Class I antiarrhythmic drug effects on ouabain binding to guinea pig cardiac Na+ -K+ ATPase

The notion that the inhibition of the Mg2+ -dependent ATP-hydrolytic function of the myocardial Na+ -K+ ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 microM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 microM, 25-44% at 100 microM, and 37-56% at 200 microM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 microM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+ -K+ ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.     

The effect of Na+-K+ ATPase inhibition by ouabain on histamine release from human cutaneous mast cells

Background: There are controversial reports on the effect of sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) inhibition on mast cell mediator release. Some of them have indicated that ouabain (strophanthin G), a specific Na⁺-K⁺ ATPase inhibitor, inhibited the release, whereas the others have shown that ouabain had no effect or even had a stimulatory effect on the mediator secretion. Most of these studies have utilized animal-derived mast cells. The aim of this study was to determine the effect of Na⁺-K⁺ ATPase inhibition on human skin mast cells. Methods: Unpurified and purified mast cells were obtained from newborn foreskins and stimulated by calcium ionophore A23187 (1 μM) for 30 min following a 1 hr incubation with various concentrations (10⁻⁴ to 10⁻⁸ M) of ouabain. Histamine release was assayed by enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that ouabain had no significant effect on the non-immunologic histamine release from human skin mast cells, in vitro. Conclusions: Na⁺-K⁺ ATPase inhibition by ouabain had no significant effect on the non-immunologic histamine release from human cutaneous mast cells and suggested differences between human and animal mast cells.     

Alternative reactions of ouabain with a Na+ and K+ ATPase

Alternative reactions of ouabain with Na⁺ + K⁺ ATPase are described which may be interpreted by assuming that a conformational change takes place. Each conformational form appears to be dependent upon the cationic environment. The reaction of ouabain with one form inhibits the dephosphorylation step and inhibits the binding of ATP when it reacts with another conformational form.     

Variations in myocardial CPK and Na+-K+ ATPase following long-term freezing.

The activity of the soluble enzyme CPK and the membrane bound enzyme Na⁺-K⁺ ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at ?196 °C and ?79 °C and falls off during one month storage at ?40 °, ?20 °, and 0 °C. Na⁺-K⁺ ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer.     

Tumor necrosis factor alpha down-regulates the Na+-K+ ATPase and the Na+-K+2Cl- cotransporter in the kidney cortex and medulla

The effect of TNF-alpha on the renal Na+-K+ pump and the Na+-K+2Cl- cotransporter was investigated in the rat. Animals were injected with the cytokine, and 4h later, a homogenate from the cortical and medullary tissues was prepared and used to assay the activity of the Na+-K+ ATPase and the protein expression of the pump and symporter. TNF-alpha reduced the activity and expression of the pump in both cortex and medulla, and its effect disappeared when animals were pre-treated with indomethacin, suggesting that TNF-alpha acts via PGE2. Higher levels of PGE2 were detected by enzyme immunoassay, in kidney tissues isolated from rats treated with PGE2, thus confirming this hypothesis. The cytokine also down-regulated the Na+-K+2Cl- cotransporter but this effect was not abrogated by indomethacin. PGE2, injected into animals, exerted a dose-dependent effect. Low doses did not have any effect on the two transporters in the cortex while high doses inhibited and down-regulated the pump and up-regulated the cotransporter. In the medulla low doses increased the activity and expression of the pump but down-regulated the cotransporter while high doses exerted an exactly opposite effect on the two transporters. It was concluded that the effect of TNF-alpha on the pump is mediated via PGE2 which is released at relatively high doses. The effect of the cytokine on the cotransporter is, however, independent of PGE2.     

Endogenous ouabain in renal Na+ handling and related diseases

The Na⁺ pump and its Endogenous modulator Ouabain (EO) can be considered as an ancestral enzymatic system, conserved among species ranging from Drosophila to humans, related to Na handling. In this review, we examine how EO is linked with vascular function in hypertension and if it impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium–calcium exchanger duet. Biosynthesis of EO occurs in adrenal glands and is under the control of angiotensin II, ACTH and epinephrine. Elevated concentrations of EO and in the sub-nanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure. Experimental data suggest that the Na/K-ATPase α₂-catalytic subunit causes EO-induced vasoconstriction. Finally, maneuvers that promote Na depletion, as diuretic therapy or reduced Na intake, raise the EO levels. Taken together, these findings suggest a key role for EO in body Na homeostasis.     

Effect of inhibition of Na+-K+ ATPase on the prostacyclin generation of cultured human vascular endothelial cells

Prostacyclin (PGI2) generation of cultured human vascular endothelial cells (VEC) was observed coincidentally with the increase of 45Ca net influx. Ca ionophore A23187 enhanced not only PGI2 generation and 45Ca net influx but also 45Ca efflux. PGI2 generation was completely abolished by the pretreatment with Ca++ immobilizer, TMB-8. A Na+-K+ ATPase inhibitor, ouabain increased 45Ca net influx, but decreased 45Ca efflux, and enhanced PGI2 generation. These observation indicate that PGI2 generation of VEC may be regulated by not only Ca++ but also Na+, and it was suggested that enhanced PGI2 generation by ouabain might be derived from the increased cytosolic Ca++concentration by the decreased Ca++ efflux, and it was considered to be originated from the suppression of Na+-Ca++ exchange systems by the increased intracellular Na+ concentration via inhibition of Na+-K+ ATPase activity by ouabain. Enhancement of PGI2 generation of VEC by the increased ouabain like substances (OLS) in hypertension is suspected to be beneficial on the maintenance of vascular homeostasis.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

Ischemia-reperfusion alters gene expression of Na+-K+ ATPase isoforms in rat heart

The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na(+)-K(+) ATPase isoforms. The levels of mRNA for Na(+)-K(+) ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 x 10(4)U/L) plus catalase (7.5 x 10(4)U/L), an antioxidant mixture. The levels of mRNA for the alpha(2), alpha(3), and beta(1) isoforms of Na(+)-K(+) ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the alpha(1) isoform. Pretreatment with superoxide dismutase+catalase preserved the ischemia-reperfusion-induced changes in alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, whereas the alpha(1) mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H(2)O(2) (300 microM), or a free radical generator, xanthine (2mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H(2)O(2) or xanthine/xanthine oxidase depressed the alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, but had lesser effects on alpha(1) mRNA levels. These results indicate that Na(+)-K(+) ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na(+)-K(+) ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Contractile regulation of the Na+-K+-2Cl{-} cotransporter in vascular smooth muscle

Vasoconstrictors activate theNa⁺-K⁺-2Cl cotransporter NKCC1 inrat aortic smooth muscle, but the mechanism is unknown. Efflux of⁸⁶Rb⁺ from rat aorta in response tophenylephrine (PE) was measured in the absence and presence ofbumetanide, a specific inhibitor of NKCC1. Removal of extracellularCa²⁺ completely abolished the activation of NKCC1 by PE.This was not due to inhibition of Ca²⁺-dependentK⁺ channels since blocking these channels withBa²⁺ in Ca²⁺-replete solution did not preventactivation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited70% by 75 µM ML-9, 97% by 2 µM wortmannin, and 70% by 2 mM2,3-butanedione monoxime, each of which inhibited isometric forcegeneration in aortic rings. Bumetanide-insensitive Rb⁺efflux, an indication of Ca²⁺-dependent K⁺channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to120% of normal almost completely blocked the stimulation of NKCC1 byPE without inhibiting the stimulation by hypertonic shrinkage. Weconclude that activation of theNa⁺-K⁺-2Cl cotransporter by PE isthe direct result of smooth muscle contraction throughCa²⁺-dependent activation of myosin light chain kinase.This indicates that theNa⁺-K⁺-2Cl cotransporter isregulated by the contractile state of vascular smooth muscle.