Effects of hyperprolactinemia on testosterone production in rat Leydig cells

The pathogenesis of hyperprolactinemia (hyperPRL) induced hypogonadism has been suggested to be related with a dysfunction of hypothalamus-pituitary-testis axis. While the direct inhibitory effects of prolactin (PRL) on testosterone (T) release have been demonstrated, the mechanism is still unclear. Our previous study demonstrated a diminished T release in the testicular interstitial cells (TICs) from the anterior pituitary (AP)-grafted rats as compared with the control, and the pattern was in agreement with the in vivo model. However, TICs incubation cannot totally represent the response of the Leydig cells. Therefore, a Percoll gradient purified Leydig cell model was adopted to explore the response of T release under similar challenges in this study to investigate the effects of hyperPRL on the Leydig cells per se. HyperPRL in male rats was induced by grafting rat AP under the renal capsule. The control animals were grafted with rat brain cortex tissue (CX). Six weeks after grafting, the rats were sacrificed. Either TICs or Leydig cells were isolated, respectively, for in vitro incubation and challenge. Challenge drugs included human chorionic gonadotropin (hCG, 0.05 IU/ml), steroidogenic precursors (25-OH-cholesterol, 10(-6) M; pregnenolone, 10(-6) M), forskolin (an anenylyl cyclase activator, 10(-4) M) and 8-bromo-3':5' cyclic adenosine monophosphate (cAMP) (8-Br-cAMP 10(-4) M). T released by TICs or Leydig cells was determined by radioimmunoassay. The TICs from the AP-grafted rats showed lower levels of T release than the control group while the purified Leydig cells demonstrated a reverse pattern in response to challenges of hCG, steroidogenic precursors, forskolin and 8-Br-cAMP. In hyperPRL rats, a paradoxical pattern of T release between TICs and purified Leydig cells is observed. The purified Leydig cells from AP-grafted rats demonstrated a higher level amount of T release than the control after stimulation. The phenomenon can be attributed to the change of Leydig cell sensitivity to the stimulation after the effects of chronic hyperPRL. Moreover, another possibility is the role played by other interstitial cells to modulate steroidogenesis in Leydig cells.     

Effect of experimentally induced hyperprolactinemia on growth hormone secretion

In order to study the existence of possible interrelation-ships between prolactin (PRL) and growth hormone (GH) secretions, adult male rats bearing an anterior pituitary graft under the kidney capsule since day 90 of life and their sham-operated controls were submitted to a single i.p. administration of L-dopa (50 mg/kg weight) or saline 30 days after the operation. Plasma PRL and GH levels were measured by using specific RIA methods. Dopamine (DA) and norepinephrine (NE) contents in the hypothalamus and in the in situ anterior pituitary gland were measured by using a specific radioenzymatic assay. An increase in plasma PRL levels and a decrease in plasma GH levels were shown in grafted rats. Hypothalamic contents of DA and NE were increased in these animals, while the anterior pituitary content of DA was not modified as compared to controls. The administration of a single injection of L-dopa led to decreases of plasma PRL and GH levels in both grafted and control rats, but while marked increases in hypothalamic and anterior pituitary contents of DA were shown in both groups, the hypothalamic content of NE was only increased in control animals. These data suggest that PRL and GH secretions were closely related. Dopamine could be mediating the action of PRL on GH, while NE would be less involved.     

Morphology of the epithelial cells and expression of androgen receptor in rat prostate dorsal lobe in experimental hyperprolactinemia

The effect of hyperprolactinemia on the prostate has not been well investigated. Since androgens play an important role in prostate development, growth and function, the goal of the present study was to estimate the influence of hyperprolactinemia on expression of the androgen receptor (AR) in rat epithelial cells of prostate dorsal lobe and on morphology of these cells. Studies were performed on sexually mature male Wistar rats. The experimental group rats received metoclopramide (MCP) intraperitoneally to provoke hyperprolactinemia. The control group animals were given saline in the same way. For light and electron microscopy the prostate dorsal lobes were obtained routinely. To evaluate the intensity of immunohistochemical reaction for AR in epithelial cells, the optical density was measured and computer-assisted image analysis system was used. Morphological observations of the dorsal lobe epithelial cells were carried out in transmission electron microscope. MCP caused over twofold increase in prolactin (PRL) serum levels. In rats with hyperprolactinemia, the testosterone levels (T) were twofold decreased. The intensity of immunohistochemical reaction for AR in epithelial cells of dorsal lobe in the experimental group was significantly lower than in the control group. In the dorsal lobe epithelial cells of experimental group animals, the transmission electron microscopy (TEM) revealed highly dilated RER cisternae and reduced number of microvilli on the cellular surface when compared to the control group. The results show that hyperprolactinemia in male rats causes morphological abnormalities in the dorsal lobe of prostate. The abnormalities are caused by elevated prolactin either directly or indirectly through decreased level of testosterone. Decreased expression of AR in epithelial cells of prostate dorsal lobe is likely to be caused by decreased testosterone level.     

Effects of hyperprolactinemia on the control of luteinizing hormone and follicle-stimulating hormone secretion in the male rat

Experiments were conducted to determine the effects of acute hyperprolactinemia (hyperPRL) on the control of luteinizing hormone and follicle-stimulating hormone secretion in male rats. Exposure to elevated levels of prolactin from the time of castration (1 mg ovine prolactin 2 X daily) greatly attenuated the post-castration rise in LH observed 3 days after castration. By 7 days after castration, LH concentrations in the prolactin-treated animals approached the levels observed in control animals. HyperPRL had no effect on the postcastration rise in FSH. Pituitary responsiveness to gonadotropin hormone-releasing hormone (GnRH), as assessed by LH responses to an i.v. bolus of 25 ng GnRH, was only minimally effected by hperPRL at 3 and 7 days postcastration. LH responses were similar at all time points after GnRH in control and prolactin-treated animals, except for the peak LH responses, which were significantly smaller in the prolactin-treated animals. The effects of hyperPRL were examined further by exposing hemipituitaries in vitro from male rats to 6-min pulses of GnRH (5 ng/ml) every 30 min for 4 h. HyperPRL had no effect on basal LH release in vitro, on GnRH-stimulated LH release, or on pituitary LH concentrations in hemipituitaries from animals that were intact, 3 days postcastration, or 7 days postcastration. However, net GnRH-stimulated release of FSH was significantly higher by pituitaries from hyperprolactinemic, castrated males. To assess indirectly the effects of hyperPRL on GnRH release, males were subjected to electrical stimulation of the arcuate nucleus/median eminence (ARC/ME) 3 days postcastration. The presence of elevated levels of prolactin not only suppressed basal LH secretion but reduced the LH responses to electrical stimulation by 50% when compared to the LH responses in control castrated males. These results suggest that acute hyperPRL suppresses LH secretion but not FSH secretion. Although pituitary responsiveness is somewhat attenuated in hyperprolactinemic males, as assessed in vivo, it is normal when pituitaries are exposed to adequate amounts of GnRH in vitro. Thus, the effects of hyperPRL on pituitary responsiveness appear to be minimal, especially if the pituitary is exposed to an adequate GnRH stimulus. The suppression of basal LH secretion in vivo most likely reflects inadequate endogenous GnRH secretion. The greatly reduced LH responses after electrical stimulation in hyperprolactinemic males exposed to prolactin suggest further that hyperPRL suppresses GnRH secretion.     

Morphological and immunohistochemical comparison of three rat prostate lobes (lateral, dorsal and ventral) in experimental hyperprolactinemia

The prolactin plays an important role in the regulation of growth and differentiation of prostate gland besides androgens. The goal of this study was to reveal the influence of elevated prolactin concentration on epithelial cells of prostate. We compared the morphology of epithelial cells of prostate dorsal, lateral and ventral lobes and expression of androgen receptors in these cells in rats with hyperprolactinemia and in control rats. We used sexually mature male Wistar rats. The experimental rats received metoclopramide; the control group received saline in the same way. The prostate dorsal, lateral and ventral lobes were collected routinely for light and electron microscopy. The intensity of immunohistochemical reaction of androgen receptor in epithelial cells of dorsal, lateral and ventral lobes was evaluated by measure of optical density with computer image analysis. The light and electron (transmission and scanning) microscopes were used for morphological observations. Results: In experimental rats twofold increase in prolactin and twofold decrease in testosterone found. In experimental group the expression of androgen receptor was lower in columnar epithelial cells of dorsal and ventral lobes but higher in lateral one. We observed morphological abnormalities in columnar epithelial cells of lateral and dorsal lobes. The columnar epithelial cells of ventral lobes didn't show any morphological changes in hyperprolactinemia.     

Effects of aging on growth factors gene and protein expression in the dorsal and ventral lobes of rat prostate

We hypothesize that various growth factors and their receptors gene and protein are modulated in dorsal and ventral lobes of aging prostate. To test this hypothesis, TGFbeta1, TGFbeta2 TGFbeta3, TGFbetaR-I, TGFbetaR-II, TGFalpha, EGF, EGFR, KGF and KGFR gene and protein expression were analyzed in dorsal and ventral lobes of aging rat prostates (1, 3, 6, 9, 12, 18, 24, and 28/30 months). KGF gene expression was very weak or absent in 1, 3, and 6 month old rat dorsal and ventral lobes of prostate whereas it re-expressed in 9, 12, 18, 24 and 30 month old rat prostate. All growth factors and their receptors expect KGF and EGFR were mainly localized in epithelium of ventral and dorsal lobes of aging rat prostates. EGF, TGFalpha, TGFbeta1, and TGFbetaR-I protein expression was lacking in stroma of dorsal and ventral lobes of 1, 3, 6, 9, 12/18 months old rat prostates. However, EGF, TGFbeta1 and TGFbetaR-I proteins re-expressed in stroma of 24 and 28 months old rat prostates. KGF protein expression was lacking in epithelium of dorsal and ventral lobes of all aging rat prostates. This is the first report to demonstrate differential gene and protein expression of growth factors in dorsal and ventral lobes is associated with aging rat prostate, suggesting their role in pathogenesis of prostatic diseases with aging.     

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3 alpha(beta)-hydroxysteroid:NAD(P)+ oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of Ki for their inhibition of this enzyme are similar to that of the Km for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5 alpha-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD.     

Effects of isoproterenol on cyclic-AMP metabolism in rat ventral prostate.

Beta-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase CEC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this beta-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3mg/kg, ip) partially reversed these alterations, administration of an alpha-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-(3H) AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (-cyclic-AMP/+cyclic AMP) was significantly elevated from 0.51+/0.05 to 0.95+/0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by beta-adrenergic stimulation.     

Effects of bestatin on intrauterine growth of rat fetuses

Pregnant Wistar rats were injected with bestatin, a specific inhibitor of aminopeptidase M. Placental aminopeptidase M activity was inhibited by injection of bestatin, and fetal body weight was statistically lower than that in the saline-injected or control group. Our present data suggest that placental aminopeptidase M plays an important role in fetal growth.     

Effects of estradiol on prostate epithelial cells in the castrated rat.

There is evidence that estrogens can modulate the activity of prostate epithelial cells. To determine whether estradiol can have a direct influence on rat prostate, this study examined the effects of estradiol-17beta (E(2)) administered alone or in combination with dihydrotestosterone (DHT) to castrated rats for 3 weeks on prostate binding protein (PBP) C1 mRNA expression and androgen receptor (AR) localization. PBP C1 mRNA levels were measured by semi-quantitative in situ hybridization using a (35)S-labeled cDNA probe. In intact animals, strong hybridization signal could be observed in prostate sections after 12 hr of exposure to Kodak X-Omat films. In castrated rats, no PBP C1 mRNA could be detected even with longer exposure times, an effect that was prevented by administration of DHT. E(2) administered alone induced a detectable hybridization signal, and the concomitant administration of E(2) and DHT induced an increase in PBP C1 mRNA that significantly exceeded that obtained in animals that received only DHT. In prostate epithelial cells of intact animals, AR immunostaining was restricted to the nucleus. In castrated animals the alveoli were decreased in size and the epithelial cells were atrophied. AR staining was weak and was detected in both cytoplasm and nucleus. DHT administration completely obviated the effect of castration on epithelial cell histology and on AR immunostaining distribution and intensity. Interestingly, E(2) administration alone induced moderate hypertrophy of epithelial cells compared to the histological appearance of cells in untreated castrated rats. Moreover, in E(2)-treated animals the nuclear staining was much stronger than that detected in untreated castrated rats, whereas the cytoplasmic staining was not modified by the treatment. In animals that received both DHT and E(2), the staining was similar to that seen in DHT-treated rats. These results suggest that E(2) can influence the activity of rat prostate epithelial cells by mechanisms that remain to be fully clarified.     

Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway

Epidermal growth factor (EGF), circulating in the blood, is taken up by rat liver hepatocytes by means of specific and saturable receptor- mediated endocytosis. These experiments were undertaken to determine (a) the transport pathway(s) of EGF taken up by rat liver and (b) the effects of lysosomal inhibition on its transport. 125I-EGF was injected into rat portal veins, and bile samples were collected and analyzed for both total and immunoprecipitable radioactivity. In addition, the livers were examined by electron microscopic autoradiography. Some animals received injections of chloroquine before surgery, to disrupt lysosomal function. The results indicate that most of the EGF taken up by the hepatocytes is transported to lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a pathway independent of the lysosomal system, resulting in secretion of intact EGF: (a) Both degraded and immunoprecipitable EGF are secreted into bile. (b) Immunoprecipitable radioactivity peaks at 20 min after EGF injection, whereas degradation-associated radioactivity does not peak until 40 min postinjection. (c) EGF isolated from bile is specifically taken up by isolated hepatocytes in monolayer culture, indicating that it is still recognizable by the EGF receptor. (d) When the lysosomal system is inhibited with chloroquine, secretion of degraded EGF is significantly inhibited, whereas the amount of intact EGF secreted into bile is unchanged. The utilization by liver of a dual transport process for EGF represents an unusual system of intracellular ligand processing, whose physiological significance has yet to be determined.     

转化生长因子β1对大鼠心肌细胞肥大的影响

目的：研究转化生长因子β1（TGF-β1）及其下游Smad3信号蛋白在大鼠心肌细胞肥大中的作用。方法：TGF-β1干预培养新生大鼠心肌细胞,流式细胞仪检测心肌细胞总蛋白含量。结扎大鼠腹主动脉复制心肌肥厚模型,在不同时间点处死动物,检测左室质量指数（LVM1）,RT—PCR检测TGF-β1及Smad3的mRNA表达,Westernblot检测Smad3蛋白的表达。结果：不同剂量TGF-β1均能明显增加体外培养的心肌细胞总蛋白含量,TGF-β1（3ng／ml）还增加心肌细胞Smad3 mRNA和蛋白的表达,其表达量1h达高峰,持续至TGF-β1刺激后8h。大鼠腹主动脉结扎术后3d LVMI开始上升并持续至术后28d,心肌组织中TGF-β1、Smad3的mRNA表达水平以及Smad3蛋白表达术后3d也开始上升持续至术后28d,术后14d为表达高峰（P〈0．01）。结论：TGF-β1能诱导大鼠心肌细胞肥大,其信号蛋白Smad3参与了大鼠心肌肥大的病理过程。     

Effects of norepinephrine on neonatal rat cardiocyte growth and differentiation

Summary Norepinephrine stimulates the growth in size of nondividing neonatal cardiocytes. During this time the neonatal cardiocyte is in a period of transition in which the cell can synthesize DNA and yet does not divide. Because the cell undergoes karyokinesis without cytokinesis the objective of this study was to determine whether the norepinephrine-induced growth in size of the neonatal cardiocyte was accompanied by an increase in a) the number of cardiocytes synthesizing DNA, b) the number of binucleate cardiocytes, and c) organized myofibrils. One- to four-d-old neonatal rat heart cells were isolated and placed in serum-free medium which was then supplemented with serum, norepinephrine, norepinephrine plus propranolol, or isoproterenol. After 4 d the number and size of the cells was determined using a Coulter counter. In other cultures cardiocytes were fixed on Days 0, 1, 2, and 4, and an increase in the number of binucleate cardiocytes was found in all treatment groups including controls. However, the rate of binucleation was faster in the norepinephrine group. It was also determined by proliferating cell nuclear antigen (PCNA) antibody staining that by Day 4, over 50% of the cardiocytes were in the cell cycle. The percentage of cells in which PCNA could be detected was higher in the norepinephrine and norepinephrine plus propranolol groups. Furthermore, there was a concomitant increase in the amount and organization of myofibrils in the catecholamine-treated cardiocytes. Supported in part by grant No. HL 29351 from the National Institutes of Health, by a grant from the American Heart Association and with the support of the Southeastern Pennsylvania and Pennsylvania Affiliates of the American Heart Association.     

Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin

Analogs of a potent octapeptide analog of somatostatin (SRIF)

were synthesized. Aromatic substitutions for Tyr resulted in little change in inhibitory potency on growth hormone (GH) secretion in the rat. Substitutions for Val or (D)Trp resulted in analogs with diminished activity. Substitution of (D)Nal for (d)Phe increased duration of GH inhibition. Final weights of subcutaneously implanted prostate tumors (R3327) were 41% lower in rats treated with an N-terminal 4-chloro-(D)phenylalanyl analog as compared to vehicle treated controls. The analog had no effect on testicular weight or final plasma testosterone levels. SRIF analogs may represent an alternative treatment for prostate cancer that would be free of the untoward reproductive effects of other treatments (e.g. LH-RH or castration).