J Cardiovasc Pharmacolä Volume 50, Number 4, October 2007
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Dobrydneva et al
compounds with triphenylethylethylene core and novel poly-
mer-conjugated derivatives of 4-hydroxytamoxifen. This study
provides some preliminary information into the mechanism by
which tamoxifen increases [Ca2+]i in platelets and defines the
pharmacophore responsible for tamoxifen action to stimulate
Ca2+ influx into platelets.
[Ca2+]i were performed at room temperature in a SPEX ARCM
spectrofluorometer using excitation wavelengths of 340 nm
and 380 nm and an emission wavelength of 505 nm.
Calibration was performed as previously described.14 In some
experiments, the data was expressed as 340 nm/380 nm ratios.
The level of [Ca2+]i was calculated by using the SPEX dM3000
software. To calculate the percent inhibition of thrombin or
tamoxifen by different analogs, thrombin-induced or tamox-
ifen-induced elevation of [Ca2+]i in the presence of DMSO was
compared with this in the presence of the analog. DMSO up to
1% (v/v) did not affect platelet responses. None of the
compounds used in the study affected fura-2 fluorescence.
MATERIALS AND METHODS
Materials
The following were obtained from Sigma-Aldrich,
St Louis, MO: EGTA, dimethyl sulfoxide (DMSO), tamoxifen,
4-hydroxytamoxifen, diethylstilbestrol, human thrombin, thap-
sigargin, norepinephrine, adenosine diphosphate (ADP), and
arginine vasopressin. HEPES and glucose were from Fisher
Scientific Co., Pittsburgh, PA. U-73122 and U-73343 were
from Alexis Biochemicals, San Diego, CA. ICI 182,780
(fulvestrant) (Fig. 1) was from Tocris. MER-25 (ethamoxy-
triphetol) (Fig. 1) was from Merrell Dow Research Institute.
Ethyl bromide tamoxifen (Fig.1) was generously provided by
Dr. Gregory M. Dick, Department of Cellular and Integrative
Physiology, Indiana University School of Medicine, Indian-
apolis, IN.13 Fura-2/AM was from Invitrogen, Carlsbad, CA.
Synthetic Experimental
Some of the compounds [tamoxifen N-oxide and (Z)-4-
(1,2-diphenyl-1-butenyl) phenol vinyl ether, trivial name des
dimethylamino tamoxifen; (Fig. 1)] were prepared according to
modified procedures published in the literature. The polymer
conjugates of 4-hydroxytamoxifen were synthesized by conju-
gating a 4-hydroxytamoxifen analog containing a diaminoalkyl
linker containing either 2 or 6 carbons15 to a poly(methacrylic
acid) polymer prepared as the N-hydroxysuccinimide—
activated ester.16 After coupling in dimethylformamide for
72 hours at 80°C, the solution was concentrated and treated
with either 2 M NaOH or 2 M NH4OH for 8 hours to convert
the unreacted side chains to either carboxylic acids or
carboxamides, respectively. Neutralization of the base fol-
lowed by extensive dialysis provided the conjugates. Percent
incorporation was determined by comparing the NMR
integration of the polymer methyl group peak with the peak
corresponding to the methyl group on the tamoxifen analog.
Starting materials and solvents were obtained commer-
cially from Sigma Aldrich Chemical, Milwaukee, WI. Thin
layer chromatography (TLC) was performed on Analtech silica
gel plates. Solvent system was ethyl acetate:methanol:NH4OH,
100:10:3 by volume. Melting points were uncorrected. Proton
NMR spectra were obtained on a Varian Unity 200 MHz
spectrometer in solvent CDCl3 as indicated. The chemical
shifts are reported in d values down field from tetramethylsilane
(TMS). Singlets, doublets, triplets, and quartets are reported as
s, d, t, and q. Elemental analyses were obtained from Huffmann
Laboratories Inc., Golden, CO and were within 0.4% of the
theoretical. Mass spectra were obtained by positive electrospray
technology. Samples were dissolved in acetonitrile, an Agilent
Technologies 1100LC/MSD machine was used at Hunter
College Mass Spectrometry Facility, New York, NY.
Blood Donors and Platelet Preparation
All donors were healthy, nonsmoking volunteers (age,
20 to 60 years). Venous blood was collected into 1/10 volume
of (74.8 mM sodium citrate, 38.1 mM citric acid, and 123 mM
dextrose pH 6.4; Baxter Healthcare Corp.). The blood was
centrifuged at 250 3 g for 10 minutes at room temperature to
obtain platelet rich plasma (PRP). The PRP was centrifuged
at 550 3 g for 12 minutes to sediment the platelets. The plate-
lets were then suspended in a modified Tyrode’s physiological
salt solution (NaCl, 145 mM; KCl, 4 mM; MgSO4, 1 mM;
Na2HPO4, 0.5 mM; Na/HEPES, 10 mM; glucose, 6 mM; pH
7.4) containing 1.0 mM EGTA to prevent spontaneous
aggregation during the various experimental manipulations.
Platelet Loading With Fura-2 and
Measurement of [Ca2+]i
Intracellular free calcium ([Ca2+]i) was measured using
the fluorescent dye fura-2. Platelets were incubated with cell
permeant fura-2/AM (2 mM) for 1 hour at room temperature.
Excess fura-2/AM was removed by centrifugation (500 3 g for
10 min), and the platelets were suspended in a modified
Tyrode’s buffer, without added EGTA. Aliquots of platelet sus-
pension (0.5 mL) were added to 1.0-mL aggregometer tubes
containing a Teflon-coated stirrer bar (CHRONO-LOG,
Havertown, PA). To measure intracellular Ca2+ mobilization,
no Ca2+ was added to the platelet suspension; test compounds
were added at approximately 10 seconds. To evaluate Ca2+
influx, at approximately 30 seconds before [Ca2+]i measure-
ments were performed, Ca2+ was added to the buffer to a final
concentration of 2.0 mM, then test compounds dissolved in
DMSO (various concentrations in 2.5 mL) were added. In
some cases, thrombin was added after test compounds were
added to evaluate their ability to either inhibit or potentiate the
ability of thrombin to increase [Ca2+]i. The measurements of
Preparation of Tamoxifen-N-oxide
Tamoxifen N-oxide (Fig. 1) was synthesized by the
following procedure. Tamoxifen, (0.3715 g, 0.001 mol) was
taken in 30 mL of methanol and stirred with warming at 50°C
to 60 °C for 3 hours with 3 mL of H2O2 (35 % by weight) and
left overnight. The reaction mixture was processed by chilling
and adding water. A white precipitate (0.165 g, 43% yield)
was obtained by filtration; mp 81°C to 82°C. Mass spectrum
by electrospray showed a peak, m/e 388 calculated for
C26H29NO2. Proton NMR spectrum (CDCl3) showed a mul-
tiplet at d 7.32 -7.05 (10 H, aromatic); a pair of doublets
were obtained, centered at 6.75 and 6.51 (J = 8.8 Hz, 4H,
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q 2007 Lippincott Williams & Wilkins