1975 Volume 51 Issue 12 Pages 1033-1042
Norethindrone (ENT), which is a representative in estrane series of progestogen, is not only strongly progestational but also estrogenic and in some cases, antiestrogenic.
To understand progestational effect and antiestrogenic effect, the interactions of ENT on estrogen and progestogen receptors were studied in the uterine cytosol of white female rabbit. The 274,200 × G supernatant of uterine homogenate was used as cytosol. 3H-Estradiol, 3H-Progesterone, 3H-ENT or cold ENT were incubated with uterine cytosol at 4°C for 2 hours.
Results are as follows :
1. Sucrose gradient centrifugation [5-20% linear and 40,000 rpm (159,200 × G) for 16 hours at 4°C] :
ENT was bound to estrogen 8S receptor in immature rabbit uterus (Fig. 2 & 3), and to progestogen 8S receptor in estrogen primed rabbit uterus (Fig. 5).
2. Kinetic study, determined by dextran coated charcoal (0.001 % dextran and 0.1 % charcoal) : (1) In the uterine cytosol of immature rabbit, 3H-estradiol-receptor binding was observed with Kd≅3.6 × 10-9M and it was revealed that ENT was a competitive inhibitor to this binding with Ki≅2.6 × 10-6M, as in Fig. 6. (2) 8S component, obtained by centrifugation of uterine cytosol (Fig. 1) in estrogen primed rabbit, binds 3H-progesterone with Kd≅8.1 × 10-10M and Bm (maximal binding sites) ≅5.0 × 10-8M/ mg of protein, and ENT was a competitive inhibitor in this binding with Ki≅2.3 × 10-9M (Fig. 7 & 8). 3H-ENT-8S binding was demonstrated with Kd≅1.1 × 10-9M and Bm≅8.7 × 10-8M/mg of cytosol protein (Fig. 8).
These results indicate : (a) ENT is bound to both estrogen and progestogen receptors in 8S macromolecules of uterine cytosol, (b) competitive inhibition of ENT to these bindings indicated that ENT is bound to these receptors at the steroid binding sites where estradiol and progesterone bind to, (c) ENT has much more affinity to progestogen receptor (Ki≅2.3 × 10-9M) than to estrogen receptor (Ki≅2.6 × 10-6M), (d) while ENT is bound to progestogen and estrogen receptors at the same time, Bm of ENT (8.7 × 10-8M/ mg of cytosol protein) is more than Bm of progesterone (5.0 × 10-9M/mg of cytosol protein), and Kd of ENT (1.1 × 10-9M) was less than Ki of ENT (2.3 × 10-9M) in the binding to progesterone-receptor.
Biologically, while ENT is bound to progestogen-receptor with high affinity and to estrogen receptor with low affinity, ENT is actually progestational in low dose and antiestrogenic in high dose but the anti-estrogenicity seems to be incomplete in vivo as ENT may be metabolized to a potent estrogenic compound, ethinyl estradiol.
