Is decreased imipramine binding by platelet membranes in major depression associated with plasma autoantibodies to imipramine binding sites?

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Peabody CA, Minkoff JR, Davies HD, Winograd anterior pituitary function test in normal subjects. CH, Yesavage J, Tinklenberg JR (1986): ThyJ Clin Endocrinol Metab 60:623-630. rotropin-releasing hormone stimulation test ~md Sunderland T, Tariot PN, Mueller EA, Newhouse Alzheimer's disease. 8iolPsychiatry 21:553-556. PA, Murphy DL, Cohen RM (1985): TRH stimRe RN, Kourides IA, Ridgway BD, Weinttaub BD, ulation test in dementia of ~he ,Mzheimertype and Maloof F (1976): The effect of .~bacocotticoid elderly controls. Psychiatry Res 16:269-275. administration of human pituitary secretion of thyrotropin and prolaetin. J Clin Endocrinol Metab Thomas DR, Hailwood R, Harris B, Williams PA, Scanlon MF, John R (1987): Thyroid status in 43:338-346. senile dementia of the Alzhehner type (SDAT). Reisberg B, Ferris SH, de Leon MJ, Crook T (1982): Acta P~chiatr Scend 76:158-163. The Global Deterioration Scale (GDS): An instrument for the assessment of primary, degener- Wehrenberg WB, Baird A, Ying S-Y, Rivier C, Ling N, Guillemin R (1984): Multiple stimulation of ative dementia. Am 3 Psychiatry 139:1136--1139. the adenohypophysis by combinations of hypoSheldon WR, DeBold CR, Evans WS, et a| (1985): thalamie releasing factors. Endocrinology Rapid sequential intravenous administration of four 114:1995-2001. hypothalamic releasing hormones as a corr,bined

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Is Decreased m pra ne Binding by Platelet Membranes in Major Depression Associated with Plasma Autoantibedies to Irnipramine Binding Sites? iyad Alosachie, James B. Peter, Hiroshi Tsuchihashi, David L. Knight, and Chmles B. Nemeroff

Introduction Decreases in 3H-imipramine binding sites on wi~.hout a significant change in the ligand affinity (Kd) are found in some pap l a t e l e t s (P'max)

From S~ciaky Labor~tories, Inc. Santa Monica, CA (I.A., J.B.P.,H.T.); and the Departments of Psychiatry (O.LK., C.B.N.) and Phanr~acology (C.B.N.). Duke Unwersity Medical Center, Durham, NC. Supported by NIMH Grant No. MA-40159. Address reFrint reqt:ests to lyad Alosachie. M. ~., Ph.D., Specialty Laboratories. l,c.. 2211 Mmhigan Avenue. Santa Monica, CA 9O4O4-3900. Received September 14, 1'989; revised February 17, i990.

© i990 Society of Biological Psychiatry

tients with major depression (Suranyi-Cadotte et ai i984; Pau! et al. 1981;.Raisman et M. 1981; Nemeroff et al. 1988). The mechanism underlying the reduction in binding site density is not understood. It;,q autoantibodies binding to a va,riety of specific recep:.o~, ~-c k~',-,~ ~ be iml~3rtant in the pathogenesis of several d~seases, including Addison's disease, Gzaves' disease. myasthenia gra~.is~ a~,d insulin-resistant diabetes meiiitus (Blecher 1984). Accordingly, we studied the effects of IgG from untreated patients with major depression on ~H-imipramime binding on platelets from normal volunteer~;. " "

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BIOL PSYCHIATRY 1990;28:365-368

concentrations of 3H-imipramine (0.25 riM-10 nM) were used for saturation isotherms in duFor the purpose of this study, six samples of plicates. platelet-free plasma were chosen from the preIgG was added in 100 Izl of the assay buffer viously published study (Nemeroff et al. 1988). with or without desipramine (Sigma Chemical, These samples were those of major depression St. Louis, MO) (50 pd~lfinal concentration), for patients, free of psychotropic drugs for at least the determination of nonspecific binding. Plate7 days bel'ore cci~in-,dblood ~,anples were drawn. lets (100 gtg protein) were added in 100 ~tl of These vatients Lad shown the most significant the assay buffer. Final assay volume was 250 reduction in the number of platelet 3H-imipraIxl. The binding reaction was initiated either by mine binding sites in the above-mentioned study. the addition of platelets in the first set of exSix other platelet-free plasma samples from periments or by the addition of 3H-imipramine healthy individuals with normal platelet 3H-imin the second set of experiments. Tubes were ipramin,, binding site density served as controls. then incubated for 60 min at ice-bath temperatare. The reaction was terminated by dilution with 5 ml of ice-cold assay buffer and rapid IgG Isolation filtration unt~r vacuum through presoaked All plasma samples were first desalted using Whatman GF/B glass tiber filters (Whatman InAmicon Centriprep-10 (Amicon Company, ternational Ltd., Maidstone, England). The illDanvers, MA). IgG was isolated by DEAE af- ters were then washed three times with 5 ml of finity gel-blue column chromatography (Bio-Rad ice-cold assay buffer. Retained 3H-imipramine Laboratory, Richmond, CA).The purified IgG radioactivity was determined by liquid scinti!fractions were concentrated by Amicon, Curt- lation spectrometry. Specific binding of 3H-imtrip, p-10 Concentrators, and reconstituted in ipramine to platelet membranes was calculated phosphate-buffered saline, pH 8 at a final con- as the difference between total binding and noncentration of 1000 mg/dl as determined by rate specific binding. Scatchard analysis was used to nephelometry (Beckman Array Systems, Brad, obtain the Bm~, and the Kd for the tritiated imCA). ipramine. Linear regression of Scatchard plots was used to determine the correlation coefficient

Mateda!s and Methods

r.

Binding Assay Platelet membranes for the binding assay were prepared using the procedure described by Nemeroff et al. (1988). Membrane protein concentration was adjusted to 1 mg/ml. Binding of 3HimJpraTaine by membranes from normal plztelets w~s determined in the absence and in the presence of IgG from depressed patients and/or controls (IgG final concentration 1 mg/ml). In the first get of experiments, IgG was added directly to the assay tube. in the second set of experiments, platelets were preincubated "-':"IgG for I hr at 37°C with cor,tiauoas ~haking prior to the binding assay which was then carried out at ice-bath temperature. °H-imipramine (specific activity 46.7 Cb~mmol)(New England Nuclear, Boston, .~,.A .A . ~ wvs diluted in the assay buffer. In each binding assay, seven different • l ltli

Statistical Analysis Results are expressed as mean -4- ~tandard e~or of the mean (SEM). Statistical comparison of t_he Bmax and K,~ values of the binding of 3H-imipramine by platelets between the different groups was by Student's t-test, p values <0.05 were considered significant.

Results Scatchard plots of 3H-im~pramine binding to platelets from no~mal controls were linear (r > 0.90), and showed a single noninteracting site in each case (Table 1). The mean Bmax for the six control preparations of I ~ was 1111.5 __. 60.5 fmol/mg protein, and the mean ga was

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Table 1. Comparison of the Effect of lgG Between Control and Depressed Patients on the 3H-lmipramine Binding IgG (normel) Control

lgG (normal;

lgG (depressed)

Bnma

Assay (no.)

Bmaxa

K a~

r

Bmaxa

Ka'b

r

Bnmxa

Kdb

r

I. 2. 3. 4. 5. 6. Mean: SEM:

1010.9 1163.9 1277. I 1274.4 1014.8 927.9 I ! 1! .5 60.5

2.07 2.46 1.03 1.04 2.04 2. ~2 1.79 0.25

0.94 0.94 0.94 0.92 0.92 0.98

1015.6 1080. I 1160.8 1051.3 1079.8

2.69 2.85 3.43 2.86 2.87

0.99 0.99 0.99 0.97 0.99

I 110.5 1015.4 1006.8 I 115.3 1135.8

3.48 2.80 2.63 1.90 1.89

0.99 0.97 0.99 0.98 0 95

1077.5 23.9

2.94 O. 13

1076.5 27.2

2.52 0.30

/Ca./'

lgG (depressed) r

-Rr~xa

Ka3'

r

2.28 2.42 1.72 2.01 ! .93 2.03 2.13 O. 13

0.98 0.93 0.97 0.93 0.97 0.91

{I hr preincubation at 37°C) 869.2 1070.5 994.3 1247.3 1134.6 1048.3 1060.6 52.2

2.04 1.87 2.01 2.69 2.43 2.49 2.26 O. 13

O.b9 0.99 0.98 0.96 0.97 0.91

1104. ! 880.5 951.6 ! 127.2 954.6 I Iq1.8 1029. ! 42.8

°Bronx values expressed in f m o l / m g protein. b£~ values expressed in nM.

1.79 -_4-_ 0.25 nM. When purified IgG was added directly to the assay mixture without prior incubation with platelet membranes, the mean Bma,, for imipramine in the presence of control IgG (1077.5 +_ 23.9 fmoL'mg protein) was no*, s-;gnificantly different from the average (1076.5 _+ 27.2 fmol/mg protein) found in the presence of IgG from depressed patients. The Kd values in the presence of ~ontrol IgG (2.94 _+ 0.13 nM) and IgG from depressed patients (2.52 _+ 0.30 nM) were not s~gnificantly different (Table l). In order to facilitate the postulated association of IgG reactive with the imipramine b~nding sites and to slow its d~ssociation, the effect of the IgG prepara~,lons were also studied after prei~cabat'cn wi;.h the normal platei<,~t membranes at 3~°C for 1 hr followed by 0--4°C for 1 hr. Under these circumstance~, which were designed to maximize ~ s i b i ! i ties for detection of the putative autoantibodies, the mean Bmax in the presence of control IgG (1060.6 _ 52.2 fmol/mg of protein) was not significantly different from that of the depressed patients (1029.1 _+ 42.8 fmol/mg of protein). Also, Kd values of these experiments in the presence ~f control IgG (2.26 _+ 0.13 nM) were not sit2nificantly different from those obtained in the, presence of IgG from der~:essed patients (2.13 =~: 0~13 nM) (Table J )

Discussion A variety of abnormalities of inur~une function have been described i,, patients with various forms of depression (Mendlewicz and Sevy 1986). Taking into consideration that our patients were among those with the greatest decreases in imipramine binding by platelets (Nemeroff et al. 1988), the results suggest that autoantibodies are not involved in the decrease of imipramine binding by platelets. We cannot, of course, rule out the possibility that all of the putative antibody in the blood of those patients was absorbed in vivo to the imipramine binding sites of theft platelets. There is, however, no precedent for such comrlcte absorption. As our patient's sample size was lim:ted, we also cannot exclude the possibility that the study of a larger number of patients might yield evidence of a subtype in which a significant presence of autoantibodies could be detected. We, l~,owever, found no evidence for such a group. Recently we have ol;~erved increases in the plasma concentrations of u r a c i d glycoprote':n, a purgative endogenous inhibitor of the 3H-imi~ pramine binding site, in drug-free depressed patientq when compared to age- and gender-matched controls (Nemeroff et al. 1990). This may underlie the reduction in ~H-imipramine binding in depression~

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We are grateful to Nancy Campman and Marion Logan for excellent secretarialassistance.

References Blecher M (1984): Receptors, antibodies, and disease. Clin Chem 30:1137-1156. Mendlewicz J, Sevy S (1986): Genetic and immunological factors in affective disorders and schizophrenia. Prog Brain Res 65:1-14. NemeroffCB, Knight DL, Krishnan KRR, et al (I988): Marked reduction in the number of platelet-tritiated imipfamine binding sites in geriatric depression. Arch Gen Psychiatry 45:919-923. Nememff CB, Krh~hnan KRR, Blazer DG, Knight DL, Benjamin D, Meyerson LR (1990): Elevated

Bri,'.f Reports

plasma concentrations of at-acid glycoprotein: A putative endogenous inhibitor of the 3H-imipramine site in depressed patients. Arch Gen Psychiatry (in press). Paul SM, Rehavi M, Skolnick P, Ballenger JC, Goodwin FK (1981): Depressed patients have decreased binding of tritiated i,-nipramine to platelet semtonin "transporter." Arch Gen Psychiatry 38:1315-1317. Raisman R, Sechter D, Briley MS, Zarifian E, Langer SZ (1981): High affinity 3H imipramine binding in platelets from untreated and depressed patients compared to healthy volunteers. Psychopharmacology 75:368-37 !. Suranyi-Cadotte B, Quirion R, McQuade P, et al (1984): Platelet 3H imipramine binding: ,~ statedependent-marker in depression. Prog Neurops:~o.hopharmacol Biol Psychiatry 8:737-741.