Beatrice M. Anner

Inhibition of HIV-1 infection by zinc group metal compounds

Thirty-seven metal compounds were examined for inhibitory activities against infection with human immunodeficiency virus type 1 (HIV-1). Zinc group metal compounds, namely, zinc acetate, zinc chloride, zinc nitrate, cadmium acetate and mercury chloride, showed anti-HIV-1 activities. Cadmium and mercury compounds at 1-10 microg/ml and zinc compounds at 100 microg/ml strongly inhibited HIV-1 infection, although the cadmium, mercury and zinc compounds had severe cytotoxities at 100, 100 and 1000 microg/ml, respectively. They inhibited transcription of HIV-1 RNA and HIV-1 production at concentrations at which they did not affect the growth of HIV-1-producing cells. They had little effect on syncytium formation resulting from cocultivation of uninfected with HIV-1-producing cells. Nor did they affect HIV-1 DNA synthesis following HIV-1 infection. The metal compounds may owe their anti-HIV-1 effects to inhibition of HIV-1 DNA to RNA transcription, rather than inhibition of the adsorption, penetration or reverse transcription step of HIV-1 infection.

Potent and reversible interaction of silver with pure Na,K-ATPase and Na,K-ATPase-liposomes

The Na,K-ATPase (EC 3.6.1.37) is the receptor for cardioactive steroids, the only specific inhibitors known at the present time for this unique membrane bound transport system. We report here that silver is the most rapid and potent inhibitor of isolated Na,K-ATPase ever described. Inhibition of Na,K-ATPase activity by silver is immediate and strikingly distinct from other inhibitors: addition of 1 mM of cysteine or DMPS reactivates the silver blocked-enzyme immediately. The results reveal that silver interacts with Na,K-ATPase and inhibits differently by an on-off mechanism involving most likely a few critical sulfhydryl groups. Inhibition of Na-K transport by silver has been demonstrated also in an artificial membrane, e.g., in liposomes reconstituted with pure Na,K-ATPase performing active transport. Silver inhibits the active 86Rb transport mediated by the pure Na,K-ATPase molecule. The Na,K-ATPase contained in the liposomes was labeled specifically with 110mAg and appeared to bind two silver ions. Taken together, the results show that the mechanism of silver interaction with Na,K-ATPase might be different from other metals, for instance, mercury. The unique action mechanism of silver suggests a fundamental role of a few critical sulfhydryl groups for Na,K-transport.

Cysteine protects Na,K-ATPase and isolated human lymphocytes from silver toxicity

Metal-binding proteins are important components of retroviruses such as human immunodeficiency virus (HIV). Therefore, metals could be used as antiviral agents. However, most metals are toxic for humans with the exception of silver which is toxic only to prokaryotic cells and viruses. In addition, HIV infection causes a decrease in body cysteine. We formed a complex of silver and cysteine, named silver-cysteine. Healthy human lymphocytes were incubated with silver-nitrate or silver-cysteine. Negligible cell survival was seen at 50 microM silver-nitrate. However, in presence of 1 mM cysteine, the viability remained unaffected up to 1 mM of silver. Further, silver inhibition of isolated Na,K-ATPase was easily reversed by cysteine. Thus, non-toxic silver-cysteine could be used as an anti-viral and cysteine-replenishing agent.

Role of cell membrane Na,K-ATPase for survival of human lymphocytes in vitro

Lymphocytes are primordial immune cells with variable life times. Besides genetic programming, extracellular factors interacting with cell surface receptors might alter cell survival. We investigated whether the activity of the membrane-embedded Na,K-ATPase (EC 3.6.1.37) or sodium pump (NKA) plays a role for cell survival since this ubiquitous system establishes the vital transmembrane Na and K gradients as well as the resulting high intracellular K/Na ratio required for macromolecule synthesis; furthermore, the system exposes an extracellular inhibitory receptors for cardioactive steroids and palytoxin. Isolated human lymphocytes were incubated in vitro and their viability assessed by exclusion of trypan blue. Various incubation conditions were compared; in RPMI-1640 medium cell viability was preserved for 30 h at 37 °C. Externally added ouabain, a hydrophilic cardioactive steroid, blocked the [86Rb]potassium uptake at nanomolar concentrations. Despite pump inhibition ouabain did not alter lymphocyte survival, even at 10 mM for 30 h. By contrast, the hydrophilic toxin palytoxin, the most potent animal poison described so far, killed all cells within 2 h at 10 nM; this toxin is known to act via the sodium pump and to provoke deadly cation-leaks by unmasking a channel component. Intracellular Na increased and K decreased as measured by atomic absorption spectrometry in presence of palytoxin; cell swelling was seen by electron microscopy. Ouabain protected the cells from the toxic effect of palytoxin. The results reveal a pivotal role of NKA integrity for lymphocyte survival.

Preparation of Na,K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na,K-transport capacity to the ATPase activity

A microprocedure for the preparation of Na,K-ATPase-containing liposomes with a minimal starting material (200 microgram) of purified Na,K-ATPase is presented. Phosphatidylcholine is added gradually to cholate-solubilized Na,K-ATPase of various concentrations and the lipid-induced decrease in enzyme activity is monitored. After removal of the detergent by dialysis, the transport parameters of the resulting Na,K-ATPase-liposomes are established by a microassay. By relating the transport properties to the Na,K-ATPase activity preset before dialysis, a procedure is developed which allows to prepare standardized Na,K-ATPase-liposomes with predictable transport properties.

Chelation of mercury by ouabain-sensitive and ouabain-resistant renal Na,K-ATPase

The SH-reactive HgCl2 inhibits the Na,K-ATPase activity potently in a manner antagonized only partially by EDTA or cysteine; solely dimercaprol, a dithiol antidote for mercury, blocks the HgCl2 effects entirely as confirmed also by 203Hg-binding experiments. The results reveal the presence of a chelating component in pure Na,K-ATPase with an affinity for mercury superior to EDTA. The mercury-sensitivity of the Na,K-ATPase is not related to the ouabain-sensitivity. This criterion will be useful for the distinction between ouabain-like and mercury-like inhibitors from body fluids and tissues.

On the kinetics of the na:k exchange in the initial and final phase of sodium pump activity in liposomes

Abstract Purified Na,K-ATPase is incorporated into phosphatidylcholine-liposomes. The reconstituted, inside-out sodium pump activity is divided into an initial phase which follows the addition of ATP, and into a final phase where the internal K ions have been replaced by Na ions, and no net cation flux occurs. The kinetics of the Na:K exchange in the initial transport phase seems to be transiently influenced by the Na:K concentration ratio in the solution. Depending on the Na and K concentrations present, the coupling ratio varies from 0.8Na:1K to 1.8Na:1K. The uneven coupling ratio is a transient phenomenon because it soon approaches a ratio close to 1Na:1K. Indeed, the pump exchanges one internal K ion for i external Na ion until the internal K-pool is exhausted and the final pump phase is reached. Thus, the kinetics of the ATP-induced Na:K exchange in Na,K-ATPaseliposomes appears to be regulated by the initial Na:K concentration ratio, as well as by the limiting cation pool.

Interaction of hypothalamic Na,K-ATPase inhibitor with isolated human peripheral blood mononuclear cells.

A ligand for the digitalis receptor located on the membrane-embedded Na,K-ATPase (NKA; EC 3.6.1.37) has been isolated from bovine hypothalamus (hypothalamic inhibitory factor; HIF) and identified as isomeric ouabain (Tymiaket al, 1993,Proc. Natl. Acad. Sci.90: 8189–8193). In analogy to cardioactive steroids (CS) derived from plants or from toad, HIF inhibits the Na/K-exchange process and the ATPase activity of isolated Na,K-ATPase although by a different molecular action mechanism. In the present work we show that, as plant-derived ouabain, HIF inhibits86Rb-uptake by isolated human lymphocytes with an IC50 of about 20 nM; above this concentration HIF reduces cell viability in contrast to ouabain. The decrease in cell viability by excess HIF is accompanied by discrete morphological alterations (mitochondrial swelling) visible by transmission electron microscopy of ultra-thin sectioned peripheral blood mononuclear cells. Taken together the results show that the hypothalamic NKA inhibitor blocks NKA of isolated human lymphocytes with high potency at nanomolar concentrations without toxicity; concentrations exceeding the ones required to block86Rb-uptake reduce cell viability, probably due to leak formation across the NKA molecule. Thus, lymphocytes constitute a potential target for HIF action and by their altered NKA status a possible messenger between the nervous and the immune system.

Na,K-ATPase characterized in artificial membranes. 1. Predominant conformations and ion-fluxes associated with active and inhibited states

The Na,K-ATPase (NKA) system is the receptor for the cardioactive steroids of plant or animal origin. It is not yet known whether passive ion fluxes traverse the inactivated receptor and thereby contribute to the hormonal, pharmacological or toxic actions of these compounds. To look for putative passive ion-fluxes across the ouabain-NKA complex, we incorporated it into the artificial membrane of liposomes. Since this synthetic membrane is virtually impermeable to Na and K ions, the hypothetical ion-fluxes mediated by the NKA can be determined. E2-forms and E2-ouabain-forms of purified NKA were incorporated, in parallel, into separate liposome preparations and the permeability of the resulting E2-liposomes and E2-ouabain-liposomes to K, Na and Ca ions was compared. The E2-liposomes expressed a typical K-permeability which was not observed in the E2-ouabain-liposomes; the latter showed a slightly higher Na-permeability and a similar Ca-permeability as compared to the former. Thus, ouabain does not induce leaks for K or Ca ions in the NKA molecule.

Characterisation of tissue-specific oligosaccharides from rat brain and kidney membrane preparations enriched in Na+,K+-ATPase.

The organ-specific nature of the glycosylation of Na+,K+-ATPase-enriched preparations from kidney and brain tissues has earlier been indicated by the use of lectin-staining techniques. Na+,K+-ATPase is ubiquitous and abundant, and subject to upregulation during cell-division and in certain pathological conditions. Lectins specific for the different carbohydrates displayed by the Na+,K+-ATPases may, therefore, be useful carriers/mediators in tissue-specific targeting. N-linked oligosaccharides purified from Na+,K+-ATPase-enriched preparations from rat brain and kidney were consequently characterised in detail in this study using weak anion exchange and normal phase HPLC (combined with serial glycosidase digestions) and matrix-assisted laser desorption/ionisation mass spectrometry. The oligomannose series of glycans were most abundant in the brain tissue preparation and this contrasted with the renal-associated oligosaccharides that were dominated by families of tetra-antennary glycans (with/without a core fucose) with up to four lactosaminylglycan residues in either branched or linear formation.