Na,K-ATPase Acts as a Beta-Amyloid Receptor Triggering Src Kinase Activation.

Beta-amyloid (Aß) has a dual role, both as an important factor in the pathology of Alzheimer's disease and as a regulator in brain physiology. The inhibitory effect of Aß42 oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer's disease. Still, the physiological role of the monomeric form of Aß42 interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aß42 monomer, triggering Src kinase activation. The co-localization of Aß42 with α1- and ß1-subunits of Na,K-ATPase, and Na,K-ATPase with Src kinase in SH-SY5Y neuroblastoma cells, was observed. Treatment of cells with 100 nM Aß42 causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aß42 with α1ß1 Na,K-ATPase isozyme leads to activation of Src kinase associated with the enzyme. Notably, prevention of Na,K-ATPase:Src kinase interaction by a specific inhibitor pNaKtide disrupts the Aß-induced Src kinase activation. Stimulatory effect of Aß42 on Src kinase was lost under hypoxic conditions, which was similar to the effect of specific Na,K-ATPase ligands, the cardiotonic steroids. Our findings identify Na,K-ATPase as a Aß42 receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aß42 in the nervous system.

Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function.

By maintaining the Na(+) and K(+) transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aß) at the early stages of Alzheimer's disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aß(1-42) forms a tight (Kd of 3 µM), enthalpy-driven equimolar complex with α1ß1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aß(1-42) is localized in the "gap" between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aß(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aß(1-42) level. However prolonged increase of Aß(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

N-Nitrosamine-{cis-Re[CO]2}(2+) cobalamin conjugates as mixed CO/NO-releasing molecules.

Mixed CO/NO-releasing molecules were prepared by conjugation of the 17-electron rhenium dicarbonyl cis-[Re(CO)2Br4](2-) complex to N-nitrosamine modified cyanocobalamin (B12) bio-vectors. The species were fully characterized by standard analytical techniques, including X-ray crystallography for cyanocobalamin-5'-O-pyrazine and () and its N-pyrazine nitrosylated derivative (). The N-nitrosamine B12 derivatives are able to liberate low NO doses in buffer solutions and appear to be "activated" towards NO release if in contact with cultured cells. Coordination of the cis-[Re(CO)2Br4](2-) complex on the axial cyanide of B12 allows for the combined loss of CO and NO from the conjugates. The mixed CO/NO-releasing molecules show cytoprotection in an ischemia-reperfusion model but no significant enhanced synergistic effects over the relative NORMs and CORMs building constituents.

Live-fibroblast IR imaging of a cytoprotective PhotoCORM Activated with Visible Light.

Carbon monoxide releasing molecules (CORMs) are an emerging class of pharmaceutical compounds currently evaluated in several preclinical disease models. There is general consensus that the therapeutic effects elicited by the molecules may be directly ascribed to the biological function of the released CO. It remains unclear, however, if cellular internalization of CORMs is a critical event in their therapeutic action. To address the problem of cellular delivery, we have devised a general strategy which entails conjugation of a CO-releasing molecule (here a photoactivated CORM) to the 5'-OH ribose group of vitamin B12. Cyanocobalamin (B12) functions as the biocompatible water-soluble scaffold which actively transports the CORM against a concentration gradient into the cells. The uptake and cellular distribution of this B12-photoCORM conjugate is demonstrated via synchrotron FTIR spectromicroscopy measurements on living cells. Intracellular photoinduced CO release prevents fibroblasts from dying under conditions of hypoxia and metabolic depletion, conditions that may occur in vivo during insufficient blood supply to oxygen-sensitive tissues such as the heart or brain.

Epo and non-hematopoietic cells: what do we know?

The hematopoietic growth factor erythropoietin (Epo) circulates in plasma and controls the oxygen carrying capacity of the blood (Fisher. Exp Biol Med (Maywood) 228:1-14, 2003). Epo is produced primarily in the adult kidney and fetal liver and was originally believed to play a role restricted to stimulation of early erythroid precursor proliferation, inhibition of apoptosis, and differentiation of the erythroid lineage. Early studies showed that mice with targeted deletion of Epo or the Epo receptor (EpoR) show impaired erythropoiesis, lack mature erythrocytes, and die in utero around embryonic day 13.5 (Wu et al. Cell 83:59-67, 1995; Lin et al. Genes Dev. 10:154-164, 1996). These animals also exhibited heart defects, abnormal vascular development as well as increased apoptosis in the brain suggesting additional functions for Epo signaling in normal development of the central nervous system and heart. Now, in addition to its well-known role in erythropoiesis, a diverse array of cells have been identified that produce Epo and/or express the Epo-R including endothelial cells, smooth muscle cells, and cells of the central nervous system (Masuda et al. J Biol Chem. 269:19488-19493, 1994; Marti et al. Eur J Neurosci. 8:666-676, 1996; Bernaudin et al. J Cereb Blood Flow Metab. 19:643-651, 1999; Li et al. Neurochem Res. 32:2132-2141, 2007). Endogenously produced Epo and/or expression of the EpoR gives rise to autocrine and paracrine signaling in different organs particularly during hypoxia, toxicity, and injury conditions. Epo has been shown to regulate a variety of cell functions such as calcium flux (Korbel et al. J Comp Physiol B. 174:121-128, 2004) neurotransmitter synthesis and cell survival (Velly et al. Pharmacol Ther. 128:445-459, 2010; Vogel et al. Blood. 102:2278-2284, 2003). Furthermore Epo has neurotrophic effects (Grimm et al. Nat Med. 8:718-724, 2002; Junk et al. Proc Natl Acad Sci U S A. 99:10659-10664, 2002), can induce an angiogenic phenotype in cultured endothelial cells and is a potent angiogenic factor in vivo (Ribatti et al. Eur J Clin Invest. 33:891-896, 2003) and might enhance ventilation in hypoxic conditions (Soliz et al. J Physiol. 568:559-571, 2005; Soliz et al. J Physiol. 583, 329-336, 2007). Thus multiple functions have been identified breathing new life and exciting possibilities into what is really an old growth factor.This review will address the function of Epo in non-hematopoietic tissues with significant emphasis on the brain and heart.

17 e- rhenium dicarbonyl CO-releasing molecules on a cobalamin scaffold for biological application.

Cyanocobalamin (B(12)) offers a biocompatible scaffold for CO-releasing 17-electron dicarbonyl complexes based on the cis-trans-[Re(II)(CO)(2)Br(2)](0) core. A Co-C≡N-Re conjugate is produced in a short time and high yield from the reaction of [Et(4)N](2)[Re(II)Br(4)(CO)(2)] (ReCORM-1) with B(12). The B(12)-Re(II)(CO)(2) derivatives show a number of features which make them pharmaceutically acceptable CO-releasing molecules (CORMs). These cobalamin conjugates are characterized by an improved stability in aqueous aerobic media over the metal complex alone, and afford effective therapeutic protection against ischemia-reperfusion injury in cultured cardiomyocytes. The non-toxicity (at µM concentrations) of the resulting metal fragment after CO release is attributed to the oxidation of the metal and formation in solution of the ReO(4)(-) anion, which is among the least toxic of all of the rare inorganic compounds. Theoretical and experimental studies aimed at elucidating the aqueous chemistry of ReCORM-1 are also described.

CO releasing properties and cytoprotective effect of cis-trans-[Re(II)(CO)2Br2L2]n complexes.

The carbon monoxide (CO) releasing properties of a series of rhenium(II)-based complexes of general formula cis-trans-[Re(II)(CO)(2)Br(2)L(2)](n) and cis-trans-[Re(II)(CO)(2)Br(2)N[intersection]N] (where L = monodentate and N[intersection]N = bidentate ligand) are reported. Complexes evaluated in this study were obtained from direct ligand substitution reactions of the cis-[Re(II)(CO)(2)Br(4)](2-) synthon (2) recently described. (1) All molecules have been fully characterized. The solid-state structures of the cis-trans-[Re(II)(CO)(2)Br(2)L(2)] (with L = N-methylimidazole (3), benzimidazole (4) and 4-picoline (5)) and the cis-trans-[Re(II)(CO)(2)Br(2)N[intersection]N] (with N[intersection]N = 4,4'-dimethyl-2,2'-bipyridine (8) and 2,2'-dipyridylamine (11)) adducts are presented. The release of CO from the cis-trans-[Re(II)(CO)(2)Br(2)L(2)](n) complexes was assessed spectrophotometrically by measuring the conversion of deoxymyoglobin (Mb) to carbonmonoxy myoglobin (MbCO). Only compounds bearing monodentate ligands were found to liberate CO. The rate of CO release was found to be pH dependent with half-lives (t(1/2)) under physiological conditions (25 degrees C, 0.1 M phosphate buffer, and pH 7.4) varying from ca. 6-43 min. At lower pH values, the time required to fully saturate Mb with CO liberated from the metal complexes gradually decreased. Complex 2 and the cis-trans-[Re(II)(CO)(2)Br(2)(Im)(2)] adduct (with Im = imidazole (6)) show a protective action against "ischemia-reperfusion" stress of neonatal rat ventricular cardiomyocytes in culture.

Oxygen-induced Regulation of Na/K ATPase in cerebellar granule cells.

Adjustment of the Na/K ATPase activity to changes in oxygen availability is a matter of survival for neuronal cells. We have used freshly isolated rat cerebellar granule cells to study oxygen sensitivity of the Na/K ATPase function. Along with transport and hydrolytic activity of the enzyme we have monitored alterations in free radical production, cellular reduced glutathione, and ATP levels. Both active K(+) influx and ouabain-sensitive inorganic phosphate production were maximal within the physiological pO(2) range of 3-5 kPa. Transport and hydrolytic activity of the Na/K ATPase was equally suppressed under hypoxic and hyperoxic conditions. The ATPase response to changes in oxygenation was isoform specific and limited to the alpha1-containing isozyme whereas alpha2/3-containing isozymes were oxygen insensitive. Rapid activation of the enzyme within a narrow window of oxygen concentrations did not correlate with alterations in the cellular ATP content or substantial shifts in redox potential but was completely abolished when NO production by the cells was blocked by l-NAME. Taken together our observations suggest that NO and its derivatives are involved in maintenance of high Na/K ATPase activity under physiological conditions.

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.