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BIOL PSYCHIATRY 199o;28:362-365

365

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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366

Brief Reports

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 0.90), 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

Brief Reports

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367

1990;28:365-368

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