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Glucocorticoid Receptors in Lung

II. SPECIFIC BINDING OF GLUCOCORTICOIDS TO NUCLEAR COMPONENTS OF RABBIT FETAL LUNG

From the ¹ From the Departments of Experimental Medicine and Biochemistry, McGill University and the University Clinic, Royal Victoria Hospital, Montreal, Canada

The specific interaction of glucocorticoids with lung and liver nuclei of rabbit fetuses was studied in vivo and in vitro. Twenty minutes after the administration of [³H]cortisol to rabbit fetuses in utero, the concentration of radioactivity in lung nuclei is about 5 times higher than in liver nuclei. The simultaneous administration of a 250-fold excess of non-labeled cortisol almost completely inhibits the nuclear uptake of the labeled hormone. A major fraction of the radioactivity (identified as cortisol) extracted from lung and liver nuclei is bound to macromolecules excluded from Sephadex G-25.

After incubation of fetal lung and liver with [³H]cortisol or [³H]dexamethasone at 37°, it was demonstrated that both tissues contain a limited number of specific nuclear binding sites which are saturated with low hormone concentrations (1 x 10^-8 m dexamethasone and 5 x 10^-8 m cortisol). The concentrations of specific binding sites are estimated to be 6000 per nucleus in the lung and 1500 per nucleus in the liver, assuming a homogeneous cell population. The ability of various steroids to block the nuclear uptake of [³H]cortisol and [³H]dexamethasone in fetal lung correlates well with their glucocorticoid potency with the exception of cortisone and 21-deoxycortisol. The effect of cortisone is due to the ability of the fetal lung, under the conditions used, to convert efficiently cortisone to cortisol. The nuclear glucocorticoid-binding macromolecule of fetal lung appears to be a protein since the complex is destroyed by Pronase and p-chloromercuribenzoate but not only by nucleases.

Competition studies indicate that in the fetal lung both [³H]cortisol and [³H]dexamethasone interact with the same nuclear sites, but the steroid-protein complexes differ in stability. The [³H]cortisol-protein complex is very unstable and dissociates rapidly at 0°. In contrast, the [³H]dexamethasone-protein complex is relatively stable, and it was characterized by sucrose density gradient centrifugation and gel filtration on Sephadex G-200. The complex has a sedimentation coefficient of 4 to 5 S in the presence or absence of 0.4 m KCl. Upon gel filtration through Sephadex G-200 bound [³H]dexamethasone in lung nuclear extracts is resolved into two fractions. The first fraction represents heavy complexes (probably aggregate) which are excluded from the gel. The second and major fraction of bound [³H]-dexamethasone is associated with proteins which are retained by the gel and have a molecular weight of about 150,000.

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