321
Biochimica et Biophysica Acta, 585 (1979) 321--332 © Elsevier/North-Holland Biomedical Press
BBA 28900
T R A N S P A N C R E A T I C T R A N S P O R T OF DIGESTIVE ENZYME
LOIS D. ISENMAN and S.S. ROTHMAN
Department of Physiology, University of California, San Francisco, San Francisco, CA 94143 (U.S.A.) (Received November 3rd, 1978)
Key words: Pancreatic transport; Digestive enzyme; Secretion
Summary When porcine a-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [3H]leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase by a b o u t 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, n o t only of the increased secretion of unlabeled enzyme, b u t also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition o f amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath b y a b o u t 20 times; it did n o t reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested b y the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrypsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction o f the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.
322
Introduction Equilibrating fluxes of digestive enzyme have been demonstrated across the zymogen granule membrane [1], the lumenal plasma membrane [2], and even the basolateral membrane [3,4] of the pancreatic acinar cell. These and other observations have led to the proposal that digestive enzyme in zymogen granules (and perhaps other intracellular storage pools) is in an equilibrium state with its own species in the cytoplasm and that the cytoplasmic pool of digestive enzyme is the immediate precursor pool for secretion across the apical (and basolateral) membrane of the acinar cell [5]. This has been termed the 'equilibrium' theory because it proposes that the transport of these proteins across 'secretory' membranes is an equilibrium-dependent process in that their movement is bidirectional and concentration dependent. The discovery of a basolateral permeability to digestive enzyme, prompted Liebow and one of us (S.S.R.) to suggest an 'enteropancreatic circulation' of digestive enzyme, akin to the enterohepatic circulation of bile salts, in which secreted digestive enzyme is absorbed into the blood from the intestine and recycled through the pancreas [4,6]. The evidence suggested a cellular transpancreatic route. The current study assesses the capacity of the pathway by examining the effect on ductal secretion of the addition of a substantial amount of amylase or chymotrypsinogen to the medium bathing the whole rabbit pancreas in culture. The results demonstrate a substantial flux of digestive enzyme across the acinar cell and confirm this prediction of the enteropancreatic circulation hypothesis. Also, as predicted by the equilibrium theory, the system appears to be responsive to mass action and enzyme entering the cell via the basolateral membrane apparently mixes with endogenous enzyme pools prior to secretion. Methods
Biological preparation. Glands were obtained from male, New Zealand white rabbits (2--3 kg b o d y weight). After an overnight fast (14--18 h), the animals were anesthetized with a mixture of allobarbital (0.34 mmol/kg b o d y weight), urethan (3.1 mmol/kg), and m o n o e t h y l u r e a (3.2 mol/kg), administered intraperitoneally. The pancreatic duct was cannulated in situ after a laparotomy, the pancreas removed from the animal, and m o u n t e d in a Plexiglass chamber by techniques described elsewhere [7]. The gland was bathed by 400 ml of HCOjbuffered Krebs-Henseleit medium [8] enriched with 5.5 mM glucose and a mixture of a m i n o acids [9]. The medium was gassed with 95% O2 and 5% CO2 and its temperature maintained at 30°C. The functional characteristics of this biological preparation have been extensively studied and are the subject of numerous publications [10--14]. Experimental procedure. After 1 h of incubation, the bathing medium was changed and control samples collected at either 10- or 20-min intervals (as specified) for an additional hour. At this time, either porcine a-amylase (Sigma, Type VI-A), the same material repurified (as described below), or 5 times crystallized bovine chymotrypsinogen A (Worthington Chemical Co.) was added to the bathing medium at specified concentrations. Secretion was then collected
323 at 10-min intervals for 1 h and at 15-rain intervals for an addition hour. Samples were assayed for either amylase activity alone or both chymotrypsinogen and amylase activity. In a number of preparations, 4 mCi L-[4,5-3H]leucine (specific radioactivity, 62 Ci/mmol) (Schwarz Mann Co.) was added to the batching medium and the gland was incubated in the radioactive leucine for 4 h before exogenous amylase was added. During this time, secretion was collected at hourly intervals for 3 h, followed by a 50 rain and then a 10 min collection. When exogenous amylase was added, secretion was collected at 10-min intervals for 1 h and at 15-min intervals during the subsequent hour. The incorporation of [3H]leucine into secreted amylase was followed by purifying amylase in all samples by glycogen precipitation as described below, and measuring its 3H content. In a number of experiments, after 2 h of incubation in chymotrypsinogen, acetyl-~-methylcholine chloride (1.0 mg/100 ml bath fluid) was added to the bathing medium and secretion collected at 10-rain intervals for an additional hour. Analytical techniques, s-Amylase activity was measured by following the hydrolysis of an amylose substrate labeled covalently with Remazol Brilliant Blue R (Amylose Azure, Calbiochem) [15]. To 0.5 ml of diluted secretion, equivalent to 1--2 pl of undiluted secretion, 4.5 ml of the substrate in 0.02 M sodium phosphate buffer (pH 7.0) containing 0.05 M NaC1 was added. Samples were incubated in a shaking water bath at 37°C and the reaction terminated at 15 min by the addition of 2 ml 0.1 N acetic acid. Samples were then filtered through No. 2 Whatman filter paper and the absorbance of the filtrate read at 595 nm. The absorbance was compared to a standard curve made to 2 times crystallized porcine ~-amylase (506 I.U./mg) (Worthington Chemical Co.). Chymotrypsinogen was measured after the activation of a small sample of secretion (10--50 pl) in 0.05 M sodium phosphate buffer (pH 6.0) containing 0.5 mg/ml enteropeptidase (EC 3.4.21.9) (Miles Laboratories) which was incubated for 30 rain at 37°C. Immediately following incubation, the activation mixture was added to 1.0 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 3.0 ml of the substrate (80 mM suspension of aeetyl tyrosine ethyl ester in 30% methanol) and the initial rate of liberation of H* measured at pH 7.4 using a pH-stat technique. The presence of c h y m o t r y p t i c activity was related to sample size linearly over the range used. The protein content of samples was estimated using the Folin phenol reagent
[161. Amylase was purified from secretion by precipitation with shellfish glycogen [17]. 25--50 ul of juice (containing between approximately 25 and 315 pg of amylase) was added to 1 ml of 40% ethanol at 4°C, and then 50 ~l of 0.2 M sodium phosphate buffer (pH 8.0), 50 pl of 2% shellfish glycogen and 70 pl 95% ethanol were added in sequence and the mixture agitated for 5 min at 4°C. The mixture was centrifuged at 2000 × g for 6 min and the pellet resuspended in 1 ml of 40% cold ethanol containing 0.01 M sodium phosphate buffer (pH 8.0). The wash procedure was repeated three times and the final pellet suspended in 1 ml H20. 100 pg of amylase was added to control samples which contained smaller amounts of amylase to roughly equate the a m o u n t of
324 amylase being purified between control and experimental samples. Over the range of amylase sample size u s e d , the yield of amylase recovered by glycogen precipitation was between 75 and 100% and no systematic variation in yield was observed. This purification technique increased amylase specific activity by between 5- and 10-fold depending upon the specific activity of amylase in the original secretory sample. Porcine a-amylase (Sigma, Type VLA) was repurified by a similar procedure. 750 mg of amylase was added to 1 1 of 0.01 M sodium phosphate buffer (pH 8.0) in 40% ethanol at room temperature and then cooled to 4°C. All the following procedures were carried o u t at 4°C. 1 g shellfish glycogen was added followed by the volume of 95% ethanol necessary to return the ethanol concentration to 40%. The mixture was agitated for 5 min and then centrifuged at 5000 X g for 5 min. The pellet was resuspended first in one volume of the buffered ethanol solution, respun and then suspended again in 0.5 volume of the same solution for a final spin. The final pellet was suspended in 10 ml of KrebsHenseleit medium and left at room temperature for 1 h. The procedure produced a yield of almost 100% of the added amylase and specific activity was increased by approximately 5-fold. Amylase accounted for some 20% of the protein in the original material, the rest presumably being primarily other digestive enzymes. We estimated chymotrypsinogen contamination in the original material, and assuming that its specific activity is roughly the same as 5 times purified bovine chymotrypsinogen (Worthington Chemical Co.), chymotrypsinogen accounted for a b o u t 10% of its protein content. Chymotrypsinogen contamination of the repurified amylase was only a b o u t 0.07%. Results
The effect o f a-amylase added to the bathing medium on amylase secretion When 1 mg/ml amylase (Sigma, Type VI-A; containing approximately 1/3 protein by weight and amylase at a concentration o f 67 t~g/ml) was added to the medium bathing the in vitro pancreas, the amylase content of ductal secretion increased rapidly and dramatically. The response peaked approximately 20 min after the enzyme was added at an average amylase o u t p u t of 11 times the unstimulated (basal) level (Fig. 1), or 15 + 3 (S.E.) times basal when each preparation was normalized to its own control. This effect was equivalent to approximately 20% of the amylase ductal o u t p u t at the peak of a maximum cholinergic response for this in vitro system. The amylase content in ductal secretion returned towards its original level over time, although bath amylase concentration remained essentially unchanged. When the dose of exogenous amylase was varied, 0.1, 0.2, as compared to 1.0 mg/ml o f the Sigma material, the response did n o t increase linearly with concentration, as predicted for a non-saturating system, b u t appeared to reach a maximum value between 0.2 and 1.0 mg/ml (Table I). When repurified amylase was used instead of the crude material, the response was smaller, although still quite substantial (6.1 ± 1.6 (S.E.) times prior control values (n = 4), or a b o u t half the magnitude of the response produced by the crude material). Thus, amylase secretion apparently was in part stimulated by other molecules in the crude amylase. We tested for the possibility that small
325
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F i g . 1. E f f e c t o f t h e a d d i t i o n o f 1 m g / m l a - a m y l a s e t o t h e b a t h o n t h e d u c t a l s e c r e t i o n o f a m y l a s e . D a t a p r e s e n t e d as m e a n a m y l a s e o u t p u t / 1 0 rain (+-S.E.). V a l u e at t i m e z e r o is o u t p u t f o r 1 0 rain c o n t r o l p e r i o d prior t o a d d i t i o n o f a - a m y l a s e . N = 1 1 , 1 3 , 1 3 , 1 2 , 1 2, 1 1 , 9 , 8, 5, a n d 5 f r o m 0 t o 1 0 5 rain.
compounds were responsible by dialyzing the Sigma material against KrebsHenseleit medium for 24 h at 4°C before adding it to the pancreas preparation. The secretory response was undiminished as a result of dialysis. Therefore, that portion of the amylase response not elicited by amylase itself was apparently due to the presence of other large molecules, probably other digestive enzymes, in the mixture. Evidence relating to this point will be presented below. The effect o f chymotrypsinogen added to the bath on the secretion of chymotrypsinogen When 1 mg/ml of a relatively pure chymotrypsinogen (5 times crystallized bovine chymotrypsinogen A, Worthington Chemical Co.) (amylase contamination based on an assumed specific activity equal to 2 times crystallized porcine a-amylase (specific activity, 506 I.U./mg) was less than 0.004%) was added to the medium, chymotrypsinogen output in ductal secretion increased. The response developed more slowly than when amylase was added and peaked at 60 min (Fig. 2). When each experiment was normalized to its own control, for the peak period chymotrypsinogen output was approximately 7.5 -+ 2.5 (S.E.)
TABLE
I
THE EFFECT OF VARYING CONCENTRATIONS BIT P A N C R E A S IN V I T R O O N T H E S E C R E T I O N
OF AMYLASE IN T H E M E D I U M OF AMYLASE INTO THE DUCT
BATHING
RAB-
Amylase used was Sigma Type VI-A. [Amylase]bath (mg/ml)
0.1 0.2 1.0
n
3 3 10
Amylase output at peak period (10--20 rain) (#g/10 rain) Observed
Predicted for a non-saturating system
300 * 550 950
(300) 600 3000
* T h i s v a l u e is s o m e 5 t i m e s basal a m y l a s e o u t p u t w h i c h a v e r a g e d 6 4 _+ 1 2 ( S . E . ) in t h e 1 0 rain c o n t r o l p e r i o d prior t o t h e a d d i t i o n o f a m y l a s e t o t h e m e d i u m ( n = 1 6 ) .
326
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F i g . 2. E f f e c t o f t h e a d d i t i o n o f 1 m g / m l c h y m o t r y p s i n o g e n A to the bath on the ductal secretion of chymotrypsinogen. Data are mean chymotrypsinogen o u t p u t / l O r a i n (-+S.E.) m i n u s t h e m e a n b a s a l s e c r e t o r y o u t p u t o f 2 . 6 6 # t o o l s u b s t r a t e s p l i t / r a i n p e r 1 0 r a i n ( w h i c h is t h e v a l u e f o r t h e 1 0 r a i n c o n t r o l p e r i o d prior to the addition of chymotrypsinogen A (time zero on the graph)). N = 9 from 0 to 99 rain; 6 at 105 rain; and 7 at 120 min.
times the unstimulated control output (Fig. 6). The magnitude of the response was equal to approximately 30% of the chymotrypsinogen output at the peak of a maximum cholinergic response for this in vitro system (Fig. 5).
The effect of exogenous a-amylase on the secretion of endogenous amylase To determine if it was the enzyme that was added to the medium that appeared in secretion, we labeled endogenous digestive enzyme pools with radioactive leucine for 4 h prior to the addition of the exogenous enzyme and followed the specific radioactivity of secreted amylase. Transpancreatic transport of the unlabeled, exogenous enzyme should lower the specific radioactiv-
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Fig. 3. E f f e c t o f t h e a d d i t i o n o f :[ m g / m l u n l a b e l e d c~-amylase t o t h e b a t h a f t e r a 4 h i n e u h a t i o n w i t h [3H]leucine, on the specific radioactivity of amylase in ductal secretion. [3H]beucine was added at time z e r o . S p e c i f i c a c t i v i t y i s e x p r e s s e d a s t h e r a t i o o f m e a n a m y l a s e c p m (n = 8 4 2 4 0 r a i n a n d p o s t - s t i m u l u s p o i n t s a s i n F i g . 4 ) t o m e a n a m y l a s e o u t p u t ( n = 8 o r g r e a t e r f o r all o u t p u t p o i n t s e x c e p t a t 3 3 0 a n d 3 4 5 w h e r e n = 5).
327
ity of amylase in secretion. When amylase was added to the bath, the specific radioactivity of this e n z y m e in secretion did drop precipitously. 10 min after the addition of unlabeled enzyme to the medium, the specific radioactivity of amylase in ductal secretion was reduced by 82%; and by 60 min it was only 7% of control values (Fig. 3). The specific radioactivity remained depressed for the duration of the experiment, despite the fact that over time amylase o u t p u t fell back toward control levels (Fig. 1 and 3). Thus, even as the response declined, about 90% of the secreted amylase was unlabeled and apparently of exogenous origin. In addition, the presence of amylase in the bath altered the secretion of endogenous amylase. Labeled amylase secretion increased transiently (by 72% on the average) and was followed by a sustained inhibition (Fig. 4). The transient increase was quite variable and ranged from +0 to +250%, accounting for the large standard error seen for these periods (Fig. 4). By 60 min, amylase radioactivity in secretion had declined to 28% -+ 6 (S.E.) of prior control levels where it stayed for the remainder of the experiment (Fig. 4). Inhibition of labeled amylase secretion was not seen when either 0.1 or 0.2 mg/ml amylase was used and only a slight inhibition was observed when amylase was present at a concentration of 0.3 mg/ml (concentrations refer to weight of Sigma, Type VI-A amylase) (Table II) * The inhibition of endogenous amylase secretion by the addition of amylase to the bathing medium suggests competitive transport inhibition between exogenous and endogenous e n z y m e and hence, along with earlier studies [4], suggests that the enzyme moves through the acinar cell. The fact that enzyme movement is through the acinar cell rather than via paracellular shunts is also supported by the observation that the addition of amylase to the bathing medium increased the duct to bath concentration difference for this enzyme. If a diffusional equilibration through paracellular shunts were all that were involved, the concentration difference should have been reduced. At the peak of the response, the amylase concentration in the duct averaged 6463 I.U./ml (with a range of 1457--18 155 I.U./ml), whereas bath amylase was approximately 61 I.U./ml (Table III). This concentration difference (6402 I.U./ml) is 14 times that of the control situation (455 I.U./ml; range 46--1822 I.U./ml) * It has b e e n s h o w n b y l a b e l i n c o r p o r a t i o n s t u d i e s t h a t t h e r e are m u l t i p l e i n t r a c e l l u l a r p o o l s o f digestive e n z y m e w i t h i n t h e a c i n a r cell. W h e n t h e in v i t r o r a b b i t p a n c r e a s is s t i m u l a t e d w i t h a c h o l i n e r g i c a g o n i s t a f t e r a 2 h i n c u b a t i o n w i t h [ 3 H ] l e u c i n e , t h e specific r a d i o a c t i v i t y of digestive e n z y m e in s e c r e t i o n d r o p s p r e c i p i t o u s l y , i n d i c a t i n g t h e p r e f e r e n t i a l s e c r e t i o n o f old o r u n l a b e l e d p r o t e i n . A similar b u t less d r a m a tic e f f e c t c a n b e seen a t 4 h, a n d in o n e p u b l i s h e d e x a m p l e [ 2 2 ] , t h e specific r a d i o a c t i v i t y o f t o t a l protein was r e d u c e d b y 35% w i t h c h o l i n e r g i c s t i m u l a t i o n at this t i m e , a n d specific r a d i o a c t i v i t y r e t u r n e d to p r e - s t i m u l u s levels b y 6 0 m i n . I t is possible t h a t t h e results d e s c r i b e d h e r e are d u e t o a similar p h e n o m e n o n , i.e. t h e a d d i t i o n o f a - a m y l a s e to t h e b a t h s t i m u l a t e s t h e release of less h i g h l y l a b e l e d e n d o g e n o u s stores. This s e e m s u n l i k e l y f o r t h e f o l l o w i n g reasons: (1) a m y l a s e a d d e d t o t h e b a t h at 4 h p o s t [ 3 H ] l e u c i n e p r o d u c e d a m u c h l a r g e r d e c r e a s e in specific r a d i o a c t i v i t y ( 9 5 % ) t h a n a cholinerRic d r u g a d d e d a t t h e s a m e t i m e ; (2) u n l i k e c h o l i n e r g i c s t i m u l a t i o n , t h e d e p r e s s i o n in specific r a d i o a c t i v i t y was s u s t a i n e d o v e r t i m e in t h e c o n t i n u e d p r e s e n c e o f t h e e n z y m e ; (3) t h e a m o u n t of l a b e l e d p r o t e i n in secretion after cholinergic stimulation at 4 h only decreased very transiently, and t he n increased b y over an order of m a g n i t u d e within 10 min. In contrast, amylase added to the bath p r o d u c e d a transient increase a n d a s t e a d y - s t a t e i n h i b i t i o n o f a b o u t 70% (Fig. 4); (4) i n c u b a t i n g a g l a n d f o r 4 h w i t h a c h o l i n e r g i c s t i m u l a n t a l o n g w i t h [ 3 H ] l e u c i n e in o r d e r t o d e p l e t e cold p o o l s did n o t c h a n g e t h e p a t t e r n o f t h e r e s p o n s e w h e n e x o g e n o u s a m y l a s e was a d d e d , i.e. s u s t a i n e d i n h i b i t i o n o f l a b e l e d a m y l a s e s e c r e t i o n was still seen.
328 100
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Fig. 4. E f f e c t o f the a d d i t i o n o f 1 m g / m l C~-amylase to t h e b a t h on the a m o u n t o f r a d i o a c t i v e a m y l a s e in ductal s e c r e t i o n . D a t a are e x p r e s s e d as m e a n c p m in a m y l a s e / 1 0 rain (-+S.E.). V a l u e at t i m e z e r o is c p m in a m y l a s e in s e c r e t i o n for 10 rain c o n t r o l prior t o t h e a d d i t i o n of ~ - a m y l a s e to the b a t h and 2 3 0 - - 2 4 0 m i n a f t e r t h e a d d i t i o n o f [ 3 H ] l e u c i n e (see Fig. 3). N = 8 - - 4 0 rain; 7 at 5 0 a n d 6 0 m i n ; a n d 3 at 75, 90, and 1 0 5 rain.
(Table III), or about 20 times control periods when each experiment was paired to its own prior control (Table III). A cellular route is also suggested by experiments in which a cholinergic drug was added to the bathing medium 2 h after the addition o f chymotrypsinogen A. Under these circumstances, the chymotrypsinogen response to the cholinergic drug was 3.3 times greater than that seen when the cholinergic agent was added in the absence of chymotrypsinogen (Fig. 5).
The effect o f exogenous amylase and chymotrypsinogen on the secretion o f other enzymes The addition of crude amylase (Sigma, Type VI-A) to the bathing medium stimulated the secretion of chymotrypsinogen, as well as amylase (Fig. 6), and the increase was of the same magnitude. Indeed, overall protein secretion was also increased in similar proportions which suggests that the secretion of other T A B L E II P E R C E N T A G E C H A N G E I N S E C R E T I O N O F L A B E L E D A M Y L A S E 6 0 rain A F T E R T H E A D D I T I O N OF V A R Y I N G C O N C E N T R A T I O N S OF E X O G E N O U S A M Y L A S E TO T H E MEDIUM M e a s u r e m e n t s w e r e m a d e 5 h a f t e r t h e a d d i t i o n o f [ 3 H ] l e u c i n e . A m y l a s e u s e d w a s Sigma T y p e VI-A. [ Amylase ] bath (mg/ml)
P e r c e n t a g e c h a n g e in l a b e l e d a m y l a s e s e c r e t i o n , 60 m i n a f t e r the a d d i t i o n o f a m y l a s e t o t h e b a t h i n g m e d i u m (relative t o P r e t r e a t m e n t c o n t r o l , - - 1 0 r a i n )
0.1 0.2 0.3 1.0
+58 +41 --16 --72 *
* See Fig. 4.
329
TABLE III T H E E F F E C T OF 1 m g / m l E X O G E N O U S A M Y L A S E ( S I G M A T Y P E V I - A ) ON [ A M Y L A S E ] d u c t [AMYLASE]bath" Experiment
1 2 3 4 5 6 7 8 9 10 11 12 S.E.
Amylase concentration difference (duct--bath) (I.U./ml) A. No a m y l a s e ( 1 0 rain p r i o r to + a m y l a s e )
B. A m y l a s e a d d e d (peak values)
+a~nylase/--amylase (B/A)
364 131 506 430 556 273 1822 146 46 349 759 86
4 1 6 6 7 8 11 1 1 4 18 6
190 862 315 011 833 136 577 457 558 043 155 416
11.5 14.2 12.5 14.0 14.1 29.8 6.4 10.1 34.0 11.6 23.9 74.6
6 463 1 374
21.4 5.4
455 138
enzymes was augmented as well (evidence n o t presented here). The removal of other digestive enzymes from the impure amylase by its repurification did n o t eliminate this effect and roughly parallel, although diminished, secretion o f the t w o enzymes was still observed even though only very small amounts of chymotrypsinogen were added to the bathing medium under these conditions (see Methods) (for the period of peak response when repurified amylase was added, the increase over controls was 7.5 times for amylase and 9.5 times for chymotrypsinogen in one experiment, and 7.9 times for amylase and 5.3 times for chymotrypsinogen in another). Similarly, when the relatively pure chymotrypsinogen was added, the secretion of amylase was augmented in a fashion roughly equivalent to that seen for chymotrypsinogen (a maximum o u t p u t of some 6--7 fold control rates; Fig. 6). Thus, the addition of one enzyme to the medium apparently stimulated the secretion of other enzymes and in a grossly parallel manner. Since the Sigma VI-A amylase is contaminated with other enzymes, this is probably the reason why the crude amylase evoked a larger amylase response than the repurified amylase itself. While the outputs of the two enzymes were more or less parallel when means were compared, a more detailed analysis of the experimental data indicated that the responses were not identical, it could be seen that the addition of amylase to the medium altered the distribution of points, curiously skewing the data towards a more chymotrypsinogen-dominant secretion (Fig. 7). The broken line in Fig. 7 represents one standard deviation either plus or minus the mean slope for control data and includes approximately 2/3 of the control points. On the other hand, 64% of the amylase-treated points were greater than 1 S.D. above the mean line, i.e. were chymotrypsinogen dominant relative to controls. The +amylase points (i.e. amylase added to the bath) also show a
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Fig. 5. E f f e c t o f a e e t y l - ~ - m e t h y l c h o l i n e c h l o r i d e (1 r a g / 1 0 0 m l b a t h v o l u m e ) o n e h y m o t r y p s i n o g e n o u t p u t w i t h a n d w i t h o u t p r i o r e x p o s u r e (2 b) to 1 m g / m l b o v i n e c h y m o t r y p s i n o g e n A in b a t h i n g m e d i u m . A c e t y l - f l - m e t h y l c h o l i n e c h l o r i d e w a s a d d e d a t t i m e z e r o . N = 5 to 40 rain; 3 at 40 a n d 50 m i n i a n d 4 at 50 and 60 rain for prior exposure condition. N = 7 for control data. The difference between chymotryps i n o g e n p r e - t r e a t e d a n d c o n t r o l g r o u p s w a s P < 0 . 0 1 at 0 - - 1 0 r a i n ( t w o - t a i l e d t-test), a n d P < 0 . 0 5 at 1 0 - 20 rain ( M a n n - W h i t n e y r a n k t e s t ) . Fig. 6. E f f e c t o f a d d i n g 1 m g / m l a - a m y l a s e to t h e b a t h o n t h e s e c r e t i o n o f e h y m o t r y p s i n o g e n a n d a m y lase, a n d t h e e f f e c t o f a d d i n g 1 m g / m l c h y m o t r y p s i n o g e n A to t h e b a t h o n t h e s e c r e t i o n o f a m y l a s e a n d c h y m o t r y p s i n o g e n . D a t a are n o r m a l i z e d f o r e a c h i n d i v i d u a l e x p e r i m e n t to a 10 r a i n c o n t r o l p e r i o d p r i o r to t h e a d d i t i o n o f e i t h e r a - a m y l a s e or c h y m o t r y p s i n o g e n A to t h e b a t h i n g m e d i u m . V a l u e s at t h i s t i m e ( t i m e z e r o ) h a v e b e e n s e t e q u a l to 100. N = 9 to 9 0 r a i n ; 6 at 1 0 5 r a i n ; a n d 7 at 1 2 0 rain.
40 ~
#'3o " -
g~
:,
~
!~
m ~ 6~°Io
". /"/'I'/./~/~ o•
•
..
j ~ - O
L 7-
~ .
•
200
4
400
i
I
t
600 800 IO00 Amylase Output (pg)
i
1200
_
_
1400
F i g . 7. E f f e c t o f a d d i n g 1 m g / m l C~-amylase t o t h e b a t h o n t h e r e l a t i v e p r o p o r t i o n s o f c h y m o t r y p s i n o g e n a n d a m y l a s e i n s e c r e t i o n . T h e c o n t r o l l i n e is c a l c u l a t e d f r o m 1 2 4 i n d i v i d u a l d a t a p o i n t s a n d i n c l u d e s d a t a from both time-paired parallel controls and the first hou~ of control secretion for the experimental glands. ...... , one standard deviation ± the mean slope for control points. Experimental points are from 0 to 5 0 r a i n a f t e r t h e a d d i t i o n o f a m y l a s e t o t h e b a t h a n d a r e f r o m s e v e n e x p e r i m e n t s . All o u t p u t s a r e f o r 1 0 rain sampling periods.
33!
much greater spread and when the variance about the two calculated functions (+amylase and control) was compared, the difference was considerable 2 2 (Samylase = 14.3 ; S c o n t r o l = 2.7; F = 5.3; P < 0.01). Thus, the addition of enzyme to the bath greatly reduced the covariance between the secretion of the two enzymes (Fig. 7). This change in covariance between the secretion of different enzymes is similar to that reported in other circumstances [18,19], and indicates that even though the average response was roughly parallel for the two enzymes, their secretion became more disconnected or independent of each other after amylase was added. Therefore, the parallelism does not indicate linked or obligatorily coupled transport such as in a vesicular release mechanism [20,21]. Discussion Amylase added to the medium bathing the rabbit pancreas in vitro produced a dramatic increase in amylase secretion, and likewise, the addition of chymotrypsinogen to the medium increased chymotrypsinogen secretion. To determine if it was the enzyme added to the medium t h a t was recovered in secretion, we incorporated labeled amino acids into endogenous amylase pools prior to the addition of exogenous amylase. We reasoned t h a t if the augmented secretion were of exogenous origin, then the specific radioactivity of secretion would decrease substantially and remain suppressed as long as the exogenous enzyme was in the bath. This was observed; the specific radioactivity of amylase in secretion was reduced by about 90% at the apparent steady state. In addition, adding a substantial a m o u n t of exogenous enzyme should inhibit the secretion of endogenous enzyme, if both exogenous and endogenous material mix and are transported into the duct by a c o m m o n mechanism and if the enzyme is present in the pre-secretory pool at concentrations which saturate or nearly saturated the transport system. The addition of exogenous amylase produced a sustained depression in the absolute secretion of labeled enzyme (by 72% on the average), while total amylase secretion into the duct was increased by about an order of magnitude. Furthermore, as we would expect if endogenous and exogenous amylase compete for exit sites, the inhibition of endogenous amylase secretion was not seen when relatively low concentrations of exogenous amylase were added, even though there was a substantial response in terms of amylase o u t p u t (Table II). The presence of a saturable transport mechanism is also shown by the observation t h a t varying the concentration of amylase in the medium did not produce a linear change in transpancreatic transport. That is, at the highest concentration of bath amylase, amylase o u t p u t was only about 1/3 of that predicted if bath concentration and secret o r y flux were linearly related (Table I). The overall secretory response was n o t maintained at maximal levels but decreased over time, despite the continued presence of enzyme in the medium at a constant concentration. The transient nature of the overall response may be the result of a transport-related substrate becoming depleted over time or a regulatory adaptation, but it can also be explained in terms of equilibrating fluxes if a back-flux from duct to cell develops slowly. This latter interpretation is consistent with the observation t h a t the inhibition of labeled amylase
332 secretion was maintained over time despite the declining overall secretory output (Figs. 1 and 4) which would be the case if the efflux remained maximal despite a decreased net flux. The observation that the addition of one enzyme to the bathing medium stimulated the secretion of other enzymes cannot wholly be accounted for by contamination of the added enzyme with other enzyme species. Relatively pure amylase and chymotrypsinogen both elicited secretory responses for the other enzyme roughly equivalent in time course and magnitude to that of their own transpancreatic transport (Fig. 6). Indeed, the chymotrypsinogen response to the addition of amylase was much greater and occurred more rapidly than the chymotrypsinogen response to the addition of chymotrypsinogen itself. Thus, there appears to be a kind of entrainment in the responses for the two enzymes which is not predicted by the equilibrium hypothesis in its simplest form, i.e. as in diffusion, the transport of different molecular species should segregate independently. On the other hand, this entrainment does not appear to be the result of the linked transport of the enzymes [20,21]. To the contrary, the addition of amylase decreased the covariance or relatedness of their secretion. Thus, the augmented secretion of secondary endogenous molecules appears to be an independent event, stimulated in some manner by the uptake and secretion of the exogenous enzyme. Acknowledgements This work was supported by Research Grant AM16990 and Training Grant GM00927 from the National Institutes of Health. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
L i e b o w , C. a n d R o t h m a n , S.S. ( 1 9 7 6 ) Biochim. Biophys. A c t a 4 5 5 , 2 4 1 - - 2 5 3 L i e b o w , C. a n d R o t h m a n , S.S. ( 1 9 7 4 ) Am. J. Physiol. 2 2 6 , 1 0 7 7 - - 1 0 8 1 I s e n m a n , L.D. a n d R o t h m a n , S.S. ( 1 9 7 7 ) Proc. Natl. Aead. Sci. U.S. 74, 4 0 6 8 - - 4 0 7 2 L i e b o w , C. a n d R o t h m a n , S.S. ( 1 9 7 5 ) Science 1 8 9 , 4 7 2 - - 4 7 4 R o t h m a n , S.S. ( 1 9 7 5 ) Science 190, 7 4 7 - - 7 5 3 Goetze, H. a n d R o t h m a n , S.S. ( 1 9 7 5 ) N a t u r e 2 5 7 , 6 0 7 - - 6 0 9 R o t h m a n , S.S. ( 1 9 6 4 ) N a t u r e 2 0 4 , 8 4 - - 8 5 Krebs, H.A. a n d Henseleit, K. ( 1 9 3 2 ) Z. Physiol. Chem. 2 1 0 , 3 3 - 6 6 Eagle, H. ( 1 9 5 9 ) Science 1 3 0 , 4 3 2 - - 4 8 7 R o t h m a n , S.S. a n d B r o o k s , F.P. ( 1 9 6 5 ) Am. J. Physiol. 2 0 8 , 1 1 7 1 - - 1 1 7 6 R e b e r , H.A. a n d Wolf, C.J. ( 1 9 6 8 ) Am. J. Physiol. 215, 3 4 - - 4 0 R i d d e r s t a p , A.S. a n d B o n t i n g , S.L. ( 1 9 6 9 ) A m . J. Physiol. 2 1 7 , 1 7 2 1 - - 1 7 2 4 S w a n s o n , C.H. a n d S o l o m o n , A.K. ( 1 9 7 3 ) J. Gen. Physiol. 6 2 , 4 0 7 - 4 2 9 Welch, R.W. a n d L i t t m a n , A. ( 1 9 7 4 ) J. Appl. Physiol. 37, 2 3 5 - - 2 3 8 R i n d e r k n e e h t , H., Wilding, P. a n d H a v e r b a c k , B.J. ( 1 9 6 7 ) E x p e r i e n t i a 23, 8 0 5 L o w r y , O.H., R o s e b r o u g h , N.J., Farr, A.L. a n d Randall, R.J. ( 1 9 5 1 ) J. Biol. Chem. 193, 2 6 5 - - 2 7 5 L o y t e r , A. a n d S c h r a m m , M. ( 1 9 6 2 ) Biochirn. Biophys. A c t a 65, 2 0 0 - - 2 0 6 Adelson, J.W. a n d R o t h m a n , S.S. ( 1 9 7 5 ) Am. J. Physiol. 2 2 9 , 1 6 8 0 - - 1 6 8 6 R o t h m a n , S.S. ( 1 9 7 6 ) A m . J. Physiol. 2 3 1 , 1 8 4 7 - - 1 8 5 1 J a m i e s o n , J.D. a n d Palade, G.E. ( 1 9 7 7 ) In: I n t e r n a t i o n a l Cell Biology (Brinkley, B.R. a n d Porter, K.R., eds.), pp. 3 0 8 - - 3 1 7 , R o c k e f e l l e r University Press, New Y o r k 21 Palade, G.E. ( 1 9 7 5 ) Science 1 8 9 , 3 4 7 - - 3 5 8 22 R o t h m a n , S.S. a n d I s e n m a n , L.D. ( 1 9 7 4 ) Am. J. Physiol. 2 2 6 , 1 0 8 2 - - 1 0 8 7
Biochimica et Biophysica Acta, 585 (1979) 321--332 © Elsevier/North-Holland Biomedical Press
BBA 28900
T R A N S P A N C R E A T I C T R A N S P O R T OF DIGESTIVE ENZYME
LOIS D. ISENMAN and S.S. ROTHMAN
Department of Physiology, University of California, San Francisco, San Francisco, CA 94143 (U.S.A.) (Received November 3rd, 1978)
Key words: Pancreatic transport; Digestive enzyme; Secretion
Summary When porcine a-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [3H]leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase by a b o u t 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, n o t only of the increased secretion of unlabeled enzyme, b u t also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition o f amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath b y a b o u t 20 times; it did n o t reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested b y the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrypsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction o f the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.
322
Introduction Equilibrating fluxes of digestive enzyme have been demonstrated across the zymogen granule membrane [1], the lumenal plasma membrane [2], and even the basolateral membrane [3,4] of the pancreatic acinar cell. These and other observations have led to the proposal that digestive enzyme in zymogen granules (and perhaps other intracellular storage pools) is in an equilibrium state with its own species in the cytoplasm and that the cytoplasmic pool of digestive enzyme is the immediate precursor pool for secretion across the apical (and basolateral) membrane of the acinar cell [5]. This has been termed the 'equilibrium' theory because it proposes that the transport of these proteins across 'secretory' membranes is an equilibrium-dependent process in that their movement is bidirectional and concentration dependent. The discovery of a basolateral permeability to digestive enzyme, prompted Liebow and one of us (S.S.R.) to suggest an 'enteropancreatic circulation' of digestive enzyme, akin to the enterohepatic circulation of bile salts, in which secreted digestive enzyme is absorbed into the blood from the intestine and recycled through the pancreas [4,6]. The evidence suggested a cellular transpancreatic route. The current study assesses the capacity of the pathway by examining the effect on ductal secretion of the addition of a substantial amount of amylase or chymotrypsinogen to the medium bathing the whole rabbit pancreas in culture. The results demonstrate a substantial flux of digestive enzyme across the acinar cell and confirm this prediction of the enteropancreatic circulation hypothesis. Also, as predicted by the equilibrium theory, the system appears to be responsive to mass action and enzyme entering the cell via the basolateral membrane apparently mixes with endogenous enzyme pools prior to secretion. Methods
Biological preparation. Glands were obtained from male, New Zealand white rabbits (2--3 kg b o d y weight). After an overnight fast (14--18 h), the animals were anesthetized with a mixture of allobarbital (0.34 mmol/kg b o d y weight), urethan (3.1 mmol/kg), and m o n o e t h y l u r e a (3.2 mol/kg), administered intraperitoneally. The pancreatic duct was cannulated in situ after a laparotomy, the pancreas removed from the animal, and m o u n t e d in a Plexiglass chamber by techniques described elsewhere [7]. The gland was bathed by 400 ml of HCOjbuffered Krebs-Henseleit medium [8] enriched with 5.5 mM glucose and a mixture of a m i n o acids [9]. The medium was gassed with 95% O2 and 5% CO2 and its temperature maintained at 30°C. The functional characteristics of this biological preparation have been extensively studied and are the subject of numerous publications [10--14]. Experimental procedure. After 1 h of incubation, the bathing medium was changed and control samples collected at either 10- or 20-min intervals (as specified) for an additional hour. At this time, either porcine a-amylase (Sigma, Type VI-A), the same material repurified (as described below), or 5 times crystallized bovine chymotrypsinogen A (Worthington Chemical Co.) was added to the bathing medium at specified concentrations. Secretion was then collected
323 at 10-min intervals for 1 h and at 15-rain intervals for an addition hour. Samples were assayed for either amylase activity alone or both chymotrypsinogen and amylase activity. In a number of preparations, 4 mCi L-[4,5-3H]leucine (specific radioactivity, 62 Ci/mmol) (Schwarz Mann Co.) was added to the batching medium and the gland was incubated in the radioactive leucine for 4 h before exogenous amylase was added. During this time, secretion was collected at hourly intervals for 3 h, followed by a 50 rain and then a 10 min collection. When exogenous amylase was added, secretion was collected at 10-min intervals for 1 h and at 15-min intervals during the subsequent hour. The incorporation of [3H]leucine into secreted amylase was followed by purifying amylase in all samples by glycogen precipitation as described below, and measuring its 3H content. In a number of experiments, after 2 h of incubation in chymotrypsinogen, acetyl-~-methylcholine chloride (1.0 mg/100 ml bath fluid) was added to the bathing medium and secretion collected at 10-rain intervals for an additional hour. Analytical techniques, s-Amylase activity was measured by following the hydrolysis of an amylose substrate labeled covalently with Remazol Brilliant Blue R (Amylose Azure, Calbiochem) [15]. To 0.5 ml of diluted secretion, equivalent to 1--2 pl of undiluted secretion, 4.5 ml of the substrate in 0.02 M sodium phosphate buffer (pH 7.0) containing 0.05 M NaC1 was added. Samples were incubated in a shaking water bath at 37°C and the reaction terminated at 15 min by the addition of 2 ml 0.1 N acetic acid. Samples were then filtered through No. 2 Whatman filter paper and the absorbance of the filtrate read at 595 nm. The absorbance was compared to a standard curve made to 2 times crystallized porcine ~-amylase (506 I.U./mg) (Worthington Chemical Co.). Chymotrypsinogen was measured after the activation of a small sample of secretion (10--50 pl) in 0.05 M sodium phosphate buffer (pH 6.0) containing 0.5 mg/ml enteropeptidase (EC 3.4.21.9) (Miles Laboratories) which was incubated for 30 rain at 37°C. Immediately following incubation, the activation mixture was added to 1.0 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 3.0 ml of the substrate (80 mM suspension of aeetyl tyrosine ethyl ester in 30% methanol) and the initial rate of liberation of H* measured at pH 7.4 using a pH-stat technique. The presence of c h y m o t r y p t i c activity was related to sample size linearly over the range used. The protein content of samples was estimated using the Folin phenol reagent
[161. Amylase was purified from secretion by precipitation with shellfish glycogen [17]. 25--50 ul of juice (containing between approximately 25 and 315 pg of amylase) was added to 1 ml of 40% ethanol at 4°C, and then 50 ~l of 0.2 M sodium phosphate buffer (pH 8.0), 50 pl of 2% shellfish glycogen and 70 pl 95% ethanol were added in sequence and the mixture agitated for 5 min at 4°C. The mixture was centrifuged at 2000 × g for 6 min and the pellet resuspended in 1 ml of 40% cold ethanol containing 0.01 M sodium phosphate buffer (pH 8.0). The wash procedure was repeated three times and the final pellet suspended in 1 ml H20. 100 pg of amylase was added to control samples which contained smaller amounts of amylase to roughly equate the a m o u n t of
324 amylase being purified between control and experimental samples. Over the range of amylase sample size u s e d , the yield of amylase recovered by glycogen precipitation was between 75 and 100% and no systematic variation in yield was observed. This purification technique increased amylase specific activity by between 5- and 10-fold depending upon the specific activity of amylase in the original secretory sample. Porcine a-amylase (Sigma, Type VLA) was repurified by a similar procedure. 750 mg of amylase was added to 1 1 of 0.01 M sodium phosphate buffer (pH 8.0) in 40% ethanol at room temperature and then cooled to 4°C. All the following procedures were carried o u t at 4°C. 1 g shellfish glycogen was added followed by the volume of 95% ethanol necessary to return the ethanol concentration to 40%. The mixture was agitated for 5 min and then centrifuged at 5000 X g for 5 min. The pellet was resuspended first in one volume of the buffered ethanol solution, respun and then suspended again in 0.5 volume of the same solution for a final spin. The final pellet was suspended in 10 ml of KrebsHenseleit medium and left at room temperature for 1 h. The procedure produced a yield of almost 100% of the added amylase and specific activity was increased by approximately 5-fold. Amylase accounted for some 20% of the protein in the original material, the rest presumably being primarily other digestive enzymes. We estimated chymotrypsinogen contamination in the original material, and assuming that its specific activity is roughly the same as 5 times purified bovine chymotrypsinogen (Worthington Chemical Co.), chymotrypsinogen accounted for a b o u t 10% of its protein content. Chymotrypsinogen contamination of the repurified amylase was only a b o u t 0.07%. Results
The effect o f a-amylase added to the bathing medium on amylase secretion When 1 mg/ml amylase (Sigma, Type VI-A; containing approximately 1/3 protein by weight and amylase at a concentration o f 67 t~g/ml) was added to the medium bathing the in vitro pancreas, the amylase content of ductal secretion increased rapidly and dramatically. The response peaked approximately 20 min after the enzyme was added at an average amylase o u t p u t of 11 times the unstimulated (basal) level (Fig. 1), or 15 + 3 (S.E.) times basal when each preparation was normalized to its own control. This effect was equivalent to approximately 20% of the amylase ductal o u t p u t at the peak of a maximum cholinergic response for this in vitro system. The amylase content in ductal secretion returned towards its original level over time, although bath amylase concentration remained essentially unchanged. When the dose of exogenous amylase was varied, 0.1, 0.2, as compared to 1.0 mg/ml o f the Sigma material, the response did n o t increase linearly with concentration, as predicted for a non-saturating system, b u t appeared to reach a maximum value between 0.2 and 1.0 mg/ml (Table I). When repurified amylase was used instead of the crude material, the response was smaller, although still quite substantial (6.1 ± 1.6 (S.E.) times prior control values (n = 4), or a b o u t half the magnitude of the response produced by the crude material). Thus, amylase secretion apparently was in part stimulated by other molecules in the crude amylase. We tested for the possibility that small
325
8°° I 700 [ "~ 600 I
sooI •~- 400
~3
• ~ 300 v~
200
100 0
L 10
20
. 30
. 40. .50 . 60 . . 70.
80
90
100 1 0
TIME (rain)
F i g . 1. E f f e c t o f t h e a d d i t i o n o f 1 m g / m l a - a m y l a s e t o t h e b a t h o n t h e d u c t a l s e c r e t i o n o f a m y l a s e . D a t a p r e s e n t e d as m e a n a m y l a s e o u t p u t / 1 0 rain (+-S.E.). V a l u e at t i m e z e r o is o u t p u t f o r 1 0 rain c o n t r o l p e r i o d prior t o a d d i t i o n o f a - a m y l a s e . N = 1 1 , 1 3 , 1 3 , 1 2 , 1 2, 1 1 , 9 , 8, 5, a n d 5 f r o m 0 t o 1 0 5 rain.
compounds were responsible by dialyzing the Sigma material against KrebsHenseleit medium for 24 h at 4°C before adding it to the pancreas preparation. The secretory response was undiminished as a result of dialysis. Therefore, that portion of the amylase response not elicited by amylase itself was apparently due to the presence of other large molecules, probably other digestive enzymes, in the mixture. Evidence relating to this point will be presented below. The effect o f chymotrypsinogen added to the bath on the secretion of chymotrypsinogen When 1 mg/ml of a relatively pure chymotrypsinogen (5 times crystallized bovine chymotrypsinogen A, Worthington Chemical Co.) (amylase contamination based on an assumed specific activity equal to 2 times crystallized porcine a-amylase (specific activity, 506 I.U./mg) was less than 0.004%) was added to the medium, chymotrypsinogen output in ductal secretion increased. The response developed more slowly than when amylase was added and peaked at 60 min (Fig. 2). When each experiment was normalized to its own control, for the peak period chymotrypsinogen output was approximately 7.5 -+ 2.5 (S.E.)
TABLE
I
THE EFFECT OF VARYING CONCENTRATIONS BIT P A N C R E A S IN V I T R O O N T H E S E C R E T I O N
OF AMYLASE IN T H E M E D I U M OF AMYLASE INTO THE DUCT
BATHING
RAB-
Amylase used was Sigma Type VI-A. [Amylase]bath (mg/ml)
0.1 0.2 1.0
n
3 3 10
Amylase output at peak period (10--20 rain) (#g/10 rain) Observed
Predicted for a non-saturating system
300 * 550 950
(300) 600 3000
* T h i s v a l u e is s o m e 5 t i m e s basal a m y l a s e o u t p u t w h i c h a v e r a g e d 6 4 _+ 1 2 ( S . E . ) in t h e 1 0 rain c o n t r o l p e r i o d prior t o t h e a d d i t i o n o f a m y l a s e t o t h e m e d i u m ( n = 1 6 ) .
326
80
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E
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~=
6.0
z~
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4.0
~®~E
3.o
~-
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o_JL 10
20
.
.
30
.
.
40
.
.
.
.
.
.
.
50 60 70 TIME [rnin)
.
.
80
.
.
.
90 100 110
F i g . 2. E f f e c t o f t h e a d d i t i o n o f 1 m g / m l c h y m o t r y p s i n o g e n A to the bath on the ductal secretion of chymotrypsinogen. Data are mean chymotrypsinogen o u t p u t / l O r a i n (-+S.E.) m i n u s t h e m e a n b a s a l s e c r e t o r y o u t p u t o f 2 . 6 6 # t o o l s u b s t r a t e s p l i t / r a i n p e r 1 0 r a i n ( w h i c h is t h e v a l u e f o r t h e 1 0 r a i n c o n t r o l p e r i o d prior to the addition of chymotrypsinogen A (time zero on the graph)). N = 9 from 0 to 99 rain; 6 at 105 rain; and 7 at 120 min.
times the unstimulated control output (Fig. 6). The magnitude of the response was equal to approximately 30% of the chymotrypsinogen output at the peak of a maximum cholinergic response for this in vitro system (Fig. 5).
The effect of exogenous a-amylase on the secretion of endogenous amylase To determine if it was the enzyme that was added to the medium that appeared in secretion, we labeled endogenous digestive enzyme pools with radioactive leucine for 4 h prior to the addition of the exogenous enzyme and followed the specific radioactivity of secreted amylase. Transpancreatic transport of the unlabeled, exogenous enzyme should lower the specific radioactiv-
E: 9o o
8o
O Z0 ~ :- 70 ~> ~ ._o
=~
6o
o_~o ~o ~UJO
u~u~ ~ " 6~
40
20 ,
i
J
0
0 TIME (min)
Fig. 3. E f f e c t o f t h e a d d i t i o n o f :[ m g / m l u n l a b e l e d c~-amylase t o t h e b a t h a f t e r a 4 h i n e u h a t i o n w i t h [3H]leucine, on the specific radioactivity of amylase in ductal secretion. [3H]beucine was added at time z e r o . S p e c i f i c a c t i v i t y i s e x p r e s s e d a s t h e r a t i o o f m e a n a m y l a s e c p m (n = 8 4 2 4 0 r a i n a n d p o s t - s t i m u l u s p o i n t s a s i n F i g . 4 ) t o m e a n a m y l a s e o u t p u t ( n = 8 o r g r e a t e r f o r all o u t p u t p o i n t s e x c e p t a t 3 3 0 a n d 3 4 5 w h e r e n = 5).
327
ity of amylase in secretion. When amylase was added to the bath, the specific radioactivity of this e n z y m e in secretion did drop precipitously. 10 min after the addition of unlabeled enzyme to the medium, the specific radioactivity of amylase in ductal secretion was reduced by 82%; and by 60 min it was only 7% of control values (Fig. 3). The specific radioactivity remained depressed for the duration of the experiment, despite the fact that over time amylase o u t p u t fell back toward control levels (Fig. 1 and 3). Thus, even as the response declined, about 90% of the secreted amylase was unlabeled and apparently of exogenous origin. In addition, the presence of amylase in the bath altered the secretion of endogenous amylase. Labeled amylase secretion increased transiently (by 72% on the average) and was followed by a sustained inhibition (Fig. 4). The transient increase was quite variable and ranged from +0 to +250%, accounting for the large standard error seen for these periods (Fig. 4). By 60 min, amylase radioactivity in secretion had declined to 28% -+ 6 (S.E.) of prior control levels where it stayed for the remainder of the experiment (Fig. 4). Inhibition of labeled amylase secretion was not seen when either 0.1 or 0.2 mg/ml amylase was used and only a slight inhibition was observed when amylase was present at a concentration of 0.3 mg/ml (concentrations refer to weight of Sigma, Type VI-A amylase) (Table II) * The inhibition of endogenous amylase secretion by the addition of amylase to the bathing medium suggests competitive transport inhibition between exogenous and endogenous e n z y m e and hence, along with earlier studies [4], suggests that the enzyme moves through the acinar cell. The fact that enzyme movement is through the acinar cell rather than via paracellular shunts is also supported by the observation that the addition of amylase to the bathing medium increased the duct to bath concentration difference for this enzyme. If a diffusional equilibration through paracellular shunts were all that were involved, the concentration difference should have been reduced. At the peak of the response, the amylase concentration in the duct averaged 6463 I.U./ml (with a range of 1457--18 155 I.U./ml), whereas bath amylase was approximately 61 I.U./ml (Table III). This concentration difference (6402 I.U./ml) is 14 times that of the control situation (455 I.U./ml; range 46--1822 I.U./ml) * It has b e e n s h o w n b y l a b e l i n c o r p o r a t i o n s t u d i e s t h a t t h e r e are m u l t i p l e i n t r a c e l l u l a r p o o l s o f digestive e n z y m e w i t h i n t h e a c i n a r cell. W h e n t h e in v i t r o r a b b i t p a n c r e a s is s t i m u l a t e d w i t h a c h o l i n e r g i c a g o n i s t a f t e r a 2 h i n c u b a t i o n w i t h [ 3 H ] l e u c i n e , t h e specific r a d i o a c t i v i t y of digestive e n z y m e in s e c r e t i o n d r o p s p r e c i p i t o u s l y , i n d i c a t i n g t h e p r e f e r e n t i a l s e c r e t i o n o f old o r u n l a b e l e d p r o t e i n . A similar b u t less d r a m a tic e f f e c t c a n b e seen a t 4 h, a n d in o n e p u b l i s h e d e x a m p l e [ 2 2 ] , t h e specific r a d i o a c t i v i t y o f t o t a l protein was r e d u c e d b y 35% w i t h c h o l i n e r g i c s t i m u l a t i o n at this t i m e , a n d specific r a d i o a c t i v i t y r e t u r n e d to p r e - s t i m u l u s levels b y 6 0 m i n . I t is possible t h a t t h e results d e s c r i b e d h e r e are d u e t o a similar p h e n o m e n o n , i.e. t h e a d d i t i o n o f a - a m y l a s e to t h e b a t h s t i m u l a t e s t h e release of less h i g h l y l a b e l e d e n d o g e n o u s stores. This s e e m s u n l i k e l y f o r t h e f o l l o w i n g reasons: (1) a m y l a s e a d d e d t o t h e b a t h at 4 h p o s t [ 3 H ] l e u c i n e p r o d u c e d a m u c h l a r g e r d e c r e a s e in specific r a d i o a c t i v i t y ( 9 5 % ) t h a n a cholinerRic d r u g a d d e d a t t h e s a m e t i m e ; (2) u n l i k e c h o l i n e r g i c s t i m u l a t i o n , t h e d e p r e s s i o n in specific r a d i o a c t i v i t y was s u s t a i n e d o v e r t i m e in t h e c o n t i n u e d p r e s e n c e o f t h e e n z y m e ; (3) t h e a m o u n t of l a b e l e d p r o t e i n in secretion after cholinergic stimulation at 4 h only decreased very transiently, and t he n increased b y over an order of m a g n i t u d e within 10 min. In contrast, amylase added to the bath p r o d u c e d a transient increase a n d a s t e a d y - s t a t e i n h i b i t i o n o f a b o u t 70% (Fig. 4); (4) i n c u b a t i n g a g l a n d f o r 4 h w i t h a c h o l i n e r g i c s t i m u l a n t a l o n g w i t h [ 3 H ] l e u c i n e in o r d e r t o d e p l e t e cold p o o l s did n o t c h a n g e t h e p a t t e r n o f t h e r e s p o n s e w h e n e x o g e n o u s a m y l a s e was a d d e d , i.e. s u s t a i n e d i n h i b i t i o n o f l a b e l e d a m y l a s e s e c r e t i o n was still seen.
328 100
oz so
~
60
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20
;
20 20 6`0 80 ~;o TIME (rain)
Fig. 4. E f f e c t o f the a d d i t i o n o f 1 m g / m l C~-amylase to t h e b a t h on the a m o u n t o f r a d i o a c t i v e a m y l a s e in ductal s e c r e t i o n . D a t a are e x p r e s s e d as m e a n c p m in a m y l a s e / 1 0 rain (-+S.E.). V a l u e at t i m e z e r o is c p m in a m y l a s e in s e c r e t i o n for 10 rain c o n t r o l prior t o t h e a d d i t i o n of ~ - a m y l a s e to the b a t h and 2 3 0 - - 2 4 0 m i n a f t e r t h e a d d i t i o n o f [ 3 H ] l e u c i n e (see Fig. 3). N = 8 - - 4 0 rain; 7 at 5 0 a n d 6 0 m i n ; a n d 3 at 75, 90, and 1 0 5 rain.
(Table III), or about 20 times control periods when each experiment was paired to its own prior control (Table III). A cellular route is also suggested by experiments in which a cholinergic drug was added to the bathing medium 2 h after the addition o f chymotrypsinogen A. Under these circumstances, the chymotrypsinogen response to the cholinergic drug was 3.3 times greater than that seen when the cholinergic agent was added in the absence of chymotrypsinogen (Fig. 5).
The effect o f exogenous amylase and chymotrypsinogen on the secretion o f other enzymes The addition of crude amylase (Sigma, Type VI-A) to the bathing medium stimulated the secretion of chymotrypsinogen, as well as amylase (Fig. 6), and the increase was of the same magnitude. Indeed, overall protein secretion was also increased in similar proportions which suggests that the secretion of other T A B L E II P E R C E N T A G E C H A N G E I N S E C R E T I O N O F L A B E L E D A M Y L A S E 6 0 rain A F T E R T H E A D D I T I O N OF V A R Y I N G C O N C E N T R A T I O N S OF E X O G E N O U S A M Y L A S E TO T H E MEDIUM M e a s u r e m e n t s w e r e m a d e 5 h a f t e r t h e a d d i t i o n o f [ 3 H ] l e u c i n e . A m y l a s e u s e d w a s Sigma T y p e VI-A. [ Amylase ] bath (mg/ml)
P e r c e n t a g e c h a n g e in l a b e l e d a m y l a s e s e c r e t i o n , 60 m i n a f t e r the a d d i t i o n o f a m y l a s e t o t h e b a t h i n g m e d i u m (relative t o P r e t r e a t m e n t c o n t r o l , - - 1 0 r a i n )
0.1 0.2 0.3 1.0
+58 +41 --16 --72 *
* See Fig. 4.
329
TABLE III T H E E F F E C T OF 1 m g / m l E X O G E N O U S A M Y L A S E ( S I G M A T Y P E V I - A ) ON [ A M Y L A S E ] d u c t [AMYLASE]bath" Experiment
1 2 3 4 5 6 7 8 9 10 11 12 S.E.
Amylase concentration difference (duct--bath) (I.U./ml) A. No a m y l a s e ( 1 0 rain p r i o r to + a m y l a s e )
B. A m y l a s e a d d e d (peak values)
+a~nylase/--amylase (B/A)
364 131 506 430 556 273 1822 146 46 349 759 86
4 1 6 6 7 8 11 1 1 4 18 6
190 862 315 011 833 136 577 457 558 043 155 416
11.5 14.2 12.5 14.0 14.1 29.8 6.4 10.1 34.0 11.6 23.9 74.6
6 463 1 374
21.4 5.4
455 138
enzymes was augmented as well (evidence n o t presented here). The removal of other digestive enzymes from the impure amylase by its repurification did n o t eliminate this effect and roughly parallel, although diminished, secretion o f the t w o enzymes was still observed even though only very small amounts of chymotrypsinogen were added to the bathing medium under these conditions (see Methods) (for the period of peak response when repurified amylase was added, the increase over controls was 7.5 times for amylase and 9.5 times for chymotrypsinogen in one experiment, and 7.9 times for amylase and 5.3 times for chymotrypsinogen in another). Similarly, when the relatively pure chymotrypsinogen was added, the secretion of amylase was augmented in a fashion roughly equivalent to that seen for chymotrypsinogen (a maximum o u t p u t of some 6--7 fold control rates; Fig. 6). Thus, the addition of one enzyme to the medium apparently stimulated the secretion of other enzymes and in a grossly parallel manner. Since the Sigma VI-A amylase is contaminated with other enzymes, this is probably the reason why the crude amylase evoked a larger amylase response than the repurified amylase itself. While the outputs of the two enzymes were more or less parallel when means were compared, a more detailed analysis of the experimental data indicated that the responses were not identical, it could be seen that the addition of amylase to the medium altered the distribution of points, curiously skewing the data towards a more chymotrypsinogen-dominant secretion (Fig. 7). The broken line in Fig. 7 represents one standard deviation either plus or minus the mean slope for control data and includes approximately 2/3 of the control points. On the other hand, 64% of the amylase-treated points were greater than 1 S.D. above the mean line, i.e. were chymotrypsinogen dominant relative to controls. The +amylase points (i.e. amylase added to the bath) also show a
330
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Fig. 5. E f f e c t o f a e e t y l - ~ - m e t h y l c h o l i n e c h l o r i d e (1 r a g / 1 0 0 m l b a t h v o l u m e ) o n e h y m o t r y p s i n o g e n o u t p u t w i t h a n d w i t h o u t p r i o r e x p o s u r e (2 b) to 1 m g / m l b o v i n e c h y m o t r y p s i n o g e n A in b a t h i n g m e d i u m . A c e t y l - f l - m e t h y l c h o l i n e c h l o r i d e w a s a d d e d a t t i m e z e r o . N = 5 to 40 rain; 3 at 40 a n d 50 m i n i a n d 4 at 50 and 60 rain for prior exposure condition. N = 7 for control data. The difference between chymotryps i n o g e n p r e - t r e a t e d a n d c o n t r o l g r o u p s w a s P < 0 . 0 1 at 0 - - 1 0 r a i n ( t w o - t a i l e d t-test), a n d P < 0 . 0 5 at 1 0 - 20 rain ( M a n n - W h i t n e y r a n k t e s t ) . Fig. 6. E f f e c t o f a d d i n g 1 m g / m l a - a m y l a s e to t h e b a t h o n t h e s e c r e t i o n o f e h y m o t r y p s i n o g e n a n d a m y lase, a n d t h e e f f e c t o f a d d i n g 1 m g / m l c h y m o t r y p s i n o g e n A to t h e b a t h o n t h e s e c r e t i o n o f a m y l a s e a n d c h y m o t r y p s i n o g e n . D a t a are n o r m a l i z e d f o r e a c h i n d i v i d u a l e x p e r i m e n t to a 10 r a i n c o n t r o l p e r i o d p r i o r to t h e a d d i t i o n o f e i t h e r a - a m y l a s e or c h y m o t r y p s i n o g e n A to t h e b a t h i n g m e d i u m . V a l u e s at t h i s t i m e ( t i m e z e r o ) h a v e b e e n s e t e q u a l to 100. N = 9 to 9 0 r a i n ; 6 at 1 0 5 r a i n ; a n d 7 at 1 2 0 rain.
40 ~
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F i g . 7. E f f e c t o f a d d i n g 1 m g / m l C~-amylase t o t h e b a t h o n t h e r e l a t i v e p r o p o r t i o n s o f c h y m o t r y p s i n o g e n a n d a m y l a s e i n s e c r e t i o n . T h e c o n t r o l l i n e is c a l c u l a t e d f r o m 1 2 4 i n d i v i d u a l d a t a p o i n t s a n d i n c l u d e s d a t a from both time-paired parallel controls and the first hou~ of control secretion for the experimental glands. ...... , one standard deviation ± the mean slope for control points. Experimental points are from 0 to 5 0 r a i n a f t e r t h e a d d i t i o n o f a m y l a s e t o t h e b a t h a n d a r e f r o m s e v e n e x p e r i m e n t s . All o u t p u t s a r e f o r 1 0 rain sampling periods.
33!
much greater spread and when the variance about the two calculated functions (+amylase and control) was compared, the difference was considerable 2 2 (Samylase = 14.3 ; S c o n t r o l = 2.7; F = 5.3; P < 0.01). Thus, the addition of enzyme to the bath greatly reduced the covariance between the secretion of the two enzymes (Fig. 7). This change in covariance between the secretion of different enzymes is similar to that reported in other circumstances [18,19], and indicates that even though the average response was roughly parallel for the two enzymes, their secretion became more disconnected or independent of each other after amylase was added. Therefore, the parallelism does not indicate linked or obligatorily coupled transport such as in a vesicular release mechanism [20,21]. Discussion Amylase added to the medium bathing the rabbit pancreas in vitro produced a dramatic increase in amylase secretion, and likewise, the addition of chymotrypsinogen to the medium increased chymotrypsinogen secretion. To determine if it was the enzyme added to the medium t h a t was recovered in secretion, we incorporated labeled amino acids into endogenous amylase pools prior to the addition of exogenous amylase. We reasoned t h a t if the augmented secretion were of exogenous origin, then the specific radioactivity of secretion would decrease substantially and remain suppressed as long as the exogenous enzyme was in the bath. This was observed; the specific radioactivity of amylase in secretion was reduced by about 90% at the apparent steady state. In addition, adding a substantial a m o u n t of exogenous enzyme should inhibit the secretion of endogenous enzyme, if both exogenous and endogenous material mix and are transported into the duct by a c o m m o n mechanism and if the enzyme is present in the pre-secretory pool at concentrations which saturate or nearly saturated the transport system. The addition of exogenous amylase produced a sustained depression in the absolute secretion of labeled enzyme (by 72% on the average), while total amylase secretion into the duct was increased by about an order of magnitude. Furthermore, as we would expect if endogenous and exogenous amylase compete for exit sites, the inhibition of endogenous amylase secretion was not seen when relatively low concentrations of exogenous amylase were added, even though there was a substantial response in terms of amylase o u t p u t (Table II). The presence of a saturable transport mechanism is also shown by the observation t h a t varying the concentration of amylase in the medium did not produce a linear change in transpancreatic transport. That is, at the highest concentration of bath amylase, amylase o u t p u t was only about 1/3 of that predicted if bath concentration and secret o r y flux were linearly related (Table I). The overall secretory response was n o t maintained at maximal levels but decreased over time, despite the continued presence of enzyme in the medium at a constant concentration. The transient nature of the overall response may be the result of a transport-related substrate becoming depleted over time or a regulatory adaptation, but it can also be explained in terms of equilibrating fluxes if a back-flux from duct to cell develops slowly. This latter interpretation is consistent with the observation t h a t the inhibition of labeled amylase
332 secretion was maintained over time despite the declining overall secretory output (Figs. 1 and 4) which would be the case if the efflux remained maximal despite a decreased net flux. The observation that the addition of one enzyme to the bathing medium stimulated the secretion of other enzymes cannot wholly be accounted for by contamination of the added enzyme with other enzyme species. Relatively pure amylase and chymotrypsinogen both elicited secretory responses for the other enzyme roughly equivalent in time course and magnitude to that of their own transpancreatic transport (Fig. 6). Indeed, the chymotrypsinogen response to the addition of amylase was much greater and occurred more rapidly than the chymotrypsinogen response to the addition of chymotrypsinogen itself. Thus, there appears to be a kind of entrainment in the responses for the two enzymes which is not predicted by the equilibrium hypothesis in its simplest form, i.e. as in diffusion, the transport of different molecular species should segregate independently. On the other hand, this entrainment does not appear to be the result of the linked transport of the enzymes [20,21]. To the contrary, the addition of amylase decreased the covariance or relatedness of their secretion. Thus, the augmented secretion of secondary endogenous molecules appears to be an independent event, stimulated in some manner by the uptake and secretion of the exogenous enzyme. Acknowledgements This work was supported by Research Grant AM16990 and Training Grant GM00927 from the National Institutes of Health. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
L i e b o w , C. a n d R o t h m a n , S.S. ( 1 9 7 6 ) Biochim. Biophys. A c t a 4 5 5 , 2 4 1 - - 2 5 3 L i e b o w , C. a n d R o t h m a n , S.S. ( 1 9 7 4 ) Am. J. Physiol. 2 2 6 , 1 0 7 7 - - 1 0 8 1 I s e n m a n , L.D. a n d R o t h m a n , S.S. ( 1 9 7 7 ) Proc. Natl. Aead. Sci. U.S. 74, 4 0 6 8 - - 4 0 7 2 L i e b o w , C. a n d R o t h m a n , S.S. ( 1 9 7 5 ) Science 1 8 9 , 4 7 2 - - 4 7 4 R o t h m a n , S.S. ( 1 9 7 5 ) Science 190, 7 4 7 - - 7 5 3 Goetze, H. a n d R o t h m a n , S.S. ( 1 9 7 5 ) N a t u r e 2 5 7 , 6 0 7 - - 6 0 9 R o t h m a n , S.S. ( 1 9 6 4 ) N a t u r e 2 0 4 , 8 4 - - 8 5 Krebs, H.A. a n d Henseleit, K. ( 1 9 3 2 ) Z. Physiol. Chem. 2 1 0 , 3 3 - 6 6 Eagle, H. ( 1 9 5 9 ) Science 1 3 0 , 4 3 2 - - 4 8 7 R o t h m a n , S.S. a n d B r o o k s , F.P. ( 1 9 6 5 ) Am. J. Physiol. 2 0 8 , 1 1 7 1 - - 1 1 7 6 R e b e r , H.A. a n d Wolf, C.J. ( 1 9 6 8 ) Am. J. Physiol. 215, 3 4 - - 4 0 R i d d e r s t a p , A.S. a n d B o n t i n g , S.L. ( 1 9 6 9 ) A m . J. Physiol. 2 1 7 , 1 7 2 1 - - 1 7 2 4 S w a n s o n , C.H. a n d S o l o m o n , A.K. ( 1 9 7 3 ) J. Gen. Physiol. 6 2 , 4 0 7 - 4 2 9 Welch, R.W. a n d L i t t m a n , A. ( 1 9 7 4 ) J. Appl. Physiol. 37, 2 3 5 - - 2 3 8 R i n d e r k n e e h t , H., Wilding, P. a n d H a v e r b a c k , B.J. ( 1 9 6 7 ) E x p e r i e n t i a 23, 8 0 5 L o w r y , O.H., R o s e b r o u g h , N.J., Farr, A.L. a n d Randall, R.J. ( 1 9 5 1 ) J. Biol. Chem. 193, 2 6 5 - - 2 7 5 L o y t e r , A. a n d S c h r a m m , M. ( 1 9 6 2 ) Biochirn. Biophys. A c t a 65, 2 0 0 - - 2 0 6 Adelson, J.W. a n d R o t h m a n , S.S. ( 1 9 7 5 ) Am. J. Physiol. 2 2 9 , 1 6 8 0 - - 1 6 8 6 R o t h m a n , S.S. ( 1 9 7 6 ) A m . J. Physiol. 2 3 1 , 1 8 4 7 - - 1 8 5 1 J a m i e s o n , J.D. a n d Palade, G.E. ( 1 9 7 7 ) In: I n t e r n a t i o n a l Cell Biology (Brinkley, B.R. a n d Porter, K.R., eds.), pp. 3 0 8 - - 3 1 7 , R o c k e f e l l e r University Press, New Y o r k 21 Palade, G.E. ( 1 9 7 5 ) Science 1 8 9 , 3 4 7 - - 3 5 8 22 R o t h m a n , S.S. a n d I s e n m a n , L.D. ( 1 9 7 4 ) Am. J. Physiol. 2 2 6 , 1 0 8 2 - - 1 0 8 7
