AMERICAN JOURNAL OF PEIYsJOLOGY
Vol. 228, No, 6, June 1975.
Printed in U.S.A.
Enzyme
secretion
S. S. ROTHMAN
De&zrtment of Physiology,
in the absence of zymogen
University
of California,
San Francisco, California
digestive
granulation;
enzyme;
protein
pancreas;
transport
secretion
granule;
acinar
cell;
de-
EARLIER
REPORTS
(2, 7, 11, 28) suggest that the pancreas
responds substantially
to stimuli that elicit protein secretion
even when the acinar cells appear
to be devoid of zymogen
granules,
the enzyme-containing
secretion granule
of the
pancreas. The present study was undertaken
to reexamine
this observation
and to place it in a more quantitative
framework by comparing
the secretion of digestive enzymes to the
number of zymogen granules within the acinar cell, as estimated from the relative cell volume occupied
by electronopaque granules during and after progressive degranulation
produced
by repeated injections of a cholinergic
stimulus.
METHODS
Pre/mation Df ruts for stud? of ;bancreatic secretion in situ. Adult
wlale rats from the Holtzman
Company
were anesthetized
with 0.7 ml of Dial with urethan solution per kilogram
body
weight (Ciba Pharmaceutical
Company)
after an overnight
fast of between 18 and 22 h (23) Pure pancreatic
secretion
was collected from the common
bile duct after excluding
bile by ligating
the duct close to the liver (23). Thirty
to
sixty minutes were allowed
for stabilization
of the system
and washout of bile from the duct and tubing+ Secretion was
collected at hourly intervals unless otherwise stipulated.
The
first collection
period was a control (0 h), the second, third,
and fourth periods (1 h, 2 h, 3 h) were each initiated
by a
subcutaneous
injection
of 0.8 mg/kg body wt of methacho1828
94143
line chloride
(MCh) . The fifth period (4 h) was a final control collection.
Untreated
rats were studied over the same
time course. In another group of animals, 1 mCi “H-labeled
leucine (30 Ci/mmol)
was injected intravenously
at the time
of the third methacholine
injection
(3 h), and the rate of
appearance
of labeled protein was monitored
in secretion
for 70 min. In these animals, 0.5 h after the third injection,
a fourth MCh injection
was administered
at the same dose.
Collection of pancreatic tissue fur microscopic. MCh was administered at hourly intervals
either 1, 2, or 3 times to rats
fasted prior to study as described above. Animals
were sacrificed
by spinal section after light etherization
and the
glands were excised 30 min after the injection
(either 1, 2,
or 3). Control animals were injected with 0.9 % NaCl. Tissue
samples were fixed in 1.3 % formaldehyde,
0.3 % potassium
dichromate,
and 2 G/oglutaraldehyde,
and then stained with
1 % uranyl acetate followed
by 0.02 % lead citrate
(27).
Sections were cut, after being embedded
in epoxy resin, for
examination
under the light microscope.
Chemical techniques. Trypsinogen
and chymotrypsinogen
were estimated in the secretion from the esterase activities of
their active forms against 26.0 mM p-toluenesulfonyl-L-arginine methyl ester or 8*0 mM N-acetyl-L-tyrosine
ethyl
ester, respectively,
as determined
from the initial reaction
velocity of substrate hydrolysis. The reaction was followed by
titrating
the acid end product with 0.1 N NaOH to maintain the pH in the reaction vessel constant (15, 18, 23) ; the
amount of NaOH
titrated
is the molar equivalent
of the
amount of substrate split. All samples for both reactions had
linear reaction velocities for the duration
of the assay. Enteropeptidase
(EC 3.4.4.8) (Calbiochem,
grade B) was used
to initiate
the activation
of both enzymes.
It was present
in the reaction mixture in excess of the amount required
to
produce maximal
activation
of trypsinogen
and chymotrypsinogen at 37°C within 30 min for the largest sample tested.
The activity of each sample was measured
immediately
after activation.
Sample size was chosen to be within
the
narrowest
possible measurement
range.
Protein was measured using the Folin phenol reagent (12)
and the volume of secretion was determined
gravimetrically.
Labeled protein was estimated after precipitation
of samples
of secretion in 20 % trichloroacetic
acid (TCA)+ The precipitate was collected and washed with 20 % TCA containing 10 mll/l: leucine on 0*22-pm cellulose-ester
filters. The
filters were then placed in scintillation
vials for counting
by
liquid scintillation
spectroscopy
(22).
Quantitative stereology. The relative cell volume occupied by
zymogen granules was determined
at the times specified in
the text with the use of a random point-count
method (26)
on 8 X 10 inch photographs
of microscopic
sections at a
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on April 13, 2017
RoTHK4N,
S. S. En<yme secretion in the absence of zymogen granules.
228(6):
1828-1834.
Am.
J. Physiol.
19 75 .-Pure
pancreatic
juice
was collected
from
the cannulated
common
bile duct of
anesthetized
rats studied
after an overnight
fast. Digestive
enzyme
secretion
was folIowed
in these animals
during
and after
the
progressive
degranulation
of acinar
cells produced
by sequentially
applied
cholinergic
stimuli.
The
kinetics
of degranulation,
a
progressive
decrease
in the number
of zymogen
granules
in
acinar cells, was estimated
from the relative
cell volume
occupied
by electron-opaque
granules
at various
times using a random
pointcount
stereological
technique
to examine
tissue sections.
Three
hourly
injections
of methacholine
chloride
were
sufficient
to
produce
the almost
complete
disappearance
of electron-opaque
granules
from
secretory
cells. Greatly
augmented
enzyme
secretion was still1 observed
in their absence,
7-25 times greater
than
control
values : -IO-fold
for protein
output
overall,
-7-fold
for
trypsinogen,
and -25-fold
for chymotrypsinogen.
Secretion
in
the absence of zymogen
granules
is discussed
relative
to exocytosis
and
three-compartment
(intracellular
storage,
cytoplasm,
and
duct lumen)
secretory
models.
granules
SECRETION
OF
DIGESTIVE
1829
ENZYhIE
l
RESULTS
Enqjme secretion in response to multiple injections of methacholine
chloride. Enzyme output after a second hourly injection
of
MCh was approximately
40 % less than the initial response
to a standard dose of the drug (Fig. l), The response to a
third injection
was still less, being about one-third
of the
output initially
seen (Fig. 1). Even though the response to
MCh decreased
with additional
injections,
a substantial
response was still observed, from approximately
7 to more
than 20 times the time-paired
(Table 1) or sequential
(Fig.
1) controls, depending
on the parameter
being considered
for the hour following
the third injection. A fourth MCh injection, given 30 min after the third, produced
a response of
approximately
equal magnitude
to that seen following
the
previous
(third)
injection
(Fig. Z), about one-third
the
maximal
or initial response.
The secretory responsiveness
was different or “nonparal10 for diRerent
measures (Table
1) : for protein
overall,
about a IO-fold increase; for trypsinogen,
only about 7 times
control; for chymotrypsinogen,
about 25 times the unstimulated output.
This is consistent
with a growing
body of
evidence which demonstrates
short-term,
nonparallel
or enzyme-selective
secretion by the acinar cell (see 1, 16-18, 20,
23-25, for example).
of Pancreatic acinar cell in resfwnse to multiple
Degranulation
injections of methacholine chloride. One-half hour after the initial
injection
of MCh our estimate indicates that the acinar cells
I I
El
IQ
69*/L
0%
I%
Total granules
remaining
9
250
Iii
5
2
Tota.l granules
lost
4
3
2
I
i
0
0
2
I
Time
3
4
0
I Hours)
1. Chymotrypsinogen,
trypsinogen,
and
protein
output
in
loline
chloride-treated
rats. &ur
0 (h 0) is preinjection
control
and h 4 is postinjection
control.
Hours I, 2, and 3 were each initiated
with
a subcutaneous
injection
of 0.8 mg methacholine
chloride
per
kilogram
body
weight.
Values
in circles
and rectangles
were
taken
from
Fig. 6 and refer
to number
of zymogen
granules
in tissue
at end
of h 0 and at beginning
of h 4 (circles),
or percent
of control
number
(lOOyO)
lost d uring
hours
subsequent
to methacholine
chloride
injection
(rectangles).
At time 0, 100%
refers
to percentage
of cell volume
occupied
by zymogen
granules
after an overnight
fast and before
administration
of cholinergic
drug.
Enzyme
outputs
are means
+ SE
for 6 rats. For each period,
1st bar is for chymotrypsinogen,
2nd bar is
for trypsinogen,
and 3rd bar is for protein.
FIG.
metha
TABLE 1. Enzyme out@4t in res@we to methacholine
chloride from degranulated cells
-.
Enzyme
Protein,
m&h
Trypsinogen,
pmoles
substrate
split
min-l/h
--.--
Output,
l
Control*
Methacholine
0.23
2.83
Et
+
.06-f
.32
0.89
6.51
+
&
.18
1.04
Values
are means
h SE. n = six animals
degranulated
cells
had ~7~yG
of the control
Secretion
was measured
for the hour
following
choline
chloride
injection.
* Time-paired
unstimulated.
U/h
Chymotrypsinogen,
pmoles substrate
split mmin-l/h
-
3.45
89.20
in each
number
the
control
&
zt
.67
13.75
group.
The
of granules.
third
methagroup
at 3 h,
contained
only about 50 % of their fasting content of zymogen granules (F ig. 3). By 0.5 h after the third injection,
only
about 10 % of the original
number
of granules
remained
(Fig. 4, A and B). Approximately
90 % of the zymogen granules were Yost” in 3 h, about two-thirds
of them within the
1st h as a result of a single injection
of MCh. After the secretory response to the final injection
waned, the secretory
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on April 13, 2017
magnification
of approximately
X 2,000. The measurements
were made by placing a translucent
overlay over each photograph which contained
a random
pattern of 735 dots of
about one-fourth
to one-half the radius of the zymogen granules in the photograph.
The overlay was divided into 10
longitudinal
strips. The number of dots superimposed
over
granules, nuclei, and the rest of the cell were counted separately for each longitudinal
strip. The longitudinal
divisions of the overlay served to make the measurement
less
prone to counting
error by dividing
the field into smaller
divisions as well as by providing
a convenient
base for the
statistical
evaluation
of the measurement;
each column
sampled from 1 to 11 cells, as estimated by the number
of
nuclei. The number of nuclei counted were used to estimate
the number of cells and may either overestimate
the actual
number,
in that binucleate
cells are relatively
common in
this tissue, or underestimate
it, in that granules over cytoplasmic areas that lacked nuclei were counted. Only granule
profiles of distinctly
different opacity from the background
were counted,
i.e., relatively
electron-dense
profiles. This
included
the so-called condensing vacuoles. The past history
of the section being counted was not known by the person
doing the counting.
Using a similar
stereological
technique,
Kramer
and
Geuze (8) estimated the relative cell volume occupied
by
zymogen granules in fasted rats at 23.6 % as compared
to
our value (mean & SE) of 24 IIZ 1.6 70. They also examined
the kinetics of degranulation
as a function of a series of pilocarpine injections
and observed rapid degranulation
with a
pattern quite similar to that shown in Fig. 3 (viz., 11 1 %
and 1.6 70 after 2 h and two
after 1 h and one injection,
injections
(8)).
1830
S. S. ROTHMAN
rate decreasing
back to unstimulated
levels (Fig. 1), the
relative
volume of the acinar cell occupied
by zymogen
granules increased toward control values (to 100 %, or about
24 % of cell volume),
relatively rapidly
at first (from 10 to
about 40 % of control values in 1 h for the data in Fig. 3).
Relationship between degranulation and enzyme output.The rate
of disappearance
of zymogen granules, and presumably
their
contents as well, as estimated from the relative volume of the
cell that they occupy, was veryrapid
relative to the observed
decline in protein secretion over time with continued
methacholine chloride
administration
(Figs. 1 and 2). When the
decrease in the relative cell volume occupied
by zymogen
granules for each hour of methacholine
stimulation
is plotted
against the hourly output of trypsinogen,
chymotrypsinogen, or total protein,
this nonlinear
relationship
can be
DISCUSSION
0 bb'
0 120
130
140
150
Time
160
(Minutes)
170
180
'
190
FIG. 2. LIethacholine
chloride-augmented
protein
secretion
in tissue containing
very
few zymogen
granules.
At time 140 min, only
7y0
01 original
number
of granules
remained
(see Fig. 6). Two injections
of methacholine
chloride
at the same dose (0.8 mg/kg
body
wt) were
given
prior
to 2 injections
shown
in this figure.
Values
are means
=t SE
for 6 rats.
Response
to 4th
injection
of methacholine
chloride
was
approximately
1/3rd
as large
as maximum
response
to 1st injection.
Unstimulated
protein
secretion
for hour
from
120 to 180 min
was
0.04 =t 0.01 mg/lO
min
(see Table
1).
[ Methacholine
chloride
Secretion of digestive enqme in absence of cymogen granules. AS
earlier (2, 7, 11, 28) as well as more recent studies (6) suggest, greatly augmented
digestive enzyme secretion occurs
in the almost complete
absence of the enzyme-containing,
electron-opaque
zymogen granule* The continued
secretion
of enzyme in the apparent
absence of zymogen granules is a
prediction
of the hypothesis that digestive enzymes can be
secreted directly from the cytoplasm by their transfer across
the plasma membrane,
being derived under these conditions
from intracellular
enzyme stores other than zvmogen granules (16-l 9, 2 1, 22). However,
in and of itself this observation does not require such a mechanism,
and continued
augmented secretion in the absence of zymogen granules could
still be accounted
for by an exocytotic
process of either a
different type or of altered kinetics, both of which have recently been suggested (different
tvpe (6), altered kinetics
(8)). However,
neither evidence nor circumstance
supports
these exocvtotic
exnlanations.
1
l--l--i
FIG. 3. Effect
of methacholine
chloride
on percentage
of cell volume
occupied
by zymogen
granules
as determined
by a random
point-count
method.
Number
of nuclei
counted
for each bar
is 384, 2 14, 262, 244, 499, 281, 189, 69,
247,
and
15 1 starting
from
left.
Bars
placed
between
hours
are for samples
taken
at every
0.5 h. Error
bars show
SE of measurement
for cells with varying
numbers
of
zymogen
granules
(see
METHODS).
0
I
2
3
4
Time
5
(hours)
6
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on April 13, 2017
clearly seen (Fig. 5) (y = aebx : trypsinogen,
r = .9995,
P < 0.01; chymotrypsinogen,
r = .998, P < 0.0 1; protein,
r = -999, P < 0.01). It can also be seen that the zymogen
granules account for only about 60 % of the total enzyme
secreted during the 3-h period, on the assumption
that the
rate of granule loss is substantially
greater than the formation of new granules during
the administration
of methacholine chloride
(see below for further discussion).
EJect of degranulation on time of appearance of labeled protein in
secretion. New or labeled proteins do not appear in the secretion collected from fasted, anesthetized
rats in substantial
amounts until a minimum
of about 40-60 min after the injection of a radioactive
amino acid (Fig. 6). Little difference
was seen in this characteristic
among rats after three sequential hourly injections
of methacholine
chloride,
in which
case labeled enzyme in the secretion also started to increase
at about 40-60 min postinjection,
although in lesser amounts
than in the controls (Fig. 6). It should be noted that small
amounts of labeled protein were collected in the secretion
as early as 10 min after injection of the radioisotope,
but the
amount of this rapidly
appearing
labeled protein
was not
increased by repeated methacholine
injections.
SECRETION
OF DIGESTIVE
ENZYME
1831
of rat pancreatic
The emergence of a pool of zymogen granules or other vesicles capable of exocytosis that turn over rapidly, one of two
potential
types of kinetic alteration
that can be considered,
does not seem possible, since only an insubstantial
amount of
newly synthesized
protein appears in secretion from zymogen granule-depleted
glands during
the 1st h after the
injection
of a radioactive
amino acid (Fig. 6). This means
that the number of exocytotic interactions
in 1 h cannot be
substantially
greater than the number
of vesicles, of whatever variety, within
the cells at the beginning
of that hour
and is, in all likelihood,
less. A second potential
kinetic alteration,
an increase in the enzyme concentration
of individual
granules
of sufficient
magnitude
to account for
secretion, seems unlikely
as well. It requires that granules
in actively secreting glands contain enzymes in much higher
concentrations
than granules from unstimulated
glands after
an overnight fast, conditions
that produce maximum
enzyme
storage.
In the absence of these kinetic changes, the possibility
that secretion from zymogen granule-depleted
cells can be
quantitatively
accounted
for by the exocytosis of the contents of another
type of vesicle also seems unlikely.
The
prime candidate
for such a role is a small vesicle (6, 8), of
the order of l/lOth
the diameter
of the zymogen granule
(about 0.1 pm) seen in zymogen
granule-depleted
cells.
Such a small-vesicle
system poses two problems.
First, since
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on April 13, 2017
FIG. 4. Micrographs
tissue after an overnight
fast (A), and
after 3 injections
of methacholine
chloride subsequent
to fast (B). Tissue was
removed
0.5 h after 3rd injection
in
treated
condition.
Inserts
are areas of
larger section of tissue, each showing
an
acinus.
1832
S. S. RQTHMAN
Chymotrypsinogen
0
I
100
40
80
I
r
output,
ymolas
120
I
160
substrate
split-
240
200
1
min-hr(+)
280
1
320
1
360
I
Totat protein output in response
to 3 sequential
MCh injections
Non-zymogen
I
1
I
I
I
I
1
I
2
3
4
5
6
7
8
9
Protein
FIG. 5. Relationship
between degranulation
and chymotrypsinogen,
trypsinogen,
and protein
outputs. Points displayed
are taken from data
7r
?
0
IO
2’0
30
Time
40
(Minutes)
50
60
70
80
FIG. 6. Appearance
of labeled protein
in secretion
after intravenous
injection
of labeled
amino acids. Open circles are values from untreated
rats (n = 3). Closed circles are values from rats given 3 injections of methacholine
chloride
(MCh)
prior to addition
of label and
another
methacholine
injection
0.5 h subsequent
to it (n = 6 for +4
MCh).
the volume of a sphere is a cubic function
of its radius, a
vesicle of I/ 10th the radius of the zymogen granule would
have to be present in 1,000 times the number
to carry the
same amount of matter at the same intravesicular
concentration.
Of course, the number
required
would
increase
geometrically
if even smaller vesicles are considered.
Second,
the surface-to-volume
ratio increases as the vesicle radius
decreases and therefore
requires
a substantial
increase in
the amount of membrane
needed to transfer a given amount
of material,
10 times as much for the considered
example.
I
IO
output,
I
II
mglhr
I
12
(@)
pools
I
I
I
I
I
I
I
A
13
14
I5
16
17
I8
I9
20
in Figs. 2 and 6. Equations
for curves
down of intracellular
sources is shown
are of form y = a@. A breakonly for protein
secretion.
There is no indication
at present that this small vesicle, or
any other vesicle for that matter, is present in sufficient number in granule-depleted
cells to account for secretion, even
if it were clear that they contained
digestive enzyme and
were capable of undergoing
an exocytotic
event.
Kinetics of degranulation. There is even doubt about the role
of exocytosis in the secretion of zymogen granule
contents
themselves. Exocytosis is thought to account for the movement (secretion) of their contents from an intracellular
compartment
(granule)
to an extracellular
compartment
(duct
lumen),
en masse, by virtue of the formation
of a direct
connection
between the two compartments;
viz., a hole is
formed between the two compartments
as the result of a
specialized fusion of their adjacent, membranous
boundaries.
process, in which
As such, exocytosis is a “mass-transport”
matter is moved without
regard to the nature of the transported molecule. The secretion of granule contents by such
a process would be characterized
by a proportional
linear
decrease in the number of granules in the secretory cell, as
long as the rate of secretion
of granule
contents is substantially greater than the rate of new granule formation
or
filling.
This is the case in the presence of a stimulant
of
pancreatic
protein
secretion such as the cholinergic
drug
used in the present experiments.
However, under these conditions this proportionality
was not seen. When ccloss” of
granules per hour was plotted against enzyme secretion for
each hour of a 3-h sequence of methacholine
chloride
injections, the two measures were found to be highly nonlinear
with respect to each other (Fig. 5). In the apparent
absence
of kinetic alterations,
as discussed above, this nonlinear
relationship
can be expIained
only by the interposition
of a
third compartment,
presumably
the cytoplasm,
in the se-
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.2 on April 13, 2017
I
1
b
granule intracellular
(38.7 %)
SECRETION
OF
1833
ENZYME
DIGESTIVE
membrane
fuse remains quite fragile despite the assertions
of its importance,
no less its existence+ Such evidence as
does exist is based primarily
on electron-microscope
images
which have been interpreted
as demonstrating
not membrane-membrane
fusion itself but apparent
geometric
sequelas to such an event. While this transport
mechanism
may well exist and certainly
has great appeal due to its directness, simplicity,
and analogy to other processes such as
phagocytosis,
its quantitative
importance
in the normal
process of protein secretion has not yet been demonstrated.
Finally,
a final-common-step,
or membrane-transport
or
three-compartment
(referring to intracellular-compartment,
cytoplasm,
and duct lumen),
model is wholly
consistent
with a variety of experiments,
on both the pancreas and other
systems, in which autoradiographic
and cell-fractionation
techniques
have been used to seek proof for the exocytosis
hypothesis
(see ref. 4 for a recent review of these studies on
the pancreas).
These studies show relatively
clearly that
there is a sequential
accumulation
of newly synthesized digestive enzyme
within
defined
subcellular
fractions
or
specific areas of the acinar cell after a pulse of labeled amino
acid is applied to the system. But since these techniques,
as
used, measure the accumulation
of secretory
protein
in
various parts of the cell and not the actual flow or flux from
compartment
to compartment,
they give us no indication
of I) the number of compartments
involved in the secretion
process (only the nature of compartments
that accumulate
protein),
or 2) the magnitude
of enzyme Auxes via one or
another presumed
pathway.
The
author
thanks
MS, Margaret
Coppe
excellent
technical
assistance.
The
microscopic
studies
were
done
Susumo
Ito of the Department
of Anatomy,
This work
was supported
by National
AM15672,
AM10455,
and AM16990.
and
Ms.
Lois
Isenman
for
their
Received
for
publication
10 April
in
collaboration
with
Dr.
Harvard
Medical
School.
Institutes
of Health
Grants
1974.
REFERENCES
1. ADELSON,
secretion
1087-1089,
2.
3.
4.
5.
J. W., AND S. S. ROTHMAN.
Selective
pancreatic
enzyme
due to a new peptide
called
chymodenin.
Science
183 :
1974.
ALMEIDA,
A. L., AND M. I. GROSSMAN. Experimental
production
of pancreatitis
with
ethionine.
Gastroenterology
20 : 554-577,
1952.
BLOOM, W., AND D. W, FAWCETT. A Textbook
of Histology
(9th ed).
Saunders
: Philadelphia,
1968, p. 99-l 00.
JAMIESON, J. D. The secretory
process
in the pancreatic
exocrine
cell : morphological
and biochemical
aspects.
In : Secretin,
Cholecystokznin,
Pancreozymin
and Gas&in,
edited
by J. E. Jorpes
and
V. Mutt.
Berlin
: Springer-Verlag,
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of an
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the nonlinear,
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by multiple
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that are discordant
with
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simply by hypothesizing
that the movement of enzyme across
the cell membrane
into the duct system is from the cytoplasm, and that such movement
is a final common step in
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intracellular
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pancreatic secretion or zymogen granule. However, evidence
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as a result of a process in which granule membrane
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