K-ATPase Inhibition Research Articles

Effect of photobiomodulation therapy on neuronal injuries by ouabain: the regulation of Na, K-ATPase; Src; and mitogen-activated protein kinase signaling pathway

BackgroundTo determine whether photobiomodulation (PBM) rescued the disruption of Na+/Ca2+ homeostasis and mitochondrial membrane potential by ouabain; the Na, K-ATPase inhibitor. For PBM in this study, a 660 nm LED array was used at energy densities of 0.78, 1.56, 3.12, 6.24, and 9.36 J/cm2.ResultsHCN-2 neuronal cells treated with ouabain showed loss of cell polarity, disrupted cell morphology, and decreased cell viability, which were improved after PBM treatment. We found that ouabain-induced Na, K-ATPase inhibition promoted activation of downstream signaling through Src, Ras, and mitogen-activated protein kinase (MAPK), which were suppressed after PBM treatment. This provided evidence of Na, K-ATPase α-subunit inactivation and intracellular Ca2+ increase. In response to ouabain, we observed activation of Src and MAPK by Na, K-ATPase, decreased mitochondrial membrane potential, and Na+-dependent Ca2+ increases, which were restored by PBM treatment.ConclusionsThis study demonstrated that Na+/K+ imbalance could be regulated by PBM treatment in neuronal cells, and we suggest that PBM is a potential therapeutic tool for Na, K-ATPase targeted neuronal diseases.

Tracking protein function with sodium multi quantum\xa0spectroscopy in a 3D-tissue culture based on microcavity arrays

The aim of this study was to observe the effects of strophanthin induced inhibition of the Na-/K-ATPase in liver cells using a magnetic resonance (MR) compatible bioreactor. A microcavity array with a high density three-dimensional cell culture served as a functional magnetic resonance imaging (MRI) phantom for sodium multi quantum (MQ) spectroscopy. Direct contrast enhanced (DCE) MRI revealed the homogenous distribution of biochemical substances inside the bioreactor. NMR experiments using advanced bioreactors have advantages with respect to having full control over a variety of physiological parameters such as temperature, gas composition and fluid flow. Simultaneous detection of single quantum (SQ) and triple quantum (TQ) MR signals improves accuracy and was achieved by application of a pulse sequence with a time proportional phase increment (TQTPPI). The time course of the Na-/K-ATPase inhibition in the cell culture was demonstrated by the corresponding alterations of sodium TQ/SQ MR signals.

Istaroxime, a first in class new chemical entity exhibiting SERCA-2 activation and Na–K-ATPase inhibition: a new promising treatment for acute heart failure syndromes?

Acute heart failure syndromes (AHFS) are associated with the rapid onset of heart failure (HF) signs and symptoms. Hospitalizations for AHFS continue to rise and are associated with significant mortality and morbidity. Several pharmacological agents are currently approved for the treatment of AHFS, but their use is associated with an increase in short-term mortality. There is a need for new agents that can be given in the acute setting with increased efficacy and safety. Istaroxime is a unique agent with both inotropic and lusitropic properties which is currently being studied for the treatment of AHFS. Istaroxime inhibits the sodium-potassium adenosine triphosphatase (ATPase) and stimulates the sarcoplasmic reticulum calcium ATPase isoform 2 (SERCA-2) thereby improving contractility and diastolic relaxation. Early data from human studies reveal that istaroxime decreases pulmonary capillary wedge pressure (PCWP) and possibly improves diastolic function without causing a significant change in heart rate (HR), blood pressure, ischemic or arrhythmic events. Most commonly reported side effects were related to gastrointestinal intolerance and were dose related. In conclusion, istaroxime is a novel agent being investigated for the treatment of AHFS whose mechanism of action and cellular targets make it a promising therapy. Further studies with longer infusion times in patients with hypotension are required to confirm its efficacy and safety.

Marine toxins and the cytoskeleton

One of the most striking features of marine toxins is that many of them produce diarrhea. This effect has been attributed to an effect on the cytoskeleton, with the consequent disorganization of the adherent cells on the intestinal cell barrier leading to an excess of liquid. But this has never been fully proven, as many neurotoxins show diarrheic effects that could be explained by an effect on the innervation system rather than on the epithelial intestinal cells. There is a wide diversity of mechanisms of action for marine toxins: phosphatase inhibition (okadaic acid), phosphodiesterase activation (yessotoxin), Na ⁄K-ATPase inhibition (palytoxin), Na channel blockage (saxitoxin, ciguatoxin, brevetoxin, conotoxin), nicotinic receptor antagonism (gymnodimin), mitogen-activated protein kinase activation (azaspiracid), Ca channel opening (maitotoxin), and K blockade (gambierol, conotoxin). Despite this complex array of modes of action, only pectenotoxins are known to target specifically the actin filaments. But regardless of the mechanism of action, most of the toxin groups have profound effects on cytoskeletal dynamics. The relevance of this is difficult to clarify, as the cytoskeleton is a common transduction step for many signals, but the effect of phycotoxins on the cytoskeleton is becoming a useful tool with which to investigate the toxicity and the mechanism of action of the toxins. This minireview series is intended to bring together a comprehensive set of data on four of the best recently studied toxin groups. Okadaic acid and their analogs, the dinophysistoxins, have been known for years as phosphatase inhibitors. They are very common worldwide, and important both economically, as a major cause of loss for shellfish producers, and scientifically, as okadaic acid is often used in research as a phosphatase inhibitor. Okadaic acid has been considered to be a very selective drug, in a similar fashion as initially thought for phorbol esters and protein kinase C, and its mode of action has clear implications for the dynamics of actin reorganization, but recent evidence indicates that phosphatases may not be the only targets of these toxins. The diarrhea caused by okadaic acid and analogs is so important that in the marine toxins field, members of the group are called diarrheic shellfish poisons. Palytoxin is well known to modify the function of the Na ⁄K-ATPase, but does not compete with ouabain, and its specific binding site has not yet been identified. The complexities of this compound and its many analogs make the group unique [largest nonrepetitive (peptide or sugar) molecule in nature], and also very interesting from a food safety perspective, as this group of compounds has been recently identified in Europe as a new risk phenomenom. Azaspiracids are currently by far the most complex toxin group and, 10 years after the first document reporting their presence, the mechanism of action remains elusive. Their complex transduction pathway is tightly linked to the cytoskeleton, and deserves a review. Finally, pectenotoxins are clearly associated with actin. Their action has been reported to involve a 1 : 1 binding with actin. However, the affinity of this binding is undefined as yet, and there are many unresolved questions regarding the implications of this effect for cell structure. Of all the marine toxin groups, okadaic acid, palytoxin, azaspiracids and pectenotoxins seem to be the most intriguing and interesting with regard to their effects on the cystoskeleton, and the four minireviews in this series describe what is known to date.

Cardioprotection by Ouabain and Digoxin in Perfused Rat Hearts

This work was aimed at determining the cardioprotective effect of digitalis glycosides in rat heart, and to relate it with Na, K-ATPase inhibition and ERK1/2 activation. Isolated working rat hearts were perfused in the presence of ouabain or digoxin, which were used at concentrations ranging from 10 to 10 M. The hearts were then subjected to 30 minutes of global normothermic ischemia followed by 120 minutes of retrograde reperfusion; irreversible tissue injury was determined on the basis of triphenyltetrazolium chloride staining. Significant cardioprotection was observed with 10 M and 10 M ouabain (ischemic injury averaged 7.0 +/- 3.5% and 8.3 +/- 0.6% versus 37.3 +/- 2.0% in controls, P < 0.01 in each case). Hearts treated with digoxin showed decreased ischemic injury at 10 M and 10 M (18.0 +/- 1.5% and 14.2 +/- 1.0%, P < 0.01 versus control in both cases). In parallel experiments, ERK2 phosphorylation was increased by 10 to 10 M ouabain, while ERK1 and ERK2 phosphorylation was increased by 10 to 10 M digoxin. The cardioprotective effect was not related to Na, K-ATPase inhibition, since Rbuptake was not significantly different between control and treated hearts.

Vasotab, a vasoactive peptide from horse flyHybomitra bimaculata(Diptera, Tabanidae) salivary glands

Horse flies feed from superficial haematomas and probably rely heavily on the pharmacological properties of their saliva to find blood. Here we describe the first evidence of vasodilators in horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary gland extract and clone and express one of the active peptides (termed vasotab). Physiological tests using crude salivary gland extracts and reverse-phase HPLC fractions demonstrated positive inotropism in isolated rat hearts, vasodilatation of coronary and peripheral vessels, and Na, K-ATPase inhibition. One of the vasoactive fractions was analysed by N-terminal Edman degradation and a 47-amino-acid sequence obtained. A full-length cDNA encoding the peptide was cloned from a phage library using degenerate primer PCR and the peptide expressed in insect cells. A 20-amino-acid signal sequence precedes the mature 56-amino-acid vasotab peptide, which is a member of the Kazal-type protease inhibitor family. The peptide has a unique 7-amino-acid insertion between the third and fourth cysteine residues. The recombinant peptide prolonged the action potential and caused positive inotropism of isolated rat heart myocytes, and may be an ion channel modulator.

Defective Renal Dopamine D1 Receptor Function Contributes to Hyperinsulinemia-Mediated Hypertension

Hyperinsulinemia is reported to play a role in hypertension, as abnormalities in blood pressure regulation and sodium handling exist in diabetes mellitus. Kidney dopamine promotes sodium excretion via the activation of renal D1 receptors. Because there is a close relationship between renal D1 receptor function and sodium excretion, it is hypothesized that a defect in this mechanism may contribute to decreased sodium excretion and hypertension during hyperinsulinemia. Renal D1 receptor function was studied in insulin-induced hypertension in male Sprague Dawley rats. Insulin pellets were implanted subcutaneously for controlled insulin release for three weeks; sham rats served as a control. Compared to control rats, insulin pellets increased plasma insulin levels by eight fold and decreased blood glucose by 40%. Insulin also caused a 22 mmHg increase in mean arterial blood pressure compared to control animals. The intravenous infusion of SKF-38393, a D1 receptor agonist, increased sodium excretion in control rats, but SKF-38393 failed to produce natriuresis in hyperinsulinemic animals. Renal proximal tubules from hyperinsulinemic rats had a reduced D1 receptor number, defective receptor-G protein coupling, and blunted SKF-38393 induced Na, K-ATPase inhibition. Insulin seems to reduce D1 receptor expression and coupling to the G-protein, leading to a reduced D1 receptor-mediated Na, K-ATPase inhibition, and a diminished natriuretic response to SKF-38393. These phenomena could account for sodium retention and hypertension associated with hyperinsulinemia.

ELEVATION OF [NA+]I PROTECTS ENDOTHELIAL CELLS FROM APOPTOSIS TRIGGERED BY [3H]-DECAY-INDUCED DNA DAMAGE

Objective: Endothelial cell damage contributes to vascular remodeling seen in hypertension and other cardiovascular diseases. Previously, we reported that elevation of the [Na+]i/[K+]i ratio protects vascular smooth muscle cells against apoptosis. This study examines the role of intracellular monovalent cations in apoptosis of porcine aorta endothelial cells (PAEC). Design and Methods: Apoptosis in cultured freshly-isolated PAEC was estimated by MTT assay and caspase-3 activity. [14C]-urea available space and atomic absorption spectrophotometry were employed to measure cell volume and intracellular Na and K content, respectively. Results: 24-hr treatment of PAEC with 5 uCi/ml of [3H]-thymidine led to 2-fold attenuation of MTT staining and 10-fold elevation of caspase-3 activity. Apoptosis in [3H]-thymidine-treated cells was abolished by nonradioactive thymidine, the pan-caspase inhibitor z-VAD.fmk and by Na,KATPase inhibition in K+ free medium. Incubation of PAEC in K+ free medium led to 10-fold elevation of Na + i and to 8-fold decrease of K + i. Inhibition of the Na,K-ATPase in K + free Na + depleted medium resulted in the same loss of K + i, but led to modest (2-fold) elevation of Na + i and did not protect PAEC against apoptosis. Conclusions: Extensive DNA labeling with [3H]-thymidine is sufficient to induce apoptosis in PAEC. Elevation of [Na+]i protects PAEC from apoptosis triggered by [3H]-decay-induced DNA damage at a step upstream of caspase-3. These data should be considered under analysis of the involvement of endogenous Na+,K+ ATPase inhibitors and other potent Na + i raising compounds in endothelial cell survival and vascular remodeling seen in hypertension.

Salinity Dependence of Vanadium Toxicity against the Brackish Water Hydroid Cordylophora caspia

Vanadium, an abundant metal, enters the environment through natural rock weathering or by combustion of oil products. A third pathway is the leaching of vanadium-rich building materials. Stones made from steel industry residual slags, so-called slag stones, contain rather large amounts of vanadium. The increasing use of these slag stones in riverbank reinforcement has therefore led to increased interest in the toxicity of vanadium to aquatic organisms. The aim of the study was to determine the toxicity of vanadium to the brackish water hydroid Cordylophora caspia and the effect of vanadium on the membrane-bound enzyme Na, K-ATPase at various salinities. EC50 values for population growth inhibition were determined from 1.74 to 7.96 mg L−1 vanadium, depending on salinity. The maximum inhibition of population growth by vanadium was observed at low salinities. Correspondingly, maximum Na, K-ATPase inhibition was also measured at low salinities and decreased with increasing salinity. The present study suggests that the observed inhibition of population growth of C. caspia caused by vanadium-contaminated rearing water is due to the vanadium-induced inhibition of phosphatases.

The Role of Na, K-Atpase Inhibitors in Hypertension and End-Stage Renal Disease

To review the role of Na,K-ATPase inhibitors in the pathogenesis of essential hypertension and hypertension associated with end-stage renal disease. MEDLINE search, 1966 to 1997. There is a suggestive physiologic and epidemiologic relationship between Na,K-ATPase inhibition and hypertension. However, clearance data cannot support the hypothesis that differential metabolism of this family of compounds explains the improved hypertensive control seen in patients on peritoneal dialysis compared to those on hemodialysis. As a result of the complex methodologies involved, it is unclear whether Na,K-ATPase inhibitors play a significant role in the hypertension of end-stage renal disease in general and peritoneal dialysis in particular.

Cordil reversibly inhibits the Na,K-ATPase from outside of the cell membrane. Role of K-dependent dephosphorylation.

Cordil-LND796 is a new cardiotonic glycoside under development. In rat brain microsomes where three isoforms of the Na,K-ATPase with differential affinities for cardiac glycosides have been identified, Cordil had higher affinity for the alpha 3 (IC50 = 0.02 microM) than for the alpha 2 (IC50 = 0.6 microM) and the alpha 1 (IC50 = 30 microM) isozymes. Cordil is potentially a selective inhibitor for both alpha 2 and alpha 3 Na,K-ATPase isoforms. Using inside out vesicles we have shown that Cordil binds to and inhibits Na,K-ATPase at an extracellular site. The dissociation kinetic rates (k-1) from the ATPase and the phosphatase activity (K-dependent dephosphorylation) of the Na,K-ATPase were similar for Cordil. Despite these similarities to ouabain comparison of the kinetics of the Na,K-ATPase inhibition by ouabain and Cordil revealed marked differences in their association rates (k+1 = 0.7 l mol-1 min-1 and k+1 = 6 x 10(-3) l mol-1 min-1 respectively) and their dissociation rates (k-1 = 1.3 +/- 0.2 x 10(-4) s-1 and k-1 = 69 +/- 7 x 10(-4) s-1 respectively). Both binding association and dissociation rates were enhanced for Cordil. These data are compatible with a stabilizing effect of Cordil on the E2P conformational state of Na,K-ATPase.

Effects of digitoxin and hypokalaemia on pancreatic NaHCO3secretion and pancreatic Na, K-ATPase activity

To study the role played by Na,K-ATPase in the pancreatic secretion of NaHCO3, experiments were performed in 20 anaesthetized, secretin-infused pigs (3.0 clinical units X kg b. wt. X h-I). The relationship between pancreatic NaHCO3 secretion and arterial pH was obtained before and during Na,K-ATPase inhibition by digitoxin and hypokalaemia. Na,K-ATPase activity in pancreatic tissue homogenate averaged 5.45 (5.02-6.68) mumol Pi X mg X protein X h-I. Retrograde injection of 0.5 ml 1.4 X 10(-4) mol X l-I digitoxin into pancreatic ducts reduced pancreatic Na,K-ATPase activity by 3I(I8-47)%, while intra-arterial injection of 0.2 mg X kg b. wt-I digitoxin reduced pancreatic Na,K-ATPase activity by 50(45-56)%. Digitoxin and hypokalaemia reduced the rate of pancreatic NaHCO3 and shifted the normal, proportional relationship between NaHCO3 secretion and arterial pH towards higher pH. Hypokalaemia reduced Na,K-ATPase activity and NaHCO3 secretion in proportion. These effects indicate that Na,K-ATPase helps to sustain the requisite electrochemical potential gradients for driving H+ ions, and hence HCO-3 ions, out of secretory cells.

Cardiac glycosides, calcium and the release of neurotransmitter from peripheral noradrenergic nerves

1. The requirement for external Ca (Cao) of neurotransmitter release evoked by cardenolides has been investigated in canine saphenous vein. 2. Basal efflux of3H-compounds from saphenous veins pre-loaded with3H-noradrenaline was the same in the absence as in the presence of Cao; Cao is not required for basal efflux of neurotransmitter. 3. Efflux of3H-compounds was increased by cardenolides. Both ACS and ouabain caused a similar maximum net efflux of3H suggesting that each evokes release from the same pool of3H compounds. 4. The similarity of the effects obtained with cardenolides to those obtained during exposure of saphenous vein preparations to potassium-free media suggests that3H-efflux is the result of Na, K-ATPase inhibition. 5. With ACS (ca. EC50) the net efflux of3H-compounds early (<60 min) in the release period was greater in the absence of Cao than in its presence whereas at longer times the reverse was true; net efflux was less in the absence of Cao than in its presence. The difference in the3H-efflux pattern was paralleled qualitatively throughout by efflux of3H-noradrenaline. 6. The ACS-evoked efflux of3H-compounds in the presence and absence of Cao derives from sympathetic, noradrenergic nerves;3H present in extraneuronal tissues was not released by the cardenolide. 7. With ouabain ( EC50): the total efflux of3H was less in the absence of Cao than in its presence. 8. In the presence of Cao the Ca-channel blocker VP reduced the total3H-efflux evoked by ouabain (>EC50), but did not reduce that evoked by ouabain (<EC50). 9. It is concluded that cardenolide evoked neurotransmitter efflux resulting from Na,K-ATPase inhibition is not dependent upon Cao although its presence, and subsequent entry via VP sensitive channels, does increase the magnitude of the efflux caused by higher concentrations of cardenolides. Whether a component of that efflux evoked by lower concentrations of cardenolides, (or higher concentrations over short time periods) depends on internal Ca stores or is independent of the cation, is not yet clear.

Influence of 16β formylation on Na, K-ATPase inhibition by cardiac glycosides

The inhibitory effect of formylated cardiac steroids (gitaloxin and its derivatives) on guinea-pig heart Na, K-ATPase was compared to that of other cardiac steroids with various hydroxy substituents. The decreasing order of potency of aglycones at equilibrium was as follows: gitaloxigenin greater than digitoxigenin greater than ouabagenin greater than digoxigenin greater than gitoxigenin greater than diginatigenin. This sequence was different to the sequence of drugs hydrophobic character. The compounds with hydroxy groups in the vicinity of the lactone ring (gitoxigenin, diginatigenin) were less potent than the hydrophilic compound ouabagenin. We propose that intramolecular bounding between 16 beta-OH and the lactone ring contributes to the relatively low potency of gitoxigenin and diginatigenin. The formylation of 16 beta-OH increased the potency of gitoxigenin by a factor of 41. The formylated compound (gitaloxigenin) was 5-fold more potent than digitoxigenin. The 3 beta-glycosylation of digoxigenin lead to pseudo-irreversible inhibitors of Na, K-ATPase. The half-time to achieve the equilibrium (for 5 mumol/l) was equal to 54 s, 90 s and 108 s respectively for digoxigenin monodigitoxoside, digoxin and desacetyllanatoside C. However, at equilibrium the three glycosides were equipotent, suggesting the existence of steric effects at the sugar site of the receptor. The sequence of potency observed for monodigitoxosides, monodigitalosides and tridigitoxosides after 60 min incubation was similar to that observed for the corresponding aglycones. These results suggest that the strongly negative inductive group 16 beta-OCHO is tightly bound to Na, K-ATPase, possibly to the same receptor site than that which is thought forming hydrogen and ionic bonds with the lactone ring. They show that the high toxicity of gitaloxin in guinea-pig heart is likely due to its high potency as Na, K-ATPase inhibitor.

Separate and combined effects of ouabain and extracellular potassium on renin secretion from rat renal cortical slices.

1. Renin secretion of rat renal cortical slices was measured as a function of extracellular K and ouabain concentrations in the incubation medium.2. A sigmoid relationship was found between renin secretion and log K concentration over the range 1.0-4.0 mM. Secretion was maximal at about 2.25 mM-K and half-maximal at about 1.43 mM-K.3. In media containing 4.0 mM-K, ouabain at 10(-8), 10(-7), and 10(-6)M did not affect renin secretion. Higher concentrations of ouabain inhibited secretion. A sigmoid relationship was found between% inhibition of secretion and log ouabain concentration (10(-6)-10(-3)M). Inhibition was half-maximal at 2.3 x 10(-5)M and complete at 10(-3)M-ouabain.4. Lowering extracellular K concentration from 4.0 to 2.25 mM shifted the dose-effect curve of ouabain to the left. At 2.25 mM-K, inhibition of renin secretion was half-maximal at 10(-5)M-ouabain.5. The inhibitory effect of 2 x 10(-5)M-ouabain (twice the dose for 50% inhibition) in media containing 2.25 mM-K was nearly identical to the combined effect of lowering K to 1.43 mM (the concentration required for 50% inhibition) and adding 10(-5)M-ouabain. This observation suggests that ouabain and low extracellular K act at a common site, presumably on Na, K-ATPase activity, to inhibit renin secretion.6. Neither 10(-3)M-ouabain nor K-free medium inhibited renin secretion when the concentration of free Ca in the medium was lowered to < 10(-8)M. Therefore it is proposed that as a result of Na, K-ATPase inhibition, (a) intracellular Na increases, (b) intracellular Ca increases via Na-Ca exchange, provided that extracellular Ca exceeds 10(-8)M, and that (c) Ca accumulation, in some unknown manner, inhibits renin secretion from rat renal cortical slices.