Makoto Yono

Effect of insulin treatment on tissue size of the genitourinary tract in BB rats with spontaneously developed and streptozotocin-induced diabetes

To examine the differences between spontaneous and streptozotocin (STZ)-induced diabetes, four parallel studies were performed; three studies of diabetes-prone BB (BBDP/Wor) rats maintained for 8, 16, and 32 weeks and one study of STZ-injected diabetes-resistant BB (BBDR/Wor) rats maintained for 32 weeks. Each diabetic study has three groups of rats: a control group; a euglycemic group, which received sufficient amounts of insulin; and a hyperglycemic group, which received a suboptimal dose of insulin. The extent of tissue weight changes was generally shown to be less dramatic in the euglycemic diabetic than in the hyperglycemic diabetic rats. STZ-induced diabetes increased the bladder weight more dramatically (up to 3-fold) than did spontaneous diabetes (up to 2-fold). Furthermore, a significant decrease in the size of the adrenal gland (20%) and testis (10%) is observed only with spontaneous diabetes, whereas a significant decrease in the size of the ventral prostate (30%) is observed only with STZ-induced diabetes, although the serum testosterone levels are similar in both groups. Our data demonstrate that there are differences in the effect of insulin treatment on the tissue size of the genitourinary tract between spontaneously developed and streptozotocin-induced diabetes in BB rats.

Quantification of endothelins, their receptors, and endothelin-converting enzyme mRNAs in rat genitourinary tract using real-time RT-PCR

INTRODUCTION Quantification of mRNA expression is essential for the assessment of endothelin (ET) receptor-mediated mechanisms. Recently, a novel technique for the determination of mRNA expression, termed real-time reverse transcription polymerase chain reaction (RT-PCR), has been developed. We therefore applied real-time PCR using SYBR Green I to quantify ET-1, ET-3, ET-converting enzyme-1 (ECE-1), and ETA and ETB receptor subtype mRNA expression in the rat genitourinary tract. METHODS The cDNA was synthesized by RT of RNA extracted from the rat bladder, ventral prostate, dorsolateral prostate, and vas deferens. All steps subsequent to the RT reaction were carried out by the thermal cycler/detector and computer-assisted programs processed a quantitative result. RESULTS Designing optimal primer sequences that minimized primer-dimer formation and adjusting annealing temperatures that prevented nonspecific product amplification have made it possible to identify a single peak in the melt curve and to obtain an appropriate standard curve for each gene transcript. In our experiments, input cDNA levels as low as 100 copies of the product could be detected. DISCUSSION We demonstrated that significant quantitative variations existed in the expression levels of ET-1, ET-3, ECE-1, and ETA and ETB receptor subtype mRNAs within a tissue and between different regions of the genitourinary tract and that the predominant expression of ETs and their receptor mRNAs in all tissues studied were ET-1 and the ETA receptor subtype, respectively.

Developmental changes in the functional, biochemical and molecular properties of rat bladder endothelin receptors

The effect of aging on functional, biochemical, anatomical and molecular properties of endothelin (ET) receptors in bladder smooth muscle of the 3-week-, 3-month- and 22-month-old rats was examined using isolated muscle bath techniques, radioligand binding on membrane particulates and slide mounted tissue sections, and real-time reverse transcription polymerase chain reaction (RT-PCR). ET-1 induced significantly larger contractile responses in bladder dome muscle strips from 3-week- than from 3-month- and 22-month-old rats. The expression level of total ET receptors, determined by saturation binding experiments with [125I]ET-1, was higher in detrusor from 3-week- than 22-month-old rats. Inhibition studies with BQ123, a selective ETA receptor antagonist, indicated the predominance of the ETA receptor subtype and a similar proportion of ETA to ETB receptor subtypes in the rat detrusor at all ages studied. Autoradiographic data support the age-dependent decrease in the density of ET receptors and also indicate that the ETA receptor subtype is primarily located in the smooth muscle layer, whereas the ETB receptor subtype is located in both the urothelial and smooth muscle layers. Determined by real-time RT-PCR, ET 1, ET-3, ECE-1 and ET receptor subtype (ETA and ETB) mRNAs were shown to be higher in bladders of 3-week- compared to 3-month- or 22-month-old rats. This study indicates age-dependent alterations in the ET receptor system at both gene transcript and protein levels in the Fischer rat detrusor.

Age-Related Alterations in the Biochemical and Functional Properties of the Bladder in Type 2 Diabetic GK Rats

Previous studies have demonstrated that experimental type 1 diabetes induced by streptozotocin causes alterations in the biochemical and functional properties of several receptor systems in the rat bladder. However, the exact mechanism involved in the pathophysiology of voiding dysfunction in type 2 diabetic patients is unknown. Because the GK rat is a widely accepted genetically determined rodent model for human type 2 diabetes, we investigated diabetes-induced changes in the bladder smooth muscle of the GK rats at several time points. Male GK rats and age-matched Wistar rats, as controls, were maintained for 4, 8, 16, and 32 weeks. Contractile responses to KCl, carbachol, ATP, and electrical field stimulation (EFS) were measured by using the isolated muscle bath techniques. Acetylcholine (ACh) release induced by EFS from bladder muscle strips was measured by using high-performance liquid chromatography coupled with a microdialysis procedure. Maximum contractile responses to carbachol and ATP, the release of ACh, and tissue sorbitol levels were similar in bladders from GK and control rats until 8 weeks of age. At 16 weeks of age, however, the contractile responses to carbachol and ATP, and tissue sorbitol levels were increased, and the EFS-induced ACh release was decreased in GK rats compared with controls. Although the maximum contractile responses to EFS were unchanged until 16 weeks of age, they were decreased in 32-week-old GK rats, compared with controls. Our data indicate the presence of age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats.

Differential effects of diabetes induced by streptozotocin and that develops spontaneously on prostate growth in Bio Breeding (BB) rats

We investigated molecular changes in the response to insulin in prostates of spontaneously developed (Bio Breeding) and streptozotocin (STZ)-induced diabetic rats that received sufficient amounts (euglycemic group), or suboptimal doses (hyperglycemic group) of insulin for 32 weeks, using Affymetrix GeneChip analysis of gene expression. Alterations in gene expression levels identified by microarray analysis, having potential biological relevance to prostate growth, were verified by real-time reverse transcription polymerase chain reaction (RT-PCR). A significant decrease in the weight of ventral prostate was observed in the hyperglycemic STZ-induced but not spontaneously developed diabetic group. Microarray analysis revealed that gene expression profiles were distinctly different in each region of the prostate, and that hyperglycemic diabetes in spontaneously developed and STZ-diabetic rats was associated with differential changes in the prostatic expression levels of 856 genes, of which 35 were related to cell growth, proliferation and death. RT-PCR data verified significant differences in the mRNA expression levels of Igfbp6, Tieg, and Clu between euglycemic and hyperglycemic groups, whereas expression levels of these genes in control and euglycemic diabetic groups were not significantly different. In ventral prostate, the mRNA expression levels of Igfbp6 and Tieg were significantly higher in the hyperglycemic STZ-induced diabetic than in the hyperglycemic spontaneously diabetic BBDP/Wor rats. Our data demonstrate that the diabetes induced by STZ in the BBDR/Wor rats affects prostate growth and the molecular response to insulin differently than that observed in BBDP/Wor rats that develop diabetes spontaneously.

Identification of potential therapeutic targets in hypertension-associated bladder dysfunction

To investigate differential gene expression profiles in the bladder of spontaneously hypertensive rat (SHR), as the underlying mechanisms involved in hypertension‐associated bladder dysfunction remain to be clarified.

Age-Related Changes in the Properties of the Endothelin Receptor System at Protein and mRNA Levels in the Rat Vas Deferens

As age-related changes occur in the properties of the endothelin (ET) receptor system in several mammalian tissues, and as there are significant amounts of functional ET receptors in the vas deferens, we investigated the age-related changes in the ET receptor system at the protein and mRNA levels in the rat vas deferens. The ET system was investigated in the vasa deferentia of 3 weeks, 3 months and 22 months old rats. ET receptors were characterized and quantified at the protein level by radioligand receptor binding, and gene transcript levels of ET-1, ET-3, ET converting enzyme-1 (ECE-1), and ETA and ETB receptor subtypes were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). The results of radioligand receptor binding assays demonstrate that there is a higher density of total ET receptors in the vas deferens of 3 weeks old rats than in 3 months and 22 months old rats, and that the predominant ET receptor is of the ETA subtype in all three ages. Real-time RT-PCR data show that the predominant mRNA expression of ETs and their receptors in all age groups studied are ET-1 and the ETA receptor subtype, respectively. Furthermore, ET-1, ET-3, ECE-1, and ETA and ETB receptor subtype mRNAs are expressed at higher levels in the 3 weeks old rats as compared with the other two age groups. These results demonstrate the presence of age-related changes in the properties of the ET receptor system at both protein and mRNA levels in the rat vas deferens.

Short‐ and long‐term effects of silodosin, a selective α1A‐adrenoceptor antagonist, on ejaculatory function in rats

To investigate the short‐ and long‐term effects of silodosin, a selective α1A‐adrenoceptor antagonist, on spontaneous seminal emission by isolated rats and on the properties of α1‐adrenoceptor subtypes in the rat seminal vesicle, as silodosin produces a relatively high incidence rate of abnormal ejaculation and chronic administration of receptor antagonists causes an up‐regulation in the targeted receptor.

Regulatory effect of castration on endothelins, their receptors and endothelin‐converting enzyme in rat seminal vesicle

To investigate the effects of castration on the expression of endothelins (ETs), ET receptors and ET converting enzyme‐1 (ECE‐1) in the rat seminal vesicle (RSV).