Inhibition Of Na/K-ATPase Activity Research Articles

A previously undescribed phenylethanoid glycoside from Callicarpa kwangtungensis Chun acts as an agonist of the Na/K-ATPase signal transduction pathway

The new concept that Na/K-ATPase acts as a receptor prompted us to look for new ligands from Callicarpa kwangtungensis Chun. Using column chromatography, an undescribed phenethyl alcohol glycoside, callicarpanoside A, and an undescribed benzyl alcohol glycoside, callicarpanoside B, along with twelve known polyphenols were isolated from Callicarpa kwangtungensis Chun. All the isolated compounds were evaluated for their Na/K-ATPase (NKA) inhibitory activities. Using our NKA technology platform-based screening assay protocols, callicarpanoside B was identified as an undescribed Na/K-ATPase agonist. In particular, the newly identified benzyl alcohol glycoside was found to bind NKA and activate the receptor NKA/Src complex, resulting in the activation of protein kinase cascades. These cascades included extracellular signal-regulated kinases and protein kinase C epsilon, as well as NKA α1 endocytosis at nanomolar concentrations. Unlike the class of cardiotonic steroids, callicarpanoside B showed less inhibition of NKA activity and caused less cellular toxicity. Moreover, callicarpanoside B was found to bind NKA at a different site other than the cardiotonic steroids binding site. Thus, we have identified an undescribed NKA α1 agonist that may be used to enhance the physiological processes of NKA α1 signaling.

Enhancement in cellular Na+K+ATPase activity by low doses of peroxynitrite in mouse renal tissue and in cultured HK2 cells.

In the normal condition, endogenous formation of peroxynitrite (ONOOˉ) from the interaction of nitric oxide and superoxide has been suggested to play a renoprotective role. However, the exact mechanism associated with renoprotection by this radical compound is not yet clearly defined. Although ONOOˉ usually inhibits renal tubular Na+K+ ATPase (NKA) activity at high concentrations (micromolar to millimolar range [μM–mM], achieved in pathophysiological conditions), the effects at lower concentrations (nanomolar range [nM], relevant in normal condition) remain unknown. To examine the direct effect of ONOOˉ on NKA activity, preparations of cellular membrane fraction from mouse renal tissue and from cultured HK2 cells (human proximal tubular epithelial cell lines) were incubated for 10 and 30 min each with different concentrations of ONOOˉ (10 nmol/L–200 μmol/L). NKA activity in these samples (n = 5 in each case) was measured via a colorimetric assay capable of detecting inorganic phosphate. At high concentrations (1–200 μmol/L), ONOOˉ caused dose‐dependent inhibition of NKA activity (−3.0 ± 0.6% and −36.4 ± 1.4%). However, NKA activity remained unchanged at 100 and 500 nmol/L ONOOˉ concentration, but interestingly, at lower concentrations (10 and 50 nmol/L), ONOOˉ caused small but significant increases in the NKA activity (3.3 ± 1.1% and 3.1 ± 0.6%). Pretreatment with a ONOOˉ scavenger, mercaptoethylguanidine (MEG; 200 μmol/L), prevented these biphasic responses to ONOOˉ. This dose‐dependent biphasic action of ONOO − on NKA activity may implicate that this radical compound helps to maintain sodium homeostasis either by enhancing tubular sodium reabsorption under normal conditions or by inhibiting it during oxidative stress conditions.

Enhancement of cellular Na+K+ATPase activity in the mouse renal tissue in‐vitro with low concentration of peroxynitrite

Endogenous production of peroxynitrite (ONOO°−) in normal condition has been suggested to play a renoprotective role. However, the exact mechanism associated with this protection is not yet clearly defined. ONOO°− at higher concentrations (μM to mM; in pathology) can inhibit cellular Na+K+ATPase (NKA) activity. However, the effect of ONOO°− on NKA activity at low conc. (nM; which is physiologically more relevant) remains unknown. To examine the hypothesis that ONOO°− at low conc. enhances NKA activity, preparations of cellular membrane fraction from mouse renal tissue (n=5 in each case) were incubated for 10 min each with different conc. of ONOO°− (10 nM to 200 μM). NKA activity in these samples was measured via a colorimetric assay capable of detecting inorganic phosphate. At high conc. (1–200 μM), ONOO°− caused dose‐dependent inhibition of NKA activity (−3.0±0.6 % to – 36.4±1.4 %) as reported previously. However, NKA activity remained unchanged at 100 and 500 nM ONOO°− conc., but interestingly, at lower conc.(10 and 50 nM), ONOO°− caused a small but significant increases in the NKA activity (3.3±1.1 % & 3.1±0.6 %). These responses were prevented at preparations co‐incubated with ONOO°− scavenger, mercapto ethyl guanadine (200 μM). These data indicate that NKA activity remains unaffected or rather increased at the physiological conc. of ONOO°− that facilitates the reno‐protective role for this oxidant molecule.

PTH and DA regulate Na-K ATPase through divergent pathways.

Parathyroid hormone (PTH) and dopamine (DA) inhibit Na-K ATPase activity and sodium-phosphate cotransport in proximal tubular cells. We previously showed that PTH and DA inhibit phosphate transport in opossum kidney (OK) cells through different signaling pathways. Therefore, we hypothesized that PTH and DA also inhibit Na-K ATPase through divergent pathways. We measured PTH and DA inhibition of Na-K ATPase activity in the presence of inhibitors of signaling pathways. PTH and DA inhibited Na-K ATPase in a biphasic manner, the early inhibition through protein kinase C (PKC)- and phospholipase A(2) (PLA(2))-dependent pathways and the late inhibition through protein kinase A- and PLA(2)-dependent pathways. Inhibition of extracellular signal-regulated kinase (ERK) activation blocked early and late inhibition of Na-K ATPase by PTH but not by DA. Pertussis toxin blocked early and late inhibition by DA but not by PTH. Treatment with DA, but not PTH, resulted in an early downregulation of basolateral membrane expression of the alpha-subunit, whereas total cellular expression remained constant for both agonists. We conclude that PTH and DA regulate Na-K ATPase by different mechanisms through activation of divergent pathways.

Cardiac NaK ATPase activity during positive inotropic and toxic actions of ouabain

In order to define pharmacological actions of ouabain in the dog heart, ouabain uptake and subcellular distribution and its effect on NaK ATPase (Mg 2+ dependent, Na +−K +-activated adenosinetriphosphate phosphohydrolase, E.C. 3.6.1.3), have been investigated in 21 open-chest dogs. A continuous infusion of ouabain (0.036 μg/kg/min) after a loading dose (20 μg/kg) produced a relatively constant plasma concentration of approximately 10 −8 (6 ng/ml) ouabain, which induced a sustained positive inotropic response for the 300 min experimental period. In these hearts much greater binding of ouabain was noted in the NaK ATPase and microsomal fractions than in other myocardial fractions. No statistically significant inhibition of NaK ATPase activity was noted. Doubling the loading and infusion doses of ouabain raised the plasma level of ouabain to approximately 3 × 10 −8 M and produced various types of arrhythmia within an hour, which persisted for the rest of the 5 h experimental period. Under this experimental protocol there was a significant inhibition of NaK ATPase activity and increased binding of ouabain to this enzyme. This study does not support the hypothesis that there is a causal relationship between inotropic response to ouabain and NaK ATPase inhibition. It was concluded that NaK ATPase inhibition might be causally related to the development of ouabain toxicity.

Effect of diuretics on renal NaK-ATPase and adenyl cyclase.

Contradictory results on the role of NaK-ATPase as the diuretic receptor are reported in the literature. In own experiments the effects of 1 mM ouabain and 0.2 mM ethacrynic acid on fractional sodium reabsorption and oxygen consumption was investigated in an isolated perfused kidney system. NaK-ATPase was measured in basal-lateral membranes of kidney cortex tubular cells. Combination of ouabain and ethacrynic acid resulted in a complete inhibition of fractional sodium reabsorption at only a 46% inhibition of NaK-ATPase activity. The complete inhibition of sodium reabsorption at only a partial inhibition of NaK-ATPase probably is caused by additional metabolic effects of ethacrynic acid. It is suggested that NaK-ATPase is not the only diuretic receptor. — Another enzyme possibly involved in the control of sodium transport and water permeability is the renal adenyl cyclase. 1 mM furosemide and 1 mM ethacrynic acid inhibited the enzyme in homogenates of the kidney cortex and inner medulla, while amiloride was without any effect. In the cortex the furosemide induced inhibition of adenyl cyclase can be reversed by 1 U parathyroid hormone and 0.1 mM isoproterenol. The ethacrynic acid effect on adenyl cyclase was not influenced by isoproterenol. In the inner medulla 1 mU antidiuretic hormone but not isoproterenol antagonized the furosemide inhibition of adenyl cyclase. Antidiuretic hormone and isoproterenol exerted no effect on the ethacrynic acid inhibition of adenyl cyclase. It is suggested that furosemide and ethacrynic acid act at different sites of the adenyl cyclase or at a mixture of adenyl cyclases with different sensitivities.

Studies of sodium-potassium activated adenosine triphosphatase: VII—inhibition by erythrophleum alkaloids

Erythrophleine and cassaine, two alkaloids occurring in the bark, leaves, and seeds of the genus Erythrophleum, were shown to be potent inhibitors of the digitalissensitive Na-K activated adenosine triphosphatase (Na-K ATPase) of rabbit brain, kidney, and ciliary body and cat choroid plexus. At 10 −4 M concentration, erythrophleine inhibited Na-K ATPase activity in all four tissues completely, while cassaine at 10 −4 M inhibited Na-K ATPase activity in the same tissues-an average of 86 per cent. Neither of these compounds caused significant inhibition of Mg-activated ATPase activity. The inhibition curves for erythrophleine and cassaine in rabbit brain had the same general shape as the curve for ouabain. The negative logarithms of the molar inhibitor concentration at which 50 per cent inhibition of Na-K ATPase activity occurs were 6.58 for erythrophleine, 5.96 for ouabain, and 5.28 for cassaine. All three compounds caused a stimulation of approximately 6 per cent at about 1 300 of their halfaximal inhibition concentrations. Increasing the K concentration in the assay medium from 0 to 40 mM caused 75 per cent reversal of the inhibition of rabbit brain Na-K ATPase by 5 × 10 −7 M erythrophleine as well as by 5 × 10 −7 M ouabain. No reversal was obtained of the Na-K ATPase inhibition by 10 −4 M erythrophleine and ouabain. It is concluded that the similarity of the effects of the erythrophleum alkaloids and the digitalis glycosides on the Na-K ATPase system may explain the remarkable similarity between these two groups of compounds in pharmacological properties and in inhibitory effects on active cation transport.