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at a volume of 5 ml/kg. Compounds were formulated in
methylcellulose 0.5%. Vehicle-treated controls rats
received the same volume of vehicle. Rats were sacrificed
by decapitation at various times after dosing (0, 10, 30,
120 minutes). Blood samples were collected in Sarstedt Z
tubes, allowed to clot for 20 minutes and centrifuged (3000
rpm, Jouan CR422) at 4°C. Serums were removed and
stored at -20°C until assayed. Serum PTH was quantified
according to the provider’s instructions using a rat IRMA
kit (Immutopics, 50-2000).
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15. The effect starts to be significant at 10 minutes post
dose, even with a drop in the control group at 10
minutes possibly due to the effect of stress on PTH level
after oral gavage. At 30 minutes and 2 hours, the
vehicle group is back to normal range and PTH level
remains very low in the treated group with compound
46.
12. The human parathyroid cell Ca2+ receptor cDNA was
subcloned into the mammalian expression vector PECE
(Ref. 1). The luciferase reporter was subcloned into the
mammalian expression vector pGL3basic (Promega).
Resistance to neomycin (pSV2-neo) and resistance to
puromycin (pSG5-puro) were used as selection markers.
All these plasmids were simultaneously transfected into
CHO cells by calcium phosphate precipitation. Transfected
cells were grown in F12 medium containing 7.5% foetal
bovine serum, 100 U/ml penicillin and 100 µg/ml
streptomycin (as 1% Pen-Strep, BioWithaker), neomycin
(750 µg/ml) and puromycin (5 µg/ml). Neomycin and
puromycin resistant colonies were subcloned and assayed
for activation against a range of calcium concentration.
Clone 8-5-5 was used to assess the effects of compounds
on [Ca2+]i. This stably transfected cell line is termed ET8-
5-5.
16. Deprez, P.; Temal, T.; Jary, H.; Auberval, M.; Lively,
S.; Guédin, D.; Vevert, J.-P. Bioorg. Med. Chem. Lett.
2013, 23, submitted.
For measurements of [Ca2+]i, the cells were recovered
from tissue culture flasks by brief treatment with Trypsin-
EDTA (Invitrogen; containing 0.53 mM EDTA•4Na in
HBSS) and then seeded in Culture treated 96-well plates
(Greiner) at 50K cells per well in the growth media (same
as above, except neomycin 400 µg/ml). Cells were grown
in 37°C TC incubator for 24 hours. The culture medium
was then removed and replaced with F12 medium, 1%
Pen-Strep for an overnight foetal bovine serum starvation
in 37°C TC incubator. Then the starvation medium was
removed and replaced with a test buffer (20 mM HEPES
pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5.5 mM
Glucose,
supplemented with
2
g/L lysosyme and 0.3 mM CaCl2)
range of test compound
a
concentrations crossed against a superadded range of
CaCl2 concentrations. The cells were incubated with the
test compounds for 5 hours in 37°C TC incubator. Then
the test buffer was discarded, and cells were added with
the substrate for Luciferase from SteadyLite Kit (Perkin-
Elmer).The luminescence was recorded.
13. in vivo mesurements : Overnight- fasted male Sprague-
Dawley rats (200-220g BW, CERJ, France) were orally
dosed by gavage with calcimimetics (30 mg/kg) or vehicle