Jamshid Latifpour

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.

Age-dependent Alterations in beta-adrenergic Responsiveness of Rat Detrusor Smooth Muscle

The relaxant effects of norepinephrine (NE, 10(-7) to 10(-4) M.) and isoproterenol (ISO, 10(-9) to 10(-4) M.) on maximal KCl-induced tonic contractions and the relaxant effects of ISO on contractions induced by electrical field stimulation (EFS) were measured in detrusor muscle strips obtained from 22-25 day, 90-95 day and 22-month-old male Fischer 344 rats. The maximum relaxant response to NE and ISO on KCl-induced tonic contractions decreased significantly with increasing age. The ED50 values for ISO, but not NE, increased with age. The maximum relaxant response to ISO on EFS-induced contractions also was reduced significantly in the old bladders. The relaxation effects of forskolin (10(-6) to 3 x 10(-5) M.), dibutyryl cyclic AMP (DBcAMP, 10(-4) to 3 x 10(-3) M.) and cholera toxin (10 micrograms/ml.) were examined on maximal KCl-induced contractions of the muscle strips obtained from the three age groups. The relaxant responses to forskolin decreased significantly with increasing age, whereas DBcAMP relaxed the muscle strips from the three age groups equally. Cholera toxin (10 micrograms) attenuated KCl-induced phasic contractions, and this effect was impaired in the aged rat detrusor. The density of beta-adrenergic receptors, as determined by radioligand binding with [125I]iodopindolol ([125I]-PIN) decreased with increasing age. These data demonstrate an age-related decrease in the responsiveness of the bladder detrusor to beta-adrenergic stimulation that may be related to the decreased density of beta-adrenergic receptors and decreased cyclic AMP (cAMP) production.

Regional differences in the density and subtype specificity of endothelin receptors in rabbit urinary tract

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.

Experimental diabetes-induced regression of the rat prostate is associated with an increased expression of transforming growth factor-β

PURPOSE Transforming growth factor-beta (TGF-beta), 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-beta 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-beta alterations at the mRNA and protein levels in the diabetic rat prostate. MATERIALS AND METHODS The expression of TGF-beta1 and TGF-beta2 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-beta1, and mRNA level of TGF-beta2 were up-regulated in the diabetic rat prostate. Insulin-treatment normalized changes observed in prostatic weight and serum testosterone levels, and reversed the alterations in the TGF-beta1 and TGF-beta2 expression at the gene transcript and protein levels to control levels. Immunohistochemical studies demonstrated that TGF-beta1 is localized to prostatic stromal cells, whereas TGF-beta2 is located in both epithelial and stromal cells. CONCLUSION These results suggest that TGF-beta1 and TGF-beta2 may be involved in the diabetes-induced regression of the prostate gland.

Evidence for the Presence of Regional Differences in the Subtype Specificity of Muscarinic Receptors in Rabbit Lower Urinary Tract

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: atropine > 4-DAMP > methoctramine > pirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.

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.

The reversal effect of insulin on diabetes-induced alterations in beta adrenergic and muscarinic receptors in rat prostate.

Previous studies from our laboratory demonstrated that 8 weeks after the induction of diabetes by the administration of streptozotocin (STZ) there was a downregulation of beta adrenergic and muscarinic cholinergic receptors in rat prostate, and that early insulin treatment (started 3 days after the onset of diabetes) prevented these alterations from occurring. In the present study, the effects of later insulin treatment (started 8 weeks after the onset of diabetes) on the reversibility of diabetes-induced alterations in beta adrenergic and muscarinic receptors in rat prostate were investigated. Three groups of rats were maintained for 16 weeks: 1) diabetics, 2) insulin-treated diabetics (subcutaneously injected with 5 to 8 U per day starting 8 weeks after the onset of diabetes) and 3) age matched controls. Binding studies with [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB) showed a significantly lower density of beta adrenergic and muscarinic cholinergic receptors in the diabetic rat prostate than in prostate from either controls or insulin-treated diabetic animals. Inhibition of [3H]DHA binding by isoproterenol, a beta adrenergic agonist, and binding of [3H]QNB by carbachol, a muscarinic agonist, indicated the presence of low and high affinity agonist binding sites for each receptor. The relative proportion of high affinity to total binding sites as well as the low and high affinity constants were similar in all groups. These data indicate that insulin treatment, begun 8 weeks after the onset of diabetes, can reverse the diabetes-induced downregulation of both beta adrenergic and muscarinic cholinergic receptors in STZ-diabetic rat prostates.

Differential Regulation of Bladder β-Adrenergic and Muscarinic Cholinergic Receptors in Experimental Diabetes

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.

Reversibility of Diabetes- and Diuresis-Induced Alterations in Rat Bladder Dome Muscarinic Receptors

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.

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.