The highly purified, molybdate-stabilized '8 -9-S' form of the chick oviduct progesterone receptor complexed with [3H]progesterone (Specific activity z 10-12 nmol bound steroid/mg protein) was prepared according to the three-step method Eur. J. Biochem. 127,71-791 and 50 -90 pg samples were injected into a goat and a rabbit. Specific antibodies were observed as early as 2 weeks following the first booster injection and increased considerably 12 weeks later after further booster injections. Results were similar in the two animals, with higher titers observed in the goat. Antibodies cross-reacted with the 'transformed' 4-S form of the chick oviduct progesterone receptor. They also cross-reacted with molybdate-stabilized 8 -9-S progesterone receptors of mammalian species (obtained from MCF-7 human breast cancer cells and from rabbit and mouse uteri), suggesting some evolutionary conservation of steroid hormone receptors. No cross-reaction was observed with chick oviduct oestradiol receptor and with chick plasma transcortin (a non-receptor progesterone-binding protein).The chick oviduct progesterone receptor has been extensively studied, but reports of its purification (review in [I]) have not been followed by successful immunological studies [2]. Anti-receptor antibodies have been obtained against partially purified oestradiol receptor from calf uterus [3, 41, rat liver glucocorticosteroid receptor [5, 61, and progesterone receptor from rabbit uterus [7]. Some of these antibodies have been used for receptor immuno-assay [8] and immuno-cytological studies [9 -1 I]. We have recently achieved the purification to apparent homogeneity of a molybdate-stabilized '8-9-S' form of the chick oviduct progesterone receptor [12-141 and have obtained, after injection of the receptor into a goat and a rabbit, antibodies which cross-react with the progesterone receptor from mammalian species. MATERIALS AND METHODS ReagentsTritiated progesterone, ([2,4,6,7-3H]progesterone, 96 Ci/ mmol), [3H]dexamethasone (1 10 Ci/mmol) and t3H]ORG 2058 (45 Ci/mmol) were from the Radiochemical Centre, Amersham (England). The synthetic radioactive progestin [3H]-R 5020 (87 Ci/mmol) was from New England Nuclear. Their purity was verified by thin-layer chromatography. Non-radioactive steroids were obtained from Roussel-Uclaf, Romainville, except ORG 2058 (a gift from Organon, Oss, The Netherlands).They were > 95 % pure.All chemicals were reagent grade and obtained from Merck (Darmstadt). Dialysis tubing was from Thomas Lab Co. Philadelphia, PA.

In previous studies it was shown that bovine GH (bGH) suppressed and glucagon stimulated the level of 24- and 30- to 34-kilodalton insulin-like growth factor-binding proteins (IGFBPs) in the media of cultured rat hepatocytes. In the present study we have evaluated the regulation of IGFBP-1 gene expression in primary rat hepatocyte cultures. Glucagon produced a dose-dependent stimulation of hepatocyte IGFBP-1 messenger RNA (mRNA), attaining levels 2- to 6-fold greater than control at a glucagon concentration of 100 ng/ml. GH inhibited the accumulation of IGFBP-1 mRNA in a dose-dependent manner producing, 40-70% inhibition at 50 ng/ml. The effect of glucagon was comparable to and additive with dexamethasone (1 microM). The addition of 3-isobutyl-1-methylxanthine (100 microM) and (Bu)2cAMP (100 microM) augmented IGFBP-1 mRNA levels 5- to 6-fold. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (300 nM) was found to inhibit IGFBP-1 mRNA levels by 40-50%. The inhibitory effect of bGH on IGFBP-1 mRNA levels was abolished after preincubation with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (10 microM) for 24 h, whereas glucagon's stimulatory effect was unaffected. The addition of staurosporine (500 nM) and H-7 (1 mM) abolished the inhibitory effect of GH but also significantly inhibited the stimulatory effect of glucagon, a result consistent with these agents acting on both protein kinase C (PKC) and PKA. In the presence of 10 micrograms/ml cycloheximide, IGFBP-1 gene expression was superinduced by bGH, whereas the effect of glucagon was uninfluenced. Thus the inhibitory action of GH involves, in part, the activation of PKC. Glucagon's stimulatory effect seems to involve the activation of PKA. The inhibitory effect of bGH on IGFBP-1 gene expression may require the continuing synthesis of one or more labile protein(s).

Activation (transformation) of the chick oviduct progesterone receptor was found to be induced at 0 degrees C by heparin free in solution as well as by chromatography on a column of heparin linked to acrylamide/agarose. The transformed molecule displayed properties of the activated form of [3H]progesterone-receptor complex obtained by heat treatment or by high ionic strength: smaller size (s20,w = 3.9 S, Stokes radius = 5.2 nm), lower rate of dissociation (t 1/2 approx. 50 h at 0 degrees C compared with approx. 20 h for the 'native' form) and increased binding to phosphocellulose. In all cases, molybdate was an effective inhibitor of transformation and stabilized a large 'native' form (s20,w = 7.9 S, Stokes radius = 7.6 nm). Transformation by neither KCl nor heparin depended on the presence of ligand bound to the receptor, and the properties of the receptor molecule produced by treatment of ligand-free receptor with high ionic strength or with heparin were identical with those of the activated progesterone-receptor complex, demonstrating that receptor activation can be obtained experimentally in the absence of hormone. Our data are compatible with a model in which activation implies separation of the 4 S units, which compose the approx. 8 S 'native' form.

In previous studies it was shown that bovine GH (bGH) and glucagon, when individually added to primary rat hepatocyte cultures, modestly stimulated IGF-I mRNA levels 1.8- to 2.5-fold, but when combined, synergized to stimulate IGF-I mRNA levels by 10- to 12-fold. In the present study we have explored further the mechanism of this effect in primary rat hepatocyte cultures. Like glucagon, the addition of 3-isobutyl-1-methylxanthine (100 microM) or (Bu)2cAMP (150 microM) augmented IGF-I mRNA levels 1.8- to 2.0-fold, but when combined with bGH (50 ng/ml), they augmented levels up to 12-fold. The half-life of IGF-I mRNA, determined by incubating hepatocytes with actinomycin-D was 12 h. Although bGH did not affect the decay rate, glucagon (100 ng/ml) or (Bu)2cAMP (100 microM) reduced the rate of loss by about 70%. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate minimally stimulated IGF-I mRNA levels 1.2- to 1.4-fold, but displayed no synergism when added with bGH, glucagon, or (Bu)2cAMP. The stimulatory effect of bGH plus glucagon was inhibited 80% after preincubation with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (10 microM) for 24 h. The addition of staurosporine, sphingosine, or H-7 [1-(5-isoquinolinyl sulfonyl)2-methyl piperazine] inhibited the stimulatory effect of bGH plus glucagon on hepatocyte IGF-I mRNA by 80%, 90%, and 85%, respectively. Preincubation with cycloheximide (10 micrograms/ml) blocked the synergistic effect of bGH plus either glucagon or (Bu)2cAMP by 65-80%. The effect of glucagon, mediated via the activation of adenylate cyclase, involves in part the posttranscriptional stabilization of IGF-I mRNA levels. The effect of GH, mediated in part by the activation of protein kinase-C, appears to be at the level of transcription. The synergistic augmentation of hepatocyte IGF-I mRNA levels by GH and glucagon involves the activation of PKA and PKC, but also appears to require the synthesis of one or more protein(s).