82
Biochimica et Biophysica Acta, 392 ( 1 9 7 5 ) 8 2 - - 9 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 2 7 6 3 9
E F F E C T OF A D R E N E R G I C AGENTS ON a-AMYLASE RELEASE AND ADENOSINE 3',5'-MONOPHOSPHATE ACCUMULATION IN RAT PAROTID TISSUE SLICES
F R E D R. B U T C H E R , J A M E S A. G O L D M A N a n d M A R K N E M E R O V S K I
Division o f Biological and Medical Sciences, Brown University, Providence, R.I. 02912
(U.S.A.) (Received O c t o b e r ] 5th, 1 9 7 4 )
Summary The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20--30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both a-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of p o t e n c y and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a fll -adrenergic mechanism. At the lowest concentrations tested, each of the adrenergic agonists stimulated significant a-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased a-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of a-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of a-amylase release by isoproterenol. Stimulation of a-amylase release by phenylephrine was only partially blocked by either a- or fl-adrenergic blocking agents, whereas stimulation of Abbreviations: Monobutyryl cyclic AMP, N6-monobutyryl adenosine 3',5'-monophosphate; dibutyryl cyclic AMP, N6,02 '-dibutyryl adenosine 3',5'-monophosphate.
83 adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and aamylase release. However, phenoxybenzamine also potentiated the stimulation of a-amylase release by N6,02'-dibutyryl adenosine 3',5'-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using a-adrenergic blocking agents as tools for investigation of a- and ~-adrenergic antagonism.
Introduction a-Adrenergic stimulation of the rat parotid gland is associated with enhanced potassium and water efflux and with a slight stimulation of a-amylase release [1,2]. In contrast, fi-adrenergic agonists cause a marked release of aamylase, but have little or no effect on potassium or water efflux [1,3]. Schramm and coworkers [4,5] have proposed that the effect of fi-adrenergic agents on a-amylase release is mediated by changes in the intracellular concentration of cyclic AMP [6]. Evidence for this role of cyclic AMP includes the following: (a) dibutyryl and m o n o b u t y r y l cyclic AMP enhance a-amylase release from parotid slices [6,7] ; (b) partially purified plasma membranes from parotid tissue contain an adenylate cyclase which is activated by epinephrine [4] ; (c) the stimulation of adenylate cyclase activity and a-amylase release by norepinephrine is blocked by propranolol, a ~-adrenergic antagonist [4]; and (d) epinephrine causes an intracellular accumulation of cyclic AMP [1]. Intracellular mediation of a-adrenergic stimulation is less well characterized. The present studies were undertaken to further investigate the effects of both a- and ~-adrenergic agents on rat parotid tissue slices in vitro. Adrenergic stimulation of a-amylase release and intracellular cyclic AMP accumulation was classified pharmacologically with the aid of various adrenergic agonists and antagonists. The relationship between these responses was explored, and the concept developed that large increases in the intracellular level of cyclic AMP are not necessary for stimulus-secretion coupling in this tissue. Materials and Methods Female Sprague-Dawley CD rats (Charles River Breeding Laboratories, Wilmington, Mass.), 7--9 weeks old, fed ad libitum, were used for all experiments. Since some drugs have been shown to stimulate a-amylase release from parotid tissue by releasing endogenous stores of catecholamines [8], all rats were injected subcutaneously, 18 h prior to use, with 40 pg of reserpine per 100 g body weight (400 #g/ml of 50% dimethylsulfide, v/v). Such pretreatment effectively depleted parotid catecholamine stores. Norepinephrine levels in the glands were 120 + 20 ng/mg protein (mean + S.E.) for a control group of three individual animals and 2.0 + 0.5 ng/mg protein for a group treated with reserpine. Norepinephrine levels were determined by the m e t h o d of Crout [9] following homogenization of the glands in cold 0.4 M HC104.
84 Parotid glands from at least two rats were removed and prepared at 37°C according to the procedure of Babad et al. [7]. Slices were incubated in stoppered, plastic vials containing Krebs-Ringer-bicarbonate buffer with O:/CO2 (95 : 5, v/v) as the gas phase. Incubation volumes were 2.0 ml for studies of a-amylase release and 1.0 ml for studies of cyclic AMP levels. At the end of the incubation periods for a-amylase release, the tissue slices were separated from the buffer by filtration through nylon gauze fitted over the tops of the plastic vials. Tissue slices in each vial were then homogenized in 5.0 ml of 0.02 M potassium phosphate/6.7 mM NaC1, pH 6.9. The a-amylase activity of the homogenate and incubation buffer was assayed according to Bernfeld [10]. The total amount of a-amylase was derived by summing the content of a-amylase remaining in the tissue slices plus that released into the incubation buffer. All data for release of a-amylase are given as the percent of the total a-amylase released into the incubation buffer. Cyclic AMP from tissue slice and medium was determined as described previously [11]. In none of the experiments reported here was there a disproportionate effect on the release of cyclic AMP into the medium. All data are expressed as the average of individual paired experiments which have been repeated at least three times. For determination of adenylate cyclase activity, parotid glands were homogenized in 0.3 M sucrose/0.05 mM EDTA, pH 7.5, and centrifuged at 150 X g for 10 min. The pellet was discarded and the supernatant was centrifuged at 10 000 X g for 10 min. The pellet obtained was washed one time with 50 mM Tris--HC1/0.05 mM EDTA, pH 7.5, and resuspended in 0.3 M sucrose/0.05 mM EDTA, pH 7.5. The adenylate cyclase assay was conducted at 37°C for 10 min in a total volume of 100 pl which contained (in final concentrations) 3.0 mM ATP, 4.0 mM MgCl:, 1.0 mM theophylline, 40 mM Tris--HC1, 0.1 mM EDTA, 0.1% (w/v) bovine serum albumin, 13 mM creatine phosphate, and 0.18 unit of creatine phosphokinase at a final pH of 7.5. The reaction was terminated by boiling for 2 min. The cyclic AMP formed was assayed by the procedure of Brown et al. [12]. Cyclic AMP standards were assayed in the presence of the same amount of reagents from the adenylate cyclase assay as the sample aliquots. Under the conditions of the assay it was not necessary to purify the cyclic AMP before assaying. D (--)~pinephrine bitartrate, D (--}-norepinephrine hydrochloride, D (---)phenylephrine hydrochloride, (+_)-isoproterenol, atropine, reserpine, theophylline, dibutyryl cyclic AMP, creatine phosphate, and creatine phosphokinase were products of the Sigma Chemical Co. (St. Louis, Mo.). Salbutamol was a gift from Aliens and Hansbury, Ltd, (Ware, England). (--)- and (+)-propranolol were gifts from Ayerst Laboratories, Incorp. (New York, N.Y.). Phentolamine was obtained from CIBA (Ardsley, N.Y.) and phenoxybenzamine was from Smith, Kline and French Laboratories (Philadelphia, Pa.). Results
The time course for changes in intracellular cyclic AMP concentration after the addition of either isoproterenol or norepinephrine (final concentration 15 pM) was investigated. Cyclic AMP accumulated rapidly, and the levels reached a maximum by 5 to 10 min after the addition of either drug (Fig. 1).
85
.-
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10
15 Minutes
20
25
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F i g . 1. T i m e c o u r s e o f t h e e f f e c t s o f i s o p r o t e r e n o l a n d n o r e p i n e p h r i n e o n c y c l i c A M P a c c u m u l a t i o n in r a t parotid tissue slices. Tissue slices from three rats were incubated with the catecholamines, 15 ~M, for the indicated times.
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Concentration, uM (log scale) F i g . 2. T h e e f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s o f a d r e n e r g i c a g o n i s t s o n s - a m y l a s e r e l e a s e a n d c y c l i c A M P accumulation in tissue slices of rat parotid. Tissue slices from six rats were prepared and divided into two lots. After addition of the drugs to the indicated concentrations, slices from one lot were incubated for 45 rain prior to determination of s-amylase release, and slices from the other lot were incubated for 10 min prior to determination of cyclic AMP. Basal value for s-amylase release was 3.3% and that for cyclic AMP was 11.1 pmol/mg protein.
86 From our own studies (not shown) and those of others [ 1 , 1 3 ] , the effect of catecholamines on cyclic AMP appears to slightly precede or coincide with their earliest detectable effect on a-amylase release. The effects of isoproterenol, norepinephrine, epinephrine, salbutamol, and phenylephrine on cyclic AMP accumulation and ~-amylase release are shown in Fig. 2. Isoproterenol was much more effective than any of the other four adrenergic agonists in promoting both a-amylase release and cyclic AMP accumulation. Norepinephrine and epinephrine were equally potent in their effects on these two parameters. Salbutamol and phenylephrine were least effective. At the lowest concentrations employed, all of the catecholamines elevated a-amylase release without a measurable stimulation of cyclic AMP accumulation. Salbutamol and phenylephrine markedly stimulated enzyme release, but produced only a very small or no increment in cyclic AMP levels. The time course for changes in cyclic AMP levels were the same for the highest and lowest agonist concentrations (not shown). In order to magnify any slight effect of salbutamol or phenylephrine on cyclic AMP accumulation, these drugs were added in the presence of theophylline, an inhibitor of both low and high Km phosphodiesterase activities in parotid (Butcher, F.R., unpublished). In the presence of 1.0 mM theophylline, a stimulatory effect of 15 pM salbutamol on cyclic AMP levels was clearly discernible (Table I). The effect of salbutamol on cyclic AMP was blocked by (--)-propranolol and by practolol and less effectively by the weaker ~3-antagonist, (+)-propranolol. The ~-adrenergic antagonists that blocked the effect of salbutamol on cyclic AMP accumulation also blocked its effect on a-amylase TABLE I T H E E F F E C T O F f l - A D R E N E R G I C B L O C K I N G A G E N T S ON T H E S T I M U L A T I O N OF C Y C L I C AMP A C C U M U L A T I O N A N D (~-AMYLASE R E L E A S E BY S A L B U T A M O L I n E x p t 1, for d e t e r m i n a t i o n o f c y c l i c A M P levels, t i s s u e slices f r o m t w o rats w e r e p r e p a r e d a n d i n c u b a t e d for 1 0 rain in t h e p r e s e n c e o f t h e b l o c k i n g a g e n t s a n d t h e o p b y l l i n e (1.0 raM), a f t e r w h i c h t i m e s a l b u t a m o l (15 #M) w a s a d d e d for an a d d i t i o n a l 1 0 rain. I n E x p t 2, for d e t e r m i n a t i o n o f a - a m y l a s e release, tissue slices f o m t w o rats w e r e p r e p a r e d a n d i n c u b a t e d for 1 0 rain w i t h t h e b l o c k i n g a g e n t s , a f t e r w h i c h t i m e s a l b u t a m o l w a s a d d e d for an a d d i t i o n a l 4 5 m i n . Experiment number
Additions
C y c l i c A M P ( p m o l / m g p r o t e i n ) in p r e s e n c e o f 1.0 m M theophylline
Basal
+ S a l b u t a m o l (15 ~zM)
None ( + ) - P r o p r a n o l o l , 1.0 #M ( - ) - P r o p r a n o l o l , 1.0 pM P r a c t o l o l , 10 pM
11.5 12.1 11.7 10.8
± ± ± ±
1.2 1.9 1.3 0.7
Additions
Percent a-amylase released Basal
None ( + ) - P r o p r a n o l o l , 1.0 DM ( - ) - P r o p r a n o l o l , 1.0 pM P r a c t o l o l , 10 pM
4.5±0.8 3.9±0.5 4.7±0.1 5.0±0.5
30.8 20.0 12.9 12.1
+ ± ± +
1.6 0.5 1.6 0.4
+ S a l b u t a r a o l (15 # M ) 34.6±0.3 32.121.0 4.9±0.7 12.4±0.8
87
release (Table I). In contrast to the elevation of cyclic AMP levels by salbutamol and theophylline, phenylephrine and theophylline at several concentrations and time points were without effect (not shown). The agonists used in the studies shown in Fig. 2 were also examined for their effects on adenylate cyclase (Fig. 3). It was observed that the drugs had the same relative order of potencies for their stimulation of adenylate cyclase as for their stimulation of ~-amylase release and cyclic AMP accumulation. The adenylate cyclase preparations appeared about as sensitive to low levels of catecholamines as did the intact slices. The high sensitivity of the adenylate cyclase preparations to agonists was surprising in that the sensitivity of adenylate cyclase to agonists is usually decreased by homogenization. It was also apparent that salbutamol and phenylephrine stimulated adenylate cyclase activity, whereas they had only slight or no detectable effect on intracellular accumulation of cyclic AMP. The stimulation of adenylate cyclase by phenylephrine was particularly striking in view of our finding that phenylephrine had no detectable effect on cyclic AMP accumulation even in the presence of theophylline. The effects of the fi-adrenergic antagonist propranolol on the stimulation of cyclic AMP accumulation and s-amylase release by isoproterenol were also examined (Fig. 4). Since it has been reported that propranolol has both specific and non-specific effects [14], the (+)- and (--)-isomers of propranolol were employed in the present study. The (--)-isomer is a strong competitive fi-adrenergic antagonist, whereas the (+)-isomer is a weak competitive fl-adrenergic antagonist. However, both isomers are equipotent on a molar basis as nonspecific antagonists [ 1 4 ] . Thus, by using both (+)- and C-)-propranolol at the appropriate concentrations, it is possible to distinguish between the specific and non-specific effects. At all concentrations tested, (--)-propranolol inhibited the stimulation of ~-amylase release by isoproterenol to a greater extent than did (+)-propranolol. The same was true for the effects of (+}- and (--)-proprano-
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Fig. 3. The effects of different concentrations o f adrenergic agonists on activation o f adenylate cyclase a c t i v i t y f r o m r a t p a r o t i d t i s s u e . T h e c o n d i t i o n s o f i n c u b a t i o n a r e d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e a m o u n t o f p r o t e i n u s e d p e r a s s a y w a s 2 4 0 ~tg.
88
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Fig. 4. E f f e c t o f (+)- a n d ( - - ) - p r o p r a n o l o l c o n c e n t r a t i o n on t h e s t i m u l a t i o n of C~-amylase release a n d cyclic AMP a c c u m u l a t i o n b y i s o p r o t e r e n o l in tissue slices o f r a t p a r o t i d . Tissue slices f r o m six rats w e r e p r e p a r e d a n d d i v i d e d into t w o lots. One lot was used f o r d e t e r m i n a t i o n o f s - a m y l a s e release a n d t h e o t h e r for d e t e r m i n a t i o n o f cyclic AMP a c c u m u l a t i o n . T h e slices w e r e i n c u b a t e d in t h e p r e s e n c e of t h e i n d i c a t e d c o n c e n t r a t i o n s o f p r o p r a n o l o l f o r 20 m i n b e f o r e a d d i t i o n o f i s o p r o t e r e n o l ( 1 0 pM). A f t e r a d d i t i o n of i s o p r o t e r e n o l , slices w e r e i n c u b a t e d for 4 5 rain p r i o r to d e t e r m i n a t i o n o f s - a m y l a s e release a n d for 10 mill p r i o r t o d e t e r m i n a t i o n of cyclic AMP.
lol on the stimulation of cyclic AMP accumulation by isoproterenol. However, at high concentrations of two isomers, (+)-propranolol markedly inhibited stimulation of cyclic AMP accumulation by isoproterenol, whereas stimulation of a-amylase release was only slightly inhibited. Fig. 4 also demonstrates that the effect of isoproterenol on cyclic AMP accumulation was much more sensitive to inhibition b y propranolol than was the effect on a-amylase release. The data presented thus far would indicate that the effects of adrenergic agonists on cyclic AMP accumulation and a-amylase release could be classified as/3-adrenergic responses. However, it is known that epinephrine and norepinephrine have both ~-adrenergic and a-adrenergic agonist properties [15]. To evaluate the action of the a-adrenergic agonists on a-amylase release, t w o approaches were used. First, the effects of phenylephrine on cyclic AMP accumulation, a-amylase release, and adenylate cyclase activity were examined. This drug has been classified as predominantly an a-adrenergic agonist [15]. Second, the effects of isoproterenol and norepinephrine on cyclic AMP accumulation and a-amylase release were studied in the presence of a-adrenergic antagonists. Phenylephrine, at relatively high concentrations, stimulated a-amylase release {Fig. 2, Table II). The effect was almost completely blocked by (--)propranolol and was partially antagonized by (+)-propranolol and by phentolamine, an a-antagonist. Atropine had no effect. It would appear that part of the effect of phenylephrine on a-amylase release could be classified as fl-
89 TABLE
II
THE EFFECT OF ADRENERGIC AND CHOLINERGIC OF a-AMYLASE RELEASE BY PHENYLEPHRINE
BLOCKING
AGENTS
ON THE STIMULATION
Tissue slices f r o m t w o rats w e r e p r e p a r e d a n d i n c u b a t e d 1 0 r a i n in t h e p r e s e n c e o f b l o c k i n g a g e n t s , after w h i c h t i m e p h e n y l e p h r i n e w a s a d d e d for an a d d i t i o n a l 4 5 m i n .
Additions
None (+)-Propranolol, 2.0 pM (-)-Propranolol, 2.0 pM Phentolamine, 10 pM Atropine, 5.0 pM
Percent ~-amylase released Basal
+Phenylephrine (25 #M)
8.6 7.2 8.1 7.5 8.5
26.5 17.4 12.9 20.4 27.8
-+ 0 . 4 -+ 1 . 7 + 1.2 + 2.1 + 0.8
+ 0.9 + 0 -+ 1 . 2 -+ 0 . 1 + 1.5
adrenergic and part as a-adrenergic. Since 2 pM (+)-propranolol had no nonspecific effects on the stimulation of a-amylase release by isoproterenol {Fig. 4), it is likely that (+)-propranolol partially blocked the effect of phenylephrine as a result of a weak competitive fl-adrenergic antagonist action. Phenylephrine did not have a detectable effect on cyclic AMP accumulation, even in the presence of theophylline, which stands in marked contrast to the stimulatory effect of phenylephrine on adenylate cyclase (Fig. 3, Table III). Phenylephrine is generally considered to be an a-adrenergic agonist, but appeared to stimulate a-amylase release in part by a fl-adrenergic mechanism (Table II). Since stimulation of adenylate cyclase by adrenergic agents is classified as a fl-adrenergic response, the effect of phenylephrine on adenylate cyclase from parotid tissue was investigated. It can be seen in Table III that the stimulation of adenylate cyclase by phenylephrine was blocked by (--)propranolol and not by phentolamine. It has been proposed that the actions of a- and fl-adrenergic agonists are opposed to one another [16,17], resulting in an apparent antagonism between the actions of the two types of agonists. If such antagonism exists, inhibition of the a-agonist activity of an agent that has both a- and fl-adrenergic agonist properties should enhance the fl-agonist activity of that drug. In support of this idea, the a-adrenergic antagonists phenoxybenzamine and phentolamine enTABLE
III
THE EFFECT OF ADRENERGIC BLOCKING CYCLASE ACTIVITY BY PHENYLEPHRINE
AGENTS
ON THE STIMULATION
OF ADENYLATE
A d e n y l a t e c y e l a s e p r e p a r a t i o n is d e s c r i b e d u n d e r Materials a n d M e t h o d s . T h e a m o u n t o f p r o t e i n u s e d p e r assay was 190/~g. Additions
None (-)-Propranolol, 10 pM Phentolamine
p m o l c y c l i c A M P f o r m e d ( P m o l • m g p r o t e i n -1 • r a i n - 1 ) Basal
+Phenylephrine
4 . 0 -+ 0 . 4 3 . 5 +- 0 . 1 4.2 + 0.5
2 7 . 4 -+ 0 . 5 7.3 + 1.1 2 5 . 8 -+ 2 . 8
(25 #M)
9O
Amylase +Phenoxybenzamine
411
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Narepinephrlne, uM (lag scalel Fig. 5. E f f e c t o f a - a d r e n e r g i c b l o c k i n g a g e n t s on t h e s t i m u l a t i o n of a - a m y l a s e release a n d cyclic AMP a c c u m u l a t i o n b y n o r e p i n e p h r i n e in tissue slices of r a t p a r o t i d . Tissue slices w e r e p r e p a x e d f r o m t w o g r o u p s of t h r e e rats. T h e slices f r o m o n e g r o u p w e r e used for d e t e r m i n a t i o n s of a - a m y l a s e release a n d slices f r o m t h e o t h e r w e r e used for d e t e r m i n a t i o n s of cyclic AMP. I n b o t h cases t h e slices w e r e i n c u b a t e d w i t h e i t h e r p h e n o x y b e n z a m i n e (10 ~zM) o r p h e n t o l a m i n e (10 pM) 1 5 m i n b e f o r e t h e a d d i t i o n of n o r e p i n e p h r i n e to t h e i n d i c a t e d c o n c e n t r a t i o n s . A f t e r a d d i t i o n of n o r e p i n e p h r i n e , i n c u b a t i o n s w e r e c o n t i n u e d for 45 re.in for d e t e r m i n a t i o n of a - a m y l a s e release a n d 10 rain for d e t e r m i n a t i o n of cyclic AMP a c c u m u l a t i o n . Basal v a l u e f o r a - a m y l a s e release was 6.5% a n d t h a t for cyclic AMP was 14.1 p m o l / m g p r o t e i n .
hanced the effect of norepinephrine on cyclic AMP accumulation and a-amylase release (Fig. 5). However, the same effects of phenoxybenzamine and phentolamine were observed for the stimulation of a-amylase release and cyclic AMP accumulation by isoproterenol (Fig. 6). Isoproterenol is believed to have only minimal a-adrenergic agonist activity; however, it should be remembered that the relative degree of a- and fi-adrenergic agonist activity for a given drug is a function of the tissue under study [15]. Batzri et al. [1] reported that phentolamine did not potentiate the effect of epinephrine on the levels of cyclic AMP in rat parotid tissue slices. However, in the presence of phentolamine, the level of cyclic AMP remained elevated for a longer period of time after addition of epinephrine. For this reason, the cyclic AMP levels shown in Figs 5 and 6 were determined at the time of maximal increase of the cyclic AMP level following the addition of norepinephrine or isoproterenol. In each instance this was the 10-min time point (Fig. 1). Non-specific effects of phenoxybenzamine may be indicated since this c o m p o u n d also enhanced the action of dibutyryl cyclic AMP on a-amylase release (Fig. 7). In the same experiment, phentolamine was without effect. Since these results suggest that the effects of phenoxybenzamine and phentolamine might be non-specific, caution should be used in interpreting evidence concerning a- and fi-adrenergic interactions.
91
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Biochimica et Biophysica Acta, 392 ( 1 9 7 5 ) 8 2 - - 9 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 2 7 6 3 9
E F F E C T OF A D R E N E R G I C AGENTS ON a-AMYLASE RELEASE AND ADENOSINE 3',5'-MONOPHOSPHATE ACCUMULATION IN RAT PAROTID TISSUE SLICES
F R E D R. B U T C H E R , J A M E S A. G O L D M A N a n d M A R K N E M E R O V S K I
Division o f Biological and Medical Sciences, Brown University, Providence, R.I. 02912
(U.S.A.) (Received O c t o b e r ] 5th, 1 9 7 4 )
Summary The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20--30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both a-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of p o t e n c y and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a fll -adrenergic mechanism. At the lowest concentrations tested, each of the adrenergic agonists stimulated significant a-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased a-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of a-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of a-amylase release by isoproterenol. Stimulation of a-amylase release by phenylephrine was only partially blocked by either a- or fl-adrenergic blocking agents, whereas stimulation of Abbreviations: Monobutyryl cyclic AMP, N6-monobutyryl adenosine 3',5'-monophosphate; dibutyryl cyclic AMP, N6,02 '-dibutyryl adenosine 3',5'-monophosphate.
83 adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and aamylase release. However, phenoxybenzamine also potentiated the stimulation of a-amylase release by N6,02'-dibutyryl adenosine 3',5'-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using a-adrenergic blocking agents as tools for investigation of a- and ~-adrenergic antagonism.
Introduction a-Adrenergic stimulation of the rat parotid gland is associated with enhanced potassium and water efflux and with a slight stimulation of a-amylase release [1,2]. In contrast, fi-adrenergic agonists cause a marked release of aamylase, but have little or no effect on potassium or water efflux [1,3]. Schramm and coworkers [4,5] have proposed that the effect of fi-adrenergic agents on a-amylase release is mediated by changes in the intracellular concentration of cyclic AMP [6]. Evidence for this role of cyclic AMP includes the following: (a) dibutyryl and m o n o b u t y r y l cyclic AMP enhance a-amylase release from parotid slices [6,7] ; (b) partially purified plasma membranes from parotid tissue contain an adenylate cyclase which is activated by epinephrine [4] ; (c) the stimulation of adenylate cyclase activity and a-amylase release by norepinephrine is blocked by propranolol, a ~-adrenergic antagonist [4]; and (d) epinephrine causes an intracellular accumulation of cyclic AMP [1]. Intracellular mediation of a-adrenergic stimulation is less well characterized. The present studies were undertaken to further investigate the effects of both a- and ~-adrenergic agents on rat parotid tissue slices in vitro. Adrenergic stimulation of a-amylase release and intracellular cyclic AMP accumulation was classified pharmacologically with the aid of various adrenergic agonists and antagonists. The relationship between these responses was explored, and the concept developed that large increases in the intracellular level of cyclic AMP are not necessary for stimulus-secretion coupling in this tissue. Materials and Methods Female Sprague-Dawley CD rats (Charles River Breeding Laboratories, Wilmington, Mass.), 7--9 weeks old, fed ad libitum, were used for all experiments. Since some drugs have been shown to stimulate a-amylase release from parotid tissue by releasing endogenous stores of catecholamines [8], all rats were injected subcutaneously, 18 h prior to use, with 40 pg of reserpine per 100 g body weight (400 #g/ml of 50% dimethylsulfide, v/v). Such pretreatment effectively depleted parotid catecholamine stores. Norepinephrine levels in the glands were 120 + 20 ng/mg protein (mean + S.E.) for a control group of three individual animals and 2.0 + 0.5 ng/mg protein for a group treated with reserpine. Norepinephrine levels were determined by the m e t h o d of Crout [9] following homogenization of the glands in cold 0.4 M HC104.
84 Parotid glands from at least two rats were removed and prepared at 37°C according to the procedure of Babad et al. [7]. Slices were incubated in stoppered, plastic vials containing Krebs-Ringer-bicarbonate buffer with O:/CO2 (95 : 5, v/v) as the gas phase. Incubation volumes were 2.0 ml for studies of a-amylase release and 1.0 ml for studies of cyclic AMP levels. At the end of the incubation periods for a-amylase release, the tissue slices were separated from the buffer by filtration through nylon gauze fitted over the tops of the plastic vials. Tissue slices in each vial were then homogenized in 5.0 ml of 0.02 M potassium phosphate/6.7 mM NaC1, pH 6.9. The a-amylase activity of the homogenate and incubation buffer was assayed according to Bernfeld [10]. The total amount of a-amylase was derived by summing the content of a-amylase remaining in the tissue slices plus that released into the incubation buffer. All data for release of a-amylase are given as the percent of the total a-amylase released into the incubation buffer. Cyclic AMP from tissue slice and medium was determined as described previously [11]. In none of the experiments reported here was there a disproportionate effect on the release of cyclic AMP into the medium. All data are expressed as the average of individual paired experiments which have been repeated at least three times. For determination of adenylate cyclase activity, parotid glands were homogenized in 0.3 M sucrose/0.05 mM EDTA, pH 7.5, and centrifuged at 150 X g for 10 min. The pellet was discarded and the supernatant was centrifuged at 10 000 X g for 10 min. The pellet obtained was washed one time with 50 mM Tris--HC1/0.05 mM EDTA, pH 7.5, and resuspended in 0.3 M sucrose/0.05 mM EDTA, pH 7.5. The adenylate cyclase assay was conducted at 37°C for 10 min in a total volume of 100 pl which contained (in final concentrations) 3.0 mM ATP, 4.0 mM MgCl:, 1.0 mM theophylline, 40 mM Tris--HC1, 0.1 mM EDTA, 0.1% (w/v) bovine serum albumin, 13 mM creatine phosphate, and 0.18 unit of creatine phosphokinase at a final pH of 7.5. The reaction was terminated by boiling for 2 min. The cyclic AMP formed was assayed by the procedure of Brown et al. [12]. Cyclic AMP standards were assayed in the presence of the same amount of reagents from the adenylate cyclase assay as the sample aliquots. Under the conditions of the assay it was not necessary to purify the cyclic AMP before assaying. D (--)~pinephrine bitartrate, D (--}-norepinephrine hydrochloride, D (---)phenylephrine hydrochloride, (+_)-isoproterenol, atropine, reserpine, theophylline, dibutyryl cyclic AMP, creatine phosphate, and creatine phosphokinase were products of the Sigma Chemical Co. (St. Louis, Mo.). Salbutamol was a gift from Aliens and Hansbury, Ltd, (Ware, England). (--)- and (+)-propranolol were gifts from Ayerst Laboratories, Incorp. (New York, N.Y.). Phentolamine was obtained from CIBA (Ardsley, N.Y.) and phenoxybenzamine was from Smith, Kline and French Laboratories (Philadelphia, Pa.). Results
The time course for changes in intracellular cyclic AMP concentration after the addition of either isoproterenol or norepinephrine (final concentration 15 pM) was investigated. Cyclic AMP accumulated rapidly, and the levels reached a maximum by 5 to 10 min after the addition of either drug (Fig. 1).
85
.-
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5
10
15 Minutes
20
25
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F i g . 1. T i m e c o u r s e o f t h e e f f e c t s o f i s o p r o t e r e n o l a n d n o r e p i n e p h r i n e o n c y c l i c A M P a c c u m u l a t i o n in r a t parotid tissue slices. Tissue slices from three rats were incubated with the catecholamines, 15 ~M, for the indicated times.
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Concentration, uM (log scale) F i g . 2. T h e e f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s o f a d r e n e r g i c a g o n i s t s o n s - a m y l a s e r e l e a s e a n d c y c l i c A M P accumulation in tissue slices of rat parotid. Tissue slices from six rats were prepared and divided into two lots. After addition of the drugs to the indicated concentrations, slices from one lot were incubated for 45 rain prior to determination of s-amylase release, and slices from the other lot were incubated for 10 min prior to determination of cyclic AMP. Basal value for s-amylase release was 3.3% and that for cyclic AMP was 11.1 pmol/mg protein.
86 From our own studies (not shown) and those of others [ 1 , 1 3 ] , the effect of catecholamines on cyclic AMP appears to slightly precede or coincide with their earliest detectable effect on a-amylase release. The effects of isoproterenol, norepinephrine, epinephrine, salbutamol, and phenylephrine on cyclic AMP accumulation and ~-amylase release are shown in Fig. 2. Isoproterenol was much more effective than any of the other four adrenergic agonists in promoting both a-amylase release and cyclic AMP accumulation. Norepinephrine and epinephrine were equally potent in their effects on these two parameters. Salbutamol and phenylephrine were least effective. At the lowest concentrations employed, all of the catecholamines elevated a-amylase release without a measurable stimulation of cyclic AMP accumulation. Salbutamol and phenylephrine markedly stimulated enzyme release, but produced only a very small or no increment in cyclic AMP levels. The time course for changes in cyclic AMP levels were the same for the highest and lowest agonist concentrations (not shown). In order to magnify any slight effect of salbutamol or phenylephrine on cyclic AMP accumulation, these drugs were added in the presence of theophylline, an inhibitor of both low and high Km phosphodiesterase activities in parotid (Butcher, F.R., unpublished). In the presence of 1.0 mM theophylline, a stimulatory effect of 15 pM salbutamol on cyclic AMP levels was clearly discernible (Table I). The effect of salbutamol on cyclic AMP was blocked by (--)-propranolol and by practolol and less effectively by the weaker ~3-antagonist, (+)-propranolol. The ~-adrenergic antagonists that blocked the effect of salbutamol on cyclic AMP accumulation also blocked its effect on a-amylase TABLE I T H E E F F E C T O F f l - A D R E N E R G I C B L O C K I N G A G E N T S ON T H E S T I M U L A T I O N OF C Y C L I C AMP A C C U M U L A T I O N A N D (~-AMYLASE R E L E A S E BY S A L B U T A M O L I n E x p t 1, for d e t e r m i n a t i o n o f c y c l i c A M P levels, t i s s u e slices f r o m t w o rats w e r e p r e p a r e d a n d i n c u b a t e d for 1 0 rain in t h e p r e s e n c e o f t h e b l o c k i n g a g e n t s a n d t h e o p b y l l i n e (1.0 raM), a f t e r w h i c h t i m e s a l b u t a m o l (15 #M) w a s a d d e d for an a d d i t i o n a l 1 0 rain. I n E x p t 2, for d e t e r m i n a t i o n o f a - a m y l a s e release, tissue slices f o m t w o rats w e r e p r e p a r e d a n d i n c u b a t e d for 1 0 rain w i t h t h e b l o c k i n g a g e n t s , a f t e r w h i c h t i m e s a l b u t a m o l w a s a d d e d for an a d d i t i o n a l 4 5 m i n . Experiment number
Additions
C y c l i c A M P ( p m o l / m g p r o t e i n ) in p r e s e n c e o f 1.0 m M theophylline
Basal
+ S a l b u t a m o l (15 ~zM)
None ( + ) - P r o p r a n o l o l , 1.0 #M ( - ) - P r o p r a n o l o l , 1.0 pM P r a c t o l o l , 10 pM
11.5 12.1 11.7 10.8
± ± ± ±
1.2 1.9 1.3 0.7
Additions
Percent a-amylase released Basal
None ( + ) - P r o p r a n o l o l , 1.0 DM ( - ) - P r o p r a n o l o l , 1.0 pM P r a c t o l o l , 10 pM
4.5±0.8 3.9±0.5 4.7±0.1 5.0±0.5
30.8 20.0 12.9 12.1
+ ± ± +
1.6 0.5 1.6 0.4
+ S a l b u t a r a o l (15 # M ) 34.6±0.3 32.121.0 4.9±0.7 12.4±0.8
87
release (Table I). In contrast to the elevation of cyclic AMP levels by salbutamol and theophylline, phenylephrine and theophylline at several concentrations and time points were without effect (not shown). The agonists used in the studies shown in Fig. 2 were also examined for their effects on adenylate cyclase (Fig. 3). It was observed that the drugs had the same relative order of potencies for their stimulation of adenylate cyclase as for their stimulation of ~-amylase release and cyclic AMP accumulation. The adenylate cyclase preparations appeared about as sensitive to low levels of catecholamines as did the intact slices. The high sensitivity of the adenylate cyclase preparations to agonists was surprising in that the sensitivity of adenylate cyclase to agonists is usually decreased by homogenization. It was also apparent that salbutamol and phenylephrine stimulated adenylate cyclase activity, whereas they had only slight or no detectable effect on intracellular accumulation of cyclic AMP. The stimulation of adenylate cyclase by phenylephrine was particularly striking in view of our finding that phenylephrine had no detectable effect on cyclic AMP accumulation even in the presence of theophylline. The effects of the fi-adrenergic antagonist propranolol on the stimulation of cyclic AMP accumulation and s-amylase release by isoproterenol were also examined (Fig. 4). Since it has been reported that propranolol has both specific and non-specific effects [14], the (+)- and (--)-isomers of propranolol were employed in the present study. The (--)-isomer is a strong competitive fi-adrenergic antagonist, whereas the (+)-isomer is a weak competitive fl-adrenergic antagonist. However, both isomers are equipotent on a molar basis as nonspecific antagonists [ 1 4 ] . Thus, by using both (+)- and C-)-propranolol at the appropriate concentrations, it is possible to distinguish between the specific and non-specific effects. At all concentrations tested, (--)-propranolol inhibited the stimulation of ~-amylase release by isoproterenol to a greater extent than did (+)-propranolol. The same was true for the effects of (+}- and (--)-proprano-
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Fig. 3. The effects of different concentrations o f adrenergic agonists on activation o f adenylate cyclase a c t i v i t y f r o m r a t p a r o t i d t i s s u e . T h e c o n d i t i o n s o f i n c u b a t i o n a r e d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e a m o u n t o f p r o t e i n u s e d p e r a s s a y w a s 2 4 0 ~tg.
88
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Fig. 4. E f f e c t o f (+)- a n d ( - - ) - p r o p r a n o l o l c o n c e n t r a t i o n on t h e s t i m u l a t i o n of C~-amylase release a n d cyclic AMP a c c u m u l a t i o n b y i s o p r o t e r e n o l in tissue slices o f r a t p a r o t i d . Tissue slices f r o m six rats w e r e p r e p a r e d a n d d i v i d e d into t w o lots. One lot was used f o r d e t e r m i n a t i o n o f s - a m y l a s e release a n d t h e o t h e r for d e t e r m i n a t i o n o f cyclic AMP a c c u m u l a t i o n . T h e slices w e r e i n c u b a t e d in t h e p r e s e n c e of t h e i n d i c a t e d c o n c e n t r a t i o n s o f p r o p r a n o l o l f o r 20 m i n b e f o r e a d d i t i o n o f i s o p r o t e r e n o l ( 1 0 pM). A f t e r a d d i t i o n of i s o p r o t e r e n o l , slices w e r e i n c u b a t e d for 4 5 rain p r i o r to d e t e r m i n a t i o n o f s - a m y l a s e release a n d for 10 mill p r i o r t o d e t e r m i n a t i o n of cyclic AMP.
lol on the stimulation of cyclic AMP accumulation by isoproterenol. However, at high concentrations of two isomers, (+)-propranolol markedly inhibited stimulation of cyclic AMP accumulation by isoproterenol, whereas stimulation of a-amylase release was only slightly inhibited. Fig. 4 also demonstrates that the effect of isoproterenol on cyclic AMP accumulation was much more sensitive to inhibition b y propranolol than was the effect on a-amylase release. The data presented thus far would indicate that the effects of adrenergic agonists on cyclic AMP accumulation and a-amylase release could be classified as/3-adrenergic responses. However, it is known that epinephrine and norepinephrine have both ~-adrenergic and a-adrenergic agonist properties [15]. To evaluate the action of the a-adrenergic agonists on a-amylase release, t w o approaches were used. First, the effects of phenylephrine on cyclic AMP accumulation, a-amylase release, and adenylate cyclase activity were examined. This drug has been classified as predominantly an a-adrenergic agonist [15]. Second, the effects of isoproterenol and norepinephrine on cyclic AMP accumulation and a-amylase release were studied in the presence of a-adrenergic antagonists. Phenylephrine, at relatively high concentrations, stimulated a-amylase release {Fig. 2, Table II). The effect was almost completely blocked by (--)propranolol and was partially antagonized by (+)-propranolol and by phentolamine, an a-antagonist. Atropine had no effect. It would appear that part of the effect of phenylephrine on a-amylase release could be classified as fl-
89 TABLE
II
THE EFFECT OF ADRENERGIC AND CHOLINERGIC OF a-AMYLASE RELEASE BY PHENYLEPHRINE
BLOCKING
AGENTS
ON THE STIMULATION
Tissue slices f r o m t w o rats w e r e p r e p a r e d a n d i n c u b a t e d 1 0 r a i n in t h e p r e s e n c e o f b l o c k i n g a g e n t s , after w h i c h t i m e p h e n y l e p h r i n e w a s a d d e d for an a d d i t i o n a l 4 5 m i n .
Additions
None (+)-Propranolol, 2.0 pM (-)-Propranolol, 2.0 pM Phentolamine, 10 pM Atropine, 5.0 pM
Percent ~-amylase released Basal
+Phenylephrine (25 #M)
8.6 7.2 8.1 7.5 8.5
26.5 17.4 12.9 20.4 27.8
-+ 0 . 4 -+ 1 . 7 + 1.2 + 2.1 + 0.8
+ 0.9 + 0 -+ 1 . 2 -+ 0 . 1 + 1.5
adrenergic and part as a-adrenergic. Since 2 pM (+)-propranolol had no nonspecific effects on the stimulation of a-amylase release by isoproterenol {Fig. 4), it is likely that (+)-propranolol partially blocked the effect of phenylephrine as a result of a weak competitive fl-adrenergic antagonist action. Phenylephrine did not have a detectable effect on cyclic AMP accumulation, even in the presence of theophylline, which stands in marked contrast to the stimulatory effect of phenylephrine on adenylate cyclase (Fig. 3, Table III). Phenylephrine is generally considered to be an a-adrenergic agonist, but appeared to stimulate a-amylase release in part by a fl-adrenergic mechanism (Table II). Since stimulation of adenylate cyclase by adrenergic agents is classified as a fl-adrenergic response, the effect of phenylephrine on adenylate cyclase from parotid tissue was investigated. It can be seen in Table III that the stimulation of adenylate cyclase by phenylephrine was blocked by (--)propranolol and not by phentolamine. It has been proposed that the actions of a- and fl-adrenergic agonists are opposed to one another [16,17], resulting in an apparent antagonism between the actions of the two types of agonists. If such antagonism exists, inhibition of the a-agonist activity of an agent that has both a- and fl-adrenergic agonist properties should enhance the fl-agonist activity of that drug. In support of this idea, the a-adrenergic antagonists phenoxybenzamine and phentolamine enTABLE
III
THE EFFECT OF ADRENERGIC BLOCKING CYCLASE ACTIVITY BY PHENYLEPHRINE
AGENTS
ON THE STIMULATION
OF ADENYLATE
A d e n y l a t e c y e l a s e p r e p a r a t i o n is d e s c r i b e d u n d e r Materials a n d M e t h o d s . T h e a m o u n t o f p r o t e i n u s e d p e r assay was 190/~g. Additions
None (-)-Propranolol, 10 pM Phentolamine
p m o l c y c l i c A M P f o r m e d ( P m o l • m g p r o t e i n -1 • r a i n - 1 ) Basal
+Phenylephrine
4 . 0 -+ 0 . 4 3 . 5 +- 0 . 1 4.2 + 0.5
2 7 . 4 -+ 0 . 5 7.3 + 1.1 2 5 . 8 -+ 2 . 8
(25 #M)
9O
Amylase +Phenoxybenzamine
411
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Narepinephrlne, uM (lag scalel Fig. 5. E f f e c t o f a - a d r e n e r g i c b l o c k i n g a g e n t s on t h e s t i m u l a t i o n of a - a m y l a s e release a n d cyclic AMP a c c u m u l a t i o n b y n o r e p i n e p h r i n e in tissue slices of r a t p a r o t i d . Tissue slices w e r e p r e p a x e d f r o m t w o g r o u p s of t h r e e rats. T h e slices f r o m o n e g r o u p w e r e used for d e t e r m i n a t i o n s of a - a m y l a s e release a n d slices f r o m t h e o t h e r w e r e used for d e t e r m i n a t i o n s of cyclic AMP. I n b o t h cases t h e slices w e r e i n c u b a t e d w i t h e i t h e r p h e n o x y b e n z a m i n e (10 ~zM) o r p h e n t o l a m i n e (10 pM) 1 5 m i n b e f o r e t h e a d d i t i o n of n o r e p i n e p h r i n e to t h e i n d i c a t e d c o n c e n t r a t i o n s . A f t e r a d d i t i o n of n o r e p i n e p h r i n e , i n c u b a t i o n s w e r e c o n t i n u e d for 45 re.in for d e t e r m i n a t i o n of a - a m y l a s e release a n d 10 rain for d e t e r m i n a t i o n of cyclic AMP a c c u m u l a t i o n . Basal v a l u e f o r a - a m y l a s e release was 6.5% a n d t h a t for cyclic AMP was 14.1 p m o l / m g p r o t e i n .
hanced the effect of norepinephrine on cyclic AMP accumulation and a-amylase release (Fig. 5). However, the same effects of phenoxybenzamine and phentolamine were observed for the stimulation of a-amylase release and cyclic AMP accumulation by isoproterenol (Fig. 6). Isoproterenol is believed to have only minimal a-adrenergic agonist activity; however, it should be remembered that the relative degree of a- and fi-adrenergic agonist activity for a given drug is a function of the tissue under study [15]. Batzri et al. [1] reported that phentolamine did not potentiate the effect of epinephrine on the levels of cyclic AMP in rat parotid tissue slices. However, in the presence of phentolamine, the level of cyclic AMP remained elevated for a longer period of time after addition of epinephrine. For this reason, the cyclic AMP levels shown in Figs 5 and 6 were determined at the time of maximal increase of the cyclic AMP level following the addition of norepinephrine or isoproterenol. In each instance this was the 10-min time point (Fig. 1). Non-specific effects of phenoxybenzamine may be indicated since this c o m p o u n d also enhanced the action of dibutyryl cyclic AMP on a-amylase release (Fig. 7). In the same experiment, phentolamine was without effect. Since these results suggest that the effects of phenoxybenzamine and phentolamine might be non-specific, caution should be used in interpreting evidence concerning a- and fi-adrenergic interactions.
91
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