- Email: [email protected]
Altered platelet protein kinase C activity in bipolar affective disorder, manic episode
.~20
BIOL PSYCHIATRY 1993~33:520-525
Altered Platelet Protein Kinase C Activity in Bipolar Affective Disorder, Manic Episode Eitan Friedman, Hoau-Yan-Wang, Douglas Levinson, Thomas A. Connell, and Hardeep Singh
Protein kinase C (PKC) activity and PKC translocation in response to serotonin were investigated in platelets obtained from bipolar affective disorder subjects before and during lithium treatment. Ratios of platelel membrane-bound to cytosolic PKC activities were elevated in the manic subjects. In addition, serotonin-elicited platelet PKC translocation was found to be enhanced in those subjects. Lithium treatment for up to 2 weeks resulted in a reduction in cytosolic and membrane-associated PKC activities and in an attenuated PKC translocation in response to serotonin. These preliminary results suggest that alteration in platelet PKC is associated with the manic phase of bipolar illness. The results also suggest that lithium treatment reduces the sensitivity of platelets to PKC translocation induced by activation of serotonin-2 receptors.
Key Words:
Protein kinase C, platelet, bipolar affective disorder, mania, lithium, serotonin
Introduction Bipolar disorder is unique among the affective illnesses in its presentation of a past or current manic episode, which is characterized by euphoria and/or an irritable mood, grandiose :a . . . . .v. ., .~. m ~ u., speech, increased motor, social, ,,,ea~, and/or sexual activity, and loss of sleep. The disorder has a lifetime prevalence of approximately 1% in the industrialized nations and a modal age of onset of 30 years (Chamey and Weissman 1988). Twin, adoption, and family studies have demonstrated that genetic factors play a significant role in the biology of bipolar disorders (Vande-berg et al 1986). The mode of inheritance remains unclear, and gene or genes associated with bipolar disorders have not been identified (Kelso et al 1989).
From the Medical College of Pennsylvania, Philadelphia, PA. Address reprint requests to E. Friedman, Phi), Division of Neurochemistry, Depar'ments of Psychiatry and Pharmacology, Medical College of Pennsylvania~-PP1, 3200 Henry Avenue, Philadelphia, PA 19129. Receive' May 4, 1991; revised December 19, 1992. © 1993 Society of Biological Psychiatry
The pathophysiology of bipolar disorders is not understood, bi,t its symptoms can be best treated with lithium ion. The monoaminergic neuronal systems that employ the neurotransmitters norepinephfine, acetylcholine, and serotonin have been most frequently implicated in the disease process as well as in the therapeutic action of lithium ion (Gershon and Yuwiler 1960). Recent investigations in our laboratory have demonstrated that lithium inhibits PKCactivated serotonin release (Wang and Friedman 1989). This effect of the ion may be attributed to its inhibition of stimulation-induced PKC translocation from the cytosolic fraction to the membranous fraction of brain tissue (Wang and Friedman 1989). These studies suggest that PKC may be the site of action of lithium in treatment of bipolar disorders. In earlier work we have demonstrated that the activation of 5-HT2 receptors in both platelets or brain cortical slices results in the translocation of PKC to the respective membranous fractions of the tissue homogenates (Wang and Friedman 1990 a,b). In the present communication, the blood platelet serves as a model system 0006-3223/93/$06.00
I~IOLeSYCHt~TRY
Piatelet PKC in Mania
for the investigation of PKC in humans. We report on platelet PKC and its subcellular distribution in patients with bipolar affective disorder, manic episode, and on the effect of lithium treatment on PKC activity in platelets from bipolar manic patients undergoing treatment with the drug. Methods
Experimental Subjects The study was conducted on two groups of consenting inpatients at The Eastern Pennsylvania Psychiatric Institute. The first group of subjects consisted of 12 patients (eight women and four men) with a mean age of 35 -2.6 years. The subjects were interviewed by a research psychiatrist based on the Schedule for Affective Disorders and Schizophrenia (SADS, Endicott and Spitzer 1978).On the basis of these interviews, manic disorder was diagnosed by Research Diagnostic Criteria (RDC, Spitzer et al 1978), and bipolar disorder (manic episode) by DSMIII-R. Prior to initiation of treatment with lithium carbonate, blood samples were collected from all patients for measurement of platelet PKC (Wang and Friedman 1990b). The patients were treated with lithium carbonate in dosages that varied between 900 and 2100 rag/day in order to maintain effective blood lithium levels of 0.6--1.4 mEq/L. Blood samples used for the protein kinase C experiments were collected on days 7 and 14 of lithium treatment. The second group of subjects was comprised of nine patients (5 women and 4 men) with a mean age of 32 ___ 2.3 years and DSM-III-R and RDC diagnosis of acute exacerbation of chronic schizophrenia. Prior to initiation of treatment with neuroleptics, blood samples were collected from all patients for measurement of platelet PKC. Normal control subjects were laboratory personnel, office personnel, and students who reported having no history of psychiatric illness; psychiatric diagnostic interviews were not condrifted, however. The control subjects (10 women, 5 men) had a mean age of 35 +-- 2.5 years.
1993;33:520-525
521
Disrupter for 1 min and centrifuged at 25,000 g for 15 min, as previously described (Friedman and Wang 1989). The supernatant was applied onto DE52 anion exchange columns, and enzyme activity was measured in the eluant (cytosolic PKC). The pellet was solubilized on ice for 1 hr in 200 Vd of the above buffer coniaw,ing 1% Nonident P-40 diluted with 1.8 ml of sucrose/Tris HCi buffer, and centrifuged at 25,000 g for 15 rain. The supematant was applied onto DE2 columns, enzyme was eluted and PKC activity determined (membrane-bound). The columns were washed with 4 ml of buffer followed by 0.5 m! of bt~tfer containing 0. ! M NaCI and the enzyme was eluted with an additional 1.5 ml of buffer/0.1 mmol/L NaCI and used immediately for PKC activity determination. The final assay mixture (volume 250 Ixl) consisted of 5 mmol/NaCl, 0.025 mmol/l, ethyleneglycoi-bis-(13-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 0.2 mmol/L ethylenediamine tetraacetic acid (EDTA), 24 mmol/L Tris HCI, 0.015% 2-mercaptoethanol, 30 p.g/ml leupeptin, 0.01 mmol/L phenylmethylsulfonyl fluoride, 0.25 mg/ml histone type III (lysine-rich, Sigma), 1.2 mmol/CaCl2, 20 Ixg PS, 8.1 mmoFL pborbol 12-myristate, 13-acetate (PMA), 10 mmol/L MgAc2, and 30 ixmol/L [~/_32p] adenosine triphosphate (ATP). Assay mixtures were preincubated at 30°C for 5 min, and phosphorylation was initiated by addition of 25 p.l of column eluates and incubated for 1 min at 30°(:. Reactions were terminated by transferring 25 I~1 aliquots of the incubate to a 1 x 2 cm phosphocellulose strip (Whatman p81) and immersed in 75 mmol/L phosphoric acid (10 ml per sample). The strips were swirled gently for 2 min, the phosphoric acid decanted, and the phosphocellulose strips washed two additional times in phosphoric acid. After drying in air, the radioactivity was determined by liquid scinillation counting (LKB 1214 RACKBETA). Enzyme blanks were run in each experiment. The protein phosphorylation reaction was found to be linear for up to 3 min under the conditions used. Protein was determined by the method of Lowry et al (1951).
Statistical Analysis Assay of Protein Kinase C Activity and Translocation Platelets were isolated and resuspended in 0.32 mmol/L sucrose/5 mmol/L (N-[Z-hydroxyethyl]piper~ine-N'-[2'ethanesulfonic acid (HEPES) (pH 7.5). Aliquots were incubated for 10 min at 37°C with 0-100 p.mol/L serotonin in a total volume of 1 ml. Following incubation, the reaction was stopped by adding 1 ml of 0.32 nnnol/L sucrose/20 mmol/L "Iris HCI (pH 7.5) containing 2 mmol/L EGTA, 50 I~g/ml leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride and 0.1% 2-mercaptoethanol. The diluted reaction mixture was sonicated in a Kontes Micro Cell
Dose-lresponse relationships and groups differences were assessed by two-factor analysis of variance. The effect of lithium treatment was analyzed by multivariate analysis of variance (MANOVA) with repeat measures for the duration of treatment factor (SPSS procedure, MANOVA SPSS I,e., Chicago, III.).
Results The membrane-to-cytosolic distribution of platelet PKC activity in control, manic, and schizophrenic subjects is summarized in Figure 1. Platelet membranous to cytosolic
522
BIOL PSYCHIATRY 1993;33:5~"t~525
~"
4.0
~
3.6
41 111 I~
S.O
o
8.6
W 0 (..+
z
E. Friedman et al
CONTROL BIPOLAR, MANIA SCHIZOPHRENIA
II i
1.5
o m i
m
1
1.0
0.§
:m 0.0
0
1.0
0.5
10
100
Figure 1. Distribution of PKC in platelets of bipolar (mania), schizophrenic, and control subjects. Blood platelets obtained from bipolar patients, manic episode, schizophrenic patients, and control subjects were subdivided into 5 separate tubes that were respectively incubated for 10 rain with the indicated concentrations of 5-HT added in vitro. Platelet cytosolic and membrane-bound PKC activities were measured and ratios of enzyme activities in membranes/cytosol were calculated. Significant 5-HT dose-response relationships (p < 0.001) and differences among the diagnostic groups (p < 0.001) were observed. At 0.5 I~moi~L 5-HT, for instance, the bipolar group showed a significandy greater response than either the control or schizophrenic groups (p < 0.05, Newman-Keuls).
[5-ST] (.M)
enzyme ratios were elevated in the manic subjects when
activities were found in the schizophrenic subjects when compared to those o f controls. Incubation o f platelets with serotonin elicits a dose-related translocation o f platelet PKC activity (Figures 1, 3). The response o f platelets to serotonin was markedly enhanced in the bipolar manic subjects when compared either to control or to the schizophrenic
compared to either age- and scx-mm,_:n~u . . . . . . co~-~trols or to schizophrenic subjects. The data also indicated that this increase in ratio was entirely caused by an elevation in membrane-bound enzyme (Figure 2A). In contrast, no differences in cytosolic or membrane-associated platelet PKC
[--7
"~ 4,0
A
Q
CYTOSOL MgMBRANE-BOUND
[
B
o h w
3.0
3.
--T..
n --o
2.0 -
~
1.0 -
it
r.1 kt 0.0
Nil
I
1I
CONTROL
MANIA
MANIA
MANIA/IWK M&NIk/BWK [
LITHIUM - - I
Figure 2. Effect of lithium treatment on platelet cytosol and membrane PKC activities. Platelets obtained from control subjects (n -- 15) or from bipolar subjects (n -- 12) before and during lithium treatment were prepared and PKC activities were measured in cytosolic and membranous fractions following partial purification on DE 52 columns. Each bar represents the mean _ SEM. Platelet PKC activities in control and manic subjects prior to lithium treatment (A) were compared. Significantly higher PKC values were observed in the membranous fraction of the manic group (p < 0.001, Student's t-test). The effect of lithium treatment in the manic group is depicted in B. For the four subjects who completed the 2 weeks of treatment, a significant reduction in cytosolic and membrane-associated enzyme activities was noted, as determined by MANOVA with repeat measures on the duration of treatment factor (p < 0.001).
Platelet P K C
in Mania
BIOL FSYCI.]IATRY
523
1993;33:520-525
groups. Enzyme translocation in response to serotonin in the schizophrenic group did not differ from that in the control subjects. The effect of lithium treatment on platelet PKC activity is shown in Figure 2B. Enzyme activity was found to be reduced in both cytosolic and membrane-bound fractions during the 2 weeks of lithium treatment. In addition, the responsiveness of platelets to serotonin was reduced in bipolar patients undergoing lithium treatment (Figure 3). The reductions in both enzyme activity and serotonin-induced translocation were related to the duration of lithium treatment.
Discussion The present results demonstrate that hipolar manic patients have high platelet PKC activity, and that a greater proportion of the cellular enzymatic activity is found in association with the membranous tissue fraction. The results also indicate that the responsiveness of platelets obtained from manic subjects to serotonin is enhanced. In previous
• O.
~" C
studies we have shown that stimulation of cell-surface serotonin receptors in brain or platelets, as well as direct PKC stimulation with phorbol esters, induces translocation of PKC from the cytosolic to the particulate fraction (Wang and Friedman 1989; Wang and Friedman 1990b). In both platelets and brain, serotonin-stimulated PKC translocation was found to be mediated through 5-HT2 receptors (Wang and Friedman 1990a,b). Previous workers have noted increases in 5-HT2 receptor densities both in platelets of suicidal patients and in brains from suicides (Stanley and Mann 1983; Biegon et al 1990). The present finding of enhanced responsiveness to serotonin in bipolar subjects may be a reflection of supersensitive platelet 5-HT2 receptors caused by an increase in 5-HT2 receptor density or by other changes in signal transduction. However, the presence in bipolar subjects of both an increase in PKC activity as well as a distribution of PKC, which favors the particulate cell fraction, suggests that additional factors may contribute to the results presented. Alteration in basal platelet PKC in patients with this disease may be caused
Baseline 1 - w k Li t r e a t m e n t 2-wk Li t r e a t m e n t 5.0
3.o
I
4.5
2.5
4.0
E 3.5
E
2.O 3.0
O.
>
2.5
1.5
,,t,,,U 0
u
g
...........
. . . . . . . . . . . t ...........
...........
2.0
1.0
1.5
0 f-
"
=
1.0 0.5
MEMBRANE
CYTOSOL
0.5
0 0.0
0.0
I
I
I
I
0.5
1.0
10.0
100.0
[S-HT] ( ~ . )
0.0
I
I
I
I
0.5
1.0
10.0
100.0
0.0
[S-HT] ( , . )
Figure 3. Effect of lithium treatment on platelet cytosol and membrane-associated PKC activities. Blood platelets were prepared from bipolar patients prior to (baseline, n = 12) and following 1 (n = 7) or 2 weeks (n = 4) of lithium administration. Platelets were incubated with the indicated doses of 5-HT for i0 rain, and PKC activities in cytosolic and membranous fractions were determined. Each data point represents the mean --- SEM. The data obtained from the four subjects who completed the 2-week lithium treatment were subjected to MANOVA with the duration-of-treatment as the repeat measure factor. Significant reductions in 5-HT-induced cha~ges in cytosolic (p < 0.001) and in membrane-PKC activities (p < 0.001) were observed.
524
E. Friedman et al
BIOL PSYCHIATRY 1993;33:520--525
by differences in enzyme protein as well as in the susceptibility of PKC to inactivation by proteolysis. In addition, other factors that °re involved in the translocation of PKC such as Ca 2 +, membrane lipids or other structural membrane changes may also play a role in mediating the change in platelet PKC distribution (Wolf et al 1985). Lithium treatment in bipolar manic subjects appears to lower both cytosolic and membrane-associated platelet PKC activities. Drug treatment also reduced the sensitivity of platelets to serotonin stimulation in a time-related manner, similar to its known course associated with its clinical response. The decreased serotonin receptor sensitivity after lithium may be caused by changes in 5-HT2 receptor binding, as previous investigators have described reduced ligand binding at this receptor site after chronic lithium treatment in rats (Treiser et al 1981; Closse and Jaton 1984; Goodwin et al 1986; Tanimoto et al 1983). However, supersensitivity of the serotonin platelet receptor was described in patients during lithium treatment (Wood et al 1985). Furthermore, in previous studies in rat brain we have noted that lithium ion inhibits PKC translocation in response to phorbol esters, which is not dependent on 5HT receptor stimulation (Wang and Friedman 1989) but is rather a direct effect on the enzyme. These considerations, therefore, suggest that the effects of lithium described here are distal to the receptor. There are several limitations to these preliminary data in addition to the small sample sizes. Only the manic state has been studied; it would be useful to be able to compare
manic, depressed, and euthymic states longitudinally (which will be difficult to study in the medication-free state) or at least cross-sectionally. Also, the control group was not interviewed for psychiatric diagnosis; however, this would be expected to decrease rather than increase differences between manic a~d control subjects, as untreated or unacknowledged affective disorders are relatively common in the population. Despite these potential problems, significant between-group differences were seen here. The data presented suggest that an abnormality in cellular PKC distribution and/or in the mecb~-.",ism for its activation may be associated with the manic episode in bipolar affective disorder. The results also show that lithium treatment can reduce both cytosolic and membrane-associated PKC as well as decrease platelet sensitivity to 5-HT-induced enzyme translocation. Taken together, these prelimir, a_"y results implicate PKC-mediated phosphorylation both in the biology o f bipolar illness and in the pharmacology of lithium ion. Further studies are needed to elucidate (1) whether this finding is specific to mania or whether it is associated with other behavioral states in bipolar illness and (2) the biological cause that is responsible for this alteration in platelet PKC distribution. This work was supportedin part by USPHSgrant MH 45166. The authors would like to acknowledge Dr. Ashiwel Undie for his assistance and discussion of the statistical analyses and Ms. Janie Rickards' capable help in the preparation of this manuscript.
References Biegon A, Grispoon A, Blumenfield B, Bleich A, Apter A, Mester R ( 1990): Increased serotonin 5-HT~_receptor binding on blood platelets of suicidal men. Psychopharmacology 100:165-167. Charney EA, Weissman MM (1988): Epidemiology of depressive anu mnamHL,byuuromc. Ill ,aeoIgota~ A, Cancro R (eds), Depression and Mania. New York: Elsevier, pp 26-52. Closse A, Jaton AL (1984): Investigation of the influence of lithium upon the down-regulation of serotonin2 receptor in rat frontal cortex induced by long-term treatment with debenzepin, an antidepressant without appreciable affinity to serotonin2 receptors. Naunyn Schmiedebergs Arch Pharmacot 326:291-293. Endicott .1, Spitzer RL ( 1978): A diagnostic interview: The schedule for affective disorders and schizophrenia, Arch Gen Psychiatry 35:837-844. . _
J
.
.
.
.
.
.
.
.
Friedman E, Wang HY ( 1989): The effect of age on brain cortical protein 'i~llla~, ~ -- C ,~,u--~i~s mcdiaiion of scrotznin release. J Ne,trochem 52( I ): 187-192. Gershon S, Yuwiler A (1960): Lithium ion: A specific pharmacological approach to the treatment of mania. J Neuropsychiat !:229-241. Goodwin GM, De Souza R.I, Wood AJ, Green AR (1986): Lith-
ium decreases 5-HT~a and 5-HT2 receptors and alpha2-adrenoreceptor mediated function in mice. Psychopharmacology 90:482--487. Kelso JR, Ginns El, Egeland JA et al (1989): Re-evaluation of the linkage relationship between chromosome 1 lp loci and the gone for bipolar affective disorder in the Old Order Amish. Nature 342:238-243. Lowry OH, Rosebrough N J, Fair AL, Randall ILl (1951 ): Protein measurement with the Folin phenol reagent. J Biol Chem 249:265-275. Spitzer RL, Endicott J, Robins E (1978): Research diagnostic criteria: Rationale and reliability. Arch Gen P~-ychiatry 35:773782. Stanley MM, Mann JJ ( 1983): Increased serotonin-2 binding sites in frontal cortex or suicide victims. Lancet 1:214-216. Tanimoto K, Maeda K, Terada T (1983): Inhibitory effect of lithium on neuroleptic and serotonin receptors in rat brain. Brain Res 265:148-151. Treiser SL, Cascia CS, O'Donohue TL, Thoa TL, Jacobowitz DM, Kellar KJ (1981): Lithium increases serotonin release and decreases serotonin receptors in the hippocampus. Science 213:1529-1531.
Platelet PKC in Mania
Vandenberg SG, Singer SM, Pauls DL (1986): The Heredity of Behavior Disorders in Adults and Children. New York: Plenum. Wang HY, Friedman E (1989): Lithium inhibition of protein kinase C activation-induced serotonin release. Psychopharmacology 99:213-218. Wang HY, Friedman E (1990a): Central 5-hydroxylryptamine receptor-linked protein kinase C translocation: A functional postsynaptic signal transduction. Mol Pharmacol 37:75-79.
eIOLPSYCHIATRY 1993:33:520-525
525
Wang HY, Friedman E (1990b): Protein kinase C translocation in human blood platelet. Life Sci 47:1419-1425. Wolf M, Cuatrecasas P, Sahyoun N (1985): Interaction of protein kinase C with membranes is regulated by C a * ' , phorbol esters and ATP. J Biol Chem 2fiO:15718-15722. Wood K, Swade C, Abou-Saleh MT, Coppen A ( 1985): Apparent supersensitivity of platelet 5-I-[1"receptors in lithium-treated patients. J Affective Disord 8:69-72.
BIOL PSYCHIATRY 1993~33:520-525
Altered Platelet Protein Kinase C Activity in Bipolar Affective Disorder, Manic Episode Eitan Friedman, Hoau-Yan-Wang, Douglas Levinson, Thomas A. Connell, and Hardeep Singh
Protein kinase C (PKC) activity and PKC translocation in response to serotonin were investigated in platelets obtained from bipolar affective disorder subjects before and during lithium treatment. Ratios of platelel membrane-bound to cytosolic PKC activities were elevated in the manic subjects. In addition, serotonin-elicited platelet PKC translocation was found to be enhanced in those subjects. Lithium treatment for up to 2 weeks resulted in a reduction in cytosolic and membrane-associated PKC activities and in an attenuated PKC translocation in response to serotonin. These preliminary results suggest that alteration in platelet PKC is associated with the manic phase of bipolar illness. The results also suggest that lithium treatment reduces the sensitivity of platelets to PKC translocation induced by activation of serotonin-2 receptors.
Key Words:
Protein kinase C, platelet, bipolar affective disorder, mania, lithium, serotonin
Introduction Bipolar disorder is unique among the affective illnesses in its presentation of a past or current manic episode, which is characterized by euphoria and/or an irritable mood, grandiose :a . . . . .v. ., .~. m ~ u., speech, increased motor, social, ,,,ea~, and/or sexual activity, and loss of sleep. The disorder has a lifetime prevalence of approximately 1% in the industrialized nations and a modal age of onset of 30 years (Chamey and Weissman 1988). Twin, adoption, and family studies have demonstrated that genetic factors play a significant role in the biology of bipolar disorders (Vande-berg et al 1986). The mode of inheritance remains unclear, and gene or genes associated with bipolar disorders have not been identified (Kelso et al 1989).
From the Medical College of Pennsylvania, Philadelphia, PA. Address reprint requests to E. Friedman, Phi), Division of Neurochemistry, Depar'ments of Psychiatry and Pharmacology, Medical College of Pennsylvania~-PP1, 3200 Henry Avenue, Philadelphia, PA 19129. Receive' May 4, 1991; revised December 19, 1992. © 1993 Society of Biological Psychiatry
The pathophysiology of bipolar disorders is not understood, bi,t its symptoms can be best treated with lithium ion. The monoaminergic neuronal systems that employ the neurotransmitters norepinephfine, acetylcholine, and serotonin have been most frequently implicated in the disease process as well as in the therapeutic action of lithium ion (Gershon and Yuwiler 1960). Recent investigations in our laboratory have demonstrated that lithium inhibits PKCactivated serotonin release (Wang and Friedman 1989). This effect of the ion may be attributed to its inhibition of stimulation-induced PKC translocation from the cytosolic fraction to the membranous fraction of brain tissue (Wang and Friedman 1989). These studies suggest that PKC may be the site of action of lithium in treatment of bipolar disorders. In earlier work we have demonstrated that the activation of 5-HT2 receptors in both platelets or brain cortical slices results in the translocation of PKC to the respective membranous fractions of the tissue homogenates (Wang and Friedman 1990 a,b). In the present communication, the blood platelet serves as a model system 0006-3223/93/$06.00
I~IOLeSYCHt~TRY
Piatelet PKC in Mania
for the investigation of PKC in humans. We report on platelet PKC and its subcellular distribution in patients with bipolar affective disorder, manic episode, and on the effect of lithium treatment on PKC activity in platelets from bipolar manic patients undergoing treatment with the drug. Methods
Experimental Subjects The study was conducted on two groups of consenting inpatients at The Eastern Pennsylvania Psychiatric Institute. The first group of subjects consisted of 12 patients (eight women and four men) with a mean age of 35 -2.6 years. The subjects were interviewed by a research psychiatrist based on the Schedule for Affective Disorders and Schizophrenia (SADS, Endicott and Spitzer 1978).On the basis of these interviews, manic disorder was diagnosed by Research Diagnostic Criteria (RDC, Spitzer et al 1978), and bipolar disorder (manic episode) by DSMIII-R. Prior to initiation of treatment with lithium carbonate, blood samples were collected from all patients for measurement of platelet PKC (Wang and Friedman 1990b). The patients were treated with lithium carbonate in dosages that varied between 900 and 2100 rag/day in order to maintain effective blood lithium levels of 0.6--1.4 mEq/L. Blood samples used for the protein kinase C experiments were collected on days 7 and 14 of lithium treatment. The second group of subjects was comprised of nine patients (5 women and 4 men) with a mean age of 32 ___ 2.3 years and DSM-III-R and RDC diagnosis of acute exacerbation of chronic schizophrenia. Prior to initiation of treatment with neuroleptics, blood samples were collected from all patients for measurement of platelet PKC. Normal control subjects were laboratory personnel, office personnel, and students who reported having no history of psychiatric illness; psychiatric diagnostic interviews were not condrifted, however. The control subjects (10 women, 5 men) had a mean age of 35 +-- 2.5 years.
1993;33:520-525
521
Disrupter for 1 min and centrifuged at 25,000 g for 15 min, as previously described (Friedman and Wang 1989). The supernatant was applied onto DE52 anion exchange columns, and enzyme activity was measured in the eluant (cytosolic PKC). The pellet was solubilized on ice for 1 hr in 200 Vd of the above buffer coniaw,ing 1% Nonident P-40 diluted with 1.8 ml of sucrose/Tris HCi buffer, and centrifuged at 25,000 g for 15 rain. The supematant was applied onto DE2 columns, enzyme was eluted and PKC activity determined (membrane-bound). The columns were washed with 4 ml of buffer followed by 0.5 m! of bt~tfer containing 0. ! M NaCI and the enzyme was eluted with an additional 1.5 ml of buffer/0.1 mmol/L NaCI and used immediately for PKC activity determination. The final assay mixture (volume 250 Ixl) consisted of 5 mmol/NaCl, 0.025 mmol/l, ethyleneglycoi-bis-(13-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 0.2 mmol/L ethylenediamine tetraacetic acid (EDTA), 24 mmol/L Tris HCI, 0.015% 2-mercaptoethanol, 30 p.g/ml leupeptin, 0.01 mmol/L phenylmethylsulfonyl fluoride, 0.25 mg/ml histone type III (lysine-rich, Sigma), 1.2 mmol/CaCl2, 20 Ixg PS, 8.1 mmoFL pborbol 12-myristate, 13-acetate (PMA), 10 mmol/L MgAc2, and 30 ixmol/L [~/_32p] adenosine triphosphate (ATP). Assay mixtures were preincubated at 30°C for 5 min, and phosphorylation was initiated by addition of 25 p.l of column eluates and incubated for 1 min at 30°(:. Reactions were terminated by transferring 25 I~1 aliquots of the incubate to a 1 x 2 cm phosphocellulose strip (Whatman p81) and immersed in 75 mmol/L phosphoric acid (10 ml per sample). The strips were swirled gently for 2 min, the phosphoric acid decanted, and the phosphocellulose strips washed two additional times in phosphoric acid. After drying in air, the radioactivity was determined by liquid scinillation counting (LKB 1214 RACKBETA). Enzyme blanks were run in each experiment. The protein phosphorylation reaction was found to be linear for up to 3 min under the conditions used. Protein was determined by the method of Lowry et al (1951).
Statistical Analysis Assay of Protein Kinase C Activity and Translocation Platelets were isolated and resuspended in 0.32 mmol/L sucrose/5 mmol/L (N-[Z-hydroxyethyl]piper~ine-N'-[2'ethanesulfonic acid (HEPES) (pH 7.5). Aliquots were incubated for 10 min at 37°C with 0-100 p.mol/L serotonin in a total volume of 1 ml. Following incubation, the reaction was stopped by adding 1 ml of 0.32 nnnol/L sucrose/20 mmol/L "Iris HCI (pH 7.5) containing 2 mmol/L EGTA, 50 I~g/ml leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride and 0.1% 2-mercaptoethanol. The diluted reaction mixture was sonicated in a Kontes Micro Cell
Dose-lresponse relationships and groups differences were assessed by two-factor analysis of variance. The effect of lithium treatment was analyzed by multivariate analysis of variance (MANOVA) with repeat measures for the duration of treatment factor (SPSS procedure, MANOVA SPSS I,e., Chicago, III.).
Results The membrane-to-cytosolic distribution of platelet PKC activity in control, manic, and schizophrenic subjects is summarized in Figure 1. Platelet membranous to cytosolic
522
BIOL PSYCHIATRY 1993;33:5~"t~525
~"
4.0
~
3.6
41 111 I~
S.O
o
8.6
W 0 (..+
z
E. Friedman et al
CONTROL BIPOLAR, MANIA SCHIZOPHRENIA
II i
1.5
o m i
m
1
1.0
0.§
:m 0.0
0
1.0
0.5
10
100
Figure 1. Distribution of PKC in platelets of bipolar (mania), schizophrenic, and control subjects. Blood platelets obtained from bipolar patients, manic episode, schizophrenic patients, and control subjects were subdivided into 5 separate tubes that were respectively incubated for 10 rain with the indicated concentrations of 5-HT added in vitro. Platelet cytosolic and membrane-bound PKC activities were measured and ratios of enzyme activities in membranes/cytosol were calculated. Significant 5-HT dose-response relationships (p < 0.001) and differences among the diagnostic groups (p < 0.001) were observed. At 0.5 I~moi~L 5-HT, for instance, the bipolar group showed a significandy greater response than either the control or schizophrenic groups (p < 0.05, Newman-Keuls).
[5-ST] (.M)
enzyme ratios were elevated in the manic subjects when
activities were found in the schizophrenic subjects when compared to those o f controls. Incubation o f platelets with serotonin elicits a dose-related translocation o f platelet PKC activity (Figures 1, 3). The response o f platelets to serotonin was markedly enhanced in the bipolar manic subjects when compared either to control or to the schizophrenic
compared to either age- and scx-mm,_:n~u . . . . . . co~-~trols or to schizophrenic subjects. The data also indicated that this increase in ratio was entirely caused by an elevation in membrane-bound enzyme (Figure 2A). In contrast, no differences in cytosolic or membrane-associated platelet PKC
[--7
"~ 4,0
A
Q
CYTOSOL MgMBRANE-BOUND
[
B
o h w
3.0
3.
--T..
n --o
2.0 -
~
1.0 -
it
r.1 kt 0.0
Nil
I
1I
CONTROL
MANIA
MANIA
MANIA/IWK M&NIk/BWK [
LITHIUM - - I
Figure 2. Effect of lithium treatment on platelet cytosol and membrane PKC activities. Platelets obtained from control subjects (n -- 15) or from bipolar subjects (n -- 12) before and during lithium treatment were prepared and PKC activities were measured in cytosolic and membranous fractions following partial purification on DE 52 columns. Each bar represents the mean _ SEM. Platelet PKC activities in control and manic subjects prior to lithium treatment (A) were compared. Significantly higher PKC values were observed in the membranous fraction of the manic group (p < 0.001, Student's t-test). The effect of lithium treatment in the manic group is depicted in B. For the four subjects who completed the 2 weeks of treatment, a significant reduction in cytosolic and membrane-associated enzyme activities was noted, as determined by MANOVA with repeat measures on the duration of treatment factor (p < 0.001).
Platelet P K C
in Mania
BIOL FSYCI.]IATRY
523
1993;33:520-525
groups. Enzyme translocation in response to serotonin in the schizophrenic group did not differ from that in the control subjects. The effect of lithium treatment on platelet PKC activity is shown in Figure 2B. Enzyme activity was found to be reduced in both cytosolic and membrane-bound fractions during the 2 weeks of lithium treatment. In addition, the responsiveness of platelets to serotonin was reduced in bipolar patients undergoing lithium treatment (Figure 3). The reductions in both enzyme activity and serotonin-induced translocation were related to the duration of lithium treatment.
Discussion The present results demonstrate that hipolar manic patients have high platelet PKC activity, and that a greater proportion of the cellular enzymatic activity is found in association with the membranous tissue fraction. The results also indicate that the responsiveness of platelets obtained from manic subjects to serotonin is enhanced. In previous
• O.
~" C
studies we have shown that stimulation of cell-surface serotonin receptors in brain or platelets, as well as direct PKC stimulation with phorbol esters, induces translocation of PKC from the cytosolic to the particulate fraction (Wang and Friedman 1989; Wang and Friedman 1990b). In both platelets and brain, serotonin-stimulated PKC translocation was found to be mediated through 5-HT2 receptors (Wang and Friedman 1990a,b). Previous workers have noted increases in 5-HT2 receptor densities both in platelets of suicidal patients and in brains from suicides (Stanley and Mann 1983; Biegon et al 1990). The present finding of enhanced responsiveness to serotonin in bipolar subjects may be a reflection of supersensitive platelet 5-HT2 receptors caused by an increase in 5-HT2 receptor density or by other changes in signal transduction. However, the presence in bipolar subjects of both an increase in PKC activity as well as a distribution of PKC, which favors the particulate cell fraction, suggests that additional factors may contribute to the results presented. Alteration in basal platelet PKC in patients with this disease may be caused
Baseline 1 - w k Li t r e a t m e n t 2-wk Li t r e a t m e n t 5.0
3.o
I
4.5
2.5
4.0
E 3.5
E
2.O 3.0
O.
>
2.5
1.5
,,t,,,U 0
u
g
...........
. . . . . . . . . . . t ...........
...........
2.0
1.0
1.5
0 f-
"
=
1.0 0.5
MEMBRANE
CYTOSOL
0.5
0 0.0
0.0
I
I
I
I
0.5
1.0
10.0
100.0
[S-HT] ( ~ . )
0.0
I
I
I
I
0.5
1.0
10.0
100.0
0.0
[S-HT] ( , . )
Figure 3. Effect of lithium treatment on platelet cytosol and membrane-associated PKC activities. Blood platelets were prepared from bipolar patients prior to (baseline, n = 12) and following 1 (n = 7) or 2 weeks (n = 4) of lithium administration. Platelets were incubated with the indicated doses of 5-HT for i0 rain, and PKC activities in cytosolic and membranous fractions were determined. Each data point represents the mean --- SEM. The data obtained from the four subjects who completed the 2-week lithium treatment were subjected to MANOVA with the duration-of-treatment as the repeat measure factor. Significant reductions in 5-HT-induced cha~ges in cytosolic (p < 0.001) and in membrane-PKC activities (p < 0.001) were observed.
524
E. Friedman et al
BIOL PSYCHIATRY 1993;33:520--525
by differences in enzyme protein as well as in the susceptibility of PKC to inactivation by proteolysis. In addition, other factors that °re involved in the translocation of PKC such as Ca 2 +, membrane lipids or other structural membrane changes may also play a role in mediating the change in platelet PKC distribution (Wolf et al 1985). Lithium treatment in bipolar manic subjects appears to lower both cytosolic and membrane-associated platelet PKC activities. Drug treatment also reduced the sensitivity of platelets to serotonin stimulation in a time-related manner, similar to its known course associated with its clinical response. The decreased serotonin receptor sensitivity after lithium may be caused by changes in 5-HT2 receptor binding, as previous investigators have described reduced ligand binding at this receptor site after chronic lithium treatment in rats (Treiser et al 1981; Closse and Jaton 1984; Goodwin et al 1986; Tanimoto et al 1983). However, supersensitivity of the serotonin platelet receptor was described in patients during lithium treatment (Wood et al 1985). Furthermore, in previous studies in rat brain we have noted that lithium ion inhibits PKC translocation in response to phorbol esters, which is not dependent on 5HT receptor stimulation (Wang and Friedman 1989) but is rather a direct effect on the enzyme. These considerations, therefore, suggest that the effects of lithium described here are distal to the receptor. There are several limitations to these preliminary data in addition to the small sample sizes. Only the manic state has been studied; it would be useful to be able to compare
manic, depressed, and euthymic states longitudinally (which will be difficult to study in the medication-free state) or at least cross-sectionally. Also, the control group was not interviewed for psychiatric diagnosis; however, this would be expected to decrease rather than increase differences between manic a~d control subjects, as untreated or unacknowledged affective disorders are relatively common in the population. Despite these potential problems, significant between-group differences were seen here. The data presented suggest that an abnormality in cellular PKC distribution and/or in the mecb~-.",ism for its activation may be associated with the manic episode in bipolar affective disorder. The results also show that lithium treatment can reduce both cytosolic and membrane-associated PKC as well as decrease platelet sensitivity to 5-HT-induced enzyme translocation. Taken together, these prelimir, a_"y results implicate PKC-mediated phosphorylation both in the biology o f bipolar illness and in the pharmacology of lithium ion. Further studies are needed to elucidate (1) whether this finding is specific to mania or whether it is associated with other behavioral states in bipolar illness and (2) the biological cause that is responsible for this alteration in platelet PKC distribution. This work was supportedin part by USPHSgrant MH 45166. The authors would like to acknowledge Dr. Ashiwel Undie for his assistance and discussion of the statistical analyses and Ms. Janie Rickards' capable help in the preparation of this manuscript.
References Biegon A, Grispoon A, Blumenfield B, Bleich A, Apter A, Mester R ( 1990): Increased serotonin 5-HT~_receptor binding on blood platelets of suicidal men. Psychopharmacology 100:165-167. Charney EA, Weissman MM (1988): Epidemiology of depressive anu mnamHL,byuuromc. Ill ,aeoIgota~ A, Cancro R (eds), Depression and Mania. New York: Elsevier, pp 26-52. Closse A, Jaton AL (1984): Investigation of the influence of lithium upon the down-regulation of serotonin2 receptor in rat frontal cortex induced by long-term treatment with debenzepin, an antidepressant without appreciable affinity to serotonin2 receptors. Naunyn Schmiedebergs Arch Pharmacot 326:291-293. Endicott .1, Spitzer RL ( 1978): A diagnostic interview: The schedule for affective disorders and schizophrenia, Arch Gen Psychiatry 35:837-844. . _
J
.
.
.
.
.
.
.
.
Friedman E, Wang HY ( 1989): The effect of age on brain cortical protein 'i~llla~, ~ -- C ,~,u--~i~s mcdiaiion of scrotznin release. J Ne,trochem 52( I ): 187-192. Gershon S, Yuwiler A (1960): Lithium ion: A specific pharmacological approach to the treatment of mania. J Neuropsychiat !:229-241. Goodwin GM, De Souza R.I, Wood AJ, Green AR (1986): Lith-
ium decreases 5-HT~a and 5-HT2 receptors and alpha2-adrenoreceptor mediated function in mice. Psychopharmacology 90:482--487. Kelso JR, Ginns El, Egeland JA et al (1989): Re-evaluation of the linkage relationship between chromosome 1 lp loci and the gone for bipolar affective disorder in the Old Order Amish. Nature 342:238-243. Lowry OH, Rosebrough N J, Fair AL, Randall ILl (1951 ): Protein measurement with the Folin phenol reagent. J Biol Chem 249:265-275. Spitzer RL, Endicott J, Robins E (1978): Research diagnostic criteria: Rationale and reliability. Arch Gen P~-ychiatry 35:773782. Stanley MM, Mann JJ ( 1983): Increased serotonin-2 binding sites in frontal cortex or suicide victims. Lancet 1:214-216. Tanimoto K, Maeda K, Terada T (1983): Inhibitory effect of lithium on neuroleptic and serotonin receptors in rat brain. Brain Res 265:148-151. Treiser SL, Cascia CS, O'Donohue TL, Thoa TL, Jacobowitz DM, Kellar KJ (1981): Lithium increases serotonin release and decreases serotonin receptors in the hippocampus. Science 213:1529-1531.
Platelet PKC in Mania
Vandenberg SG, Singer SM, Pauls DL (1986): The Heredity of Behavior Disorders in Adults and Children. New York: Plenum. Wang HY, Friedman E (1989): Lithium inhibition of protein kinase C activation-induced serotonin release. Psychopharmacology 99:213-218. Wang HY, Friedman E (1990a): Central 5-hydroxylryptamine receptor-linked protein kinase C translocation: A functional postsynaptic signal transduction. Mol Pharmacol 37:75-79.
eIOLPSYCHIATRY 1993:33:520-525
525
Wang HY, Friedman E (1990b): Protein kinase C translocation in human blood platelet. Life Sci 47:1419-1425. Wolf M, Cuatrecasas P, Sahyoun N (1985): Interaction of protein kinase C with membranes is regulated by C a * ' , phorbol esters and ATP. J Biol Chem 2fiO:15718-15722. Wood K, Swade C, Abou-Saleh MT, Coppen A ( 1985): Apparent supersensitivity of platelet 5-I-[1"receptors in lithium-treated patients. J Affective Disord 8:69-72.