A. M. Palmer et al. / Bioorg. Med. Chem. 16 (2008) 1511–1530
1529
1976, 97, 401–414; Berglindh, T.; Obrink, K. J. Acta
Acknowledgments
Physiol. Scand. 1976, 96, 150–159). After the gastric
glands were washed several times, they were suspended
in Krebs-Henseleit solution containing 2 mg/ml rabbit
serum albumin and 2 mg/ml glucose. Glands were incu-
bated for 30 min at 37ꢁC in a shaker bath (200 osc/min)
in the presence of 0.125 lM 14C-AP (113 lCi/lmol) at
pH 7.4. Glands were stimulated with 1 mM dibutyryl
cAMP in absence or presence of the corresponding
inhibitor (concentration range 3 nM–100 lM). The reac-
tion was stopped by centrifugation (10 s at 20,000g).
After centrifugation, the accumulation of 14C-AP in
the glands was calculated as follows: radioactivity was
measured in an aliquot of the supernatant (200 ll) and
in the precipitate after dissolution in 1 ml of 1 N NaOH.
In order to calculate the amount of protein, the Eppen-
dorf tubes were weighed empty, with protein (wet
weight) and with freeze-dried protein (dry weight). This
ratio of supernatant and pellet protein radioactivity was
used to calculate the accumulation of 14C-AP in the
glands. The inhibitor concentration required to achieve
50% inhibition (IC50) of 14C-AP accumulation was
determined by fitting the equation for the expected inhi-
bition pattern to the data points.
We are grateful to Ms. C. Jecke, Mr. B. Grobbel,
Ms. M. David, Ms. I Hartig-Beecken and Ms. B. Bo¨sse-
necker for their technical assistance.
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