Selective inhibition of multimolecular forms of human serum and placental oxytocinase activity by prostaglandins and cyclic GMP

Prostaglandins

Leukotrienes

and Medicine

14:

105-111,

1984

SELECTIVE INHIBITION OF MULTIMOLECULAR FORMS OF HUMAN SERW AND PLACENTAL OXYTOCINASE ACTIVITY BY PROSTAGLANDINS AND CYCLIC GHF’ Ashim C. Roy, Michael Yeang, Sri R. Kottegoda and Shan S. Ratnam Department of Obstetrics and Gynaecology, National University of Singapore, Kandang Kerbau Hospital, Singapore 0821 (Reprint requests to ACR) ABSTRACT Ultrogel acrylamide-agarose chromatography was employed for fractionation of oxytocinase isoenzymes from serum of pregnant women and from human placenta. Using S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) as substrates, three activity peaks (PI, PII, PIII) from placenta, and one peak (SI) from serum were identified. SI coincided with PII, and with all isoenzvmes the hydrolysis of LN was greater than that of BCN. Prostaglandins E and F inhibited all oxytocinases, more potently the hydrolysl 4’0:2 LN th& BCN and at pH 6.2 than at pH 6.8. Although cyclic GMP and its 8-bromo derivative similarly inhibited these isoenzymes except PIII, they were considerably more effective against the hydrolysis of BCN than LN. INTRODUCTION Using S-benzyl-L-cysteine-p-nitroanilide(BCN) and L-leucine-pnitroanilide (LN) as substrates, we have recently demonstrated (l-4) a pH dependent inhibition of serum oxytocinase (EC 3.4.11.3) activity from pregnant women by prostaglandins (PGs) and cyclic GMP (cGMP). These and related studies (also ref. 5 & 6) suggest that the oxytocic action of PGs could be mediated, at least partly, by their ability to maintain or elevate oxytocin levels in the uterus through the inhibition of oxytocinase activity. There is also a possibility that cGMP may be involved in the humoral events controlling parturition. The placenta is widely regarded as the source of human serum oxytocinase activity, and it is well known that the enzyme exists in multimolecular forms (7-17). This report is concerned with studies on the selective inhibition of these isoenzymes by PGs and cGMP.

105

MATERIALS AND METHODS Ultrogel AcA 22 was purchased from LKB, Sweden; sodium azide from Merck, West Germany; theophylline and bovine serum albumin (BSA) from Sigma Chemical Company, USA. All other materials were obtained as stated elsewhere (1,3). Prostaglandins E and E were solubilized in absolute ethanol. The final concentrate *bn of e?hanol in the incubate was 0.4%. All other test compounds were dissolved in the assay buffer (phosphate, 0.1 M, pH 6.2 or 6.8). Blood samples were drawn from a forearm vein of healthy pregnant women between 30 and 40 weeks of gestation with normal fetal growth and without any medication in the preceding week. The samples were allowed to clot and then centrifuged at 1700 g and 4°C for 10 minutes. The sera were separated, pooled and kept at -2O'C until analysed. The normal full-term human placenta was placed on ice immediately after delivery. Within 10 minutes of collection, the membranes were trimned and approximately 5 rnncubes of tissue were cut out and washed thoroughly in chilled (4'C) physiological saline (9 g NaCl/l), and blotted dry. The tissue (ca. 45 g) was then homogenized at 4'C in 50 ml buffer (0.01 M phosphate, pH 7.0; sucrose 0.25 M) using a hand homogenizer. The homogenate was filtered through a cheese-cloth and centrifuged at 1700 g and 4°C for 10 minutes. The supernatant, referred to as placental homogenate, was immediately used for chromatography on acrylamide-agarose Ultrogel AcA 22 columns. Pooled serum from pregnant women was chromatographed similarly. Fractions in each peak were pooled, BSA added to a concentration of 0.052, and stored at -2O'C until assayed. Oxytocinase activity was determined as described elsewhere (3). Protein was estimated according to the method of Lowry and associates (18). Results of inhibition studies were analysed by Students' t test for independent samples. RESULTS Gel filtration of placental homogenate and maternal serum Elution patterns of the enzyme(s) hydrolyzing BCN and LN substrates separately after Ultrogel filtration of the placental homogenate and serum pool are shown in Fig. 1. Various parameters of the gel filtration are given in Table I. Placental homogenate produced three distinct hydrolytic peaks in the eluate between 45 ml and 70 ml (PI), 100 ml and 120 ml (PII), and 125 ml and 155 ml (PIII), respectively. Using either substrate, PI> PIII> PII, but the hydrolysis of LN was greater than that of BCN. However, serum showed only one hydrolytic peak (SI) coinciding with PI1 between 95 ml and 130 ml of the eluate.

106

Sodium azide has been TABLE I I used at 0.02% concentration as a preservative in gel chromatoParameters of Ultrogel AcA 22 graphy (17). We have reported chromatography (19) a pH independent significant inhibition of all oxytociColumn dimensions: 2.5 x 35 cm nase peaks by this agent at Total gel volume: 172 ml this concentration with BCN as Sample placental homogenate:12 m substrate. Therefore, sodium volume [maternal serum: 5m azide was not used as preservaFraction volume: 5 ml tive in this study. Triton XElution rate: 12 ml/h 100 could not be used for soluEluting medium: phosphate buffer bilizing placental enzymes bec(0.01 M, pH 7.0) ause it also significantly inh- 1 Temperature: 4°C I ibited both serum and placental oxytocinases at the required concentration (0.3%) (unpublished). Preliminary studies (unpublished) showed gross instability of placental isoenzymes during incubation. This difficulty was overcome by the addition of BSA (0.05%) to the incubate.

PEAK

1

FIG. 1

I

‘ 3

PEIK III

I 1 g F:0. Y ;: E 2 .

SERU4

I

0 0

50

ml ELUTION

VOLLM

MO

1% lmlt

107

Ultrogel AcA 22 elution patterns of placental homogenate and maternal serum. Parameters of gel filtration are given in Table I. The hydrolysis of 500 PM S-benzyl-L-cysteine-p-nitroanilide (BcN) or L-leucine-p-nitroanilide (LN) by the eluates was assayed at 37°C for 30 minutes as described previously (3). Bovine serum albumin (BSA) was added to the assay mixtures, and its final concentration was 0.05% and 0.002% for placental and serum isoenzymes, respectively. Open circles represent BCN hydrolysis at pH 6.8: closed circles, LN hydrolysis at pH 7.4; and dotted lines, protein concentration.

Inhibition by PGs and cGMP of serum and placental oxytocinases Prostaglandins and cGMP were tested for their inhibitory effect on the hydrolysis of BCN and LN by maternal serum and placental oxytocinase activity peaks at pH 6.2 and pH 6.8. Their 50% inhibitory concentrations (IC ) are given in Table II. PGs Eh, E$ and F2 significantly inhibit.28 all oxytocinase peaks, more p ten ly the fiydrolysis of LN than BCN (P< 0.001) and at pH 6.2 than pH 6.8 (P
108

II

336 + 54

*15i

158 222

248 f 16

DGF 2a

cGMP

3-Br cGMP *14i

189 6

+$

432 + 18

167 + 40

103 2 26

+ 1388

PI1

NE

NE

256 + 35

196 A 14

k 235 31

PI11

BCN

1429 2351

f 579 16

f 535 21

596 + 49

397 f 49

k 412 lg

PI

915 +100

688 + 95

581 f 19

+ 489 13

SI

+ 732 19

NE

NE

623 t 74 1714 k415

337 k 27

464 + 51

$j;;

i;;;

32 5

23 5 2 f

k 2G

SI

423 f 18

PI11 + 261 27

+437 18

PI1

pH 6.8

I

52 2

62 ?r 17

54 f 5

f ?t

PI1

t;;;

>2000

>2000 ,200o

+

37 k 9

+ “2

PI

pH 6.2

+

NE

NE

28 2

43 + 10

* “i

PI11

184 k 22

187 f 15

klii

SI

217 k 14

227 + 13

*‘ii

PI

310 2 12

367 f 42

k2i66

PI1

pH 6.8

2000

J2000

>2000

>2000 >2000 ,200O

LN

NE

NE

64 * 12

106 + 11

f ii

PI11

Hydrolysis of the substrates was determined as described elsewhere (3) with the following exceptions: the final concentration of SI, PI, PI1 and PI11 in 0.5 ml incubate was 0.02, 0.02, 0.05 and 0.02 ml, respectively; at pH 6.2 LN was incubated with the isoenzymes for 30 minutes; respective incubates of maternal serum and placental isoenzymes contained 0.002% and 0.05% bovine serum albumin (BSA). The results were expressed as the mean (+ SD) of duplicate incubations from at least three separate experiments. ICso, 50% inhibitory concentration; NE, no effect.

k

86 f 18

104 k 16

2

‘GE

+ 101 24

PI

+ 114 13

SI

pH 6.2

jGE1

Inhibitor

I

Inhibitory effects of various compounds on the hydrolysis of S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) by human maternal serum (SI) and placental (PI, PI1 and PIII) oxytocinases at pH 6.2 and pH 6.8.

TABLE

cance of these differential inhibitions, and studies on human uterine oxytocinases are in progress. ACKNOWLEDGEMENTS We wish to thank Madam Tan Suan Mian and Madam Ho Lai Meng for technical assistance; Singapore Turf Club and the Endocrine & Metabolic Society of Singapore for financial support. REFERENCES 1.

Roy AC, Yeang M and Karim SMM. Inhibition of serum oxytocinase activity by prostaglandins. Prostaglandins Med 6: 577-587, 1981.

2.. Roy AC, Yeang M and Karim SMM. Inhibition by prostaglandins of serum oxytocinase activity. Eighth International Congress of Pharmacology, Tokyo, Abstract 668, 19-24 July, 1981. 3.

Roy AC, Yeang M and Karim SMM. pH dependent inhibition of serum oxytocinase activity by prostaglandins and cyclic GMP. Prostaglandins, Leukotrienes Med 8: 173-179, 1982.

4.

Yeang M, Roy AC and Kottegoda SR. Kinetic studies on the inhibition of oregnancy serum oxytocinase activity by prostaglandins and cyclic GMP. IRCS Med Sci 11: 831, 1983.

5.

Roy AC and Karim SMM. Review: significance of the inhibition by prostaglandins and cyclic GMP of oxytocinase activity in human pregnancy and labour. Prostaglandins 25: 55-70, 1983.

6.

Roy AC and Karim SMM. Review: interaction between oxytocin and prostaglandins in reproduction. Singapore J Obstet Gynaecol 14: 5-15, 1983.

7.

Page EW, Titus MA, Mohun G and Glendening MB. The origin and distribution of oxytocinase. Am J Obstet Gynecol 82: 1090-1095, 1961.

8.

Sjoholm I and Yman L. Electrophoretic studies on oxytocinase (cystine aminopeptidase). Relation between electrophoretically found "isozymes". Acta Pharm Suecica 3: 389-396, 1966.

9.

James NT. Histochemical demonstration of oxytocinase in the human placenta. Nature 210: 1276-1277, 1966.

10.

Kleiner H and Brouet-Yager M. Separation of L-cystinyl-di-8naphthylamide hydrolase ("oxytocinase") isoenzymes by acrylamide gel electrophoresis of human pregnancy sera. Clin Chim Acta 40: 177-180, 1972.

11.

Oya M, Yoshino M and Mizutani S. Appearance of placental aminopeptidase isozyme during the course of pregnancy. Acta Obstet Gynaecol Jap 22: 49-50, 1975.

12.

Mizutani S, Yoshino M and Oya M. Placental and non-placental leucine aminopeptidases during normal pregnancy. Clin Biochem 9: 16-18, 1976.

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13.

Ota T, Chen CH-J and Robinson JC. Pregnancy-associated aminopeptidases of man and monkey. Acta Obstet Gynaecol Jap 23: 101-104, 1976.

14.

Spellacy WN, Usategui-Gomez M and Fernandez de Castro A. Plasma human placental lactogen, oxytocinase, and placental phosphatase in normal and toxemic pregnancies. Am J Obstet Gynecol 127: lo16, 1977.

15.

Lampelo S and Vanha-Perttula T. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta. J Reprod Fertil 56: 285-296, 1979.

16.

Lampelo S and Vanha-Perttula T. Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of human serum during pregnancy. J Reprod Fertil 58: 225-235, 1980.

17.

Kleiner H, Dictus-Vermeulen C, May-Cocriamont C, Brouet-Yager M, Popowski A, Mosselmans R and Graff G. Human placental oxytocinase and its relationship to pregnancy plasma oxytocinase. Clin Chim Acta 101: 113-123, 1980.

18.

Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275,

1951.

19.

Yeang M, Roy AC and Karim SMM. Selective inhibition of oxytocinase isoenzymes by prostaglandins, cyclic GMP, L-methionine and sodium azide. Third Southeast Asian and Western Pacific Regional Meeting of Pharmacoloqists, Bangkok, Thailand, Abstract No 15, p 96, 25-28 May, 1982.

20.

Roy AC. Biochemical studies on the mode of action of disodium cromoglycate and related substances. PhD Thesis, Brunel University, England, 1975.

21.

Kleiner H and Schram E. Separation des isozymes hydrolysant la L-leucyl-B-naphthylamidepar electrophorese verticale en gel d'acrylamide. Clin Chim Acta 14: 377-385, 1966.

22.

Ryden G. Cystine aminopeptidase and oxytocinase activity in pregnancy. A comparative study in human and rat tissues. Acta Obstet Gynecol Stand 45 (Suppl 3): l-105, 1966.

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