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Evidence for a peripheral dopaminergic mechanism in the antihypertensive action of lergotrile
Life Sciences, Vol. 27, pp. 349-354 Printed in the U.S.A.
Pergamon Press
EVIDENCE FOR A PERIPHERAL DOPAMINERGIC MECHANISM IN THE ANTIHYPERTENSIVE ACTION OF LERGOTRILE i Alan
F. Sved and John D. Fernstrom
L a b o r a t o r y of B r a i n and Metabolism Department of N u t r i t i o n and Food Science Massachusetts Institute of Technology Cambridge, Massachusetts 02139 (Received in final form May 27, 1980)
SUMMARY Lergotrile ( 0 . 5 mg/kg, i . p . ) lowered blood p r e s s u r e significantly in s p o n t a n e o u s l y h y p e r t e n s i v e r a t s . This effect was a n t a g o n i z e d by p r e t r e a t m e n t w i t h h a l o p e r i d o l , pimozide, or domperidone. In n o r m o t e n s i v e r a t s , administration of h a l o p e r i d o l or domperidone r a p i d l y i n c r e a s e d serum p r o l a c t i n levels. H a l o p e r i d o l also i n c r e a s e d s t r i a t a l l e v e l s of d i h y d r o x y p h e n y l a c e t i c acid and h o m o v a n i l l i c acid; however, domperidone did n o t , which c o n f i r m s t h a t this latter b l o c k e r p r o b a b l y acts p r i m a r i l y as a p e r i p h e r a__~l dopamine a n t a g o n i s t . Taken t o g e t h e r , these data suggest t h a t l e r g o t r i l e lowers blood p r e s s u r e in h y p e r t e n s i v e r a t s m a i n l y by s t i m u l a t i n g p e r i p h e r a l dopamine r e c e p t o r s . Lergotrile, bromocriptine, and pergolide each lower blood p r e s s u r e when a d m i n i s t e r e d to e x p e r i m e n t a l animals or humans ( 1 - 6 ) . These e r g o t derivatives are a l s o potent dopaminergic agonists (7-9). I t has t h e r e f o r e been suggested t h a t they e x e r t t h e i r hypot e n s i v e and a n t i h y p e r t e n s i v e e f f e c t s by s t i m u l a t i n g dopamine receptors (1-6). Such dopamine r e c e p t o r s could be l o c a t e d anywhere in the body: on neural elements w i t h i n the b r a i n ( i 0 , I i ) ; in the p e r i p h e r y , on postganglionic s y m p a t h e t i c neurons ( 1 2 ) ; or on smooth muscle of the v a s c u l a r bed ( 1 3 ) . We t r i e d to d e t e r m i n e whether l e r g o t r i l e lowers blood p r e s s u r e by s t i m u l a t i n g dopamine r e c e p t o r s , and i f so, whether these r e c e p t o r s are l o c a t e d i n s i d e or o u t s i d e the c e n t r a l nervous system. Our results indicate that lergotrile p r o b a b l y does lower blood p r e s s u r e by s t i m u l a t i n g p e r i p h e r a l dopamine r e c e p t o r s . Materials
and Methods
Male s p o n t a n e o u s l y h y p e r t e n s i v e (SHR) r a t s (300-350 g; r e s t i n g blood p r e s s u r e , 180-210 mm Hg) of the Okamoto s t r a i n ( C h a r l e s R i v e r iThese s t u d i e s were s u p p o r t e d in p a r t by g r a n t s from the N a t i o n a l Institute of Mental Health and the N a t i o n a l A e r o n a u t i c s and Space Administration. A . F . S . holds a f e l l o w s h i p from the NIMH; J . D . F . h o l d s a Research S c i e n t i s t Development Award from the NIMH. 0024-3205/300349-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd
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Breeding L a b o r a t o r i e s , W i l m i n g t o n , MA) were housed 3-4 per cage in our animal f a c i l i t i e s . Food ( C h a r l e s R i v e r Rat, Mouse, and Hamster M a i n t e n a n c e F o r m u l a ) and w a t e r were p r o v i d e d ad l i b i t u m . The room was l i g h t e d (300 pw/cm 2, V i t a - L i t e ; D u r o - T e s t C o r p . , North Bergen, NJ) 14 hours per day, and the ambient t e m p e r a t u r e was m a i n t a i n e d at 22°C. Arterial p r e s s u r e was e s t i m a t e d by the t a i l - c u f f method ( 1 4 ) , u s i n g a N a r c o - B i o s y s t e m s pneumatic p u l s e t r a n s d u c e r ( N a r c o - B i o s y s tems, Houston, TX). The a n i m a l s were warmed at 37°C f o r 20 m i n u t e s b e f o r e each r e a d i n g . E i g h t measurements per animal were made at each t i m e p o i n t , and the averaged v a l u e was taken as the blood p r e s sure. A l l SHR r a t s were a c c l i m a t e d to t h i s p r o c e d u r e f o r 4 days b e f o r e the e x p e r i m e n t s , to m i n i m i z e s t r e s s - i n d u c e d f l u c t u a t i o n s in blood p r e s s u r e . SHR r a t s were used in s e v e r a l d i f f e r e n t experim e n t s ; however, at l e a s t 7 days i n t e r v e n e d between e x p e r i m e n t s . 250
N o r m o t e n s i v e male Sprague-Dawley r a t s ( C h a r l e s R i v e r ) , w e i g h i n g g, were used in b i o c h e m i c a l and n e u r o e n d o c r i n e s t u d i e s . Serum TABLE I A t t e n u a t i o n by dopamine a n t a g o n i s t s of lergotrile's antihypertensive action in s p o n t a n e o u s l y h y p e r t e n s i v e r a t s
Treatment
Blood p r e s s u r e (mm Hg) Before After
Change in pressure (mm H~)
Experiment I Vehicle-Vehicle Vehicle-Lergotrile Haloperidol-Vehicle Haloperidol-Lergotrile
191 197 187 190
+ ¥ T ¥
3 5 3 6
191 129 176 170
+ ¥ ¥ ¥
3 3* 3** 5**
0 -68 -11 -20
+ T ¥ T
3 6* 4 5**
Experiment 2 Vehicle-Vehicle Vehicle-Lergotrile Pimozide-Vehicle Pimozide-Lergotrile
194 187 190 191
+ ¥ ¥ ¥
4 3 4 4
193 139 186 176
+ T ~ T
5 3* 5 3**
-I -48 -4 -15
+ ¥ ¥ T
3 3* 3 3**
Experiment 3 Vehicle-Vehicle Vehicle-Lergotrile Domperidone-Vehicle Domperidone-Lergotrile
182 180 185 182
+ T T T
6 5 2 3
182 131 185 167
+ T ¥ ¥
3 3* 3 2**
0 -49 0 -15
+ T ~ ¥
4 3* 2 5**
Groups of 5 SHR r a t s r e c e i v e d h a l o p e r i d o l (2 m g / k g ) , p i m o z i d e (2 m g / k g ) , domperidone (2 m g / k g ) , or v e h i c l e i.p. after baseline blood pressures (before) were recorded. Lergotrile ( 0 . 5 mg/kg) or i t s v e h i c l e was administered 30 m i n u t e s later, and blood pressures (after) were taken again I hour t h e r e a f t e r . Data are p r e s e n t e d as means + SEM. *P < 0 . 0 5 , c h a n g e - i n blood p r e s s u r e d i f f e r s signific a n t l y from t h a t e x p e r i e n c e d by c o n t r o l rats (vehiclevehicle). **P < 0 . 0 5 , change in blood p r e s s u r e d i f f e r s signific a n t l y from t h a t e x p e r i e n c e d by e i t h e r c o n t r o l r a t s or rats receiving lergotrile alone ( v e h i c l e - l e r g o t r i l e ) .
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prolactin l e v e l s were measured using a d o u b l e - a n t i b o d y radioimmunoassay ( R I A ) , w i t h m a t e r i a l s p r o v i d e d by the N a t i o n a l I n s t i t u t e s of H e a l t h Rat P i t u i t a r y Program. Rat p r o l a c t i n RP-I served as the reference standard. Striatal l e v e l s of d i h y d r o x y p h e n y l a c e t i c acid (DOPAC) and h o m o v a n i l l i c acid (HVA) were measured by e l e c t r o c h e m i cal d e t e c t i o n , a f t e r s e p a r a t i o n by h i g h - p r e s s u r e l i q u i d chromatography (HPLC) ( 1 5 ) . T i s s u e s were homogenized in 0 . I M p e r c h l o r i c a c i d , and the r e s u l t i n g homogenates c e n t r i f u g e d . The s u p e r n a t a n t s were e x t r a c t e d with ether, and the e t h e r phases were removed, placed in clean t u b e s , and l y o p h i l i z e d . Ether r e s i d u e s were subsequently reconstituted in a c e t a t e b u f f e r (50 mM, pH 5 . 0 ) , and i n jected onto the HPLC column (pBondapak C18; Waters A s s o c i a t e s , Milford, MA). The m o b i l e phase was 50 mM a c e t a t e b u f f e r (pH 5 . 0 ) and the column was run at 1.5 m l / m i n u t e at room t e m p e r a t u r e . Ret e n t i o n times f o r DOPAC, HVA, and v a n i l l i c acid ( i n t e r n a l standard added at time of h o m o g e n i z a t i o n ) were 4 . 6 , 1 2 . 4 , and 10.4 m i n u t e s , respectively. Lergotrile mesylate ( 2 - c h l o r o - 6 - m e t h y l - 8 B - a c e t o n i t r i l e ) was provided by the Eli L i l l y Company ( I n d i a n a p o l i s , IN). Domperidone was a g i f t from Janssen Pharmaceuticals (Beerse, Belgium). Haloperidol and pimozide w e r e supplied by McNeil Laboratories (Fort Washington, P A ) . L e r g o t r i l e was dissolved in water; the dopamine antagonists were dissolved in warm, d i l u t e acetic acid. All drugs were administered i n t r a p e r i t o n e a l l y to rats in a volume of 2 ml/kg. The data were analyzed by analysis of variance and the Neuman-Keuls t e s t (16). Results As we had a n t i c i p a t e d , administration of l e r g o t r i l e to SHR rats markedly reduced blood pressure 1 hour l a t e r (Table I ) . This a n t i hypertensive action of l e r g o t r i l e was blocked considerably by pretreatment with a dopamine antagonist, e i t h e r h a l o p e r i d o l , pimozide, or domperidone (Table I ) . Normotensive rats i n j e c t e d with e i t h e r haloperidol or domperidone showed s i g n i f i c a n t l y increased serum p r o l a c t i n levels 90 minutes l a t e r (Table I I ) . Administration of h a l o p e r i d o l , but not of domperidone, s i g n i f i c a n t l y elevated s t r i a t a l DOPAC and HVA levels (Table I I ) . TABLE I I Effects of haloperidol or domperidone i n j e c t i o n on serum prolaction and s t r i a t a l DOPAC and HVA levels Striatal
Treatment Vehicle Haloperidol Domperidone
DOPAC (~g/g)
HVA (~g/g)
1.02 + 0.05 4.74 + 0.36* 1.10 ¥ 0.06
0.71 + 0.05 3.26 ~- 0 . 1 9 " 0.73 T 0.07
Serum prolactin (ng/ml)
2 + 56 +53 +-
1 5* 8*
Groups of 6 normotensive rats received haloperidol (2 mg/kg, i . p . ) , domperidone (2 mg/kg, i . p . ) , or vehicle and were k i l l e d 90 minutes l a t e r . D a t a are presented as means + SEM. *P < OT01 d i f f e r s s i g n i f i c a n t l y from vehicle l e v e l s .
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Discussion These r e s u l t s confirm t h a t l e r g o t r i l e reduces blood pressure in SHR r a t s [as p r e v i o u s l y reported ( 1 , 6 ) ] . They also demonstrate that lergotrile's a n t i h y p e r t e n s i v e e f f e c t is markedly a t t e n u a t e d by p r i o r a d m i n i s t r a t i o n of h a l o p e r i d o l or pimozide, c e n t r a l l y a c t i n g dopamine a n t a g o n i s t s ( 1 7 , 1 8 ) , or domperidone, a dopamine a n t a g o n i s t t h a t acts only in the p e r i p h e r y ( 1 9 , 2 0 ) . Since domperidone e f f e c t i v e l y antagonized the l e r g o t r i l e - i n d u c e d r e d u c t i o n in blood pressure, the most s t r a i g h t f o r w a r d interpretation of the f i n d i n g s is that lergotrile reduces blood pressure by s t i m u l a t i n g p e r i p h e r a l dopamine r e c e p t o r s . Domperidone was chosen f o r t h i s study because of r e p o r t s t h a t in v i v o i t blocks dopamine r e c e p t o r s , but only in the p e r i p h e r y . The f o l l o w i n g in v i t r o f i n d i n g s suggest t h a t domperidone is a dopamine a n t a g o n i s t : i t [ a ] competes f o r J H - h a l o p e r i d o l and ~H-spiperone binding s i t e s ( 1 9 ) , [ b ] blocks the s t i m u l a t i o n of s t r i a t a l adenylate cyclase e l i c i t e d by dopamine ( 1 9 ) , [ c ] antagonizes apomorphine-induced suppression of p r o l a c t i n release from c u l t u r e d pituitary cells (21), and [ d ] antagonizes the dopamine-induced r e l a x a t i o n of guinea pig stomach smooth muscle (22). The f o l l o w i n g o b s e r v a t i o n s support the hypothesis t h a t domperidone acts in v i v o to block dopamine r e c e p t o r s only in the p e r i p h e r y . Domperidone (administered i~traperitoneally or o r a l l y ) causes no r e d u c t i o n in the number of ~H-spiperone binding s i t e s in b r a i n (19) [whereas h a l o p e r i d o l and pimozide do have t h i s e f f e c t ( 1 9 , 2 3 ) ] . Domperidone, u n l i k e c e n t r a l l y a c t i n g dopamine a n t a g o n i s t s ( 2 4 , 2 5 ) , does not increase brain dopamine t u r n o v e r , as r e f l e c t e d by i t s i n a b i l i t y to increase s t r i a t a l DOPAC and HVA l e v e l s ( r e f . 19, Table I I ) . Lastly, radiolabeled domperidone, unlike other dopamine a n t a g o n i s t s , p e n e t r a t e s i n t o the brain only very p o o r l y a f t e r i t s p a r e n t e r a l a d m i n i s t r a t i o n (19). The increments in s t r i a t a l DOPAC and HVA l e v e l s produced by haloperidol (Table I I ) are s i m i l a r to those observed by others ( 2 4 , 2 6 ) ; the absence of such changes a f t e r domperidone i n j e c t i o n is also c o n s i s t e n t with other r e p o r t s (19) [and supports the n o t i o n t h a t domperidone does not act as a c e n t r a l dopamine a n t a g o n i s t ] . Both h a l o p e r i d o l and domperidone elevated serum p r o l a c t i n l e v e l s , to values c o n s i s t e n t l y observed a f t e r blockade of dopamine receptors (27,28). The a b i l i t y of these agents to e l e v a t e serum p r o l a c t i n l e v e l s is c o n s i s t e n t with the notion t h a t they block p e r i p h e r a l dopamine r e c e p t o r s , since the dopamine r e c e p t o r s t h a t mediate i n h i b i t i o n of p r o l a c t i n release l i e o u t s i d e the b l o o d - b r a i n b a r r i e r (29,30). Our s t u d i e s were performed in normotensive r a t s , because of the extreme expense of doing such s t u d i e s in SHR r a t s ; t h e r e was no reason to suspect t h a t the drugs would produce d i f f e r e n t e f f e c t s in d i f f e r e n t rat s t r a i n s . Also, the tlme chosen f o r measurements and the drug doses used were based on published r e p o r t s of peak e f f e c t s f o r such agents ( 1 8 , 2 4 , 2 5 , 2 6 ) . Domperidone's apparent a b i l i t y to block p e r i p h e r a l but not central dopamine r e c e p t o r s provides a useful pharmacologic t o o l f o r investigating the anatomic s i t e s of a c t i o n of dopamine a g o n i s t s . Since domperidone blocks lergotrile's antihypertensive action, these data support the h y p o t h e s i s t h a t l e r g o t r i l e reduces blood pressure by s t i m u l a t i n g peripheral dopamine r e c e p t o r s . Another study suggests t h a t a s i m i l a r drug, b r o m o c r i p t i n e , may decrease
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blood pressure by stimulating peripheral dopamine receptors (31). Bromocriptine was more effective in lowering blood pressure when administered intravenously rather than into the cerebral ventricles or vertebral a r t e r y . These i n v e s t i g a t o r s , and others, also found that dopamine antagonists blocked the drug's e f f e c t , which supports the notion that bromocriptine actually lowers b l o o d pressure by stimulating a dopamine receptor (5,31,32). I f l e r g o t r i l e (and other dopamine agonist/ergot derivatives) lower blood pressure by stimulating peripheral dopamine receptors, there are at least two possible sites for such receptors. First, they may reside within certain vascular beds [ e . g . , the renovasculature and mesenteric arteries (13)], presumably on smooth muscle, since pharmacologic stimulation of dopamine receptors in these vascular beds causes vasodilation (13). This p o s s i b i l i t y seems somewhat remote, however, at least for bromocriptine, since i t is a poor agonist for vascular dopamine receptors (33). Moreover, such a mechanism of action seems u n l i k e l y , since reduction in blood pressure by such a route would be expected to e l i c i t a reflex increase in sympathetic tone; t h i s has not yet been observed, at least for bromocriptine (5,31). Second, dopamine agonists might lower blood pressure by decreasing norepinephrine release from sympathetic nerve terminals. These agents would probably act by stimulating dopamine receptors located on postganglionic sympathetic neurons (12). R e p o r t s that bromocriptine administration reduces both blood and urine norepinephrine levels (34,35) would tend to support such a hypothesis. Bromocriptine has also been shown to decrease norepinephrine release f r o m sympathetic nerve terminals a f t e r direct n e r v e stimulation (31,32), as had previously been demonstrated to be an action of other dopamine agonists (36). Further studies now seem warranted to locate these peripheral dopamine receptors. Since l e r g o t r i l e appears to reduce blood pressure by a peripheral action, one f r u i t f u l avenue of research might be the development of dopamine agonists that do not penetrate into the central nervous system. Such agents might ol-o-w-er blood pressure e f f e c t i v e l y , while not e l i c i t i n g any of the known side effects of c e n t r a l l y acting dopamine agonists (37). A new class of c l i n i c a l l y useful antihypertensive agents might thus be invented. References 1. A.F. SVED and J.O. FERNSTROM, J. Pharm. Pharmacol. 31 814-817 (1979). 2. K.O. STUMPE, R. KOLLOCH, M. HIGUCHI, F. KRUCK and H. VETTER, Lancet 2 211-213 (1977). 3. S.B. KA~E, K.M. SHAW and E.S. ROSS, Lancet 1 1176-1177 (1976). 4. J.K. GREENACRE, P . F . TEYCHENNE, A. PETRIE, D.B. CALNE, P.N. LEIGH and J.L. REID, Br. J. Clin. Pharmacol. 3 571-574 (1976). 5. B.J. CLARK, G. CHOLTYSIK and E. FLUCKIGER, Ac~a Endocrinol. 88 (Suppl. 216) 75-81 (1978). 6. T.T. YEN, N.B. STAMM and J.A. CLEMENS, Life Sci. 25 209-216 (1979). 7. L. LEMBERGER, Fed. Proc. 37 2176-2180 (1978). 8. M. GOLDSTEIN, J.Y. LEW, S. NAKAMURA, A.F. BATTISTA, A. LIEBERMAN and K. FUXE, Fed. Proc. 37 2202-2206 (1978). 9. K. FUXE, B.B. FREDHOLM, S.O. OGREN~--L.F. AGNATI, T. HOKFELT and J.A. GUSTAFSSON, Fed. Proc. 37 2181-2191 (1978).
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Pergamon Press
EVIDENCE FOR A PERIPHERAL DOPAMINERGIC MECHANISM IN THE ANTIHYPERTENSIVE ACTION OF LERGOTRILE i Alan
F. Sved and John D. Fernstrom
L a b o r a t o r y of B r a i n and Metabolism Department of N u t r i t i o n and Food Science Massachusetts Institute of Technology Cambridge, Massachusetts 02139 (Received in final form May 27, 1980)
SUMMARY Lergotrile ( 0 . 5 mg/kg, i . p . ) lowered blood p r e s s u r e significantly in s p o n t a n e o u s l y h y p e r t e n s i v e r a t s . This effect was a n t a g o n i z e d by p r e t r e a t m e n t w i t h h a l o p e r i d o l , pimozide, or domperidone. In n o r m o t e n s i v e r a t s , administration of h a l o p e r i d o l or domperidone r a p i d l y i n c r e a s e d serum p r o l a c t i n levels. H a l o p e r i d o l also i n c r e a s e d s t r i a t a l l e v e l s of d i h y d r o x y p h e n y l a c e t i c acid and h o m o v a n i l l i c acid; however, domperidone did n o t , which c o n f i r m s t h a t this latter b l o c k e r p r o b a b l y acts p r i m a r i l y as a p e r i p h e r a__~l dopamine a n t a g o n i s t . Taken t o g e t h e r , these data suggest t h a t l e r g o t r i l e lowers blood p r e s s u r e in h y p e r t e n s i v e r a t s m a i n l y by s t i m u l a t i n g p e r i p h e r a l dopamine r e c e p t o r s . Lergotrile, bromocriptine, and pergolide each lower blood p r e s s u r e when a d m i n i s t e r e d to e x p e r i m e n t a l animals or humans ( 1 - 6 ) . These e r g o t derivatives are a l s o potent dopaminergic agonists (7-9). I t has t h e r e f o r e been suggested t h a t they e x e r t t h e i r hypot e n s i v e and a n t i h y p e r t e n s i v e e f f e c t s by s t i m u l a t i n g dopamine receptors (1-6). Such dopamine r e c e p t o r s could be l o c a t e d anywhere in the body: on neural elements w i t h i n the b r a i n ( i 0 , I i ) ; in the p e r i p h e r y , on postganglionic s y m p a t h e t i c neurons ( 1 2 ) ; or on smooth muscle of the v a s c u l a r bed ( 1 3 ) . We t r i e d to d e t e r m i n e whether l e r g o t r i l e lowers blood p r e s s u r e by s t i m u l a t i n g dopamine r e c e p t o r s , and i f so, whether these r e c e p t o r s are l o c a t e d i n s i d e or o u t s i d e the c e n t r a l nervous system. Our results indicate that lergotrile p r o b a b l y does lower blood p r e s s u r e by s t i m u l a t i n g p e r i p h e r a l dopamine r e c e p t o r s . Materials
and Methods
Male s p o n t a n e o u s l y h y p e r t e n s i v e (SHR) r a t s (300-350 g; r e s t i n g blood p r e s s u r e , 180-210 mm Hg) of the Okamoto s t r a i n ( C h a r l e s R i v e r iThese s t u d i e s were s u p p o r t e d in p a r t by g r a n t s from the N a t i o n a l Institute of Mental Health and the N a t i o n a l A e r o n a u t i c s and Space Administration. A . F . S . holds a f e l l o w s h i p from the NIMH; J . D . F . h o l d s a Research S c i e n t i s t Development Award from the NIMH. 0024-3205/300349-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd
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Breeding L a b o r a t o r i e s , W i l m i n g t o n , MA) were housed 3-4 per cage in our animal f a c i l i t i e s . Food ( C h a r l e s R i v e r Rat, Mouse, and Hamster M a i n t e n a n c e F o r m u l a ) and w a t e r were p r o v i d e d ad l i b i t u m . The room was l i g h t e d (300 pw/cm 2, V i t a - L i t e ; D u r o - T e s t C o r p . , North Bergen, NJ) 14 hours per day, and the ambient t e m p e r a t u r e was m a i n t a i n e d at 22°C. Arterial p r e s s u r e was e s t i m a t e d by the t a i l - c u f f method ( 1 4 ) , u s i n g a N a r c o - B i o s y s t e m s pneumatic p u l s e t r a n s d u c e r ( N a r c o - B i o s y s tems, Houston, TX). The a n i m a l s were warmed at 37°C f o r 20 m i n u t e s b e f o r e each r e a d i n g . E i g h t measurements per animal were made at each t i m e p o i n t , and the averaged v a l u e was taken as the blood p r e s sure. A l l SHR r a t s were a c c l i m a t e d to t h i s p r o c e d u r e f o r 4 days b e f o r e the e x p e r i m e n t s , to m i n i m i z e s t r e s s - i n d u c e d f l u c t u a t i o n s in blood p r e s s u r e . SHR r a t s were used in s e v e r a l d i f f e r e n t experim e n t s ; however, at l e a s t 7 days i n t e r v e n e d between e x p e r i m e n t s . 250
N o r m o t e n s i v e male Sprague-Dawley r a t s ( C h a r l e s R i v e r ) , w e i g h i n g g, were used in b i o c h e m i c a l and n e u r o e n d o c r i n e s t u d i e s . Serum TABLE I A t t e n u a t i o n by dopamine a n t a g o n i s t s of lergotrile's antihypertensive action in s p o n t a n e o u s l y h y p e r t e n s i v e r a t s
Treatment
Blood p r e s s u r e (mm Hg) Before After
Change in pressure (mm H~)
Experiment I Vehicle-Vehicle Vehicle-Lergotrile Haloperidol-Vehicle Haloperidol-Lergotrile
191 197 187 190
+ ¥ T ¥
3 5 3 6
191 129 176 170
+ ¥ ¥ ¥
3 3* 3** 5**
0 -68 -11 -20
+ T ¥ T
3 6* 4 5**
Experiment 2 Vehicle-Vehicle Vehicle-Lergotrile Pimozide-Vehicle Pimozide-Lergotrile
194 187 190 191
+ ¥ ¥ ¥
4 3 4 4
193 139 186 176
+ T ~ T
5 3* 5 3**
-I -48 -4 -15
+ ¥ ¥ T
3 3* 3 3**
Experiment 3 Vehicle-Vehicle Vehicle-Lergotrile Domperidone-Vehicle Domperidone-Lergotrile
182 180 185 182
+ T T T
6 5 2 3
182 131 185 167
+ T ¥ ¥
3 3* 3 2**
0 -49 0 -15
+ T ~ ¥
4 3* 2 5**
Groups of 5 SHR r a t s r e c e i v e d h a l o p e r i d o l (2 m g / k g ) , p i m o z i d e (2 m g / k g ) , domperidone (2 m g / k g ) , or v e h i c l e i.p. after baseline blood pressures (before) were recorded. Lergotrile ( 0 . 5 mg/kg) or i t s v e h i c l e was administered 30 m i n u t e s later, and blood pressures (after) were taken again I hour t h e r e a f t e r . Data are p r e s e n t e d as means + SEM. *P < 0 . 0 5 , c h a n g e - i n blood p r e s s u r e d i f f e r s signific a n t l y from t h a t e x p e r i e n c e d by c o n t r o l rats (vehiclevehicle). **P < 0 . 0 5 , change in blood p r e s s u r e d i f f e r s signific a n t l y from t h a t e x p e r i e n c e d by e i t h e r c o n t r o l r a t s or rats receiving lergotrile alone ( v e h i c l e - l e r g o t r i l e ) .
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prolactin l e v e l s were measured using a d o u b l e - a n t i b o d y radioimmunoassay ( R I A ) , w i t h m a t e r i a l s p r o v i d e d by the N a t i o n a l I n s t i t u t e s of H e a l t h Rat P i t u i t a r y Program. Rat p r o l a c t i n RP-I served as the reference standard. Striatal l e v e l s of d i h y d r o x y p h e n y l a c e t i c acid (DOPAC) and h o m o v a n i l l i c acid (HVA) were measured by e l e c t r o c h e m i cal d e t e c t i o n , a f t e r s e p a r a t i o n by h i g h - p r e s s u r e l i q u i d chromatography (HPLC) ( 1 5 ) . T i s s u e s were homogenized in 0 . I M p e r c h l o r i c a c i d , and the r e s u l t i n g homogenates c e n t r i f u g e d . The s u p e r n a t a n t s were e x t r a c t e d with ether, and the e t h e r phases were removed, placed in clean t u b e s , and l y o p h i l i z e d . Ether r e s i d u e s were subsequently reconstituted in a c e t a t e b u f f e r (50 mM, pH 5 . 0 ) , and i n jected onto the HPLC column (pBondapak C18; Waters A s s o c i a t e s , Milford, MA). The m o b i l e phase was 50 mM a c e t a t e b u f f e r (pH 5 . 0 ) and the column was run at 1.5 m l / m i n u t e at room t e m p e r a t u r e . Ret e n t i o n times f o r DOPAC, HVA, and v a n i l l i c acid ( i n t e r n a l standard added at time of h o m o g e n i z a t i o n ) were 4 . 6 , 1 2 . 4 , and 10.4 m i n u t e s , respectively. Lergotrile mesylate ( 2 - c h l o r o - 6 - m e t h y l - 8 B - a c e t o n i t r i l e ) was provided by the Eli L i l l y Company ( I n d i a n a p o l i s , IN). Domperidone was a g i f t from Janssen Pharmaceuticals (Beerse, Belgium). Haloperidol and pimozide w e r e supplied by McNeil Laboratories (Fort Washington, P A ) . L e r g o t r i l e was dissolved in water; the dopamine antagonists were dissolved in warm, d i l u t e acetic acid. All drugs were administered i n t r a p e r i t o n e a l l y to rats in a volume of 2 ml/kg. The data were analyzed by analysis of variance and the Neuman-Keuls t e s t (16). Results As we had a n t i c i p a t e d , administration of l e r g o t r i l e to SHR rats markedly reduced blood pressure 1 hour l a t e r (Table I ) . This a n t i hypertensive action of l e r g o t r i l e was blocked considerably by pretreatment with a dopamine antagonist, e i t h e r h a l o p e r i d o l , pimozide, or domperidone (Table I ) . Normotensive rats i n j e c t e d with e i t h e r haloperidol or domperidone showed s i g n i f i c a n t l y increased serum p r o l a c t i n levels 90 minutes l a t e r (Table I I ) . Administration of h a l o p e r i d o l , but not of domperidone, s i g n i f i c a n t l y elevated s t r i a t a l DOPAC and HVA levels (Table I I ) . TABLE I I Effects of haloperidol or domperidone i n j e c t i o n on serum prolaction and s t r i a t a l DOPAC and HVA levels Striatal
Treatment Vehicle Haloperidol Domperidone
DOPAC (~g/g)
HVA (~g/g)
1.02 + 0.05 4.74 + 0.36* 1.10 ¥ 0.06
0.71 + 0.05 3.26 ~- 0 . 1 9 " 0.73 T 0.07
Serum prolactin (ng/ml)
2 + 56 +53 +-
1 5* 8*
Groups of 6 normotensive rats received haloperidol (2 mg/kg, i . p . ) , domperidone (2 mg/kg, i . p . ) , or vehicle and were k i l l e d 90 minutes l a t e r . D a t a are presented as means + SEM. *P < OT01 d i f f e r s s i g n i f i c a n t l y from vehicle l e v e l s .
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Discussion These r e s u l t s confirm t h a t l e r g o t r i l e reduces blood pressure in SHR r a t s [as p r e v i o u s l y reported ( 1 , 6 ) ] . They also demonstrate that lergotrile's a n t i h y p e r t e n s i v e e f f e c t is markedly a t t e n u a t e d by p r i o r a d m i n i s t r a t i o n of h a l o p e r i d o l or pimozide, c e n t r a l l y a c t i n g dopamine a n t a g o n i s t s ( 1 7 , 1 8 ) , or domperidone, a dopamine a n t a g o n i s t t h a t acts only in the p e r i p h e r y ( 1 9 , 2 0 ) . Since domperidone e f f e c t i v e l y antagonized the l e r g o t r i l e - i n d u c e d r e d u c t i o n in blood pressure, the most s t r a i g h t f o r w a r d interpretation of the f i n d i n g s is that lergotrile reduces blood pressure by s t i m u l a t i n g p e r i p h e r a l dopamine r e c e p t o r s . Domperidone was chosen f o r t h i s study because of r e p o r t s t h a t in v i v o i t blocks dopamine r e c e p t o r s , but only in the p e r i p h e r y . The f o l l o w i n g in v i t r o f i n d i n g s suggest t h a t domperidone is a dopamine a n t a g o n i s t : i t [ a ] competes f o r J H - h a l o p e r i d o l and ~H-spiperone binding s i t e s ( 1 9 ) , [ b ] blocks the s t i m u l a t i o n of s t r i a t a l adenylate cyclase e l i c i t e d by dopamine ( 1 9 ) , [ c ] antagonizes apomorphine-induced suppression of p r o l a c t i n release from c u l t u r e d pituitary cells (21), and [ d ] antagonizes the dopamine-induced r e l a x a t i o n of guinea pig stomach smooth muscle (22). The f o l l o w i n g o b s e r v a t i o n s support the hypothesis t h a t domperidone acts in v i v o to block dopamine r e c e p t o r s only in the p e r i p h e r y . Domperidone (administered i~traperitoneally or o r a l l y ) causes no r e d u c t i o n in the number of ~H-spiperone binding s i t e s in b r a i n (19) [whereas h a l o p e r i d o l and pimozide do have t h i s e f f e c t ( 1 9 , 2 3 ) ] . Domperidone, u n l i k e c e n t r a l l y a c t i n g dopamine a n t a g o n i s t s ( 2 4 , 2 5 ) , does not increase brain dopamine t u r n o v e r , as r e f l e c t e d by i t s i n a b i l i t y to increase s t r i a t a l DOPAC and HVA l e v e l s ( r e f . 19, Table I I ) . Lastly, radiolabeled domperidone, unlike other dopamine a n t a g o n i s t s , p e n e t r a t e s i n t o the brain only very p o o r l y a f t e r i t s p a r e n t e r a l a d m i n i s t r a t i o n (19). The increments in s t r i a t a l DOPAC and HVA l e v e l s produced by haloperidol (Table I I ) are s i m i l a r to those observed by others ( 2 4 , 2 6 ) ; the absence of such changes a f t e r domperidone i n j e c t i o n is also c o n s i s t e n t with other r e p o r t s (19) [and supports the n o t i o n t h a t domperidone does not act as a c e n t r a l dopamine a n t a g o n i s t ] . Both h a l o p e r i d o l and domperidone elevated serum p r o l a c t i n l e v e l s , to values c o n s i s t e n t l y observed a f t e r blockade of dopamine receptors (27,28). The a b i l i t y of these agents to e l e v a t e serum p r o l a c t i n l e v e l s is c o n s i s t e n t with the notion t h a t they block p e r i p h e r a l dopamine r e c e p t o r s , since the dopamine r e c e p t o r s t h a t mediate i n h i b i t i o n of p r o l a c t i n release l i e o u t s i d e the b l o o d - b r a i n b a r r i e r (29,30). Our s t u d i e s were performed in normotensive r a t s , because of the extreme expense of doing such s t u d i e s in SHR r a t s ; t h e r e was no reason to suspect t h a t the drugs would produce d i f f e r e n t e f f e c t s in d i f f e r e n t rat s t r a i n s . Also, the tlme chosen f o r measurements and the drug doses used were based on published r e p o r t s of peak e f f e c t s f o r such agents ( 1 8 , 2 4 , 2 5 , 2 6 ) . Domperidone's apparent a b i l i t y to block p e r i p h e r a l but not central dopamine r e c e p t o r s provides a useful pharmacologic t o o l f o r investigating the anatomic s i t e s of a c t i o n of dopamine a g o n i s t s . Since domperidone blocks lergotrile's antihypertensive action, these data support the h y p o t h e s i s t h a t l e r g o t r i l e reduces blood pressure by s t i m u l a t i n g peripheral dopamine r e c e p t o r s . Another study suggests t h a t a s i m i l a r drug, b r o m o c r i p t i n e , may decrease
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blood pressure by stimulating peripheral dopamine receptors (31). Bromocriptine was more effective in lowering blood pressure when administered intravenously rather than into the cerebral ventricles or vertebral a r t e r y . These i n v e s t i g a t o r s , and others, also found that dopamine antagonists blocked the drug's e f f e c t , which supports the notion that bromocriptine actually lowers b l o o d pressure by stimulating a dopamine receptor (5,31,32). I f l e r g o t r i l e (and other dopamine agonist/ergot derivatives) lower blood pressure by stimulating peripheral dopamine receptors, there are at least two possible sites for such receptors. First, they may reside within certain vascular beds [ e . g . , the renovasculature and mesenteric arteries (13)], presumably on smooth muscle, since pharmacologic stimulation of dopamine receptors in these vascular beds causes vasodilation (13). This p o s s i b i l i t y seems somewhat remote, however, at least for bromocriptine, since i t is a poor agonist for vascular dopamine receptors (33). Moreover, such a mechanism of action seems u n l i k e l y , since reduction in blood pressure by such a route would be expected to e l i c i t a reflex increase in sympathetic tone; t h i s has not yet been observed, at least for bromocriptine (5,31). Second, dopamine agonists might lower blood pressure by decreasing norepinephrine release from sympathetic nerve terminals. These agents would probably act by stimulating dopamine receptors located on postganglionic sympathetic neurons (12). R e p o r t s that bromocriptine administration reduces both blood and urine norepinephrine levels (34,35) would tend to support such a hypothesis. Bromocriptine has also been shown to decrease norepinephrine release f r o m sympathetic nerve terminals a f t e r direct n e r v e stimulation (31,32), as had previously been demonstrated to be an action of other dopamine agonists (36). Further studies now seem warranted to locate these peripheral dopamine receptors. Since l e r g o t r i l e appears to reduce blood pressure by a peripheral action, one f r u i t f u l avenue of research might be the development of dopamine agonists that do not penetrate into the central nervous system. Such agents might ol-o-w-er blood pressure e f f e c t i v e l y , while not e l i c i t i n g any of the known side effects of c e n t r a l l y acting dopamine agonists (37). A new class of c l i n i c a l l y useful antihypertensive agents might thus be invented. References 1. A.F. SVED and J.O. FERNSTROM, J. Pharm. Pharmacol. 31 814-817 (1979). 2. K.O. STUMPE, R. KOLLOCH, M. HIGUCHI, F. KRUCK and H. VETTER, Lancet 2 211-213 (1977). 3. S.B. KA~E, K.M. SHAW and E.S. ROSS, Lancet 1 1176-1177 (1976). 4. J.K. GREENACRE, P . F . TEYCHENNE, A. PETRIE, D.B. CALNE, P.N. LEIGH and J.L. REID, Br. J. Clin. Pharmacol. 3 571-574 (1976). 5. B.J. CLARK, G. CHOLTYSIK and E. FLUCKIGER, Ac~a Endocrinol. 88 (Suppl. 216) 75-81 (1978). 6. T.T. YEN, N.B. STAMM and J.A. CLEMENS, Life Sci. 25 209-216 (1979). 7. L. LEMBERGER, Fed. Proc. 37 2176-2180 (1978). 8. M. GOLDSTEIN, J.Y. LEW, S. NAKAMURA, A.F. BATTISTA, A. LIEBERMAN and K. FUXE, Fed. Proc. 37 2202-2206 (1978). 9. K. FUXE, B.B. FREDHOLM, S.O. OGREN~--L.F. AGNATI, T. HOKFELT and J.A. GUSTAFSSON, Fed. Proc. 37 2181-2191 (1978).
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