Luiz O. Leiria

Role of PKC and CaV1.2 in Detrusor Overactivity in a Model of Obesity Associated with Insulin Resistance in Mice

Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM) in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC) and Cav1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Cav1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001–100 µM), α,β-methylene ATP (1–10 µM), KCl (1–300 mM), extracellular Ca2+ (0.01–100 mM) and phorbol-12,13-dibutyrate (PDBu; 0.001–3 µM) all produced greater DSM contractions in obese mice, which were fully reversed by the Cav1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM) also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Cav1.2 channel protein expression was not modified in any experimental group. Our findings show that Cav1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.

Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice.

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Obesity and asthma: beyond TH2 inflammation

Obesity is a major risk factor for asthma. Likewise, obesity is known to increase disease severity in asthmatic subjects and also to impair the efficacy of first-line treatment medications for asthma, worsening asthma control in obese patients. This concept is in agreement with the current understanding that some asthma phenotypes are not accompanied by detectable inflammation, and may not be ameliorated by classical anti-inflammatory therapy. There are growing evidences suggesting that the obesity-related asthma phenotype does not necessarily involve the classical T(H)2-dependent inflammatory process. Hormones involved in glucose homeostasis and in the pathogeneses of obesity likely directly or indirectly link obesity and asthma through inflammatory and non-inflammatory pathways. Furthermore, the endocrine regulation of the airway-related pre-ganglionic nerves likely contributes to airway hyperreactivity (AHR) in obese states. In this review, we focused our efforts on understanding the mechanism underlying obesity-related asthma by exploring the T(H)2-independent mechanisms leading to this disease.

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels.

BACKGROUND AND PURPOSE Diabetic 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 Ca2+ influx through L‐type voltage‐operated Ca2+ channels (L‐VOCC).

The Soluble Guanylyl Cyclase Activator BAY 60-2770 Ameliorates Overactive Bladder in Obese Mice

PURPOSE Activators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder. MATERIALS AND METHODS C57BL/6 male mice fed for 10 weeks with standard chow or a high fat diet were treated with 1 mg/kg BAY 60-2770 per day for 2 weeks via gavage. Cystometric evaluations were done and responses to contractile agents in isolated bladders were determined. RESULTS Obese mice showed an irregular micturition pattern characterized by significant increases in voiding and nonvoiding contractions, which were normalized by BAY 60-2770. Carbachol, KCl and CaCl2 produced concentration dependent contractions in isolated bladder strips, which were markedly greater in obese than in lean mice. BAY 60-2770 normalized bladder contractions in the obese group. A 78% increase in reactive oxygen species generation in the bladder tissue of obese mice was observed, which was unaffected by BAY 60-2770. Treatment with BAY 60-2770 generated a tenfold increase in cyclic guanosine monophosphate in the bladders of obese mice without affecting the nucleotide level in the lean group. Protein expression of the soluble guanylyl cyclase α1 and β1 subunits was decreased 40% in the bladder tissue of obese mice but restored by BAY 60-2770. CONCLUSIONS Two-week BAY 60-2770 therapy increased cyclic guanosine monophosphate and rescued expression of the soluble guanylyl cyclase α1 and β1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction.

Soluble Guanylyl Cyclase (sGC) Degradation and Impairment of Nitric Oxide-Mediated Responses in Urethra from Obese Mice: Reversal by the sGC Activator BAY 60-2770

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 μM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of β1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and Aims Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested. Methods Human washed platelet aggregation and adhesion assays, as well as flow cytometry for αIIbβ3 integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte). Results BAY 60-2770 (0.001–10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca2+ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca2+ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced αIIbβ3 activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770. Conclusion The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and αIIbβ3 activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.

Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum

Impairment of nitric oxide (NO)-mediated cavernosal relaxations in middle age contributes to erectile dysfunction. However, little information is available about the alterations of sympathetic neurotransmission and contraction in erectile tissue at middle age. This study aimed to evaluate the alterations of the contractile machinery associated with tyrosine hydroxylase (TH) in rat corpus cavernosum (RCC) at middle age, focusing on the role of superoxide anion. Male Wistar young (3.5-mo) and middle-aged (10-mo) rats were used. Electrical-field stimulation (EFS)- and phenylephrine-induced contractions were obtained in RCC strips. Levels of reactive-oxygen species (ROS) and TH mRNA expression, as well as protein expressions for α₁/β₁-subunits of soluble guanylyl cyclase (sGC), in RCC were evaluated. The neurogenic contractile responses elicited by EFS (4-32 Hz) were greater in RCC from the middle-aged group that was accompanied by elevated TH mRNA expression (P < 0.01). Phenylephrine-induced contractions were also greater in the middle-aged group. A 62% increase in ROS generation in RCC from middle-aged rats was observed. The mRNA expression for the α₁A-adrenoceptor remained unchanged among groups. Protein levels of α₁/β₁-sGC subunits were decreased in RCC from the middle-aged compared with young group. The NADPH oxidase inhibitor apocynin (85 mg·rat(-1)·day(-1), 4 wk) fully restored the enhanced ROS production, TH mRNA expressions, and α₁/β₁-subunit sGC expression, indicating that excess of superoxide anion plays a major role in the sympathetic hyperactivity and hypercontractility in erectile tissue at middle age. Reduction of oxidative stress by dietary antioxidants may be an interesting approach to treat erectile dysfunction in aging population.

Insulin relaxes bladder via PI3K/AKT/eNOS pathway activation in mucosa: unfolded protein response-dependent insulin resistance as a cause of obesity-associated overactive bladder

•  Metabolic syndrome, obesity and insulin resistance are risk factors for overactive bladder, but little is known about the impact of insulin resistance on bladder functioning. •  We aimed to investigate the insulin effects in murine and human bladder, and whether its defective action contributes to the bladder dysfunction associated with obesity. •  Under physiological conditions, insulin relaxes urinary bladder through the release of nitric oxide from the mucosal layer, and this mechanism is impaired in bladders from insulin‐resistant obese mice. •  The defective insulin action in bladder mucosa from obese mice is due to endoplasmic reticulum stress, which remarkably contributes to the bladder overactivity in obesity conditions. •  Our results enable a better understanding of the mechanism of action of insulin in the urinary bladder and how its defective action in mucosa contributes to bladder dysfunction in conditions of obesity‐associated insulin resistance.

Therapy with resveratrol attenuates obesity-associated allergic airway inflammation in mice

Obesity and insulin resistance have been associated with deterioration in asthma outcomes. High oxidative stress and deficient activation of AMP-activated protein kinase (AMPK) have emerged as important regulators linking insulin resistance and inflammation. This study aimed to evaluate the effects of resveratrol on obesity-associated allergic pulmonary inflammation. Male C57/Bl6 mice fed with high-fat diet to induce obesity (obese group) or standard-chow diet (lean group) were treated or not with resveratrol (100mg/kg/day, two weeks). Mice were sensitized and challenged with ovalbumin (OVA). At 48h thereafter, bronchoalveolar lavage fluid was performed, and lungs collected for morphological studies and Western blot analysis. Treatment of obese mice with resveratrol significantly reduced hyperglycemia and insulin resistance, as well as the body measures (body mass, fat mass, % fat, and body area). OVA-challenge promoted a higher increase in pulmonary eosinophil infiltration in obese compared with lean mice, which was nearly abrogated by resveratrol treatment. Resveratrol markedly increased the phosphorylated AMPK expression in lung tissues of obese compared with lean mice. Resveratrol reduced the p47phox expression and reactive-oxygen species (ROS) production, and elevated the superoxide dismutase (SOD) levels in lung tissues of obese mice. The increased pulmonary levels of TNF-α and inducible nitric oxide synthase (iNOS) in obese mice were also normalized after resveratrol treatment. In lean mice, resveratrol failed to affect the levels of fasting glucose, p47phox, ROS levels, TNF-α, iNOS and phosphorylated AMPK. Resveratrol exhibits protective effects in obesity-associated lung inflammation that is accompanied by local AMPK activation and antioxidant property.