Pancreatic phospholipase A2 from the small intestine is a secretin-releasing factor in rats

James P. Li, Ta-Min Chang, David Wagner, and William Y. Chey

Konar Center for Digestive and Liver Diseases, University of Rochester Medical Center, Rochester, New York 14624


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A secretin-releasing activity exists in the upper small intestine and pancreatic juice in the rat and the dog. Group I pancreatic phospholipase A2 (PLA2) in canine pancreatic juice and porcine pancreatic PLA2 stimulate the release of secretin from both STC-1 cells and a secretin-producing cell (S cell)-enriched preparation isolated from rat duodenal mucosa. We investigated the distribution and release of pancreatic PLA2-like immunoreactivity in the gastrointestinal tract and the role of PLA2 on the release of secretin and pancreatic exocrine secretion in response to duodenal acidification in anesthetized rats. PLA2-like immunoreactivity was detected in the mucosa throughout the gastrointestinal tract. High concentrations of PLA2 were found in both the small intestine and the pancreas. Duodenal acidification significantly increased the release of PLA2 from the upper small intestine (385% over basal secretion). Intravenous infusion of an anti-PLA2 serum (anti-PLA2) dose-dependently inhibited the release of secretin and pancreatic exocrine secretion in response to duodenal acid perfusion. Preincubation of the concentrate of intestinal acid perfusate (10-fold) from donor rats with the anti-PLA2 significantly suppressed its stimulation of secretin release and pancreatic exocrine secretion in recipient rats. We conclude that pancreatic PLA2 also functions as a secretin-releasing factor in the small intestine that mediates acid-stimulated release of secretin in rats.

pancreatic secretion; anti-phospholipase A2 serum


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SECRETIN IS A major gut hormone that regulates pancreatic water and bicarbonate secretion (9, 10, 14). Secretin is released from S cells of the upper small intestine in response to luminal stimuli, including gastric acid, fatty acid, bile salts, and other dietary elements (10). Some neuropeptides and neurotransmitters, such as pituitary adenylate cyclase-activating polypeptide (PACAP; see Ref. 27), gastrin-releasing peptide (GRP; see Ref. 30), or serotonin (29), also stimulate or are involved in the release of secretin. It has been reported that secretin-releasing peptides exist in both the upper small intestine (31) and pancreatic juice (32) to mediate the release of endogenous secretin in the dog (42) and rat (32). Chang et al. (8) have purified two secretin-releasing factors (SRFs) from canine pancreatic juice. Both are 14-kDa polypeptides that are structurally homologous to canine pancreatic phospholipase A2 (PLA2). These two secretin-releasing peptides (8) and purified porcine pancreatic PLA2 (4) were found to stimulate secretin release from STC-1 cells and S cell-enriched cell preparations isolated from rat duodenal mucosa. However, it has not been studied whether the secretin-releasing peptide activity in the small intestine is attributed to PLA2.

It has become evident that PLA2 are a heterogeneous family of lipolytic enzymes that can be classified into at least five subtypes of mammalian origin (12). Group I, II, and III PLA2 are low-molecular-weight PLA2 of 14 kDa referred to as secretory, extracellular enzymes. Group IV PLA2 are high-molecular-weight PLA2 of 60-110 kDa referred to as cytosolic PLA2, whereas bee venom PLA2 constitute the group V enzyme. Recent data have indicated that PLA2 play a number of important roles in cells and tissues (13). Pancreatic PLA2 belongs to the group I PLA2 and plays a central role in the digestion of dietary and biliary phospholipids. It is also involved in stimulation of cell growth (15), contraction of smooth muscle (41), and regulation of progesterone (39) and prostaglandin (44) release. We hypothesized that PLA2 may function as a modulator of intestinal endocrine cells to play an important role in stimulation of secretin release in the rat.

The aim of the present study was to investigate the distribution of phospholipase A2-like immunoreactivity (PLA2-LI) in the gastrointestinal tract and the pancreas, the release of PLA2 from the upper small intestine and pancreas after duodenal perfusion of acid, the effect of a specific anti-PLA2 serum (anti-PLA2) on pancreatic exocrine secretion and the release of secretin in response to duodenal acidification, and the effect of the antibody on SRF activity in duodenal acid perfusate in anesthetized rats.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation. Male Sprague-Dawley rats weighing between 220 and 280 g were fasted for 24 h with free access to drinking water before surgery. Under anesthesia with intraperitoneal injection of 25% urethane at a dose of 120 mg/100 g body wt, a midline abdominal incision was made. A polyethylene tube (ID 3.0 mm, OD 4.0 mm) for duodenal infusion was inserted in the proximal duodenum 5 mm distal to the pylorus through the stomach followed by ligation of the pylorus. A jugular vein catheter was prepared with a polyethylene tube (PE-50, ID 0.58 mm, OD 0.96 mm). The tube was kept patent by infusion of 0.15 M NaCl at a rate of 1 ml/h. In the donor-recipient study, a 20-cm upper small intestinal loop was made by placing an additional cannula (ID 3.0 mm, OD 4.0 mm) in the jejunum 15 cm distal to the ligament of Treitz for collection of the perfusate. A polyethylene tube (PE-10, ID 0.28, OD 0.61 mm) was inserted in the pancreatic duct via the ampulla for collection of pancreatic juice. A second PE-10 tube was inserted in the bile duct proximal to the pancreatic duct for diversion of bile to the exterior. The abdominal wound was covered by a piece of wet gauze soaked with isotonic saline.

Experimental design. Experiments were performed 30 min after surgery. After 90-min collection of basal pancreatic secretion, 0.02 N HCl in saline was infused intraduodenally at a rate of 4.5 ml/h for 60 min in five rats. To study the effect of anti-PLA2 on acid-induced pancreatic exocrine secretion and the release of secretin, anti-PLA2 at 0.1, 0.3, and 0.5 ml/rat was injected via the jugular vein 30 min before duodenal infusion of acid in five rats each. Normal rabbit serum (NRS) was injected intravenously at 0.5 ml/rat before intraduodenal infusion of 0.02 N HCl began in five rats as controls. The antibody used in this experiment was the same as the one used for RIA for PLA2 described below.

In the donor-recipient study, the upper small intestinal loop was washed with 40 ml of warm 0.15 M NaCl followed by perfusion of the loop with 0.02 N HCl at 0.3 ml/min for 1.5 h while both bile and pancreatic juice were diverted. The acid-perfusate was continuously collected from the jejunal cannula in an ice-chilled beaker and then centrifuged at 3,000 g and 4°C for 25 min. The supernatant solution was lyophilized and concentrated 10-fold, adjusted to pH 7.0, and then incubated with anti-PLA2 or NRS (1:10) at 37°C for 30 min. The materials were further filtered through an Amicon PM-10 membrane (Grace, Danvers, MA) to remove the antibody and antibody-antigen complex. The filtrate was reinfused in the upper small intestine of recipient rats after a 90-min collection of basal pancreatic secretion.

To determine the release of PLA2-LI by duodenal acidification, an aliquot of 2 ml of the perfusate collected during the basal period or duodenal acid perfusion was mixed with 0.1 ml of 2% BSA in saline and stored at -20°C before determination of PLA2-LI by RIA. To determine if PLA2 has different susceptibility in the duodenal lumen during saline and acid perfusion, porcine pancreatic PLA2 at a concentration of 1 ng/ml was added to perfusion solution of saline or acid. After a 30-min perfusion with saline alone in the basal period, PLA2 in saline, HCl alone, or PLA2 with or without HCl was perfused intraduodenally in three groups of three rats each as described above. The perfusate was collected during basal and experimental periods in 30-min intervals. The perfusate was treated and stored as described below for RIA of PLA2.

To determine the distribution of PLA2 in the gastrointestinal tract, five rats were killed by cervical dislocation after 24 h of fasting. The stomach, duodenum, jejunum-ileum, colon, and pancreas were immediately taken and washed separately with 0.15 M NaCl at 4°C. Each tissue was homogenized in 5 vol of 10 mM Tris · HCl, pH 7.4, at 4°C using a Polytron at maximum output for 30 s. The homogenate was centrifuged at 108,000 g for 1 h at 4°C. The supernatant solution was stored at -70°C for RIA of PLA2.

RIA of PLA2. RIA of PLA2 was carried out using a specific anti-PLA2 serum raised in our laboratory. The antiserum was raised in New Zealand White rabbits by immunization with purified porcine pancreatic PLA2 (4) emulsified in Freund's adjuvant and boosted monthly with the enzyme emulsified in Freund's incomplete adjuvant (5, 6). After five booster injections, the titer of the antibody increased to 1:106. The antibody is specific for PLA2-I and has no cross-reaction with any known gastrointestinal regulatory peptides or islet hormones, including rat secretin, CCK-8, human gastrin-17-I, glucagon, porcine GRP, insulin, rat pancreatic polypeptide, vasoactive intestinal peptide, and PACAP-38. The competitive tracer-binding curves with standard PLA2 and some of these peptides are shown in Fig. 1. The anti-PLA2 serum also had no cross-reaction with reptilian and bee venom PLA2 (all obtained from Sigma), as shown in Fig. 2. An RIA method was developed using purified porcine pancreatic PLA2 as the standard. Purified PLA2 (5 µg) was radioiodinated using the chloramine T-catalyzed method (19). The labeled enzyme was then purified by gel filtration through a Sephadex G-50 superfine column (1.3 × 58 cm) run in 50 mM sodium phosphate, pH 7.5, containing 0.15 M NaCl, 0.5% BSA, and 0.02% NaN3. Purified 125I-labeled PLA2 had a specific activity of 260.6 µCi/nmol. The sample or standard was incubated with anti-PLA2 (at 1:106 dilution) in 50 mM sodium phosphate, pH 7.0, containing 0.1% BSA and 0.02% NaN3 at 4°C for 48 h. 125I-PLA2 (5,000 counts/min) was then added, and the reaction mixture was further incubated for 48 h at 4°C. The reaction mixture was then mixed with 30 µg of bovine IgG followed with 1.4 mg of insoluble protein A (Sigma) in a final volume of 2 ml. The final reaction mixture was incubated further for 1 h with occasional mixing and then was centrifuged at 1,650 g for 30 min. The supernatant solution (containing free PLA2 counts) and the pellet (containing antibody-bound PLA2 counts) were separated, and both were counted in a Wallac model 1271 gamma counter with automatic data reduction using the RIACALC software provided by the manufacturer. The assay has a minimum detection limit of 10 pg PLA2 and intra- and interassay variations of 7 and 9%, respectively.


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Fig. 1.   Standard curve and cross-reaction of anti-phospholipase A2 (PLA2) antibody with some gastrointestinal hormones. B/B0, bound-to-free ratio; VIP, vasoactive intestinal peptide, Gas, gastrin; rSec, rat secretin.



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Fig. 2.   Cross-reaction of anti-PLA2 antibody with other PLA2. The results of cross-reactivity of porcine pancreatic PLA2 are compared with those of reptilian and bee venom PLA2. The antibody did not cross-react with nonmammalian secretory PLA2.

Determination of pancreatic secretion and plasma concentration of secretin. Pancreatic juice was collected continuously by inserting the pancreatic duct cannula in a glass micropipette (Drummond Scientific, San Francisco, CA) in 30-min intervals. The volume of the fluid retained in the micropipette was determined by measuring the length of fluid filling the micropipette (3.85 µl/cm), as described previously (28, 31). After the measurement of the fluid volume, the content of the micropipette was blown in a 0.2-ml microcentrifuge tube using a rubber bulb. A sample of 10 µl was immediately taken to determine the bicarbonate concentration using a 965 Carbon Dioxide Analyzer (Ciba-Corning Diagnostics, Halstead Essex, UK). At the end of each experiment, blood samples were drawn immediately from the aorta and collected in heparinized glass tubes at 4°C. Plasma was obtained and stored at -70°C. Plasma secretin level was measured by the RIA method described previously (6).

Data analyses. All values were expressed graphically as means ± SE. The percentage (%) increase in pancreatic secretion over the basal value was calculated by comparison between the values obtained in the last 60-min treatment period and the last 60-min basal period. The statistical differences in these data were analyzed using one-way ANOVA. Tukey's post hoc test was applied for multiple comparisons of the means. A difference between two means with P values of <0.05 is regarded as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Distribution and release of PLA2 in the gastrointestinal tract and the pancreas. RIA of PLA2 in the tissue extracts indicated that PLA2 distributed throughout the gastrointestinal tract and the pancreas (Table 1). The contents of PLA2 in the pancreas and the small intestine were similar, although PLA2 in the duodenum was slightly less than that in the jejunum-ileum and the pancreas. PLA2 in the stomach and the colon was much less compared with that in the pancreas and the small intestine.

                              
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Table 1.   Pancreatic PLA2-like immunoreactivity in the gastrointestinal tissue and the pancreas of rats

Perfusion of the upper small intestinal loop with 0.02 N HCl significantly increased the concentration of PLA2 in the intestinal perfusate (from 0.42 ± 0.04 to 1.49 ± 0.63 ng/ml, n = 5, P < 0.05; Fig. 3). When PLA2 in saline was perfused intraduodenally, there was no time-dependent decrease in PLA2 immunoreactivity in the perfusate. As shown in Table 2, the average concentration of PLA2-LI of the nine samples collected was 1.05 ± 0.14 ng/ml, which was 0.93 ng/ml above the average basal concentration, indicating a 93% recovery of the exogenous PLA2. Similarly, the average concentration of PLA2-LI found in the perfusate of PLA2 with or without HCl (2.08 ± 0.20 ng/ml) was 0.98 ng/ml over that of the HCl control, corresponding to a 98% recovery of exogenous PLA2. Although acid perfusion did not influence the PLA2 concentration in pancreatic juice (Fig. 3), the output of PLA2 was significantly increased because the volume of pancreatic fluid, as shown in Fig. 4, was markedly elevated (72% over basal secretion) in response to acid.


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Fig. 3.   Release of pancreatic PLA2 from the upper small intestine (A) and the pancreas (B) in response to duodenal acidification. Basal, basal PLA2 level after 24 h of fasting; HCl, 0.02 N HCl (4.5 ml/h id). Upper small intestinal acid perfusate and pancreatic juice were continuously collected for 60 min during acid perfusion. PLA2 levels were determined by RIA. Values are means ± SE from 5 rats in each group. *P < 0.05 compared with basal values.


                              
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Table 2.   Stability of porcine pancreatic PLA2 perfused through the duodenum



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Fig. 4.   Effect of PLA2 antibody on acid-stimulated pancreatic secretion. Pancreatic PLA2 antibody at 0.1, 0.3, and 0.5 ml/rat or normal rabbit serum (NRS) at 0.5 ml/rat was injected iv 30 min before infusion of 0.02 N HCl id for 1.5 h. Pancreatic exocrine secretion in the last 60 min was compared with basal secretion in 60 min. A: volume. B: bicarbonate output. Values are means ± SE from 5 rats in each group. *P < 0.05 and **P < 0.01 vs. basal pancreatic secretion.

Effect of anti-PLA2 on pancreatic exocrine secretion and the release of secretin in response to duodenal acidification. Basal pancreatic secretion of fluid (17.6 ± 3.6 µl/30 min) and bicarbonate (0.54 ± 0.14 µeq/30 min) was stable during the study period. Duodenal infusion of 0.02 N HCl significantly increased pancreatic secretion of fluid (73.2 ± 10.7% over basal secretion, P < 0.01) and bicarbonate output (144.1 ± 27.8% over basal, P < 0.01). Intravenous injection of anti-PLA2 at 0.1, 0.3, and 0.5 ml did not influence basal pancreatic exocrine secretion (data not shown) but dose-dependently inhibited the acid-stimulated pancreatic fluid and bicarbonate secretion (Fig. 4). The plasma level of secretin also decreased in a dose-related manner after anti-PLA2 administration (Fig. 5). NRS did not influence either pancreatic secretion or the release of secretin (Figs. 4 and 5).


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Fig. 5.   Effect of PLA2 antibody on acid-stimulated release of secretin. Pancreatic PLA2 antibody at 0.1, 0.3, and 0.5 ml/rat or NRS at 0.5 ml/rat was injected iv 30 min before infusion of 0.02 N HCl id for 1.5 h. Values are means ± SE from 5 rats in each group. *P < 0.05 and **P < 0.01 vs. basal plasma concentration of secretin.

Pancreatic secretion and release of secretin in response to intestinal acid perfusate with and without preincubation with anti-PLA2 in recipient rats. Intraduodenal administration of intestinal acid perfusate increased pancreatic secretion of fluid volume (72.2 ± 11.3% over basal secretion) and bicarbonate output (140.7 ± 28.9% over basal) and caused an elevation of plasma secretin concentration to 6.2 ± 1.2 pM. To test if PLA2-LI present in the duodenal acid perfusate contributes to this SRF activity (30), the perfusate was preincubated with either NRS or anti-PLA2 and then ultrafiltrated. As shown in Fig. 6, intraduodenal perfusion in the recipient rats of the donor acid perfusate, which was preincubated with NRS, resulted in an increase in plasma secretin concentration to 5.6 ± 0.8 pM and an increase of pancreatic secretion of fluid volume by 61.5 ± 7.6% and bicarbonate output by 108.7 ± 12.7% over basal. These increases were not significantly different from those produced by the untreated perfusate described above. In contrast, the acid perfusate preincubated with anti-PLA2 failed to elevate plasma secretin concentration (1.5 ± 0.6 pM) and produced only a small increase in pancreatic secretion of fluid volume (10.1 ± 5.1% over basal) and bicarbonate output (44.0 ± 19.0% over basal) in the recipient rats. Thus anti-PLA2-treated perfusate elicited less fluid volume and bicarbonate output by 81.2 ± 8.9 and 62.0 ± 9.9%, respectively, than NRS-treated perfusate.


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Fig. 6.   Release of secretin and pancreatic secretion in response to upper small intestinal acid perfusate preincubated with anti-PLA2 in recipient rats. The upper small intestine was perfused with 0.02 N HCl at 0.3 ml/min for 1.5 h while bile and pancreatic juice were diverted in donors. The acid perfusate was concentrated 10-fold and incubated with anti-PLA2 or NRS (1:10) at 37°C for 30 min. The materials were filtered through a PM-10 Amicon membrane to remove anti-PLA2 and reinfused into the recipient rats. A: volume. B: bicarbonate output. C: secretin. Values are means ± SE from 5 rats in each group. *P < 0.05 and **P < 0.01 vs. basal pancreatic secretion or basal plasma concentration of secretin.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study have indicated that pancreatic PLA2 is distributed in the small intestine mucosa and may function as an SRF during duodenal acidification. Our previous studies (28, 31) have shown that duodenal acidification in the rat elicited the release of an SRF activity in the duodenal lumen that can be recovered from the acid perfusate. Infusion of a concentrate of ultrafiltrated duodenal acid perfusate in the duodenum of a recipient rat stimulates the release of secretin and pancreatic exocrine secretion of fluid and bicarbonate. We have demonstrated in the present study that pancreatic PLA2-LI is present in the luminal perfusate, and its concentration is increased upon duodenal acidification in the rat. Because exogenous PLA2 in saline or acid perfused intraduodenally did not result in different recovery of PLA2-LI (Table 2), the increase in PLA2-LI concentration must be the result of an increase in the release of endogenous PLA2. Immunoneutralization of PLA2 with a specific anti-PLA2 serum result in a significant inhibition of the increases of plasma secretin concentration and pancreatic exocrine secretion of fluid and bicarbonate elicited by duodenal acidification. Pretreatment of the concentrated perfusate from donor rats with anti-PLA2 resulted in a substantial decrease in the SRF activity of the perfusate. The observation suggests that a PLA2-LI in the acid perfusate is a constituent of the SRF activity and thus corroborates well our previous observations that canine (8) and porcine (4) pancreatic PLA2 stimulate secretin release from secretin-producing cells. The source of the pancreatic PLA2-like SRF appeared to be in the upper small intestine as pancreatic juice was diverted in both donor and recipient rats in the present study. This view is supported by the findings that pancreatic PLA2 is widely distributed in the intestine and is released in the duodenal lumen upon acid perfusion during pancreatic juice diversion. It should be noted that the presence of pancreatic PLA2 in nonpancreatic tissues, including the stomach and the intestine, has been documented previously (25, 37, 40, 45). mRNA for PLA2-I has been detected in the mucosa of human ileum (34) and guinea pig stomach (47). Moreover, luminal secretion of PLA2 activity has been documented in the rat small intestine (1, 2) and guinea pig gastric juice (45). It should be noted that PLA2-LI in the intestinal acid perfusate could not be derived from gastric juice as the pylorus was ligated in the present study. In addition, it is unlikely that the detection of PLA2-like immunoreactivity in the intestinal lumen was the result of mucosal damage because we infused a diluted acid (0.02 N HCl) so that, during acid infusion, the luminal pH was maintained between 3.0 and 4.0 because of duodenal bicarbonate secretion (unpublished observation). Moreover, we have previously shown by electron microscopy in dogs that duodenal perfusion with 0.1 N HCl does not cause mucosal damage (7).

The mechanism of action of pancreatic PLA2 as an SRF is not clear at present. Although a small amount of IgG has been shown to be transported across human jejunal mucosa (22), it is unlikely that a sufficient amount of anti-PLA2 is transported to the intestinal lumen to inhibit the release of secretin and exocrine pancreatic secretion upon duodenal acidification. An alternative explanation is that PLA2 may also be released and acts locally at the basolateral interstitial space of the intestinal mucosa. Our previous study (28) indicated that the release and action of SRF during duodenal acidification are neurally mediated, depending on the vagal afferent pathway. It is possible that, in addition to the release in intestinal lumen, PLA2 is also released locally and acts on mucosal vagal afferent fibers to stimulate secretin release. The presence of a PLA2-specific receptor (11, 26) and action of secretory PLA2 in neurons has been documented (37). On the other hand, luminally released PLA2 may penetrate the intestinal mucosa and acts on S cells directly and/or indirectly through the vagal afferent pathway. The neural action of PLA2, either locally released or derived from the lumen, would then be sensitive to anti-PLA2 that reaches the lamina propria in the intestinal mucosa where mucosal afferent nerve fibers are found. However, these modes of action by PLA2 remain hypothetical and may be tested in our future study.

It is not unusual for pancreatic PLA2 to be involved in the regulation of secretin release. PLA2 are a family of lipolytic enzymes that release fatty acid specifically from the sn-2 position of phospholipids and are classified into several groups according to their primary structures (12, 13, 46). Several important functions have been identified for various PLA2, including phospholipid digestion and metabolism, host defense, and signal transduction (13). Pancreatic PLA2 is a group I PLA2 and a well-defined digestive enzyme. The results of recent studies have indicated that this enzyme is also present in nonpancreatic tissues (36, 40) and is involved in regulation of other cellular functions, including secretion of progesterone (39), prostaglandin production (44), gene expression of type II PLA2 (24), stimulation of smooth muscle contraction (23, 41), and stimulation of cell proliferation (15-17). Thus our observations that pancreatic PLA2 stimulates secretin release from secretin-producing cells (4, 8) and may function as an SRF (present study) have provided an addition to the functional list of this enzyme. It is not surprising, therefore, that pancreatic PLA2 is involved in the regulation of secretin release.

It should be noted that, based on the results of this study, we could not rule out the possibility that other SRF exist and also play a role in acid-stimulated release of secretin. This is quite analogous to that of CCK-releasing factors in regulation of the release of CCK (35, 38). Two luminal CCK-releasing factors, luminal CCK-releasing factor (43) and diazepam-binding inhibitor (18), have been purified, sequenced, and shown to release CCK and elevate pancreatic protein secretion through a CCK-dependent mechanism. In addition, monitor peptide isolated from rat pancreatic juice (21) also stimulated the release of CCK from CCK-producing cells (3, 33) and from rats in vivo (20, 21). We have attempted to isolate SRF from rat intestinal acid perfusate. So far, we have identified a few fractions possessing SRF activity that have no cross-reaction with PLA2 antibody and have chromatographic properties different from that of PLA2 (unpublished observation). Therefore, it is likely that SRF also exists in multiple forms to mediate acid-stimulated secretin release.

In summary, the results of the present study demonstrate that pancreatic PLA2 is distributed throughout the gastrointestinal tract and was abundant in the pancreas and the small intestine. Duodenal infusion of diluted acid stimulated the release of PLA2 from the upper small intestine to elicit the secretion of secretin, which was abolished by intravenous administration of anti-PLA2. SRF activity in duodenal acid perfusate from donor rats was also completely suppressed by preincubation with anti-PLA2. These results strongly suggested that pancreatic PLA2 in the small intestine is an SRF in regulation of the release of secretin and pancreatic exocrine secretion in response to duodenal acidification in the rat.


    ACKNOWLEDGEMENTS

We are grateful to Laura Braggins and Frank Roth for technical assistance.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-25962.

Address for reprint requests and other correspondence: W. Y. Chey, Rochester Institute for Digestive Diseases and Sciences, 222 Alexander St., Ste. 3100, Rochester, NY 14607.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 April 2000; accepted in final form 10 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arcuni, J, Wang L, Franson RC, and Sonnino RE. Biochemical alterations in rat Thiry-Vella fistulas. J Invest Surg 13: 95-101, 2000[ISI][Medline].

2.   Acruni, J, Wang L, Yousef K, Chiu S, Mikkelson K, Franson RD, and Sonnino RE. Secretory event in intestinal grafts during preservation ischemia. J Surg Res 84: 233-239, 1999[ISI][Medline].

3.   Bouras, EP, Misukonis MA, and Liddle RA. Role of calcium in monitor peptide-stimulated cholecystokinin release from perifused intestinal cells. Am J Physiol Gastrointest Liver Physiol 262: G791-G796, 1992[Abstract/Free Full Text].

4.   Chang, T-M, Chang CH, Wagner DR, and Chey WY. Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells. J Biol Chem 274: 10758-10764, 1999[Abstract/Free Full Text].

5.   Chang, T-M, and Chey WY. Radioimmunoassay of secretin, vasoactive intestinal polypeptide and motilin. In: Gastrointestinal Hormones, edited by Glass BJ.. New York: Raven, 1980, p. 797-817.

6.   Chang, T-M, and Chey WY. Radioimmunoassay of secretion: a critical review and current status. Dig Dis Sci 25: 529-552, 1980[ISI][Medline].

7.   Chang, TM, Chey WY, Lee KY, and Choi BH. Immunoreactive secretin in canine duodenal juice is biologically active (Abstract). Gastroenterology 80: 1122, 1981.

8.   Chang, T-M, Lee KY, Chang CH, Li P, Song Y, Roth FL, and Chey WY. Purification of two secretin-releasing peptides structurally related to phospholipase A2 from canine pancreatic juice. Pancreas 19: 401-405, 1999[ISI][Medline].

9.   Chey, WY. Hormonal control of pancreatic exocrine secretion. In: The Pancreas: Biology, Pathobiology, and Diseases, edited by Go VLW. New York: Raven, 1993, p. 403-424.

10.   Chey, WY, and Chang TM. Secretin. In: Handbook of Physiology. The Gastrointestinal System. Neural and Endocrine Biology, , edited by Makhlouf GM.. New York: Oxford Univ. Press, 1989, p. 359-402.

11.   Cupillard, L, Mulherkar R, Gomez N, Kadam S, Valentin E, Lazdunski M, and Lambeau G. Both group IB and group IIA secreted phospholipase A2 are natural ligands of the mouse 180-kDa M-type receptor. J Biol Chem 274: 7043-7051, 1999[Abstract/Free Full Text].

12.   Dennis, EA. Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem 269: 13057-13060, 1994[Free Full Text].

13.   Dennis, EA, Rhee SG, Billah MM, and Hannun YA. Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J 5: 2068-2077, 1991[Abstract/Free Full Text].

14.   Doyle, HR, Lluis F, and Rayford PL. Secretin. In: Gastrointestinal Endocrinology, edited by James CT.. New York: McGraw-Hill, 1987, p. 203-233.

15.   Hanada, K, Kinoshita E, Itoh M, Hirata M, Kajiyama G, and Sugiyama M. Human pancreatic phospholipase A2 stimulates the growth of human pancreatic cancer cell line. FEBS Lett 373: 85-87, 1995[ISI][Medline].

16.   Hanasaki, K, and Arita H. Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution. J Biol Chem 267: 6414-6420, 1992[Abstract/Free Full Text].

17.   Hara, S, Kudo I, Komatani T, Takahashi K, Nakatani Y, Natori Y, and Inoue K. Human pancreatic phospholipase A2 stimulates the growth of human pancreatic cancer cell line. FEBS Lett 313: 85-87, 1995.

18.   Herzig, KH, Schon I, Tatemoto K, Ohe Y, Li Y, Folsch UR, and Owyang C. Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine. Proc Natl Acad Sci USA 93: 7927-7932, 1996[Abstract/Free Full Text].

19.   Hunter, WM, and Greenwood FC. Preparation of iodine 131-labeled human growth hormone of high specific activity. Nature 194: 495-496, 1962[ISI].

20.   Iwai, K, Fukuoka S-I, Fushiki T, Kodaira T, and Ikei N. Elevation of plasma CCK concentration after intestinal administration of a pancreatic enzyme secretion-stimulating peptide purified from rat bile-pancreatic juice: analysis with N-terminal region specific radioimmunoassay. Biochem Biophys Res Commun 136: 701-706, 1986[ISI][Medline].

21.   Iwai, K, Fukuoka S, Fushiki T, Tsujikawa M, Hirose M, Tsunasawa S, and Akiyama F. Purification and sequencing of a trypsin-sensitive cholecystokinin-releasing peptide from rat pancreatic juice. Its homology with pancreatic secretory trypsin inhibitor. J Biol Chem 262: 8956-8959, 1987[Abstract/Free Full Text].

22.   Jonard, PP, Rambaud JC, Dive C, Vaerman JP, Galian A, and Delacroix DL. Secretion of immunoglobulins and plasma proteins from the jejunal mucosa. Transport rate and origin of polymeric immunoglobulin A. J Clin Invest 74: 525-535, 1984[ISI][Medline].

23.   Kanemasa, T, Arimura A, Kishino J, Ohtani M, and Arita H. Contraction of guinea pig lung parenchyma by pancreatic type phospholipase A2 via specific binding site. FEBS Lett 303: 217-220, 1992[ISI][Medline].

24.   Kishino, J, Ohara O, Nomura K, Kramer RM, and Arita H. Pancreatic-type phospholipase A2 induces group II phospholipase A2 expression and prostaglandin biosynthesis in rat mesangial cells. J Biol Chem 269: 5092-5098, 1994[Abstract/Free Full Text].

25.   Kortesuo, PT, Hietaranta AJ, Jamia M, Hirsimaki P, and Nevalainen TJ. Rat pancreatic phospholipase A2. Purification, localization, and development of an enzyme immunoassay. Int J Pancreatol 13: 111-118, 1993[ISI][Medline].

26.   Lambeau, M, and Lazdunski M. Receptors for growing family of secreted phospholipases A2. Trends Pharmacol Sci 20: 162-170, 1999[ISI][Medline].

27.   Lee, ST, Lee KY, Li P, Coy DH, Chang T-M, and Chey WY. Pituitary adenylate cyclase-activating peptide stimulates rat pancreatic secretion via secretin and cholecystokinin releases. Gastroenterology 114: 1054-1060, 1998[ISI][Medline].

28.   Li, P, Chang T-M, and Chey WY. Neuronal regulation of the release and action of secretin-releasing peptide and secretin. Am J Physiol Gastrointest Liver Physiol 269: G305-G312, 1995[Abstract/Free Full Text].

29.   Li, P, Chang T-M, and Chey WY. 5-Hydroxytryptamine (5-HT) receptors mediated the release and action of secretin on pancreatic secretion induced by duodenal acidification in rats (Abstract). Gastroenterology 114: G4740, 1998.

30.   Li, P, Chang T-M, Coy DH, and Chey WY. Gastrin-releasing peptide (GRP) stimulates release of secretin in anesthetized rats (Abstract). Gastroenterology 108: A985, 1995[ISI].

31.   Li, P, Lee KY, Chang T-M, and Chey WY. Mechanism of acid-induced release of secretin in rats. Presence of a secretin-releasing peptide. J Clin Invest 86: 1474-1479, 1990[ISI][Medline].

32.   Li, P, Song Y, Lee KY, Chang T-M, and Chey WY. A secretin-releasing peptide exists in dog pancreatic juice. Life Sci 66: 1307-1316, 2000[ISI][Medline].

33.   Liddle, RA, Misukonis MA, Pacy L, and Balber AE. Cholecystokinin cells purified by fluorescence-activated cell sorting respond to monitor peptide with an increase in intracellular calcium. Proc Natl Acad Sci USA 89: 5147-5151, 1992[Abstract].

34.   Lilja, I, Smedh K, Olaison G, Sjodahl R, Tagesson C, and Gustafson-Svard C. Phospholipase A2 gene expression and activity in histologically normal ileal mucosa and in Crohn's ileitis. Gut 37: 380-385, 1995[Abstract].

35.   Lu, L, Louie D, and Owyang C. A cholecystokinin releasing peptide mediates feedback regulation of pancreatic secretion. Am J Physiol Gastrointest Liver Physiol 256: G430-G435, 1989[Abstract/Free Full Text].

36.   Matsuda, Y, Owgawa M, Shibata T, Nakaguchi K, Nishijima J, Wakasugi C, and Mori T. Distribution of immunoreactive pancreatic phospholipase A2 (IPPL-2) in various human tissues. Res Commun Chem Pathol Pharmacol 58: 281-284, 1987[ISI][Medline].

37.   Matsuzawa, A, Murakami M, Atsumi G, Imai K, Prados P, Inoue K, and Kudo I. Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion. Biochem J 328: 701-709, 1996[ISI].

38.   Miyasaka, K, Guan D, Liddle RA, and Green GM. Feedback regulation by trypsin: evidence for intraluminal CCK-releasing peptide. Am J Physiol Gastrointest Liver Physiol 257: G175-G181, 1989[Abstract/Free Full Text].

39.   Nomura, K, Fujita H, and Arita H. Gene expression of pancreatic-type phospholipase A2 in rat ovaries: stimulatory action on progesterone release. Endocrinology 135: 603-609, 1994[Abstract].

40.   Sakata, T, Nakamura E, Tsuruta Y, Tamaki M, Teraoka H, Tojo H, Ono T, and Okamoto M. Presence of pancreatic type phospholipase A2 mRNA in rat gastric mucosa and lung. Biochim Biophys Acta 1007: 124-126, 1989[ISI][Medline].

41.   Sommers, CD, Bobbit JL, Bemis KG, and Snyder DW. Porcine pancreatic phospholipase A2-induced contractions of guinea pig lung pleural strips. Eur J Pharmacol 216: 87-96, 1992[ISI][Medline].

42.   Song, Y, Li P, Lee KY, Chang T-M, and Chey WY. Canine pancreatic juice stimulates the release of secretin and pancreatic secretion in the dog. Am J Physiol Gastrointest Liver Physiol 277: G731-G735, 1999[Abstract/Free Full Text].

43.   Spannagel, AW, Green GM, Guan D, Liddle RA, Faull K, and Reeve JR, Jr. Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion. Proc Natl Acad Sci USA 93: 4415-4420, 1996[Abstract/Free Full Text].

44.   Tohkin, M, Kishino J, Ishizaki J, and Arita H. Pancreatic-type phospholipase A2 stimulates prostaglandin synthesis in mouse osteoblastic cells (MC3T3-E1) via a specific binding site. J Biol Chem 268: 2865-2871, 1993[Abstract/Free Full Text].

45.   Tojo, H, Ying Z, and Okamoto M. Purification and characterization of guinea pig gastric phospholipase A2 of the pancreatic type. Eur J Biochem 215: 81-90, 1993[Abstract].

46.   Verheij, HM, Slotboom AJ, and de Haas GH. Structure and function of phospholipase A2. Rev Physiol Biochem Pharmacol 91: 92-203, 1981.

47.   Ying, Z, Tojo H, Nonaka Y, and Okamoto M. Cloning and expression of phospholipase A2 from guinea pig gastric mucosa, its induction by carbachol and secretion in vivo. Eur J Biochem 215: 91-97, 1993[Abstract].


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