Interaction of Progestins with Steroid Receptors in Human Uterus

Blochem. J. (1978) 176,531-539Printed in Great Britain

Interaction of Progestins with Steroid Receptors in Human Uterus

By AlTAN KASID, KAMAL BUCKSHEE, VEERA HINGORANIand KESHO R. LAUMAS

Departments ofReproductive Biology and Obstetrics and Gynaecology,All India Institute ofMedical Sciences, New Delhi-1 10016, India

(Received 23 May 1978)

Norethindrone (17fi-hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one) and norethindroneacetate (17fl-acetoxy-19-nor-17a-pregn-4en-20-yn-3-one) interfered to a varying degree,by competitive inhibition, with the binding of progesterone and oestradiol to respectivecytoplasmic receptors in the human uterus. Progesterone binding to 4S macromoleculewas saturable and co-specific for progestins. Competitors like norgestrel (17f-hydroxy-18-methyl-19-nor-17a-pregn-4en-20-yn-3-one), 19-norprogesterone, medroxyprogesteroneacetate (17a-acetoxy-6a-methylpregn-4ene-3,20-dione) and compound R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) possessed higher binding affinities for theprogestin receptor. The dissociation constant (Kd) for the progesterone-receptor inter-action was 0.6-1.6nM and the receptor concentration ranged between 6600 and 8200 sites/cell. Norethindrone and norethindrone acetate competed for the progesterone receptorwith inhibition constants (KM) of 6.8 and 72nM respectively. Gradient displacement andcompetitive-receptor assays indicated that norethindrone acetate-binding affinity forprogestin receptor was approximately one-tenth that of norethindrone and progesterone.The progestins also inhibited oestradiol binding to 4.6S oestrogenic receptor by 8-12%,involving interaction at the oestradiol-binding site with a calculated K, value of0.5-0.8gM.The competitive interaction ofprogestins with steroid receptors may be of putative import-ance in explaining the progestin action at the target site.

Steroid-hormone receptors are the primaryrecognizing and regulatory molecules, functioning as

biological transducers at the target site. Since uterinesteroid-hormone receptors are indispensably impli-cated in the action of progesterone and oestradiol(Baulieu, 1975; Janne et al., 1975; O'Malley &Schrader, 1976), any alteration in the receptor-binding properties by other compounds could modu-late the processes controlled by these hormones.Previous work has shown that synthetic progestinslike norethindrone (17fi-hydroxy-19-nor-17a-pregn-4-en-20-yn-3-one) and norethindrone acetate (17f,-acetoxy-19-nor-17a-pregn-4-en-20-yn-3-one) bindspecifically to auterine macromoleculewith character-istics similar to the progesterone receptor (Laumas &Kasid, 1977). These findings prompted us to investi-gate whether binding of these progestins occurred atthe progesterone-binding site (with inhibition kineticsof competitive type), or that the progestins interactedby binding at some other site on the receptor mole-cule. Further, these progestins have also been reportedto interfere with the binding of oestradiol to theoestrogenic target tissues in rat (Van Kordelaar et al.,1975). However, no quantitative data are available.As progestin-receptor interaction may be significantin the understanding of progestin action and conse-quently the pharmacological manipulation of cellularfunctioning, this interaction needed further eluci-

Vol. 176

dation. The present work embodies the qualitativeand quantitative evaluation of the interaction ofnorethindrone and norethindrone acetate with thereceptor proteins for progesterone (pregn-4-ene-3,20-dione) and oestradiol (oestra-1,3,5(10)-triene-3,17.8-diol) in the human uterus.

Materials and Methods

Radioactive steroid

[15,16-3H]Norethindrone (sp. radioactivity 57Ci/mmol) and [15,16_3H]norethindrone acetate (sp.radioactivity 57Ci/mmol) were gifts from ScheringA.-G. (Berlin, West Germany) and purified on LH-20columns with toluene/methanol (17: 3, v/v) as solvent.[1,2-3H]Progesterone (sp. radioactivity 48Ci/mmol)and [6,7-3H]oestradiol-17fi (sp. radioactivity 4OCi/mmol) were obtained from New England Nuclear(Boston, MA, U.S.A.). The radiochemical purity ofthe steroids was confirmed by t.l.c. with chloroform/acetone (9:1, v/v) as solvent for progesterone andchloroform/ethyl acetate (4: 1, v/v) for oestradiol.

Human uterine tissueNormal uterine tissue was obtained from women

undergoing hysterectomy operations because of thefollowing conditions: myomata uteri, adenomyosis

531

A. KASID, K. BUCKSHEE, V. HINGORANI AND K. R. LAUMAS

and prolapsus uteri. The subjects were in the repro-ductive-age group (26-40 years) with normal men-strual cycles and without any hormone treatment.Immediately after hysterectomy, the uterus was cutlongitudinally, adequate samples were taken forhistological examination, and the pieces of tissuewere chilled on ice. Further processing of samplescommenced immediately at 0-20C.

Preparation ofcytosolfractionsAfter thorough washing, the uterine tissue was

finely minced and homogenized in buffer A (0.01 M-Tris/HCl/0.OOl5mM-disodium EDTA, pH7.4), con-taining 0.012mM-thioglycerol, 20% (v/v) glyceroland 1 M-cortisol (1 1fl,17a,21-trihydroxypregn-4-ene-3,20-dione). The homogenate was centrifuged atlO50OOg for 1 h at 4°C to obtain the cytosol (super-natant) fraction. For binding assays, the cytosol wasdiluted with buffer A (10mg of protein/ml of cytosol)and used fresh.

Sucrose-density-gradient centrifugationCytosol was analysed for binding by centrifugation

on linear 5-20% (w/v) sucrose gradients, prepared inbuffer A containing 10% glycerol with a Beckmangradient former. A sample (200,p) of cytosol wasincubated with a lOnM solution of 3H-labelledsteroid in the presence or absence of a 100-fold excessof unlabelled steroid for I h at 0°C and layered on topof the gradients. Centrifugation was carried out at50000rev./min for 15h at 4°C in an SW 56 rotor.Bovine serum albumin (4.4S) and human y-globulin(7.1 S) were run as reference proteins in the estimationofsedimentation coefficients by the method of Martin& Ames (1961). Fractions (2 drops; 130,u1) werecollected and A280 was recorded by using a ISCOgradient fractionator.

Quantification of3H-labelled steroid-binding sites anddetermination ofdissociation constant (Kd) (Scatchardanalysis)

Cytosol samples (100l1) were incubated withincreasing concentrations of 3H-labelled steroid(0.2-12.5nM) for 12-16h at 0°C. The samples wereassayed for receptor-bound radioactivity by usingthe dextran/charcoal adsorption technique (Koren-man, 1975). The binding data were analysed asdescribed by Scatchard (1949), and the correction forspecific binding was made as described by Chamness& McGuire (1975). For each assay, the relationshipbetween the bound to free ratio and the concentrationbound (nM) was subjected to linear regressionanalysis by using a Hewlett-Packard model 9810Acalculator. The instrument was programmed to

provide a correlation coefficient (r), slope (repre-senting I/lKd) and the concentration of binding sites(pmol/mg of protein), where the number of bindingsites per receptor molecule was assumed to be one.Receptor concentration per cell was obtained fromthe concentration per mg ofDNA by the expression(mol of receptor-binding sites/mg of DNA) x (mg ofDNA/cell) x Avogadro's number. DNA content wasassumed to be 6pg/cell as the diploid amount ofDNAin human cells (Vendrely, 1955).

Non-specific (non-saturable) binding was measuredin parallel incubation mixtures where a 100-fold molarexcess of the respective unlabelled steroid was added.It was subtracted from the total binding to obtain thespecific saturable binding.

Determination of the type of inhibition (Lineweaver-Burk plots)

Cytosol samples were incubated with increasingconcentrations (0.25-lOnM) of 3H-labelled steroidalone, and also in the presence of a constant concen-tration of inhibitor (competitor). The assay protocolswere as follows: set A, cytosol labelled with [3H]pro-gesterone with norethindrone and norethindroneacetate as competitors; set B, cytosol complexed with[3H]norethindrone with progesterone and nor-ethindrone acetate as competitors; set C, cytosollabelled with [3H]norethindrone acetate with pro-gesterone and norethindrone as competitors; set D,cytosol labelled with [3H]oestradiol with norethin-drone and norethindrone acetate as competitors.

After equilibrium was attained (12-16h), thesamples were assayed for receptor-bound radio-activity by the dextran/charcoal technique. At eachexperimental point, control tubes with only labelledsteroids at different concentrations were incubatedto give the uncompeted values. All values werecorrected for blank. The double-reciprocal plots ofthe specific-binding data were adapted from classicLineweaver-Burk (1934) analysis. The best-fittedlines were drawn by the least-squares method. Thedissociation constant (Kd) was calculated either fromthe slope and ordinate intercept or from the negativeabscissa intercept. The number of binding sites wereinterpreted from the ordinate intercept.To increase the specificity, all incubation solutions

with labelled progestins also contained non-labelled1 jM-oestradiol. Similarly all incubation solutionswith labelled oestradiol also contained unlabelled1 ,M-progesterone.

Determination of inhibition constants (KL)The calculated K1 values of various progestins were

obtained either by using the equation for competitiveinhibition or directly from Dixon plots (Dixon &Webb, 1960).

1978

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PROGESTIN-STEROID RECEPTOR INTERACTION IN HUMAN UTERUS

Determination ofrelative binding affinityThe relative binding affinities of various steroids

was evaluated by their ability to compete with saturat-ing amounts of any one of the following 3H-labelledsteroids, progesterone, norethindrone or norethin-drone acetate, for binding to cytosol progestinreceptor.Samples (100,ul) of cytosol were added to assay

tubes containing 0.5 nM 3H-labelled steroid in100ul of buffer A and lOOp1 of buffer A alone orcontaining various concentrations (0.01-100ng) ofunlabelled steroid competitors. The separation ofbound from free radioactive steroid was carried out at0-2°C by using the dextran/charcoal adsorptiontechnique.Each assay point was determined in duplicate. All

values were corrected for blanks (incubation solutionswithout cytosol treated with dextran/charcoal).A plot of percentage of radioactivity bound (c.p.m.)against the logarithm of the mass of the competingligand added gave a competition curve for eachligand. The molar concentration of each competitorrequired to decrease the binding of 3H-labelled pro-gestin by 50% was calculated from these plots. Theeffectiveness of the competitor was calculated asdescribed by Smith et al. (1974).

Protein and DNA determination

Protein was determined by the method of Lowryet al. (1951) with bovine serum albumin as standard.DNA was determined by the diphenylamine methodas described by Burton (1956) with calf thymus DNAas standard.

Results

Mediation by progestins of the displacement of 3H-labelled steroidfrom receptor sites on densitygradients

[3H]Progesterone displacement. When cytosol waslabelled in vitro with [3H]progesterone (lOnM) andcentrifuged on a density gradient (Fig. 1) the peak ofradioactivity was invariably localized in the 4.1-4.3 Sregion. More than 90 % of the total binding measuredwas stereospecific for progestins. Unlabelled pro-gestins, when added simultaneously, decreasedprogesterone binding, although with varying degreeof efficiency. Nevertheless, as the concentration ofunlabelled progestins increased, the binding of [3H]-progesterone decreased in the 4S region, indicatingthat the binding was saturable. Binding of [3H]pro-gesterone was completely abolished when a 100-foldexcess of progestin was added.

[3H]Norethindrone/norethindrone acetate displace-ment. The sedimentation profile of cytosol proteinscomplexed with [3H]norethindrone or [3H]nor-ethindrone acetate (results not shown) demon-

Vol. 176

strated the complex sedimenting with a mean S valueof 4.2±0.2. The binding of [3H]progestin was com-pletely inhibited by concomitant addition of a 100-fold molar excess of potent progestins. However,oestradiol and cortisol showed no displacement ofprogestin binding.

[3H]Oestradiol displacement. As shown in Fig. 2,[3H]oestradiol was specifically bound to a 4.6Sprotein. Unlabelled oestradiol (100-fold excess)distinctly displaced the [3H]oestradiol from thebinding sites. However, norethindrone and nor-ethindrone acetate (100-fold excess) inhibited [3H]-oestradiol binding to the extent of 8-12% only.

Competitive interaction ofprogestins with 3H-labelledsteroid-receptor complex

Determination of binding constants (Scatchardanalysis). The concentration of the specific-bindingsites with corresponding dissociation constants (Kd)

c).t;

ci0

x

0I 3

00

Fraction no.Fig. 1. Competitive displacement of[3H]progesterone bound

to cytoplasmic receptor (sucrose-gradient analysis)Uterine cytosol was incubated with [3H]progesterone(lOnM, 2h, 0C) alone (e) or together with nor-ethindrone (lOnM, *; lOOnM, v; IpM, A) or nor-ethindrone acetate (lOnM, U; lOOnM, [1; 1pM, 0).The samples were analysed for receptor-bound[3H]progesterone on linear 5-20% (w/v) sucrosegradients by centrifugation at 50000rev./min for 15hat 2-40C. The broken line shows the migration ofcytosol protein after centrifugation. Arrows denotepositions of bovine serum albumin (BSA) and humany-globulin (HG). The horizontal arrow indicates thedirection of sedimentation.

533


for progesterone-, norethindrone- and norethindroneacetate-receptor interaction are summarized inTable 1. The binding in the competed tubes (with a100-fold molar excess of the test steroid) was desig-nated as non-specific and the difference between thenon-competitive (without a 100-fold molar excess)and competitive binding was defined as specific orreceptor binding. The number of receptor bindingsites was almost the same for all the progestins, butthe K4 values were 8-10 times higher for norethin-drone acetate as compared with norethindrone andprogesterone.

Competitive inhibition of progesterone binding bynorethindrone/norethindrone acetate (Lineweaver-Burk plots). Binding of progesterone in the presenceof norethindrone and norethindrone acetate has beengraphically (double-reciprocal plots) represented inFigs. 3(a) and 3(b) respectively. The progestins causedno inhibition of progesterone binding to non-specificsites, but there was significant inhibition ofprogester-one binding to the specific or displaceable sites,particularly at lower progesterone concentration.However, this effect tended to decrease at higherconcentrations of progesterone. Since the interactionwith the ordinate remained essentially constant (atthe maximum-binding points) in the presence of thecompeting steroids, the patterns were for typicalcompetitive inhibition. The calculated number ofprogesterone-binding sites was 260fmol/mg ofprotein (6600 molecules/cell) and the Kd valuewas in the range 0.61-0.63nM. Conversely, whenuterine proteins were labelled with different concen-trations of either pH]norethindrone (results notshown) or [3H]norethindrone acetate in the presenceof indicated concentrations of unlabelled pro-gesterone (Fig. 4a) or unlabelled norethindrone(Fig. 4b), a typical competitive inhibition was con-sistently observed. The concentration of progestinreceptor was calculated to be about 6000 sites/cell.

Determination of inhibition constants (K,) (Dixonplots). The K, values of norethindrone (Fig. Sa) andnorethindrone acetate (Fig. Sb) for progesteronereceptor were 6.8 and 72nM respectively. In other

41

,

x0xX

0 10 20 30Fraction no.

Fig. 2. Competitive displacement of[3Hoestradiol bound tocytoplasmic receptor (sucrose-gradient analysis)

Uterine cytosol was incubated with [3H]oestradiol(5nM, 2h, 0°C) alone (e) or together with a 100-foldexcess of norethindrone (N), norethindrone acetate(A) or oestradiol (o). The samples were analysed on5-20% sucrose gradients (SOOOOrev./min, 1Sh,2-4°C). Arrows denote positions of bovine serumalbumin (BSA) and human y-globulin (HG). Thehorizontal arrow indicates the direction of sedimen-tation.

Table 1. Comparison ofthe binding constants ofuterine cytosol receptorsfor different steroidsCytosol samples were incubated with increasing concentrations (0.2-12.Snm) of 3H-labelled steroid for 12h at 0-2°Cand assayed for bound steroid by the dextran/charcoal adsorption technique. Protein normalized Scatchard plots wereprepared and the concentration of binding sites and equilibrium dissociation constant (Kd) were calculated by using theequation [bound/free] = 1/K, x [(binding sites)-(bound)]. Results are Mans S.E.M. for the numbers oftissue samples(from day 13-15 of menstrual cycle) processed, indicated in parentheses.

Receptor-binding site concentration

SteroidNorethindrone (5)Norethindrone

acetate (9)Progesterone (5)Oestradiol (3)

(fmol/mg ofcytosol protein)

331±102280± 67

326± 83265± 60

(molecules/cell*)8340± 25707060+ 1690

8200± 20906680±1500

* DNA content assumed to be 6pg/cell.

1978

(K4) (nM)1.3 ±0.3

12.9±3.11.6±0.40.5±0.1

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t 0 1 2 3 4 t 0 1 2 3 4-1/Kd -l/Kd

1/[Progesteronel (nM-1)

Fig. 3. Competitive inhibition of (3H]progesterone binding to uterine cytosol receptor by norethindrone (a) and norethindroneacetate (b)

The cytosol was incubated with increasing concentrations (0.25-lOnM) of [3H]progesterone alone (i= 0) or togetherwith the indicated concentration of inhibitor (i). The specific bound radioactivity was analysed by the dextran/charcoaladsorption technique. The double-reciprocal plots represent the best-fitted lines drawn by linear regression analysis.

100 100063 b)10I 0.53n w46

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=0.21,m80e cyo si - 0.22qnm

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0 .~~~1=0i=0

140- 40-

020 - 20-

tO0 2 4 6 t0 2 4 6-i//Cd -1//Cd

l/[Norethindrone acetate] (nm'1)

Fig. 4. Competitive inhibition byprogesterone (a) and norethindrone (b) of[3H]norethindrone acetate binding to uterine cytosolreceptor

The cytosol was incubated with increasing concentrations (0.2-10nm) of [3HJnorethindrone acetate alone (i = 0) ortogether with indicated concentrations ofinhibitor(i. Specifically bound [3HJprogestin was determined by the dextran/charcoal adsorption technique. The best-fitted lines were drawn by linea regression analysis.

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535


20

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121m ,,.1-- o 8-O 8

_A

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l- 1In -1 ln

-Ki 0 4 8 12 16 -K; 0 100 200300400[Norethindronel (nM) [Norethindrone acetate]

(nM)

Fig. 5. Determination of inhibition constants of norethin-drone (a) and norethindrone acetate (b) for the progesterone

receptor of uterine cytosolUterine cytosol was incubated with [3H]progesteroneat either 0.5nM (A) or l.5nM (e) in the absence orpresence of various concentrations of inhibitors. Thespecifically bound [3H]progesterone was analysed bythe dextran/charcoal technique. The horizontal linedrawn at I/n, where n = 260fmol/mg of protein,represents the number of progesterone-binding sitesin the uterine cytosol.

experiments, where cytosol receptor was complexedwith [3H]norethindrone acetate, the K1 values were0.28nM for norethindrone (Fig. 6a) and 0.24nM forprogesterone (Fig. 6b), indicating high competitiveefficiencies of norethindrone and progesteronecompared with norethindrone acetate for binding toprogestin receptor.

Interaction ofprogestins with oestradiol receptor

Fig. 7 represents the double-reciprocal plots of[3H]oestradiol binding in the absence or presence of(a) norethindrone and (b) norethindrone acetate. Theplots suggested competitive inhibition of oestradiolbinding by progestins, with average Ki values of0.5 ,UM for norethindrone and 0.8Up for norethindroneacetate. The specific number of oestradiol-bindingsites in the absence of progestin was approx. 7000sites/cell and theKd value of oestradiol interaction wascalculated to be 0.61-0.64nM.

Ligandspecificityof[3H]progestin-receptor interactionThe ligand affinities for progestin-binding proteins

have been summarized in Table 2. For each competi-tor, relative binding affinity was indexed by itsability to compete separately with saturating amountsof [3H]progesterone, [3H]norethindrone or [3H]nor-ethindrone acetate (relative affinity in each case takenas 100). Most of the steroids tested exhibited similarrelative competitive indices for progesterone- andnorethindrone-binding proteins. Potent progestinscompetedwith an affinity either higher than [norgestrel(171J-hydroxy- 18 -methyl- 19-nor- 17a-pregn-4-en-

-._100 (a) 100 (b)

'o80 -80-

O0 60 -60-

40 ~~~~40

0~~~~~~~2---i/- --- --1/n-

-Kj 0 0.2 0.4 0.6 -Kj 0 0.2 0.4 0.6[Norethindrone] (nM) [Progesterone] (nM)

Fig. 6. Determination of inhibition constants of nor-ethindrone (a) and progesterone (b) for norethindrone

acetate receptor of uterine cytosolUterine cytosol was incubated with [3H]norethin-drone acetate at either 0.3 nm (A) or 1.8nM (M) in theabsence or presence of various concentrations ofinhibitors. Other details were similar to those given inthe legend to Fig. 5 (n = 240fmol/mg of progestin).

O 12-

0 10

la6

0I

g4

m 2-

to0- 1/Kd

(a)

i=o

- (b)

0S=0

1 2 3 4ft 0 1 2 3 4-1/Kd

1/[Oestradiol] (nM-')

Fig. 7. Inhibition of[3H]oestradiol binding by norethindrone(a) and norethindrone acetate (b) in the human uterine

cytosolThe cytosol was incubated with increasing concentra-tions of [3H]oestradiol (0.2-5nM) alone (i =0) ortogether with different concentrations (200nM, A;500nM, o) of progestins. Specifically bound[3H]oestradiol was determined by the dextran/charcoal adsorption technique. The best-fitted lineswere drawn by using the method of least squares.

20-yn-3-one), 19-norprogesterone, medroxyproges-terone acetate (17a - acetoxy -6- methylpregna -4,6-diene-3,20-dione)] or comparable with [compoundR5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione), megestrol acetate (17a-acetoxy-6-methyl-pregna-4,6-diene-3,20-dione)] those for progesteroneand norethindrone. Compound R2323 (13-ethyl-17-hydroxy-18,19-dinor-17a-pregna-4,9,1 1-trien- 20-yn-one), chlormadinone acetate (17a-acetoxy-6-chloro-pregna-4,6-diene-3,20-dione) and 17a-acetoxyprog-esterone competed with relative affinity valuesranging between 40 and 60. Steroids like 5a-pregnane-3,20-dione, 11fi-hydroxyprogesterone, norethynodrel(17a-ethynyl-17-hydroxyoestr-5(10)-en-3-one)andl9-

1978

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Relative binding Relative bindingaffinity ofENT-R affinity ofENTA-R

Fig. 8. Correlation of binding affinities of progesteronereceptor with norethindrone receptor (A) and progesteronereceptor with norethindrone acetate receptor (e) in human

uterine cytosolEach point represents triplicate assays. The best-fitted lines were drawn by linear regression analysis.Abbreviations used: P-R, progesterone receptor;ENT-R, norethindrone receptor; ENTA-R, nor-ethindrone acetate receptor.

nortestosterone (17fi-hydroxy-19-norandrost-4-en-3-one) had relative affinity varying between 16 and 32.However, the relative affinity of various competitorsfor norethindrone acetate receptor indicated poorreceptor affinity of the progestin. All potent pro-gestins were 4-18 times more effective than nor-ethindrone acetate in binding to the uterine progestinreceptor. Oestrogens, testosterone and cortisolshowed little or no competition for progestinreceptors.

Fig. 8 illustrates the correlation of relative bindingaffinities of progesterone receptor with norethindronereceptor and progesterone receptor with norethin-drone acetate receptor. High correlation within thereceptor affinities was noted. The correlation coeffi-cients (r) between the receptor specificities were asfollows: progesterone against norethindrone, r =

0.98; progesterone against norethindrone acetate,r= 0.95. The difference between the two correlationsobserved was not significant (P < 0.05).

Discussion

The present results convincingly demonstratethat in the human uterus progestins like norethin-drone and norethindrone acetate are bound to theprogesterone-recognition site, but with varyingdegrees of inhibition and binding constants. Froip

this significant steroid-protein interaction, the actionof different progestins in the human uterus via acommon receptor is conceivable. Further, theseprogestins were also found to interfere with thebinding of oestradiol to oestradiol receptor.

Studies on density-gradient centrifugation indi-cated that the uterine receptor, which migrated as a4.1-4.3 S macromolecule, bound progestins ingeneral rather than the physiological ligand proges-terone. Previous work by McGuire& DeDella (1971),Murugesan & Laumas (1973), Verma & Laumas(1973), Terenius (1974), Kontula et al. (1975) andFeil et al. (1976), demonstrating progestin-mediateddisplacement of progesterone binding to uterinereceptor, supports our observations. However, nodata are available to further characterize the type ofinhibition obtained with the progestins. Our resultsinvolving competitive-receptor assays, which havebeen evaluated by the double-reciprocal plots,unequivocally demonstrate the existence of a com-petitive mechanism between the progestins andprogesterone for the same binding site on the receptor.The interpretation becomes more plausible in that aputative intracellular receptor has been characterizedfor norethindrone and norethindrone acetate, withphysicochemical and kinetic behaviour very similarto that of uterine progesterone receptor (Laumas &Kasid, 1977). Further, these progestins also inhibitedoestradiol binding to oestrogenic receptor, but onlyto the extent of 8-12%. The decrease in the uptake of[3H]oestradiol in the presence of norethynodrel(Laumas et al., 1972), chlormadinone acetate (Rosneret al., 1972) and norethindrone/norethindrone acetate(Van Kordelaar et al., 1975) has been well demon-strated. In the present study, this progestin-oestradiolinteraction was investigated at the receptor level,giving adequate attention to the specific and non-specific binding. It appeared that these progestinsinhibited oestradiol binding by competitively modu-lating the receptor activity.

Receptor affinity is of prime importance in studiesof kinetics and biological proffle of the drugs. Thedisplacement efficiency on density gradients and therelative binding data with [3H]progesterone, [3H]nor-ethindrone or p3H]norethindrone acetate to tag theprogestin receptor suggested that norethindrone hadreceptor-binding affinity comparable with progester-one, but markedly higher than norethindrone acetate.The kinetic constants (Kd and K,) further indicatedthat norethindrone acetate affinity for progestinreceptor was approximately one-tenth that of nor-ethindrone or progesterone, which is contrasted bythe uniformity in receptor concentration (7060-8340molecules/cell; Table 1) for all the three progestins,when quantified concurrently in the cytosol samples.Probably, the discrepancy on the lack of correlationof receptor-binding affinity and the high biologicalpotency of norethindrone acetate could be explained

1978

538

PROGESTIN-STEROID RECEPTOR INTERACTION IN HUMAN UTERUS 539

by the low dissociation rate of progestin-receptorinteraction (t- 110-130min; A. Kasid & K. R.Laumas, unpublished work) and a high conversioninto norethindrone (with strong receptor affinity).Ligand specificity data yielded some valuable

information on the structural requirements for effi-cient binding to progestin receptor, and supportedthe earlier observations by Liao et al. (1973), Smithet al. (1974) and Kontula et al. (1975). In general a3-oxo-4-ene structure is required for effective binding.Modifications of the planar structures of the A/Bring of steroid decreased the binding significantly.The orientation of the 17fl-hydroxy group and as aconsequence its hydrogen-bond geometry and thedirectional interaction of the 17-ethynyl group weredirectly linked to the binding mechanism of progestinon the uterine receptor. The absence of a C-19 methylgroup (19-norsteroids) facilitated the binding con-siderably. However, the introduction ofan additional18-methyl group in d-norgestrel showed dramaticenhancement in the binding. The esterification of the17a-hydroxy function leads to a substantial increasein the binding affinity. However, the introduction ofan acetate group at the 171i-position disrupted thesteroid binding considerably.The conclusive evidence in the present paper that

the progestins bind to the progesterone-binding siteon the receptor, and also inhibit the oestradiol binding,is of considerable significance. It is well known thatoestradiol prepares the uterus for progesteroneaction,and progesterone is necessary for the implantation.Studies with a single silastic implant (Bhatnagaret al., 1 975; Srivastava & Laumas, 1975) indicatethat norethindrone acetate released in microquantities does not appear to interfere with ovulationin the earlier months, but suppresses ovulation duringthe later months ofuse. The contraceptive effect, whenovulation is not suppressed, could probably resultfrom the progestin action at some other site, likeuterus. Thus the interaction of these progestins withthe receptors for progesterone and oestradiol, eitherby simple competition or by change in the relativespatial orientation at the active site, would change thereceptor availability, leading to modulation ofhormone-dependent changes. This would criticallyinfluence uterine 'receptivity' to hormonal stimuli andand thus account for the contraceptive action.

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Acta 317, 403-419

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Interaction of Progestins with Steroid Receptors in Human Uterus