ABSTRACT
By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Benzodiazepinones/metabolism , Pancreas/metabolism , Receptors, Cholecystokinin/metabolism , Sincalide/metabolism , Amylases/metabolism , Animals , Benzodiazepinones/pharmacology , Carbachol , Cholecystokinin/antagonists & inhibitors , Devazepide , Dose-Response Relationship, Drug , In Vitro Techniques , Male , Pancreas/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Cholecystokinin/agonists , Receptors, Cholecystokinin/antagonists & inhibitors , Signal Transduction , Sincalide/pharmacologyABSTRACT
Examining the actions of L-364,718 on rat pancreatic acini, we found that L-364,718 causes persistent inhibition of cholecystokinin (CCK)-8-stimulated enzyme secretion in acini that were first incubated with L-364,718, washed repeatedly, and then reincubated with CCK-8. This inhibition is maximal after as little as 5 s of first incubation with L-364,718, is unaltered by reducing the temperature of the first incubation from 37 to 4 degrees C and is specific for CCK-8 in that carbachol-stimulated enzyme secretion is unaltered. The inhibitory potency of L-364,718 added to the first incubation followed by washing and reincubation with CCK-8 is nearly the same as when L-364,718 is added together with CCK-8 in the same incubation. The persistent inhibitory action of L-364,718 is not attributable to residual free L-364,718 in the bulk phase of the second incubation medium. In addition, L-364,718 does not cause persistent inhibition by binding irreversibly to CCK receptors because the binding reaction is completely reversible and the persistent inhibition can be surmounted with appropriate concentrations of CCK-8. When acini are first incubated with L-364,718 and washed repeatedly, approximately 0.2% of the original L-364,718 remains trapped in a microenvironment within the acini. This trapping presumably results in a sufficiently high concentration of L-364,718 to produce its persistent, albeit surmountable inhibition.
Subject(s)
Amylases/metabolism , Benzodiazepinones/pharmacology , Cholecystokinin/antagonists & inhibitors , Pancreas/enzymology , Sincalide/pharmacology , Animals , Atropine/pharmacology , Benzodiazepinones/metabolism , Carbachol/pharmacology , Devazepide , Kinetics , Male , Pancreas/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Cholecystokinin/metabolismABSTRACT
In the course of examining the actions of the cholecystokinin octapeptide (CCK-8) on pancreatic acini we found that CCK-8 can stimulate release of the large-molecular-weight cytoplasmic protein, lactate dehydrogenase (LDH) by as much as 6-fold. CCK-8-stimulated LDH release is mediated by a CCK-preferring receptor, detectable at 100 pM CCK-8, maximal at 100 nM CCK-8, constant for up to 30 min, reversible, not desensitized, and dependent on oxidative metabolism and incubation temperature but not on calcium in the extracellular medium. This action of CCK-8 is blocked by inhibitors of protein kinases, staurosporine, H-7, H-8 and H-9, but not by calmodulin antagonists, chlorpromazine, trifluoperazine or W-7. This action of CCK-8 on LDH release is not reproduced by TPA, 8Br-cAMP or A23187. Thus, it appears to be mediated by a previously uncharacterized protein kinase or an isoform of protein kinase C that is not maximally stimulated by TPA.
