Cholecystokinin Inhibits Phosphatidylcholine Synthesis Via a Ca2+ ...

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The effects of cholecystokinin (CCK) and other pan- creatic secretagogues on phosphatidylcholine (PC) syn- thesis were studied in isolated rat pancreatic acini.

THEJOURNAL OF BIOLOGICAL CHEMISTRY IC:

Vol. 266, No. 33, Issue of November 25, pp. 22246-22253,1991 Printed in U.S. A .

1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Cholecystokinin Inhibits Phosphatidylcholine Synthesis Via a Ca2+-Calmodulin-dependentPathway inIsolated Rat Pancreatic Acini A POSSIBLEMECHANISMFOR

DIACYLGLYCEROL ACCUMULATION* (Received for publication, March 11, 1991)

Takashi MatozakiS, ChoitsuSakamoto, Hogara Nishisaki, Toshiya Suzuki, Ken Wada, Kohei Matsuda, Osamu Nakano, Yoshitaka Konda, Munehiko Nagao, and Masato Kasuga From the Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan

The effects of cholecystokinin (CCK) and other pan- both the reduction of choline uptake into acini and the creatic secretagogueson phosphatidylcholine (PC) syn- inhibition of CTP:phosphocholine cytidylyltransferase thesis were studied in isolated rat pancreatic acini. activity. Furthermore, the results suggest the possibilWhen acini were incubated with [3H]choline in the ity that the activation of Ca”-calmodulin-dependent presence of 1 nM CCK-octapeptide (CCKS) for 60 min, kinase in response to CCK may phosphorylate cytithe incorporations of [3H]choline into both water-sol- dylyltransferase thereby decreasing this enzyme activuble choline metabolitesand PC in acini were reducedity in pancreatic acinarcells. By contrast, the activaby CCKS to 74 and 41% of control, respectively. Pulse- tion of CAMP-dependent protein kinase or protein kichase study revealed thatCCKS reduced both the dis- nase C may not be involvedin CCK-induced inhibition appearance of phosphocholine and the synthesis of PC. of PC synthesis in acini. The inhibition byCCK of PC Other Ca2+-mobilizingsecretagoguessuch as car- synthesis may contribute to the sustained accumulation bamylcholine, bombesin, and Ca2+ ionophore A23187 of diacylglycerol in pancreatic acinar cells. also reduced PC synthesis to the same extent as did CCKS. When combined with 1 nM CCKS, A23187 or carbamylcholine did not further inhibit PC synthesis. Furthermore, W-7 or W-5, a calmodulin antagonist, Cholecystokinin (CCK)’ and acetylcholine are the major reversed the inhibitionby CCK8 of PC synthesis, sug- physiological stimulants for digestive enzyme secretion from gesting that a CaZ+-calmodulin-dependentpathway pancreatic acinar cells (1).The mechanism underlying stimmay be involved in CCK-induced inhibition of PC syn- ulus-secretion coupling of these pancreatic secretagogues has thesis in acini. By contrast, neither CAMP-dependent been explored extensively. It is well established that CCK or secretagogues suchas secretin and dibutyryl CAMPnor cholinergic agents stimulate the activation of phospholipase a phorbol ester had any effect on PC synthesisin acini. Staurosporine or H-7, a protein kinaseC inhibitor, did C, which catalyzes PIP, breakdown (2, 3) to produce both not affect the inhibitionby CCK of PC synthesis. The DAG and 1,4,5-IP3 (4, 5 ) . DAG is known to be the presumed analysis of enzyme activity involved in PC synthesis physiological activator of protein kinase C (4,5 ) and 1,4,5via CDP-choline pathway showed thatCCK treatment IP, initiates a rapid release of Ca2+ froman intracellularCa2+ of acini reduced CTP:phosphocholine cytidylyltrans- store (4). Activation of protein kinase C by synthetic DAG or ferase activity in both cytosolic and particulate frac- TPA and Ca2+mobilization induced by Ca‘+ ionophore have tion, a finding consistent with the delayed disappear- been shown to stimulate digestive enzyme release in a synerance of phosphocholine induced byCCK in pulse-chase gistic manner (6, 7). Because of the importance of polyphosstudy. By contrast, CCK treatment of acini did not phoinositide breakdown as the initial intracellular event after alter the activitiesof choline kinase and phosphocho- the binding of CCK or acetylcholine to its receptor, studies line transferase in acini. The extent of inhibition by have accumulated on the metabolism of this membrane phosCCK of cytidylyltransferaseactivity became much pholipid and inositol phosphates in pancreatic acinar cells (1larger when subcellular fractions of acini were pre- 4,8). However, little detailed information is available on the pared in the presence of phosphatase inhibitors. In relationship between actions of pancreatic hormones and the addition, W-7 reversed the inhibitory effect of CCK metabolism of other phospholipids such as PC in pancreatic treatment on cytidylyltransferase activity in acini. acinar cells. Recently, it has been proposed that not only When acini were labeled with [3H]myristi~ acid and (9), P E (10) and PA (9, polyphosphoinositides but also PC chased, CCK8 (1 nM) reduced the synthesis of t3H] 11) or their metabolites could play an important role in the myristic acid-labeled PC to 27%of control after a 60min chase period. This inhibition of PC synthesis in’ The abbreviations usedare: CCK, cholecystokinin;CCK8, duced by CCK was accompanied by a delayed disap- COOH-terminal CCK-octapeptide; PC, phosphatidylcholine; DAG, pearance of [“H]diacylglycerol, theradioactivity of sn-1,2-diacylglycerol; PI, phosphatidylinositol;PA, phosphatidic acid; which was 225% of control at 60 min. These results PIP, phosphatidylinositol4-phosphate; PIP2, phosphatidylinositol indicate that CCK inhibits PC synthesis by inducing 4,5-bisphosphate; PE, phosphatidylethanolamine; PS, phosphatidyl9

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence should be sent.

serine; TG, triacylglycerol; protein kinase C, Ca2+-activated, phospholipid-dependent protein kinase; protein kinase A, cyclic AMPdependent protein kinase; TPA, 12-0-tetradecanoylphorbol-13-acetate; IP:3,inositol trisphosphate; HEPES,4-(2-hydroxyethyl)-l-piperazineethansulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)]tetracetic acid HR, HEPES-bufferedRinger’s solution.

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Cholecystokinin and PC Synthesis in Pancreatic Acini actions of hormones or mitogens in a number of cell types (9).

In the present study, therefore, we studied the effects of CCK and other pancreatic secretagogues on PC synthesis in rat pancreatic acini. CCK inhibited PC synthesis in aciniby inducing a decrease in both total choline uptake into acini and the activity of cytidylyltransferase, a key regulatory enzyme of the CDP-choline pathway(12, 13). The inhibition of PC synthesis induced by CCK or other Ca2+-mobilizing agonists isprobably mediated by the Ca2+-calmodulin-dependent pathway. Furthermore, experiments using acini labeled with ["Hlmyristic acid indicate that the inhibition by CCK of PC synthesis results in the accumulation of DAG which is not incorporated into PC. EXPERIMENTALPROCEDURES

Materials Synthetic CCK8 was a gift from the Squibb Research Institute, Princeton, NJ. The following were purchased chromatographically purified collagenasefromCooperBiomedicalInc.; PC, oleic acid, soybean trypsin inhibitor, ATP, CTP, cholinechloride, phosphocholine,CDP-choline,dithiothreitol,TPA, A23187, dibutyrylCAMP, fatty acid-free bovine serum albumin, staurosporine, sodium vanadate,and lipid standards fromSigma; Merk Silica Gel 60 high performance thinlayer chromatography plates and NaF from Nakarai Chemicals;H-7, W-5,andW-7fromSeikagaku Kogyo Co.; ['HI choline chloride (75-85 Ci/mmol) and ['4C]phosphocholine (50 Ci/ mmol) from Amersham Corp.; ['Hlmyristic acid (39.3 Ci/mmol) and ['4C]CDP-choline (40-60 Ci/mmol) from Du Pont-New England Nuclear; Centriprep-10 from Amicon Corp.; bovine serum albumin from Miles Laboratories, Elkhart, IN; Bio-Rad protein assay reagent from Bio-Rad; secretin andbombesin from Peptide Institute Inc., Osaka. Methods Isolated Pancreatic Acini-Isolated rat pancreatic acini were prepared by enzymatic digestion with collagenase of pancreas obtained from male Wistar rats as described previously (14). For all experiments, acini were suspended in HEPES-buffered Ringer's solution (HR) containing (in mM) 10 NaHEPES, 129 NaCI, 4.7 KCI, 0.58 MgC12, 1 Na,HP04, 1.28 CaCI,, 11.1glucose, and essential aminoacid solution neutralized with NaOH. ThisH R buffer was supplemented with 0.5% bovine serum albumin and 0.02% soybean trypsin inhibitor, gased with 100% O,, and adjusted to pH7.4. Pulse and Chase Studies-In pulse studies, acini were incubated with 2pCi/ml['Hlcholinefor the indicated time at 37 "C in the presence or absence of CCKS. The incubations were terminated by centrifugation a t 10,000 X g in a microcentrifuge for 15 s, and pellets were washed once with fresh HR buffer. Labeled acini suspended in 1 ml of water were extracted with 3 ml of a ch1oroform:methanol:HCI (1:2:0.02, v/v) mixture followed by the addition of 1 ml of chloroform and 1 ml of water. After centrifugation at 1,000 X g for 15 min, both the aqueous phase and organic phase were removed and counted ina liquid scintillation counter. Samples from the aqueous phase with standards added were processed by TLC for analysis of choline metabolites using the solvent system methanol:0.9% NaCI:NH,OH (50:505, v/v) (15). The standards were visualized by exposure of the plates toiodine vapor. Areascontaining choline, phosphocholine, and CDP-choline were scraped and transferred to scintillationvials containing 1 ml of 0.1 N NaOH. After 12 h the mixtures were acidified with 0.1 ml of 1.5 N acetic acid, and the radioactivitywas measured with a liquid scintillationcounteraftertheaddition of 10 ml of scintillation fluid (16). ["HICholine-labeled phospholipids were also separated from the organic phase by TLC using the solvent system ch1oroform:methanol:acetic acidwater (50:308:4, v/v) (17). Areas on the TLC plate corresponding to authentic PC, sphingomyelin, and lysophosphatidylcholine were scraped, and the radioactivity was determined after the addition of l ml of water to the scintillation vial prior to adding 10ml of scintillation fluid. The pulse-chase study was performed by first incubating acini with 2 pCi/ml ['Hlcholine or 5 pCi/ml ['Hlmyristic acid for 30 min. The labeling of acini with ['Hlmyristic acid was performed in HR buffer containing 0.5% fatty acid-free bovine serum albumin. Labeled acini were washed twice with fresh HR buffer and then further incubated

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with or withoutsecretagogues for the indicatedtime. The incubations were terminated by centrifugation in the microcentrifuge, and pelleted acini were extractedwith 3 ml of ch1oroform:methanol:HCI a (1:2:0.02, v/v) mixture. [3H]Choline-labeled PC and choline metabolites were analyzed as described above. For the separation of phospholipids from ['H]myristic acid-labeled acini, PIP, PIP,, PA, and P E were separated on potassium oxalate-impregnated TLC plates using ch1oroform:methanol:acetone:acetic acidwater (40:15:15:12:8, v/v) as a solvent system (18)PC, PI, PS, lyso-PC, and sphingomyelin were separated with the solvent system ch1oroform:methanol:acetic acidwater (50:30:8:4, v/v). Neutral lipids from acini were also separated using the solvent system hexane:ethyl ether:acetic acid (7030:2, v/v) (19). For labeling aciniwith ['H]choline forlongerperiods,freshly prepared acini were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.02% soybean trypsin inhibitor. Acini from one pancreas were suspended in 50-75 ml of culture medium and incubated with2 pCi/ml ['H]choline in a six-well plastic dish at 37 "C under an air atmosphere containing5% CO,. When acini were labeled under this condition the incorporation of [3H]choline into phospholipid of acini reacheda plateauafter 18-24 h (datanot shown). Analysis of phospholipid extracted from these labeled acini by TLC showed that 95 k 2% ( n = 3) of the label was PC. For the determination of PCdegradation, labeled pancreaticacini were washed twice with fresh HR buffer, and then 1-ml aliquotsof labeled acinar suspension were incubated with or without1 nM CCK8 for the indicated time a t 37 "C. The incubations were terminated by centrifugation a t 10,000 X g, and 0.8 ml of each supernatant and pellet suspended in 1 ml of water was extracted with 3 ml of the chloroform:methanol(1:2) mixture. After the additionof 1 ml of chloroform and 1 ml of water, the aqueous phase extracted from medium and organic phase extracted from cells were counted to determine the release of ["Hlcholine metabolites into the medium and ["HIPC in acini, respectively. Assuy for Enzyme Activity-After acini were incubated with 1 nM CCK8 for 60 min, the acini were washed twice with ice-cold saline and suspended in 2 ml of ice-cold homogenization buffer containing 10 mM Tris-HC1 (pH 7.4),154 mM NaCI, 1 mM benzamidine, and 0.1 phenylmethylsulfonyl fluoride. In some experiments0.1 mM vanadate and 1 mM NaF were also included in the homogenization buffer. All subsequent procedures were performed a t 4 "C. Acini were homogenized by 50 strokes of a tight fitting Dounceglass homogenizer. This homogenate was centrifuged a t 500 X g, for 10 min, and the pellet was homogenized again and spun. The supernatants were combined andthen centrifuged at 100,000 X g for 60min.Theresultant supernatant andpellet were designed as thecytosolic and particulate fractions, respectively. The cytosol was concentrated further with Centriprep-10as recommended by the supplier, andtheprotein concentrations of both cytosolic and particulate fraction were determined by usingBio-Rad protein assay reagent. Reactions were carried out for 20 min a t 37 "C andused a boiled enzyme as a blank. Choline kinase activity was assayed as described previously (20). Briefly, the reaction mixture contained 100 mM Tris-HC1 (pH 8.0), 10 mM MgCI,, 10 mM ATP, 0.2 mM dithiothreitol, 0.25 mM ["HI choline chloride, and 0.2-0.3 mg of protein in a final volume of 0.1 ml. CTP:phosphocholine cytidylyltransferase activity was assayed by the methods asdescribed previously (21). The reaction mixture contained (in a total volume of 0.1 ml) 50 mM Tris-HC1 (pH7.4), 20 mM MgC12, 5 mM CTP, 1.5 mM ['4C]phosphocholine, and 0.4 mg of protein. For choline kinase and cytidylyltransferase assay reactions were terminated by placing the reaction tube into a boiling water bath for 2 min, and a standard containing choline, phosphocholine, and CDP-choline was added. Samples were analyzed by TLC using the solvent system methanol:0.9% NaCI:NH:IOH (50:50:5, v/v). Phosphocholine transferase activity was measured by incubating 50 mM Tris-HC1 (pH 7.4), 10 mM MgCla, 0.4 mM ["CICDP-choline, 0.08 mM 1,2-diacylglycerol, and 0.4 mg of protein in a total volume in 0.8 ml as described previously (22). Diacylglycerol was dried under an N2 stream and resuspended in the incubation mixture by sonication for 10 min. The reaction was terminated by the addition of 3 ml of a chloroform:methanol:HC1 (1:2:0.02, v/v) mixture. The organic phase separated asdescribed above was dried under nitrogen inliquid scintillation vials, and the radioactivitywas measured. In the experiment examining theeffects of Ca2+ on cytidylyltransferaseactivity, different free Cas+ concentrations were obtained by altering the Ca"

Cholecystokinin and

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PC Synthesis Pancreatic in Acini

to EGTA ratio using a computer program as described previously (23). The results presented are the means k S.E. of three ormore experiments unless otherwise stated.Statistical analysis was performed by Student's t test.

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RESULTS

Effects of CCK on ['HICholine Incorporation into PC in Rat Pancreatic Acini-The effects of CCK on ['Hlcholine incorporation to PC in acini were first examined by incubation of acini with 2 pCi/ml ['Hlcholine in the presence or absence of 1 nM CCK8 for the indicated time. CCK8 reduced the incorporation of ["Hlcholine into both water-soluble choline metabolites and PC in acini (Fig. 1).CCK8 reduced the uptake of ['Hlcholine into choline metabolites to 73.7 & 2.3% of control ( n = 3) at 60 min. The analysis of choline metabolites extracted from acini after 60-min a incubation by TLC showed that 85 k 3% of the label ( n = 3) was phosphocholine; choline and CDP-choline were 14 k 2% and 0.4 & 0.2%, respectively. Furthermore, 1 nM CCK8 reduced the synthesis of [3H]PC to 41.4 f 1.0% of control ( n = 4) at 60 min. Analysis of phospholipid extracted from acini labeled for 60 min by TLC showed that 97 & 2% of label ( n = 3) was PC (lyso-PC, 0.5 f 0.2%; sphingomyelin, 2.5 k 0.3%). Since the extentof CCK-induced reduction of PC synthesis was much larger than that of the uptake of labeled choline into water-soluble choline metabolites, it is likely that CCK may regulate another level of PC synthesis pathway in pancreatic acini. Therefore, the effects of CCK on PC synthesis were evaluated further ina pulse-chase experiment. After acini were pulsed with [3H]choline for 30 min and washed with fresh HR buffer, labeled acini were incubated further with or without 1 nM CCK8 for the indicated time. As shown in Fig. 2, incontrolacini the radioactivity of either[3H] choline or [3HH]phosphocholine decreased in a time-dependent manner while the radioactivity of CDP-choline was not significantly changed. Accompanying these changes, atime-

TIME (

rnin )

FIG. 2. Pulse-chase study on the metabolism of [3H]choline in pancreatic acini and the effects of CCK. Acini were pulsed with 2 pCi/ml [3H]choline for 30 min and resuspended in medium without radiolabel. Labeled acini were then chased with (0)or without (0)1nM CCK8 for the indicated times. The radioactivity in [3H] choline metabolites and [3H]PC was determined by TLC. A, choline; B, phosphocholine; C, CDP-choline; D, PC. The radioactivity of [3H] PC observed at the beginning of the chase period was subtracted. Each value was determined in duplicate, and the result shown is representative of three separate experiments.

SECRETAGOGUES ( log M )

FIG. 3. Concentration-dependent inhibition of PC synthesis induced by Ca2+-mobilizingsecretagogues in acini. Acini pulsed with 2 pCi/ml [3H]choline were resuspended and incubated with increasing concentrations of CCK8 (O),carbamylcholine (CCH) (O), and the Ca2*ionophore A23187 (A) for 60 min. [3H]Choline-labeled PC was then extracted, and its radioactivity was determined. From each value the radioactivity observed at zero time was subtracted. Values were expressed as thepercentage of control value and are the mean f S.E. of three separate experiments.

20

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0

30

60

eo

120

TIME ( min )

FIG. 1. The effects of CCK on the incorporation of [3H] choline into water-soluble choline metabolites and PC in acini. Pancreatic acini were incubated with 2 pCi/ml [3H]choline in the presence ( 0 )or absence (0)of 1 nM CCK8 for the indicated times. Labeled acini were washed twice, and the radioactivity incorporated into total choline metabolites and PC was measured. Each value was determined in duplicate, and the result shown is representative of three separate experiments.

dependent increase in the radioactivity of [3H]PC was observed, indicating the conversion from ['Hlcholine metabolites to ['HIPC via a CDP-choline pathway (12, 13). When acini were chased with 1 nM CCK8, CCK inhibited the synthesis of ['HIPC to 36.8 & 2.0% of control ( n = 10) at 60 min (Fig. 2). In addition, the inhibition of PC synthesis induced by CCK was accompanied by a significant delayed disappearance of ['H]phosphocholine in acini. The radioactivity of phosphocholine in CCK-treated acini at 60 min was 144 f 8% of control ( n = 3). By contrast, the amount of label in either choline or CDP-choline in acini was not significantly altered by CCK treatment (Fig. 2). Theseresults suggest that CCK may inhibit PC synthesis not only by reducing choline uptake into acini but also by affecting the formation of CDPcholine from phosphocholine (12, 13). When labeled acini were incubated with increasing concentrations of CCK8 for 60 min, CCK reduced PC synthesis in a concentration-dependent manner with the half-maximal and the maximal effects observed at 100 PM and 1 nM CCK8, respectively (Fig. 3). Effects of Various Pancreatic Secretagogues on PC Synthesis

Cholecystokinin and

PC Synthesis in

Pancreatic Acini

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TABLEI1 in Acini-Since carbamylcholine,bombesin, and the Ca2+ Effects of calmodulin antagonists or protein kinase C inhibitors on ionophore A23187 are considered to stimulate digestive enCCK-induced PC inhibition of PC synthesis in acini zyme release from pancreatic acini via an intracellular mechAcini pulsed with 2 pCi/ml [3H]cholinefor 30 min were incubated anism similar to that of CCK (l),we next examined the or without 1 nM CCK8 for 60 min in the presence or absence of effects of these pancreatic secretagogues on PC synthesis in with various inhibitors. [3H]Choline-labeled PC was then extracted, and acini. As shown in Fig. 3, carbamylcholineinhibited PC the radioactivity was determined. For each value the radioactivity synthesis in a concentration-dependent manner. 100 p~ car- determined at the beginning of the chase period was subtracted. bamylcholine inhibited PC synthesis 44.0 to +. 2.0%of control Values were expressed as thepercentage of control value and are the ( n = 5) at 60 min; this maximal effect was similar to that of mean f S.E. for the number of experiments shown. Neither calmodCCK. The Ca2+ ionophoreA23187, which stimulate amylase ulin antagonists nor protein kinase C inhibitors alone significantly release by mobilizing Ca2+(l),also inhibited PC synthesis in affect PC synthesis in control acini (data not shown). No. of [3H]PC a concentration-dependent fashion, and3 p~ A23187 reduced Agents experiments synthesis P C synthesis to46.4 f 3.6% of control ( n = 4) at 60 min (Fig. % of control 3). In addition,100 nM bombesin reduced PC synthesis (Table CCK8 (1 nM) 5 37.6 f 2.3 I). When combined with 1nM CCK8, which showeda maximal 4 47.0 f 2.0 CCK8 + W-7 (10 pM) inhibition of PC synthesis, neither 100 p~ carbamylcholine 4 CCKS + W-7 (100 pM) 90.4 k 3.6 nor 3 p~ A23187 induced a n additionalinhibition of PC CCK8 + W-5 (10 p M ) 3 38.7 f 3.9” synthesis(TableI), suggesting that these Ca2+-mobilizing 3 52.0 rt 4.0 CCKS + W-5 (100 p M ) secretagogues inhibit PC synthesis by the same mechanism. 38.5 rt 4.0” 3 CCK8 + staurosporine (100 nM) CCK has been considered to stimulate protein kinaseC acti3 CCK8 + H-7 (100 p M ) 39.5 f 3.0” vation (1, 6, 7); however, neither 100 nM nor 1 p M TPA, a NS uersus 1 nM CCK8. potent activatorof protein kinaseC (5), had anyeffect on PC synthesisinacini a t 60 min(TableI).Furthermore,no significant change in PC synthesis was observed when labeled induced amylase release from pancreatic acini (26), did not of PC synthesisinduced by CCK8 (Table acini were incubated with 100 nM TPA for 0-120 min (data affect the inhibition not shown). Secretin and dibutyryl CAMP, which stimulate 11). In addition, 100 p~ H-7, another protein kinaseC inhibamylase release via a CAMP-dependent pathway (l),had no itor (27), also had noeffect on CCK-induced inhibitionof PC effect on PC synthesis (Table I). These data, therefore, sug- synthesis (Table 11). Thus, the resultssuggest that the Ca2+gest thatCCK and carbamylcholinemay inhibit PC synthesis calmodulin dependent pathway may be involved in this inhibition. in pancreatic aciniby a Ca2+-dependent mechanism. Studies were also performed to determine whether CCK Reversal of CCK-induced Inhibition of P C Synthesis by stimulated the degradationof PC by using acini labeled with Calmodulin Antagonists but Not by Protein Kinase C Inhibi[3H]choline for 24 h to equilibrium as described under “Methtors-To investigate further the mechanism underlying CCKods.” When these labeled acini were incubated with 1 nM induced inhibition of PC synthesis, the effects of calmodulin CCK8 for 60 min, CCK failed to induce an increase in the antagonists on the reduction of PC synthesis induced by CCK [3H]choline metabolites released in themedium or a decrease in aciniwere studied. As shown in Table 11, W-7, a calmodulin in [3H]PC in acini when compared with control acini (data antagonist (24), reversed CCK8-inducedinhibition of PC not shown). Thus, PC breakdownmay not contribute to the synthesis in a concentration-dependent manner. In addition, inhibition of PC synthesisinduced by CCK in acini. W-5, another calmodulin antagonist (24), also reversed the Effects of CCK on the Enzyme Activity via the CDP-Choline inhibitory effect of CCK8 on PC synthesis although W-5 was Pathway-To determine whether the treatment of CCK alters much weaker than W-7 in these experiments (Table 11). On the activitiesof enzymes involved in the denouo synthesis of the other hand, 100 nM staurosporine, a protein kinase C PC via the CDP-choline pathway, the activities of enzymes inhibitor (25) that has been shown to inhibit CCKor TPA- prepared from control and CCK-treated acini were assayed.

Since the enzymes involved in the CDP-choline pathway of PC synthesis in pancreatic acinar cells have not been studied Effects of various secretagogueson rH]choline-hbeled PC previously we determined the apparent K,,, and V,,, values of synthesis in pancreatic acini each enzyme. Cholinekinaseactivityandphosphocholine Acini pulsed with 2 pCi/ml [3H]cholinefor 30 min were incubated with or without various secretagogues for 60 min. [3H]Choline-labeled transferase activity were detected only in the cytosolic fracPC was then extracted, and the radioactivity was determined. For tion and the particulate fraction, respectively. The K, and Vmaxof choline kinase forATP were 1.55 f 0.09 mM and 3.42 each value the radioactivity determined at thebeginning of the chase period was subtracted. Values were expressed as the percentage of f 0.37 nmol/min/mg protein ( n = 4), respectively. The K, control value and are the mean f S.E. for the number of experiments and V,,, of phosphocholine transferase for DAG were 0.015 shown. f 0.007 mM and 1.35 f 0.38 nmol/min/mg protein ( n = 3), No. of [3H]PC respectively. The K, and V,,, of cytidylyltransferase in a Secretagogues experiments synthesis cytosolic fraction for C T P were 0.44 f 0.07 mM and 0.78 f % of control 0.13 nmol/min/mg protein (n = 4), respectively. The K, and V,,, of cytidylyltransferase in a particulate fraction for CTP CCK8 (1nM) 10 36.8 f 2.0 Carbamylcholine (100 p ~ ) 5 44.0 f 2.0 were 0.37 -+ 0.05 mM and 0.62 -t 0.10 nmol/min/mg protein 4 A23187 (3 p M ) 46.4 f 3.6 ( n = 4), respectively. These apparent K, and Vmaxvalues of CCK8 (1 nM) + carbamylcholine (100 p M ) 3 37.7 f 3.9 each enzyme were similar to those observed in othercell types 3 38.0 -C 4.0 CCK8 (1nM) + A23187 (3 p M ) (12, 13). When acini were treated with 1 nM CCK8 for 60 3 57.3 f 6.4 Bombesin (10 nM) min, choline kinase activity and phosphocholinetransferase 102.0 f 3.5” 3 TPA (100 nM) activity were notsignificantly changed by CCK (data not 3 98.5 & 5.3” TPA (1 p M ) 3 102.4 f 4.3“ Secretin (10 nM) shown). By contrast, CCK treatment of acini reduced cyti3 97.5 f 4.8” Dibutyryl CAMP (100 p ~ ) dylyltransferase activities in both cytosolic and particulate Not significant versus control. fractions (Table 111, Experiment 1). These results indicate TABLEI

Cholecystokinin Synthesis PCand

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TABLE I11 Effects of CCK on the activity of cytidylyltransferase in pancreatic acini Acini were incubated with or without 1 nM CCK8 for 60 min in the presence or absence of 100 p~ W-7, and then both cytosolic and particulate fractions were prepared in the homogenizing buffer with (Experiment 2) or without (Experiment 1) 0.1 mM vanadate and 1 mM NaF as described under "Methods." The results presented are the mean -t S.E. of three orfour separate experiments. Values in parentheses indicate enzyme activities expressed as the percentage of control. activity Cytidylyltransferase Specific

nmolfminfmgprotein

Experiment 1 Cytosolic Control ( n = 4) 0.73 rtr 0.05 CCK8 ( n = 4) 0.41 rtr 0.06" (56) Particulate Control ( n = 4) 0.57 rtr 0.06 CCK8 ( n = 4) 0.43 k 0.04b(75) Experiment 2 Cytosolic 0.70 k 0.06 Control ( n = 4) CCK8 ( n = 4) 0.29 rtr 0.04' (41) CCK8 + W-7 ( n = 4) 0.65 rtr 0.06 Particulate 0.55 -+ 0.05 Control ( n = 4) CCK8 ( n = 4) 0.28 -+ 0.04"(47) CCK8 + W-7 ( n = 4) 0.50 rtr 0.06 a Significantly different than control ( p < 0.005). Significantly different than control ( p < 0.05). Significantly different than control ( p < 0.0005).

that CCK treatment results in the inhibition of activity of cytidylyltransferase, which catalyzes formation of CDP-choline from phosphocholine (12, 13), a finding consistent with a delayed disappearance of phosphocholine induced by CCK in the pulse-chase study. Furthermore, when cytosolic and particulate fractions were prepared in the presence of 0.1 mM vanadate and 1 mM NaF, both of which are known to be phosphatase inhibitors (28, 29), the extent of inhibition by CCK of cytidylyltransferase activity was much larger than that observed without adding phosphatase inhibitors to the homogenizing buffer (Table 111, Experiments 1 and 2). Since calmodulin antagonist reversed the CCK-induced inhibition of PC synthesis, the activity of cytidylyltransferase in acini was assayed when acini were treated with 1 nM CCK8 in the presence of 100 PM W-7. As shown in Table I11 (Experiment 2), the inhibition of cytidylyltransferase activity byCCK treatment was also blocked by W-7. These resultssuggest the possibility that CCKmay phosphorylate cytidylyltransferase by the activation of Ca2+-calmodulin-dependent kinase, thereby decreasing the activity of this enz$me. On the other hand, itis possible that theelevated concentration of cytosolic Ca2+induced by CCK (1) may regulate the activity of cytidylyltransferase in pancreatic acini. We therefore examined whether or not Ca2+ would change this enzyme activity directly. However, when the activity of cytidylyltransferase prepared from cytosol was assayed in thepresence of various concentrations of free ca2+(1 nM-1 mM), there was no significant change in the enzyme activity in the presence of 1 nM-100 g M free Ca2+ (datanotshown),and 1 mM Ca2+ reduced enzyme activity to 91% ( n = 2) of control. Effects of CCK on [3H]Myristic Acid Incorporation into PC i n Pancreatic Acini-SinceCCK reduced PC formation in acini labeled with [3H]choline,we next examined whether the inhibitory effect of CCK on PC formation is also observed when acinar phospholipid is labeled with radiolabeled fatty

in Pancreatic Acini acid. To isolate changes induced by CCK in PC and other lipids maximally, acini were labeled with [3H]myri~ti~ acid because myristic acid has been shown to be preferentially incorporated into PC over other phospholipids (16,30). Acini were labeled with 5 pCi/ml [3H]myristic acid for 30 min, washed, and incubated further with or without 1 nM CCK8 for the indicated time. In control acini the incorporation of [3H]myristi~acid into PC increased, and the radioactivity in [3H]DAG decreased in atime-dependent manner (Fig. 4), indicating the formation of PC from DAG and CDP-choline (12, 13). Analysis of lipids extracted from labeled acini by TLC showed that labeled myristic acid was not incorporated selectively into PC (63 k 4% of total radioactivity incorporated to lipids, n = 4) (Table IV) in contrast to other cell types (16, 30). However, CCK8 reduced the incorporation of labeled myristic acid into PC in acini (Fig. 4); 1 nM CCK8 inhibited the formation of [3H]myristi~acid-labeled PC to 26.6 f 4.7% ( n = 4) of control at 60 min. Furthermore, CCK induced a significant delayed disappearance of labeled DAG (Fig. 4). The radioactivity of 3H in DAG in CCK-treated acini was 225 k 20% ( n = 4) of control at 60 min. Table IV shows the effects of CCK on the distribution of 3H among lipid metabolites of acini prelabeled with [3H]myristic acid. In addition to the changes induced by CCK8 in PC and DAG, treatment of labeled acini with 1 nM CCK8 resulted in a significant increase in the radioactivity of PA and TG when compared with control (PA, 441 +- 30%;TG, 215 -+ 23%, n = 4) (Table IV). The radioactivity of [3H]PE was also increased by CCK treatment to 134 f 19% of control ( n = 4) although this increase induced by CCK wasnot statisticallysignificant. DISCUSSION

In the present study, we have shown clearly that CCK inhibits PC synthesis via the CDP-choline pathway (12, 13)

U l

I

14

2

2L.!?EIl 0

30

60

90

TIME ( rnin ) FIG. 4. Effects of CCK on the incorporation of [3H]myristi~ acid into PC and DAG in pancreatic acini. Acini were labeled with 5 pCi/ml [3H]myristic acid for 30 min. Labeled acini were resuspended and then incubated with (0)or without (0)1 nM CCK8 for the indicated times. [3H]Myristic acid-labeled PC and DAG were extracted and analyzed by TLC. The radioactivity of [3H]PC at zero time was subtracted from each value, Values are the mean of duplicate determinations, and the data shown are representative of three separate experiments.

Control

Cholecystokinin Synthesis PCand TABLE IV Effects of CCK on the distributionof 3Hin lipids of acini prelabeled with PHImyristic acid After acini were labeled with 5 pCi/ml [3H]myristic acid for 30 min, labeled acini were incubated further withor without 1 nM CCK8 for 60 min. Lipids were extracted at the beginning and the end of incubation period and the distribution of the radioactivity of 3H in cell lipids were determined by TLC as described under “Methods.” Values shown are the mean of closely agreeing duplicate determinations, and the result shown is representative of four separate experiments. Radioactivity of 3H in lipids

in Pancreatic Acini

22251

(25) that has been shown to inhibit CCK-or TPA-stimulated amylase release (26), did not affect CCK-induced inhibition of PC synthesis as well as H-7, another protein kinase C inhibitor (27). Therefore, although CCK has been shown to stimulate both protein kinase C activation and Ca2+mobilization (l), the present resultssuggest strongly that theCCK may cause the inhibition of PC synthesis through a Ca2+calmodulin-dependent pathway in pancreatic acini, although the activation of either protein kinase C or protein kinase A may not be involved in CCK-induced inhibition of PC synthesis in acini. The CDP-choline pathway has been known to be a major Lipids CCK8 0 min at 60 at 60 pathway of PC synthesis, and the enzymes comprising this min min pathway havebeen studied extensively (12,13). In the present study, CCK treatment of acini specifically reduced the activity cpm of CTP:phosphocholine cytidylyltransferasein both cytosolic 27,822 PC 24,708 32,724 190 PI + PS 177 180 and particulate fractions; yet neither choline kinase activity 1,138 1,273 PE 881 nor phosphocholine transferase activitywas affected by CCK 136 136 82 Sphingomyelin treatment. This result corresponds well with data in the pulse108 87 83 Lyso-PC chase study, which shows inhibition of PC formation accom1,123 306 607 PA panied by a delayed disappearance of phosphocholine in acini 57 47 50 PIP treated with CCK. When subcellular fractions of acini were 8 12 15 PIP2 prepared in the presence of vanadate and NaF and enzyme the 1,620 754 1,810 DAG activities were then assayed, the extent of inhibition of cyti14,170 12,370 TG 10,806 dylyltransferase by CCK treatment was much larger as com440 432 Free fatty acid 755 pared with that observed without the additionof phosphatase 56 54 48 Monoglyceride inhibitors to the homogenizing solution. Furthermore, when acini were treated with CCK in the presence of W-7, the in pancreatic acini. The pulse study using [3H]choline has reduction of cytidylyltransferase activityinduced by CCK was shown that CCK reduced both the total uptake of labeled also reversed. These results strongly suggest the possibility choline into acini and the incorporation of labeled choline that the activation of Ca”-calmodulin-dependent kinase in into PC. Furthermore, pulse-chase experiments demonstrated responseto CCKmay phosphorylate cytidylyltransferase that CCK induced inhibition of [3H]PC formationwhich was thereby decreasing this enzyme activity in pancreatic acini. accompanied with a delayed disappearance of [3H]phospho- Thus, the prevention by phosphatase inhibitors of dephoscholine in acini, indicating thatCCK may affect the conver- phorylation of cytidylyltransferase during the preparationof sion from phosphocholine to CDP-choline thereby inhibiting acinar subcellular fraction may lead to increase the extentof PC synthesis in acini. Hormonal regulation of PC synthesis inhibition by CCK of the enzyme activity which is subsehas been shown recently in other cell types (15, 31-33, 36). quently assayed. The presenceof more than oneform of Ca2+I n hepatocytes, glucagon (31), norepinephrine (32), and vacalmodulin-dependent kinase has been shown in pancreatic sopressin (33) have been shownto reduce PC synthesis.Cyclic acinar cells(38). It has recently been shown that protein AMP derivatives or phosphodiesterase inhibitors alsoreduce kinase A phosphorylates cytidylyltransferase in hepatocytes P C synthesis in hepatocytes (34), partly by reducing total (39). In addition, phosphorylation of cytidylyltransferase by choline uptake into cells (34) as observed in pancreatic acini protein kinaseA decreases the activityof cytidylyltransferase treated with CCK.This suggests that the activation of protein whereas the dephosphorylation condition increases the activkinase A may be involved in the regulation of PC synthesis ity of this enzyme (39). Further studies will be necessary to in this cell type (34). By contrast, it has been demonstrated examine whether cytidylyltransferase of acinar cells can be that hormones or TPA increases PC synthesis in HeLa cells directlyphosphorylated by Ca2+-calmodulin-dependentki(35), adipocytes (36), GH, cells (15), indicating possible in- nase in uitro. volvement of protein kinase C activation in regulation of PC It is also possible that the increased concentrationof cytosynthesis in these cell types. In pancreatic acini, not only solic Ca2+induced by CCK may directly regulate the activity CCK but also other Ca2+-mobilizing secretagogues such as of cytidylyltransferase. However, this possibility seems to be carbamylcholine and bombesin reduced PC synthesis. In ad- unlikely because the activity of cytidylyltransferase prepared dition to receptor-activated inhibition of PC synthesis, the fromacini was notsignificantlyaltered by physiological Caz+ ionophoreA23187, which has been shown to stimulate concentrations of free Ca2+ (1 nM-1 p M ) (40),and only amylase release via intracellular Ca2+ mobilization (1, 6, 7), a supraphysiological concentration of Ca2+ (1 mM) caused a reduced PC synthesis to the same extent as did CCK. The small decrease in the enzyme activity. It has been also shown concentration of CCK dependence for both the inhibition of that high concentration of calcium decreases the activity of PC synthesis and the increase of cytosolic Ca2+(37) is almost purified cytidylyltransferase of liver (41). By contrast, Sangidentical. When combined with CCK, A23187 or carbamyl- hera and Vance (42) showed that the 7 mM calcium in the choline did not cause a further inhibition of PC synthesis, medium as well as A23187 and vasopressin increasesPC indicating that these secretagogues inhibit PC synthesis via synthesisandcytidylyltransferaseactivityin hepatocytes. the same mechanism. By contrast, neither TPA nor CAMP- This seems to be a result opposite those of our study in dependent secretagogues had any effect on PC synthesis in pancreatic acini. However, Tijburg et al. (35) have also reacini. Furthermore, we have shown that calmodulin antago- ported that vasopressin inhibits PC synthesis in hepatocytes nists reverse the inhibition of PC synthesis induced by CCK. (33), a finding contrary to the report by Sanghera and Vance On the other hand, staurosporine, a protein kinaseC inhibitor (42). Sanghera and Vance (42) discussed whether the stimu-

22252

Cholecystokinin and PC Synthesis i n Pancreatic Acini

lation of cytidylyltransferase and PC synthesis by a high concentration of calcium may be partially the result of Ca2+ interacting with the plasma membrane. The CCK-induced inhibition of PC synthesis was also observed when acini were labeled with [3H]myristic acid. It has been shown previously that myristic acid is selectively incorporated into PC in other cell types (16, 30) where more than 80% of labeled myristic acid incorporated to total lipids was in PC (16, 30). Although this was not the case in pancreatic acinar cells, more than 60% of the total radioactivity of [3H]myristi~acid incorporated into lipids was observed in PC. Inaddition to a decrease in PCformation, CCK treatment of acini caused a delayed disappearance of [3H]DAGin acini. Because PC is formed from DAG and CDP-choline (12, 13), it is likely that CCK-induced inhibition of [3H]PC synthesis via the CDP-choline pathway may result inthe accumulation of [3H]DAG which is not utilized for PC synthesis in acini. CCK treatment also significantly increased 13H]PAand [3H] TG. DAG, which is not utilized for PC synthesis in CCKtreated acini, may be converted to PA by DAG kinase activation or utilized for the synthesis of TG. It has been shown recently that CCK induces a biphasic DAG production in pancreatic acini (43) by utilizing a sensitive mass assay for DAG (44). PIP2 hydrolysis is a major source of the early increase in DAG at 5 s but not of the sustained increase in DAG (43). CCK has been shown to stimulate the release of [3H]choline metabolites into the medium from acini labeled with [3H]choline for 2 h (43), suggesting the involvement of PC hydrolysis in a sustainedDAG production. However, this labeling condition did not achieve the equilibrium labeling of PC in acini because incorporation of labeled choline into PC stillincreased after 2 h of incubation. Subsequent experiments utilizing streptolysin-o-permeabilized acini have shown that [3H]choline metabolite release induced by CCK may be the redistribution of choline metabolites from cell to medium but not caused by PC hydrolysis (45). In the present study, when PC in acini were labeled with [3H]choline in the equilibrium condition, CCK did not cause an increase in radiolabeled choline metabolites in the medium or a decrease in [3H]PC in cells. Under this equilibrium condition much more label in PC was observed as compared with 2-h labeling (data notshown). If CCK really stimulated PCbreakdown in acini, a larger increase of choline metabolite release in response to CCK could be expected. We do not know exactly why CCK did not stimulate asignificant release of choline metabolites from acini labeled for 24 h. One possible explanation couldbe that the releasable pool of choline metabolites in response to CCK may diminish during longer incubation. Thus, it seems unlikely that CCK stimulates PC breakdown to accumulate DAG, and hence the precise mechanism for a sustained DAG production stimulated byCCK remains unknown at present. However, the present results raise the possibility that CCK mayaccumulate DAG by reducing the synthesis of PC from DAG and CDPcholine. It has been demonstrated recently that a cholinedeficient diet increases the mass level of DAG in the ratliver accompanied by a decrease in PC and an increase in TG, resulting in the activation of protein kinase C (46). Therefore, it is possiblethat theinhibition by CCK of PC formation may contribute to the sustained accumulation ofDAG and the subsequent activation of protein kinase C inpancreatic acini. Another interesting aspect is that the inhibitory effect of CCKon PCsynthesis maybe related to the etiology of pancreatitis. It is well known that administration of a high dose of CCK or caerulein induces acute interstitial pancreatitis (47). This CCK-induced experimental pancreatitis is a

good model for studying acute pancreatitis although the precise mechanism of how CCK causes this severe pancreatitis is not fully understood. On the other hand, acholine-deficient diet has been also known to induce severe hemorrhagic pancreatitis (48). Together, the present study suggests that both CCK and a choline-deficient diet may cause a significant decrease in the synthesis of PC, an essential element of cell membrane lipid bilayer, in the animal pancreas. It can be speculated, therefore, that the decrease in the amount of PC in cell membranes may impair the formation of zymogen vesicles or the normal fusion of zymogenvesicles to cell membranes, resulting in abnormal activation of digestive enzymes (49). REFERENCES 1. Williams, J. A., Burnhuam, D. B., and Hootman, S. R. (1989)in Handbook of Physiology: The Gastrointestinal System (Forte J. G. ed) vol. 3, pp. 419-441,American Physiology Society, Bethesda, MD 2. Streb, H.,Heslop, J. P., Irvine, R. F., Schulz, I., and Berridge, M. J. (1985)J. Biol. Chem. 260,7309-7315 3. Merritt, J. E., Taylor, C. W., Rubin, R. P., and Putney, J. W., Jr. (1986)Biochem. J . 238,825-829 4. Berridge, M. J. (1984)Biochem. J. 220,345-360 5. Nishizuka, Y. (1986)Science 233, 305-312 6. Noguchi, M., Adachi, H., Gardner, J. D., and Jensen, R. T. (1985) Am. J. Physiol. 248, G6924701 7. Pandol, S. J., and Schoeffield, M. S. (1986)J. Biol. Chem. 261, 4438-4444 8. Putney, J. W., Jr. (1987)Am. J . Physiol. 252, G149-Gl57 9. Exton, J. H. (1988)FASEB J. 2,2670-2676 10. Liscovitch, M. (1989)J. Biol. Chem. 264,1450-1456 11. Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987)J. Biol. Chem. 262,15309-15315 12. Pelech, S. L., and Vance, D. E. (1984)Biochim. Biophys. Acta 779,217-251 13. Tijburg, L. B. M., Geelen, M. J. H., and von Golde, L. M. G. (1989)Biochim. Biophys. Acta 1004, 1-19 14. Williams, J. A., Korc, M., and Dormer, R.L. (1978)Am. J. Physiol. 235, E517-E524 15. Kolesnick, R. N. (1987)J. Biol. Chem. 262,14525-14530 16. Martin, T. W., and Michaelis, K. (1989)J. Biol. Chem. 264, 8847-8856 17. Skipski, V. P., Peterson, R. F., and Barclay, M. (1964)Biochem. J. 90,374-377 18. Jolles, J., Zwiers, H., Dekker, A., Wirtz, K., and Gispen, W. H. (1981)Biochem. J. 194, 283-291 19. Slivka, S. R., Meier, K. E., and Insel, P. A. (1988)J. Biol. Chem. 263,12242-12246 20. Weinhold, P. A., and Rethy, V. B. (1974)Biochemistry 13,51355141 21. Vance, D. E., Pelech, S. L., and Choy, P. C. (1981)Methods En~ymol.71, 576-581 22. Vance, D. E., and Burke, D. C. (1974)Eur. J. Biochem. 43,327336 23. Kitagawa, M., Williams, J. A., and De Lisle, R. C. (1990)Am. J. Physiol. 269,G157-Gl64 24. Hidaka, H., Sasaki, Y., Tanaka, T., Endo, T., Ohno, S., Fujii, Y., and Nagata, T. (1981)Proc. Natl. Acad. Sci. U. S. A. 78,43544357 25. Tamaoki, T., Namoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986)Biochem. Biophys. Res. Commun. 135, 397-402 26. Verme, T. B., Velarde, R. T., Cunnungham, R. M., and Hootman, S. R. (1989)Am. J. Physiol. 257, G548-G553 27. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23,5036-5041 28. Trudel, S., Downey, G.P., Grinstein, S., and Paquet, M. R. (1990) Biochem. J. 269,127-131 29. Burnham, D. B., and Williams, J . A. (1982)J . Biol. Chem. 257, 10523-10528 30. Cabot, C. M.. Welsh, C. J., Cao,. H.,. and Chabbott, H. (1988) FESS Lett.'233,153-157 31. Geelen, M. J. H., Groener, J. E. M., de Haas, C. G. M., and van Golde, L. M. G. (1979)FEBS Lett. 105, 27-30

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