EPO and EPO-Receptor System as Potential Actionable Mechanism for the Protection of Brain and Heart in Refractory Epilepsy and SUDEP.

The most important activity of erythropoietin (EPO) is the regulation of erythrocyte production by activation of the erythropoietin receptor (EPO-R), which triggers the activation of anti-apoptotic and proliferative responses of erythroid progenitor cells. Additionally, to erythropoietic EPO activity, an antiapoptotic effect has been described in a wide spectrum of tissues. EPO low levels are found in the central nervous system (CNS), while EPO-R is expressed in most CNS cell types. In spite of EPO-R high levels expressed during the hypoxicischemic brain, insufficient production of endogenous cerebral EPO could be the cause of determined circuit alterations that lead to the loss of specific neuronal populations. In the heart, high EPO-R expression in cardiac progenitor cells appears to contribute to myocardial regeneration under EPO stimulation. Several lines of evidence have linked EPO to an antiapoptotic role in CNS and in heart tissue. In this review, an antiapoptotic role of EPO/EPO-R system in both brain and heart under hypoxic conditions, such as epilepsy and sudden death (SUDEP) has been resumed. Additionally, their protective effects could be a new field of research and a novel therapeutic strategy for the early treatment of these conditions and avoid SUDEP.

Erythropoietin Improves Cardiovascular Function in Adult Rats After Acute Hemorrhage.

Erythropoietin (EPO) has been linked to cardioprotective effects. However, its effects during the aging process are little known. We investigated the effect of EPO administration on hemodynamic parameters, cardiac function, oxidative damage, and erythropoietin receptor (EPOR) expression pattern in the hypovolemic state. EPO was administered (1000 IU/kg/3 days) and then acute hemorrhage (20% blood loss) was induced in young and adult rats. There was no difference in plasmatic EPO in either age group. The hemodynamic basal condition was similar, without alterations in renal function and hematocrit, in both age groups. After bleeding, both EPO-treated age groups had increased blood pressure at the end of the experimental protocol, being greater in adult animals. EPO attenuated the tachycardic effect. Ejection fraction and fractional shortening were higher in adult EPO-treated rats subjected to hemorrhage. In the left ventricle, young and adult EPO-treated rats subjected to bleeding showed an increased EPOR expression. A different EPOR expression pattern was observed in the adult right atrial tissue, compared with young animals. EPO treatment decreased oxidative damage to lipids in both age groups. EPO treatment before acute hemorrhage improves cardiovascular function during the aging process, which is mediated by different EPOR pattern expression in the heart tissue.

Thyroid disorders and nitric oxide in cardiovascular adaptation to hypovolemia.

This study aimed to investigate whether nitric oxide participates in the cardiovascular function and haemodynamic adaptation to acute haemorrhage in animals with thyroid disorders. Sprague-Dawley rats aged 2months old treated with T3 (hyper, 20µg/100g body weight) or 0.02% methimazole (hypo, w/v) during 28days were pre-treated with N(G) nitro-l-arginine methyl ester (L-NAME) and submitted to 20% blood loss. Heart function was evaluated by echocardiography. Measurements of arterial blood pressure, heart rate, nitric oxide synthase activity and protein levels were performed. We found that hypo decreased fractional shortening and ejection fraction and increased left ventricle internal diameter. Hyper decreased ventricle diameter and no changes in cardiac contractility. Haemorrhage elicited a hypotension of similar magnitude within 10min. Then, this parameter was stabilized at about 30-40min and maintained until finalized, 120min. L-NAME rats showed that the immediate hypotension would be independent of nitric oxide. Nitric oxide synthase inhibition blunted the changes of heart rate induced by blood loss. Hyper and hypo had lower atrial enzyme activity associated with a decreased enzyme isoform in hypo. In ventricle, hyper and hypo had a higher enzyme activity, which was not correlated with changes in protein levels. Haemorrhage induced an increased heart nitric oxide production. We concluded that thyroid disorders were associated with hypertrophic remodelling which impacted differently on cardiac function and its adaptation to a hypovolemia. Hypovolemia triggered a nitric oxide synthase activation modulating the heart function to maintain haemodynamic homeostasis. This involvement depends on a specific enzyme isoform, cardiac chamber and thyroid state.

Involvement of nitric oxide and caveolins in the age-associated functional and structural changes in a heart under osmotic stress.

Previous work done in our laboratory showed that water restriction during 24 and 72h induced changes in cardiovascular NOS activity without altering NOS protein levels in young and adult animals. These findings indicate that the involvement of NO in the regulatory mechanisms during dehydration depends on the magnitude of the water restriction and on age. Our aim was to study whether a controlled water restriction of 1 month affects cardiac function, NO synthase (NOS) activity and NOS, and cav-1 and -3 protein levels in rats during aging. Male Sprague-Dawley rats aged 2 and 16 months were divided into 2 groups: (CR) control restriction (WR) water restriction. Measurements of arterial blood pressure, heart rate, oxidative stress, NOS activity and NOS/cav-1 and -3 protein levels were performed. Cardiac function was evaluated by echocardiography. The results showed that adult rats have greater ESV, EDV and SV than young rats with similar SBP. Decreased atria NOS activity was caused by a reduction in NOS protein levels. Adult animals showed increased cav-1. Water restriction decreased NOS activity in young and adult rats associated to an increased cav-1. TBARS levels increased in adult animals. Higher ventricular NOS activity in adulthood would be caused by a reduction in both cav. Water restriction reduced NOS activity and increased cav in both age groups. In conclusion, our results indicated that dehydration modifies cardiac NO system activity and its regulatory proteins cav in order to maintain physiological cardiac function. Functional alterations are induced by the aging process as well as hypovolemic state.

Caveolinas y modulación del sistema del óxido nítrico en el hipotiroidismo según avanza la edad / Age-related Influence of Hypothyroidism on Caveolins and Nitric Oxide Modulation

Introducción El hipotiroidismo y la edad impactan sobre la producción de óxido nítrico (NO) cardíaco y renal. Las caveolinas, moduladores negativos de la actividad enzimática de la NO sintetasa (NOS), se afectan con ambos factores. Objetivos Evaluar la implicación de las caveolinas (cav) en la modulación de la actividad de la NOS cardíaca y renal en animales hipotiroideos adultos. Material y métodos Se utilizaron ratas macho Sprague-Dawley eutiroideas e hipotiroideas [metimazol 0,02% (v/v) en el agua de bebida durante 28 días]. Los animales fueron sacrificados para extraer el corazón y los riñones. Resultados La actividad de la NOS en la aurícula derecha disminuyó con la edad y el hipotiroidismo. La expresión de cav-1 aumentó con la edad y el hipotiroidismo. La actividad de la NOS en el ventrículo izquierdo aumentó con el avance de la edad y el hipotiroidismo. La expresión de ambas caveolinas disminuyó en los grupos adulto e hipotiroideo. En la médula renal, el hipotiroidismo disminuyó la actividad de la NOS en jóvenes y la aumentó en adultos. La expresión de cav-1 disminuyó con la edad y en jóvenes hipotiroideos. Los niveles proteicos de cav-3 disminuyeron en animales adultos hipotiroideos. Conclusiones El hipotiroidismo impacta sobre la actividad de la NOS y de sus moduladores, las caveolinas, en el sistema cardiovascular y renal. El hipotiroidismo intensifica los efectos del avance de la edad en ambos sistemas.(AU)

Introduction Hypothyroidism and age impact on cardiac and renal nitric oxide (NO) production. Caveolins, which are negative modulators of NO synthase (NOS) activity, are affected by both factors. Objectives The aim of this study was to evaluate caveolin (CAV) participation in the modulation of renal and cardiac NOS activity in adult hypothyroid animals. Methods Euthyroid and hypothyroid [methimazole 0.02% (v/v) in the drinking water during 28 days] male Sprague-Dawley rats were used. Animals were sacrificed to remove the heart and kidneys. Results Right atrial NOS activity decreased with age and hypothyroi-dism. Caveolin-1 expression increased with age and hypothyroidism. Conversely, left ventricular NOS activity increased with aging and hypothyroidism and the expression of both CAV isoforms decreased in adult and hypothyroid groups. In the renal medulla, hypothyroidism reduced NOS activity in young and raised it in adult animals and CAV-1 expression decreased with age and in hypothyroid young animals. Caveolin-3 protein levels decreased in adult hypothyroid animals. Conclusions Hypothyroidism impacts on NOS activity and on that of its modulators, caveolins, in the cardiovascular and renal systems. Hypothyroidism enhances the effects of aging in both systems.(AU)

Caveolinas y modulación del sistema del óxido nítrico en el hipotiroidismo según avanza la edad / Age-related Influence of Hypothyroidism on Caveolins and Nitric Oxide Modulation

Introducción El hipotiroidismo y la edad impactan sobre la producción de óxido nítrico (NO) cardíaco y renal. Las caveolinas, moduladores negativos de la actividad enzimática de la NO sintetasa (NOS), se afectan con ambos factores. Objetivos Evaluar la implicación de las caveolinas (cav) en la modulación de la actividad de la NOS cardíaca y renal en animales hipotiroideos adultos. Material y métodos Se utilizaron ratas macho Sprague-Dawley eutiroideas e hipotiroideas [metimazol 0,02% (v/v) en el agua de bebida durante 28 días]. Los animales fueron sacrificados para extraer el corazón y los riñones. Resultados La actividad de la NOS en la aurícula derecha disminuyó con la edad y el hipotiroidismo. La expresión de cav-1 aumentó con la edad y el hipotiroidismo. La actividad de la NOS en el ventrículo izquierdo aumentó con el avance de la edad y el hipotiroidismo. La expresión de ambas caveolinas disminuyó en los grupos adulto e hipotiroideo. En la médula renal, el hipotiroidismo disminuyó la actividad de la NOS en jóvenes y la aumentó en adultos. La expresión de cav-1 disminuyó con la edad y en jóvenes hipotiroideos. Los niveles proteicos de cav-3 disminuyeron en animales adultos hipotiroideos. Conclusiones El hipotiroidismo impacta sobre la actividad de la NOS y de sus moduladores, las caveolinas, en el sistema cardiovascular y renal. El hipotiroidismo intensifica los efectos del avance de la edad en ambos sistemas.

Introduction Hypothyroidism and age impact on cardiac and renal nitric oxide (NO) production. Caveolins, which are negative modulators of NO synthase (NOS) activity, are affected by both factors. Objectives The aim of this study was to evaluate caveolin (CAV) participation in the modulation of renal and cardiac NOS activity in adult hypothyroid animals. Methods Euthyroid and hypothyroid [methimazole 0.02% (v/v) in the drinking water during 28 days] male Sprague-Dawley rats were used. Animals were sacrificed to remove the heart and kidneys. Results Right atrial NOS activity decreased with age and hypothyroi-dism. Caveolin-1 expression increased with age and hypothyroidism. Conversely, left ventricular NOS activity increased with aging and hypothyroidism and the expression of both CAV isoforms decreased in adult and hypothyroid groups. In the renal medulla, hypothyroidism reduced NOS activity in young and raised it in adult animals and CAV-1 expression decreased with age and in hypothyroid young animals. Caveolin-3 protein levels decreased in adult hypothyroid animals. Conclusions Hypothyroidism impacts on NOS activity and on that of its modulators, caveolins, in the cardiovascular and renal systems. Hypothyroidism enhances the effects of aging in both systems.

Nitric oxide and AQP2 in hypothyroid rats: a link between aging and water homeostasis.

OBJECTIVE: Hypothyroid state and aging are associated with impairment in water reabsorption and changes in aquaporin water channel type 2 (AQP2). Nitric oxide (NO) is involved in AQP2 trafficking to the apical plasma membrane in medullary collecting duct cells. The purpose of this study was to investigate whether aging and hypothyroidism alter renal function, and whether medullary NO and AQP2 are implicated in maintaining water homeostasis. MATERIALS/METHODS: Sprague-Dawley rats aged 2 and 18months old were treated with 0.02% methimazole (w/v) during 28days. Renal function was examined and NO synthase (NOS) activity ([(14)C (U)]-L-arginine to [(14)C (U)]-L-citrulline assays), NOS, caveolin-1 and -3 and AQP2 protein levels were determined in medullary tissue (Western blot). Plasma membrane fraction and intracellular vesicle fraction of AQP2 were evaluated by Western blot and immunohistochemistry. RESULTS: A divergent response was observed in hypothyroid rats: while young rats exhibited polyuria with decreased medullary NOS activity, adult rats exhibited a decrease in urine output with increased NOS activity. AQP2 was increased with hypothyroidism, but while young rats exhibited increased AQP2 in plasma membrane, adult rats did so in the cytosolic site. CONCLUSIONS: Hypothyroidism contributes in a differential way to aging-induced changes in renal function, and medullary NO and AQP2 would be implicated in maintaining water homeostasis.

Contribution of caveolin-1 to ventricular nitric oxide in age-related adaptation to hypovolemic state.

Our previous results have shown that hypovolemic state induced by acute hemorrhage in young anesthetized rats triggers heterogeneous and dynamic nitric oxide synthase (NOS) activation, modulating the cardiovascular response. Involvement of the nitric oxide pathway is both isoform-specific and time-dependent. The aim of the present study was to investigate changes in activity and protein levels of the different NOS forms, changes in the abundance of caveolin-1 during hypovolemic state and caveolin-1/eNOS association using young and middle-aged rats. Therefore, we studied (i) changes in NOS activity and protein levels and (ii) caveolin-1 abundance, as well as its association with endothelial NOS (eNOS) in ventricles from young and middle-aged rats during hypovolemic state. We used 2-month (young) and 12-month (middle-aged) old male Sprague-Dawley rats. Animals were divided into two groups (n=14/group): (a) sham; (b) hemorrhaged animals (20% blood loss). With advancing age, we observed an increase in ventricle NOS activity accompanied by a decrease in eNOS and caveolin-1 protein levels, but increased inducible NOS (iNOS). We also observed that aging is associated with caveolin-1 dissociation from eNOS. Myocardia from young and middle-aged rats subjected to hemorrhage-induced hypovolemia exhibited an increase in NOS activity and protein levels with a reduction in caveolin-1 abundance, accompanied by a greater dissociation between eNOS and its regulatory protein. Further, an increase in iNOS protein levels after blood loss was observed only in middle-aged rats. Our evidence suggests that aging and acute hemorrhage contribute to the development of upregulation in NOS activity. Our findings demonstrate that specific expression patterns of ventricular NOS isoforms, alterations in the amount of caveolin-1 and caveolin-1/eNOS interaction are involved in aged-related adjustment to hypovolemic state.

Salt-induced downregulation of renal aquaporins is prevented by losartan.

AIMS: The purpose of this study was to investigate the expression of aquaporin-1 (AQP-1) and aquaporin-2 (AQP-2) in the renal tubule of rats fed with a high-salt diet and its modulation by the AT1 receptor blocker losartan. MAIN METHODS: The experiments were performed in four groups of rats fed for 3 weeks with the following diets: regular rat chow (NS); high-salt (8% NaCl) chow (HS), NS plus losartan (NS-L) and HS plus losartan (HS-L). Losartan (40 mg x kg(-1)) was administered in the drinking water. Systolic blood pressure (SBP) and renal function were evaluated. The intrarenal levels of angiotensin II (Ang II), TGF-ß(1), α-smooth muscle actin (α-SMA), endothelial nitric oxide synthase (eNOS), AQP-1 and AQP-2 were determined by immunohistochemistry. AQP-1 and AQP-2 protein levels were measured by western blot analysis. KEY FINDINGS: A high-sodium diet downregulated AQP-1 and AQP-2 expression levels in the proximal tubule and collecting duct, respectively. The high-sodium diet also induced Ang II, TGF-ß(1) and α-SMA overexpression and decreased eNOS expression in the renal cortex and medulla. Losartan increased the diuresis and natriuresis, favoring urinary sodium concentration. Additionally, losartan prevented the profibrogenic response, decreasing Ang II, TGF-ß(1) and α-SMA levels and normalizing AQP-2 expression in the HS-L group. AQP-1 expression was upregulated by losartan in both the NS-L and HS-L groups. SIGNIFICANCE: These results show that increased intrarenal Ang II in rats fed with a high-salt diet downregulates renal AQP-1 and AQP-2 expressions. In addition, although losartan increased diuresis and natriuresis, it prevented the downregulation of aquaporins, favoring urinary sodium concentration.

Diastolic function during hemorrhagic shock in rabbits.

Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.

Shock hemorrágico: óxido nítrico en ratas anestesiadas y no anestesiadas / Hemorrhagic Shock: Nitric Oxide in Anesthetized and Non Anesthetized Rats

Antecedentes En un trabajo previo mostramos que el estado hipovolémico inducido por una pérdida aguda de sangre se acompaña de una activación dinámica, heterogénea y dependiente del tiempo de la óxido nítrico sintetasa (NOS) cardíaca. Este sistema estaría involucrado en las alteraciones hemodinámicas que se observan luego de la depleción de volumen sanguíneo. Objetivo El objetivo del presente trabajo fue evaluar la participación del sistema del óxido nítrico (NO) mitocondrial en la respuesta adaptativa del sistema cardiovascular ante un shock hipovolémico en ratas anestesiadas y no anestesiadas. Material y métodos El estudio se llevó a cabo con cuatro grupos de animales (n = 7 por grupo): grupo A, ratas control anestesiadas; grupo C, ratas control no anestesiadas; grupo AH, ratas anestesiadas sometidas a una hemorragia (20% de la volemia) y grupo CH, ratas no anestesiadas sometidas a una hemorragia. Se evaluaron el consumo de oxígeno, la actividad funcional de la NOS mitocondrial (mtNOS) y la producción mitocondrial de NO. Resultados No se observaron diferencias significativas entre los valores de control respiratorio en los distintos grupos estudiados. La actividad funcional de la mtNOS fue menor en el grupo AH respecto del grupo A (12 ± 2 y 19 ± 1, respectivamente). Este efecto fue de menor magnitud cuando la hemorragia se provocó en animales no anestesiados (17 ± 1 y 20 ± 1, respectivamente). La producción mitocondrial de NO disminuyó en los grupos sometidos a una pérdida aguda de sangre, tanto no anestesiados como anestesiados, respecto de los animales controles. Conclusiones El sistema del NO mitocondrial estaría involucrado en la respuesta de adaptación del sistema cardiovascular frente a la depleción aguda de volumen. Esta participación dependería del grado de anestesia del animal.

Background We have previously demonstrated that hypovolemia induced by acute bleeding is accompanied by a dynamic, heterogenous and time-dependent activation of the cardiac nitric oxide synthase (NOS). This system might be involved in the hemodynamic anomalies observed after blood volume depletion. Objective To assess the role of the mitochondrial nitric oxide (NO) system in the adaptive response of the cardiovascular system in anesthetized and non anesthetized rats under hypovolemic shock. Material and Methods Animals were divided in four groups (n=7 animals per group): Group A, anesthetized control rats; group C, non anesthetized control rats; group AB, anesthetized rats subjected to bleeding (20% of blood volume), and group CB, non anesthetized rats subjected to bleeding. Oxygen consumption, functional activity of mitochondrial NOS (mtNOS) and mitochondrial production of NO were assessed. Results There were no significant differences in the values of respiratory parameters among the different study groups. Group AB had less functional activity of mtNOS compared to group A (12±2 and 19±1, respectively). This effect was even lower in non anesthetized animals subjected to bleeding (17±1 and 20±1, respectively). Mitochondrial production of NO decreased in anesthetized and non anesthetized animals with acute bleeding compared to controls. Conclusions Mitochondrial NO system might be involved in the adaptive response of the cardiovascular system under acute volume depletion, depending on the animal's degree of anesthesia.

Programación temprana de alteraciones en el sistema del óxido nítrico renal y vascular inducidas por la deficiencia de cinc / Early Programming of Alterations in Renal and Vascular Nitric Oxide System Induced by Zinc-Related Deficiencies

Introducción Numerosos estudios mostraron que la deficiencia nutricional durante la vida fetal y posnatal predisponen al desarrollo de patologías en la vida adulta, como la hipertensión arterial y las enfermedades renales. La distribución ubicua del cinc y sus propiedades químicas determinan su esencialidad en los sistemas biológicos. Objetivos Evaluar si las alteraciones renales y cardiovasculares en la vida adulta inducidas por la restricción moderada de cinc durante la vida fetal, la lactancia y/o el crecimiento se asocian con cambios en el sistema del óxido nítrico. Material y métodos Ratas Wistar hembra recibieron durante la preñez hasta el destete de las crías una dieta control o una baja en cinc. Luego del destete, las crías macho se asignaron al azar a dos grupos que recibieron una dieta control o una baja en cinc durante 60 días. Resultados Los resultados mostraron que el aporte insuficiente de cinc durante el crecimiento previo y/o posterior al destete indujo un aumento de la presión arterial y una disminución del volumen de filtrado glomerular en la vida adulta, asociados con una disminución del sistema del óxido nítrico renal y vascular. Además, el bajo aporte de este mineral durante la vida fetal indujo un peso menor al nacer, que se correlacionó en forma negativa con la presión arterial en la vida adulta. Conclusiones Este trabajo brinda evidencias importantes que sugieren que el aporte inadecuado de cinc durante el crecimiento prenatal y posnatal constituye un factor de riesgo cardiovascular y renal, dado que induce alteraciones en la regulación de la presión arterial y en la función renal en el individuo adulto.

Background Several studies have reported that nutritional deficiencies during fetal and postnatal life predispose to the development of diseases such as hypertension and renal disorders during adulthood. The ubiquitous distribution of zinc and its chemical properties determine their essentiality in the biological systems. Objectives To assess whether renal and cardiovascular alterations induced by moderate zinc restriction during fetal life, lactation period and/or growth are associated with changes in the nitric oxide system. Material and Methods Female Wistar rats received low zinc diet or control diet from the beginning of pregnancy up to weaning. After weaning, male offspring were randomly fed with low zinc diet or control diet for 60 days. Results Zinc deficiency through pre-weaning and post-weaning growth induced increase in blood pressure and reduced glomerular filtration volume in adult life; these findings were associated with reductions in renal and vascular nitric oxide system. In addition, low zinc intake during intrauterine life induced low birth weight offspring which had a negative correlation with blood pressure in adulthood. Conclusions Zinc deficiency during prenatal and postnatal growth constitutes a risk factor for cardiovascular and kidney diseases as it induces alterations in blood pressure and renal function regulation in adult life.

Cardiac mitochondrial nitric oxide: a regulator of heart rate?

Alterations in autonomic control and myocardial nitric-oxide (NO) production are likely linked to the development and progression of heart dysfunction. By focusing on heart rate, the complexity of the actions of NO at distinct levels throughout the autonomic nervous system and its relationship with other regulators can be demonstrated. Given the multiple and opposing actions of NO on cardiac control, it is difficult to interpret a response after a global intervention in the NO system. The diversity of intracellular pathways activated by NO, and their differing sensitivities to different levels of NO, might account for some aspects of reported specific but opposite effects. We discuss factors that might contribute to this diversity of actions. A proper elucidation of the effects of NO on metabolic pathways and on energy generation could lead to novel therapeutic strategies aimed at the early treatment of heart dysfunction.

Respuesta hipotensora al tratamiento agudo con péptido natriurético auricular: su relación con la expresión y la actividad de la óxido nítrico sintetasa cardíaca en ratas espontáneamente hipertensas / Hypotensive Response to Acute Treatment with Atrial Natriuretic Peptide: Its Relationship with Cardiac Nitric Oxide Synthase Expression and Activity in Spontaneously Hypertensive Rats

Introducción El péptido natriurético auricular (ANP) y el óxido nítrico (NO) aumentan la diuresis y la natriuresis y disminuyen el tono vascular. Previamente demostramos que el NO está involucrado en el efecto hipotensor del ANP en ratas normotensas. Objetivo Estudiar el efecto del ANP sobre la presión arterial media (PAM) y el sistema del NO en ratas espontáneamente hipertensas (SHR) y Wistar Kyoto (WKY) y la participación de la isoforma inducible de la NO-sintasa (iNOS). Material y métodos Protocolo 1: los animales fueron infundidos con solución salina (0,05 ml/min) o con ANP (0,2 µg/kg/min) durante 1 hora. Se determinaron: PAM y nitritos y nitratos urinarios (NOx). Se extrajo el corazón y se determinaron la actividad, con L-[U14C]-arginina, y la expresión (Western blot) de iNOS y NOS endotelial (eNOS). Protocolo 2: luego del agregado de ANP (1 µM), cANP(4-23) (agonista NPR-C,1µM) o aminoguanidina (inhibidor de iNOS, 1 µM) se determinó la actividad de la NOS en la aurícula derecha y en el ventrículo izquierdo de SHR y WKY. Resultados La infusión con ANP disminuyó la PAM y aumentó los NOx en ambos grupos. La actividad NOS fue mayor en SHR y se incrementó con la infusión de ANP. Se observaron niveles proteicos mayores para eNOS e iNOS en SHR, que no se modificaron con ANP. La actividad basal de iNOS fue mayor en SHR. En la aurícula, el ANP sólo interactuaría con el NPR-C para activar la NOS y en el ventrículo también participarían los receptores NPR-A/B. El desarrollo y/o el mantenimiento de la hipertensión en este modelo experimental involucraría alteraciones en la interacción entre ambos sistemas, ANP y NO.

Background Atrial natriuretic peptide (ANP) and nitric oxide (NO) increase diuresis and natriuresis and reduce vascular tone. We have previously demonstrated that NO is involved in ANP hypotensive effect in normotensive rats. Objective To assess the effect of ANP on mean blood pressure (MBP) and on NO system in spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY), and the role of the inducible isoform of nitric oxide synthase (iNOS). Material and Methods Protocol 1: animals were instilled with saline solution (0.05 ml/min) or with ANP (0.2 µg/kg/min) for an hour. MBP and urinary nitrites and nitrates (NOx) were assessed. The heart was extracted and iNOS and endothelial iNOS (eNOS) activity (with L-[U14C]-arginine) and expression (Western blot) were determined. Protocol 2: after adding ANP (1 µM), cANP(4-23) (NPR-C agonist, 1µM) or aminoguanidine (iNOS inhibitor, 1 µM) NOS activity in the right atrium and left ventricle of SHR and WKY was determined. Results Instillation with ANP reduced MBP and increased NOx in both groups. NOS activity was greater in SHR, and increased with the instillation of ANP. In SHR, greater eNOS and iNOS protein levels were observed, which were not modified by ANP. iNOS basal activity was greater in SHR. In the atrium, ANP interacts only with NPR-C in order to activate NOS, and NPR-A/B receptors would also take part in the ventricle. In this experimental model, the development and maintenance of hypertension could involve alterations in the interaction between both systems, ANP and NO.

Nitric oxide synthases are involved in the modulation of cardiovascular adaptation in hemorrhaged rats.

AIM: Nitric oxide has been implicated in the cardiovascular adaptation to hemorrhagic shock. Our aim was to study the potential role of nitric oxide synthases (NOS) in the cardiovascular response in hemorrhagic hypotension produced experimentally in anesthetized rats. METHODS: Groups of animals (n = 14, per group): (a) normotensive; (b) hypovolemic (20% blood loss); (c) normotensive and pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME); (d) hypovolemic and pretreatment with L-NAME. RESULTS: L-NAME restored the hypotension induced by hemorrhage. Blood loss decreased heart rate in the first stage increasing at 60 and 120 min. L-NAME blunted this effect. Right atria and left ventricle histochemical NOS activities increased at 60 and 120 min (atria 8% and 24%, respectively; ventricle 21% and 45%, respectively). This activity increased 17% in smooth muscle at 120 min. Heart endothelial NOS protein levels increased in heart at 60 min being attenuated at 120 min. Inducible NOS protein levels raised significantly in right atria, left ventricle and aorta at 120 min. CONCLUSION: Hemorrhagic shock induced a time-dependent and specific NOS activation modulating cardiovascular function. The inhibition of nitric oxide system appears to prevent the acceleration of heart rate during late phases after acute hypovolemic state induced by blood loss.

Autonomic regulation of pacemaker activity: role of heart nitric oxide synthases.

In autonomic-blocked rats treated with NG-nitro-L-arginine methyl ester (L-NAME, 7.5 mg/kg), heart rate increased 18% and mean arterial pressure increased 48%. Thyroidectomy, along with autonomic blockade, hampered the chronotropic response but did not modify the effect on blood pressure. After 150 min of autonomic blockade, the experimental end point, total nitric oxide (NO) production by heart NO synthases (NOS) decreased 61%: from 54 to 21 nmol NO.min-1.g heart-1. Mitochondrial NOS (mtNOS) and sarcoplasmic reticulum endothelial NOS activities decreased 74% and 52%, respectively. Mitochondria isolated from whole heart showed a well-coupled oxidative phosphorylation with high respiratory control and ADP-to-O ratios, decreased mtNOS activity (55-60%), and decreased mtNOS protein expression (70%). Immunohistochemistry with anti-inducible NOS antibody linked to gold particles localized mtNOS at the inner mitochondrial membranes. Histochemical right atrial NOS (NADPH-diaphorase) decreased 55% after heart denervation. The effects of autonomic denervation on the NO system were partially prevented by thyroidectomy performed simultaneously with autonomic blockade. Western blot analysis indicated a very rapid mtNOS protein turnover (half time=120 min) with a process of protein expression that was upregulated by thyroidectomy and a degradation process that was downregulated by the autonomic nervous system. The observations suggest that NO-mediated pathways contribute to pacemaker heart activity, likely through the NO steady-state levels in the right atrium and the whole heart.

Moderate zinc deficiency influences arterial blood pressure and vascular nitric oxide pathway in growing rats.

There is an increasing interest in the involvement of trace elements such as zinc in the pathogenesis of cardiovascular diseases. This study was designed to examine whether moderate zinc deficiency during growth influences blood pressure (BP) and vascular nitric oxide (NO) pathway. Three-week-old weaned male Wistar rats were randomly divided into two dietary groups and fed either a moderately zinc-deficient diet (zinc content 9 mg/kg; n = 12) or a control diet (zinc content 30 mg/kg; n = 12) for 60 d. The following were measured: systolic BP, nitrates and nitrites urinary excretion, urinary chemiluminescence intensity, NADPH-diaphorase activity in the thoracic aorta and intestinal arterioles, and NO synthase (NOS) catalytic activity using L-[U14C]-arginine as substrate in the thoracic aorta. Zinc deficiency during growth induced an increase in BP from day 30 of the experimental period, leading to hypertension on day 60. Animals that were fed the zinc-deficient diet had lower urinary excretion levels of nitrates and nitrites and higher intensity of spontaneous luminescence on day 60. At the end of the experiment, zinc-deficient rats showed decreased NADPH diaphorase activity in endothelium and smooth muscle of the thoracic aorta and intestinal arterioles and decreased activity of NOS in thoracic aortic tissue. An imbalance in zinc bioavailability during postnatal and growing periods may be may be a risk factor in development of cardiovascular alterations in adult life. The mechanisms involved may include an impaired vascular NO system as a result of decreased NOS activity and higher systemic oxidative stress.