RESUMEN

OBJECTIVE: To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. DESIGN: We present the results of a randomised, double-blinded, placebo-controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 75 patients allocated 1:1 from 11 April to 30 August 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The primary endpoints were the need for supplemental oxygen, time of hospitalisation, need for admission and length of stay in intensive care unit and death rate. RESULTS: Seventy-two patients (36 for placebo and 36 for colchicine) completed the study. Median (and IQR) time of need for supplemental oxygen was 4.0 (2.0-6.0) days for the colchicine group and 6.5 (4.0-9.0) days for the placebo group (p<0.001). Median (IQR) time of hospitalisation was 7.0 (5.0-9.0) days for the colchicine group and 9.0 (7.0-12.0) days for the placebo group (p=0.003). At day 2, 67% versus 86% of patients maintained the need for supplemental oxygen, while at day 7, the values were 9% versus 42%, in the colchicine and the placebo groups, respectively (log rank; p=0.001). Two patients died, both in placebo group. Diarrhoea was more frequent in the colchicine group (p=0.26). CONCLUSION: Colchicine reduced the length of both, supplemental oxygen therapy and hospitalisation. The drug was safe and well tolerated. Once death was an uncommon event, it is not possible to ensure that colchicine reduced mortality of COVID-19. TRIAL REGISTRATION NUMBER: RBR-8jyhxh.

Asunto(s)

Tratamiento Farmacológico de COVID-19 , Colchicina/administración & dosificación , Tiempo de Internación , Terapia por Inhalación de Oxígeno , SARS-CoV-2/genética , Índice de Severidad de la Enfermedad , Adulto , Anciano , COVID-19/mortalidad , COVID-19/virología , Colchicina/efectos adversos , Diarrea/inducido químicamente , Método Doble Ciego , Femenino , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Resultado del Tratamiento

RESUMEN

Lipids govern vital cellular processes and drive physiological changes in response to different pathological or environmental cues. Lipid species can be roughly divided into structural and signalling lipids. The former is essential for membrane composition, while the latter are usually oxidized lipids. These mediators provide beneficial effects against cardiometabolic diseases (CMDs), including fatty-liver diseases, atherosclerosis, thrombosis, obesity, and Type 2 diabetes. For instance, several oxylipins were recently found to improve glucose homeostasis, increase insulin secretion, and inhibit platelet aggregation, while specialized pro-resolving mediators (SPMs) are able to ameliorate CMD by shaping the immune system. These lipids act mainly by stimulating GPCRs. In this review, we provide an updated and comprehensive overview of the current state of the literature on signalling lipids in the context of CMD. We also highlight the network encompassing the lipid-modifying enzymes and the lipid-binding GPCRs, as well as their interactions in health and disease.

Asunto(s)

Aterosclerosis , Diabetes Mellitus Tipo 2 , Humanos , Lípidos , Obesidad , Unión Proteica

RESUMEN

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.

Asunto(s)

Betacoronavirus/fisiología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Trampas Extracelulares/fisiología , Neumonía Viral/inmunología , Neumonía Viral/virología , Células A549 , Adulto , Enzima Convertidora de Angiotensina 2 , COVID-19 , Muerte Celular , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/patología , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Células HeLa , Humanos , Masculino , Activación Neutrófila , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/sangre , Neumonía Viral/patología , SARS-CoV-2 , Serina Proteasas/metabolismo , Succión , Tráquea/inmunología

RESUMEN

Adipose tissue exerts multiple vital functions that critically maintain energy balance, including storing and expending energy, as well as secreting factors that systemically modulate nutrient metabolism. Since lipids are the major constituents of the adipocytes, it is unsurprising that the lipid composition of these cells plays a critical role in maintaining their functions and communicating with other organs and cells. In both positive and negative energy balance conditions, lipids and free fatty acids secreted from adipocytes exert either beneficial or detrimental effects in other tissues, such as the liver, pancreas and muscle. The way the adipocytes communicate with other organs tightly depends on the nature of their lipidome composition. Notwithstanding, the lipidome composition of the adipocytes is affected by physiological factors such as adipocyte type, gender and age, but also by environmental cues such as diet composition, thermal stress and physical activity. Here we provide an updated overview on how the adipose tissue lipidome profile is shaped by different physiological and environmental factors and how these changes impact the way the adipocytes regulate whole-body energy metabolism.

Asunto(s)

Metabolismo Energético/genética , Lipidómica , Lípidos/genética , Termogénesis/genética , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Humanos , Hígado/metabolismo , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología

RESUMEN

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.

Asunto(s)

Antioxidantes/uso terapéutico , Asma/tratamiento farmacológico , Eosinófilos/fisiología , Pulmón/efectos de los fármacos , Obesidad/tratamiento farmacológico , Neumonía/tratamiento farmacológico , Estilbenos/uso terapéutico , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Progresión de la Enfermedad , Pulmón/patología , Ratones , Ratones Endogámicos , Resveratrol

RESUMEN

Obesity is a major risk factor for asthma, which is characterized by airway hyperreactivity (AHR). In obesity-associated asthma, AHR may be regulated by non-TH2 mechanisms. We hypothesized that airway reactivity is regulated by insulin in the CNS, and that the high levels of insulin associated with obesity contribute to AHR. We found that intracerebroventricular (ICV)-injected insulin increases airway reactivity in wild-type, but not in vesicle acetylcholine transporter knockdown (VAChT KD(HOM-/-)), mice. Either neutralization of central insulin or inhibition of extracellular signal-regulated kinases (ERK) normalized airway reactivity in hyperinsulinemic obese mice. These effects were mediated by insulin in cholinergic nerves located at the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA), which convey parasympathetic outflow to the lungs. We propose that increased insulin-induced activation of ERK in parasympathetic pre-ganglionic nerves contributes to AHR in obese mice, suggesting a drug-treatable link between obesity and asthma.

Asunto(s)

Tronco Encefálico/enzimología , Hiperreactividad Bronquial/complicaciones , Neuronas Colinérgicas/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hiperinsulinismo/complicaciones , Sistema de Señalización de MAP Quinasas , Animales , Hiperreactividad Bronquial/enzimología , Hiperreactividad Bronquial/fisiopatología , Broncoconstricción , Neuronas Colinérgicas/patología , Dieta Alta en Grasa , Activación Enzimática , Hiperinsulinismo/enzimología , Hiperinsulinismo/fisiopatología , Inflamación/patología , Inyecciones Intraventriculares , Insulina/metabolismo , Cloruro de Metacolina , Ratones Endogámicos C57BL , Ratones Obesos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Receptor de Insulina/metabolismo

RESUMEN

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.

Asunto(s)

Tejido Adiposo/inmunología , Adiposidad , Asma/etiología , Modelos Biológicos , Obesidad/fisiopatología , Células Th2/inmunología , Adiponectina/sangre , Adiponectina/metabolismo , Tejido Adiposo/metabolismo , Resistencia de las Vías Respiratorias , Animales , Asma/sangre , Asma/inmunología , Asma/metabolismo , Humanos , Leptina/sangre , Leptina/metabolismo , Obesidad/sangre , Obesidad/inmunología , Obesidad/metabolismo , Células Th2/metabolismo

RESUMEN

INTRODUCTION: Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases. AIM: This study aimed to evaluate the effects of 2-week oral intake with BAY 60-2270 on a murine model of obesity-associated ED. METHODS: C57BL/6 male mice were fed for 12 weeks with standard chow or high-fat diet. Lean and obese mice were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks). MAIN OUTCOME MEASURES: Measurements of intracavernosal pressure (ICP), along with acetylcholine (10(-9) to 10(-5) M) and electrical field stimulation (EFS; 4-10 Hz)-induced corpus cavernosum relaxations in vitro, were obtained. Levels of cyclic guanosine monophosphate (cGMP), reactive oxygen species (ROS), and sGC protein expressions in cavernosal tissues were measured. RESULTS: Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P < 0.05). Two-week therapy with BAY 60-2770 fully reversed the decreased ICP in obese group. Acetylcholine-induced cavernosal relaxations were 45% lower (P < 0.001) in obese mice, which were fully restored by BAY 60-2770 treatment. Likewise, the EFS-induced relaxations in obese mice were restored by BAY 60-2770. Basal cGMP content in erectile tissue was 68% lower (P < 0.05) in obese mice, an effect normalized by BAY 60-2770. Levels of ROS were 52% higher (P < 0.05) whereas protein expression of α1 sGC subunit was reduced in cavernosal tissue of obese mice, both of which were normalized by BAY 60-2770. In lean group, BAY 60-2770 did not significantly affect any functional, biochemical, or molecular parameter analyzed. CONCLUSIONS: Two-week therapy with BAY 60-2770 restores the erectile function in obese mice that is associated with reduced ROS levels, up-regulation of α1 sGC subunit, and increased cGMP levels in the erectile tissue.

Asunto(s)

Benzoatos/administración & dosificación , Compuestos de Bifenilo/administración & dosificación , Activadores de Enzimas/administración & dosificación , Disfunción Eréctil/tratamiento farmacológico , Disfunción Eréctil/fisiopatología , Hidrocarburos Fluorados/administración & dosificación , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , GMP Cíclico/metabolismo , Dieta Alta en Grasa/efectos adversos , Disfunción Eréctil/enzimología , Disfunción Eréctil/etiología , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/complicaciones , Erección Peniana/efectos de los fármacos , Pene/irrigación sanguínea , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , Regulación hacia Arriba

RESUMEN

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 ß1 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.

Asunto(s)

Benzoatos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Activadores de Enzimas/uso terapéutico , Guanilato Ciclasa/metabolismo , Hidrocarburos Fluorados/uso terapéutico , Óxido Nítrico/metabolismo , Obesidad/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Uretra/efectos de los fármacos , Animales , Benzoatos/administración & dosificación , Compuestos de Bifenilo/administración & dosificación , Relación Dosis-Respuesta a Droga , Activación Enzimática , Activadores de Enzimas/administración & dosificación , Hidrocarburos Fluorados/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Relajación Muscular/efectos de los fármacos , Tono Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Músculo Liso/metabolismo , Obesidad/complicaciones , Obesidad/enzimología , Obesidad/fisiopatología , Especies Reactivas de Oxígeno/metabolismo , Guanilil Ciclasa Soluble , Uretra/enzimología , Uretra/metabolismo , Vejiga Urinaria Hiperactiva/enzimología , Vejiga Urinaria Hiperactiva/etiología , Vejiga Urinaria Hiperactiva/fisiopatología , Vejiga Urinaria Hiperactiva/prevención & control

RESUMEN

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 ß1 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 ß1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction.

Asunto(s)

Benzoatos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Activadores de Enzimas/uso terapéutico , Guanilato Ciclasa/efectos de los fármacos , Hidrocarburos Fluorados/uso terapéutico , Obesidad/epidemiología , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Animales , Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Western Blotting , Hidrocarburos Fluorados/farmacología , Síntomas del Sistema Urinario Inferior/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno , Vejiga Urinaria Hiperactiva/epidemiología , Vejiga Urinaria Hiperactiva/prevención & control

RESUMEN

We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration-response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1-100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.

Asunto(s)

Insulina/fisiología , Óxido Nítrico Sintasa de Tipo III/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Vejiga Urinaria/fisiología , Adolescente , Adulto , Animales , Dieta Alta en Grasa , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Membrana Mucosa/metabolismo , Relajación Muscular/efectos de los fármacos , Obesidad/fisiopatología , Respuesta de Proteína Desplegada , Vejiga Urinaria Hiperactiva/fisiopatología , Adulto Joven

RESUMEN

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 Ca(v)1.2 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 Ca(v)1.2 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,ß-methylene ATP (1-10 µM), KCl (1-300 mM), extracellular Ca(2+) (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 Ca(v)1.2 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 Ca(v)1.2 channel protein expression was not modified in any experimental group. Our findings show that Ca(v)1.2 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.

Asunto(s)

Canales de Calcio Tipo L/metabolismo , Resistencia a la Insulina , Obesidad/complicaciones , Obesidad/fisiopatología , Proteína Quinasa C/metabolismo , Vejiga Urinaria Hiperactiva/complicaciones , Vejiga Urinaria Hiperactiva/fisiopatología , Adiposidad/efectos de los fármacos , Amlodipino/farmacología , Animales , Peso Corporal/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Cloruro de Calcio/farmacología , Carbacol/farmacología , Modelos Animales de Enfermedad , Epidídimo/efectos de los fármacos , Epidídimo/patología , Epidídimo/fisiopatología , Técnicas In Vitro , Insulina/farmacología , Masculino , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Contracción Muscular/efectos de los fármacos , Obesidad/enzimología , Tamaño de los Órganos/efectos de los fármacos , Forbol 12,13-Dibutirato/farmacología , Cloruro de Potasio/farmacología , Delgadez/complicaciones , Delgadez/fisiopatología , Vejiga Urinaria Hiperactiva/enzimología

RESUMEN

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 α(IIb)ß(3) integrin activation and Western blot for α1 and ß1 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 Ca(2+) levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca(2+) levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced α(IIb)ß(3) 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 ß1 sGC subunits, which were prevented by BAY 60-2770. CONCLUSION: The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca(2+) levels and α(IIb)ß(3) 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.

Asunto(s)

Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Plaquetas/efectos de los fármacos , GMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Guanilato Ciclasa/metabolismo , Hidrocarburos Fluorados/farmacología , Activación Plaquetaria/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Plaquetas/citología , Plaquetas/enzimología , Calcio/metabolismo , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/efectos de los fármacos , Guanilil Ciclasa Soluble