Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way.

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na+ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na+ + K+-ATPase and the ouabain-insensitive, K+-insensitive, and furosemide-sensitive Na+-ATPase, which participate in the active Na+ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na+-ATPase activity (C1P50 ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na+-ATPase. Conversely, we did not observe any effect on the Na+ + K+-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na+-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.

Anti-parasitic effect of the diuretic and Na+-ATPAse inhibitor furosemide in cutaneous leishmaniasis.

Leishmania amazonensis promastigotes are known to express furosemide (Lasix®)-sensitive P-type membrane Na+-ATPase. In the present study, furosemide activity was studied in intracellular amastigotes and infected BALB/c mice to investigate its efficacy in cutaneous leishmaniasis (CL). Intracellular parasites, but not macrophages, were found to be sensitive to killing by furosemide (IC50 = 87 µ m vs CC50 ≫ 1000 µ m, respectively). Although furosemide did not induce nitric oxide production or intracellular pH changes in infected macrophages, it led to a significant reactive oxygen species (ROS) burst. Freshly isolated tissue parasites expressed a high degree of Na+-ATPase activity that decreased with culture, indicative of a higher enzyme expression in amastigotes than in promastigotes. Both intraperitoneal and oral treatment of L. amazonensis-infected mice with furosemide dosages equivalent to that prescribed as a diuretic significantly reduced the parasite's growth compared with the situation in untreated mice. Combination with oral furosemide increased the efficacy and safety of intraperitoneal treatment with sodium stibogluconate (SSG). To summarize, furosemide control of intracellular leishmanial growth by means of parasite Na+-ATPase inhibition, and macrophage ROS activation may help explain its sole and SSG-combined therapeutic effect against murine CL.

Is angiotensin-(3-4) (Val-Tyr), the shortest angiotensin II-derived peptide, opening new vistas on the renin-angiotensin system?

Angiotensin-(3-4) (Ang-(3-4) or Val-Tyr) is the shorter angiotensin (Ang) II-derived peptide, formed through successive hydrolysis that culminates with the release of Val-Tyr as a dipeptide. It is formed both in plasma and in kidney from Ang II and Ang III, and can be considered a component of the systemic and organ-based renin-angiotensin system. It is potently antihypertensive in humans and rats, and its concerted actions on proximal tubule cells culminate in the inhibition of fluid reabsorption, hyperosmotic urinary excretion of Na+. At the renal cell signaling level, Ang-(3-4) counteracts Ang II-type 1 receptor-mediated responses by acting as an allosteric enhancer in Ang II-type 2 receptor populations that target adenosine triphosphate-dependent Ca2+ and Na+ transporters through a cyclic adenosine monophosphate-activated protein kinase pathway.

ANG-(3-4) inhibits renal Na+-ATPase in hypertensive rats through a mechanism that involves dissociation of ANG II receptors, heterodimers, and PKA.

The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study 1)investigates whether ANG-(3-4) modulates ouabain-resistant Na(+)-ATPase resident in proximal tubule cells and 2) verifies whether its possible action on pumping activity, considered the fine tuner of Na(+) reabsorption in this nephron segment, depends on blood pressure. ANG-(3-4) inhibited Na(+)-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto (WKY) rats or on Na(+)-K(+)-ATPase. PD123319 (10(-7) M) and PKA(5-24) (10(-6) M), an AT2 receptor (AT2R) antagonist and a specific PKA inhibitor, respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10(-8) M) completely blocked stimulation of Na(+)-ATPase induced by 10(-10) M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10(-10) M ANG II and dissociate to a very low dimerization state upon addition of 10(-8) M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased urinary Na(+) concentration and urinary Na(+) excretion with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus the influence of ANG-(3-4) on Na(+) transport and its hypotensive action depend on receptor association and on blood pressure.

Solubilization and partial characterization of ouabain-insensitive Na+-ATPase from basolateral plasma membranes of the instestinal epithelial cells

It has been proposed that intestinal sodium transport is mediated by two different active mechanisms: the ouabain-sensitive Na+/K+-ATPase and ouabain-insensitive Na+-ATPase. In order to determine the optimum conditions to solubilize the membrane-bound Na+-ATPase of enterocyte, basolateral plasma membranes were solubilized using different amounts of octyl glucoside (O.G), Tween 20, octaethylene glycol monododecyl ether (C12E8), and polyoxyethylene 9-lauryl ether (C12E9). Solubilized fractions were assayed for protein concentration and ATPase activity and characterized by electrophoresis analysis. Optimal solubilization of Na+-ATPase was obtained after mixing of 1 mg of basolateral plasma membrane with 1.5 mg of C12E9. Under these conditions, C12E9 solubilized over 60% membrane protein and Na+- and Na+/K+- ATPases activities were recovered over 80% in the soluble fraction without inactivation. In addition, when 25 % glycerol and 2 mM ATP were added, the solubilized Na+-ATPase was stable after 3 days at 4°C. The C12E9-solubilized Na+-ATPase presented the following kinetic characteristics: 1) is only stimulated by the Na+ salt, 2) K0.5 for Na+= 4.62 ± 0.06 mM, 3) is similarly stimulated by the Na+ salt of different anions, 4) optimal pH= 7.0, 5) inhibited by furosemide (IC50= 0.52 ± 0.10 nm). These kinetic properties of the solubilized Na+-ATPase were similar to those described to the native membrane-bound enzyme. This work reports for the first time, solubilization and characterization of a fully active and stable Na+-ATPase from basolateral plasma membranes of enterocyte using C12E9.

Ha sido propuesto que el transporte intestinal del sodio es mediado por dos mecanismos: la ATPasa de Na+/K+, inhibida por ouabaina y la ATPasa de Na+ la cual es insensible a la ouabaina y es inhibida por la furosemida. Con la finalidad de determinar las condiciones óptimas para solubilizar la ATPasa de Na+ del enterocito, membranas plasmáticas laterobasales fueron solubilizadas utilizando diferentes detergentes, octyl glucoside, Tween 20, C12E8 y C12E9. La solubilización de la ATPasa de Na+ y de la ATPasa de Na+/K+ fue óptima después de mezclar 1 mg de membranas con 1,5 mg de C12E9. El C12E9 solubilizó más del 60% de las proteinas de membranas y las ATPasas de Na+ y Na+/K+ fueron recuperadas en un 80% en la fracción soluble. Adicionalmente, cuando glicerol al 25 % y ATP 2 mM fueron utilizados, la ATPasa de Na+ fue estable despues de 3 dias. La ATPasa de Na+ soluble demostró las siguientes características cinéticas: 1) es específicamente estimulada por sales de Na+; 2) K0.5 para Na+= 4.62 ± 0.1 mM; 3) es estimulada por todas las sales de Na+, 4) pH óptimo= 7.0; 5) es inhibida por furosemida (IC50= 0,52 ± 0,10 nm). Las características cinéticas de la enzima solubilizada fueron similares a las descritas para la forma de la enzima unida a la membrana. Este trabajo demuestra la solubilización y caracterización de la ATPasa Na+ a partir de membranas laterobasales del enterocito usando C12E9.