Targeting Cell Surface Receptors
A R T I C L E S
3%/methanol 2%. This gave 0.6 g (51%) of 1-[3-(2-aminoethyl)-1H-
indol-5-yloxyl]-3,6-dioxa-8-(4-methoxybenzylthio)octane as a pale yel-
low oil.
siently transfected into HeLa cells and grown at 37° in a 5% CO2
humidified incubator. Cells were maintained in complete medium
containing Dulbecco’s minimal essential media (DMEM), 250 mg/L
G418sulfate, 10% fetal bovine serum, 2 mM l-glutamine, 100 units/
mL penicillin, and 100 µg/mL streptomycin and grown on poly(D-
lysine)-coated 24-well plates. Cells were plated at a density of 100 000
cells/well and allowed to grow to a confluency for 2 days before
assaying. The cells were assayed for tritiated serotonin transport in
Krebs-Ringer-HEPES assay buffer as previously described.32 Briefly,
cells were washed in assay buffer (120 nM NaCl, 4.7 mM KCl, 2.2
mM CaCl2, 1.2 mM MgSO4 1.2 mM KH2PO4 10 mM HEPES, pH
7.4), followed by a 10-min preincubation in 37 °C assay buffer
containing 0.18% glucose. Cells were incubated in assay buffer for 10
min at 37 °C with varying concentrations of unconjugated or conjugated
nanocrystals with 20 nM tritiated serotonin (110 Ci/mmol), 100 µM
pargyline, and 100 µM ascorbic acid. Assays were terminated by
washing three times with ice-cold assay buffer. The cells were dissolved
in OptiPhase scintillation fluid, and accumulated tritiated serotonin was
quantified by liquid scintillation counting using a Wallac Microbeta
plate reader. Specific uptake was determined by subtracting data from
experiments in which cells were treated with 0.5 µM paroxetine, a high-
affinity SERT antagonist. The estimated half-maximal (EC50) values
were derived using a nonlinear least-squares curve fit (Kaleidagraph,
Synergy Software). Experiments were performed in duplicate or
triplicate and repeated in two or more separate assays. Means were
compared using a two-sided Student t-test (Graph-Pad InStat for
MacIntosh).
Electrophysiology Measurements. Xenopus Oocyte Electrophysi-
ology. Plasmids (pBluescript-SKII) containing the human serotonin
receptor 5HT3 or hSERT cDNA were linearized with NotI and
transcribed with the mMessage Machine T7 In Vitro Transcription Kit
(Ambion, Austin, TX). The hSERT coding region is flanked 5′ by alfalfa
mosaic virus and 3′ by Xenopus â-globin UTRs. Stage V and VI oocytes
were harvested, defolliculated, and injected with 40 ng of 5HT3 or 24
ng of hSERT cRNA.33-35 The oocytes were maintained at 18 °C for
18 days, in Ringer’s media (in mM: 96 NaCl, 2 KCl, 5 MgCl2, 5 Hepes;
pH 7.6) supplemented with 0.6 mM CaCl2, 5% dialyzed horse serum
(GIBCO-BRL/ Life Technology), 50 µg/mL tetracycline, 100 µg/mL
streptomycin, and 550 µg/mL sodium pyruvate.
Electrophysiologoical experiments were performed in the modified
cut-open oocyte voltage clamp configuration.36 A key element of this
technique is that ∼10% of the total oocyte surface is electrically isolated,
so that the transmembrane potential can be controlled, while both faces
of the membrane are also accessible for treatment with experimental
solutions. The apparatus was purchased from Dagan Corp. (Min-
neapolis, MN), and voltage protocols were applied with a Dagan CA-
1B amplifier. Data were digitized with a DigiData 1200 Interface (Axon
Instruments, Foster City, CA) and collected by a PC running pClamp
v. 7.0 software (Axon Instruments). The external membrane of the
oocyte was superfused at ∼1 mL/min with Ringer’s media containing
100 µM CaCl2, 100 µM pargyline, 100 µM ascorbic acid, 200 µM
niflumic acid, and the concentration of serotonin or LSNAC indicated.
The internal face of the oocyte was perfused at 20-80 µL/min with
internal oocyte solution (in mM: 50 KCl, 5 NaCl, 50 KOH, 70
methanesulfonic acid, 10 Hepes, 2 MgCl2, 0.1 EGTA; pH 7.4). CaCl2
was added such that the final free Ca concentration was 50 nM. Oocytes
were first clamped to -60 mV until a stable baseline was achieved,
∼10 min. For 5HT3, the external serotonin or LSNAC perfusion was
then begun, holding the voltage clamped and acquiring data at 10 Hz.
1-[3-(2-Aminoethyl)-1H-indol-5-yloxy]-3,6-dioxa-8-mercaptooc-
tane. 1-[3-(2-Aminoethyl)-1H-indol-5-yloxyl]-3,6-dioxa-8-(4-methoxy-
benzylthio)octane (0.6 g, 0.0014 mol) was dissolved in trifluoroacetic
acid (15 mL) and cooled to 0 °C. Anisole (1.5 mL) and mercury(II)
acetate (0.516 g, 0.0016 mol) were added, and the mixture was stirred
at 0° C for 2 h. The solution was evaporated, and the resulting solid
was washed with diethyl ether (3 × 50 mL). After air-drying, the solid
was dissolved in glacial acetic acid (25 mL) and hydrogen sulfide was
bubbled through the solution for 30 min. Mercuric sulfide was removed
by filtration, and the solution was evaporated to dryness.30 The resulting
oil was dissolved in dichloromethane and washed with sodium
bicarbonate solution (1 M, 1 × 20 mL). The solution was dried over
magnesium sulfate, filtered, and evaporated. This gave 0.2 g (39%) as
a pale yellow oil: 1H NMR (CDCl3) δ 2.10 (br s, NH2), 2.60 (t, J )
6.45 Hz, 2H), 2.78 (t, J ) 6.84 Hz, 2H), 2.90 (t, J ) 6.15 Hz, 2H),
3.42-3.66 (m, 6H), 3.82 (t, J ) 5.13 Hz, 2H), 4.11 (t, J ) 4.68 Hz,
2H), 6.74 (d, 1 ArH), 6.78 (d, 1 ArH), 6.89 (s, 1 ArH), 6.94 (d, 1
ArH), 7.15 (d, NH); m/z 324.39 [M+], 325.09 [M1+].
Synthesis of Core/Shell Nanocrystals and the Serotonin-Linker
Arm-Nanocrystal Conjugates. TOPO-coated CdSe/ZnS core/shell
nanocrystals (75-Å mean diameter) were synthesized according to the
method of Dabbousi et al.3 from 30-Å cores. TOPO-coated core/shells
had a fluorescent quantum yield of 38%. To attach the serotonin ligand
to the core/shells, the TOPO ligands were first exchanged with pyridine
at 60 °C (60 mg of core/shells in 10 mL of pyridine, stir for 24 h). The
serotonin ligands were dissolved in dicloromethane and added to the
pyridine solution (60 °C). After 2 h, the nanocrystals were cooled to
room temperature and precipitated with hexanes. As covering the entire
surface of the nanocrystal with the serotonin ligand would introduce
stearic interactions between the ligands and interfere with ligand-SERT
interactions, we added mercaptoacetic acid, a short, water-soluble
coligand, to the surface of the nanocrystal. The serotoin-linker arm-
conjugated nanocrystals (LSNACs) were dissolved in equal volumes
of DMF (1 mL) and mercaptoacetic acid (1 mL), and the resultant
mixure was stirred for 24 h at room temperature. Two molar equivalents
of potassium tert-butoxide was then added to this solution to neutralize
the mercaptoacetic acid. The LSNACs were then collected by cen-
trifugation, washed four times with methanol (20 mL), and dried under
reduced pressure. The fluorescent quantum yield of the LSNCs drops
to 3%.
HPLC Measurements. HPLC measurements were performed to
ensure that free serotonin was not present in SNAC solutions or
generated in cell assays. The analytical details for serotonin measure-
ments were previously described by Lindsey.31 The HPLC system
consists of a Waters 501 pump, 712 WISP autosampler, 464 electro-
chemical detector, and Millennium 32 data system. The HPLC column
used was a catecholamine column (3.9 × 100 mm) (Catalog No. C5100)
from Chromosystems. The lower detection limit for serotonin was 2.5
nM.
Rutherford Backscattering Spectroscopy. Rutherford backscat-
tering spectroscopy (RBS) was performed to estimate the extent of
ligand coverage of the LSNACs. An iodinated derivative of the
serotonin ligand was prepared such that iodine could be used as a marker
in the RBS experiment. RBS on nanocrystals was previously de-
scribed.25 In this experiment, samples were prepared by dropping 0.2
mL of a concentrated iodinated LSNACs/pyridine solution onto a 1-cm2
graphite substrate and wicking off excess solution. To obtain the I and
Zn ratios, the individual peaks are integrated and normalized by the
square of their atomic numbers.
(31) Lindsey, J. W. J. Toxicol. EnViron. Health A 1998, 54, 421-429
(32) Barker, E. L.; Perlman, M. A.; Adkins, E. M.; Houlihan, W. J.; Pristupa,
Z. B.; Niznik, H. B.; Blakely, R. D. J. Biol. Chem. 1998, 273, 19459-
19468.
Serotonin Transport Measurements. Human and Drosophila
serotonin transporters (hSERT and dSERT, respectively) were tran-
(33) Goldin, A.L. Methods Enzymol. 1992, 207, 266-279.
(34) Stuhmer, W. Methods Enzymol. 1998, 293, 280-300.
(35) Elsner, H.-A.; Honck, H.-H.; Willmann, F.; Kreienkamp H.-J.; Iglauer, F.
Comp. Med. 2000, 50, 206-211.
(30) Gordon, E.; Godfrey, J.; Delaney, N.; Asaad, M.; Von Langen, D.;
Cushman, D. J. Med. Chem. 1988, 31, 2199-2211.
(36) Costa, A. C. S.; Patrick, J. W.; Dani, J. A. Biophys. J. 1994, 67, 395-401.
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