Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.

Mechanisms involved in the vasorelaxant effects produced by the acute application of amfepramone in vitro to rat aortic rings

Amfepramone (diethylpropion) is an appetite-suppressant drug used for the treatment of overweight and obesity. It has been suggested that the systemic and central activity of amfepramone produces cardiovascular effects such as transient ischemic attacks and primary pulmonary hypertension. However, it is not known whether amfepramone produces immediate vascular effects when applied in vitro to rat aortic rings and, if so, what mechanisms may be involved. We analyzed the effect of amfepramone on phenylephrine-precontracted rat aortic rings with or without endothelium and the influence of inhibitors or blockers on this effect. Amfepramone produced a concentration-dependent vasorelaxation in phenylephrine-precontracted rat aortic rings that was not affected by the vehicle, atropine, 4-AP, glibenclamide, indomethacin, clotrimazole, or cycloheximide. The vasorelaxant effect of amfepramone was significantly attenuated by NG-nitro-L-arginine methyl ester (L-NAME) and tetraethylammonium (TEA), and was blocked by removal of the vascular endothelium. These results suggest that amfepramone had a direct vasorelaxant effect on phenylephrine-precontracted rat aortic rings, and that inhibition of endothelial nitric oxide synthase and the opening of Ca2+-activated K+ channels were involved in this effect.

Alterações vasculares em ratos obesos por dieta rica em gordura: papel da Via L-arginina/NO Endotelial / Vascular alterations in high-fat diet-obese rats: role of Endothelial L-arginine/NO Pathway / Alteraciones vasculares en ratones obesos por dieta rica en grasa: papel de la vía L-arginina/NO endotelial

FUNDAMENTO: Mecanismos subjacentes a anormalidades vasculares na obesidade ainda não estão completamente esclarecidos. OBJETIVO: Foi avaliada a via do óxido nítrico/L-arginina na resposta vascular de ratos obesos por dieta rica em gordura, enfocando as células endoteliais e do músculo liso. MÉTODOS: Ratos com 30 dias de vida foram divididos em 2 grupos: controle (C) e obeso (OB, ratos sob dieta rica em gordura por 30 semanas). Após 30 semanas, foram registrados o peso corporal, índice de adiposidade, pressão arterial e perfis metabólicos e endócrinos dos animais. Foram obtidas curvas para noradrelanina na ausência e presença de inibidor de óxido nítrico sintase (L-NAME, 3x10-4M) em aorta torácica intacta e com desnudamento em ratos C e OB. RESULTADOS: As medidas de peso corporal, índice de adiposidade, leptina e insulina aumentaram nos ratos OB, enquanto a pressão arterial permaneceu inalterada. A obesidade também produziu tolerância à glicose e resistência à insulina. A reatividade à noradrenalina da aorta intacta foi similar em ratos C e OB. A presença de L-NAME produziu um aumento similar nas respostas máximas, mas um desvio maior à esquerda das respostas nas aortas intactas dos ratos C em relação aos ratos OB [EC50 (x10-7M): C = 1,84 (0,83-4,07), O = 2,49 (1,41-4,38); presença de L-NAME C = 0,02 (0,01-0,04)*, O = 0,21 (0,11-0,40)*†,*p < 0,05 vs controle respectivo,†p < 0,05 vs controle mais L-NAME, n = 6-7]. Nenhum dos protocolos alterou a reatividade à noradrenalina de aortas com desnudamento. CONCLUSÃO: A obesidade induzida por dieta rica em gordura promove alterações metabólicas e vasculares. A alteração vascular envolveu uma melhora da via endotelial L-arginina/NO provavelmente relacionada à hiperinsulinemia e hiperleptinemia induzidas por dieta. A maior resistência aos efeitos do L-NAME na aorta de ratos obesos diz respeito a menor vulnerabilidade de indivíduos obesos na presença de patologias associadas que causam danos à atividade do sistema NO.

BACKGROUND: Mechanisms underlying vascular abnormalities in obesity remain to be completely clarified. OBJECTIVE: L-arginine/nitric oxide pathway was evaluated on vascular response of high-fat diet-obese rats, focusing on endothelial and smooth muscle cells. METHODS: 30-day-old rats were divided in two groups: control (C) and obese (OB, high-fat diet for 30 weeks). After 30 weeks, body weight, adiposity index, blood pressure, and metabolic and endocrine profiles of the animals were recorded. Curves to noradrenaline were obtained in absence and presence of nitric oxide synthase inhibitor (L-NAME, 3x10-4M) on intact and denuded thoracic aorta from C and OB rats. RESULTS: Body weight, adiposity index, leptin and insulin levels were increased in OB, while blood pressure was unchanged. Obesity also produced glucose tolerance and insulin resistance. Reactivity to noradrenaline of intact aorta was similar in C and OB rats. L-NAME presence produced a similar increase in maximal responses, but a higher leftward shift of noradrenaline responses in intact aorta from C than in OB rats [EC50 (x10-7M): C = 1.84 (0.83-4.07), O = 2.49 (1.41-4.38); L-NAME presence C = 0.02 (0.01-0.04)*, O = 0.21 (0.11-0.40)*†,*p < 0.05 vs respective control, †p < 0.05 vs control plus L-NAME, n = 6-7]. None of the protocols altered the reactivity to noradrenaline of denuded aortas. CONCLUSION: High-fat diet-induced obesity promotes metabolic and vascular alterations. The vascular alteration involved an endothelial L-arginine/NO pathway improvement was probably correlated to diet-induced hyperinsulinemia and hyperleptinemia. The greater resistance to L-NAME effects in aorta of obese rats raises concerns about the lower cardiovascular vulnerability of obese individuals in the presence of associated pathologies that impair NO-system activity.

FUNDAMENTO: Los mecanismos subyacentes a las anormalidades vasculares en la obesidad todavía no están completamente aclarados. OBJETIVO: Se evaluó la vía del óxido nítrico/L-arginina en la respuesta vascular de ratones obesos por dieta rica en grasa, concentrándonos en las células endoteliales y en el músculo liso. MÉTODOS: Ratones con 30 días de vida que fueron divididos en 2 grupos: control (C) y obeso (OB, ratones bajo dieta rica en grasa durante 30 semanas). Después de 30 semanas, fueron registrados el peso corporal, el índice de adiposidad, la presión arterial y los perfiles metabólicos y endocrinos de los animales. Fueron obtenidas las curvas para noradrelanina en ausencia y en presencia del inhibidor de óxido nítrico sintasa (L-NAME, 3x10-4M), en la aorta torácica intacta y con denudación de los ratones C y OB. RESULTADOS: Las medidas de peso corporal, índice de adiposidad, leptina e insulina aumentaron en los ratones OB, mientras que la presión arterial permaneció inalterada. La obesidad también produjo una tolerancia a la glucosa y una resistencia a la insulina. La reactividad a la noradrenalina de la aorta intacta fue similar en los ratones C y OB. La presencia de L-NAME generó un aumento similar en las respuestas máximas, pero una desviación mayor a la izquierda de las respuestas en las aortas intactas de los ratones C con relación a los ratones OB [EC50 (x10-7M): C = 1,84 (0,83-4,07), O = 2,49 (1,41-4,38); presencia de L-NAME C = 0,02 (0,01-0,04)*, O = 0,21 (0,11-0,40)*†,*p < 0,05 vs control respectivo, †p < 0,05 vs control más L-NAME, n = 6-7]. Ninguno de los protocolos alteró la reactividad a la noradrenalina de las aortas con denudación. CONCLUSIÓN: La obesidad inducida por dieta rica en grasa genera alteraciones metabólicas y vasculares. La alteración vascular conllevó a una mejoría de la vía endotelial L-arginina/NO tal vez relacionada con la hiperinsulinemia e hiperleptinemia inducidas por dieta. La mayor resistencia a los efectos del L-NAME en la aorta de ratones obesos, se refiere a una menor vulnerabilidad de individuos obesos en presencia de patologías asociadas que causan daños a la actividad del sistema NO.

Beneficial effects of angiotensin II AT1 blocker on cardiovascular adverse remodeling due to nitric oxide synthesis blockade / Efectos Beneficiosos del Bloqueador AT1 de la Angiotensina II en la Remodelación Cardiovascular Adversa Causada por el Bloqueo de la Síntesis de Óxido Nítrico

Se estudiaron con herramientas morfológicas, los efectos de diferentes dosis de Losartan sobre el remodelamiento cardiovascular, en ratas deficientes en óxido nítrico. 30 ratas Wistar, con 15 semanas de edad, fueron separadas en 6 grupos. control (C), L-NAME (LN), y 4 grupos en que administró LN junto con Losartan, en diferentes dosis (1, 5, 20 y 40 mg/kg/dia). El L-NAME fue administrado durante 9 semanas y la administración de Losartan se inició en la segunda semana de experimentación. Se estudiaron el corazón, la parte torácica de la aorta y la arteria mesentérica craneal, con microscopía de luz y estereología. La presión arterial (PA) aumentó desde la primera semana de administración de L-NAME. El tratamiento con Losartan, en las dosis de 20 y 40 mg/kg/día, fue eficiente para reducir la PA después de la séptima semana de tratamiento. El remodelamiento cardiaco adverso en el grupo LN se caracterizó por intensa fibrosis intersticial, disminución de la microvascularización miocárdica e hipertrofia y consecuente pérdida de cardiomiocitos. La estructura de la pared de la aorta (densidad por área de núcleos de células musculares lisas y densidad de superficie de lamelas), y la relación media/luz de la arteria mesentérica craneal, también fueron muy alteradas por la administración de L-NAME. Sólo en una dosis igual o mayor que 20 mg/kg/día, el Losartan tuvo efecto benéfico tratando estas alteraciones. En conclusión, tanto el corazón como la pared arterial de ratas deficientes en óxido nítrico, presentan un proceso de remodelamiento acentuado, y éste es eficientemente tratado con Losartan en diferentes dosis. La eficiencia del tratamiento con Losartan en el modelo de bloqueo de la síntesis de óxido nítrico se correlaciona con el efecto hipotensor de la droga, principalmente en las dosis más elevadas.

We studied with morphological tools the effects of different doses of Losartan upon the cardiovascular remodeling in nitric oxide deficient rats. At 15 weeks of age, thirty Wistar rats were separated in six groups: control (C), L-NAME (LN), and four groups were LN was given plus Losartan at different doses (1, 5, 20 and 40 mg/kg/day). The L-NAME was given for 9 weeks, the Losartan administration starting on the 2nd week of experiment. We studied the heart, thoracic aorta and superior mesenteric artery with light microscopy and stereology. The blood pressure (BP) increased since the first week of L-NAME administration, the Losartan treatment at doses of 20 and 40 mg/kg/day was efficient to reduce BP after the 7th week of treatment. The cardiac adverse remodeling in the LN group was characterized by intense interstitial fibrosis, impairment of the myocardial microvascularization, cardiomyocyte hypertrophy and consequent loss of cardiomyocytes. The aortic wall structure (density per area of smooth muscle cell nuclei and surface density of lamellae), and the superior mesenteric artery media/lumen ratio were also strongly affected by L-NAME administration. Only in the dose equal or higher than 20 mg/kg/day Losartan showed beneficial effects treating these alterations. In conclusion, both the heart and the arterial wall of NO deficient rats suffer a marked adverse remodeling process that is efficiently treated by a dose-dependent Losartan administration. The efficiency of Losartan treatment in this model of NO synthesis blockade correlates with the hypotensor effect of the drug mainly in the high dose treatment.

Elevated Contractile Responses to Acetylcholine in Organ Cultured Rabbit Carotid Artery

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum-free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.