Expression studies on NA+/K+-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K+-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35‰ salinities and maintained for 20 days to observe long term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5‰ compared to 25‰ (isosmotic salinity) and 0.48 folds lower at 35‰. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5‰ the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25‰ and 35‰ NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25‰. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5‰ measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.

Hypertensive effects of the iv administration of picomoles of ouabain

Ouabain, an endogenous digitalis compound, has been detected in nanomolar concentrations in the plasma of several mammals and is associated with the development of hypertension. In addition, plasma ouabain is increased in several hypertension models, and the acute or chronic administration of ouabain increases blood pressure in rodents. These results suggest a possible association between ouabain and the genesis or development and maintenance of arterial hypertension. One explanation for this association is that ouabain binds to the α-subunit of the Na+ pump, inhibiting its activity. Inhibition of this pump increases intracellular Na+, which reduces the activity of the sarcolemmal Na+/Ca2+ exchanger and thereby reduces Ca2+ extrusion. Consequently, intracellular Ca2+ increases and is taken up by the sarcoplasmic reticulum, which, upon activation, releases more calcium and increases the vascular smooth muscle tone. In fact, acute treatment with ouabain enhances the vascular reactivity to vasopressor agents, increases the release of norepinephrine from the perivascular adrenergic nerve endings and promotes increases in the activity of endothelial angiotensin-converting enzyme and the local synthesis of angiotensin II in the tail vascular bed. Additionally, the hypertension induced by ouabain has been associated with central mechanisms that increase sympathetic tone, subsequent to the activation of the cerebral renin-angiotensin system. Thus, the association with peripheral mechanisms and central mechanisms, mainly involving the renin-angiotensin system, may contribute to the acute effects of ouabain-induced elevation of arterial blood pressure.

A key role for Na+/K+-ATPase in the endothelium-dependent oscillatory activity of mouse small mesenteric arteries

Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

Distrés respiratorio. Nuevas perspectivas en el manejo del edema pulmonar / [Respiratory distress. New Perspectives to lung edema treatment]

Acute respiratory distress syndrome (ARDS) is a life threatening condition associated with great morbidity and mortality. it is characterized initially by accumulation of fluid in the alveolar space that impairs alveolar oxygen exchange. Eventually, this syndrome leads to multiorgan failure. Therefore, rapid edema clearance has generally been associated with better outcome in patients with acute respiratory distress syndrome. Clearance of alveolar fluid is driven predominantly by active Na+ transport out of the alveolar space, mediated by increased apical Na(+)-channel and Na-K-ATPase activity. It has been demonstrated that increases in Na-K-ATPase in response to catecholamines in the alveolar epithelium are associated with increased lung edema clearance. The cellular mechanisms involve the recruitment of new Na-K-ATPase molecules to the plasma membrane from intracellular organelles. It also appears that adenovirus-mediated Na-K-ATPase gene transfer and increased Na-K-ATPase expression may provide an alternative and efficient pathway for transient increase in alveolar fluid reabsorption and resolution of pulmonary edema.

Sodium pumps in the Malpighian tubule of Rhodnius sp

Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.

Cell Biology of H+ Transport in Epithelia

Cell homeostasis of H+ ions has been an object of wide interest in the last two decades, which has led to extended knowledge about a considerable number of membrane transport mechanisms responsible for keeping cell pH within physiological limits. Among these mechanisms the most important are Na+/H+ exchange, the vacuolar H+-ATPase, the H+-K+-ATPase, Cl-/HCO3 - exchange and Na+/HCO3 - cotransport. The present review covers both cellular function and molecular aspects of these transporters, starting from a discussion of the methods used for the determination of cell pH and epithelial H+ transport, and analysing their molecular constitution, cloning and known isoforms, as well as their functional role in the maintenance of cell pH and epithelial transport.

Na, K-ATPase: a molecular target for Leptospira interrogans endotoxin

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1) GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC5O: 120-220 mug protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 mug protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ñ SEM; control: 23.8 ñ 1.8; GLP, 88 mug protein/ml: 8.2 ñ 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.

Consecuencias de la inhibición de la bomba de sodio en neuronas y su relevancia en la fisiopatología del daño celular producido por isquemia anóxica / Consecuences of sodium pump inhibition in neurons, and its relevance in the physiopathology of cellular damage by anoxic ischemia

Se estudió el efecto que sobre la conductancia total de la membrana (Gm)tiene la alteración del gradiente electroquímico del Na+ en neuronas de Helix aspersa. La sustitución del Na+ extracelular por un catión impermeante (glucamina) produjo una disminución inicial de Gm (4.6 ñ 2.9 por ciento), seguida de un sostenido aumento de la misma (30.3 ñ 10.5 por ciento), acompañado por una hiperpolarización de la membrana. Se sugiere que la eliminación del Na+ extracelular provoca la inversión del intercambiador Na+/Ca++, produciendo un aumento en la concentración intracelular de calcio iónico, [Ca++]i, que incrementa la permeabilidad de la membrana al K+. La inhibición de la bomba de Na+ con mabaína (lmM), también produjo un aumento en Gm, coincidente con un eflujo de K+ previamente descrito. Se modeló matemáticamente la conducta del intercambiador Na+/Ca++, encontrándose que aumentos modestos en la [Na+]i producen cambios, significativos en la [Ca++]i, congruentes con resultados previos. Se postula que la inhibición de la bomba de Na+ provoca un aumento en la [Ca++]i que dispara la permeabilidad al K+. La salida de K+ (junto con aniones y agua), produce una disminución del volumen celular acuoso. Se sugiere que al inhibir la bomba de Na+, el Ca++ entra a las neuronas vía intercambiador Na+/Ca++ y canales de Ca++. Los resultados se discuten en relación a los cambios que ocurren en el tejido nervioso durante isquemia anóxica.

Inmunocaracterización enzimática del endotelio capilar cerebral / Enzymatic immunocharacterization of brain capillary endothelium

La barrera hematoencefálica formada principalmente por los microvasos cerebrales, limita y controla el movimiento de iones y solutos entre la sangre y el cerebro. La enzima Na+K+ATSasa constituye una de las más importantes bombas de membrana, dispuesta para mantener las composiciones iónicas intra y extracelulares. esta bomba ha sido localizada en la membrana abluminal de distintos epitelios y se ha determinado que su funcionalidad depende de la fosforilación en residuos de aspartato. Por otra parte, la enzima aspartato quinasa (AK), desempeña el papel de fosforilar ácido aspártico, formando un compuesto altamente inestable. En trabajos anteriores, hemos inmunodetectado esa quinasa, asociada tanto a membranas como al citoplasma, en células de diferentes tejidos. En este estudio, basado en la inmunodetección de las enzimas por anticuerpos policlonales específicos, en secciones ultrafinas de cerebro de rata, hemos notado una asociación en la ubicación de Na+/K+APTasa y AK, en la membrana luminal de los endotelios de los capilares cerebrales. También, hemos observado vesículas en el citoplasma de los vasos, que tienen una reacción positiva al anticuerpo de AK marcado con peroxidasa. La presencia de Na+/K+APTasa en la membrana luminal y abluminal del endotelio de los microvasos cerebrales, indica una falta de polaridad de esta enzima. El análisis de las observaciones sugiere que ambas enzimas podrían estar funcionalmente relacionadas

Molecular isoforms of the sodium pump in brain

La Na+, K+ -ATPase es un elemento básico en toda célula que mantiene gradientes transmembranales de Na+ y K+. Cuando esté integrado en una membrana artificial, la Na+, K+-ATPasa es capaz de trasladar activamente Na+ y K+, asi establece su identidad con la bien conocida bomba de sodio. recientemente, tres isoformas de la retina, tales como retinitis pigmentosa. En este estudio se examinó la síntesis de las isoformas alfa-1 y alfa-2 en retinas de ratas pretratadas con citrato intraocular...(AU) subunidad catalítica han sido identificadas en el cerebro de la retina, tales como retinitis pigmentosa. En este estudio se examinó la síntesis de las isoformas alfa-1 y alfa-2 en retinas de ratas pretratadas con citrato intraocular...(AU) rata. La elucidación de los procesos que gobiernan su biosíntesis es esencial al comprender las alteraciones fisiológicas que acompañan la diabetes, intoxicación con esteroides cardiotónicos y enfermedades degenerativas de la retina, tales como retinitis pigmentosa. En este estudio se examinó la síntesis de las isoformas alfa-1 y alfa-2 en retinas de ratas pretratadas con citrato intraocular...