Interaction of the anti-oestrogen, nafoxidine hydrochloride, with the soluble nuclear oestradiol-binding protein in chick liver

Abstract

Nafoxidine hydrochloride (Upjohn, 11100A)injected with oestradiol into immature chicks inhibits the hormone-induced increase in [3H]oestradiol-binding activity in salt extracts of liver nuclei as well as the subsequent production by liver of egg-yolk phosphoprotein. Substantial inhibition of both oestradiol-induced responses is seen when nafoxidine is given in a dose approximately equimolar with that of oestradiol. In vitro nafoxidine competitively inhibits binding of [3H]oestradiol in nuclear extracts. The Ki for the inhibition is 43 nM, which indicates an affinity of nafoxidine for the binding protein about 4% of that of oestradiol. The inhibitory action of nafoxidine in vivo thus is more potent than the relative binding affinity determined in vitro might indicate. One possible explanation is that the primary site of nafoxidine action is at a point proximal to nuclear receptor interaction. Nafoxidine injected alone into the chick does not induce phosphoprotein synthesis, but it does increase [3H]oestradiol-binding activity in extracts of liver nuclei to a limited extent. No differences in the properties of the oestradiol-binding activity in extracts from nafoxidine-treated chicks or from oestradiol-treated chicks were detected. Chick liver cytosol does not contain detectable high-affinity oestradiol-binding activity. A low-affinity oestradiol-binding component with a sedimentation coefficient of 3.5S was found, but it was unaffected by treatment of chicks with earlier nafoxidine or oestradiol. The results suggest a difference in the mechanism of oestradiol action in the chick liver and in the widely studied rat uterus, on which the usual model for oestradiol action is largely based.

A high-affinity oestrogen-binding protein in cockerel liver cytosol

“…At 10-fold excess, oestradiol-17a and oestriol are only 50% as potent as the stronger oestrogens. These relative affinities are similar for the soluble nuclear oestrogen receptor in cockerel liver (Lazier & Alford, 1977).…”

“…Regardless of whether or not complete recovery of high-affinity oestrogen-binding sites from cockerel liver cytosol is presently being achieved, it is clear that a binding activity that has many properties of a typical oestrogen receptor can be detected, and that earlier speculation that the mode of action of oestrogen in avian liver was independent of a cytosol receptor mechanism is not justified (Sheridan, 1975;Gschwendt, 1975a;Lazier & Alford, 1977). The dynamics of regulation of receptor concentrations in avian liver may differ somewhat from that in other target tissues.…”

“…In avian liver, there seems to be initial translocation of the small number of 1979 cytoplasmic sites to the nucleus, followed by marked nuclear receptor accumulation by a mechanism involving either induced synthesis and/or stabilization of the protein (Schneider & Gschwendt, 1977). Non-steroidal anti-oestrogens appear to be more purely antagonists of oestrogen action in liver than in uterus, possibly because they block the mechanism of nuclear receptor accumulation in the former tissue (Gschwendt, 1975b;Lazier & Alford, 1977), but permit a substantial increase in nuclear receptor in the latter tissue (Clark et al, 1974). A further consideration with regard to the role of cytoplasmic oestrogen receptor in avian liver is that very high doses of oestradiol in propylene glycol are necessary to stimulate a prolonged increase in nuclear oestrogen-receptor concentration and apparently consequent vitellogenin production (Lazier, 1975;Joss et al, 1976;Schneider & Gschwendt, 1977;Burns et al, 1978).…”

“…Oestrogen induction of the synthesis of egg-yolk protein (vitellogenin) by avian or amphibian liver is a model for the study of oestrogen action which has the particular advantage that highly specific transcriptional changes can be measured accurately in both the primary and the secondary responses (Tata, 1976;Deeley et al, 1977;Baker & Shapiro, 1978). We have been studying oestrogen receptors in avian liver with the long-term goal of understanding their role in the regulation of the vitellogenic response (Lazier, 1975(Lazier, , 1978Lazier & Alford, 1977).…”

“…Liver nuclei from oestrogen-treated immature chickens, roosters or laying hens contain a substantial concentration 'of specific, high-affinity oestradiolbinding activity generally considered to be 'receptor' (Mester & Baulieu, 1972;Gschwendt & Kittstein, 1974;Lazier, 1975;Joss et al, 1976;Lebeau et al, 1977). The prediction that such oestrogen-receptor sites would be found in the liver cytosol of untreated immature chicks or roosters could not be substantiated by using conventional techniques (Gschwendt, 1975a;Lazier & Alford, 1977;Lebeau et al, 1977). However, Gschwendt (1977) has characterized a highaffinity oestrogen-binding protein in embryonicchicken liver cytosol.…”