Possible senescence associated change in the predominant &alpha -Na+/K+ ATP-ase isoform in the renal cortex of the rat

With aging the kidney exhibits progressive deterioration, with a decrease in renal function. Most of the filtered Na+ is actively reabsorbed in the proximal tubules through different transporters located in apical membrane. This process is possible because basolateral Na+/K+-ATP-ase generates electrochemical conditions necessary for energetically favorable Na+ transport. The α-subunit is the catalytic domain of Na+/K+-ATP-ase. There are three isoforms of the α/subunit present in rat kidney. The present study was undertaken to examine the expression pattern of rat α-Na+/K+-ATP-ase during senescence. We tested the impact of aging on mRNA expression of α-Na+/K+-ATP-ase in cortex and medulla of aged Wistar rats. We observed a significant expression decrease in mRNA levels and a possible change of isoform in the cortex of aged animals. These expression changes observed for αsubunit could be contributing to affect the renal function in conditions of water and salt stress.

Con el avance de la edad los riñones exhiben un deterioro funcional progresivo con disminución de la función renal. La mayor parte del sodio (Na+) filtrado es reabsorbido activamente en los túbulos proximales a través de diferentes transportadores ubicados en la membrana apical. Este proceso es posible por la existencia de la Na+/K+-ATP-asa basolateral, que genera las condiciones electroquímicas necesarias para que el transporte de Na+ sea energéticamente favorable. La subunidad αde la Na+/K+-ATP-asa es el dominio catalítico de la enzima. Existen tres isoformas de subunidad α, que están presentes en el riñón de la rata. En este trabajo se examinan los patrones de expresión de la α-Na+/K+-ATP-asa durante la senescencia. Se estudió así si el aumento de la edad incidía en la expresión del ARNm de la α-Na+/K+-ATP-asa en corteza y médula renal de ratas Wistar senescentes. Se observó una disminución en la expresión del ARNm de la subunidad αy un posible cambio de isoforma predominante en la corteza de los animales senescentes. Los cambios observados para la expresión de la subunidad αpodrían contribuir a afectar la función renal en condiciones de estrés hídrico y salino.

La segunda bomba de sodio: de la proteína al gen / The second sodium pump: from protein to gene

En la membrana laterobasal del epitelio del intestino delgado y del túbulo renal proximal han sido descrito dos mecanismos diferentes de transporte activo primario de Na+: (1) uno dependiente de K+, sensible a la ouabaina e insensible a la furosemida, correspondiente a la bomba de Na+/K+; y (2) otro independiente de K+, insensible a la ouabaina pero inhibida por furosemida, el cual es referido como la segunda bomba de sodio. De igual modo, dos actividades ATPásicas, dependientes de Mg2+, estimuladas por Na+ e inhibidas por vanadato, han sido descritas en esta membrana: (1) una dependiente de K+, sensible a la ouabaina e insensible a la furosemida, correspondiente a la ATPasa de Na+/K+; y (2) otra independiente de K+, insensible a la iuabaina pero inhibida por furosemida, la cual ha sido denominada como la ATPasa de Na+. Dadas las similitudes bioquímicas, se considera que la bomba de Na+/K+ y la segunda bomba de sodio están asociadas con las ATPasas de Na+/K+ y de Na+, respectivamente, como una entidad bioquímica única. No obstante, no había sido posible la separación óptima de ambas enzimas. Recientemente, se logró solubilizar ambas ATPasas utilizando un detergente no-iónico (C12E9), preservando sus actividades, y purificar la ATPasa de Na+ por selección negativa a través de una cromatografía de afinidad con Concanavalina-A-sefarosa. La ATPasa de Na+ esta constituida por dos subunidades: una subunidad alfa de 98 KDa y una subunidad beta de 50 KDa. La subunidad alfa fue parcialmente secuenciada por espectrometría de masa en serie, identificándose tres péptidos que están presentes en la subunidad alfa1 de la ATPasa de Na+/K+. La formación de intermediarios fosforilados durante el ciclo de reacción de la ATPasa de Na+, así como su dependencia de Mg2+ y sensibilidad a vanadato, identifican a esta enzima como integrante de las ATPasas tipo P.

Basolateral plasma membranes of small intestine and proximal renal tubule present two active Na+-transportmechanisms: (1) The Na+/K+-pump, which depends on K+, is inhibited by ouabain but insensitive to furosemide and (2) The Second sodium pump, which is K+-independent, insensitive to ouabain but inhibited by furosemide. Thse two transport mechanisms have been associated with two different Mg2+-dependent Na+-ATPases, inhibited by vanadate: (1) The K+-dependent Na+/K+-ATPase, sensitive to ouabain and insensitive to furosemide, and (2) The K+ independent, Na+-ATPase, which is inhibitable by furosemide and insensitiveto ouabain. There exist multiple biochemical and functional evidences indicating that these two ATPases are different but only recently it has been possible to identify the Na+-ATPase as a unique biochemical entity. Both ATPases can be solubilized in an active form using C12E9 as detergent and separated by exclusion chromatography in sepharose 6-B and affinity chromatography in concanavalinA-sepharose. The Na+-ATPase is constituted by two sub-units: an alpha subunit of 98 KDa and a beta subunit of 50 KDa. The alpha subunit was partially sequenced by Tandem Mass Spectrometry, evidenced three peptides that are also present in the alpha1 subunit of the Na+/K+-ATPase. Na+-ATPase is Mg2+-dependent, inhibited by vanadate and forms phosphorylated intermediate during its reaction cycle ATP, indicating that it si a P-type ATPase. These facts induced us to design degenerated primers against the most preserves motifs present in these ATPases and to intent the cloning of the Na+-ATPase. Thus, we identified a cDNA for a new P-type ATPase probably related with this enzyme.

Life-long food restriction prevents renal membrane lipid deposition and lowers renal work in rats.

Renal cortical brush-border (BBM), basolateral membrane (BLM), and medullary plasma membrane (mPM) preparations were analyzed to assess the effects of life-long food restriction in aged rats on membrane lipid content. Young male Fischer 344 x Brown-Norway F1 rats consumed food ad libitum (young AL) or were food-restricted (FR, 60% of AL consumption) for either 6 weeks (young FR) or until the age of 30 months old (old FR). Senescent FR rats had 50 per cent decreases in fractional excretion of Na and K (p < 0.001) as compared with the young AL rats. Long-term FR reduced phosphate and titratable acid excretion by 80 per cent (p < 0.001). These values were not significantly different from those observed in young rats during 6 weeks of FR. Food restriction decreased renal Na, K-ATPase activity by 50 per cent (p < 0.001) in both old and young FR animals. Reduction of food intake, in old and young rats, decreased all BBM phospholipid concentrations (phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) by 50 per cent than in the AL rats (p < 0.001). In BLM, chronic FR resulted only in lower phosphatidylcholine concentration (by 21%, p < 0.05) while phosphatidylethanolamine was increased approximately 80 per cent (p < 0.001). Total phospholipid content in mPM was progressively decreased by 23 per cent (p < 0.05) in the young FR group to be 55 per cent (p < 0.001) in the old FR rats. Cholesterol content was reduced in BBM and mPM by 38 per cent and 25 per cent (p < 0.05), respectively, during long-term FR. Both total phospholipid and cholesterol contents detected in mPM of the old FR rats were significantly lower than those obtained from the young FR animals (by 42%, p < 0.001 and 12%, p < 0.05, respectively). Plasma glucose, blood urea nitrogen, and body weight maintained at significantly lower levels during chronic FR. That life-long FR could prevent renal membrane lipid deposition and could lower renal work may explain the mechanisms that FR can delay the onset and diminish the severity of age-associated renal diseases.

Tryptophan and tyrosine catabolic pattern in neuropsychiatric disorders.

Catabolism of tryptophan and tyrosine in relation to the isoprenoid pathway was studied in neurological and psychiatric disorders. The concentration of trytophan, quinolinic acid, kynurenic acid, serotonin and 5-hydroxyindoleacetic acid was found to be higher in the plasma of patients with all these disorders; while that of tyrosine, dopamine, epinephrine and norepinephrine was lower. There was increase in free fatty acids and decrease in albumin (factors modulating tryptophan transport) in the plasma of these patients. Concentration of digoxin, a modulator of amino acid transport, and the activity of HMG CoA reductase, which synthesizes digoxin, were higher in these patients; while RBC membrane Na+-K+ ATPase activity showed a decrease. Concentration of plasma ubiquinone (part of which is synthesised from tyrosine) and magnesium was also lower in these patients. No morphine could be detected in the plasma of these patients except in MS. On the other hand, strychnine and nicotine were detectable. These results indicate hypercatabolism of tryptophan and hypocatabolism of tyrosine in these disorders, which could be a consequence of the modulating effect of hypothalamic digoxin on amino acid transport.

Effect of diabetes mellitus on epididymal enzymes of adult rats.

Diabetes mellitus caused significant reduction in serum testosterone and accessory sex glands weight. The sperm content of epididymal regions also decreased. Among the epididymal regions, the cauda epididymidal tissue alone showed significant reduction in Na(+)-K+ ATPase activity. However, Mg2+ ATPase activity was lowered in caput epididymidis only. Specific activity of Ca2+ ATPase significantly decreased in caput and cauda epididymides. All three ATPases decreased significantly in caput epididymidal spermatozoa leaving cauda epididymidal spermatozoa unaffected. Specific activity of alkaline phosphatase was suppressed in caput epididymidis and in the spermatozoa collected from caput and cauda epididymides, while the acid phosphatase was unaffected. In general, the results are suggestive of definite influence of diabetes on epididymal phosphatases which is region specific. Diabetes induced decrease in phosphatases may have an impact on secretory and absorptive functions of epididymis and thus on sperm maturation.