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The effect of autonomic and psychotropic drugs on adrenaline reversal in the rat uterus
European Journal o f Pharmacology, 32 ( 1 9 7 5 ) 2 2 1 - - 2 2 6 © N o r t h - H o l l a n d Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
THE E F F E C T OF AUTONOMIC AND PSYCHOTROPIC D R U G S ON A D R E N A L I N E R E V E R S A L IN THE RAT UTERUS ANNE TOTHILL
Department o f Pharmacology and Therapeutics, The Middlesex Hospital Medical School, Cleveland Street, London, W1, and The Institute o f Obstetrics and Gynaecology, Hammersmith Hospital, Du Cane Road, London W12 OHS, England Received 2 D e c e m b e r 1 9 7 4 , revised MS received 7 March 1975, a c c e p t e d 13 March 1975
A. T O T H I L L , The effect o f autonomic and psychotropic drugs on adrenaline reversal in the rat uterus, E u r o p e a n J. P h a r m a c o l . 32 ( 1 9 7 5 ) 2 2 1 - - 2 2 6 . D e s i p r a m i n e a n d o t h e r p s y c h o t r o p i c a n d a u t o n o m i c drugs were tested for t h e i r ability to p r e v e n t t h e m o t o r r e s p o n s e of t h e r a t u t e r u s to a d r e n a l i n e in vitro. T h o s e agents which have b e e n s h o w n to p r e v e n t c a t e c h o l a m i n e u p t a k e i n t o s t o r e s m o s t readily reversed t h e m o t o r r e s p o n s e ; t h o s e w h i c h h a d little or n o effect o n catechola m i n e storage were usually w i t h o u t e f f e c t o n t h e m o t o r r e s p o n s e to a d r e n a l i n e . T h e m o t o r r e s p o n s e of the rat u t e r u s to a d r e n a l i n e is due to p r o s t a g l a n d i n E2. Agents b l o c k i n g t h e m o t o r r e s p o n s e p r e v e n t e d t h e release of p r o s t a g l a n d i n a n d did n o t b l o c k t h e a c t i o n of p r o s t a g l a n d i n o n t h e uterus. Since all the effective agents also i n t e r f e r e d with u p t a k e with c a t e c h o l a m i n e stores it seems likely t h a t passage of c a t e c h o l a m i n e in and o u t of storage d e p o t s is involved w i t h t h e s y n t h e s i s o f p r o s t a g l a n d i n s .
Motor response to adrenaline
Prostaglandin
1. Introduction Noradrenaline, adrenaline and isoprenaline inhibit the spontaneous contractions of the isolated rat uterus at all stages of the oestrous cycle (Tothill, 1967a). With the uterus from an oestrogen-dominated or late pregnant rat, during inhibition produced by any of these agents, the addition of adrenaline results in rhythmic contractions (Tothill, 1967a). It was suggested (Tothill, 1967b) that during catecholamine inhibition a substance accumulated which was released by addition of adrenaline and caused this motor response. Further work has shown the substance to be prostaglandin E2 (Tothill et al., 1971). The m o t o r response to adrenaline was prevented by a number of agents which block the actions of adrenaline on a-receptors (Tothill, 1967a). Phenoxybenzamine in low doses was
Rat uterus
P s y c h o t r o p i c drugs
always effective in this respect, but phentolamine only prevented the motor effect in much higher doses, which on their own caused contraction of the preparation. Of the ergot alkaloids which are known to block a-receptors, ergotamine was completely ineffective and dihydroergotamine only marginally active, whilst a mixture of dihydroergotamine, dihydroergocryptine and dihydroergocristine completely blocked the motor response to adrenaline. The activities of antagonists did n o t appear to parallel their a-blocking activities and the possibility of another mode of action was considered. None of the agents which blocked the motor response are known to prevent the action of released prostaglandin on the uterus (Horton, 1969) but most of them inhibited the uptake of catecholamines into rat heart tissue stores (Iversen, 1967). In the present work the efficacy
222 of other agents known to block uptake into these catecholamine stores in preventing the motor response of the rat uterus to adrenaline was investigated to see whether these two actions are always associated, or whether the evidence would suggest that there are other sites of action.
2. Materials and methods 2.1. General The experiments were carried out on 365 Sprague--Dawley female albino rats weighing a b o u t 200 g. The following groups were used: (1) Induced oestrus, prepared according to Tothill (1967a); (2) Natural dioestrus, the functional state of the animals was assessed with vaginal smears taken on the day of the experiment; (3) Pregnant, the rats were mated and the finding of a vaginal plus was designated as day 1. Animals were used on days 20--22. In the case of day 22, they were used before parturition behaviour began. Strips of pregnant or horns of non-pregnant uterus were set up as previously described (Tothill, 1967a,b). Phenol red (17 mg/litre) was added to the medium so that continuous observation of pH was possible. 2.2. Detection o f prostaglandin E2 released by adrenaline Tothill (1967a) found that a motor response to adrenaline could be obtained from strips of uteri from animals in late pregnancy and horns from animals in induced oestrus, but n o t from horns from animals in natural dioestrus. The motor response was shown to be due to release of prostaglandin E2 (Tothill et al., 1971). In addition it was found that, although not releasing prostaglandin E2 in response to adrenaline, the dioestrous horn responsed to added prostaglandin E2 (Tothill et al., 1971). A dioestrous horn may therefore be used to test for prostaglandin E2 in bath fluid which contains adrenaline, and is as sat-
A. TOTHILL isfactory a test system as the rat fundal strip. The strip of uterus to be tested and a dioestrous horn were set up in separate organ baths. Isoprenaline (100 ng/ml) was added to both preparations and 3 min later, without washing, adrenaline (100 ng/ml). After 7 min the fluid was drained from both baths and that from the strip to be tested was transferred to the bath containing the dioestrous horn. In these circumstances, the production of a series of rhythmical contractions by this horn indicated the release of prostaglandin E 2 from the test horn. 2. 3. Drugs The drugs which were used fall into the following groups. Catecholamines: adrenaline acid tartrate; isoprenaline sulphate. Monoamine oxidase inhibitors: iproniazid sulphate (Marsilid, Roche Products Limited); nialamide (Niamid, Pfizer Limited); phenelzine sulphate (Nardil, Wander Laboratories); tranylcypromine sulphate (Parnate, Smith, Kline and French Laboratories Limited). Dibenzazepine derivatives: imipramine hydrochloride (Geigy U.K. Limited); desimipramine hydrochloride (Geigy U.K. Limited). Phenothiazine derivatives: chlorpromazine hydrochloride (Largactil, May and Baker Limited); promazine hydrochloride (Sparine, Wyeth, John and Brother Limited). fi-Adrenergic blocking agents: pronethalol hydrochloride (Alderlin, Imperial Chemical Industries Limited, Pharmaceutical Division). Catecholamine depleting hypotensives: guanethedine sulphate (Burroughs Wellcome & Co.); bethanidine sulphate (Burroughs Wellcome & Co.). Others: diethylstilboestrol. 3. Results 3.1. Prevention o f the motor response to adrenaline by drugs affecting catecholamine store mechanisms 3.1.1. Induced oestrus When isoprenaline (100 ng/ml) was added
DRUGS AND ADRENALINE REVERSAL
to a spontaneously contracting uterine horn from a rat in induced oestrus, inhibition of spontaneous contractions occurred immediately and the preparation was still quiescent after about 10 min. Adrenaline (100 ng/ml) was then added and a typical motor effect was observed (fig. la). Having obtained a satisfactory motor response to adrenaline the preparation was washed and allowed to recover. The experi-
0
•
•
•
b
•
•
•
•
Fig. 1. Induced oestrous uterus. (a) 1st marker, isoprenaline, 100 ng/ml; 2nd marker, adrenaline, 100 ng/ml added without washing the preparation; 3rd marker, wash. (b) 1st marker, desipramine, 100 ng/ ml; 2rid marker, isoprenaline, 100 ng/ml; 3rd marker, adrenaline, 100 ng/ml and 4th marker, wash. Time marker 5 rain.
223
ment was repeated in the presence of a blocking agent using the same uterine horn. In fig. l b desipramine (100 ng/ml) was used as the blocking agent. As before, inhibition took place with isoprenaline (100 ng/ml) and adrenaline (100 ng/ml) was added after the same time interval as in the first experiment. No motor effect was now obtained although the preparation was observed for a further 10 min (fig. lb). After washing out the bath, spontaneous activity recovered immediately. Desipramine has a long lasting action and it was not possible to wash out its effects completely and demonstrate recovery of the m o t o r response in all preparations after the use of this drug, but recovery was demonstrated whenever possible. The effects of this and other blocking agents tested in this way for activity in preventing the motor response of the uterus from rats in induced oestrus to adrenaline are shown in table 1. They are compared with the activity of the same agents in blocking uptake of noradrenaline into stores in the rat heart, as described by Iversen (1967). In general the drugs which were most effective in preventing the motor response showed the greatest activity in uptake blockade.
3.1.2. Late pregnancy (20--22 days) Similar experiments were performed with uterine strips obtained from animals in late pregnancy. Since the motor response is most readily and rapidly obtained in this state adrenaline (100 ng/ml) was added after the preparation had been in contact with isoprenaline for only 3 min; otherwise the procedure was the same. The same agents used in induced oestrus preparations (table 1} were tested for their ability to prevent the motor response to adrenaline in the late pregnant rat uterus. The results were similar to those shown in table 1 except that approximately 10 times the dose of each drug was required to produce complete block of the motor response. The higher dose needed could be accounted for since prostaglandins are in greater abundance in late pregnancy (Tothill, unpublished observation).
224
A. TOTHILL
TABLE 1 Agent
Dose (pg/ml)
Motor effect*
Extent of blocking of catecholamine uptake into stores (heart)** • % (ug/ml)
Phenoxybenzamine Dihydroergotamine Bromolysergic acid Desipramine Hydergine
0.1"** 0.1"** 0.1--0.5"** 0.1--1.0 0.1--0.5"**
5/6 2/5 5/6 6/6 4/5
91.5 (10) --81--92.5 (0.1--1) --
Chlorpromazine Tranylcypromine Imipramine Promazine Guanethedine Bethanidine Ergotamine
1.0 1.0 1.0 1.0 1.0 1.0 1.0"**
5/5 5/5 5/5 5/5 2/5 2/5 0/5
88.5 (10) 88 (10) 75 ( 1 )
10.0"** 10.0
5/6 4/4
66 60
2/5 0/5 0/5
69 (10) 3.5 (10) nil (10)
Phentolamine Pronethalol Phenelzine Nialamide Iproniazid
100.0 100.0 100.0
78.5 (20)
(10) (50)
* Numerator (number of uteri not giving motor effect): denominator (number of uteri tested). ** Iversen, 1967. *** Tothill, 1967a.
3.2. Mode o f action o f catecholamine uptake blocking drugs in preventing the motor response to adrenaline In previous w o r k (Tothill et al., 1 9 7 1 ) , it has been s h o w n t h a t prostaglandin E~ is released in association with the m o t o r response o f the rat u t e r u s to adrenaline. Desipramine and o t h e r agents which p r e v e n t the m o t o r response were e x a m i n e d to see if t h e y prev e n t e d the release o f prostaglandin f r o m the tissue (see Materials and m e t h o d s ) or antagonized t h e e f f e c t o f released prostaglandin o n t h e uterus. Since t h e u t e r u s f r o m an animal in the d i o e s t r o u s state does n o t release prostaglandin in response to adrenaline or isoprenaline s t i m u l a t i o n , this p r e p a r a t i o n was used for tests f o r t h e presence o f prostaglandin. The e x p e r i m e n t was carried o u t with desip r a m i n e as the blocking agent. A strip o f u t e r u s f r o m a p r e g n a n t animal was set u p in
an organ b a t h and t r e a t e d with isoprenaline ( 1 0 0 ng/ml) f o l l o w e d b y adrenaline ( 1 0 0 ng/ml). T w o m i n u t e s later the b a t h fluid was t e s t e d o n a d i o e s t r o u s u t e r i n e h o r n which had b e e n t r e a t e d with desipramine (100 ng/ml) f o l l o w e d b y isoprenaline and t h e n adrenaline. A m o t o r e f f e c t o c c u r r e d (fig. 2a). This result i n d i c a t e d t h a t prostaglandin released f r o m the p r e g n a n t strip was n o t p r e v e n t e d f r o m acting b y desipramine. In a f u r t h e r e x p e r i m e n t the p r e g n a n t strip was t r e a t e d with desipramine and t h e n witho u t washing, isoprenaline ( 1 0 0 ng/ml) and t h e n adrenaline ( 1 0 0 ng/ml) were added. 2 min later the c o n t e n t s o f this b a t h were added to one containing a dioestrous horn which had b e e n t r e a t e d with isoprenaline ( 1 0 0 n g / m l ) and adrenaline ( 1 0 0 ng/ml), b u t n o desipramine. No m o t o r e f f e c t o c c u r r e d (fig. 2b). It can be c o n c l u d e d t h a t desipramine prevents t h e f o r m a t i o n or release of
DRUGS AND ADRENALINE REVERSAL
225
aline. It therefore appears that all the agents tested which block the m o t o r response to adrenaline do so by preventing the release or synthesis of prostaglandin rather than by antagonising its effects.
4. Discussion
•
• I
• ,I
Fig. 2. Dioestrous uterus. (a) 1st marker, desipramine, 100 ng/ml; 2nd marker, isoprenaline, 100 ng/ml; 3rd marker, bath fluid substituted from that bathing a pregnant horn which had been treated similarly; 4th marker, wash. The arrow indicates a space of 35 rain. (b) Control to show that a motor response cannot be elicited from a dioestrous horn first by addition of adrenaline. 1st marker, isoprenaline, 100 ng/ml; 2rid marker, adrenaline, 100 ng/ml; 3rd marker, wash.
prostaglandin b u t n o t its action after it has been released. These experiments were repeated with all the blocking agents used in this present study. Results similar to those obtained with desipramine were demonstrated for all the agents which prevented the m o t o r response to adren-
The motor response of the rat uterus to adrenaline has been shown to be prevented by a variety of agents. These drugs differ widely in structure and appear to have little in common. When all their properties were compared it was found that, on the whole, those which were highly effective in preventing uptake of catecholamines into stores were also effective in preventing the motor response, whereas those which had little or no effect on catecholamine uptake were also ineffective in blocking the motor response. Those agents which block the motor response to adrenaline prevent the release of prostaglandin E2 and do not antagonize its action when it is released. Ramwell and Shaw (1971) have shown that in production of endogenous prostaglandin by stimulation of the tissue by drugs or other means, more prostaglandin is released than can initially be extracted. It follows that their conditions of prostaglandin release involve not only release of any stores that may be present but also stimulation of prostaglandin synthesis. The synthesis of prostaglandin appears to involve five distinct steps {Lands et al., 1971) during which cyclization and insertion of atoms of oxygen take place. The drugs which have been found to block the motor response to adrenaline in the present work may be interfering with the synthesis of prostaglandin E2 b y acting as antioxidants. Kunze and Vogt (1971} have shown that activation of phospholipase A or the elimination of barriers preventing its access to phospholipids stimulate the formation of prostaglandins, and the motor response blockers may interfere with these mechanisms. On the other hand, the degree of parallelism found between the activities of all these chemically divergent drugs in blocking
226
the motor response to adrenaline with their activities in interfering with uptake into catecholamine stores makes it seem possible that the passage of catecholamines in and out of storage depots may be linked with the synthesis of prostaglandins.
References Horton, E.W., 1969, Hypotheses on physiological roles of prostaglandins, Physiol. Rev. 49,122. Iversen, L.L., 1967, The Uptake and Storage of Noradrenaline (Cambridge University Press).
A. TOTHILL Kunze, H. and W. Vogt, 1971, Significance of phospholipase A, Ann. N.Y. Acad. Sci. 180, 123. Lands, W., R. Lee and W. Smith, 1971, Factors regulating the biosynthesis of various prostaglandins, Ann. N.Y. Acad. Sci. 180, 107. Ramwell, P. and J. Shaw, 1971, The biological significance of the prostaglandins, Ann. N.Y. Acad. Sci. 180, 10. Tothill, A.U., 1967a, Investigation of adrenaline reversal in the rat uterus by induction of resistance to isoprenaline, Brit. J. Pharmacol. 29, 291. Tothill, A.U., 1967b, Motor effect of adrenaline on the rat uterus, Nature (London) 213, 1230. Tothill, A.U., L. Rathbone and E. Willman, 1971, Relation between prostaglandin E2 and adrenaline reversal in the rat uterus, Nature (London) 233, 56.
THE E F F E C T OF AUTONOMIC AND PSYCHOTROPIC D R U G S ON A D R E N A L I N E R E V E R S A L IN THE RAT UTERUS ANNE TOTHILL
Department o f Pharmacology and Therapeutics, The Middlesex Hospital Medical School, Cleveland Street, London, W1, and The Institute o f Obstetrics and Gynaecology, Hammersmith Hospital, Du Cane Road, London W12 OHS, England Received 2 D e c e m b e r 1 9 7 4 , revised MS received 7 March 1975, a c c e p t e d 13 March 1975
A. T O T H I L L , The effect o f autonomic and psychotropic drugs on adrenaline reversal in the rat uterus, E u r o p e a n J. P h a r m a c o l . 32 ( 1 9 7 5 ) 2 2 1 - - 2 2 6 . D e s i p r a m i n e a n d o t h e r p s y c h o t r o p i c a n d a u t o n o m i c drugs were tested for t h e i r ability to p r e v e n t t h e m o t o r r e s p o n s e of t h e r a t u t e r u s to a d r e n a l i n e in vitro. T h o s e agents which have b e e n s h o w n to p r e v e n t c a t e c h o l a m i n e u p t a k e i n t o s t o r e s m o s t readily reversed t h e m o t o r r e s p o n s e ; t h o s e w h i c h h a d little or n o effect o n catechola m i n e storage were usually w i t h o u t e f f e c t o n t h e m o t o r r e s p o n s e to a d r e n a l i n e . T h e m o t o r r e s p o n s e of the rat u t e r u s to a d r e n a l i n e is due to p r o s t a g l a n d i n E2. Agents b l o c k i n g t h e m o t o r r e s p o n s e p r e v e n t e d t h e release of p r o s t a g l a n d i n a n d did n o t b l o c k t h e a c t i o n of p r o s t a g l a n d i n o n t h e uterus. Since all the effective agents also i n t e r f e r e d with u p t a k e with c a t e c h o l a m i n e stores it seems likely t h a t passage of c a t e c h o l a m i n e in and o u t of storage d e p o t s is involved w i t h t h e s y n t h e s i s o f p r o s t a g l a n d i n s .
Motor response to adrenaline
Prostaglandin
1. Introduction Noradrenaline, adrenaline and isoprenaline inhibit the spontaneous contractions of the isolated rat uterus at all stages of the oestrous cycle (Tothill, 1967a). With the uterus from an oestrogen-dominated or late pregnant rat, during inhibition produced by any of these agents, the addition of adrenaline results in rhythmic contractions (Tothill, 1967a). It was suggested (Tothill, 1967b) that during catecholamine inhibition a substance accumulated which was released by addition of adrenaline and caused this motor response. Further work has shown the substance to be prostaglandin E2 (Tothill et al., 1971). The m o t o r response to adrenaline was prevented by a number of agents which block the actions of adrenaline on a-receptors (Tothill, 1967a). Phenoxybenzamine in low doses was
Rat uterus
P s y c h o t r o p i c drugs
always effective in this respect, but phentolamine only prevented the motor effect in much higher doses, which on their own caused contraction of the preparation. Of the ergot alkaloids which are known to block a-receptors, ergotamine was completely ineffective and dihydroergotamine only marginally active, whilst a mixture of dihydroergotamine, dihydroergocryptine and dihydroergocristine completely blocked the motor response to adrenaline. The activities of antagonists did n o t appear to parallel their a-blocking activities and the possibility of another mode of action was considered. None of the agents which blocked the motor response are known to prevent the action of released prostaglandin on the uterus (Horton, 1969) but most of them inhibited the uptake of catecholamines into rat heart tissue stores (Iversen, 1967). In the present work the efficacy
222 of other agents known to block uptake into these catecholamine stores in preventing the motor response of the rat uterus to adrenaline was investigated to see whether these two actions are always associated, or whether the evidence would suggest that there are other sites of action.
2. Materials and methods 2.1. General The experiments were carried out on 365 Sprague--Dawley female albino rats weighing a b o u t 200 g. The following groups were used: (1) Induced oestrus, prepared according to Tothill (1967a); (2) Natural dioestrus, the functional state of the animals was assessed with vaginal smears taken on the day of the experiment; (3) Pregnant, the rats were mated and the finding of a vaginal plus was designated as day 1. Animals were used on days 20--22. In the case of day 22, they were used before parturition behaviour began. Strips of pregnant or horns of non-pregnant uterus were set up as previously described (Tothill, 1967a,b). Phenol red (17 mg/litre) was added to the medium so that continuous observation of pH was possible. 2.2. Detection o f prostaglandin E2 released by adrenaline Tothill (1967a) found that a motor response to adrenaline could be obtained from strips of uteri from animals in late pregnancy and horns from animals in induced oestrus, but n o t from horns from animals in natural dioestrus. The motor response was shown to be due to release of prostaglandin E2 (Tothill et al., 1971). In addition it was found that, although not releasing prostaglandin E2 in response to adrenaline, the dioestrous horn responsed to added prostaglandin E2 (Tothill et al., 1971). A dioestrous horn may therefore be used to test for prostaglandin E2 in bath fluid which contains adrenaline, and is as sat-
A. TOTHILL isfactory a test system as the rat fundal strip. The strip of uterus to be tested and a dioestrous horn were set up in separate organ baths. Isoprenaline (100 ng/ml) was added to both preparations and 3 min later, without washing, adrenaline (100 ng/ml). After 7 min the fluid was drained from both baths and that from the strip to be tested was transferred to the bath containing the dioestrous horn. In these circumstances, the production of a series of rhythmical contractions by this horn indicated the release of prostaglandin E 2 from the test horn. 2. 3. Drugs The drugs which were used fall into the following groups. Catecholamines: adrenaline acid tartrate; isoprenaline sulphate. Monoamine oxidase inhibitors: iproniazid sulphate (Marsilid, Roche Products Limited); nialamide (Niamid, Pfizer Limited); phenelzine sulphate (Nardil, Wander Laboratories); tranylcypromine sulphate (Parnate, Smith, Kline and French Laboratories Limited). Dibenzazepine derivatives: imipramine hydrochloride (Geigy U.K. Limited); desimipramine hydrochloride (Geigy U.K. Limited). Phenothiazine derivatives: chlorpromazine hydrochloride (Largactil, May and Baker Limited); promazine hydrochloride (Sparine, Wyeth, John and Brother Limited). fi-Adrenergic blocking agents: pronethalol hydrochloride (Alderlin, Imperial Chemical Industries Limited, Pharmaceutical Division). Catecholamine depleting hypotensives: guanethedine sulphate (Burroughs Wellcome & Co.); bethanidine sulphate (Burroughs Wellcome & Co.). Others: diethylstilboestrol. 3. Results 3.1. Prevention o f the motor response to adrenaline by drugs affecting catecholamine store mechanisms 3.1.1. Induced oestrus When isoprenaline (100 ng/ml) was added
DRUGS AND ADRENALINE REVERSAL
to a spontaneously contracting uterine horn from a rat in induced oestrus, inhibition of spontaneous contractions occurred immediately and the preparation was still quiescent after about 10 min. Adrenaline (100 ng/ml) was then added and a typical motor effect was observed (fig. la). Having obtained a satisfactory motor response to adrenaline the preparation was washed and allowed to recover. The experi-
0
•
•
•
b
•
•
•
•
Fig. 1. Induced oestrous uterus. (a) 1st marker, isoprenaline, 100 ng/ml; 2nd marker, adrenaline, 100 ng/ml added without washing the preparation; 3rd marker, wash. (b) 1st marker, desipramine, 100 ng/ ml; 2rid marker, isoprenaline, 100 ng/ml; 3rd marker, adrenaline, 100 ng/ml and 4th marker, wash. Time marker 5 rain.
223
ment was repeated in the presence of a blocking agent using the same uterine horn. In fig. l b desipramine (100 ng/ml) was used as the blocking agent. As before, inhibition took place with isoprenaline (100 ng/ml) and adrenaline (100 ng/ml) was added after the same time interval as in the first experiment. No motor effect was now obtained although the preparation was observed for a further 10 min (fig. lb). After washing out the bath, spontaneous activity recovered immediately. Desipramine has a long lasting action and it was not possible to wash out its effects completely and demonstrate recovery of the m o t o r response in all preparations after the use of this drug, but recovery was demonstrated whenever possible. The effects of this and other blocking agents tested in this way for activity in preventing the motor response of the uterus from rats in induced oestrus to adrenaline are shown in table 1. They are compared with the activity of the same agents in blocking uptake of noradrenaline into stores in the rat heart, as described by Iversen (1967). In general the drugs which were most effective in preventing the motor response showed the greatest activity in uptake blockade.
3.1.2. Late pregnancy (20--22 days) Similar experiments were performed with uterine strips obtained from animals in late pregnancy. Since the motor response is most readily and rapidly obtained in this state adrenaline (100 ng/ml) was added after the preparation had been in contact with isoprenaline for only 3 min; otherwise the procedure was the same. The same agents used in induced oestrus preparations (table 1} were tested for their ability to prevent the motor response to adrenaline in the late pregnant rat uterus. The results were similar to those shown in table 1 except that approximately 10 times the dose of each drug was required to produce complete block of the motor response. The higher dose needed could be accounted for since prostaglandins are in greater abundance in late pregnancy (Tothill, unpublished observation).
224
A. TOTHILL
TABLE 1 Agent
Dose (pg/ml)
Motor effect*
Extent of blocking of catecholamine uptake into stores (heart)** • % (ug/ml)
Phenoxybenzamine Dihydroergotamine Bromolysergic acid Desipramine Hydergine
0.1"** 0.1"** 0.1--0.5"** 0.1--1.0 0.1--0.5"**
5/6 2/5 5/6 6/6 4/5
91.5 (10) --81--92.5 (0.1--1) --
Chlorpromazine Tranylcypromine Imipramine Promazine Guanethedine Bethanidine Ergotamine
1.0 1.0 1.0 1.0 1.0 1.0 1.0"**
5/5 5/5 5/5 5/5 2/5 2/5 0/5
88.5 (10) 88 (10) 75 ( 1 )
10.0"** 10.0
5/6 4/4
66 60
2/5 0/5 0/5
69 (10) 3.5 (10) nil (10)
Phentolamine Pronethalol Phenelzine Nialamide Iproniazid
100.0 100.0 100.0
78.5 (20)
(10) (50)
* Numerator (number of uteri not giving motor effect): denominator (number of uteri tested). ** Iversen, 1967. *** Tothill, 1967a.
3.2. Mode o f action o f catecholamine uptake blocking drugs in preventing the motor response to adrenaline In previous w o r k (Tothill et al., 1 9 7 1 ) , it has been s h o w n t h a t prostaglandin E~ is released in association with the m o t o r response o f the rat u t e r u s to adrenaline. Desipramine and o t h e r agents which p r e v e n t the m o t o r response were e x a m i n e d to see if t h e y prev e n t e d the release o f prostaglandin f r o m the tissue (see Materials and m e t h o d s ) or antagonized t h e e f f e c t o f released prostaglandin o n t h e uterus. Since t h e u t e r u s f r o m an animal in the d i o e s t r o u s state does n o t release prostaglandin in response to adrenaline or isoprenaline s t i m u l a t i o n , this p r e p a r a t i o n was used for tests f o r t h e presence o f prostaglandin. The e x p e r i m e n t was carried o u t with desip r a m i n e as the blocking agent. A strip o f u t e r u s f r o m a p r e g n a n t animal was set u p in
an organ b a t h and t r e a t e d with isoprenaline ( 1 0 0 ng/ml) f o l l o w e d b y adrenaline ( 1 0 0 ng/ml). T w o m i n u t e s later the b a t h fluid was t e s t e d o n a d i o e s t r o u s u t e r i n e h o r n which had b e e n t r e a t e d with desipramine (100 ng/ml) f o l l o w e d b y isoprenaline and t h e n adrenaline. A m o t o r e f f e c t o c c u r r e d (fig. 2a). This result i n d i c a t e d t h a t prostaglandin released f r o m the p r e g n a n t strip was n o t p r e v e n t e d f r o m acting b y desipramine. In a f u r t h e r e x p e r i m e n t the p r e g n a n t strip was t r e a t e d with desipramine and t h e n witho u t washing, isoprenaline ( 1 0 0 ng/ml) and t h e n adrenaline ( 1 0 0 ng/ml) were added. 2 min later the c o n t e n t s o f this b a t h were added to one containing a dioestrous horn which had b e e n t r e a t e d with isoprenaline ( 1 0 0 n g / m l ) and adrenaline ( 1 0 0 ng/ml), b u t n o desipramine. No m o t o r e f f e c t o c c u r r e d (fig. 2b). It can be c o n c l u d e d t h a t desipramine prevents t h e f o r m a t i o n or release of
DRUGS AND ADRENALINE REVERSAL
225
aline. It therefore appears that all the agents tested which block the m o t o r response to adrenaline do so by preventing the release or synthesis of prostaglandin rather than by antagonising its effects.
4. Discussion
•
• I
• ,I
Fig. 2. Dioestrous uterus. (a) 1st marker, desipramine, 100 ng/ml; 2nd marker, isoprenaline, 100 ng/ml; 3rd marker, bath fluid substituted from that bathing a pregnant horn which had been treated similarly; 4th marker, wash. The arrow indicates a space of 35 rain. (b) Control to show that a motor response cannot be elicited from a dioestrous horn first by addition of adrenaline. 1st marker, isoprenaline, 100 ng/ml; 2rid marker, adrenaline, 100 ng/ml; 3rd marker, wash.
prostaglandin b u t n o t its action after it has been released. These experiments were repeated with all the blocking agents used in this present study. Results similar to those obtained with desipramine were demonstrated for all the agents which prevented the m o t o r response to adren-
The motor response of the rat uterus to adrenaline has been shown to be prevented by a variety of agents. These drugs differ widely in structure and appear to have little in common. When all their properties were compared it was found that, on the whole, those which were highly effective in preventing uptake of catecholamines into stores were also effective in preventing the motor response, whereas those which had little or no effect on catecholamine uptake were also ineffective in blocking the motor response. Those agents which block the motor response to adrenaline prevent the release of prostaglandin E2 and do not antagonize its action when it is released. Ramwell and Shaw (1971) have shown that in production of endogenous prostaglandin by stimulation of the tissue by drugs or other means, more prostaglandin is released than can initially be extracted. It follows that their conditions of prostaglandin release involve not only release of any stores that may be present but also stimulation of prostaglandin synthesis. The synthesis of prostaglandin appears to involve five distinct steps {Lands et al., 1971) during which cyclization and insertion of atoms of oxygen take place. The drugs which have been found to block the motor response to adrenaline in the present work may be interfering with the synthesis of prostaglandin E2 b y acting as antioxidants. Kunze and Vogt (1971} have shown that activation of phospholipase A or the elimination of barriers preventing its access to phospholipids stimulate the formation of prostaglandins, and the motor response blockers may interfere with these mechanisms. On the other hand, the degree of parallelism found between the activities of all these chemically divergent drugs in blocking
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the motor response to adrenaline with their activities in interfering with uptake into catecholamine stores makes it seem possible that the passage of catecholamines in and out of storage depots may be linked with the synthesis of prostaglandins.
References Horton, E.W., 1969, Hypotheses on physiological roles of prostaglandins, Physiol. Rev. 49,122. Iversen, L.L., 1967, The Uptake and Storage of Noradrenaline (Cambridge University Press).
A. TOTHILL Kunze, H. and W. Vogt, 1971, Significance of phospholipase A, Ann. N.Y. Acad. Sci. 180, 123. Lands, W., R. Lee and W. Smith, 1971, Factors regulating the biosynthesis of various prostaglandins, Ann. N.Y. Acad. Sci. 180, 107. Ramwell, P. and J. Shaw, 1971, The biological significance of the prostaglandins, Ann. N.Y. Acad. Sci. 180, 10. Tothill, A.U., 1967a, Investigation of adrenaline reversal in the rat uterus by induction of resistance to isoprenaline, Brit. J. Pharmacol. 29, 291. Tothill, A.U., 1967b, Motor effect of adrenaline on the rat uterus, Nature (London) 213, 1230. Tothill, A.U., L. Rathbone and E. Willman, 1971, Relation between prostaglandin E2 and adrenaline reversal in the rat uterus, Nature (London) 233, 56.