Effect of m-calpain in PKCα-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain.

There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate, PKC activity using an assay kit and assay of Na+/K+ATPase activity spectrophotometrically. We purified m-calpain and PKCα by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCα by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCα (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCα in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCα increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2+]i-mCalpain-PKCα signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.

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 low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 ìg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 ± 5.1 to 150 ± 4.7; DAP: 93.7 ± 7.7 to 116 ± 3.5 mmHg; P<0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

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.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group) underwent left coronary artery ligation (infarcted, Inf) or sham-operation (Sham). Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e) and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05). The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05), while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

Quando linfócitos são ativados por lectinas mitogênicas apresentam mudanças do potencial de membrana, elevação das concentrações citoplasmáticas de cálcio, proliferação e/ou morte celular induzida por ativação (AICD). Concentrações baixas de ouabaína (um inibidor da Na,K-ATPase) suprimem a proliferação induzida por mitógenos e aumentam a morte celular. Para entender os mecanismos envolvidos, uma série de parâmetros foram avaliados usando sondas fluorescentes e citometria de fluxo. A adição de 100nM de ouabaína para culturas de linfócitos de sangue periférico ativadas por fitohemaglutinina (PHA) não modificou o aumento de expressão do receptor Fas ou de seu ligante FasL induzida pelo mitógeno. No entanto, o tratamento com ouabaína potenciou a apoptose induzida por um anticorpo anti-Fas funcionando como agonista. Um sinergismo entre ouabaína e PHA também foi observado com relação à despolarização da membrana plasmática. Com relação à membrana mitocondrial, PHA por si só não produziu despolarização, mas quando as células foram também expostas à ouabaina uma dissipação do potencial foi observado, mas isso foi um evento tardio. É possível que o efeito inibitório da ouabaína em linfócitos de sangue periférico ativados envolva a potencialização de alguns aspectos do processo apoptótico e reflita uma exacerbação do mecanismo de AICD.

Biochemical profile of erythrocyte membrane of jaundiced neonates.

Studies in newborn humans have demonstrated alteration in the lipid, phospholipid and cholesterol content when compared with age-matched control. Membrane bound (Na+ + K+)ATPase activity is found to be significantly increased in jaundiced neonates. Alteration in membrane permeability characteristics in jaundiced neonates causes severe microenvironmental changes in red blood cell profile.

Papel de la adenosina sobre la acción antiarrítmica de los digitálicos / Role of adenosine on the antiarrhythmic action of digitalis

La acción de la adenosina sobre el periodo refractario funcional de los tejidos auriculares y en la cancelación del flutter auricular, es semejante a la de los digitálicos. Tal hecho sugiere la posible existencia de una participación adenílica en la acción digitálica. Por ello medimos las concentraciones plasmáticas de adenosina al infundir ouabaína, e investigamos el efecto del digitálico sobre el periodo refractario de tejidos auriculares y sobre el flutter en condiciones de inhibición colinérgico y bloqueo purinérgico. En perros, se administró ouabaína hasta inducir fibrilación ventricular. Se obtuvo sangre del seno coronario y de la vena femoral, midiendo la adenosina por cromatagrafía de líquidos. El periodo refractario se midió con la ténica del estímulo extra acoplado. El flutter se indujo al estimular el haz internodal posterior. Los resultados muestran que la concentración de adenosina se elevó más de 100 por ciento en plama de seno coronario; el efecto de la ouabaína sobre el periodo refractario no se modificó con la inhibición colinérgica, pero sí fue inhibido por aminofilina; el digitálico modificó su acción en la cancelación del flutter en presencia de aminofilina. Conclusión: en la acción antiarrítmica de los digitálicos parece participar un componente adenílico que resulta de la liberación de adenosina del corazón

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the alpha-subunit of Na+, K+ -ATPase inhibiting the Na+ -pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension.

Electrolyte disturbances due to ouabain sensitive sodium potassium pump in erythrocytes of children with sepsis.

The possible mechanism of hyponatraemia in septicaemic children was studied by measuring the intracellular red cell sodium in relation to ouabain sensitive Na(+)-K(+) pump by flame photometry. Hyponatraemia and hyperkalaemia were observed in most of the patients. There was a marked elevation in serum sodium levels and a significant reduction in serum potassium levels on recovery following therapy. The alteration in the distribution of electrolytes between plasma and erythrocytes resulted in significantly high levels of sodium and low levels of potassium within the erythrocytes of septicaemic patients which normalized on recovery. The ouabain sensitive sodium efflux rate and ouabain sensitive efflux rate constant were significantly decreased in the membranes of erythrocytes of septicaemic patients which also normalized on recovery. Our findings suggest that it is the intrinsic alterations in the transport capacity of Na(+)-K+ pump which could account for the rise in intracellular erythrocyte sodium and fall in intracellular potassium contents in septicaemic children.

Decrease in the ratio of high to low affinity isozymes of (Na+ +K+)-ATPase during the development of rat cardiac ventricles

The ratio of (Na++K+)-ATPase (EC 3.6.1.3.) isoforms with high and low affinity for cardiac glycosides was studied in heart preparations from neonatal, 3-month and 6-month old Wistar rats. Biphasic ouabain inhibition curves of (Na++K+)-ATPase activity indicated that the relative contribution of the high-affinity process decreased from 34 percent at 9 days to 23 percent at 3 months and to 10 percent at 6 months. Scatchard plots for [3H]ouabain binding were curvillinear and indicated that the relative contribution of the high-affinity sites (Kd = 0.1-0.25 µM) decreased by about one-half between 3 months (19-24 percent, N = 2) and 6 months (9-11 percent, N = 2)

Myocardial mitogenic effect of erythropoietin through the activation of Na+-K+-ATPase activity

La eritropoyetina ha sido descripta clásicamente como el principal factor de crecimiento que promueve la proliferación y diferenciación de las células de la progenic eritroidea. En este trabajo hemos observado una acción modulatoria, dosis dependiente, de la eritropoyetina humana recombinante (rHuEpo) sobre la proliferación de un cultivo primario de miocardiocitos de rata. La rHuEpo en bajas concentraciones (0,1-1U/ml) estimula el crecimiento celular, pero en altas concentraciones (3-10 U/ml) lo inhibe. La acción mitogénica de la hormona tiene correlación con la actividade de Na+-K+-ATPasa microsomal cardíaca de modo tal que concentraciones de rHuEpo que incrementan el crecimiento celular estimulan la actividad de paranitrofenilfosfatasa (pNPfasa) mientras que concentraciones que inhiben la actividad enzimática bloquean la acción mitogénica de la hormona. Más aún, ouabaina 10**-5M, concentración que inhibe la actividade de Na+-ATPasa, impide la acción estimulatoria que ejerce la rHuEpo sobre la proliferación celular. A su vez, la rHuEpo mientras estimula la actividad de Na+-K+-ATPasa microsomal cardíaca, es capaz de modificar la acción contráctil de la ouabaina sobre aurículas aisladas de ratas neonatas de modo tal que 1 U/ml de EpoHur produjo un incremento de la acción no-tóxica del glicósido cardíaco, atenuando y retrasando el comienzo del efecto tóxico de la ouabaina. Estos resultados muestran que la rHuEpo presenta un efecto no hematopoyético sobre el miocardio, asociado con la actividad de Na+-K+-ATPasa cardíaca, que regula el crecimiento de miocardiocitos en eultivo y la acción biológica de los glicósidos cardíacos sobre la aurícula aislada de rata neonata

Altered kinetic attributes of Na(+)+K(+)-ATPase activity in kidney, brain and erythrocyte membranes in alloxan-diabetic rats.

Effects of alloxan-diabetes on kinetic attributes of Na(+)+K(+)-ATPase were examined in rat kidney, brain and erythrocyte membranes. The enzyme activity decreased significantly from 60-80% in the three membrane systems as a result of diabetic state. Kinetic analysis revealed that Km of the enzyme increased by 5- and 10-fold respectively in the kidney and brain membranes while registering a 50-60% decrease in Vmax. Ouabain binding studies revealed that in the kidney membranes the I50 value increased by 150-fold in diabetic animals with a significant decrease in number of ouabain molecules bound; at concentrations beyond 10(-7) M de-binding of ouabain occurred. For the brain membranes the I50 values for ouabain increased even more significantly (2000-fold increase) without any change in Hill coefficient for ouabain binding. Glycosylation studies revealed that its extent was highest for the brain and least for the kidney membranes which correlated to some extent with the I50 and Km values but not with Vmax. The results thus suggest that glycosylation in critical domains of the membrane and/or enzyme structure may play an important regulatory role. Physiological significance of these findings is discussed.

Effects of Na+, K(+)-pump inhibitors on acetylcholine-induced relaxation in the rabbit aorta

The purpose of this study was to investigate the effects of inhibitors of the Na+, K(+)-pump and membrane depolarizing agents on endothelium-dependent acetylcholine-induced relaxation in the rabbit thoracic aorta. Aortic rings were prepared from the rabbit descending thoracic aorta and the contractility of the ring was measured in various conditions such as application of ouabain, exposure to K(+)-free Krebs-Henseleit solution and high K+. Ouabain or exposure to K(+)-free Krebs-Henseleit solution inhibited acetylcholine or sodium nitroprusside-induced relaxation. KCl also inhibited the acetylcholine or sodium nitroprusside-induced relaxation. These results suggest that the Na+, K(+)-pump may play a role in endothelium-dependent acetylcholine-induced relaxation.

Effect of tityustoxin and quabain on protein phosphorylation in a crude synaptosome fraction

1. The effect of titiyustoxin (TsTX) and ouabain on the incorporation of 32P into a protein of the same apparent molecular weight as synapsin I it described. 2. Tityustoxin-stimulated protein phosphorylation in a crude synaptosome fraction increased up to a concentration of 3.0 micron time of 15 s. 3. Trifluoperazizne (100 micronM) inhibited, while trifluoperazine sulphoxide (100 micronM) did not alter the effect on the protein phosphorylation induced by tityustoxin. 4. Unlike tityutixin, ouabain (100 micronM) hada no effect on protein phosphorylation even after incubation up to 20 min. 5. Ouabain at at 10 micronM, a concentration having no effect on rates of respiration and ATP hydrolysis in brain cortical slices, also had no effect on protein phosphorylation

Effect of inhibition of lens membrane Na+-K+ ATPase by ouabain pre-treatment--an in vitro study.

The precise factors responsible for the cataract formation are not known. The role of Na+-K+-ATPase in maintaining ionic concentration of lens and lens membrane permeability and its involvement in the formation of cataracts has been of recent interest. Thus the present study was undertaken to study the effect of pre-treatment with Ouabain, a known Na-K-ATPase inhibitor, on the intra-lenticular ionic concentration and the rule of lens membrane permeability in cataractogenesis. Fresh goat lenses were used for the experimental work. Isolated lens culture technique was used. The electrolytes were estimated before and 24 hours after Ouabain pre-treatment. The electrolyte pattern showed significant changes after pre-treatment with Ouabain. Lens sodium ion concentration increased significantly with a concommitant decrease in potassium ion concentration. Intra-lenticular chloride concentration also showed a significant increase as compared to control. Calcium and magnesium ion concentrations also showed slight increase after the inhibition of Na+-K+-ATPase system by Ouabain.

The effects of ouabain on the ERG c-wave

ID clarify the effects of ouabain on the ERG c-wave, isolated chick retinas were exposed to different concentrations of ouabain and the results noted. Although the c-wave was abolished at a highe. dose of ouabain (10(-4)M), its amp1itude increased in the presence of ouabain at a concentration of 10(-7)M, which was within the range of clinical use of the cardiac glycoside. On the other hand, the standing potential of the retina did not change appreciably until 10(-6)M and then decreased gradually at higher concentrations.In the presence of 10(-4)M ouabain, the concentration which completely blorked Na-K-ATPase, both the c-wave and the standing potential were almost abolished. These phenomena were more conspicuous when ouabain was applied to the vitreous side rather than the choroidal side. In the presence of 10(-7)M ouabain, the light sensitivity of the retina was elevated to 0.5 log unit and the maximum response increased about 30%. This may be a sign of visual complications of ouabain, such as metachromatopsia.

Comparação dos efeitos eletrocardiográficos de doses inotrópicas eqüipotentes de amrinone e de ouabaína em corações isolados de cobaia / Comparison of the electrocardiographic effects of equipotent inotropic doses of amrinone and ouabain in isolated hearts of guinea pigs

Os efeitos eletrocardiográficos de doses de amrinone (A) e de ouabaína (O) capazes de desenvolver açöes inotrópicas eqüipotentes foram estudados em coraçöes isolados de cobaia mantidos em perfusäo de Langendorff, nutridos por soluçäo de Krebs-Henseleit. Um baläo de látex, em comunicaçäo com um transdutor de pressäo, era mantido dentro do ventrículo esquerdo, que contraía isovolumetricamente. Nestas condiçöes, a pressäo desenvolvida durante as contraçöes é o indicador do inotropismo cardíaco. Elétrodos epicárdicos possibilitavam o registro do eletrocardiograma. Foi verificado que a concentraçäo de A (140 mg/l) necessária para elevar a pressäo desenvolvida (PD) em 100% é cerca de 200 vezes maior que a dose de O (0,65 mg/l). Para doses eqüipotentes. A provocou discreta taquicardia (195 + ou - 25 bpm para 220 + ou - 25 bpm; p < 0,05) e raras extra-sístoles. Sob infusäo ocom O, näo houve variaçäo de freqüência cardíaca (183 + ou - 32 bpm para 180 + ou - 27 bpm; ns) e ocorreu retardo da conduçäo A-V do estímulo (PRi passou de 0,08 + ou - 0,03 para 0,12 + ou - 0,04; p < 0,01). Em todas as preparaçöes infundidas com O ocorreram extra-sístoles freqüentes seguidas de arrtmias mais complexas (fibrilaçäo ventricular, taquicardia paroxística supraventricular). Estes resultados estäo de acordo com relatos da literatura de que A pode exercer efeitos inotrópicos equivalentes aos de digitálicos, sem as arrtmias habitualmente provocadas pelos glicosídeos

Effect of glucagon on arrhythmias induced by coronary artery occlusion and ouabain in dogs.

The effect of glucagon in arrhythmias induced by coronary artery occlusion and ouabain was studied in dogs. Intravenous administration of glucagon (50 microgram/kg) to 6 dogs with more than 70% ectopic activity after coronary artery occlusion, resulted in significant (P less than 0.01) decrease in ectopics and increase in heart rate. Infusion of glucagon (2.5 microgram/kg/min) for 30 min caused complete elimination of ectopic activity during infusion period. In another series of 7 experiments, glucagon failed to abolish the ouabain-induced ectopic beats. In fact the hormone itself caused a significant (P less tha 0.01) increase in ectopic activity and heart rate. However, in 7 dogs with complete heart block produced after ouabain conversion to normal sinus rhythm was observed after glucagon.