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The mechanism of enzyme secretion by the cell
A R C H I V E S OF B I O C H E M I S T R Y AND B I O P H Y S I C S
104, 58-66 (1964)
The Mechanism of Enzyme Secretion by the Cell II. Secretion of Amylase and Other Proteins by Slices of Rat Parotid Gland 1' 2 AVNER
B D O L A H , ~ R U T H B E N - Z V I 3 AND M I C H A E L
SCHRAMM
From the Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel Received J u n e 3, 1963 R a t p a r o t i d gland slices in K r e b s - R i n g e r b i c a r b o n a t e m e d i u m secrete amylase and D N a s e a t a n e n h a n c e d r a t e in response to epinephrine. A high p o t a s s i u m concentration (60 mM) effectively replaced epinephrine as a s t i m u l a n t of secretion. T h e process was d e p e n d e n t on oxygen, was i n h i b i t e d b y D N P a n d cyanide, b u t not b y inhibitors of glycolysis. W h e n epinephrine was t h e s t i m u l a n t , secretion was also d e p e n d e n t on a minimal c o n c e n t r a t i o n of potassium (5 mM). Slices lost 90% of t h e i r p o t a s s i u m cont e n t in 75 m i n u t e s when i n c u b a t e d in a medium free of t h i s ion. Once e x h a u s t e d of p o t a s s i u m t h e slices no longer showed e n h a n c e d enzyme secretion w h e n e p i n e p h r i n e was added. O t h e r ions of the K r e b s - R i n g e r b i c a r b o n a t e m e d i u m did not a p p e a r essential for protein secretion. Amylase was secreted at a c o n s t a n t specific a c t i v i t y t h r o u g h o u t a n i n c u b a t i o n of 3 hours when 78% of t h e enzyme a n d 53% of the t o t a l p r o t e i n of t h e slice appeared in the medium. D u r i n g early periods of i n c u b a t i o n t h e a m o u n t of enzyme secreted into the m e d i u m was e q u i v a l e n t to the a m o u n t lost from t h e intracellular zymogen granules. Loss of amylase from t h e soluble s u p e r n a t a n t f r a c t i o n of t h e slice was observed only after t h e zymogen granules h a d released a b o u t 80% of t h e i r initial amylase content. The n u m b e r of granules appeared to r e m a i n almost c o n s t a n t during secretion a l t h o u g h t h e i r size a n d c o n t r a s t was changed. I t is therefore t e n t a t i v e l y concluded t h a t only t h e c o n t e n t s of t h e granules are t r a n s p o r t e d out of t h e cell. INTRODUCTION
thesis and its secretion beyond the cell membrane. It appeared likely that gland slices in a defined medium would serve well for the investigation of these processes. Hokin and his collaborators had already demonstrated that secretion by slices of various exocrine glands could be enhanced by specific agents which are known to be effective in vivo (4-6). In the present work factors governing enzyme secretion by the parotid gland slice in correlation to changes in the intracellular distribution of the enzyme are described. Some of the observations were recorded in a short note (7).
Previous studies had established that rat parotid gland produces, stores, and secretes large amounts of a-amylase (1-3). It was also shown that the enzyme synthesized in the ribosomes is rapidly released from these structures and accumulates in the membrane-bound zymogen granules and in a soluble fraction (3). As yet there is little information concerning the mechanism of enzyme transport from the site of syni This work was s u p p o r t e d b y grants from the N a t i o n a l Science F o u n d a t i o n (G-22153) a n d the N a t i o n a l I n s t i t u t e s of H e a l t h , U n i t e d States Public H e a l t h Service (AI. 03426-03). 2 P a r t I has been published in Biochim. Biophys. Acta 50, 102 (1961). 3 E x p e r i m e n t s reported in this c o m m u n i c a t i o n are p a r t of a thesis to be s u b m i t t e d in partial fulfilment of the requirements for the P h . D . degree.
MATERIALS AND METHODS ENZYME ACTIVITY Amylase was assayed according to the m e t h o d used b y Bernfeld (8), enzyme units and specific a c t i v i t y being defined as n o t e d in a previous com58
E N Z Y M E S E C R E T I O N B Y SLICES OF P A R O T I D G L A N D m u n i c a t i o n (2). Deoxyribonuclease was assayed b y measuring t h e f o r m a t i o n of acid soluble deoxynucleotides with the diphenylamine reagent (9). The r a t p a r o t i d gland enzyme was found to be Mg ++ dependent. I n c u b a t i o n was carried out at 30~ in 0.07 M p h o s p h a t e buffer, p H 6.5, in the presence of 0.017 M MgSO4. A u n i t of D N a s e is defined as t h a t a m o u n t t h a t converts I rag. equivalent of D N A to acid soluble deoxynucleotides in 20 minutes. Specific a c t i v i t y is expressed as units per milligram protein. ANALYTICAL ~/~ETHOD S P r o t e i n was d e t e r m i n e d according to t h e m e t h o d of Lowry et al. (10). P o t a s s i u m was determined b y flame p h o t o m e t r y . The s t a n d a r d s also c o n t a i n e d N a + in amounts roughly equivalent to those present in t h e samples to be determined. Since t h e slices swell during p r e p a r a t i o n and incubation, it was decided to express t h e relative a m o u n t of K + on t h e basis of initial protein r a t h e r t h a n wet weight. I n i t i a l p r o t e i n was calculated from the a m o u n t s secreted plus t h a t remaining finally in the slice. Values are given as seq. K + per milligram initial protein. PREPARATION OF SLICES Glands were removed from s t a r v e d a d u l t rats as previously described (2) a n d k e p t on ice in the medium to be used for the experiment. E a c h gland of a b o u t 80 mg. fresh weight was cut with a scalpel on wet filter p a p e r into 10-20 slices. F o r each exp e r i m e n t slices were p r e p a r e d from 8 glands a t least a n d were all pooled. A b o u t 300 mg. of slices were t r a n s f e r r e d to a 50-ml. E r l e n m e y e r flask conraining 10 ml. of cold medium. This ratio of slices to volume of medium a n d to t h e size of the gas phase was kept c o n s t a n t when larger a m o u n t s of tissue were i n c u b a t e d in a single flask. T h e flask was gassed for 5 minutes w i t h a suitable gas mixture and stoppered. INCUBATION AND PREPARATION FOR ANALYSES Slices were p r e i n c u b a t e d for 15 minutes at 37~ with shaking (200 strokes per minute). I t was found t h a t d u r i n g this period the specific a c t i v i t y of amylase secreted was lower t h a n t h a t of t h e homogenate of t h e slice. T h e p r e i n c u b a t i o n medium was therefore removed b y d e c a n t a t i o n or filtration on filter paper and discarded unless otherwise specified. The slices were t r a n s f e r r e d to flasks containing the a p p r o p r i a t e cold experim e n t a l mixtures. After gassing, t h e flasks were i n c u b a t e d for various time periods u n d e r the conditions specified above. At t h e end of the experim e n t the medium was immediately s e p a r a t e d from
59
the slices a n d k e p t for analysis. The slices were homogenized (2) in 0.02 M p h o s p h a t e buffer, p H 6.9, when only amylase and protein were m e a s u r e d , in distilled water when potassium assays were also. required, a n d in 0.25 M sucrose when it was int e n d e d to isolate subcellular fractions b y differential centrifugation. MEDIA FOR INCUBATION K r e b s - R i n g e r b i c a r b o n a t e ( K R B ) m e d i u m (11) and K r e b s medium I (12) were used with a gas phase of 5% CO2, 95% Oz. K r e b s - R i n g e r p h o s p h a t e (11) was used w i t h oxygen as the gas phase since with air t h e secretion rate was m u c h lower. STIMULANTS OF SECRETION The epinephrine c o n c e n t r a t i o n used was 10-~ M. Increasing the c o n c e n t r a t i o n to 10-4 M did n o t produce a stronger effect within 60 minutes of i n c u b a t i o n . Since epinephrine was readily oxidized, a fresh a m o u n t (10-5 M) was added a f t e r each h o u r w h e n e v e r maximal secretion rates were required. F o r electric s t i m u l a t i o n , a c u r r e n t of square waves was applied to the medium in which t h e slices were freely floating (13). T h e frequency was 200 cycles per second with a c u r r e n t of 6 ma. a n d a peak voltage of 15 v. CALCULATION OF PER CENT SECRETION The a m o u n t of m a t e r i a l released into t h e m e d i u m during all phases of the experiment plus t h e a m o u n t finally remaining w i t h i n the slice was defined as 100%. T h e q u a n t i t y of material secreted during a n y time i n t e r v a l is thus expressed as p e r cent of total. I t was found t h a t t h e per cent of amylase and protein secreted b y the slice was indep e n d e n t , within a wide range, of the a b s o l u t e a m o u n t of slices per flask. Therefore t h e a m o u n t of slices placed in each flask was n o t accurately p r e d e t e r m i n e d b y weighing. The secretion r a t e as t e s t e d in duplicate experiments could be d e t e r mined w i t h a n over-all accuracy of 4-7%. ~V[ATERI&LS E p i n e p h r i n e chloride in ampules of 1/1000 was purchased from Hillel Co., Israel. C a r b a m y l choiin, eserin sMicylate, a n d pilocarpin hydrochloride were of U S P grade. Bovine pancreatic D N a s e was a 1X crystallized p r e p a r a t i o n from W o r t h i n g t o n Co. All o t h e r chemicals were of anMyticul grade. RESULTS When rats starved for 20 hours were fed and sacrificed after 30 minutes, the parofid
60
BDOLAH, BEN-ZVI AND SCHRAMM
glands showed a 30-80% loss of amylase and a 20-60 % loss of protein as compared to the unfed control group. Iniection of pilocarpin (about 2.5 mg./100 g.) to the starved animals caused a similar rate of secretion (cf. refs. 1 and 3). The slice system as demonstrated in the present work attained secretion rates of the same order of magnitude as those observed in the intact animal. EFFECT OF SECRETION STIMULANTS AND METABOLIC INHIBITORS Various a g e n t s k n o w n to s t i m u l a t e secretion in vivo were tried i n the slice s y s t e m i n order to choose one which would c o n s i s t e n t l y cause high rates of e n z y m e TABLE I STIMULATORY E F F E C T OF VARIOUS AGENTS ON AMYLASE SECRETION BY THE SLICE a
Expt.
II
III
IV
Stimulant added
Specific activity Amylase of amylase secreted (units/rag. protein) (% of total) in in slices medium
None Carbamylcholin Epinephrine
10 19 40
550 540 420
630 670 750
None Carbamylcholin Epinephrine Carbamylcholin + epinephrine
16 22 37
530 470 430
760 700 770
33
450
780
None Eserin Carbamylcholin Pilocarpin Pitocarpin + earbamylcholin
12 21 20 21
400 400 440 420
500 670 740 700
22
440
750
16 28 35
450 390 390
630 680 680
41
360
690
None Electric pulses Epinephrine Epinephrine -lelectric pulses
secretion. T a b l e I shows t h a t all a g e n t s tested, i n c l u d i n g electric pulses, increased the secretion rate above t h a t of the control. T h e specific a c t i v i t y of a m y l a s e secreted was always higher t h a n t h a t of the e n z y m e r e m a i n i n g w i t h i n the slice, i n d i c a t i n g specificity i n p r o t e i n secretion. T h e effect of a c o m b i n a t i o n of two s t i m u l a n t s was n o t a d d i t i v e a n d was u s u a l l y n o t m u c h higher t h a n t h a t of the more p o t e n t s t i m u l a n t applied alone. E p i n e p h r i n e c o n s i s t e n t l y showed the highest activity. I n presence of this a g e n t the rate of secretion was u s u a l l y more t h a n twice as high as t h a t of the control i n spite of the fact t h a t the a b s o l u t e rate varied ( T a b l e I). E p i n e p h r i n e has therefore b e e n r o u t i n e l y used i n all subs e q u e n t experiments. I n a d d i t i o n to a m y l a s e the slice also secreted D N a s e . T h e l a t t e r e n z y m e h a d a subcellular d i s t r i b u t i o n a l m o s t identical to t h a t of amylase, large a m o u n t s b e i n g
Slices containing on the average 4000 amylase units per flask were incubated for 60 minutes. The medium was Krebs medium I in expt. I and KRB in expts. II-IV. Pilocarpin was 4 X 10-4 M; other stimulants were 10-5 M.
TABLE II E F F E C T OF ~NHIBITORS AND LACK OF OXYGEN ON D N A s E AND AMYLASE SECRETION a
Expt.
II
Additions to medium
Enzyme secreted (% of total) Amylase
DNase
None Epinephrine Epinephrine, iodoacetate b Epinephrine, KCN Epinephrine, DNP Epinephrine, N~c
16 33
18 34
33 16 12 17
-13 11 15
None n-Ethylmaleimide Epinephrine Epinephrine, n-ethylmaleimide Epinephrine, DNP
22 20 50
----
50 25
---
" Slices containing about 4000 amylase units and 700 DNase units per flask were incubated for 60 minutes. Krebs medium I was used in expt. I and KRB medium in expt. II. The concentration of inhibitors was 10-3 M. b Slices were kept in the cold in presence of the inhibitor for 30 minutes prior to incubation at 37~ c The gas phase was 95% N2,5% CO2.
ENZYME SECRETION BY SLICES OF PAROTID GLAND located in the zymogen granules. The specific activity of the D N a s e secreted was in the same range as t h a t of the zymogen granule fraction, about 120 units per milligram protein. A commercial preparation of crystalline bovine pancreatic DNase, assayed under the same conditions, had a specific activity of 750. Evidently, rat parotid gland contains and secretes considerable amounts of D N a s e together with amylase. Table I I shows t h a t the enhanced rate of D N a s e and amylase secretion caused b y epinephrine was absolutely dependent on oxygen. Cyanide and 2,4-dinitrophenol brought the enhanced secretion down to the level obtained in absence of epinephrine while inhibitors of glycolysis were without effect. The factors responsible for the basal rate of secretion or leakage in absence of epinephrine have not been identified. EFFECTS OF MEDIUM COMPONENTS The substrates present in Krebs medium I (ghlcose, pyruvate, fumarate, and glutamate) and each one of these tested separately were without effect on the secretion rate for the first 60 minutes and were therefore omitted in experiments of short duration. Insulin (3 gg. per milliliter) added with glucose also had no effect on the secretion rate. The slice apparently contains sufficient amounts of endogenous substrate. When the requirements for the inorganic components of the K R B medium were investigated, it was found t h a t Ca ++ , Mg ++, and inorganic phosphate could be omitted without causing a marked drop in the rate of amylase secretion. Bicarbonate and CO2 appeared also not to be required since Krebs-Ringer phosphate could replace the K R B medium. A potent inhibitor of carbonic anhydrase, acetazolamide (10 -4 M), was without effect on the secretion of amylase. The secretion rate was, however, strikingly dependent on potassium. I t had already been demonstrated t h a t in the absence of this ion, epinephrine stimulated secretion only to a slight extent (7). These findings prompted a study of the potassium content of the slice and its effect on secretion. I t
61
was found t h a t the fresh gland contained 0.3-0.6 /~eq. K + per milligram protein (40-70 meq. K + per kilogram fresh weight). During preparation of slices (15 minutes) in cold K R B , some K + leaks out and the ratio drops to 0.2-0.3 geq. K + per milligram initial protein. This value then remained fairly constant when slices were incubated a t 37~ in the above medium, with or without epinephrine or even when storage in the cold was continued for another 30 minutes. However, the slices were almost completely depleted of K + when incubated in a K+-free medium (Table I I I ) . Such depleted preparations released protein at a somewhat higher rate t h a n normal slices and showed no response when epinephrine was added. Secretion could no longer be effectively enhanced b y epinephrine even when the depleted slices were again incubated in presence of K + (Table IV). Control over secretion is apparently lost only when the slice is almost exhausted of K +. A decrease of K + to the level of 0.1 ~eq. per milligram initial protein caused b y Ouabain (10-s M) in K R B medium did not affect the ability of the slices to respond to epinephrine. Another effect of K + became apparent when media containing high concentrations of this ion were tested. Raising the K + TABLE I I I R E L E A S E OF POTASSIUM FROM SLICES INCUBATED IN A P O T A S S I U M - F R E E M E D I U M a Time (rain.)
K + released into medium (% of total)
K + remaining in slice (#eq./mg. initial protein)
0 15 45 75
0 47 72 91
0.23 0.13 0.06 0.04
Slices were prepared in KRB and softly wiped on filter paper. A sample was removed for analysis of K + and protein content at zero time. The remaining slices were directly transferred into art incubation vessel at 37~ containing a KRB medium from which K + was omitted. At times indicated in the table a sample of slices was removed for analysis and the remainder transferred to fresh K+-free medium. K + released was measured on aliquots of incubation medium.
62
BDOLAH, B E N - Z V I A N D S C H R A M M T A B L E IX: EFFECT OF POTASSIUM D E P L E T I O N ON THE SECRETION OF AMYLASEa K+ in incubation medium
Slice preparation
Preincubated without K +
0 min.c
30 rain.
-+ +
-+ -+
29 31 25 31
<0.05 <0.05 <0.05 <0.05
<0.05 <0.05 0.15 0.13
+ +
-+
19 40
0.27 0.27
0.27 0.24
-
P r e i n c u b a t e d with K +
K+ (~q./mg. initia[ protein)
Amylase secreted (% of total) b
Epinephrine
-
a I n c u b a t i o n media were with or w i t h o u t K +, other components being those of K R B medium. To d e p l e t e slices of K +, these were p r e i n c u b a t e d for 75 minutes w i t h o u t K +, during which time 36% of t h e amylase was secreted. The control slices were preincubated for 75 minutes with K + and secreted 30% of t h e i r amylase content. b The amylase remaining in the slice at the end of the preincubation period is t a k e n as 100%. A f t e r preincubation. TABLE V ]?OT&SSIUM AT HIGH CONCENTRATION AS A STIMULANT OF AMYLASE
~7o
SECRETION a
Expt.
Specific activity Amylase of amylase secreted (units/mg. protein) (% of total) in in slices medium
Additions
[K+] (mM)
Epinephrine
5 16 59 117 5
22 20 47 46 49
360 400 310 300 280
620 580 700 730 670
9 24 40 59 140
25 27 38 62 50
390 350 320 230 310
760 750 770 870 870
5 140 140
16 35 13
390 340 400
680 810 550
II
III DNP
8O
a Slices were incubated for 60 minutes. The medium was K R B in which K + replaced Na + at c o n c e n t r a t i o n s shown in the Table. D N P was 10-3 M.
concentration to 60 m M or above enhanced the secretion rate to a level equal to that achieved by epinephrine in the normal K R B medium (Table V). Concentrations in the
6o
o 50
~30 "-
20
.o'"
//
4j
..o. . . . . . . . .
.... 9
~0 1
2
3
Time (hr,) F l a . i . K i n e t i c s of amylase and protein secretion during a 3-hour incubation period. The medium was K R B which also contained 10 m M pyruvate. Slices were transferred to fresh medium, with or without epinephrine, after each hour. The used media were kept for analysis. - - , amylase secreted; . . . . , protein secreted. O, with epinephrine; O , without epinephrine.
range of 20-60 m M had a partial effect. When epinephrine was added to media containing a high K + concentration no further increase of the secretion rate occurred (7). Thus a K + concentration of 60 m M acted as a stimulant of secretion equal in potency to epinephrine. The enhancement of secretion by K + appeared
63
E N Z Y M E S E C R E T I O N B Y SLICES OF P A R O T I D G L A N D
to be due to a true metabolic effect since it was abolished by D N P (Table V). While respiration in brain slices is stimulated by a high K + / C a ++ ratio (14), the effect of K + on secretion as described above appeared to be due to the absolute K + concentration. When the Ca ++ Concentration was raised from 2.5 to 20 m M in presence of 60 m M K +, the secretion rate remained undiminished. Ammonium ion which can replace K + in the transport system of the erythrocyte (15) could not support enzyme secretion by the parotis slice at concentrations equivalent to K +. Enhanced secretion was not observed when the medium contained 60 m M NH4 + instead of K +. In addition, slices which were slightly depleted of K + by a 15-minute incubation in the absence of this ion did not respond to epinephrine when subsequently incubated in a medium in which 5 m M NH4 + replaced K +. Such depicted slices responded normally to the stimulant when 5 m M K + was present.
T A B L E VI ZYMOGEN G R A N U L E S AS THE SOURCE OF AMYLASE FOR SECRETION a
Relative distribution oI amylase (%) In slices
Time (min.)
0 30 60 120
Zymogen granules
Supernatant
In extracellular medium
70 30 14 11
30 33 32 27
0 42 58 63
a T h r e e flasks c o n t a i n i n g slices (about 18,000 amylase u n i t s per flask) in K R B m e d i u m w i t h e p i n e p h r i n e were i n c u b a t e d as described u n d e r Materials and Methods. E p i n e p h r i n e was not replenished d u r i n g the experiment. A t each t i m e period one flask was removed for analysis. T h e homogenate of the slices was subjected to centrifugation a t 2100g for 10 minutes to yield a crude zymogen granule f r a c t i o n a n d a s u p e r n a t a n t fraction. T h e relative a m o u n t s of amylase in these subcellular fractions a n d in the extracellular m e d i u m are shown in the Table.
TABLE VII R E L A T I V E CONSTANCY OF THE N U M B E R OF ZYMOGEN G R A N U L E S D U R I N G SECRETION OF T H E I R AMYLASE CONTENT a
Expt.
Time (rain.)
Number X 101~
I II lII
0 180 0 120 0 30
"Membrane" protein
Granules
3.8 3.3 5.5 3.3 5.6 5.1
Amylase in granules
~ob
rag.
%b
100 87 100 60 100 91
3.3 3.7 4.3 4.0 2.4 2.4
100 112 100 93 100 100
Units
17,000 2600 15,100 2500 10,100 2500
~ob
100 15 100 17 100 25
Slices from 12 glands of 6 s t a r v e d r a t s served for each experiment. A t zero time, before p r e i n c u b a tion, half of the slices were homogenized in 0.25 M sucrose. The fraction s e d i m e n t e d b y 5-minute centrifugation a t 250 g was washed once. T h e p r e c i p i t a t e was discarded a n d t h e s u p e r n a t a n t was added to t h e rest of t h e homogenate. T h e zymogen granule f r a c t i o n was t h e n isolated b y e e n t r i f u g a t i o n a t 2100 g for 10 minutes. Zymogen granule " m e m b r a n e s " were p r e p a r e d b y h y p o t o n i c shock as previously described (2). The remaining slices were i n c u b a t e d w i t h epinephrine a f t e r 15 m i n u t e s p r e i n c u b a t i o n . To s u b s t r a t e was added to t h e K R B medium. A t t h e end of t h e i n c u b a t i o n period t h e slices were reved from t h e medium, homogenized, a n d f r a c t i o n a t e d as described for t h e sample at zero time. T h e t f e n t s of t o t a l amylase secreted into t h e m e d i u m were 58, 61, a n d 48 for expts. I, I I , a n d I I I , respeeZ I n expt. I e p i n e p h r i n e was n o t replenished d u r i n g i n c u b a t i o n . micr3,gen granules were counted in t h e conventional blood c o u n t c h a m b e r with a phase contras~ p e r ali~ e- Two to four aliquots of each sample were examined. A t least 500 granules were c o u n t e d ~8%. ~. T h e n u m b e r calculated from different aliquots of t h e same sample varied in t h e range of b Values ,
zero time are a r b i t r a r i l y defined as 100~.
64
BDOLAH, BEN-ZVI AND SCHRAMM
KINETICS OF AMYLASE SECRETION AND CHANGES IN THE INTRACELLULAR DISTRIBUTION O f THE ENZYME
When slices were incubated in the presence of epinephrine, 78% of the total amylase and 53 % of the total protein were secreted during 3 hours (Fig. 1). The specific activity of the amylase released into the medium remained nearly constant (about 800 units per milligram protein) throughout the incubation period while that of the slice decreased from 520 at zero time to 260 at the end of 3 hours. Figure 1 also shows that secretion in presence of epinephrine followed roughly first order kinetics. The secretion rate of the control without stimulant was much lower and declined further more rapidly than the experimental system. Incubation in presence of stimulant could often be prolonged up to 5 hours resulting in the secretion of 90% of the amylase content of the slice without a noticeable drop in the specific activity of the enzyme secreted. A study of the intracellular distribution of the enzyme during secretion revealed a precipitous drop in amylase content of the zymogen granules equivalent to the amount of enzyme secreted into the extracellular medium (Table VI). Only after the zymogen granules lost about 80% of their amylase content did the supernatant fraction show a loss of this enzyme. The extreme loss of amylase from the zymogen granule fraction during secretion was not accompanied by a parallel decrease in the absolute number of granules. When the granule fraction retained only 15-25 % of its initial amylase content, 60-90% of the granules were still present (Table VII). The granules after secretion appeared much smaller and less dense, especially in experiments of long duration (Expts. I and II). Difficulties in detecting all the granules after secretion may account for the apparent decrease in their number. It was indeed found that the water insoluble "membrane" protein of the granule fraction remained unchanged during secretion (Table VII).
DISCUSSION The rat parotid gland slice described in the present work demonstrates the major features of the secreting gland as it operates in the living animal. Physiological stimulants such as epinephrine cause an enhanced and relatively specific secretion of amylase and other proteins at a rate comparable to that observed i n vivo. Energy generated by oxidative processes seems to be the driving force in the transport of these proteins in the cell and across the cell border. Indeed, oxidation is probably a limiting factor since secretion by the slice in presence of oxygen proceeded at a higher rate than in presence of air. The striking acceleration of amylase secretion by high potassium concentrations in the medium might also be ascribed to the stimulating effect of this ion on oxidative energy yielding processes (14, 16). Whether the inability of potassium depleted slices to respond to epinephrine is due to a defect in the generation of energy cannot as yet be decided. It should, however, be noted that the failure to show enhanced secretion occurs only after the slices are almost completely exhausted of their internal potassium by incubation in a potassium-free medium. Other tissues rarely show such extreme and rapid losses of this ion. However, the parotid gland is known to secrete potassium (17), and it is therefore possible that the slice continues to secrete this ion even when it is unable to regain it from the medium because of extreme dilution. The slice system was found to maintain specific secretion up to a stage when it is almost exhausted of secretory proteins. Changes in the subcellular structures of the cell and in the distribution of secretory proteins as they occur with the progress of secretion may therefore be readily detected and measured. During the first phases secretion, the zymogen granules are sh~ to lose their amylase content rapidly :n~ the amount of enzyme in the supe~ant" fraction of the gland remains cr n . ~ Preliminary experiments indicate ~e~::o f ing the first minutes a transient i .-~ amylase in the supernatant e~orapan~r
ENZYME SECRETION BY SLICES OF PAROTID GLAND loss of the enzyme from the zymogen granules. These findings could mean that the enzymes which are released by the granules pass through the supernatant pool into the medium. The supernatant fraction might thus represent to a large extent the enzymes which have already been transported beyond the cell membrane into the lumen and ducts of the gland. I t is indeed hard to visualize that the extracellular space of the gland would ever be free of secreted proteins. Although the supernatant fraction would also contain amylase released from zymogen granules and other cell structures damaged during fractionation, it is quite possible that some part of it is indeed true soluble intracellular amylase. While the zymogen granules secrete their protein contents, the membranes of these structures apparently remain inside the cell. The evidence on which this conclusion is based is mainly that the number of granules changed only slightly dulfing the period when most of the amylase and other proteins of the granule fraction were secreted. The fraction counted is not homogeneous and probably contains mitochondria and some nuclei. I t is also not known whether new granules could have been formed during the period of the experiment. These reservations apply equally to the findings that the total "'membrane" protein of the granule fraction remained constant during secretion. It should be observed that the amount of "membrane" protein per granule varies by a factor of about 2.5 between the different experiments presented in Table VII. T h e cause of this variation is not clear. With the above noted reservations in mind, it is quite probable that the membranes of the zymogen granules remain inside the cell and may serve repeatedly in the secretion process. Classic histological studies indicated that the granules disappear from the cell during secretion (18). It is however quite possible that the empty granules were not detected on the background of the whole cell section because of their smaller size and lack of contrast. Keller and Cohen (19) and also Greene (20) have demonstrated that the digestive
65
enzymes appear in the pancreatic juice in the same proportions as found in the intracellular zymogen granules. The present work shows that the parotid gland slice secretes DNase and amylase into the medium in the same relative amounts as found within the slice. Furthermore, it is shown that the ratio of amylase to other proteins secreted remains constant up to the stage when the slice is almost depleted of amylase. I t therefore seems likely that the different proteins secreted derive from a common pool and that the gland has no means of withholding one digestive enzyme while secreting others. ACKNOWLEDGMENT The experiments were carried out with the most valuable assistance of Mrs. Sarah Eldar. REFERENCES 1. SCI-INEYER,L. H., AND SCttNEYER, C. A., Ann. N. Y. Acad. Sci. 85, 189 (1960). 2. SCttRAMM, M., AND DANON, D., Biochim. Biophys. Acta 50, 102 (1961). 3. GROMET-ELHANAN, Z., AND WINNICK, T., Biochim. Biophys. Acta 85, 69 (1963). 4. HOKIN, L. E., Biochem. J. 48, 320 (1951). 5. HOKIN, L. E., AND SHERWIN,A. L., J. Physiol. London 135, 18 (1957). 6. EGGMAN, L. D., AND HOKIN, L. E., J. Biol. Chem. 235, 2569 (1960). 7. BDOLAn, A., AND SCHRAMM, M., Biochem. Biophys. Res. Communs. 8, 266 (1962). 8. BERNFELD,P., in "Methods in Enzymology"
(S. P. Colowick and N. O. Kaplan, eds.), Vol. 1, p. 149. Academic Press, New York, 1955. 9. McDoNALD, M. R., in "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.), Vol. 2, p. 437. Academic Press, New York, 1955. I0. Lowaz, O. H., ROSEBROUGH, N. J., FARR,
A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 11. COHEN, P. P., in "Manometric Methods" (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.) 3rd ed., p. 149. Burgess Publishing Co., Minneapolis, 1957. 12. KREBS, H. A., Biochim. Biophys. Acta 4, 249 (195o). 13. MCILWAIN,H., Biochem. J. 50, 132 (1952).
66
BDOLAH, BEN-ZVI AND SCHRAMM
14. USSING, H. H., in "The Alkali Metal Ions in Biology, Handbuch der experimentellen Pharmakologie" (O. Eiehler and A. Farah, eds.), Vol. 13, p. 24. Springer Verlag, Berlin, 1960. 15. POST, R. L., AND JOLLY, P. C., Biochim. Biophys. Acta 25, 118 (1957). 16. HEALD, P. J., Biochem. J. 57,673 (1954). 17. BURGEN, A. S. V., AND EMMELIN, N. G.,
"Physiology of the Salivary Glands," p. 148. Edward Arnold Ltd., London, 1961. 18. BABKIN, B. P., "Secretory Mechanism of the Digestive Glands," 2nd ed., p. 23. Harper (Hoeber), New York, 1950. 19. KELLER, P. J., AND COHEN, E., J. Biol. Chem. 236, 1407 (1961). 20. GREENE, L. J., HIRS, C. H. W., AND P&LADE, G. E., J. Biol. Chem. 238, 2054 (1963).
104, 58-66 (1964)
The Mechanism of Enzyme Secretion by the Cell II. Secretion of Amylase and Other Proteins by Slices of Rat Parotid Gland 1' 2 AVNER
B D O L A H , ~ R U T H B E N - Z V I 3 AND M I C H A E L
SCHRAMM
From the Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel Received J u n e 3, 1963 R a t p a r o t i d gland slices in K r e b s - R i n g e r b i c a r b o n a t e m e d i u m secrete amylase and D N a s e a t a n e n h a n c e d r a t e in response to epinephrine. A high p o t a s s i u m concentration (60 mM) effectively replaced epinephrine as a s t i m u l a n t of secretion. T h e process was d e p e n d e n t on oxygen, was i n h i b i t e d b y D N P a n d cyanide, b u t not b y inhibitors of glycolysis. W h e n epinephrine was t h e s t i m u l a n t , secretion was also d e p e n d e n t on a minimal c o n c e n t r a t i o n of potassium (5 mM). Slices lost 90% of t h e i r p o t a s s i u m cont e n t in 75 m i n u t e s when i n c u b a t e d in a medium free of t h i s ion. Once e x h a u s t e d of p o t a s s i u m t h e slices no longer showed e n h a n c e d enzyme secretion w h e n e p i n e p h r i n e was added. O t h e r ions of the K r e b s - R i n g e r b i c a r b o n a t e m e d i u m did not a p p e a r essential for protein secretion. Amylase was secreted at a c o n s t a n t specific a c t i v i t y t h r o u g h o u t a n i n c u b a t i o n of 3 hours when 78% of t h e enzyme a n d 53% of the t o t a l p r o t e i n of t h e slice appeared in the medium. D u r i n g early periods of i n c u b a t i o n t h e a m o u n t of enzyme secreted into the m e d i u m was e q u i v a l e n t to the a m o u n t lost from t h e intracellular zymogen granules. Loss of amylase from t h e soluble s u p e r n a t a n t f r a c t i o n of t h e slice was observed only after t h e zymogen granules h a d released a b o u t 80% of t h e i r initial amylase content. The n u m b e r of granules appeared to r e m a i n almost c o n s t a n t during secretion a l t h o u g h t h e i r size a n d c o n t r a s t was changed. I t is therefore t e n t a t i v e l y concluded t h a t only t h e c o n t e n t s of t h e granules are t r a n s p o r t e d out of t h e cell. INTRODUCTION
thesis and its secretion beyond the cell membrane. It appeared likely that gland slices in a defined medium would serve well for the investigation of these processes. Hokin and his collaborators had already demonstrated that secretion by slices of various exocrine glands could be enhanced by specific agents which are known to be effective in vivo (4-6). In the present work factors governing enzyme secretion by the parotid gland slice in correlation to changes in the intracellular distribution of the enzyme are described. Some of the observations were recorded in a short note (7).
Previous studies had established that rat parotid gland produces, stores, and secretes large amounts of a-amylase (1-3). It was also shown that the enzyme synthesized in the ribosomes is rapidly released from these structures and accumulates in the membrane-bound zymogen granules and in a soluble fraction (3). As yet there is little information concerning the mechanism of enzyme transport from the site of syni This work was s u p p o r t e d b y grants from the N a t i o n a l Science F o u n d a t i o n (G-22153) a n d the N a t i o n a l I n s t i t u t e s of H e a l t h , U n i t e d States Public H e a l t h Service (AI. 03426-03). 2 P a r t I has been published in Biochim. Biophys. Acta 50, 102 (1961). 3 E x p e r i m e n t s reported in this c o m m u n i c a t i o n are p a r t of a thesis to be s u b m i t t e d in partial fulfilment of the requirements for the P h . D . degree.
MATERIALS AND METHODS ENZYME ACTIVITY Amylase was assayed according to the m e t h o d used b y Bernfeld (8), enzyme units and specific a c t i v i t y being defined as n o t e d in a previous com58
E N Z Y M E S E C R E T I O N B Y SLICES OF P A R O T I D G L A N D m u n i c a t i o n (2). Deoxyribonuclease was assayed b y measuring t h e f o r m a t i o n of acid soluble deoxynucleotides with the diphenylamine reagent (9). The r a t p a r o t i d gland enzyme was found to be Mg ++ dependent. I n c u b a t i o n was carried out at 30~ in 0.07 M p h o s p h a t e buffer, p H 6.5, in the presence of 0.017 M MgSO4. A u n i t of D N a s e is defined as t h a t a m o u n t t h a t converts I rag. equivalent of D N A to acid soluble deoxynucleotides in 20 minutes. Specific a c t i v i t y is expressed as units per milligram protein. ANALYTICAL ~/~ETHOD S P r o t e i n was d e t e r m i n e d according to t h e m e t h o d of Lowry et al. (10). P o t a s s i u m was determined b y flame p h o t o m e t r y . The s t a n d a r d s also c o n t a i n e d N a + in amounts roughly equivalent to those present in t h e samples to be determined. Since t h e slices swell during p r e p a r a t i o n and incubation, it was decided to express t h e relative a m o u n t of K + on t h e basis of initial protein r a t h e r t h a n wet weight. I n i t i a l p r o t e i n was calculated from the a m o u n t s secreted plus t h a t remaining finally in the slice. Values are given as seq. K + per milligram initial protein. PREPARATION OF SLICES Glands were removed from s t a r v e d a d u l t rats as previously described (2) a n d k e p t on ice in the medium to be used for the experiment. E a c h gland of a b o u t 80 mg. fresh weight was cut with a scalpel on wet filter p a p e r into 10-20 slices. F o r each exp e r i m e n t slices were p r e p a r e d from 8 glands a t least a n d were all pooled. A b o u t 300 mg. of slices were t r a n s f e r r e d to a 50-ml. E r l e n m e y e r flask conraining 10 ml. of cold medium. This ratio of slices to volume of medium a n d to t h e size of the gas phase was kept c o n s t a n t when larger a m o u n t s of tissue were i n c u b a t e d in a single flask. T h e flask was gassed for 5 minutes w i t h a suitable gas mixture and stoppered. INCUBATION AND PREPARATION FOR ANALYSES Slices were p r e i n c u b a t e d for 15 minutes at 37~ with shaking (200 strokes per minute). I t was found t h a t d u r i n g this period the specific a c t i v i t y of amylase secreted was lower t h a n t h a t of t h e homogenate of t h e slice. T h e p r e i n c u b a t i o n medium was therefore removed b y d e c a n t a t i o n or filtration on filter paper and discarded unless otherwise specified. The slices were t r a n s f e r r e d to flasks containing the a p p r o p r i a t e cold experim e n t a l mixtures. After gassing, t h e flasks were i n c u b a t e d for various time periods u n d e r the conditions specified above. At t h e end of the experim e n t the medium was immediately s e p a r a t e d from
59
the slices a n d k e p t for analysis. The slices were homogenized (2) in 0.02 M p h o s p h a t e buffer, p H 6.9, when only amylase and protein were m e a s u r e d , in distilled water when potassium assays were also. required, a n d in 0.25 M sucrose when it was int e n d e d to isolate subcellular fractions b y differential centrifugation. MEDIA FOR INCUBATION K r e b s - R i n g e r b i c a r b o n a t e ( K R B ) m e d i u m (11) and K r e b s medium I (12) were used with a gas phase of 5% CO2, 95% Oz. K r e b s - R i n g e r p h o s p h a t e (11) was used w i t h oxygen as the gas phase since with air t h e secretion rate was m u c h lower. STIMULANTS OF SECRETION The epinephrine c o n c e n t r a t i o n used was 10-~ M. Increasing the c o n c e n t r a t i o n to 10-4 M did n o t produce a stronger effect within 60 minutes of i n c u b a t i o n . Since epinephrine was readily oxidized, a fresh a m o u n t (10-5 M) was added a f t e r each h o u r w h e n e v e r maximal secretion rates were required. F o r electric s t i m u l a t i o n , a c u r r e n t of square waves was applied to the medium in which t h e slices were freely floating (13). T h e frequency was 200 cycles per second with a c u r r e n t of 6 ma. a n d a peak voltage of 15 v. CALCULATION OF PER CENT SECRETION The a m o u n t of m a t e r i a l released into t h e m e d i u m during all phases of the experiment plus t h e a m o u n t finally remaining w i t h i n the slice was defined as 100%. T h e q u a n t i t y of material secreted during a n y time i n t e r v a l is thus expressed as p e r cent of total. I t was found t h a t t h e per cent of amylase and protein secreted b y the slice was indep e n d e n t , within a wide range, of the a b s o l u t e a m o u n t of slices per flask. Therefore t h e a m o u n t of slices placed in each flask was n o t accurately p r e d e t e r m i n e d b y weighing. The secretion r a t e as t e s t e d in duplicate experiments could be d e t e r mined w i t h a n over-all accuracy of 4-7%. ~V[ATERI&LS E p i n e p h r i n e chloride in ampules of 1/1000 was purchased from Hillel Co., Israel. C a r b a m y l choiin, eserin sMicylate, a n d pilocarpin hydrochloride were of U S P grade. Bovine pancreatic D N a s e was a 1X crystallized p r e p a r a t i o n from W o r t h i n g t o n Co. All o t h e r chemicals were of anMyticul grade. RESULTS When rats starved for 20 hours were fed and sacrificed after 30 minutes, the parofid
60
BDOLAH, BEN-ZVI AND SCHRAMM
glands showed a 30-80% loss of amylase and a 20-60 % loss of protein as compared to the unfed control group. Iniection of pilocarpin (about 2.5 mg./100 g.) to the starved animals caused a similar rate of secretion (cf. refs. 1 and 3). The slice system as demonstrated in the present work attained secretion rates of the same order of magnitude as those observed in the intact animal. EFFECT OF SECRETION STIMULANTS AND METABOLIC INHIBITORS Various a g e n t s k n o w n to s t i m u l a t e secretion in vivo were tried i n the slice s y s t e m i n order to choose one which would c o n s i s t e n t l y cause high rates of e n z y m e TABLE I STIMULATORY E F F E C T OF VARIOUS AGENTS ON AMYLASE SECRETION BY THE SLICE a
Expt.
II
III
IV
Stimulant added
Specific activity Amylase of amylase secreted (units/rag. protein) (% of total) in in slices medium
None Carbamylcholin Epinephrine
10 19 40
550 540 420
630 670 750
None Carbamylcholin Epinephrine Carbamylcholin + epinephrine
16 22 37
530 470 430
760 700 770
33
450
780
None Eserin Carbamylcholin Pilocarpin Pitocarpin + earbamylcholin
12 21 20 21
400 400 440 420
500 670 740 700
22
440
750
16 28 35
450 390 390
630 680 680
41
360
690
None Electric pulses Epinephrine Epinephrine -lelectric pulses
secretion. T a b l e I shows t h a t all a g e n t s tested, i n c l u d i n g electric pulses, increased the secretion rate above t h a t of the control. T h e specific a c t i v i t y of a m y l a s e secreted was always higher t h a n t h a t of the e n z y m e r e m a i n i n g w i t h i n the slice, i n d i c a t i n g specificity i n p r o t e i n secretion. T h e effect of a c o m b i n a t i o n of two s t i m u l a n t s was n o t a d d i t i v e a n d was u s u a l l y n o t m u c h higher t h a n t h a t of the more p o t e n t s t i m u l a n t applied alone. E p i n e p h r i n e c o n s i s t e n t l y showed the highest activity. I n presence of this a g e n t the rate of secretion was u s u a l l y more t h a n twice as high as t h a t of the control i n spite of the fact t h a t the a b s o l u t e rate varied ( T a b l e I). E p i n e p h r i n e has therefore b e e n r o u t i n e l y used i n all subs e q u e n t experiments. I n a d d i t i o n to a m y l a s e the slice also secreted D N a s e . T h e l a t t e r e n z y m e h a d a subcellular d i s t r i b u t i o n a l m o s t identical to t h a t of amylase, large a m o u n t s b e i n g
Slices containing on the average 4000 amylase units per flask were incubated for 60 minutes. The medium was Krebs medium I in expt. I and KRB in expts. II-IV. Pilocarpin was 4 X 10-4 M; other stimulants were 10-5 M.
TABLE II E F F E C T OF ~NHIBITORS AND LACK OF OXYGEN ON D N A s E AND AMYLASE SECRETION a
Expt.
II
Additions to medium
Enzyme secreted (% of total) Amylase
DNase
None Epinephrine Epinephrine, iodoacetate b Epinephrine, KCN Epinephrine, DNP Epinephrine, N~c
16 33
18 34
33 16 12 17
-13 11 15
None n-Ethylmaleimide Epinephrine Epinephrine, n-ethylmaleimide Epinephrine, DNP
22 20 50
----
50 25
---
" Slices containing about 4000 amylase units and 700 DNase units per flask were incubated for 60 minutes. Krebs medium I was used in expt. I and KRB medium in expt. II. The concentration of inhibitors was 10-3 M. b Slices were kept in the cold in presence of the inhibitor for 30 minutes prior to incubation at 37~ c The gas phase was 95% N2,5% CO2.
ENZYME SECRETION BY SLICES OF PAROTID GLAND located in the zymogen granules. The specific activity of the D N a s e secreted was in the same range as t h a t of the zymogen granule fraction, about 120 units per milligram protein. A commercial preparation of crystalline bovine pancreatic DNase, assayed under the same conditions, had a specific activity of 750. Evidently, rat parotid gland contains and secretes considerable amounts of D N a s e together with amylase. Table I I shows t h a t the enhanced rate of D N a s e and amylase secretion caused b y epinephrine was absolutely dependent on oxygen. Cyanide and 2,4-dinitrophenol brought the enhanced secretion down to the level obtained in absence of epinephrine while inhibitors of glycolysis were without effect. The factors responsible for the basal rate of secretion or leakage in absence of epinephrine have not been identified. EFFECTS OF MEDIUM COMPONENTS The substrates present in Krebs medium I (ghlcose, pyruvate, fumarate, and glutamate) and each one of these tested separately were without effect on the secretion rate for the first 60 minutes and were therefore omitted in experiments of short duration. Insulin (3 gg. per milliliter) added with glucose also had no effect on the secretion rate. The slice apparently contains sufficient amounts of endogenous substrate. When the requirements for the inorganic components of the K R B medium were investigated, it was found t h a t Ca ++ , Mg ++, and inorganic phosphate could be omitted without causing a marked drop in the rate of amylase secretion. Bicarbonate and CO2 appeared also not to be required since Krebs-Ringer phosphate could replace the K R B medium. A potent inhibitor of carbonic anhydrase, acetazolamide (10 -4 M), was without effect on the secretion of amylase. The secretion rate was, however, strikingly dependent on potassium. I t had already been demonstrated t h a t in the absence of this ion, epinephrine stimulated secretion only to a slight extent (7). These findings prompted a study of the potassium content of the slice and its effect on secretion. I t
61
was found t h a t the fresh gland contained 0.3-0.6 /~eq. K + per milligram protein (40-70 meq. K + per kilogram fresh weight). During preparation of slices (15 minutes) in cold K R B , some K + leaks out and the ratio drops to 0.2-0.3 geq. K + per milligram initial protein. This value then remained fairly constant when slices were incubated a t 37~ in the above medium, with or without epinephrine or even when storage in the cold was continued for another 30 minutes. However, the slices were almost completely depleted of K + when incubated in a K+-free medium (Table I I I ) . Such depleted preparations released protein at a somewhat higher rate t h a n normal slices and showed no response when epinephrine was added. Secretion could no longer be effectively enhanced b y epinephrine even when the depleted slices were again incubated in presence of K + (Table IV). Control over secretion is apparently lost only when the slice is almost exhausted of K +. A decrease of K + to the level of 0.1 ~eq. per milligram initial protein caused b y Ouabain (10-s M) in K R B medium did not affect the ability of the slices to respond to epinephrine. Another effect of K + became apparent when media containing high concentrations of this ion were tested. Raising the K + TABLE I I I R E L E A S E OF POTASSIUM FROM SLICES INCUBATED IN A P O T A S S I U M - F R E E M E D I U M a Time (rain.)
K + released into medium (% of total)
K + remaining in slice (#eq./mg. initial protein)
0 15 45 75
0 47 72 91
0.23 0.13 0.06 0.04
Slices were prepared in KRB and softly wiped on filter paper. A sample was removed for analysis of K + and protein content at zero time. The remaining slices were directly transferred into art incubation vessel at 37~ containing a KRB medium from which K + was omitted. At times indicated in the table a sample of slices was removed for analysis and the remainder transferred to fresh K+-free medium. K + released was measured on aliquots of incubation medium.
62
BDOLAH, B E N - Z V I A N D S C H R A M M T A B L E IX: EFFECT OF POTASSIUM D E P L E T I O N ON THE SECRETION OF AMYLASEa K+ in incubation medium
Slice preparation
Preincubated without K +
0 min.c
30 rain.
-+ +
-+ -+
29 31 25 31
<0.05 <0.05 <0.05 <0.05
<0.05 <0.05 0.15 0.13
+ +
-+
19 40
0.27 0.27
0.27 0.24
-
P r e i n c u b a t e d with K +
K+ (~q./mg. initia[ protein)
Amylase secreted (% of total) b
Epinephrine
-
a I n c u b a t i o n media were with or w i t h o u t K +, other components being those of K R B medium. To d e p l e t e slices of K +, these were p r e i n c u b a t e d for 75 minutes w i t h o u t K +, during which time 36% of t h e amylase was secreted. The control slices were preincubated for 75 minutes with K + and secreted 30% of t h e i r amylase content. b The amylase remaining in the slice at the end of the preincubation period is t a k e n as 100%. A f t e r preincubation. TABLE V ]?OT&SSIUM AT HIGH CONCENTRATION AS A STIMULANT OF AMYLASE
~7o
SECRETION a
Expt.
Specific activity Amylase of amylase secreted (units/mg. protein) (% of total) in in slices medium
Additions
[K+] (mM)
Epinephrine
5 16 59 117 5
22 20 47 46 49
360 400 310 300 280
620 580 700 730 670
9 24 40 59 140
25 27 38 62 50
390 350 320 230 310
760 750 770 870 870
5 140 140
16 35 13
390 340 400
680 810 550
II
III DNP
8O
a Slices were incubated for 60 minutes. The medium was K R B in which K + replaced Na + at c o n c e n t r a t i o n s shown in the Table. D N P was 10-3 M.
concentration to 60 m M or above enhanced the secretion rate to a level equal to that achieved by epinephrine in the normal K R B medium (Table V). Concentrations in the
6o
o 50
~30 "-
20
.o'"
//
4j
..o. . . . . . . . .
.... 9
~0 1
2
3
Time (hr,) F l a . i . K i n e t i c s of amylase and protein secretion during a 3-hour incubation period. The medium was K R B which also contained 10 m M pyruvate. Slices were transferred to fresh medium, with or without epinephrine, after each hour. The used media were kept for analysis. - - , amylase secreted; . . . . , protein secreted. O, with epinephrine; O , without epinephrine.
range of 20-60 m M had a partial effect. When epinephrine was added to media containing a high K + concentration no further increase of the secretion rate occurred (7). Thus a K + concentration of 60 m M acted as a stimulant of secretion equal in potency to epinephrine. The enhancement of secretion by K + appeared
63
E N Z Y M E S E C R E T I O N B Y SLICES OF P A R O T I D G L A N D
to be due to a true metabolic effect since it was abolished by D N P (Table V). While respiration in brain slices is stimulated by a high K + / C a ++ ratio (14), the effect of K + on secretion as described above appeared to be due to the absolute K + concentration. When the Ca ++ Concentration was raised from 2.5 to 20 m M in presence of 60 m M K +, the secretion rate remained undiminished. Ammonium ion which can replace K + in the transport system of the erythrocyte (15) could not support enzyme secretion by the parotis slice at concentrations equivalent to K +. Enhanced secretion was not observed when the medium contained 60 m M NH4 + instead of K +. In addition, slices which were slightly depleted of K + by a 15-minute incubation in the absence of this ion did not respond to epinephrine when subsequently incubated in a medium in which 5 m M NH4 + replaced K +. Such depicted slices responded normally to the stimulant when 5 m M K + was present.
T A B L E VI ZYMOGEN G R A N U L E S AS THE SOURCE OF AMYLASE FOR SECRETION a
Relative distribution oI amylase (%) In slices
Time (min.)
0 30 60 120
Zymogen granules
Supernatant
In extracellular medium
70 30 14 11
30 33 32 27
0 42 58 63
a T h r e e flasks c o n t a i n i n g slices (about 18,000 amylase u n i t s per flask) in K R B m e d i u m w i t h e p i n e p h r i n e were i n c u b a t e d as described u n d e r Materials and Methods. E p i n e p h r i n e was not replenished d u r i n g the experiment. A t each t i m e period one flask was removed for analysis. T h e homogenate of the slices was subjected to centrifugation a t 2100g for 10 minutes to yield a crude zymogen granule f r a c t i o n a n d a s u p e r n a t a n t fraction. T h e relative a m o u n t s of amylase in these subcellular fractions a n d in the extracellular m e d i u m are shown in the Table.
TABLE VII R E L A T I V E CONSTANCY OF THE N U M B E R OF ZYMOGEN G R A N U L E S D U R I N G SECRETION OF T H E I R AMYLASE CONTENT a
Expt.
Time (rain.)
Number X 101~
I II lII
0 180 0 120 0 30
"Membrane" protein
Granules
3.8 3.3 5.5 3.3 5.6 5.1
Amylase in granules
~ob
rag.
%b
100 87 100 60 100 91
3.3 3.7 4.3 4.0 2.4 2.4
100 112 100 93 100 100
Units
17,000 2600 15,100 2500 10,100 2500
~ob
100 15 100 17 100 25
Slices from 12 glands of 6 s t a r v e d r a t s served for each experiment. A t zero time, before p r e i n c u b a tion, half of the slices were homogenized in 0.25 M sucrose. The fraction s e d i m e n t e d b y 5-minute centrifugation a t 250 g was washed once. T h e p r e c i p i t a t e was discarded a n d t h e s u p e r n a t a n t was added to t h e rest of t h e homogenate. T h e zymogen granule f r a c t i o n was t h e n isolated b y e e n t r i f u g a t i o n a t 2100 g for 10 minutes. Zymogen granule " m e m b r a n e s " were p r e p a r e d b y h y p o t o n i c shock as previously described (2). The remaining slices were i n c u b a t e d w i t h epinephrine a f t e r 15 m i n u t e s p r e i n c u b a t i o n . To s u b s t r a t e was added to t h e K R B medium. A t t h e end of t h e i n c u b a t i o n period t h e slices were reved from t h e medium, homogenized, a n d f r a c t i o n a t e d as described for t h e sample at zero time. T h e t f e n t s of t o t a l amylase secreted into t h e m e d i u m were 58, 61, a n d 48 for expts. I, I I , a n d I I I , respeeZ I n expt. I e p i n e p h r i n e was n o t replenished d u r i n g i n c u b a t i o n . micr3,gen granules were counted in t h e conventional blood c o u n t c h a m b e r with a phase contras~ p e r ali~ e- Two to four aliquots of each sample were examined. A t least 500 granules were c o u n t e d ~8%. ~. T h e n u m b e r calculated from different aliquots of t h e same sample varied in t h e range of b Values ,
zero time are a r b i t r a r i l y defined as 100~.
64
BDOLAH, BEN-ZVI AND SCHRAMM
KINETICS OF AMYLASE SECRETION AND CHANGES IN THE INTRACELLULAR DISTRIBUTION O f THE ENZYME
When slices were incubated in the presence of epinephrine, 78% of the total amylase and 53 % of the total protein were secreted during 3 hours (Fig. 1). The specific activity of the amylase released into the medium remained nearly constant (about 800 units per milligram protein) throughout the incubation period while that of the slice decreased from 520 at zero time to 260 at the end of 3 hours. Figure 1 also shows that secretion in presence of epinephrine followed roughly first order kinetics. The secretion rate of the control without stimulant was much lower and declined further more rapidly than the experimental system. Incubation in presence of stimulant could often be prolonged up to 5 hours resulting in the secretion of 90% of the amylase content of the slice without a noticeable drop in the specific activity of the enzyme secreted. A study of the intracellular distribution of the enzyme during secretion revealed a precipitous drop in amylase content of the zymogen granules equivalent to the amount of enzyme secreted into the extracellular medium (Table VI). Only after the zymogen granules lost about 80% of their amylase content did the supernatant fraction show a loss of this enzyme. The extreme loss of amylase from the zymogen granule fraction during secretion was not accompanied by a parallel decrease in the absolute number of granules. When the granule fraction retained only 15-25 % of its initial amylase content, 60-90% of the granules were still present (Table VII). The granules after secretion appeared much smaller and less dense, especially in experiments of long duration (Expts. I and II). Difficulties in detecting all the granules after secretion may account for the apparent decrease in their number. It was indeed found that the water insoluble "membrane" protein of the granule fraction remained unchanged during secretion (Table VII).
DISCUSSION The rat parotid gland slice described in the present work demonstrates the major features of the secreting gland as it operates in the living animal. Physiological stimulants such as epinephrine cause an enhanced and relatively specific secretion of amylase and other proteins at a rate comparable to that observed i n vivo. Energy generated by oxidative processes seems to be the driving force in the transport of these proteins in the cell and across the cell border. Indeed, oxidation is probably a limiting factor since secretion by the slice in presence of oxygen proceeded at a higher rate than in presence of air. The striking acceleration of amylase secretion by high potassium concentrations in the medium might also be ascribed to the stimulating effect of this ion on oxidative energy yielding processes (14, 16). Whether the inability of potassium depleted slices to respond to epinephrine is due to a defect in the generation of energy cannot as yet be decided. It should, however, be noted that the failure to show enhanced secretion occurs only after the slices are almost completely exhausted of their internal potassium by incubation in a potassium-free medium. Other tissues rarely show such extreme and rapid losses of this ion. However, the parotid gland is known to secrete potassium (17), and it is therefore possible that the slice continues to secrete this ion even when it is unable to regain it from the medium because of extreme dilution. The slice system was found to maintain specific secretion up to a stage when it is almost exhausted of secretory proteins. Changes in the subcellular structures of the cell and in the distribution of secretory proteins as they occur with the progress of secretion may therefore be readily detected and measured. During the first phases secretion, the zymogen granules are sh~ to lose their amylase content rapidly :n~ the amount of enzyme in the supe~ant" fraction of the gland remains cr n . ~ Preliminary experiments indicate ~e~::o f ing the first minutes a transient i .-~ amylase in the supernatant e~orapan~r
ENZYME SECRETION BY SLICES OF PAROTID GLAND loss of the enzyme from the zymogen granules. These findings could mean that the enzymes which are released by the granules pass through the supernatant pool into the medium. The supernatant fraction might thus represent to a large extent the enzymes which have already been transported beyond the cell membrane into the lumen and ducts of the gland. I t is indeed hard to visualize that the extracellular space of the gland would ever be free of secreted proteins. Although the supernatant fraction would also contain amylase released from zymogen granules and other cell structures damaged during fractionation, it is quite possible that some part of it is indeed true soluble intracellular amylase. While the zymogen granules secrete their protein contents, the membranes of these structures apparently remain inside the cell. The evidence on which this conclusion is based is mainly that the number of granules changed only slightly dulfing the period when most of the amylase and other proteins of the granule fraction were secreted. The fraction counted is not homogeneous and probably contains mitochondria and some nuclei. I t is also not known whether new granules could have been formed during the period of the experiment. These reservations apply equally to the findings that the total "'membrane" protein of the granule fraction remained constant during secretion. It should be observed that the amount of "membrane" protein per granule varies by a factor of about 2.5 between the different experiments presented in Table VII. T h e cause of this variation is not clear. With the above noted reservations in mind, it is quite probable that the membranes of the zymogen granules remain inside the cell and may serve repeatedly in the secretion process. Classic histological studies indicated that the granules disappear from the cell during secretion (18). It is however quite possible that the empty granules were not detected on the background of the whole cell section because of their smaller size and lack of contrast. Keller and Cohen (19) and also Greene (20) have demonstrated that the digestive
65
enzymes appear in the pancreatic juice in the same proportions as found in the intracellular zymogen granules. The present work shows that the parotid gland slice secretes DNase and amylase into the medium in the same relative amounts as found within the slice. Furthermore, it is shown that the ratio of amylase to other proteins secreted remains constant up to the stage when the slice is almost depleted of amylase. I t therefore seems likely that the different proteins secreted derive from a common pool and that the gland has no means of withholding one digestive enzyme while secreting others. ACKNOWLEDGMENT The experiments were carried out with the most valuable assistance of Mrs. Sarah Eldar. REFERENCES 1. SCI-INEYER,L. H., AND SCttNEYER, C. A., Ann. N. Y. Acad. Sci. 85, 189 (1960). 2. SCttRAMM, M., AND DANON, D., Biochim. Biophys. Acta 50, 102 (1961). 3. GROMET-ELHANAN, Z., AND WINNICK, T., Biochim. Biophys. Acta 85, 69 (1963). 4. HOKIN, L. E., Biochem. J. 48, 320 (1951). 5. HOKIN, L. E., AND SHERWIN,A. L., J. Physiol. London 135, 18 (1957). 6. EGGMAN, L. D., AND HOKIN, L. E., J. Biol. Chem. 235, 2569 (1960). 7. BDOLAn, A., AND SCHRAMM, M., Biochem. Biophys. Res. Communs. 8, 266 (1962). 8. BERNFELD,P., in "Methods in Enzymology"
(S. P. Colowick and N. O. Kaplan, eds.), Vol. 1, p. 149. Academic Press, New York, 1955. 9. McDoNALD, M. R., in "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.), Vol. 2, p. 437. Academic Press, New York, 1955. I0. Lowaz, O. H., ROSEBROUGH, N. J., FARR,
A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 11. COHEN, P. P., in "Manometric Methods" (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.) 3rd ed., p. 149. Burgess Publishing Co., Minneapolis, 1957. 12. KREBS, H. A., Biochim. Biophys. Acta 4, 249 (195o). 13. MCILWAIN,H., Biochem. J. 50, 132 (1952).
66
BDOLAH, BEN-ZVI AND SCHRAMM
14. USSING, H. H., in "The Alkali Metal Ions in Biology, Handbuch der experimentellen Pharmakologie" (O. Eiehler and A. Farah, eds.), Vol. 13, p. 24. Springer Verlag, Berlin, 1960. 15. POST, R. L., AND JOLLY, P. C., Biochim. Biophys. Acta 25, 118 (1957). 16. HEALD, P. J., Biochem. J. 57,673 (1954). 17. BURGEN, A. S. V., AND EMMELIN, N. G.,
"Physiology of the Salivary Glands," p. 148. Edward Arnold Ltd., London, 1961. 18. BABKIN, B. P., "Secretory Mechanism of the Digestive Glands," 2nd ed., p. 23. Harper (Hoeber), New York, 1950. 19. KELLER, P. J., AND COHEN, E., J. Biol. Chem. 236, 1407 (1961). 20. GREENE, L. J., HIRS, C. H. W., AND P&LADE, G. E., J. Biol. Chem. 238, 2054 (1963).