3H-Imipramine Binding in Panic Disorder Patients and ... - Europe PMC

Comparative Study of Platelet 3H-Paroxetine and 3H-Imipramine Binding in Panic Disorder Patients and Healthy Controls Gabor Faludi, M.D., Ph.D. I, Kornelia Tekes, Ph.D.2, Laszl6 T6thfalusi, Dr. Univ.2 ISemmelweis Medical University, Department of Psychiatry, Budapest, Balassa, Hungary. 2Semmelweis Medical University, Department of Pharmacodynamics, Budapest, Xagyvkrad, Hungary. Submitted: July 22, 1993 Accepted: October 18, 1993

High affinity 3H-paroxetine and 3H-imipramine binding sites were simultaneously studied in platelets of29 untreated patients with panic disorder and 12 healthy controls. The maximum number of binding sites (Bmax) was found to be significantly lower in the panic patients compared to the controls using either ligand. No difference in the Kd values between the groups of subjects was found. The disturbance ofserotonin neurotransmission in panic disorder- decrease in Bm. values may be either a consequence or a reason of serotonergic dysfunction. -

Key Words: panic disorder, serotonin, paroxetine, imipramine, binding, platelet

INTRODUCTION

Platelet serotonin transporter labeled with 3H-imipramine Not only 3H-IMI binding to brain tissue but also DMI (3H-IMI) has often been used as a biological marker in sensitive 3H-IMI binding on platelets has been reported as psychiatric disorders since platelets are considered a peri- being heterogenous in nature (Biessen et al 1988; Hrdina pheral model for central serotonergic neurons (Pletscher 1989). Furthermore, it was shown that DMI sensitive 3H-IMI 1988). The number of specific high-affinity binding sites for

binding also includes sites unrelated to 5HT uptake (Hrdina 1984; Demushkin et al 1987) and DMI displaces 3H-IMI from both sites. Recently, the non-tricyclic selective serotonin uptake inhibitors, citalopram (D'Amato et al 1987), indalpine (Benavides et al 1985) or paroxetine (PAR) have been used to label the sodium-dependent 5HT-transporter differences or clinical factors since the effects of circadian or (Langer 1987). seasonal variations as well as of age and gender on B. are PAR apparently binds to a single population of binding minimized in the recently published studies. However, non- sites on the macromolecular complex of the 5HT-transporter selectivity of IMI for the serotonin uptake site may also be that is identical in neural and platelet membranes (Mellerup involved in the contradictory results. and Plenge 1986). However, the binding sites for 3H-IMI and 3H-PAR appear to be located on two different domains Address reprint requests to: Dr. Gabor Faludi, Semmelweis (Mellerup et al 1985). In spite of the fact that specific binding of 3H-PAR is well Medical University, Department of Psychiatry, u6, 1083 Budapest Balassa, Hungary. defined both in human brain tissue (Backstrom et al 1989)

3H-IMI on platelets of depressed patients was found to be significantly lower compared to healthy controls in several but not all reports (WHO Collaborative Study 1990). Conflicting results of the B. of 3H-IMI binding on platelets of depressive patients may be caused by either methodological

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and platelets (Mellerup et al 1983; Langer 1987; Segonzac et al 1987) there are only few and contradictory data on platelet PAR binding in psychiatric disorders, especially in panic disorder. When 3H-PAR binding to platelet membranes in patients suffering from unipolar depression was compared to healthy controls, no difference in either the Kd or the B. was found (D'haenen et al 1988). However, decreased density of both 3H-IMI and 3H-PAR binding sites was reported in putamen of patients with Parkinson's disease (Raisman et al 1986). Clorimipramine treatment was found to rapidly reduce 3H-IMI, but not 3H-PAR binding in human platelets (Plenge et al 1986). In our present study both DMI sensitive 3H-IMI and fluoxetine sensitive 3H-PAR binding on platelet membranes of drug-free panic disorder patients and of healthy controls was measured in order to gain more information on the serotonin uptake process in panic disorder. As the proposed two different binding sites for IMI and PAR may involve two differentially modulated domains of the 5HT transporter, IMI and PAR binding was measured simultaneously.

METHODS

Subjects Patients were recruited from outpatients of the Department of Psychiatry, Semmelweis University of Medicine. Twenty-nine patients who suffered from panic disorder (20 females and nine males) and 12 healthy subjects (seven females and five males) participated in the study. All were informed on the objective of the trial and gave their informed consent. Diagnoses were established on the basis of the DSM-III-R criteria. Fifteen of the patients were considered to have panic disorder with agoraphobia (11 females and four males). Additional inclusion criteria were duration of illness (at least three months) and having no major physical illness or other psychiatric illness including alcohol dependency. The mean age (± SD) of the patients was 32.3 ± 6.8 and that of the controls 29.6 ± 7.3 years. The mean frequency of panic attacks per weeks were 6.3 ± 2.4. The panic group was formed of patients for whom this was the first contact with a psychiatrist and they were not taking any medication. Control subjects were healthy volunteers recruited from the medical staff and were also drug-free prior to the study (not taking even oral contraceptives). Blood samples (40 ml) were taken approximately ten hours after last food intake at 8:00 am. Membrane preparation

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particle counter (Laborscale). Thereafter, PRP was processed as described by Segonzac et al (1987). The membrane suspension was stored for no longer than three to five days at - 70C. Protein content was measured according to Peterson (1977).

3H-Paroxetine binding The binding of 3H-paroxetine was measured as described by Segonzac et al (1987). Briefly, to 200 p1 dilutions (seven concentrations between 0.02 nM and 1.28 nM) of 3Hparoxetine (NEN, spec. act. 1058.2 GBq/mmol) 800 p1 buffer (Tris-HCl 50 mM, NaCl 120 mM and KCI 5 mM, pH 7.4) was added and the reaction was started by the addition of 1 ml membrane suspension. Incubation was run for 180 minutes at 37°C, stopped by the addition of 5 ml ice-cold buffer. The samples were then immediately filtered on Whatman GF/F filters under mild vacuum. The filters were washed three times with 5 ml ice-cold buffer, dried at room temperature, then placed in 5 ml toluene (PPO 0.5%, POPOP 0.01%), and after one night extraction radioactivity was measured by liquid scintillation counting (Beckman LS 9000). Specific binding was calculated from the difference between the dpm values measured in samples incubated in the presence or absence of 20 jM fluoxetine.

3H-Imipramine binding The method of Segonzac et al (1987) was used. Briefly, to 50 p1 dilutions of 3H-imipramine (NEN, spec. act. 2.1 TBq/mmol,7 concentrations between 0.31 nM and 10 nM) 100 gl buffer (Tris-HCl 50 mM, NaCl 120 mM, KCI 5 mM, pH 7.4) was added and the reaction was started by 1000 ul membrane suspension (about 800 jg to 100 ,ug protein /ml). Incubation was run for 60 minutes at 0'C, stopped by the addition of 3 ml ice-cold buffer, then samples were filtered on Whatman GF/F filters under mild vacuum. The filters were washed three times with 3 ml ice-cold buffer, dried at room temperature, and radioactivity was measured in toluene (PPO 0.5%, POPOP 0.01%). The difference between the cpm values measured in the presence and absence of 100 FM desipramine was regarded as specific binding.

Analysis The maximum number of binding sites (B.) and the equilibrium dissociation constant (Kd) were calculated by non-linear regression fitting to a one binding site model using LIGAND program. Comparisons between means were performed using unpaired Student's t-test.

Venous blood was collected in plastic tubes containing RESULTS anticoagulant (1 ml of 93 mM trisodium citrate, 213 mM citric acid and 111 mM glucose per 10 ml blood), then gently 3H-IMI and 3H-PAR binding to platelet membranes mixed. After centrifugation at room temperature for 20 minCurves of 3H-IMI binding defined with 100 jM DMI and utes at 200 rpm, the platelet-rich plasma (PRP) was carefully drawn offand platelet count was determined by an automatic that of fluoxetine sensitive 3H-PAR binding were saturable

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Table 1

3H-imipramine and 3H-paroxetine binding to platelet membranes from untreated patients suffering from panic disorder and healthy controls Paroxetine Imipramine

Controls (n = 12) Patients (n = 29) mean ± S.E.M.; a = p < 0.01

Kd (nM) 0.89 ± 0.06 0.98 ± 0.07

and fitted to a single site model in both groups of subjects. The mean values of the maximal number of binding sites (B.) for 3H-IMI were significantly lower (F 39,t = 3.123 1, p < 0.01) in panic patients than in the controls. Regarding individual B. values, overlap between the groups was considerable. The Kd values did not differ significantly (F 39, t = 1.5346, ns). The B. values for fluoxetine sensitive 3H-PAR binding were found also significantly decreased in patients suffering from panic disorder (F 39,t = 3.3830, p < 0.01) compared to the controls. No significant differences were seen between Kd values in panic patients and controls (F 39, t = 1.4684, ns). It is worth mentioning that Kd values for 3H-IMI binding were in an order of magnitude higher than those of 3H-PAR binding. Data obtained are summarized in Table 1. When correlation between B.a values and age, sex or body weight was calculated, no significant correlations were found.

DISCUSSION To our knowledge, simultaneous study of 3H-paroxetine and 3H-imipramine binding on platelet membranes of patients suffering from panic disorder has not been reported so far. In our present study we have found that patients with panic disorder exhibit a decrease in B.l values of DMI sensitive 3H-IMI binding to platelet membranes as compared to healthy controls. Our results are in accordance with the report of Lewis et al (1985) showing decreased Bma of 3H-imipramine binding in patients with agoraphobia and also of Marazziti et al (1988) who found significantly lower B. of 3H-IMI binding in panic disorder patients compared to healthy controls. However, in four other studies (Innis et al 1987; Uhde et al 1987; Nutt and Fraser 1987; Schneider et al 1987), no difference between controls and patients with panic dist,rder was found. It is difficult to explain these contradictory results. The selection of patients or methodological aspects might be the most likely (there was a relatively high number of patients with agoraphobia in this study who had never been treated). However, Kd values found in our study (less than 1 nM) indicate that conditions of the binding were near to optimal and the potential effect of previous medication was also excluded.

Bn, (fmol/mg protein) 1076.5 ± 110.7 580.1 ± 89.2a

Kd (nM) 0.088 ± 0.002 0.090 ± 0.01

Bm (fmol/mg protein) 1136.6 ± 84.6 615.5 ± 101.4a

DMI sensitive IMI binding sites in platelets similar to human and rat brain (Backstrom and Marcusson 1987), were shown to involve two classes of binding sites (Hrdina 1984; Demushkin et al 1987): a 5HT insensitive site which is unrelated to 5HT uptake site and a 5HT sensitive site which is closely related to the 5HT uptake site. The proportion of DMI defined IMI binding that is unrelated to 5HT uptake site in human platelets was reported to be about 30% (Hrdina 1989), however, the nature of this site is unknown. Decreased platelet 3H-IMI binding was also found in adolescent females with anorexia nervosa (Weizman et al 1986a), in enuretic children and adolescents (Weizman et al 1985) and in obsessive-compulsive patients (Weizman et al 1986b). However, in the WHO collaborative study (1990) validity of platelet 3H-IMI binding as a biological marker for endogenous depression was not supported. 3H-paroxetine binding was reported to be a more reliable radioligand for the 5HT uptake site both on human platelets (Langer 1987; Mellerup et al 1985) and in brain tissue (Backstrom et al 1989). Binding sites are apparently identical in neuronal and platelet membranes and apparently paroxetine binds to a single population of binding sites. Because of the much lower proportion of non-specific binding, the density of 5HT binding sites can be estimated more accurately using 3H-PAR as ligand. In this study, significantly lower B. of 3H-PAR binding was found in patients with panic disorder who had never been treated compared to healthy controls. There was no difference in Kd values between the groups. The kinetic parameters in the control group were comparable to those reported by others (Langer 1987; Mellerup et al 1985; Klompenhouwer et al 1990). The significant difference in B. values between the groups of subjects may indicate functionally disturbed 5HT uptake process in panic disorder since the effect of season and gender on B.a (Klompenhouwer et al 1990) was excluded. Serotonergic dysfunction in panic disorder has been suggested (Coplan et al 1992), however, the nature of this dysregulation is unknown. If platelets can be considered as a peripheral model of central serotonergic neurons (Pletscher 1988), a decreased number of 3H-PAR binding sites on platelet membranes may be an indication of decreased 5HT

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reuptake in the CNS of panic disorder patients. However, a Hrdina PD (1984) Differentiation of two components of specific 3H-imipramine binding in rat brain. Eur J Phardecreased number of 3H-PAR binding sites is only one of the macol 102:481-488. a it can be either measures of the serotonergic function and consequence or a reason of dysregulation in the 5HTergic Hrdina PD (1989) Imipramine binding sites in brain and platelets: role in affective disorders. Int J Clin Pharmacol function. Res 9:119-122. Further studies on serotonin release, functioning of preand postsynaptic serotonergic receptors and also of regula- Innis RB, Charney DS, Heninger, GR (1987) Differential 3H-imipramine platelet binding in patients with panic tion of 5HT reuptake process is needed to evaluate the and depression. Psychiatry Res 21:33-43. disorder meaning of the lowered number of 3H-PAR binding sites on platelet membrane ofuntreated panic disorder patients in this Klompenhouwer JL, Fekkes D, van Hust AM, Moleman P, Pepplinkhuizen L, Mulder PGH (1990) Seasonal variastudy. tions in binding of 3H-paroxetine to blood platelets in volunteers: indications for a gender difference. healthy ACKNOWLEDGEMENTS Biol Psychiat 28:509-517. Langer SZ (1987) The imipramine binding site in depression. This study was supported by OTKA Grant No 1116 and IST Atlas of Science, Pharmacology 1: 143-146. No 1130. The authors thank Lilly Research Laboratories Lewis DA, Noyes R, Coryell W et al (1985) Tritiated imipfor kindly donating fluoxetine. The excellent technical assisramine binding to platelets is decreased in patients with tance of Zita Prymula is appreciated. agoraphobia. Psychiatry Res 16:1-9. Marcusson J, Tiger G (1988) 3H-imipramine binding of protein nature in human platelets: inhibition by 5-HT and 5-HT uptake inhibitors J Neurochem 50:1032-1036. REFERENCES Marazziti D, Rotondo A, Placidi GF, Perugi G, Cassano GB, Pacific GM (1988) Imipramine binding in platelets of Backstrom IT, Marcusson JO (1987) 5-Hydroxytryptaminepatients with panic disorder. Pharmacopsychiat 21:47-49. sensitive 3H-imipramine binding of protein nature in the Mellerup ET, Plenge P, Engelstoft M (1983) High affinity human brain. I Characteristics. Brain Res 425:128-136. binding of 3H-paroxetine and 3H-imipramine to human Backstrom I, Bergstrom M, Marcusson J (1989) High affinity platelet membranes. Eur J Pharmacol 96:303-30?. 3H-paroxetine binding to serotonin uptake sites in human Mellerup ET, Plenge P, Nielsen M (1985) Size determination brain tissue. Brain Res 486:261-268. of binding polymers for 3H-imipramine and 3H-paroxetB6navides J, Savaki HE, Malgouris C, Laplace C, ine in human platelet membranes. Eur J Pharmacol Margelidon C, Daniel M, Courteix J, Uzan A, Gueremy 106:411-413. C, Le Fur G (1985) Quantitative autoradiography of 3H- Mellerup ET, Plenge P (1986) High affinity binding of 3Hindalpine binding sites in the rat brain. I Pharmacological paroxetine and 3H-imipramine to rat neuronal memcharacterization. J Neurochem 45:514-520. branes. Psychopharmacol 89:436-439. Biessen EAL, Norder JA, Horn AS, Robillard GT (1988) Nutt DJ, Fraser S (1987) Platelet binding studies in panic Evidence for the existence of at least two different binding disorder. J Affect Dis 12:7-11. sites for 5-HT reuptake inhibitors within the 5-HT Peterson GL (1977) A simplification of the protein assay reuptake system from human platelets. Biochem Pharmethod of Lowry et al, which is more generally applicamacol 37:3959-3966. ble. Anal Biochem 83:346-356. Coplan JD, Gorman JM, Klein DF (1992) Serotonin related Plenge P, Mellerup ET, Rafaelson OJ (1986) Chlorimipramfunctions in panic-anxiety: a critical overview. Neuine - but not imipramine - rapidly reduces imipramine ropsychopharmcol 6:189-199. - but not paroxetine - binding in human platelets. Clin D'Amato RJ, Largent BL, Snowman AM, Snyder SH (1987) Neuropharmacol 9:(Suppl 4) 113-115. Selective labeling of serotonin uptake sites in rat brain by Pletscher A (1988) Platelets as models: use and limitations. 3H-citalopram contrasted to labeling of multiple binding Experientia 44:152-155. sites by 3H-imipramine. J Pharmacol Exp Ther 242:364Raisman R, Cash R, Agid Y (1986) Parkinson's disease: 371. decreased density of 3H-imipramine and 3H-paroxetine D'haenen H, De Waele M, Leysen JE (1988) Platelet 3Hin putamen. Neurology 36:556-560 sites binding paroxetine binding in depressed patients. Psychiatry Res Schneider Munjack D, Severson JA et al (1987) Platelet LS, 26:11-17. in generalized anxiety disorder, binding 3H-imipramine Demushkin VP, Fomenko AM, Plyashkevich VG, Shchurin with panic attacks. Biol and disorder, agoraphobia panic MR, Brusov OS (1987) High- and low-affinity 3H-imip22: 59-66. Psychiatry, ramine binding sites on human platelets: separate determination and involvement of sulphur-containing bonds. Segonzac A, Schoemaker H, Langer SZ (1987) Temperature dependence of drug interaction with the platelet EurJ Pharmacol 140:171-178.

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