Dysregulation of corticosterone secretion in streptozotocin-diabetic rats: modulatory role of the adrenocortical nitrergic system.

An increased activity of the hypothalamo-pituitary-adrenal axis resulting in exaggerated glucocorticoid secretion has been repeatedly described in patients with diabetes mellitus and in animal models of this disease. However, it has been pointed out that experimental diabetes is accompanied by a decreased glucocorticoid response to ACTH stimulation. Because previous studies from our laboratory demonstrate the involvement of nitric oxide (NO) in the modulation of corticosterone production, present investigations were designed to evaluate 1) the impact of streptozotocin (STZ)-induced diabetes on the adrenocortical nitrergic system and 2) the role of NO in the modulation of adrenal steroidogenesis in STZ-diabetic rats. Four weeks after STZ injection, increased activity and expression levels of proteins involved in L-arginine transport and in NO synthesis were detected, and increased levels of thiobarbituric acid reactive species, carbonyl adducts, and nitrotyrosine-modified proteins were measured in the adrenocortical tissue of hyperglycemic rats. An impaired corticosterone response to ACTH was evident both in vivo and in adrenocortical cells isolated from STZ-treated animals. Inhibition of NO synthase activity resulted in higher corticosterone generation in adrenal tissue from STZ-treated rats. Moreover, a stronger inhibition of steroid output from adrenal cells by a NO donor was observed in adrenocortical Y1 cells previously subjected to high glucose (30 mM) treatment. In summary, results presented herein indicate an inhibitory effect of endogenously generated NO on steroid production, probably potentiated by hyperglycemia-induced oxidative stress, in the adrenal cortex of STZ-treated rats.

Levonorgestrel antagonism on estrogen-induced pituitary tumors is mediated by progesterone receptors.

Using both IN VITRO and IN VIVO approaches, we studied the antagonism exerted by the synthetic progestin levonorgestrel on estrogen-induced prolactinomas, considering that levonorgestrel shows partial androgenic properties and that androgens inhibit estrogen-induced prolactin synthesis and secretion. In the tumors, binding of estrogens to their receptors was competed neither by progesterone receptor ligands nor by androgen receptor ligands, ruling out direct inhibitory effects of these drugs on tumor development. Progestin binding was competed by the progesterone receptor agonists progesterone and levonorgestrel, by the antagonist mifepristone, and also by the androgen dihydrotestosterone, whereas the androgen receptor antagonist hydroxyflutamide was a weak competitor. In addition, both progesterone receptor and androgen receptor ligands competed for binding to androgen receptors. In primary cultures of pituitary tumors, levonorgestrel decreased prolactin secretion, an effect that was blocked by mifepristone but not by hydroxyflutamide. IN VIVO results indicated that levonorgestrel inhibition of both estrogen-induced pituitary weight increment and hyperprolactinemia was reduced by mifepristone, whereas flutamide was unable to block levonorgestrel effects. Our results suggest that even when an interaction of levonorgestrel with androgen receptors in the tumors is possible, the antagonistic effects of levonorgestrel on tumor development and functionality are mediated by progesterone receptors.

Gender differences in the expression of galanin and vasoactive intestinal peptide in oestrogen-induced prolactinomas of Fischer 344 rats.

We have previously described a sexual dimorphism in oestrogen-induced anterior pituitary tumorigenesis in Fischer 344 rats, with female tumours averaging twice the size of those of males. Neonatal androgenization of female Fischer 344 rats with 100 micro g of testosterone propionate reverted that effect, causing a 'male-like' phenotype. The peptides galanin and vasoactive intestinal peptide (VIP) are possible mediators of oestrogen effects on the anterior pituitary, including hyperprolactinemia and lactotroph proliferation. To further extend our previous findings, we investigated the expression of galanin and VIP in the anterior pituitary of control and oestrogenized male, female and neonatally androgenized female Fischer 344 rats. At 3 months of age, rats were deprived of their gonads and divided into control and diethylstilbestrol (DES)-treated groups. In the anterior pituitary of control rats, galanin and VIP immunoreactive cells were absent. However, in DES-treated rats, pituitaries from normal ovariectomized females showed higher number of galanin and VIP positive cells than pituitaries from neonatally androgenized ovariectomized females and gonadectomized males. This pattern correlated with changes in anterior pituitary weight and serum prolactin. Our study suggests that sexual differences in oestrogen-induced pituitary tumorigenesis could be due to the differential expression of galanin and VIP. Furthermore, our data support the fact that neonatal exposure to androgens, as in normal males and androgenized females, may condition the response of the pituitary gland to oestrogens in adult life.

Progestin regulation of galanin and prolactin gene expression in oestrogen-induced pituitary tumours.

Galanin is a peptide widely distributed in the hypothalamic-pituitary axis. In the female rat pituitary, galanin is mainly present in lactotrophs, where it regulates their secretion and proliferation. Galanin expression is increased in oestrogen-induced prolactinomas, and it has been proposed that oestrogen effects on lactotroph function and proliferation could be mediated by galanin. Previous studies from our laboratory demonstrated that the synthetic progestin levonorgestrel antagonizes pituitary tumorigenesis of rats given oestrogen, reducing the number of proliferating cells and increasing cell death by nonapoptotic mechanism(s). To elucidate the role of galanin in levonorgestrel effects on the tumours, we examined galanin and prolactin mRNA and peptide expression in prolactinomas of rats receiving the progestin. Levonorgestrel reduced the pituitary weight and serum prolactin concentrations in oestrogen-treated rats. Galanin mRNA expression (determined by in situ hybridization), and the number of galanin expressing cells (determined by immunocytochemistry) were also reduced by the progestin in tumour-bearing rats. However, neither prolactin mRNA content, nor the number of prolactin-expressing cells, were modified by levonorgestrel treatment of oestrogen-receiving rats. The present study suggests that levonorgestrel controls pituitary growth by diminishing galanin expression. In contrast, changes in serum prolactin concentration seem to be more related to the reduction in tumour size, since the reduction in galanin expression was not large enough to regulate prolactin mRNA expression or the percentage of lactotrophs.

Sexual dimorphism in diethylstilbestrol-induced prolactin pituitary tumors in F344 rats.

Female F344 rats treated chronically with diethylstilbestrol (DES) develop prolactin (PRL)-producing pituitary tumors. These tumors are larger in female than in male rats. To investigate gender differences in DES-induced pituitary tumor formation, we employed female and male rats and neonatally androgenized females, which received 100 microg of testosterone propionate (TP) after birth. At 3 months of age, all rats were deprived of their gonads and divided into control and DES-treated groups. Forty days after beginning treatment, control pituitary weight and serum PRL were similar in gonadectomized males (GDX), ovariectomized females (OVX) and androgenized-ovariectomized females (OVX + TP), but weight of DES-induced tumors was 2.5-fold higher and serum PRL 5.6-fold higher in OVX + DES than in GDX + DES or OXV + TP + DES (p<0.001). At the pituitary level, nuclear estrogen receptors (NE(2)R) amounted to >100 fmol/mg DNA in all rats receiving DES. However, NE(2)R were lower in OVX + DES (101.3+/-9.0 fmol/mg DNA) than in GDX + DES (174.6 +/-16.8; p<0.05) and in OXV + DES + TP (150.3+/-27.7; p<0.05). A similar profile was found for cytosolic progestin receptors. Using electron microscopy (EM), hyperplasia/hypertrophy of lactotropes was found in all DES-stimulated pituitaries. However, tumors of OVX + DES rats were enriched in hyperstimulated typical lactotropes, i.e., cells with high rate of hormonal synthesis, processing and secretion. Instead, tumors from GDX + DES and OVX + TP + DES rats were a mixture of typical and atypical lactotropes, i.e. a cell subpopulation with refractory secretory response and a few gonadotropes. In agreement with these data, immunoreactive pituitary PRL was lower in OVX + DES than in OVX + TP + DES and GDX + DES groups. Thus, differences in the sensitivity to DES, serum and tumor PRL, NE(2)R and progestin receptors between estrogenized female rats on one side and male and TP-androgenized females on the other, may by due in part to heterogeneity of cell populations. Our data further suggest that neonatal hypothalamic exposure to androgens, as in normal males or androgenized females with masculinization of hypothalamic centers, may condition the response to DES stimulation later in life.

Antagonism by progesterone of diethylstilbestrol-induced pituitary tumorigenesis in Fischer 344 rats: effects on sex steroid receptors and tyrosine hydroxylase mRNA.

It is known that chronic exposure of F344 rats to diethylstilbestrol (DES) induces prolactin (PRL)-secreting pituitary tumors composed of proliferating mammotropic cells. In the present work, we studied the effects of progesterone (P4) on several parameters stimulated in the pituitary tumors (DES-T), such as nuclear estrogen receptors (NE2R), cytosolic progestin receptors (CP4R) and serum PRL. Additionally, we have measured in hypothalamus the mRNA levels for tyrosine hydroxylase (TH), the rate-limiting enzyme for synthesis of dopamine, the main PRL-inhibitory factor. We found that pellet implantation of P4 during 1 month significantly reduced weight, ligand binding to NE2R and CP4R and serum PRL in the tumorous glands. Reductions in sex steroid receptor binding were due to changes in Bmax without changes in Kd, as observed after Scatchard plot analysis. Receptor binding data, therefore, suggests a pituitary site of action of P4. TH mRNA expression was studied in tuberoinfundibular dopaminergic (TIDA) neurons by in situ hybridization techniques employing a 35S-labeled oligonucleotide probe. Mean number of grains/cell decreased significantly in DES-T, an effect partly reversed by P4 treatment. Frequency histograms were constructed by plotting the number of cells versus the number of grains/cell and examined by x2 test and analysis of residuals. We found that DES-T presented significantly more cells with less grains whereas in control glands, P4-treated rats and DES-T receiving P4, cells with a higher grain number prevailed. These results suggest that in addition to a direct pituitary effect, P4 may also antagonize DES-induced tumorigenesis acting on mRNA for TH and presumably on the activity of TIDA neurons of the hypothalamus. The use of DES-T as a model for hyperprolactinemia may allow further assessment of P4 effects in pituitary adenomas in humans.

The 21-aminosteroid U-74389F increases the number of glial fibrillary acidic protein-expressing astrocytes in the spinal cord of control and Wobbler mice.

1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Given in vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [3H]dexamethasone binding when added in vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.