Prostaglandins Leukotrienes and Essential 0 Lawman Group UK Ltd l!W.
Fatty Acids
(1992) 46, 3!bt6
Increased Turnover of Platelet Phosphatidylinositol in Schizophrenia J. K. Yao, P. Yasaei* and D. P. van Kammen Department of Veterans Affairs Medical Center, and Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, PA 1.5206, USA (Reprint requests to JKY) ABSTRACT. The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in (Yadrenergic receptor function in the platelets of schizophrenic patients. Little is known, however, about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [%I]myoinositol or [3%]orthophosphate into resting and activated platelets of normal controls and schizophrenic patients with and without neuroleptic treatment. After 5 h incubation at 37”C, the majority of [‘Hlmyoinositol was incorporated into platelet PI. Following thrombin-induced platelet activation, there was rapid formation of %-labeled inositol phosphates (IPs) with inositol monophosphate (IPI) being the most abundant product. The thrombin-induced formation of platelet IPs was found .significantly hiiher in both haloperidol-stabilized and drug-free schizophrenics than in normal control subjects. When platelets were prelabeled with [32P]orthophosphates, thrombin-induced formation of phosphatidic acid (PA) was also significantly higher in haloperidol-stabilized schizophrenics than in normal controls. It is thought that thrombin-induced platelet activation is mediated through hydrolysis of polyphosphoinositides (poly-PI). The present data thus may reflect an increased signal transduction in schizophrenia, which is mediated through neuroleptic-regulated inositol phospholipid hydrolysis.
INTRODUCTION
a model in which abnormal regulation of signal transduction could lead to the episodic accumulation of biologically active transducers or second messengers. Because of lithium’s ability to inhibit inositol-1-phosphatase (2,3), the sensitivity of those receptor systems which utilize PI may be altered by prolonged exposure to lithium (4). This is of particular relevance to the therapeutic effects of lithium on manic-depressive illness (5) and on selected patients with schizophrenia (6, 7). Although previous studies have reported many defects of cellular processes in schizophrenia, little is known about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. Platelets not only show a similarity to serotonergic pre- and postsynaptic membranes (8,9), but also are comparable to catecholaminergic neurons (10). Thus, platelet membrane provides us a convenient model of a neurosecretory cell to study membranerelated functions, e.g. signal transduction mechanisms, in psychiatric disorders. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [3H]inositol or [ 3%‘]ortho-
Previous studies have related schizophrenia to an excess of dopaminergic activity, to a reduced synthesis of prostaglandins, to a viral infection, and to a disturbance of autoimmune function. Although the dopamine hypothesis is among the most widely studied conceptualizations, none of the above hypotheses can fully explain the etiology of schizophrenia due to its heterogeneity. The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in a+adrenergic receptor function in the platelets of schizophrenic patients. Recently, Lachman and Papolos (1) have proposed
* Current address: Food and Drug Administration HFF 423, Room 1030, 200 C Street SW, Washington, DC 20204, USA
Date received 8 April 1991 Date accepted 3 September 1991 39
40
Prostaglandins Leukotrienes
and Essential Fatty Acids
phosphate into resting and activated platelets of normal controls and schizophrenic patients. A preliminary report of this work was presented at the 21st Annual Meeting of the American Society for Neurochemistry, Phoenix, Arizona, 6 March 1990 (11).
MATERIALS AND METHODS Subjects All patients were admitted to the Schizophrenia Research Unit of the Highland Drive Department of Veterans Affairs Medical Center in Pittsburgh, PA, and had been treated on a long-term basis with neuroleptics prior to admission. After giving informed consent for all procedures, 23 physically healthy, male schizophrenic patients (mean age f SD, 37 f 8 years) participated in this study. Patients were diagnosed according to DSM-IIIR criteria. The Schedule for Affective Disorders and Schizophrenia was used to obtain the data needed for the diagnosis of schizophrenia. All patients had received neuroleptic treatment for at least 3 months prior to entry into the study. Neuroleptic treatment was converted to an equipotent dose of haloperidol prior to the blood drawing, if the patients were not already being treated with haloperidol. The patients were stabilized on the lowest therapeutic dosage to avoid having to give anticholinergic medication. The use of anticholinergic agents on a PRN basis was kept to a minimum and was not used within 2 weeks of blood drawing. After maintenance on a constant, optimal haloperidol dose for at least 2 weeks, a 100 ml fasting blood sample was drawn from these patients. Neuroleptic wash-out was then achieved by the substitution of an equal number of identicallooking placebo capsules for up to 6 weeks. Patients were rated daily by the nursing staff using the Bunney-Hamburg (BH) Psychosis Rating Scale (12), and weekly using the BH and the Brief Psychiatric Rating Scale (BPRS) Psychosis Subscale by clinicians who were blind to the patients’ medication status. The psychotic exacerbation criteria determining relapse was defined as an increase of 3 or more points in the psychosis item on the nurses’ daily BH scale over a period of at least 3 days as compared to the mean psychosis rating from the test week prior to the drug withdrawal period. Those patients who relapsed were eliminated from the study and returned to neuroleptic treatment. A second blood sample was drawn from the same patient following a 5-week drug ‘wash-out’ period. During the study, all patients were maintained on a low-monoamine, caffeine-restricted, and alcoholfree diet. Patients did not receive any sleeping
medication, antianxiety drugs, or any other medication during the time they were in the study. A healthy control population matched for age, sex, and race of studied patients was screened for psychiatric illness with the use of the SADS interview and Minnesota Multiphasic Personality Inventory (MMPI). Prestudy evaluations included a complete medical history, physical examination, serum lipid analysis, and any other blood or laboratory tests as indicated by the history and physical examination. Subjects who had a history or any clinical evidence of hepatic disorders, endocrine or metabolic diseases associated with abnormal plasma lipid profiles or prolonged treatment with lithium were excluded from the study. Patients with obesity, current alcohol or other drug abuse or dependence were also excluded. Exclusion criteria for the control subjects included the same medical disorders as for the schizophrenic group.
Preparation of samples Platelets were prepared from fresh blood with 0.15 volume of anticoagulant citrate dextrose (ACD: 85 mM trisodium citrate, 111 mM dextrose, and 71 mM citric acid) according to the procedure described by Siess et al (13). Platelet-rich plasma (PRP), pH 6.4, was obtained by centrifugation at 200 x g for 15 min. To prevent platelet activation, PGI* (5 rig/ml) was added to PRP (14) before isolating platelets by centrifugation at 850 x g for 15 min. The final platelet precipitates were gently resuspended to one-fifth their original volume, using Tris-saline buffer (5 mM D-glucose, 0.15 M NaCl, 0.02 M Tris-HC1 pH 7.4):platelet poor plasma (1: 1, v/v). Protein concentration from an aliquot of platelets dissolved in SDS solution was determined by the modified Lowry method (15). The platelet count was determined using a hemacytometer chamber.
Labeling of platelets The resuspended platelets from 1 and 5 mL of PRP were prelabeled with 300 oCi [32P]orthophosphate and 100 &i of [3H]inositol for 2 and 5 h at 37°C respectively. Following incubation, each sample was immediately washed four times with Tris-saline buffer containing PG12 (400 ng/mL). The final platelet pellet was then resuspended in Tris-saline buffer (without PG12) equal to one-fifth the original volume of PRP, and then placed into aggregometer tubes at 37°C for 3 min prior to stimulation. Another aliquot of prelabeled platelet suspension was used for lipid extraction.
Increased Turnover of Platelet Phosphatidylinositol
Platelet activation The mechanisms
by which thrombin stimulated the formation of phosphatidic acid (PA) were dependent on the concentration of the platelet stimuli used (13). Immediately after labeling, an aliquot of prelabeled platelets was stimulated by thrombin (2 units/ml), which formation of PA was insensitive to inhibition by indomethacin. Following platelet stimulation for various time periods, the reaction was stopped by addition of 3.75 volumes of chloroform/methanol/cone. HCl (1:2:0.02) (16).
41
in, Schizophrenia
(45: 35: 10, by vol.) (18). Separation of total labeled lipids into nonpolar lipid and phospholipid subclasses by Sep-Pak silica cartridge and HPTLC has been described previously (19). To ensure the accuracy and resolution of radioactivity distribution, fluorography was used to identify the labeled lipids according to the procedure described previously (19, 20). Quantification of fluorograms was carried out with an LKB Ultrascan Laser Densitometer and Apple IIe computer.
RESULTS Extraction of radiolabeled phosphates
lipids and inositol
The method used for extraction of labeled compounds was essentially described by Watson et al (16). For extraction of polyphosphatidylinositols, the labeled samples were added to 0.75 mL of CHCls, 1.0 mL of 2 M KCl, and 5.0 mL of CHC13 : CH s0H (2: 1, v/v). Water-soluble inositol phosphates were extracted with 3.78 mL of 1.5 mL of H20, and 5.0 mL of chloroform, CHCls : CH s0H (2: 1, v/v). Each extract was then washed three times with Folch’s upper phase (CH30H:H20:CHC13, 48:47:3, by vol.). Analysis
of labeled compounds
The procedure used for separation of inositol phosphates on Dowex 1 anion exchange column was similar to that described by Berridge (17). The water-soluble extracts were applied to a pre-washed poly-prep column (0.8 x 4.0 cm) containing Dowex1 (x8; formate form; Bio-Rad, Anaheim, CA, USA). The water soluble components were eluted with: (a) 15 mL of distilled water; (b) 5 mL of 5 mM disodium tetraborate; (c) 15 mL of 5 mM disodium tetraborate and 60 mM ammonium formate, pH 4.75; (d) 15 mL of 250 mM ammonium formate, pH 4.75; (e) 20 mL of 600 mM ammonium formate, pH 4.75; and (f) 15 mL of 1.5 M ammonium pH 4.75. Under these conditions, formate, glycerolphosphoinositol (GPI), IPi, IP2, and IP3 were recovered in fractions (c), (d), (e) and (f), respectively. The eluant was collected in 1 mL portions, using an automated fractional collector (Bio-Rad). A 0.5 mL portion of each fraction was taken for liquid-scintillation counting (Beckman, Model LS 5801) using Ready Safe scintillation cocktail (Beckman). Quenching was determined by calculation of the H number, and corrections for 32P isotope decay were routinely employed. Separation of inositol phospholipids was carried out with oxalate-impregnated Silica gel G-25 TLC of plates using a solvent system consisting ammonium hydroxide chloroform/methanol/4N
Incorporation platelets
of [‘Hlmyoinositol
into human
To demonstrate metabolic turnover of inositol phospholipids to inositol phosphates (IP), membrane phosphatidylinositol (PI) was first labeled by incorporation of [3H]myoinositol into platelet suspension. There was a linear relationship between rate of incorporation and incubation time for at least 3 h. In resting platelets, the majority of 3Hlabeling was found in the phospholipid fraction. The mean incorporation of [3H]myoinositol into platelet phospholipids was significantly higher (p < 0.01) in haloperidol-stabilized schizophrenics than in normal controls (Table). There was no significant difference between haloperidol-stabilized and drug-free Table lipids
Incorporation
Subjects
of [%I]myoinositol into human platelet
Sex Age
N
‘H-labeled lipids
p*
x lO*dpn(mg protein/h
Normal controls Schizophrenics Haloperidolstabilized Drug-free
M
33f6
12
4.83 f 2.30
M
37+8
16
9.92 + 5.78
Fatty Acids
(1992) 46, 3!bt6
Increased Turnover of Platelet Phosphatidylinositol in Schizophrenia J. K. Yao, P. Yasaei* and D. P. van Kammen Department of Veterans Affairs Medical Center, and Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, PA 1.5206, USA (Reprint requests to JKY) ABSTRACT. The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in (Yadrenergic receptor function in the platelets of schizophrenic patients. Little is known, however, about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [%I]myoinositol or [3%]orthophosphate into resting and activated platelets of normal controls and schizophrenic patients with and without neuroleptic treatment. After 5 h incubation at 37”C, the majority of [‘Hlmyoinositol was incorporated into platelet PI. Following thrombin-induced platelet activation, there was rapid formation of %-labeled inositol phosphates (IPs) with inositol monophosphate (IPI) being the most abundant product. The thrombin-induced formation of platelet IPs was found .significantly hiiher in both haloperidol-stabilized and drug-free schizophrenics than in normal control subjects. When platelets were prelabeled with [32P]orthophosphates, thrombin-induced formation of phosphatidic acid (PA) was also significantly higher in haloperidol-stabilized schizophrenics than in normal controls. It is thought that thrombin-induced platelet activation is mediated through hydrolysis of polyphosphoinositides (poly-PI). The present data thus may reflect an increased signal transduction in schizophrenia, which is mediated through neuroleptic-regulated inositol phospholipid hydrolysis.
INTRODUCTION
a model in which abnormal regulation of signal transduction could lead to the episodic accumulation of biologically active transducers or second messengers. Because of lithium’s ability to inhibit inositol-1-phosphatase (2,3), the sensitivity of those receptor systems which utilize PI may be altered by prolonged exposure to lithium (4). This is of particular relevance to the therapeutic effects of lithium on manic-depressive illness (5) and on selected patients with schizophrenia (6, 7). Although previous studies have reported many defects of cellular processes in schizophrenia, little is known about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. Platelets not only show a similarity to serotonergic pre- and postsynaptic membranes (8,9), but also are comparable to catecholaminergic neurons (10). Thus, platelet membrane provides us a convenient model of a neurosecretory cell to study membranerelated functions, e.g. signal transduction mechanisms, in psychiatric disorders. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [3H]inositol or [ 3%‘]ortho-
Previous studies have related schizophrenia to an excess of dopaminergic activity, to a reduced synthesis of prostaglandins, to a viral infection, and to a disturbance of autoimmune function. Although the dopamine hypothesis is among the most widely studied conceptualizations, none of the above hypotheses can fully explain the etiology of schizophrenia due to its heterogeneity. The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in a+adrenergic receptor function in the platelets of schizophrenic patients. Recently, Lachman and Papolos (1) have proposed
* Current address: Food and Drug Administration HFF 423, Room 1030, 200 C Street SW, Washington, DC 20204, USA
Date received 8 April 1991 Date accepted 3 September 1991 39
40
Prostaglandins Leukotrienes
and Essential Fatty Acids
phosphate into resting and activated platelets of normal controls and schizophrenic patients. A preliminary report of this work was presented at the 21st Annual Meeting of the American Society for Neurochemistry, Phoenix, Arizona, 6 March 1990 (11).
MATERIALS AND METHODS Subjects All patients were admitted to the Schizophrenia Research Unit of the Highland Drive Department of Veterans Affairs Medical Center in Pittsburgh, PA, and had been treated on a long-term basis with neuroleptics prior to admission. After giving informed consent for all procedures, 23 physically healthy, male schizophrenic patients (mean age f SD, 37 f 8 years) participated in this study. Patients were diagnosed according to DSM-IIIR criteria. The Schedule for Affective Disorders and Schizophrenia was used to obtain the data needed for the diagnosis of schizophrenia. All patients had received neuroleptic treatment for at least 3 months prior to entry into the study. Neuroleptic treatment was converted to an equipotent dose of haloperidol prior to the blood drawing, if the patients were not already being treated with haloperidol. The patients were stabilized on the lowest therapeutic dosage to avoid having to give anticholinergic medication. The use of anticholinergic agents on a PRN basis was kept to a minimum and was not used within 2 weeks of blood drawing. After maintenance on a constant, optimal haloperidol dose for at least 2 weeks, a 100 ml fasting blood sample was drawn from these patients. Neuroleptic wash-out was then achieved by the substitution of an equal number of identicallooking placebo capsules for up to 6 weeks. Patients were rated daily by the nursing staff using the Bunney-Hamburg (BH) Psychosis Rating Scale (12), and weekly using the BH and the Brief Psychiatric Rating Scale (BPRS) Psychosis Subscale by clinicians who were blind to the patients’ medication status. The psychotic exacerbation criteria determining relapse was defined as an increase of 3 or more points in the psychosis item on the nurses’ daily BH scale over a period of at least 3 days as compared to the mean psychosis rating from the test week prior to the drug withdrawal period. Those patients who relapsed were eliminated from the study and returned to neuroleptic treatment. A second blood sample was drawn from the same patient following a 5-week drug ‘wash-out’ period. During the study, all patients were maintained on a low-monoamine, caffeine-restricted, and alcoholfree diet. Patients did not receive any sleeping
medication, antianxiety drugs, or any other medication during the time they were in the study. A healthy control population matched for age, sex, and race of studied patients was screened for psychiatric illness with the use of the SADS interview and Minnesota Multiphasic Personality Inventory (MMPI). Prestudy evaluations included a complete medical history, physical examination, serum lipid analysis, and any other blood or laboratory tests as indicated by the history and physical examination. Subjects who had a history or any clinical evidence of hepatic disorders, endocrine or metabolic diseases associated with abnormal plasma lipid profiles or prolonged treatment with lithium were excluded from the study. Patients with obesity, current alcohol or other drug abuse or dependence were also excluded. Exclusion criteria for the control subjects included the same medical disorders as for the schizophrenic group.
Preparation of samples Platelets were prepared from fresh blood with 0.15 volume of anticoagulant citrate dextrose (ACD: 85 mM trisodium citrate, 111 mM dextrose, and 71 mM citric acid) according to the procedure described by Siess et al (13). Platelet-rich plasma (PRP), pH 6.4, was obtained by centrifugation at 200 x g for 15 min. To prevent platelet activation, PGI* (5 rig/ml) was added to PRP (14) before isolating platelets by centrifugation at 850 x g for 15 min. The final platelet precipitates were gently resuspended to one-fifth their original volume, using Tris-saline buffer (5 mM D-glucose, 0.15 M NaCl, 0.02 M Tris-HC1 pH 7.4):platelet poor plasma (1: 1, v/v). Protein concentration from an aliquot of platelets dissolved in SDS solution was determined by the modified Lowry method (15). The platelet count was determined using a hemacytometer chamber.
Labeling of platelets The resuspended platelets from 1 and 5 mL of PRP were prelabeled with 300 oCi [32P]orthophosphate and 100 &i of [3H]inositol for 2 and 5 h at 37°C respectively. Following incubation, each sample was immediately washed four times with Tris-saline buffer containing PG12 (400 ng/mL). The final platelet pellet was then resuspended in Tris-saline buffer (without PG12) equal to one-fifth the original volume of PRP, and then placed into aggregometer tubes at 37°C for 3 min prior to stimulation. Another aliquot of prelabeled platelet suspension was used for lipid extraction.
Increased Turnover of Platelet Phosphatidylinositol
Platelet activation The mechanisms
by which thrombin stimulated the formation of phosphatidic acid (PA) were dependent on the concentration of the platelet stimuli used (13). Immediately after labeling, an aliquot of prelabeled platelets was stimulated by thrombin (2 units/ml), which formation of PA was insensitive to inhibition by indomethacin. Following platelet stimulation for various time periods, the reaction was stopped by addition of 3.75 volumes of chloroform/methanol/cone. HCl (1:2:0.02) (16).
41
in, Schizophrenia
(45: 35: 10, by vol.) (18). Separation of total labeled lipids into nonpolar lipid and phospholipid subclasses by Sep-Pak silica cartridge and HPTLC has been described previously (19). To ensure the accuracy and resolution of radioactivity distribution, fluorography was used to identify the labeled lipids according to the procedure described previously (19, 20). Quantification of fluorograms was carried out with an LKB Ultrascan Laser Densitometer and Apple IIe computer.
RESULTS Extraction of radiolabeled phosphates
lipids and inositol
The method used for extraction of labeled compounds was essentially described by Watson et al (16). For extraction of polyphosphatidylinositols, the labeled samples were added to 0.75 mL of CHCls, 1.0 mL of 2 M KCl, and 5.0 mL of CHC13 : CH s0H (2: 1, v/v). Water-soluble inositol phosphates were extracted with 3.78 mL of 1.5 mL of H20, and 5.0 mL of chloroform, CHCls : CH s0H (2: 1, v/v). Each extract was then washed three times with Folch’s upper phase (CH30H:H20:CHC13, 48:47:3, by vol.). Analysis
of labeled compounds
The procedure used for separation of inositol phosphates on Dowex 1 anion exchange column was similar to that described by Berridge (17). The water-soluble extracts were applied to a pre-washed poly-prep column (0.8 x 4.0 cm) containing Dowex1 (x8; formate form; Bio-Rad, Anaheim, CA, USA). The water soluble components were eluted with: (a) 15 mL of distilled water; (b) 5 mL of 5 mM disodium tetraborate; (c) 15 mL of 5 mM disodium tetraborate and 60 mM ammonium formate, pH 4.75; (d) 15 mL of 250 mM ammonium formate, pH 4.75; (e) 20 mL of 600 mM ammonium formate, pH 4.75; and (f) 15 mL of 1.5 M ammonium pH 4.75. Under these conditions, formate, glycerolphosphoinositol (GPI), IPi, IP2, and IP3 were recovered in fractions (c), (d), (e) and (f), respectively. The eluant was collected in 1 mL portions, using an automated fractional collector (Bio-Rad). A 0.5 mL portion of each fraction was taken for liquid-scintillation counting (Beckman, Model LS 5801) using Ready Safe scintillation cocktail (Beckman). Quenching was determined by calculation of the H number, and corrections for 32P isotope decay were routinely employed. Separation of inositol phospholipids was carried out with oxalate-impregnated Silica gel G-25 TLC of plates using a solvent system consisting ammonium hydroxide chloroform/methanol/4N
Incorporation platelets
of [‘Hlmyoinositol
into human
To demonstrate metabolic turnover of inositol phospholipids to inositol phosphates (IP), membrane phosphatidylinositol (PI) was first labeled by incorporation of [3H]myoinositol into platelet suspension. There was a linear relationship between rate of incorporation and incubation time for at least 3 h. In resting platelets, the majority of 3Hlabeling was found in the phospholipid fraction. The mean incorporation of [3H]myoinositol into platelet phospholipids was significantly higher (p < 0.01) in haloperidol-stabilized schizophrenics than in normal controls (Table). There was no significant difference between haloperidol-stabilized and drug-free Table lipids
Incorporation
Subjects
of [%I]myoinositol into human platelet
Sex Age
N
‘H-labeled lipids
p*
x lO*dpn(mg protein/h
Normal controls Schizophrenics Haloperidolstabilized Drug-free
M
33f6
12
4.83 f 2.30
M
37+8
16
9.92 + 5.78
