295

Biochimica et Biophysica Acta, 583 (1979) 295--308 © Elsevier/North-Holland Biomedical Press

BBA 28840

AMYLASE RELEASE F R O M R A T P A R O T I D GLANDS I. G E N E R A L CHARACTERISTICS

J.C. K U S E K

Department of Pharmacology, University of Illinois,College of Medicine, Chicago, IL (U.S.A.) (Received August 29th, 1978) Key words: Amylase release; Stimulus-secretion coupling; Ca2+;Energy dependence; Cyclic AMP; (Parotid gland)

Summary The involvement of calcium, ATP, and cyclic AMP-dependent protein kinase activity in the release of amylase from rat parotid glands was examined. Pretreatment of the glandular tissue in 11.25 mM Ca 2÷ medium potentiated the secretory responses to: dibutyryl cyclic AMP, elevation of the extracellular K ÷ concentration, reduction of the tC concentration, La 3÷, and caffeine. Uncoupling of oxidative phosphorylation blocked release induced b y dibutyryl cyclic AMP, K ÷, and reduction of IF, b u t had no effect on La 3÷, caffeine or tolbutamide~stimulated release. Inhibition of cyclic AMP-dependent protein kinase activity blocked only dibutyryl cyclic AMP-induced release and did not inhibit the responses to K ÷, reduction of IF or caffeine. The loss of lactate dehydrogenase was used to access the integrity of the tissue during amylase release. No significant increase in the release of lactate dehydrogenase was observed during the secretory responses to: dibutyryl cyclic AMP, La 3÷, caffeine, or tolbutamide. Triton X-100 and ethanol increased the efflux o f both amylase and lactate dehydrogenase. The differential involvement o f Ca 2÷, ATP, and cyclic AMP-dependent protein kinase activity in amylase release induced by the various secretagogues suggests that three types of reactions are involved in the release of amylase. Introduction The physiologic secretion of proteins stored in the secretory granules of salivary glands involves a series of cellular events initiated by neural or horA b b r e v i a t i o n : Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

296 monal stimulation, and culminating in the fusion of the granular and plasma membranes and the release of the granular contents by exocytosis [1,2]. Although the intermediary events in the process of stimulus-secretion coupling have not been fully delineated, two intracellular messengers, cyclic 3',5'-adenosine monophosphate (cyclic AMP) and calcium, appear to be involved. Cyclic AMP acts as an intracellular messenger mediating/3-adrenergic effects in tissues [ 3 ]. Amylase release from rat parotid glands is strongly stimulated by agents with f~-agonist activity [4--7] and by dibutyryl cyclic AMP [7--9]. Tolbutamide, an inhibitor of a cyclic AMP-dependent protein kinase activity, reduces the secretory response to/Ladrenergic stimulation [10]; and theophyllin, an inhibitor of phosphodiesterase, stimulates amylase release and potentiates the response to fl-adrenergic stimulation [8]. The release of amylase in response to fl-agonists does not appear to require extracellular calcium [2,4,11,12]. Extracellular Ca 2÷ is required for stimulation of parotid glands by a-adrenergic agonists, cholinergic agonists, and undecapeptides. These substances produce K ÷ efflux and cause a slight increase in amylase release [2,13--17]. A-23187, an ionophore which facilitates calcium movement across membranes, mimics the effects of a-adrenergic and cholinergic agonists and undecapeptides in the presence of extracellular Ca2÷ [18,19]. It has been postulated that calcium acts as the intracellular second messenger mediating the responses to a-adrenergic and cholinergic agonists and undecapeptides, while cyclic AMP is the second messenger in ~-adrenergic responses [20]. However, depletion of tissue calcium by prolonged incubation with a calcium-chelating agent reduces the secretory response to dibutyryl cyclic AMP [21]. Although such drastic treatment is open to multiple interpretations, the suggestion that intraceUular calcium may play a role in H-induced release has yet to be resolved. Furthermore, the point of involvement of cyclic AMP in the process of amylase release is not known. The objective of the experiments reported in this communication was to examine the involvement of calcium, cyclic AMP, and ATP in the release of amylase from rat parotid glands in an attempt to clarify their roles and identify intermediate events in the process of stimulus-secretion coupling. A superfusion technique was used which allowed determination of the rate of release, and which facilitated the determination of concentration-response curves to stimulants of release. Methods Male Holtzman rats, 200--300 g, were fasted overnight but allowed water ad libitum. T h e animals were anesthetized with 50 mg/kg sodium pentobarbital, intraperitoneaUy, and exanguinated by severing the arch of the aorta. The parotid glands were removed bilaterally, placed in oxygenated superfusion medium, cleaned of connective tissue, and cut into slices of 0.5--1 mm thickness and 2--4 mm 2 area. Slices were randomly distributed and equilibrated by superfusion for 45 min prior to the start of an experiment. The superfusion apparatus consisted of multiple tissue holders, each with a manifold for changing the superfusion medium which was delivered at a rate of 1 ml/min via a 12~hannel peristaltic

297 pump. Since the spontaneous rate of amylase release and the magnitude of stimulated release varied among preparations, some tissue samples (usually three) served as controls, and a similar number of samples were used as experimentals. The composition of the basic superfusion medium was: 140 mM Na ÷, 5 mM K ÷, 1 mM Mg2+, 1.25 mM Ca 2÷, 149.5 mM CI-, 5 mM Hepes, 5 mM Bistris, and 10 mM glucose. In experiments in which the K ÷ concentration was increased from 5 to 105 mM, the Na ÷ concentration was reduced from 140 to 40 mM. Glucose was omitted from the medium when the effect of dinitrophenol was tested. Addition of drugs was made directly to the medium except for isoproterenol which was infused into the medium at the base of the manifold via a calibrated syringe pump. The media were oxygenated b y bubbling with 100% 02. All experiments were performed at 21--22°C. The pH of the medium was 6.5 in all experiments unless otherwise noted. Reduction of the temperature and the pH of the medium reduced the rate of amylase release, thus allowing for more lengthy procedures, e.g. cumulative concentration-response curves, or functional loading of intracellular pools of calcium during stimulated secretion [22]. The amylase activity of the tissue and post-tissue superfusion medium was determined b y the saccharogenic m e t h o d o f Gindler [23], modified to accomm o d a t e smaller sample volumes. Duplicate 10-~l samples were incubated at r o o m temperature with 0.2 ml borohydride-reduced starch substrate (1.0%, w/v) for 5--10 min. The reaction was terminated b y addition of 0.2 ml o f color reagent: 1.7% 3,5-dinitrosalicyclic acid, 1.55% NaOH, and 2.62% KOH. Color was developed b y heating in a dry block for 10 min at 95°C. After cooling, 1.1 ml distilled water was added, and the intensity of color was measured at 500 nm. Amylase activity, expressed as units, was calculated as the maltose equivalents formed per min. The rate of amylase release from the tissue was calculated according to the following equation: units amylase in effluent × flow rate sample volume (ml) total units amylase (tissue and.released)

100

-

% released min

Tissue samples were homogenized at 0--4°C in 20 ml 5 mM Hepes buffer (pH 7.0) for 1 min using a Super Dispax Tissumizer at maximum speed. The homogenate was appropriately diluted and assayed for amylase activity. Neither the amylase assay nor the enzyme activity was affected b y any of the drugs used to alter the rate of amylase release. Cumulative concentration-response curves were determined b y increasing the drug concentration in 0.5 log increments every 40 min. Effluent samples (1 ml) were collected at 10-min intervals during the entire course o f the experiment and analyzed for amylase activity. In calcium-loading experiments, the initial equilibration with 1.25 mM Ca 2÷ medium was followed b y a 60 min equilibration in which half of the tissue samples were exposed to 11.25 mM Ca 2÷ medium and half to control (1.25 mM Ca 2÷) medium. Cumulative concentrationresponse curves were then performed with corresponding Ca 2+ concentrations. Dinitrophenol pretreatment consisted of a 60 min equilibration o f the tissue

298 samples with 1 mM dinitrophenol medium, followed b y exposure to stimulants of release in medium containing dinitrophenol. Batch incubation was used in experiments in which amylase and lactate dehydrogenase release were compared. Slices from the glands of 2 or 3 rats were prepared and equilibrated for 60 min in 300 ml of control medium {composition was identical to the superfusion medium with the exception that the total buffer concentration was increased in 40 mM to minimize pH changes during incubation). Equilibrated slices were washed with control medium and randomly distributed into 50-ml Erlenmeyer flasks containing 25 ml of experimental medium. All media were continuously oxygenated with 100% 02. Tissues were incubated at 21--22°C for 60 min with gentle agitation, and 5 ml of medium was removed and used to determine amylase and lactate dehydrogenase release. The tissue was homogenized in the remaining 20 ml of medium, and subjected to t w o rapid freeze-thaw cycles in order to disrupt the secretory vesicles. The homogenate was centrifuged at 100 000 X g for 45 min, and the supernatant assayed for enzyme activities. Enzyme release, expressed as the percent of the original activity, was calculated as follows: % Released =

(25 X units/ml medium) X 100 (units/ml homogenate) + (5 X units/ml medium)

Lactate dehydrogenase activity was determined by following the rate of disappearance of NADH at 340 nm. The assay consisted of: sample plus control medium (final volume 1.3 ml), 0.1 mM NADH and 0.7 mM pyruvate. A unit of activity was defined as 1 mmol NADH oxidized/min per ml sample. Each experiment was replicated at least three times with similar results. All data are expressed as the means _+ S.D. Student's t-test was used to determine the significance of the difference between t w o means. The effects of secretagogues on lactate dehydrogenase loss was examined b y one way analysis of variance, and the means were compared b y the sequential variant of the Q method [24]. Dibutyryl cyclic AMP, caffeine, NADH, pyruvate, propranolol, isoproterenol, Bistris and Hepes were obtained from Sigma Chemical Co. (St. Louis, MO). Dinitrophenol was purchased from Calbiochem (Los Angeles, CA). Tolbutamide was a gift of the Upjohn Co. (Kalamazoo, MI). LaC13 was obtained from K and K Laboratories (Plalnview, NY). Reagents for the amylase assay were purchased from Pierce Chemical Co. {Rockford, IL). Sodium pentobarbital solution was a product of A b b o t t Laboratories (North Chicago, IL). Results

Preparation stability The superfused parotid slice preparation exhibited stable rates of spontaneous amylase release for over 2 h. Although there was a general tendency for the spontaneous rates to decrease slightly with time, the rates after 120 min were n o t significantly different from those obtained immediately following the equilibration period {time 0). The secretory responses of the preparation to 10 -s M isoproterenol were similar after 30 and 120 min of control superfusion {Fig.

1).

299 0.16c::

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120

140

160

Time (min) Fig. I . P r e p a r a t i o n s t a b i l i t y . Fottr tissue s a m p l e s w e r e e q u i l i b r a t e d b y s u p e r f u s l o n w i t h c o n t r o l m e d i u m for 45 min. Following equilibration, effluent was collected (1-ml samples) at lO-min intervals for 30 m i n i n o r d e r t o d e t e r m i n e t h e s p o n t a n e o u s rate o f release. A t t h e first a r r o w , e x p o s u r e o f t w o tissue s a m p l e s t o i s o p r o t e r e n o l ( 1 0 -5 M) w a s i n i t i a t e d and t h e r e s p o n s e w a s f o l l o w e d for 4 0 rain. T h e r e m a i n i n g t w o s a m p l e s w e r e s u p e r f u s e d w i t h c o n t r o l m e d i u m for a n a d d i t i o n a l 9 0 m i n ; and at t h e s e c o n d a r r o w a n isop r o t e r e n o l infusion was initiated.

Amylase release appeared to occur in two phases. The rate of release rapidly increased during the first 30 min of exposure to stimulants of secretion, and this phase was followed by a very slow increase in the rate of release which was observable for an additional 50--70 min (data not shown). This pattern was consistently observed with all stimulants of release. The values for stimulated amylase release reported in this communication were obtained following the initial rapid increase in release.

Calcium loading Calcium loading was accomplished by superfusion o f the preparations for 60 min with 11.25 mM Ca2÷ medium prior to exposure to stimulants of release. Calcium loading had minimal effects on the spontaneous rates of amylase release. The concentration-response curves of dibutyryl cyclic AMP, caffeine, and lanthanum were shifted to the left following calcium loading (Fig. 2). The highest concentration of caffeine used in the present experiments was 30 mM, and this concentration did n o t yield maximal responses in either control or calcium-loaded tissues (Fig. 2B). The secretory response to La 3÷ was maximal at a concentration of 1 mM, and higher concentrations resulted in submaximal stimulation. Both the ascending and descending limb (supramaximal La 3÷ concentration) were shifted to lower La 3÷ concentrations by calcium loading, b u t the magnitude of the maximal response was not affected (Fig. 2C). Calcium loading also potentiated the secretory responses to reduction of the H ÷ concentration or elevation of the K ÷ concentration of the medium (Table I). Alterations of the I-F concentration of the medium produced concentrationdependent changes in amylase release which were reversible (Fig. 3).

Lanthanum The effect of La 3÷ on the response of the preparation to other stimulants was examined. Submaximal La 3÷ concentrations (0.3 mM) caused an elevation of the concentration-response curves to dibutyryl cyclic AMP (Fig. 4A) and

300

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Fig. 2. C o n c e n t r a t i o n - r e s p o n s e c u r v e s to d i b u t y r y l cyclic AMP (A), c a f f e i n e (B), a n d L a 3+ (C), a n d t h e e f f e c t o f c a l c i u m l o a d i n g . P r e p a r a t i o n s w e r e d i v i d e d i n t o six tissue s a m p l e s a n d e q u i l i b r a t e d b y s u p e r f u sion f o r 4 5 rain w i t h c o n t r o l m e d i u m . T h e initial e q u i l i b r a t i o n w a s f o l l o w e d b y a s e c o n d e q u i l i b r a t i o n p e r i o d ( 6 0 m i n ) in w h i c h t h r e e s a m p l e s w e r e s u p e r f u s e d w i t h 1 1 . 2 5 m M Ca 2+ m e d i u m (4) a n d t h r e e w i t h 1 . 2 5 m M Ca 2+ m e d i u m (e). E a c h s a m p l e w a s t h e n e x p o s e d f o r 4 0 rain p e r i o d s t o p r o g r e s s i v e l y h i g h e r conc e n t r a t i o n s o f s t i m u l a n t in m e d i u m w i t h a Ca 2+ c o n c e n t r a t i o n c o r r e s p o n d i n g t o t h e s e c o n d e q u i l i b r a t i o n . S a m p l e s (1 ml) o f e f f l u e n t w e r e c o l l e c t e d a t 10-rain i n t e r v a l s a n d a s s a y e d f o r a m y l a s e a c t i v i t y in o r d e r to insure c o m p l e t i o n of t h e r a p i d p h a s e of t h e r e s p o n s e . A m y l a s e release is e x p r e s s e d as t h e p e r c e n t of t o t a l tissue c o n t e n t r e l e a s e d p e r rain. V a l u e s are m e a n s -+ S.D.

caffeine (Fig. 4B). However, the curves were similar in shape to the controls. A secretory response to 10 -s M isoproterenol was observable in the presence o f 0.3 mM La 3÷. The increment of isoproterenol-stimulated release (stimulated minus spontaneous release) of the controls was 0.093 + 0 . 0 0 6 %/min, and the increment o f stimulation in the presence o f 0.3 mM La 3÷ was 0 . 0 7 8 ± 0 . 0 1 6 %/min (mean -+ S.D.).

301 TABLE I EFFECT

OF CALCIUM L O A D I N G ON A M Y L A S E R E L E A S E

P r e p a r a t i o n s w e r e d i v i d e d i n t o six tissue s a m p l e s w h i c h were e q u i l i b r a t e d by s u p e r f u s i o n w i t h c o n t r o l m e d i u m f o r 4 5 rain. T h r e e s a m p l e s w e r e t h e n c a l c i u m l o a d e d b y a 6 0 rain s u p e r f u s i o n w i t h 1 1 . 2 5 m M Ca 2+ m e d i u m w h i l e t h e o t h e r t h r e e s a m p l e s w e r e s u p e r f u s e d w i t h 1 . 2 5 m M Ca 2+ m e d i u m . S p o n t a n e o u s r e f e r s to the r a t e of a m y l a s e release f o l l o w i n g t h e s e c o n d a r y e q u i l i b r a t i o n w i t h e i t h e r 1 . 2 5 o r 1 1 . 2 5 m M Ca 2+. S a m p l e s w e r e t h e n s w i t c h e d to s t i m u l a n t - c o n t a i n i n g m e d i u m w i t h Ca 2+ c o n c e n t r a t i o n s c o r r e s p o n d ing t o the s e c o n d a r y e q u i l i b r a t i o n . E f f l u e n t (1 m l ) was c o l l e c t e d a t 10-rain i n t e r v a l s a n d a s s a y e d for a m y l a s e a c t i v i t y . V a l u e s given are t h e r a t e s of release f o l l o w i n g 4 0 rain of e x p o s u r e t o s t i m u l a n t a n d are e x p r e s s e d as t h e p e r c e n t of t o t a l tissue a m y l a s e r e l e a s e d p e r rain. A m y l a s e release (%Imin) 1 . 2 5 m M Ca 2+

1 1 . 2 5 m M Ca 2+

M e a n +_ S.D.

M e a n ± S.D.

Change

Change

(%) A. S p o n t a n e o u s 105 mM K +

0,043 + 0.004 0 . 1 4 8 _+ 0 . 0 1 3

B. S p o n t a n e o u s p H 7.5

0.031 ± 0,005 0.112 + 0.010

(%)

+224

0.047 + 0.004 0.192 + 0.009 *

+309

+261

0.033 + 0,004 0.151 + 0.016 *

+358

* S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o r r e s p o n d i n g r a t e of release in tissues e q u i l i b r a t e d w i t h 1.25 m M Ca 2+, P < 0.05.

B. A.

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