a simplified method to quantify dysregulated tyrosine transport in schizophrenia
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Schizophrenia Research xxx (2013) xxx–xxx
SCHRES-05587; No of Pages 6
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A simplified method to quantify dysregulated tyrosine transport in schizophrenia
Rodolfo Bongiovanni a, Sherry Leonard b, George E. Jaskiw a,c,⁎a Psychiatry Service, Louis Stokes Cleveland DVAMC, Cleveland, OH 44106, United Statesb Department of Psychiatry, University of Colorado Denver, Aurora, CO 80045, United Statesc Department of Psychiatry, Case Western Reserve University, Cleveland, OH 44106, United States
⁎ Corresponding author at: Psychiatry Service, LouCleveland, OH 44106, United States. Tel.: +1 216 791 380
E-mail addresses: [email protected] ([email protected] (S. Leonard), george.jaskiw
0920-9964/$ – see front matter. Published by Elsevier B.Vhttp://dx.doi.org/10.1016/j.schres.2013.08.041
Please cite this article as: Bongiovanni, R., et a(2013), http://dx.doi.org/10.1016/j.schres.20
a b s t r a c t
a r t i c l e i n f oArticle history:
Received 29 April 2013Received in revised form 26 August 2013Accepted 27 August 2013Available online xxxxKeywords:TyrosineSchizophreniaFibroblastClozapineHaloperidolChlorpromazine
Background: Schizophrenia is associated with altered tyrosine transport across plasma membranes. This is typi-cally demonstrated bymeasuring the uptake of radiolabeled tyrosine in cultured human fibroblasts. Our primarygoal was to determine whether tyrosine uptake could be characterized using unlabeled tyrosine. A secondarygoal was to assess the effect of antipsychotic drugs added during the incubation.Method: Epithelium-derived fibroblast cultures were generated from patients with schizophrenia (n = 6) andage-matched controls (n = 6). Cells between cycles 8–12 were exposed to an amino acid free medium for60 min and then for 1 min to media containing tyrosine (0.008–1.0 mM). Amino acid levels were measuredand Michaelis–Menten parameters determined. Uptake of tyrosine (0.5 mM) was also measured in controlcells after antipsychotic drugs were introduced during the depletion or uptake phases.Results: Tyrosine uptake was sodium-independent. The maximal transport velocity (Vmax) was significantlylower in patients with schizophrenia than in controls (p b 0.01). The transporter affinity (Km) did not differbetween the groups. Tyrosine uptake was differentially affected (p b 0.001) by inclusion of 10−4 M haloperidol,
chlorpromazine or clozapine during different periods of incubation.Conclusion:Dysregulated tyrosine kinetics in schizophrenia can be readily studiedwithout the use of radiolabeledtracers. The data also indicate that tyrosine uptake may be subject to complex pharmacological effects.Published by Elsevier B.V.
1. Introduction
Dysregulated tyrosine transport across plasma membranes is oneof the most consistently demonstrated biomarkers in schizophrenia(Hagenfeldt et al., 1987; Wiesel et al., 1994; Ramchand et al., 1996;Flyckt et al., 2001). The abnormal tyrosine transport is maternallyinherited (Flyckt et al., 2011), evident in antipsychotic drug naïvepatients (Flyckt et al., 2001) and associated with greater cognitive dys-function (Wiesel et al., 2005). Since common amino acid transportersare present throughout the body (Verrey et al., 2004), most data onaberrant tyrosine kinetics have been derived from cultures of humanfibroblasts. Without exception, the maximal velocity (Vmax) of tyrosineuptake has been found to be lower in schizophrenia (Hagenfeldtet al., 1987; Wiesel et al., 1994; Ramchand et al., 1996; Flyckt et al.,2001). Some groups have also reported a higher transporter affinity(Ramchand et al., 1996; Flyckt et al., 2001). Despite these highly con-sistent data, the link between abnormal tyrosine transport and thepathophysiology of schizophrenia remains to be fully defined. This
is Stokes Cleveland DVAMC,0x5538; fax: +1 216 707 7900.. Bongiovanni),@va.gov (G.E. Jaskiw).
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l., A simplifiedmethod to qua13.08.041
process could be accelerated by simplifications in the methodologyfor measuring tyrosine transport.
In the standard approach, fibroblasts are cultured over severalcycles, incubated for a short period in an amino acid free medium todeplete their internal stores and then briefly (1 min) exposed to variousconcentrations of radiolabeled tyrosine (Hagenfeldt et al., 1987). Theintracellular content of radiolabeled tyrosine is then used to derive clas-sical Michaelis–Menten parameters (Hagenfeldt et al., 1987; Wieselet al., 1994; Ramchand et al., 1996; Flyckt et al., 2001). While the useof a radiolabel allows measurement of trace quantities of tyrosine,the cost and logistics of using radioactive materials can be limitingin some laboratories. Given improvements in analytic techniques foramino acids (Bongiovanni et al., 2001), we posited that unlabeled tyro-sine could be used equallywell to generate kinetic parameters. The test-ing of this hypothesis was the main purpose of the current study.
The pharmacological sensitivity of tyrosine transport in man is alsoincompletely characterized. One study showed that high concentrationsof haloperidol or chlorpromazine present only during the 1 min uptakephase markedly inhibited tyrosine uptake (Wiesel et al., 1994). Sincethen, a longer antipsychotic drug exposure (≥3 h) has been reportedto affect the transport of other classes of amino acids (Marchesi et al.,2006). Accordingly, our secondary aim was to determine whether tyro-sine uptake parameters could be affected by a longer exposure to anti-psychotic drugs. If dysregulated tyrosine transport mediates symptoms
ntify dysregulated tyrosine transport in schizophrenia, Schizophr. Res.
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of schizophrenia, then pharmacologic agents that normalize tyrosineuptake in fibroblasts should prove to be therapeutic. In that case, fibro-blast cultures could provide a valuable in vitro model for developingand evaluating new drug candidates.
2. Method
2.1. Subjects
Postmortem tissue was collected at autopsy following family con-sent under a study exempt from human subject approval. The DenverSchizophrenia Center Brain Bank was initiated in 1980. Human post-mortem brain and matching skin fibroblasts were collected, followingdonation by the family. Subjects were identified by local coroners andthe family contacted by Schizophrenia Center staff. In the currentstudy, subjects with HIV or hepatitis B were excluded. The culturing offibroblasts through several generations was used to attenuate medica-tion and cause of death effects.
All subjects were Caucasian males and included controls (n = 6)with no history of mental illness and patients with schizophrenia(n = 6). Diagnosis (DSM-IV) wasmade by two board certified psychia-trists on the basis of medical records as well as family and physicianinterviews. The groups did not differ significantly in age (control52.17 ± 10.40 yr; schizophrenia 62.33 ± 16.40 yr), or in postmorteminterval (control 14.50 ± 5.90 h; schizophrenia, 17.00 ± 6.58 h).Med-ication information (Table 1)was collected from the chart. Two patientswere on a single antipsychotic drug at the time of death (olanzapineor fluphenazine) and one was on multiple antipsychotic drugs(chlorpromazine, trifluoperazine, haloperidol). The medication statusof the other three patients is not known.
2.2. Fibroblast cultures
Skin fibroblasts were cultured from cadavers as described (Canastaret al., 2012). Briefly, the skin was swabbed with betadine and alcoholbefore harvesting. Skin (approximately 1 cm2) was dissected fromthe upper and inner arm of the deceased and immediately placedin media with antibiotics (DMEM/F12 1:1, 15% fetal calf serum, 2 mML-glutamine, penicillin/streptomycin) and brought to the laboratory.The tissue was dissected into small pieces with a sterile scalpel and for-ceps and placed into a Corning T-25 flask. Medium, 2.0 ml was slowlydripped onto the tissue in the flask. Cultures were placed in a 37 °C,5% CO2 incubator overnight. After 24 h, 4.0 ml of additional media wasadded slowly. Medium was changed twice weekly. Fibroblast growth isusually seen in 7–14 days. Cells were passaged once and then frozen inmedia with 20% fetal calf serum and 10% DMSO at −80 °C. Afterthawing, individual cell lines were cultured in 75 cm2 plastic (Costar®)flasks in a humidified atmosphere of 10% CO2 at 37 °C. The cells werefrequently checked for mycoplasma and other bacterial contamination.
Table 1Subject Characteristics.
Age (yr) Dx COD PMI (h) Drugs
53 CON Acute disease 17.0 Thyroxine, antilipidemic72 CON Heart failure 18.5 Digoxin, vancomycin, hydrocortisone,45 CON Acute disease 15.0 Furosemide, sertraline, codeine, prochl51 CON Renal 6.0 Sevelamer, phenytoin, warfarin, amlod43 CON Trauma 21.5 None49 CON Cardiac 9.0 Alcohol63 SZ Cardiac 18.0 Digoxin, procainamide, isosorbide, cap38 SZ Suicide 11.0 Cannabis, alcohol59 SZ Acute disease 19.5 Chlorpromazine, trifluoperazine, halop57 SZ Heart failure 18.0 Fluphenazine, insulin68 SZ Heart failure 8.5 Amitriptyline, risperidone, venlafaxine89 SZ Heart failure 27.0 Olanzapine, sertraline, trazodone
CON (control), SZ (schizophrenia), Diagnosis (Dx), cause of death (COD), postmortem interval
Please cite this article as: Bongiovanni, R., et al., A simplifiedmethod to qua(2013), http://dx.doi.org/10.1016/j.schres.2013.08.041
Primary cultures between the 8th and 12th passages were then frozenin 10% dimethyl sulfoxide in Dulbecco minimal essential medium with10% fetal calf serum and stored in liquid nitrogen for later use.
Culture reagents were purchased from Sigma-Aldrich: Dulbecco'sModified Eagle Medium (DMEM), insulin/transferrin/selenium sup-plement (ITS) (1 mg/ml/0.55 mg/ml/0.5 μg/ml respectively), fibro-blast growth factor (25 μg/ml), fetal bovine serum (FBS), penicillin/streptomycin (10,000 U/ml/10,000 μg/ml respectively), phosphatebuffer saline (PBS) with 0.1% glucose, L-glutamine 200 mM. The cul-ture medium was made by adding the following to 500 ml DMEM:penicillin/streptomycin 5 ml, L-glutamine 5 ml, ITS 5 ml, fibroblastgrowth factor, FBS 100 ml. Thus the culture medium contained FBS(17%, vol/vol), penicillin (85 U/ml), streptomycin (85 μg/ml), gluta-mine (1.7 mM), fibroblast growth factor (45 ng/ml), and ITS (insulin8.5 μg/ml, transferrin 5 μg/ml, selenium 4.5 ng/ml).
2.3. Amino acid depletion and tyrosine uptake
Following the cluster-tray method (Gazzola et al., 1981; Hagenfeldtet al., 1987) approximately 4 × 104 cells were seeded into each well(4.8 cm2, 12/tray) of a multiwell tray (Corning® CellBIND® Surface).Two equivalent trays were set up for each cell line and cultured to con-fluence in 5 days. A partial depletion of intracellular endogenous aminoacid stores was achieved by incubating the fibroblasts in an amino acid-free medium (Gazzola et al., 1980). The cells grown to confluence werewashed with 2 ml of phosphate buffered saline (PBS) containing 1 g/lglucose and incubatedwith an additional 2 ml PBS for onehour at 37 °C.
The PBS incubation mediumwas then removed and tyrosine uptakemeasured during a 1 min incubation at 37 °C in 0.5 ml of PBS containinga known concentration of tyrosine. Uptake was terminated rapidly byplacing the trayon ice andwashing twicewith ice-cold PBS. The residualPBS was removed from the wells, 250 μl of 5% trichloroacetic acid TCAwas added and the trays incubated for an additional 30 min at roomtemperature. The acid soluble extract from each well was transferredto a 1 ml tube for measurement of free amino acids. The cells in eachwell were then digested by the addition of 250 μL of 1 N NaOH andthen incubation for 30 min. Protein quantities were assayed directly inthe tray utilizing a modified Bio-Rad method (Bradford, 1976).
Since the concentrations of glucose and TCA used differ across pub-lished studies (Gazzola et al., 1980;Hagenfeldt et al., 1987),we conducteda preliminary experiment. Glucose and TCA concentrations were variedand large neutral amino acid levels measured in the PBS removed at theend of the 60 min incubation as well as in the acid soluble fraction subse-quently extracted with TCA. In a second experiment, only glucose levelswere varied and the 1 min uptake of 0.5 mM tyrosine measured. Insofaras tyrosine uptake in fibroblasts is sodium-independent (Hagenfeldtet al., 1987), we conducted a confirmatory study, comparing tyrosine up-take (0.063–1.5 mM) in regular PBS and in a medium in which sodium
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ntify dysregulated tyrosine transport in schizophrenia, Schizophr. Res.
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was replaced by equimolar choline. Following this, we examined uptakecomparing PBS and 11 concentrations of tyrosine (0.008–1.0 mM).
2.4. Antipsychotic drug effects
In a separate series of experiments, haloperidol, chlorpromazine(Sigma-Aldrich) or clozapine (Novartis) was dissolved in the PBS mediaused during the 60 min depletion and/or during the 1 min uptakephase. The medium used during the 1 min uptake phase also containedtyrosine (0.5 mM). Confluent cells in a 12 well tray were assigned toone of three groups: Group 1— no drug exposure, Group 2— drug expo-sure during 60 min depletion period and during the 1 min uptake phase,Group 3— drug exposure only during the 1 min uptake phase. Only cellsfrom controls were tested in this fashion.
2.5. Amino acid analysis
The amino acids of interest weremeasured using an improved HPLCmethod of analysis (Bongiovanni et al., 2001). Briefly, the HPLC systemconsisted of an all phase C18 column 4.6 × 100 mm, an LC-ECD operat-ed at a relative potential of 0.75 V to a Ag/AgCl reference electrodeand a delivery system that included a temperature controlled (4 °C)autosampler. The mobile phase (0.5 ml/min) consisted of 75% aque-ous 0.133 mM Na2HPO4 and 25% methanol adjusted to pH 6.8 witho-phosphoric acid. To detect amino acids, a derivatizing agent wasused (OPA-S); 10 mg o-phthaldehyde and 30 mg sodium sulfite dilutedto 5.0 ml with 0.1 M sodium carbonate at pH 10.4. The reaction tubecontaining (sequentially added) 10 μl of sample, 10 μl of norvaline(1 μg/ml; internal standard), 10 μl sodium carbonate, and 5 μl of OPA-S was incubated for 5 min and then diluted to 75 μl with mobilephase. Sample derivatives are stable for at least 24 h at 4 °C. A volumeof 10 μl was injected on the column.
2.6. Calculation of kinetic parameters
Kinetic parameters Vmax (nmol min−1 mg-protein−1), Km (μM), Kd
(μl min−1 mg-protein−1) were calculated (GraphPad) utilizing a mod-ified Michaelis−Menton equation νo = ((Vmax [S]) / (Km + [S])) +Kd[S] where νo is the initial transport velocity, [S] is the concentrationand Kd is the non-saturable component (Segel, 1976). Kd was estimatedas the slope of the line from a plot of tyrosine uptake against the 3highest tyrosine concentrations in the uptake medium. Net uptakeover 1 min (νo)was determined by subtracting tyrosine levels in blanks(no tyrosine in uptake medium) from those in cells exposed to varyingtyrosine concentrations (0.008–1.0 mM) and correcting for Kd[S]. Km
and Vmax values for healthy controls and patients with schizophreniawere compared by unpaired two-tail Student's t-tests.
3. Results
3.1. Chromatography
In studieswhere all the amino acidswere to be quantified, a run timeof 50 min proved more than adequate (Fig. 1A). In samples depleted ofall amino acids and then incubated for 1 min with varying concentra-tions of tyrosine, peaks other than tyrosine were markedly attenuated.With additional adjustments, this permitted the run time to be loweredto 15 min without any interference from late peaks (Fig. 1B).
3.2. Glucose and TCA effects
A 60 min incubation in amino acid free PBS containing 0.1% glucosefollowed by extraction with 5% TCA showed that large neutral aminoacids were lowered by 60–80% (Table 2). The degree of depletion wasvirtually the same for other combinations of glucose/TCA (1%/5%;1%/10%) (data not shown). In a second set of studies, cells exposed
Please cite this article as: Bongiovanni, R., et al., A simplifiedmethod to qua(2013), http://dx.doi.org/10.1016/j.schres.2013.08.041
for 1 min to tyrosine 0.5 mM (in PBS) after a 60 min incubation inamino acid free PBS containing glucose. 0.1%, 4.5% or 10%, had atotal tyrosine content of 9.74 ± 1.02, 10.41 ± 0.94 and 10.25 ±0.71 (mean ± SD) respectively (extraction with 5% TCA). These werenot significantly different (one way ANOVA, p b 0.6, F(2,11) = 0.6).For the remainder of the study, the incubation PBS contained glucose0.1% and the acid soluble fraction was extracted with 5% TCA.
3.3. Sodium independence
Tyrosine uptake in regular PBS was compared to that in a sodium-free medium. A two-way ANOVA with tyrosine concentration (0.063–1.5 mM) as one factor and sodium content as a second factor showedno sodium effect (F(1,6) = 0.9, p = 0.9) or tyrosine × sodium interac-tion (F(5,6) = 0.2, p = 0.9). Furthermore, uptake was not significantlydifferent between PBS and the sodium-free medium (p N 0.05 withBonferroni correction) at any tyrosine concentration (Fig. 2).
3.4. Kinetic parameters
Baseline amino acid levels (controls mean ± SD: tyrosine 11.04 ±1.24, valine 11.13 ± 1.51, isoleucine 14.72 ± 1.45, leucine 15.66 ±1.44, phenylalanine 10.95 ± 2.39, tryptophan 0.80 ± 0.11 nmol mg-protein−1) were not significantly different between controls andpatients (patient data not shown). While Vmax was significantly lowerin patients than in controls, Km and Kd values did not differ betweenthe groups (Table 3).
3.5. Antipsychotic drug effects
When chlorpromazine or haloperidol (0.1 mM) was present in themedium throughout, that is during the 60 min depletion incubationand 1 min uptake period, the uptake of tyrosine was significantly in-creased relative to the no-drug condition (Fig. 3). However, uptake oftyrosine was not affected when cells were exposed to chlorpromazineor haloperidol (0.1 mM) only during the 1 min uptake period. In con-trast, clozapine (0.1 mM) significantly lowered the rate of tyrosineuptakewhen the drugwas present only during the 1 min uptake period(Fig. 3).
4. Discussion
4.1. Validation of the experimental approach
Consistent with our hypothesis, altered tyrosine kinetics in schizo-phrenia could be demonstrated in human fibroblasts without the useof radiolabeled tyrosine. In addition, antipsychotic drug effects not evi-dent during a 1 min exposure became apparent during a 61 min drugexposure. These data are best understood in light of methodologicallimitations and previous studies.
Free intracellular amino levels are dynamically determined by sev-eral simultaneous processes, mainly transport and protein synthesis.Depletion of the amino acid pool, by incubation in an amino acid freemedium, slows amino acid fluxes and simplifies evaluation of theirbasic activity (Gazzola et al., 1980). The standard 60 min incubation inamino acid free medium (Hagenfeldt et al., 1987; Wiesel et al., 1994;Ramchand et al., 1996; Flyckt et al., 2001) typically lowers intracellulartyrosine by ~56% (Hagenfeldt et al., 1987), highly comparable to the61% depletion we achieved (Table 2). The depleted cells are thenexposed to a known concentration of radiolabeled tyrosine for 1 min(Hagenfeldt et al., 1987; Wiesel et al., 1994; Ramchand et al., 1996;Flyckt et al., 2001), a period during which uptake is linear (Gazzolaet al., 1980).
Taking into account intracellular tyrosine levels, the rate of influx(Hagenfeldt et al., 1987) and current analytic techniques (Bongiovanniet al., 2001) we posited that another approach would work equally
ntify dysregulated tyrosine transport in schizophrenia, Schizophr. Res.
Fig. 1. (A) Chromatogram of standard solution containing known amounts of amino acids. (B) Chromatogram of acid soluble contents of cells incubated for 60 min in an amino acidfree medium and then exposed for 1 min to tyrosine (mobile phase organic composition adjusted to 30%). (ILE — isoleucine, LEU — leucine, LYS — lysine, NORVAL — norvaline, PHE —
phenylalanine, TRP — tryptophan, TYR — tyrosine, VAL — valine,).
4 R. Bongiovanni et al. / Schizophrenia Research xxx (2013) xxx–xxx
well. During the first 1 min of uptake, most amino acids that enter thedepleted cell remain as free intracellular amino acids and do not appre-ciably leave the cell or become committed to protein synthesis (Gazzolaet al., 1980). Analogously, protein catabolism during such a short periodshould not significantly affect the free intracellular amino acid pool.Under these conditions, the net change in intracellular unlabeled free
Table 2Amino acid levels after 60 min incubation in amino acid free medium.
nmol mg-protein−1 TYR VAL ILE
TCA fraction 3.957 (0.427) 1.464 (0.276) 1.1 (0.PBS fraction 6.277 (0.434) 7.083 (0.359) 2.901 (Total 10.234 8.547 4.001PBS (% Total) 61% 83% 73%
TCA (trichloroacetic acid) fraction = intracellular fraction. PBS fraction = medium fraction. ILexpressed as mean (±SD).
Please cite this article as: Bongiovanni, R., et al., A simplifiedmethod to qua(2013), http://dx.doi.org/10.1016/j.schres.2013.08.041
tyrosine levels should constitute a valid index of tyrosine uptake. Weexposed parallel groups of cells from the same tray either to an aminoacid-free medium (blanks) or to one containing known concentrationsof unlabeled tyrosine. The calculated difference in free tyrosine levelsbetween blanks and other groups was deemed to be the net tyrosineuptake. The uptake was sodium-independent (Fig. 2). One limitation
LEU PHE TRP
263) 2.059 (0.459) 5.988 (1.097) 0.662 (0.120)0.287) 4.213 (0.318) 13.816 (1.684) 1.012 (0.049)
6.272 19.804 1.67467% 70% 60%
E — isoleucine, LEU — leucine, PHE — phenylalanine, TYR — tyrosine, VAL — valine. Data
ntify dysregulated tyrosine transport in schizophrenia, Schizophr. Res.
Fig. 2. Tyrosine (TYR) uptake in cells incubated for 60 min in an amino acid free mediumand then exposed for 1 min to different concentrations of TYR either in regular PBS ormedium in which sodium was replaced by equimolar choline. There were no significantdifferences between the groups (mean ± SD).
Fig. 3. Uptake of tyrosine (TYR) (0.5 mM) in fibroblasts exposed to drugs (10−4 M) for1 min (uptake) or 61 min (depletion + uptake). (***p b 0.001 relative to control, unpairedt-test). (CLZ — clozapine, CPZ — chlorpromazine, HAL — haloperidol). Tyrosine uptake(μmole/min/mg-protein) in controls for CPZ (12.56 ± 1.01), CLZ (9.39 ± 1.02) and HAL(10.41 ± 2.09) did not differ significantly.
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of our study is that we did not conduct parallel uptake studies usingradiolabeled tyrosine, considered to be the golden standard until now.However, our kinetic parameters (Table 3) were highly comparable tothose previously generated using radiolabeled tyrosine and as expected,Vmax was lower in fibroblasts from patients with schizophrenia (Gazzolaet al., 1980; Hagenfeldt et al., 1987; Wiesel et al., 1994; Ramchand et al.,1996; Flyckt et al., 2001). This comparability supports the validity of ourapproach.
Our study also demonstrates that dysregulated tyrosine transportcan be detected in fibroblast cultures generated from skin biopsiesharvested post-mortem.
4.2. Drug effects
The original study on tyrosine transport, in schizophrenia examinedthe effects of several classes of psychotropic drugs (10−9–10−3 M)on the 1 min uptake phase (Wiesel et al., 1994). At a concentrationof 1 mM, the first-generation antipsychotic drugs haloperidol and chlor-promazine completely suppressed tyrosine uptake (Wiesel et al., 1994).More recently, aspartate uptake in human fibroblasts was inhibited by3–48 h exposure to chlorpromazine or clozapine in the 0.05 mM range(Marchesi et al., 2006). While the shortest effective exposure was notdetermined, there was no effect when drugs were present only duringthe 1 min uptake phase (Marchesi et al., 2006). For that reason, we com-pared three groups of cells. Group 1 hadno antipsychotic drug exposure.Group 2 was exposed to antipsychotic drugs (0.1 mM) from the begin-ning of the depletion phase to the end of the uptake period (61 mintotal) and Group 3 drug was exposed to antipsychotic drugs duringthe uptake period only (1 min).
All the drug effects were time-dependent. A 61 min exposure butnot 1 min exposure to the first-generation antipsychotic drugs haloper-idol or chlorpromazine (0.1 mM), increased tyrosine uptake (Fig. 3).This would be consistent with a sub-acute interaction between thedrugs and processes regulating the intracellular pool of free tyrosine.
Table 3Kinetic parameters for tyrosine transport.
Controls Schizophrenia
Km 40.0 ± 22.0 18.9 ± 6.0Vmax 14.2 ± 0.81 11.3 ± 0.72⁎⁎
Kd 1.32 ± 0.57 1.53 ± 0.27
Km (μM), Vmax (μmol min−1 mg-protein−1), Kd (μl min−1 mg-protein−1).⁎⁎ p b 0.01, unpaired t-test.
Please cite this article as: Bongiovanni, R., et al., A simplifiedmethod to qua(2013), http://dx.doi.org/10.1016/j.schres.2013.08.041
In contrast, tyrosine uptake was lowered when the second-generationdrug clozapine (0.1 mM) present only during the 1 min uptake phase.There was no effect, however, when clozapine was present during theincubation and uptake phases (Fig. 3). This suggests an acute interactionbetween tyrosine transport processes and clozapine that then attenu-ates within 60 min.
The pharmacological data have several heuristic limitations. First,changes in the intracellular pool over 1 min are largely determined bytransport (Gazzola et al., 1980). In comparison, a 60 min exposure to an-tipsychotic drugs is sufficient to affect protein synthesis (Cumming et al.,1998). Second, we examined a single drug concentration (0.1 mM). Itwas an order of magnitude below the lowest effective concentration re-ported previously (Hagenfeldt et al., 1987) but still above usual therapeu-tic serum levels (haloperidol 0.05 μM, chlorpromazine 0.1 μM, clozapine1.0 μM)(VanPutten et al., 1991;Ulrich et al., 1998;Mauri et al., 2007). Onthe other hand, in vivo tissue accumulation of antipsychotic drugs canexceed therapeutic serum concentrations by 10–30 fold (Kornhuberet al., 2006). Third, antipsychotic drug effects were examined only incells from controls. Whether cells from patients with schizophreniashow a differential antipsychotic drug response profile remains to be de-termined. Limitations notwithstanding it is noteworthy that clozapinehaddifferent effects thanhaloperidol. Although clozapinewas introducedclinically over 40 years ago, it still retains the distinction of being the onlyagent with consistently demonstrated superior antipsychotic efficacycompared to other first and second-generation antipsychotic drugs(Essali et al., 2009; McEvoy et al., 2006). Future investigations shouldexamine the effect of a wider range of antipsychotic drug doses anddrug types in fibroblasts harvested from controls as well as patientswith schizophrenia. Similarly, we examined drug effects on tyrosine up-take at a single tyrosine concentration (0.5 mM). It would be of particularinterest to conduct full uptake studies that generate Km or Vmax to deter-mine whether any drugs ‘normalize’ kinetic parameters in fibroblastsfrom patients with schizophrenia. Aberrant tyrosine transport has beenlinked to cognitive deficits both in patients (Wiesel et al., 2005) and innon-affected mothers (Flyckt et al., 2011). Insofar as cognitive deficitsstrongly determine functional outcome in schizophrenia (Green, 1996),tyrosine dysregulationmay both point to an endophenotype of the illnessand provide an assay for testing potential therapeutic agents.
4.3. Conclusions and implications
Since common transporters move tyrosine across cell membranesthroughout the body (Verrey et al., 2004), our data confirm that schizo-phrenia is associatedwith a generalized dysregulation of tyrosine trans-port (Hagenfeldt et al., 1987;Wiesel et al., 1994; Ramchand et al., 1996;
ntify dysregulated tyrosine transport in schizophrenia, Schizophr. Res.
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Flyckt et al., 2001). The precise link between the pathophysiology ofschizophrenia and a lower Vmax for tyrosine transport is not known. Ifmaternal inheritance (Flyckt et al., 2011) is confirmed it could implicateeither an X-linked gene or mitochondrial DNA. The main membranetransporters for tyrosine, however, are known (Vumma et al., 2008)and none are located on the X chromosome. The possibility that an X-linked gene affects tyrosine transport indirectly, however, cannot beprecluded. It has been suggested that aberrant tyrosine kinetics maycontribute to dopamine dysregulation (Hagenfeldt et al., 1987). In pa-tients with schizophrenia, the atypical tyrosine transport in fibroblastsis associated with attenuated competition between serum tyrosine andother amino acids for transport across the blood brain barrier (Wieselet al., 1999). The predicted result would be wider than expected fluctu-ations in brain availability of tyrosine. As the obligatory precursor fordopamine, tyrosine can affect brain dopamine synthesis (Bongiovanniet al., 2008; Bongiovanni et al., 2012; Brodnik et al., 2012) and dopamineefflux in vivo (Jaskiw and Bongiovanni, 2004; Jaskiw et al., 2004, 2005,2008). Brain regional dopamine dysregulation remains the most con-sistently replicated in vivo neurochemical finding in schizophrenia(Howes et al., 2012).
In summary, tyrosine kinetics in human fibroblasts can be studiedwithout the use of radiolabeled tyrosine. Tyrosine transport shows amore complex response to antipsychotic drugs than was previouslyappreciated. These findings deserve to be evaluated in other cell sys-tems. Extension of our technique to cultures of individual-specifichuman neural cells (Benitez-King et al., 2011) or to cells modeling dis-crete molecular features of schizophrenia should facilitate delineationof underlying pathophysiology and promote the development of newtreatments for the disorder.
Role of funding sourceThis researchwas supported byMERIT grant 1 I01 BX000381-01 awarded to GEJ from
the Office of Research and Development, Medical Research Service of the Departmentof Veterans Affairs (DVAMC) as well as a Research Initiative Grant to GEJ from VISN 10DVAMC. No funding source played any role in the collection, analysis, interpretation orpublication of data.
ContributorsGEJ and RB designed the protocol. SL harvested and banked the tissue and conducted
the chart review. RB collected the data. GEJ wrote the first draft of the manuscript. Allauthors contributed to, reviewed and approved the final manuscript.
Conflict of interestThe authors confirmed that there are no known conflicts of interest associated with
this publication and there has been no significant financial support for this work that couldhave influenced its outcome.
AcknowledgmentThe authors thank Tosha Vann for her technical assistance in maintaining the cell
cultures.
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