the of biological chemistry vol. 266, pp. ic: inc. printed ... · the journal of biological...

THE JOURNAL OF BIOLOGICAL CHEMISTRY IC: 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Cholecystokinin Inhibits Phosphatidylcholine Synthesis Via a Ca2+-Calmodulin-dependent Pathway in Isolated Rat Pancreatic Acini A POSSIBLE MECHANISM FOR DIACYLGLYCEROL ACCUMULATION*

(Received for publication, March 11, 1991)

Takashi MatozakiS, Choitsu Sakamoto, 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 pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H]choline in the presence of 1 nM CCK-octapeptide (CCKS) for 60 min, the incorporations of [3H]choline into both water-soluble choline metabolites and PC in acini were reduced by CCKS to 74 and 41% of control, respectively. Pulse- chase study revealed that CCKS reduced both the disappearance of phosphocholine and the synthesis of PC. Other Ca2+-mobilizing secretagogues such as carbamylcholine, bombesin, and Ca2+ ionophore A23187 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, reversed the inhibition by CCK8 of PC synthesis, suggesting that a CaZ+-calmodulin-dependent pathway may be involved in CCK-induced inhibition of PC synthesis in acini. By contrast, neither CAMP-dependent secretagogues such as secretin and dibutyryl CAMP nor a phorbol ester had any effect on PC synthesis in acini. Staurosporine or H-7, a protein kinase C inhibitor, did not affect the inhibition by CCK of PC synthesis. The analysis of enzyme activity involved in PC synthesis via CDP-choline pathway showed that CCK treatment of acini reduced CTP:phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction, a finding consistent with the delayed disappearance of phosphocholine induced by CCK in pulse-chase study. By contrast, CCK treatment of acini did not alter the activities of choline kinase and phosphocholine transferase in acini. The extent of inhibition by CCK of cytidylyltransferase activity became much larger when subcellular fractions of acini were prepared in the presence of phosphatase inhibitors. In addition, W-7 reversed the inhibitory effect of CCK treatment on cytidylyltransferase activity in acini.

When acini were labeled with [3H]myristi~ acid and chased, CCK8 (1 nM) reduced the synthesis of t3H] myristic acid-labeled PC to 27% of control after a 60- min chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [“H]diacylglycerol, the radioactivity of which was 225% of control at 60 min. These results indicate that CCK inhibits PC synthesis by inducing

* 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.

9

$ T o whom correspondence should be sent.

both the reduction of choline uptake into acini and the inhibition of CTP:phosphocholine cytidylyltransferase activity. Furthermore, the results suggest the possibility that the activation of Ca”-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acinar cells. By contrast, the activation of CAMP-dependent protein kinase or protein kinase C may not be involved in CCK-induced inhibition of PC synthesis in acini. The inhibition by CCK of PC synthesis may contribute to the sustained accumulation of diacylglycerol in pancreatic acinar cells.

Cholecystokinin (CCK)’ and acetylcholine are the major physiological stimulants for digestive enzyme secretion from pancreatic acinar cells (1). The mechanism underlying stim- ulus-secretion coupling of these pancreatic secretagogues has been explored extensively. I t is well established that CCK or cholinergic agents stimulate the activation of phospholipase C, which catalyzes PIP, breakdown (2, 3) to produce both DAG and 1,4,5-IP3 (4, 5 ) . DAG is known to be the presumed physiological activator of protein kinase C (4, 5 ) and 1,4,5- IP, initiates a rapid release of Ca2+ from an intracellular Ca2+ store (4). Activation of protein kinase C by synthetic DAG or TPA and Ca2+ mobilization induced by Ca‘+ ionophore have been shown to stimulate digestive enzyme release in a syner- gistic manner (6, 7). Because of the importance of polyphos- phoinositide breakdown as the initial intracellular event after the binding of CCK or acetylcholine to its receptor, studies have accumulated on the metabolism of this membrane phospholipid and inositol phosphates in pancreatic acinar cells (1- 4, 8). However, little detailed information is available on the relationship between actions of pancreatic hormones and the metabolism of other phospholipids such as PC in pancreatic acinar cells. Recently, it has been proposed that not only polyphosphoinositides but also PC (9), PE (10) and PA (9, 11) or their metabolites could play an important role in the

’ The abbreviations used are: CCK, cholecystokinin; CCK8, COOH-terminal CCK-octapeptide; PC, phosphatidylcholine; DAG, sn-1,2-diacylglycerol; PI, phosphatidylinositol; PA, phosphatidic acid; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PS, phosphatidyl- serine; TG, triacylglycerol; protein kinase C, Ca2+-activated, phospholipid-dependent protein kinase; protein kinase A, cyclic AMP- dependent protein kinase; TPA, 12-0-tetradecanoylphorbol-13-ace- tate; IP:3, inositol trisphosphate; HEPES, 4-(2-hydroxyethyl)-l-piper- azineethansulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)]te- tracetic acid HR, HEPES-buffered Ringer’s solution.

22246

Cholecystokinin and PC Synthesis in Pancreatic Acini 22247

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 acini by 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 ago- nists is probably 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.

EXPERIMENTAL PROCEDURES

Materials

Synthetic CCK8 was a gift from the Squibb Research Institute, Princeton, NJ. The following were purchased chromatographically purified collagenase from Cooper Biomedical Inc.; PC, oleic acid, soybean trypsin inhibitor, ATP, CTP, choline chloride, phosphocholine, CDP-choline, dithiothreitol, TPA, A23187, dibutyryl CAMP, fatty acid-free bovine serum albumin, staurosporine, sodium vanadate, and lipid standards from Sigma; Merk Silica Gel 60 high performance thin layer chromatography plates and NaF from Nakarai Chemicals; H-7, W-5, and W-7 from Seikagaku 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 Nu- clear; Centriprep-10 from Amicon Corp.; bovine serum albumin from Miles Laboratories, Elkhart, IN; Bio-Rad protein assay reagent from Bio-Rad; secretin and bombesin 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,, 11.1 glucose, and essential amino acid solution neutralized with NaOH. This HR buffer was supplemented with 0.5% bovine serum albumin and 0.02% soybean trypsin inhibitor, gased with 100% O,, and adjusted to pH 7.4.

Pulse and Chase Studies-In pulse studies, acini were incubated with 2 pCi/ml ['Hlcholine for 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 in a 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 to iodine vapor. Areas containing choline, phosphocholine, and CDP-choline were scraped and transferred to scintillation vials 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 radioactivity was measured with a liquid scintillation counter after the addition 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 10 ml 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

with or without secretagogues for the indicated time. The incubations were terminated by centrifugation in the microcentrifuge, and pelleted acini were extracted with 3 ml of a ch1oroform:methanol:HCI (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 acini with ['H]choline for longer periods, 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 with 2 pCi/ml ['H]choline in a six-well plastic dish at 37 "C under an air atmosphere containing 5% CO,. When acini were labeled under this condition the incorporation of [3H]choline into phospholipid of acini reached a plateau after 18-24 h (data not 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 PC degradation, labeled pancreatic acini were washed twice with fresh HR buffer, and then 1-ml aliquots of labeled acinar suspension were incubated with or without 1 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 addition of 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 experiments 0.1 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 Dounce glass 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 and then centrifuged at 100,000 X g for 60 min. The resultant supernatant and pellet were designed as the cytosolic and particulate fractions, respectively. The cytosol was concentrated further with Centriprep-10 as recommended by the supplier, and the protein concentrations of both cytosolic and particulate fraction were determined by using Bio-Rad protein assay reagent. Reactions were carried out for 20 min a t 37 "C and used 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 as described previously (21). The reaction mixture contained (in a total volume of 0.1 ml) 50 mM Tris-HC1 (pH 7.4), 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).

50 mM Tris-HC1 (pH 7.4), 10 mM MgCla, 0.4 mM ["CICDP-choline, Phosphocholine transferase activity was measured by incubating

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 sonica- tion 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 as described above was dried under nitrogen in liquid scintillation vials, and the radioactivity was measured. In the experiment examining the effects of Ca2+ on cytidylyltransferase activity, different free Cas+ concentrations were obtained by altering the Ca"

22248 Cholecystokinin and PC Synthesis in Pancreatic Acini to EGTA ratio using a computer program as described previously (23).

The results presented are the means k S.E. of three or more experiments unless otherwise stated. Statistical analysis was performed by Student's t test.

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 a 60-min 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 extent of 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 in a 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, in control acini 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, a time-

20 -

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.

{loo

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) 1 nM 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+-mobilizing secretagogues 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 the percentage of control value and are the mean f S.E. 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). These results suggest that CCK may inhibit PC synthesis not only by reducing choline uptake into acini but also by affecting the formation of CDP- choline 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


in Acini-Since carbamylcholine, bombesin, and the Ca2+ ionophore A23187 are considered to stimulate digestive enzyme release from pancreatic acini via an intracellular mechanism similar to that of CCK (l), we next examined the effects of these pancreatic secretagogues on PC synthesis in acini. As shown in Fig. 3, carbamylcholine inhibited PC synthesis in a concentration-dependent manner. 100 p~ carbamylcholine inhibited PC synthesis to 44.0 +. 2.0% of control (n = 5) at 60 min; this maximal effect was similar to that of CCK. The Ca2+ ionophore A23187, which stimulate amylase release by mobilizing Ca2+ (l), also inhibited PC synthesis in a concentration-dependent fashion, and 3 p~ A23187 reduced PC synthesis to 46.4 f 3.6% of control ( n = 4) at 60 min (Fig. 3). In addition, 100 nM bombesin reduced PC synthesis (Table I). When combined with 1 nM CCK8, which showed a maximal inhibition of PC synthesis, neither 100 p~ carbamylcholine nor 3 p~ A23187 induced an additional inhibition of PC synthesis (Table I), suggesting that these Ca2+-mobilizing secretagogues inhibit PC synthesis by the same mechanism. CCK has been considered to stimulate protein kinase C activation (1, 6, 7); however, neither 100 nM nor 1 p M TPA, a potent activator of protein kinase C (5), had any effect on PC synthesis in acini a t 60 min (Table I). Furthermore, no significant change in PC synthesis was observed when labeled acini were incubated with 100 nM TPA for 0-120 min (data not shown). Secretin and dibutyryl CAMP, which stimulate amylase release via a CAMP-dependent pathway (l), had no effect on PC synthesis (Table I). These data, therefore, suggest that CCK and carbamylcholine may inhibit PC synthesis in pancreatic acini by a Ca2+-dependent mechanism.

Reversal of CCK-induced Inhibition of P C Synthesis by Calmodulin Antagonists but Not by Protein Kinase C Inhibi- tors-To investigate further the mechanism underlying CCK- induced inhibition of PC synthesis, the effects of calmodulin antagonists on the reduction of PC synthesis induced by CCK in acini were studied. As shown in Table 11, W-7, a calmodulin antagonist (24), reversed CCK8-induced inhibition of PC synthesis in a concentration-dependent manner. In addition, W-5, another calmodulin antagonist (24), also reversed the inhibitory effect of CCK8 on PC synthesis although W-5 was much weaker than W-7 in these experiments (Table 11). On the other hand, 100 nM staurosporine, a protein kinase C inhibitor (25) that has been shown to inhibit CCK- or TPA-

TABLE I Effects of various secretagogues on rH]choline-hbeled PC

synthesis in pancreatic acini Acini pulsed with 2 pCi/ml [3H]choline for 30 min were incubated

with or without various secretagogues for 60 min. [3H]Choline-labeled PC was then extracted, and the radioactivity was determined. For each value the radioactivity determined at the beginning of the chase period was subtracted. Values were expressed as the percentage of control value and are the mean f S.E. for the number of experiments shown.

Secretagogues No. of [3H]PC experiments synthesis

% of control CCK8 (1 nM) 10 36.8 f 2.0 Carbamylcholine (100 p ~ ) A23187 (3 p M )

5 44.0 f 2.0

CCK8 (1 nM) + carbamylcholine (100 p M ) 3 37.7 f 3.9 4 46.4 f 3.6

CCK8 (1 nM) + A23187 (3 p M ) 3 38.0 -C 4.0 Bombesin (10 nM) 3 57.3 f 6.4 TPA (100 nM) 3 102.0 f 3.5” TPA (1 p M ) 3 98.5 & 5.3” Secretin (10 nM) 3 102.4 f 4.3“ Dibutyryl CAMP (100 p ~ ) 3 97.5 f 4.8”

Not significant versus control.

TABLE I1 Effects of calmodulin antagonists or protein kinase C inhibitors on

CCK-induced PC inhibition of PC synthesis in acini Acini pulsed with 2 pCi/ml [3H]choline for 30 min were incubated

with or without 1 nM CCK8 for 60 min in the presence or absence of various inhibitors. [3H]Choline-labeled PC was then extracted, and the radioactivity was determined. For each value the radioactivity determined at the beginning of the chase period was subtracted. Values were expressed as the percentage of control value and are the mean f S.E. for the number of experiments shown. Neither calmodulin antagonists nor protein kinase C inhibitors alone significantly affect PC synthesis in control acini (data not shown).

Agents No. of [3H]PC experiments synthesis

% of control CCK8 (1 nM) 5 37.6 f 2.3

4 47.0 f 2.0 90.4 k 3.6 38.7 f 3.9”

3 52.0 rt 4.0 CCK8 + staurosporine (100 nM) 3 CCK8 + H-7 (100 p M ) 3

38.5 rt 4.0” 39.5 f 3.0”

NS uersus 1 nM CCK8.

CCK8 + W-7 (10 pM) CCKS + W-7 (100 pM) 4 CCK8 + W-5 (10 pM) 3 CCKS + W-5 (100 p M )

induced amylase release from pancreatic acini (26), did not affect the inhibition of PC synthesis induced by CCK8 (Table 11). In addition, 100 p~ H-7, another protein kinase C inhibitor (27), also had no effect on CCK-induced inhibition of PC synthesis (Table 11). Thus, the results suggest that the Ca2+- calmodulin dependent pathway may be involved in this inhibition.

Studies were also performed to determine whether CCK stimulated the degradation of PC by using acini labeled with [3H]choline for 24 h to equilibrium as described under “Meth- ods.” When these labeled acini were incubated with 1 nM CCK8 for 60 min, CCK failed to induce an increase in the [3H]choline metabolites released in the medium or a decrease in [3H]PC in acini when compared with control acini (data not shown). Thus, PC breakdown may not contribute to the inhibition of PC synthesis induced by CCK in acini.

Effects of CCK on the Enzyme Activity via the CDP-Choline Pathway-To determine whether the treatment of CCK alters the activities of enzymes involved in the de nouo synthesis of PC via the CDP-choline pathway, the activities of enzymes 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 previously we determined the apparent K,,, and V,,, values of each enzyme. Choline kinase activity and phosphocholine transferase activity were detected only in the cytosolic fraction and the particulate fraction, respectively. The K, and Vmax of choline kinase for ATP were 1.55 f 0.09 mM and 3.42 f 0.37 nmol/min/mg protein (n = 4), respectively. The K, and V,,, of phosphocholine transferase for DAG were 0.015 f 0.007 mM and 1.35 f 0.38 nmol/min/mg protein ( n = 3), respectively. The K, and V,,, of cytidylyltransferase in a cytosolic fraction for CTP were 0.44 f 0.07 mM and 0.78 f 0.13 nmol/min/mg protein (n = 4), respectively. The K, and V,,, of cytidylyltransferase in a particulate fraction for CTP were 0.37 -+ 0.05 mM and 0.62 -t 0.10 nmol/min/mg protein (n = 4), respectively. These apparent K, and Vmax values of each enzyme were similar to those observed in other cell types (12, 13). When acini were treated with 1 nM CCK8 for 60 min, choline kinase activity and phosphocholinetransferase activity were not significantly changed by CCK (data not shown). By contrast, CCK treatment of acini reduced cytidylyltransferase activities in both cytosolic and particulate fractions (Table 111, Experiment 1). These results indicate

22250 Cholecystokinin and PC Synthesis in Pancreatic Acini 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 or four separate experiments. Values in parentheses indicate enzyme activities expressed as the percentage of control.

Cytidylyltransferase Specific activity

nmolfminfmgpro- tein

Experiment 1 Cytosolic

Control ( n = 4) CCK8 ( n = 4)

Control (n = 4) 0.57 rtr 0.06 CCK8 (n = 4) 0.43 k 0.04b (75)

0.73 rtr 0.05 0.41 rtr 0.06" (56)

Particulate

Experiment 2 Cytosolic

Control ( n = 4) 0.70 k 0.06 CCK8 ( n = 4) 0.29 rtr 0.04' (41) CCK8 + W-7 ( n = 4) 0.65 rtr 0.06

Particulate Control ( n = 4) 0.55 -+ 0.05 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 by CCK treatment was also blocked by W-7. These results suggest the possibility that CCK may phosphorylate cytidylyltransferase by the activation of Ca2+-calmodulin-dependent kinase, thereby decreasing the activity of this enz$me. On the other hand, it is possible that the elevated 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 the presence 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+ (data not shown), 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-Since CCK 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

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 a time-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 was not statistically significant.

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.


TABLE IV Effects of CCK on the distribution of 3H in 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 with or 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

Lipids Control CCK8 0 min at 60 at 60

min min

PC PI + PS PE

Lyso-PC Sphingomyelin

PA PIP PIP2

DAG TG Free fatty acid Monoglyceride

24,708 177 881

82 83

607 50 15

1,810 10,806

755 48

cpm 32,724

180 1,138

136 87

306 47

8

754 12,370

432 54

27,822 190

1,273 136 108

1,123 57 12

1,620 14,170

440 56

in pancreatic acini. The pulse study using [3H]choline has shown that CCK reduced both the total uptake of labeled choline into acini and the incorporation of labeled choline into PC. Furthermore, pulse-chase experiments demonstrated that CCK induced inhibition of [3H]PC formation which was accompanied with a delayed disappearance of [3H]phosphocholine in acini, indicating that CCK may affect the conversion from phosphocholine to CDP-choline thereby inhibiting PC synthesis in acini. Hormonal regulation of PC synthesis has been shown recently in other cell types (15, 31-33, 36). In hepatocytes, glucagon (31), norepinephrine (32), and vasopressin (33) have been shown to reduce PC synthesis. Cyclic AMP derivatives or phosphodiesterase inhibitors also reduce PC synthesis in hepatocytes (34), partly by reducing total choline uptake into cells (34) as observed in pancreatic acini treated with CCK. This suggests that the activation of protein kinase A may be involved in the regulation of PC synthesis in this cell type (34). By contrast, it has been demonstrated that hormones or TPA increases PC synthesis in HeLa cells (35), adipocytes (36), GH, cells (15), indicating possible involvement of protein kinase C activation in regulation of PC synthesis in these cell types. In pancreatic acini, not only CCK but also other Ca2+-mobilizing secretagogues such as carbamylcholine and bombesin reduced PC synthesis. In addition to receptor-activated inhibition of PC synthesis, the Caz+ ionophore A23187, which has been shown to stimulate amylase release via intracellular Ca2+ mobilization (1, 6, 7), reduced PC synthesis to the same extent as did CCK. The concentration of CCK dependence for both the inhibition of PC synthesis and the increase of cytosolic Ca2+ (37) is almost identical. When combined with CCK, A23187 or carbamylcholine did not cause a further inhibition of PC synthesis, indicating that these secretagogues inhibit PC synthesis via the same mechanism. By contrast, neither TPA nor CAMP- dependent secretagogues had any effect on PC synthesis in acini. Furthermore, we have shown that calmodulin antagonists reverse the inhibition of PC synthesis induced by CCK. On the other hand, staurosporine, a protein kinase C inhibitor

(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 results suggest strongly that the CCK 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 pathway of PC synthesis, and the enzymes comprising this pathway have been studied extensively (12,13). In the present study, CCK treatment of acini specifically reduced the activity of CTP:phosphocholine cytidylyltransferase in both cytosolic and particulate fractions; yet neither choline kinase activity nor phosphocholine transferase activity was affected by CCK treatment. This result corresponds well with data in the pulse- chase study, which shows inhibition of PC formation accompanied by a delayed disappearance of phosphocholine in acini treated with CCK. When subcellular fractions of acini were prepared in the presence of vanadate and NaF and the enzyme activities were then assayed, the extent of inhibition of cytidylyltransferase by CCK treatment was much larger as compared with that observed without the addition of phosphatase inhibitors to the homogenizing solution. Furthermore, when acini were treated with CCK in the presence of W-7, the reduction of cytidylyltransferase activity induced by CCK was also reversed. These results strongly suggest the possibility that the activation of Ca”-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acini. Thus, the prevention by phosphatase inhibitors of dephosphorylation of cytidylyltransferase during the preparation of acinar subcellular fraction may lead to increase the extent of inhibition by CCK of the enzyme activity which is subse- quently assayed. The presence of more than one form of Ca2+- calmodulin-dependent kinase has been shown in pancreatic acinar cells (38). It has recently been shown that protein kinase A phosphorylates cytidylyltransferase in hepatocytes (39). In addition, phosphorylation of cytidylyltransferase by protein kinase A decreases the activity of cytidylyltransferase whereas the dephosphorylation condition increases the activity of this enzyme (39). Further studies will be necessary to examine whether cytidylyltransferase of acinar cells can be directly phosphorylated by Ca2+-calmodulin-dependent kinase in uitro.

It is also possible that the increased concentration of cytosolic Ca2+ induced by CCK may directly regulate the activity of cytidylyltransferase. However, this possibility seems to be unlikely because the activity of cytidylyltransferase prepared from acini was not significantly altered by physiological concentrations of free Ca2+ (1 nM-1 p M ) (40), and only a supraphysiological concentration of Ca2+ (1 mM) caused a small decrease in the enzyme activity. It has been also shown that high concentration of calcium decreases the activity of purified cytidylyltransferase of liver (41). By contrast, Sang- hera and Vance (42) showed that the 7 mM calcium in the medium as well as A23187 and vasopressin increases PC synthesis and cytidylyltransferase activity in hepatocytes. This seems to be a result opposite those of our study in pancreatic acini. However, Tijburg et al. (35) have also re- ported that vasopressin inhibits PC synthesis in hepatocytes (33), a finding contrary to the report by Sanghera and Vance (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. In addition to a decrease in PC formation, CCK treatment of acini caused a delayed disappearance of [3H]DAG in 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 in the accumulation of [3H]DAG which is not utilized for PC synthesis in acini. CCK treatment also significantly increased 13H]PA and [3H] TG. DAG, which is not utilized for PC synthesis in CCK- treated 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 sensi- tive 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 sustained DAG production. However, this labeling condition did not achieve the equilibrium labeling of PC in acini because incorporation of labeled choline into PC still increased after 2 h of incubation. Subsequent experiments utilizing streptolysin-o-per- meabilized 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 not shown). If CCK really stimulated PC breakdown 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 a significant release of choline metabolites from acini labeled for 24 h. One possible explanation could be that the releasable pool of choline metabolites in response to CCK may diminish during longer incubation. Thus, it seems unlikely that CCK stimu- lates PC breakdown to accumulate DAG, and hence the precise mechanism for a sustained DAG production stimulated by CCK remains unknown at present. However, the present results raise the possibility that CCK may accumulate DAG by reducing the synthesis of PC from DAG and CDP- choline. It has been demonstrated recently that a choline- deficient diet increases the mass level of DAG in the rat liver accompanied by a decrease in PC and an increase in TG, resulting in the activation of protein kinase C (46). Therefore, it is possible that the inhibition by CCK of PC formation may contribute to the sustained accumulation of DAG and the subsequent activation of protein kinase C in pancreatic acini.

Another interesting aspect is that the inhibitory effect of CCK on PC synthesis may be 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, a choline-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 zymogen vesicles to cell membranes, resulting in abnormal activation of digestive enzymes (49).

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