Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or beta-endorphin in female rats.

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.

Binding of Cu2+, Zn2+, and Ni(2+)-GnRH complexes with the rat pituitary receptor.

Complex of copper with the gonadotropin-releasing hormone, GnRH, competed more efficiently for the GnRH receptor than native GVRH, while complexes of nickel with GnRH and zinc with GnRH had slightly lower affinity. Copper ion added to the incubation mixture inhibited the buserelin binding to the receptor.

Receptor binding and conformational properties of bovine and ovine prolactins after chemical modification of the two tryptophan residues.

The two tryptophan residues in ovine and bovine prolactins were modified by reaction with 2-nitrophenylsulfenyl chloride in 75% formic acid. These derivatives exhibited an important loss of receptor affinity (less than 1% of the native hormones) to a rabbit mammary gland preparation. To a lesser degree, they also lost their binding affinity to specific guinea pig antibodies as detected by radioimmunoassay. The chemical modifications induced a change in the folding of the polypeptide chain, which in itself could be partly or totally responsible for the loss of biological or binding activities. This conformational change has been analyzed by circular dichroism and by prediction of secondary structures from the amino acid sequence using the method of Garnier et al. (Garnier, J., Osguthorpe, D.J. and Robson, B. (1978) J. Mol. Biol. 120, 97--120). The comparison of predicted prolactin and somatotropin structures revealed almost identical alpha-helix, turns and coil regions with an overall content of 67% alpha-helix, 5% beta-sheet, 17% turn and 11% of aperiodic structures. These values were close to those obtained from circular dichroism. The conformational change of the chemically modified hormones as compared to native folding, can be described as a partial loss of alpha-helical structure and an increase in beta-sheet content.

Biosynthesis of the luteinizing hormone releasing hormone (LH-RH) in the rat hypothalamus.

The biosynthesis of LH-RH was followed and evaluated in the hypothalamus of intact and castrated female rats after the infusion into the 3rd cerebral ventricle of L-3H-Glycine and its incorporation into the synthetized hormone. Incorporation of 3H-Gly into the LH--RH molecule that means biosynthesis of this hormone was found 30 min after the infusion of labeled amino acid. The incorporation rate in castrated animals was 2 times higher than in intact ones. Estradiol-17 beta only slightly depressed the LH--RH biosynthesis rate in castrated animals. Pretreatment with cycloheximide or puromycin decreased biosynthesis rate of the hormone by 50%. On the basis of cycloheximide and puromycin action it is suggested that the ribosomes are involved in the biosynthesis of LH--RH.