Two Functionally Distinct Cholecystokinin Receptors ...

THE JOURNAL OF BIOLOGICAL CHEMLWRY B 1990 by The American Society for Biochemistry

Vol. 265, No. 11, Issue of April 15, pp. 6247~6254,199O Printed m U.S. A.

and Molecular Biology, Inc.

Two Functionally Distinct Cholecystokinin Receptors Show Different Modes of Actions on Ca2’ Mobilization and Phospholipid Hydrolysis in Isolated Rat Pancreatic Acini STUDIES

USING

A

NEW

Takashi MatozakiS, John A. Williams

CHOLECYSTOKININ

Burkhard

From the Departments of Physiology §Centre de Pharmacologic-Endocrinologie,

ANALOG,

Gbke,

JMV-180*

Yasuhiro

and Internal 34094

Tsunoda,

Marc

(Received

for publication,

Rodriguez§,

Jean

University of Michigan, Cedex. France

Medicine, Montpellier

Ann

Arbor,

November

Martinez&

Michigan

48109

10, 1989)

and and

the

phorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCKS. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis. By contrast, the occupancy of low affinity state of receptors by CCK induces phosphatidyl-inositol 4,5-bisphosphate hydrolysis leading to both IP3 formation and an early peak of DAG and a large transient increase in [Ca2+]i.

* This work was supported by National Institutes of Health DK 41122 and DK 41225 and the Deutsche Forschungsgemeinshaft. The costs of publication of this article were defrayed in part payment of page charges. This article must therefore be marked “adoertisement” in accordance with 18 U.S.C. Section solely to indicate this fact. j To whom correspondence should be addressed: Dept. of ology, 7744 Medical Science II, University of Michigan, Ann MI 48109-0622.

’ The abbreviations used are: CCK, cholecystokinin; CCKB, COOH-terminal CCK-octapeptide; DAG, sn-1,2-diacylglycerol; protein kinase C, Ca*+-activated, phospholipid-dependent protein kiiase; PIP*, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; 1,4,5-IP3, inositol 1,4,5-trisphosphate; [Caz+lL, cytosolic free calcium concentration: JMV-180. JMV-180 (Boc-Tvr(SO;)-Nle-GlvTrp-Nle-Asp-2-phenylethyleste;); EGTA, [&hylenkbis(oðylenenitrilo)]tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethansulfonic acid.

Grants by the hereby 1734 PhysiArbor,

Earlier studies of the binding of radioactive cholecystokinin (CCK)’ to pancreatic acini indicated two different classes of CCK-binding sites with high and low affinity for CCK (l-3). CCK also possesses both stimulatory and inhibitory effects on amylase release depending on its concentrations (l-5). It has been proposed that the occupancy of high affinity sites of receptors by low concentrations of CCK, usually less than 100 PM, is closely correlated to stimulation of amylase release and that occupancy of low affinity sites by higher concentrations of CCK, greater than 100 PM, is correlated to the suppression of amylase release (1, 2). The stimulatory action of CCK on amylase secretion is believed mediated by the mobilization of intracellular Ca*+ (6-9). It has been shown that CCK stimulates PIP, breakdown (10,ll) to produce both DAG and 1,4,5IPB (12, 13), the latter of which causes Ca*+ release from an intracellular Ca2+ store (12). Activation of protein kinase C by DAG and Ca2+ mobilization induced by 1,4,5-IP3 have been proposed to act in a synergistic fashion in the stimulation of pancreatic enzyme secretion (8, 14-16). However, it has not yet been clarified how the occupancy of these two distinct classes of CCK receptors by CCK is related to the generation of intracellular second messengers. Recently, a new heptapeptide analog of CCK, JMV-180,

6247

Downloaded from www.jbc.org by guest, on April 25, 2012

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO:)-Nle-Gly-Trp-Nle-Asp-2phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCKS) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCKS on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCKS was reduced. Measurement of 46Ca2+ efflux and cytosolic free calcium concentration ([Ca”‘]i) by the fluorescence of fura-a-loaded acini in a stirred cuvette showed that JMV- 180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCKS. When [Ca2+]i of individual fura-2loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 j.bM) induced repetitive transient [Ca2+li spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCKS (1 nM) caused a large increase in [Ca2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 ~.LM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+lj and I,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV- 180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCKS. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phos-

6248

CCK Receptors and Second Messengers

EXPERIMENTAL

PROCEDURES

Materials

Synthetic CCK8 was a gift from the Squibb Research Institute, Princeton, NJ. CCK-derived peptide (JMV-180) was synthesized as previously described (17, 18). The following were purchased: chromatographically purified collagenase from Cooper Biomedical, Malvern, PA, soybean trypsin inhibitor, ATP, dithiothreitol, 12-O-tetradecanoylphorbol-13-acetate; DETAPAC, diethylenetriaminepentaacetic acid, lipid standards, carbamylcholine, octyl-/3-n-glucoside from Sigma; Escherichia coli DAG kinase from Lipidex, Middleton, WI; bovine cardiolipin from Avanti Polar Lipids Inc., Pelham, AL; Merck Silica Gel high performance thin layer chromatography plate from VWR Scientific; Chicago; [y-32P]ATP (3000 Ci/mmbl), my0-[2-~H] inositol (14.3 Ci/mmol). “CaCl, (20 Ci/mmol). 13Hlcholine chloride (75-85 Gi/mmol) and’i,4,5-IP; ‘assay’ kit from Amersham Corp.; bovine serum albumin from Miles Laboratories, Elkhart, IN; minimal essential media amino acid 50 X concentrated from GIBCO; Bio-Rad protein assay reagent from Bio-Rad; fura-Z/AM and BAPTA/AM from CA.

Molecular

Probes,

Eugene,

OR;

VIP

from

Bachem,

Torrance,

Methods Preparations

of Isolated

Rat

Pancreatic

Acini-Isolated

rat

pan-

creatic acini were prepared by enzymatic digestion with collagenase of pancreas obtained from male Sprague-Dawley rats as previously described (3,5, 22). For all experiments, acini were suspended in HR solution containing (in mM) 10 sodium HEPES, 128 NaCl, 4.7 KCl, 0.58 MgCl*, 1 Na,HP04, 1.28 CaClz, 11.1 glucose, and an essential amino acid solution neutralized with NaOH. This HR buffer was supplemented with 0.5% bovine serum albumin and 0.02% soybean trypsin inhibitor, gassed with 100% 01, and adjusted to pH 7.4. Amylase Release-Acini were preincubated with or without 50 PM BAPTA/AM for 30 min, washed once, and resuspended in fresh HR solution without CaC12. Two-ml aliquots were then distributed into 25-ml polycarbonate Erlenmeyer flasks and incubated with secretagogues-in the presence of 1.28mM CaCIZ or without added CaC& plus 0.1 mM EGTA at 37 “C for the indicated time. The incubation was terminated by centrifugation for 15 s in a microcentrifuge, and amylase released into the supernatant was assayed by use of procion yellow starch as substrate (21). Amylase release was expressed as the percentage of the total content of amylase in the acini at the beginning of the incubation. In each experiment, duplicate or triplicate flasks were used to determine amylase release.stimulated by each treatment. Assay for 45Ca2’ Efflur--‘5Ca2+ efflux from acini was measured by a slight modification of the method described previously (22). Acini were prelabeled with 2 &i/ml 45CaC12 for 1 h in HR solution at 37 “C, centrifuged, washed once with ice-cold HR solution, and resuspended

in fresh medium. Two-ml aliquots of acinar suspension were then distributed into 25-ml Erlenmeyer flasks and incubated with CCK8 or JMV-180 for 6 min at 37 ‘C. After the termination of incubation by centrifugation of 1 ml of acinar suspension for 15 s in a microcentrifuge, the radioactivity in 0.5 ml of the supernatant was measured by a liquid scintillation counter. 45CaZ+ efflux was expressed as the percentage of the total radioactivity in the acini at the beginning of the incubation. Measurement of [Ca”+/i-[Ca2+]; was determined by monitoring changes in the fluorescence of the Ca’+-sensitive indicator, fura-2, in both populations of acinar cells in suspension and individual acinar cells as previously reported (23, 24). Briefly, isolated acini were incubated with 2 FM fura-P/AM at 37 ‘C! in 10 ml of HR solution for 20 min. The acini were then washed with fresh HR solution, resuspended, and kept at room temperature until use. All experiments utilized a dual wavelength modular fluorometer system (Spex Fluorolog 2) coupled to a Nikon Diaphot inverted microscope (24). For the measurement of furafluorescence in the cell populations, 2 ml of acinar suspension were transferred to a cuvette, and fluorescence was recorded at 37 “C under continuous stirring. To measure fluorescence of individual acinar cells, acini were placed on a glass coverslip mounted on the bottom of a chamber (O.l-ml volume) kept at 37 “C. Single acinar cells were optically isolated using a pinhole diaphragm stopped down to an optical diameter of 10 pm. Acini were superfused with HR solution containing 0.05% bovine serum albumin at a flow rate of 1 ml/min. The excitation light at 340 and 380 nm was alternately selected via a rotating chopper mirror. Emitted light was monitored at an emission wavelength of 510 nm in the cuvette studies and using a 480 nm cut off filter in the microscope (24). As previously described (23, 24), the fluorescence ratio (340 nm/380 nm) was calculated and the ratio converted to [Ca’+]i by using the formula (25): [Ca’+]; = Kd ((R - R,,,)/(R,., - R)) FJF, Kd = 224 nM. For microspectrofluorometry, Rmin, R,.., and F,/F, were determined with an external standard (24); a value for the Kd of 224 nM (25) was used. For cuvette studies intracellular furawas released with digitonin and Rmin and R,., determined by adding EGTA or Ca2+ (24). Assay for ZPn Production in A&i-The measurement of IPB production was performed by both [3H]inositol labeling of acini followed by anion exchange column separation and by use of a 1,4,5-IP3 radioreceptor assay. For the measurement of 3H-labeled IP,, acini were prelabeled with 20 &i/ml [3H]inositol for 90 min at 37 “C. The labeled acini were washed twice with fresh HR solution containing 10 mM LiCl and suspended in the same solution. Aliquots (0.6 ml) of these acinar suspensions were incubated with JMV-180 or CCK8 for 90 s at 37 “C and the incubation terminated by the addition of an equal volume of ice-cold 20% trichloroacetic acid. After centrifugation of the precipitate at 1000 x g for 15 min, 0.9 ml of the supernatants were washed five times with 5 volumes of water-saturated diethyl ether, neutralized with 1 M KHC03, and mixed with 2.5 ml of water. Each sample was then processed by the method described by Berridge (26) for the analysis of inositol phosphates. The radioreceptor assay was carried out as described previously (27). Briefly, 0.6-ml aliquots of acinar suspension was incubated with CCK analogs at 37 “C for 5 s but without LiCl, and inositol phosphates were extracted as mentioned above. 100 ~1 of these extracts or known amounts of authentic 1,4,5-IP3 from 0.19 to 25 pmol were then incubated with a bovine adrenal IPa-binding protein preparation in the presence of 0.01 &i of [3H]1,4,5-IP3 in 10 mM Tris buffer, pH 9.0, for 20 min at 4 “C. Bound and free labeled 1,4,5-IP3 were then separated by centrifugation at 1000 x g for 5 min, and the radioactivity associated with the pellet was counted in a liquid scintillation counter. The 1,4,5-IP3 content of the extract, and thus of the acini used to prepare the extract was determined by comparing the extent of the inhibition of [3H]1,4,5-IP3 binding with that observed with known amounts of authentic 1,4,5-IP3. DAG Assay-DAG levels in crude lipid extracts of acini were measured bv the method of Preiss et al. (28) as previously described (27). After “0.8-ml aliquots of acinar suspension were incubated in tubes with CCK analogs for the indicated time at 37 “C, the incubation was stopped by the addition of 3 ml of chloroform/methanol mixture (1:2, v/v). Mixed micelles were prepared by solubilizing each dried crude lipid extract in 20 pl of 7.5% octyl-P-D-glucoside and 5 mM cardiolipin in 1 mM diethylenetriaminepentaacetic acid. The mixed micelles were then reacted with 10 ~1 of diacylglycerol kinase and 10 mM [T-~*P]ATP for 30 min at 25 “C as described (27), and the reaction was terminated by extraction with chloroform/methanol. The lower phase was dried and subjected to the thin layer chromatography on Silica-Gel 60 high performance thin layer chromatogra-


has been synthesized (17, 18) and shown to possess the same efficacy for amylase release from rat pancreatic acini as CCK8 but without any inhibition of amylase release at supramaximal concentrations (18-20). In addition, this analog appears to interact as an agonist with only one class of receptors on rat pancreatic acini of which occupancy by CCK analogs is coupled to the stimulatory mechanism for amylase release (20). Furthermore, JMV-180 also interacts with the low affinity state of CCK receptors as a functional antagonist (18-20). Thus, this new CCK analog should be a useful tool in studying the intracellular mediators activated by CCK as only some of actions of CCK are present, and these are presumed to be mediated by only one class of CCK receptors. In the present study, therefore, we studied the relationship between amylase release, phospholipid turnover, and Ca*+ mobilization caused by both CCK8 and JMV-180. Both the actions of JMV-180 and CCK8 were dependent on an increase in [Ca2+li as they could be blocked by the Ca2+ chelator BAPTA loaded into acini. However, JMV-180 induced repetitive oscillations in [Ca*+]; similar to low concentrations of CCK8 without the large transient increase in [Ca’+]i induced by high concentrations of CCK8. In addition, JMV-180 had little effect on 1,4,5IP3 production but increased DAG levels probably in part from phosphatidylcholine hydrolysis.

6249

CCK Receptors and Second Messengers phy plates developed with chloroform/acetone/methanol/acetic acid/ water (50:20:15:10:5, v/v), followed by autoradiography, and liquid scintillation counting. The amount of DAG present in the original sample was calculated from the amount of [32P]phosphatidic acid produced, the sample volume, and the specific activity of the [r-“‘PI ATP. All data points were assayed in duplicate and expressed as nanomoles per milligram of acinar protein which was measured by using a Coomassie Blue dye protein assay reagent. PC Hydrolysis Assay-The experiments measuring [3H]choline metabolite release were carried out as previously reported (27). Briefly, acini were prelabeled with 5 &i/ml [3H]choline chloride for 90 min at 37 “C. Labeled acini were then washed twice with fresh HR buffer, then l-ml aliquots of labeled acinar suspension were incubated with CCK analogs for 30 min at 37 “C. The incubations were terminated by centrifugation at 10,000 X g in a microcentrifuge for 15 s and 0.8 ml of each supernatant was extracted with 3 ml of chloroform/ methanol (1:2) mixture. After the addition of 1 ml of chloroform and 1 ml of water followed by centrifugation, radioactivity in the aqueous phase was evaluated to determine the release of [3H]choline metabolites. Statistical Analysis-The results presented are the means + SE. of three or more experiments unless otherwise stated. The statistical analysis was performed by analysis of variance and Student’s t test.

22

0

B 5 x z 2

0

basal

I EZ

CCKB 100 pM JMV-160 100nM

6 4

x

0 Medikn Cd’ BAPTA

+ -

-

-

+ +

+

RESULTS

release, we examined the effect of preloading acini with the calcium chelator BAPTA (29, 30). It has been reported that during the first 5-min period of agonist stimulation, amylase release is dependent on intracellular Ca2+ mobilization while latter secretion depends on the presence of extracellular Ca2+ (6, 8, 9). Therefore, acini were incubated with CCK analogs for 5 min in order to maximally isolate the role of Ca2+ mobilization. Acini were incubated with 100 pM CCK or 100 nM

JMV-180,

concentrations

of these CCK analogs

known to

induce maximal stimulation of amylase release (N-20) in both the presence of 1.28 mM CaC12 or its absence plus 0.1 mM EGTA; amylase release induced by either JMV-180 or CCK8 was similar for the two different conditions of Ca2+ in the medium (Fig. 1A). This result indicates that the action of both JMV-180 and CCK8 on amylase release during the first 5-min period of stimulation is independent of extracellular Ca2+ concentration. Basal amylase release was not changed by the preloading with BAPTA for 30 min. However, the preloading of acini with BAPTA reduced both 100 nM JMV180 or 100 pM CCK8-stimulated amylase release in the presence of 1.28 mM CaC& (Fig. L4). In the absence of CaC12, neither JMV-180 or CCK8-stimulated amylase release from BAPTA-loaded acini was significantly further reduced as compared with the values obtained in the presence of 1.28 mM CaC12(Fig. 1A). As shown in Fig. lB, BAPTA preloading of acini did not change amylase release stimulated by 100 nM 12-0-tetradecanoylphorbol-13-acetate, an activator of protein kinase C (13) or 100 nM vasoactive intestinal peptide, an agent acting via CAMP (9, 31), indicating that the inhibition by BAPTA of JMV-180- or CCK8-stimulated amylase release is most likely due to Ca2+ chelation and is not a nonspecific or toxic effect.

BAPTA

+

FIG. 1. Effect of preloading of acini with BAPTA on CCK analog-stimulated amylase release in the presence and absence of Ca” (A) and on amylase release stimulated by various pancreatic secretagogues (B). A, acini were preloaded by incubation with 50 pM BAPTA/AM for 30 min. Acini were then washed

and suspended in fresh HR solution without added Car&. The incubation with CCK analogs was carried out for 5 min in the presence of 1.28 mM CaClz or no added Cat& plus 0.1 mM EGTA. Each value is the mean f SE. from four separate experiments. B, acini preloaded with or without BAPTA were incubated with secretagogues for 30 min in the presence of 1.28 mM CaClz (HR) or no added CaC12 plus 0.1 mM EGTA. Each value is the means f SE. from four separate experiments. *, not statistically significant; P > 0.05, uersus without BAPTA.

tion-dependent manner (Fig. 2A). The lowest concentration of JMV-180 was 10 nM and a maximal effect on 45Ca2+efflux was observed at 300 nM. However, the maximal effect of JMV-180

was only

two-thirds

that

stimulated

with

1 nM

CCK8. A similar concentration dependence for the increase of [Ca2+]i in response to CCK8 and JMV-180 was obtained when fura- loaded acini were used and [Ca’+]i measured fluorometrically in a stirred cuvette (Fig. 2B). The lowest effective concentration of JMV-180 on [Ca2+]i was 30 nM JMV-180; 1 pM JMV-180 maximally increased [Ca2+]i from 98 f 3 nM (n = 42) to 510 + 47 nM (n = 4), a value which was 58% of the maximal response obtained at 1 nM CCK8. Thus, concentrations of JMV-180 less than 30 nM showed little or no effect on Ca2+ mobilization when 45Ca2’efflux and [Ca2+li were determined in a population of cells even though amylase release is known to be stimulated (18, 20). To further evaluate the effect of JMV-180 on Ca2+ mobilization in acini, [Ca’+]i was measured in individual fura-2loaded acinar cells by microspectrofluorometry. Recent studies by us have shown this technique to resolve oscillatory [Ca2+li increases in rat acini in response to CCK8 at concentrations as low as 1 PM, the threshold for amylase release Effects of JMV-180 and CCKB on the Ca2+ Mobilization in from rat acini (24). As shown in Fig. 3, JMV-180 over the Acini-Since preloading of acini with BAPTA reduced amylase release stimulated by either CCK8 or JMV-180, Ca2+ concentration range of 1 nM to 1 j,tM induced repetitive, transient [Ca2+li spikes (Ca2+ oscillations). In two experimobilization is likely involved in the actions of both these ments 10 pM JMV-180 gave results similar to those shown for CCK analogs. Therefore, in the following set of experiments the effect of JMV-180 on intracellular Ca*+ mobilization was 1 PM. The lowest effective concentration of JMV-180 (1 nM) evaluated by three different ways. First, when 4sCa2+ efflux which induced Ca2+ oscillation corresponded to that which was measured as an indicator of mobilization of stored Ca2+, stimulates amylase release (18-20). At concentrations greater both JMV-180 and CCKS stimulated ‘Ya*+ in a concentrathan 10 nM a small sustained increase in [Ca”+]; upon which


Effect of JMV-180 and CCKB on Amy&e Release from Pancreatic Acini: Inhibition by Intracellular Ca2+ ChelationTo determine whether an increase in intracellular free Ca2+ is necessary for the action of JMV-180 to increase amylase


6250

the Ca’+ oscillations were superimposed was also observed. A lag time of l-2 min before Ca2+ oscillations appeared was observed at l-10 nM JMV-180. The amplitude of the Ca2+ oscillations was dependent on the concentration of JMV-180: the amplitude of spikes increased from 72 nM (1 nM) to 227 nM (1 pM) (Table I). The frequency of Ca*+ oscillations was constant at 1.8-2.l/min over the range of 10 nM-1 pM JMV180 (Table I). - so4

A

-10 -9 -6 CCK analog (log M)

-7

-6

on %a*+ efflux (A) A, acini prelabeled with 2 &X/ml %a*+ were incubated with CCK analogs for 6 min. Values are the mean + S.E. from three separate experiments. B, acini loaded with fura- were suspended in fresh HR solution, and 2-ml aliquots were incubated in a stirred cuvette. Changes in peak value of [Ca*+], over a single tracing are expressed as a function of the concentration of CCK analog. Each value is the mean & SE. of four to five separate experiments. and

FIG. 2. Effects [Ca’+]< (B)

of JMV-180 in a population

and CCKS of acini.

Joe-

200.

termked by dual crospectrofluorometry ing with fura-2.

representative observations.

wavelength after

miload-

Each plot shown is of four to five separate

“O ?,

“1

II

au+IW

Juv-loo

loonu

0 0

5

10

15

* 20

IjaM

0 0

TIME ( min )

5

10

IS

20


CCK analog ( log M )

In previous studies (18-20), JMV-180 was found to reverse the inhibition of amylase release induced by high concentration of CCK8. Since the inhibition of amylase release has been correlated with the occupancy of a low affinity state of the CCK receptor by high concentrations of CCK (1,2), JMV180 may act as a functional antagonist for the low affinity state of CCK receptors (18-20). Application of 1 pM JMV180 followed by 1 nM CCK8 blocked the large transient increase in [Ca*+]i observed in control cells (Fig. 4A). HOWever, the subsequent application of 10 pM carbamylcholine caused a large transient increase of [Ca2+]i even in the presence of JMV-180 (Fig. 4A). A low concentration of CCK8 also induced Ca2+ oscillations (Fig. 4B). When the concentration of CCK8 was increased, a large transient increase of [Ca’+]i occurred followed by a small sustained elevation of [Ca’+]; without oscillations (Fig. 4B). These results suggest that JMV-180 in addition to inducing Ca2+ oscillations acts as an antagonist to block the effect of high concentrations of CCKS to induce a large transient increase of [Ca’+]i. Effect of JMV-180 and CCKB on IP3 Production in AciniSince JMV-180 was found to induce oscillations in [Ca*+]i similar to low concentrations of CCK8 but without the response pattern induced by high concentrations of CCK8, the effect of JMV-180 on IP3 production was examined and compared with that of CCK8. When [3H]inositol-labeled acini were incubated for 90 s with increasing concentrations of JMV-180, concentrations less than 1 pM failed to stimulate measurable IPs production in acini, and a 1 pM concentration induced only a very small increase (Fig. 5A). In contrast, CCK8 at concentrations above 1 nM induced a large increase of IPs. The concentration dependence of total [3H]inositol phosphate production in response to either CCK8 or JMV180 was similar to that of [3H]IP3 production. Since the [3H] inositol labeling assay measures all isomers of IP3, the 1,4,5IPs content was measured by a 1,4,5-IP3 receptor binding assay. A similar concentration-dependent increase in 1,4,5IPs in response to both CCK8 and JMV-180 was obtained as that obtained with [3H]inositol labeling was used (Fig. 5B). JMV-180 at concentrations less than 10 pM showed no increase in 1,4,5-IP3 content in acini and 10 pM increased 1,4,5-

CCK Receptors

and Second

6251

Messengers

TABLE I Amplitude

and frequency

of Co”

oscillations

induced

by JMV-I80

Each value is expressed as the mean f SE. for the number of separate experiments indicated in parenthesis. JMV Amplitude Frequency

10 (n = 5)

n.44 12 f 14 195 11

100 (n = 4) 1000 (n = 4)

221 f 41

IZM

1 (n = 4)

per 1.2 1.9 1.8 2.1

161 f 9

min c 0.1 rt 0.1 + 0.2

2 0.1

0

;

-12

-11

-10

-9

-8

-7

-6

-5

CCK analog (log M)

-0

5

10

7-w FIG. 4. Blockage induce

a large

increase

by

JMV-180 in [Caz’]i

15

20

(min)

of the effect of CCKS in individual aeinar cells.

of increasing production

concentrations acini. A,

of

JMV-180

acini labeled with 20 &i/ml [3H]inositol were stimulated with CCK analogs for 90 s. ‘HLabeled inositol phosphates were extracted and separated as described under “Experimental Procedures.” Values are the mean f SE. from three separate experiments. B, after the incubation of acini with CCK analogs for 5 s, 1,4,5-IP3 was extracted and assayed using competitive binding assay. Each point was determined in duplicate and the mean + SE. calculated from four separate experiments. *, P < 0.05 uers’suswithout CCK analogs. CCK8

on

IP3

in

to A,

JMV-180 (1 PM) induces [Ca”], oscillations and blocks the effect of CCKS (1 nM) but not carbamylcholine (CCh) (10 PM) to induce a large transient increase in [Ca”+],. B, CCKB (10 PM) induces [Ca’+]i oscillations similar to JMV-180 but does not block the subsequent effect of 1 nM CCKS. IP, content only from 5.1 + 1.6 pmol/mg of protein (basal) (n = 4) to 13.2 + 2.0 pmol/mg of protein (n = 4), a value much smaller than that obtained with CCKS at a concentration of 1 nM (51.5 + 6.4 pmol/mg, n = 4). Since JMV-180 blocked the effect of high concentrations of CCK8 on the large transient increase in [Ca”] i in individual acinar cells, we also determined whether JMV-180 would block the effect of CCK8 on 1,4,5-IP3 production. When combined with 1 PM JMV-180, the effect of 1 nM CCK8 to increase 1,4,5-IP3 was completely blocked, and 1,4,5-IP3 production induced by 10 nM CCK8 was markedly reduced (Fig. 6). In contrast, no change in IP, production stimulated by 1 mM carbamylcholine was observed in the presence of 1 pM JMV-180. Effect of JMV-180 on CCKB on Diacylglycerol Formation in Acini-Activation of protein kinase C by DAG is believed to be involved in the action of CCK on amylase release (8,9,1416). Therefore, we examined whether JMV-180 stimulates DAG formation in acini. When acini were incubated with 1 fiM JMV-180, DAG formation reached a plateau between 1530 min but without a significant increase in DAG until 60 s (Fig. 7). By contrast, as shown recently (27) 10 nM CCK8 caused a biphasic stimulation of DAG formation; an early increase of DAG peaked at 5-10 s and a later increase peaking at 15-30 min (Fig. 7). Treatment of acini with increasing concentrations of JMV-180 for 30 min resulted in a concentration-dependent increase of DAG with a lowest significant stimulation at 3 nM JMV-180 (Fig. 8). 100 nM JMV-180

9

tam1

CCK

Ill&4

CCK

10

“Y

nl CCh

IrnY

FIG. 6. Reversal of CCKS-stimulated 1,4,5-IP3 production Acini were incubated with or without 1 PM JMV-180 in the presence of 1 nM CCKB, 10 nM CCK8, and 1 mM carbamylcholine (0%). IP3 was extracted and measured with competitive binding assay. Values are the mean + SE. of three separate experiments. by JMV-180.

maximally stimulated DAG formation from 1.03 f 0.05 nmol/ mg of protein (basal) to 1.42 k 0.07 nmol/mg of protein (n = 6). High concentrations of CCK8 (more than 1 nM) caused much larger increases of DAG when compared with JMV-180 (Fig. 8). Effect of JMV-180 and CCKB on Phosphatidylcholine Hydrolysis in Acini-Since JMV-180 had at most a small effect on PIP2 breakdown to produce IP3, it seems likely that JMV180 stimulated DAG formation in acini by an alternate mechanism. Therefore, we examined whether JMV-180 stimulates the release of phosphorylated choline as an indicator of PC hydrolysis. PC has recently been demonstrated by this technique and others to be a source of DAG in a variety of cell types (32) including pancreatic acini (27). When [3H]cholinelabeled acini were incubated with different concentrations of JMV-180, release of [3H]choline metabolites occurred in a concentration-dependent fashion (Fig. 9). JMV-180 at the concentration of 100 nM induced a maximal stimulation of [3H]choline metabolite release to 178 2 7% (n = 4); this value


FIG. 5. Effects and

6252


*.Oi r

JMV-180

was not different than the maximal response obtained with 1 nM CCK8 (190 f ll%, n = 4). The lowest effective concentration of JMV-180 on 13H]choline metabolite release was 1 nM JMV-180, the threshold concentration for increasing DAG. Analysis of the [3H]choline metabolites by thin layer chromatography showed 91 f 1% of the 3H released by JMV180 was phosphorylated choline.

1 @4

DISCUSSION

k

0 5

s

15

30

45

60

TIME ( min )

0.61

’ 0

’

15

’

’ 30

’

*

fi 60

TIME ( set )

FIG. 8. Concentration-dependent increase of DAG in acini stimulated by JMV-180 and CCKS. Acini were incubated with CCK analogs for 30 min after which DAG was extracted and assayed. Each value is the mean + S.E. from four separate experiments. *, P < 0.05 versus without CCK analogs.

FIG. 9. Concentration dependence of JMV-180 and CCKS stimulation of [‘HIcholine metabolite release from [‘H]choline-labeled acini. Acini labeled with [3H]choline were incubated with CCK analogs for 30 min. [3H]Choline metabolites released into the medium were extracted, and the radioactivity of the aqueous phase was determined. The radioactivity observed at zero time was subtracted from each value and the net release expressed as percentage of the control release. Data shown are the mean + S.E. from four separate experiments.


FIG. 7. Effects of JMV-180 or CCKS on DAG formation in rat pancreatic acini. Acini were incubated with 1 pM JMV-180,lO nM CCKS, or without CCK analogs for the indicated times. DAG was extracted and assayed as described under “Experimental Procedures.” Each value is expressed as the mean f SE. from three separate experiments. *, P < 0.01 versa.9 time 0. **, P < 0.01 versrhs 5 s of 10 nM CCKO stimulation.

In this study, we have shown that the CCK analog JMV180, which stimulates amylase release without any inhibition at its supramaximal concentration has different actions on intracellular Ca*+ mobilization and phospholipid hydrolysis when compared with CCK8. Both CCK8 and JMV-180 stimulate amylase release to the same extent although in our experiments JMV-180 is about lOOO-fold weaker in potency (20). The present results clearly show that the action of JMV180 to induce amylase release can be blocked by loading with the Ca*+ chelator BAPTA. The ability of JMV-180 to induce normal amylase release in medium lacking Ca*+ also indicates that the action of JMV-180 is independent of extracellular Ca*+. Hence if Ca*+ is important in the action of JMV-180 it must be mobilized from intracellular stores. That this does in fact take place was shown by studies measuring *‘Ca’+ efflux and average levels of [Caz+lt using fura-2. However, the maximal response obtained with JMV-180 in both assays was twothirds of that with CCK8. By utilizing microspectrofluorometry of fura- in individual acinar cells, the mode of action of JMV-180 on [Ca*+]i was found to be the induction of Ca*+ oscillations. Similar Ca*+ oscillations are the predominate response of rat acini to l-30 pM CCK8 as recently reported (24). The amplitude of Ca2+ oscillations induced by JMV-180 is similar to that of CCK8 but the frequency of JMV-180-induced CaZf oscillations is slightly greater than that induced by CCK8 (JMV-180, 1.8P.l/min; CCKS, 1.5/min (24)). By contrast, higher concentrations of CCK8 (more than 100 PM) cause a large transient increase of [Ca’+]i followed by a small sustained increase of [Ca*+]i without oscillations. Thus, it is clear that JMV-180 or low concentrations of CCK8, which stimulate amylase release, have different effects on [Ca”]i from that of the high concentration of CCK8. The lowest effective concentration of JMVwas 1 nM, a concentration 180 to induce Ca*+ oscillations which corresponds to the lowest effective concentration of JMV-180 for amylase release (18-20). Similarly, the lowest concentration of CCKS inducing Ca2+ oscillations (1 PM) corresponds to the lowest concentration inducing amylase release (24). These results suggest, therefore, that Ca2+ oscillation behavior caused by JMV-180 or low concentration of CCKS might be responsible for the stimulatory mechanism for amylase release from acini. Thus, it seems likely that a large increase of [Ca2+li is not necessary to stimulate amylase release. In the stirred cuvette experiment, JMV-180 at concentrations less than 30 nM, which stimulate amylase release, failed to induce a detectable increase in [Ca2+]i. The inability to detect a rise in [Ca”+]; at low concentrations of JMV-180 (l-10 nM) in a population of acini probably results from asynchronous Ca*+ oscillations that are averaged and hidden in the signal noise. This discrepancy has also been noted for CCK (24). It has been proposed that PIP2 hydrolysis producing IP3 is involved in CCK-stimulated Ca2+ mobilization in acini (911). However, in the present study when the total cellular IPS production was measured, neither JMV-180 or low concentrations of CCK8 stimulated measurable IP3 production in acini by either of two methods of analysis. A minimal increase of


been shown here that JMV-180 stimulates the release of [3H] choline metabolites, predominantly phosphorylcholine, indicating the possible activation of phospholipase C specific for PC which would produce DAG. Hormone-stimulated hydrolysis of PC as a source of DAG has been reported recently in a number of cell types (40-42). Therefore, these present results suggest that PC hydrolysis may be involved in JMV180-stimulated DAG production and that it might be more important for the increase of DAG rather than PIP2 breakdown when pancreatic acini are stimulated by physiological concentrations of CCK to cause amylase secretion. Acknowledgment-We agement.

thank

E. Stuenkel

for

advice

and

encour-

REFERENCES 1. Sankaran, H., Goldline, I. D., Bailey, A., Licko, V., and Williams, J. A. (1982) Am. J. Physiol. 242, G250-257 2. Sankaran, H., Goldfine, I. D., Deveney, C. W., Wong, E. Y., and Williams. J. A. (1980) J. Biol. Chem. 255. 1849-1853 3. Williams, J: A., Bailey, A. C., and Roach, E. (i988) Am. J. Physiol. 254, G513-521 4. Jensen, R. T., Lemp, G. F., and Gardner, J. D. (1980) Proc. N&l. Acad. Sci. U. S. A. 77, 2079-2083 5. Williams, J. A., Korc, M., and Dormer, R. L. (1978) Am. J. Physiol. 235, E517-524 6. Williams, J. A. (1980) Am. J. Physiol. 238, G269-279 7. Ochs, D. L., Korenbrot, J. I., and Williams, J. A. (1985) Am. J. Ph.ysiol. 249, G389-398 8. Pandol, S. J., Schoeffield, M. S., Sachs, G., and Muallem, S. (1985) J. Biol. Chem. 260.10081-10086 9. Williams, J. A., and Hootman, S. R. (1986) in Exocrine Pancreas: Biology, Pathology and Diseases (Go, V. L. W., Gardner, J. D., Brooks. F. P.. Lebenthal. E., DiMasno, E. P., and Scheele. G. A., eds j pp. 123-139, Raven Press, New York’ 10. Streb, H., Heslop, J. P., Irvine, R. F., Shultz, I., and Berridge, M. J. (1985) J. Biol. Chem. 260,7309-7315 11. Merritt, J. E., Taylor, C. W., Rubin, R. P., and Putney, J. W., Jr. (1986) Biochzm. J. 238,825-829 12. Berridge, M. J. (1984) Biochem. J. 220, 345-360 13. Nishizuka, Y. (1986) Science 233,305-312 14. Noguchi, M., Adachi, H., Gardner, J. D., and Jensen, R. T. (1985) Am. J. Physiol. 246, G692-701 15. Merritt, J. E., and Rubin, R. P. (1985) Biochem. J. 230,151-159 16. Burnham, D. P., Munowitz, P., Hootman, S. R., and Williams, J. A. (1986) Biochem. J. 235, 125-131 17. Fulcrand, C., Rodriguez, M., Galas, M. C., Lignon, M. F., Laur, J., Aumelas, A., and Martinez, J. (1988) Int. J. Peptide Protein Res. 32,384-395 18. Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez. J. (1988) Am. J. Physiol. 254. Gi76-182 19. Stark, H. A., Sharp, C. M., Sutliff, V. E., Martinez, J., Jensen, R. T., and Gardner, J. D. (1989) Biochim. Biophys. Acta 1010, 145-150 20. Matozaki, T., Martinez, J., and Williams, J. A. (1989) Am. J. Physiol. 257, G594-600 21. Jung, D. H. (1980) Clin. Chem. Acta 100, 7-11 22. Otsuki. M., and Williams, J. A. (1982) Am. J. Phvsiol. 243. G285-2S6 23. Stuenkel, E., Tsunoda, Y., and Williams, J. A. (1989) Biochem. Biophys. Res. Commun. 168.863-869 24. Tsunoda, Y., Stuenkel, E., and Williams, J. A. (1990) Am. J. Physiol.: Cell Physiol. 258, C147-155 25. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260,3440-3450 26. Berridge, M. J. (1983) Biochem. J. 212,849-858 27. Matozaki, T., and Williams, J. A. (1989) J. Biol. Chem. 264, 14729-14734 28. Preiss, J., Loomis, C. R., Bishop. W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261.8597-8600 29. Tsien, R. Y. (1981) Nature 290,527-528 ’ 30. Dormer, R. L. (1984) Biochem. Biophys. Res. Commun. 119,876883 31. Collen, M. J., Sutliff, V. E., Pan, G. Z., and Gardner, J. D. (1985)


1,4,5-IP3 has also been reported when rat pancreatic acini were stimulated with low concentrations of caerulein (11). The recent measurement of total inositol phosphates in rat pancreatic acini also showed little increase in response to JMV-180 (33). In contrast to results with low concentrations of CCK8 and all concentrations of JMV-180, high concentrations of CCK8 caused a large increase in IP3 production. The concentrations of CCK (more than 100 PM) which stimulated a large increase in IP3 correspond well with those causing a transient large increase of [Ca*+]i consistent with IP3 being responsible for the large transient increase of [Ca’+];. JMV180 also blocked the action of high concentrations of CCK8 on both the [Ca’+], increase and IPS production. It has been shown that JMV-180 blocks the inhibition of amylase release caused by high concentration of CCK8 (18-20). Thus, JMV may act as an agonist when it interacts with the high affinity site of CCK receptor but act as a functional antagonist for the low affinity state of CCK receptors (18-20). The present results, therefore, indicate that the action of CCK8 on IPS generation which induces a large transient increase in [Ca*+]i may be mediated by the occupancy of low affinity state of CCK receptors. Furthermore, the occupancy of high affinity state of CCK receptors by JMV-180 or physiological concentrations of CCK may be related to the Ca*+ oscillation mode with minimal stimulation of PIP2 breakdown. Recently, it was reported that a non-hydrolyzable guanine nucleotide analog inhibits the binding of labeled CCK8 to pancreatic acinar cell membranes but not the binding of labeled CCK analog, D-Tyr-Gly-[(Nle28S31)CCK-26-32]-phenethyl ester which is similar to JMV-180 (34). These authors suggested that a novel pathway not involving a guanine nucleotide-binding protein mediates CCK action. A guanine nucleotide-binding protein has been shown to be involved in CCK-stimulated PIP2 breakdown to produce IP, in pancreatic acinar cells (35-37). These results coupled with our observation that JMV-180 has little effect on IP3 production raise the possibility that the mechanism for induction of Ca*+ oscillations may be different from that for IPa-induced large transient Ca*+ mobilization in pancreatic acini. However, it has recently been reported that inclusion of IP, in a micropipette can induce oscillations in Ca*+-activated Cl- currents in mouse pancreatic acinar cells (38). Therefore, it is possible that only a small and possibly compartmentalized increase in IP3 is necessary to induce [Ca*+]i oscillations. Since it is not yet possible to measure the local concentration or change of IP3 at the single cell level, further efforts will be necessary to clarify the mechanism of Ca*+ oscillation induced by JMV180 or low concentrations of CCK in acini. The activation of protein kinase C by DAG is also believed to play an important role in CCK-stimulated amylase release (8, 9, 14-16). In this study, we have shown that JMV-180 stimulates an increase in DAG in acini, suggesting that the activation of protein kinase C might be involved in JMV-180 stimulated amylase release. Although the possibility that the activation of protein kinase C could play an inhibitory role for amylase release (39) has been previously proposed, it is clear here that DAG formation occurred when amylase release was stimulated. In contrast to the biphasic time course of DAG increase stimulated by higher concentrations of CCK8, JMV-180 caused a monophasic and sustained stimulation of DAG without an early increase. We have recently presented data indicating that PIP, breakdown is a major source of the early increase in DAG but not of the sustained increase in DAG (27). Thus, the limited effect of JMV-180 on IP3 production corresponds with the lack of effect of JMV-180 to induce an early phase of DAG formation. Furthermore, it has

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