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BACKGROUND AND PURPOSEDiabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca 2+ influx through L-type voltage-operated Ca 2+ channels (L-VOCC). EXPERIMENTAL APPROACHMale C57BL/6 mice were injected with streptozotocin (125 mg·kg -1 ). Four weeks later, contractile responses to carbachol, a,b-methylene ATP, KCl, extracellular Ca 2+ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M2/M3 receptors, purine P2X1 receptors and L-VOCC in the bladder was determined. KEY RESULTSDiabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, a,b-methylene ATP, KCl, extracellular Ca 2+ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M3 receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M2 and P2X1 receptors remained unchanged. CONCLUSIONS AND IMPLICATIONSDiabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca 2+ influx through L-VOCC.

IntroductionHydrochlorothiazide has a negative influence on penile erection but little is known about the mechanism(s) involved.AimsTo characterize the effects of this diuretic on mouse corpus cavernosum (CC) smooth muscle in vitro and ex vivo.MethodsCC strips of C57BL/6 mice (12–16 weeks old) were mounted in organ baths containing Krebs-Henseleit solution and tissue reactivity was evaluated. Expression of genes encoding diuretic targets and enzymes involved in penile erection were evaluated by polymerase chain reaction.Main Outcome MeasuresStimulation-response curves to phenylephrine (10 nmol/L–100 μmol/L) or to electrical field stimulation (1–32 Hz) were constructed, with or without hydrochlorothiazide. Strips of CC from mice after long-term hydrochlorothiazide treatment (6 mg/kg/day for 4 weeks) with or without amiloride (0.6 mg/kg/day for 4 weeks) in vivo also were studied. Nitric oxide and Rho-kinase pathways were evaluated.ResultsHydrochlorothiazide (100 μmol/L) increased the maximum response to phenylephrine by 64% in vitro. This effect was unaffected by the addition of indomethacin (5 μmol/L) but was abolished by N(ω)-nitro-L-arginine methyl ester (100 μmol/L). Hydrochlorothiazide (100 μmol/L) potentiated electrical field stimulation-induced contraction in vitro, but not ex vivo. Long-term treatment with hydrochlorothiazide increased the maximum response to phenylephrine by 60% and resulted in a plasma concentration of 500 ± 180 nmol/L. Amiloride (100μmol/L) caused rightward shifts in concentration-response curves to phenylephrine in vitro. Long-term treatment with hydrochlorothiazide plus amiloride did not significantly increase the maximum response to phenylephrine (+13%). Reverse transcriptase polymerase chain reaction did not detect the NaCl cotransporter in mouse CC. Hydrochlorothiazide did not change Rho-kinase activity, whereas amiloride decreased it in vitro and ex vivo (approximately 18% and 24% respectively). A 40% decrease in Rock1 expression also was observed after long-term treatment with hydrochlorothiazide plus amiloride.ConclusionHydrochlorothiazide potentiates contraction of smooth muscle from mouse CC. These findings could explain why diuretics such as hydrochlorothiazide are associated with erectile dysfunction.

Introduction The antihypertensive effects of thiazide diuretics such as hydrochlorothiazide are commonly associated with erectile dysfunction. The association of hydrochlorothiazide/amiloride is not associated with erectile dysfunction. The hypothesis is that amiloride has beneficial effect in penile erection and, therefore, counterbalances the hydrochlorothiazide-induced disruptive effect. Aim To investigate the effects of amiloride and its analogues hexamethylamiloride and benzamil on rat isolated corpus cavernosa (CC) and intracavernous pressure (ICP) in anaesthetized rats. Methods Rat isolated CC were incubated with amiloride, hexamethylamiloride, and benzamil (10 and 100 μmol/L each), followed by phenylephrine, potassium chloride, and electrical field stimulation (EFS). Their effect on the relaxant responses to EFS and sodium nitroprusside were also determined. Oral (30 mg/kg) and intraperitoneal (3 mg/kg) treatments with amiloride were also investigated on nerve-evoked ICP. Main Outcome Measures In vitro functional studies and in vivo ICP measurement on rat CC were performed. Additionally, phosphodiesterase type V isoform A1 activity and the mRNA expressions of Na+/H+ pump, epithelial sodium channel exchangers (ENaC) channels (α-, β- and γ subunits) and Na+/Ca2+ exchangers were evaluated in CC tissues. Results Amiloride and its analogues significantly reduced the phenylephrine-, potassium chloride–, and EFS-induced CC contractions, which were not changed by nitro-L-arginine methyl ester (100 μmol/L) or indomethacin (6 μmol/L). In phenylephrine-precontracted CC tissues, amiloride itself caused concentration-dependent relaxation and significantly increased the EFS-induced relaxation. Oral and intraperitoneal treatment with amiloride significantly increased the ICP. Phosphodiesterase type V isoform A1 activity was not affected by amiloride. Na+/H+ pump, ENaC, and Na+/Ca2+ exchanger mRNA expressions were all detected in rat CC tissues. Clinical Implication Amiloride analogues may have therapeutic potential for erectile dysfunction. Strength & Limitations The interesting effect of amiloride in penile erection was observed in both in vitro and in vivo methods. The evidence at the moment is restricted to rat CC. Conclusion Amiloride reduces in vitro CC contractility and enhances erectile function after oral and intraperitoneal administration, possibly via inhibition of ENaC.

3 e E f e i t o d a r e p o s i ç ã o d a i n s u l i n a n a s a l t e r a ções funcionais e morfológicas do corpo cavernoso, no diabetes induzido por estreptozotocina em camundongos / Fernanda Del Grossi Ferraz Carvalho. --Campinas, SP : [s.n.], 2012. O r i e n t a d o r : G i l b e r t o D e N u c c i . T e s e ( D o u t o r a d o ) -U n i v e r s i d a d e E s t a d u a l d e Campinas, Faculdade de Ciências Médicas.