Harry E. Gray

MOUSE BRAIN TYROSINE HYDROXYLASE AND GLUTAMIC ACID DECARBOXYLASE FOLLOWING TREATMENT WITH ADRENOCORTICOTROPHIC HORMONE, VASOPRESSIN OR CORTICOSTERONE

The activities of tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) from several mouse brain regions were assayed following repeated administration of adrenocorticotrophic hormone (ACTH), lysine vasopressin (LVP) or corticosterone. Although similar treatments with ACTH have been shown to result in changes of catecholamine turnover and GABA content, no changes in the activity of either TH or GAD were observed in any brain region. Likewise LVP had no effect on either enzyme. Since the assays for TH were performed with concentrations of tyrosine and tetrahydrobiopterin cofactor below their respective Michaelis constants, this suggests that the changes of catecholamine turnover are not mediated by changes of TH activity. Twice daily corticosterone adrninistration for four days increased TH activity in the hypothalamus but not in any other brain region.

Hydrodynamic and biochemical correlates of the activation of the glucocorticoid-receptor complex

Possible changes in the size and shape of the glucocorticoid-receptor complex (GRC) following activation remain poorly documented, due to the lability and possible activation of the receptor during the determination of these hydrodynamic parameters. In the present study molybdate was used to stabilize the GRC, thus preventing these uncontrolled transformations. Cytosol prepared from mouse whole brains was incubated for 18 h at 0-2 degrees C with [3H]triamcinolone acetonide (+/- molybdate). Activation was then initiated by incubation at 22 degrees C for variable times and quenched at 0 degree C by adding molybdate. The Stokes radius and sedimentation coefficient of the GRC declined from 77 A and 9.2 S before activation to 58 A and 3.8 S after activation. These measurements remained consistent after recycling GRC between sedimentation and gel filtration procedures and correspond to a 3-fold reduction in the relative molecular mass. The loss and formation of the 297 and 92 kDa species, respectively, after different durations of activation correlated nearly perfectly with increased binding of GRC to DNA-cellulose (DNA-C). The observed size change also correlated well with decreased adsorption to DEAE-cellulose filters (DE-81) and increased adsorption to glass fiber filters (GF/C). The increased adsorption to GF/C may reflect an increase in hydrophobicity which, with extended durations of activation, leads to increased aggregation and reduced binding to DNA-C, but not to a change in adsorption to DE-81. We propose that during activation the 297 kDa form of the GRC splits to form a 92 kDa species that displays an increased affinity for DNA.

Estrogen-induced sexual receptivity and localization of 3H-estradiol in brains of female mice: Effects of 5α-reduced androgens, progestins and cyproterone acetate

Sexual receptivity induced in ovariectomized CD-1 mice with chronic daily administration of estradiol benzoate (E2 B) was blocked by concurrent administration of the 5 alpha-reduced androgen, dihydrotestosterone (DHT). Receptivity was restored in these females with progesterone-, but not with dihydroprogesterone-priming 6 hr prior to testing. Delaying the DHT injections until 12 hr after the E2 B injections greatly reduced its inhibitory properties. Receptivity in E2 B-primed females was also blocked by concurrent treatment with cyproterone acetate and 3 alpha-, but not 3 beta-adrostanediol. Pretreatment with DHT, or 3 alpha- or 3 beta-androstanediol failed to consistently affects 3H-estradiol accumulation in crude nuclear and supernatant fractions from brain and pituitary.

Regional and subcellular [3H]estradiol localization in selected brain regions and pituitary of female mice: Effects of unlabeled estradiol and various anti-hormones

Groups of ovariectomized mice were pretreated for 30 min with either vehicle, unlabeled estradiol benzoate or one of the following anti-hormones: cyproterone acetate, CI-628 or CN-69, 725-27. One hour after an I.V. injection of [3H]estradiol the levels of toluence extractable radioactivity retained in the crude nuclear and cytosol fractions of selected brain regions, pituitary and plasma were determined by liquid scintillation spectrometry. The major findings were: (1) In vehicle treated animals [3H]estradiol uptake in the pituitary greatly exceeded that in all brain regions. Within the brain uptake was greatest in the preoptic anterior hypothalamus (POA-AH) and medial basal hypothalamus (MBH) samples. Nuclear uptake exceeded cytosol uptake only in the pituitary and POA-AH and MBH samples. (2) Estradiol benzoate pretreatment greatly reduced nuclear, and to a lesser extent cytosol [3H]estradiol uptake in pituitary, POA-AH and MBH samples. Estradiol benzoate pretreatment also reduced nuclear uptake in the dorsal middle hypothalamus (DMH). (3) The anti-androgen cyproterone acetate, previously shown to have anti-estrogenic effects in female sexual behavior tests, was found to have no effect on nuclear or cytosol [3H]estradiol uptake in brain and pituitary. (4) The anti-estrogen CI-628 was found to reduce nuclear [3H]estradiol uptake in the POA-AH and pituitary samples. It also reduced cytosol [3H]estradiol uptake in the pituitary sample. The anti-estrogen CN-69, 725-27 produced a much greater inhibition of nuclear [3H]estradiol uptake than CI-628 in the POA-AH, MBH, DMH, dorsal posterior hypothalamus and pituitary samples. This anti-estrogen also reduced cytosol [3H]estradiol uptake in the POA-AH, MBH and pituitary samples. (5) A preliminary chromatographic analysis of vehicle control samples indicated that 70-98% of the nuclear radioactivity in target regions such as POA-AH, MBH and pituitary was iso-polar with authentic estradiol, while less than 50% of the radioactivity in plasma and cortex behaved as estradiol.

Protein synthesis and amnesia: studies with emetine and pactamycin.

Two antibiotic inhibitors of protein synthesis, emetine and pactamycin, have been tested for their effects on cerebral and peripheral protein synthesis and amnesia. Peripherally administered emetine but not pactamycin inhibited cerebral protein synthesis, although this inhibition was lower than that observed with cycloheximide or anisomycin. Pactamycin had a lesser effect on adrenal protein synthesis than emetine. This was reflected in the ability of emetine but not pactamycin to block ACTH-induced corticosteroidogenesis. Anisomycin and cycloheximide caused amnesia in a passive avoidance task, whereas pactamycin and emetine did not. These results are inconsistent with the amnesia being due to inhibition of protein synthesis in a peripheral organ. They are also inconsistent with the amnesia being due to the suppression of an adrenocortical response as previously suggested. No obvious correlation between amnesia and the mechanism of protein synthesis was observed. The most parsimonious explanation is that inhibition of cerebral protein synthesis is necessary for amnesia.

Kinetic analyses of activation-induced changes in the hydrodynamic and surface properties of the glucocorticoid-receptor complex in mouse brain

Unactivated, molybdate-stabilized, [3H]triamcinolone acetonide-labeled, glucocorticoid receptors from mouse whole brain were activated by removal of the molybdate and incubation at 22°C for 1.5 to 24 min and then rapidly quenched at 0°C with molybdate. The loss of the 9.2 S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8 S (activated) form displayed first-order kinetics with a half-time of less than two min. The increase in the 3.8 S form correlated nearly perfectly with an increased binding to DNA-cellulose, and with a decreased and increased adsorption to DEAE-cellulose and glass fiber filters, respectively. The changes in adsorption to these filters, which occurred at a faster rate than did the changes in binding to DNA-C, are thought to reflect an increase in the relative number of positive charges and hydrophobic groups on the surface of the activated complex.

Apparent involvement of protein synthesis and LH-RH in the inhibition by dihydrotestosterone of estrogen-induced sexual receptivity in the ovariectomized mouse

Abstract Sexual receptivity in ovariectomized CD-1 mice induced by chronic daily injections of estradiol benzoate (E 2 B) was inhibited in a dose-dependent fashion by daily injections of dihydrotestosterone (DHT) given 4 or 5 hr prior to the E 2 B injections. Administration of the decapeptide LH-RH 6–8 hr before behavioral testing, or large doses of the protein synthesis inhibitor anisomycin given concurrently with the DHT for 4 days, produced a significant partial restoration of receptivity in these E 2 B+DHT treated mice.

Estrogen Increases Hypothalamic and Pituitary Polyamine Levels in Ovariectomized Rats

The diamine putrescine and the polyamines spermidine and spermine are ubiquitously distributed in animal tissues. Although the details of their physiological role are unclear, increasing evidence strongly associates high polyamine levels with tissue growth in vivo and in vitro (e.g., Janne et al., 1978). Polyamines have been particularly linked to the regulation of nucleic acid and protein synthesis and protein and membrane function (Bachrach, 1973; Raina and Janne, 1975, Janne et al., 1978). In their peripheral nonneural target tissues, trophic hormones intensely and rapidly stimulate the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, leading to increased polyamine levels usually followed by marked increases in tissue growth (Cohen et a]., 1970; Bachrach, 1973, Raina and Janne, 1975). The demonstration by Oka and co-workers that the induction of casein synthesis in mammary gland explants by combined treatment with insulin, cortisol, and prolactin requires spermidine synthesis, and that spermidine could substitute for the cortisol requirement in the stimulation of casein synthesis (Oka and Perry, 1974; Oka et al., 1977), and also the recent demonstration by Igarishi et al. (1978) that spermidine could substitute for testosterone in the stimulation of protease activity in the rat submaxillary gland, strongly suggest that a major effect of these steroid hormones in vivo may be to stimulate polyamine biosynthesis. Steroid hormones exert important organizational and growth-promoting influences on the developing nervous system (see Naftolin and Brawer, 1977, for review) and have profound neurochemical, neuroendocrine, and behavioral effects on the adult nervous system and pituitary (see McEwen et al., 1979, for review). Since nucleic acid and protein synthesis are required for at least some of the central nervous and pituitary actions of sex steroids (Jackson, 1975; Quadagno et al., 1976; Gray and Luttge, 1978; Maurer, 1979), we have investigated the effects of estrogen on polyamine levels in the pituitary, cerebral cortex, and those brain regions of the adult female rat brain that contain the highest densities of estrogen-concentrating cells (Pfaff and Keiner, 1973).

Pentylenetetrazol: inhibitory avoidance behavior, brain seizure activity, and [3H]lysine incorporation into brain proteins of different mouse strains.

The effects of pentylenetetrazol (PTZ,Metrazol) on step-through inhibitory avoidance behavior, brain seizure activity, and Z 3 H]lysine incorporation into brain proteins were examined in Swiss/ICR and C57B1/6J mice. Immediate post-training administration of PTZ (60 mg/kg) resulted in significant retention deficits in both strains of mice when tested 24 hr later. In Swiss/ICR mice, subconvulsive doses of PTZ (50 mg/kg) were sufficient to produce amnesia, and there was no correlation between the presence or absence of convulsions and the induction of amnesia. Electrographic recordings from the cerebral cortex and hippocampus indicated that behavorial convulsions always accompanied sustained high-voltage spiking activity in the hippocampus and cortex. Both convulsive and nonconvulsive doses of PTZ inhibited the incorporation of Z 3 H]lysine into brain proteins during the first 10 min after training and PTZ administration. There was no correlation between the presence or absence of convulsions and the degree of inhibition of Z 3 H]lysine incorporation in the brains of Swiss/ICR mice receiving 50 mg/kg of PTZ. PTZ also inhibited Z 3 H]lysine incorporation into liver proteins. In C57B1/6J mice administered a convulsive dose of PTZ, the inhibition of Z 3 H]lysine incorporation showed the following regional distribution: cortex > diencephalon + striatum > hippocampus ⋍ brainstem. Thus, PTZ both produced amnesia and inhibited the incorporation of Z 3 H]lysine into brain proteins in the absence of gross electrographic abnormalities, but neither of these effects was related to the presence or absence of convulsions.