Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.     

Regulation of acetylated tubulin/Na+,K+-ATPase interaction by L-glutamate in non-neural cells: involvement of microtubules

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na+,K+-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes with L-glutamate induces dissociation of the acetylated tubulin/Na+,K+-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells. As in the case of astrocytes, the effect of L-glutamate is mediated by its transporters and not by specific receptors. In COS cells, the effect of L-glutamate was reversed by its elimination from culture medium, provided that d-glucose was present. The effect of L-glutamate was not observed when Na+ was replaced by K+ in the incubation medium. The ionophore monensin, in the presence of Na+, had the same effect as L-glutamate. Treatment of cells with taxol prevented the dissociating effect of L-glutamate or monensin. Nocodazole treatment of intact cells or isolated membranes dissociated the acetylated tubulin/Na+,K+-ATPase complex. The dissociating effect of nocodazol does not require Na+. These results indicate a close functional relationship among Na+,K+-ATPase, microtubules, and L-glutamate transporters, and a possible role in cell signaling pathways.     

High glucose/High Lipids impair vascular adiponectin function via inhibition of caveolin-1/AdipoR1 signalsome formation

Reduced levels of adiponectin (APN) contribute to cardiovascular injury in the diabetic population. Recent studies demonstrate elevated circulating APN levels are associated with endothelial dysfunction during pre-diabetes, suggesting the development of APN resistance. However, mechanisms leading to, and the role of, vascular APN resistance in endothelial dysfunction remain unidentified. The current study determined whether diabetes cause endothelial APN resistance, and by what mechanisms. Under high glucose/high lipids (HG/HL), APN-stimulated nitric oxide production by HUVEC was decreased, phosphorylation of eNOS, AMPK, and Akt was attenuated (P<0.01), and APN's anti-TNFα effect was blunted (P<0.01). APN receptor expression remained normal, whereas Cav1 expression was reduced in HG/HL cells (P<0.01). The AdipoR1/Cav1 signaling complex was dissociated in HG/HL cells. Knock-down of Cav1 inhibited APN's anti-oxidative and anti-inflammatory actions. Conversely, preventing HG/HL-induced Cav1 downregulation by Cav1 overexpression preserved APN signaling in HG/HL cells. Knock-in of a wild type Cav1 in Cav1 knock-down cells restored caveolae structure and rescued APN signaling. In contrast, knock-in of a mutated Cav1 scaffolding domain restored caveolae structure, but failed to rescue APN signaling in Cav1 knock-down cells. Finally, AdipoR1/Cav1 interaction was significantly reduced in diabetic vascular tissue, and the vasorelaxative response to APN was impaired in diabetic animals. The current study demonstrates for the first time the interaction between AdipoR1 and Cav1 is critical for adiponectin-mediated vascular signaling. The AdipoR1/Cav1 interaction is adversely affected by HG/HL, due largely to reduced Cav1 expression, supporting a potential mechanism for the development of APN resistance, contributing to diabetic endothelial dysfunction.     

Benzoxazinone-thiosemicarbazones as antidiabetic leads via aldose reductase inhibition: Synthesis,biological screening and molecular docking study

Aldose reductase is an important enzyme in the polyol pathway, where glucose is converted to fructose, and sorbitol is released. Aldose reductase activity increases in diabetes as the glucose levels increase, resulting in increased sorbitol production. Sorbitol, being less cell permeable tends to accumulate in tissues such as eye lenses, peripheral nerves and glomerulus that are not insulin sensitive. This excessive build-up of sorbitol is responsible for diabetes associated complications such as retinopathy and neuropathy. In continuation of our interest to design and discover potent inhibitors of aldo-keto reductases (AKRs; aldehyde reductase ALR1 or AKR1A, and aldose reductase ALR2 or AKR1B), herein we designed and investigated a series of new benzoxazinone-thiosemicarbazones (3a-r) as ALR2 and ALR1 inhibitors. Most compounds exhibited excellent inhibitory activities with IC₅₀ values in lower micro-molar range. Compounds 3b and 3l were found to be most active ALR2 inhibitors with IC₅₀ values of 0.52 ± 0.04 and 0.19 ± 0.03 μM, respectively, both compounds were more effective inhibitors as compared to the standard ALR2 inhibitor (sorbinil, with IC₅₀ value of 3.14 ± 0.02 μM).     

Reconstitution of detergent-solubilized Na,K-ATPase and formation of two-dimensional crystals

Very pure, detergent-solubilized Na,K-ATPase from dog or lamb kidneys has been successfully reconstituted at high protein-to-lipid weight ratios. Studies have been conducted to establish the orientation of the Na,K-ATPase molecules in the reconstituted membranes and to assess the functional activity and the conformational state of the reconstituted enzyme. Results indicate that reincorporation of the Na,K-ATPase molecules in the lipid bilayer is unidirectional and that the reconstituted enzyme retains its functional and structural integrity. Two-dimensional crystals have been induced in these preparations by vanadate ions. The arrays, with a dimeric structure in the unit cell, have a morphology similar to that of the crystals that had previously formed in the native membranes. Filtered images show that in projection, the molecule had an asymmetrical mass distribution, which at the resolution of 2.5 nm is identical to that of the earlier crystals. These sheets, although small, represent the first crystals of Na, K-ATPase to be formed by reconstitution. We expect that optimization of the reconstitution and crystallization parameters will lead to larger and better-ordered sheets, suitable for electron crystallography.     

Short-term NO synthase inhibition and the Na+-binding properties of cardiac Na,K-ATPase

It is known that hypertension is accompanied by increased [Na+]i. The functional properties of Na,K-ATPase, which transports the Na+ out and K+ into myocardial cells during the relaxation phase, were investigated in the left ventricle (LV), septum (SV) and the right ventricle (RV) of anesthetized dogs with moderate acute blood pressure elevation elicited by short-term (4-hour) NO synthase inhibition. The NO-insufficiency was induced by administration of an L-arginine analogue, the N(G)-nitro-L-arginine methyl ester (L-NAME). Concerning the function of Na,K-ATPase under the conditions of lowered NO synthesis, we focused our attention to the binding of Na+ to the enzyme molecule. Activation of the enzyme by increasing Na+ concentrations revealed significant changes in both the maximal velocity (Vmax) and the affinity for Na+ (K(Na)) in all investigated heart sections. The Vmax increased by 27% in LV, by 87% in SV and by 58% in RV. The K(Na) value increased by 86% in LV, by 105% in SV and by 93% in RV, indicating an apparent decrease in the sensitivity of the Na+-binding site in the Na,K-ATPase molecule. This apparently decreased pump affinity for Na+ together with the increase of Vmax suggest that, during the short-term inhibition of NO synthesis, the Na,K-ATPase is capable of extruding the excessive Na+ from the myocardial cells more effectively at higher [Na+]i, as compared to the Na,K-ATPase of control animals.     

High levels of glucose induce apoptosis in cardiomyocyte via epigenetic regulation of the insulin-like growth factor receptor

Diabetic hyperglycemia result in cardiovascular complications, but the mechanisms by which high levels of glucose (HG) cause diabetic cardiomyopathy are not known. We investigate whether HG-induced repression of insulin-like growth factor 1 receptor (IGF-1R) mediated by epigenetic modifications is one potential mechanism. We found that HG resulted in decreased IGF-1 receptor (IGF-1R) mRNA levels, and IGF-1R protein when compared with H9C2 rat cardiomyocyte cells incubated in normal glucose. HG also induced apoptosis of H9C2 cells. The effects of HG on reduced expression of IGF-1R and increased apoptosis were blocked by silencing p53 with small interference RNA but not by non-targeting scrambled siRNA. Moreover, HG negatively regulated IGF-1R promoter activity as determined by ChIP analysis, which was dependent on p53 since siRNA-p53 attenuated the effects of HG on IGF-1R promoter activity. HG also increased the association of p53 with histone deacetylase 1 (HDAC1), and decreased the association of acetylated histone-4 with the IGF-1R promoter. Furthermore, HDAC inhibitor relieved the repression of IGF-1R following HG state. These results suggest that HG-induced repression of IGF-1R is mediated by the association of p53 with the IGF-1R promoter, and by the subsequent enhanced recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF-1R promoter-p53 complex. In conclusion, our data demonstrate that HG decreases expression of IGF-1R and decreases the association of acetylated histone-4 with the IGF-1R promoter. These studies may help delineate the complex pathways regulating diabetic cardiomyopathy, and have implications for the development of novel therapeutic strategies to prevent diabetic cardiomyopathy by epigenetic regulation of IGF-1R.     

Short-term NO synthase inhibition and the ATP affinity of cardiac Na,K-ATPase

It was previously shown that 4 hours lasting inhibition of nitric oxide synthesis by administration of an L-arginine analogue, the A(G)-nitro-L-arginine methyl ester (L-NAME) changed the affinity of the Na-binding site of Na,K-ATPase thus resulting in elevation of enzyme activity especially at higher concentrations of sodium. Using the same experimental model, we focused our attention in the present study to the question of binding of ATP to the enzyme molecule in the left ventricle (LV), ventricular septum (S) and the right ventricle (RV) of the dog heart. Activation of the enzyme by increasing concentrations of ATP revealed a significant increase of the Vmax only in septum (by 38 %). The K(M) increased significantly in septum (by 40 %) and in left ventricle (by 56 %) indicating an altered sensitivity of the ATP-binding site of Na,K-ATPase in the hearts of NO-deficient animals. The alterations of Na,K-ATPase in its ability to bind and hydrolyze ATP are localized to the tissue surrounding the cavity of the left ventricle.     

The relipidation of delipidated Na,K-ATPase. An analysis of complex formation with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine.

Enzymatically inactive, delipidated Na,K-ATPase from dogfish rectal glands was titrated with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine. The process of relipidation has the following characteristic properties. Enzymatic activities reappear independently of each other: first the phosphatase, then the ATPase. The properties of the phosphatase regenerated depend on the ratio of lipid/protein used; the ATPase seems to be independent of this ratio. The simplest model that is consistent with the above results and with the shapes of the titration curves, has the following requirements. Firstly, the enzyme is composed of two subunits that, as far as lipid binding is concerned, are identical and independent of each other. Secondly, lipid adds onto the enzyme as preformed clumps of 25 molecules of phosphatidylcholine or 18 molecules of phosphatidylethanolamine. Thirdly, each subunit binds two clumps of lipid, and binding shows positive cooperativity. Fourthly, when either subunit becomes saturated with lipid, the enzyme exhibits one form of phosphatase. Fifthly, when both subunits are saturated with lipid, the enzyme exhibits a second form of phosphatase and ATPase. The data and their analysis according to this model lead to the suggestion that Na,K-ATPase is a functional dimer, the interaction between subunits being influenced by the Na+ and K+ concentrations in the medium: K+ favouring the functional independence of the subunits and Na+ favouring their functional interaction.     

Similarities and differences between organic cation inhibition of the Na,K-ATPase and PMCA

The effects of three classes of organic cations on the inhibition of the plasma membrane Ca pump (PMCA) were determined and compared to inhibition of the Na pump. Quaternary amines (tetramethylammonium, tetraethylammonium, and tetrapropylammonium, TMA, TEA, and TPA, respectively) did not inhibit PMCA. This is not to imply that PMCA is inherently selective against monovalent cations because guanidine and tetramethylguanidine inhibited PMCA by competing with Ca(2+). The divalent organic cation, ethyl diamine, inhibited PMCA but was not competitive with Ca(2+). In contrast, propyl diamine did compete with Ca(2+) and was about 10-fold more potent than butyl diamine in inhibiting PMCA. For the Na pump, both TEA and TPA inhibited, but TMA did not. TEA, guanidine, and tetramethylguanidine inhibition was competitive with Na(+) for ATPase activation and with K(+) for pNPPase activation, both of which are cytoplasmic substrate cation effects. Thus, these findings are consistent with TEA, guanidine, and tetramethylguanidine inhibiting from the cytoplasmic side of the Na pump; in contrast, we have previously shown that TPA did not inhibit from the cytoplasmic side. The divalent alkane diamines ethyl, propyl, and butyl diamine all inhibited the Na pump and all competed at the intracellular surface. The order of potency was ED > PD > BD consistent with an optimal size for binding; similarly, for the quaternary amines TMA is apparently too small to make appropriate contacts, and TPA is too large. Homology models based upon the high-resolution SERCA structure are included to contextualize the kinetic observations.     

Inhibition of aldose reductase by dietary antioxidant curcumin: Mechanism of inhibition, specificity and significance

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. In this study we show that curcumin inhibits ALR2 with an IC₅₀ of 10 μM in a non-competitive manner, but is a poor inhibitor of closely-related members of the aldo-keto reductase superfamily, particularly aldehyde reductase. Results from molecular docking studies are consistent with the pattern of inhibition of ALR2 by curcumin and its specificity. Moreover, curcumin is able to suppress sorbitol accumulation in human erythrocytes under high glucose conditions, demonstrating an in vivo potential of curcumin to prevent sorbitol accumulation. These results suggest that curcumin holds promise as an agent to prevent or treat diabetic complications.     

Alpha-synuclein overexpression increases phospho-protein phosphatase 2A levels via formation of calmodulin/Src complex

Alpha-synuclein (α-Syn) is the principal protein component of Lewy bodies, a pathological hallmark of Parkinson’s disease (PD). This protein may regulate protein phosphatase 2A (PP2A) activity, although the molecular mechanisms for α-Syn-mediated regulation of PP2A and the potential neuroprotective actions of PP2A against PD-associated pathology remain largely unexplored. We found that α-Syn gene overexpression in SK-N-SH cells and primary neurons led to PP2A/C phosphorylation at Y307, a known target of Src kinase, and consequent phosphatase inhibition. In addition, phospho-activated Src (p-Y416 Src, pSrc) was higher in SK-N-SH cells and primary neurons overexpressing α-Syn. Thus, α-Syn may promote Src activation and PP2A inactivation, leading to hyperphosphorylation of proteins. Immunoprecipitation revealed higher calmodulin/Src complex formation in α-Syn-overexpressing cells and α-Syn transgenic mice. A TUNEL apoptosis assay and an MTT cell viability assay demonstrated that the PP2A activator C₂-ceramide protected neurons against α-Syn-induced cell injury. Buffering the Ca²⁺ elevations induced by α-Syn overexpression ameliorated the cytotoxicity of α-Syn. Our findings define a potential molecular mechanism for α-Syn-mediated regulation of PP2A through formation of the calmodulin/Src complex, activation of Src, and Src-mediated phospho-inhibition of PP2A. Overexpression of α-Syn may lead to neurodegeneration in PD in part by suppressing the endogenous neuroprotective activity of PP2A.     

Regulation of intracellular creatine in erythrocytes and myoblasts: Influence of uraemia and inhibition of Na,K-ATPase

The regulation of intracellular creatine concentration in mammalian cells is poorly understood, but is thought to depend upon active sodium-linked uptake of creatine from extracellular fluid. In normal human erythrocytes, creatine influx into washed cells was inhibited by 40 per cent in the absence of extracellular sodium. In washed cells from uraemic patients, sodium-independent creatine influx was normal, whereas the sodium-dependent component of creatine influx was 3·3 times higher than normal, possibly relecting the reduced mean age of uraemic erythrocytes. In spite of this, the intracellular creatine concentration was no higher than normal in uraemic erythrocytes, implying that some factor in uraemic plasma in vivo inhibits sodium-dependent creatine influx. Both in normal and uraemic erythrocytes, the creatine concentration was 10 times that in plasma, and the concentration in the cells showed no detectable dependence on that in plasma, suggesting that the intracellular creatine concentration is controlled by an active saturable process. Active sodium-dependent accumulation of creatine was also demonstrated in L6 rat myoblasts and was inhibited when transport was measured in the presence of 10^?4M ouabain or digoxin, implying that uptake was driven by the transmembrane sodium gradient. However, when creatine influx was measured immediately after ouabain or digoxin had been washed away, it was higher than in control cells, suggesting that Na,K-ATPase and/or sodium-linked creatine transport are up-regulated when treated with inhibitors of Na,K-ATPase.     

Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation,MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling

Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α₂ isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK_1/2 and PI3K/AKT; interaction between EGF receptor, α₁, and α₂ isoforms of Na,K-ATPase, Src, ERK_1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR⁸⁴⁵ and EGFR¹⁰⁶⁸; (ii) ammonia-induced ERK_1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK_1/2 phosphorylation; (iv) ammonia stimulates association between the α₁ Na,K-ATPase isoform, Src, EGF receptor, ERK_1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK_1/2, but in response to 3 mM ammonia it does not appear until after 12 h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.     

Efficacy of glucose, ouabain and an aldose reductase inhibitor on 2-[3H] myo-inositol uptake by human, rat and rabbit erythrocytes

Myo-inositol uptake by erythrocytes from humans, rabbits and rats was studied with an isotope technique. In human erythrocytes, the inhibitory effect on myo-inositol uptake was stronger with glucose than with ouabain. However, an aldose reductase inhibitor (ONO-2235, 100 microM) or insulin (200 microU/ml) failed to correct the decrease in myo-inositol uptake in packed RBC, produced by either 10 mM glucose or 2mM ouabain. Ten mM ouabain had an inhibitory effect on myo-inositol uptake in all species, but an inhibitory effect was not observed with 20 mM glucose in rabbit erythrocytes. The results suggest that myo-inositol uptake by erythrocytes may be dependent on the active transport system via sodium-ATPase and that erythrocytes may not be a suitable model to monitor the possible effect of an aldose reductase inhibitor on myo-inositol concentrations in other tissues concerned with diabetic complications.     

Kinetics of ternary complex formation between dihydrofolate reductase, coenzyme, and inhibitors

The kinetics of ligand binding to dihydrofolate reductase from Lactobacillus casei (MTX/R) to form the ternary enzyme-inhibitor-coenzyme complex have been investigated by the stopped-flow fluorescence technique. The fluorescence changes observed when coenzymes or inhibitors bind to the binary complex of the enzyme with the complementary ligand occur in a single fast phase. Under pseudo-first-order conditions the reaction traces could be fitted with precision to a single-exponential decay, and apparent bimolecular rate constants in the range 2 x 10(6) to 3 x 10(7) M-1s-1 have been measured assuming a bimolecular-unimolecular model. The kinetic constants obtained suggest that prior binding of an inhibitor to the enzyme may, to a minor extent, interfere with coenzyme binding but the rates of inhibitor binding seem to be unaffected by the presence of a bound coenzyme. Dissociation rate constants appear to be less than 1 s-1 which suggests that both coenzymes and inhibitors are tightly bound in the ternary complex. An investigation of the effects of pH on the kinetics of ternary complex formation indicated the involvement of ionizable groups in ligand binding, but this shows some ligand dependence. The rates of ligand bindings to form the ternary complex are fairly high, but it is unlikely that these associations are diffusion controlled because their measured activation energies of 7.8-14.5 kcal mol-1 are higher than expected from reactions whose rates are limited by diffusion in aqeous solution.