Structural requirements of cholecystokinin analogs to activate
different second messengers, ca2+ signaling and pancreatic amylase
secretion.
Tsunoda, Yasuhiro, Hitoshi Yoshida, and Chung Owyang.
Department of Internal Medicine, University of Michigan, Ann Arbor,
MI 48109
APStracts 2:0244G, 1995.
We previously demonstrated that, in rat pancreatic acinar cell, the
high affinity cholecystokinin (CCK) receptor agonist, JMV-180,
utilizes the phospholipase A2 (PLA2)/arachidonic acid cascade to
mediate Ca2+ oscillations and amylase secretion. In contrast, the low
affinity CCK receptor utilizes the Gq/phospholipase
Cbeta1(PLCbeta1)/phosphoinositol pathway. Thus, a single receptor may
couple to at least two different signal transduction systems. In this
study, we investigated the structural requirements of CCK analogs to
activate different intracellular pathways. CCK-analogs such as CCK-8
(Met28,31; EC50=0.4 pM), CCK-7 (Met28,31; 0.7 pM) and NONA
(Thr28/Nle31; 5 pM) caused a biphasic amylase secretion. Reduction of
amylase secretion occurred with high doses of these peptides
(>100 pM). In contrast, CCK-5 (Met31; EC50=20,000 pM), JMV-180
(Nle28,31; 1500 pM) and OPE (Nle28,31; 200 pM) cause a monophasic
amylase secretion. CCK-8, but not JMV-180, increased protein kinase C
(PKC) activities in a transient and concentration-dependent manner.
The PKC activator phorbol ester significantly inhibited an increase
in myoinositol 1,4,5-triphosphate levels induced by CCK-8 and
abolished monophasic amylase secretion induced by OPE. CCK-8, CCK-7
and NONA caused either Ca2+ oscillations (<100 pM) or large Ca2+
transients (>100 pM). In contrast, both JMV-180 and OPE evoked
Ca2+ oscillations even in high doses. Ca2+ signaling modes induced by
CCK-5 were intermediate types between CCK-8 and JMV-180. Both CCK-8
and CCK-7-stimulated Ca2+ spikes were inhibited by the PLC inhibitor
U-73122, but not by the PLA2 inhibitor ONO-RS-082. The action of CCK
-5 was only partially sensitive to the PLC inhibitor. In contrast,
JMV-180 and OPE-stimultaed Ca2+ oscillations were inhibited by the
PLA2 inhibitor, but not by the PLC inhibitor. NONA was sensitive to
both PLC and PLA2 inhibitors. Although JMV 180 differs from CCK-8 by
having a ASP-2phenylethylester rather than an ASP-phenylalanine
amide, it is unlikely that these differences in the carboxyl terminal
are important in determining which second messenger systems will be
activated. This is because CCK-5 (Phe33-CONH2) cause monophasic
amylase secretion and Ca2+ oscillation in a manner similar to those
induced by JMV-180 (2-phenylethylester). Meanwhile NONA (Phe33-CONH2)
appeared to activate both PLC and PLA2 pathways. The actions of all
CCK analogs were abolished by L-364,718, indicating mediation by CCKA
receptors. Based on the amino acid sequence of the carboxy terminal
of CCK analogs, it appears that Met or Thr in the position of 28th of
the carboxy terminal of CCK peptides is crucial to cause reduction of
amylase secretion evoked by high doses of CCK peptides, probably
through an increase of PKC activities. Met28,31 (CCK-8/CCK-7) is
important to activate the low affinity CCK receptor linked to the PLC
cascade. If the analog contains only Met31 but not Met28 (CCK-5), it
is only partially sensitive to the PLC pathway but the Ca2+ signaling
and amylase secretory patterns seem to be similar to JMV-180/OPE.
Nle28,31 (JMV-180/OPE) is critical to stimulate the high affinity CCK
receptor coupled to the PLA2 pathways, Ca2+ oscillation and
monophasic amylase secretion. If the analog contains only Nle31 but
not Nle28 (NONA), it may activate both the PLC and PLA2 pathways but
the Ca2+ signaling and amylase secretory patterns seem to be similar
to CCK-8/CCK-7. Therefore, depending on the agonist use, the CCKA
receptor activation in pancreatic acini may result in the
differential involvement of second messenger systems, Ca2+ signal
transduction and amylase secretion. Key amino acids for this
differentiation are Met28 (or Thr28) for PLC pathways and Nle28 for
PLA2 pathways.
Received 26 August 1994; accepted in final form 24 October 1995.
APS Manuscript Number G326-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 December 95