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Electronic Theses, Treatises and Dissertations
The Graduate School
2004
The Initiating Mechanism of Premature
Trypsin Activation in Pancreatitis
Kai Yang
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THE FLORIDA STATE UNIVERSITY
COLLEGE OF ARTS AND SCIENCES
THE INITIATING MECHANISM OF PREMATURE TRYPSIN
ACTIVATION IN PANCREATITIS
By
KAI YANG
A Thesis submitted to the
Department of Biological Science
in partial fulfillment of the
requirements for the degree of
Master of Science
Degree Awarded:
Summer Semester, 2004
The members of the Committee approve the Thesis of Kai Yang defended on 21
April, 2004.
Wei-Chun Chin
Professor Co - Directing Thesis
George Bates
Professor Co - Directing Thesis
Thomas Keller
Committee Member
Laura Keller
Committee Member
Approved:
Timothy S.Moerland, Chair, Department of Biological Science
The Office of Graduate Studies has verified and approved the above named
committee members.
ii
ACKNOWLEDGEMENTS
I would like to express my thanks and appreciation to my advisor, Dr. WeiChun Chin for his continuous encouragement and kind help in my research and
study. When I was in trouble, he gave me a chance to continue my study. I am
also very grateful to Dr. George Bates, my co-major professor for his help.
Without their support, I never would have finished my master degree in the
department of biological science. I also wish to thank Dr. Thomas Keller and Dr.
Laura Keller for their contribution as my committee members.
I also would like to express my love and gratitude to my family for their
support, patient, kindness, inspiration and love. Without them, I cannot recover
from the trouble.
iii
TABLE OF CONTENTS
List of Figures
....................................................................................
v
..........................................................................................
vi
INTRODUCTION....................................................................................
1
MATERIALS AND METHODS................................................................
6
RESULTS
....................................................................................
11
DISCUSSION
....................................................................................
16
REFERENCES ....................................................................................
34
BIOGRAPHICAL SKETCH ....................................................................
40
Abstract
iv
LIST OF FIGURES
Figure 1: Illustration of the validation of the deconvolution program ..... .........21
Figure 2: Effect of incubation time on trypsin activity in zymogen granules.....22
Figure 3: Effect of increasing [Ca2+] in the intracellular solution on [Ca2+]G ,
[pH]G and trypsin activity in zymogen granules ...................................... .........23
Figure 4: Effect of increasing [Ca2+] in the intracellular solution on the [K+] in
isolated mouse pancreatic zymogen granules ...................................... .........24
Figure 5: Ca2+/K+ ion exchange in zymogen granules ........................... .........25
Figure 6: H+/K+ ion exchange in zymogen granules .............................. .........26
Figure 7: The effect of [Ca2+]G alone on trypsinogen autoactivation in zymogen
granules
.................................................................................... .........27
Figure 8: The effect of [pH]G alone on trypsinogen autoactivation in zymogen
granules
.................................................................................... .........28
Figure 9: The effect of both increased [Ca2+]G and decreased [pH]G on
trypsinogen autoactivation in zymogen granules ................................... .........29
Figure 10: The effect of TEA (20 mM and 10 mM) on [Ca2+]G, [pH]G and trypsin
activity in zymogen granules .................................................................. .........30
Figure 11: The effect of apamin (100 nM) on [Ca2+]G , [pH]G and trypsin activity
in zymogen granules ............................................................................. .........31
Figure 12: The effect of charybdotoxin (10 nM) on [Ca2+]G , [pH]G and trypsin
activity in zymogen granules .................................................................. .........32
Figure 13: Model of the dynamics of H+ and Ca2+ inside zymogen granules. ..33
v
ABSTRACT
Under normal physiological conditions, trypsin remains inactive as trypsinogen
inside the pancreas. Upon entering the small intestine, trypsinogen is converted to
active trypsin. Acute pancreatitis is caused by premature activation of trypsinogen and
the digestion of the pancreas. Up to now the exact initiating mechanism of this
premature activation is still not clear. In these experiments, pH fluctuations, Ca2+
concentration changes and trypsin activity inside pancreatic zymogen granules were
monitored. The effects of possible pharmacological inhibitors were also assessed. The
results show that a sustained increase of Ca2+ in the cytosol can trigger K+ influx into
zymogen granules (ZGs) via a Ca2+-activated K+ channel (ASKCa). This influx of K+ then
mobilizes bound Ca2+ by Ca2+/K+ ion-exchange to increase free Ca2+ concentration in
the ZGs and also mobilizes bound H+ by H+/K+ ion-exchange to decrease the pH in the
ZGs. Both the increase of free Ca2+ concentration and the decrease of pH in the ZGs
will facilitate trypsinogen autoactivation and stabilize active trypsin. Moreover these
investigations show that the ASKCa in the membrane of ZGs may be a small
conductance Ca2+-activated K+ channel (SKCa channel), because it can be activated by
300 nM [Ca2+] and inactivated by apamin (100 nM) and TEA (20 mM).
vi
INTRODUCTION
Pancreatitis is an inflammation of the pancreas associated with pancreas
damage (Mergener & Baillie, 1998). There are an estimated 50,000 to 80,000
cases in the United States each year. Nearly 20% of them develop life-threatening
complications, such as renal or respiratory failure. The overall mortality is 5-10%
and may increase to 35% with complications (Mergener & Baillie, 1998; NIH,
2001). Moreover pancreatic inflammation is a risk factor for pancreatic cancer,
which is the fourth leading cause of cancer death in US with extremely poor
prognosis (Farrow & Evers, 2002). People with alcohol abuse, the human
immunodeficiency virus (HIV) infection or hereditary genetics defects are at high
risk to develop acute pancreatitis (Steinberg & Tenner, 1994; NIH, 2001).
Pancreatitis includes chronic pancreatitis and acute pancreatitis. In acute
pancreatitis the duration of inflammation that can result in the damage of the
pancreas is usually short and reversible, while in chronic pancreatitis the
inflammation is mild but it lasts for a long time. Most cases of pancreatitis are
caused either by alcohol abuse or by gallstones. Investigations find that alcohol
can sensitize the receptors of agonists in the membrane of the pancreatic acinar
cells. Alcohol stimulates the zymogen activation process induced by agonists, but
it has no effect alone (Lu et al, 2002). It has long been hypothesized that in
gallstone pancreatitis, gallstones block the bile ducts and bile flows back into the
pancreatic ductal system. This hypothesis has been challenged by some
investigators who favor pancreatic ductal hypertension as the main reason for
gallstone pancreatitis (Steer et al, 1988). In this review, gallstones can block
pancreatic ducts and result in ductal hypertension, so small ducts are ruptured
΁
and digestive enzymes will be released, leading to acute pancreatitis. Other less
common triggering factors include nicotine (Chowdhury et al, 2002) and
hereditary genetics defects (Whitcomb et al, 1996).
The development of acute pancreatitis consists of three phases: an initiating
phase, an intra-acinar cell phase such as cell injury, and an extra-acinar cell
phase including local and systemic inflammation such as pulmonary and renal
failure. The earliest events in pancreatitis include the loss of secretory cell polarity
and the appearance of intracellular vacuoles. These phenomena are all
associated with the abnormal activation of digestive enzymes (Steer, 1999;
Raraty et al., 1999).
The pancreatic acinar cell can synthesize many digestive enzymes such as
trypsinogen, proelastase and procarboxypeptidase. These proteins are produced
in the rough endoplasmic reticulum (RER) and modified by the Golgi complex.
Digestive enzymes existing primarily as inactive zymogens are packaged in
condensing vesicles at the trans side of the Golgi and carried towards the plasma
membrane. At the plasma membrane, these vesicles will form the mature ZGs.
When stimulated, those ZGs fuse with the plasma membrane and digestive
enzymes are released.
Many pancreatic digestive enzymes exist as inactive zymogens in the
pancreatic acinar cells. When entering the small intestine, trypsinogen is
converted to trypsin by the brush border hydrolase enterokinase, which is
normally only found in the small intestine (Gorelick et al., 1992). Under normal
physiological conditions, trypsinogen has only a small amount of enzymatic
΂
activity. But in pancreatitis, trypsinogen is prematurely activated in pancreas. The
activated trypsin then activates the other pancreatic zymogens. It also can
activate acinar cells, duct cells and imflammatory cells via the trypsin receptor,
namely protease activated receptor isoform 2 (PAR-2) (Hirota et al., 2003), and
result in the cascade reactions and damage the pancreas.
In cells, [Ca2+] is a very crucial messenger for many cellular processes, such
as cell growth, muscle contraction, transcription factor activation, apoptosis and
cell secretion (Berridge, 1997, 1998; Berridge and Lipp, 2000; Berridge et al.,
2003). Pancreatic acinar cells are an excellent model for the study of Ca2+ signals
because they can maintain their structural and functional polarity after even
isolation. Resting free Ca2+ concentration in the cytosol ([Ca2+]C) of the pancreatic
acinar cells is about 100 nM. This Ca2+ gradient is controlled by a plasma
membrane Mg2+-dependent Ca2+-ATPase and a Na+-Ca2+ cotransporter (Kribben
et al, 1983; Bayerdorffer et al, 1985). Agonists binding to the receptors on the
plasma membrane in the basolateral region of the acinar cell (Rosenzweig et al.,
1983) will result in the formation of inositol 1,4,5-trisphosphate (IP3) and cyclic
ADP-ribose (cADPr) from phosphatidylinositol 4,5-bisphosphate (PIP2) and
nicotinamide adenine dinucleotíde (NAD+) respectively. These messengers
diffuse through the cytosol and induce Ca2+ release from intracellular stores by
interacting with IP3 receptors and ryanodine receptors on the stores such as
vesicles or ER. Vesicles in the apical region of the cell are much more sensitive to
IP3 and cADPr than stores in other regions of the cell, so the increase in Ca2+
concentration in the intracellular solution ([Ca2+]i) initiates exocytosis and the
release of digestive enzymes from the apical region of the acinar cells. Sustained
rise of [Ca2+]C is found to be associated with the intracellular activation and
release of digestive enzymes from ZGs inside the pancreas (Raraty et al., 2000).
΃
Many studies have also provided evidence that the normal process of Ca2+
signaling is disrupted early in acute pancreatitis (Raraty et al., 2000; Kruger et al.,
2000). Further studies show that administration of Ca2+ channel blockers can
reduce the acute pancreatitis induced by agonists (Raraty et al., 1999).
Low lumenal pH inside ZGs is also very important. It is involved in protein
sorting, protein processing, regulation of protein-protein interaction and
biogenesis of the secretory granules (Urbe et al., 1997; Laine and Lebel, 1999).
The pH gradient is precisely regulated along the secretory pathway. Under normal
physiological conditions, the pH in the ER is 7.5, decreases to 6 in the Golgi and
5.5 in the secretory granules. Studies have shown that the pH in the lumen of ZGs
is precisely regulated by equilibrium between H+ leakage and H+ influx across the
ZG membrane (Demaurex, 2002). The H+ influx is mainly carried out by active H+
V-ATPases. The exact mechanism of H+ leakage is not clear, but it is driven by the
transmembrane pH gradient. The transmembrane pH gradient decreases
progressively from the ER to the Golgi to the ZGs while the activity of H+
V-ATPases in the membrane increases, thus ZGs are more acidic than Golgi and
ER (Wu et al., 2001; Schapiro and Grinstein, 2000; Tompkins et al., 2002;
Demaurex, 2002). Low pH in the lumen of ZGs is also required for the activation
of trypsinogen. For example, premature protease activation induced by hormone
stimulation is blocked when ZGs are neutralized by exposure to weak
cell-permeable bases (Lerch et al., 1991).
Inside the secretory granules of most cells, there is a pair of countercharged
chemical moieties: a polyanioic polymeric matrix and a cation or polycation. The
cation or polycation may counteract the repulsion force within the polyanioic
matrix and stabilize it. In the presence of other cations, these cations will compete
΄
with bound Ca2+ and other cations bound to the polyanioic polymer matrix and
result in the release of bound cations (Fernandez et al., 1991; Marszalek et al.,
1997; Espinosa et al., 2002; Reigada et al., 2003).
Although it is widely believed that the premature activation of trypsin plays an
initiating role in the pathogenesis of pancreas, the exact mechanism of premature
activation is still not clear. K+ ion channels seem to exist in the membranes of
secretory granules for K+ influx (Nguyen et al., 1998; Quezada et al., 2001; Chin
et al., 2002; O’Rourke et al., 1994). Studies on goblet and mast cells indicate that
K+ influx via ASKCa can exchange with bound Ca2+ and bound H+ of the granular
polyanionic matrices. Recent investigations find that this K+ ion-exchange
mechanism is also highly relevant to the pathophysiology of acute pancreatitis
that is initiated by abnormal elevated [Ca2+]C (Quezada et al., 2001; Thevenod,
2002). The experiments in this thesis test the hypothesis that a sustained
increase of free [Ca2+]C can trigger K+ influx into ZGs via ASKCa channels. This
influx of K+ then exchanges with bound Ca2+ through Ca2+/K+ ion-exchange to
increase free Ca2+ concentration in ZGs ([Ca2+]G), and also exchanges with bound
H+ through H+/K+ ion-exchange to decrease the pH in ZGs ([pH]G). Both the
increase of free [Ca2+] and the decrease of pH in ZGs can facilitate trypsinogen
autoactivation and stabilize trypsin activity leading to the premature activation of
digestive enzymes. In addition, the properties of the ASKCa channels in the
membranes of ZGs are investigated here.
΅
MATERIALS AND METHODS
Validation of the optical deconvolution software
15 µm FocalCheck fluorescent microspheres (Molecular Probes, Eugene,
Oregon) were used for validation of the optical deconvolution software. Their
outermost portion is stained by fluorescent dye. So when viewed with a
fluorescence microscope, it appears as a fluorescent ring of varying diameters
depending on the focal plane (Fig 1). The microspheres in suspension were
diluted with distilled water and observed with by an Olympus IX70 inverted
epifluorescence microscope. Different focal plane were selected and fluorescence
images were recorded. The final step was the deconvolution of these images
using optical deconvolution software Image – Pro Plus (version 4.5)
(MediaCybernetics corporation, San Diego, CA).
Data calibration and analysis
Normal ZGs are round and 1 µm in diameter. When collecting data, any ZGs
that were not round, or that were larger or smaller than normal, were excluded
from the analysis. Moreover, BZiPAR was used as a marker for ZGs because only
in ZGs, can BZiPAR be cleaved and emit fluorescence. Using these criteria, any
fluorescent granules of abnormal shape or size were excluded from analysis.
Fluorescence images were taken and deconvoluted by software. After
deconvolution, fluorescence intensity was measured using the same software.
Each point in the figures of my thesis corresponds to the mean + SD of more than
Ά
30 granules from three separate experiments.
Isolation of ZGs from pancreatic acinar cells and measurement of
trypsin activity, [Ca2+]G, [H+]G and K+ concentration in the ZG ([K+]G)
Mice were sacrificed by exposure to CO2 according to a protocol approved by
Florida State University Animal Care Committee. The pancreas of normal mice
(Jackson Laboratory, Bar Harbor, ME) was digested with 1000 unit / ml
collagenase (Sigma-Aldrich corporation, St. Louis, MO) for 10 minutes at 38°C.
After digestion, the pancreatic acinar cells were collected by centrifugation. Then
cells were suspended in Ca2+-free Hanks’ solution (pH =7.2) and centrifuged at
3,000 rpm for 5 minutes. These wash procedures were performed for at least
three times to remove the collagenase. Then cells were loaded with dyes in
Ca2+-free Hanks’ solution. Labeled cells were transferred to an intracellular
solution (140 mM potassium glutamate, 20 mM Tris, 5 mM MgSO4, 10 mM
2-Morpholinoethanesulfonic acid sodium salt (MES), and 2 mM EGTA, at room
temperature, pH 7.3) and centrifuged at 3,000 rpm for 5 minutes to remove any
excess dye. These wash procedures were also performed at least three times.
Subsequently, the cells were lysed by brief sonication and ZGs were separated by
centrifugation at 10,000 rpm for 5 min. The granules were suspended and
centrifuged again. And then they were suspended in intracellular solution and
allowed to settle and attach for 120 min at 37°C on poly-lysine-coated chambers.
The [Ca2+] in the intracellular solution was varied from 100 nM to 700 nM. The
chambers were mounted and kept at 37°C on the thermoregulated stage of the
Olympus IX70 inverted epifluorescence microscope. Fluorescence of BZiPAR in
the granules was monitored.
·
To investigate the change of trypsin activity in ZGs, 10 µM rhodamine 110, bis
-(CBZ –L– isoleucyl-L- prolyl-L-arginine amide) dihydrochloride (BZiPAR) (λexcitation
= 498nm, λemission = 521 nm) (Molecular Probes, Eugene, Oregon) was used.
Isolated acinar cells were loaded with BZiPAR for 20 minutes at 37°C and ZGs
were isolated. BZiPAR is a specific substrate for the serine protease trypsin and
becomes fluorescent after clevage of the two oligopeptide side chains.
To investigate the change of [Ca2+] in ZGs, 5 µM (Kd = 20 µM,
excitation
= 549 nm,
emission
= 576 nm) (Molecular Probes, Eugene, Oregon) was
used. Isolated acinar cells were loaded with Calcium Orange-5N-AM for 20
minutes at 37°C and ZGs were isolated (Nguyen et al., 1998; Quezada et al.,
2001; Chin et al., 2002).
To investigate the change of [H+] in ZGs, 1 µM LysoSensor Blue DND-167
(pKa = 5.1,
excitation
= 373 nm,
emission
= 425 nm) (Molecular Probes, Eugene,
Oregon) was used. Isolated acinar cells were loaded with LysoSensor Blue
DND-167 for 30 minutes at 37°C and ZGs were isolated.
To investigate the change of [K+] in ZGs, 5 µM PBFI- AM (Kd = 10 mM,
λexcitation=340 nm, λemission=505 nm) (Molecular Probes, Eugene, Oregon) was
used. Isolated acinar cells were loaded with PBFI for 20 minutes at 37°C and ZGs
were isolated.
H+/K+ ion exchange in ZGs
Έ
To investigate the H+/K+ ion exchange properties of the ZG matrix,
LysoSensor Blue DND-167 was used to monitor changes in [pH]G and BZiPAR
was used to monitor changes in typsin activity in ZGs. Isolated ZGs loaded with
LysoSensor Blue DND-167 and BZiPAR were placed in intracellular buffer with 10
µM valinomycin (a K+ ionophore) (Calbiochem, San Diego, California) to
equilibrate K+ across ZG membrane (Chin et al., 2002). [K+] in the intracellular
buffer was increased from 0 mM to 140 mM. Ionic strength and osmolarity were
kept constant by adjusting the concentration of NMG in the intracellular solution.
Ca2+/K+ ion exchange in ZGs
ZGs were loaded with 5 µM Calcium Orange 5N-AM to monitor the changes
of [Ca2+]G and 10 µM BZiPAR to monitor the changes of trypsin activity in ZGs.
Then K+ was equilibrated across ZG membrane in intracellular buffer with 10 µM
valinomycin (K+ ionophore) (Nguyen et al., 1998; Quezada et al., 2001) was used
to. The [K+] in the intracellular buffer was varied from 0 mM to 140 mM. Ionic
strength and osmolarity were kept constant by adjusting the concentration of
NMG in the intracellular solution.
The effect of [Ca2+]G alone on the trypsinogen autoactivation in ZGs
Isolated ZGs were loaded with BZiPAR and LysoSensor Blue DND-167 to
monitor trypsin activity in ZGs and [pH]G simultaneously, 10 µM ionomycin (Ca2+
ionophore) (Calbiochem, San Diego, California) was used to equilibrate the Ca2+
between the ZG membrane. [Ca2+]G was varied from 10 µM to 100 µM.
The effect of [H+]G alone on the trypsinogen autoactivation in ZGs
Ή
Isolated ZGs were loaded with BZiPAR and Calcium Orange 5N-AM to
monitor trypsin activity in ZGs and [Ca2+]G simultaneously, 10 µM carbonyl
cyanide
4-(trifluoromethoxy)phenylhydrazone
(FCCP)
(a
H+
ionophore)
(Sigma-Aldrich corporation, St. Louis, MO) was used to equilibrate the H+
between the ZG membrane. [H+]G varied from 5.5 to 4.5.
Measurement of trypsin activity in ZGs with different [Ca2+]G and [H+]G
Isolated ZGs loaded with BZiPAR were placed in intracellular buffer with 10
µM ionomycin and 10 µM FCCP to equilibrate Ca2+ and H+ across ZG membrane.
[Ca2+]G was increased and [H+]G was decreased by adjusting the Ca2+
concentration and pH in the intracellular buffer. The corresponding changes of
trypsinogen autoactivation were monitored with the fluorescence of BZiPAR.
Effects of K+-channel blockers
In separate preparations, apamin (100 nM) (a blocker for SKCa channel)
(Sigma-Aldrich corporation, St. Louis, MO), tetraethylammonium chloride (TEA)
(10 mM and 20 mM) (a general K+ channel blocker) (Sigma-Aldrich corporation,
St. Louis, MO) and charybdotoxin (10 nM) (a toxin for big conductance Ca2+ –
activated K+ channel (BKca channel)) (Sigma-Aldrich corporation, St. Louis, MO)
were used to block the ASKCa channels in the membrane of ZGs.
N-Methyl-D-glucamine (NMG) (Sigma-aldrich corporation, St. Louis, MO) was
used to replace K+ in the K+-free intracellular solution.
΁΀
RESULTS
The effect of incubation time on trypsin activity in ZGs
Figure 2 shows that the length of time the ZGs were incubated (from 10 to
240 minutes in intracellular solution) had no effect on trypsin activity in the
granules. This result doesn’t resemble to a typical enzymatic reaction with
time-dependent characteristics, but may be explained as follows. First, the
activation of trypsin and cleavage of BZiPAR occur quickly (Leytus et al., 1983;
Boonacker et al., 2001; Kawabata et al., 1988; Schaller et al., 2000). It usually
takes only 200 seconds (Raraty et al., 2000; Leytus et al., 1984). In my
experiments, the ZGs were washed several times before the fluorescence was
recorded, and this would allow sufficient time for trypsin activation and the
cleavage of BZiPAR to reach equilibrium. Second, other factors such as the
leakage of dye, the inhibition of active trypsin by the pancreatic secretory trypsin
inhibitor (PSTI), and the degradation of active trypsin can prevent the further
increase of fluorescence in ZGs. It has previously been shown that BZiPAR
fluorescence due to trypsin cleavage equilibrated quickly in ZGs. As shown in
Figure 2, here, and elsewhere in this thesis. (Raraty et al., 2000),
The increase of [Ca2+]G, [H+]G and trypsin activity in ZGs when [Ca2+]i is
increased
Figure 3a, b and c shows that the resting [Ca2+]G was 9.10 + 0.97 µM (mean
΁΁
+ SD, n=40) and the resting [pH]G of ZGs was 5.47 + 0.05 (mean + SD, n=40).
These results are consistent with previous data (Lebel et al., 1988). Because
trypsin activity directly could not be measured, the fluorescence intensity of the
BZiPAR was used to represent trypsin activity. The resting intraluminal trypsin
activity was 829.03 + 14.73 (mean + SD, n=40). By using BZiPAR, LysoSensor
DND-167 and Calcium Orange 5N-AM, trypsin activity in ZGs, [Ca2+]G and [pH]G
could be monitored simultaneously. As the [Ca2+]i was increased from 50 nM to
700 nM, trypsin activity increased 1.4-fold, the [Ca2+]G increased 4.5-fold, and the
[pH]G decreased from 5.5 to 4.5. Under normal physiological conditions, the
resting free [Ca2+] in the intracellular solution was 100 nM and [Ca2+]G, [H+]G and
trypsin activity in ZGs were low. Figure 3 demonstrates that when [Ca2+]i was
increased from 50 nM to 300 nM, the [Ca2+]G, [H+]G and trypsin activity in ZGs
were slightly elevated. But when [Ca2+]i was elevated to 400 nM, the [Ca2+]G, [H+]G
and trypsin activity in ZGs increased sharply. When [Ca2+]i became 500 nM, the
[Ca2+]G, [H+]G and trypsin activity in ZGs peaked. The threshold for the increase in
[Ca2+]G, [H+]G and trypsin activity in ZGs was around 300 nM [Ca2+]i.
Effect of [Ca2+]i on the K+ influx into ZGs
Figure 4 shows that the resting [K+]G was 38.38 + 6.16 mM (mean + SD,
n=30). As the [Ca2+]i was increased, [K+]G increased about 3-fold to 107.89 + 7.03
mM. When [Ca2+]i was increased from 50 nM to 300 nM, the [K+]G increased
slightly. But when [Ca2+]i was elevated to 400 nM, [K+]G increased sharply. The
[K+]G peaked When [Ca2+]i was 500 nM, The threshold of [K+]i increase occurred
around 300 nM [Ca2+], just the same as that seen for the increase in trypsin
activity, [Ca2+]G and [H+]G.
΁΂
Ca2+/K+ exchange and H+/K+ exchange in ZGs
In this experiment, the [K+]G was varied from 0 mM to 140 mM. Figure 5
shows that increasing [K+]G results in elevation of [Ca2+]G and trypsin activity in
ZGs. When [K+]G increased from 0 mM to 50 mM, there was no change in [Ca2+]G
or trypsin activity in ZGs, but when [K+]G increased from 50 mM to 100 mM,
[Ca2+]G was elevated about 4-fold and trypsin activity in ZGs was elevated about
1.3-fold. The threshold of the [Ca2+]G increase was similar with that for activation
of trypsin in ZGs and occurred around 50 mM [K+].
Figure 6 demonstrates that when the [K+]G was increased,
[H+]G and trypsin
activity in ZGs were also elevated. When [K+]G increased from 50mM to 100 mM,
[H+]G was decreased from 5.5 to 4.5 and trypsin activity in ZGs was elevated
about 1.3-fold. The threshold of [H+]G increase was similar with that of trypsin
activity in ZGs and [Ca2+]G increase.
The effect of [Ca2+]G alone and [H+]G alone on trypsinogen autoactivation
in ZGs
Figure 7 shows that when [Ca2+]G was increased from 10 µM to 70 µM,
trypsin activity increased and reached its maximum at 50 µM. When [Ca2+]G
increased further to 70 µM, trypsin activity remained steady or even decrease
slightly. Note that the maximum trypsin activity induced by [Ca2+]G alone was
lower than that induced by the [Ca2+]G and [H+]G together (Figure 9). Moreover
when [Ca2+]G was increased, [H+]G remained around its baseline.
Figure 8 demonstrates that as [pH]G was decreased, trypsin activity in ZGs
΁΃
continued increased, but [Ca2+]G wasn’t changed. The maximum trypsin activity
induced by [Ca2+]G alone was lower than that induced by [Ca2+]G and [H+]G
together.
Both the increase of [Ca2+]G and the decrease of [pH]G induce
trypsinogen autoactivation in ZGs
To investigate the effect of [pH]G and [Ca2+]G together on trypsinogen
activation in ZGs, isolated ZGs were loaded with BZiPAR, then ionomycin and
FCCP were used to equilibrate the Ca2+ and H+ across the ZG membrane. [Ca2+]G
was increased from 5 µM to 50 µM at pH 4.5, pH 5.0 and pH 5.5 respectively, As
shown in Fig 9, when the [Ca2+]G was increased, trypsin activity in ZGs also
increased. Moreover, decreased [pH]G also enhanced the activation ability of
[Ca2+]G on trypsinogen autoactivation. But only under the combined effects of
increased [Ca2+]G and decreased [pH]G, did trypsinogen autoactivation reach its
maximum.
The requirement of K+ influx for trypsinogen autoactivation in ZGs
To investigate the requirement of K+ influx for trypsinogen autoactivation in
ZGs, NMG was used to replace the K+ in the intracellular solution. The ionic
strength of the intracellular solution was kept constant. When ZGs were
suspended in K+-free medium, with increasing [Ca2+]i there was no change in
[Ca2+]G, [H+]G, trypsin activity in ZGs. ( Figure 3 and Figure 4).
Characterization of K+ channels in the membrane of ZGs
΁΄
TEA (10 mM) (Figure 10) and charybdotoxin (10 nM) (Figure 12) had no
impact on [Ca2+]G, [H+]G and trypsin activity in ZGs with an increased [Ca2+]i in the
intracellular solution. The changes of [Ca2+]G, [H+]G and trypsin activity in ZGs
were similar with that in the absence of TEA (10 mM) or charybdotoxin (10 nM).
But TEA (20 mM) (Figure 10) could completely block these K+ channels, although
the [Ca2+]i was increased in the intracellular solution, [Ca2+]G, [H+]G and trypsin
activity in ZGs were not changed. Apamin (100 nM) (Figure 11) could also abolish
the changes of [Ca2+]G, [H+]G and trypsin activity in ZGs by inhibiting the K+
channels.
΁΅
DISCUSSION
It has been demonstrated that the premature activation of trypsin is a critical
event in acute pancreatitis (Lerch and Gorelick, 2000). Under physiological
conditions only a small amount of active trypsin exists in the pancreatic acinar cell.
In order to prevent cell injury induced by active trypsin, some protective
mechanisms must exist in the pancreas. These mechanisms include: 1. digestive
enzymes being synthesized and secreted as inactive zymogens. 2. the presence
of trypsin inhibitors, which can inhibit the activity of digestive enzymes. 3. the
presence of proteases that can destroy other already active proteases also
present in the pancreas. 4. the pH in ZGs is not suitable for the activation of most
digestive enzymes. PSTI is synthesized in the acinar cells of the pancreas to
block trypsin activity by binding to its active sites (Gorelick et al., 1992). PSTI also
blocks further activation of pancreatic cells via the trypsin receptor, PAR-2 and
prevents the enzyme cascade reaction (Hirota et al., 2003). But the inhibitory
ability of PSTI is not strong, it can only inhibit 5% of the potential trypsin activity
and its inhibitory ability is lost in acidic pH. So in the presence of high [Ca2+] and
low pH, the inhibitory ability of PSTI can be easily overwhelmed (Gorelick et al.,
1992). Proteases that can specifically degrade the active enzyme may be an
additional safety mechanism. Some studies show that a thiol protease may play
an important role in the degradation of trypsin, but the identity of this thiol
protease and the degradation site of trypsin are not clear (Gorelick et al., 1992).
This protease might be calpain or some lysosomal hydrolase (Gorelick et al.,
1992).
Normally pancreatic zymogens are only activated when they reach the small
΁Ά
intestine and are cleaved by enterokinase. But in vitro, trypsinogen can undergo
autoactivation without enterokinase. Although this autoproteolysis occurs at a low
rate, it increases at acidic pH and in the presence of sustained elevated [Ca2+]
(Gorelick et al., 1999). Trypsinogen can also be activated by other hydrolases,
such as cathepsin B. Optimum conditions for this activation require an acidic pH
(Lerch et al., 1999).
Several recent studies support the central role of trypsinogen activation in the
onset of acute pancreatitis. Most patients with hereditary pancreatitis have a
R122H mutation in the gene encoding cationic trypsinogen. The mutant trypsin is
more resistant to degradation by thiol proteases and is active longer than its
wild-type counterpart (Whitcomb et al., 1996). Studies with knock – out - mice
lacking cathepsin B, a lysosomal hydrolase that can convert trypsinogen to trypsin,
also support the importance of trypsinogen activation in pancreatitis. After
administration of the agonist caerulein, trypsin activity of the cathepsin B-deficient
mice is more than 80% lower than that of wild-type mice. (Halangk et al., 2000).
Ca2+ and low pH are both required for the premature enzyme activation.
Some studies have demonstrated that Ca2+ can stabilize trypsin. The presence of
Ca2+ significantly delays the trypsin-induced trypsinogen activation. Ca2+ also
delays trypsin-induced trypsin degradation, allowing trypsin activity to persist
much longer after trypsin is activated (Colomb et al., 1979; Figarella et al., 1988).
Although high Ca2+ concentration is required for premature protease activation, it
is not sufficient to induce this process alone. Low pH in the lumen of ZGs is also
required for the activation of enzyme and plays an important role in autoactivation
of trypsinogen (Lerch et al., 1991).
΁·
The experiments in this thesis showed that although low pH alone (Figure 7)
and elevated [Ca2+]G alone could induce trypsin activity (Figure 8), only the
combination of low pH and elevated [Ca2+]G could result in the maximum trypsin
activity (Figure 9). In these experiments, The increase of [Ca2+]G caused by
K+/Ca2+ exchange was associated with an increase in [H+]G caused by K+/H+
exchange. The increase in [Ca2+]G was not sufficient to mobilize the bound H+
from the matrix of ZGs (Figure 7) and vice verse (Figure 8). But H+ had significant
effect on K+/Ca2+ exchange, it could increase the opportunity of K+ releasing Ca2+
from the matrix and accelerate the K+/Ca2+ exchange (Chin et al., 2002).
Condensed polyanionic polymeric networks, and one or more cations or
polycations are omnipresent in many secretory granules (Nguyen et al., 1998;
Quezada et al., 2001; Chin et al., 2002). These networks are important for
condensation and sorting of the secretory products. Within mucin granules, the
polyanion is mucin itself and the cations are Ca2+ and H+. Within chromaffin
granules, the polyanion is chromogranin A, it is associated with cations H+, ATP
and catecholamines (Winkler and Westhead, 1980). For torpedo synaptic vesicles,
the polyanion is intravesicular proteoglycan matrix, which binds to acetylcholine
(Ach) and ATP (Reigada et al., 2003). In presence of other cations, bound cations
will be released from the polymeric matrix by ionic exchange. Because many
small transmitters, secretory products and ions are stored within the polymeric
networks of secretory granules, this ion-exchange mechanism can control the
release of ions and hormones (Fernandez et al., 1991; Marszalek et al., 1997).
Less is known about the components of the polymeric network in ZGs of
pancreatic acinar cells. This condensed polyanionic polymeric network may
consist of sulfated glycoproteins and/or proteoglycans with bound Ca2+ and H+.
΁Έ
De Lisle et al (1994) found the most abundant component is mucin, which is a
300-kDa glycoprotein with sulfated O-linked carbohydrate chains. The major
glycoprotein of the ZG membrane, GP2, links with the proteoglycans of the
submembranous matrix and helps keep them tightly associated with the
membrane (De Lisle, 1994; 2002; Schmidt et al., 2000).
ASKCa channels have three different major types: BKCa, intermediate
conductance Ca2+ – activated K+ channel (IKCa) and SKCa. They are distinguished
by their very different single-channel conductances. They also have different
voltage, pharmacological sensitivities, and different functional roles. Different
types of ASKCa channels have different sensitivities to changes in [Ca2+]. BKCa is
activated by [Ca2+] in the µM range, while SKCa channels are activated by
between 200 nM and 700 nM [Ca2+]. The Ca2+-dependence of IKCa channels is
somewhere between BKCa and SKCa channels (Latorre et al., 1989). Previous
research demonstrated that ASKCa channels exist in the membranes of secretory
granules of mast cells and goblet cells (Nguyen et al, 1998; Quezada et al., 2001;
Chin et al., 2002). My data on the Ca2+ – dependence of K+ uptake in ZGs of
pancreatic acinar cells were consistent with the presence of SKca channels. K+
uptake by the granules was promoted by 300 nM [Ca2+], and was blocked by the
SKCa–specific blocker apamin (100 nM) (Figure 11) and the ASKCa channel
blocker TEA (20 mM) (Figure 10). It was not blocked by BKCa–specific toxin
charybdotoxin (10nM) (Figure 12).
Further, my experiments showed that trypsin
activation in the granules depended on K+ influx, since replacing K+ in the
intracellular medium with the nonpermeant cation NMG blocked Ca2+-induced
trypsin activation.
Taken together, my data suggest the model shown in Figure 13 for the
΁Ή
premature activation of trypsin in pancreatitis. The autoactivation of trypsin in ZGs
requires the coordinated interaction of two molecular components: one is a
polyanionic matrix that functions as an ion exchanger, the other is Ca2+-activated
K+ channel that imports K+ into ZGs. When [Ca2+] is increased in the cytosol, it
causes opening of the ASKCa channels and K+ will move into the lumen of ZGs.
The increase in [K+]G will mobilize bound Ca2+ by Ca2+/K+ ion-exchange thus
increasing the free Ca2+ concentration in ZGs, it will also mobilize bound H+ by
H+/K+ ion-exchange and decrease the pH in ZGs. Both the elevated [Ca2+]G and
decreased [pH]G not only facilitate the autoactivation of trypsinogen to active
trypsin but also stabilize trypsin activity leading to the premature activation of
digestive enzymes in pancreatic acinar cells and possible acute pancreatitis
(Figure. 13).
To date, much research on pancreatitis has focused on understanding and
limiting the inflammatory reaction that follows the initiating of pancreatitis.
However understanding the early molecular mechanisms initiating acute
pancreatitis may also be important. Understanding potential factors initiating
acute pancreatitis and their activation mechanisms should facilitate the
development of targeted therapies.
΂΀
1
2
3
1
3
2
Figure 1. Illustration of the validation of the deconvolution program. This algorithm
successfully excluded the out-of –focus fluorescence in the images, leaving only
fluorescence originating within the shallow depth of field of the objective. The
diameter of the fluorescent ring (or disk) seen was dependent on the depth of the
optical focal plane.
΂΁
Figure 2. Effect of incubation time on trypsin activity in ZGs. The granules were
allowed to settle and attach for different incubation times in the intracellular
solution containing 100 nM, 400 nM and 700 nM Ca2+ respectively. As the
incubation time went on, trypsin activity in ZGs did not change. Each point
corresponds to the mean + SD of more than 30 granules from three separate
experiments.
΂΂
a
b
c
Figure 3. Effect of increasing [Ca2+]i on the [Ca2+]G, [pH]G and trypsin activity in
ZGs. a. Increasing [Ca2+]i resulted in the increase of [Ca2+]G. b. Increasing [Ca2+]i
resulted in the increase of [H+]G. c. Increasing [Ca2+]i resulted in the increase of
trypsin activity in ZGs. But in the absence of K+, [Ca2+]G , trypsin activity in ZGs
and [pH]G were not changed. Each point corresponds to the mean + SD of 40
granules from three separate experiments.
΂΃
Figure 4. Effect of increasing [Ca2+]i on the [K+]G. Increasing [Ca2+]i resulted in an
increase of [K+]G, but in the absence of K+, [K+]G was not changed. Each point
corresponds to the mean + SD of more than 30 granules from three separate
experiments.
΂΄
a
b
Figure 5. Ca2+/K+ ion exchange in ZGs. The isolated ZGs were equilibrated in
intracellular buffer containing 10 µM valinomycin. The inflow of K+ to the lumen via
the valinomycin resulted in an increase in [Ca2+]G and trypsin activity in ZGs. Each
point corresponds to the mean + SD of more than 30 granules from three
separate experiments.
΂΅
a
b
Figure 6. H+/K+ ion exchange in ZGs. The isolated ZGs were equilibrated in
intracellular buffer containing 10 µM valinomycin. The inflow of K+ to the lumen
resulted in an decrease in [pH]G and activation of trypsin activity in ZGs. Each
point corresponds to the mean + SD of more than 30 granules from three
separate experiments.
΂Ά
a
b
Figure 7. The effect of [Ca2+]G alone on trypsinogen autoactivation in ZGs. The
isolated ZGs were equilibrated in intracellular buffer containing 10 µM ionomycin.
Increased [Ca2+]G was not sufficient to mobilize bound H+ from matrix networks
but it could increase trypsin activity in ZGs. Each point corresponds to the mean +
SD of more than 30 granules from three separate experiments.
΂·
a
b
Figure 8. The effect of [pH]G alone on trypsinogen autoactivation in ZGs. The
isolated ZGs were equilibrated in intracellular buffer containing 10 µM FCCP. Low
pH was not sufficient to mobilize the bound Ca2+ from matrix networks but it did
increase trypsin activity in ZGs. Each point corresponds to the mean + SD of more
than 30 granules from three separate experiments.
΂Έ
Figure 9. The effect of both increased [Ca2+]G and decreased [pH]G on
trypsinogen autoactivation in ZGs. The isolated ZGs were equilibrated in
intracellular buffer containing 10 µM FCCP and 10 µM ionomycin. Both increased
[Ca2+]G and decreased [pH]G triggered the activation of trypsin, but only under the
combined effect of increased [Ca2+]G and decreased [pH]G, was trypsin activity the
highest. Each point corresponds to the mean + SD of more than 30 granules from
three separate experiments.
΂Ή
a
b
c
Figure 10. The effect of TEA (20 mM and 10 mM) on the [Ca2+]G, [pH]G and trypsin
activity in ZGs. 10 mM TEA did not inhibit the increase of [Ca2+]G , trypsin activity
in ZGs or the decrease of [pH]G, but in the presence of 20 mM TEA, the increase
of [Ca2+]G, trypsin activity in ZGs and [pH]G was inhibited,. Each point
corresponds to the mean + SD of more than 30 granules from three separate
experiments.
΃΀
a
b
c
Figure 11. The effect of apamin (100 nM) on [Ca2+]G, [pH]G and trypsin activity in
ZGs. 100 nM apamin fully inhibited the increase of [Ca2+]G , trypsin activity in ZGs
and the decrease in [pH]G when the [Ca2+]i was increased. Each point corresponds
to the mean + SD of more than 30 granules from three separate experiments.
΃΁
a
b
c
Figure 12. The effect of charybdotoxin (10 nM) on [Ca2+]G, [pH]G and trypsin
activity in ZGs. Charybdotoxin did not inhibit the increase of [Ca2+]G, trypsin
activity in ZGs or the decrease of [pH]G when the [Ca2+]i was increased. Each point
corresponds to the mean + SD of more than 30 granules from three separate
experiments.
΃΂
[Ca2+]C ↑
Cytosol
Autoactivation of Trypsinogen
(+)
(+)
(+)
Ca2+
H+
K+
K+
KCa
Zymogen
Matrix
ZG Granular Lumen
Figure 13. Model of the dynamics of H+ and Ca2+ inside ZGs. Two pools of cations
are found inside ZGs, a pool bound to the matrix of ZGs and a pool of free ionized
cations. The changes of [Ca2+]G and [H+]G require the functional interaction of the
ZG polyanionic matrix with K+, acting as H+/K+ ion exchanger and Ca2+/K+
ion-exchanger. ASKCa channels are also located in the membrane of ZGs, these
channels are SKCa channels and can be activated by [Ca2+] (300 nM) and
inactivated by apamin (100 nM) and TEA (20 mM). When the [Ca2+]i is increased,
it will open the these K+ channels in the membrane of ZGs. The influx of K+ will
trigger the H+/K+ exchange and Ca2+/K+ exchange, thus increasing the free Ca2+
concentration and H+ concentration in ZGs. Both the increase of [Ca2+]G and the
decrease of [pH]G can facilitate trypsinogen autoactivation and stabilize trypsin
activity.
΃΃
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΃Ή
BIOGRAPHICAL SKETCH
The author was born in China. Before he came to Florida State University, he
received a B.S. in cellular and developmental biology program from the college of
life sciences, Wuhan University, Wuhan, Hubei, P.R.C. He also received his M.S.
in Genetics program from the college of life sciences, Wuhan University, Wuhan,
Hubei, P.R.C.
In the summer of 2001, the author enrolled at Florida State University. His
research is focused on the initial mechanism of trypsin premature activation in
pancreatitis.
΄΀