Porcine Pancreatic Phospholipase A2 Stimulates Secretin Release from Secretin-producing Cells*

Ta-min ChangDagger , Cecilia H. Chang, David R. Wagner, and William Y. Chey

From the Konar Center for Digestive and Liver Diseases, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipases A2 (PLA2)1 are a family of enzymes that catalyze hydrolysis of ester bond at sn-2 position of phospholipids producing fatty acids and lysophospholipids. These enzymes exist either in extracellular secretions or intracellularly in the cell cytosol or other organelles (1-4). The cytosolic forms, including an 85-kDa enzyme and other smaller forms, are believed to function as effector enzymes in various receptor-mediated signal transduction cascades that involve the release of arachidonic acid as the second messenger. The secretory PLA2s are classified into three major subtypes according to their primary structural homology with PLA2s purified from snake venom (1-4). Those from the Elapidae and Hydrophidae are grouped as type I and those from Crotalidae and Viperidae as type II. The bee venom PLA2, which has no sequence homology with either types of snake PLA2s, but has a similar structural organization for calcium ion binding and catalytic domains (4), is classified as Type III. Both type I and type II PLA2s are 14-18-kDa proteins that are dependent on Ca2+ in milimolar concentration for enzymatic activity. In mammalian species, both type I and type II secretory PLA2s of 14 kDa (1-4) and a 60-kDa form from bovine seminal plasma (5) have been isolated. Pancreatic PLA2 is a type I enzyme secreted into the pancreatic juice. Non-pancreatic PLA2 isolated from other tissues and tissue fluids, including the one isolated from human synovial fluid of rheumatoid arthritis, are type II enzymes. However, it has become clear recently that type I PLA2 is also present in other tissues, including kidney, small intestine, spleen, lung, and stomach (6, 7).

Along with function as an extracellular enzyme, type I PLA2 has been shown to elicit receptor-mediated cellular responses, including stimulation of prostaglandin (8) and steroid hormone (9) secretion, cell proliferation (10-12), and vascular smooth muscle contraction (13). Specific receptors for secretory PLA2 have been cloned (14, 15). Receptor binding and receptor-mediated action by type I PLA2 is not dependent on Ca2+ nor PLA2's enzymatic activity (16, 17). Thus, type I PLA2 appears to have a cell-mediated function independent of its enzymatic activity.

We have shown previously in the rat (18) that acid-induced release of secretin from secretin cells in the upper intestinal mucosa is mediated by a lumenally active secretin-releasing factor (SRF). Similarly, canine pancreatic juice has also been shown to possess an SRF activity (19). Our attempt to isolate SRF from canine pancreatic juice has led to the purification of two SRFs of 14 kDa whose N-terminal sequences were identical to that of canine pancreatic PLA2 (20). This observation suggests that pancreatic PLA2 may function as a modulator of intestinal endocrine cells besides its function as a digestive enzyme. In the present report we will present evidence that pancreatic PLA2 indeed possesses secretin-releasing activity by acting directly on secretin-producing cells. This action is independent of its enzymatic activity and appears to involve a receptor-mediated signal cascade.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Porcine pancreatic PLA2 was obtained from Sigma or Boehringer Mannheim. Diltiazem, 4beta -12-tetradecanoylphorbol-13-acetate (beta -TPA), 3-isobutyl-1-methylxanthine, 4-bromophenacyl bromide (BPB), HPLC grade trifluoroacetic acid, and pertussis toxin were purchased from Sigma. HPLC grade water and acetonitrile were obtained from Fisher. Percoll and 1,2-bis-(S-decanoyl)-1,2-dithio-sn-glycero-3-phosphocholine were obtained from Amersham Pharmacia Biotech and Molecular Probes, Inc., Eugene, OR, respectively. Streptomycin, penicillin, and gentamycin sulfate were obtained from Flow Laboratories, McLean, VA. All the tissue culture ware and media were purchased from Life Technologies, Inc.

Purification of Porcine Pancreatic PLA2-- Porcine pancreatic PLA2 suspended in ammonium sulfate solution was centrifuged in an Eppendorff microcentrifuge for 2 min at 4 °C. The supernatant solution was removed, and the pellet corresponding to 2.5 mg of protein was dissolved in 16% acetonitrile, 0.1% trifluoroacetic acid and injected into a Vydac 218TP semipreparative column (7.5 × 250 mm). The column was linked to an ISCO single pump HPLC system (ISCO, Omaha, NE) consisting of a model 2360 gradient former, a model 2350 pump, and a V4 absorbance detector that were controlled by an IBM personal computer. The column was pre-equilibrated with 20% elution solvent B and then eluted with a gradient of 30-54% solvent B at an increment rate of 0.3%/min followed by 54-100% solvent B at 2.3%/min at a flow rate of 2.5 ml/min. The elution solvents were: solvent A, 0.1% trifluoroacetic acid and solvent B, 80% acetonitrile/0.09% trifluoroacetic acid. The elution profile was monitored by absorbance at 215 nm. The major peaks were collected and dried in vacuo in a Speed Vac (Savant Instrument, Inc., Farmingdale, NY). Each peak was rechromatographed to obtain a homogenous single peak and tested for PLA2 enzymatic activity, secretin-releasing activity, and molecular mass determination.

Inactivation of Enzymatic Activity of Purified PLA2 with 4-Bromophenacyl Amide-- Two mg of purified PLA2 were dissolved in 0.5 ml Dulbecco's phosphate-buffered saline and then incubated with 0.1 mM BPB at room temperature in the dark for 18 h. The reaction mixture was then passed through a Sephadex G25 column (0.9 × 25 cm) equilibrated in the same phosphate-buffered saline to collect the protein peak. The concentration of the BPB-treated enzyme was determined by protein assay as described below.

Cell Culture-- STC-1 cells of a murine intestinal neuroendocrine tumor cell line that secrete secretin (21) were maintained in monolayer cultures in 24-well plates as described previously (22, 23).

Preparation of Secretin Cell-enriched Cell Preparation from Rat Duodenal Mucosa-- Mucosal cells enriched in secretin-containing endocrine cells were prepared by collagenase digestion of rat duodenal mucosa followed by centrifugation in a discontinuous Percoll density gradient according to a method described previously (24).

Studies of the Release of Secretin from STC-1 and Rat Mucosal Cell Preparation-- The release of secretin from STC-1 or rat mucosal cell preparation was studied as described previously (24). Briefly, monolayers of STC-1 cells were incubated in the presence or absence of porcine pancreatic PLA2 at various concentrations or other agents in Earle's balanced salt solution containing 10 mM Hepes, pH 7.4, 5 mM sodium pyruvate, 2 mM L-glutamine, 0.01% soybean trypsin inhibitor, and 0.2% bovine serum albumin under 95% air, 5% CO2 at 37 °C for 60 min or various time periods as specified. The plate was chilled on ice, and an aliquot of the medium was then removed for assay of secretin using a specific radioimmunoassay as described (24, 25). In some experiments the cells were preincubated with diltiazem (10 µM) for 30 min or either pertussis toxin (10 ng/ml) or beta -TPA (0.1 µM) for 10 h before incubation of the cells with PLA2 and assay of secretin release. Rat S cell-enriched preparation was suspended at 0.5-1.0 × 106 cells/ml in Hanks' balanced salt solution containing Hepes, L-glutamine, pyruvate, soybean trypsin inhibitor, and bovine serum albumin as described above for Earle's balanced salt solution. The cell suspension was incubated in the absence or presence of varying concentrations of PLA2 or other test agents under 95% O2, 5% CO2 with gentle gyration in a water bath at 37 °C for 30 min. The cell suspension was then centrifuged at 500 × g for 5 min at 4 °C, and an aliquot of the supernatant was removed for assay of secretin. The cell pellet was extracted with 0.1 N HCl and centrifuged at 17,100 × g and 4 °C for 30 min. The supernatant solution was lyophilized and used for assay of the cellular content of secretin. The data were calculated as the amount of secretin-like immunoreactivity (in femtomoles) released per milligram of cell protein or in percentage of total cellular content. The effect of PLA2 was determined by comparing the amount of secretin released in the presence of PLA2 and that of the corresponding control. Stimulation of secretin release was expressed as percentage over the corresponding control. All data were presented as mean ± S.E.

Measurement of Cellular cAMP and Inositol 1,4,5-Triphosphate Contents-- Intracellular cAMP content was measured by radioimmunoassay using the assay kit obtained from Biomedical Technologies, Inc., Stoughton, MA, as described previously (22). Cellular content of inositol 1,4,5-triphosphate was determined by a radioreceptor assay using an assay kit purchased from NEN Life Science Products as described (24).

Other Determinations-- Amino acid sequence analysis was carried out by Edman degradation using an Applied Biosystem 477A automated sequencer located in the Department of Dental Research, University of Rochester School of Medicine and Dentistry. Mass spectra of protein and peptides were determined by Core Laboratory, Louisiana State University Medical Center, New Orleans, LA using a Matrix-assisted Laser Desorption/Ionization Time-of-Flight Delayed Extraction Mass Spectrometer (PerSeptive Biosystems Inc., Cambridge, MA). Cellular protein was determined by the bicinchoninic acid method using the assay kit provided by Pierce using crystalline bovine serum albumin as a standard. Phospholipase A2 activity was determined by the microplate assay method of Reynolds et al. (26).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterogeneity of Commercial Porcine Pancreatic PLA2-- Pancreatic PLA2 obtained from both Sigma and Boehringer Mannheim stimulated the release of secretin from STC-1 cells. However, upon reverse phase HPLC they were resolved into several components. As shown in Fig. 1A, Sigma PLA2 was resolved into more than 11 peaks. Of the 11 major peaks designated according to the order of their retention time, Peaks 1, 5, 6, 9, 10, and 11 were devoid of secretin-releasing activity, while Peaks 2, 3, 4, 7, and 8 contained various secretin-releasing activities. As shown in Fig. 1B, Boehringer Mannheim PLA2 was less heterogeneous but was still resolved into four major peaks. Of these, Peaks 2 and 4 had the same retention time as those of the corresponding peaks derived from Sigma PLA2; whereas Peaks 3a and 5a were eluted ahead of Peaks 3 and 5, respectively. Although Peak 4 was broad in Fig. 1B, it was eluted as a single peak upon rechromatography. These four peaks were also active to stimulate secretin release. All the bioactive peaks from both sources except for Peak 8 were purified to homogeneity by rechromatography. As examples, using the same gradient of 34-54% solvent B at a rate of 0.4%/min as shown in Fig. 2, Peak 2 was purified as a single peak at 18.1 min, Peak 3 at 20.5 min, Peak 4 at 21.6 min, and Peak 7 at 36.1 min, respectively.


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Fig. 1.   Fractionation of commercial porcine pancreatic phospholipase A2 by reverse phase high performance liquid chromatography. A, 2.5 mg of Sigma porcine pancreatic PLA2 was chromatographed on a Vydac semipreparative C18 column as described under "Experimental Procedures." B, 2.5 mg of Boehringer Mannheim porcine pancreatic PLA2 was chromatographed under the same conditions as in A. The numbers denote the major peaks according to the order of their elution times. Peak 3a is designated as the peak eluted behind Peak 2 but ahead of Peak 3 in A and Peak 5a as the peak eluted after Peak 4 but ahead of Peak 5.


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Fig. 2.   Purification of Sigma porcine pancreatic PLA2 peaks by rechromatography. Peaks 2, 3, 4, and 7 of Sigma pancreatic PLA2 were rechromatographed by using the same gradient of 34-54% solvent B at 0.4%/min. The tracings from top to bottom represent the chromatograms of Peaks 2, 3, 4, and 7, respectively. The number next to each peak denotes the elution time of the peak.

Characterization of Purified Peaks with Secretin-releasing Activity-- The purified peaks were analyzed for molecular mass, PLA2 activity, and secretin-releasing activity in STC-1 cells. As summarized in Table I, Peaks 2 and 4 from both sources had the highest secretin-releasing and enzymatic activities. Their molecular masses were 13,969-14,001 Da, which were the same within experimental error as 13,982 Da calculated from the amino acid sequence of porcine pancreatic PLA2 (27). The mass spectra of Peaks 2 and 4 are shown in Fig. 3, A and B, respectively. In each spectrum, the lower m/z peak represented the molecule of PLA2 with two net charges. The N-terminal amino acid sequences of these two peaks were determined. The results indicated that except for the blanked cycles at cystine residues, their N-terminal 30 residues were identical to that of porcine pancreatic PLA2 (Table II). Peak 5a has a molecular mass of 13,956 Da and is high in both enzymatic and secretin-releasing activities. Peaks 3 and 3a that had molecular masses lower than porcine PLA2 by 500 and 900 Da, respectively, were lower in both enzymatic activity and secretin-releasing activity than the above mentioned peaks. On the other hand, Peaks 7 and 8 with molecular masses of 8,213 and 17,503 Da, respectively, had very little enzymatic activity but had secretin-releasing activities comparable with those of Peaks 3 and 3a. It should be mentioned that the calculated PLA2 activities of these two peaks were not significantly different from the variation observed in the substrate blank of the enzyme assay. In addition, the mass spectrum of Peak 8 also indicated the presence of a minor component (about 15%) with molecular mass of 17,035 Da.

                              
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Table I
Properties of purified PLA2 peaks with secretin-releasing activity
Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim (BM) was purified as described under "Experimental Procedures." The retention time (RT) of each peak obtained in Fig. 1, relative molecular mass, PLA2 enzymatic activity (PLA2 activity), and secretin-releasing activity (SR activity) at 0.1 µM determined after purification are summarized.


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Fig. 3.   Mass spectra of purified Peaks 2 and 4 of Sigma PLA2. A, Peak 2; B, Peak 4.

                              
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Table II
N-terminal amino acid sequence analysis of PLA2 Peaks 2 and 4 
Purified PLA2 Peaks 2 and 4 were subjected to sequence analysis by automated Edman degradation as described under "Experimental Procedures." The yield of the designated amino acid residues rounded to the full picomole values are given below.

All five active peaks derived from Sigma PLA2 and all four main peaks from Boehringer Mannheim exhibited concentration-dependent stimulation of secretin release in STC-1 cells. The results of these dose-response studies are shown in Fig. 4, A and B, respectively. The results shown in Fig. 4A indicated that Peaks 2 and 4 from Sigma were more than 10 times as potent as Peaks 3, 7, and 8. Also at the same concentration of 1 µM, Peaks 2 and 4 were about two to three times as effective as the other three peaks. The half-maximal dose of Peak 2 (400 nM) was slightly higher than that of Peak 4 (100 nM). Similarly, Peaks 2 and 4 from Boehringer Mannheim were about 10-20 times as potent as Peaks 5a and 3a (Fig. 4B). Since Peak 4 was the predominant and most potent fraction to stimulate secretin release, it was used in the subsequent studies.


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Fig. 4.   Concentration-dependent stimulation of secretin release from STC-1 cells by various PLA2 peaks. STC-1 cells were incubated with 0 or 1 nM to 5 µM of PLA2 peaks purified from Sigma PLA2 (A, n = 8) or from Boehringer Mannheim PLA2 (B, n = 8) for 1 h at 37 °C. The amount of secretin-like immunoreactivity released was then determined and compared with that of the control without PLA2. The symbols in A denote the data of: Peak 2 (open circles), Peak 3 (open squares); Peak 4 (open triangles), Peak 7 (filled circles), and Peak 8 (filled triangles). The symbols in B denote the data of: Peak 2 (open circles), Peak 3a (filled circles), Peak 4 (open triangles), and Peak 5a (filled triangles). Each data point represents mean ± S.E. of six experiments. * and ** indicate significant stimulation over the basal (control) with p < 0.05 and p < 0.01, respectively.

The effect of Peak 4 on secretin release from STC-1 cells was also time-dependent. Thus, addition of Peak 4 (0.5 µM) to STC-1 cells resulted in a continuous stimulation of secretin release for 60 min, reaching 130% increase over basal secretion at 15 min and 250% at 60 min.

Although the secretin-releasing activity was highest in the fractions with the highest PLA2 enzymatic activity, stimulation of secretin release did not appear to depend on its enzymatic activity. Thus, as shown in Fig. 5, pretreatment of Peak 4 with 4-bromophenacyl bromide resulted in inhibition of its enzymatic activity by 95% (from 5.3 units/mg to 0.3 units/mg). The treated enzyme, BPB-PLA2, at 0.1 µM stimulated secretin release by 337 ± 49% over basal, which was not significantly different from 283 ± 11% stimulated by the untreated enzyme.


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Fig. 5.   Effect of 4-bromophenacyl bromide on enzymatic and secretin-releasing activities of purified PLA2. Purified PLA2 (Sigma, Peak 4) was pretreated with 4-BPB as described under "Experimental Procedures." The enzymatic activity of the treated enzyme (BPB-PLA2) was then compared with the untreated enzyme in A. The secretin-releasing activity of BPB-PLA2 was compared with that of the untreated enzyme in B by incubating with STC-1 cells at 0.1 µM for 60 min as described in Fig. 4. The data in A represent the average of two assays. The data in B represent the average of four experiments.

The Effect of Purified PLA2 on Secretin Release from Rat Mucosal S Cell Preparation-- Purified Peak 4 from Sigma also stimulated secretin release from an S cells-enriched preparation isolated from rat small intestinal mucosa. As shown in Fig. 6, the purified fraction stimulated the release of secretin from isolated rat mucosal cells concentration-dependently and was more than 10 times as active as the unfractionated PLA2.


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Fig. 6.   Effect of porcine pancreatic PLA2 on secretin release from rat S cell-enriched preparation. Rat mucosal cells were incubated with 1 nM to 1 µM of unfractionated (circles) or purified PLA2 (squares) for 30 min at 37 °C to determine the amount of secretin released as described under "Experimental Procedures." * depicts significant increase in secretin release over the basal with p < 0.05 (n = 4).

Cellular Mechanism of Stimulation of Secretin Release by PLA2 in STC-1 Cells-- We studied if any signal transduction pathway mediated the action of PLA2 on secretin release. Incubation of STC-1 cells with PLA2 did not increase cellular level of cAMP over a period of 30 min (data not shown), although the cells responded well to pituitary adenylate cyclase activating polypeptide which increased cellular cAMP level from 31.3 ± 2.1 pmol/mg of cell protein to a peak level of 94.5 ± 3.4 pmol/mg of protein at 2 min. PLA2 also did not affect the cellular content of inositol 1,4,5-triphosphate over a period of 10 min (data not shown), although the cells responded well to bombesin which increased inositol 1,4,5-triphosphate level from 5.4 ± 1.6 pmol/mg of cell protein to 51.3 ± 9.6 and 54.9 ± 12.4 pmol/mg of cell protein at 15 and 30 s, respectively. These two neuropeptides had been shown to stimulate secretin release through the generation of the corresponding second messengers (24). However, as shown in Table III, when STC-1 cells were incubated with PLA2 in the presence of an L-type calcium ion channel blocker, diltiazem (10 µM), the stimulatory effects of PLA2 on secretin release at 50 and 500 nM were inhibited by 47%. Down-regulation of protein kinase C by pretreatment of the cells with 0.1 µM beta -TPA also resulted in a significant inhibition (~50%) of PLA2-stimulated secretin release. A similar inhibition was observed when incubation of STC-1 cells with PLA2 was carried out in the presence of 1 µM staurosporine, a protein kinase C-selective inhibitor (data not shown). Moreover, the stimulatory effects of PLA2 at these two concentrations decreased significantly (38 and 49%, respectively) after pretreatment of STC-1 cells with pertussis toxin (10 ng/ml, 10 h).

                              
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Table III
Effects of protein kinase C down-regulation, pertussis toxin, and an L-type calcium ion channel blocker on PLA2-stimulated secretin release from STC-1 cells
STC-1 cells were preincubated with either 0.1 µM beta -TPA or 10 ng/ml PTX at 37 °C for 10 h or 10 µM diltiazem for 30 min and then incubated with or without PLA2 as described under "Experimental Procedures." The extent of stimulation of secretin release over the corresponding control was then determined. The data represent mean ± S.E. of n experiments as indicated. The number in parentheses indicates percentage inhibition by each treatment as compared to the corresponding control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The result of the present study provides strong evidence that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity. Thus, commercially available porcine pancreatic PLA2 contained several peaks of secretin-releasing activity. Among the active peaks, those with the highest enzymatic activity, the same molecular weight and N-terminal amino acid sequence as porcine pancreatic PLA2 were the most potent fractions. Among the resolved peaks, Peak 4 with the highest enzymatic activity and identical Mr with PLA2 probably represented the native enzyme. Peak 2 and Peak 5a, with a slightly higher and lower Mr than Peak 4, respectively, were probably genetic variants of PLA2 with reduction of both enzymatic activity and potency of secretin-releasing activity. Peaks 3 and 3a had a significantly lower molecular weight than that of the native enzyme and about 50% enzymatic activity, and substantially decreased potency in secretin-releasing activity could be partially degraded product of PLA2 that lost a few amino acid residues. All these peaks were found to cross-react well (>10%) with an anti-PLA2 serum raised against purified Peak 4 (data not shown), suggesting that they are all antigenically related to PLA2. On the other hand, the relationship between Peak 7 and PLA2 cannot be clearly discerned at present due to its lack of enzymatic activity and absence of amino acid sequence data. Peak 8 does not appear to be related to the enzyme as it lacked enzymatic activity, and both its major and minor components had relative molecular masses greater than that of prepro-PLA2 (16,278.5 Da), calculated from the amino acid sequence deduced from the cDNA coding sequence of porcine PLA2 precursor (27). Moreover, both of these peaks did not appear to contain the antigenic determinant of PLA2, since they had very low cross-reaction with the anti-PLA2 serum (<0.2%) that could be due to a small contamination of the enzyme.

The effect of PLA2 on secretin release does not appear to depend on its enzymatic activity, as inactivation of the enzymatic activity with 4-bromophenacyl bromide did not diminish its secretin-releasing activity. This observation suggested that the release of secretin elicited by PLA2 was not due to membrane damage and leakage of the hormone nor due to enzymatic release of fatty acids that are known to be stimulants of secretin release (25). The fact that we did not observe any increase in trypan blue inclusion after treatment of STC-1 cell with PLA2 (data not shown) appeared to support the former argument. Since PLA2 also stimulated the release of secretin from rat mucosal cells, this secretory response to PLA2 apparently is a common property of secretin-producing cells rather than a result of tumorigenic transformation.

The results of recent studies have revealed that pancreatic PLA2 is also present in other organs, including the spleen, lung, kidney, small and large intestine, and stomach (6, 7). In addition to function as a digestive enzyme, pancreatic PLA2 has also been shown to regulate cellular function, including cell proliferation (10-12), vascular smooth muscle contraction (13), stimulation of prostaglandin production and type II PLA2 gene expression (8, 28), and secretion of a steroid hormone (9). Many of these actions of pancreatic PLA2 have been shown to occur through mediation by a specific receptor independent of its enzymatic activity. Moreover, specific receptor for PLA2 has been cloned (14, 15) and shown to bind PLA2 independent of its enzymatic activity (16, 17). It is likely that PLA2 stimulates secretin release through a similar receptor. In the present study we have also observed that stimulation of secretin release by PLA2 from STC-1 cells is partially inhibited by PTX, an L-type Ca2+ channel blocker, and by down-regulation of protein kinase C activity or a protein kinase C inhibitor. These observations suggest that the action of PLA2 on secretin release may be mediated in part by activation of a PTX-sensitive G protein, the L-type Ca2+ channel and protein kinase C. However, the relationships among these three elements of signal cascade remain to be studied.

It should be noted that the release of both secretin and cholecystokinin (CCK) from the upper small intestinal mucosa is subject to feedback inhibition by pancreatic juice (29-32). This effect has been shown to be due to inactivation of the corresponding lumenal releasing factors for these hormones by pancreatic proteases. Indeed, two lumenally active CCK-releasing factors, lumenal CCK-releasing factor and diazepam-binding inhibitor, have been isolated and shown to release CCK when given to the intestinal lumen (33, 34). In addition, another CCK-releasing factor, monitor peptide, which is a variant form of pancreatic Kazal-type trypsin inhibitor, has been isolated from rat pancreatic juice (35). Therefore, it is not surprising that another protein from the pancreatic juice, PLA2, is found to have secretin-releasing activity. Indeed, we have found recently that, in conscious dogs, intraduodenal administration of fresh canine pancreatic juice in the interdigestive state resulted in a significant increase in pancreatic secretion of fluid and bicarbonate as well as plasma secretin concentration (36). Although more studies are needed to define its possible physiological role on the release of secretin and pancreatic secretion, pancreatic PLA2 in the duodenal lumen may exert a stimulatory action on pancreatic secretion of fluid and bicarbonate in the interdigestive or the fasting state by releasing secretin from the duodenum. This contention is supported by the observation that intravenous administration of a polyclonal antibody specific for PLA2 (Peak 4 of the present study) resulted in marked inhibition of pancreatic exocrine secretion of fluid and bicarbonate and the release of secretin in response to duodenal acidification in rats (37). In addition, when the duodenal acid perfusate, which contains a secretin-releasing factor activity (18), was collected from donor rats, preincubated with the anti-PLA2 antibody and then ultrafiltrated to remove the antibody-antigen complex, the secretin-releasing factor activity in the filtrate disappeared (37). These observations suggest that PLA2 is released from the upper small intestinal mucosa to mediate the release of secretin in response to duodenal acidification. Thus, given that pancreatic PLA2 is secreted into the intestinal lumen and present in the gastrointestinal mucosa, it is tempting to speculated that the enzyme may also participate in a regulatory function in the release of other gut hormones.

    ACKNOWLEDGEMENT

We thank Laura Braggins for her excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DDK 25962.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.

Dagger To whom correspondence should be addressed: Konar Center for Digestive and Liver Diseases, University of Rochester Medical Center, P. O. Box 646, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-2858; Fax: 716-756-8481; E-mail: ta-min_chang{at}urmc.rochester.edu.

    ABBREVIATIONS

The abbreviations used are: PLA2, phospholipase A2; CCK, cholecystokinin; HPLC, high performance liquid chromatography; PTX, pertussis toxin; SRF, secretin-releasing factor; beta -TPA, 4beta -12-tetradecanoylphorbol-13-acetate; BPB, 4-bromophenacyl bromide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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