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Platelet [3H]paroxetine binding in panic disorder
joumal of AFFEcTm
DISORDERS
ELSEVIER
Journal of Affective Disorders 33 (1995) 117- 122
Platelet Kay P. Maguire
[
“Hlparoxetine
*, Trevor
binding in panic disorder
R. Norman, Maria Apostolopoulos, Graham D. Burrows
Fiona K. Judd,
Department of Psychiatry, University of Melbourne, Austin Hospital, Heidelberg 3084, Victoria, Australia
Received 16 May 1994; revised 20 September 1994; accepted 22 September 1994
Abstract Platelet [3H]paroxetine binding was measured in 20 patients (14 females, six males) with panic disorder normal controls (14 females, six males). For patients with panic disorder, II,,, was 1987 k 1135 fmol/mg (mean + SD) compared with control B,,, values of 1626 + 611 fmol/mg protein. The mean K, value patients was 0.076 5 0.056 nM and for the controls was 0.083 + 0.033 nM. ANCOVA indicated no significant of sex. diagnosis (panic disorder vs. normal controls) or season of sampling on either K, or B,,,. The data no evidence for an abnormality of the platelet uptake mechanism in panic disorder. Keywords:
Panic disorder;
Paroxetine
binding;
Platelet;
1. Introduction
The biological basis of panic disorder remains unknown. Hypotheses implicating dysfunction of the GABAergic and adrenergic systems have been proposed as possible important aetiological factors. More recently, evidence for dysfunction within the serotonergic system has also been suggested as significant in the genesis of panic. This is supported by the empirical findings from animal, drug treatment and platelet studies together with the results of specific neuroendocrine challenge tests (Coplan et al., 1992; Sheehan et al., 1993 ).
* Corresponding
author.
and 20 protein for the effect provide
Serotonin
Preclinical and animal studies have suggested a role for serotonin (5HT) in anxiety although both increased and decreased function have been reported as relevant (Coplan et al., 1992). Important evidence for the involvement of 5HT in panic disorder comes from clinical drug treatment studies. Although the mode of action of medications used as anti-panic agents is unknown, most affect 5HT function. In particular, agents, such as the tricyclic antidepressants, inhibit 5HT re-uptake and also induce a decrease in 5HT, receptor number on chronic administration (Peroutka and Snyder, 1980) in common with other drugs, such as the monoamine oxidase inhibitors. More importantly, the newer specific serotonin re-uptake inhibitors (SSRIs) have been found to be effective in panic disorder (Sheehan et al., 1993).
0165-0327/95/$09.50 0 1995 El sevier Science B.V. All rights reserved SSDI 0165-0327(94)00081-6
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of Affectiue Disorders 33 (19951 I 17-122
More convincing evidence for abnormalities of the 5HT system in panic disorder comes from neuroendocrine challenge tests in patients. The specific 5HT agonists m-chlorophenyl piperazine, MK 212, dl-fenfluramine and d-fenfluramine and their effect on prolactin and/or cortisol release have been investigated in panic disorder. We have found an increased prolactin response to dl-fenfluramine in panic patients when compared with controls (Apostolopoulos et al., 1993). Using the more specific agonist d-fenfluramine, we found a greater prolactin increase in patients than controls (Judd et al., 1994) in agreement with other studies of 5HT agonists in panic disorder (Kahn et al., 1988; Targum and Marshall, 1989). Due to the inaccessibility of the brain, peripheral models, such as the platelet, have been widely used in psychiatric research. There are many physiological similarities between platelets and neurons, such as (Y* receptors, 5HT, receptors, an active uptake site for 5HT and calcium-dependent release of 5HT (Leonard, 1991). Further validity of the platelet model comes from the observation that the structure of the human platelet 5HT transporter is identical with that found in human brain (Lesch et al., 1993). Platelets have an ion-dependent active uptake system for 5HT. We have studied this transport system and found a significant increase in maximal uptake (I&,,> of 5HT by the platelet in panic patients in comparison with normal controls (Norman et al., 1986). In a replication study, a similar increase in Vm, was observed (Norman et al., 1989a). In contrast, Pecknold et al. (1988) found a significant decrease in V,,, for panic patients compared with controls as did Butler et al. (1992). Den Boer and Westenberg (1990) found no difference in platelet 5HT uptake between panic disorder and controls. Another approach to studying the 5HT transporter has been to measure [3H]imipramine binding to platelet membranes. Some studies have found lower maximal number of binding sites (B,,,) in panic patients compared with controls (Lewis et al., 1985; Marazzitti et al., 1989). However, the majority of [ 3H]imipramine-binding studies (Innis et al., 1987; Nutt and Fraser, 1987;
Uhde et al., 1987; Schneider et al., 19871, including our own (Norman et al., 1989b), have not found any significant difference in B,,, between panic patients and controls. Although imipramine and drugs which inhibit 5HT uptake have been suggested to bind to the platelet 5HT transporter, a functional relationship between parallel measurements of both 5HT uptake by platelets and L3Hlimipramine binding to platelet membrane has not been demonstrated (Raisman et al., 1982; Wood et al., 1983; Halbreich et al., 1991). This lack of correlation possibly occurs because [“Hlimipramine binds to two sites on the platelet membrane but only one site is related to the 5HT transporter (Hrdina, 1989). Recently, [“Hlparoxetine has been shown to bind only to the 5HT transporter (Plenge and Mellerup, 1985). We have measured platelet 5HT uptake and [“Hlparoxetine binding to platelet membranes from normal volunteers and found a significant correlation between the V,,, of platelet 5HT uptake and the B,,, of [3H]paroxetine binding (Maguire et al., 1993). It would appear that [3Hlparoxetine is a better ligand for the study of the platelet 5HT transporter. In the present study, we have investigated [ 3H]paroxetine binding to platelet membranes from 20 patients diagnosed with panic disorder and 20 healthy nonpsychiatric controls.
2. Methods 2.1. Subjects
Patients with panic disorder were included in the study if they met DSM-III-R criteria for panic disorder/agoraphobia and had more than one panic attack per week, were male or female, aged 18-65 years and had been drug-free for > 2 weeks. Patients were excluded from the study if physically unwell, if there was a life-time history of alcohol or drug abuse or if they suffered from any other psychiatric disorder. Longitudinal comorbidity as in panic disorder/depression was allowed but current co-morbidity was not. A healthy medication-free control group drawn from the general population was studied for com-
K.P. Muguire et al. /Journul of Affective Disorder.v33 (I 995) 117-122
parison. Volunteers were screened by clinical interview for current or past psychiatric illness and were excluded from the study if they met criteria for any psychiatric disorder. None had somatic illness as judged by physical examination and none had a history of drug abuse or were currently using medications. All subjects gave written informed consent for the study which was approved by the Austin Hospital Medical Research Committee.
I IY
sis using the Ligand computer program. Intraassay variation was + 4% for f?,,;,, and f 10% for K,. Interassay variation was + 10% for B,,;,, and + 15% for K,. Statistical analysis of the data was carried out using SuperANOVA for the Macintosh. ANCOVA was used to determine the influence of age, sex, season of sampling and diagnosis (panic disorder vs. controls) on K, and B,,,;,,. Correlations between parameters were examined using Pearson’s product-moment correlations.
2.2. Blood sampling Blood (30 ml> was taken into tubes containing EDTA (final concentration OX-l.5 mg/ml) between O&30 and lo:30 with the subject supine for at least 30 min following insertion of a butterfly needle. Blood was centrifuged at 200 x g for 20 min at room temperature and the platelet-rich plasma removed. Platelets were precipitated by centrifugation at 2200 x g for 20 min and washed with huffer (50 mM Tris, 3 mM EDTA, pH 7.0), reprecipitated and stored at -70°C until analysed. 2.3. I’aroxetine-binding assay Platelets were thawed, washed with buffer (70 mM Tris, pH 7.5) and platelet membranes prepared and [‘Hlparoxetine binding performed as described previously (Maguire et al., 1993) with minor modifications. Briefly, the binding of [“Hlparoxetine (0.1-2.0 nM, 25 Ci/mmol) to platelet membrane in the presence or absence of 10 PM citalopram was measured after a 2-h incubation at 20°C. After addition of ice-cold buffer, the samples were rapidly filtered through GF/B filters using a Brandel M-24R cell harvester, the filters washed 3 X and the radioactivity determined by liquid scintillation spectrometry. Protein concentration of the diluted platelet suspension was measured using the Bio-Rad kit. 2.4. I)ata analysis The maximal number of binding sites (B,,,, expressed in fmol/mg protein) and equilibrium dissociation constant (K,, expressed in nM) for each subject were calculated by Scatchard analy-
3. Results 20 panic patients (14 females, six males) of mean age 33 + 10 (-t SD> years entered the study. The mean B,,,, for the patient group was 1987 ) 1135 fmol/mg protein and the mean K,, was 0.076 + 0.056 nM. 20 controls (14 females, six males) of mean age 27 f 7 years were also studied. The mean B,,;,, for the control group was 1626 -t 611 fmol/mg protein and the mean K, was 0.083 + 0.033 nM. Individual values for both B,,, and K, are presented in Fig. I. ANCOVA indicated, after adjusting for age, no significant effect of sex (partial F = 2.176, df = 1,33, P = 0.1496), diagnosis (partial F = 0.761, df = 1,33, P = 0.3894) or month of sampling (partial F = 0.5 14, df = 3,33. P =z 0.6754) on K,, values. After adjusting for age. ANCOVA indicated no significant effect on B,,;,, of sex (partial F = 0.408, df = 1,33, P = 0.5274) season (partial F = 2.492, df = 3,33. P = 0.0772) or diagnosis (partial F = 1.554, df = 1,33. P ==0.2213). There was a significant difference in age between the two groups (Welch’s I = _ 3.243, P = 0.0027), however, there were no correlations between age and either B,,, or K,, in either the patient or control groups (B,;,,: controls r= - 0.0874, P = 0.7142, patients r = 0.1398, P = 0.5566; K,: controls r = - 0.0691, P =: 0.7721, patients r = 0.3338, P = 0.1504).
4. Discussion Serotonin dysfunction has been implicated in panic disorder. Conflicting results have been ob-
K.P. Maguire et al. /Journal
120
of Affective Disorders 33 (1995) II 7-122
We found no significant difference in either between the panic disorder patients and the control group, taking into account age, sex and season of sampling. This is in contrast to a recent report by Faludi et al. (1994) who found a significant decrease in B,,, for 29 panic patients when compared with 12 healthy controls. One reason for the conflicting findings may be the fact that the latter study included only panic disorder patients who had not been previously treated. Both studies excluded concurrent psychiatric disorders but the effects of longitudinal comorbidity cannot be excluded in either study. Decreased [ 3H]paroxetine binding has also been observed in depressed patients (Nemeroff, 1992) and in patients with posttraumatic stress disorder (Arora et al., 1993). In contrast, no difference in [3H]paroxetine binding between a normal control group and patients with major depressive disorder was reported in two studies (D’Haenen et al., 1988; Lawrence et al., 1993). Increased [3H]paroxetine binding has been reported in one study of alcoholic patients (Mellerup et al., 1993) but Daoust et al. (1991) found no difference between alcoholics and controls. Comorbidity of these disorders with panic is common and, thus, may confound reported results. Given the previously demonstrated correlation between platelet 5HT uptake and [3H]paroxetine binding in normal controls, the present findings do not support our original observation of increased 5HT uptake in panic disorder. On the other hand, platelet 5HT uptake and t3H]paroxetine binding may not be correlated in panic disorder and the increased 5HT uptake may be an indication of hyperactivity of the serotonergic system as suggested by Kahn et al. (1991). The finding of decreased platelet [ 3H]paroxetine binding in panic disorder by Faludi et al. (1994) does not concur with this hypothesis but does agree with suggestions of decreased 5HT function in anxiety by Coplan et al. (1992). Thus, the role of 5HT in panic disorder remains controversial. Further studies are required in a larger group of patients with panic disorder and matched controls of both platelet 5HT uptake and [3H]paroxetine binding to clarify the status of the 5HT transporter in this disorder. B max or K,
4000 .g if! Q
1
m
3000 -
E
0
.z 2 E m
2000
-
1000
-
: 0
’ PANIC
CONTROLS
Fig. 1. Individual values for B,,, group and normal controls.
and
Kd for panic
disorder
tained from studies of platelet 5HT uptake and from platelet [3H]imipramine binding. This may be due to methodological and patient population differences between the studies. [3H]paroxetine binding is a more specific ligand for the platelet 5HT transporter and has been shown to correlate with 5HT uptake (Maguire et al., 1993). Studies of platelet [ 3H]paroxetine binding may, thus, clarify the status of the 5HT transporter in panic disorder.
K.P. Maguire et al. /Journal
of Affective Disorders 33 (1995) I 17-122
Acknowledgements
We would like to thank the National Health and Medical Research Council of Australia for financial support and H. Lundbeck A/S for the kind donation of citalopram.
References Apostolopoulos, M., Judd, F.K., Burrows, G.D. and Norman, T.R. (1993) Prolactin response to d[-fenfluramine in panic disorder. Psychoneuroendocrinology 18, 337-342. Arora, R.C., Fichtner, C.G., O’Connor, F. and Crayton, J.W. (19Y3) Paroxetine binding in the blood platelets of posttraumatic stress disorder patients. Life Sci. 53, 919-928. Butler, J., O’Halloran, A. and Leonard, B.E. (1992) The Galway study of panic disorder: II Changes in some periphcral markers of noradrenergic and serotonergic function in DSM-111-R panic disorder. J. Affect. Disord. 26, x9- 100. Coplan. J.D., Gorman, J.M. and Klein, D.F. (1992) Serotonin related functions in panic-anxiety: a critical overview. Neuropavchopharmacology 6, 189-200. Daoust. M., Lhuintre, J.P., Ernouf, E., Breton, P. and Bouly, P. (1991) Ethanol intake and “H-serotonin uptake II: a study in alcoholic patients using platelet 3H-paroxetine bindmg. Life Sci. 48, 1977-1983. D’Haenen, H., De Waele, M. and Leysen, J.E. (1988) Platelet ‘H-paroxetine binding in depressed patients. Psychiatr. Res. 26, 11-17. Den Boer, J.A. and Westenberg, H.G.M. (1990) Serotonin function in panic disorder: a double blind placebo controlled study with fluvoxamine and ritanserin. Psychopharmacology 102, 85-94. Faludi, G., Tekes, K. and Tothfalusi, L. (1994) Comparative studv of platelet ‘H-paroxetine and ‘H-imipramine binding m panic disorder patients and healthy controls. J. Psychiatry Neurosci. 19, 109-113. Halbreich, U., Rojansky, N., Zander, K.J. and Barkai, A. (1941) Influence of age, sex and diurnal variability on imipramine receptor binding and serotonin uptake in platelets of normal subjects. J. Psychiatr. Res. 25, 7-18. Hrdina. P.D. (1989) Differences between sodium-dependent and desipramine defined ‘H-imipramine binding in intact human platelets. Biol. Psychiatry 25, 576-584. Innis. R.B., Charney. D.S. and Heninger, G.R. (1987) Differentl&d ‘H-imipramine platelet binding in patients with panic disorder and depression. Psychiatr. Res. 21, 33-41. Judd, F K., Apostolopoulos, M., Burrows, G.D. and Norman, T.R (1994) Serotonergic function in panic disorder: endocl-ine responses to d-fenfluramine. Prog. Neuro Psychopharmacol. Biol. Psychiatry 18, 329-337. Kahn, RX, Anis, G.M., Wetzler, S. and van Praag, H.M. (19xX) Neuroendocrine evidence for the 5HT receptor
121
hypersensitivity in panic disorder. Psychopharmacology 96, 360-369. Kahn, R.S., Kalus, O., Wetzler. S. and Van Praag, H.M. (1991) The role of serotonin in the regulation of anxiety. In S.L. Brown and H.M. van Praag (Eds.), The Role of Serotonin in Psychiatric Disorders. Brunner Mazel, New York, NY. pp. 129-160. Lawrence, K.M.. Falkowski, J., Jacobson, R.R. et al. (1993) Platelet _5-HT uptake sites in depression: three concurrent measures using “H-imipramine and ‘H-paroxetine. Psychopharmacology, 110, 235-239. Leonard. B.E. (1991) Blood cells as models of neurons: studies in the affective disorders. J. Coil. Physicians Surg. 20, 282-284. Lesch, K.P., Wolozin, B.L., Murphy, D.L and Riedener. P. (1993) Primary structure of the human platelet serotonin uptake site: identity with the brain serotonin transporter. J. Neurochem. 60, 2319-2322. Lewis, D.A., Noyes, R., Coryell, W. and Clancy, J. (1985) Tritiated imipramine binding is decreased in patients with agoraphobia. Psychiatr. Res. 16, 1-9. Maguire, K.. Tuckwell, V., Pereira, A., Dean, B. and Singh, B. (1993) Significant correlation between “C-5-HT uptake by and ‘H-paroxetine binding to platelets from healthy volunteers. Biol. Psychiatr. 34, 356-350. Marazzitti, D., Placidi, G.F., Cassano, G.B. and Akiskal. H.S. (1989) Lack of specificity of reduced platelet imipramine binding in different psychiatric conditions. Psychiatr. Res. 30, 21-29. Mellerup, E.T., Beth. P., Lauriston, L., Lunde, M. and Plenge, P. (1993) Platelet paroxetine binding in alcoholics. Alcohol Alcoholism 27, 603-606. Nemeroff, C.B. (1992) The presynaptic serotonin uptake site in depression. Clin. Neuropharmacol. 15. 347-348A. Norman, T.R., Judd, F.K., Burrows. G.D. and McIntyre, I. (1989a) Platelet serotonin uptake in panic disorder patients: a replication study. Psychiatr. Res. 30. 63-68. Norman. T.R., Sartor, D.M., Judd, F.K., Burrows, G.D.. Gregory, M. and McIntyre, I. (1989b) Platelet serotonin uptake and ‘H-imipramine binding in panic disorder. J. Affect. Disord. 17. 77-81. Norman, T.R., Judd, F.K., Gregory, M., James, R.H.. Kimber, N.M., McIntyre, 1. and Burrows, G.D. (1986) Platelet serotonin uptake in panic disorder. J. Affect. Disord. 11. 69-72. Nutt, D.J. and Fraser, S. (1987) Platelet binding studies in panic disorder. J. Affect. Disord. 12, 7-11. Pecknold, J.C., Suranyi-Cadotte, B.. Chang. H. and Nair, N.P.V. (1988) Serotonin uptake in panic disorder and agoraphobia. Neuropsychopharmacology 1. 173-176. Peroutka, S.J. and Snyder, S.H. (1980) Long term antidepressant treatment decreases spiroperodol-labelled serotonin receptor binding. Science 210, 8X-90. Plenge, P. and Mellerup, E.T. (1985) Antidepressive drugs can change the affinity of ‘H-imipramine and ‘H-paroxetine binding to platelet and neuronal membranes. Eur. J. Pharmacol. 119, 1-8.
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of Affectice Disorders 33 (1995) II 7-122
Raisman. R., Briley, M.S., Bouchami, F., Sechter, D., Zarifian, E. and Langer, S.Z. (1982): “H-imipramine binding and serotonin uptake in platelets from untreated patients and control volunteers. Psychopharmacology 77, 332-335. Schneider, L., Munjack, D., Severson, J. and Palmer, R. (1987) Platelet OH-imipramine binding in generalised anxiety disorder, panic disorder and agoraphobia with panic attacks. Biol. Psychiatr. 22, 59-66. Sheehan, D.V., Raj, B.A., Trehan, R.R. and Knapp, E.L. (1993) Serotonin in panic disorder and social phobia. Int. J. Clin. Psychopharmacol. 852, 63-77.
Targum, S.D. and Marshall, L.E. (1989) Fenfluramine provocation of anxiety in patients with panic disorder. Psychiatr. Res. 28, 295-306. Uhde, T., Berrettini, W., Roy-Byrne, P., Boulenger, J.-P. and Post, R.M. (1987) Platelet “H-imipramine binding in patients with panic disorder. Biol. Psychiatr. 22, 52-58. Wood, P.L., Suranyi-Cadotte, B.E., Nair, N.P.V., LaFaille, F. and Schwartz, G. 11983) Lack of association between “Iiimipramine binding sites and uptake of serotonin in control, depressed and schizophrenic patients. Neuropharmacology 22, 1211-1214.
DISORDERS
ELSEVIER
Journal of Affective Disorders 33 (1995) 117- 122
Platelet Kay P. Maguire
[
“Hlparoxetine
*, Trevor
binding in panic disorder
R. Norman, Maria Apostolopoulos, Graham D. Burrows
Fiona K. Judd,
Department of Psychiatry, University of Melbourne, Austin Hospital, Heidelberg 3084, Victoria, Australia
Received 16 May 1994; revised 20 September 1994; accepted 22 September 1994
Abstract Platelet [3H]paroxetine binding was measured in 20 patients (14 females, six males) with panic disorder normal controls (14 females, six males). For patients with panic disorder, II,,, was 1987 k 1135 fmol/mg (mean + SD) compared with control B,,, values of 1626 + 611 fmol/mg protein. The mean K, value patients was 0.076 5 0.056 nM and for the controls was 0.083 + 0.033 nM. ANCOVA indicated no significant of sex. diagnosis (panic disorder vs. normal controls) or season of sampling on either K, or B,,,. The data no evidence for an abnormality of the platelet uptake mechanism in panic disorder. Keywords:
Panic disorder;
Paroxetine
binding;
Platelet;
1. Introduction
The biological basis of panic disorder remains unknown. Hypotheses implicating dysfunction of the GABAergic and adrenergic systems have been proposed as possible important aetiological factors. More recently, evidence for dysfunction within the serotonergic system has also been suggested as significant in the genesis of panic. This is supported by the empirical findings from animal, drug treatment and platelet studies together with the results of specific neuroendocrine challenge tests (Coplan et al., 1992; Sheehan et al., 1993 ).
* Corresponding
author.
and 20 protein for the effect provide
Serotonin
Preclinical and animal studies have suggested a role for serotonin (5HT) in anxiety although both increased and decreased function have been reported as relevant (Coplan et al., 1992). Important evidence for the involvement of 5HT in panic disorder comes from clinical drug treatment studies. Although the mode of action of medications used as anti-panic agents is unknown, most affect 5HT function. In particular, agents, such as the tricyclic antidepressants, inhibit 5HT re-uptake and also induce a decrease in 5HT, receptor number on chronic administration (Peroutka and Snyder, 1980) in common with other drugs, such as the monoamine oxidase inhibitors. More importantly, the newer specific serotonin re-uptake inhibitors (SSRIs) have been found to be effective in panic disorder (Sheehan et al., 1993).
0165-0327/95/$09.50 0 1995 El sevier Science B.V. All rights reserved SSDI 0165-0327(94)00081-6
118
K.P. Maguire et al. /Journal
of Affectiue Disorders 33 (19951 I 17-122
More convincing evidence for abnormalities of the 5HT system in panic disorder comes from neuroendocrine challenge tests in patients. The specific 5HT agonists m-chlorophenyl piperazine, MK 212, dl-fenfluramine and d-fenfluramine and their effect on prolactin and/or cortisol release have been investigated in panic disorder. We have found an increased prolactin response to dl-fenfluramine in panic patients when compared with controls (Apostolopoulos et al., 1993). Using the more specific agonist d-fenfluramine, we found a greater prolactin increase in patients than controls (Judd et al., 1994) in agreement with other studies of 5HT agonists in panic disorder (Kahn et al., 1988; Targum and Marshall, 1989). Due to the inaccessibility of the brain, peripheral models, such as the platelet, have been widely used in psychiatric research. There are many physiological similarities between platelets and neurons, such as (Y* receptors, 5HT, receptors, an active uptake site for 5HT and calcium-dependent release of 5HT (Leonard, 1991). Further validity of the platelet model comes from the observation that the structure of the human platelet 5HT transporter is identical with that found in human brain (Lesch et al., 1993). Platelets have an ion-dependent active uptake system for 5HT. We have studied this transport system and found a significant increase in maximal uptake (I&,,> of 5HT by the platelet in panic patients in comparison with normal controls (Norman et al., 1986). In a replication study, a similar increase in Vm, was observed (Norman et al., 1989a). In contrast, Pecknold et al. (1988) found a significant decrease in V,,, for panic patients compared with controls as did Butler et al. (1992). Den Boer and Westenberg (1990) found no difference in platelet 5HT uptake between panic disorder and controls. Another approach to studying the 5HT transporter has been to measure [3H]imipramine binding to platelet membranes. Some studies have found lower maximal number of binding sites (B,,,) in panic patients compared with controls (Lewis et al., 1985; Marazzitti et al., 1989). However, the majority of [ 3H]imipramine-binding studies (Innis et al., 1987; Nutt and Fraser, 1987;
Uhde et al., 1987; Schneider et al., 19871, including our own (Norman et al., 1989b), have not found any significant difference in B,,, between panic patients and controls. Although imipramine and drugs which inhibit 5HT uptake have been suggested to bind to the platelet 5HT transporter, a functional relationship between parallel measurements of both 5HT uptake by platelets and L3Hlimipramine binding to platelet membrane has not been demonstrated (Raisman et al., 1982; Wood et al., 1983; Halbreich et al., 1991). This lack of correlation possibly occurs because [“Hlimipramine binds to two sites on the platelet membrane but only one site is related to the 5HT transporter (Hrdina, 1989). Recently, [“Hlparoxetine has been shown to bind only to the 5HT transporter (Plenge and Mellerup, 1985). We have measured platelet 5HT uptake and [“Hlparoxetine binding to platelet membranes from normal volunteers and found a significant correlation between the V,,, of platelet 5HT uptake and the B,,, of [3H]paroxetine binding (Maguire et al., 1993). It would appear that [3Hlparoxetine is a better ligand for the study of the platelet 5HT transporter. In the present study, we have investigated [ 3H]paroxetine binding to platelet membranes from 20 patients diagnosed with panic disorder and 20 healthy nonpsychiatric controls.
2. Methods 2.1. Subjects
Patients with panic disorder were included in the study if they met DSM-III-R criteria for panic disorder/agoraphobia and had more than one panic attack per week, were male or female, aged 18-65 years and had been drug-free for > 2 weeks. Patients were excluded from the study if physically unwell, if there was a life-time history of alcohol or drug abuse or if they suffered from any other psychiatric disorder. Longitudinal comorbidity as in panic disorder/depression was allowed but current co-morbidity was not. A healthy medication-free control group drawn from the general population was studied for com-
K.P. Muguire et al. /Journul of Affective Disorder.v33 (I 995) 117-122
parison. Volunteers were screened by clinical interview for current or past psychiatric illness and were excluded from the study if they met criteria for any psychiatric disorder. None had somatic illness as judged by physical examination and none had a history of drug abuse or were currently using medications. All subjects gave written informed consent for the study which was approved by the Austin Hospital Medical Research Committee.
I IY
sis using the Ligand computer program. Intraassay variation was + 4% for f?,,;,, and f 10% for K,. Interassay variation was + 10% for B,,;,, and + 15% for K,. Statistical analysis of the data was carried out using SuperANOVA for the Macintosh. ANCOVA was used to determine the influence of age, sex, season of sampling and diagnosis (panic disorder vs. controls) on K, and B,,,;,,. Correlations between parameters were examined using Pearson’s product-moment correlations.
2.2. Blood sampling Blood (30 ml> was taken into tubes containing EDTA (final concentration OX-l.5 mg/ml) between O&30 and lo:30 with the subject supine for at least 30 min following insertion of a butterfly needle. Blood was centrifuged at 200 x g for 20 min at room temperature and the platelet-rich plasma removed. Platelets were precipitated by centrifugation at 2200 x g for 20 min and washed with huffer (50 mM Tris, 3 mM EDTA, pH 7.0), reprecipitated and stored at -70°C until analysed. 2.3. I’aroxetine-binding assay Platelets were thawed, washed with buffer (70 mM Tris, pH 7.5) and platelet membranes prepared and [‘Hlparoxetine binding performed as described previously (Maguire et al., 1993) with minor modifications. Briefly, the binding of [“Hlparoxetine (0.1-2.0 nM, 25 Ci/mmol) to platelet membrane in the presence or absence of 10 PM citalopram was measured after a 2-h incubation at 20°C. After addition of ice-cold buffer, the samples were rapidly filtered through GF/B filters using a Brandel M-24R cell harvester, the filters washed 3 X and the radioactivity determined by liquid scintillation spectrometry. Protein concentration of the diluted platelet suspension was measured using the Bio-Rad kit. 2.4. I)ata analysis The maximal number of binding sites (B,,,, expressed in fmol/mg protein) and equilibrium dissociation constant (K,, expressed in nM) for each subject were calculated by Scatchard analy-
3. Results 20 panic patients (14 females, six males) of mean age 33 + 10 (-t SD> years entered the study. The mean B,,,, for the patient group was 1987 ) 1135 fmol/mg protein and the mean K,, was 0.076 + 0.056 nM. 20 controls (14 females, six males) of mean age 27 f 7 years were also studied. The mean B,,;,, for the control group was 1626 -t 611 fmol/mg protein and the mean K, was 0.083 + 0.033 nM. Individual values for both B,,, and K, are presented in Fig. I. ANCOVA indicated, after adjusting for age, no significant effect of sex (partial F = 2.176, df = 1,33, P = 0.1496), diagnosis (partial F = 0.761, df = 1,33, P = 0.3894) or month of sampling (partial F = 0.5 14, df = 3,33. P =z 0.6754) on K,, values. After adjusting for age. ANCOVA indicated no significant effect on B,,;,, of sex (partial F = 0.408, df = 1,33, P = 0.5274) season (partial F = 2.492, df = 3,33. P = 0.0772) or diagnosis (partial F = 1.554, df = 1,33. P ==0.2213). There was a significant difference in age between the two groups (Welch’s I = _ 3.243, P = 0.0027), however, there were no correlations between age and either B,,, or K,, in either the patient or control groups (B,;,,: controls r= - 0.0874, P = 0.7142, patients r = 0.1398, P = 0.5566; K,: controls r = - 0.0691, P =: 0.7721, patients r = 0.3338, P = 0.1504).
4. Discussion Serotonin dysfunction has been implicated in panic disorder. Conflicting results have been ob-
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We found no significant difference in either between the panic disorder patients and the control group, taking into account age, sex and season of sampling. This is in contrast to a recent report by Faludi et al. (1994) who found a significant decrease in B,,, for 29 panic patients when compared with 12 healthy controls. One reason for the conflicting findings may be the fact that the latter study included only panic disorder patients who had not been previously treated. Both studies excluded concurrent psychiatric disorders but the effects of longitudinal comorbidity cannot be excluded in either study. Decreased [ 3H]paroxetine binding has also been observed in depressed patients (Nemeroff, 1992) and in patients with posttraumatic stress disorder (Arora et al., 1993). In contrast, no difference in [3H]paroxetine binding between a normal control group and patients with major depressive disorder was reported in two studies (D’Haenen et al., 1988; Lawrence et al., 1993). Increased [3H]paroxetine binding has been reported in one study of alcoholic patients (Mellerup et al., 1993) but Daoust et al. (1991) found no difference between alcoholics and controls. Comorbidity of these disorders with panic is common and, thus, may confound reported results. Given the previously demonstrated correlation between platelet 5HT uptake and [3H]paroxetine binding in normal controls, the present findings do not support our original observation of increased 5HT uptake in panic disorder. On the other hand, platelet 5HT uptake and t3H]paroxetine binding may not be correlated in panic disorder and the increased 5HT uptake may be an indication of hyperactivity of the serotonergic system as suggested by Kahn et al. (1991). The finding of decreased platelet [ 3H]paroxetine binding in panic disorder by Faludi et al. (1994) does not concur with this hypothesis but does agree with suggestions of decreased 5HT function in anxiety by Coplan et al. (1992). Thus, the role of 5HT in panic disorder remains controversial. Further studies are required in a larger group of patients with panic disorder and matched controls of both platelet 5HT uptake and [3H]paroxetine binding to clarify the status of the 5HT transporter in this disorder. B max or K,
4000 .g if! Q
1
m
3000 -
E
0
.z 2 E m
2000
-
1000
-
: 0
’ PANIC
CONTROLS
Fig. 1. Individual values for B,,, group and normal controls.
and
Kd for panic
disorder
tained from studies of platelet 5HT uptake and from platelet [3H]imipramine binding. This may be due to methodological and patient population differences between the studies. [3H]paroxetine binding is a more specific ligand for the platelet 5HT transporter and has been shown to correlate with 5HT uptake (Maguire et al., 1993). Studies of platelet [ 3H]paroxetine binding may, thus, clarify the status of the 5HT transporter in panic disorder.
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of Affective Disorders 33 (1995) I 17-122
Acknowledgements
We would like to thank the National Health and Medical Research Council of Australia for financial support and H. Lundbeck A/S for the kind donation of citalopram.
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