Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G Protein, Gα_s^*,♦

Gradual Accumulation of Antidepressant Drugs in Plasma Membrane Microdomains Is Independent of SERT

Although there are many potential targets for monoamine-centric drugs, none offer an explanation for the hysteresis (6–8 weeks) between initiation of therapy and clinical efficacy. C6 glioma cells were used in these experiments because they do not express monoamine transport proteins (Fig. 1), yet sustained treatment with antidepressant drugs translocates Gα_s from lipid rafts to non-raft regions of the plasma membrane (7, 8, 20). Furthermore, glia may contribute to both the etiology and the treatment of depression (21, 22).

One binding site for many of the antidepressant drugs (e.g. tricyclic antidepressants and SSRIs) is the serotonin reuptake transport protein (SERT). To determine whether SERT expression affects the redistribution of Gα_s, C6 cells stably expressing Gα_s-GFP were engineered to also express SERT.

Gα_s-GFP is identical in its activation by GPCR and activation of adenylyl cyclase with wild type Gα_s (23). When kept to a moderate level of expression (2–3× endogenous Gα_s), Gα_s-GFP is transparent to cellular physiology, allowing a window on its movements in response to treatment with antidepressants (Gα_s moves out of lipid rafts) (24).

Gα_s-GFP translocation from lipid rafts in response to escitalopram, as measured by fluorescence recovery after photobleaching (FRAP), does not improve upon expression of hSERT (Fig. 1, B and C, respectively). Moreover, HEK cells do not respond to chronic stimulation with escitalopram, nor when stably transfected with hSERT (Fig. 1, D and E, respectively). These data suggest that some additional cellular component is responsible for the redistribution of Gα_s, and we hypothesized that the antidepressant drugs accumulate, gradually, in lipid rafts.

Gradual Accumulation of Antidepressant Drugs in Plasma Membrane Microdomains Correlates with Gα_s Subcellular Redistribution

Due to our earlier observations that Gα_s translocates from rafts after extended exposure to antidepressants (8), and the observations by Rupprecht and colleagues (25), we hypothesized that antidepressants preferentially associate with lipid rafts, and that all monoamine-centric antidepressants share this property. We assessed the accumulation of representative drugs from each antidepressant class: MAO inhibitor (phenelzine), tricyclic antidepressant (desipramine, imipramine, and amitriptyline), and SSRI (escitalopram/inactive stereoisomer R-citalopram and fluoxetine), as well as the atypical antipsychotic aripiprazole, which has some independent antidepressant properties, and the atypical antipsychotic, olanzapine, which does not (26). We expected that antidepressants would gradually accumulate in raft fractions of C6 cells over time to mediate the translocation of Gα_s out of the lipid raft and that the non-antidepressant compounds tested would not have this property.

Lipid raft fractions were isolated via sucrose density gradient, and the UV-visible spectrum was taken for each fraction. Each antidepressant absorbs at a characteristic wavelength for which measurements were normalized to protein content in the sample and the -fold change was calculated relative to treatment-naive or untreated controls. The accumulation of drugs over time in lipid raft fractions of the plasma membrane appears to be a class-specific mechanism (e.g. SSRIs and MAO inhibitors) (Fig. 2A). Olanzapine, an antipsychotic lacking primary antidepressant properties (27), appears to accumulate as well, which may be due to its highly hydrophobic nature (Fig. 2B). However, analysis of raft fractions by GC-MS revealed that accumulation of drugs in rafts is independent of hydrophobicity, as olanzapine and aripiprazole do not accumulate.

Escitalopram, and its therapeutically inactive enantiomer, R-citalopram, were selected for further investigation. To parallel the experiments by Eisensamer et al. (25), escitalopram was added to sucrose density gradients prepared from membrane fractions. Escitalopram, but not R-citalopram, associated with lipid raft fractions of the plasma membrane (Fig. 2C). To minimize background measurements as much as possible for this method of detection, we normalized the readings to protein (280 nm) and subtracted the control absorbance. These measurements were then corroborated via mass spectrometry.

GC-MS is sensitive and selective, due in large part to the separation efficiency achieved in the analysis of small molecules. C6 cells were treated for 72 h with 10 μm of antidepressant, and the lipid raft fraction was extracted for determination of drug presence. Analysis of the total ion chromatograms (TICs) of lipid raft extractions showed that only phenelzine, fluoxetine, and escitalopram accumulated in lipid rafts after 72 h of treatment (Fig. 3); R-citalopram, imipramine, amitriptyline, aripiprazole, and olanzapine did not accumulate, although with the exception of R-citalopram, each of these drugs shows an “antidepressant signature” in translocating Gα_s from lipid rafts. Base peaks of the molecular ion profile from each TIC elution profile were matched to the National Institute of Standards and Technology (NIST) database to identify the drug presence among membrane fractions.

The accumulation of escitalopram, fluoxetine, and phenelzine, but not R-citalopram or olanzapine, parallels their capacity to mediate the movement of Gα_s from lipid rafts (20). Because neither the tricyclic antidepressants (desipramine, imipramine, and amitriptyline) nor aripiprazole accumulate in rafts, this phenomenon may be class-specific for antidepressants. Furthermore, accumulation is independent of the lipophilicity of the compound (Fig. 2B).

Gradual Accumulation of Escitalopram in Plasma Membrane Microdomains Is Time- and Concentration-dependent

The finding that antidepressants, but not other related drugs, demonstrated gradual accumulation, along with the stereospecificity of antidepressant accumulation, led us to select escitalopram for closer analysis. Tracking the accumulation of escitalopram in the lipid raft fraction derived from C6 cells with GC-MS analysis revealed that escitalopram accumulates in a concentration- and time-dependent manner. Detectable accumulation occurred following 1 μm treatments for 72 h or 100 nm treatments for 120 h and at 24-, 48-, and 72-h treatments with 10 μm escitalopram (Fig. 4). Treatment with 10 μm antidepressant for 72 h is a standard assay condition (8) and parallels doses used in rat studies (7, 28). However, these drugs translocate Gα_s at concentrations as low as 50 nm over the same period (29).

These measurements are consistent with the drug time and dose required for Gα_s translocation from rafts, measured either directly (8) or by FRAP (20). Moreover, stereo specificity and the fact that escitalopram does not accumulate over time in HEK cells indicates a specific target (Fig. 4C). Furthermore, antidepressant-mediated redistribution of Gα_s independent of SERT suggests a molecular drug-binding target that is distinct from the monoamine transport system. Only a protein target(s) could account for both the enantioselectivity as well as the lack of hydrophobic contribution toward the gradual accumulation of SSRIs in lipid rafts.

Chemicals

DMEM, fetal bovine serum, trypsin, and penicillin/streptomycin were purchased from Sigma-Aldrich. Cell culture flasks were from NUNC (VWR International, West Chester, PA). Escitalopram and R-citalopram were kindly provided from H. Lundbeck A/S, Copenhagen, Denmark. Desipramine hydrochloride and olanzapine were purchased from Tocris Bioscience, Ellisville, MO. Phenelzine sulfate, fluoxetine hydrochloride, amitriptyline hydrochloride, imipramine hydrochloride, and aripiprazole were purchased from Sigma-Aldrich.

Drug Treatments

C6 cells were cultured in DMEM, 4.5 g of glucose/liter, 10% newborn calf serum (HyClone Laboratories, Logan, UT), 100 mg ml⁻¹ bacteriostatic penicillin-streptomycin at 37 °C in humidified 5% CO₂ atmosphere to a confluence of ∼40% before drug treatments were begun. Treatment with 10 μm for 72 h is a standard assay condition (8); however, the effects of these drugs in this cellular system can be observed at concentrations as low as 50 nm after 72 h (20). Culture media and drug were changed daily, and no apparent change in cell morphology was observed during treatment. Before assay, cells were rinsed twice with pre-warmed 1× PBS to remove debris and wash away unbound drugs.

Lipid Raft Isolation

Cells were washed and harvested in ice-cold 1× PBS. Lipid raft fractions were prepared as described previously (38). Briefly, C6 cells were extracted in 1 ml of ice-cold lysis buffer (10 mm HEPES, pH 7.4; 150 mm NaCl; 1 mm DTT; 0.25 m sucrose; 1% Triton X-100; protease inhibitor cocktail). Following a 30-min incubation on ice, the lysates were homogenized, and 1 ml was gently mixed (v/v) with ice-cold 80% (w/v) sucrose in TME (10 mm Tris-HCl; 1 mm MgCl₂; 1 mm EDTA, pH 7.5; 1 mm DTT; protease inhibitors), and then loaded in the bottom of a centrifuge tube. Samples were overlaid by syringe and fine needle with 1 ml each of 30% sucrose, 15% sucrose, and finally 5% sucrose. Sucrose gradients were spun at 40,000 rpm in an SW55-Ti rotor in a Beckman ultracentrifuge at 4 °C overnight for 16–18 h. Lipid rafts exist between 5 and 15% sucrose layers as opaque white clusters. Raft fractions were collected and sucrose was removed via sequential mixing (v/v) in wash buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm DTT) and centrifugation at 21,000 rpm at 4 °C for 20 min (∼4–5 times) until a pellet emerged. Lipid raft pellets were reconstituted in 0.5 ml of TME buffer. Protein content was determined by absorbance at 280 nm on a NanoDrop UV-visible spectrophotometer.

Accumulation of Antidepressants Measured by UV-visible Spectrophotometer

UV absorbance of antidepressants was used to determine their association with membrane fractions similar to before (25), but in the absence of HPLC purification. C6 cells treated (72 h) with 10 μm escitalopram, R-citalopram, fluoxetine, desipramine, phenelzine, or olanzapine were extracted by Triton X-100/114, and the cytosolic, non-raft membrane, and lipid raft fractions were analyzed by UV absorbance and normalized to protein content (λ = 280 nm).

Furthermore, 500-μl sucrose density gradient fractions were incubated with a final concentration of 10 μm escitalopram or R-citalopram. S- and R-citalopram absorbance (λ = 238 nm) in each fraction was assessed before and after incubation, measurements were normalized to protein, and the -fold change was reported.

Drug Hydrophobicity

Partition coefficients of drugs were determined as described previously (39) in a 1:1 v/v octanol to double-distilled H₂O, and the UV-visible spectrum was recorded for each phase. Absorbances are as follows: phenelzine (256 nm), desipramine (252 nm), imipramine (295 nm), amitriptyline (262 nm), fluoxetine (226 nm), citalopram (238 nm), aripiprazole (298 nm), or olanzapine (270 nm).

Gas Chromatography Mass Spectrometry (GC-MS)

Analyses were performed using an Agilent HP-6890 gas chromatograph, equipped with an Agilent 19091S-602 HP-1MS capillary column (25 m, 0.20 mm, 0.33 μm, 7-inch cage), and interfaced with an Agilent HP-5973 mass-selective detection spectrometer equipped with a Single Flame Ionization Detector, single 100-psi EPC Split/Splitless Injection Ports, 7673C-6890 Auto Sampler: 6890 Control Electronics, 6890 Injector, 100 Position Tray, and 6890 Mounting Bracket. Helium was used as the carrier gas at 1.0 ml min⁻¹ in corrected constant flow mode. Primary oven temperature was programmed at 70 °C for 2 min and increased at 20 °C for min⁻¹ to 230 °C where it was held for 10 min. The front inlet thermal modulator was set to 20 °C higher relative to the primary oven and 18.91 psi. Constant flow injection of 1 μl was used, and inject split mode was changed to Splitless. The injector, transfer line, and ion source temperatures were maintained at 250, 280, and 230 °C, respectively, throughout each analysis. Data acquisition was performed in the full scan mode from m/z 50 to 550 with an acquisition rate of 20 Hz. Molecular ion profiles were matched against the standard mass spectral database of the NIST.

Accumulation of Antidepressants Measured by GC-MS

The accumulation of antidepressants in lipid rafts and non-raft membranes of C6 glioma cells was measured via GC/MS to accompany results obtained via increases in the UV absorbance spectrum for escitalopram as opposed to R-citalopram. C6 cells were treated (72 h) with 10 μm escitalopram, R-citalopram, fluoxetine, desipramine, imipramine, amitriptyline, aripiprazole, phenelzine, or olanzapine. More elaborate concentration and temporal measurements were restricted to escitalopram. The accumulation of increasing concentrations, from 10 nm to 10 μm, of escitalopram over 72 h, as well as temporally from 3 to 120 h with 10 μm escitalopram was measured in lipid raft and non-raft membrane; R-citalopram served as the control.

Cells were collected, and membranes were fractionated into Triton X-100-soluble and Triton X-114-soluble fractions. Extraction of accumulated antidepressant drugs in lipid rafts (Triton X-114 fraction) may be assessed on large volume samples as described previously (40), but is not appropriate for the small volumes used here. Membrane fractions were chloroform-methanol precipitated as described previously (41), and the water, chloroform, and methanol phases vacuum were centrifuged to recover accumulated drug. Desiccant was dissolved into 1 ml of methanol, and then injected directly onto a GC Capillary Column (Agilent J&W HP-1ms), interfaced with an Agilent HP-5973 mass-selective detection spectrometer. Finally, quantitation of accumulated drug was performed by the internal standard method. Peak-area ratios were determined for the controls. Drug concentrations were calculated from the standard curve values. All data were acquired and analyzed by Agilent software, Enhanced G1701BA ChemStation version B.00.00. Molecular ion profiles were matched against the standard mass spectral database of the NIST.

FRAP

C6 glioma cells stably expressing hSERT were a kind gift from Dr. Kim Neve, Oregon Health & Science University (OHSU). These cells were further transfected with GFP-Gα_s, and cells expressing the fluorescent construct were selected with G418. Cells were plated on glass microscopy dishes and treated with 10 μm escitalopram or desipramine for 3 days. For imaging, drug was washed out for 1 h prior, and medium was replaced with low serum (2.5% newborn calf serum) phenol red-free DMEM to limit fluorescent background. Temperature was maintained at 37 °C using a PeCon temperature-controlled stage during imaging. Imaging utilized a Zeiss LSM 710 confocal microscope at 512 × 512 resolution with an open pinhole to maximize signal but minimize photobleaching. One hundred fifty data points, ∼300 ms apart (including 10 pre-bleach values), were measured for each cell. Zeiss Zen software was used to calculate FRAP recovery half-time utilizing a one-phase association fit, correcting for total photobleaching of the analyzed regions.

Western Blotting

Western blots were conducted according to standard protocols with a rabbit polyclonal anti-SERT (1:1,000; EMD Millipore, Billerica, MA, catalogue number AB9726), rabbit polyclonal anti-caveolin 1 (1:10,000; BD Biosciences, catalogue number 610059), and rabbit monoclonal anti-β-tubulin (1:5,000; made in-house).

Statistical Analysis

All measurements are presented as the mean (a minimum of n = 3) ± S.E. Calculation error was propagated throughout each calculation. We further subjected each data set to statistical analyses using GraphPad Prism (version 5.0), using a one-way analysis of variance followed by a post hoc Student's t test (two groups) or Dunnett's t test (multiple groups) (95% confidence interval).