = 10 and 2.38 PM, n = 10).
competition curve (fig. 2) or, equivalently, a decrease in Ki (fig. 3) after 3 weeks treatment with AMI, DMI, or IPR. The presence of 150 mM NaCl in the binding mixtures was found to significantly reduce Ki in all treatment groups; this effect was most striking in the control group and was found to largely eliminate the antidepressant effect (fig. 3, table 1). The slope of the competition curves, which pro-
on agonist
affinity
were
vides an estimate of the Hill coefficient (Hill, 1910), was also evaluated using probit analysis. The slight decrease in slope seen in membranes from antidepressant-treated animals (fig. 2, table 1) failed to reach statistical significance. Hill coefficients for phenylephrine/[3H]prazosin competition curves obtained in the present study are in agreement with those reported (Glossmann and Hornung, 1981). The increase in agonist affinity
TABLE
2
Effect of chronic antidepressant treatment on phenylephrine competition for [ 3H]prazosin binding sites in cerebral cortical membranes. Values for Ki and slope represent mean&S.E.M. for 2 separate experiments (with triplicate replications). Overall analysis of variance showed a significant effect of drug pretreatment on Ki (F3.,* 7.18 P < 0.05). However slope was not significantly altered by drug pretreatment (F3,,2 3.24, NS). Individual treatment group differences from control were assessed with Duncan’s multiple range statistic (Duncan, 1955).
Fig. 3. Effect of chronic antidepressant treatment on K, values for inhibition of [3H]prazosin binding by phenylephrine. Values indicate mean ( f S.E.M.), Ki from three separate experiments, each with S-10 animals per treatment group. Statistical analysis of these data is presented in table 1. 0 No addition, w 150 mM NaCl.
SAL AM1 DMI IPR
Ki (PM)
Slope (A probit/A
3.56 f 0.49 2.54+0.14 a 1.47 f 0.25 b 1.36 f 0.25 b
0.88 0.97 0.76 0.81
B P < 0.05. b P < 0.01.
f 0.05 f 0.07 f 0.03 kO.05
log (PE))
39 TABLE
3
Effect of acute antidepressant treatment on phenylephrine competition for [3H]prazosin binding site in thalamic membranes. Values for Ki and slope represented mean f S.E.M. for quadruplicate replications from thalamic tissue pooled from 6 rats per treatment group. Overall analysis of variance showed no significant differences for drug pretreatment on either Ki (F4,,6 0.75, NS) or slope (F4 ,6 3.54, NS). Data were analyzed by probit procedure as descibed in Methods.
SAL AMI DMI IPR
Ki (PM)
Slope (Aprobit/Alog
5.00*0.47 5.11 kO.89 3.94k0.48 4.84 f 0.67
0.84*0.05 0.74 f 0.05 0.87 f 0.03 0.95 f 0.05
(PE))
for [3H]prazosin was not restricted to the thalamic membranes in TCA treatments. Significant decreases in the Ki for phenylephrine were also seen in cerebral cortical membranes prepared from animals treated for 3 weeks with AMI, DMI, or IPR (table 2). As in the thalamic membranes from TCA treated animals, no significant changes in the slope of the phenylephrine competition curves were seen in cerebral cortical membranes from antidepressant-treated rats (table 2). In contrast, in thalamic membranes prepared from animals receiving a single (acute) injection of TCA 1 h prior to sacrifice, no statistically significant alteration in phenylephrine affinity was observed (table 3).
4. Discussion This study shows that chronic but not acute treatment of rats with tricyclic antidepressants enhances the ability of an agonist, phenylephrine, to compete for antagonist sites labelled with [3H]prazosin. This alteration, which was found to occur without any change in the density or antagonist affinity of [ 3H]prazosin sites, suggests that a-adrenoceptors become more ‘agonist preferring’ after antidepressant treatment. This interpretation would be consistent with previous physiological (Menkes et al., 1980; Menkes and Aghajanian, 1981) and behavioral (Davis et al., 1981; Maj et al., 1979, 1980) studies showing a-adrenergic supersensitivity after chronic antidepressants.
As in these latter studies, the present result was obtained with drugs of differing acute effects on central noradrenergic systems; in particular, AMI, DMI, and IPR have markedly different affinities for a,-adrenoceptors (UPrichard et al., 1978; Tang and Seeman, 1980). This consideration makes it unlikely that direct interaction of these drugs with the receptor can account for the comparable shift in agonist affinity seen following chronic treatment. Furthermore, the acute effects of the TCAs were tested in thalamic membranes from animals receiving a single injection of drug. In these studies, acute administration of TCAs failed to affect phenylephrine affinity in contrast to the enhanced affinity for phenylephrine observed after chronic treatment with the same drugs. Since brain levels of TCAs are higher 1 h after a single dose than 24 h following a chronic daily sequence (Vetulani et al., 1976), the presence of residual drug cannot explain the agonist affinity changes observed after chronic drug administration. The possibility that enhanced agonist affinity is related to the physiological supersensitivity observed previously is strengthened by several considerations. First, prazosin is a remarkably efficaselective antagonist of cu,-adrenoceptor cious, mediated activations of neurons in the dorsal lateral geniculate nucleus (Menkes et al., 1981; Rogawski and Aghajanian, 1982). Thus it is likely that thalamic [3H]prazosin binding is of physiological relevance. Second, recent autoradiographic studies (Unnerstall et al., 1982) have indicated that [3H]prazosin sites are concentrated in various regions of the thalamus, including the dorsal lateral geniculate nucleus, paralleling the density of noradrenergic input (Fuxe, 1965). Third, increased agonist affinity has also been observed in cerebral cortical membranes from chronic TCA treated animals, thus this effect may be relevant to chronic TCA effects in other brain areas. Finally, the present results are consistent with peripheral studies of a,-adrenoceptor regulation, in which denervation supersensitivity has been found to correlate with enhanced agonist affinity for [3H]WB4101 sites (Colucci et al., 1981; Hata et al., 1981). As has been reported previously, the presence of 150 mM NaCl in the binding assay increases the affinity of phenylephrine for [ ‘Hlprazosin sites
40
(Glossmann and Hornung, 1981). This in vitro modulation by sodium ions is retained in membranes from TCA treated rats. Interestingly, high salt largely eliminates Ki differences between antidepressant treated and control membranes for phenylephrine (fig. 3). However, the possibile physiological significance of these in vitro sodium ion effects is obscure. While the receptor site itself is presumably located on the external surface of the membrane, it is not clear whether the cation-sensitive site is associated with the extracellular surface or the cytosol, since homogenization would disrupt these distinctions. In addition, in contrast to phenylephrine, some non-selective (Yagonists appear to have reduced affinity for [ 3H]prazosin sites in the presence of 150 mM NaCl (Glossmann and Hornung, 1981). The results of this study suggest a mechanism whereby a,-adrenoceptors become functionally supersensitive with tricyclic treatment. This finding may have relevance for the noradrenergic hypothesis of depression (Schildkraut, 1965; Bunney and Davis, 1965) which posits a functional deficit of noradrenaline in the disease. On the other hand based on studies of noradrenaline-stimulated cyclic AMP accumulation and /3-adrenoceptor density (Banerjee et al., 1977), Sulser and co-workers (Sulser et al., 1978) have suggested the opposite: that antidepressant treatment decreases noradrenaline P-receptor sensitivity and thereby remedies a noradrenergic hyperactivity in depression. Taken together, these findings suggest that tricyclic antidepressants may induce reciprocal changes in (Yand fi-adrenergic sensitivity (cf. Charney et al., 198 1; Maggi et al., 1980). While clinical neuroendocrine studies have indicated a-adrenergic subsensitivity in certain depressives (Matussek et al., 1980; Checkley et al., 1981), it remains to be determined whether regulation of cr- or /3-adrenoceptors (or both) is related to the therapeutic action of antidepressants in man.
Acknowledgements We thank Edward A. Wakeman, Annette Zimnewicz and Nancy Margiotta for technical help, and Leslie Fields for typing the manuscript. Supported by USPHS Grants MH-
25642, MH-17871, MH-14459, a grant from the Esther A. and Joseph Klingenstein Fund to D.W.G.. and the State of Connecticut.
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