1546
C. Dallanoce et al. / Bioorg. Med. Chem. 7 (1999) 1539±1547
contractions were measured isometrically (Basile Mod.
7080 Italy) and recorded on a two-channel recorder
(Basile Mod.7070). Throughout the experiments 1 mM
yohimbine was added to the buer solution to block
a2-adrenoceptors.
Acknowledgements
We thank G. Domenichini for his excellent technical
assistance. Financial support from MURST (Ministero
della Ricerca Scienti®ca e Tecnologica), Rome is grate-
fully acknowledged.
Guinea pig left atrium. Heart was rapidly dissected and
right and left atria were separated out. As described by
Eglen et al.,25 left atrium was suspended under 0.5 g
tension in a 20 mL organ bath and modi®ed Krebs±
Henseleit buer solution (mM composition: NaCl
118.9, KCl 4.6, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25,
MgSO4.7H2O 1.2, glucose 11.1) gassed with carbogen
(95% O2±5% CO2) at 32ꢀC, served as bath ¯uid. The
tissue was electrically-paced by rectangular submaximal
impulses (2 Hz, 5 ms, 5 V) by using platinum electrodes.
After a 30 min stabilization period, inotropic responses
were measured as changes in isometric tension.
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Protocols. In all the experiments complete concentra-
tion±response curves to the compounds under study
were obtained in a cumulative fashion by increasing the
concentration of the agonists by 0.3 log units (in the
range from 0.03 nM to 100 mM) following the method
described by Van Rossum.29 Oxotremorine and oxo-
tremorine-M were used as reference drugs and McN-A-
343 as a conventional M1 agonist. Derivative 9a was
also investigated as antagonist against oxotremorine in
guinea pig atria and McN-A-343 in rabbit vas deferens
(in the range 10±100 mM).
The apparent potency of the agonists was expressed
as their pD2 value ( log of the molar concentration
which gives 50% of the maximum response) calculated
by linear regression using the least squares method.
Intrinsic activity (a) was determined by comparing the
maximum response to the reference drugs with that to
the test compounds. In the antagonism studies, the
pKB values were calculated as indicated by Furchgott30
and pA2 values were obtained by Schild analysis.31
All the data are presented as means SEM (n=8)
and signi®cant dierences were assessed by use of Stu-
dent's t test, with a value of P<0.05 being considered
signi®cant.
In vivo experiments. All the experiments were performed
between 9 and 12 a.m. and the drugs, dissolved in saline,
were given intraperitoneally at dierent doses to groups
of eight mice in a volume of 1 mL/100 g body weight.
The animals had access to food and water ad libitum
before the experiment. Following drugs or saline
administration, tremor was scored as present or not
present during a period of 10 min. ED50 values (i.e. the
doses required to produce tremor in 50% of the ani-
mals) were calculated by means of probit analysis.32
26. Barocelli, E.; Chiavarini, M.; Ballabeni, V.; Bordi, F.;
Impicciatore, M. Br. J. Pharmacol. 1993, 108, 393.