A novel, quantitative bio-assay for cholecystokinin type-1 receptor activity in the anaesthetised rat.
BACKGROUND
Cholecystokinin type-1 (CCK(1)) receptors mediate many of the physiological functions of CCK including delay of gastric emptying, pancreatic enzyme secretion, intestinal motility and gallbladder contractility. Existing in-vivo assays for the quantitative measurement of CCK(1) receptor mediated function are generally variable, limited in precision and require a relatively large number of animals to obtain statistically meaningful data. We found that they did not provide robust pharmacokinetic-pharmacodynamic data for profiling compounds acting at these receptors. Accordingly, here we describe a novel rat duodenal contractility assay that addresses these problems.


METHODS
Rats were anaesthetised and a saline-filled balloon was inserted through the body of the stomach and secured in the duodenum approximately 1 cm from the pyloric sphincter for measurement of intra-lumenal pressure. Studies were performed to determine a dose, rate and frequency of administration of CCK8S that produced a readily quantifiable response.


RESULTS
Initial experiments revealed that sustained exposure to CCK8S resulted in the rapid development of tachyphylaxis. After investigating different dosing paradigms, it was found that pulsatile delivery of CCK8S (intravenous infusion for 1 min every 10 min) produced a readily quantifiable contractile response that did not exhibit tachyphylaxis. The assay response output was defined as the number of contractions >5 mm Hg over baseline. The contractions were blocked in a dose-dependent manner by intravenous bolus injections of the CCK(1) receptor antagonists, dexloxiglumide (2 and 20 micromol/kg), and devazepide (3-100 nmol/kg) but not by the CCK(2) receptor antagonist gastrazole (10 micromol/kg).


CONCLUSIONS
A novel, simple, high quality assay for the quantification of the in-vivo activity of CCK(1) receptor ligands is described. CCK8S delivered by pulsatile intravenous infusion to anesthetized rats produced a burst of contractile activity of the duodenum mediated by CCK(1) receptors. This activity was highly reproducible and sustained for more than 3 h providing an assay that circumvents problems associated with agonist-induced tachyphylaxis.
Freedman
Jamie M
JM
Physiological Systems, Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.
Barrett
Terrance D
TD
Shankley
Nigel P
NP
D000758:Anesthesia / Q000379:methods*; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004386:Duodenum / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043886:Receptor, Cholecystokinin A / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012844:Sincalide / Q000494:pharmacology
D043886:Receptor, Cholecystokinin A; D012844:Sincalide