C. Trapella et al. / Bioorg. Med. Chem. 14 (2006) 692–704
703
experiments were performed as previously described.41
The mouse vas deferens and guinea pig ileum tissues
were suspended in 5 mL organ baths containing Krebs
solution. For mouse vas deferens experiments the bath
temperature was set at 33 ꢁC and the Krebs solution
was Mg2+ free, while for guinea pig ileum experiments
the bath temperature was set at 37 ꢁC. The tissues were
stimulated through two platinum ring electrodes with
supramaximal rectangular pulses of 1 ms duration and
0.05 Hz frequency. The resting tension was maintained
at 0.3 g. The electrically evoked contractions were mea-
sured isotonically by means of a Basile strain gauge
transducer and recorded with a PC-based acquisition
system (Autotrace, RCS, Florence, Italy). After an
equilibration period of about 1 h, the contractions in-
duced by electrical field stimulation were stable. At this
time, cumulative concentration–response curves to N/
OFQ or J-113397 derivatives were performed (0.5 log
unit steps). When tested as antagonists the compounds
were added to the Krebs solution 15 min before per-
forming the concentration–response curve to N/OFQ.
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9.2. Receptor binding and stimulation of GTPcS binding
to human recombinant receptors
The binding of Trap-101 to recombinant human NOP,
MOP, DOP, and KOP expressed in Chinese hamster
ovary cells was assessed by the displacement of either
a fixed concentration of [3H]N/OFQ (for NOP) or
[3H]diprenorphine (for MOP, DOP, and KOP) and
increasing concentrations of Trap-101 as previously de-
scribed.54 Upstream functional antagonism of Trap-101
was assessed in a standard GTPc35S binding protocol54
using 3, 30, and 300 nM Trap-101 against N/OFQ.
The pharmacological terminology adopted in this study
is in line with IUPHAR recommendations:55 the agonist
potencies are given as pEC50 = the negative logarithm to
base 10 of the molar concentration of an agonist that
produces 50% of the maximal possible effect. Antagonist
potencies have been evaluated using the Gaddum Schild
equation: pA2 = log ((CRÀ1)/[antagonist]) assuming a
slope value equal to unity. In receptor binding studies,
the concentration of displacer producing 50% displace-
ment was corrected for the competing mass of radiolabel
to yield Ki according to Cheng and Prusoff.56
Acknowledgments
We thank Professor M. Recanatini for critical reading of
the manuscript and we are very grateful to Professor G.
P. Pollini for useful and stimulating discussions. This
work was financially supported by the University of
Ferrara (60% grant to S.S. and G.C.), the Italian Minis-
try of University (FIRB 2001 grant to D.R. and S.S.),
and by the National Institute of Health (RO1HL71212
grant to D.R.).
20. Marti, M.; Stocchi, S.; Paganini, F.; Mela, F.; De Risi, C.;
CaloÕ, G.; Guerrini, R.; Barnes, T. A.; Lambert, D. G.;
Beani, L.; Bianchi, C.; Morari, M. Br. J. Pharmacol. 2003,
138, 91–98.
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M. Neuroscience 2002, 109, 349–358.
22. Jennings, E. A. Neuroreport 2001, 12, 645–648.
23. Vaughan, C. W.; Connor, M.; Jennings, E. A.; Marinelli,
S.; Allen, R. G.; Christie, M. J. J. Physiol. 2001, 534, 849–
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