Short-loop feedback mechanism of luteinizing hormone: LH stimulated hypothalamic L-cystine arylamidase to inactivate LH-RH in the rat hypothalamus.

The effect of the intravenous administration of several hormones on L-cystine arylamidase in the hypothalamus of the rat was examined. The injection of 10 mug LH into adult male and female rats was followed within 90 min by an increase of the enzyme activity by 50%, while infantile animals were not affected. When prepuberal rats were pretreated sc with testosterone and progesterone. The iv injection of 0.1 mug oestradiol-17beta into female, and of 0.5 mug testosterone into male intact mature rats also resulted in an increase of hypothalamic enzyme activity. The maximal increase in enzyme activity was seen 16 h after steroid treatment. As it had previously been shown that L-cystine arylamidase inactivates LH-RH, it may be assumed that this enzyme is involved in the short-loop feedback of LH. This assumption is based on the observation that an elevation of plasma LH brings about an activation of the enzyme system, which subsequently leads to an increased inactivation of LH-RH in the hypothalamus. This mechanism seems to depend on the presence of certain plasma levels of oestrogen in female, and of testosterone in male animals.     

Stimulation by oestradiol of soluble-protein synthesis in rat hypothalamus

The effect of oestradiol treatment on protein synthesis in the cytosol fraction (10⁵ g supernatant) from overiectomized mature and developing female rat hypothalami were studied. Results obtained on adults by double-label technique and SDS polyacrylamide gel or cellogel electrophoresis show that, as in the uterus, oestradiol-induced protein synthesis (IP) occurred in the cytosol fraction of the hypothalamus. The molecular weight of IP was about 40 000 dalton. During postnatal development, IP was also observed in cytosol fractions from the hypothalami of female rats 14, 21 and 28 days old. No clear-cut effect of oestradiol was found in the 7-day-old animals.     

Sex differences in the regulation of oxytocin receptors by ovarian steroids in the ventromedial hypothalamus of the rat.

The facilitation of sexual receptivity by oxytocin (OT) in female rats is related to the regulation of oxytocin receptors (OTR) by ovarian steroids in the ventromedial nuclei (VMN) of the hypothalamus. In a previous study, we have shown that estradiol benzoate (EB) causes a twofold increase in OTR binding in the VMN. Progesterone (P) then modulates levels of the estrogen-induced OTR and increases the area occupied by the receptors by acting on the neuronal membrane. In the present study, we compared the effects of EB and P on OTR binding between males and females. In both sexes, EB increased the density of OTR and the area covered by the receptors at the level of the medial and caudal VMN. In estrogen-primed females, P further increased OTR levels in the medial VMN and the area covered by OTR at the level of the caudal VMN. By contrast, P did not modulate OTR binding in estrogen-primed males. Thus, the behavioral insensitivity of male rats to ovarian hormones, in particular to P, may be related to sex differences affecting the modulation of OTR binding.     

Evaluation of changes in the secretion of corticotrophin releasing activity using the isolated rat hypothalamus incubated in vitro

The secretion of corticotrophin releasing activity (CRA) from the isolated rat hypothalamus incubated in vitro was investigated under various conditions of incubation and of pretreatment of donor animals providing hypothalami. Media from hypothalamic incubations were assayed for CRA by a validated double in-vitro bioassay technique which differentiates CRA from vasopressin. A circadian rhythm was found in the secretion of CRA in vitro from isolated hypothalami obtained from animals killed at different times of the day. Secretion of CRA increased significantly at 19.00 h (dusk) compared with the secretion rate at 07.00 h, in synchrony with a rise in plasma corticosterone levels. In addition, both plasma corticosterone concentrations and CRA secretion in vitro were higher at 07.00 h than at 19.00 h after exposure of the donor animals to a reversed light cycle for 7-10 days. Hypothalami obtained from animals chronically treated with betamethasone in the drinking water showed a diminished secretion of CRA in vitro. Exposure of untreated animals to ether vapour for 2 min immediately before death significantly increased the subsequent secretion of CRA in vitro. Ether exposure did not significantly affect the secretion of CRA in vitro from hypothalami of betamethasone-treated rats. There was a close correlation between plasma corticosterone levels and in-vitro CRA release after these treatments. The results suggest that the secretion of CRA examined in this way is a phenomenon which can reflect the changes which occur in the activity of the hypothalamo-pituitary-adrenal system in vivo during the 24-h cycle, after glucocorticoid treatment and after ether stress.     

Stimulation of aromatase activity in the fetal rat gonads by cAMP and FSH

The gonads from 17- to 21-day-old fetal rats were cultured in vitro in the presence of [3H]testosterone and in the presence or absence of cAMP or FSH, and estrone and estradiol formed were measured by double isotopic dilution and recrystallization to constant specific activity. Estrogen synthesis by testes was stimulated by both cAMP and FSH as early as at 18 days of gestation. FSH did not enhance aromatase activity in ovaries, although cAMP did. It is remarkable that FSH controls estrogen synthesis in the testis earlier than in the ovary.     

Stimulation of aromatase activity in the fetal rat testis by cyclic AMP and FSH

The effect of dibutyryl cyclic AMP and FSH on oestrogen biosynthesis was investigated in testes from 18- to 21-day-old fetal rats cultured in vitro in the presence of tritiated testosterone. Oestrone and oestradiol concentrations were measured by determination of constant specific activity after isotopic dilution. Dibutyryl cyclic AMP and FSH markedly stimulated the conversion of testosterone into both oestrone and oestradiol at all stages studied. Oestradiol synthesis was stimulated by two- to sevenfold, while stimulation of oestrone synthesis was even greater. The results demonstrate that the aromatase enzyme system of the fetal rat testis responds to cyclic AMP and FSH.     

Stimulation of ornithine decarboxylase activity by insulin in developing rat brain

Insulin administered ip or intracisternally (ic) increased the activity of ornithine decarboxylase (ODC) in whole brains and brain parts of neonatal rats. Maximal stimulation of activity occurred 4-5 h after ip administration. At the highest doses, insulin stimulated ODC activity by up to 5- and 8-fold after ip and ic injection, respectively. The same amount of insulin given ic caused greater increases in activity than when given ip. Insulin stimulated ODC activity in 2-day-old and in 17- to 60-day-old rats but not in 5- or 9-day old neonates or 80-day-old adults. When insulin-induced hypoglycemia was prevented by giving dextrose, the stimulation of ODC activity was approximately the same as that in animals receiving insulin without dextrose. This indicates that insulin-induced stimulation of brain ODC activity was not caused by insulin-induced hypoglycemia or physiological responses to hypoglycemia. Since ODC is considered an indicator of growth stimulation, these results suggest that insulin or insulin-like peptides have a role in the regulation of brain development.     

Relationship between erythrocyte volume and sodium transport in the Milan hypertensive rat and age-dependent changes

The relationship between differences in red blood cell (RBC) volume and ion transport across the erythrocyte cell membrane were investigated in the Milan Hypertensive (MHS) and Milan Normotensive (MNS) rat strains, under different experimental conditions and during ageing. The results obtained indicate that: the difference in Na+/K+ cotransport between MHS and MNS disappear when the RBC volume of the two strains becomes equal under hypotonic swelling; MHS RBCs are osmotically more fragile than those of MNS, probably because of a different membrane structure rather than a different amount of membrane surface, and the smaller volume and the lower Na+ content of MHS RBCs are maintained throughout the life span, while Na+/K+ pump activity and Na+/K+ cotransport undergo age-dependent changes, related to the development of hypertension. All these findings suggest that a primary abnormality of the cell membrane structure of MHS, probably located in the cytoskeleton, is responsible for the cell functional alterations that we previously demonstrated to be genetically associated with MHS hypertension.     

Regulation by gonadal steroids of the mRNA encoding for a type I receptor for TGF-beta in the female rat hypothalamus

We have recently shown that the mRNA encoding for a type I receptor for transforming growth factor beta and activin - named B1 - is expressed in hypothalamic areas implicated in gonadotropin-releasing hormone regulation, particularly in estrogen-receptive regions. In the present study, we examined whether ovarian steroids may regulate expression of B1 mRNA in the hypothalamus. Comparing relative levels of B1 mRNA expression in ovariectomized (OVX), OVX + estradiol-treated, and OVX + estradiol + progesterone-treated female rats, we observed that estrogen significantly (p < 0.05) downregulated B1 mRNA levels in the anteroventral periventricular nucleus (by 12.5%), medial preoptic nucleus (by 27.5%), and arcuate nucleus (by 29.5%). In contrast, no effects of gonadal steroids were observed in the median preoptic nucleus. We next examined whether cells expressing B1 mRNA may be direct targets for the action of estrogen. Using an in situ hybridization coupled to immunohistochemical labeling, we found that many B1-mRNA-expressing cells also exhibited estrogen receptor alpha immunoreactvity in anteroventral periventricular nucleus, medial preoptic nucleus, and arcuate nucleus. Taken together, these results reveal that estrogen may directly modulate expression of B1 mRNA in the hypothalamus and support the idea that transforming growth factors beta play an important role in the hypothalamic control of gonadotropin-releasing hormone function.     

Sex steroids stimulate the activity of hypothalamic arylamidases in the rat

The effect of treatment of rats with ethinyl estradiol, testosterone, and progesterone on the measurement of hypothalamic arylamidase activity by synthetic substrates was studied. A method which uses L-alpha-amino acid-p-nitroanalides instead of beta-naphthylamides for measuring arylamidase activity is presented. The advantages of this technique are a shortening of reaction time and a molar extinction coefficient of p-nitroaniline which is twice as high as that of the dye made when beta-naphthylamine is used. There was a sex-dependent response in the activity of hypothalamic arylamidases to treatment of the rats with the sex steroids. In female rats, the injection of 7 mcg ethinyl estradiol was followed by a significant rise of L-cystine and L-glutamic acid arylamidase activity 16 hours later. In male rats, the injection of testosterone and of ethinyl estradiol brought about a general stimulation of all enzyme activities, indicating that this enzyme system responds to steroid treatment of the animals with a sex-specific pattern.     

Age-related changes in growth hormone releasing factor and somatostatin in the rat hypothalamus

Distribution and staining intensities of growth hormone releasing factor (GR (GRF) and somatostatin (SRIF) were examined in young (3 months of age) and old (24 months of age) male rats of Sprague-Dawley strain, using the PAP immunocytochemical procedure. Some animals of each age group were intraventricularly injected with colchicine to demonstrate immunoreactive neuronal perikarya. GRF-immunoreactive intensities of old rats were markedly reduced in the median eminence as compared with those of young rats. No remarkable difference could be detected between SRIF immunoreactivities in the young and old animals, since intensive SRIF immunoreactivities were found in the external layer of the median eminence of both groups of animals. Between two age groups injected with colchicine, we also found no difference in the distribution and staining intensities of immunoreactive perikarya of GRF and SRIF in the hypothalamus and also detected no significant difference in total neuron numbers of each peptide. These findings suggest that the synthesis and/or release of GRF in GRF-containing neurons are decreased, though GRF-containing neurons themselves remain alive and have the capacity to synthesize GRF.     

Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.     

Stimulation by alpha-adrenergic mechanisms of the secretion of growth hormone-releasing factor (GRF) from perifused rat hypothalamus

The possible involvement of adrenergic mechanisms in regulating the secretion of growth hormone (GH)-releasing factor (GRF) from the rat hypothalamus was examined in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated GRF release from the hypothalamus. The infusion of clonidine (10(-4) M), an alpha 2-adrenergic stimulant, resulted in an increase in the spontaneous release of GRF. In the presence of propranolol (10(-5) M), a beta-adrenergic blocking agent, clonidine (10(-5) and 10(-4) M) stimulated GRF release more prominently in a dose-related manner, whereas propranolol (10(-5) and 10(-4) M) by itself did not affect the spontaneous GRF release. The stimulatory effect of clonidine (10(-4) M) on GRF release in the presence of propranolol was inhibited by yohimbine (10(-4) M), an alpha 2-adrenergic blocking agent. These findings suggest that alpha 2-adrenergic mechanisms play a role in stimulating GRF release from the hypothalamus in rats.