Effects of 5HT uptake inhibitors on the pressor response to 5HT in the pithed rat. The significance of the 5HT blocking property

European Journal o f Pharmacology, 43 (1977) 209--215

209

© Elsewer/North-Holland Blomedmal Press

EFFECTS OF 5HT UPTAKE INHIBITORS ON THE PRESSOR RESPONSE TO 5HT IN THE PITHED RAT. THE SIGNIFICANCE OF THE 5HT BLOCKING PROPERTY

ERLING N PETERSEN, SVEN-OLLE OLSSON and RICHARD F SQUIRES

Ferrosan Research Laboratories, Sydmarken 1--5, DK-2860 Soeborg, Denmark and Malmo, Sweden Received 10 November 1976, rewsed MS received 21 January 1977, accepted 31 January 1977

E N PETERSEN, S -O OLSSON and R F SQUIRES, Effects o f 5HT uptake mhlbztors on the pressor response to 5HT m the p~thed rat The slgn~fzcance o f the 5HT blockmg property, European J Pharmacol 43 (1977) 209-215 A study was made of the effects of several serotonm (5HT) uptake mhlbltors on 5HT-mduced pressor responses m pithed rats, 5HT uptake into rat brain synaptosomes and 5HT-mduced contractions of rat ileum m vitro All drugs except deslmlpramme were potent uptake mhlbltors (ICs0 < 10 -7 M) Femoxetme, chlonmlpramme, lmlpramme and demmlpramme all inhibited 5HT-mduced contractmns of the rat ileum m wtro and the pressor response to 5HT m vlvo FG 7051, FG 7052 and dexchlorphemramme were weak 5HT antagomsts on the rat ileum but potentmted the pressor responses to 5HT, the most potent uptake inhibitor, FG 7051, was the strongest potentmtor These results suggest that uptake mhlblhon is important for this p o t e n h a t m n It is concluded that 5HT uptake mhlbltors with potent 5HT receptor blocking properties antagonize the pressor response to 5HT and mask the p o t e n t m h o n due to uptake mhtblhon D-receptor blockade

Antidepressants

Potentiation

1. Introduction Studms using several vascular systems suggest the emstence of different types of vascular 5HT receptors There seem to be at least 2 receptor types for 5HT m the carotid bed, one medmtmg constrmtlon and the other medmtmg vasodflatatmn (Saxena et a l , 1971, Vargaftlg and Lefort, 1974, Schonbaum et a l , 1975) C o n s t n c t m n is generally d o m m a n t and dilator effects hrst appear after selechve blockade of constrmtor effects e.g. by ergotamme (Vargaftlg and Lefort, 1974) Some analogms to the a- and p-effects of adrenahne therefore seem to exist for vascular 5HT receptors Fozard and Leach (1968) have characterized the cardmvascular system of the pithed rat as a dlbenzyhne sensitive (D-receptor) system m whmh 5HT mainly produces pressor responses

Serotonm

Cardiovascular system

due to low basal vascular tonus Intravenously rejected 5HT has a half-hfe m plasma of 1--2 m m (Thomas and Vane, 1967), whmh mdmates that uptake and storage of 5HT is a relatively rapid process. In blood, 5HT is mamly taken up by, and stored m, platelets and most of the 5HT found m various organs Is probably contained m blood platelets (Vohcer et a l , 1970) Trmychc antidepressant drugs have been shown m vitro and m VlVO to inhibit the uptake of 5HT into blood platelets (Marshall et al., 1960; T o d n c and Talt, 1969). After i v administration of 5HT to an ammal pretreated with a 5HT uptake inhibitor one might expect higher concentrahons of free 5HT m plasma for a longer tnne than m untreated control anunals Thus pretreatment with an uptake inhibitor should potentiate the cardmvascular effects of 5HT, and this has indeed been

210 d e m o n s tr ated f o r cocaine (Fozard, 1969) However, in addltmn to blocking uptake of 5HT into blood platelets, some trlcychc antidepressants block the pressor response to 5HT (M¢ller Nmlsen et a l , 1966), gwmg rise to dual effects of these drugs on the cardiovascular responses to 1 v 5HT To elucidate these dual effects on blood pressure chlorlmlpramme, lmlpramlne, deslmlpramme, dexchlorphemramme, femoxetme (FG 4963, Buus Lassen et a l , 1975), FG 7051 and FG 7052 (optmal antipodes of a structural analogue of FG 4963), drugs which differ with respect to their relative potencies in blocking 5HT uptake and D-receptors have been tested in the pithed rat preparation

2. Materials and m e t h o d s

2 1 Blood pressure experiments Male Wlstar rats weighing 250- - 350 g were pithed according to the m e t h o d of Shlpley and Tilden (1947) and ventilated artificially by a Palmer electromcally controlled ammal ventilator PE 50 catheters were inserted into one jugular vein and one carotid artery and f o r some experiments, in b o t h carotid arteries In most experiments, 2 1 v mjectmns of 5 pg 5HT/kg were given 10 m m apart to estabhsh a control response taken as the mean of these 2 Injections These 5HT rejections were repeated 10 and 30 m m after the test drug was g,ven Both the increase in systolic pressure and the pressor area of the mean blood pressure response were recorded, the latter by means o f an electronic integrator Changes were calculated as percent of the cont r ol response Each rat received only one dose of the test drug and 5 rats were used per dose In some experiments dose--response curves to low, non-desensitizing, doses of 5HT were made with and w i t h o u t drugs in the same animal In these experiments the time interval b e t w e e n 5HT rejections was 5 m m

EN PETERSENETAL

2 2 Antagontsm of 5 H T o n rat deum The experiments were carrmd out as desc n b e d earlier by Buus Lassen et al (1975)

2 3 Inhtbztmn tosomes

of 5HT uptake by synap-

ICs0 values were determined using synaptosomes from rat forebram (Sqmres, 1974)

2 4 Substances 5HT creat m m e sulfate, c h l o n m l p r a m m e HC1, lmlpramme HC1, deslmlpramme HC1, fem o x e t m e HC1, d e x c h l o r p h e m r a m m e maleate, FG 7051 and FG 7052 bot h as malemates were used The doses used refer to the salts

2 5 Stattstws Statlstmal evaluation was p e r i o r m e d using Student's t-test (one-tailed)

3. Results

3 1 Blood pressure experzments Fig I shows that, within a single rat, reproduclble dose--response curves for 5HT can be constructed during the experimental period Both the systohc increase and the area under the mean blood pressure curve remain stable w i t h o u t drug t r e a t m e n t Fig 1 and 2 show the curves after t r e a t m e n t with chlonmlpramme, a p o t e n t blocker of b o t h 5HT uptake and D-receptors, and F G 7 0 5 1 , a p o t e n t upt ake inhibitor with weak D-receptor blockmg propertms These curves d e m o n s t r a t e that b o t h t he systohc increase and the area of the 5HT response are affected by 5HT upt ake mh~bltors, sometimes m di fferent dlrectmns FG 7051 inhibited the systohc response to 10 pg 5HT/kg 1 v b u t did n o t affect this response to 5HT given mtra-arterlally (1 a ) Mean pressor responses to 5HT were prolonged after b o t h 1 v and 1 a rejections, this effect

P R E S S O R R E S P O N S E T O 5HT A F T E R 5HT U P T A K E INHIBITORS

I

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211

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Fig 1 D o s e - - r e s p o n s e c u r v e s f o r 1 v 5 H T b e f o r e d r u g s ~ a n d a f t e r I v s a h n e or c h l o m m l p r a m m e 1 m g / k g iv A A V a l u e s are mean-+ S E M of 5 e x p e m m e n t s B P = blood pressure a , p < 0 0 5 , b , p < 0 0 1 , c , p < 0 001

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Fig 2 D o s e - - r e s p o n s e c u r v e s f o r i v a n d 1 a 5 H T b e f o r e F G 7 0 5 1 ~ and after FG 7051 1 mg/kg 1v V a l u e s are m e a n +- S E M o f 5 e x p e m m e n t s BP = b l o o d p r e s s u r e a, p < 0 0 5 , b, p < 0 0 1 , c, p < 0 0 0 1

212

EN

PETERSENETAL

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0005

0 mg/kg

~5_ H _ _T- - - J _ 0,005

5HT 0,005

- CI-IMI ~ 5 0

5 HI T 0005

5 HI T mg/kg 0,005

Fig 3 The e f f e c t o f F G 7 0 5 1 0 2 m g / k g l v on the b l o o d pressure r e s p o n s e ~o 5 pg 5HT/kg i v m t h e p i t h e d rat

Fig 4 The e l f e c t o f c h l o n m l p r a m m e 5 m g / k g 1 v on the b l o o d pressure r e s p o n s e to 5 / l g 5 H T / k g l v m the p i t h e d rat N o t e , o n l y t h e pressor part o f the r e s p o n s e Is i n t e g r a t e d

a p p e a r s as a s l g m f i c a n t l y increased area, e s p e c m l l y a f t e r l o w d o s e s (fig 2) Fig 2 also s h o w s t h a t t h e systolic r e s p o n s e to 5 H T u n d e r c o n t r o l c o n d i t i o n s was slgmhc a n t l y higher a f t e r i v t h a n a f t e r 1 a rejections T h e i n t e g r a t e d r e s p o n s e s are v e r y similar b u t s h g h t l y larger a f t e r 10 p g / k g 1 v With F G 7 0 5 1 , t h e s y s t o h c p r e s s o r r e s p o n s e to 5 H T increased significantly m o r e a f t e r 1 a t h a n a f t e r i v m j e c t m n o f 5 H T T h e integrated r e s p o n s e was u n a f f e c t e d b y t h e r o u t e o f a d m l m s t r a t l o n T h e s e results suggest t h a t the pattern of 5HT metabohsm, excretion,

d i s t r i b u t i o n a n d storage was stmllar a f t e r 1 a a n d 1 v r e j e c t i o n a n d t h a t , d u r i n g a single pass, t h e lungs r e m o v e o n l y a small p a r t o f t h e rejected 5HT T w o r e p r e s e n t a t i v e e x p e r i m e n t s (figs 3 and 4) s h o w t h a t t h e r e s p o n s e s to 5 H T a d m i n i s t e r e d 10 and 30 m m a f t e r t h e drugs were usually q u a h t a t l v e l y a n d q u a n t i t a t i v e l y s l m d a r In a f e w cases t h e r e s p o n s e s to 5 H T were d i f f i c u l t to d e t e r m i n e 10 m l n a f t e r high d o s e s o f a d r u g w i t h a p r e s s o r e f f e c t o f its o w n F o r this r e a s o n o n l y t h e e f f e c t s on 5 H T r e s p o n s e s 30 m l n a f t e r d r u g a d m i n i s t r a t i o n

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Fig 5 The e f f e c t o f u p t a k e l n h l b l t o r s o n t h e m a x i m a l s y s t o h c b l o o d pressure increase and o n the i n t e g r a t e d r e s p o n s e o f t h e m e a n b l o o d pressure r e s p o n s e to 5 p g 5 H T / k g i v 30 m m a f t e r t h e drugs The e f f e c t is e x p r e s s e d as p e r c e n t o f m e a n c o n t r o l r e s p o n s e (see fig 3) D e x c h l o r p h e m r a m m e is a b b r e v i a t e d DCP Values are m e a n _+ SEM of5expenments a,p< 005,b,p< 001,c,p< 0001

PRESSOR RESPONSE TO 5HT AFTER 5HT UPTAKE INHIBITORS

213

TABLE 1 In vitro concentrations (ICs0) giving 50% mhlb,tlon of uptake into synaptosomes and of ileum contractions The numbers of experiments are g~ven m parentheses Test substance

Uptake of 5HT into synaptosomes from rat forebram (M)

Rat ileum contractmns produced by 5HT (M)

FG7051 FG7052 Femoxetme Chlorlmlpramme Imlpramme Desn~mpramme Dexchlorphemramme

3× 2× 8x 6× 3× 3x 2×

5× 1x 2× 1× 3X 1× 2x

10 - 8 10 - 7 10 - 8 10 - 8 10 - 7 10 - 6 10 - 7

(5) (3) (11) (14) (13) (17) (3)

are d e s c r i b e d T h e drugs t e s t e d can b e d l w d e d into two groups o n e w i t h a p p a r e n t Dr e c e p t o r b l o c k m g p r o p e r t i e s including chlor l m l p r a m m e , ~m~pramme, d e s l m ~ p r a m m e and femoxetlne, and another with 5HT potentinting p r o p e r t m s , including d e x c h l o r p h e n l r a m m e , F G 7 0 5 1 a n d F G 7 0 5 2 . F G 7 0 5 2 and demmlpramme were both weak D-receptor b l o c k e r s , b u t o n l y F G 7 0 5 2 was a p o t e n t upt a k e i n h i b i t o r and p r o b a b l y b e c a u s e o f this fell in t h e p o t e n t i a t i n g g r o u p A p l a t e a u (ceiling) e f f e c t seems to exist f o r F G 7 0 5 1 , whmh produces the same response, about a seven-fold increase m t h e i n t e g r a t e d r e s p o n s e , m d o s e s r a n g m g f r o m 0 2 t o 5 m g / k g (fig 5)

3 2 Antagonmm of 5HT on rat ileum C h l o n m l p r a m m e , ,m~pramlne, d e m m l p r a mine and femoxetme antagomzed 5HT-mduced c o n t r a c t m n s o f t h e r a t i l e u m with ICs0 b e t w e e n 1 0 - 6 - - 1 0 -s M. F G 7 0 5 2 , F G 7051 a n d d e x c h l o r p h e m r a m m e h a d all ICs0/> 10 -s M (tablel) FG70515X 1 0 - 7 - - 5 × 10 - 6 M even s h g h t l y p o t e n t i a t e d t h e 5 H T - m d u c e d contractions No lntrmsm effects of any of t h e drugs w e r e o b s e r v e d

3 3 Inh~b~tzon tosomes

to

of

5HT

uptake

by synap-

All drugs e x c e p t d e m m l p r a m m e w e r e f o u n d be s t r o n g u p t a k e m h l b , t o r s w i t h ICs0

10 - 5 10 - 5 10 - 6 10 - 6 10 -6

10 - 5 10 - 5

(3) (3) (3) (3) (3) (3) (3)

between 3 X 1 0 - s - - 3 X 10 -7 M, b e i n g t h e s t r o n g e s t (table 1)

F G 7051

4. D m c u s s m n Thin w o r k clearly s h o w s t h a t several p o t e n t 5 H T u p t a k e m h l b l t o r s (e g c h l o n m l p r a m m e , l m l p r a m m e a n d f e m o x e t m e ) are s t r o n g b l o c k ers o f t h e p r e s s o r r e s p o n s e s to 1 v 5 H T m t h e p i t h e d rat, an e f f e c t w h m h seems t o parallel t h e i r 5 H T a n t a g o m s t l c e f f e c t o n t h e isolated rat ileum Although the inhibition of 5HT uptake probably mcreases the concentration o f 5 H T m p l a s m a f o r a longer t i m e , thin d o e s not appear suffmmnt to overcome the 5HT receptor blockade C h l o n m l p r a m l n e , l m l p r a m l n e and f e m o x e t l n e s e e m to e f f e c t i v e l y b l o c k t h e 5 H T r e c e p t o r s m e d i a t i n g vascular c o n s t r i c t i o n , an effect whmh apparently cannot be overcome by 5HT uptake mhlblUon The mechanism by whmh dexchlorphemramme, FG 7051 and FG 7 0 5 2 p o t e n t i a t e t h e pressor e f f e c t s o f i v 5 H T is n o t e n t m e l y clear, b u t u p t a k e inhibit , o n s e e m s to be I m p o r t a n t , b e c a u s e t h e m o s t p o t e n t u p t a k e i n h i b i t o r , F G 7 0 5 1 , is also t h e s t r o n g e s t 5 H T p o t e n t m t o r O t h e r f a c t o r s cont m b u t m g to 5 H T p o t e n t m t l o n c o u l d b e sensltmatmn of 5HT receptors medmtlng vasocons t n c t m n or b l o c k a d e o f 5 H T r e c e p t o r s medla t m g v a s o d f l a t a t l o n F G 7 0 5 1 itself has a p r e s s o r e f f e c t lasting less t h a n 30 m m w h i c h

214 p r o b a b l y c a n n o t a c c o u n t for its 5 H T p o t e n tinting e f f e c t (fig 3) In the rat ileum, FG 7 0 5 1 in low c o n c e n t r a t i o n s p o t e n t m t e d the response to 5 H T b u t e x h i b i t e d n o lntrmsm e f f e c t at a n y c o n c e n t r a t i o n B l o c k a d e o f 5 H T r e c e p t o r s m e d m t m g v a s o d f i a t a t m n c a n n o t be e x c l u d e d at p r e s e n t a n d remains an interesting possibility T h e use o f s y n a p t o s o m e s instead o f b l o o d platelets s e e m e d justified smce the active 5 H T u p t a k e m e c h a m s m m t h e t w o systems a p p e a r to be very mmllar if n o t identical ( T u o m l s t o , 1974) A pressor r e s p o n s e can be described b y m e a n s of t h e systolic, m e a n or diastolic b l o o d pressure merease or o f t h e integrated response o f each of these p a r a m e t e r s T h e i n t e g r a t e d r e s p o n s e is a p r o d u c t o f b o t h t h e m a x i m a l increase and the d u r a t m n o f the response A r e d u c e d m a x i m a l response t o g e t h e r with a prolonged duration of the response may p r o d u c e an u n c h a n g e d integrated r e s p o n s e as m a y be seen with c h l o n m l p r a m m e (fig 1) The m a x i m a l systolic increase is s o m e t i m e s o n l y m a i n t a i n e d f o r s e c o n d s and is p r o b a b l y m o r e i n f l u e n c e d b y d i f f e r e n t speeds o f reject i o n and o t h e r , less c o n t r o l a b l e variables t h a n is the i n t e g r a t e d response This m i g h t explain w h y F G 7051 u n i f o r m l y p o t e n t i a t e d t h e dose - - i n t e g r a t e d r e s p o n s e curves to 1 a and 1 v 5HT, b u t m s o m e e x p e r i m e n t s inhibited the systolic response to 10 pg 5 H T / k g 1 v S o m e n e w selective 5 H T u p t a k e mhlbytors o f the p h e n y l p l p e r l d m e t y p e r e p r e s e n t e d b y f e m o x e t m e , b l o c k e d the pressor r e s p o n s e t o 5 H T while others, r e p r e s e n t e d b y F G 7 0 5 1 and F G 7 0 5 2 , p o t e n t i a t e d it M a n y a u t h o r s have p r o p o s e d a role for 5 H T m migraine ( A n t h o n y et a l , 1 9 6 9 , S a x e n a et a l , 1 9 7 1 , S]aastad, 1 9 7 5 , Fog-M¢ller, 1 9 7 6 ) 5 H T u p t a k e m h l b l t o r s m a y c h a n g e the storage of, and vascular r e s p o n s e to, 5 H T to such an e x t e n t t h a t an investigation o f their effects m migraine m i g h t be justified It is c o n c e i v a b l e t h a t a r e d u c t m n m releaseable platelet 5 H T (Marshall et a l , 1 9 6 0 , Yates et a l , 1 9 6 3 ) , or a p o t e n t i a t i o n o f the c o n s t r i c t i n g e f f e c t o f 5 H T o n cranial vessels (Saxena, 1 9 7 1 ) m i g h t have

E N PETERSEN ET AL a beneficial e f f e c t m migraine What slgmflc a n c e these differential effects o f 5HT u p t a k e inhlbltors o n vascular 5 H T r e c e p t o r s m i g h t have m migraine t h e r a p y is at p r e s e n t unknown The p h e n y l p l p e r i d m e s described here m a y be valuable tools for the s t u d y o f migraine

Acknowledgements The authors wish to thank Mrs A L Svarer for skllful technical assistance and Mrs Solvelg Bonde for preparing the manuscript

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PRESSOR RESPONSE TO 5HT AFTER 5HT UPTAKE INHIBITORS Shlpley, R E and J H Tilden, 1947, A pithed rat preparation suitable for assaying pressor substances, Proc Soc Exptl Biol 6 4 , 4 5 3 Sjaastad, O , 1975, The slgnlfmance of blood serotonm levels m migraine, Acta Neurol Skandmav 51,200 Squires, R F , 1974, Effects of noradrenahne pump blockers on its uptake by synaptosomes from several brain regions, additional evidence for dopamme terminals m frontal cortex, J Pharm Pharmacol 2 6 , 3 6 4 Thomas, D P and J R Vane, 1967, 5-Hydroxytryptamme m the clrculatmn of the dog, Nature (London) 2 1 6 , 3 3 5 Todrlck, A and A C Talt, 1969, The mhlbltmn of human platelet 5-hydroxytryptamme uptake by trmychc antldepresmve drugs The relation

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between structure and potency, J Pharm Pharmacol 21,751 Tuomlsto, J , 1974, A new modification for studying 5-HT uptake by blood platelets a re-evaluation of trlcychc antidepressants as uptake mhlbltors, J Pharm Pharmacol 26, 92 Vargaftlg, B B and J Lefort, 1974, Pharmacological evidence for a vasodllator receptor to serotonm in the nasal vessels of the dog, European J Pharmacol 25, 216 Vohcer, L , M A Beaven and W D Reid, 1970, The accumulation of H3-serotomn m platelet-free rats, Pharmacology 4, 203 Yates, C M , A Todrmk and A C Talt, 1963, Aspects of the chnlcal chemistry of desmethyhmlpramme in man, J Pharm Pharmacol 1 5 , 4 3 2