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Evidence that deprenyl, a type B monoamine oxidase inhibitor, is an indirectly acting sympathomimetic amine
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Biochemical Pharmacology, Vol. 27, pp. 1591-1595. (~ Pergamon Press Ltd. 1978. Printed in Great Britain.
EVIDENCE THAT DEPRENYL, A TYPE B MONOAMINE O X I D A S E I N H I B I T O R , IS AN I N D I R E C T L Y A C T I N G SYMPATHOMIMETIC AMINE LANCE L. SIMPSON*']" Department of Pharmacology, College of Physicians and Surgeons, Columbia University. New York, NY, U.S.A.
(Received 27 June 1977: accepted 14 September 1977) Abstract--Various doses of /-deprenyl were tested for their abilities to increase blood pressure or heart rate in animals pretreated with a ganglionic blocking agent. The use of a ganglionic blocker (chlorisondamine) ensured that deprenyl-induced responses were mediated by the peripheral autonomic nervous system, and that these responses were not influenced by the central nervous system. It was found that /-deprenyl had a modest ability to increase blood pressure and a marked ability to increase heart rate. The ability of/-deprenyl to increase heart rate was diminished or abolished by propranolol, reserpine, and chemical sympathectomy (6-hydroxydopamine). Deprenyl-induced responses were negligibly affected by adrenalectomy. These data suggest that /-deprenyl is an indirectly acting sympathomimetic amine whose responses are mediated by norepinephrine in postganglionic sympathetic neurons. An additional finding was that desmethylimipramine, a known blocker of the norepinephrine pump, antagonized the effects of/-deprenyl. This finding suggests that/-deprenyl enters sympathetic neurons via the membrane pump. The ability of/-deprenyl to enter sympathetic neurons and evoke release of endogenous amines was not accompanied by a significant loss of tissue (heartl norepinephrine.
T h e r e is b o t h b i o c h e m i c a l a n d p h a r m a c o l o g i c a l evid e n c e to suggest t h a t t h e e n z y m e m o n o a m i n e oxid a s e ( M A O ) e x i s t s in multiple f o r m s (see r e v i e w s in Refs. 1-3). P e r h a p s t h e m o s t c o m p e l l i n g e v i d e n c e a r i s e s f r o m t h e finding t h a t t h e r e are s e l e c t i v e l y a c t i n g i n h i b i t o r s of M A O . S t u d i e s with t h e s e inhibitors h a v e led to the s u g g e s t i o n t h a t M A O e x i s t s in at least t w o f o r m s . F o r s a k e of simplicity, t h e s e forms (isoenzymes?) have been labeled type A and t y p e B. In t e r m s of s e l e c t i v e i n h i b i t i o n , it h a s b e e n f o u n d that clorgyline is a p r e f e r e n t i a l i n h i b i t o r of the t y p e A e n z y m e [4], a n d d e p r e n y l is a p r e f e r e n t i a l i n h i b i t o r of the t y p e B e n z y m e [5]. T h e e x i s t e n c e of multiple f o r m s of M A O c o u l d m e a n t h a t the individual species h a v e a u n i q u e localization or a u n i q u e f u n c t i o n . A n u m b e r of investigators h a v e e x a m i n e d this possibility b y a d m i n i s tering o n e or t h e o t h e r of t h e m a j o r i n h i b i t o r s (i.e. clorgyline or deprenyl), and then monitoring evoked c h a n g e s in the n e r v o u s s y s t e m (e.g. tissue levels of c a t e c h o l a m i n e s ) or in b e h a v i o r (e.g. s p o n t a n e o u s m o t o r activity). In e s s e n c e , t h e s e i n v e s t i g a t o r s h a v e u s e d M A O i n h i b i t o r s as p h a r m a c o l o g i c a l tools. A n e x a m i n a t i o n of the literature r e v e a l s t h a t this a p p r o a c h is b e i n g used in s e v e r a l l a b o r a t o r i e s (see r e v i e w in Ref. 6). T h e v a l u e of u s i n g M A O i n h i b i t o r s as p h a r m a cological tools is b a s e d o n t h e p r e m i s e t h a t t h e s e d r u g s are specific in t h e i r m e c h a n i s m s of action. T h e * Supported in part by National Heart and Lung Institute Program Project Grant HL 12738 and by a grant from Warner-Lambert Co. + Address reprint requests to: Dr. Lance L. Simpson, Department of Pharmacology, College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032. 1591
p u r p o s e o f the p r e s e n t s t u d y is to r e p o r t t h a t , i n s o f a r as d e p r e n y l is c o n c e r n e d , such specificity m a y be elusive. D a t a are p r e s e n t e d w h i c h s h o w that deprenyl, in a d d i t i o n to b e i n g a t y p e B inhibitor, is also an indirectly a c t i n g s y m p a t h o m i m e t i c a m i n e . This finding h a s i m p l i c a t i o n s for i n t e r p r e t i n g the effects of d e p r e n y l o n t h e n e r v o u s s y s t e m a n d on behavior. MATERIALS AND METHODS
Animals. Male W i s t a r rats ( C h a r l e s R i v e r Breeding L a b o r a t o r i e s , W i l m i n g t o n , M A ) weighing bet w e e n 200 a n d 275 g w e r e u s e d in t h e s e studies. A n i m a l s w e r e s u b j e c t e d to v a r i o u s b i o c h e m i c a l or c a r d i o v a s c u l a r p r o c e d u r e s that are d e s c r i b e d below. Recording of blood pressure and heart rate. T h e t e c h n i q u e s used for r e c o r d i n g c a r d i o v a s c u l a r resp o n s e s h a v e b e e n d e s c r i b e d in detail [7, 8]. In brief, the p r o c e d u r e s w e r e as follows. Blood p r e s s u r e was m o n i t o r e d t h r o u g h a c a n n u l a (PE50) installed in the left c a r o t i d artery. B l o o d p r e s s u r e r e c o r d i n g s w e r e o b t a i n e d b y using a p r e s s u r e t r a n s d u c e r ( H e w l e t t P a c k a r d 1280B) a n d p r e s s u r e amplifier ( H e w l e t t P a c k a r d 8805C) c o n n e c t e d to a t h e r m a l r e c o r d i n g s y s t e m ( H e w l e t t P a c k a r d 7754A). H e a r t rate was m o n i t o r e d with t w o s u b d e r m a l e l e c t r o d e s (active leads) in the c h e s t wall a n d o n e s u b d e r m a l e l e c t r o d e ( r e f e r e n c e lead) in a h i n d limb. H e a r t rate r e c o r d i n g s w e r e o b t a i n e d b y c o u p l i n g a rate c o m p u t e r ( H e w l e t t P a c k a r d 8812A) with a b i o e l e c t r i c amplifier ( H e w l e t t P a c k a r d 881 IA), b o t h of w h i c h were c o n n e c t e d to the r e c o r d i n g s y s t e m . During experiments on blood pressure and heart rate, a n i m a l s w e r e a n e s t h e t i z e d ( p e n t o b a r b i t a l s o d i u m , 50 mg/kg, i.p.). In a d d i t i o n , a n i m a l s were
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pretreated with a muscarinic cholinergic blocking agent (atropine, 25 mg/kg, i.p.) and with a ganglionic blocking agent (chlorisondamine, 1.0 mg/kg, i.p.). These p r e t r e a t m e n t s ensured that all tonic efferent activity and all reflex activity w e r e eliminated. This in turn served to 'isolate' the a u t o n o m i c nervous system f r o m the influences of the central nervous system. Adrenalectomy and sympathectomy. The adrenal glands were r e m o v e d under ether anesthesia. Bilateral incisions were made through the skin and muscle wall o v e r l y i n g the kidneys. The adrenals were located and r e m o v e d by teasing. A f t e r a d r e n a l e c t o m y , skin and muscle were closed with surgical staples.The operation was rapid ( ~ 10 min) and p r o d u c e d little bleeding. A f t e r the operation, animals were maintained on a normal diet plus saline (0.154 M NaCI) that contained t e r r a m y c i n (~ 0.5 g/l). Animals w e r e used 1 w e e k after adrenalectomy. A separate group of animals was chemically s y m p a t h e c t o m i z e d with 6 - h y d r o x y d o p a m i n e (6OHDA). A dose of 50 mg/kg was given on clay 1, and an additional dose of 100 mg/kg was given on day 2. Studies on e v o k e d c a r d i o v a s c u l a r r e s p o n s e s or on tissue levels of norepinephrine were c o n d u c t e d on day 4 . 6 - O H D A was administered in a saline vehicle (0.154 M NaCI) that contained ascorbic acid (0.01 M). The mixture was prepared immediately prior to use. Animals were injected via the tail vein.
Tissue levels of norepinephrine (ME) and uptake of radioactive norepinephrine [3H]I-NE). E n d o g e n o u s N E was assayed by the t r i h y d r o x y i n d o l e method[9] as subsequently modified by Shellenberger and Gordon[10]. Animals were sacrificed by decapitation, and hearts w e r e rapidly r e m o v e d , washed and blotted dry. A f t e r being weighed, tissues were h o m o g e n i z e d in iced 0.5 N perchloric acid and then centrifuged (5000 r e v / m i n , 10 min, Fisher clinical centrifuge). The supernatant was applied to an alumina c o l u m n (pH adjusted to ~ 8.6), and three washes (distilled water) were p e r f o r m e d to r e m o v e n o n a d s o r b e d substances. N E was subsequently eluted with acetic acid (0.5 N). The acid eluate was analyzed for N E spectrofluorimetrically. Data were c o r r e c t e d for r e c o v e r y . U p t a k e of radioactive N E into heart was studied after the administration (femoral vein) of tracer doses (0.15/~g/kg) of [aH]/-NE (saline vehicle) to rats. Fifteen min after [3H]/-NE injection, animals were decapitated. Their hearts w e r e r e m o v e d rapidly and treated identically to hearts a n a l y z e d for e n d o g e n o u s N E . [:~HI/-NE was assayed by liquid scintillation s p e c t r o m e t r y . Data were c o r r e c t e d for recovery. Drugs. The drugs used w e r e : / - d e p r e n y l HCI (gift f r o m W a r n e r - L a m b e r t C o . , Morris Plains, N J), reserpine (gift f r o m Smith Kline & F r e n c h Laboratories, Philadelphia, PAL d e s m e t h y l i m i p r a m i n e HCI (gift f r o m U S V Pharmaceutical C o r p . , T u c k a h o e , NY), chlorisondamine chloride (gift f r o m Ciba Pharmaceutical Co., Summit, N J), [3H]/-norepinephrine (5.85 C i / m - m o l e , N e w England N u c l e a r , Boston, MA),/-norepinephrine bitartrate (Winthrop Laboratories, N e w York, NY) and d,l-propranolol HC1,
6 - h y d r o x y d o p a m i n e H B r and atropine (Sigma Chemical Co., St. Louis, MO). All drug dosages are e x p r e s s e d as the free base. Data. Individual data points on each figure represent the mean +_ standard deviation (S.D.) of at least four observations. Likewise, data referred to in the text represent the mean _+ S.D. S t a t e m e n t s on statistical analyses (P values) refer to use of the S t u d e n t ' s t-test. RESULTS
Effect of I-deprenyl on blood pressure and on heart rate. Individual groups of rats In = 4 or more) r e c e i v e d one of several doses of /-deprenyl, after which either blood pressure or heart rate responses were monitored. The doses of l-deprenyl that were tested ranged f r o m 0.1 to 10.0mg/kg (i.v.). As shown in Fig. 1, I-deprenyl e v o k e d both pressor and tachycardiac responses. Figure I also shows that heart rate responses w e r e much more pronounced. Accordingly, the effects of /-deprenyl on fladrenergic transmission were e x a m i n e d in detail. fl-Adrenergie activity. Figure 1 indicates that the EDso for deprenyl-induced responses is about 2.0 mg/kg. W h e n this dose was administered to animals in = 5), the average heart rate response was 87 _+ 9 beats/rain (bpm). Pretreatment of animals In = 4) with d,/-propranolol (I.0 mg/kg, i.v., 30 rain) essentially abolished this r e s p o n s e ( < 5 bpm). Furt h e r m o r e , the administration of d,/-propranolol ( 1.0 mg/kg, i.v.) 15 rain after I-deprenyl (2.0 mg/kg, i.v.) caused t a c h y c a r d i a to disappear. Interaction with reserpine. The role of e n d o g e n ous c a t e c h o l a m i n e s in deprenyl-induced responses was assessed by using reserpine. W h e n administered at a dose of 1.0 mg/kg (i.p., 16-20 hr), reser-
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DOSE (mg/kg) Fig. I. Average change (A) in mean arterial pressure and the average change in heart rate evoked by various doses of/-deprenyl. Each point on the figure represents the mean response (_+S.D.) obtained from four or more animals. Each animal received only one injection (i.v.) of/-deprenyl. Heart rate responses were much more pronounced than blood pressure responses. Nevertheless, the EDs0for both responses was about 2.0 mg/kg. A dose of 10 mg/kg was the maximum close practical for testing: higher doses tended to be toxic.
Indirectly acting amine pine caused m a r k e d depletion of cardiac N E . The values obtained in control and in reserpinized animals were, r e s p e c t i v e l y , 806 _+ 74 and 83 ± 32 ng/g. The difference is statistically significant (P < 0.001). W h e n animals (n = 4) w e r e pretreated with reserpine (as above), r e s p o n s e s to l-deprenyl (2.0 mg/kg, i.v.) were greatly attenuated. The r e s p o n s e s in control and in reserpinized animals w e r e , respectively, 93 _+ 12 and 9.5-+6.7 bpm. The difference is statistically significant (P < 0.001). Adrenalectomy. The striking effect of catecholamine depletion on d e p r e n y l - i n d u c e d responses could implicate either the adrenal glands or postganglionic sympathetic neurons. A putative role for the adrenals was tested by administering l-deprenyl (2.0 mg/kg, i.v.) to a d r e n a l e c t o m i z e d animals (n = 6). The a v e r a g e r e s p o n s e e v o k e d in these animals was 71 ± 15bpm. In m a t c h e d controls, the e v o k e d r e s p o n s e was 86 ± 6 bpm. This difference does not attain statistical significance. Sympathectomy. l-Deprenyl (2.0 mg/kg, i.v.) was administered to animals in = 4 ) pretreated with 6 - O H D A . In such animals, d e p r e n y l - i n d u c e d heart rate r e s p o n s e s were 3.3 ± 4.4 bpm, i.e. essentially nil. In related e x p e r i m e n t s , tissue levels of cardiac N E were d e t e r m i n e d in control animals and in symp a t h e c t o m i z e d animals (n = 4 each). The r e s p e c t i v e values were 754 ± 126 and 181 ± 103 ng/g. The difference is statistically significant (P < 0.001). Temporal aspects of 1-deprenyl activity. Regardless of w h e t h e r l-deprenyl was administered to control animals or to e x p e r i m e n t a l animals ( a d r e n a l e c t o m y , s y m p a t h e c t o m y or drug pretreatment), e v o k e d responses attained a peak within
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Fig. 2. Per cent inhibition of [:~H]I-NE uptake into heart. Various doses of desmethylimipramine (O-----O) or of ldeprenyl iCY---O)were administered to rats in = 4 or more per data point), after which [3H]I-NE was administered (i.v.). Uptake of radioactive NE was compared in experimental animals (desmethylimipramine or l-deprenyl pretreatment) and in control animals. Per cent inhibition of uptake was calculated from the equation: % Inhibition = 100 - [(uptakee,p/uptakeco,) x 100]. Desmethylimipramine was more potent than l-deprenyl in blocking uptake of [all]l-HE. (Note: These data should not be used to make quantitative comparisons relating to drug affinity for the norepinephrine pump. The reader is reminded that the experiments were conducted in vivo, and thus the data are a function of both affinity for the pump and disposition of drug.)
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A Con,,o, 0
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I - NOREPINEPHRINE
I - DEPRENYL
I - NOREPINEPHRINE
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a= 50 0
DEPRENYL
Fig. 3. Effect of desmethylimipramine (DMI) on responses to I-norepinephrine and to l-deprenyl. In panel A are illustrated the responses to a series of injections of Inorepinephrine (0.1 /zg/kg, i.v., 10-rain intervals) and a single injection of l-deprenyl (2.0 mg/kg, i.v.) to a control animal. In panel B are illustrated the same types of responses in an animal pretreated (30 min) with DMi (0.3 mg/kg, i.p.). Note that DMI increased the magnitude and duration of responses to l-norepinephrine, but it decreased the magnitude of responses to l-deprenyl. 10-15 min. In the present study, no attempt was made to relate response duration to blood levels of drug. H o w e v e r , preliminary o b s e r v a t i o n s indicated that half-decay times for e v o k e d responses (e.g. 2.0 mg/kg) were greater than 2 hr.
Interaction with desmethylimipramine (DMI). Various doses of D M I (i.p., 30 rain) w e r e tested for their ability to block uptake of [:~H]I-NE into heart. The ID~o for D M I - i n d u c e d blockade of uptake was b e t w e e n 0.1 and 0.3 mg/kg (Fig. 2). A dose of 0.3 mg/kg was used in subsequent studies. Animals were prepared for recording of heart rate responses. A control group of animals r e c e i v e d a series of injections of I-NE (0.1 /~g/kg, i.v.), after which a single injection of l-deprenyl (2.0 mg/kg, i.v.) was given. An experimental group of animals was treated identically, e x c e p t that DMI (0.3 mg/kg, i.p.) was given as a p r e t r e a t m e n t (30 min). A representative e x p e r i m e n t is illustrated in Fig. 3. In control animals, responses to I-NE a v e r a g e d about 50 bpm, and responses to l-deprenyl a v e r a g e d about 92 bpm. A f t e r D M I p r e t r e a t m e n t , responses to I-NE w e r e increased in magnitude (85 bpm) and in duration. By contrast, responses to l-deprenyl w e r e much reduced (24 bpm). Both the increase in r e s p o n s i v e n e s s to I-NE and the d e c r e a s e in responsiveness to l-deprenyl were statistically significant (P < 0.01). Tissue levels o[ NE and uptake of [aHII-NE. lD e p r e n y l was tested at several doses (0.1 to 10.0 mg/kg, i.v., 30 min) for its ability to increase or d e c r e a s e heart levels of NE. At no dose did Ideprenyl alter e n d o g e n o u s levels of NE. On the other hand, l-deprenyl did have an effect on uptake of e x o g e n o u s N E . W h e n administered 15 min prior to [~H]I-NE (0.15/~g/kg, i.v.), l-deprenyl (i.v.) produced a d o s e - d e p e n d e n t blockade of N E uptake into heart (Fig. 2). DISCUSSION
Drugs that affect b e h a v i o r are usually e x a m i n e d " for their effects on the peripheral a u t o n o m i c ner-
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v o u s s y s t e m . S u c h studies are t h o u g h t to be app r o p r i a t e for a v a r i e t y of r e a s o n s , o n e of w h i c h is particularly g e r m a n e to this report. By s t u d y i n g the a c t i o n s of a d r u g o n the a u t o n o m i c n e r v o u s s y s t e m , o n e might learn s o m e t h i n g t h a t w o u l d apply to the c e n t r a l n e r v o u s s y s t e m (CNS). T h i s i n f o r m a t i o n could lead to a n u n d e r s t a n d i n g of the cellular e v e n t s that c u l m i n a t e in a d r u g - i n d u c e d b e h a v i o r a l response. Unlike m o s t p s y c h o a c t i v e drugs, I-deprenyl has r e c e i v e d only limited a t t e n t i o n in t e r m s of its autonomic effects. T h i s lack of a t t e n t i o n must be v i e w e d in the c o n t e x t that I-deprenyl h a s b e e n used as a p h a r m a c o l o g i c a l tool to s t u d y the role of C N S a m i n e s [6]. It h a s also b e e n used to t r e a t d e p r e s s i v e illness [ 11 ] a n d to treat P a r k i n s o n ' s d i s e a s e [ 12]. All of t h e s e u s e s are linked to the ability o f / - d e p r e n y l to inhibit t y p e B M A O . H o w e v e r . the p r e s e n t s t u d y d e m o n s t r a t e s that /-deprenyl is not solely an M A O inhibitor. An e x a m i n a t i o n of the a u t o n o m i c effects o f / - d e p r e n y l i n d i c a t e s that the drug is an indirectly acting s y m p a t h o m i m e t i c a m i n e . As Fig. 1 illustrates, I-deprenyl has a m a r k e d ability to i n c r e a s e h e a r t rate, but it h a s only a m o d e s l ability to i n c r e a s e b l o o d p r e s s u r e . In light of t h e s e findings, a t t e n t i o n h a s b e e n f o c u s e d on t h e h e a r t rate effects of t h e drug. In w o r k to be r e p o r t e d e l s e w h e r e (J. Bilezikian, p e r s o n a l c o m m u n i c a t i o n ) , it h a s b e e n f o u n d t h a t /-deprenyl is not a fl-agonist. O n the o t h e r h a n d , d e p r e n y l - i n d u c e d t a c h y c a r d i a c a n be d i m i n i s h e d or a b o l i s h e d by p r o p r a n o l o l . This s e q u e n c e of findings s u g g e s t s that the effects of l-deprenyl are m e d i a t e d by e n d o g e n o u s catec h o l a m i n e s . T h e fact that r e s e r p i n e p r e t r e a t m e n t nearly a b o l i s h e s r e s p o n s e s to /-deprenyl f u r t h e r implicates e n d o g e n o u s a m i n e s . I f / - d e p r e n y l w e r e an indirectly a c t i n g a m i n e , i.e. a n a g e n t that e v o k e s the r e l e a s e of e n d o g e n o u s c a t e c h o l a m i n e s , t h e n e i t h e r a d r e n a l e c t o m y or symp a t h e c t o m y s h o u l d a l t e r r e s p o n s e s . In reality, a d r e n a l e c t o m y h a d o n l y a negligible effect. By c o n t r a s t , s y m p a t h e c t o m y h a d a striking effect. O f the v a r i o u s p r o c e d u r e s that w e r e used, 6 - O H D A i n d u c e d s y m p a t h e c t o m y was the m o s t effective in d i m i n i s h i n g r e s p o n s e s to /-deprenyl. T h i s finding indicates that postganglionic sympathetic neurons are the s o u r c e of c a t e c h o l a m i n e s t h r o u g h w h i c h /-deprenyl e v o k e s r e s p o n s e s . T h e r e are actually several p o s s i b l e m e c h a n i s m s by w h i c h / - d e p r e n y l might act. T h r e e of t h e s e m e c h a n i s m s are w o r t h y of c o n s i d e r a t i o n . F i r s t , / - d e p r e n y l might diffuse a c r o s s the n e u r o n a l m e m b r a n e . O n c e inside the n e r v e e n d i n g , it might e v o k e t r a n s m i t t e r release. S e c o n d , I-deprenyl m i g h t be a s u b s t r a t e for the m e m b r a n e p u m p t h a t c a p t u r e s N E a n d r e l a t e d c o m p o u n d s a n d t r a n s p o r t s t h e m into the n e r v e . O n c e again, /-deprenyl might act i n t r a n e u r o n a l l y to e v o k e t r a n s m i t t e r release. T h i r d , l-deprenyl might act mainly o n the e x t e r n a l s u r f a c e of the n e r v e m e m b r a n e as a n i n h i b i t o r of the N E p u m p . In so doing, /-deprenyl w o u l d inhibit t h e m a j o r m e c h a n ism f o r i n a c t i v a t i n g N E , a n d t h u s i n c r e a s e the a m o u n t of N E a v a i l a b l e to i n t e r a c t with p o s t j u n c tional r e c e p t o r s . A d e c i s i o n a m o n g t h e s e t h r e e possibilities c a n be m a d e by u s i n g a k n o w n i n h i b i t o r of the N E p u m p ,
n a m e l y , D M I . If the first possibility were true, then /-deprenyl a n d D M I s h o u l d act synergistically. N E r e l e a s e d by /-deprenyl w o u l d be p r o t e c t e d f r o m i n a c t i v a t i o n by DMI. If the s e c o n d possibility w e r e true, t h e n / - d e p r e n y l s h o u l d be a n t a g o n i z e d by DM 1. B l o c k a d e of the N E p u m p w o u l d p r e v e n t I-deprenyl f r o m r e a c h i n g its site of action. If the third possibility were true, t h e n I-deprenyl a n d DMI s h o u l d act additively. T h e t w o d r u g s would act jointly to i n a c t i v a t e the p u m p a n d t h e r e b y i n c r e a s e availability of N E . T h e d a t a illustrated in Fig. 3 leave little d o u b l a b o u t the most plausible c o n c l u s i o n . W h e n DMI was c o m b i n e d with the ED.~o for /-deprenyl, res p o n s e s to the latter were n o t a b l y a n t a g o n i z e d . This o c c u r r e d in the p r e s e n c e of clear e v i d e n c e that DMI was b l o c k i n g the N E p u m p (Fig. 2) and that DMI was p o t e n t i a t i n g the a c t i o n s of N E (Fig. 3). It app e a r s that /-deprenyl e n t e r s p o s t g a n g l i o n i c syrnp a t h e t i c n e u r o n s via the N E p u m p , a n d that it s u b s e q u e n t l y e v o k e s the release of n e u r o n a l catec h o l a m i n e s . T h e i m p o r t a n c e of passive diffusion a c r o s s the m e m b r a n e or b l o c k a d e of the m e m b r a n e p u m p must be minimal. A q u e s t i o n that h a s not b e e n a n s w e r e d is that of w h y / - d e p r e n y l e x e r t s g r e a t e r effects on s y m p a t h e t i c n e u r o n s in h e a r t as o p p o s e d to t h o s e in v a s c u l a t u r e . A t e n t a t i v e e x p l a n a t i o n could be the dual possibility that: (a) /-deprenyl has a relatively low affinity for the N E p u m p (see Fig. 2 a n d Ref. 13), a n d I b ) s y m p a t h e t i c n e r v e s in heart m a y h a v e a more avid u p t a k e m e c h a n i s m than s y m p a t h e t i c n e r v e s in vasculature. T h e m a t t e r r e q u i r e s f u r t h e r study. A n o t h e r q u e s t i o n of note p e r t a i n s to the C N S . T h e d a t a in this report deal e x c l u s i v e l y with the p e r i p h e r a l n e r v o u s s y s t e m . It is not k n o w n w h e t h e r l-deprenyl is an indirectly acting a m i n e in the brain. H o w e v e r , as interest in /-deprenyl m o u n t s , and as the f r e q u e n c y of its use as an i n v e s t i g a t i o n a l tool i n c r e a s e s , studies to d e t e r m i n e w h e t h e r /-deprenyl is a n indirectly acting a m i n e in the brain must be u n d e r t a k e n . In the a b s e n c e of such w o r k , the neurological a n d b e h a v i o r a l effects of /-deprenyl m a y be difficult to explain. Acknowledg, e m e n t - - T h e author is pleased to express him indebtedness to John Aletta and Gene Miskin for their diligent work and for their keen observations.
REFERENCES I. M. Sandier and M. B. H. Youdim. Pharma~. Rev. 24, 33t (1972). 2. E. Usdin (Ed.), Neuropsychopharma('ology oj Monoamines and Their Regulatory Enzymes. Raven Press, NY (1974). 3. Monamine Oxidase and Its Inhibition, Ciba Foundation Symposium 39. Elsevier, Amsterdam (1976). (1976). 4. J. P. Johnston, Bio('hern. Pharma('. 17, 1285 (1968). 5. A. J. Christmas, C. J. Coulson, D. R. Maxwell and D. Riddell, Br. J. Pharmac. 45,490 (1972). 6. N. H. Neff and J. A. Fuentes, in M o n o a m i n e Oxidase and Its Inhibition, Ciba Foundation Symposium 3% p. 000. Elsevier, Amsterdam (1976). 7. D. A. Gilder, W. Fain and L. L. Simpson, J. Pharmac. exp. Ther. 198, 255 (1976). 8. L. L. Simpson. Biochem. Pharmac. 26, 1315 (1977L
lndireclly acting amine 9. A. H. A n t o n and D. F. Sayre. J. Pharmac. exp. Ther. 138, 36(I (1962). 10. M. K. Shellenberger and J. H. Gordon, Analyt. Biochem. 39, 356 (1971). 1 I. L. Tringer, G. Halts and E. Varga, Societas Pharmacologica Hungarica. V Conferentia Hungarica Pro Therapia et Investigatione in Pharmacologia (1971 I.
~ P. 2 7 1 1
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12. L. Ambrozi, W. Birkmayer, P. Riederer and M. B. H. Youdim, Br. J. Pharmac. 58, 423P (1976). 13. C. Braestrup, H. A n d e r s e n and A. Randrup, Eur. J. Pharmac. 34, 181 (1975).
Biochemical Pharmacology, Vol. 27, pp. 1591-1595. (~ Pergamon Press Ltd. 1978. Printed in Great Britain.
EVIDENCE THAT DEPRENYL, A TYPE B MONOAMINE O X I D A S E I N H I B I T O R , IS AN I N D I R E C T L Y A C T I N G SYMPATHOMIMETIC AMINE LANCE L. SIMPSON*']" Department of Pharmacology, College of Physicians and Surgeons, Columbia University. New York, NY, U.S.A.
(Received 27 June 1977: accepted 14 September 1977) Abstract--Various doses of /-deprenyl were tested for their abilities to increase blood pressure or heart rate in animals pretreated with a ganglionic blocking agent. The use of a ganglionic blocker (chlorisondamine) ensured that deprenyl-induced responses were mediated by the peripheral autonomic nervous system, and that these responses were not influenced by the central nervous system. It was found that /-deprenyl had a modest ability to increase blood pressure and a marked ability to increase heart rate. The ability of/-deprenyl to increase heart rate was diminished or abolished by propranolol, reserpine, and chemical sympathectomy (6-hydroxydopamine). Deprenyl-induced responses were negligibly affected by adrenalectomy. These data suggest that /-deprenyl is an indirectly acting sympathomimetic amine whose responses are mediated by norepinephrine in postganglionic sympathetic neurons. An additional finding was that desmethylimipramine, a known blocker of the norepinephrine pump, antagonized the effects of/-deprenyl. This finding suggests that/-deprenyl enters sympathetic neurons via the membrane pump. The ability of/-deprenyl to enter sympathetic neurons and evoke release of endogenous amines was not accompanied by a significant loss of tissue (heartl norepinephrine.
T h e r e is b o t h b i o c h e m i c a l a n d p h a r m a c o l o g i c a l evid e n c e to suggest t h a t t h e e n z y m e m o n o a m i n e oxid a s e ( M A O ) e x i s t s in multiple f o r m s (see r e v i e w s in Refs. 1-3). P e r h a p s t h e m o s t c o m p e l l i n g e v i d e n c e a r i s e s f r o m t h e finding t h a t t h e r e are s e l e c t i v e l y a c t i n g i n h i b i t o r s of M A O . S t u d i e s with t h e s e inhibitors h a v e led to the s u g g e s t i o n t h a t M A O e x i s t s in at least t w o f o r m s . F o r s a k e of simplicity, t h e s e forms (isoenzymes?) have been labeled type A and t y p e B. In t e r m s of s e l e c t i v e i n h i b i t i o n , it h a s b e e n f o u n d that clorgyline is a p r e f e r e n t i a l i n h i b i t o r of the t y p e A e n z y m e [4], a n d d e p r e n y l is a p r e f e r e n t i a l i n h i b i t o r of the t y p e B e n z y m e [5]. T h e e x i s t e n c e of multiple f o r m s of M A O c o u l d m e a n t h a t the individual species h a v e a u n i q u e localization or a u n i q u e f u n c t i o n . A n u m b e r of investigators h a v e e x a m i n e d this possibility b y a d m i n i s tering o n e or t h e o t h e r of t h e m a j o r i n h i b i t o r s (i.e. clorgyline or deprenyl), and then monitoring evoked c h a n g e s in the n e r v o u s s y s t e m (e.g. tissue levels of c a t e c h o l a m i n e s ) or in b e h a v i o r (e.g. s p o n t a n e o u s m o t o r activity). In e s s e n c e , t h e s e i n v e s t i g a t o r s h a v e u s e d M A O i n h i b i t o r s as p h a r m a c o l o g i c a l tools. A n e x a m i n a t i o n of the literature r e v e a l s t h a t this a p p r o a c h is b e i n g used in s e v e r a l l a b o r a t o r i e s (see r e v i e w in Ref. 6). T h e v a l u e of u s i n g M A O i n h i b i t o r s as p h a r m a cological tools is b a s e d o n t h e p r e m i s e t h a t t h e s e d r u g s are specific in t h e i r m e c h a n i s m s of action. T h e * Supported in part by National Heart and Lung Institute Program Project Grant HL 12738 and by a grant from Warner-Lambert Co. + Address reprint requests to: Dr. Lance L. Simpson, Department of Pharmacology, College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032. 1591
p u r p o s e o f the p r e s e n t s t u d y is to r e p o r t t h a t , i n s o f a r as d e p r e n y l is c o n c e r n e d , such specificity m a y be elusive. D a t a are p r e s e n t e d w h i c h s h o w that deprenyl, in a d d i t i o n to b e i n g a t y p e B inhibitor, is also an indirectly a c t i n g s y m p a t h o m i m e t i c a m i n e . This finding h a s i m p l i c a t i o n s for i n t e r p r e t i n g the effects of d e p r e n y l o n t h e n e r v o u s s y s t e m a n d on behavior. MATERIALS AND METHODS
Animals. Male W i s t a r rats ( C h a r l e s R i v e r Breeding L a b o r a t o r i e s , W i l m i n g t o n , M A ) weighing bet w e e n 200 a n d 275 g w e r e u s e d in t h e s e studies. A n i m a l s w e r e s u b j e c t e d to v a r i o u s b i o c h e m i c a l or c a r d i o v a s c u l a r p r o c e d u r e s that are d e s c r i b e d below. Recording of blood pressure and heart rate. T h e t e c h n i q u e s used for r e c o r d i n g c a r d i o v a s c u l a r resp o n s e s h a v e b e e n d e s c r i b e d in detail [7, 8]. In brief, the p r o c e d u r e s w e r e as follows. Blood p r e s s u r e was m o n i t o r e d t h r o u g h a c a n n u l a (PE50) installed in the left c a r o t i d artery. B l o o d p r e s s u r e r e c o r d i n g s w e r e o b t a i n e d b y using a p r e s s u r e t r a n s d u c e r ( H e w l e t t P a c k a r d 1280B) a n d p r e s s u r e amplifier ( H e w l e t t P a c k a r d 8805C) c o n n e c t e d to a t h e r m a l r e c o r d i n g s y s t e m ( H e w l e t t P a c k a r d 7754A). H e a r t rate was m o n i t o r e d with t w o s u b d e r m a l e l e c t r o d e s (active leads) in the c h e s t wall a n d o n e s u b d e r m a l e l e c t r o d e ( r e f e r e n c e lead) in a h i n d limb. H e a r t rate r e c o r d i n g s w e r e o b t a i n e d b y c o u p l i n g a rate c o m p u t e r ( H e w l e t t P a c k a r d 8812A) with a b i o e l e c t r i c amplifier ( H e w l e t t P a c k a r d 881 IA), b o t h of w h i c h were c o n n e c t e d to the r e c o r d i n g s y s t e m . During experiments on blood pressure and heart rate, a n i m a l s w e r e a n e s t h e t i z e d ( p e n t o b a r b i t a l s o d i u m , 50 mg/kg, i.p.). In a d d i t i o n , a n i m a l s were
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pretreated with a muscarinic cholinergic blocking agent (atropine, 25 mg/kg, i.p.) and with a ganglionic blocking agent (chlorisondamine, 1.0 mg/kg, i.p.). These p r e t r e a t m e n t s ensured that all tonic efferent activity and all reflex activity w e r e eliminated. This in turn served to 'isolate' the a u t o n o m i c nervous system f r o m the influences of the central nervous system. Adrenalectomy and sympathectomy. The adrenal glands were r e m o v e d under ether anesthesia. Bilateral incisions were made through the skin and muscle wall o v e r l y i n g the kidneys. The adrenals were located and r e m o v e d by teasing. A f t e r a d r e n a l e c t o m y , skin and muscle were closed with surgical staples.The operation was rapid ( ~ 10 min) and p r o d u c e d little bleeding. A f t e r the operation, animals were maintained on a normal diet plus saline (0.154 M NaCI) that contained t e r r a m y c i n (~ 0.5 g/l). Animals w e r e used 1 w e e k after adrenalectomy. A separate group of animals was chemically s y m p a t h e c t o m i z e d with 6 - h y d r o x y d o p a m i n e (6OHDA). A dose of 50 mg/kg was given on clay 1, and an additional dose of 100 mg/kg was given on day 2. Studies on e v o k e d c a r d i o v a s c u l a r r e s p o n s e s or on tissue levels of norepinephrine were c o n d u c t e d on day 4 . 6 - O H D A was administered in a saline vehicle (0.154 M NaCI) that contained ascorbic acid (0.01 M). The mixture was prepared immediately prior to use. Animals were injected via the tail vein.
Tissue levels of norepinephrine (ME) and uptake of radioactive norepinephrine [3H]I-NE). E n d o g e n o u s N E was assayed by the t r i h y d r o x y i n d o l e method[9] as subsequently modified by Shellenberger and Gordon[10]. Animals were sacrificed by decapitation, and hearts w e r e rapidly r e m o v e d , washed and blotted dry. A f t e r being weighed, tissues were h o m o g e n i z e d in iced 0.5 N perchloric acid and then centrifuged (5000 r e v / m i n , 10 min, Fisher clinical centrifuge). The supernatant was applied to an alumina c o l u m n (pH adjusted to ~ 8.6), and three washes (distilled water) were p e r f o r m e d to r e m o v e n o n a d s o r b e d substances. N E was subsequently eluted with acetic acid (0.5 N). The acid eluate was analyzed for N E spectrofluorimetrically. Data were c o r r e c t e d for r e c o v e r y . U p t a k e of radioactive N E into heart was studied after the administration (femoral vein) of tracer doses (0.15/~g/kg) of [aH]/-NE (saline vehicle) to rats. Fifteen min after [3H]/-NE injection, animals were decapitated. Their hearts w e r e r e m o v e d rapidly and treated identically to hearts a n a l y z e d for e n d o g e n o u s N E . [:~HI/-NE was assayed by liquid scintillation s p e c t r o m e t r y . Data were c o r r e c t e d for recovery. Drugs. The drugs used w e r e : / - d e p r e n y l HCI (gift f r o m W a r n e r - L a m b e r t C o . , Morris Plains, N J), reserpine (gift f r o m Smith Kline & F r e n c h Laboratories, Philadelphia, PAL d e s m e t h y l i m i p r a m i n e HCI (gift f r o m U S V Pharmaceutical C o r p . , T u c k a h o e , NY), chlorisondamine chloride (gift f r o m Ciba Pharmaceutical Co., Summit, N J), [3H]/-norepinephrine (5.85 C i / m - m o l e , N e w England N u c l e a r , Boston, MA),/-norepinephrine bitartrate (Winthrop Laboratories, N e w York, NY) and d,l-propranolol HC1,
6 - h y d r o x y d o p a m i n e H B r and atropine (Sigma Chemical Co., St. Louis, MO). All drug dosages are e x p r e s s e d as the free base. Data. Individual data points on each figure represent the mean +_ standard deviation (S.D.) of at least four observations. Likewise, data referred to in the text represent the mean _+ S.D. S t a t e m e n t s on statistical analyses (P values) refer to use of the S t u d e n t ' s t-test. RESULTS
Effect of I-deprenyl on blood pressure and on heart rate. Individual groups of rats In = 4 or more) r e c e i v e d one of several doses of /-deprenyl, after which either blood pressure or heart rate responses were monitored. The doses of l-deprenyl that were tested ranged f r o m 0.1 to 10.0mg/kg (i.v.). As shown in Fig. 1, I-deprenyl e v o k e d both pressor and tachycardiac responses. Figure I also shows that heart rate responses w e r e much more pronounced. Accordingly, the effects of /-deprenyl on fladrenergic transmission were e x a m i n e d in detail. fl-Adrenergie activity. Figure 1 indicates that the EDso for deprenyl-induced responses is about 2.0 mg/kg. W h e n this dose was administered to animals in = 5), the average heart rate response was 87 _+ 9 beats/rain (bpm). Pretreatment of animals In = 4) with d,/-propranolol (I.0 mg/kg, i.v., 30 rain) essentially abolished this r e s p o n s e ( < 5 bpm). Furt h e r m o r e , the administration of d,/-propranolol ( 1.0 mg/kg, i.v.) 15 rain after I-deprenyl (2.0 mg/kg, i.v.) caused t a c h y c a r d i a to disappear. Interaction with reserpine. The role of e n d o g e n ous c a t e c h o l a m i n e s in deprenyl-induced responses was assessed by using reserpine. W h e n administered at a dose of 1.0 mg/kg (i.p., 16-20 hr), reser-
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10
DOSE (mg/kg) Fig. I. Average change (A) in mean arterial pressure and the average change in heart rate evoked by various doses of/-deprenyl. Each point on the figure represents the mean response (_+S.D.) obtained from four or more animals. Each animal received only one injection (i.v.) of/-deprenyl. Heart rate responses were much more pronounced than blood pressure responses. Nevertheless, the EDs0for both responses was about 2.0 mg/kg. A dose of 10 mg/kg was the maximum close practical for testing: higher doses tended to be toxic.
Indirectly acting amine pine caused m a r k e d depletion of cardiac N E . The values obtained in control and in reserpinized animals were, r e s p e c t i v e l y , 806 _+ 74 and 83 ± 32 ng/g. The difference is statistically significant (P < 0.001). W h e n animals (n = 4) w e r e pretreated with reserpine (as above), r e s p o n s e s to l-deprenyl (2.0 mg/kg, i.v.) were greatly attenuated. The r e s p o n s e s in control and in reserpinized animals w e r e , respectively, 93 _+ 12 and 9.5-+6.7 bpm. The difference is statistically significant (P < 0.001). Adrenalectomy. The striking effect of catecholamine depletion on d e p r e n y l - i n d u c e d responses could implicate either the adrenal glands or postganglionic sympathetic neurons. A putative role for the adrenals was tested by administering l-deprenyl (2.0 mg/kg, i.v.) to a d r e n a l e c t o m i z e d animals (n = 6). The a v e r a g e r e s p o n s e e v o k e d in these animals was 71 ± 15bpm. In m a t c h e d controls, the e v o k e d r e s p o n s e was 86 ± 6 bpm. This difference does not attain statistical significance. Sympathectomy. l-Deprenyl (2.0 mg/kg, i.v.) was administered to animals in = 4 ) pretreated with 6 - O H D A . In such animals, d e p r e n y l - i n d u c e d heart rate r e s p o n s e s were 3.3 ± 4.4 bpm, i.e. essentially nil. In related e x p e r i m e n t s , tissue levels of cardiac N E were d e t e r m i n e d in control animals and in symp a t h e c t o m i z e d animals (n = 4 each). The r e s p e c t i v e values were 754 ± 126 and 181 ± 103 ng/g. The difference is statistically significant (P < 0.001). Temporal aspects of 1-deprenyl activity. Regardless of w h e t h e r l-deprenyl was administered to control animals or to e x p e r i m e n t a l animals ( a d r e n a l e c t o m y , s y m p a t h e c t o m y or drug pretreatment), e v o k e d responses attained a peak within
I00
14J :X:
80 60
z
40
"" ,,-r-
20
o
0,01
0,1 1,0 DOSE ( m g / k g )
I0
Fig. 2. Per cent inhibition of [:~H]I-NE uptake into heart. Various doses of desmethylimipramine (O-----O) or of ldeprenyl iCY---O)were administered to rats in = 4 or more per data point), after which [3H]I-NE was administered (i.v.). Uptake of radioactive NE was compared in experimental animals (desmethylimipramine or l-deprenyl pretreatment) and in control animals. Per cent inhibition of uptake was calculated from the equation: % Inhibition = 100 - [(uptakee,p/uptakeco,) x 100]. Desmethylimipramine was more potent than l-deprenyl in blocking uptake of [all]l-HE. (Note: These data should not be used to make quantitative comparisons relating to drug affinity for the norepinephrine pump. The reader is reminded that the experiments were conducted in vivo, and thus the data are a function of both affinity for the pump and disposition of drug.)
1593
A Con,,o, 0
DMI
I - NOREPINEPHRINE
I - DEPRENYL
I - NOREPINEPHRINE
I-
a= 50 0
DEPRENYL
Fig. 3. Effect of desmethylimipramine (DMI) on responses to I-norepinephrine and to l-deprenyl. In panel A are illustrated the responses to a series of injections of Inorepinephrine (0.1 /zg/kg, i.v., 10-rain intervals) and a single injection of l-deprenyl (2.0 mg/kg, i.v.) to a control animal. In panel B are illustrated the same types of responses in an animal pretreated (30 min) with DMi (0.3 mg/kg, i.p.). Note that DMI increased the magnitude and duration of responses to l-norepinephrine, but it decreased the magnitude of responses to l-deprenyl. 10-15 min. In the present study, no attempt was made to relate response duration to blood levels of drug. H o w e v e r , preliminary o b s e r v a t i o n s indicated that half-decay times for e v o k e d responses (e.g. 2.0 mg/kg) were greater than 2 hr.
Interaction with desmethylimipramine (DMI). Various doses of D M I (i.p., 30 rain) w e r e tested for their ability to block uptake of [:~H]I-NE into heart. The ID~o for D M I - i n d u c e d blockade of uptake was b e t w e e n 0.1 and 0.3 mg/kg (Fig. 2). A dose of 0.3 mg/kg was used in subsequent studies. Animals were prepared for recording of heart rate responses. A control group of animals r e c e i v e d a series of injections of I-NE (0.1 /~g/kg, i.v.), after which a single injection of l-deprenyl (2.0 mg/kg, i.v.) was given. An experimental group of animals was treated identically, e x c e p t that DMI (0.3 mg/kg, i.p.) was given as a p r e t r e a t m e n t (30 min). A representative e x p e r i m e n t is illustrated in Fig. 3. In control animals, responses to I-NE a v e r a g e d about 50 bpm, and responses to l-deprenyl a v e r a g e d about 92 bpm. A f t e r D M I p r e t r e a t m e n t , responses to I-NE w e r e increased in magnitude (85 bpm) and in duration. By contrast, responses to l-deprenyl w e r e much reduced (24 bpm). Both the increase in r e s p o n s i v e n e s s to I-NE and the d e c r e a s e in responsiveness to l-deprenyl were statistically significant (P < 0.01). Tissue levels o[ NE and uptake of [aHII-NE. lD e p r e n y l was tested at several doses (0.1 to 10.0 mg/kg, i.v., 30 min) for its ability to increase or d e c r e a s e heart levels of NE. At no dose did Ideprenyl alter e n d o g e n o u s levels of NE. On the other hand, l-deprenyl did have an effect on uptake of e x o g e n o u s N E . W h e n administered 15 min prior to [~H]I-NE (0.15/~g/kg, i.v.), l-deprenyl (i.v.) produced a d o s e - d e p e n d e n t blockade of N E uptake into heart (Fig. 2). DISCUSSION
Drugs that affect b e h a v i o r are usually e x a m i n e d " for their effects on the peripheral a u t o n o m i c ner-
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L . L . SIMPSON
v o u s s y s t e m . S u c h studies are t h o u g h t to be app r o p r i a t e for a v a r i e t y of r e a s o n s , o n e of w h i c h is particularly g e r m a n e to this report. By s t u d y i n g the a c t i o n s of a d r u g o n the a u t o n o m i c n e r v o u s s y s t e m , o n e might learn s o m e t h i n g t h a t w o u l d apply to the c e n t r a l n e r v o u s s y s t e m (CNS). T h i s i n f o r m a t i o n could lead to a n u n d e r s t a n d i n g of the cellular e v e n t s that c u l m i n a t e in a d r u g - i n d u c e d b e h a v i o r a l response. Unlike m o s t p s y c h o a c t i v e drugs, I-deprenyl has r e c e i v e d only limited a t t e n t i o n in t e r m s of its autonomic effects. T h i s lack of a t t e n t i o n must be v i e w e d in the c o n t e x t that I-deprenyl h a s b e e n used as a p h a r m a c o l o g i c a l tool to s t u d y the role of C N S a m i n e s [6]. It h a s also b e e n used to t r e a t d e p r e s s i v e illness [ 11 ] a n d to treat P a r k i n s o n ' s d i s e a s e [ 12]. All of t h e s e u s e s are linked to the ability o f / - d e p r e n y l to inhibit t y p e B M A O . H o w e v e r . the p r e s e n t s t u d y d e m o n s t r a t e s that /-deprenyl is not solely an M A O inhibitor. An e x a m i n a t i o n of the a u t o n o m i c effects o f / - d e p r e n y l i n d i c a t e s that the drug is an indirectly acting s y m p a t h o m i m e t i c a m i n e . As Fig. 1 illustrates, I-deprenyl has a m a r k e d ability to i n c r e a s e h e a r t rate, but it h a s only a m o d e s l ability to i n c r e a s e b l o o d p r e s s u r e . In light of t h e s e findings, a t t e n t i o n h a s b e e n f o c u s e d on t h e h e a r t rate effects of t h e drug. In w o r k to be r e p o r t e d e l s e w h e r e (J. Bilezikian, p e r s o n a l c o m m u n i c a t i o n ) , it h a s b e e n f o u n d t h a t /-deprenyl is not a fl-agonist. O n the o t h e r h a n d , d e p r e n y l - i n d u c e d t a c h y c a r d i a c a n be d i m i n i s h e d or a b o l i s h e d by p r o p r a n o l o l . This s e q u e n c e of findings s u g g e s t s that the effects of l-deprenyl are m e d i a t e d by e n d o g e n o u s catec h o l a m i n e s . T h e fact that r e s e r p i n e p r e t r e a t m e n t nearly a b o l i s h e s r e s p o n s e s to /-deprenyl f u r t h e r implicates e n d o g e n o u s a m i n e s . I f / - d e p r e n y l w e r e an indirectly a c t i n g a m i n e , i.e. a n a g e n t that e v o k e s the r e l e a s e of e n d o g e n o u s c a t e c h o l a m i n e s , t h e n e i t h e r a d r e n a l e c t o m y or symp a t h e c t o m y s h o u l d a l t e r r e s p o n s e s . In reality, a d r e n a l e c t o m y h a d o n l y a negligible effect. By c o n t r a s t , s y m p a t h e c t o m y h a d a striking effect. O f the v a r i o u s p r o c e d u r e s that w e r e used, 6 - O H D A i n d u c e d s y m p a t h e c t o m y was the m o s t effective in d i m i n i s h i n g r e s p o n s e s to /-deprenyl. T h i s finding indicates that postganglionic sympathetic neurons are the s o u r c e of c a t e c h o l a m i n e s t h r o u g h w h i c h /-deprenyl e v o k e s r e s p o n s e s . T h e r e are actually several p o s s i b l e m e c h a n i s m s by w h i c h / - d e p r e n y l might act. T h r e e of t h e s e m e c h a n i s m s are w o r t h y of c o n s i d e r a t i o n . F i r s t , / - d e p r e n y l might diffuse a c r o s s the n e u r o n a l m e m b r a n e . O n c e inside the n e r v e e n d i n g , it might e v o k e t r a n s m i t t e r release. S e c o n d , I-deprenyl m i g h t be a s u b s t r a t e for the m e m b r a n e p u m p t h a t c a p t u r e s N E a n d r e l a t e d c o m p o u n d s a n d t r a n s p o r t s t h e m into the n e r v e . O n c e again, /-deprenyl might act i n t r a n e u r o n a l l y to e v o k e t r a n s m i t t e r release. T h i r d , l-deprenyl might act mainly o n the e x t e r n a l s u r f a c e of the n e r v e m e m b r a n e as a n i n h i b i t o r of the N E p u m p . In so doing, /-deprenyl w o u l d inhibit t h e m a j o r m e c h a n ism f o r i n a c t i v a t i n g N E , a n d t h u s i n c r e a s e the a m o u n t of N E a v a i l a b l e to i n t e r a c t with p o s t j u n c tional r e c e p t o r s . A d e c i s i o n a m o n g t h e s e t h r e e possibilities c a n be m a d e by u s i n g a k n o w n i n h i b i t o r of the N E p u m p ,
n a m e l y , D M I . If the first possibility were true, then /-deprenyl a n d D M I s h o u l d act synergistically. N E r e l e a s e d by /-deprenyl w o u l d be p r o t e c t e d f r o m i n a c t i v a t i o n by DMI. If the s e c o n d possibility w e r e true, t h e n / - d e p r e n y l s h o u l d be a n t a g o n i z e d by DM 1. B l o c k a d e of the N E p u m p w o u l d p r e v e n t I-deprenyl f r o m r e a c h i n g its site of action. If the third possibility were true, t h e n I-deprenyl a n d DMI s h o u l d act additively. T h e t w o d r u g s would act jointly to i n a c t i v a t e the p u m p a n d t h e r e b y i n c r e a s e availability of N E . T h e d a t a illustrated in Fig. 3 leave little d o u b l a b o u t the most plausible c o n c l u s i o n . W h e n DMI was c o m b i n e d with the ED.~o for /-deprenyl, res p o n s e s to the latter were n o t a b l y a n t a g o n i z e d . This o c c u r r e d in the p r e s e n c e of clear e v i d e n c e that DMI was b l o c k i n g the N E p u m p (Fig. 2) and that DMI was p o t e n t i a t i n g the a c t i o n s of N E (Fig. 3). It app e a r s that /-deprenyl e n t e r s p o s t g a n g l i o n i c syrnp a t h e t i c n e u r o n s via the N E p u m p , a n d that it s u b s e q u e n t l y e v o k e s the release of n e u r o n a l catec h o l a m i n e s . T h e i m p o r t a n c e of passive diffusion a c r o s s the m e m b r a n e or b l o c k a d e of the m e m b r a n e p u m p must be minimal. A q u e s t i o n that h a s not b e e n a n s w e r e d is that of w h y / - d e p r e n y l e x e r t s g r e a t e r effects on s y m p a t h e t i c n e u r o n s in h e a r t as o p p o s e d to t h o s e in v a s c u l a t u r e . A t e n t a t i v e e x p l a n a t i o n could be the dual possibility that: (a) /-deprenyl has a relatively low affinity for the N E p u m p (see Fig. 2 a n d Ref. 13), a n d I b ) s y m p a t h e t i c n e r v e s in heart m a y h a v e a more avid u p t a k e m e c h a n i s m than s y m p a t h e t i c n e r v e s in vasculature. T h e m a t t e r r e q u i r e s f u r t h e r study. A n o t h e r q u e s t i o n of note p e r t a i n s to the C N S . T h e d a t a in this report deal e x c l u s i v e l y with the p e r i p h e r a l n e r v o u s s y s t e m . It is not k n o w n w h e t h e r l-deprenyl is an indirectly acting a m i n e in the brain. H o w e v e r , as interest in /-deprenyl m o u n t s , and as the f r e q u e n c y of its use as an i n v e s t i g a t i o n a l tool i n c r e a s e s , studies to d e t e r m i n e w h e t h e r /-deprenyl is a n indirectly acting a m i n e in the brain must be u n d e r t a k e n . In the a b s e n c e of such w o r k , the neurological a n d b e h a v i o r a l effects of /-deprenyl m a y be difficult to explain. Acknowledg, e m e n t - - T h e author is pleased to express him indebtedness to John Aletta and Gene Miskin for their diligent work and for their keen observations.
REFERENCES I. M. Sandier and M. B. H. Youdim. Pharma~. Rev. 24, 33t (1972). 2. E. Usdin (Ed.), Neuropsychopharma('ology oj Monoamines and Their Regulatory Enzymes. Raven Press, NY (1974). 3. Monamine Oxidase and Its Inhibition, Ciba Foundation Symposium 39. Elsevier, Amsterdam (1976). (1976). 4. J. P. Johnston, Bio('hern. Pharma('. 17, 1285 (1968). 5. A. J. Christmas, C. J. Coulson, D. R. Maxwell and D. Riddell, Br. J. Pharmac. 45,490 (1972). 6. N. H. Neff and J. A. Fuentes, in M o n o a m i n e Oxidase and Its Inhibition, Ciba Foundation Symposium 3% p. 000. Elsevier, Amsterdam (1976). 7. D. A. Gilder, W. Fain and L. L. Simpson, J. Pharmac. exp. Ther. 198, 255 (1976). 8. L. L. Simpson. Biochem. Pharmac. 26, 1315 (1977L
lndireclly acting amine 9. A. H. A n t o n and D. F. Sayre. J. Pharmac. exp. Ther. 138, 36(I (1962). 10. M. K. Shellenberger and J. H. Gordon, Analyt. Biochem. 39, 356 (1971). 1 I. L. Tringer, G. Halts and E. Varga, Societas Pharmacologica Hungarica. V Conferentia Hungarica Pro Therapia et Investigatione in Pharmacologia (1971 I.
~ P. 2 7 1 1
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12. L. Ambrozi, W. Birkmayer, P. Riederer and M. B. H. Youdim, Br. J. Pharmac. 58, 423P (1976). 13. C. Braestrup, H. A n d e r s e n and A. Randrup, Eur. J. Pharmac. 34, 181 (1975).