The ascorbic acid (AA) concentration in anterior pituitary and blood plasma was measured by the Roe-Kuether method in control rats and rats treated with oestradiol benzoate alone, methylene blue alone and with both oestradiol and methylene blue. We have found that methylene blue alone caused a significant drop in hypophyseal both AA and plasma AA concentrations. Methylene blue treatment prevented the increase in plasma AA concentration in oestradiol benzoate-treated rats.
Male rats received estradiol benzoate in a long acting microcrystalline suspension (1 mg/rat i.m., twice a week), methylene blue (MB) 0.5 % in the food and the combination of estradiol and MB. After three weeks, MB partially inhibited the growth response of the anterior pituitary to estradiol and it partially inhibited the increase of cAMP content in anterior pituitary. The increase of anterior pituitary cGMP content was not modified by MB, neither the ratio cAMP/cGMP in the anterior pituitary which, however, decreased after estradiol. This decrease was not modified by MB. On the other hand, the prolactin (PRL) increase in the blood after estradiol was inhibited by MB, although the prolactin content in the anterior pituitary was not. Methylene blue alone did not change blood prolactin concentration, but it unexpectedly elevated blood thyroxine levels and this effect was partially inhibited by simultaneous estradiol treatment.
In three experiments (2 on famales,l on males),we determined the blood flow in the tibia and the distal part of the femur, together with cardiac output (by meansi of 85Sr-microspheres),tibial bone density and tibial ash weight related to bone volume. We found that 1) the bone blood flow always fell significantly after oestradiol benzoate, 2) no change occurred after norethisterone in doses corresponding to those of oestradiol benzoate, but the blood flow showed a tendency to fall after doses one order higher (it decreased significantly in one case only), 3) the density of the tibia and tibial ash weight related to bone volume rose nonsignificantly after oestradiol benzoate, but fell (mostly statistically significantly) after norethisterone. The lowering of the bone mineral indexes in rat bones after norethisterone is a surprising and potentially significant finding requiring further verification.
Quinidine-like immunoreactivity (Abbott TDx Quinidine fluorescence polarization immunoassay) was measured in blood serum of control rats and rats exposed to hypoxia in a hypobaric chamber (7 500 m for one hour). The mean "quinidine" levels (mean±S.D.) were 0.159±0.058 and 0.260±0.110 («mol quinidinq/1 serum), respectively (p<0.01).
Argyrophil nucleolar organizer regions (AgNORs) were morphometrically evaluated in rat anterior pituitaries of control and oestrogenized rats using an automatized (LCJCIA M — Laboratory Universal Computer Image Analysis Micro) and a non-automatized approaches to investigate not only the sensitivity and effectiveness of the automatized procedure but also the morphology of AgNORs in oestrogen-induced rat anterior pituitary hyperplasia. In the experimental oestrogen-induced rat anterior pituitary hyperplasia, the image analysis system LUCIA M was compared to a non-automatized morphometric procedure and proved to be very efficient and yielded analogical results. The AgNOR morphology in the oestrogenized pituitaries was characterized by an increase of the total AgNOR area and clustering of AgNORs in the large nucleoli.
Pituitary hyperplasia as well as proliferation of the endometrium are typical responses to estrogen administration in rodents. Both insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) have been implicated as paracrine mediators and amplifiers of estrogen action in the rodent uterus. The auto/paracrine role of IGF-I, EGF, their receptors and IGF binding proteins in pituitary proliferation has not yet been solved. Here we have used a semi-quantitative reverse transcription polymerase chain reaction (RT PCR) assay to demonstrate the changes in IGF-I mRNA and EGF mRNA abundance in the proliferating male rat pituitary in response to estradiol benzoate (EB; 1 mg/kg b.w. twice weekly i.m. for 3 weeks) and modifying effect of drugs antagonizing the pituitary enlargement — antiestrogen tamoxifen (TAM, 5 mg/kg b.w. daily) and also the dopaminergic agonist terguride (TER, 0.66 mg/kg b.w. daily, routinely used for the treatment of prolactinomas). In three separate experiments, EB induced a 2.2-2.5 fold increase in pituitary weight. The abundance of IGF-I and EGF mRNAs in pituitaries of EB-treated animals did not differ from the controls in two experiments and in the third series with the most marked pituitary hyperplasia mRNAs of both growth factors were even significantly decreased. Antiestrogen TAM administered with EB partially blocked the EB-induced proliferation and significantly stimulated IGF-I mRNA (p = 0.003) and EGF mRNA (p = 0.023) expression, while EB or TAM alone did not stimulate mRNAs of the studied growth factors. Significant antiproliferative effect of dopaminergic agonist TER on EB-induced pituitary proliferation (p = 0.006) was accompanied with decreased IGF-I mRNA (p = 0.025), but not EGF mRNA abundance. Our results suggest that the estrogen-induced pituitary proliferation is independent of the local expression of IGF-I and EGF mRNAs.
The administration of oestrogens increases the hepatic synthesis and plasma level of ceruloplasmin both in man and laboratory animals. Methylene blue, an oxidizing agent and inhibitor of soluble guanylate cyclase, is widely used to block the effects of endothelium-derived relaxing factor (nitric oxide). We describe the inhibitory effect of methylene blue on the increase of ceruloplasmin plasma level in rats during oestradiol treatment.
An increase in bone blood flow (BBF) was observed in rats after castration whereas a decrease in BBF occurred after oestradiol or testosterone. The possible participation of prostaglandins in these changes was demonstrated. The present results show that the endothelium-derived relaxing factor, i. e. nitric oxide (EDRF-NO), might play a role in these hormonal actions on BBF. Until now, almost nothing is known about the possible action of NO on bone circulation. Methylene blue (MB) as a substance blocking EDRF-NO was administered to sham-operated or oophorectomized (OOX) female rats. We determined local blood flow (85Sr-microsphere uptake), cardiac output, blood pressure, heart rate, density of the tibia and ash weight, as well as 24-h incorporation of 45Ca and 3H-proline into the tibia. The administration of MB (0.5 % in the food for 4 weeks) significantly lowered both 85Sr- microsphere uptake and blood flow values in the tibia and distal femur of sham-operated and OOX rats. MB lowered cardiac output and blood pressure to the same extent, indicating no change in the vascular resistance. After the administration of MB (0.1 % in the food), 85Sr-microsphere uptake decreased significantly in the tibia of OOX females while no significant change was found in soft tissues. Bone density and ash weight were significantly lower in OOX rats and in sham-operated rats after MB treatment. Finally, the 24-h incorporation of both 45Ca and 3H-proline decreased significantly in OOX females after MB administration (0.04 % in the food). It can be concluded that 1) MB lowers BBF, suggesting the participation of EDRF-NO in BBF regulation, 2) MB does not influence or may even suppress cardiac output and blood pressure in high dosage, 3) MB lowers 24-hour incorporation of 45Ca and 3H-proline into the tibia of OOX rats, which is in agreement with the circulatory effect, 4) MB lowers bone density and ash weight of the tibia in non-castrated female rats. The effects of MB observed in our experiments partially differ from those of arginine-derived blocking agents. This requires further elucidation.
Adipocyte hormone leptin (OB protein) is considered to be an "adiposity signal" regulating body weight homeostasis and energy balance. We have previously reported that oestrogens (oestradiol-benzoate) significantly decrease the body weight in male rats, increase anterior pituitary and serum levels of the intracellular messenger cAMP, which activates cAMP-dependent protein kinase A , their targets include hormone-sensitive lipase and they influence the brain sympathetic system. The present study tested our hypothesis that oestrogens could influence serum leptin levels in male mice. We found that chronic administration of oestradiol-benzoate significantly attenuated serum levels of leptin, in the dependence on the duration of its administration, and simultaneously decreased body weight. We suppose that oestrogens affect leptin levels interacting with the signal transmission system of cAMP, possibly at the genome level. Our observations that the food consumption of mice with simultaneously decreased body weight and levels of serum leptin support the idea that there exists a satiety factor that counters the effect of low leptin.
Male and female rats were given oestradiol benzoate (1 mg s.c. twice a week for 3 weeks) and/or sodium nitroprusside (SN), a donor of nitric oxide (NO), which was administered in their food in amounts of 0.2 or 0.6 mg/rat/day. Neither oestradiol-induced hypertrophy of the hypophysis, nor the serum prolactin (PRL) level, was affected by the simultaneous administration of SN. The PRL content of the hypophysis rose after oestradiol in the males, but the increase was again uninfluenced by the simultaneous administration of SN and the cAMP content of the hypophysis - raised after oestradiol - was likewise unaffected. The amount of cGMP in the hypophysis after oestradiol rose only in males. Both the serum and the hypophyseal prolactin level were found to be correlated to the cAMP and the cGMP content of the hypophysis. It was found that the simultaneous administration of SN together with oestradiol slightly reduced the increase in the cGMP content of the hypophysis elicited with oestradiol treatment only.