Thássio Ricardo Ribeiro Mesquita

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R−/−), B2- (B2R−/−) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R−/−). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R−/− presented similar level of vascular dysfunction as found in B1R−/− or B2R−/− mice. In accordance, aortic rings from B1R−/− or B2R−/− mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R−/− and B2R−/− mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R−/− or B2R−/− mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.

Increased Nitric Oxide Bioavailability and Decreased Sympathetic Modulation Are Involved in Vascular Adjustments Induced by Low-Intensity Resistance Training

Resistance training is one of the most common kind of exercise used nowadays. Long-term high-intensity resistance training are associated with deleterious effects on vascular adjustments. On the other hand, is unclear whether low-intensity resistance training (LI-RT) is able to induce systemic changes in vascular tone. Thus, we aimed to evaluate the effects of chronic LI-RT on endothelial nitric oxide (NO) bioavailability of mesenteric artery and cardiovascular autonomic modulation in healthy rats. Wistar animals were divided into two groups: exercised (Ex) and sedentary (SED) rats submitted to the resistance (40% of 1RM) or fictitious training for 8 weeks, respectively. After LI-RT, hemodynamic measurements and cardiovascular autonomic modulation by spectral analysis were evaluated. Vascular reactivity, NO production and protein expression of endothelial and neuronal nitric oxide synthase isoforms (eNOS and nNOS, respectively) were evaluated in mesenteric artery. In addition, cardiac superoxide anion production and ventricle morphological changes were also assessed. In vivo measurements revealed a reduction in mean arterial pressure and heart rate after 8 weeks of LI-RT. In vitro studies showed an increased acetylcholine (ACh)-induced vasorelaxation and greater NOS dependence in Ex than SED rats. Hence, decreased phenylephrine-induced vasoconstriction was found in Ex rats. Accordingly, LI-RT increased the NO bioavailability under basal and ACh stimulation conditions, associated with upregulation of eNOS and nNOS protein expression in mesenteric artery. Regarding autonomic control, LI-RT increased spontaneous baroreflex sensitivity, which was associated to reduction in both, cardiac and vascular sympathetic modulation. No changes in cardiac superoxide anion or left ventricle morphometric parameters after LI-RT were observed. In summary, these results suggest that RT promotes beneficial vascular adjustments favoring augmented endothelial NO bioavailability and reduction of sympathetic vascular modulation, without evidence of cardiac overload.

Cardioprotective Action of Ginkgo biloba Extract against Sustained β-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway

Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic β-adrenergic receptor stimulation (β-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M2) and downregulation of β1-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M2/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba.

Endothelium adjustments to acute resistance exercise are intensity-dependent in healthy animals

AIMS We evaluated the acute effects of different intensities of resistance exercise over endothelium-dependent vasodilatation, eNOSser1177 phosphorylation level and endothelial production of NO in superior mesenteric artery of healthy rats. MAIN METHODS Groups: control (Ct), resistance exercise in the intensities of 30% (Ex30%), 50% (Ex50%) and 70% (Ex70%) of the maximal load established by the maximal repetition test (1RM). Exercise protocol: 15 sets of 10 repetitions. The rings of mesenteric artery were mounted in an isometric system or were prepared for further implementation of Western blot and DAF-FM techniques. KEY FINDINGS The maximal response of the relaxation induced by insulin was not altered in the animals of the Ex30% group when compared to the Ct group. However, the animals of the Ex50% and Ex70% groups presented an increase in this response when compared to the Ct group. The eNOSser1177 phosphorylation levels showed an increase in Ex50% and Ex70% groups when compared to the Ct (1.6-fold and 3.3-fold, respectively). In the endothelial production of NO, it was observed that the Ex30% group did not show alteration in the NO production when compared to the Ct group. On the other hand, the animals exercised in the Ex50% and Ex70% groups showed increase in the NO synthesis when compared to the animals in the Ct group. SIGNIFICANCE Our results suggest that the magnitude of these vascular endothelium adjustments is strongly related to the increase of the resistance exercise intensity from the intensity of 50% of 1 RM.

Resistance exercise mediates remote ischemic preconditioning by limiting cardiac eNOS uncoupling

BACKGROUND Currently viewed as a complementary non-pharmacological intervention for preventing cardiac disorders, long-term aerobic training produces cardioprotection through remote ischemic preconditioning (RIPC) mechanisms. However, RIPC triggered by acute exercise remains poorly understood. Although resistance exercise (RE) has been highly recommended by several public health guidelines, there is no evidence showing that RE mediates RIPC. Hence, we investigated whether RE induces cardiac RIPC through nitric oxide synthase (NOS)-dependent mechanism. METHODS AND RESULTS Acute RE at 40% of the maximal load augmented systemic nitrite levels, associated with increased cardiac eNOS phosphorylation, without affecting nNOS activity. Using an experimental model of myocardial infarction (MI) through ischemia-reperfusion (IR), RE fully prevented the loss of cardiac contractility and the extent of MI size compared to non-exercised (NE) rats. Moreover, RE mitigated aberrant ST-segment and reduced life-threatening arrhythmias induced by IR. Importantly, inhibition of NOS abolished the RE-mediated cardioprotection. After IR, NE rats showed increased cardiac eNOS activity, associated with reduced dimer/monomer ratio. Supporting the pivotal role of eNOS coupling during MI, non-exercised rats displayed a marked generation of reactive oxygen species (ROS) and oxidative-induced carbonylation of proteins, whereas RE prevented these responses. We validated our data demonstrating a restoration of physiological ROS levels in NE + IR cardiac sections treated with BH4, a cofactor oxidatively depleted during eNOS uncoupling, while cardiac ROS generation from exercised rats remained unchanged, suggesting no physiological needs of supplemental eNOS cofactors. CONCLUSION Together, our findings strongly indicate that RE mediates RIPC by limiting eNOS uncoupling and mitigates myocardial IR injury.

Effects of a Single Bout of Resistance Exercise in Different Volumes on Endothelium Adaptations in Healthy Animals

Background Resistance exercise (RE) has been recommended for patients with cardiovascular diseases. Recently, a few studies have demonstrated that the intensity of a single bout of RE has an effect on endothelial adaptations to exercise. However, there is no data about the effects of different volumes of RE on endothelium function. Objective The aim of the study was to evaluate the effects of different volumes of RE in a single bout on endothelium-dependent vasodilatation and nitric oxide (NO) synthesis in the mesenteric artery of healthy animals. Methods Male Wistar rats were divided into three groups: Control (Ct); low-volume RE (LV, 5 sets x 10 repetitions) and high-volume RE (HV, 15 sets x 10 repetitions). The established intensity was 70% of the maximal repetition test. After the exercise protocol, rings of mesenteric artery were used for assessment of vascular reactivity, and other mesenteric arteries were prepared for detection of measure NO production by DAF-FM fluorescence. Insulin responsiveness on NO synthesis was evaluated by stimulating the vascular rings with insulin (10 nM). Results The maximal relaxation response to insulin increased in the HV group only as compared with the Ct group. Moreover, the inhibition of nitric oxide synthesis (L-NAME) completely abolished the insulin-induced vasorelaxation in exercised rats. NO production showed a volume-dependent increase in the endothelial and smooth muscle layer. In endothelial layer, only Ct and LV groups showed a significant increase in NO synthesis when compared to their respective group under basal condition. On the other hand, in smooth muscle layer, NO fluorescence increased in all groups when compared to their respective group under basal condition. Conclusions Our results suggest that a single bout of RE promotes vascular endothelium changes in a volume-dependent manner. The 15 sets x 10 repetitions exercise plan induced the greatest levels of NO synthesis.

NITRIC OXIDE AND CA2+ DYNAMICS IN CARDIOMYOCYTES: INFLUENCE OF EXERCISE CAPACITY

Introduccion: La capacidad intrinseca para el ejercicio aerobico esta relacionada con el inotropismo cardiaco. Por otro lado, todavia se desconoce la contribucion del oxido nitrico (ON) como mensajero intracelular sobre la dinamica del Ca2+ en ratones con diferentes capacidades intrinsecas para el ejercicio. Objetivo: Evaluar si el ON modula diferencialmente la variacion transitoria intracelular de Ca2+ y las liberaciones espontaneas de Ca2+ (sparks) en cardiomiocitos de ratones con diferentes capacidades intrinsecas para el ejercicio. Metodos: Ratones machos Wistar fueron seleccionados como desempeno estandar (DE) y alto desempeno (AD), de acuerdo con la capacidad de ejercicio hasta la fatiga, medida a traves del test de fuerza progresiva en la caminadora o cinta electrica. Los cardiomiocitos de los ratones fueron utilizados para determinar el transito intracelular y sparks de Ca2+ evaluados en microscopio confocal. Para estimar la contribucion del ON fue utilizado un inhibidor de sintesis del ON (L-NAME, 100 µM). Los datos fueron analizados a traves de un ANOVA two-way seguido de un post-test Tukey y presentados como promedios ± EPM. Resultados: Los cardiomiocitos de ratones AD mostraron aumento en la amplitud de la variacion transitoria de Ca2+ en comparacion con los DE. Asi mismo, el L-NAME incremento la amplitud transitoria de Ca2+ solamente en ratones DE. No se encontraron diferencias en la constante del tiempo de decaimiento de la variacion transitoria ( t ) de Ca2+ en cardiomiocitos de ratones DE e AD. Todavia, la administracion de L-NAME mostro una reduccion en el t en cardiomiocitos de ambos los grupos. Cardiomiocitos de ratones AD presentaron menor amplitud y frecuencia de sparks de Ca2+ en comparacion al grupo DE. La administracion de L-NAME incremento la amplitud de sparks de Ca2+ en cardiomiocitos del grupo AD. Conclusion: El ON modula la variacion de Ca2+ y sparks de Ca2+ en cardiomiocitos de ratones con diferentes capacidades intrinsecas para el ejercicio.

ÓXIDO NÍTRICO Y DINAMICA DE CA2+ EN CARDIOMIOCITOS: INFLUENCIA DE LA CAPACIDAD DE EJERCÍCIO

Introduccion: La capacidad intrinseca para el ejercicio aerobico esta relacionada con el inotropismo cardiaco. Por otro lado, todavia se desconoce la contribucion del oxido nitrico (ON) como mensajero intracelular sobre la dinamica del Ca2+ en ratones con diferentes capacidades intrinsecas para el ejercicio. Objetivo: Evaluar si el ON modula diferencialmente la variacion transitoria intracelular de Ca2+ y las liberaciones espontaneas de Ca2+ (sparks) en cardiomiocitos de ratones con diferentes capacidades intrinsecas para el ejercicio. Metodos: Ratones machos Wistar fueron seleccionados como desempeno estandar (DE) y alto desempeno (AD), de acuerdo con la capacidad de ejercicio hasta la fatiga, medida a traves del test de fuerza progresiva en la caminadora o cinta electrica. Los cardiomiocitos de los ratones fueron utilizados para determinar el transito intracelular y sparks de Ca2+ evaluados en microscopio confocal. Para estimar la contribucion del ON fue utilizado un inhibidor de sintesis del ON (L-NAME, 100 µM). Los datos fueron analizados a traves de un ANOVA two-way seguido de un post-test Tukey y presentados como promedios ± EPM. Resultados: Los cardiomiocitos de ratones AD mostraron aumento en la amplitud de la variacion transitoria de Ca2+ en comparacion con los DE. Asi mismo, el L-NAME incremento la amplitud transitoria de Ca2+ solamente en ratones DE. No se encontraron diferencias en la constante del tiempo de decaimiento de la variacion transitoria ( t ) de Ca2+ en cardiomiocitos de ratones DE e AD. Todavia, la administracion de L-NAME mostro una reduccion en el t en cardiomiocitos de ambos los grupos. Cardiomiocitos de ratones AD presentaron menor amplitud y frecuencia de sparks de Ca2+ en comparacion al grupo DE. La administracion de L-NAME incremento la amplitud de sparks de Ca2+ en cardiomiocitos del grupo AD. Conclusion: El ON modula la variacion de Ca2+ y sparks de Ca2+ en cardiomiocitos de ratones con diferentes capacidades intrinsecas para el ejercicio.

ÓXIDO NÍTRICO E DINÂMICA DE CA2+ EM CARDIOMIÓCITOS: INFLUÊNCIA DA CAPACIDADE DE EXERCÍCIO

Introduccion: La capacidad intrinseca para el ejercicio aerobico esta relacionada con el inotropismo cardiaco. Por otro lado, todavia se desconoce la contribucion del oxido nitrico (ON) como mensajero intracelular sobre la dinamica del Ca2+ en ratones con diferentes capacidades intrinsecas para el ejercicio. Objetivo: Evaluar si el ON modula diferencialmente la variacion transitoria intracelular de Ca2+ y las liberaciones espontaneas de Ca2+ (sparks) en cardiomiocitos de ratones con diferentes capacidades intrinsecas para el ejercicio. Metodos: Ratones machos Wistar fueron seleccionados como desempeno estandar (DE) y alto desempeno (AD), de acuerdo con la capacidad de ejercicio hasta la fatiga, medida a traves del test de fuerza progresiva en la caminadora o cinta electrica. Los cardiomiocitos de los ratones fueron utilizados para determinar el transito intracelular y sparks de Ca2+ evaluados en microscopio confocal. Para estimar la contribucion del ON fue utilizado un inhibidor de sintesis del ON (L-NAME, 100 µM). Los datos fueron analizados a traves de un ANOVA two-way seguido de un post-test Tukey y presentados como promedios ± EPM. Resultados: Los cardiomiocitos de ratones AD mostraron aumento en la amplitud de la variacion transitoria de Ca2+ en comparacion con los DE. Asi mismo, el L-NAME incremento la amplitud transitoria de Ca2+ solamente en ratones DE. No se encontraron diferencias en la constante del tiempo de decaimiento de la variacion transitoria ( t ) de Ca2+ en cardiomiocitos de ratones DE e AD. Todavia, la administracion de L-NAME mostro una reduccion en el t en cardiomiocitos de ambos los grupos. Cardiomiocitos de ratones AD presentaron menor amplitud y frecuencia de sparks de Ca2+ en comparacion al grupo DE. La administracion de L-NAME incremento la amplitud de sparks de Ca2+ en cardiomiocitos del grupo AD. Conclusion: El ON modula la variacion de Ca2+ y sparks de Ca2+ en cardiomiocitos de ratones con diferentes capacidades intrinsecas para el ejercicio.