Mammalian bombesin receptors are coupled to multiple signal
transduction pathways in pancreatic acini
Hirokazu
Nishino,
Yasuhiro
Tsunoda, and
Chung
Owyang
Department of Internal Medicine, University of Michigan, Ann Arbor,
Michigan 48109
 |
ABSTRACT |
We investigated
the structural requirements for bombesin (BB)-like peptides to
stimulate amylase secretion in rat pancreatic acini and examined the
responsible intracellular signal transduction pathways. The
tetradecapeptide BB-(1
14) was a full agonist, whereas the
heptapeptide BB-(8
14) did not evoke amylase secretion. The mammalian
BB analog neuromedin C decapeptide [NMC-(5
14)] was as
potent as BB-(1
14) in stimulating amylase secretion, suggesting that
Gly5-Asn6-His7
(or Gln7) of the COOH-terminal
decapeptide are essential amino acids for full biological activity. BB
and NMC equipotently stimulated
D-myo-inositol 1,4,5-trisphosphate production, which was inhibited by the
phospholipase C (PLC) inhibitor U-73122. BB and NMC also stimulated
protein tyrosine kinase (PTK) activities. The half-maximal effective
concentration (EC50) for
NMC-activated PTK was 2 log units less than the
EC50 for BB-activated PTK. NMC was
10-34 times more potent than BB in increasing leukotriene
C4 (an index of arachidonic acid
production). The production of leukotriene
C4 was inhibited by the
phospholipase A2
(PLA2) inhibitor ONO-RS-082. NMC
is structurally homologous to BB-(5
14) except that
Gln7 in BB is replaced by
His7 in NMC. Therefore,
substitution of Gln7 for
His7 may alter the signal
transduction systems to include the PTK and
PLA2 pathways. U-73122 inhibited
Ca2+ spiking and amylase secretion
induced by NMC and BB. However, the PTK inhibitor genistein and the
PLA2 inhibitor ONO-RS-082 inhibited secretion induced by NMC but not that induced by BB. In
contrast to nonmammalian BB receptors, which primarily use the PLC
pathway, the rat BB receptor is linked to three different signal
transduction systems: PLC, PTK, and
PLA2 pathways.
phospholipase C; phospholipase
A2; protein tyrosine kinases; calcium; amylase
 |
INTRODUCTION |
BOMBESIN (BB) is a tetradecapeptide originally isolated
from the skin of the frog, Bombina
bombina (1, 10). The mammalian homolog of BB,
gastrin-releasing peptide (GRP), is a 27-amino-acid BB-like peptide
that was isolated from porcine gastric tissues and other mammalian
species (15, 22, 30, 33). Its active fragment neuromedin C
[GRP-(18
27), or NMC-(5
14)] was extracted from porcine
spinal cord (25). These mammalian peptides possess the same ability as
the entire BB molecule to function as neurotransmitters, paracrine
hormones, and growth factors (32, 43, 45).
The BB receptor was recently cloned. It is a member of the
heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptor superfamily, and it is composed of seven transmembrane domains
with 384 and 390 amino acids for the BB type 2 and type 1 receptors,
respectively (3, 34). The BB receptor possesses 53% sequence
similarity to the tachykinin receptor (19, 34). GRP and NMC
preferentially bind to the BB receptor type 2, whereas the BB subfamily
and mammalian-type neuromedin B [NMB-(5
14)] bind to the
BB type 1 receptor (22, 44). Although the distribution of these
receptor subtypes varies in different tissues and species (16), the BB
type 2 receptor is predominant in rat pancreatic acinar cells (31).
Most studies suggest that BB and its analogs act through a G protein to
increase phosphatidylinositol turnover (16). Other studies indicate
that BB receptors are coupled to the protein tyrosine kinase (PTK)
cascades in some cell types (6, 13, 17, 50, 51). In pancreatic acini,
the BB receptor appears to be coupled to the
Gq protein-phospholipase
C
1 pathway to produce
intracellular second messengers,
D-myo-inositol
1,4,5-trisphosphate (IP3) and
1,2-diacylglycerol (DAG) (21, 28).
IP3 releases Ca2+ from intracellular pools, and
DAG activates protein kinase C translocation from the cytosol to the
plasma membrane (4, 27). This suggests that the BB and cholecystokinin
(CCK) receptors are coupled to similar intracellular messengers (47).
Both peptides induce amylase secretion, but at supramaximal
concentrations only CCK inhibits amylase secretion (12). Moreover,
intracellular DAG levels stimulated by BB are considerably less than
those stimulated by CCK-8, although
IP3 levels stimulated by both
agonists are similar (21, 28). This may explain the monophasic amylase secretion induced by BB (28).
Recent studies suggest that the CCK receptors in pancreatic acini may
be coupled to different signal transduction systems depending on the
receptor affinity states. The high- and low-affinity CCK receptors
appear to be coupled to different signal transduction pathways; the
high-affinity CCK receptor is linked to the phospholipase A2
(PLA2) pathway, and the
low-affinity CCK receptor to the phospholipase C (PLC) cascade (20, 37,
39, 49). The CCK receptor may also be coupled to the PTK pathways (8,
18, 41). This PTK pathway may regulate the receptor-operated
Ca2+ entry mechanism (41).
It is conceivable that the BB receptors in pancreatic acini may be
coupled to multiple signal transduction pathways. The aim of this study
was to investigate the structural requirements for BB-like peptides to
activate BB receptors in rat pancreatic acini and to examine the
possibility that these receptors are coupled to multiple signal
transduction pathways.
 |
METHODS |
Materials.
Human GRP, porcine NMC, porcine NMB, and neuromedin K (NMK) were
obtained from Sigma Chemical (St. Louis, MO). BB-(1
14), BB-(8
14), BB(9
14), and BB-(11
14) were from Research Plus
(Bayonne,NJ). U-73122
[1-(6-{[(17
)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione], U-73343
[1-(6-{-[(17
)-3-methoxyestra-1,3,5(10)-trien-17yl]amino}hexyl)2,5-pyrrolidinedione], and ONO-RS-082
[2-(p-amylcinnamoyl)-amino-4-chlorobenzoic
acid] were purchased from Biomol (Plymouth Meeting, PA).
Genistein (4',5,7-trihydroxyisoflavone) was obtained from
GIBCO-BRL (Grand Island, NY), and fura 2-AM was from Molecular Probes
(Eugene, OR).
Isolation of pancreatic acinar cells and measurements of
intracellular
Ca2+
concentration and amylase secretion.
Isolated rat pancreatic acini were prepared by collagenase digestion
with pancreata obtained from male Sprague-Dawley rats (40). Acini were
suspended in a physiological salt solution (PSS). The PSS contained
0.1% bovine serum albumin, 0.1 mg soybean trypsin inhibitor, and in
(mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1 NaH2PO4,
10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), Eagle's minimum essential amino acid neutralized with
NaOH, 2 L-glutamine, and 5.5 D-glucose and was adjusted to pH
7.35 and equilibrated with 100%
O2.
The intracellular Ca2+
concentration
([Ca2+]i)
measurements in individual pancreatic acini were performed as described
previously (40). In brief, isolated acini were incubated with 2 µM
fura 2-AM at 37°C in 10 ml PSS for 30 min. All experiments were
done using a dual excitation wavelength (340/380 nm emitted at 505 nm)
modular fluorometer system (SPEX) coupled to a Nikon Diaphot inverted
microscope (×40). Isolated acini placed on a cover glass and
mounted on a closed chamber were superfused from a reservoir (1 ml/min). A fluorescence ratio was converted to
[Ca2+]i
according to in vitro calibration with an external standard and 25 µM
fura 2 potassium salt.
Amylase secretion studies were performed using acini that were
preincubated for 30 min in 40 ml PSS, washed twice by centrifugation (50 g), and resuspended in 40 ml
fresh PSS. Aliquots were distributed into flasks and incubated with
reagents for 60 min at 37°C. The incubation was terminated by
centrifugation [10,000 revolutions/min (rpm)] for 30 s at
4°C in a Microfuge (1 ml × 2 in 20 groups). The amylase
released into the supernatant and remaining in the pellet was assayed,
using Procion yellow starch as a substrate. Amylase secretion was
expressed as the percentage of the total content in each sample.
Measurement of IP3.
The radioimmunoassay (RIA) of IP3
was performed as previously described
(IP3
3H assay system; Amersham,
Arlington Heights, IL) (20). In brief, 0.5-ml aliquots of the acinar
suspension (2 × 106
cells/PSS) were incubated with reagents at 37°C for various time intervals. Incubation was stopped by adding chilled trichloroacetic acid (TCA) (0.125 ml) to obtain a final TCA concentration of 10%. After sonicating for 30 s and allowing the cell suspension to settle
for 30 min at 4°C, whole fractions were centrifuged (10,000 rpm)
for 10 min at 4°C. The resultant supernatant (600 µl) was extracted with 3 ml water-saturated diethyl ether three times, gassed
with nitrogen, and then titrated to pH 7.5 with
NaHCO3. The extracts (100-µl
samples) were then incubated with a bovine adrenal
IP3-binding protein (100 µl),
0.007 µCi
[3H]IP3
(100 µl) (1:1, water/ethanol), and 100 µl of 0.1 M
tris(hydroxymethyl)aminomethane (Tris) buffer (pH 9) containing 4 mM
EDTA and 4 mg/ml bovine serum albumin for 15 min at 4°C. Bound and
free labeled IP3 were separated by
centrifugation (10,000 rpm) for 3 min. The pellets were solubilized in
10 ml scintillant (Cytoscint; ICN, Costa Mesa, CA), and the radioactivity was counted in a liquid scintillation counter.
IP3 in the cells was expressed as
picomoles per milligram of total protein. Protein was measured using a
Bio-Rad assay system (Hercules, CA) (300 µg in each tube).
Measurement of PTK activities.
For measurement of PTK activities, RIA was performed using a PTK assay
system (GIBCO-BRL), as previously described (41). In brief, aliquots
(0.5 ml) of the acinar cell suspension (2 × 106 cells/PSS) were incubated with
reagents for 3 min at 37°C. The incubation was stopped with 1 ml
chilled PSS, and the aliquots were immediately centrifuged (10,000 rpm)
for 40 s at 4°C in a Microfuge. The supernatant was removed, and
the resultant pellet was resuspended in 50 µl chilled 50 mM HEPES
buffer (pH 7.4) containing (in mM) 50
-glycerophosphate, 25 NaF, 150 NaCl, 20 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 15 MgCl2, 1 dithiothreitol (DTT), 1% Triton X-100, 25 µg/ml leupeptin, and 25 µg/ml aprotinin. The suspension was immediately frozen in liquid
nitrogen and stored at
70°C overnight. Each suspension was
thawed and sonicated for 30 s at 4°C. The sonicates were vortexed
and allowed to settle for 10 min at 4°C and then centrifuged
(10,000 rpm) for 15 min at 4°C. Supernatants (10-µl samples, 50 µg protein) were incubated in 12.5 µl of substrate solution
containing 1 mM RR-SRC peptide substrate (29). The substrate solution
also contained 60 mM HEPES, 20 mM
MgCl2, 40 µM EDTA, 0.2 mM DTT,
50 µg/ml bovine serum albumin, 0.3% (vol/vol) Nonidet P-40, 140 µM
sodium orthovanadate, 120 µM ATP, and 1 µCi of
[
-32P]ATP.
For the control reactions, supernatants (10-µl samples of cell
extracts) were incubated with 12.5 µl substrate solution without
RR-SRC peptide. After incubation for 30 min at 37°C, the reaction
was stopped with 20 µl of ice-cold 10% TCA. All samples were placed
on ice for 10 min and then centrifuged (10,000 rpm) for 10 min at
4°C. Supernatant (21.25 µl from each tube) was removed and
spotted onto separate phosphocellulose disks. Each disk was placed in a
scintillation vial containing 10 ml of 5% acetic acid. Subsequent to
mixing at room temperature for 10 min, the washing reagent was decanted
and each paper was mixed with 10 ml of 1% acetic acid for 10 min
followed by 10 ml distilled water for 10 min at room temperature after
decanting 1% acetic acid. After the distilled water was decanted, 10 ml of scintillant (Cytoscint) were added to each vial and the
radioactivity remaining on each binding paper was counted in a liquid
scintillation counter. Nonspecific binding of
[32P]ATP to the
binding paper without the substrate was subtracted from each control
sample. PTK activities were expressed as picomoles per minute per
milligram protein of the cell extract.
Measurements of leukotriene C4.
Leukotriene C4
(LTC4) was measured by RIA using
the LTC4-specific
3H assay system (Amersham).
Aliquots (0.5 ml) of the acinar cell suspension (2 × 106 cells/PSS) were incubated with
reagents at 37°C for various time intervals. At each time interval,
the incubation was stopped with 1 ml chilled PSS and suspensions were
immediately centrifuged (10,000 rpm) for 40 s at 4°C in a
Microfuge. The supernatant was removed, and the pellet was immediately
resuspended in 0.5 ml chilled 50 mM Tris · HCl buffer
(pH 7.5) containing 5 mM EDTA, 10 mM EGTA, 0.3% (wt/vol)
-mercaptoethanol, 10 mM benzamidine, 50 µg/ml phenylmethylsulfonyl
fluoride, and 1% Triton X-100 and frozen by liquid nitrogen. The
suspension was stored at
70°C overnight, thawed, and
sonicated for 30 s at 4°C. The sonicates were centrifuged (10,000 rpm) for 15 min at 4°C to obtain soluble fractions in the
supernatant. Supernatants (100-µl samples) were incubated with
LTC4 antibody (100 µl), which
was solubilized in 0.1 M phosphate buffer (pH 7.2) including 0.1%
gelatin, 0.001% thimerosal, and 0.01 µCi of
[3H]LTC4
(100 µl in ethanol/water/acetic acid, 60:40:1) for 16 h at 8°C.
Bound and free labeled LTC4 were
separated by centrifugation (10,000 rpm) for 3 min at 4°C with 0.5 ml dextran-coated charcoal in phosphate-buffered saline. The
supernatant was solubilized in 10 ml scintillant (Cytoscint), and the
radioactivity was counted in a liquid scintillation counter.
LTC4 levels were expressed as
picograms per milligram protein of the cell extract.
LTC4 has little cross-reactivity
with other arachidonic acid (AA) metabolites: 5% with
LTD4, 0.5% with leukotriene
E4, and <0.001% with other metabolites.
 |
RESULTS |
Evidence that
Gly5-Asn6-His7
(or Gln7) of BB-like peptides are key amino
acids for full biological activity.
The structure of the BB analogs used in this study is shown in Fig.
1. The basal amylase secretion was 9.2 ± 0.3% of total/60 min (n = 31)
in dispersed rat pancreatic acini. As shown in Fig. 2, frog BB, porcine NMC, human GRP, and
porcine NMB each caused a dose-dependent and monophasic amylase
secretion with peak increases of 3.2- to 3.6- fold over basal at the
supramaximal dose (100 nM). The half-maximal effective concentrations
(EC50) for BB, NMC, GRP, and NMB
were 0.2, 0.1, 0.2, and 3 nM, respectively. The tetradecapeptide
BB-(1
14) was a full agonist, whereas the heptapeptide BB-(8
14),
which has the same COOH-terminal seven amino acids as BB, GRP, and NMC,
did not evoke amylase secretion at any dose (1 pM-100 nM). This
indicates that the COOH-terminal heptapeptide is not sufficient for
biological activity. The shorter BB peptides [e.g., BB-(9
14)
and BB-(11
14)] did not increase amylase secretion over basal.
Because the BB receptor has sequence similarity with the tachykinin
receptor (19, 34), effects of NMK (neurokinin B), which is an agonist
of tachykinin receptors, were examined. NMK did not evoke amylase
secretion, suggesting the absence of tachykinin receptors in pancreatic
acini. However, the decapeptide NMC-(5
14) was as potent as BB-(1
14)
in stimulating amylase secretion, suggesting that
Gly5-Asp6-His7
(or Gln7) of the BB-like
peptides are critical amino acids for full biological activity.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Structures of frog bombesin (BB), human and porcine gastrin-releasing
peptide (GRP), porcine neuromedin C (NMC), and porcine neuromedin B
(NMB) are shown. The x in human GRP represents a 13-amino acid segment
consisting of Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys. The
x in porcine GRP represents a 13-amino acid segment consisting of
Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys. Note that in all
BB analogs the COOH terminus of Met is amidated
(Met14-NH2)
(1, 10, 15, 22, 24, 25, 30, and 33).
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
Amylase secretion from dispersed rat pancreatic acini induced by
bombesin analogs. BB [half-maximal effective concentration
(EC50) = 0.2 nM], GRP
(EC50 = 0.2 nM), NMC
(EC50 = 0.1 nM), and NMB
(EC50 = 3 nM) evoked
dose-dependent monophasic amylase secretion. BB-(8 14), BB-(9 14),
and BB-(11 14) and the decapeptide neuromedin K (NMK)
(Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2)
did not evoke amylase secretion over basal. Data are means ± SE
from 4-8 separate experiments (n = 3-9 for each point). US, unstimulated cells (basal).
|
|
BB and NMC increase IP3 levels, PTK
activities, and AA metabolite production.
We further investigated signal transduction pathways used by the
nonmammalian and mammalian BB analogs. The basal
IP3 was 1.50 ± 0.95 pmol/mg
protein (n = 8). BB (10 nM,
nonmammalian) and NMC (100 nM, mammalian) equipotently stimulated
intracellular IP3 production with
a peak increase of 5.8 (n = 8) and 5.3-fold (n = 4) over basal, respectively, after 15 s of cell stimulation. These
responses induced by BB and NMC (10-100 nM) were abolished by
pretreatment of acini with the PLC inhibitor U-73122 (5 µM) [2.30 ± 1.29 pmol/ng protein for BB (n = 4) and 0.95 ± 0.48 pmol/ng protein for NMC (n = 4)].
BB and NMC dose dependently stimulated PTK activities with peak
increases of 3.8-fold over basal after 3 min of cell stimulation. Basal
PTK activity was 0.25 ± 0.06 pmol · min
1 · mg
protein
1
(n = 6) (Fig.
3A). The
EC50 for NMC-activated PTK was 2 log units less than the EC50 for
BB-activated PTK. Peak PTK levels for both BB (100 nM) and NMC (100 nM)
were attained after 1 min and sustained for up to 10 min after cell
stimulation (Fig. 3B).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Protein tyrosine kinase (PTK) activities stimulated by BB and NMC.
A: PTK activities at 3 min after cell
stimulation with either BB or NMC. P < 0.05, F = 4.53, n = 16, and SD = 0.46 between NMC and BB (1 and 10 nM) by one-way analysis of
variance. B: time course of PTK
activities after cell stimulation with either BB (100 nM) or NMC (100 nM). Data represent means ± SE from 4 separate experiments
(n = 3-8 for each point).
|
|
LTC4 and
15-hydroxyeicosatetraenoic acid (HETE), but not prostaglandins, are
major metabolites of AA in pancreatic acini (38). They are
produced by activation of the 15-lipoxygenase pathway (2). RIA provides
a more quantitative measurement than direct release studies of AA. For
these reasons, we measured intracellular LTC4 levels
([LTC4]i)
as an index of AA production. Basal
[LTC4]i was 23.23 ± 16.88 pg/mg protein (n = 8). As shown in Fig.
4A, BB (30 nM) and NMC (30 nM) stimulated
[LTC4]i
with increases of 5.0- and 38.1-fold over basal, respectively, after 3 min of cell stimulation. These increases in
[LTC4]i
induced by BB and NMC were significantly inhibited by pretreatment of
acini with the PLA2 inhibitor
ONO-RS-082 (10 µM) for 10 min. Our time course studies showed that
NMC (100 nM) was 10-34 times more potent than BB (100 nM) in
increasing
[LTC4]i
at various time intervals (Fig. 4B),
suggesting that NMC, the mammalian type of BB analog, preferentially activates the PLA2 pathway to
produce AA and its metabolites.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Intracellular leukotriene C4
concentration
([LTC4]i)
stimulated by BB and NMC. A:
[LTC4]i
was measured 3 min after cell stimulation with either BB (30 nM) or NMC
(30 nM) in the presence or absence of the phospholipase
A2
(PLA2) inhibitor ONO-RS-082 (10 µM). Acini were pretreated with ONO-RS-082 for 10 min before cell
stimulation and further incubated with secretagogues for 3 min. Data
represent means ± SE from 3 separate experiments
(n = 4-10 for each column).
B: time course of
[LTC4]i
after cell stimulation with either BB (100 nM) or NMC (100 nM). Data
represent means ± SE from 3 separate experiments
(n = 3-6 for each point).
|
|
Effects of inhibitors of PLC, PTK, and
PLA2 on
Ca2+ spiking
induced by BB and NMC.
As described in the previous section, BB and NMC equipotently
stimulated IP3 production in
pancreatic acini. To further investigate the biological significance of
the BB receptor-coupled PLC-IP3 pathway, we examined the effects of PLC inhibitor on
Ca2+ spiking stimulated by BB and
NMC in fura 2-loaded individual rat pancreatic acini. Reports have
shown that in rabbit pancreatic acinar cells, the PLC inhibitor U-73122
(3-10 µM) alone caused an increase in
[Ca2+]i
(46). However, in individual rat pancreatic acini, application of
U-73122 (5 and 10 µM) for 5-10 min caused no change in basal [Ca2+]i
in six of six cells (Fig.
5A,
inset). Similarly, application of
the PTK inhibitor genistein (100 µM) and the
PLA2 inhibitor ONO-RS-082 (10 µM) also did not change basal
[Ca2+]i
in nine of nine cells. U-73122, genistein, and ONO-RS-082 were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of
0.01-0.1%. At these concentrations, DMSO did not produce a change
in
[Ca2+]i.
U-73122 (5 µM) transiently inhibited the sustained
[Ca2+]i
plateau induced by low doses of BB (1-10 nM) in four of four cells
(Fig. 5A). BB at lower
concentrations (e.g., 0.1 nM) caused a small sustained
[Ca2+]i
increase (or Ca2+ oscillations),
but this was not inhibited by U-73122 in four of five cells. The
sustained
[Ca2+]i
plateau induced by higher concentrations of BB (100 nM) was inhibited
by U-73122 in 75% of cells (6 of 8 cells) (Fig.
5B). U-73122 inhibited the
Ca2+ oscillations induced by NMC
(0.3 nM) in four of five cells and the sustained plateau evoked by NMC
(100 nM) in three of four cells. The effects of U-73122 were not caused
by fura 2 quenching or other artificial elements, as
[Ca2+]i
was measured by the 340/380 nm ratio, and it was observed that U-73122
symmetrically changed both fluorescences (Fig.
5B,
inset).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of PLC inhibitor U-73122 on BB-stimulated
Ca2+ spiking modes.
A is representative of 9 different
individual pancreatic acini and B is
representative of 8. A
(inset): U-73122 (10 µM, ; 5 µM, ), genistein (100 µM, ), and ONO-RS-082 (10 µM, ) did not change basal
intracellular Ca2+ concentration
([Ca2+]i)
in 15 different cells. B
(inset): 340 and 380 nm fluorescences are depicted; ,
application of U-73122.
|
|
Both BB and NMC stimulated PTK activities. Although the function of BB
receptor-coupled PTK in stimulus-secretion coupling of pancreatic acini
still remains to be clarified, our previous results suggested that the
Ca2+ entry mechanism coupled to
the low-affinity CCK receptor activation may be regulated by the PTK
pathways (41). If similar observations occur with BB receptor
activation, one would expect that the PTK inhibitor would inhibit the
extracellular
[Ca2+]-dependent and
sustained
[Ca2+]i
plateau stimulated by BB and NMC. We showed that genistein (100 µM)
eliminated the sustained plateau and
Ca2+ oscillations induced by low
doses of BB (0.1-10 nM) in 75% of cells examined (6 of 8 cells)
(Fig.
6A).
Genistein (100 µM) eliminated the sustained
[Ca2+]i
plateau evoked by high doses of BB (100 nM) in three of six cells (Fig.
6B). In separate studies, we showed
that genistein inhibited NMC (0.3-1 nM)-stimulated
Ca2+ oscillations and the
sustained plateau induced by NMC (100 nM) in three of four cells (Fig.
6, C and
D). In all cases, genistein symmetrically changed 340 and 380 nm fluorescences (Fig. 6,
insets).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of the PTK inhibitor genistein on bombesin- and NMC-stimulated
Ca2+ spiking modes.
A is representative of 8 different
individual acini; B,
C, and
D are each representative of 4. Insets: 340 and 380 nm fluorescences. , Application of
genistein.
|
|
Because NMC increased
[LTC4]i,
the effects of the PLA2 inhibitor
on Ca2+ spiking during NMC
stimulation were examined and compared with that of BB. The
[Ca2+] oscillation
evoked by low doses of BB (0.1-10 nM) was not inhibited by
ONO-RS-082 (10 µM) in 11 of 13 cells. The sustained
[Ca2+]i
plateau elicited by BB (100 nM) was not significantly affected by
ONO-RS-082 (10 µM) in four of four cells. In two of four cells, ONO-RS-082 (10 µM) inhibited
Ca2+ oscillations stimulated by
NMC (0.3-10 nM) (Fig.
7A). The
inhibitory effects of ONO-RS-082 on
Ca2+ spiking were apparent with
NMC (100 nM) stimulation. In three of three cells, ONO-RS-082 abolished
the sustained
[Ca2+]i
plateau (Fig. 7B). Note that after
removal of ONO-RS-082 from the superfusion medium, a new and large
[Ca2+]i
transient was observed in the presence of NMC. This was not observed
with BB. Therefore, ONO-RS-082 was more potent in inhibiting Ca2+ spiking induced by NMC than
that induced by BB. This is consistent with the data showing that NMC
is 10-34 times more potent than BB in producing
LTC4. In all cases, ONO-RS-082
symmetrically changed 340 and 380 nm fluorescences (Fig. 7,
insets). These results indicate that
U-73122 effectively inhibits the actions evoked by BB and NMC, whereas
genistein and ONO-RS-082 effectively inhibit
Ca2+ spiking evoked by NMC.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of the PLA2 inhibitor
ONO-RS-082 (10 µM) on NMC-stimulated Ca2+
spiking modes. A is
representative of 4 different individual acini and
B is representative of 3. Insets: 340 and 380 nm fluorescences
are depicted. , Application of ONO-RS-082. Note that a large
[Ca2+]i
transient stimulated by NMC was observed after removal of ONO-RS-082
from the superfusion medium (B).
|
|
Effects of inhibitors of PLC, PTK, and
PLA2 on amylase secretion induced by BB and
NMC.
U-73122 (5-7 µM) significantly reduced amylase secretion induced
by high doses of BB (10-100 nM) (Fig.
8A). In
contrast, U-73343 (10 µM), an inactive analog of U-73122, did not
inhibit but enhanced amylase secretion stimulated by BB. Similarly,
U-73122 (5 µM) significantly inhibited amylase secretion stimulated
by NMC (1-100 nM) (Fig. 8B).
U-73343 (10 µM), as a negative control, did not inhibit the NMC
action. Genistein (100-300 µM) did not have a significant effect
on amylase secretion stimulated by BB at any dose (0.1-100 nM),
but it significantly inhibited the action of NMC (0.1-1 nM) (Fig.
9). Similarly, ONO-RS-082 (10-30 µM)
did not alter amylase secretion induced by BB, but it significantly inhibited amylase secretion induced by both low (0.1 nM) and high (100 nM) concentrations of NMC (Fig. 10).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of U-73122 on BB- and NMC-stimulated amylase secretion. Acini
were pretreated with either U-73122 (5-7 µM) or its inactive
analog U-73343 (10 µM) for 10 min before cell stimulation. Data
represent means ± SE from 6 separate experiments
(n = 4-10 for each point).
* P < 0.05 and
** P < 0.01 by two-tailed
unpaired t-test (U-73122 vs.
control).
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Effects of genistein on BB- and NMC-stimulated amylase secretion. Acini
were pretreated with genistein (100-300 µM) for 10 min before
cell stimulation. Data represent means ± SE from 7 separate
experiments (n = 4-6 for each
point). * P < 0.05 by
two-tailed unpaired t-test (genistein
vs. control).
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 10.
Effects of ONO-RS-082 on BB- and NMC-stimulated amylase secretion.
Acini were pretreated with ONO-RS-082 (10-30 µM) for 10 min
before cell stimulation. Data represent means ± SE from 7 separate
experiments (n = 4-6 for each
point). * P < 0.05 and
** P < 0.01 by unpaired
t-test (ONO-RS-082 vs. control).
|
|
 |
DISCUSSION |
Erspamer et al. (10) first observed that crude methanol extracts of
amphibian skin produced various actions on vascular smooth muscle
cells, exocrine and endocrine secretion, and renal circulation and
function. This extract contained the tetradecapeptide BB (1).
Subsequently, a structurally related GRP and NMC were isolated from the
gastric tissue and spinal cord of pigs and other mammalian species (15,
22, 25, 30, 33). Currently, 13 BB-like peptides have been isolated from
different amphibian species and grouped into three subfamilies
according to their COOH-terminal tripeptide (9). BB and alytesin have
the COOH-terminal His-Leu-Met-NH2, the ranatensins and litorin have the COOH-terminal
His-Phe-Met-NH2, and the
phyllolitorins have Ser-Leu (or
Phe)-Met-NH2. A shorter form of
ranatensins, known as NMB, has also been isolated from the porcine
spinal cord (24). GRP and NMC bind to the type 2 BB receptor, whereas
NMB preferentially binds to the type 1 BB receptor (44). Note that an
-amidated COOH-terminal heptapeptide is conserved and shared by
amphibian, avian, and mammalian BB-related peptides including BB, GRP,
and NMC. Studies of the structural activities of smooth muscle
preparations suggest that the COOH-terminal heptapeptide contains the
minimal segment associated with biological activity and that the
COOH-terminal nonapeptide of BB is as potent as the natural BB (5).
Moreover, the
-amidated COOH-terminal methionine residue appears to
be essential for the binding of BB and GRP to the high-affinity cell
surface receptors and for the initiation of biological response (26).
It is well recognized that BB, GRP, and NMC cause amylase secretion
through the BB type 2 receptor in mammalian pancreatic acinar cells
(14, 31). Our study confirmed this observation in rat pancreatic acini;
NMC was 30-fold more potent in stimulating amylase secretion than NMB.
We also showed that in rat pancreatic acini, the
-amidated
COOH-terminal heptapeptide BB-(8
14) or shorter peptides
[e.g., BB-(9
14) and BB-(11
14)] did not evoke amylase
secretion, indicating that the BB heptapeptide is not sufficient for
biological activity. Because the decapeptide NMC-(5
14), BB-(1
14),
and GRP-(1
27) were equipotent in stimulating amylase secretion,
Gly5-Asp6-His7
(or Gln7) of the COOH-terminal
decapeptide must be critical amino acids for full biological activity
of the BB analogs. This possibility is supported by the observation
that the heptapeptide of GRP [GRP-(21
27) or GRP-(8
14), see
Fig. 1] had no effect on the binding of
125I-GRP to the murine pancreatic
membranes, whereas the octapeptide AcGRP [GRP-(7
14), see Fig.
1] completely inhibited
125I-GRP binding
(11). This indicates the importance of
His7 for receptor binding.
In this study, we showed that the major difference between frog BB and
mammalian NMC relates to their abilities to activate the
PLA2 pathway: NMC was 10-34
times more potent than BB in increasing intracellular
[LTC4]i,
an index of AA production. The only difference between BB and NMC from
the 5th to the 14th position is that the basic amino acid
His7 in NMC is replaced by a
nonionized (at the neutral pH), but polar, amino acid
Gln7 in BB, which suggests that
His7 is also a key amino acid for
recognizing the PLA2 pathway in mammalian pancreatic acinar cells. Note that only one amino acid difference has also been observed between the mouse BB receptors and
Swiss 3T3 cell GRP receptors:
Arg309 at the seventh
transmembrane domain in mice is
replaced by His309 in Swiss 3T3
cells (3, 34). It has been suggested that
Asp87 in the second hydrophobic
transmembrane domain may play an important role in ligand binding (34,
35). We predict that Asp, which is a negatively charged amino acid, may
preferentially form an ionic bond with
His7, a positively charged amino
acid.
Previous studies have clearly demonstrated that activation of the BB
receptor evokes phosphatidylinositol turnover by activating the G
protein and PLC, resulting in the production of
IP3 and DAG (16).
IP3 stimulates
Ca2+ release from intracellular
stores, and DAG activates protein kinase C translocation from the
cytosol to the plasma membrane (4, 27). Without exception, BB also
increases IP3 and DAG levels to
elicit intracellular Ca2+ release
and PKC activation in pancreatic acini (21, 28). We confirmed these
observations by showing that frog BB and porcine NMC equipotently
increased IP3 concentration, which
was inhibited by the PLC inhibitor U-73122. We also
demonstrated that U-73122 inhibited
Ca2+ spiking and amylase secretion
elicited by BB and NMC. However, we found that higher concentrations of
BB and NMC (10-100 nM) were required for sufficient production of
IP3 and that the inhibitory actions of U-73122 on
[Ca2+]i
levels and amylase secretion were observed only if high doses of BB and
NMC were used. This suggests that other intracellular signal
transduction pathways may be involved in mediating amylase secretion
stimulated by BB-like ligands.
Our study demonstrated that in rat pancreatic acinar cells, both BB and
NMC stimulated PTK activity, which was measurable by RIA using RR-SRC
peptide as a substrate. Previous studies using a specific
antiphosphotyrosine antibody demonstrated that BB induced the
phosphorylation of 120-, 115-, 90-, and 75-kDa proteins in Swiss 3T3
cells (6, 17, 50, 51). These are probably nonreceptor PTKs. BB has also
been shown to stimulate oncogenes (e.g.,
c-myc and
c-fos) via a PTK pathway in this
cell line (16). Our study showed that the
EC50 for NMC-activated PTK was 2 log units less than the EC50 for
BB-activated PTK. Furthermore, the
Ca2+ spiking and amylase secretion
stimulated by NMC appear to be more sensitive to the PTK inhibitor
genistein than the Ca2+ spiking
and amylase secretion evoked by BB. This suggests that the BB receptor
on rat pancreatic acini is probably coupled to the PTK pathway. Note
that in contrast to the PLC pathway, which requires high doses of BB
analogs for its activation, the PTK cascades appear to be activated by
lower concentrations of NMC. We and others (8, 18, 41) have reported
that activation of the CCKA
receptor in the rat pancreatic acini resulted in the tyrosyl
phosphorylation of 105- and 85-kDa and pp60src
proteins. Although the precise biological functions of
these PTKs are still unknown, these nonreceptor PTKs may mediate
extracellular Ca2+-dependent
pancreatic enzyme secretion (41).
Our study also demonstrated that the BB receptor in rat pancreatic
acini is coupled to the PLA2
cascade. PLA2 is a
Ca2+-dependent esterase capable of
catalyzing the hydrolysis of fatty-acid ester bonds at the
sn-2 position of glycerophospholipid
(42). Evidence suggests that the cytosolic 85-kDa
PLA2, which produces AA by
catalyzing phosphatidylcholine and phosphatidylethanolamine, plays a
major role in intracellular signal transduction (2, 7, 42). AA produced
via this cascade acts as a second messenger to release
Ca2+ from intracellular stores in
several cells, including pancreatic acinar cells (36, 37, 48).
Recently, we demonstrated that the high-affinity CCK receptors are
coupled to the PLA2-AA pathway to
mediate Ca2+ oscillation and
amylase secretion in rat pancreatic acini (37, 39). We also showed that
a melittin-derived PLA2-activating protein elicits Ca2+ oscillation
and amylase secretion accompanied by an increase in the intracellular
AA metabolite HETE (38). Similar observations were made with activation
of the BB receptors on rat pancreatic acini; both BB and NMC stimulated
the production of the AA metabolite LTC4. These findings are similar
to those reported in work with Swiss 3T3 cells, which showed that BB
caused a rapid release of AA and prostaglandin
E2 via the cyclooxygenase
pathways, mediating a mitogenic response (23). These metabolic pathways
of AA are different from those in pancreatic acini (i.e., lipoxygenase
and cytochrome P-450 pathways) (38,
39). Note that NMC was more potent than BB in increasing
[LTC4]i
and that the PLA2 inhibitor ONO-RS-082 inhibited amylase secretion evoked by NMC but not that by
BB, suggesting that the BB receptor in rat pancreatic acini is also
coupled to the PLA2 cascade. In
contrast to the PLC pathway, the
PLA2 pathway may be activated by
low and high doses of NMC, since the actions of NMC at 0.1 and 100 nM
were inhibited by the PLA2
inhibitor ONO-RS-082.
We conclude that in contrast to the nonmammalian BB receptor, which
primarily uses the PLC pathway, the rat BB receptor is linked to three
different signal transduction systems. Activation by the mammalian
BB-like peptide NMC results in activation of the PLC, PTK, and
PLA2 pathways, evoking
Ca2+ spiking and amylase
secretion. It appears that His at the 7th position of BB analogs is a
key amino acid to activate the PTK and
PLA2 pathways.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants R01-DK-32830 and 5P 30-DK-34933.
 |
FOOTNOTES |
Address for reprint requests: C. Owyang, 3912 Taubman Center, Univ. of
Michigan, Ann Arbor, MI 48109-0362.
Received 4 November 1996; accepted in final form 7 December 1997.
 |
REFERENCES |
1.
Anastasi, A.,
V. Erspamer,
and
M. Bucci.
Isolation and structure of bombesin and alytesin: two analogous active peptides from the skin of the European amphibians Bombina and Alytes.
Experientia
27:
166-167,
1971[Medline].
2.
Axelrod, J.,
R. M. Burch,
and
C. L. Jelsema.
Receptor-mediated activation of phospholipase A2 via GTP-binding proteins: arachidonic acid and its metabolites as second messengers.
Trends Neurosci.
11:
117-123,
1988[Medline].
3.
Battey, J. F.,
J. M. Way,
M. H. Corjay,
H. Shapira,
K. Kusano,
R. Harkins,
J. M. Wu,
T. Slattery,
E. Mann,
and
R. I. Feldman.
Molecular cloning of the bombesin/gastrin releasing peptide receptor from Swiss 3T3 cells.
Proc. Natl. Acad. Sci. USA
88:
395-399,
1991[Abstract].
4.
Berridge, M.,
and
R. F. Irvine.
Inositol trisphosphate as a novel second messenger in cellular signal transduction.
Nature
312:
315-321,
1984[Medline].
5.
Broccardo, M.,
G. F. Erspamer,
P. Melchiorri,
L. Negri,
and
R. de Castiglione.
Relative potency of bombesin-like peptides.
Br. J. Pharmacol.
55:
221-227,
1975[Abstract].
6.
Cirillo, D. M.,
G. Gaudino,
L. Naldini,
and
P. M. Comoglio.
Receptor for bombesin with associated tyrosine kinase activity.
Mol. Cell. Biol.
6:
4641-4649,
1986[Medline].
7.
Clark, J. D.,
L.-L. Lin,
R. W. Kriz,
C. S. Ramesha,
L. A. Sultzman,
A. Y. Lin,
N. Milona,
and
J. L. Knopf.
A novel arachidonic acid-selective PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP.
Cell
65:
1043-1051,
1990.
8.
Duan, R.-D.,
A. C. C. Wagner,
D. I. Yule,
and
J. A. Williams.
Multiple inhibitory effects of genistein in stimulus-secretion coupling in rat pancreatic acini.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G303-G310,
1994[Abstract/Free Full Text].
9.
Erspamer, G. F.,
C. Severini,
V. Erspamer,
P. Melchiorri,
G. delle Fave,
and
T. Nakajima.
Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes.
Regul. Pept.
21:
1-11,
1988[Medline].
10.
Erspamer, V.,
G. F. Erspamer,
M. Inselvini,
and
L. Negri.
Occurrence of bombesin and alytesin in extracts of the skin of three European discoglossid frogs and pharmacological actions of bombesin on extravascular smooth muscle.
Br. J. Pharmacol.
45:
333-348,
1972[Medline].
11.
Fanger, B. O.,
A. C. Wade,
and
A. R. Cardin.
Characterization of the murine pancreatic receptor for gastrin releasing peptide and bombesin.
Regul. Pept.
32:
241-251,
1991[Medline].
12.
Gardner, J. D.,
and
R. T. Jensen.
Secretagogue receptors on pancreatic acinar cells.
In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 1109-1127.
13.
Gaudino, G.,
D. Cirillo,
L. Naldini,
P. Rossino,
and
P. M. Comoglio.
Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small lung carcinomas.
Proc. Natl. Acad. Sci. USA
85:
2166-2170,
1988[Abstract].
14.
Jensen, R. T.,
T. Moody,
C. Part,
J. E. Rivier,
and
J. D. Gardner.
Interaction of bombesin and litorin with specific membrane receptors on pancreatic acinar cells.
Proc. Natl. Acad. Sci. USA
75:
6139-6143,
1978[Abstract].
15.
Lebacq-Verheyden, A. M.,
G. Krystal,
O. Sartor,
J. Way,
and
J. F. Battey.
The rat prepro gastrin releasing peptide gene is transcribed from two initiation sites in the brain.
Mol. Endocrinol.
2:
556-563,
1988[Abstract].
16.
Lebacq-Verheyden, A. M.,
J. Trepel,
E. A. Sausville,
and
J. F. Battey.
Bombesin and gastrin releasing peptide: neuropeptides, secretagogues, and growth factors.
In: Peptide Growth Factors and Their Receptors. II., edited by M. B. Sporn,
and A. B. Roberts. New York: Springer-Verlag, 1991, chapt. 21, p. 71-124.
17.
Leeb-Lundberg, L. M. F.,
and
X.-H. Song.
Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group in Swiss 3T3 cells.
J. Biol. Chem.
266:
7746-7749,
1991[Abstract/Free Full Text].
18.
Lutz, M. P.,
S. L. Sutor,
R. T. Abraham,
and
L. J. Miller.
A role for cholecystokinin-stimulated protein tyrosine kinase phosphorylation in regulated secretion by the pancreatic acinar cell.
J. Biol. Chem.
268:
11119-11124,
1993[Abstract/Free Full Text].
19.
Masu, Y.,
K. Nakayama,
H. Tamaki,
Y. Harada,
M. Kuno,
and
S. Nakanishi.
cDNA cloning of bovine substance-K receptor through oocyte expression system.
Nature
329:
836-838,
1987[Medline].
20.
Matozaki, T.,
B. Goke,
Y. Tsunoda,
M. Rodriguez,
J. Martinez,
and
J. A. Williams.
Two functionally distinct cholecystokinin receptors show different modes of actions on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini.
J. Biol. Chem.
265:
6247-6254,
1990[Abstract/Free Full Text].
21.
Matozaki, T.,
W.-Y. Zhu,
Y. Tsunoda,
B. Goke,
and
J. A. Williams.
Intracellular mediators of bombesin action on rat pancreatic acinar cells.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G858-G864,
1991[Abstract/Free Full Text].
22.
McDonald, T. J.,
H. Jornvall,
G. Nilsson,
M. Vagne,
M. Ghatei,
S. R. Bloom,
and
V. Mutt.
Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue.
Biochem. Biophys. Res. Commun.
90:
227-233,
1979[Medline].
23.
Millar, J. B.,
and
E. Rozengurt.
Arachidonic acid release by bombesin.
J. Biol. Chem.
265:
19973-19979,
1990[Abstract/Free Full Text].
24.
Minamino, N.,
K. Kangawa,
and
H. Matsuno.
Neuromedin B: a novel bombesin-like peptide identified in porcine spinal cord.
Biochem. Biophys. Res. Commun.
114:
541-548,
1983[Medline].
25.
Minamino, N.,
K. Kangawa,
and
H. Matsuno.
Neuromedin C: a bombesin-like peptide identified in porcine spinal cord.
Biochem. Biophys. Res. Commun.
119:
14-20,
1984[Medline].
26.
Moody, T. W.,
C. B. Pert,
J. Rivier,
and
M. R. Brown.
Bombesin: specific binding to rat brain membranes.
Proc. Natl. Acad. Sci. USA
75:
5372-5376,
1978[Abstract].
27.
Nishizuka, Y.
The role of protein kinase C in cell surface signal transduction and tumor promotion.
Nature
308:
693-698,
1984[Medline].
28.
Pandol, S. J.,
and
M. S. Schoeffield.
1,2-Diacylglycerol, protein kinase C, and pancreatic enzyme secretion.
J. Biol. Chem.
261:
4438-4444,
1986[Abstract/Free Full Text].
29.
Pike, C. J.,
B. Gallis,
J. E. Casnellie,
P. Bornstein,
and
E. G. Krebs.
Epidermal growth factor stimulates the phosphorylation of synthetic tyrosine-containing peptides by A431 cell membranes.
Proc. Natl. Acad. Sci. USA
79:
1143-1147,
1982.
30.
Reeve, J. R., Jr.,
J. H. Walsh,
P. Chew,
B. Clark,
D. Hawke,
and
J. E. Shively.
Amino acid sequences of three bombesin-like peptides from canine intestine extracts.
J. Biol. Chem.
258:
5582-5588,
1983[Abstract/Free Full Text].
31.
Sekar, C.,
N. Vermura,
O. H. Coy,
B. I. Hirschowitz,
and
E. J. Dickinson.
Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide coupled receptor, but are differentially regulated by guanine nucleotides.
Biochem. J.
280:
163-169,
1991[Medline].
32.
Spindel, E. R.
Mammalian bombesin-like peptides.
Trends Neurosci.
9:
130-133,
1986.
33.
Spindel, E. R.,
W. W. Chin,
J. Price,
L. H. Rees,
G. M. Besser,
and
J. F. Habener.
Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.
Proc. Natl. Acad. Sci. USA
81:
5699-5703,
1984[Abstract].
34.
Spindel, E. R.,
E. Giladi,
P. Brehm,
R. H. Goodman,
and
T. P. Segerson.
Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin releasing peptide receptor.
Mol. Endocrinol.
4:
1956-1963,
1990[Abstract].
35.
Strader, C. D.,
I. S. Sigal,
R. B. Register,
M. R. Candelore,
E. Rands,
and
R. A. Dixon.
Identification of residues required for ligand binding to the beta-adrenergic receptor.
Proc. Natl. Acad. Sci. USA
84:
4384-4388,
1987[Abstract].
36.
Tsunoda, Y.
Receptor-operated Ca2+ signaling and crosstalk in stimulus secretion coupling.
Biochim. Biophys. Acta
1154:
105-156,
1993[Medline].
37.
Tsunoda, Y.,
and
C. Owyang.
Differential involvement of phospholipase A2/arachidonic acid and phospholipase C/phosphoinositol pathways during cholecystokinin receptor activated Ca2+ oscillations in pancreatic acini.
Biochem. Biophys. Res. Commun.
194:
1194-1202,
1993[Medline].
38.
Tsunoda, Y.,
and
C. Owyang.
A newly cloned phospholipase A2-activating protein elicits Ca2+ oscillations and pancreatic amylase secretion via mediation of G protein beta/phospholipase A2/arachidonic acid cascades.
Biochem. Biophys. Res. Commun.
203:
1716-1724,
1994[Medline].
39.
Tsunoda, Y.,
and
C. Owyang.
High-affinity cholecystokinin receptors are coupled to phospholipase A2 pathways to mediate pancreatic amylase secretion.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G435-G444,
1995[Abstract/Free Full Text].
40.
Tsunoda, Y.,
E. L. Stuenkel,
and
J. A. Williams.
Oscillatory mode of calcium signaling in rat pancreatic acinar cells.
Am. J. Physiol.
258 (Cell Physiol. 27):
C147-C155,
1990[Abstract/Free Full Text].
41.
Tsunoda, Y.,
Y. Yoshida,
L. Africa,
G. J. Steil,
and
C. Owyang.
Src kinase pathways in extracellular Ca2+-dependent pancreatic enzyme secretion.
Biochem. Biophys. Res. Commun.
227:
876-884,
1996[Medline].
42.
Van den Bosch, H.
Intracellular phospholipase A.
Biochim. Biophys. Acta
604:
191-246,
1980[Medline].
43.
Walsh, J. H.,
H. C. Wong,
and
G. J. Dockray.
Bombesin-like peptides in mammals.
Federation Proc.
38:
2315-2319,
1979[Medline].
44.
Watson, S., and A. Girdlestone. Receptor and ion channel
nomenclature. Trends Pharmacol. Sci.
16, Suppl.: 1-73, 1995.
45.
Wharton, J.,
J. M. Polak,
S. R. Bloom,
M. A. Ghatei,
E. Solcia,
M. R. Brown,
and
A. G. E. Pearse.
Bombesin-like immunoreactivity in the lung.
Nature
273:
769-770,
1978[Medline].
46.
Willems, P. H. G. M.,
F. H. M. M. Van de Put,
R. Engbersen,
R. R. Bosch,
H. J. M. Van Hoof,
and
J. J. H. H. M. de Pont.
Induction of Ca2+ oscillations by selective, U73122-mediated, depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells.
Eur. J. Physiol.
427:
233-243,
1994.[Medline]
47.
Williams, J. A.,
and
G. T. Blevins, Jr.
Cholecystokinin and regulation of pancreatic acinar cell function.
Physiol. Rev.
73:
701-723,
1993[Free Full Text].
48.
Wolf, B. A.,
W. R. J. Turk,
W. R. Sherman,
and
M. L. McDaniel.
Intracellular Ca2+ mobilization by arachidonic acid.
J. Biol. Chem.
261:
3501-3511,
1986[Abstract/Free Full Text].
49.
Yule, D. I.,
and
J. A. Williams.
U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.
J. Biol. Chem.
267:
13830-13835,
1992[Abstract/Free Full Text].
50.
Zachary, I.,
J. Gil,
W. Lehmann,
J. Sinnett-Smith,
and
E. Rozengurt.
Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells.
Proc. Natl. Acad. Sci. USA
88:
4577-4581,
1991[Abstract].
51.
Zachary, I.,
J. Sinnett-Smith,
and
E. Rozengurt.
Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin in intact cells.
J. Biol. Chem.
266:
24126-24133,
1991[Abstract/Free Full Text].
AJP Gastroint Liver Physiol 274(3):G525-G534
0193-1857/98 $5.00
Copyright © 1998 the American Physiological Society