PROSTAGLANDINS
PROSTACYCLIN ( PGI2) IN PREGNANT BUMAN UTERUS. Omini C., Folco G.C., PasargiklianR?, Fano M. and Berti F. Institute of Pharmacology and Pharmacognosy,School of Pharmacy, University of Milan, 20129 Gilan, Italy. *II- Clinic of Obstetric and Gynecology, School of Medicine, University of Milan, 20100 Milan, Italy. ABSTRACT Prostacyclin lowers the tonus and reduces the spontaneousmotility of isolated pregnant humanmyometrium. This effect seems to be related to cyclic-AMP accumulation,since PG12 increase's the formation of this cyclic nucleotide in incubatedminces of pregnant and non-pregnant uterus. The ability of this tissue to generate a labile substancewhich inhibits platelets aggregation,has been demost'ratedand discusse'd. INTRODUCTION In a previous paper (l), we reported that prostacyclin (PG12.) inhibits spontaneousmotility of iso'latednormal human myometrium and specificallyreduces the spasmus of this tissue induced by prostaglandinFti(PGF2&). A possible implication arising from these pharmacologicalobservationsis that PGI could play a role in 2. ins,'provided that modulating the oxitocic activity of prost'aglan generation of prostacyclin could take place in myometrium during pregnancy. Recently Williams et al. (2) were able to demostrate that PG12 formation in rat uterus increases considerablyat the end of gesti tion, suggesting thus 'an'endocrine-like"function of uterus in maintaining adequate blood supply for the developing foetus. In order to provide more substantial and less'speculativeevi dence on the physiological importance of myometrial prostacyclin, we have studied the ability of this tissue to generate this substance also in connectionwith its 'relaxingactivity. METHODS Effect of PGI, and 6-oxo-PGFldonisolated human uterus.'Specimens of pregnant and non-pregnant uterus, were obtained from multiparas (25-35 years old) in connection with surgery, hysterectomy for prolapsus and during cesarean section for delivery.
JANUARY 1979VOL. 17 NO. 1
113
PROSTAGLANDINS
Longitudinal myometrial strips (pregnant)were superfusedwith oxigenated Tyrode solution at 37'C. and at the flowing rate of 10 ml/min. as previously described (1). Prostacyclin and its metabolite 6-oxo-PGFWwere given at different concentrationafter allowing the tissue a suitable period of equilibration. Generation of prostacyclin-likematerial from pregnant and nonpregnant human uterus "in vrtro". Pieces 'ofpregnant and non pregnant myometrium were incubated for a period of 2 and 15 minutes in Ca++-free Krebs solution (pH 8,O) to give 20 mg of tissue in 0.1 ml of medium. The supernatant of the incubationmixture, (O,l0.2 ml) was directly assayed for prostacyclin-likeactivity. Rat stomach strips (RSS), circular fibres of rabbit stomach RbSS), and strips of rabbit coeliac artery (RbCA)weresuperfused in cascade with Krebs solution (37") at a flow rate of 10 ml /min. according to Moncada et al. (31. The bioassay tissues were bathed with a mixture of antagonists according to Gilmore (4), and in order to prevent the endogenous proetaglandinsgeneration, indomethacin (10 pg/ml) was also added.
*
Platelet aggregation.Aggregation was studied using rabbit washedplatelets '(RbWP)following the method described by Hamberg et al.(S) Irreversible aggregationwas induced using thrombin (2-4 U,I,/ ml.), and monitored using a Born aggregometer. Aliquots (0.1 ml ) of the supernatant from the reaction mixture mentioned above, were added to RbWP I min. prior to the proaggregazoryagent. When antiaggregatory activity was detected, the incubation sample was allowed to stand for 15 min. at room temperature in order to cause complete degradation of PG12 and its effect on aggregation retested. In other experimentsBrachidonic acid (5 ng /ml ) and PGG2 (1 pg ,/ml) were added to the reaction mixture in order to increase prostacyclin like activity. Cyclic-AMP determination.Thecapacity of PG12 to increase levels of cyclic-adenosine-3'-5'monophosphate (CAMP),was studied "in vitro" in pregnant and non-pregnanthuman uterus. Ihe minced tissue was incu bated in Tyrode solution (37') for 5 min.. Cyclic-AMP, purified according to Mao and Guidotti (6), was measured following the method described by Gilman (7), using a commerciallyavailable kit from the RadiochemicalCenter Amersham. RRSULTS PGI and 6-oxo-PGFbAon isolated human pregnant uterus. Superfusion ofrsolated human pregnant uterus with prostacyclin Induces a decrease of the frequency of the spontaneous contractionsand lowers the basal tonus. This effect is dose dependent and it is already evident at the
114
JANUARY 1979 VOL. 17 NO. 1
PROSTAGLANDINS
concentrationof 2 ng / ml of PGI2. In those preparationswhere the spontaneousmotility was absent, the relaxing activity of prostacyclinwas still present. Even the stable metabolite of PG12, 6-0x0-PGF ,causes similar effects but the dose required is at least lo-$0 times higher (Fig. 1). As previously observed in non pregnant uterus (l), this relaxing effect is not abolished by /J-adrenergicblockade. The tocolitic effect of prostacyclin is tentatively explained by an increase formation of cyclic-AMP in the myometrial cells. In fact PG12, as shown in table NO.1, is able to induce accumulation of the cyclic nucleotide in both pregnant and non pregnant uterus.
Cyclic-AMP pmol/mg prot. PG12 Non pregnant-uterus
Pregnant uterus
0.63 + 0.15
1.72 _+0.21
1O-5 M
1.66 ,+0.09
4.03 + 0.38
1O-4 M
3.25 _+0.19
4.97 2 0.34
Table No. 1 - Prostacyclin increases cyclic-AMP formation in incubated (5 min.) minces of human mycmetrial tissue. The figures represent the mean values of at least 6 experiments f S.E..
Prostacyclin-likeactivity generation. The injection of 0,2 ml of myometrial incubation fluid on the bioassay system causes a profile of activity which is similar to that obtained with PGE2 (Fig. No 2). In these experimental conditions the bank of tissue in cascade does not allow to evidentiate prostacyclin-likeactivity. This could be due to a concomitant formation of PGE2 which masks the biological
JANUARY
1979VOL.17NO.l
115
PROSTAGLANDINS
I
PGF2,
PGl2
20 ng/mr
100 ng/ml
PGE,
PGi2
2 ng/ml
100 ng/ml
6-oxo-PGF,,
400
ng/ml
Fig. No. 1 - Prostacyclin (PGIz) and its metabolite (6-oxo-PGF$ reduce spontaneousmotility and tonus of isolated human pregnant uterus,
116
JANUARY 1979 VOL. 17 NO. 1
PROSTAGLANDINS
T 3 cm
PGE2 25ng
PGlz 1OOng
HU 2’
HU 15
HU+PGG~ 2’
HU+ PGG2 15’
PGG, 200ng
Fig. NO.2 - Bioassay of incubationmedia from pregnant human uterus (HU) Aliquots (0.2 ml] of incubate were injected over the tissues in cascade. In some samples PGG2 (1 pg/ml) was added to the reaction mixture. The responses of incubation medium were compared with that of PGE2,PG12 and PGG2.
JANUARY 1979VOL. 17 NO. 1
117
PROSTAGLANDINS
activity
of PGI2. Furthermore the addition of prostaglandin endoperoxide PGG2 with the aim to evidentiate the increased to the reaction mixture, production of prostacyclin from the tissue, was unsuccesful . However the simultaneous assay for anti-aggregatory activity gave better results. Aliquote of 0.1 ml of the incubation medium exert anti-aggr2 gatory activity which is increased when arachidonic acid was added obtained in to the reaction mixture (Fig. NO. 3). From the results different experiments we observed that 0.1 ml of fluid display an anti-aggregatory activity approximately similar to that of 5 ng of of the samples for 2 or 15 min. non detectable PGI . After incubation activity was monitored, The inhibition dif$ erence in antiaggregatory of thrombin-induced aggregation was almost absent after retesting the samples 15 min. later.
Thrombin
2 UI 1’
7
-
i
Pig.
118
Control
No 3 - Anti-aggregatory activity generated by 0.1 ml of super natant from 200 mg pregnant human uterus incubated in 1 ml of calcium-free solution pH 8 for 2 min, at 37OC.
JANUARY
1979 VOL. 17 NO. 1
PROSTAGLANDINS
DISCUSSION The results reported above clearlydemonstratethe ability of pregnant and non-pregnant human myometrium to generate a labile substance which inhibits thrombin-inducedplatelet aggregation. The properties exibited by this substance are suggestive of it being prostacyclin. Similar resuits were obtained by Myatt and Elder (8) on normal human placenta, and the importance of these fiz dings has been enphasized particularly in relation to the maintenance of the circulation of the foetal-placentaunit. Our proposal that also the human uterus could represent another source of prostacyclin-likematerial can bear some physio logical importance, expecially considering the pharmacological activity of exogenous PG12 and its metabolite on isolated strips of pregnant uterus. In fact, the relaxing activity exerted mainly by PGI on this organ, seems to indicate that prostacyclin formation may deiermine the type of motility of the myometrium and modulate the stimulatory activity of prostaglandinsand other hormones during pregnancy. The lowering of uterine tonus and contractilityinduced by PG12 could be explained by an increased accumulation in myometrial fibers of c&R', This hypothesis is strongly favoured by our results using minces of human-myometrium,As far as the mechanism of action is concerned, it is likely that PG12 stimulates adenyl-cyclaseactivity as already shown in platelets by Gorman et al. (9); however a cyclicAMP phosphodiesteraseblockade by PG12 in human uterus, cannot be ruled out, ACRNOWLEDGEXENTS We would like to thank Dr. J.R. Vane (WellcomeResearch Laboratory, England) for generous gift of PGIz and 6-oxo-PGFl* and Dr. L. Campio (The Upjohn Company, Italy) for prostaglandins. Skilled technical assistence of Mr. Giuseppe Brunelli and Mr. Giuseppe Rossoni is gratefully acknowledged. REFERENCES
1) Omini C., Pasargiklian R., Folco G.C., Fano M. and Berti F. Pharmacologicalactivity of PG12 and its metabolite 6-oxo-PGFld, on human uterus and fallopian tubes. ProstaglandinsIs: 1045, 1978
21 Williams R.I., Dembinska-KiecA,, Zmuda A, and Gryglewski R.J. Prostacyclin formation by myometrial and decidual fractions of the pregnant rat uterus. Prostaglandins-15: 343, 1978
JANUARY 1979VOL. 17 NO. 1
119
PROSTAGLANDINS
3) Moncada S,, Mugridge R,G., and Whittle B.J,R, The differentialresponse of a novel bioassay tissue, the rabbit transverse stomach-stripto prostacyclin (PGI2) and other prostaglandins. Br. J. Pharmacol.63: 451,1977 4) Gilmore N., Vane J.R. and Wyllie J,H, Prostaglandinsrelease'dby the spleen, Nature 218 :1135,1968 5) Hamberg M., Svensson J., Wakabayashi I., Samuelsson B, Isolation and structure of two prost'aglandin endoperoxides that cause'plateletaggregation, Proc. Nat. Acad. Sci. USA71:345,1974 6) Mao C.C. and Guidotti A, Simultaneousisolation of adenosine 3'-St-cyclicmonophosphate (CAMP) and guanosine 3'-5'-cvclic monophosphate (cGMP) in small tissue samples. Anal, Biochem, 2: 63.1974 7) Gilman A. A protein binding assay for adenosine 3'-5'-cyclicmonophosphate. Proc. Nat, Acad. Sci. USAE: 305, 1970 8) Myatt. L., Elder M.G, Inhibition of platelet aggregation by a placental substancewith prostacyclin-likeactivity. Nature 268: 159, 1977 9) Gorman R.R., Bunting S. and Miller 0,V. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins2: 377, 1977
Z&proved g/13/78
120
JANUARY
1979 VOL. 17 NO. 1
PROSTACYCLIN ( PGI2) IN PREGNANT BUMAN UTERUS. Omini C., Folco G.C., PasargiklianR?, Fano M. and Berti F. Institute of Pharmacology and Pharmacognosy,School of Pharmacy, University of Milan, 20129 Gilan, Italy. *II- Clinic of Obstetric and Gynecology, School of Medicine, University of Milan, 20100 Milan, Italy. ABSTRACT Prostacyclin lowers the tonus and reduces the spontaneousmotility of isolated pregnant humanmyometrium. This effect seems to be related to cyclic-AMP accumulation,since PG12 increase's the formation of this cyclic nucleotide in incubatedminces of pregnant and non-pregnant uterus. The ability of this tissue to generate a labile substancewhich inhibits platelets aggregation,has been demost'ratedand discusse'd. INTRODUCTION In a previous paper (l), we reported that prostacyclin (PG12.) inhibits spontaneousmotility of iso'latednormal human myometrium and specificallyreduces the spasmus of this tissue induced by prostaglandinFti(PGF2&). A possible implication arising from these pharmacologicalobservationsis that PGI could play a role in 2. ins,'provided that modulating the oxitocic activity of prost'aglan generation of prostacyclin could take place in myometrium during pregnancy. Recently Williams et al. (2) were able to demostrate that PG12 formation in rat uterus increases considerablyat the end of gesti tion, suggesting thus 'an'endocrine-like"function of uterus in maintaining adequate blood supply for the developing foetus. In order to provide more substantial and less'speculativeevi dence on the physiological importance of myometrial prostacyclin, we have studied the ability of this tissue to generate this substance also in connectionwith its 'relaxingactivity. METHODS Effect of PGI, and 6-oxo-PGFldonisolated human uterus.'Specimens of pregnant and non-pregnant uterus, were obtained from multiparas (25-35 years old) in connection with surgery, hysterectomy for prolapsus and during cesarean section for delivery.
JANUARY 1979VOL. 17 NO. 1
113
PROSTAGLANDINS
Longitudinal myometrial strips (pregnant)were superfusedwith oxigenated Tyrode solution at 37'C. and at the flowing rate of 10 ml/min. as previously described (1). Prostacyclin and its metabolite 6-oxo-PGFWwere given at different concentrationafter allowing the tissue a suitable period of equilibration. Generation of prostacyclin-likematerial from pregnant and nonpregnant human uterus "in vrtro". Pieces 'ofpregnant and non pregnant myometrium were incubated for a period of 2 and 15 minutes in Ca++-free Krebs solution (pH 8,O) to give 20 mg of tissue in 0.1 ml of medium. The supernatant of the incubationmixture, (O,l0.2 ml) was directly assayed for prostacyclin-likeactivity. Rat stomach strips (RSS), circular fibres of rabbit stomach RbSS), and strips of rabbit coeliac artery (RbCA)weresuperfused in cascade with Krebs solution (37") at a flow rate of 10 ml /min. according to Moncada et al. (31. The bioassay tissues were bathed with a mixture of antagonists according to Gilmore (4), and in order to prevent the endogenous proetaglandinsgeneration, indomethacin (10 pg/ml) was also added.
*
Platelet aggregation.Aggregation was studied using rabbit washedplatelets '(RbWP)following the method described by Hamberg et al.(S) Irreversible aggregationwas induced using thrombin (2-4 U,I,/ ml.), and monitored using a Born aggregometer. Aliquots (0.1 ml ) of the supernatant from the reaction mixture mentioned above, were added to RbWP I min. prior to the proaggregazoryagent. When antiaggregatory activity was detected, the incubation sample was allowed to stand for 15 min. at room temperature in order to cause complete degradation of PG12 and its effect on aggregation retested. In other experimentsBrachidonic acid (5 ng /ml ) and PGG2 (1 pg ,/ml) were added to the reaction mixture in order to increase prostacyclin like activity. Cyclic-AMP determination.Thecapacity of PG12 to increase levels of cyclic-adenosine-3'-5'monophosphate (CAMP),was studied "in vitro" in pregnant and non-pregnanthuman uterus. Ihe minced tissue was incu bated in Tyrode solution (37') for 5 min.. Cyclic-AMP, purified according to Mao and Guidotti (6), was measured following the method described by Gilman (7), using a commerciallyavailable kit from the RadiochemicalCenter Amersham. RRSULTS PGI and 6-oxo-PGFbAon isolated human pregnant uterus. Superfusion ofrsolated human pregnant uterus with prostacyclin Induces a decrease of the frequency of the spontaneous contractionsand lowers the basal tonus. This effect is dose dependent and it is already evident at the
114
JANUARY 1979 VOL. 17 NO. 1
PROSTAGLANDINS
concentrationof 2 ng / ml of PGI2. In those preparationswhere the spontaneousmotility was absent, the relaxing activity of prostacyclinwas still present. Even the stable metabolite of PG12, 6-0x0-PGF ,causes similar effects but the dose required is at least lo-$0 times higher (Fig. 1). As previously observed in non pregnant uterus (l), this relaxing effect is not abolished by /J-adrenergicblockade. The tocolitic effect of prostacyclin is tentatively explained by an increase formation of cyclic-AMP in the myometrial cells. In fact PG12, as shown in table NO.1, is able to induce accumulation of the cyclic nucleotide in both pregnant and non pregnant uterus.
Cyclic-AMP pmol/mg prot. PG12 Non pregnant-uterus
Pregnant uterus
0.63 + 0.15
1.72 _+0.21
1O-5 M
1.66 ,+0.09
4.03 + 0.38
1O-4 M
3.25 _+0.19
4.97 2 0.34
Table No. 1 - Prostacyclin increases cyclic-AMP formation in incubated (5 min.) minces of human mycmetrial tissue. The figures represent the mean values of at least 6 experiments f S.E..
Prostacyclin-likeactivity generation. The injection of 0,2 ml of myometrial incubation fluid on the bioassay system causes a profile of activity which is similar to that obtained with PGE2 (Fig. No 2). In these experimental conditions the bank of tissue in cascade does not allow to evidentiate prostacyclin-likeactivity. This could be due to a concomitant formation of PGE2 which masks the biological
JANUARY
1979VOL.17NO.l
115
PROSTAGLANDINS
I
PGF2,
PGl2
20 ng/mr
100 ng/ml
PGE,
PGi2
2 ng/ml
100 ng/ml
6-oxo-PGF,,
400
ng/ml
Fig. No. 1 - Prostacyclin (PGIz) and its metabolite (6-oxo-PGF$ reduce spontaneousmotility and tonus of isolated human pregnant uterus,
116
JANUARY 1979 VOL. 17 NO. 1
PROSTAGLANDINS
T 3 cm
PGE2 25ng
PGlz 1OOng
HU 2’
HU 15
HU+PGG~ 2’
HU+ PGG2 15’
PGG, 200ng
Fig. NO.2 - Bioassay of incubationmedia from pregnant human uterus (HU) Aliquots (0.2 ml] of incubate were injected over the tissues in cascade. In some samples PGG2 (1 pg/ml) was added to the reaction mixture. The responses of incubation medium were compared with that of PGE2,PG12 and PGG2.
JANUARY 1979VOL. 17 NO. 1
117
PROSTAGLANDINS
activity
of PGI2. Furthermore the addition of prostaglandin endoperoxide PGG2 with the aim to evidentiate the increased to the reaction mixture, production of prostacyclin from the tissue, was unsuccesful . However the simultaneous assay for anti-aggregatory activity gave better results. Aliquote of 0.1 ml of the incubation medium exert anti-aggr2 gatory activity which is increased when arachidonic acid was added obtained in to the reaction mixture (Fig. NO. 3). From the results different experiments we observed that 0.1 ml of fluid display an anti-aggregatory activity approximately similar to that of 5 ng of of the samples for 2 or 15 min. non detectable PGI . After incubation activity was monitored, The inhibition dif$ erence in antiaggregatory of thrombin-induced aggregation was almost absent after retesting the samples 15 min. later.
Thrombin
2 UI 1’
7
-
i
Pig.
118
Control
No 3 - Anti-aggregatory activity generated by 0.1 ml of super natant from 200 mg pregnant human uterus incubated in 1 ml of calcium-free solution pH 8 for 2 min, at 37OC.
JANUARY
1979 VOL. 17 NO. 1
PROSTAGLANDINS
DISCUSSION The results reported above clearlydemonstratethe ability of pregnant and non-pregnant human myometrium to generate a labile substance which inhibits thrombin-inducedplatelet aggregation. The properties exibited by this substance are suggestive of it being prostacyclin. Similar resuits were obtained by Myatt and Elder (8) on normal human placenta, and the importance of these fiz dings has been enphasized particularly in relation to the maintenance of the circulation of the foetal-placentaunit. Our proposal that also the human uterus could represent another source of prostacyclin-likematerial can bear some physio logical importance, expecially considering the pharmacological activity of exogenous PG12 and its metabolite on isolated strips of pregnant uterus. In fact, the relaxing activity exerted mainly by PGI on this organ, seems to indicate that prostacyclin formation may deiermine the type of motility of the myometrium and modulate the stimulatory activity of prostaglandinsand other hormones during pregnancy. The lowering of uterine tonus and contractilityinduced by PG12 could be explained by an increased accumulation in myometrial fibers of c&R', This hypothesis is strongly favoured by our results using minces of human-myometrium,As far as the mechanism of action is concerned, it is likely that PG12 stimulates adenyl-cyclaseactivity as already shown in platelets by Gorman et al. (9); however a cyclicAMP phosphodiesteraseblockade by PG12 in human uterus, cannot be ruled out, ACRNOWLEDGEXENTS We would like to thank Dr. J.R. Vane (WellcomeResearch Laboratory, England) for generous gift of PGIz and 6-oxo-PGFl* and Dr. L. Campio (The Upjohn Company, Italy) for prostaglandins. Skilled technical assistence of Mr. Giuseppe Brunelli and Mr. Giuseppe Rossoni is gratefully acknowledged. REFERENCES
1) Omini C., Pasargiklian R., Folco G.C., Fano M. and Berti F. Pharmacologicalactivity of PG12 and its metabolite 6-oxo-PGFld, on human uterus and fallopian tubes. ProstaglandinsIs: 1045, 1978
21 Williams R.I., Dembinska-KiecA,, Zmuda A, and Gryglewski R.J. Prostacyclin formation by myometrial and decidual fractions of the pregnant rat uterus. Prostaglandins-15: 343, 1978
JANUARY 1979VOL. 17 NO. 1
119
PROSTAGLANDINS
3) Moncada S,, Mugridge R,G., and Whittle B.J,R, The differentialresponse of a novel bioassay tissue, the rabbit transverse stomach-stripto prostacyclin (PGI2) and other prostaglandins. Br. J. Pharmacol.63: 451,1977 4) Gilmore N., Vane J.R. and Wyllie J,H, Prostaglandinsrelease'dby the spleen, Nature 218 :1135,1968 5) Hamberg M., Svensson J., Wakabayashi I., Samuelsson B, Isolation and structure of two prost'aglandin endoperoxides that cause'plateletaggregation, Proc. Nat. Acad. Sci. USA71:345,1974 6) Mao C.C. and Guidotti A, Simultaneousisolation of adenosine 3'-St-cyclicmonophosphate (CAMP) and guanosine 3'-5'-cvclic monophosphate (cGMP) in small tissue samples. Anal, Biochem, 2: 63.1974 7) Gilman A. A protein binding assay for adenosine 3'-5'-cyclicmonophosphate. Proc. Nat, Acad. Sci. USAE: 305, 1970 8) Myatt. L., Elder M.G, Inhibition of platelet aggregation by a placental substancewith prostacyclin-likeactivity. Nature 268: 159, 1977 9) Gorman R.R., Bunting S. and Miller 0,V. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins2: 377, 1977
Z&proved g/13/78
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JANUARY
1979 VOL. 17 NO. 1
