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Because hypertension related alterations occur in the properties of α(1)-adrenoceptor in several mammalian tissues and hypertension may impact ejaculatory function, we investigated hypertension related alterations in the functional, biochemical and molecular properties of α(1)-adrenoceptor in the rat seminal vesicle and vas deferens. Spontaneous seminal emission in male spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats was studied during the 3-day observation period. The characteristics of α(1)-adrenoceptor in the seminal vesicle and epididymal and prostatic portion of vas deferens of the two strains were determined using an isolated muscle bath, radioligand receptor binding and real-time reverse transcription-polymerase chain reaction techniques. SHRs had significantly higher serum testosterone than WKY rats. However, the daily mean number of ejaculatory plugs emitted and their dry weight in SHRs were significantly lower than those in WKY rats. Although there was no significant difference in the properties of α(1)-adrenoceptor in the prostatic portion of vas deferens between SHRs and WKY rats, the maximum contractile responses to phenylephrine, total α(1)-adrenoceptor density and expression of α(1A)-adrenoceptor mRNA were significantly higher in the seminal vesicle and epididymal portion of vas deferens of SHRs vs. WKY rats. Our data demonstrate the presence of hypertension related alterations in serum testosterone and in α(1)-adrenergic responsiveness of the rat seminal vesicle and vas deferens and suggest that ejaculatory function in SHRs does not mirror these hypertension related alterations.

Aims Although doxazosin, but not nifedipine, can partially prevent a decrease in urogenital expression of nitric oxide synthase (NOS) in spontaneously hypertensive rats (SHRs), the mechanisms involved in the regulated expression of NOS are not known. Therefore, we identified differential gene expression profiles in SHRs to elucidate the molecular mechanisms regulating urogenital expression of NOS. Main methods SHRs and normotensive Wistar–Kyoto (WKY) rats received doxazosin (30 mg/kg/day) or nifedipine (30 mg/kg/day) orally for 4 weeks. Microarray expression data of key transcripts were verified by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Key findings RT-PCR data, in accord with the microarray analysis, indicated that untreated SHRs had lower mRNA expression levels of cAMP responsive element binding protein 1 (Creb1) in the pelvic ganglion and vascular endothelial growth factor A (Vegfa) and kinase insert domain protein receptor (Kdr) in the penis, and higher mRNA expression levels of brain derived neurotrophic factor and neurotrophin 3 (Ntf3) in the bladder and Ntf3, Rho-kinases (Rock1 and Rock2) and caveolin 1 (Cav1) in the penis than untreated WKY rats. In SHRs, doxazosin and nifedipine caused a significant decrease in penile expression of Rock1 and Rock2, whereas the differential alterations in urogenital expression of Creb1, Vegfa, Kdr and Cav1 were attenuated by treatment with doxazosin, but not nifedipine. Significance Our data suggest that differential alterations in the expression of several genes related to pathways that mediate NOS expression in the urogenital tissues of SHRs, which can be attenuated by doxazosin treatment, may play an important role in regulating urogenital expression of NOS.

OBJECTIVE 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. MATERIALS AND METHODS Rats were treated with two doses (0.1 and 3 mg/kg/day) of silodosin orally for 3 or 30 days. Spontaneous seminal emission was studied during the 3-day observation period before completing treatment. The expression levels of α1A, α1B and α1D-adrenoceptor mRNAs in the rat seminal vesicle and prostate were quantified by real-time reverse transcription-polymerase chain reaction using SYBR Green I. RESULTS The administration of two doses of silodosin for 3 or 30 days caused a significant dose-dependent reduction in the number of ejaculatory plugs and in their dry weight. However, in rats receiving the low dose of silodosin the inhibitory effect of the drug on spontaneous seminal emission diminished significantly with chronic usage over time. Although short-term administration of silodosin did not affect expression levels of any α1-adrenoceptor subtype mRNAs in the rat seminal vesicle and prostate, long-term administration of silodosin caused a significant up-regulation in the mRNA expression of α1A-adrenoceptor in a tissue-dependent manner. CONCLUSION Silodosin-induced up-regulation of α1A-adrenoceptor mRNA in the rat seminal vesicle might indicate potential differences in the inhibitory effect of this drug on ejaculatory function with chronic usage over time.

Because age dependent differences occur in the incidence of ejaculatory dysfunction with alpha(1)-adrenoceptor antagonists used to treat lower urinary tract symptoms secondary to benign prostatic hyperplasia, we investigated age related changes in the functional, biochemical and molecular properties of alpha(1)-adrenoceptor in the rat seminal vesicle and vas deferens. The characteristics of alpha(1)-adrenoceptor in the seminal vesicle and epididymal and prostatic portion of vas deferens of 3 and 22-month-old rats were determined using an isolated muscle bath, radioligand receptor binding and real-time reverse transcription-polymerase chain reaction techniques. Old rats had significantly higher body weight and lower testosterone than young rats. Although there was no significant age dependent difference in the properties of alpha(1)-adrenoceptor in the prostatic portion of vas deferens, the maximum contractile responses to phenylephrine, total alpha(1)-adrenoceptor density and mRNA expression of all 3 alpha(1)-adrenoceptor subtypes were significantly lower in the seminal vesicle and epididymal portion of vas deferens of 22 vs 3-month-old rats. Age dependent differences in the molecular, biochemical and functional properties of alpha(1)-adrenoceptors in the rat seminal vesicle and vas deferens may indicate potential differences in the response to alpha(1)-adrenoceptor antagonists with aging.

We compared the effects of two alpha(1)-adrenoceptor antagonists with different selectivity for the alpha(1)-adrenoceptor subtypes, prazosin and naftopidil, on pelvic blood flow and nitric oxide synthase (NOS) levels in the spontaneously hypertensive rat (SHR). SHRs and normotensive Wistar-Kyoto (WKY) rats were distributed initially in four groups: group 1 received prazosin, a subtype nonselective alpha(1)-adrenoceptor antagonist (2 mg/kg/day); group 2 received naftopidil, a selective alpha(1A/D)-adrenoceptor antagonist (10 mg/kg/day); group 3 received cyclazosin, a selective alpha(1B)-adrenoceptor antagonist (5 mg/kg/day); and group 4 received the vehicle orally for 4 weeks. Pelvic blood flow was determined by using a fluorescent microsphere infusion technique. Expression levels of nNOS and eNOS mRNAs in the rat genitourinary tissues were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR) using SYBR Green I. The characteristics of alpha(1)-adrenoceptors in the rat iliac artery were determined by using radioligand receptor binding and real-time RT-PCR techniques. Untreated SHRs had lower blood flow to the ventral prostate, dorsolateral prostate, urinary bladder, and penis and lower mRNA expression levels of nNOS in the bladder and penis and eNOS in the penis than untreated WKY rats. Naftopidil had no significant effects on blood flow and NOS levels, whereas administration of prazosin and cyclazosin to the SHR caused a significant increase in blood flow to each tissue studied and a significant increase in expression levels of these genes. The density of total alpha(1)-adrenoceptors was significantly higher in iliac arteries of untreated SHRs than those of untreated WKY rats. RT-PCR data indicated that alpha(1B)-adrenoceptor mRNA was the significantly predominant gene transcript in iliac arteries of untreated SHRs. Our data show that prazosin, but not naftopidil, causes differential alterations in NOS levels in the SHR genitourinary tract, which could be due to increased pelvic blood flow resulting from inhibiting the vascular alpha(1B)-adrenoceptor. These findings may provide insight into the beneficial effects of subtype nonselective alpha(1)-adrenoceptor antagonists on prostate, bladder, and penile function, when used to treat symptoms of benign prostatic hyperplasia and elevated blood pressure.

This urodynamic double-blind crossover study in women with stress urinary incontinence (SUI) was performed to evaluate the effect of duloxetine, a serotonin and noradrenaline reuptake inhibitor, on urethral function and to determine an appropriate method for making go/no-go decisions in drug development. The urethral pressure profiles at rest and during coughing were measured before and 6 h after single oral administration of placebo or 40 mg duloxetine in 10 women aged 36 to 53 years with SUI (蠅1 episode/wk). Compared with placebo, duloxetine significantly increased the mean and maximal urethral closure pressures at rest calculated over the entire urethra. In the distal third of the urethra, during coughing, duloxetine significantly increased the mean closure pressure and the change from baseline in the maximal amplitudes of the negative closure pressure spikes compared with placebo. Our data indicate the facilitatory effects of duloxetine on both smooth muscle contractions and reflex contractions of the...

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.

We investigated molecular changes that occurred during chronic administration of doxazosin, an α1-adrenoceptor (AR) antagonist, using Affymetrix GeneChip analysis of gene expression. Rats were treated with doxazosin (4 mg/kg/day subcutaneously, supplemented with 4 mg/kg/day orally) for 12 weeks. Labeled cRNA was prepared and the subsequent hybridization to rat 230A arrays was performed. The alterations in gene expression levels of candidate genes identified by microarray analysis with potential biological relevance were verified by real-time reverse transcription polymerase chain reaction (RT-PCR) using SYBR Green I. Doxazosin treated rats had significantly heavier prostates compared to control rats. Microarray analysis revealed that chronic doxazosin treatment caused changes in the expression levels of 625 genes, of which 39 were related to cell death, necrosis, growth, proliferation and G-protein signalling pathways in the rat prostate. Furthermore, RT-PCR experiments, in accord with the microarray analysis, indicated that chronic doxazosin treatment caused an up-regulation in the mRNA expression level of clusterin, an antiapoptotic mediator, and epiregulin, a mitogen, in the ventral and dorsolateral prostate, respectively. These findings, that demonstrate chronic doxazosin administration causes significant changes in the expression of several hundred genes in the rat prostate, may provide insight into the long-term efficacy of α1-AR antagonists in the treatment of benign prostatic hyperplasia.

OBJECTIVE 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). MATERIALS AND METHODS Sprague-Dawley rats (3 months old) were surgically castrated or sham-operated, and then killed 7 days after surgery. Biochemical and pharmacological properties and the location of ET receptors in the RSV were determined by a series of binding experiments with [125I]ET-1, using membrane particulates and slide-mounted frozen sections of RSV. Expression levels of ETA and ETB receptor subtypes, ET-1, ET-3 and ECE-1 mRNAs were assessed by relative multiplex reverse-transcription polymerase chain reaction (RT-PCR). RESULTS The density of total ET receptors increased significantly in the seminal vesicle of the castrated rat. The predominance of the ETA receptor subtype in the RSV did not change with castration. Autoradiographic studies showed the presence of ET receptors on the smooth muscle and epithelium of the RSV. In addition, RT-PCR showed an up-regulation in the expression of ETA and ETB receptor subtypes, ET-1 and ECE-1 mRNAs in the seminal vesicle of the castrated rat. However, castration caused no significant change in the expression levels of ET-3 mRNA. CONCLUSION These findings suggest a regulatory role for testosterone in the expression of the ET receptor system in the RSV.

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 ET A and ET B 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 ET A and ET B 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 ET A receptor subtype, respectively.

We previously have shown that experimental diabetes in rats causes prostatic involution, reduces serum testosterone levels, and causes an upregulation in prostatic endothelin (ET) receptors. Furthermore, insulin treatment normalizes these changes (Saito et al., Mol Cell Biochem 210:1-12, 2000). Since experimental diabetes-induced reduction in serum testosterone may be a factor in the alteration of the ET receptors and of prostatic growth, we investigated the effect of castration, another means of involuting the prostate and decreasing serum testosterone levels, on the expression of ET receptors in ventral and dorsolateral regions of the rat prostate.Three-month-old Sprague-Dawley rats were surgically castrated or sham operated, and then killed on the 7th post-operative day. Biochemical and pharmacological properties, and localization of ET receptors in the rat prostate, were determined by performing a series of binding experiments with [(125)I]ET-1 and by light microscopy autoradiography, respectively. The expression levels of ET-1, ET-3, ET receptor subtypes and endothelin converting enzyme-1 (ECE-1) mRNAs were assessed by relative multiplex reverse transcription polymerase chain reaction (RT-PCR). The total density of ET receptors increases 3.7-fold in the ventral and 2.1-fold in the dorsolateral regions of the castrated rat prostate compared to sham operated animals. Castration causes a 2.4-fold increase in the density of alpha(1)-adrenoceptors (alpha(1)-ARs) in the ventral region of the prostate, but no change in the density of alpha(1)-ARs in the dorsolateral region of the rat prostate. The predominant ET receptor subtype in the rat prostate is the ETA subtype, which is mainly located in the prostatic stroma. In addition, RT-PCR data show an upregulation in the expression of ETB receptor subtype, ET-1 and ECE-1 mRNA in both regions, and a downregulation in the expression of ETA receptor subtype mRNA in the dorsolateral region of the castrated rat prostate. There is no change in the expression of ET-3 mRNA in either region. Castration does not cause significant changes in the pharmacological properties of prostatic ET receptors, i.e., the predominance of ETA receptors in either region of the prostate, or the expression of ETA receptor subtype mRNA in the ventral region of the castrated rat prostate. These results suggest the existence of a region/lobe-specific regulatory role for testosterone in the expression of the ET receptor system in the rat prostate.

Objective To assess fibroblast growth factor-2 (FGF2/bFGF), which is important in the development and maintenance of the normal prostate and in the development of human benign prostatic hyperplasia (BPH) and prostatic carcinoma, in an animal model of experimentally induced diabetes. Materials and methods Using Western blotting and immunohistochemical analyses, the expression of FGF2 in prostates from several groups of rats was investigated. Rats had diabetes for 8 or 16 weeks (induced by intravenous injection with 65 mg/kg streptozotocin); rats were also treated with insulin (starting 8 weeks after the induction of diabetes, for 8 weeks), and two further groups acted as age-matched control rats. Immunohistochemical markers for smooth muscle (α-actin) and epithelium (cytokeratin) were used to distinguish different cell types in adjacent prostatic sections. Results Diabetic rats had smaller prostates and lower serum testosterone levels than their controls; insulin treatment of diabetic rats increased prostatic size and testosterone levels. As shown by Western blotting, diabetes caused greater FGF2 expression than in controls, whereas reverse-transcriptase polymerase chain reaction studies showed similar levels of prostatic FGF-2 mRNA in all groups. Immuno-histochemical studies showed that FGF-2 was expressed in both stromal and epithelial components of the rat prostate. Furthermore, although the expression of FGF2 was higher in epithelial than stromal cells in control prostates, it was distributed uniformly in the diabetic prostate. Conclusion The differences in the level of expression and pattern of distribution of FGF2 suggests a potential role for FGF2 in the changes observed in prostatic growth in diabetic rats.

Objective To examine the effect of age on the biochemical and functional properties, and regional distribution of endothelin (ET) receptors in the rabbit renal pelvis. Materials and methods The properties of ET receptors in 6-week-old and 6-month-old male rabbit renal pelves were examined using isolated muscle-bath and radioligand receptor-binding techniques. Results ET-1 caused a significant increase in the contractile force in muscle strips from all regions of the renal pelvis from both age groups, with the following rank order: upper = middle > lower. The magnitude of the ET-1-induced contractile responses were similar in the lower pelvic regions in both ages, but the responses in the upper and middle regions were significantly greater in younger rabbits. ET-1 increased the frequency of spontaneous activity in a concentration-dependent manner in the upper and middle pelvic regions in both age groups, with significantly smaller ED50 values in the younger than in the older rabbits. In both age groups the lower pelvic region lacked spontaneous activity. The density of total ET receptors was higher in the upper and middle regions of the renal pelvis than in the lower renal pelvis of both ages, with the density in the upper and middle regions being greater in older than in younger rabbits. In all regions, ET subtype selective compounds inhibited [125I]ET-1, binding consistent with the predominance of the ETA receptor subtype, except in the lower region of the older rabbits, in which the densities of ETA and ETB subtypes were similar. In all regions, the younger renal pelvis contained a higher proportion of ETA receptors than in older tissues. Light microscopic autoradiographic data indicated the presence of ETA and ETB receptors in smooth muscle and epithelial cells, respectively. Conclusion These data indicate the presence of regional differences in the density of ET receptors and in the contractile responses to ET-1 in rabbit renal pelvis, and that although older rabbit renal pelvis contains more total ET receptors than younger renal pelvis, the latter had a higher portion of the ETA receptor subtype and the younger tissues were more responsive to ET-1.

As there are significant amounts of endothelin (ET) receptors in the mammalian urinary tract, we investigated the pharmacological properties and localization of ET receptors in the rabbit lower urinary tract as a function of age.The characteristics of ET receptors in bladder dome, trigone and urethra of 6 weeks and 6 months old male rabbits were determined using muscle bath and autoradiographic techniques.ET-1 produces significant contractile responses in smooth muscle strips from bladder dome, trigone, and urethra in both 6 weeks and 6 months old rabbits. Although there was no significant difference in the maximum contractile response of urethral muscle strips to ET-1 between 6 weeks and 6 months old rabbits, the maximum responses to ET-1 were higher in both bladder dome and trigone of 6 weeks than 6 months old rabbits. A selective ETA receptor antagonist, BQ 610, shifted the concentration response curve to ET-1 to the right without decreasing maximal contractile responses in all regions from both age groups, whereas a selective ETB receptor antagonist, IRL 1038, had no significant effect on the contractile response in these tissues. Autoradiographic studies indicate that both ET receptor subtypes are expressed in bladder dome, trigone, and urethra with the ETA subtype being located only in the smooth muscle layers and the ETB subtype being located in both the urothelial and smooth muscle layers.Our data indicate the presence of region- and age-dependent differences in the contractile properties of ET receptors in the male rabbit lower urinary tract. Although both ETA and ETB receptor subtypes are present in the smooth muscle layers, the ETA receptor is the sub-type that is primarily involved in the mediation of contractions.

Purpose: Transforming growth factor-β (TGF-β), a potent inhibitor of cell growth, plays an important role in the androgen-dependent processes of the prostate through a complex network of growth factors. TGF-β expression in the prostate is under negative regulatory control of androgen. As experimental diabetes causes a regression of the prostate and decrease in serum testosterone levels in rats, we examined TGF-β alterations at the mRNA and protein levels in the diabetic rat prostate.Materials and Methods: The expression of TGF-β1 and TGF-β2 and their respective mRNAs in prostates from streptozotocin (STZ)-induced diabetic, insulin-treated diabetic and age-matched control rats were investigated, using relative multiplex RT-PCR, semi-quantitative Western blotting, and immunohistochemistry.Results: Induction of diabetes caused a significant reduction in prostatic weight and in serum testosterone levels in rats. Both mRNA and protein levels of TGF-β1, and mRNA level of TGF-β2 were up-regulated in the diabetic...

Streptozotocin (STZ)-induced diabetes causes an upregulation in the expression of endothelin (ET) receptors in the rat prostate (Eur J Pharmacol 310:197, 1996). We examined the effects of insulin treatment, started 8 weeks after the induction of diabetes, on the expression and distribution of ET receptors and their respective mRNAs in the rat prostate. The densities, pharmacological properties and distribution of ET receptors in the rat prostate were examined using radioligand receptor binding and autoradiographic studies, and gene expression of ET receptors was evaluated utilizing the reverse transcription-polymerase chain reaction (RT-PCR). STZ-injected rats had smaller prostates and reduced serum testosterone levels than control and insulin treated diabetic animals. ET receptor density was shown to be significantly higher in the prostate from diabetic rats than those from either control or insulin treated diabetic animals. The pharmacological profile of prostatic ET receptors was similar in all groups (approximately 80% ETA; 20% ETB subtype). ET receptors were predominantly localized to the prostatic stroma. Induction of diabetes increased the expression of mRNA levels of ETA and ETB receptors, and insulin treatment reversed this upregulation to control levels. These results indicate that (1) ET receptor subtypes are expressed in the rat prostate as transcription and translation products; (2) insulin can normalize the diabetes-induced upregulation in the expression of ET receptors and their respective mRNAs; and (3) diabetes-induced regression of the prostate may involve an alteration in ET receptors.

As there are significant amounts of functional endothelin receptors in the mammalian urinary tract, we examined the effect of experimental diabetes on the expression of endothelin receptors and their mRNAs in the rat bladder dome. The density of endothelin receptors in the rat bladder dome was higher (8 and 16 weeks following the onset of diabetes) than in age-matched controls. Insulin treatment, started 8 weeks after the induction of diabetes, partially reversed the endothelin receptor alterations. The pharmacological profile of the endothelin receptors in the bladder dome was similar in all groups and was consistent with the predominance of the endothelin ET A receptor subtype (ET A :ET B =approximately 4:1). Autoradiographic studies demonstrated that the endothelin receptors were located in all tissue components of the bladder, including epithelial and muscular layers. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) data indicated that diabetes increased the expression level of gene transcripts for both endothelin receptor subtypes and that insulin treatment reversed the mRNA upregulation.

Since evidence of development/age-related alterations of endothelin receptors in circulation and respiration systems has been increasing, we attempted to investigate the pharmacological characterization of endothelin receptors in neonatal, premature, and mature male rabbit lower urinary tract. The biochemical properties of ET receptors were examined in the lower urinary tracts of 1-day (neonatal)-, 6-week (premature)-, and 1-year(mature)-old male rabbits with binding technique utilizing [125I]ET-1. The rank orders of the densities (Bmax values) of endothelin receptors in the bladder dome, bladder base, and urethra of different aged rabbits were bladder dome, 1 day > 6 week vLs 1 year, bladder base, 1 day > 6 week vLs 1 year, and urethra, 1 day > 6 week > 1 year. The pharmacological profiles of these binding sites inhibited by various kinds of endothelin receptor compounds showed similar Ki values and similar proportions of endothelin receptor subtypes in the same regions of 1-day-, 6-week-, and 1-year-old animals. Our data clearly demonstrated the presence of regional difference and development-related changes in the density and subtype specificity of endothelin receptors in the lower urinary tract of the male rabbit. Neurourol. Urodynam. 19:71–85, 2000. © 2000 Wiley-Liss, Inc.

Purpose: The subtype specificity, localization and distribution of urethral alpha1-adrenoceptors were studied in the male rabbit urethra.Materials and Methods: The properties of the urethral alpha1-adrenoceptors were investigated using radioligand receptor binding and light microscopic autoradiography with [(125) I]iodo-2-[b-(4-hydroxyphenyl)-ethylaminomethyl]tetralone (HEAT), and immunohistochemistry with monoclonal anti-alpha smooth muscle actin and anti-alpha sarcomeric actin antibodies.Results: Saturation experiments with [(125) I]HEAT demonstrated the presence of significant amounts of a single high affinity binding site for alpha1 adrenoceptors in the male rabbit urethra. The pharmacological profile of the alpha1 adrenoceptors in rabbit urethra, determined by inhibition experiments with subtype selective alpha1 adrenoceptor antagonists, was characterized by the following rank order of potency of inhibition constants (Ki values): prazosin

We studied the effects of 8 weeks of streptozotocin (STZ)-induced diabetes on the density and the pharmacological properties of α1-adrenoceptors in the rat prostate using receptor-binding experiments with [125I]iodo-2[β-(4-hydroxyphenyl)-ethylaminomethyl]tetralone [125I]HEAT. Saturation experiments showed the presence of specific [125I]HEAT-binding sites in the control and diabetic rat prostate and that the induction of diabetes significantly decreased the density of [125I]HEAT-binding sites in the rat prostate. [125I]HEAT-binding sites in the prostate of both groups were inhibited by prazosin (nonselective), spiperone (α1B-selective), WB4101 and 5-methylurapidil (α1A-selective) and BMY7378 (α1D-selective) with the following rank order of Ki values: prazosin < WB4101 < 5-methylurapidil < spiperone < BMY7378, indicating a similar pharmacological profile of α1-adrenoceptor in the 2 groups.Comparing the Ki values of the rat prostate with those obtained from the rat submaxillary gland (α1A), rat spleen (α1B), rat vas deferens (α1A + α1B) and those reported for cloned α1D, indicates the predominance of the α1A + α1B or the α1A subtype in the rat prostate. The present study demonstrates that STZ-induced diabetes downregulates the expression of α1-adrenoceptor in the rat prostate, without significantly affecting the receptor subtype specificity.

To elucidate the subtype specificity of muscarinic cholinergic receptors in mediating contractile responses in the lower urinary tract, we investigated contractile and biochemical properties of muscarinic receptors in bladder dome, bladder base and urethra of the rabbit. Isometric contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists. Bladder dome, bladder base, and urethra demonstrate different characteristics in terms of efficacy and potency with respect to carbachol-induced contractile responses. Emax values are significantly larger and ED50 values are significantly smaller in bladder dome and bladder base than in urethra. Calculation of the pA2 values, the negative logarithm of the antagonist affinity constant (KB), for a series of muscarinic antagonists, i.e., atropine (nonselective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective) indicate that the carbachol-induced contractile response in bladder dome and bladder base is mediated through the M3 receptor subtype whereas the carbachol-induced contractile response in urethra is probably mediated through the M1 and/or M3 and possibly M2 subtypes. Muscarinic cholinergic antagonists inhibit [3H]quinulidinyl benzilate binding to bladder dome, bladder base and urethra with the following rank order of affinities: atropine4-DAMPmethoctraminepirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.

As there is increasing evidence that diabetes induces changes in the plasma levels of endothelins (ETs) and in the properties of ET receptors in peripheral tissues, and as there are reports indicating the presence of significant amounts of ET receptors in mammalian vasa deferentia, we studied possible alterations in ET receptor characteristics in the vasa deferentia of the following groups of rats: 8 weeks diabetic (D8), 8 weeks age-matched control (C8), 16 weeks diabetic (D16), 16 weeks diabetic-insulin-treated (started 8 weeks after the onset of diabetes) (DI16), and 16 weeks age-matched control (C16). Diabetes was induced by the i.v. injection of 65 mg/kg streptozotocin (STZ). Diabetic rats had hyperglycemia, hypoinsulinemia, glucosuria, polydipsia, and polyuria and had smaller vasa deferentia than control and diabetic-insulin-treated animals. Receptor binding experiments with [125I]ET-1 demonstrated that the densities of ET receptors in vasa deferentia from D8, C8, D16, DI16, and C16 animals were 377 +/- 11, 255 +/- 24, 315 +/- 18, 210 +/- 12, and 214 +/- 7 fmol/mg of protein, respectively. [125I]ET-1 binding to the ET receptors was inhibited by ET-1 (non-selective), BQ 610 (ETA selective), ET-3 (ETC selective), and IRL 1620 (ETB selective) with the following rank order of Ki values: ET-1BQ 610ET-3IRL 1620. The pharmacological profile of the ET receptors was similar in all groups and was consistent with the predominance of the ETA receptor subtype in the rat vasa deferentia. Our data indicate that experimental diabetes up-regulates the density of ET receptors in the rat vasa deferentia and that the receptor up-regulation is reversed by insulin treatment.

Previous studies from our laboratory demonstrated that 8 weeks of streptozocin (STZ)-induced diabetes and sucrose-fed diuresis resulted in increases in the density of muscarinic receptors in rat bladder dome and that early insulin treatment (started 3 days after the onset of diabetes) prevented the diabetes-induced upregulation (J Pharmacol Exp Ther 248:81–88, 1989; Diabetes 40: 1150–1156, 1991; J Urol 147:760–763, 1992). To determine whether diabetes- and diuresis-induced alterations in muscarinic receptors in rat bladder dome are reversible, we administered insulin (beginning 8 weeks after the onset of diabetes) or removed sucrose from drinking water of diuretic rats (beginning 8 weeks after the onset of diuresis). Five groups of rats were maintained for 16 weeks: 1) STZ-induced diabetic rats (65 mg/kg intravenously); 2) insulin-treated diabetic rats (5–8 U/day insulin subcutaneously beginning 8 weeks after the onset of diabetes); 3) sucrose-fed diuretic rats (5% sucrose in drinking water throughout 16 weeks); 4) sucrose-removed rats (sucrose withdrawn from drinking water after 8 weeks of the sucrose-induced diuretic state); and 5) age-matched control rats. Radioligand receptor binding experiments with [3H]quinuclidinyl benzilate showed an increase in the density of muscarinic receptors in bladder dome of diabetic and sucrose-fed rats compared with age-matched control rats. Removing the 5% sucrose from the drinking water of diuretic rats reversed the increased water intake and urine output, decreased the bladder hypertrophy that accompanied the diuretic state, and corrected the upregulation of the muscarinic receptors. Late insulin treatment also improved (to a lesser extent than removing the sucrose) the bladder enlargement, polydipsia, polyuria, and the muscarinic receptor upregulation in diabetic rats. Pharmacological evaluation indicated that the induction of diabetes did not affect muscarinic subtype specificity but did result in an alteration in muscarinic receptor-G protein coupling in rat bladder dome.

α(1)-Adrenoceptors regulate blood pressure, regional vascular resistance and tissue blood flow. As aging and hypertension may impact pelvic arterial blood flow resulting in bladder and penile dysfunction, we investigated effects of age and hypertension on α(1)-adrenoceptors in the major source arteries of the rat bladder and penis. Using radioligand receptor binding, real-time reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent microsphere infusion techniques, we compared 3 and 22-month-old male Fischer rats, and male normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Twenty-two-month-old rats and SHRs had significantly higher total α(1)-adrenoceptor density in the internal iliac artery and lower blood flow to the bladder and penis than 3-month-old and WKY rats, respectively. RT-PCR data showed an age and hypertension related increase in the expression of α(1B)-adrenoceptor mRNA in the internal iliac, vesical and internal pudendal arteries and a switch from α(1A) predominance in 3-month-old and WKY rats to α(1B)α(1A) in 22-month-old rats and SHRs. Our data indicate the presence of age and hypertension related alterations in vascular α(1)-adrenoceptor subtype distribution and in blood flow to the rat bladder and penis. These findings suggest that pharmacological blockade of the vascular α(1B)-adrenoceptor, which could increase pelvic blood flow, may contribute to the improvement of bladder and penile dysfunctions in animal models for aging and hypertension.

(+)-[3HPN 200-110 (a dihydropyridine calcium channel antagonist) was utilized to characterize calcium channel binding sites in rabbit bladder dome, bladder base, and urethra. Specific binding of (+)-[3H]PN 200-110 to membrane particulates was saturable, reversible, linear to protein concentration, and of high affinity. The density of (+)-[3H]PN 200-110 binding sites (Bmax values in fmol/mg of protein) and the affinity constants for (+)-[3H]PN 200-110 (KD value in pM) in urethra, bladder dome and bladder base were 64.1 ± 7.8 and 179±31; 21.9±3.0 and 213±36; and 18.8±4.2 and 140±28, respectively. Agonists and antagonists inhibited (+)-[3H]PN 200-110 binding with Ki values in the following rank order: nitrendipine < nifedipine < niguldipine ≪ Bay K 8644 ≪ verapamil. Although carbachol-induced contractile responses were 20–30 times smaller in muscle strips from urethra than from bladder base or bladder dome, KCl-induced contractions were only 3–4 times smaller in urethra than in bladder tissues. Nifedipine inhibited carbachol-induced contractions in urethra, bladder dome, and bladder base by 76%, 64%, and 60%, respectively, and completely inhibited KCl-induced contractions in all three tissues. IC50 values for nifedipine inhibition of both carbachol- and KCl-induced contractions were significantly smaller in urethra than in bladder base or bladder dome. Nitrendipine, niguldipine and verapamil inhibited urethral contractions induced by carbachol and KCl to the same degree as did nifedipine. The IC50 values, obtaines from functional studies, for calcium channel antagonists were in good agreement with Ki values obtained from binding studies. BAY K 8644, a calcium channel agonist, increased both KCl- and carbachol-induced contractions and potentiated CaCl2-induced contractions in K+-depolarizing media in urethra but not in bladder dome or bladder base. Our data indicate that the density of (+)-[3H]PN 200-110 binding sites is higher in the urethra than in the bladder dome or bladder base and that the urethra is functionally more sensitive to dihydropyridine agonists and antagonists than is either the bladder dome or bladder base.

(+)-[3H]PN 200-110 (a dihydropyridine calcium channel antagonist) binding sites were studied in ureters of 1-day (neonatal), 6-week (premature), 6-month (young) and 4.5- to 5-year (old) female rabbits. Specific binding of (+)-[3H]PN 200-110 to ureteral membrane particulates was saturable, reversible and of high affinity. The densities (Bmax) of (+)-[3H]PN 200-110 binding sites were 46.7 +/- 2.5, 22.6 +/- 2.0, 12.7 +/- 1.8 and 11.9 +/- 1.6 fmol/mg protein in 1-day, 6-week, 6-month and 4.5- to 5-year rabbit ureters, respectively. The affinity constants (KD) of the binding sites for (+)-[3H]PN 200-110 were similar in all groups. Calcium agonists and antagonists inhibited (+)-[3H]PN 200-110 binding to 1-day and 6-week rabbit ureters with the following rank order of Ki values: nitrendipine < nifedipine < BAY K 8644 < verapamil. There were no significant differences in Ki values between the neonatal and premature groups. The data demonstrate the presence of an age-related down-regulation of (+)-[3H]PN 200-110 binding sites in rabbit ureteral membrane particulates.

OBJECTIVE 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. MATERIALS AND METHODS SHR and normotensive Wistar-Kyoto (WKY) rats were distributed initially in three groups: group 1 received doxazosin (30 mg/kg/day); group 2 received nifedipine (30 mg/kg/day); and group 3 received the vehicle orally for 4 weeks. The alterations in gene expression levels of candidate genes identified by microarray analysis with potential biological relevance were verified by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS Voiding frequency was significantly higher, and mean voided volume was significantly lower in untreated SHRs than untreated WKY rats. Microarray analysis revealed that 25 of the differentially expressed genes in untreated SHRs compared to untreated WKY rats were related to Gs, Gi, Gq and G12/13 signalling, calcium handling, ion transport and smooth muscle-related genes. Furthermore, RT-PCR data, in accord with the microarray analysis, indicated that untreated SHRs had lower mRNA expression levels of Adcy2, Adcy3, Rgs2, Rgs3, Rgs4 and Arhgdia, and higher mRNA expression levels of Arhgef1, Arhgef11, Arhgef12, Geft, Rock1 and Rock2 than untreated WKY rats. The differential alterations in the micturition patterns and in the expression of several genes related to G-protein signalling pathway observed in SHRs were attenuated by treatment with doxazosin, but not nifedipine. CONCLUSION Our data suggest that differential alterations in the expression of several genes related to Gs, Gq and G12/13 signalling pathways in the SHR bladder might be important in hypertension-associated bladder dysfunction.

To determine the contribution of diuresis-induced bladder hypertrophy, which accompanies the diabetic state, on the biochemical and functional alterations observed in the diabetic bladder, we compared three experimental groups: 8-wk streptozocin (STZ)-induced diabetic rats, 8-wk sucrose-fed diuretic rats, and age-matched controls. Diabetic and sucrose-fed rats had higher water intake, higher urine output, and larger bladders than controls. Diabetic rats had lower serum insulin levels, lower body weights, and higher serum glucose levels than either control or sucrose-fed animals. Receptor binding studies with [3H]quinuclidinyl benzilate in bladder dome demonstrated an upregulation of muscarinic receptors in diabetic and sucrose-fed rats compared with controls. Parallel binding studies with [3H]dihydroalprenolol and [125I]iodopindolol showed an upregulation of β-adrenergic receptors in diabetic but not in sucrose-fed bladder domes. Carbachol induced larger contractile responses in diabetic and sucrose-fed than in control bladder dome muscle strips. isoproterenol relaxed KCl-contracted detrusor strips from both diabetic and sucrose-fed rats to a greater degree and with a higher affinity than detrusor strips from controls. Our data show that overdistension and increased workload per se contributed to the upregulation of muscarinic but not to the upregulation of β-adrenergic receptors in STZ-induced diabetes. Furthermore, the magnitude of carbachol-induced contractions correlated with muscarinic receptor upregulation, whereas the magnitude of isoproterenol-induced relaxation did not correlate with changes in the density of the β-adrenergic receptors. Thus, it appears that different regulatory mechanisms are involved in diabetes-induced alterations in muscarinic and β-adrenergic receptors in bladder dome.

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.

OBJECTIVE To examine the regional differences in the functional (pharmacological) and biochemical properties of endothelin (ET) receptors in the rabbit prostatic urethra. MATERIALS AND METHODS The properties of ET receptors in 6-month-old male rabbit prostatic urethras were examined using isolated muscle-bath and radioligand receptor-binding techniques. Using plasma membrane suspensions, saturation and inhibition experiments with [125I]ET-1 and unlabelled agonists and antagonists (ETA-selective antagonist BQ123, and ETB-selective agonist sarafotoxin 6c, STX6c) were done to determine the ET receptor densities and their subtype specificities in the different regions of the urethra. RESULTS The ETs (ET-1 and ET-3) produced significant concentration-dependent contractile responses in the smooth muscle strips from the different regions of the urethra. Although the maximum contractile responses induced by ET-1 were similar in the different regions, the maximum contractile responses induced by ET-3 were greater in the distal region than in the proximal or middle regions, suggesting that the contractile response to ET-1 is more potent than that to ET-3 in all regions, and that there are region-specific differences in the responses to ET-3 but not ET-1. Moreover, the ET-3-induced contractile response was suppressed by BQ788 (a selective antagonist of the ETB receptor) suggesting that the ETB receptor subtype contributes to the contractile responses mediated by ET-3. The ET receptors were expressed in higher concentrations in the distal than in the proximal or middle regions. BQ123 and STX6c inhibited [125I]ET-1 binding in all regions with high and low affinity constants, indicating the presence of both ETA and ETB receptor subtypes. The proportions of high-affinity binding sites for BQ123, representing ETA receptors, were ≈ 68%, 63% and 42% in the proximal, middle and distal regions, respectively. By contrast, the proportions of high-affinity binding sites for STX6c, representing ETB receptors, were ≈ 27%, 35% and 52% in the proximal, middle, and distal regions, respectively. These data indicate the presence of regional differences in the densities and subtype specificities of ET receptor subtypes, and the existence of regional differences in the rabbit prostatic urethra. CONCLUSION The results suggest regional differences in ETB receptor subtypes that mediate contractile responses to ET-3, reflecting differences in the densities and specificities of the ET receptor subtypes in the rabbit prostatic urethra.

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.

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.

Because age related changes occur in the properties of alpha(1)-adrenoceptor in several mammalian tissues and alpha(1)-adrenoceptor antagonists are extensively used to treat lower urinary tract symptoms secondary to benign prostatic hyperplasia, we investigated age related changes in the functional, biochemical and molecular properties of alpha(1)-adrenoceptor in the rat genitourinary tract.The characteristics of alpha(1)-adrenoceptor in the ventral and dorsolateral prostate, and bladder base and dome of 3 and 22-month-old rats were determined using an isolated muscle bath, radioligand receptor binding and real-time reverse transcriptase-polymerase chain reaction techniques.Old rats had significantly higher body weight, lower testosterone, a smaller ventral prostate and a larger bladder dome than young rats. Although there was no significant age dependent difference in the properties of alpha(1)-adrenoceptor in the bladder base and dome, total alpha(1)-adrenoceptor density, mRNA expression of all 3 alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) and the maximum contractile responses to phenylephrine were significantly lower in the ventral and dorsolateral prostate of 22 vs 3-month-old rats.Age related differences in the molecular, biochemical and functional properties of alpha(1)-adrenoceptors in the rat genitourinary tract may indicate potential differences in the response to alpha(1)-adrenoceptor antagonists with aging, ie a decrease in the therapeutic response in old vs young rats in the response to alpha(1)-adrenoceptor antagonists when used to treat lower urinary tract symptoms secondary to benign prostatic hyperplasia.

Although the clinical efficacy of alpha1-adrenoceptor (alpha1-AR) antagonists for the treatment of benign prostatic hyperplasia is not disputed, their mechanism of action and ability to maintain long-term effectiveness have only recently been investigated. Since it is known that chronic administration of receptor antagonists causes up-regulation in the targeted receptor, we examined the effects of chronic administration of doxazosin, a nonspecific long acting alpha1-AR antagonist, on the properties of alpha1-AR subtypes in the rat prostate.Rats were treated with doxazosin (2 or 4 mg/kg daily subcutaneously, supplemented with 4 mg/kg daily orally) for 8 or 12 weeks. Prostatic alpha1-AR properties at the protein and gene transcript levels were quantified by radioligand receptor binding and real-time reverse transcriptase-polymerase chain reaction, respectively.Treated rats that received the highest levels of doxazosin had significantly heavier prostates compared with age matched controls. After 12 weeks of treatment radioligand binding studies with radiolabeled alpha1-AR antagonist demonstrated no significant differences in the density of total alpha1-ARs in the prostate, whereas the results of real-time reverse transcriptase-polymerase chain reaction showed up-regulation in the mRNA expression levels of all 3 alpha1-AR subtypes (alpha1A, alpha1B and alpha1D) in the ventral and dorsolateral regions of the rat prostate.These data demonstrate that chronic treatment with doxazosin causes an alteration in the properties of the alpha1-AR system in the rat prostate. These findings may provide insight into the long-term effectiveness of alpha1-AR antagonists in the treatment of benign prostatic hyperplasia.

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 ET(A) and ET(B) 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 ET(A) 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 ET(A) receptor subtype, respectively. Furthermore, ET-1, ET-3, ECE-1, and ET(A) and ET(B) 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.

We have previously demonstrated that long-term administration of doxazosin, an alpha(1)-adrenoceptor (alpha(1)-AR) antagonist, causes an up-regulation in the expression of alpha(1)-AR subtype mRNAs in the rat genitourinary tract and suggested that these changes may affect long-term effectiveness of alpha(1)-AR antagonists when used to treat the lower urinary tract symptoms of benign prostatic hyperplasia. As chronic administration of alpha(1)-AR antagonists may cause similar alterations in other tissues in which alpha(1)-ARs play a physiologic role, we examined the effects of long-term administration of doxazosin on the expression of alpha(1)-AR subtype mRNAs in several rat tissues. Rats were treated with doxazosin (4 mg/kg/day subcutaneously, supplemented with 4 mg/kg/day orally) for 12 weeks. The cDNA was prepared by reverse transcription of RNA extracted from the rat kidney, heart and aorta. alpha(1A), alpha(1B) and alpha(1D)-AR mRNA expression levels were quantified by real-time reverse transcription polymerase chain reaction. The rank order of expression levels of the alpha(1)-AR mRNAs in rat tissues were: alpha(1A)-AR, kidney > heart > aorta; alpha(1B)-AR, heart > kidney > aorta; alpha(1D)-AR, aorta > kidney = heart. Chronic administration of doxazosin caused an up-regulation in the mRNA level of alpha(1A), alpha(1B) and alpha(1D)-ARs in the rat kidney, heart and aorta, respectively. Our data demonstrate that doxazosin treatment causes differential alterations in the expression of alpha(1)-AR subtype mRNAs in different rat tissues. These findings may provide insight into the long-term effects of alpha(1)-AR antagonists in the treatment of diseases involving tissues whose function is regulated by alpha(1)-ARs.

Endothelins (ETs) mediate paracrine control of vascular tone and secretion of steroids and catecholamines in the adrenal gland through two ET receptor subtypes, ETA and ETB. The differential distribution and function of these subtypes are responsible for the multiplicity of endothelin actions in this tissue. This study examines the regulatory effects of experimental diabetes on the gene expression, subtype specificity and localization of ET receptor subtypes, ET isopeptides, and endothelin-converting enzyme-1 (ECE-1) in the rat adrenal gland. The densities, pharmacological properties and distribution of ET receptor subtypes ETA and ETB in adrenal glands from streptozotocin-induced diabetic, insulin-treated diabetic and age-matched control rats were investigated, using radioligand receptor binding and autoradiographic techniques. The gene expression of ETA and ETB receptors ET-1, ET-3 and ECE-1 was evaluated using relative multiplex reverse transcription/PCR. The induction of diabetes caused a marked reduction in body weight but no significant change in adrenal gland size. The density of ET receptors was significantly increased in the diabetic rat adrenal gland, mainly because of an increase in the expression of ETB receptors. Insulin treatment normalized the diabetes-induced changes in the expression levels of ET receptor subtypes to control levels. The expression level of ET-1 mRNA was up-regulated, whereas ET-3 mRNA was down-regulated in the diabetic adrenal gland compared with the controls. The ECE-1 mRNA level in the adrenal gland was not altered by the induction of diabetes. Autoradiographic studies showed that ETA and ETB are the predominant receptor subtypes in the adrenal medulla and cortex respectively. These results suggest that ETA and ETB receptors are differentially distributed and regulated in the diabetic rat adrenal gland.

Background: The present investigation was undertaken to examine the effect of insulin treatment on diabetes-induced alterations in endothelin (ET) receptors in rat ureters. Methods: The biochemical properties of ET receptors were examined in rat ureters from the following groups: 8 weeks diabetic (D8); 8 weeks age-matched control (C8); 16 weeks diabetic (D16); 16 weeks diabetic-insulin treated (insulin started 8 weeks after the onset of diabetes) (DI16); and 16 weeks age-matched control (C16). Diabetes was induced by the i.v. injection of 65 mg/kg streptozotocin (STZ). Results: The densities of ET receptors (Bmax values), as determined by saturation experiments with [125I]-ET-1, in the ureteral plasma membranes of D8, C8, D16, DI16 and C16 were 91.7 ± 10.1, 42.1 ± 7.2, 71.1 ± 2.4, 51.5 ± 6.3 and 45.1 ± 3.3 fmol/mg of protein, respectively. [125I]-ET-1 binding to the ET receptors in rat ureteral membrane particulates was inhibited by ET-1 (non-selective), ET-3 (ETB/C selective), BQ 610 (ETA selective) and IRL 1620 (ETB selective) with the following rank order of Ki values: ET-1 < BQ 610 < ET-3 < < IRL 1620. The pharmacological profile of the ET receptors was similar in all groups examined and was consistent with the predominance of the ETA receptor subtype in the ureteral membrane particulates. The subtype specificity of ET receptors in the ureteral tissues is confirmed with inhibition data obtained from similar binding studies in cloned human ETA and ETB receptors. Conclusion: The data indicate that diabetes results in an up-regulation of ET receptors in the rat ureter, which is normalized by insulin treatment.

The subtype specificity, localization and distribution of urethral alpha1-adrenoceptors were studied in the male rabbit urethra.The properties of the urethral alpha1-adrenoceptors were investigated using radioligand receptor binding and light microscopic autoradiography with [125I]iodo-2-[b-(4-hydroxyphenyl)-ethylaminomethyl]tetralone (HEAT), and immunohistochemistry with monoclonal anti-alpha smooth muscle actin and anti-alpha sarcomeric actin antibodies.Saturation experiments with [125I]HEAT demonstrated the presence of significant amounts of a single high affinity binding site for alpha1 adrenoceptors in the male rabbit urethra. The pharmacological profile of the alpha1 adrenoceptors in rabbit urethra, determined by inhibition experiments with subtype selective alpha1 adrenoceptor antagonists, was characterized by the following rank order of potency of inhibition constants (Ki values): prazosinor = WB 4101spiperone5-methylurapidilBMY 7378. The pKi values for the rabbit urethra were correlated with the pKi values for rat spleen, submaxillary glands, and vas deferens and for those reported for cloned alpha1d receptors with correlation coefficients of 0.68, 0.929, 0.909, and 0.523, respectively.The pharmacological characterization demonstrates the predominance of alpha1A or alpha1A + alpha1B adrenoceptor subtype(s) in male rabbit urethral smooth muscle. Furthermore, the autoradiographic and immunohistochemical studies show a heterogeneous distribution of alpha1 adrenoceptors along the longitudinal axis of the urethra, within the smooth muscle fibers, with the receptors being localized more densely in the proximal than in the distal urethra.

Background: The purpose of this study was to evaluate the muscarinic receptor subtypes expressed in rat bladder smooth muscle and characterize the muscarinic receptor-coupled phosphatidylinositol (PI) hydrolysis in order to clarify the first step of bladder smooth muscle contraction. Methods: Expressions of mRNAs of muscarinic receptor subtypes were examined by Northern blot analysis. Changes in the mass of inositol 1,4,5-trisphosphate(IP3) and the inhibitory effects of muscarinic subtype specific antagonists on PI hydrolysis were determined after carbachol stimulation. Results: mRNAs of m2 and m3 genes, encoding M2 and M3 receptors, were expressed in rat bladder smooth muscle. Carbachol produced a rapid increase of IP3, which returned to the basal level within 30 seconds. 4-Diphenylacetoxyl-N-methylpiperidine methiodide (4-DAMP; M1 and M3 antagonist) strongly inhibited the PI hydrolysis, but methoctramine (M2 antagonist) partially inhibited it at 10-4 mol/L The 1C50 value for atropine was 9.5 times 10-9 mol/L, for pirenzepine 6.4 times 10-6 mol/L, and for 4-DAMP 1.5 times 10-7 mol/L. Conclusion: M2 and M3 receptors are expressed in rat urinary bladder. Only M3 receptor was involved in the production of IP3, which might induce the initial phase of contractile response in rat bladder smooth muscle after carbachol stimulation.

We investigated the binding characteristics of endothelin (ET) receptors in the ureters of rats with experimentally induced diabetes and diuresis. Receptor binding experiments demonstrated an upregulation in the expression of [125I]ET-1 binding sites in the diabetic rat ureter but not in the diuretic rat ureter. ET-1, ET-3, IRL 1620, and BQ 610 inhibited [125I]ET-binding to the rat ureter consistent with the predominance of ETA receptors in these tissues. The subtype specificity of ET receptors in ureteral tissues was confirmed with inhibition data obtained from cloned human ETA and ETB receptors.

We studied the properties of endothelin receptors in the prostate of 8-week streptozotocin-diabetic and control rats. The density of endothelin receptors, as determined by saturation experiments with [ 125 I]endothelin-1, were 95.8 ± 5.4 and 171.3 ± 16.7 fmol/mg of protein in control and diabetic rat prostates, respectively. The pharmacological profile of the endothelin receptors was similar in both groups and was consistent with the predominance of the endothelin ET A receptor subtype in the prostate. Thus, the induction of diabetes upregulates the expression of endothelin receptors in the rat prostate, but does not alter the pharmacological profile of the receptors in these tissues.

We investigated the binding characteristics of endothelin (ET) receptors in rabbit ureter, bladder dome, bladder base, and urethra and compared the observed receptor properties with those of cloned human ETA and ETB receptors expressed in Chinese hamster ovary K-1 (CHO) cells. Receptor binding experiments with [125I]ET-1 revealed the presence of a single class of specific, saturable, high affinity [125I]ET-1 binding sites in all of the regions of the studied urinary tract. The rank order of the densities (Borax values) of [125I]ET-1 binding sites was: ureter ≫bladder dome > bladder base = urethra. ET-1 and ET-2 inhibited [125I]ET-1 binding to the membrane particulates from the various regions of the urinary tract with single high affinity constants. A selective ETA receptor antagonist, BQ 123, and selective ETB agonists, ET-3 and sarafotoxin S6c (STXc), inhibited [125I] ET-1 binding to bladder dome, bladder base, and urethra with high and low affinity constants indicating the presence of both ETA and ETB receptor subtypes in these tissues. The subtype specificity of ET receptors in the rabbit tissues is confirmed with inhibition data obtained from similar binding studies in cloned human ETA and ETB receptors. The proportions of high affinity binding sites for ET-3, representing ETB receptors, were approximately 25%, 27%, and 46% in bladder dome, bladder base, and urethra, respectively. Corresponding values for STXc were approximately 17%, 28%, and 43% in bladder dome, bladder base, and urethra, respectively. In contrast to the findings for ET-3 and STXc, the proportions of high affinity binding sites for BQ 123, representing ETA receptors, in bladder dome, bladder base, and urethra were approximately 84%, 74%, and 60%, respectively. In ureter, these selective compounds inhibited [125I]ET-1 binding with either a low (ET-3 and STXc) or a high binding affinity (BQ 123), suggesting the presence of only a single receptor subtype (ETA) in this tissue. These data indicate that there are regional differences in the density and subtype specificity of ET receptors in the rabbit urinary tract.