538
G. Caliendo et al. / Bioorg. Med. Chem. 8 (2000) 533±538
30 vol (w/v) ice-cold 50 mM Tris±HCl buer (pH 7.7 at
25 ꢀC) using a Polytron PT10 (setting 5 for 20 s).
Homogenates were centrifuged twice for 10 min at
50,000 g with resuspension of the pellet in fresh buer.
The ®nal pellet was resuspended in 50 mM ice-cold
Tris±HCl containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 10 mM
pargyline (pH 7.1 at 37 ꢀC). Each assay tube contained
50 mL [3H]spiroperidol to achieve a ®nal concentration
of 0.4 nM and 900 mL resuspended membranes (3 mg
fresh tissue). The tubes were incubated for 15 min at
37 ꢀC and the incubation was terminated by rapid ®l-
tration under vacuum through Whatman GF/B glass
®ber ®lters. The ®lters were washed three times with
5 mL ice-cold 50 mM Tris±HCl buer (pH 7.7 at 25 ꢀC).
The radioactivity bound to the ®lters was measured by a
liquid scintillation counter. Speci®c [3H]spiroperidol
binding was de®ned as the dierence between binding in
the absence or in the presence of 1 mM (+)-butaclamol.
the incubation was terminated by rapid ®ltration under
vacuum through Whatman GF/B glass ®ber ®lters. The
®lters were washed three times with 5 mL ice-cold 50
mM Tris±HCl, 0.5 mM EDTA buer (pH 7.5 at 25 ꢀC).
The radioactivity bound to the ®lters was measured by a
liquid scintillation counter. Speci®c [3H]yohimbine
binding was de®ned as the dierence between binding in
the absence or in the presence of 10 mM phentolamine.
Acknowledgements
This work was supported by a grant from Regione
Campania ai sensi della L. R. 31 dicembre 1994, No. 41,
art. 3, 1ꢀ comma. The NMR spectral data were pro-
vided by Centro di Ricerca Interdipartimentale di Ana-
lisi Strumentale, Universita degli Studi di Napoli
``Federico II''. The assistance of the sta is gratefully
appreciated.
ꢀ1-Adrenergic binding assay. The procedure used in the
radioligand binding assay has been reported in detail by
Greengrass and Bremner.14 Brain cortex was homo-
genized in 30 vol (w/v) ice-cold 50 mM Tris±HCl buer,
(pH 7.2 at 25 ꢀC) using a Polytron PT10 (setting 5 for
20 s). Homogenates were centrifuged twice for 10 min at
50,000 g with resuspension of the pellet in fresh buer.
The ®nal pellet was resuspended in 50 mM ice-cold
Tris±HCl, (pH 7.4 at 25 ꢀC). Each assay tube contained
50 mL drug solution, 50 mL [3H]prazosin to achieve a
®nal concentration of 0.4 nM, and 900 mL resuspended
membranes (10 mg fresh tissue). The tubes were incu-
bated for 30 min at 25 ꢀC and the incubation was ter-
minated by rapid ®ltration under vacuum through
Whatman GF/B glass ®ber ®lters. The ®lters were
washed three times with 5 mL ice-cold 50 mM Tris±
HCl, buer (pH 7.2 at 25 ꢀC). The radioactivity bound
to the ®lters was measured by a liquid scintillation
counter. Speci®c [3H]prazosin binding was de®ned as
the dierence between binding in the absence or in the
presence of 10 mM phentolamine.
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for 20 s). Homogenates were centrifuged three times for
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fresh buer. The ®nal pellet was resuspended in 50 mM
ice-cold Tris±HCl, 0.5 mM EDTA (pH 7.5 at 25 ꢀC).
Each assay tube contained 50 mL drug solution, 50 mL
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