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The contractile response of the isolated rat uterus to noradrenaline and 5-hydroxytryptamine
EUROPEAN JOURNAL OF PHARMACOLOGY 3 (1968) 310-315. NORTH-HOLLAND PUBL. COMP., AMSTERDAM
THE CONTRACTILE RESPONSE OF THE ISOLATED RAT UTERUS TO N O R A D R E N A L I N E AND 5-HYDROXYTRYPTAMINE David M. PATON Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada Received 10 April 1968
Accepted 29 April 1968
D. M. PATON, The contractile response of the isolated rat uterus to noradrenaline and 5-hydroxytryptamine, European J. Pharmacol. 3 (1968) 310-315. Interactions of noradrenaline and 5-hydroxytryptamine with several pharmacological agents were studied on rat uterine horns at 18-20°C. Contractions in response to 5-hydroxytryptamine were antagonized by dimethylaminopropylthiocinnamanilide (DPTC) and phenoxybenzamine but not by atropine or cocaine. Noradrenaline only produced contractions consistently in the presence of propranolol; these contractions were antagonized by phenoxybenzamine but not by atropine or DPTC. Noradrenaline and 5hydroxytryptamine induced contractions were both Ca ++ dependent. Responses to both amines were markedly potentiated by short exposure to prostaglandin El . These observations favour the existence of separate excitatory receptors for 5-hydroxytryptamine and for catecholamines in the oestrogenized rat uterus. Noradrenaline 5-hydroxytryptamine Isolated rat uterus
I. INTRODUCTION
I n v e s t i g a t i o n s into the e f f e c t s of n o r a d r e n a l i n e on the r a t u t e r u s have le d to conflicting r e s u l t s ; t h e s e have b e e n r e v i e w e d r e c e n t l y ( M i l l e r , 1967). The u s u al r e s p o n s e of the r a t u t e r u s to the a m i n e is r e l a x a t i o n and inhibition of spontaneous c o n t r a c t i o n s ( M i l l e r , 1967); this effect can be p r e v e n t e d by b e t a and, a c c o r d i n g to s o m e but not all, by alpha a d r e n e r g i c r e c e p t o r blockade (Rudzik and M i l l e r , 1962; L e v y and T o z z i , 1963). Noradrena2ine m ay a l s o s t i m u l a t e the r a t u t e r u s in the p r e s e n c e or a b s e n c e of beta a d r e n e r g i c r e c e p t o r blockade (Diamond and Brody, 1966a and b; Tothill, 1967); h o w e v e r not a l l w o r k e r s have been able to r e p r o d u c e t h e s e r e s u l t s (Levy and T o z z i , 1963; M i l l e r , 1967). The i n v e s t i g a t i o n s r e p o r t e d h e r e c o n c e r n the e f fe c ts of noradrena2ine and 5 - h y d r o x y t r y p t a m i n e (5-HT) on i s o l a t e d o e s t r o g e n t r e a t e d r a t u t e r i at 1 8 - 2 0 o c ; both a m i n e s w e r e studied as they app e a r to act on c e r t a i n o r g a n s through a c o m m o n r e c e p t o r (Innes, 1962). E x p e r i m e n t s w e r e d e signed to e x a m i n e the following a s p e c t s : (a) the usual r e s p o n s e s of the r a t u t e r u s to t h e s e a m i n e s ; (b) d i f f e r e n t i a t i o n of the r e c e p t o r s on which the a m i n e s act; (c) the i n f l u e n c e of d r u g s
Alpha adrenergic receptor Beta adrenergic receptor
on r e s p o n s e s to the a m i n e s ; (d) t h e i r c a l c i u m dependence and (e) t h e i r ability to p r o d u c e cont r a c t i o n s in d e p o l a r i s e d p r e p a r a t i o n s .
2. METHOD 2.1. Method f o r reco rd i n g uterine r e s p o n s e s I m m a t u r e f e m a l e W i s t a r r a t s (80-120 g) w e r e p r e p a r e d by t r e a t m e n t f o r 6-12 days with subcutaneous i n j e c t i o n s of 50-75 ~ g / d a y of diethyls t i l b o e s t r o l . The r a t s w e r e k i l l e d by a blow on the head, the u t e r i r ap i d l y r e m o v e d and s u s pended in K r e b s R i n g e r m e d i u m , a e r a t e d with 95% O2 and 5% CO2, f o r i s o t o n i c r e c o r d i n g s . A m i n i m u m of t e n s i o n was used sufficient to b a l ance the suspended weight of the t i s s u e s e g m e n t with the l e v e r and to o v e r c o m e the f r i c t i o n of the w r i t i n g a r m with the d r u m . The k y m o g r a p h d r u m sp eed was 3.6 r a m / r a i n . Each u t e r i n e s e g m e n t was suspended v e r t i c a l l y in an individual organ bath having a v o l u m e of 10 ml. E x p e r i m e n t s w e r e conducted at 1 8 - 2 0 o c ; an advantage of the low t e m p e r a t u r e u s e d is that such p r e p a r a t i o n s show l i t t l e spontaneous act i v i t y . D r u g s w e r e washed out of the bath e i t h e r when a maxima2 r e s p o n s e had been attained or 4 m i n
C O N T R A C T I L E RESPONSE OF U T E R U S TO AMINES
after a d m i n i s t r a t i o n , whichever was the sooner. An i n t e r v a l of at l e a s t 7 m i n was allowed between s u c c e s s i v e a d m i n i s t r a t i o n of agonists to the bath, except in the case of 5-HT when 10 m i n was allowed. When the effect of v a r i o u s condit i o n s on the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l ine was being studied, p r o p r a n o l o l was added to the K r e b s solution so as to achieve a final conc e n t r a t i o n of 1 /~g/ml; only u t e r i n e h o r n s which c o n t r a c t e d in r e s p o n s e to n o r a d r e n a l i n e , in the p r e s e n c e of p r o p r a n o l o l , were u s e d in such studies. K r e b s Ringer solution of the following composition was used (mM): NaC1 115.48, KC1 4.63, CaC12 2.47, MgSO 4 1.16, Na!-ICO3 21.91, NaH2PO 4 1.16, Glucose 49.20. K + - r i c h Ringer solution was of the s a m e composition except that NaC1 115.48 (raM) was s u b s t i t u t e d with an equiva l e n t amount of KC1. 2.2. Drugs and chemicals The following drugs and c h e m i c a l s were used and the c o n c e n t r a t i o n s given a r e in t e r m s of the salt u n l e s s otherwise stated: acetylcholine chloride, / - n o r a d r e n a l i n e hydrochloride, 5 - h y d r o x y t r y p t a m i n e c r e a t i n i n e sulphate, p r o s t a g l a n d i n E 1 (PGE1), a t r o p i n e sulphate, p r o p r a n o l o l hydrochloride, phenoxybenzamine hydrochloride, p h e n t o l a m i n e m e t h a n e s u l p h o n a t e , tolazoline h y d r o c h l o r i d e , 2' 3- dim e t h y l a m i n o p r o p y l t h i o c i n n a m a n i l i d e h y d r o c h l o r i d e (DPTC), cocaine hydrochloride. The following d r u g s were p r e p a r e d as de: s c r i b e d below and kept f r o z e n u n t i l use: n o r a d r e n a l i n e , 5-HT and p r o p r a n o l o l as 1 m g / m l solutions in 0.01 M h y d r o c h l o r i c acid; PGE 1 as a 1 m g / m l solution in 95% ethanol; and phenoxyb e n z a m i n e as a 1 m g / m l solution in propylene glycol. All other d r u g s were d i s s o l v e d in d i s t i l l e d water.
3. RESULTS 3.1. Effects of noradrenaline and 5-HT in Krebs Ringer medium At 1 8 - 2 0 o c spontaneous activity was u s u a l l y a b s e n t and when p r e s e n t , c o n t r a c t i o n s were infrequent. 5-HT produced c o n t r a c t i o n s in all p r e p a r a t i o n s studied. The amplitude of m a x i m a l r e s p o n s e s to 5-HT was always as g r e a t as or g r e a t e r than those produced by acetylcholine. T h e r e was no evidence of tachyphylaxis to 5-HT when s u b m a x i m a l c o n c e n t r a t i o n s were employed at 7-10 m i n i n t e r v a l s . No p r e p a r a t i o n c o n t r a c t e d r e g u l a r l y and con-
311
s i s t e n t l y in r e s p o n s e to n o r a d r e n a l i n e , at conc e n t r a t i o n s up to 2 ~ g / m l , the u s u a l r e s p o n s e being a s m a l l r e l a x a t i o n . A n u m b e r of p r e p a r a t i o n s (9 out of 26) r e s p o n d e d to n o r a d r e n a l i n e with s m a l l c o n t r a c t i o n s ; however, these r e s p o n s e s were not well s u s t a i n e d and with either r e p e a t e d a d m i n i s t r a t i o n or i n c r e a s e d c o n c e n t r a tions of n o r a d r e n a l i n e , the u t e r u s failed to cont r a c t . T h e s e n o r a d r e n a l i n e induced c o n t r a c t i o n s were of s m a l l amplitude, being always much l e s s than those produced by m a x i m a l c o n c e n t r a t i o n s of 5-HT and acetylcholine.
TOP TRACE A
B IN PRESENCE OF PROPRM'dOLOL( I,ug/ml )
NA I0O
NA 500
NA 300
BOTTOM TRACE A
NA ICO
J_Ji NA NA 51)0 I ~
NA 2 5 0 ng/ml
B IN
F~RESO~CEOF
~ l J J g l m l )
5HT 5 I
5HT 5HT I 2 I cm
_zJ 5HT 2
5HT 5
5HT I0
5HT 20
i 5r~n I
F i g . 1. T h e e f f e c t of p r o p r a n o l o l on t h e a c t i o n s of n o r a d r e n a l i n e (NA) and 5 - h y d r o x y t r y p t a m i n e (5-HT). T r a c e s f r o m i s o t o n i c r e c o r d i n g s of i s o l a t e d r a t u t e r i n e h o r n s ; t e m p e r a t u r e 1 8 - 2 0 o c ; b a t h v o l u m e 10 m l ; s u s p e n d e d in K r e b s R i n g e r m e d i u m ; c o n c e n t r a t i o n s of a g o n i s t s in n g / m l of bath; d r u m s p e e d 3.6 m m / m i n . T o p t r a c i n g : c o n t r o l r e s p o n s e s to n o r a d r e n a l i n e (A) and r e s p o n s e s to n o r a d r e n a l i n e in p r e s e n c e of p r o p r a n o l o l , 1 # g / m l (B); B o t t o m t r a c i n g : C o n t r o l r e s p o n s e s to 5 - h y d r o x y t r y p t a m i n e (A) and r e s p o n s e s to 5-hydroxytryptamine in p r e s e n c e of p r o p r a n o l o l , 1 l / g / m l (B).
312
D.M. PATON
3.2. E f f e c t s of propranolol on r e s p o n s e s to n o r adrenaline and 5-HT P r o p r a n o l o l , 1 p g / m l , r e d u c e d r e s p o n s e s to s u b m a x i m a l c o n c e n t r a t i o n s of 5-HT in 4 u t e r i n e h o r n s ; this effect could be o v e r c o m e by i n c r e a s ing the c o n c e n t r a t i o n of 5-HT (fig. 1). A l m o s t all p r e p a r a t i o n s (61 out of 71) r e sponded with a c o n t r a c t i o n to n o r a d r e n a l i n e in the p r e s e n c e of p r o p r a n o l o l , 1 p g / m l ; five u t e r ine horns did not r e s p o n d to n o r a d r e n a l i n e even in the p r e s e n c e of p r o p r a n o l o l , 2 ~ g / m l . As the c o n c e n t r a t i o n of n o r a d r e n a l i n e was i n c r e a s e d p r o g r e s s i v e l y , the c o n t r a c t i l e r e s p o n s e u s u a l ly i n c r e a s e d in magnitude, then d e c r e a s e d and f i nally the r e s p o n s e , at high c o n c e n t r a t i o n s (1-2 p g / m l ) was u s u al l y a r e l a x a t i o n . The m a x i m a l c o n t r a c t i o n to n o r a d r e n a l i n e was u s u a ll y s m a l l e r in magnitude than t h o s e p r o d u c e d by e i t h e r a c e t y l c h o l i n e or 5-HT (fig. 1). P r e p a r a t i o n s which f a i l e d to c o n t r a c t in r e sponse to n o r a d r e n a l i n e both in the a b s e n c e and p r e s e n c e of p r o p r a n o l o l , 1 or 2 ~zg/ml, always c o n t r a c t e d when ex p o s e d to 5-HT. Such p r e p a r a tions w e r e not u s e d in f u r t h e r s t u d ie s on the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l i n e . 3.3. Effect of other antagonists on r e s p o n s e s to noradrenaline and 5-HT The effect of an t a g o n is t s on c o n t r a c t i o n s to 5-HT and n o r a d r e n a l i n e was t e s t e d in the continuous p r e s e n c e of p r o p r a n o l o l , 1 /~g/ml. A t r o pine, 0.1 to 1.0 p g / m l , a b o l i s h e d c o n t r a c t i o n s to s u p r a m a x i m a l c o n c e n t r a t i o n s of a c e t y l c h o l i n e (10 /~g/ml) but, in 10 p r e p a r a t i o n s did not a l t e r c o n t r a c t i o n s to s u b m a x i m a l c o n c e n t r a t i o n s of e i t h e r 5-HT or n o r a d r e n a l i n e . The a d m i n i s t r a tion of DP TC , 200-500 n g / m l , a b o li s h e d cont r a c t i o n s to p r e v i o u s l y s u p r a m a x i m a l c o n c e n t r a tions of 5-HT but in t h e s e 15 p r e p a r a t i o n s , the c o n t r a c t i l e r e s p o n s e s to n o r a d r e n a l i n e w e r e not r e d u c e d in amplitude (fig. 2). P h e n o x y b e n z a m i n e , 0.5 to 1 g / m l for 5 m i n , a boli sh ed c o n t r a c t i o n s to both 5-HT and n o r a d r e n a l i n e . T o l a z o l i n e could not be u s e d e f f e c t i v e l y as an antagonist as it u s u a l ly m a r k e d l y i n c r e a s e d spontaneous activity. The r e s u l t s of the u s e of phe ntolam i n e, 10-25 p g / m l , as an antagonist on 12 p r e p a r a t i o n s w e r e i n c o n c l u s i v e since the ant a g o n i s t o c c a s i o n a l l y p r o d u c e d i n c r e a s e d spontaneous activity. In s o m e p r e p a r a t i o n s it app e a r e d to m e r e l y r e t a r d the onset of the cont r a c t i l e r e s p o n s e to n o r a d r e n a l i n e while in o th e r p r e p a r a t i o n s it ab o l i s h e d or r e d u c e d the cont r a c t i l e r e s p o n s e to n o r a d r e n a l i n e . In view of this it was i m p o s s i b l e to u s e p h e n t o l a m i n e to d i s t i n g u i s h definitely b e tw e e n the c o n t r a c t i l e r e -
A
B
AC 5HI NA I0 000 I00 400 Ilcm
AC 5HI NA I0000 I00 400
G
5HI I00
D
NA 400
NA 400 nglml
j 5rain I
Fig. 2. The effect of antagonists on the actions of acetylcholine (AC), 5-hydroxytryptamine (5-HT) and noradrenaline (NA). Traces from isotonic recordings of an isolated uterine horn; other details as in fig. 1; propranolol, 1 /~g/ml, present throughout. Control responses (A); responses to agonists in presence of atropine, 1 ftg/ml (B); responses to agonists in presence of DPTC. 300 ng/ml (C); responses to agonists after exposure to phenoxybenzamine, 500 ng/ml for 5 min (D). sp o n ses to 5-HT and n o r a d r e n a l i n e . The inability of phentolamine to c o n s i s t e n t l y a n t a g o n i s e the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l i n e may have been contributed to by the known ability of p r o p r a n o l o l to i n t e r f e r e with the e s t a b l i s h m e n t of an alpha r e c e p t o r blockade ( O l i v a r e s , Smith and Aronow, 1967).
3.4. Effect of calcium on r e s p o n s e s to noradrenaline and 5-HT Both a c e t y l c h o l i n e and 5-HT continued to p r o duce s m a l l c o n t r a c t i o n s in c a l c i u m - f r e e K r e b s R i n g e r m e d i u m f o r up to 90 rain; t h e s e c o n t r a c t i o n s w e r e a b o l i s h e d when Na2 EDTA, 5 raM, was added to the c a l c i u m - f r e e m e d i u m . C o n t r a c t i o n s to n o r a d r e n a l i n e , in the continuous p r e s e n c e of p r o p r a n o l o l , 1 p g / m l , d i s a p p e a r e d r ap i d l y howe v e r on e x p o s u r e to c a l c i u m - f r e e m e d i u m (1020 min (fig. 3). 3.5. Effect of p o t a s s i u m on r e s p o n s e s to noradrenaline and 5-HT In 6 p r e p a r a t i o n s d e p o l a r i s e d by KC1-Ringer m e d i u m , s m a l l c o n t r a c t i o n s w e r e p r o d u c e d by s u p r a m a x i m a l c o n c e n t r a t i o n s of both a c e t y l c h o line and 5-HT. In the p r e s e n c e of p r o p r a n o l o l , 1 o r 2 ~zg/ml, n o r a d r e n a l i n e , in c o n c e n t r a t i o n s up to 2 ~ g / m l , f a i l e d to c o n t r a c t the d e p o l a r i s e d p r e p a r a t i o n (fig. 3); all such p r e p a r a t i o n s had
CONTRACTILE RESPONSE OF UTERUS TO AMINES
until a maximal response was obtained, usually in less than 2 min and the PGE 1 was then washed o u t of t h e b a t h . S u b s e q u e n t l y t h e r e s p o n s e s t o t h e c o n t r o l c o n c e n t r a t i o n s of 5 - H T a n d n o r a d r e n a l i n e w e r e p o t e n t i a t e d f o r p r o l o n g e d p e r i o d s (up t o 120 rain) a f t e r t h i s s i n g l e e x p o s u r e to P G E 1 . T h i s p h e n o m e n o n w a s n o t o b s e r v e d in c o n t r o l h o r n s w h i c h h a d not b e e n e x p o s e d to P G E 1 (fig. 4).
TOP TRACE KREBS
Co - FREE KREBS RINGER
RINGER
I
A
AC
NA
5HT
5HT
I0000
200
IO
IO I0
5HT IO 20
I lcm
313
B
NA AC 5HT NA 5/RTAC 2OOKX)COIOO500 IOOIOOOO ng/m! 38 50 60 68 95 105 MINUTES
I 5mini
BOTTOM TRACE RESPONSES IN
KCl - RII~GER
f NA 500 I lcm
J r
~ NA 200 11300
J
f 51"1T NA PGE 5HT NA 10 20
NA NA 5HT tOO 5000 200 ng/ml
5HT NA 5HT NA 5HT NA 5HT NA 5HT 40 50 60 70 80 90 100 I10 120 MINUTES
TIME AFTER EXPOSURETO PROSTAGLANDIN
I 5mln I
Ilcm
J 5role I
Fig. 3. The effect of ions on the actions of n o r a d r e n a l ine (NA), 5-hydroxytryptamine (5-HT) and acetylcholine (AC). T r a c e s from isotonic r e c o r d i n g s of isolated r a t uterine horns; propranolol, 1 ]~g/ml p r e s e n t throughout; other details as in fig. 1. Top tracing: control r e s p o n s e s in Krebs Ringer medium, Ca ++ 1.5 raM; r e s p o n s e s in Ca ++ free Krebs Ringer m e d i um. Bottom tracing: r e s p o n s e s in KC1 Ringer medium; this p r e p a r a t i o n had contracted in r e s p o n s e to n o r a d renaline in Krebs Ringer medium.
Fig. 4. The effect of prostaglandin E 1 (PGE) on the actions of 5-hydroxytryptamine (5-HT) and n o r a d r e n aline (NA). T r a c e s from isotonic recordings of an isolated r a t uterine horn; propranolol, 1 /ag/ml, p r e s e n t throughout; other details as in fig. 1. The following concentrations of agonists were used throughout: 5-HT, 5 n g / m l ; NA 50 n g / m l ; PGE 2.5 ~tg/ml. Control r e s p o n s e s (A); r e s p o n s e s to agonists a R e r PGE had been applied to the p r e p a r a t i o n once and then r e moved (B).
c o n t r a c t e d i n r e s p o n s e to n o r a d r e n a l i n e solution.
4. DISCUSSION
in K r e b s
3.6. E f f e c t o f cocaine on r e s p o n s e s to n o r a d r e n aline and 5 - H T C o n t r a c t i o n s t o s u b m a x i m a l c o n c e n t r a t i o n s of 5-HT were not altered by cocaine, 1 or 2 pg/ml. C o n t r a c t i o n s in r e s p o n s e t o n o r a d r e n a l i n e w e r e s t u d i e d in t h e p r e s e n c e of p r o p r a n o l o l , 1 p g / m l . No c o n s i s t e n t r e s p o n s e w a s s e e n in t h e s e e x p e r iments; some were potentiated, some inhibited and some unaltered. 3.7. E f f e c t
o f prostaglandin E 1 on r e s p o n s e to noradrenaline and 5 - H T
Control responses to submaximal t i o n s of 5 - H T a n d n o r a d r e n a l i n e w e r e t h e p r e s e n c e of p r o p r a n o l o l , 1 ~ g / m l . arations were then exposed to PGE1,
concentrao b t a i n e d in The prep2.5 ~ g / m l
Catecholamines usually relax and inhibit s p o n t a n e o u s c o n t r a c t i o n s of t h e i s o l a t e d r a t u t e r u s ; t h e o r d e r of p o t e n c y f o r t h i s e f f e c t i s i s o prenaline > adrenaline > noradrenaline; beta adrenergic blockade prevents this response (Mill e r , 1967). In v i e w of t h e s e f i n d i n g s it c a n b e c o n c l u d e d t h a t t h e i n h i b i t o r y a c t i o n s of c a t e c h o l a m i n e s on r a t u t e r u s a r e m e d i a t e d t h r o u g h b e t a a d r e n e r g i c r e c e p t o r a c t i v a t i o n ( M i l l e r , 1967). It has also been reported that this inhibitory action can be blocked by alpha adrenergic receptor b l o c k i n g a g e n t s ( R u d z i k a n d M i l l e r , 1962); h o w ever, this has not been confirmed (Levy and T o z z i , 1963). T h e p r e s e n c e o r a b s e n c e of a l p h a a d r e n e r g i c r e c e p t o r s in t h e i s o l a t e d r a t u t e r u s h a s l o n g b e e n in d i s p u t e . A h l q u i s t r e c e n t l y c o n c l u d e d t h a t
314
D.M.
the r a t u t e r u s s e e m s 'to be c o n t r o l l e d by a fl r e c e p t o r . . . All other smooth m u s c l e a d r e n e r g i c r e s p o n s e s a r e b e s t d e s c r i b e d a s being controlled by a b a l a n c e between ~ and ~ a c t i v i t y ' (Ahlquist, 1966). However, v a r i o u s w o r k e r s have r e c e n t l y r e p o r t e d that r a t u t e r i may c o n t r a c t in r e s p o n s e to n o r a d r e n a l i n e in the p r e s e n c e or a b s e n c e of beta r e c e p t o r blockade; these n o r a d r e n a l i n e - i n duced c o n t r a c t i o n s could be abolished with alpha blocking agents (Diamond and Brody, 1966a and b; Tothill, 1967). This i n v e s t i g a t i o n has supported the findings of these l a t t e r w o r k e r s . However, unlike the i n v e s t i g a t i o n r e p o r t e d by Diamond and Brody, the u t e r i we employed did not c o n t r a c t c o n s i s t e n t l y in r e s p o n s e to n o r a d r e n a l ine u n l e s s p r o p r a n o l o l was p r e s e n t . This may r e f l e c t a difference in t e m p e r a t u r e employed; 18-20°C in this study as c o m p a r e d to 37°C used by Diamond and Brody (1966a). Innes has shown that 5-HT and n o r a d r e n a l i n e act on a common r e c e p t o r in c e r t a i n t i s s u e s (e.g., cat spleen; Innes, 1962). Tothill has r e cently suggested that the two a m i n e s may also act, at l e a s t p a r t i a l l y , on a common r e c e p t o r in r a t u t e r u s since both were blocked by a n u m b e r of the s a m e a n t a g o n i s t s (Tothill, 1967). The r e s u l t s r e p o r t e d here favour the existence of sepa r a t e alpha a d r e n e r g i c and 5-HT r e c e p t o r s for the following r e a s o n s : ( a ) 5 - H T c o n t r a c t e d u t e r i which failed to r e s p o n d to n o r a d r e n a l i n e even in the p r e s e n c e of p r o p r a n o l o l and (b) DPTC blocked r e s p o n s e s to 5-HT s e l e c t i v e l y . Diamond and Brody also showed that m e t h y s e r g i d e p r e v e n t e d r e s p o n s e s to 5-HT but not to n o r a d r e n a l i n e (Diamond and Brody, 1966b). In view of t h e s e cons i d e r a t i o n s it a p p e a r s justifiable to conclude that the o e s t r o g e n i s e d rat u t e r u s contains a 5-HT r e ceptor as well as alpha excitatory and beta inhibitory a d r e n e r g i c r e c e p t o r s ; beta r e c e p t o r s u s u a l l y appear to greatly outnumber alpha r e ceptors. Responses to n o r a d r e n a l i n e , in the p r e s e n c e of propranolol, were often v a r i a b l e , slow in onset and exhibited a u t o - i n h i b i t i o n with i n c r e a s i n g c o n c e n t r a t i o n s . Consequently many p r e p a r a t i o n s could not be used for c o m p a r a t i v e studies; s i m i l a r findings have been r e p o r t e d in i n v e s t i g a t i o n s u s i n g guinea pig u t e r i (Clegg, 1966). T h e s e cons i d e r a t i o n s make it e x t r e m e l y unlikely that the d i s s o c i a t i o n constant for an a n t a g o n i s t with the alpha r e c e p t o r could be a c c u r a t e l y d e t e r m i n e d . The ability of p r o p r a n o l o l to a n t a g o n i s e r e s p o n s e s to 5-HT p r e s u m a b l y r e f l e c t s an action at r e c e p t o r level s i m i l a r to that r e c e n t l y d e s c r i b e d for the alpha r e c e p t o r (Olivares et al., 1967). 5-HT elicited c o n t r a c t i o n s in d e p o l a r i s e d
PATON
p r e p a r a t i o n s and u t i l i z e d both the ' l o o s e l y ' and 'tightly' bound Ca ++ f r a c t i o n s ; in these r e s p e c t s it is s i m i l a r to acetylcholine and oxytocin (Daniel, 1963). It is difficult to draw any f i r m conc l u s i o n s f r o m the r e s u l t s obtained u s i n g n o r a d r e n a l i n e in t h e s e a l t e r e d media. The c o n t r a c t i l e r e s p o n s e s to n o r a d r e n a l i n e d i s a p p e a r e d m o r e rapidly than those to acetylcholine and oxytocin in Ca ++ free Ringer solution while n o r a d r e n a l i n e did not contract d e p o l a r i s e d p r e p a r a t i o n s . However, high K+ and Ca ++ free solutions not only reduce the r e t e n t i o n of but also r e l e a s e n o r a d r e n a l i n e f r o m t i s s u e s t o r e s (Gillis and Paton, 1967). This would r a i s e the a m i n e c o n c e n t r a t i o n in the r e g i o n of the r e c e p t o r s r e s u l t i n g in m o r e efficient competition with p r o p r a n o l o l for tL beta r e c e p t o r . However, the u s e of higher conc e n t r a t i o n s of p r o p r a n o l o l did not enable n o r a d r e n a l i n e to c o n t r a c t u t e r i in t h e s e media. Catec h o l a m i n e s have been shown p r e v i o u s l y to r e l a x d e p o l a r i s e d r a t u t e r i although it was noted that with t i m e , this ability was p r o g r e s s i v e l y r e duced (Schild, 1966). Cocaine potentiates the r e s p o n s e s of many t i s s u e s to exogenous n o r a d r e n a l i n e and also i n hibits the n o r m a l l y r a p i d i n a c t i v a t i o n of the a m i n e by uptake into a d r e n e r g i c n e r v e t e r m i n a l s ; the 'uptake theory' of h y p e r s e n s i t i v i t y is b a s e d on the supposed link between these two events ( T r e n d e l e n b u r g , 1966). Cocaine, however, did not potentiate the i n h i b i t o r y r e s p o n s e of rat u t e r i to n o r a d r e n a l i n e (Stafford, 1963) while cocaine has been r e p o r t e d both to have no effect on (Wurtman, Axelrod and P o t t e r , 1964) and to r e duce the uptake of exogenous a m i n e by rat u t e r i (Green and M i l l e r , 1966). Cocaine failed to potentiate the excitatory r e s p o n s e to n o r a d r e n a l i n e in this study; this finding does not in any s e n s e invalidate the uptake theory s i n c e (a) the uptake of n o r a d r e n a l i n e by u t e r i from adult r a t s is not a v e r y efficient p r o c e s s (Green and M i l l e r , 1966), (b) the a d r e n e r g i c i n n e r v a t i o n of the r a t u t e r u s is e s s e n t i a l l y l i m i t e d to the v a s c u l a r supply (Owman and Sjbberg, 1966), and (c) the low t e m p e r a t u r e u s e d in these studies would reduce uptake (Gillis and Paton, 1966). Consequently the c o n c e n t r a t i o n of a m i n e n e a r m y o m e t r i a l a d r e n ergic r e c e p t o r s is unlikely to be g r e a t l y reduced by a few n e r v e t e r m i n a l s some d i s t a n c e away. Cocaine at the c o n c e n t r a t i o n s u s e d in this study, has been shown p r e v i o u s l y not to a l t e r u t e r i n e r e s p o n s e s to 5-HT (Sinha and West, 1953). The modifying influence of PGE 1 was i n v e s t i gated as a possible f u r t h e r aid to a definite identification of s e p a r a t e r e c e p t o r s for 5-HT and n o r a d r e n a l i n e ; on s e v e r a l t i s s u e s , p r o s t a g l a n -
CONTRACTILE RESPONSE OF UTERUSTO AMINES dins potentiated a number of agonists but antagonised catecholamines (Clegg, 1966). In this study, both amines were markedly potentiated and this persisted for a prolonged period. Prostaglandins have been similarly shown not to inhibit the responses of a number of other tissues to noradrenaline (Clegg, 1966). The mechanism of potentiation by PGE1 is as yet unknown.
ACKNOWLEDGEMENTS T h i s work was supported by a grant f r o m the Medical R e s e a r c h Council of Canada (MA-2472) and was c a r r i e d out during the t e n u r e of a Canadian Heart Foundation R e s e a r c h Fellowship. I should like to a c knowledge: the technical a s s i s t a n c e of M i s s Alice F r y er; the gift of p r o s t a g l a n d i n E 1 f r o m Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan; and the" e n c o u r a g e m e n t and c o n s t r u c t i v e s u g g e s t i o n s of Dr. E. E. Daniel during t h i s study.
RE F E R E N C E S Ahlquist, R . P . , 1966, The a d r e n e r g i c r e c e p t o r , J. P h a r m . Sci. 55, 359. Clegg, P . C . , 1966, The effect of p r o s t a g l a n d i n on the r e s p o n s e of isolated s m o o t h m u s c l e p r e p a r a t i o n s to s y m p a t h o m i m e t i c s u b s t a n c e s , Mere. Soc. E n d o c r i noi. 14, 119. Daniel, E . E . , 1963, On the r o l e s of c a l c i u m , s t r o n t i u m and b a r i u m in contraction and excitability of r a t u t e r i n e m u s c l e , A r c h . Int. P h a r m a c o d y n . 146, 298. Diamond, J. and T. M. Brody, 1966a, Hormonal a l t e r a tion of the r e s p o n s e of the r a t u t e r u s to c a t e c h o l a m i n e s , Life Sci. 5, 2187, Diamond, J. and T. M. Brody, 1966b, Effect of c a t e c h o l a m i n e s on s m o o t h m u s c l e motility and p h o s p h o r y l a s e activity, J. P h a r m a c o l . Exptl. T h e r a p . 152, 202. Gillis, C. N. and D. M. Paton, 1966, Effect of hypot h e r m i a and anoxia on r e t e n t i o n of n o r a d r e n a l i n e by the cat p e r f u s e d h e a r t , B r i t . J . P h a r m a c o l . 26, 426.
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Gillis, C . N . and D . M . P a t o n , 1967, Cation dependence of s y m p a t h e t i c t r a n s m i t t e r r e t e n t i o n by s l i c e s of r a t v e n t r i c l e , B r i t . J. P h a r m a c o l . 29, 309. Green, R . D . and J . W . Miller, 1966, Evidence for the active t r a n s p o r t of epinephrine and n o r e p i n e p h r i n e by the u t e r u s of the r a t , J. P h a r m a e o l . Exptl. T h e r a p . 152, 42. Innes, I . R . , 1962, The action of 5 - h y d r o x y t r y p t a m i n e on a d r e n a l i n e r e c e p t o r s , Brit. J. P h a r m a c o l . 19, 427. Levy, B. and S. Tozzi, 1963, The a d r e n e r g i c r e c e p tive m e c h a n i s m of the r a t u t e r u s , J. P h a r m a c o l . Exptl. T h e r a p . 142, 178. Miller, J . W . , 1967, A d r e n e r g i c r e c e p t o r s in the m y o m e t r i u m , Ann. N.Y. Aead. Sci. 139, 788. O l i v a r e s , G . J . , N . T . S m i t h and L . A r o n o w , 1967, Effect of propranolol on a l p h a - a d r e n e r g i c blockade in the dog and isolated rabbit aortic s t r i p , Brit. J. P h a r m a c o l . 30, 240, Owman, C. and N.O. SjSberg, 1966, A d r e n e r g i c n e r v e s in the f e m a l e genital t r a c t of the rabbit, Z. Z e l l f o r s c h . 74, 182. Rudzik, A . D . and J . W . Miller, 1962, The m e c h a n i s m of u t e r i n e inhibitory action of r e l a x i n - c o n t a i n i n g ovarian e x t r a c t s , J, P h a r m a c o l . Exptl. T h e r a p . 138, 82. Schild, H.O., 1966, Calcium and the r e l a x a n t effect of i s o p r o t e r e n o l in the depolarized rat u t e r u s , P h a r m a c o l . Rev. 18, 495. Sinha, Y . K . and G . B . W e s t , 1953, The a n t a g o n i s m b e tween local a n a e s t h e t i c d r u g s and 5 - h y d r o x y t r y p t a m i n e , J. Pharmo P h a r m a c o l . 5, 370. Stafford, A., 1963, Potentiation of s o m e c a t e c h o l a m i n e s by phenoxybenzamine, guanethidine and cocaine, Brit. J. P h a r m a c o l . 21, 361. Tothill, A., 1967, Investigation of adrenaline r e v e r s a l in the r a t u t e r u s by the induction of r e s i s t a n c e to i s o p r e n a l i n e , Brit. J. P h a r m a c o l . 29, 291. T r e n d e l e n b u r g , U., 1966, M e c h a n i s m s of s u p e r s e n s i tivity and s u b s e n s i t i v i t y to s y m p a t h o m i m e t i e a m i n e s , P h a r m a c o l . Rev. 18, 629. W u r t m a n , R . J . , J . A x e l r o d and L . T . P o t t e r , 1964, The disposition of c a t e e h o l a m i n e s in the r a t u t e r u s and the effect of d r u g s and h o r m o n e s , J. P h a r m a c o l . Exptl. T h e r a p . 144, 150.
THE CONTRACTILE RESPONSE OF THE ISOLATED RAT UTERUS TO N O R A D R E N A L I N E AND 5-HYDROXYTRYPTAMINE David M. PATON Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada Received 10 April 1968
Accepted 29 April 1968
D. M. PATON, The contractile response of the isolated rat uterus to noradrenaline and 5-hydroxytryptamine, European J. Pharmacol. 3 (1968) 310-315. Interactions of noradrenaline and 5-hydroxytryptamine with several pharmacological agents were studied on rat uterine horns at 18-20°C. Contractions in response to 5-hydroxytryptamine were antagonized by dimethylaminopropylthiocinnamanilide (DPTC) and phenoxybenzamine but not by atropine or cocaine. Noradrenaline only produced contractions consistently in the presence of propranolol; these contractions were antagonized by phenoxybenzamine but not by atropine or DPTC. Noradrenaline and 5hydroxytryptamine induced contractions were both Ca ++ dependent. Responses to both amines were markedly potentiated by short exposure to prostaglandin El . These observations favour the existence of separate excitatory receptors for 5-hydroxytryptamine and for catecholamines in the oestrogenized rat uterus. Noradrenaline 5-hydroxytryptamine Isolated rat uterus
I. INTRODUCTION
I n v e s t i g a t i o n s into the e f f e c t s of n o r a d r e n a l i n e on the r a t u t e r u s have le d to conflicting r e s u l t s ; t h e s e have b e e n r e v i e w e d r e c e n t l y ( M i l l e r , 1967). The u s u al r e s p o n s e of the r a t u t e r u s to the a m i n e is r e l a x a t i o n and inhibition of spontaneous c o n t r a c t i o n s ( M i l l e r , 1967); this effect can be p r e v e n t e d by b e t a and, a c c o r d i n g to s o m e but not all, by alpha a d r e n e r g i c r e c e p t o r blockade (Rudzik and M i l l e r , 1962; L e v y and T o z z i , 1963). Noradrena2ine m ay a l s o s t i m u l a t e the r a t u t e r u s in the p r e s e n c e or a b s e n c e of beta a d r e n e r g i c r e c e p t o r blockade (Diamond and Brody, 1966a and b; Tothill, 1967); h o w e v e r not a l l w o r k e r s have been able to r e p r o d u c e t h e s e r e s u l t s (Levy and T o z z i , 1963; M i l l e r , 1967). The i n v e s t i g a t i o n s r e p o r t e d h e r e c o n c e r n the e f fe c ts of noradrena2ine and 5 - h y d r o x y t r y p t a m i n e (5-HT) on i s o l a t e d o e s t r o g e n t r e a t e d r a t u t e r i at 1 8 - 2 0 o c ; both a m i n e s w e r e studied as they app e a r to act on c e r t a i n o r g a n s through a c o m m o n r e c e p t o r (Innes, 1962). E x p e r i m e n t s w e r e d e signed to e x a m i n e the following a s p e c t s : (a) the usual r e s p o n s e s of the r a t u t e r u s to t h e s e a m i n e s ; (b) d i f f e r e n t i a t i o n of the r e c e p t o r s on which the a m i n e s act; (c) the i n f l u e n c e of d r u g s
Alpha adrenergic receptor Beta adrenergic receptor
on r e s p o n s e s to the a m i n e s ; (d) t h e i r c a l c i u m dependence and (e) t h e i r ability to p r o d u c e cont r a c t i o n s in d e p o l a r i s e d p r e p a r a t i o n s .
2. METHOD 2.1. Method f o r reco rd i n g uterine r e s p o n s e s I m m a t u r e f e m a l e W i s t a r r a t s (80-120 g) w e r e p r e p a r e d by t r e a t m e n t f o r 6-12 days with subcutaneous i n j e c t i o n s of 50-75 ~ g / d a y of diethyls t i l b o e s t r o l . The r a t s w e r e k i l l e d by a blow on the head, the u t e r i r ap i d l y r e m o v e d and s u s pended in K r e b s R i n g e r m e d i u m , a e r a t e d with 95% O2 and 5% CO2, f o r i s o t o n i c r e c o r d i n g s . A m i n i m u m of t e n s i o n was used sufficient to b a l ance the suspended weight of the t i s s u e s e g m e n t with the l e v e r and to o v e r c o m e the f r i c t i o n of the w r i t i n g a r m with the d r u m . The k y m o g r a p h d r u m sp eed was 3.6 r a m / r a i n . Each u t e r i n e s e g m e n t was suspended v e r t i c a l l y in an individual organ bath having a v o l u m e of 10 ml. E x p e r i m e n t s w e r e conducted at 1 8 - 2 0 o c ; an advantage of the low t e m p e r a t u r e u s e d is that such p r e p a r a t i o n s show l i t t l e spontaneous act i v i t y . D r u g s w e r e washed out of the bath e i t h e r when a maxima2 r e s p o n s e had been attained or 4 m i n
C O N T R A C T I L E RESPONSE OF U T E R U S TO AMINES
after a d m i n i s t r a t i o n , whichever was the sooner. An i n t e r v a l of at l e a s t 7 m i n was allowed between s u c c e s s i v e a d m i n i s t r a t i o n of agonists to the bath, except in the case of 5-HT when 10 m i n was allowed. When the effect of v a r i o u s condit i o n s on the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l ine was being studied, p r o p r a n o l o l was added to the K r e b s solution so as to achieve a final conc e n t r a t i o n of 1 /~g/ml; only u t e r i n e h o r n s which c o n t r a c t e d in r e s p o n s e to n o r a d r e n a l i n e , in the p r e s e n c e of p r o p r a n o l o l , were u s e d in such studies. K r e b s Ringer solution of the following composition was used (mM): NaC1 115.48, KC1 4.63, CaC12 2.47, MgSO 4 1.16, Na!-ICO3 21.91, NaH2PO 4 1.16, Glucose 49.20. K + - r i c h Ringer solution was of the s a m e composition except that NaC1 115.48 (raM) was s u b s t i t u t e d with an equiva l e n t amount of KC1. 2.2. Drugs and chemicals The following drugs and c h e m i c a l s were used and the c o n c e n t r a t i o n s given a r e in t e r m s of the salt u n l e s s otherwise stated: acetylcholine chloride, / - n o r a d r e n a l i n e hydrochloride, 5 - h y d r o x y t r y p t a m i n e c r e a t i n i n e sulphate, p r o s t a g l a n d i n E 1 (PGE1), a t r o p i n e sulphate, p r o p r a n o l o l hydrochloride, phenoxybenzamine hydrochloride, p h e n t o l a m i n e m e t h a n e s u l p h o n a t e , tolazoline h y d r o c h l o r i d e , 2' 3- dim e t h y l a m i n o p r o p y l t h i o c i n n a m a n i l i d e h y d r o c h l o r i d e (DPTC), cocaine hydrochloride. The following d r u g s were p r e p a r e d as de: s c r i b e d below and kept f r o z e n u n t i l use: n o r a d r e n a l i n e , 5-HT and p r o p r a n o l o l as 1 m g / m l solutions in 0.01 M h y d r o c h l o r i c acid; PGE 1 as a 1 m g / m l solution in 95% ethanol; and phenoxyb e n z a m i n e as a 1 m g / m l solution in propylene glycol. All other d r u g s were d i s s o l v e d in d i s t i l l e d water.
3. RESULTS 3.1. Effects of noradrenaline and 5-HT in Krebs Ringer medium At 1 8 - 2 0 o c spontaneous activity was u s u a l l y a b s e n t and when p r e s e n t , c o n t r a c t i o n s were infrequent. 5-HT produced c o n t r a c t i o n s in all p r e p a r a t i o n s studied. The amplitude of m a x i m a l r e s p o n s e s to 5-HT was always as g r e a t as or g r e a t e r than those produced by acetylcholine. T h e r e was no evidence of tachyphylaxis to 5-HT when s u b m a x i m a l c o n c e n t r a t i o n s were employed at 7-10 m i n i n t e r v a l s . No p r e p a r a t i o n c o n t r a c t e d r e g u l a r l y and con-
311
s i s t e n t l y in r e s p o n s e to n o r a d r e n a l i n e , at conc e n t r a t i o n s up to 2 ~ g / m l , the u s u a l r e s p o n s e being a s m a l l r e l a x a t i o n . A n u m b e r of p r e p a r a t i o n s (9 out of 26) r e s p o n d e d to n o r a d r e n a l i n e with s m a l l c o n t r a c t i o n s ; however, these r e s p o n s e s were not well s u s t a i n e d and with either r e p e a t e d a d m i n i s t r a t i o n or i n c r e a s e d c o n c e n t r a tions of n o r a d r e n a l i n e , the u t e r u s failed to cont r a c t . T h e s e n o r a d r e n a l i n e induced c o n t r a c t i o n s were of s m a l l amplitude, being always much l e s s than those produced by m a x i m a l c o n c e n t r a t i o n s of 5-HT and acetylcholine.
TOP TRACE A
B IN PRESENCE OF PROPRM'dOLOL( I,ug/ml )
NA I0O
NA 500
NA 300
BOTTOM TRACE A
NA ICO
J_Ji NA NA 51)0 I ~
NA 2 5 0 ng/ml
B IN
F~RESO~CEOF
~ l J J g l m l )
5HT 5 I
5HT 5HT I 2 I cm
_zJ 5HT 2
5HT 5
5HT I0
5HT 20
i 5r~n I
F i g . 1. T h e e f f e c t of p r o p r a n o l o l on t h e a c t i o n s of n o r a d r e n a l i n e (NA) and 5 - h y d r o x y t r y p t a m i n e (5-HT). T r a c e s f r o m i s o t o n i c r e c o r d i n g s of i s o l a t e d r a t u t e r i n e h o r n s ; t e m p e r a t u r e 1 8 - 2 0 o c ; b a t h v o l u m e 10 m l ; s u s p e n d e d in K r e b s R i n g e r m e d i u m ; c o n c e n t r a t i o n s of a g o n i s t s in n g / m l of bath; d r u m s p e e d 3.6 m m / m i n . T o p t r a c i n g : c o n t r o l r e s p o n s e s to n o r a d r e n a l i n e (A) and r e s p o n s e s to n o r a d r e n a l i n e in p r e s e n c e of p r o p r a n o l o l , 1 # g / m l (B); B o t t o m t r a c i n g : C o n t r o l r e s p o n s e s to 5 - h y d r o x y t r y p t a m i n e (A) and r e s p o n s e s to 5-hydroxytryptamine in p r e s e n c e of p r o p r a n o l o l , 1 l / g / m l (B).
312
D.M. PATON
3.2. E f f e c t s of propranolol on r e s p o n s e s to n o r adrenaline and 5-HT P r o p r a n o l o l , 1 p g / m l , r e d u c e d r e s p o n s e s to s u b m a x i m a l c o n c e n t r a t i o n s of 5-HT in 4 u t e r i n e h o r n s ; this effect could be o v e r c o m e by i n c r e a s ing the c o n c e n t r a t i o n of 5-HT (fig. 1). A l m o s t all p r e p a r a t i o n s (61 out of 71) r e sponded with a c o n t r a c t i o n to n o r a d r e n a l i n e in the p r e s e n c e of p r o p r a n o l o l , 1 p g / m l ; five u t e r ine horns did not r e s p o n d to n o r a d r e n a l i n e even in the p r e s e n c e of p r o p r a n o l o l , 2 ~ g / m l . As the c o n c e n t r a t i o n of n o r a d r e n a l i n e was i n c r e a s e d p r o g r e s s i v e l y , the c o n t r a c t i l e r e s p o n s e u s u a l ly i n c r e a s e d in magnitude, then d e c r e a s e d and f i nally the r e s p o n s e , at high c o n c e n t r a t i o n s (1-2 p g / m l ) was u s u al l y a r e l a x a t i o n . The m a x i m a l c o n t r a c t i o n to n o r a d r e n a l i n e was u s u a ll y s m a l l e r in magnitude than t h o s e p r o d u c e d by e i t h e r a c e t y l c h o l i n e or 5-HT (fig. 1). P r e p a r a t i o n s which f a i l e d to c o n t r a c t in r e sponse to n o r a d r e n a l i n e both in the a b s e n c e and p r e s e n c e of p r o p r a n o l o l , 1 or 2 ~zg/ml, always c o n t r a c t e d when ex p o s e d to 5-HT. Such p r e p a r a tions w e r e not u s e d in f u r t h e r s t u d ie s on the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l i n e . 3.3. Effect of other antagonists on r e s p o n s e s to noradrenaline and 5-HT The effect of an t a g o n is t s on c o n t r a c t i o n s to 5-HT and n o r a d r e n a l i n e was t e s t e d in the continuous p r e s e n c e of p r o p r a n o l o l , 1 /~g/ml. A t r o pine, 0.1 to 1.0 p g / m l , a b o l i s h e d c o n t r a c t i o n s to s u p r a m a x i m a l c o n c e n t r a t i o n s of a c e t y l c h o l i n e (10 /~g/ml) but, in 10 p r e p a r a t i o n s did not a l t e r c o n t r a c t i o n s to s u b m a x i m a l c o n c e n t r a t i o n s of e i t h e r 5-HT or n o r a d r e n a l i n e . The a d m i n i s t r a tion of DP TC , 200-500 n g / m l , a b o li s h e d cont r a c t i o n s to p r e v i o u s l y s u p r a m a x i m a l c o n c e n t r a tions of 5-HT but in t h e s e 15 p r e p a r a t i o n s , the c o n t r a c t i l e r e s p o n s e s to n o r a d r e n a l i n e w e r e not r e d u c e d in amplitude (fig. 2). P h e n o x y b e n z a m i n e , 0.5 to 1 g / m l for 5 m i n , a boli sh ed c o n t r a c t i o n s to both 5-HT and n o r a d r e n a l i n e . T o l a z o l i n e could not be u s e d e f f e c t i v e l y as an antagonist as it u s u a l ly m a r k e d l y i n c r e a s e d spontaneous activity. The r e s u l t s of the u s e of phe ntolam i n e, 10-25 p g / m l , as an antagonist on 12 p r e p a r a t i o n s w e r e i n c o n c l u s i v e since the ant a g o n i s t o c c a s i o n a l l y p r o d u c e d i n c r e a s e d spontaneous activity. In s o m e p r e p a r a t i o n s it app e a r e d to m e r e l y r e t a r d the onset of the cont r a c t i l e r e s p o n s e to n o r a d r e n a l i n e while in o th e r p r e p a r a t i o n s it ab o l i s h e d or r e d u c e d the cont r a c t i l e r e s p o n s e to n o r a d r e n a l i n e . In view of this it was i m p o s s i b l e to u s e p h e n t o l a m i n e to d i s t i n g u i s h definitely b e tw e e n the c o n t r a c t i l e r e -
A
B
AC 5HI NA I0 000 I00 400 Ilcm
AC 5HI NA I0000 I00 400
G
5HI I00
D
NA 400
NA 400 nglml
j 5rain I
Fig. 2. The effect of antagonists on the actions of acetylcholine (AC), 5-hydroxytryptamine (5-HT) and noradrenaline (NA). Traces from isotonic recordings of an isolated uterine horn; other details as in fig. 1; propranolol, 1 /~g/ml, present throughout. Control responses (A); responses to agonists in presence of atropine, 1 ftg/ml (B); responses to agonists in presence of DPTC. 300 ng/ml (C); responses to agonists after exposure to phenoxybenzamine, 500 ng/ml for 5 min (D). sp o n ses to 5-HT and n o r a d r e n a l i n e . The inability of phentolamine to c o n s i s t e n t l y a n t a g o n i s e the c o n t r a c t i l e r e s p o n s e to n o r a d r e n a l i n e may have been contributed to by the known ability of p r o p r a n o l o l to i n t e r f e r e with the e s t a b l i s h m e n t of an alpha r e c e p t o r blockade ( O l i v a r e s , Smith and Aronow, 1967).
3.4. Effect of calcium on r e s p o n s e s to noradrenaline and 5-HT Both a c e t y l c h o l i n e and 5-HT continued to p r o duce s m a l l c o n t r a c t i o n s in c a l c i u m - f r e e K r e b s R i n g e r m e d i u m f o r up to 90 rain; t h e s e c o n t r a c t i o n s w e r e a b o l i s h e d when Na2 EDTA, 5 raM, was added to the c a l c i u m - f r e e m e d i u m . C o n t r a c t i o n s to n o r a d r e n a l i n e , in the continuous p r e s e n c e of p r o p r a n o l o l , 1 p g / m l , d i s a p p e a r e d r ap i d l y howe v e r on e x p o s u r e to c a l c i u m - f r e e m e d i u m (1020 min (fig. 3). 3.5. Effect of p o t a s s i u m on r e s p o n s e s to noradrenaline and 5-HT In 6 p r e p a r a t i o n s d e p o l a r i s e d by KC1-Ringer m e d i u m , s m a l l c o n t r a c t i o n s w e r e p r o d u c e d by s u p r a m a x i m a l c o n c e n t r a t i o n s of both a c e t y l c h o line and 5-HT. In the p r e s e n c e of p r o p r a n o l o l , 1 o r 2 ~zg/ml, n o r a d r e n a l i n e , in c o n c e n t r a t i o n s up to 2 ~ g / m l , f a i l e d to c o n t r a c t the d e p o l a r i s e d p r e p a r a t i o n (fig. 3); all such p r e p a r a t i o n s had
CONTRACTILE RESPONSE OF UTERUS TO AMINES
until a maximal response was obtained, usually in less than 2 min and the PGE 1 was then washed o u t of t h e b a t h . S u b s e q u e n t l y t h e r e s p o n s e s t o t h e c o n t r o l c o n c e n t r a t i o n s of 5 - H T a n d n o r a d r e n a l i n e w e r e p o t e n t i a t e d f o r p r o l o n g e d p e r i o d s (up t o 120 rain) a f t e r t h i s s i n g l e e x p o s u r e to P G E 1 . T h i s p h e n o m e n o n w a s n o t o b s e r v e d in c o n t r o l h o r n s w h i c h h a d not b e e n e x p o s e d to P G E 1 (fig. 4).
TOP TRACE KREBS
Co - FREE KREBS RINGER
RINGER
I
A
AC
NA
5HT
5HT
I0000
200
IO
IO I0
5HT IO 20
I lcm
313
B
NA AC 5HT NA 5/RTAC 2OOKX)COIOO500 IOOIOOOO ng/m! 38 50 60 68 95 105 MINUTES
I 5mini
BOTTOM TRACE RESPONSES IN
KCl - RII~GER
f NA 500 I lcm
J r
~ NA 200 11300
J
f 51"1T NA PGE 5HT NA 10 20
NA NA 5HT tOO 5000 200 ng/ml
5HT NA 5HT NA 5HT NA 5HT NA 5HT 40 50 60 70 80 90 100 I10 120 MINUTES
TIME AFTER EXPOSURETO PROSTAGLANDIN
I 5mln I
Ilcm
J 5role I
Fig. 3. The effect of ions on the actions of n o r a d r e n a l ine (NA), 5-hydroxytryptamine (5-HT) and acetylcholine (AC). T r a c e s from isotonic r e c o r d i n g s of isolated r a t uterine horns; propranolol, 1 ]~g/ml p r e s e n t throughout; other details as in fig. 1. Top tracing: control r e s p o n s e s in Krebs Ringer medium, Ca ++ 1.5 raM; r e s p o n s e s in Ca ++ free Krebs Ringer m e d i um. Bottom tracing: r e s p o n s e s in KC1 Ringer medium; this p r e p a r a t i o n had contracted in r e s p o n s e to n o r a d renaline in Krebs Ringer medium.
Fig. 4. The effect of prostaglandin E 1 (PGE) on the actions of 5-hydroxytryptamine (5-HT) and n o r a d r e n aline (NA). T r a c e s from isotonic recordings of an isolated r a t uterine horn; propranolol, 1 /ag/ml, p r e s e n t throughout; other details as in fig. 1. The following concentrations of agonists were used throughout: 5-HT, 5 n g / m l ; NA 50 n g / m l ; PGE 2.5 ~tg/ml. Control r e s p o n s e s (A); r e s p o n s e s to agonists a R e r PGE had been applied to the p r e p a r a t i o n once and then r e moved (B).
c o n t r a c t e d i n r e s p o n s e to n o r a d r e n a l i n e solution.
4. DISCUSSION
in K r e b s
3.6. E f f e c t o f cocaine on r e s p o n s e s to n o r a d r e n aline and 5 - H T C o n t r a c t i o n s t o s u b m a x i m a l c o n c e n t r a t i o n s of 5-HT were not altered by cocaine, 1 or 2 pg/ml. C o n t r a c t i o n s in r e s p o n s e t o n o r a d r e n a l i n e w e r e s t u d i e d in t h e p r e s e n c e of p r o p r a n o l o l , 1 p g / m l . No c o n s i s t e n t r e s p o n s e w a s s e e n in t h e s e e x p e r iments; some were potentiated, some inhibited and some unaltered. 3.7. E f f e c t
o f prostaglandin E 1 on r e s p o n s e to noradrenaline and 5 - H T
Control responses to submaximal t i o n s of 5 - H T a n d n o r a d r e n a l i n e w e r e t h e p r e s e n c e of p r o p r a n o l o l , 1 ~ g / m l . arations were then exposed to PGE1,
concentrao b t a i n e d in The prep2.5 ~ g / m l
Catecholamines usually relax and inhibit s p o n t a n e o u s c o n t r a c t i o n s of t h e i s o l a t e d r a t u t e r u s ; t h e o r d e r of p o t e n c y f o r t h i s e f f e c t i s i s o prenaline > adrenaline > noradrenaline; beta adrenergic blockade prevents this response (Mill e r , 1967). In v i e w of t h e s e f i n d i n g s it c a n b e c o n c l u d e d t h a t t h e i n h i b i t o r y a c t i o n s of c a t e c h o l a m i n e s on r a t u t e r u s a r e m e d i a t e d t h r o u g h b e t a a d r e n e r g i c r e c e p t o r a c t i v a t i o n ( M i l l e r , 1967). It has also been reported that this inhibitory action can be blocked by alpha adrenergic receptor b l o c k i n g a g e n t s ( R u d z i k a n d M i l l e r , 1962); h o w ever, this has not been confirmed (Levy and T o z z i , 1963). T h e p r e s e n c e o r a b s e n c e of a l p h a a d r e n e r g i c r e c e p t o r s in t h e i s o l a t e d r a t u t e r u s h a s l o n g b e e n in d i s p u t e . A h l q u i s t r e c e n t l y c o n c l u d e d t h a t
314
D.M.
the r a t u t e r u s s e e m s 'to be c o n t r o l l e d by a fl r e c e p t o r . . . All other smooth m u s c l e a d r e n e r g i c r e s p o n s e s a r e b e s t d e s c r i b e d a s being controlled by a b a l a n c e between ~ and ~ a c t i v i t y ' (Ahlquist, 1966). However, v a r i o u s w o r k e r s have r e c e n t l y r e p o r t e d that r a t u t e r i may c o n t r a c t in r e s p o n s e to n o r a d r e n a l i n e in the p r e s e n c e or a b s e n c e of beta r e c e p t o r blockade; these n o r a d r e n a l i n e - i n duced c o n t r a c t i o n s could be abolished with alpha blocking agents (Diamond and Brody, 1966a and b; Tothill, 1967). This i n v e s t i g a t i o n has supported the findings of these l a t t e r w o r k e r s . However, unlike the i n v e s t i g a t i o n r e p o r t e d by Diamond and Brody, the u t e r i we employed did not c o n t r a c t c o n s i s t e n t l y in r e s p o n s e to n o r a d r e n a l ine u n l e s s p r o p r a n o l o l was p r e s e n t . This may r e f l e c t a difference in t e m p e r a t u r e employed; 18-20°C in this study as c o m p a r e d to 37°C used by Diamond and Brody (1966a). Innes has shown that 5-HT and n o r a d r e n a l i n e act on a common r e c e p t o r in c e r t a i n t i s s u e s (e.g., cat spleen; Innes, 1962). Tothill has r e cently suggested that the two a m i n e s may also act, at l e a s t p a r t i a l l y , on a common r e c e p t o r in r a t u t e r u s since both were blocked by a n u m b e r of the s a m e a n t a g o n i s t s (Tothill, 1967). The r e s u l t s r e p o r t e d here favour the existence of sepa r a t e alpha a d r e n e r g i c and 5-HT r e c e p t o r s for the following r e a s o n s : ( a ) 5 - H T c o n t r a c t e d u t e r i which failed to r e s p o n d to n o r a d r e n a l i n e even in the p r e s e n c e of p r o p r a n o l o l and (b) DPTC blocked r e s p o n s e s to 5-HT s e l e c t i v e l y . Diamond and Brody also showed that m e t h y s e r g i d e p r e v e n t e d r e s p o n s e s to 5-HT but not to n o r a d r e n a l i n e (Diamond and Brody, 1966b). In view of t h e s e cons i d e r a t i o n s it a p p e a r s justifiable to conclude that the o e s t r o g e n i s e d rat u t e r u s contains a 5-HT r e ceptor as well as alpha excitatory and beta inhibitory a d r e n e r g i c r e c e p t o r s ; beta r e c e p t o r s u s u a l l y appear to greatly outnumber alpha r e ceptors. Responses to n o r a d r e n a l i n e , in the p r e s e n c e of propranolol, were often v a r i a b l e , slow in onset and exhibited a u t o - i n h i b i t i o n with i n c r e a s i n g c o n c e n t r a t i o n s . Consequently many p r e p a r a t i o n s could not be used for c o m p a r a t i v e studies; s i m i l a r findings have been r e p o r t e d in i n v e s t i g a t i o n s u s i n g guinea pig u t e r i (Clegg, 1966). T h e s e cons i d e r a t i o n s make it e x t r e m e l y unlikely that the d i s s o c i a t i o n constant for an a n t a g o n i s t with the alpha r e c e p t o r could be a c c u r a t e l y d e t e r m i n e d . The ability of p r o p r a n o l o l to a n t a g o n i s e r e s p o n s e s to 5-HT p r e s u m a b l y r e f l e c t s an action at r e c e p t o r level s i m i l a r to that r e c e n t l y d e s c r i b e d for the alpha r e c e p t o r (Olivares et al., 1967). 5-HT elicited c o n t r a c t i o n s in d e p o l a r i s e d
PATON
p r e p a r a t i o n s and u t i l i z e d both the ' l o o s e l y ' and 'tightly' bound Ca ++ f r a c t i o n s ; in these r e s p e c t s it is s i m i l a r to acetylcholine and oxytocin (Daniel, 1963). It is difficult to draw any f i r m conc l u s i o n s f r o m the r e s u l t s obtained u s i n g n o r a d r e n a l i n e in t h e s e a l t e r e d media. The c o n t r a c t i l e r e s p o n s e s to n o r a d r e n a l i n e d i s a p p e a r e d m o r e rapidly than those to acetylcholine and oxytocin in Ca ++ free Ringer solution while n o r a d r e n a l i n e did not contract d e p o l a r i s e d p r e p a r a t i o n s . However, high K+ and Ca ++ free solutions not only reduce the r e t e n t i o n of but also r e l e a s e n o r a d r e n a l i n e f r o m t i s s u e s t o r e s (Gillis and Paton, 1967). This would r a i s e the a m i n e c o n c e n t r a t i o n in the r e g i o n of the r e c e p t o r s r e s u l t i n g in m o r e efficient competition with p r o p r a n o l o l for tL beta r e c e p t o r . However, the u s e of higher conc e n t r a t i o n s of p r o p r a n o l o l did not enable n o r a d r e n a l i n e to c o n t r a c t u t e r i in t h e s e media. Catec h o l a m i n e s have been shown p r e v i o u s l y to r e l a x d e p o l a r i s e d r a t u t e r i although it was noted that with t i m e , this ability was p r o g r e s s i v e l y r e duced (Schild, 1966). Cocaine potentiates the r e s p o n s e s of many t i s s u e s to exogenous n o r a d r e n a l i n e and also i n hibits the n o r m a l l y r a p i d i n a c t i v a t i o n of the a m i n e by uptake into a d r e n e r g i c n e r v e t e r m i n a l s ; the 'uptake theory' of h y p e r s e n s i t i v i t y is b a s e d on the supposed link between these two events ( T r e n d e l e n b u r g , 1966). Cocaine, however, did not potentiate the i n h i b i t o r y r e s p o n s e of rat u t e r i to n o r a d r e n a l i n e (Stafford, 1963) while cocaine has been r e p o r t e d both to have no effect on (Wurtman, Axelrod and P o t t e r , 1964) and to r e duce the uptake of exogenous a m i n e by rat u t e r i (Green and M i l l e r , 1966). Cocaine failed to potentiate the excitatory r e s p o n s e to n o r a d r e n a l i n e in this study; this finding does not in any s e n s e invalidate the uptake theory s i n c e (a) the uptake of n o r a d r e n a l i n e by u t e r i from adult r a t s is not a v e r y efficient p r o c e s s (Green and M i l l e r , 1966), (b) the a d r e n e r g i c i n n e r v a t i o n of the r a t u t e r u s is e s s e n t i a l l y l i m i t e d to the v a s c u l a r supply (Owman and Sjbberg, 1966), and (c) the low t e m p e r a t u r e u s e d in these studies would reduce uptake (Gillis and Paton, 1966). Consequently the c o n c e n t r a t i o n of a m i n e n e a r m y o m e t r i a l a d r e n ergic r e c e p t o r s is unlikely to be g r e a t l y reduced by a few n e r v e t e r m i n a l s some d i s t a n c e away. Cocaine at the c o n c e n t r a t i o n s u s e d in this study, has been shown p r e v i o u s l y not to a l t e r u t e r i n e r e s p o n s e s to 5-HT (Sinha and West, 1953). The modifying influence of PGE 1 was i n v e s t i gated as a possible f u r t h e r aid to a definite identification of s e p a r a t e r e c e p t o r s for 5-HT and n o r a d r e n a l i n e ; on s e v e r a l t i s s u e s , p r o s t a g l a n -
CONTRACTILE RESPONSE OF UTERUSTO AMINES dins potentiated a number of agonists but antagonised catecholamines (Clegg, 1966). In this study, both amines were markedly potentiated and this persisted for a prolonged period. Prostaglandins have been similarly shown not to inhibit the responses of a number of other tissues to noradrenaline (Clegg, 1966). The mechanism of potentiation by PGE1 is as yet unknown.
ACKNOWLEDGEMENTS T h i s work was supported by a grant f r o m the Medical R e s e a r c h Council of Canada (MA-2472) and was c a r r i e d out during the t e n u r e of a Canadian Heart Foundation R e s e a r c h Fellowship. I should like to a c knowledge: the technical a s s i s t a n c e of M i s s Alice F r y er; the gift of p r o s t a g l a n d i n E 1 f r o m Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan; and the" e n c o u r a g e m e n t and c o n s t r u c t i v e s u g g e s t i o n s of Dr. E. E. Daniel during t h i s study.
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