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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
Materials--
Porcine pancreatic PLA2 was obtained
from Sigma or Boehringer Mannheim. Diltiazem,
4
-12-tetradecanoylphorbol-13-acetate (
-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
-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).
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RESULTS |
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.
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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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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.
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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.
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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.
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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).
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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
-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 -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.
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DISCUSSION |
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.