Hacia el esclarecimiento del rol del anión cloruro en la hipertensión arterial: / Towards the Elucidation of the Role of the Chloride Anion in Arterial Hypertension:

RESUMEN Introducción: Se desconoce el papel del anión cloruro en los efectos deletéreos del consumo excesivo de sal (NaCl) y si sus efectos son independientes de la presencia del sodio. Objetivo: Demostrar que tanto una sobrecarga de cloruro como una sobrecarga de sodio en la dieta producen efectos deletéreos, en forma independiente, sobre la presión arterial sistólica (PAS), la función renal y los marcadores de estrés oxidativo en el riñón. Materiales y métodos: Ratas Wistar macho fueron divididas en cuatro grupos (n = 8/grupo) y fueron alimentadas con diferentes dietas durante tres semanas: C: control (dieta estándar), NaCl: hipersódica-hiperclórica, Na: hipersódica sin cloruro, Cl: hiperclórica sin sodio. Se determinaron la presión arterial sistólica (PAS) y la función renal y en la corteza renal, se evaluó la producción de especies reactivas del ácido tiobarbitúrico (en inglés: TBARS) y la actividad y la expresión de las enzimas superóxido dismutasa (SOD), catalasa (CAT) y glutatión peroxidasa (GPx). Resultados: Al cabo de tres semanas, la PAS aumentó (*) en los dos grupos alimentados con cloruro. La excreción fraccional de sodio y de cloruro aumentó (*) en los grupos NaCl y Na. La diuresis y los TBARS en la corteza renal aumentaron (*) con las tres dietas, sin cambios en la actividad y en la expresión de SOD y CAT. La actividad de la GPx aumentó (*) en los dos grupos que recibieron cloruro; (*p < 0,05 vs C). Conclusión: Tanto la sobrecarga de sodio como la de cloruro se asocian a mayor estado oxidativo caracterizado por un incremento en la peroxidación lipídica en la corteza renal. Sin embargo, solo el exceso de cloruro se asocia a mayor actividad de la GPx y de la hipertensión, sin cambios en la excreción urinaria de cloruros, sugiriendo un mayor estado prooxidante renal en comparación con el grupo Na.

ABSTRACT Introduction: The role of the chloride anion on the deleterious effects of excessive consumption of salt (NaCl) and whether its effects are independent each other of the presence of sodium remains to date, unknown and unclear. Objective: To demonstrate that both a chloride overload and a sodium overload in the diet produce deleterious effects, by different mechanisms, on systolic blood pressure (SBP), renal function and markers of oxidative stress in the kidney. Materials and Methods: Male Wistar rats were divided into four groups (n = 8 / group) and fed with different diets for three weeks: C: control (standard diet), and diets: NaCl: hypersodic-hyperchloric; Na: hypersodic without chloride and Cl: hyperchloric without sodium. Systolic blood pressure (SBP) and renal function were determined, and the production of thiobarbituric acid reactive species (TBARS) and the activity and expression of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes were evaluated in renal cortex tissue. Results: SBP increased (*) in the two groups fed with chloride. The fractional excretion of sodium and chloride increased (*) in the NaCl and Na groups. increased (*) in the renal cortex with the three diets. No changes were observed in the activity and expression of SOD and CAT. GPx activity increased (*) in the two groups that received chloride; (* p <0.05 vs C). Conclusion: Both sodium and chloride overload are associated with a higher oxidative state characterized by an increase in lipid peroxidation in the renal cortex. However, compared with Na group, only chloride overload is associated with higher GPx activity and hypertension without any changes in urinary chloride excretion, suggesting a higher renal pro-oxidant state in this experimental group.

Acute infusion of angiotensin II regulates organic cation transporters function in the kidney: its impact on the renal dopaminergic system and sodium excretion.

A close relationship between angiotensin II (ANG II) and the renal dopaminergic system (RDS) has been reported. Our aim was to study whether renal dopamine and ANG II can interact to modify renal sodium handling and then to elucidate the related mechanism. Anesthetized male Sprague-Dawley rats were used in experiments. ANG II, exogenous dopamine, and decynium-22 (or D-22, an isocyanine that specifically blocks electrogenic organic cation transporters, OCTs), were infused in vivo for 120 min. We analyzed renal and hemodynamic parameters, renal Na+, K+-ATPase levels, OCT activity, and urinary dopamine concentrations. We also evaluated the expression of D1 receptor, electroneutral organic cation transporters (OCTNs), and OCTs. ANG II decreased renal excretion of sodium in the presence of exogenous dopamine, increased Na+, K+-ATPase activity, and decreased the urinary dopamine concentration. D-22 treatment exacerbated the ANG II-mediated decrease in renal excretion of sodium and dopamine urine excretion but did not modify ANG II stimulation of Na+, K+-ATPase activity. The infusion of ANG II did not affect the expression of D1 receptor, OCTs, or OCTNs. However, the activity of OCTs was diminished by the presence of ANG II. Although ANG II did not alter the expression of D1 receptor, OCTs, and OCTNs in renal tissues, it modified the activity of OCTs and thereby decreased the urinary dopamine concentration, showing a novel mechanism by which ANG II decreases dopamine transport and its availability in the tubular lumen to stimulate D1 receptor. This study demonstrates a relationship between ANG II and dopamine, where both agents counteract their effects on sodium excretion.

Alteration of Renal Natriuretic Systems Is Associated with the Development of Hypertension and Precedes the Presence of Renal Damage in a Model of Metabolic Syndrome / La alteración de sistemas natriuréticos renales se asocia con el desarrollo de hipertensión arterial y precede en el tiempo a la aparición de daño renal en un modelo de síndrome metabólico

ABSTRACT: Background: The aim of this study was to determine the presence of alterations in the natriuretic systems of atrial natriuretic peptide and renal dopamine in a model of metabolic syndrome induced by fructose overload and to associate them with changes in systolic blood pressure, renal function, Na+/K+-ATPase status and microalbuminuria. Methods: Male Sprague-Dawley rats were divided into control (C) and fructose (F) groups receiving drinking water or a fructose so-lution (10% W/V), respectively, for 4, 8 and 12 weeks. L-dopa and dopamine, sodium, creatinine and albumin were measured in urine and ANP, insulin, sodium and creatinine in plasma. Systolic blood pressure was measured by indirect method and the renal activity and expression of Na+/K+-ATPase as well as the renal expression of A- and C-type natriuretic peptide receptors were assessed. results: Fructose overload was associated with a significant increase in insulinemia and systolic blood pressure levels and a decrease in urinary sodium excretion since week 4. A significant increase in L-dopa excretion and a decrease in dopamine excretion (increased urinary L-dopa/dopamine ratio) due to fructose overload were observed since week 4 with a decrease in plasma atrial natriuretic peptide at weeks 8 and 12. These changes were accompanied by increased activity and expression of Na+/ K+-ATPase, decreased A-type natriuretic peptide receptor and increased C-type natriuretic peptide receptor expression. Microalbuminuria was observed at week 12 of fructose overload.

RESUMEN: Objetivos: El objetivo del trabajo consistió en determinar la existencia de alteraciones en los sistemas natriuréticos del péptido natriurético atrial y dopamina renal en un modelo de síndrome metabólico por sobrecarga de fructosa y asociarlas con cambios en la presión arterial sistólica, función renal, estado de la Na+, K+-ATPasa y microalbuminuria. Material y Métodos: Ratas macho Sprague-Dawley fueron divididas en grupos control (C) y fructosa (F) con agua o solución de F (10%P/V) para beber durante 4, 8 y 12 semanas. En orina, se midió L-dopa y dopamina, sodio, creatinina y albúmina; y en plasma péptido natriurético atrial, insulina, sodio y creatinina. La presión arterial sistólica fue medida por método indirecto. Se midió la actividad y expresión de la Na+, K+-ATPasa así como la expresión del receptor de péptidos natriuréticos A y C renales. resultados: La sobrecarga de fructosa se asoció con el aumento de la insulinemia y la presión arterial sistólica, y con la disminución en la excreción urinaria de sodio desde la semana 4. La excreción urinaria de L-dopa se incrementó y la de dopamina disminuyó (cociente L-dopa/dopamina incrementado) por sobrecarga de fructosa desde la semana 4 y el péptido natriurético atrial plasmático se redujo en las semanas 8 y 12. Estos cambios fueron acompañados por un incremento de la actividad y expresión de la Na+, K+-ATPasa, disminución del receptor de péptidos natriuréticos A y aumento del C. La microalbuminuria se observó en la semana 12 de sobrecarga de fructosa. Conclusiones: Las alteraciones del péptido natriurético atrial y de la dopamina renal se asociaron con el desarrollo de hipertensión arterial y precedieron a la aparición de microalbuminuria, por lo que se pudo establecer una asociación temporal entre la alteración de ambos sistemas y el desarrollo de daño renal.

Effects of chronic fructose overload on renal dopaminergic system: alteration of urinary L-dopa/dopamine index correlates to hypertension and precedes kidney structural damage.

Insulin resistance induced by a high-fructose diet has been associated to hypertension and renal damage. The aim of this work was to assess alterations in the urinary L-dopa/dopamine ratio over three time periods in rats with insulin resistance induced by fructose overload and its correlation with blood pressure levels and the presence of microalbuminuria and reduced nephrin expression as markers of renal structural damage. Male Sprague-Dawley rats were randomly divided into six groups: control (C) (C4, C8 and C12) with tap water to drink and fructose-overloaded (FO) rats (FO4, FO8 and FO12) with a fructose solution (10% w/v) to drink for 4, 8 and 12 weeks. A significant increase of the urinary L-dopa/dopamine ratio was found in FO rats since week 4, which positively correlated to the development of hypertension and preceded in time the onset of microalbuminuria and reduced nephrin expression observed on week 12 of treatment. The alteration of this ratio was associated to an impairment of the renal dopaminergic system, evidenced by a reduction in renal dopamine transporters and dopamine D1 receptor expression, leading to an overexpression and overactivation of the enzyme Na+, K+-ATPase with sodium retention. In conclusion, urinary L-dopa/dopamine ratio alteration in rats with fructose overload positively correlated to the development of hypertension and preceded in time the onset of renal structural damage. This is the first study to propose the use of the urinary L-dopa/dopamine index as marker of renal dysfunction that temporarily precedes kidney structural damage induced by fructose overload.

Atrial Natriuretic Peptide Stimulates Dopamine Tubular Transport by Organic Cation Transporters: A Novel Mechanism to Enhance Renal Sodium Excretion.

The aim of this study was to demonstrate the effects of atrial natriuretic peptide (ANP) on organic cation transporters (OCTs) expression and activity, and its consequences on dopamine urinary levels, Na+, K+-ATPase activity and renal function. Male Sprague Dawley rats were infused with isotonic saline solution during 120 minutes and randomized in nine different groups: control, pargyline plus tolcapone (P+T), ANP, dopamine (DA), D-22, DA+D-22, ANP+D-22, ANP+DA and ANP+DA+D-22. Renal functional parameters were determined and urinary dopamine concentration was quantified by HPLC. Expression of OCTs and D1-receptor in membrane preparations from renal cortex tissues were determined by western blot and Na+, K+-ATPase activity was determined using in vitro enzyme assay. 3H-DA renal uptake was determined in vitro. Compared to P+T group, ANP and dopamine infusion increased diuresis, urinary sodium and dopamine excretion significantly. These effects were more pronounced in ANP+DA group and reversed by OCTs blockade by D-22, demonstrating that OCTs are implied in ANP stimulated-DA uptake and transport in renal tissues. The activity of Na+, K+-ATPase exhibited a similar fashion when it was measured in the same experimental groups. Although OCTs and D1-receptor protein expression were not modified by ANP, OCTs-dependent-dopamine tubular uptake was increased by ANP through activation of NPR-A receptor and protein kinase G as signaling pathway. This effect was reflected by an increase in urinary dopamine excretion, natriuresis, diuresis and decreased Na+, K+-ATPase activity. OCTs represent a novel target that links the activity of ANP and dopamine together in a common mechanism to enhance their natriuretic and diuretic effects.

Urodilatin regulates renal dopamine metabolism.

BACKGROUND: Sodium and water transport across renal proximal tubules is regulated by diverse hormones such as dopamine and urodilatin. We have previously reported that urodilatin stimulates extraneuronal dopamine uptake in external renal cortex by activation of the type A natriuretic peptide receptor, coupled to cyclic guanylate monophosphate signaling and protein kinase G. Moreover, urodilatin enhances dopamine-induced inhibition of Na+, K+-ATPase activity in renal tubules. The aim of the present study was to evaluate whether urodilatin could also alter renal dopamine synthesis, release, catabolism and turnover. METHODS: The effects of urodilatin on dopamine synthesis, release, catabolism and turnover were measured in samples of renal cortex from Sprague Dawley rats. RESULTS: The results indicate that urodilatin increases L-DOPA decarboxylase activity and decreases catechol-o-methyl transferase and monoamine oxidase activity. Moreover, urodilatin does not affect either dopamine basal secretion or potassium chloride-induced dopamine release in external renal cortex, and reduces amine turnover. CONCLUSIONS: Both the present results and previous findings show that urodilatin modifies dopamine metabolism in external renal cortex of rats by enhancing dopamine uptake and synthesis and by decreasing catechol-o-methyl transferase and monoamine oxidase activity and dopamine turnover. Those effects taken together may favor dopamine accumulation in renal cells and increase its endogenous content and availability. This would permit D1 receptor recruitment and stimulation and, in turn, overinhibition of Na+, K+-ATPase activity, which results in decreased sodium reabsorption. Therefore, urodilatin and dopamine enhance natriuresis and diuresis through a common pathway.

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways.

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.

Perfiles de síntesis y secreción de los péptidos natriuréticos durante la evolución de la hipertrofia cardíaca en ratas hipertensas DOCA-sal / Natriuretic Peptides Synthesis and Secretion Profiles during the Evolution of Cardiac Hypertrophy in DOCA-Salt Hypertensive Rats / Natriuretic Peptides Synthesis and Secretion Profiles during the Evolution of Cardiac Hypertrophy in DOCASalt Hypertensive Rats

Durante el desarrollo de la hipertensión arterial, las interacciones entre las sobrecargas de presión y de volumen conducen a diferentes patrones de hipertrofia cardíaca y a un aumento de los péptidos natriuréticos (PN). Los perfiles de síntesis y secreción de ANP y BNP se han investigado en modelos de hipertensión arterial; sin embargo, aún no se ha estudiado la evolución diferencial de estos perfiles durante períodos agudos y crónicos de la hipertrofia cardíaca producida por sobrecarga de volumen. Por este motivo estudiamos ratas Sprague- Dawley con el modelo DOCA-sal a las 2, 4, 6 y 12 semanas, correlacionando la evolución de dichos perfiles con la hipertrofia cardíaca y la hipertensión arterial. El grado de hipertrofia cardíaca se correlacionó positivamente con la expresión del ANP en el ventrículo izquierdo y con los niveles de ANP en plasma. La expresión del ANP aumentó a las 4 semanas de tratamiento, mientras que la de BNP se incrementó recién a las 6 semanas. Asimismo, el BNP plasmático se incrementó sólo en el grupo con 12 semanas de tratamiento, mientras que el ANP plasmático mostró un aumento a partir de las 2 semanas de tratamiento. Durante el desarrollo de la hipertrofia cardíaca producida en el modelo DOCA-sal, la síntesis y la secreción de los PN responden en forma diferencial, con incremento precoz del ANP. Además, el aumento de éste superó al de BNP en todos los grupos DOCA-sal, lo que permitiría considerar al ANP como un marcador más específico de la sobrecarga de volumen.

The interactions between pressure and volume overload that occur in hypertension lead to different patterns of cardiac hypertrophy and to increase in natriuretic peptides (NPs). The profiles of ANP and BNP synthesis and secretion have been investigated in models of hypertension; however, the different evolution of these profiles during the acute and chronic periods of pressure overload-induced cardiac hypertrophy is still unknown. For this reason, we studied DOCA-salt treated Sprague-Dawley rats at weeks 2, 4, 6 and 12 and correlated the evolution of these profiles with cardiac hypertrophy and hypertension. Cardiac hypertrophy had a positive correlation with ANP expression in the left ventricle and with ANP plasma levels. BNP expression increased after 4 weeks of treatment while ANP increased significantly after 6 weeks. In addition, BNP plasma levels increased only in the group treated for 12 weeks, while ANP plasma levels increased from week 2. NP secretion has a differential response in the early stages of the development of cardiac hypertrophy induced by the DOCA-salt model, with an early increase in ANP. As ANP levels were exceeded to those of BNP in all the DOCA-salt groups, ANP might be considered a more specific marker of volume overload.

Urodilatin increases renal dopamine uptake: intracellular network involved.

Dopamine and urodilatin promote natriuresis and diuresis through a common pathway that involves reversible deactivation of renal Na+, K+-ATPase. We have reported that urodilatin enhances dopamine uptake in outer renal cortex through the natriuretic peptide type A receptor. Moreover, urodilatin enhances dopamine-induced inhibition of Na+, K+-ATPase activity. The objective of the present work was to investigate the intracellular signals involved in urodilatin effects on dopamine uptake in renal cortex of kidney rats. We show that urodilatin-elicited increase in ³H-dopamine was blunted by methylene blue (10 µM), a non-specific guanylate cyclase inhibitor, and by phorbol-12-myristate-13-acetate (1 µM), a particulate guanylate cyclase inhibitor, but not by 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (10 µM), a specific soluble guanylate cyclase inhibitor; therefore the involvement of particulate guanylate cyclase on urodilatin mediated dopamine uptake was confirmed. Cyclic guanosine monophosphate and proteinkinase G were also implicated in the signaling pathway, since urodilatin effects were mimicked by the analog 125 µM 8-Br-cGMP and blocked by the proteinkinase G-specific inhibitor, KT-5823 (1 µM). In conclusion, urodilatin increases dopamine uptake in renal cortex stimulating natriuretic peptide type A receptor, which signals through particulate guanylate cyclase activation, cyclic guanosine monophosphate generation, and proteinkinase G activation. Dopamine and urodilatin may achieve their effects through a common pathway that involves deactivation of renal Na+, K+-ATPase, reinforcing their natriuretic and diuretic properties.

Participación de los péptidos natriuréticos en la hipertensión renovascular y su correlación con la evolución de la hipertrofia de miocardio / The Role of Natriuretic Peptides in Renovascular Hypertension and Its Correlation with the Evolution of Myocardial Hypertrophy

Durante la hipertensión arterial, las interacciones entre las sobrecargas de presión y volumen conducen a diferentes patrones de hipertrofia cardíaca y a un aumento de los péptidos natriuréticos (PN). Los perfiles de síntesis y secreción de ANP y BNP se han investigado en modelos de hipertensión arterial. Sin embargo, aún no se ha estudiado su evolución diferencial durante períodos agudos y crónicos de la hipertrofia cardíaca producida por sobrecarga de presión. Por este motivo estudiamos ratas Sprague-Dawley con el modelo 1 riñón-1 clip a las 2, 4, 6 y 12 semanas, correlacionando la evolución de dichos perfiles con la hipertrofia cardíaca y la hipertensión arterial. Observamos una correlación positiva entre la elevación de la presión arterial y el grado de hipertrofia cardíaca, presentando ambos parámetros un incremento dependiente del tiempo a partir de las 2 semanas. La expresión del BNP mostró un aumento precoz a las 2 semanas de tratamiento, mientras que el ANP se incrementó significativamente a las 6 semanas. No obstante, la expresión del ANP aumentó en forma gradual, lo que permitió su correlación con la hipertrofia y la hipertensión. En estadios tempranos del desarrollo de la hipertrofia producida por el modelo renovascular, la expresión de los PN respondería en forma diferencial, incrementándose en forma precoz el BNP. Con la evolución de la hipertrofia, la expresión del BNP deja de ser específica y el aumento de ambos PN pasa a depender y a correlacionarse con el grado de evolución de la hipertrofia cardíaca.

The interactions between pressure and volume overload that occur in hypertension lead to different patterns of cardiac hypertrophy and to increase natriuretic peptides (NPs) levels. The profiles of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) synthesis and secretion have been investigated in models of hypertension. However, the different evolution of these profiles during the acute and chronic periods of pressure overload-induced cardiac hypertrophy is still unknown. For this reason, we studied one-kidney, one clip model using Sprague-Dawley rats at weeks 2, 4, 6 and 12 and correlated the evolution of these profiles with cardiac hypertrophy and hypertension. We observed a positive correlation between blood pressure elevation and the degree of cardiac hypertrophy, with a time-dependent increase in both parameters from week 2. Levels of BNP expression showed an early increase after 2 weeks of treatment while ANP increased significantly after 6 weeks. Neverless, the increase in ANP expression was gradual, allowing its correlation with hypertrophy and hypertension. The NP expression has a differential response in the early stages of the development of hypertrophy induced by the renovascular model, with an early increase in BNP expression. Once hypertrophy develops, BNP expression is no longer specific and the increase of both NPs depends on and correlates with the degree of cardiac hypertrophy.

Angiotensin II regulation of renal dopamine uptake and Na(+),K(+)-ATPase activity.

BACKGROUND/AIMS: Angiotensin II (ANG II) decreases dopamine (DA) uptake in renal cortex activating AT(1) receptors. We investigated the signaling pathways that mediate this action and the incidence of DA-ANG II interaction on renal Na(+),K(+)-ATPase activity. METHODS: ANG II effects on [(3)H]-DA uptake and Na(+),K(+)-ATPase were measured in samples from the outer renal cortex of Sprague-Dawley rats. RESULTS: Inhibition of the phospholipase C (PLC) pathway blunted ANG II inhibitory effects on [(3)H]-DA uptake, since U-73122, 2-APB, TMB-8, chelerythrine and KN-93 (PLC, IP(3)-dependent Ca(2+) release channels, IP(3) receptors, protein kinase C and CaM kinase II inhibitors, respectively) each one blocked ANG II effects. Inhibition of adenylate cyclase pathway did not modify ANG II inhibitory effects on DA uptake. ANG II effects on [(3)H]-DA uptake were able to modify Na(+),K(+)-ATPase activity in carbidopa-treated rats. Exogenous DA decreased while ANG II increased the enzyme activity. Neither the addition of DA together with ANG II, nor the extraneuronal DA uptake blocker hydrocortisone altered ANG II stimulatory effects on Na(+),K(+)-ATPase activity, but hydrocortisone blocked the inhibitory effects of exogenous DA. CONCLUSION: Stimulation of renal AT(1) receptors by ANG II signals through the PLC pathway to inhibit extraneuronal DA uptake. DA and ANG II act through a common pathway involving reversible renal tubular Na(+),K(+)-ATPase deactivation and activation, respectively. In addition, ANG II by itself is able to stimulate renal Na(+),K(+)-ATPase activity.

Urodilatin and dopamine: a new interaction in the kidney.

Since renal natriuretic peptide urodilatin (URO) exerts similar natriuretic and diuretic actions to those of atrial natriuretic factor (ANF), we hypothesized that URO regulates renal dopamine (DA) availability, contributing to Na(+), K(+)-ATPase inhibition. URO (1-100 nM) increased (3)H-DA uptake in outer and juxtamedullar renal cortex and medulla slices from Sprague Dawley rats. Hydrocortisone blocked URO-stimulated DA uptake, demonstrating that DA uptake was extraneuronal. The natriuretic peptide receptor type A antagonist anantin blocked URO-dependent increase of (3)H-DA uptake, while the natriuretic peptide receptor type C agonist ANF 4-23-amide did not modify URO effect on DA uptake, suggesting that only natriuretic receptors type A are involved. Co-incubation of URO and ANF did not show additive effects on DA uptake. To test whether URO effect involves changes in Na(+), K(+)-ATPase activity we performed experiments in renal cortex samples of rats with DA synthesis and neuronal uptake inhibited by carbidopa and nomifensine, respectively. When endogenous DA synthesis was inhibited, URO or DA decreased Na(+), K(+)-ATPase activity. URO and DA added together, further decreased Na(+), K(+)-ATPase activity showing an additive effect on the sodium pump. Moreover, hydrocortisone reversed URO-DA over-inhibition of the enzyme, confirming that this inhibition is closely related to URO-stimulation on renal DA uptake. URO and DA could act via a common intracellular pathway to decrease sodium and water tubular reabsorption, contributing to its natriuretic and diuretic effects.

Participación del hipotálamo en la regulación de la presión arterial en un modelo de hipertensión e insulinorresistencia / Role of the hypothalamus in blood pressure regulation in a model of hypertension and insulin resistance

Objetivo: Evaluar el papel del área hipotalámica anterior en la regulación de la presión arterial en un modelo en ratas de hipertensión arterial (HTA) e insulinorresistencia. Material y métodos: Se utilizaron ratas Sprague-Dawley macho (n = 72) que fueron divididas en dos grupos: F,fructosa (10 por ciento p/v por 6 semanas) y C, grupo control. Se canuló la arteria carótida izquierda para la medición de la presión arterial media (PAM) y la frecuencia cardíaca (FC). Se colocó una sonda de microdiálisis en el área hipotalámica anterior (AHA) para la perfusión de yohimbina (10 y 100 µg/ml) o de clonidina (100 y 300 µg/ml), antagonista y agonista α2-adrenérgicos, respectivamente, y se evaluaron los cambios hemodinámicos. Resultados: Los animales del grupo F presentaron niveles mayores de presión arterial sistólica que los del grupo C (F: 131 ± 3 mm Hg versus C: 112 ± 4 mm Hg; p < 0,05). La perfusión intrahipotalámica de yohimbina indujo un incremento en la PAM en C, en tanto que no modificó los valores en F. No se encontraron cambios en la FC en ninguno de los grupos. La clonidina en dosis de 100 µg/ml indujo una disminución de la PAM sólo en F, mientras que en dosis de300 µg/ml la disminuyó en ambos grupos y fue mayor en F que en C. Sólo la clonidina en dosis de 300 µg/ml disminuyó la FC en el grupo F, sin modificar los valores en C. Conclusiones: Existiría un tono α2-adrenérgico menor en el AHA de las ratas F, que podría relacionarse con el incremento de la presión arterial presente en este grupo. Por otra parte, la respuesta exacerbada a la clonidina en F evidenciaría la existencia de una supersensibilidad de receptores adrenérgicos hipotalámicos, posiblemente como consecuencia de niveles extracelulares reducidos de noradrenalina en el AHA en este modelo de HTA e insulinorresistencia.

Signaling pathways involved in atrial natriuretic factor and dopamine regulation of renal Na+, K+ -ATPase activity.

Dopamine (DA) and atrial natriuretic factor (ANF) share a number of physiological effects. We hypothesized that ANF and the renal dopaminergic system could interact and enhance the natriuretic and diuretic effects of the peptide. We have previously reported that the ANF-stimulated DA uptake in renal tubular cells is mediated by the natriuretic peptide type-A receptor (NPR-A). Our aim was to investigate the signaling pathways that mediate ANF effects on renal 3H-DA uptake. Methylene blue (10 microM), an unspecific inhibitor of guanylate cyclase (GC), blunted ANF elicited increase of DA uptake. ODQ (10 microM) a specific inhibitor of soluble GC, did not modify DA uptake and did not reverse ANF-induced increase of DA uptake; then the participation of nitric oxide-dependent pathways must be discarded. The second messenger was the cGMP since the analogous 125 microM 8-Br-cGMP mimicked ANF effects. The specific inhibitor of the protein kinase G (PKG), KT 5823 (1 microM) blocked ANF effects indicating that PKG is involved. We examined if ANF effects on DA uptake were able to modify Na+, K+ -adenosine triphosphatase (Na+, K+ -ATPase) activity. The experiments were designed by means of inhibition of renal DA synthesis by carbidopa and neuronal DA uptake blocked by nomifensine. In these conditions renal Na+, K+ -ATPase activity was increased, in agreement with the decrease of DA availability. When in similar conditions, exogenous DA was added to the incubation medium, the activity of the enzyme tended to decrease, following to the restored availability of DA. The addition of ANF alone had similar effects to the addition of DA on the sodium pump, but when both were added together, the activity of Na(+), K(+)-ATPase was decreased. Moreover, the extraneuronal uptake blocker, hydrocortisone, inhibited the latter effect. In conclusion, ANF stimulates extraneuronal DA uptake in external cortex tissues by activation of NPR-A receptors coupled to GC and it signals through cGMP as second messenger and PKG. Dopamine and ANF may achieve their effects through a common pathway that involves reversible deactivation of renal tubular Na+, K+ -ATPase activity. This mechanism demonstrates a DA-ANF relationship involved in the modulation of both decreased sodium reabsorption and increased natriuresis.

Endobain E, a brain Na+, K+ -ATPase inhibitor, decreases norepinephrine uptake in rat hypothalamus.

The ability of an endogenous brain Na+, K+ -ATPase inhibitor, termed endobain E, to increase [3H]norepinephrine release in rat hypothalamus was previously reported. Endobain E effect on neurotransmitter uptake was studied by assaying [3H]norepinephrine uptake in rat hypothalamus preparations, to observe uptake inhibition, which reached 60% with endobain E equivalent to 100 mg fresh cerebral cortex, an effect achieved with 40 or 400 microM ouabain. Results support the proposal that endobain E behaves as an ouabain-like substance. Taken jointly results obtained on neurotransmitter release and uptake, the suggestion that endobain E may enhance norepinephrine availability in the synaptic gap and thus lead to an increase in noradrenergic activity is advanced.