Effects of doxazosin on blood flow and mRNA expression of nitric oxide synthase in the spontaneously hypertensive rat genitourinary tract

Hypertension may impact pelvic arterial blood flow resulting in reduction of nitric oxide synthase (NOS) levels. Although doxazosin, an alpha(1)-adrenoceptor antagonist, has been shown to improve erectile dysfunction as well as benign prostatic hyperplasia (BPH) and hypertension, it is not clear whether these improvements using doxazosin are primarily due to direct actions on the prostate, urinary bladder and penis, possibly via inhibition of vascular alpha(1)-adrenoceptors, or other sites of actions. Therefore, we investigated effects of doxazosin to the spontaneously hypertensive rat (SHR) on blood flow and NOS levels in the genitourinary tract. Four groups of rats were assessed: group 1, SHRs treated with doxazosin (30 mg/kg/day) for 4 weeks; group 2, SHRs treated with nifedipine (30 mg/kg/day) for 4 weeks; group 3, untreated SHRs; and group 4, untreated Wistar-Kyoto (WKY) rats. Blood flow to the ventral prostate, dorsolateral prostate, urinary bladder and penis was determined using a fluorescent microsphere infusion technique. Expression levels of nNOS and eNOS mRNAs were quantified by real-time RT-PCR using SYBR Green I. Blood flow to the ventral prostate, dorsolateral prostate, urinary bladder and penis was significantly lower in untreated SHRs than WKY rats. Treatment with doxazosin increased blood flow to each tissue studied in SHRs. RT-PCR data indicated that untreated SHRs had lower mRNA expression levels of nNOS in the bladder and penis and eNOS in the penis than WKY rats and that administration of doxazosin to the SHR caused an increase in expression levels of these genes, i.e., up-regulation of nNOS in the bladder and penis and eNOS in the penis. However, nifedipine had no significant effects on blood flow and NOS levels in the SHR genitourinary tract. Our data demonstrate that doxazosin treatment causes differential alterations in blood flow and NOS levels in the SHR genitourinary tract. These findings may provide insight into the beneficial effects of alpha(1)-adrenoceptor antagonists, on prostate, bladder and penile function, when used to treat symptoms of BPH and elevated blood pressure.     

Microarray analysis of gene expression profile in the corpus cavernosum of hypercholesterolemic rats after chronic treatment with PDE5 inhibitor

Gene expression changes in the corpus cavernosum of hypercholesterolemic rats were not fully assessed, which were not previously known to be associated with hypercholesterolemia-related erectile dysfunction (ED). To provide molecular insight into pathophysiology of hypercholesterolemia-related ED and to investigate the effects of Udenafil, a phosphodiesterase type 5 (PDE5) inhibitor, on gene expression, we performed microarray gene expression analysis via gene discovery methods using GenoCheck platinum cDNA chip (Ansan, S. Korea). Sixteen male Sprague-Dawley rats were fed 2% cholesterol diet for 5 months. Half of them were orally treated with Udenafil (20 mg/kg/day) simultaneously. Eight age-matched rats fed normal diet were served as normal control. RNA was extracted from corpus cavernosum and microarray analysis was performed. Decreased erectile responses and hypercholesterolemia were observed in hypercholesterolemic control group. In microarray analysis, 122 candidate genes were noted to be altered based on the magnitude of expression changes, which includes 44 down-regulated and 78 up-regulated genes compared with the age-matched normal controls. These changes were, however, significantly attenuated by treatment with Udenafil. Out of the 78 up-regulated genes, 8 genes were significantly decreased by the chronic treatment with Udenafil. The altered genes were cytochrome oxidase biogenesis protein OXA1, skeletal muscle myosin heavy chain, lipophilin, fast skeletal muscle isoforms beta/alpha, myosin light chain 3, cytochrome c oxidase, adipocyte fatty acid binding protein and one EST gene. In contrast, among the 44 down-regulated genes, Kruppel-like factor 5 and cyclin D1 genes were increased after the Udenafil treatment. These results provide the molecular basis for understanding the pathogenesis of hypercholesterolemia-related ED and offer clues on determining the underlying action mechanism of a PDE5 inhibitor.     

睾丸酮对大鼠前列腺上皮细胞有丝分裂方向的影响

目的探讨丙酸睾丸酮（T）对大鼠前列腺上皮细胞染色体有丝分裂方向的影响及其差异基因表达。方法 SPF级SD雄性大鼠（110~130 g）20只,随机分为2组。深麻醉下对所有大鼠进行去势手术,恢复一周后,给药组大鼠皮下注射T,每只0.5 mg,每日1次,连续30d;对照组大鼠皮下注射橄榄油0.1 mL。取前列腺。通过免疫组化、HE染色观察结果。并做基因芯片检查差异基因表达谱。结果给药组大鼠前列腺发生形态学增生。前列腺腺腔扩张,腺上皮高度明显增高,前列腺增生。且前列腺上皮细胞染色体有丝分裂的方向与基底膜平行,而对照组前列腺上皮细胞有丝分裂的方向与基底膜垂直。AR免疫组化染色后发现给药组的前列腺上皮中,均可见到AR标记的阳性细胞,而对照组均为阴性。基因芯片和RT-PCR结果：促进细胞增殖的基因如雄激素受体相关蛋白（RAN）、TGM4和Wnt通道的WNT2等基因均上调,而抑制细胞增殖的基因如负调控Wnt通道的DKK3和促进细胞凋亡的Fas等基因下调。结论 T注射后改变了前列腺上皮细胞有丝分裂的方向,Wnt和AR信号转导通路参与了细胞增殖和有丝分裂方向改变。     

Altered profile of gene expression in rat hearts induced by chronic nicotine consumption

Using a cDNA microarray technique, we analyzed the expression profile of 1081 genes in the whole heart tissue of rats. The expressions of three classes of genes encoding cellular energy metabolism enzymes, transmembrane receptors, and intracellular kinase network members were reduced by more than 2.5-fold in cardiac tissues from the rats fed with nicotine (3mg/kg/day) for 3 months. The down-regulated 11 genes included mitochondrial ATP synthase beta subunit, mitochondrial H(+) transporting ATP synthase F1 complex alpha subunit isoform 1, liver mitochondrial aldehyde dehydrogenase 2, glutathione-S-transferase mu type 2, corticotropin-releasing factor receptor 2, metabotropic glutamate receptor 2, N-methyl-D-aspartate receptor subtype 2B, muscarinic acetylcholine receptor M3, transmembrane receptor Unc5H1, glycogen synthase kinase 3alpha, and Ca(2+)/calmodulin-dependent protein kinase II beta subunit. It appears that chronic nicotine treatment affects cardiac function by modulating the expressions of genes involved in energy metabolism and signal transduction.     

Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets

The androgen receptor (AR) is expressed in the fetal testis; however, the role of AR in fetal testicular development is poorly understood. Disrupted AR activity and subsequent gene expression alterations may disturb developmental programming of the fetal testis and result in testicular abnormalities later in life. The present study was performed to examine global gene expression patterns in rat fetal testis following in utero exposure to various AR antagonists. Pregnant Sprague-Dawley rats were treated with flutamide (50 mg/kg/day), linuron (50 mg/kg/day), vinclozolin (200 mg/kg/day), p,p'-DDE (100 mg/kg/day) or corn oil vehicle by gavage daily from gestation day (GD) 12-19. Testes were isolated on GD 19, and AR immunostaining, histology, and global changes in gene expression were determined. There were no alterations in the pattern or expression level of AR and no apparent histological changes in the fetal testes in any treatment group. Microarray analysis using Dunnett's test with multiple testing correction revealed no significant gene expression alterations following exposure to flutamide, linuron, vinclozolin, and p,p'-DDE. A less stringent analysis yielded some chemical specific effects on gene expression, and these effects were further evaluated by real-time RT-PCR. Vinclozolin treatment reduced the expression of several genes involved in cholesterol biosynthesis, though the testosterone levels were unchanged in the fetal testes in any treatment group. In flutamide, linuron, and p,p'-DDE treatment groups, the expression of hemoglobin Y, beta-like embryonic chain (Hbb-y) was reduced. Myomesin 2 (Myom2) expression was increased following linuron treatment. Given the lack of a common set of genes and the absence of overt histopathology, we conclude that the fetal testis is not a major target for AR activity at this stage of development although some cell-type specific gene expression changes cannot be ruled out.     

Study of pharmacological effects of nilvadipine on RCS rat retinal degeneration by microarray analysis

In our recent study, we found that the Ca(2+) antagonist, nilvadipine caused significant preservation of photoreceptor cells in The Royal College of Surgeons (RCS) rats [Invest. Ophthalmol. Vis. Sci. 43 (2002) 919]. Here, to elucidate the mechanisms of nilvadipine-induced effects we analyzed altered gene expression of 1101 genes commonly expressed in rodent by DNA microarray analysis in the retinas of nilvadipine-treated and untreated RCS rats and SD rat. In the total number of genes, the expression of 30 genes was altered upon administration of nilvadipine to RCS rats, including several genes related to the apoptotic pathway and other mechanisms. Remarkably, neurotrophic factors, FGF-2 and Arc, known to suppress the apoptosis in the central nervous system, were up-regulated. These changes were also confirmed by real-time quantitative (Taqman) RT-PCR and Western blot analysis. Therefore, our present data suggested that administration of nilvadipine to RCS rats increases the expression of endogenous FGF-2 and Arc in retina, and potentially has a protective effect against retinal degeneration.     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

Microarray analysis of gene expression changes in mouse liver induced by peroxisome proliferator- activated receptor alpha agonists.

We used a microarray technique to investigate changes of gene expression in liver induced by two peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, a strong PPARalpha agonist, Wy-14,643, and a marketed fibrate drug, fenofibrate. The purposes of this work are: 1) to examine whether or not gene expression is altered in different ways by these two PPARalpha agonists and 2) to find genes whose expression has not been previously reported to be affected by PPARalpha agonists. Mice were treated orally with 100 mg/kg fenofibrate, or 30 mg/kg or 100 mg/kg Wy-14,643, and the liver was collected on Day 2 or 3. mRNA was extraction from liver, and subjected to microarray analysis. Previously reported induction or reduction of gene expression, e.g. genes involved in beta-oxidation and lipid metabolism, was confirmed in our study. Scatter plot analysis indicated that the changes of gene expression pattern induced by fenofibrate and Wy-14,643 were almost identical. However, expression levels of metallothionein 1 and 2 mRNAs were different: no change of hepatic metallothionein 1 and 2 mRNA expression was induced by 100 mg/kg fenofibrate on Day 2 or 3, while 30 mg/kg Wy-14,643 administration increased expression of both genes by 1.8-fold on Day 3. In addition to previously reported gene expression changes by PPARalpha agonists, we found expression changes of other genes, including cis-retinol/3alpha-hydroxysterol short chain dehydrogenase, vanin-1, RecA-like protein, and serum amyloid A (SAA) 2. Among them, the change of SAA2 mRNA level was noteworthy; it showed a decrease to as little as one-seventh. Seven-day fenofibrate pre-treatment of mice completely inhibited the acute-phase elevation of plasma SAA concentration triggered by acetaminophen challenge. This finding suggests that fenofibrate treatment may reduce plasma SAA concentration in patients with secondary amyloidosis.     

Repeated Administration of Dexamethasone Increases Phosphoinositide-Specific Phospholipase C Activity and mRNA and Protein Expression of the Phospholipase C β1 Isozyme in Rat Brain

Altered hypothalamic-pituitary-adrenal (HPA) function has been shown to be associated with changes in mood and behavior. The enzyme phosphoinositide-specific phospholipase C (PI-PLC), an important component of the PI signal transduction system, plays a major role in mediating various physiological functions. In the present study, we investigated the effects of a single dose and of repeated administration (0.5 or 1.0 mg/kg for 10 days) of dexamethasone (DEX), a synthetic glucocorticoid, on PI-PLC activity and on expression of PLC isozymes (beta1, delta1, and gamma1) in rat brain. Repeated administration of DEX (1.0 mg/kg) caused a significant increase in PI-PLC activity and in protein expression of the PLC beta1 isozyme in both membrane and cytosol fractions of cortex and hippocampus; however, the repeated administration of a smaller dose of DEX (0.5 mg/kg) caused these changes only in hippocampus but not in cortex. The increase in PLC beta1 protein was associated with an increase in its mRNA level, as measured by competitive RT-PCR. A single administration of DEX (0.5 or 1.0 mg/kg) to rats had no significant effects on PI-PLC activity or on the protein expression of PLC isozymes. These results suggest that DEX up-regulates PI-PLC in rat brain, which presumably is due to a selective increase in expression of the PLC beta1 isozyme, and that these changes in PI-PLC may be related to HPA axis-mediated changes in mood and behavior.     

High fat-induced obesity associated with insulin-resistance increases FGF-2 content and causes stromal hyperplasia in rat ventral prostate

Obesity affects sex hormone secretion, which can negatively influence prostatic structure, homeostasis, and disease. This investigation aimed to evaluate the repercussions of obesity induced by a high-fat diet on the rat prostate, with or without treatment with the aromatase inhibitor, Letrozole. Adult Wistar rats were fed a high-fat diet (20% saturated fat, O) for 15?weeks to induce obesity or received a balanced diet (4% fat, C). Then, a group of C and O rats were daily treated with Letrozole (1?mg/kg b.w. per day) for 2?weeks (CL and OL, respectively). Subsequently, ventral prostate was processed for analysis by transmission electron microscopy, immunohistochemistry, and Western blotting. Obesity decreased 70% of the testosterone plasma level. The prostate showed epithelial atrophy and dilated acini in the intermediate portion and epithelial wrinkling in the distal tips. The relative frequency of smooth muscle α-actin in the O group increased by 67%. Ultrastructurally, epithelial cells in obese animals presented altered secretory organelles, lipid droplets, and thicker subjacent fibromuscular layer. Letrozole treatment caused a partial restoration of the prostatic changes caused by obesity. Obesity increased the prostatic content of fibroblast growth factor-2 (FGF-2) by 150%, and Letrozole treatment increased this protein even more in the control and obese groups. This investigation shows that obesity provokes structural and ultrastructural changes in the epithelium of rat prostate; these changes might affect gland homeostasis and physiology. The epithelial and smooth muscle cell hyperplasia and increased FGF-2 expression observed in this experimental model of obesity/insulin-resistance might explain the high frequency of benign prostatic hyperplasia in insulin-resistant men.     

慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin的表达变化

目的:观察慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin(PV)的表达变化,为其功能的研究提供形态学依据。方法:将30只健康雄性SD大鼠随机分为吗啡依赖组和生理盐水对照组。吗啡依赖组大鼠腹膜腔注射吗啡,2次/d,起始剂量为5 mg/kg,逐日递增5mg,至第10d为50mg/kg;对照组注射同体积的生理盐水。于末次注射后动物分别存活3h、3 d和14d。用免疫组化方法和相对平均灰度值检测杏仁核内PV的表达。结果:在生理盐水处理组各存活时间点,杏仁核内PV的表达相同。和生理盐水对照组相比,3h时杏仁核内PV的表达明显增加(P<0.05)。第3d时,杏仁核内PV的表达减少,明显低于第3 h组(P<0.05)。至第14d时,PV的表达又开始增加,明显高于第3 d组(P<0.05)。结论:本结果提示慢性吗啡处理及戒断后杏仁核PV的表达具有时相特异性;这种变化在戒断早期可能主要与躯体依赖相关,而戒断晚期主要与精神依赖相关。     

Factors involved in the maintenance of luteal function in the pseudopregnant rat

Some data on a new antispermatogenic and antifertility compound, DL-6-(N-alpha-pipecolinomethyl)-5-hydroxy-indane maleate (PMHI) are presented. It was found that PMHI depressed testicular weight and interfered with spermatogenesis and fertility in the male rat. At equal doses, by weight, PMHI caused a greater reduction in testicular weight than WIN 18446, nitrofurazone, methallibure, and diethylstilbestrol, but lesser reductions in seminal vesicle and ventral prostate weights than were obtained with the 2 antigonadotropic compounds, methallibure and diethylstilbestrol. The data obtained in the experiments indicate that administration of PMHI causes sterility in male rats as a consequence of antispermatogenic activity. At low dosage levels (3 mg/kg), the action was reversible, but animals given 6.25 mg/kg/day or more remained sterile for 19 weeks following the treatment period. Doses of the compound which were effective in the male rat did not interfere with normal estrous cycles or fertility when administered to female rats.     

Metformin increases the PGC-1alpha protein and oxidative enzyme activities possibly via AMPK phosphorylation in skeletal muscle in vivo.

AMP-activated protein kinase (AMPK), which was activated by an antihyperglycemic drug metformin, has been hypothesized to mediate metabolic adaptations. The purposes of the present study were 1) to confirm whether acute metformin administration induced AMPK phosphorylation and 2) to determine whether chronic metformin treatment increased the peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein expression, glycolytic and oxidative enzyme activities, and cytochrome c and glucose transporter-4 (GLUT4) protein expressions in the rat soleus and red and white gastrocnemius muscles. The single oral administration of metformin (300 mg/kg body wt) enhanced the AMPK phosphorylation at 5 and/or 6 h after treatment. In the chronic study, rats were fed either normal chow or chow containing 1% metformin for 14 days. Metformin treatment resulted in a mean daily metformin intake of 631 mg.kg body wt(-1).day(-1). Metformin increased the PGC-1alpha content in all three muscles. Metformin increased the hexokinase activity in the white gastrocnemius, the citrate synthase activity in all three muscles, and the beta-hydroxyacyl-CoA dehydrogenase activity in the soleus. The cytochrome c protein content in the soleus muscle also increased. The GLUT4 content was unchanged by metformin. These results suggest that metformin enhances the PGC-1alpha expression and mitochondrial biogenesis possibly at least in part via AMPK phosphorylation in the skeletal muscle. Metformin has thus been proposed to possibly ameliorate insulin resistance, at least partially, by means of such metabolic effects.     

5,7-Dimethoxycoumarin prevents chronic mild stress induced depression in rats through increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels

The current study was aimed to investigate the role of 5,7-dimethoxycoumarin in the prevention of chronic mild stress induced depression in rats. The chronic mild stress rat model was prepared using the known protocols. The results from open-field test showed that rats in the chronic mild stress group scored very low in terms of crossings and rearings than those of the normal rats. However, pre-treatment of the rats with 10 mg/kg doses of 5,7-dimethoxycoumarin prevented decline in the locomotor activity by chronic mild stress. The level of monoamine oxidase-A in the chronic mild stress rat hippocampus was markedly higher. Chronic mild stress induced increase in the monoamine oxidase-A level was inhibited by pre-treatment with 10 mg/kg doses of 5,7-dimethoxycoumarin in the rats. Chronic mild stress caused a marked increase in the level of caspase-3 mRNA and proteins in rat hippocampus tissues. The increased level of caspase-3 mRNA and protein level was inhibited by treatment of rats with 5,7-dimethoxycoumarin (10 mg/kg). 5,7-Dimethoxycoumarin administration into the rats caused a marked increase in the levels of heat shock protein-70 mRNA and protein. The levels of heat shock protein-70 were markedly lower both in normal and chronic mild stress groups of rats compared to the 5,7-dimethoxycoumarin treated groups. Thus 5,7-dimethoxycoumarin prevented the chronic mild stress induced depression in rats through an increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels.     

Effect of alcohol on microsomal cortisol 4en-5 alpha-reductase in the liver of rats fed on a standard or low protein diet

Alcohol feeding (40% of total calories over a period of 9 days) increases microsomal cortisol-5 alpha-reductase activity in rat liver distinctly. It is assumed that this is an adaptive response to an increased release of cortisol caused by alcohol administration. Cortisol-5 alpha-reductase activity is decreased to one third of control values in rats fed an isocaloric, low protein diet. The response to alcohol feeding is susta ined in these animals. Phenobarbital treatment (80 mg/kg x day) stimulates 5 alpha-reduction of cortisol per g of microsomes almost twofold. The activity calculated per total liver increases 4-fold. Alcohol administration has no additional effect on cortisol-5alpha-reductase in phenobarbital-treated rats.