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µM HEPES, pH 7.4, 145 µM NaCl, 5.4 µM KCl, 1.8 µM CaCl2,
0.8 µM MgSO4, 1.0 µM NaH2PO4, 11.2 µM glucose, 20 µM LiCl)
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of ET-1 at different concentrations. Cells were then incubated
for an additional 45 min. The buffer was discarded, and the
accumulated inositol phosphates were extracted with ice cold
methanol and measured according to the method of Berridge.
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Pierce BCA assay after solubilizing the cells in 0.1 M NaOH.
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Tris base or sodium bicarbonate). Thirty minutes later
animals were challenged by intravenous bolus administration
of ET-1 (1 mg/kg, control). Ninety minutes later, when the
mean arterial pressure (MAP) had returned to baseline,
antagonist was administered by intravenous bolus injection
followed 30 min later by a second ET-1 challenge. The degree
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100 × (controlmax. pressor response
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P h a r m a cok in etics in Ra ts. All surgical procedures were
performed under aseptic conditions. Adult Sprague-Dawley
rats were anesthetized with a ketamine-based anesthetic
(containing ketamine, xylazine, and promace), and the jugular
vein was cannulated. The cannula were channeled under the
skin, exteriorized between the scapulae, and protected by a
spring tether (BioResearch, Montreal, Quebec). Rats were
allowed to recover for 48 h prior to use. The compound, 60
mg/kg, was administered at a dose of 1 mL/kg iv or 10 mL/kg
po, and serial blood samples (100 µL) were withdrawn from
the caudal vein over the next 24 h. Blood samples were
immediately centrifuged and the plasma frozen and stored at
-20 °C until assay. Samples were assayed by HPLC following
extraction into acetonitrile.
Ack n ow led gm en t. We acknowledge the support
and helpful scientific discussions with Drs. Richard A.
F. Dixon, Timothy P. Kogan, Tommy Brock, and Sey-
mour Mong. We thank Karin M. Keller for some of the
HPLC and mass spectrometry work. We thank Drs. Erik
J . Verner, Adam Kois, and Yalamoori Venkatachalap-
athi for helpful discussions.
(20) Doherty, A. M.; Patt, W. C.; Edmunds, J . J .; Berryman, K. A.;
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Cheng, X. M.; Walker, D. M.; Haleen, S. J .; Keiser, J . A.; Flynn,
M. A.; Welch, K. M.; Hallak, H.; Taylor, D. G.; Reynolds, E. E.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR, IR, and
high-resolution mass spectral data for compounds 5a -z, 5a a -
p p , 7-11, and 19a ,b (10 pages). Ordering information is given
on any current masthead page.
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