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ggastrin receptor antagonist, stimulates rat pancreatic enzyme secretion as a CCKA receptor agonist
European Journal of Pharmacology, 242 (1993) 105-108 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
105
EJP 21323
Short communication
PD 135158, a C C K J gastrin receptor antagonist, stimulates rat pancreatic enzyme secretion as a C C K A receptor agonist M i c h a e l H 6 c k e r ~, J o h n H u g h e s b, Ulrich R. F f l s c h
a
and W o l f g a n g E. S c h m i d t ~
a Gastrointestinal Unit, I. Department of Medicine, Christian-Albrechts-University, Schittenhelmstrasse 12, D-24105 Kiel, Germany, and b Parke-Davis Neuroscience Research Center, Hills Road, Cambridge CB2 2QB, UK
Received 17 May 1993, revised MS received 21 July 1993, accepted 23 July 1993
The CCKB/gastrin receptor antagonist, PD 135158 (CAM 1028), surprisingly stimulates lipase release from isolated rat pancreatic acini dose dependently in a biphasic manner, with identical efficacy but lower potency compared to cholecystokinin octapeptide (CCK-8). Half-maximal stimulation occurred at 0.6 p~M and maximal secretion was induced at 50/zM. Supramaximal concentrations decreased lipase release. Acinar lipase secretion in response to 25 pM CCK-8 or 1 gM CAM-1028 was abolished by 5/zM of the specific CCK A receptor antagonist loxiglumide (CR 1505), half-maximal inhibition was observed at 0.6 #M for CCK-8 and 0.4/xM for PD 135158. These data demonstrate that the CCK~/gastrin receptor antagonist, PD 135158, acts as a full agonist at the rat pancreatic CCK A receptor. CCKB/gastrin receptor antagonists; CCK A receptor antagonists; Loxiglumide; Pancreatic acini (isolated, rat)
1. Introduction
The regulatory peptide, cholecystokinin (CCK), mediates its biological effects in the gastrointestinal tract and in the nervous system via two receptor types, the CCK A and CCKB/gastrin receptor. CCK A receptors are present in the gallbladder, the lower oesophageal spincter, bowel, stomach, and the exocrine pancreas (Yu et al., 1990). Outside the gastrointestinal tract, CCK A receptors are present in different regions of the midbrain, the anterior pituitary and the caudal medulla (Crawley, 1991; Moran et al., 1986; Hill et al., 1990). C C K J g a s t r i n receptors are expressed on parietal cells, gastrointestinal smooth muscle, pancreatic acini and predominantly in the brain and spinal cord (Crawley, 1991; Yu et al., 1990). Both receptor types have recently been cloned and structurally characterized (Kopin et al., 1992; Wank et al., 1992). CCKB/gastrin receptors have been implicated in mediating anxiety, satiety, perception of pain and panic-like attacks (Crawley, 1991; Hughes et al., 1990). Potent and spe-
Correspondence to: Wolfgang E. Schmidt, Laboratory of Molecular Gastroenterology, Gastrointestinal Unit, I. Department of Medicine, Christian-Albrechts-University, Schittenhelmstrasse 12, D-24105 Kiel, Germany. Tel. +49-431-597-1395, fax +49-431-597-1302.
cific non-peptide receptor antagonists for the C C K A receptor, i.e. devazepide (L-364.718) and loxiglumide (CR 1505), and the CCK B/gastrin receptor (L-365.260) have been developed to characterize pharmacological and physiological effects of CCK and gastrin (Presti and Gardner, 1993; Schmidt et al., 1991a,b), Recently, the potent non-peptide CCKB/gastrin receptor antagonist, PD 135158, has been described to exert an anxiolytic action in rats and mice via specific inhibition of CCK binding to cerebral CCKB/gastrin receptors (Hughes et al., 1990; Horwell et al., 1991). The present study was designed to characterize the interaction of the CCKB/gastrin receptor antagonist, PD 135158, with peripheral rat CCK A receptors that mediate pancreatic enzyme release from isolated acini.
2. Materials and methods
2.1. CCK receptor antagonists PD 135158 (CAM 1028; 4-{[2-[[3-(1H-indol-3-yl)-2methyl- 1-oxo-2[[[1.7.7.-trimethyl-bicyclo[2.2.1]hept-2-yl oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino4-oxo-[1S-la.2/3[S* (S*)]4a]}-butanoate N-methyl-Dglucamine; bicyclo system 1S-endo) was provided by Dr. J. Hughes, Parke-Davis Neuroscience Research Center (Cambridge, UK). Loxiglumide (D,L-4-(3,4-di-
106
®
chloro-benzoylamino)-5-(N-3-methoxypropylpentylamino)-5-oxo-pentanoic acid) was supplied by Dr. L.C. Rovati, Rotta Research Laboratorium (Monza, Italy).
30-
E
2.2. Preparation of isolated acini W
Isolated rat pancreatic acini were prepared as previously described (H6cker et al., 1991). After a two-step collagenase digestion in Krebs-Ringer-HEPES buffer ( K R H buffer, p H 7.40, 37°C), the pancreas of a male Wistar rat (150-190 g) was dissociated by mild vortexing. Acini were separated from tissue debris by filtration through nylon meshes, purified by sedimentation in K R H buffer containing albumin (10 m g / m l and 40 m g / m l ) and washed 3 times.
;I/
2oi
(.)
15
_J
/
10
oi 10 - t l
3.1. Influence of CCK-8 and PD 135158 on acinar lipase release CCK-8 stimulated acinar lipase release in a typical biphasic manner. The threshold concentration for CCK-8 was 1 pM (5.6 + 0.3% lipase release), half-maximal secretion (SDs0) was observed at 8 pM, and the maximal lipase response at 100 pM CCK-8 (30.7 + 2.2% lipase release, fig. 1A). Typical supramaximal stimulation occurred at 1 nM CCK-8 (22.3 + 2.1% lipase release, fig. 1A). The C C K B / g a s t r i n receptor antagonist, PD 135158, stimulated lipase release from isolated acini in a biphasic manner with identical efficacy, but a five-order of magnitude lower potency compared to CCK-8. The threshold concentration was 10 nM (4.9 + 0.4% lipase release), half-maximal secretion (SDs0) was observed at 0.6 /xM, and the maximal secretory response was induced with 50 /zM PD 135158 (30.0 +
10 -s
10 - s
10-:'
,t0-~
[m01/l]
CCK-8 [2.5x10 -!1 Iol/l]
® 20
3. Results
J
5 _J
2.3. Lipase release from isolated acini Dispersed acini were incubated in a final incubation volume of 0.25 ml with CCK-8 (Peninsula Laboratories, Merseyside, UK) or PD 135158 in the presence or absence of loxiglumide in a shaking water bath (30 oscillations/min) for 30 min at 37°C. To exclude any contamination with CCK peptides, solutions of PD 135158 were analyzed for CCK-like immunoreactivity employing a highly specific CCK radioimmunoassay (H6cker et al., 1991). No CCK-like immunoreactivity was detected up to 0.1 mM PD 135158. Acinar lipase release was calculated as percentage of the initial acinar lipase content in each incubation vial. All determinations were performed in triplicate; intra- and interassay variance was less than 10%. The results were expressed as means + standard deviation (S.D.).
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LOXIGLUMIDE [log ,,ol/1]
CAN 1028 [10 -6 tool/I]
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LOXIGLUMIDE[1~ ~1/1] Fig.1. (A) Acinar lipase release in response to 1 pM-1 nM CCK-8 (e) or 0.1 nM-0.1 mM PD 135158 (11). Dispersed acini were incubated for 30 min at 37°C in the presence of the compounds and lipase release was determined. The results were calculated as means _+S.D. from 6-7 separate experiments. (B) Influence of increasing concentrations of the specific CCKA receptor antagonist loxiglumide on acinar lipase release in response to CCK-8. In the presence of 25 pM CCK-8, dispersed acini were incubated with 10 nM-0.1 mM loxiglumide for 30 min at 37°C and lipase release was determined. (C) Influence of increasing concentrations of loxiglumide on the acinar lipase release in response to the CCKB/gastrin receptor antagonist, PD 135158. In the presence of 1 p.M PD 135158, dispersed acini were incubated with 10 nM-0.1 mM loxiglumide for 30 rain at 37°C. The results were calculated as means+ S.D. from 5-7 separate experiments.
107
1.7% lipase release, fig. 1A). Higher concentrations of the compound decreased acinar lipase secretion. 3.2. Influence of loxiglumide on acinar lipase release stimulated with CCK-8 or PD 135158
Acinar lipase release in response to a submaximal concentration of CCK-8 (25 pM) (15.8 _+ 1.7% lipase release, fig. 1B) was dose dependently inhibited by the specific CCK A receptor antagonist, loxiglumide. Halfmaximal inhibition (ICs0) occurred at 0.6 /xM, 5 /zM loxiglumide abolished CCK-stimulated lipase release (fig. 1B). Lipase secretion that has been stimulated with a half-maximally effective concentration of 1 /zM PD 135158 (19.4 _+ 1.8% lipase release, fig. lC) was inhibited by loxiglumide in an identical manner. Half-maximal inhibition (IC50) occurred at 0.4/xM loxiglumide. With 5 /zM of the A-type receptor antagonist PD 135158-stimulated lipase release was decreased to basal levels (fig. 1C).
4. Discussion
In the present study, the CCKa/gastrin receptor antagonist, PD 135158, unexpectedly stimulated lipase release from isolated rat pancreatic acini dose dependently. The dose-response curves for lipase release had a biphasic configuration, identical to the CCK-stimulated dose-response curve. The specific CCK A receptor ~mtagonist, loxiglumide, inhibited the lipase release stimulated by CCK-8 or PD 135158 with nearly identical ICs0 values. The high antagonistic specificity of loxiglumide for A-type CCK receptors has been characterized extensively in various in vivo and in vitro studies (Presti and Gardner, 1993; Schmidt et al., 1991a,b). In rat pancreatic membranes, PD 135158 had an ICs0 for displacement of 125I-CCK-8 f r o m C C K Atype receptors of 1.2 #M, whereas in the same system the specific CCK A receptor antagonist devazepide (L364.718), had an ICs0 of 0.19 nM (Hughes et al., 1990). In our present experiments the dose range of PD 135158 that inhibited binding of 125I-CCK-8 to pancreatic C C K A receptors corresponded well to the dose-response curve for stimulation of acinar lipase release. These results demonstrate that PD 135158 exerts full CCKA-agonistic activity in isolated rat pancreatic acini. The chemically related gastrin/CCKB-type receptor antagonist, PD 136450 (CAM 1189), has been described to unexpectedly cause pancreatic hypertrophy in rats (Koop et al., 1991). We hypothesize that this effect is also mediated via activation of CCK A receptors by PD 136450. Therefore, to our knowledge, PD 135158, and probably related compounds (Horwell et al., 1991), qualify as the only CCK receptor antagonists
that behave as pure antagonists for the CCK a/gastrin receptor but possess full intrinsic agonistic activity at the CCK A receptor. Recently, a single amino acid residue of the CCK receptor has been identified which determines binding specificity for CCK A or CCK B receptor antagonists (Beinborn et al., 1993). Analyzing the molecular interaction between PD 135158 and the CCK receptors could help define the structural motifs of the CCK A and CCK B receptor which are crucial for receptor activation and signal transduction. A distinct distribution of CCK A and CCKB/gastrin receptors has been described in the central nervous system of various species (Moran et al., 1986; Hill et al., 1990; Crawley, 1991). CCKB/gastrin receptors seem to be involved in the induction of anxiety (Hughes et al., 1990; Singh et al., 1991). Low doses of PD 135158 have been demonstrated to exert anxiolytic activity in rats and mice via CCKa/gastrin receptors (Hughes et al., 1990). Although antagonists for the CCKB/gastrin receptor offer great potential for the treatment of various neurological disorders such as anxiety, care must be taken not to overlook a possible intrinsic CCK A agonistic activity of these compounds at higher doses.
Acknowledgements The authors express their gratitude to Anita Vogel for excellent technical assistance, to Dr. R. Nustede for radioimmunological determination of CCK and to Dr. L.C. Rovati, Rotta Research Laboratorium, for the gift of loxiglumide. The study was supported by grants from the Deutsche Forschungsgemeinschaft (Schm 805/4-1), the German-Israeli Foundation for Scientific Research and Development (I-79-063.2/88), the Eli Lilly European Gastroenterology Award 1990 (W.E.S.), and the Dr. Norbert-Henning-Award for Gastroenterology 1992 (W.E.S.).
References Beinborn, M., Y.-M. Lee, E.W. McBride, S.M. Quinn and A.S. Kopin, 1993, A single amino acid of the cholecystokinin/gastrin receptor determines specificity for non-peptide antagonists, Nature 362, 348. Crawley, N.C., 1991, Cholecystokinin-dopamine interactions, Trends Pharmacol. Sci. 12, 232. Hill, D.R., T.M. Shaw, W. Graham and G.N. Woodruff, 1990, Autoradiographical detection of cholecystokinin-A receptors in primate brain using 125I-Bolton-Hunter CCK-8 and 3H-MK-329, J. Neurosci. 10, 1070. H6cker, M., W.E. Schmidt, W. Creutzfeldt, A. Roy Choudhury, R. Nustede, A. Schafmayer and U.R. F61sch, 1991, Determination of plasma CCK concentrations by bioassay and specific radioimmunoassay in man. A critical evaluation, Regul. Pept. 37, 255. Horwell, D.C., J. Hughes, J.C. Hunter, M.C. Pritchard, R.S. Richardson, E. Roberts and G.N. Woodruff, 1991, Rationally designed 'dipeptoid' analogues of CCK, J. Med. Chem. 34, 404. Hughes, J., P. Boden, B. Costall, A. Domeney, E. Kelly, D.C. Horwell, J.C. Hunter, R.D. Pinnock and G.N. Woodruff, 1990,
108 Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity, Proc. Natl. Acad. Sci. USA 87, 6728. Koop, I., R. Eissele, H. Patberg, B. Rosskopf, H. Koop and R. Arnold, 1991, Wirkung eines neuen Cholezystokinin-(CCK)-BRezeptor-Antagonisten auf das Pankreas der Ratte, Z. Gastroenterol. 31, 285 (abstract). Kopin, A.S., Y.M. Lee, E.W. McBride, L.J. Miller, M. Lu, H.Y. Lin, L.F. Kolakowski and M. Beinborn, 1992, Expression and characterization of the canine parietal cell gastrin receptor, Proc. Natl. Acad. Sci. USA 89, 3605. Moran, T.H., P.H. Robinson, M.S. Goldrich and P.R. McHugh, 1986, Two brain cholecystokinin receptors: implications for behavioral actions, Brain. Res. 362, 175. Presti, M.E. and J.D. Gardner, 1993, Receptor antagonists for gastrointestinal peptides, Am. J. Physiol. 264, G399. Schmidt, W.E., W. Creutzfeldt, M. H6cker, R. Nustede, A. Roy Choudhury, A. Schleser, L.C. Rovati and U.R. F61sch, 1991a, Cholecystokinin receptor antagonist loxiglumide modulates plas-
ma levels of gastro-entero-pancreatic hormones in man. Feedback control of cholecystokinin and gastrin secretion, Eur. J. Clin. Invest. 21,501. Schmidt, W.E., W. Creutzfeldt, A. Schleser, A. Roy Choudhury, R. Nustede, M. H6cker, R. Nitsche, H. Sostmann, L.C. Rovati and U.R. F61sch, 1991b, Role of cholecystokinin in the regulation of pancreatico-biliary functions and gastrointestinal motility in man. Effect of short- and long term treatment with the novel potent receptor antagonist loxiglumide, Am. J. Physiol. 260, G197. Singh, L., A.S. Lewis, M.J. Field, J. Hughes and G.N. Woodruff, 1991, Evidence of an involvement of the brain cholecystokinin B receptor in anxiety, Proc. Natl. Acad. Sci. USA 88, 1130. Wank, S.A., J.R. Pisegna and A. De Weerth, 1992, Brain and gastrointestinal cholecystokinin receptor family: structure and functional expression, Proc. Natl. Acad. Sci. USA 89, 8691. Yu, D.H., S.C. Huang, S.A. Wank, S. Mantey, J.D. Gardner and R.T. Jensen, 1990, Pancreatic receptors for cholecystokinin: evidence for three receptor classes, Am. J. Physiol. 258, G86.
105
EJP 21323
Short communication
PD 135158, a C C K J gastrin receptor antagonist, stimulates rat pancreatic enzyme secretion as a C C K A receptor agonist M i c h a e l H 6 c k e r ~, J o h n H u g h e s b, Ulrich R. F f l s c h
a
and W o l f g a n g E. S c h m i d t ~
a Gastrointestinal Unit, I. Department of Medicine, Christian-Albrechts-University, Schittenhelmstrasse 12, D-24105 Kiel, Germany, and b Parke-Davis Neuroscience Research Center, Hills Road, Cambridge CB2 2QB, UK
Received 17 May 1993, revised MS received 21 July 1993, accepted 23 July 1993
The CCKB/gastrin receptor antagonist, PD 135158 (CAM 1028), surprisingly stimulates lipase release from isolated rat pancreatic acini dose dependently in a biphasic manner, with identical efficacy but lower potency compared to cholecystokinin octapeptide (CCK-8). Half-maximal stimulation occurred at 0.6 p~M and maximal secretion was induced at 50/zM. Supramaximal concentrations decreased lipase release. Acinar lipase secretion in response to 25 pM CCK-8 or 1 gM CAM-1028 was abolished by 5/zM of the specific CCK A receptor antagonist loxiglumide (CR 1505), half-maximal inhibition was observed at 0.6 #M for CCK-8 and 0.4/xM for PD 135158. These data demonstrate that the CCK~/gastrin receptor antagonist, PD 135158, acts as a full agonist at the rat pancreatic CCK A receptor. CCKB/gastrin receptor antagonists; CCK A receptor antagonists; Loxiglumide; Pancreatic acini (isolated, rat)
1. Introduction
The regulatory peptide, cholecystokinin (CCK), mediates its biological effects in the gastrointestinal tract and in the nervous system via two receptor types, the CCK A and CCKB/gastrin receptor. CCK A receptors are present in the gallbladder, the lower oesophageal spincter, bowel, stomach, and the exocrine pancreas (Yu et al., 1990). Outside the gastrointestinal tract, CCK A receptors are present in different regions of the midbrain, the anterior pituitary and the caudal medulla (Crawley, 1991; Moran et al., 1986; Hill et al., 1990). C C K J g a s t r i n receptors are expressed on parietal cells, gastrointestinal smooth muscle, pancreatic acini and predominantly in the brain and spinal cord (Crawley, 1991; Yu et al., 1990). Both receptor types have recently been cloned and structurally characterized (Kopin et al., 1992; Wank et al., 1992). CCKB/gastrin receptors have been implicated in mediating anxiety, satiety, perception of pain and panic-like attacks (Crawley, 1991; Hughes et al., 1990). Potent and spe-
Correspondence to: Wolfgang E. Schmidt, Laboratory of Molecular Gastroenterology, Gastrointestinal Unit, I. Department of Medicine, Christian-Albrechts-University, Schittenhelmstrasse 12, D-24105 Kiel, Germany. Tel. +49-431-597-1395, fax +49-431-597-1302.
cific non-peptide receptor antagonists for the C C K A receptor, i.e. devazepide (L-364.718) and loxiglumide (CR 1505), and the CCK B/gastrin receptor (L-365.260) have been developed to characterize pharmacological and physiological effects of CCK and gastrin (Presti and Gardner, 1993; Schmidt et al., 1991a,b), Recently, the potent non-peptide CCKB/gastrin receptor antagonist, PD 135158, has been described to exert an anxiolytic action in rats and mice via specific inhibition of CCK binding to cerebral CCKB/gastrin receptors (Hughes et al., 1990; Horwell et al., 1991). The present study was designed to characterize the interaction of the CCKB/gastrin receptor antagonist, PD 135158, with peripheral rat CCK A receptors that mediate pancreatic enzyme release from isolated acini.
2. Materials and methods
2.1. CCK receptor antagonists PD 135158 (CAM 1028; 4-{[2-[[3-(1H-indol-3-yl)-2methyl- 1-oxo-2[[[1.7.7.-trimethyl-bicyclo[2.2.1]hept-2-yl oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino4-oxo-[1S-la.2/3[S* (S*)]4a]}-butanoate N-methyl-Dglucamine; bicyclo system 1S-endo) was provided by Dr. J. Hughes, Parke-Davis Neuroscience Research Center (Cambridge, UK). Loxiglumide (D,L-4-(3,4-di-
106
®
chloro-benzoylamino)-5-(N-3-methoxypropylpentylamino)-5-oxo-pentanoic acid) was supplied by Dr. L.C. Rovati, Rotta Research Laboratorium (Monza, Italy).
30-
E
2.2. Preparation of isolated acini W
Isolated rat pancreatic acini were prepared as previously described (H6cker et al., 1991). After a two-step collagenase digestion in Krebs-Ringer-HEPES buffer ( K R H buffer, p H 7.40, 37°C), the pancreas of a male Wistar rat (150-190 g) was dissociated by mild vortexing. Acini were separated from tissue debris by filtration through nylon meshes, purified by sedimentation in K R H buffer containing albumin (10 m g / m l and 40 m g / m l ) and washed 3 times.
;I/
2oi
(.)
15
_J
/
10
oi 10 - t l
3.1. Influence of CCK-8 and PD 135158 on acinar lipase release CCK-8 stimulated acinar lipase release in a typical biphasic manner. The threshold concentration for CCK-8 was 1 pM (5.6 + 0.3% lipase release), half-maximal secretion (SDs0) was observed at 8 pM, and the maximal lipase response at 100 pM CCK-8 (30.7 + 2.2% lipase release, fig. 1A). Typical supramaximal stimulation occurred at 1 nM CCK-8 (22.3 + 2.1% lipase release, fig. 1A). The C C K B / g a s t r i n receptor antagonist, PD 135158, stimulated lipase release from isolated acini in a biphasic manner with identical efficacy, but a five-order of magnitude lower potency compared to CCK-8. The threshold concentration was 10 nM (4.9 + 0.4% lipase release), half-maximal secretion (SDs0) was observed at 0.6 /xM, and the maximal secretory response was induced with 50 /zM PD 135158 (30.0 +
10 -s
10 - s
10-:'
,t0-~
[m01/l]
CCK-8 [2.5x10 -!1 Iol/l]
® 20
3. Results
J
5 _J
2.3. Lipase release from isolated acini Dispersed acini were incubated in a final incubation volume of 0.25 ml with CCK-8 (Peninsula Laboratories, Merseyside, UK) or PD 135158 in the presence or absence of loxiglumide in a shaking water bath (30 oscillations/min) for 30 min at 37°C. To exclude any contamination with CCK peptides, solutions of PD 135158 were analyzed for CCK-like immunoreactivity employing a highly specific CCK radioimmunoassay (H6cker et al., 1991). No CCK-like immunoreactivity was detected up to 0.1 mM PD 135158. Acinar lipase release was calculated as percentage of the initial acinar lipase content in each incubation vial. All determinations were performed in triplicate; intra- and interassay variance was less than 10%. The results were expressed as means + standard deviation (S.D.).
:J
1
¸
15 w
\
10 _J u.I
5i 0
• 0
II
, -8
-
, -7
•
, -6
•
, -5
•
, -4
LOXIGLUMIDE [log ,,ol/1]
CAN 1028 [10 -6 tool/I]
© o~ ,N
°:]
\
15
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-
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0 - 8
,
-
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LOXIGLUMIDE[1~ ~1/1] Fig.1. (A) Acinar lipase release in response to 1 pM-1 nM CCK-8 (e) or 0.1 nM-0.1 mM PD 135158 (11). Dispersed acini were incubated for 30 min at 37°C in the presence of the compounds and lipase release was determined. The results were calculated as means _+S.D. from 6-7 separate experiments. (B) Influence of increasing concentrations of the specific CCKA receptor antagonist loxiglumide on acinar lipase release in response to CCK-8. In the presence of 25 pM CCK-8, dispersed acini were incubated with 10 nM-0.1 mM loxiglumide for 30 min at 37°C and lipase release was determined. (C) Influence of increasing concentrations of loxiglumide on the acinar lipase release in response to the CCKB/gastrin receptor antagonist, PD 135158. In the presence of 1 p.M PD 135158, dispersed acini were incubated with 10 nM-0.1 mM loxiglumide for 30 rain at 37°C. The results were calculated as means+ S.D. from 5-7 separate experiments.
107
1.7% lipase release, fig. 1A). Higher concentrations of the compound decreased acinar lipase secretion. 3.2. Influence of loxiglumide on acinar lipase release stimulated with CCK-8 or PD 135158
Acinar lipase release in response to a submaximal concentration of CCK-8 (25 pM) (15.8 _+ 1.7% lipase release, fig. 1B) was dose dependently inhibited by the specific CCK A receptor antagonist, loxiglumide. Halfmaximal inhibition (ICs0) occurred at 0.6 /xM, 5 /zM loxiglumide abolished CCK-stimulated lipase release (fig. 1B). Lipase secretion that has been stimulated with a half-maximally effective concentration of 1 /zM PD 135158 (19.4 _+ 1.8% lipase release, fig. lC) was inhibited by loxiglumide in an identical manner. Half-maximal inhibition (IC50) occurred at 0.4/xM loxiglumide. With 5 /zM of the A-type receptor antagonist PD 135158-stimulated lipase release was decreased to basal levels (fig. 1C).
4. Discussion
In the present study, the CCKa/gastrin receptor antagonist, PD 135158, unexpectedly stimulated lipase release from isolated rat pancreatic acini dose dependently. The dose-response curves for lipase release had a biphasic configuration, identical to the CCK-stimulated dose-response curve. The specific CCK A receptor ~mtagonist, loxiglumide, inhibited the lipase release stimulated by CCK-8 or PD 135158 with nearly identical ICs0 values. The high antagonistic specificity of loxiglumide for A-type CCK receptors has been characterized extensively in various in vivo and in vitro studies (Presti and Gardner, 1993; Schmidt et al., 1991a,b). In rat pancreatic membranes, PD 135158 had an ICs0 for displacement of 125I-CCK-8 f r o m C C K Atype receptors of 1.2 #M, whereas in the same system the specific CCK A receptor antagonist devazepide (L364.718), had an ICs0 of 0.19 nM (Hughes et al., 1990). In our present experiments the dose range of PD 135158 that inhibited binding of 125I-CCK-8 to pancreatic C C K A receptors corresponded well to the dose-response curve for stimulation of acinar lipase release. These results demonstrate that PD 135158 exerts full CCKA-agonistic activity in isolated rat pancreatic acini. The chemically related gastrin/CCKB-type receptor antagonist, PD 136450 (CAM 1189), has been described to unexpectedly cause pancreatic hypertrophy in rats (Koop et al., 1991). We hypothesize that this effect is also mediated via activation of CCK A receptors by PD 136450. Therefore, to our knowledge, PD 135158, and probably related compounds (Horwell et al., 1991), qualify as the only CCK receptor antagonists
that behave as pure antagonists for the CCK a/gastrin receptor but possess full intrinsic agonistic activity at the CCK A receptor. Recently, a single amino acid residue of the CCK receptor has been identified which determines binding specificity for CCK A or CCK B receptor antagonists (Beinborn et al., 1993). Analyzing the molecular interaction between PD 135158 and the CCK receptors could help define the structural motifs of the CCK A and CCK B receptor which are crucial for receptor activation and signal transduction. A distinct distribution of CCK A and CCKB/gastrin receptors has been described in the central nervous system of various species (Moran et al., 1986; Hill et al., 1990; Crawley, 1991). CCKB/gastrin receptors seem to be involved in the induction of anxiety (Hughes et al., 1990; Singh et al., 1991). Low doses of PD 135158 have been demonstrated to exert anxiolytic activity in rats and mice via CCKa/gastrin receptors (Hughes et al., 1990). Although antagonists for the CCKB/gastrin receptor offer great potential for the treatment of various neurological disorders such as anxiety, care must be taken not to overlook a possible intrinsic CCK A agonistic activity of these compounds at higher doses.
Acknowledgements The authors express their gratitude to Anita Vogel for excellent technical assistance, to Dr. R. Nustede for radioimmunological determination of CCK and to Dr. L.C. Rovati, Rotta Research Laboratorium, for the gift of loxiglumide. The study was supported by grants from the Deutsche Forschungsgemeinschaft (Schm 805/4-1), the German-Israeli Foundation for Scientific Research and Development (I-79-063.2/88), the Eli Lilly European Gastroenterology Award 1990 (W.E.S.), and the Dr. Norbert-Henning-Award for Gastroenterology 1992 (W.E.S.).
References Beinborn, M., Y.-M. Lee, E.W. McBride, S.M. Quinn and A.S. Kopin, 1993, A single amino acid of the cholecystokinin/gastrin receptor determines specificity for non-peptide antagonists, Nature 362, 348. Crawley, N.C., 1991, Cholecystokinin-dopamine interactions, Trends Pharmacol. Sci. 12, 232. Hill, D.R., T.M. Shaw, W. Graham and G.N. Woodruff, 1990, Autoradiographical detection of cholecystokinin-A receptors in primate brain using 125I-Bolton-Hunter CCK-8 and 3H-MK-329, J. Neurosci. 10, 1070. H6cker, M., W.E. Schmidt, W. Creutzfeldt, A. Roy Choudhury, R. Nustede, A. Schafmayer and U.R. F61sch, 1991, Determination of plasma CCK concentrations by bioassay and specific radioimmunoassay in man. A critical evaluation, Regul. Pept. 37, 255. Horwell, D.C., J. Hughes, J.C. Hunter, M.C. Pritchard, R.S. Richardson, E. Roberts and G.N. Woodruff, 1991, Rationally designed 'dipeptoid' analogues of CCK, J. Med. Chem. 34, 404. Hughes, J., P. Boden, B. Costall, A. Domeney, E. Kelly, D.C. Horwell, J.C. Hunter, R.D. Pinnock and G.N. Woodruff, 1990,
108 Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity, Proc. Natl. Acad. Sci. USA 87, 6728. Koop, I., R. Eissele, H. Patberg, B. Rosskopf, H. Koop and R. Arnold, 1991, Wirkung eines neuen Cholezystokinin-(CCK)-BRezeptor-Antagonisten auf das Pankreas der Ratte, Z. Gastroenterol. 31, 285 (abstract). Kopin, A.S., Y.M. Lee, E.W. McBride, L.J. Miller, M. Lu, H.Y. Lin, L.F. Kolakowski and M. Beinborn, 1992, Expression and characterization of the canine parietal cell gastrin receptor, Proc. Natl. Acad. Sci. USA 89, 3605. Moran, T.H., P.H. Robinson, M.S. Goldrich and P.R. McHugh, 1986, Two brain cholecystokinin receptors: implications for behavioral actions, Brain. Res. 362, 175. Presti, M.E. and J.D. Gardner, 1993, Receptor antagonists for gastrointestinal peptides, Am. J. Physiol. 264, G399. Schmidt, W.E., W. Creutzfeldt, M. H6cker, R. Nustede, A. Roy Choudhury, A. Schleser, L.C. Rovati and U.R. F61sch, 1991a, Cholecystokinin receptor antagonist loxiglumide modulates plas-
ma levels of gastro-entero-pancreatic hormones in man. Feedback control of cholecystokinin and gastrin secretion, Eur. J. Clin. Invest. 21,501. Schmidt, W.E., W. Creutzfeldt, A. Schleser, A. Roy Choudhury, R. Nustede, M. H6cker, R. Nitsche, H. Sostmann, L.C. Rovati and U.R. F61sch, 1991b, Role of cholecystokinin in the regulation of pancreatico-biliary functions and gastrointestinal motility in man. Effect of short- and long term treatment with the novel potent receptor antagonist loxiglumide, Am. J. Physiol. 260, G197. Singh, L., A.S. Lewis, M.J. Field, J. Hughes and G.N. Woodruff, 1991, Evidence of an involvement of the brain cholecystokinin B receptor in anxiety, Proc. Natl. Acad. Sci. USA 88, 1130. Wank, S.A., J.R. Pisegna and A. De Weerth, 1992, Brain and gastrointestinal cholecystokinin receptor family: structure and functional expression, Proc. Natl. Acad. Sci. USA 89, 8691. Yu, D.H., S.C. Huang, S.A. Wank, S. Mantey, J.D. Gardner and R.T. Jensen, 1990, Pancreatic receptors for cholecystokinin: evidence for three receptor classes, Am. J. Physiol. 258, G86.