ANP differentially modulates marinobufagenin-induced sodium pump inhibition in kidney and aorta

NaCl loading and plasma volume expansion stimulate 2 natriuretic systems, vasoconstrictor, digitalis-like Na/K-ATPase inhibitors and vasorelaxant ANP peptides. Several hormones, including ANP, regulate activity of the Na/K-ATPase by modulation of its phosphorylation state. We studied effects of ANP on Na/K-ATPase phosphorylation and inhibition by an endogenous sodium pump ligand, marinobufagenin, in the aorta and renal medulla from male Sprague-Dawley rats. Marinobufagenin dose-dependently inhibited the Na/K-ATPase in renal and vascular membranes at the level of higher (nanomolar) and lower affinity (micromolar) binding sites. Marinobufagenin (1 nmol/L) inhibited Na/K-ATPase in aortic sarcolemma (18%) and in renal medulla (19%). prepro-ANP 104 to 123 (ppANP) and alpha-human ANP ([alpha-hANP] both 1 nmol/L) potentiated marinobufagenin-induced Na/K-ATPase inhibition in the kidney, but reversed the effect of marinobufagenin in the aorta. Similarly, ppANP and alpha-hANP modulated the sodium pump (ouabain-sensitive (86)Rb uptake) inhibitory effects of marinobufagenin in the aorta and renal medulla. In renal medulla, ppANP and alpha-hANP induced alpha-1 Na/K-ATPase phosphorylation, whereas in aorta, both peptides dephosphorylated Na/K-ATPase. The effect of ppANP on Na/K-ATPase phosphorylation and inhibition was mimicked by a protein kinase G activator, 8-Br-PET-cGMP (10 micromol/L), and prevented by a protein kinase G inhibitor, KT5823 (60 nmol/L). Our results suggest that alpha-1 Na/K-ATPase inhibition by marinobufagenin in the kidney is enhanced via Na/K-ATPase phosphorylation by ANP, whereas in the aorta, ANP exerts an opposite effect. The concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, marinobufagenin, potentiate each other's natriuretic effects, but ANP peptides may offset the deleterious vasoconstrictor effect of marinobufagenin.     

Sodium pump failure in hypoxia and reoxygenation

Sodium pump measurements have not been previously documented in the intact heart close to the onset of irreversible injury in hypoxia and reoxygenation. In the present experiments, Rb+ uptake was measured in the presence and absence of ouabain in Langendorff-perfused rat hearts. In the presence of ouabain, Rb+ uptake was increased after 10 min hypoxia (P < 0.05 vs. controls), but not after 30 min hypoxia, suggesting transient activation of a passive pathway such as the ATP-sensitive K+ channels. Passive Rb+ uptake was normal during post-hypoxic reoxygenation. Active Rb+ uptake was reduced after 30 min hypoxia, increased on reoxygenation after 10 min hypoxia, and failed to recover on reoxygenation after 30 min hypoxia (P < 0.001, P < 0.05 and P < 0.001, respectively, vs. controls). Sodium pump failure may lead to calcium overload through sodium-calcium exchange, and may be a decisive influence in post-hypoxic reoxygenation injury.     

非小细胞肺癌钠泵亚单位表达及其与预后的关系

目的研究非小细胞肺癌组织钠泵α1、α3、β1、β2四个亚单位表达的改变及其与预后等临床特征的相关性。方法对手术获取的非小细胞肺癌组织标本采用免疫组化法进行染色,半定量分析四个亚单位表达情况及与正常肺组织间的差异。结果①与正常组织相比,人肺鳞癌组织中钠泵α1、β1亚单位表达增强(P分别为0.000和0.003);鳞癌组织中钠泵α3、β2亚单位与正常组织表达比较差异无统计学意义;②与正常组织相比腺癌组织钠泵α1、α3、β1亚单位表达增强,尤以β1亚单位增强最明显,差异有统计学意义。β2亚单位表达与正常组织相比无明显变化;③钠泵α1与β1亚单位的表达强度与肺癌患者的生存月分别呈正相关,P值分别为0.000和0.001。钠泵α3与β2亚单位表达强度与肺癌患者生存月无关(P值分别为0.604和0.126)。结论非小细胞肺癌存在钠泵亚单位表达的改变,此种改变与患者的生存时间呈一定的相关关系,可作为判断预后的辅助指标。     

Sodium pump inhibition, enhanced calcium influx via sodium-calcium exchange, and positive inotropic response in cultured heart cells

The effects of sodium pump inhibition produced by exposure to the cardiac glycosides, ouabain or dihydroouabain, or by reduction in extracellular potassium to 1.0 mM, on contractile state and sodium-calcium exchange were studied in primary monolayer cultures of chick embryo ventricular cells. Ouabain, 10(-6)M, dihydroouabain, 5 X 10(-5)M, and extracellular potassium of 1.0 mM all induced similar and prominent positive inotropic effects. These effects were accompanied, in each case, by 40-50% inhibition of the rate of active uptake of 42K and by similar increases in steady state sodium content. Stimulation of the rate of 45Ca uptake on exposure to zero extracellular sodium occurred in response to extracellular potassium (1.0 mM) or to glycoside concentrations that induced a positive inotropic effect and sodium-potassium pump inhibition. Reactivation of the sodium pump after return from 1.0 to 4.0 mM extracellular potassium was rapid and was associated with membrane hyperpolarization and slowing of spontaneous beating rate. With pump reactivation under these circumstances, the time course of disappearance of stimulation of sodium-calcium exchange on exposure to zero extracellular sodium was similar to the time course of loss of the positive inotropic effect. Under physiological conditions (4.0 mM extracellular potassium), exposure to positively inotropic but nontoxic concentrations of ouabain or dihydroouabain caused a small but consistent increase in unidirectional calcium influx, but had no discernible effect on calcium efflux. Since similar inotropic effects were produced for comparable degrees of glycoside or low extracellular potassium-induced sodium pump inhibition and increases in cellular sodium content, sodium pump inhibition rather than a glycoside-specific change in calcium binding appears to underlie the inotropic response. These findings are further consistent with the view that the primary mechanism of the positive inotropic effects of digitalis and low extracellular potassium in this experimental preparation is sodium pump inhibition resulting in increased intracellular sodium. We suggest that increased calcium influx via sodium-calcium exchange is the principal mechanism whereby increased intracellular sodium results in enhanced calcium availability to the myofibrils, but an additional effect on calcium efflux is not excluded.     

Sodium sensitive and sodium retaining hypertension

The differences between sodium sensitive and sodium retaining hypertension were theoretically considered using a water tank model of body fluid volume-blood pressure regulation. If an outlet valve is attached to a tank with a base area corresponding to the reciprocal of total peripheral resistance (TPR) and water is poured into this tank at a rate corresponding to the amount of Na+ intake, then equilibrium should be achieved at a certain water level, volume and output from the outlet, which represent mean arterial pressure (MAP), cardiac output (CO) and urinary Na+ excretion. The height of the outlet from the tank bottom and the size cross-sectional area, of the outlet correspond to the x-intercept and slope of the renal function (pressure-natriuresis) curve, respectively. In both nonsodium sensitive hypertension, due to the shift of the curve toward a higher blood pressure level (elevated height of the outlet) without change in the slope (size of the outlet), and sodium sensitive hypertension, due to the depressed slope of the curve (reduced outlet size), not only MAP (water level) but also CO (water volume) are increased, resulting in sodium retaining hypertension, if TPR (reciprocal of base area) remained unchanged, while CO is relatively unchanged, resulting in nonsodium retaining hypertension, if TPR is elevated. Thus, the MAP and its sensitivity to sodium intake is determined by the renal function curve. Since body fluid volume is determined by both the renal function curve and TPR, however, changes in TPR during the development of hypertension is a major factor in determining whether or not the body fluid volume has to change only a small amount or a large amount. Therefore, the sodium sensitivity of blood pressure and sodium retention must be considered separately.     

Acute sodium loading alters sodium pump in Caucasian hypertensive subjects

The effect of an acute saline load on the sodium pump was determined from measurements of intracellular sodium and potassium, ouabain-inhibitable sodium efflux, and the number of sodium-potassium adenosine triphosphatase (Na,K-ATPase) sites per cell (using 3H-ouabain binding) of erythrocytes from 22 hypertensive and 21 normotensive subjects before and after a 2-1 infusion of 0.9% saline over a 4-hour period. Before the infusion, ouabain-inhibitable sodium efflux was the only measured parameter that was significantly (p less than 0.025) different between hypertensive (1.65 +/- 0.21 mmol/l red blood cell [RBC]/hr) and normotensive (1.46 +/- 0.25 mmol/l RBC/hr) subjects. After the saline infusion, there was a significant (p less than 0.001) decrease in the ouabain-sensitive sodium efflux of the hypertensive (1.55 +/- 0.22 mmol/l RBC/hr) but not of the normotensive (1.48 +/- 0.43 mmol/l RBC/hr) subjects. Although the changes in intracellular sodium of the normotensive and hypertensive subjects caused by the saline infusion were not significant, the fact that the change was in opposite directions in the two groups yielded a significant (p less than 0.02) differential response. After the saline infusion there was a significant increase in intracellular potassium (p less than 0.001, paired t test) and in the 3H-ouabain-binding affinity constant (p less than 0.001, paired t test) for both hypertensive and normotensive subjects. A second-order rate constant, which is an estimate of the apparent affinity constant of the sodium pump, was calculated from the ouabain-inhibitable sodium efflux, the intracellular sodium, and the number of Na,K-ATPase sites per cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myocardial sodium pump activity in endotoxin shock

The effect of endotoxin administration on the integrity of sodium pump was studied in canine heart myocardium. Sodium pump activity was determined based on the 86Rb+ uptake by ventricular muscle strips. The results show that 4 hr following endotoxin administration the ouabain-sensitive 86Rb+ uptake was inhibited by 44-70%, and the inhibition was noncompetitive with 86Rb+ ions and was independent of Na+ concentrations. The sensitivity of sodium pump toward verapamil inhibition was unaffected by endotoxin administration, suggesting that the endotoxin-induced decrease in sodium pump activity is not mediated through alteration in either calcium or slow sodium channel. The sensitivity of 86Rb+ uptake to ouabain inhibition was decreased approximately sixfold in endotoxin-injected dogs. These data suggest that sodium pump is greatly impaired in dog hearts 4 hr after endotoxin administration. The impairment in sodium pump might have physiological significance in the development of myocardial dysfunction during endotoxic shock.     

ATP-driven sodium pump in Streptococcus faecalis.

Sodium extrusion by bacteria is generally attributed to secondary antiport of Na+ for H+ energized by the proton circulation. Streptococcus faecalis is an exception, in that sodium expulsion from intact cells requires the generation of ATP but does not depend on the protonmotive force. Unfortunately, studies with everted membrane vesicles failed to reveal the expected sodium pump; instead, the vesicles contained a conventional secondary Na+/H+ antiporter. We report here that everted membrane vesicles prepared in the presence of protease inhibitors retain an ATP-driven sodium transport system. The evidence includes the findings that (i) accumulation of 22Na+ by these vesicles is resistant to reagents that dissipate the protonmotive force but requires ATP and (ii) the vesicles contain a sodium-stimulated ATPase that is distinct from F1F0 ATPase, and whose presence is correlated with sodium transport activity. Sodium movements appear to be electroneutral and are accompanied by movement of H+ in the opposite direction. When membranes are incubated in the absence of protease inhibitors, a secondary Na+/H+ antiport activity emerges, possibly by degradation of the sodium pump. We suggest that S. faecalis expels Na+ by means of an ATP-driven primary transport system that mediates exchange of Na+ for H+. The Na+/H+ antiporter seen in earlier membrane preparation is an artefact of proteolytic degradation.     

Relations among sodium pump inhibition, Na-Ca and Na-H exchange activities, and Ca-H interaction in cultured chick heart cells

Sodium pump inhibition in cardiac muscle cells is associated with changes in intracellular sodium, calcium, and hydrogen concentrations as well as in membrane ion transport activity. We examined further the functional relations among these entities using cultured chick ventricular cells. [Ca]i and pHi were determined from fluorescence signals obtained from cells loaded with fura-2 or BCECF, respectively. Ouabain (100 microM) elevated [Ca]i eightfold and decreased pHi by 0.11 unit (a 30% increase in [H+]). In the presence of 10 microM ethylisopropylamiloride, a potent inhibitor of Na-H exchange, ouabain elevated [Ca]i 3.5-fold and reduced pHi by 0.16 unit (a 48% increase in [H+]). Exposure to sodium-free (sodium replaced with potassium) medium produced a twelvefold increase in [Ca]i and a 0.12 pH unit decrease in pHi. In cells treated with 100 microM ouabain, exposure to sodium-free (lithium) medium resulted in a 22-fold sustained increase in [Ca]i and a rapid intracellular acidification (pH 7.15 to 6.60). The effect of ouabain or sodium-free medium on pHi was abolished in calcium-free medium; addition of 1 mM Ca rapidly increased [Ca]i and decreased pHi. In cells treated with subtoxic (3 microM) or toxic (100 microM) concentrations of ouabain, initial 24Na uptake rates were significantly greater than in control cells and were significantly reduced in the presence of 10 microM ethylisopropylamiloride. We conclude that ouabain (100 microM) produces intracellular acidification as a result of sodium pump inhibition; calcium accumulation via Na-Ca exchange, and subsequent Ca-H interaction within the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium pump numbers and cation transport of lymphocytes in pregnancy-induced hypertension

Pregnancy-induced hypertension may be linked with sodium pump inhibition and an increase in vascular myocytic tone and, hence, flow impedance. All of the findings of studies on circulating plasma and blood cells are not, however, consistent with this hypothesis. We therefore assessed sodium pump numbers and cation transport in lymphocytes from 23 women with untreated pregnancy-induced hypertension, 28 normotensive pregnant women and 28 healthy non-pregnant women. We measured the maximum 3H-ouabain binding capacity to determine the sodium pump activity and the apparent dissociation constant (the reciprocal of which estimates binding affinity) by Scatchard analysis, ouabain-sensitive (pump-mediated) 86rubidium influx and ouabain-resistant (pump-independent) influx in lymphocytes in vitro. Pregnant women, whether normotensive or hypertensive, had significantly more sodium pump activity and a higher pump-mediated and pump-independent 86rubidium influx than non-pregnant women. Sodium pump activity and the pump-mediated and pump-independent 86rubidium influx all reached normal, non-pregnant levels in normotensive pregnant women 6 weeks after delivery, but remained high in women with pregnancy-induced hypertension. The normotensive and hypertensive pregnant women and non-pregnant women all had similar ouabain binding affinity. The results of our study do not support the circulating sodium pump inhibitor hypothesis in pregnancy-induced hypertension.     

Lymphocytic sodium and potassium pump function in Bartter's syndrome

Bartter's syndrome is characterized by chronic hypokalaemia, activation of the renin-angiotensin system and normal blood pressure. To investigate whether a generalized disturbance of sodium-potassium pump function might be of pathogenetic importance, lymphocytic sodium-potassium homeostasis was examined in 5 patients suffering from Bartter's syndrome. Two of the patients were treated with potassium chloride supplementation, the others were without medical treatment when studied. All were severely hypokalemic (serum potassium 2.8 +/- 0.24 mmol/l, mean +/- SEM). Lymphocyte sodium and potassium concentration (14.4 +/- 0.37 and 94.4 +/- 7.7 mmol/l, respectively), ouabain sensitive 22Na-efflux rate constant (2.68 +/- 0.25 h), and absolute ouabain sensitive efflux rate (38.16 +/- 4.2 mmol l-1 h) did not differ from matched controls. Ouabain binding capacity was 126 900 +/- 23 500 sites/cell in patients vs 50 400 +/- 17 900 in controls (p less than 0.05). In conclusion, patients with Bartter's syndrome may have an intrinsic abnormal pump function, characterized by an increased pump density and a low cation turn-over rate per pump unit.     

Circulating sodium pump inhibitors in five volume-expanded humans

BACKGROUND: We have previously reported the isolation from human placentas of an inhibitor of the sodium pump (Na/K ATP-ase) of molecular weight 370 Da, which is considered to have a dihydropyrone-substituted steroid (bufenolide) structure. OBJECTIVE: To examine if this inhibitor is present outside of the pregnant state. METHODS: We examined the plasma ultrafiltrate of patients who were clinically volume-expanded. During the period of this study five such patients were identified. One was receiving haemofiltration for acute renal failure and four were being treated by plasma exchange. High performance liquid chromatograph (HPLC) purified fractions obtained from each of these five patients inhibited the human leucocyte sodium pump in vitro. RESULTS: Each of the purified fractions that inhibited the leucocyte ATP-ase in vitro contained a compound of mass 370 Da, the same mass as that found previously in placental extracts. This inhibitory factor was absent from HPLC purified fractions of plasma ultrafiltrate obtained from fifty-five patients who were clinically normovolaemic. Negative ion mass spectrometry (MS)/MS of the inhibitory material produced the fragmentation pattern characteristic of the placenta-derived pump inhibitor in only one of the five samples. The other four samples, although having the same mass, exhibited a different fragmentation pattern. CONCLUSION: The results suggest that an inhibitor of the sodium pump, identical in mass to that obtained from human placentas, circulates in the plasma of volume-expanded patients. The fragmentation pattern observed in negative ion mass spectrometry in the majority of the volume expanded patients may represent the presence of an isomer of the sodium pump inhibitor previously described in placental material.     

Phasic vascular sodium pump changes in deoxycorticosterone-hypertensive rats

We determined the sodium pump activity, measured as ouabain-sensitive 86Rb+ uptake, in the tail arteries of rats treated with deoxycorticosterone and sodium chloride for 6, 9, 14, 28, and 50 days. Systolic blood pressures, plasma sodium, potassium, and creatinine concentrations were measured, and the body weights were recorded. Vascular sodium pump activity was suppressed (by 27%) at the 6th day of deoxycorticosterone and sodium chloride treatment, a prehypertensive state. By the 9th day, blood pressure of deoxycorticosterone-treated and sodium chloride rats had increased, but sodium pump activity was not different from that of control animals. However, increases in sodium pump activity were noted after 14 and 28 days of deoxycorticosterone and sodium chloride treatment (18 and 21%, respectively). By 28 days, a fully developed hypertension was noted. At 50 days, rats displayed lower vascular sodium pump activity (by 23%) than the controls. These rats, although hypertensive, had significantly lower systolic blood pressures than the rats treated for 28 days. They had high plasma creatinine levels, low potassium and sodium concentrations, and low body weights compared to the controls, suggesting the presence of a malignant state. Our data indicate that there are time-related changes in the vascular sodium pump activity with this type of hypertension.