Simultaneous measurement of cholecystokinin-stimulated amylase release and cholecystokinin receptor binding in rat pancreatic acini.
In the past, isolated-dispersed pancreatic acini have been used to examine either cholecystokinin-stimulated amylase release or pancreatic acinar cholecystokinin receptors. We have developed and validated a method for simultaneous measurement of synthetic cholecystokinin octapeptide-stimulated (CCK8-stimulated) pancreatic amylase release and cholecystokinin receptors. After an 18-hour fast, rats were killed and their pancreatic acini isolated. Three-milliliter aliquots of acinar suspension were incubated for 60 minutes in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Ringer buffer containing graded doses of CCK8 and a constant amount (7 pmol/L) of iodine 125-labeled CCK8 (by the Bolton-Hunter method) ([125I]BH-CCK8). A 1 ml sample was removed from each flask for determination of amylase release, and the remaining 2 ml were used to determine cholecystokinin receptor capacities and affinities. The median effective dose for amylase release was 16 pmol/L, and release was maximal at 100 pmol/L CCK8 plus 7 pmol/L [125I]BH-CCK8, a dose that released 28% +/- 3% of total cellular amylase content. High affinity (equilibrium dissociation constant of high-affinity receptors = 58 +/- 8 pmol/L, receptor density of high-affinity receptors = 4 +/- 1 fmol/mg protein) and low affinity (equilibrium dissociation constant of low-affinity receptors = 7 +/- 2 nmol/L, receptor density of low-affinity receptors = 313 +/- 108 fmol/mg protein) cholecystokinin receptors were measured. The results demonstrate that CCK8-stimulated amylase release and cholecystokinin receptor binding in pancreatic acini can be measured concurrently and that the parameters of amylase release and cholecystokinin receptor binding are strikingly similar to those previously observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Blevins
G T
GT
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock 72205.
Doi
R
R
Tangoku
A
A
Chowdhury
P
P
McKay
D
D
Rayford
P L
PL
D000681:Amylases / Q000378:metabolism*; D000818:Animals; D002766:Cholecystokinin / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000378:metabolism*
D011949:Receptors, Cholecystokinin; D002766:Cholecystokinin; D000681:Amylases