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Prostaglandins, not cyclic GMP, inhibit oxytocinase isoenzymes from human amniotic fluid in vitro
PROSTAGLANDINS
PROSTAGLANDINS, NOT CYCLIC GMP, INHIBIT OXYTOCINASE ISOENZYMES FROM HUMAN AMNIOTIC FLUID IN VITRO Ashim C Roy, Michael Yeang, Suan Mian Tan, Sri R Kottegoda and Shan S Ratnam Department of Obstetrics and Gynaecology, National University of Singapore, Kandang Kerbau Hospital, Singapore 0821 ABSTRACT
Two isoenzymes of oxytocinase (EC 3.4.11.3) a c t i v i t y were fractionated from human amniotic f l u i d samples between the 14th and 22nd weeks of gestation by Ultrogel acrylamide-agarose gel f i l t r a t i o n and p a r t i a l l y characterized. The isoenzymes were competitively inhibited by PGEz, PGE2 and PGF2mmore at pH 6.2 than at pH 6.8, whereas cyclic GMP (cGMP) and its 8-bromo derivative had no effect at either pH. The implications of these findings are discussed and i t is suggested that since the a c t i v i t y of amniotic f l u i d oxytocinases is very low or minimal at or near term, inhibition of these by prostaglandins may not have physiological significance in the i n i t i a t i o n of human parturition. INTRODUCTION Using S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) separately as substrates, we have recently demonstrated oxytocinase (EC 3.4.11.3) a c t i v i t y in the human amniotic f l u i d and observed an inverse relationship between the enzyme a c t i v i t y and gestational age (1,2). With both substrates, the enzyme a c t i v i t y , which was highest early in pregnancy, decreased exponentially to a minimum near or a few weeks before term. We have suggested that the amniotic f l u i d oxytocinase a c t i v i t y may play a significant role in pregnancy and labour. Oxytocinase is known to exist in multimolecular forms in the human serum, placenta and uterus, and we have shown a pH-dependent selective i n h i b i t i o n of these oxytocinases by prostaglandins (PGs) and cyclic GMP (cGMP) with maximum inhibition at pH 6.2 and l i t t l e or no i n h i b i t i o n at pH 7.4 (3-10). From these and related studies (13-15), i t appears that part of the oxytocic effect of PGs might be a t t r i b u t ed to their a b i l i t y to i n h i b i t oxytocinase a c t i v i t y and thereby protecting endogenous oxytocin. EndogenouscGMPmay also be involved in parturition partly by i n h i b i t i n g oxytocinase a c t i v i t y . AUGUST 1985 VOL. 30 NO. 2
255
PROSTAGLANDINS
We now present the results of our investigation on the separation, from the amniotic fluid, of multimolecular forms of oxytocinase by Ultrogel chromatography, their characterization and inhibition by PGs and cGMP. MATERIALS AND METHODS Sources of materials, preparation of solutions, assay of oxytocinase a c t i v i t y , and determination of protein and molecular weight were as stated elsewhere (10). Amniotic fluid samples obtained from normal pregnant women of 14 to 22 weeks gestation by amniocentesis (1,2) were centrifuged for 10 minutes at 1500 g and 4°C, pooled, chromatographed on acrylamide-agarose Ultrogel AcA 22 and stored at -20°C until used as described elsewhere (8,10). All calculations and statistical analyses were carried out with the aid of Magicalc (Artsci, Inc.) on a 64K Apple II Europlus microcomputer (9). An Applesoft Basic program (Kinetics Plotter and Graphics Dump) written by us enabled rapid, graphical determination of inhibition type. RESULTS Ultrogel f i l t r a t i o n patterns of the pooled amniotic fluid samples in relation to oxytocinase activity and protein concentration with (a) and without (b) 0.3% Triton X-IO0 are shown in Fig. 1. Gel f i l t r a t i o n parameters have already been described (8). Sodium azide was not used as a preservative in the gel chromatography because i t inhibits oxytocinase activity (6,8,10). Two hydrolytic peaks with elution volumes of 55 ml (void volume) and 130 ml were obtained in the absence of Triton X-IO0. Using either substrate peak I > peak I I , but in each case the hydrolysis of LN was greater than that of BCN. In the presence of Triton X-IO0, only one hydrolytic peak with elution volume of 110 ml was obtained. This may suggest that the isoenzyme eluted in the void volume was bound to cell particles or was in polymeric form. Unlike serum, placental and uterine oxytocinases (8,10), Triton X-IO0 had no significant effect on the hydrolysis of BCN and LN by the amniotic fluid enzymes at the concentration used. The molecular weight of peak I isoenzyme could not be determined because i t eluted in the void volume, but of peak II was 160,000. Their respective pH optima in 0.1M phosphate buffer were 6.8 and 6.6 with BCN as substrate, and 7.2 and 7.4 with LN. Only EDTA and Cu2+ ions inhibited the amniotic fluid isoenzymes (Table 1). EGTA, Ca 2+, Co 2+, Fe 2+, Mg2+, Mn2+, Ni 2+ and Zn2+ ions at comparable concentrations had no e f f e c t . However, i n h i b i t i o n of the isoenzymes by 50 ~M EDTA was reversed by equimolar concentrations of Cu2+, Co2+, Mn2+ and Zn 2+ ions. At higher concentrations (500-1000 uM), Co2+
256
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PROSTAGLANDINS
1.5 1.0 0.5 Z
o
~o
0
z
o
AFI
i
L
I
i
i
i
i
>~ 1.5 iii 1,0
0.5 0 i
50 100 150 200 ELUTION VOLUME (rnl)
250
Fig. i: Ultrogel AcA 22 elution patterns of amniotic fluid with (a) and without (b) 0.3% Triton X-IO0. The hydrolysis of 500 pM Sbenzyl-L-cysteine-p-nitroanilide (BCN) or L-leucine-pnitroanilide (LN) by the eluates was assayed at 37°C and pH 7.0 for 120 or 60 minutes respectively as described previously (5). Assay mixtures contained 0.05% bovine serum albumin (BSA). Open circles, solid triangles and dotted lines represent BCN and LN hydrolysis (545 nm) and protein concentration (280 nm) respectively.
Table I: Inhibition of the activity of amniotic fluid ox~ocinases by EDTA and Cu-- ions. IC50, Inhibitor
BCN
LN
AFI
AFII
AFI
AFII
EDTA
8.0 ± 1.0
5.8 ±2.4
8.2 ± 1.6
6.6 ±1.3
Cu2+
242 ± 29
308 ± 76
232 ± 25
542 ± 48
Hydrolysis of 500 pM S-benzyl-L-cysteine-p-nitroanillde (BCN) or L-leucinep-nitroanilide (LN) by the oxytocinase isoenzymes with or without chelating agents (i h preincubatlon) or divalent metal ions (without preincubation) was measured at 37°C for 5-60 min in TrisHCI (0.I M, pH 7.0) buffer as described elsewhere (5). The results are the mean (±SD) of at least three separate experiments.
AUGUST 1985 VOL. 30 NO. 2
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PROSTAGLANDINS
retained the restored a c t i v i t y , tory.
but Cu2+, Mn2+ and Zn2+ were i n h i b i -
Results of kinetic studies of the oxytocinase peaks and their i n h i b i tion by prostaglandins at pH 6.2 and pH 6.8 are given in Tables 2 and 3, respectively. The pH-independent (P>O.05) Km and Vmax values of the hydrolytic peaks were s i g n i f i c a n t l y greater with LN than BCN as substrate. PGEI, PGE2 and PGF2mcompetitively inhibited both isoenzymes at either pH without significant difference among their i n h i b i tory potencies using either substrate, but the i n h i b i t i o n was greater at pH 6.2 than at pH 6.8 (P
258
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PROSTAGLANDINS
Table 2: Kinetics of amniotlc fluid (AF) oxytocinases.
BCN pH 6.2 AFI Km ± Vmax
118 18
0.41 ±0.05
pH 6.8
AFII
AFI
124 24
±
LN
±
0.16 ±0.01
258 74
0.91 ±0.24
pH 6.2
pH 6.8
AFII
AFI
AFII
251 62
608 ± 112
657 ± 106
±
0.28 ±0.04
3.36 ±0.52
1.56 ±0.14
5.92 ±0.43
±
AFI 392 51
AFII 428 66
±
2.47 ±0.21
Km and Vmax are expressed in ~M and nmol p-nitroaniline released/min/mg protein, respectively. Each value is the mean (±SD) of at least five separate experiments. Oxytocinase activity was determined as described previously (9) using S-benzyl-Lcysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) as substrates with the following exceptions: the final concentration of the isoenzymes (without Triton X-100) in 0.5 ml incubate was 0.05 ml; substrate concentration ranged between 33.3 and 200 ~M; respective incubation times were 60 min and 30 min at 37°C; and all incubates contained 0,05% bovine serum albumin (BSA).
Table 3:
Kinetics of inhibition of amniotic fluid (AF) oxytocinases.
BCN Inhibitor
pH 6.2 AFI
AFII
AFI
130 20
±
96 II
±
±
95 25
±
95 19
102 20
±
79 13
PGE 2
PGF2m
p H 6.8
±
PGE I
LN
414 49
pH 6.2
AFII
AFI
pH 6.8
AFII
AFI
AFII
268 78
±
93 12
±
76 7
±
404 99
±
201 14
616 ± 127
505 ± 144
±
81 11
±
70 12
553 ± 130
±
431 33
591 ± 175
580 ± 137
±
98 8
±
64 10
±
601 60
±
424 I0
±
Ki is expressed in ~M. Each value is the mean (±SD) of at least three separate experiments. See Table 2 for the assay method.
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PROSTAGLANDINS
ACKNOWLEDGEMENTS We wish to thank Madam Ho Lai Meng for technical assistance. REFERENCES 1.
R o y , A.C., M. Yeang, S.M. Tan, S.R. Kottegoda and S.S. Ratnam. Oxytocinase a c t i v i t y in human amniotic f l u i d . IRCS Med. Sci. 12: 856, 1984.
2.
Roy, A.C., S.R. Kottegoda, O.A.C. Viegas and S.S. Ratnam. Oxytocinase a c t i v i t y in human amniotic f l u i d and i t s relationship to gestational age. Obstet. Gynecol. 1985; submitted.
3.
Roy, A.C., M. Yeang and S.M.M. Karim. I n h i b i t i o n of serum oxytocinase a c t i v i t y by prostaglandins. Prostaglandins Med. 6: 577587, 1981.
4.
R o y , A.C., M. Yeang and S.M.M. Karimo I n h i b i t i o n by prostaglandins of serum oxytocinase a c t i v i t y . Eighth International Congress of Pharmacology, Tokyo, July 19-24, 1981, Abstract 668.
5.
Roy, A.C., M. Yeang and S.M.M. Karim. pH dependent i n h i b i t i o n of serum oxytocinase a c t i v i t y by prostaglandins and cyclic GMP. Prostaglandins Leukotrienes Med. 8: 173-179, 1982.
6.
Yeang, M., A.C. Roy, and S.M.M. Karim. Selective i n h i b i t i o n of oxytocinase isoenzymes by prostaglandins, cyclic GMP, L-methionine and sodium azide. Third Southeast Asian and Western Pacific Regional Meeting of Pharmacologists, Bangkok, Thailand, 25-28 May, 1982. Abstract No. 15, p. 96.
7.
Yeang, M., A.C. Roy and S.R. Kottegoda. Kinetic studies on the i n h i b i t i o n of pregnancy serum oxytocinase a c t i v i t y b y prostaglandins and cyclic GMP. IRCS Med. Sci. 11: 831, 1983.
8.
Roy, A.C., M. Yeang, S.R. Kottegoda and S.S. Ratnam. Selective i n h i b i t i o n of multimolecular forms of human serum and placental oxytocinase a c t i v i t y by prostaglandins and cyclic GMP. Prostaglandins Leukotrienes Med. 14: 105-111, 1984.
260
AUGUST 1985 VOL. 30 NO. 2
PROSTAGLANDINS
9.
Roy, A.C., M. Yeang, S.M. Tan and S.R. Kottegoda. Kinetic studies on the i n h i b i t i o n by prostaglandins and cyclic GMP of multimolecular forms of human pregnancy serum and placental oxytocinase. IRCS Med. Sci. 12: 203, 1984.
10.
Roy, A.C., M. Yeang, L.M. Ho, S.R. Kottegoda and S.S. Ratnam. Multimolecular forms of oxytocinase a c t i v i t y in the human uterus and t h e i r i n h i b i t i o n by prostaglandins and c y c l i c GMP. Prostaglandins Leukotrienes Med. 1984; in press.
11.
Lampelo, S. and Vanha-Perttula, T. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of human serum during pregnancy. J. Reprod. F e r t i l . 58: 225-235, 1980.
12.
Lampelo, S. and Vanha-Perttula, T. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta. J. Reprod. F e r t i l . 56: 285-296, 1979.
13.
Roy, A.C. and S.M.M. Karim. Review: significance of the i n h i b i tion by prostaglandins and cyclic GMP of oxytocinase a c t i v i t y in human pregnancy and labour. Prostaglandins 25: 55-70, 1983.
14.
Roy, A.C. and S.M.M. Karim. Review: interaction between oxytocin and prostaglandins in reproduction. Singapore J. Obstet. Gynaecol. 14: 5-15, 1983.
15.
Roy, A.C., S . R . Kottegoda and S.S. Ratnam. Another |ook at i n i t i a t i o n of human p a r t u r i t i o n . Aust. N . Z . J . Obstet. Gynaecol. 1984; in press.
16.
Roy, A.C. Biochemical studies on the mode of action of disodium cromoglycate and related substances. Ph.D. Thesis, Brunel University, England, 1975.
Editor: M. Bygdeman
Received:4 - 1 7 - 8 5
AUGUST 1985 VOL. 30 NO. 2
Accepted:5-20-85
261
PROSTAGLANDINS, NOT CYCLIC GMP, INHIBIT OXYTOCINASE ISOENZYMES FROM HUMAN AMNIOTIC FLUID IN VITRO Ashim C Roy, Michael Yeang, Suan Mian Tan, Sri R Kottegoda and Shan S Ratnam Department of Obstetrics and Gynaecology, National University of Singapore, Kandang Kerbau Hospital, Singapore 0821 ABSTRACT
Two isoenzymes of oxytocinase (EC 3.4.11.3) a c t i v i t y were fractionated from human amniotic f l u i d samples between the 14th and 22nd weeks of gestation by Ultrogel acrylamide-agarose gel f i l t r a t i o n and p a r t i a l l y characterized. The isoenzymes were competitively inhibited by PGEz, PGE2 and PGF2mmore at pH 6.2 than at pH 6.8, whereas cyclic GMP (cGMP) and its 8-bromo derivative had no effect at either pH. The implications of these findings are discussed and i t is suggested that since the a c t i v i t y of amniotic f l u i d oxytocinases is very low or minimal at or near term, inhibition of these by prostaglandins may not have physiological significance in the i n i t i a t i o n of human parturition. INTRODUCTION Using S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) separately as substrates, we have recently demonstrated oxytocinase (EC 3.4.11.3) a c t i v i t y in the human amniotic f l u i d and observed an inverse relationship between the enzyme a c t i v i t y and gestational age (1,2). With both substrates, the enzyme a c t i v i t y , which was highest early in pregnancy, decreased exponentially to a minimum near or a few weeks before term. We have suggested that the amniotic f l u i d oxytocinase a c t i v i t y may play a significant role in pregnancy and labour. Oxytocinase is known to exist in multimolecular forms in the human serum, placenta and uterus, and we have shown a pH-dependent selective i n h i b i t i o n of these oxytocinases by prostaglandins (PGs) and cyclic GMP (cGMP) with maximum inhibition at pH 6.2 and l i t t l e or no i n h i b i t i o n at pH 7.4 (3-10). From these and related studies (13-15), i t appears that part of the oxytocic effect of PGs might be a t t r i b u t ed to their a b i l i t y to i n h i b i t oxytocinase a c t i v i t y and thereby protecting endogenous oxytocin. EndogenouscGMPmay also be involved in parturition partly by i n h i b i t i n g oxytocinase a c t i v i t y . AUGUST 1985 VOL. 30 NO. 2
255
PROSTAGLANDINS
We now present the results of our investigation on the separation, from the amniotic fluid, of multimolecular forms of oxytocinase by Ultrogel chromatography, their characterization and inhibition by PGs and cGMP. MATERIALS AND METHODS Sources of materials, preparation of solutions, assay of oxytocinase a c t i v i t y , and determination of protein and molecular weight were as stated elsewhere (10). Amniotic fluid samples obtained from normal pregnant women of 14 to 22 weeks gestation by amniocentesis (1,2) were centrifuged for 10 minutes at 1500 g and 4°C, pooled, chromatographed on acrylamide-agarose Ultrogel AcA 22 and stored at -20°C until used as described elsewhere (8,10). All calculations and statistical analyses were carried out with the aid of Magicalc (Artsci, Inc.) on a 64K Apple II Europlus microcomputer (9). An Applesoft Basic program (Kinetics Plotter and Graphics Dump) written by us enabled rapid, graphical determination of inhibition type. RESULTS Ultrogel f i l t r a t i o n patterns of the pooled amniotic fluid samples in relation to oxytocinase activity and protein concentration with (a) and without (b) 0.3% Triton X-IO0 are shown in Fig. 1. Gel f i l t r a t i o n parameters have already been described (8). Sodium azide was not used as a preservative in the gel chromatography because i t inhibits oxytocinase activity (6,8,10). Two hydrolytic peaks with elution volumes of 55 ml (void volume) and 130 ml were obtained in the absence of Triton X-IO0. Using either substrate peak I > peak I I , but in each case the hydrolysis of LN was greater than that of BCN. In the presence of Triton X-IO0, only one hydrolytic peak with elution volume of 110 ml was obtained. This may suggest that the isoenzyme eluted in the void volume was bound to cell particles or was in polymeric form. Unlike serum, placental and uterine oxytocinases (8,10), Triton X-IO0 had no significant effect on the hydrolysis of BCN and LN by the amniotic fluid enzymes at the concentration used. The molecular weight of peak I isoenzyme could not be determined because i t eluted in the void volume, but of peak II was 160,000. Their respective pH optima in 0.1M phosphate buffer were 6.8 and 6.6 with BCN as substrate, and 7.2 and 7.4 with LN. Only EDTA and Cu2+ ions inhibited the amniotic fluid isoenzymes (Table 1). EGTA, Ca 2+, Co 2+, Fe 2+, Mg2+, Mn2+, Ni 2+ and Zn2+ ions at comparable concentrations had no e f f e c t . However, i n h i b i t i o n of the isoenzymes by 50 ~M EDTA was reversed by equimolar concentrations of Cu2+, Co2+, Mn2+ and Zn 2+ ions. At higher concentrations (500-1000 uM), Co2+
256
AUGUST 1985 VOL. 30 NO. 2
PROSTAGLANDINS
1.5 1.0 0.5 Z
o
~o
0
z
o
AFI
i
L
I
i
i
i
i
>~ 1.5 iii 1,0
0.5 0 i
50 100 150 200 ELUTION VOLUME (rnl)
250
Fig. i: Ultrogel AcA 22 elution patterns of amniotic fluid with (a) and without (b) 0.3% Triton X-IO0. The hydrolysis of 500 pM Sbenzyl-L-cysteine-p-nitroanilide (BCN) or L-leucine-pnitroanilide (LN) by the eluates was assayed at 37°C and pH 7.0 for 120 or 60 minutes respectively as described previously (5). Assay mixtures contained 0.05% bovine serum albumin (BSA). Open circles, solid triangles and dotted lines represent BCN and LN hydrolysis (545 nm) and protein concentration (280 nm) respectively.
Table I: Inhibition of the activity of amniotic fluid ox~ocinases by EDTA and Cu-- ions. IC50, Inhibitor
BCN
LN
AFI
AFII
AFI
AFII
EDTA
8.0 ± 1.0
5.8 ±2.4
8.2 ± 1.6
6.6 ±1.3
Cu2+
242 ± 29
308 ± 76
232 ± 25
542 ± 48
Hydrolysis of 500 pM S-benzyl-L-cysteine-p-nitroanillde (BCN) or L-leucinep-nitroanilide (LN) by the oxytocinase isoenzymes with or without chelating agents (i h preincubatlon) or divalent metal ions (without preincubation) was measured at 37°C for 5-60 min in TrisHCI (0.I M, pH 7.0) buffer as described elsewhere (5). The results are the mean (±SD) of at least three separate experiments.
AUGUST 1985 VOL. 30 NO. 2
257
PROSTAGLANDINS
retained the restored a c t i v i t y , tory.
but Cu2+, Mn2+ and Zn2+ were i n h i b i -
Results of kinetic studies of the oxytocinase peaks and their i n h i b i tion by prostaglandins at pH 6.2 and pH 6.8 are given in Tables 2 and 3, respectively. The pH-independent (P>O.05) Km and Vmax values of the hydrolytic peaks were s i g n i f i c a n t l y greater with LN than BCN as substrate. PGEI, PGE2 and PGF2mcompetitively inhibited both isoenzymes at either pH without significant difference among their i n h i b i tory potencies using either substrate, but the i n h i b i t i o n was greater at pH 6.2 than at pH 6.8 (P
258
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PROSTAGLANDINS
Table 2: Kinetics of amniotlc fluid (AF) oxytocinases.
BCN pH 6.2 AFI Km ± Vmax
118 18
0.41 ±0.05
pH 6.8
AFII
AFI
124 24
±
LN
±
0.16 ±0.01
258 74
0.91 ±0.24
pH 6.2
pH 6.8
AFII
AFI
AFII
251 62
608 ± 112
657 ± 106
±
0.28 ±0.04
3.36 ±0.52
1.56 ±0.14
5.92 ±0.43
±
AFI 392 51
AFII 428 66
±
2.47 ±0.21
Km and Vmax are expressed in ~M and nmol p-nitroaniline released/min/mg protein, respectively. Each value is the mean (±SD) of at least five separate experiments. Oxytocinase activity was determined as described previously (9) using S-benzyl-Lcysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) as substrates with the following exceptions: the final concentration of the isoenzymes (without Triton X-100) in 0.5 ml incubate was 0.05 ml; substrate concentration ranged between 33.3 and 200 ~M; respective incubation times were 60 min and 30 min at 37°C; and all incubates contained 0,05% bovine serum albumin (BSA).
Table 3:
Kinetics of inhibition of amniotic fluid (AF) oxytocinases.
BCN Inhibitor
pH 6.2 AFI
AFII
AFI
130 20
±
96 II
±
±
95 25
±
95 19
102 20
±
79 13
PGE 2
PGF2m
p H 6.8
±
PGE I
LN
414 49
pH 6.2
AFII
AFI
pH 6.8
AFII
AFI
AFII
268 78
±
93 12
±
76 7
±
404 99
±
201 14
616 ± 127
505 ± 144
±
81 11
±
70 12
553 ± 130
±
431 33
591 ± 175
580 ± 137
±
98 8
±
64 10
±
601 60
±
424 I0
±
Ki is expressed in ~M. Each value is the mean (±SD) of at least three separate experiments. See Table 2 for the assay method.
AUGUST 1985 VOL. 30 NO. 2
259
PROSTAGLANDINS
ACKNOWLEDGEMENTS We wish to thank Madam Ho Lai Meng for technical assistance. REFERENCES 1.
R o y , A.C., M. Yeang, S.M. Tan, S.R. Kottegoda and S.S. Ratnam. Oxytocinase a c t i v i t y in human amniotic f l u i d . IRCS Med. Sci. 12: 856, 1984.
2.
Roy, A.C., S.R. Kottegoda, O.A.C. Viegas and S.S. Ratnam. Oxytocinase a c t i v i t y in human amniotic f l u i d and i t s relationship to gestational age. Obstet. Gynecol. 1985; submitted.
3.
Roy, A.C., M. Yeang and S.M.M. Karim. I n h i b i t i o n of serum oxytocinase a c t i v i t y by prostaglandins. Prostaglandins Med. 6: 577587, 1981.
4.
R o y , A.C., M. Yeang and S.M.M. Karimo I n h i b i t i o n by prostaglandins of serum oxytocinase a c t i v i t y . Eighth International Congress of Pharmacology, Tokyo, July 19-24, 1981, Abstract 668.
5.
Roy, A.C., M. Yeang and S.M.M. Karim. pH dependent i n h i b i t i o n of serum oxytocinase a c t i v i t y by prostaglandins and cyclic GMP. Prostaglandins Leukotrienes Med. 8: 173-179, 1982.
6.
Yeang, M., A.C. Roy, and S.M.M. Karim. Selective i n h i b i t i o n of oxytocinase isoenzymes by prostaglandins, cyclic GMP, L-methionine and sodium azide. Third Southeast Asian and Western Pacific Regional Meeting of Pharmacologists, Bangkok, Thailand, 25-28 May, 1982. Abstract No. 15, p. 96.
7.
Yeang, M., A.C. Roy and S.R. Kottegoda. Kinetic studies on the i n h i b i t i o n of pregnancy serum oxytocinase a c t i v i t y b y prostaglandins and cyclic GMP. IRCS Med. Sci. 11: 831, 1983.
8.
Roy, A.C., M. Yeang, S.R. Kottegoda and S.S. Ratnam. Selective i n h i b i t i o n of multimolecular forms of human serum and placental oxytocinase a c t i v i t y by prostaglandins and cyclic GMP. Prostaglandins Leukotrienes Med. 14: 105-111, 1984.
260
AUGUST 1985 VOL. 30 NO. 2
PROSTAGLANDINS
9.
Roy, A.C., M. Yeang, S.M. Tan and S.R. Kottegoda. Kinetic studies on the i n h i b i t i o n by prostaglandins and cyclic GMP of multimolecular forms of human pregnancy serum and placental oxytocinase. IRCS Med. Sci. 12: 203, 1984.
10.
Roy, A.C., M. Yeang, L.M. Ho, S.R. Kottegoda and S.S. Ratnam. Multimolecular forms of oxytocinase a c t i v i t y in the human uterus and t h e i r i n h i b i t i o n by prostaglandins and c y c l i c GMP. Prostaglandins Leukotrienes Med. 1984; in press.
11.
Lampelo, S. and Vanha-Perttula, T. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of human serum during pregnancy. J. Reprod. F e r t i l . 58: 225-235, 1980.
12.
Lampelo, S. and Vanha-Perttula, T. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta. J. Reprod. F e r t i l . 56: 285-296, 1979.
13.
Roy, A.C. and S.M.M. Karim. Review: significance of the i n h i b i tion by prostaglandins and cyclic GMP of oxytocinase a c t i v i t y in human pregnancy and labour. Prostaglandins 25: 55-70, 1983.
14.
Roy, A.C. and S.M.M. Karim. Review: interaction between oxytocin and prostaglandins in reproduction. Singapore J. Obstet. Gynaecol. 14: 5-15, 1983.
15.
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Editor: M. Bygdeman
Received:4 - 1 7 - 8 5
AUGUST 1985 VOL. 30 NO. 2
Accepted:5-20-85
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