AMERICAN JOURNAL OF PEIYsJOLOGY Vol. 228, No, 6, June 1 975. Printed in U.S.A.

Enzyme secretion in the absence of zymogen granules

S. S. ROTHMAN De&zrtment of Physiology, University of California, San Francisco, California 94143

RoTHK4N, S. S. En<yme secretion in the absence of zymogen granules. Am. J. Physiol. 228(6): 1828-1834. 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 point- count 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 secre- tion 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.

digestive enzyme; pancreas; secretion granule; acinar cell; de- granulation; protein transport

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 frame- work by comparing the secretion of digestive enzymes to the number of zymogen granules within the acinar cell, as esti- mated from the relative cell volume occupied by electron- opaque granules during and after progressive degranulation produced by repeated injections of a cholinergic stimulus.

METHODS

Pre/mation Df ruts for stud? of ;b ancreatic 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 methacho-

line chloride (MCh) . The fifth period (4 h) was a final con- trol 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 admin- istered at hourly intervals either 1, 2, or 3 times to rats fasted prior to study as described above. Animals were sac- rificed 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/o glutaraldehyde, 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-ar- ginine 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 main- tain 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. En- teropeptidase (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 chymotryp- sinogen 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 pre- cipitate was collected and washed with 20 % TCA contain- ing 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

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SECRETION OF DIGESTIVE ENZYhIE

magnification of approximately X 2,000. The measurements were made by placing a translucent overlay over each photo- graph which contained a random pattern of 735 dots of about one-fourth to one-half the radius of the zymogen gran- ules 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 sep- arately for each longitudinal strip. The longitudinal divi- sions 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 cyto- plasmic 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 pilo- carpine injections and observed rapid degranulation with a pattern quite similar to that shown in Fig. 3 (viz., 11 l 1 % after 1 h and one injection, and 1.6 70 after 2 h and two injections (8)).

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 in- jection, 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 “nonparal- 10 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 unstimu- lated output. This is consistent with a growing body of evidence which demonstrates short-term, nonparallel or en- zyme-selective secretion by the acinar cell (see 1, 16-18, 20, 23-25, for example).

Degranulation of Pancreatic acinar cell in resfwnse to multiple injections of methacholine chloride. One-half hour after the initial injection of MCh our estimate indicates that the acinar cells

I I

IQ

9

4

3

2

I

FIG.

metha 1. Chymotrypsinogen, trypsinogen, and protein output in loline chloride-treated rats. &ur 0 (h 0) is preinjection control

El 69*/L

0 % I %

Total granules remaining

Tota.l granules lost

0 I 2 3 4 Time I Hours)

1829

Iii 250 5

2

0 i 0

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 injec-

tion (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.

TABLE 1. Enzyme out@4t in res@we to methacholine chloride from degranulated cells

-. Enzyme Output, U/h

Protein, m&h Trypsinogen, pmoles substrate split

Chymotrypsinogen, pmoles substrate

l min-l/h split mmin-l/h --.-- -

Control* 0.23 Et .06-f 0.89 + .18 3.45 & .67 Methacholine 2.83 + .32 6.51 & 1.04 89.20 zt 13.75

Values are means h SE. n = six animals in each group. The degranulated cells had ~7~yG of the control number of granules. Secretion was measured for the hour following the third metha- choline chloride injection. * Time-paired control group at 3 h, unstimulated.

contained only about 50 % of their fasting content of zymo- gen 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 gran- ules 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 se- cretory response to the final injection waned, the secretory

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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 metha- choline 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, chymotrypsino- gen, or total protein, this nonlinear relationship can be

0 bb' ' 0 120 130 140 150 160 170 180 190

Time (Minutes)

FIG. 2. LIethacholine chloride-augmented protein secretion in tis- sue 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 1 chloride l--l--i

0 I 2 3 4 5 6 Time (hours)

S. S. ROTHMAN

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 forma- tion of new granules during the administration of metha- choline 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 secre- tion collected from fasted, anesthetized rats in substantial amounts until a minimum of about 40-60 min after the in- jection of a radioactive amino acid (Fig. 6). Little difference was seen in this characteristic among rats after three sequen- tial 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.

DISCUSSION

Secretion of digestive enqme in absence of cymogen granules. AS

earlier (2, 7, 11, 28) as well as more recent studies (6) sug- gest, 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 gran- ules (16-l 9, 2 1, 22). However, in and of itself this observa- tion does not require such a mechanism, and continued aug- mented 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 re- cently been suggested (different tvpe (6), altered kinetics (8)). However, neither evidence nor circumstance supports these exocvtotic exnlanations.

FIG. 3. Effect of methacholine chlo- ride on percentage of cell volume oc- cupied by zymogen granules as deter- mined 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).

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SECRETION OF DIGESTIVE ENZYME 1831

FIG. 4. Micrographs of rat pancreatic tissue after an overnight fast (A), and after 3 injections of methacholine chlo- ride 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.

The emergence of a pool of zymogen granules or other ve- sicles 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 zymo- gen 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 what- ever variety, within the cells at the beginning of that hour and is, in all likelihood, less. A second potential kinetic al- teration, an increase in the enzyme concentration of in- dividual 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 con- tents 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

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1832 S. S. RQTHMAN

Chymotrypsinogen output, ymolas substrate split- min-hr(+)

0 40 80 120 160 200 240 280 320 360 I I I 1 1 1 I

100 r

b

I I 1 I I I I 1 I I I I I I I I I I I A

1 2 3 4 5 6 7 8 9 IO II 12 13 14 I5 16 17 I8 I9 20

Protein output, mglhr (@)

FIG. 5. Relationship between degranulation and chymotrypsinogen, trypsinogen, and protein outputs. Points displayed are taken from data

7r ?

0 IO 2’0 30 40 50 60 70 80 Time (Minutes)

FIG. 6. Appearance of labeled protein in secretion after intravenous injection of labeled amino acids. Open circles are values from un- treated rats (n = 3). Closed circles are values from rats given 3 injec- tions 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 concen- tration. 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.

Totat protein output in response to 3 sequential MCh injections

Non-zymogen granule intracellular pools (38.7 %)

in Figs. 2 and 6. Equations for curves are of form y = a@. A break- down of intracellular sources is shown only for protein secretion.

There is no indication at present that this small vesicle, or any other vesicle for that matter, is present in sufficient num- ber 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 move- ment (secretion) of their contents from an intracellular com- partment (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. As such, exocytosis is a “mass-transport” process, in which matter is moved without regard to the nature of the trans- ported 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 sub- stantially 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 con- ditions 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 in- jections, 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 re- lationship can be expIained only by the interposition of a

third compartment, presumably the cytoplasm, in the se-

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SECRETION OF DIGESTIVE ENZYME 1833

cretory pathway between the zymogen granule and duct lumen. That is, one must postulate, a seemingly unlikely, parallel, nonzymogen granule-derived process that secretes digestive enzymes in increasing absolute amounts in the face of a decreasing overall output.

A three-compartment model for the secretion of zymogen granule contents is also supported by the following inde- pendent observations : I) the existence of reversible enzyme <equilibria across apparently intact membranes of isolated zymogen granules (9) and the related observation that in- dividual granules display partial and enzyme-selective emp- tying at the steady state in vitro (21), 2) the relatively rapid (within 1 h) and molecularly specific establishment of an equilibrium between digestive enzyme (labeled) in the me- #dium bathing slices of pancreatic tissue and the enzyme con- tent of both cytoplasm and zymogen granules (lo), and 3) competitive inhibition of the secretion of new (labeled) protein by the enhanced secretion of older protein, probably primarily of zymogen granule origin, which is elicited by cholinergic stimulation (22).

Mechanism of en<~rne secretion 63’ acinar cell. While continued enzyme secretion in the absence of zymogen granules and the nonlinear, depletion-secretion relationship may be ex- plained by multiple processes, exocytotic or nonexocytotic, these and many other observations that are discordant with a simple exocytosis model for secretion can be explained simply by hypothesizing that the movement of enzyme across the cell membrane into the duct system is from the cyto- plasm, and that such movement is a final common step in the secretion of digestive enzyme derived from a variety of intracellular pools.

Since Heidenhain’s time, many different kinds of evidence have indicated that digestive enzymes are stored within the pancreatic secretion or zymogen granule. However, evidence that these enzymes leave the cell directly from these granules as a result of a process in which granule membrane and cell

REFERENCES

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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 mem- brane-membrane fusion itself but apparent geometric se- quelas to such an event. While this transport mechanism may well exist and certainly has great appeal due to its di- rectness, 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 di- gestive 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 and Ms. Lois Isenman for their excellent technical assistance.

The microscopic studies were done in collaboration with Dr. Susumo Ito of the Department of Anatomy, Harvard Medical School.

This work was supported by National Institutes of Health Grants

AM15672, AM10455, and AM16990.

Received for publication 10 April 1974.

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