PROSTAGLANDINS
DUAL ACTIONS OF PROSTACYCLIN
(PGI2) ON THE RAT PREGNANT UTERUS
K. I. Williams, K. E. H. Ei-Tahir and Ewa Marcinkiewicz*. Department of Pharmacology, School of Pharmacy & Pharmacology, University of Bath, Bath BA2 7AY, England. *Present address: Department of Pharmacology, Copernicus Medical Academy, 31-351 Krakow, Grzegorzecka 16, Poland. ABSTRACT Using strips of rat pregnant uterus, treated with indomethacin to suppress spontaneous contractility, the oxytocic activity of prostacyclin was compared with other prostaglandins. A prostacyclin concentration of 32 ng/ml elicited uterine contractions in all experiments. In this respect prostacyclin was 80 ~imesmore active than 6-oxo-PGFI~ but less active than PGE 2 or PGF2~. Apart from a direct stimulant effect, prostacyclin also exhibited an indirect potentiating action. In t h r e s h o l d concentrations prostacyclin caused a 3-fold potentiation of threshold doses of oxytocin. A lesser ].5~foldpotentiation of PGF2~ was also observed. The implications of these findings in relation to prostacyclin playing a role in parturition are discussed. INTRODUCTION A large amount of evidence has accrued which s u g g e s t S that classical prostaglandins (PGs) such as PGF2~ synthesized by the uterus may play an active role in parturition / 1 /. It is only recently that uterine tissue from pregnant_and-pseudopregnat rats was shown to synthesize 6~oxo-PGFl~ / 2,3 /. By inference these preparations had generated prostacyciin as this substance rapidly decomposes to 6-oxo-PGFld / 4 /. Direct measurements later showed that the rat pregnant uterus did produce prostacyclin and that the myometrium was the major source of this activity / 5 7. As an adjunct to this study it seemed logical to investigate the oxytocic activity of prostacyclin. In this study we have used the rat pregnant uterus (treated with indomethacin to inhibit spontaneous contraction /- 6 7 ) to compare the oxytocic activities of prostacyclin? 6-oxo-PGFl@ , PGE 2 and PGF2~. Prostacyclin was also found to be able to potentiate the effects of other oxytocic substances. METHODS Female Wistar rats were killed on day 22 of pregnancy (day delivery). The uterine horns were excised, opened and the and placentae removed. The uterine horns were transferred solution at room temperature and 5 cm lengths were cut and
MAY 1979 VOL. 17 NO. 5
of foetuses to Tyrode's then
667
PROSTAGLANDINS
m o u n t e d in 75 ml organ bath. P r e p a r a t i o n s were b a t h e d at 37°C w i t h Tyrode's solution c o n t a i n i n g i n d o m e t h a c i n 20 ~g/ml. Isotonic c o n t r a c t i o n s of p r e p a r a t i o n s were recorded under a 2 g load. A 60 m i n u t e p e r i o d was allowed for spontaneity to disappear. Drugs were then added to the b a t h for 5 m i n periods with a 5 m i n interval b e t w e e n doses. F o r each drug a sub-threshold dose was determined, subsequent doses were increased in 2-fold increments until an oxytocic response was realised° In experiments to d e t e r m i n e the degree of p o t e n t i a t i o n of agonists by prostacyclin, the uterine activity during the p e r i o d of drug exposure was integrated and the area b e n e a t h the contractions calculated. A standard curve for o x y t o c i n or PGF2~ was c o n s t r u c t e d by p l o t t i n g area b e n e a t h contractions against log dose. The integrated activity elicited by the dose of agonist (x) plus p r o s t a c y c l i n was also calculated and the corresponding dose of agonist (X) read off from the standard curve. The degree of p o t e n t i a t i o n was obtained by X : x . RESULTS The oxytocic activity of p r o s t a c y c l i n was compared w i t h several other prostaglandins; the results of one such e x p e r i m e n t are shown in F i g u r e i. For each drug the t h r e s h o l d dose was determined, double this dose was then g i v e n to note the full oxytocic effects. P r o s t a c y c l i n elicited minimal uterine a c t i v i t y at 16 ng/ml b u t at 32 hg/ml sustained rhythmic c o n t r a c t i o n s w e r e noted. 6-oxo-PGFl~ was m u c h less p o t e n t than prostacyclin, a dose 80 times larger being n e e d e d to initiate a m a r k e d uterine stimn]ation. A l t h o u g h more p o t e n t than its metabolite, p r o s t a c y c l i n was found to be less p o t e n t than PGE 2 w h i c h elicited uterine c o n t r a c t i l i t y at 0.5 ng/ml or PGF2~ at 2 ng/ml. Five experiments were c a r r i e d out in total and in every e x p e r i m e n t 32 ng/ml of p r o s t a c y c l i n was n e e d e d to induce uterine stimulation. With the other PGs consistency of effect was also noted except in one e x p e r i m e n t w i t h P G F 2 ~ where 1 ng/ml caused an oxytocic effect. Apart from investigating the d i r e c t oxytocic action of p r o s t a cyclin, its ability to p o t e n t i a t e the effects of other uterine stimulants was also i n v e s t i g a t e d (See F i g u r e 2). As in the p r e v i o u s set of experiments the dose of p r o s t a c y c l i n was found w h i c h did not elicit rhythmic c o n t r a c t i l i t y (16ng/ml). The same dose level for oxytocin was also found (50 ~U/ml). However, as shown w h e n both these m i n i m a l l y - e f f e c t i v e doses were added s i m u l t a n e o u s l y to the b a t h a m a r k e d uterine s t i m u l a t i o n was noted. By comparison of the integrated area b e n e a t h the contractions w i t h those for standard doses of oxytocin, the effect was found to be e q u i v a l e n t to 125 pU/ml of oxytocin, a 2.5 fold p o t e n t i a t i o n of response. In a total of five experiments the p o t e n t i a t i o n of o x y t o c i n by prostacyclin was found to be 3.13 ± 0.67 (mean ± SEM). In the same experiments the ability of p r o s t a c y c l i n to p o t e n t i a t e the oxytocic effects of PGF2~ were also observed. T h e p o t e n t i a t i o n 1.5 ± 0.25,
668
MAY 1979 VOL. 17 NO. 5
PROSTAGLAND~S
5rain I
I
I
I
I
16
1280
32
.J 0.25
ng/ml
2560
6-OXOFI~
I2
I
l
1
I 0,5
E2
J
[
.i
r
1
2 F2=
ng/ml PG
Fig. i. Action of PGI2, 6-oxo-PGFl~, PGE 2 and PGF2~ on the isolated rat pregnat uterus. The Tyrode's solution contained indomethacin 20 ug/ml to inhibit spontaneous contractions. Bath size 75 ml, contact time 5 minutes.
MAY 1979 VOL. 17 NO. 5
669
PROSTAGLANDINS
5 min
2.5 cm
I
I
I
I Ox 5 0
PGI 2 16
I
I Ox 5 0 -IPG 12 16
I
I Ox 100
I
I Ox 2 0 0
uU/ml ng/ml
Fig. 2. The response of the isolated rat p r e g n a n t uterus to oxyto~ cin is p o t e n t i a t e d by PGI 2 (indomethacin 20 ug/ml p r e s e n t in b a t h i n g fluid). T h r e s h o l d doses to PGI 2 (16ng/ml) and oxytocin (50~U/ml) were determined. If these 2 doses are given simultaneously the o x y t o c i n response is p o t e n t i a t e d 2.5 fold (estimated by c o m p a r i s o n of the a r e a b e n e a t h the c o n t r a c t i o n w i t h standard doses of oxytocin).
was s i g n i f i c a n t l y lower than that seen w i h t oxytocin (p
DUAL ACTIONS OF PROSTACYCLIN
(PGI2) ON THE RAT PREGNANT UTERUS
K. I. Williams, K. E. H. Ei-Tahir and Ewa Marcinkiewicz*. Department of Pharmacology, School of Pharmacy & Pharmacology, University of Bath, Bath BA2 7AY, England. *Present address: Department of Pharmacology, Copernicus Medical Academy, 31-351 Krakow, Grzegorzecka 16, Poland. ABSTRACT Using strips of rat pregnant uterus, treated with indomethacin to suppress spontaneous contractility, the oxytocic activity of prostacyclin was compared with other prostaglandins. A prostacyclin concentration of 32 ng/ml elicited uterine contractions in all experiments. In this respect prostacyclin was 80 ~imesmore active than 6-oxo-PGFI~ but less active than PGE 2 or PGF2~. Apart from a direct stimulant effect, prostacyclin also exhibited an indirect potentiating action. In t h r e s h o l d concentrations prostacyclin caused a 3-fold potentiation of threshold doses of oxytocin. A lesser ].5~foldpotentiation of PGF2~ was also observed. The implications of these findings in relation to prostacyclin playing a role in parturition are discussed. INTRODUCTION A large amount of evidence has accrued which s u g g e s t S that classical prostaglandins (PGs) such as PGF2~ synthesized by the uterus may play an active role in parturition / 1 /. It is only recently that uterine tissue from pregnant_and-pseudopregnat rats was shown to synthesize 6~oxo-PGFl~ / 2,3 /. By inference these preparations had generated prostacyciin as this substance rapidly decomposes to 6-oxo-PGFld / 4 /. Direct measurements later showed that the rat pregnant uterus did produce prostacyclin and that the myometrium was the major source of this activity / 5 7. As an adjunct to this study it seemed logical to investigate the oxytocic activity of prostacyclin. In this study we have used the rat pregnant uterus (treated with indomethacin to inhibit spontaneous contraction /- 6 7 ) to compare the oxytocic activities of prostacyclin? 6-oxo-PGFl@ , PGE 2 and PGF2~. Prostacyclin was also found to be able to potentiate the effects of other oxytocic substances. METHODS Female Wistar rats were killed on day 22 of pregnancy (day delivery). The uterine horns were excised, opened and the and placentae removed. The uterine horns were transferred solution at room temperature and 5 cm lengths were cut and
MAY 1979 VOL. 17 NO. 5
of foetuses to Tyrode's then
667
PROSTAGLANDINS
m o u n t e d in 75 ml organ bath. P r e p a r a t i o n s were b a t h e d at 37°C w i t h Tyrode's solution c o n t a i n i n g i n d o m e t h a c i n 20 ~g/ml. Isotonic c o n t r a c t i o n s of p r e p a r a t i o n s were recorded under a 2 g load. A 60 m i n u t e p e r i o d was allowed for spontaneity to disappear. Drugs were then added to the b a t h for 5 m i n periods with a 5 m i n interval b e t w e e n doses. F o r each drug a sub-threshold dose was determined, subsequent doses were increased in 2-fold increments until an oxytocic response was realised° In experiments to d e t e r m i n e the degree of p o t e n t i a t i o n of agonists by prostacyclin, the uterine activity during the p e r i o d of drug exposure was integrated and the area b e n e a t h the contractions calculated. A standard curve for o x y t o c i n or PGF2~ was c o n s t r u c t e d by p l o t t i n g area b e n e a t h contractions against log dose. The integrated activity elicited by the dose of agonist (x) plus p r o s t a c y c l i n was also calculated and the corresponding dose of agonist (X) read off from the standard curve. The degree of p o t e n t i a t i o n was obtained by X : x . RESULTS The oxytocic activity of p r o s t a c y c l i n was compared w i t h several other prostaglandins; the results of one such e x p e r i m e n t are shown in F i g u r e i. For each drug the t h r e s h o l d dose was determined, double this dose was then g i v e n to note the full oxytocic effects. P r o s t a c y c l i n elicited minimal uterine a c t i v i t y at 16 ng/ml b u t at 32 hg/ml sustained rhythmic c o n t r a c t i o n s w e r e noted. 6-oxo-PGFl~ was m u c h less p o t e n t than prostacyclin, a dose 80 times larger being n e e d e d to initiate a m a r k e d uterine stimn]ation. A l t h o u g h more p o t e n t than its metabolite, p r o s t a c y c l i n was found to be less p o t e n t than PGE 2 w h i c h elicited uterine c o n t r a c t i l i t y at 0.5 ng/ml or PGF2~ at 2 ng/ml. Five experiments were c a r r i e d out in total and in every e x p e r i m e n t 32 ng/ml of p r o s t a c y c l i n was n e e d e d to induce uterine stimulation. With the other PGs consistency of effect was also noted except in one e x p e r i m e n t w i t h P G F 2 ~ where 1 ng/ml caused an oxytocic effect. Apart from investigating the d i r e c t oxytocic action of p r o s t a cyclin, its ability to p o t e n t i a t e the effects of other uterine stimulants was also i n v e s t i g a t e d (See F i g u r e 2). As in the p r e v i o u s set of experiments the dose of p r o s t a c y c l i n was found w h i c h did not elicit rhythmic c o n t r a c t i l i t y (16ng/ml). The same dose level for oxytocin was also found (50 ~U/ml). However, as shown w h e n both these m i n i m a l l y - e f f e c t i v e doses were added s i m u l t a n e o u s l y to the b a t h a m a r k e d uterine s t i m u l a t i o n was noted. By comparison of the integrated area b e n e a t h the contractions w i t h those for standard doses of oxytocin, the effect was found to be e q u i v a l e n t to 125 pU/ml of oxytocin, a 2.5 fold p o t e n t i a t i o n of response. In a total of five experiments the p o t e n t i a t i o n of o x y t o c i n by prostacyclin was found to be 3.13 ± 0.67 (mean ± SEM). In the same experiments the ability of p r o s t a c y c l i n to p o t e n t i a t e the oxytocic effects of PGF2~ were also observed. T h e p o t e n t i a t i o n 1.5 ± 0.25,
668
MAY 1979 VOL. 17 NO. 5
PROSTAGLAND~S
5rain I
I
I
I
I
16
1280
32
.J 0.25
ng/ml
2560
6-OXOFI~
I2
I
l
1
I 0,5
E2
J
[
.i
r
1
2 F2=
ng/ml PG
Fig. i. Action of PGI2, 6-oxo-PGFl~, PGE 2 and PGF2~ on the isolated rat pregnat uterus. The Tyrode's solution contained indomethacin 20 ug/ml to inhibit spontaneous contractions. Bath size 75 ml, contact time 5 minutes.
MAY 1979 VOL. 17 NO. 5
669
PROSTAGLANDINS
5 min
2.5 cm
I
I
I
I Ox 5 0
PGI 2 16
I
I Ox 5 0 -IPG 12 16
I
I Ox 100
I
I Ox 2 0 0
uU/ml ng/ml
Fig. 2. The response of the isolated rat p r e g n a n t uterus to oxyto~ cin is p o t e n t i a t e d by PGI 2 (indomethacin 20 ug/ml p r e s e n t in b a t h i n g fluid). T h r e s h o l d doses to PGI 2 (16ng/ml) and oxytocin (50~U/ml) were determined. If these 2 doses are given simultaneously the o x y t o c i n response is p o t e n t i a t e d 2.5 fold (estimated by c o m p a r i s o n of the a r e a b e n e a t h the c o n t r a c t i o n w i t h standard doses of oxytocin).
was s i g n i f i c a n t l y lower than that seen w i h t oxytocin (p
