Objectives and methods of iron chelation therapy.

Recent developments in the understanding of the molecular control of iron homeostasis provided novel insights into the mechanisms responsible for normal iron balance. However in chronic anemias associated with iron overload, such mechanisms are no longer sufficient to offer protection from iron toxicity, and iron chelating therapy is the only method available for preventing early death caused mainly by myocardial and hepatic damage. Today, long-term deferoxamine (DFO) therapy is an integral part of the management of thalassemia and other transfusion-dependent anemias, with a major impact on well-being and survival. However, the high cost and rigorous requirements of DFO therapy, and the significant toxicity of deferiprone underline the need for the continued development of new and improved orally effective iron chelators. Within recent years more than one thousand candidate compounds have been screened in animal models. The most outstanding of these compounds include deferiprone (L1); pyridoxal isonicotinoyl hydrazone (PIH) and; bishydroxy- phenyl thiazole. Deferiprone has been used extensively as a substitute for DFO in clinical trials involving hundreds of patients. However, L1 treatment alone fails to achieve a negative iron balance in a substantial proportion of subjects. Deferiprone is less effective than DFO and its potential hepatotoxicity is an issue of current controversy. A new orally effective iron chelator should not necessarily be regarded as one displacing the presently accepted and highly effective parenteral drug DFO. Rather, it could be employed to extend the scope of iron chelating strategies in a manner analogous with the combined use of medications in the management of other conditions such as hypertension or diabetes. Coadministration or alternating use of DFO and a suitable oral chelator may allow a decrease in dosage of both drugs and improve compliance by decreasing the demand on tedious parenteral drug administration. Combined use of DFO and L1 has already been shown to result in successful depletion of iron stores in patients previously failing to respond to single drug therapy, and to lead to improved compliance with treatment. It may also result in a "shuttle effect" between weak intracellular chelators and powerful extracellular chelators or exploit the entero-hepatic cycle to promote fecal iron excretion. All of these innovative ways of chelator usage are now awaiting evaluation in experimental models and in the clinical setting.

Pathophysiology of iron overload.

In thalassemia, iron overload is the joint outcome of excessive iron absorption and transfusional siderosis. While iron absorption is limited by a physiologic ceiling of about 3 mg/d, plasma iron turnover in thalassemia may be 10 to 15 times normal, caused by the wasteful, ineffective erythropoiesis of an enormously expanded erythroid marrow. This outpouring of catabolic iron exceeds the iron-binding capacity of transferrin and appears in plasma as non-transferrin-plasma iron (NTPI). The toxicity of NTPI is much higher than of transferrin-iron as judged by its ability to promote hydroxyl radical formation resulting in peroxidative damage to membrane lipids and proteins. In the heart, this results in impaired function of the mitochrondrial respiratory chain and abnormal energy metabolism manifested clinically in fatal hemosiderotic cardiomyopathy. Ascorbate increases the efficacy of iron chelators by expanding the intracellular chelatable iron pool, but, at suboptimal concentrations is a pro-oxidant, enhancing the catalytic effect of iron in free radical formation. NTPI is removed by i.v. DFO in a biphasic manner and reappears rapidly upon cessation of DFO, lending support to the continuous, rather than intermittent, use of chelators. Unlike DFO and other hexadentate chelators, bidentate chelators such as L1 may produce incomplete intermediate iron complexes at suboptimal drug concentrations.

Fluorescence analysis of the labile iron pool of mammalian cells.

The labile iron pool (LIP) of cells constitutes a cytosolic fraction of iron which is accessible to permeant chelators and contains the cells' metabolically and catalytically reactive iron. LIP is maintained by a balanced movement of iron from extra- and intracellular sources. We describe here an approach for tracing LIP levels in living cells based on the fluorescent probe calcein (CA). This probe binds Fe(II) rapidly, stoichiometrically, and reversibly while forming fluorescence-quenched CA-Fe complexes. Cells are loaded with CA via its acetomethoxy precursor CA-AM, attaining 1-10 microM intracellular concentrations and retaining full viability. LIP is defined here operationally as the sum of "free" and CA-bound iron of the cell. The method for assessing LIP is based on the measurement of: (a) the total intracellular concentration of CA in CA-loaded cells ([CA]1), which is estimated from fluorimetric measurements of CA in a given suspension of cells, the number of cells, and the cell volume; (b) the intracellular [CA-Fe], the concentration of [CA] bound to metals (> 95% iron), which is assessed from the relative rise in fluorescence (delta F) elicited by addition of highly permeant and high-affinity binding chelators such as salicyladehyde-isonicotinoyl-hydrazone (SIH) and the value of [CA]1; and (c) the "free" cell iron concentration [Fe(II)], which is computed from the experimentally determined values of CA-Fe(II)'s dissociation constant (Kd) in various cell lines grown in suspension (Kd = 0.22 +/- 0.01 microM). The value of cellular LIP is defined as the sum of [CA-Fe] and [Fe]. It is derived from the experimental determination of [CA]1 and [CA-Fe] and from calculation of [Fe] by application of the mass law equation using the Kd value of [CA-Fe]. The estimated values of LIP for resting erythroid and myeloid cells are in the range of 0.2-1.5 microM. The values varied commensurately with cell iron loads and iron chelator treatment. The method provides a simple, noninvasive tool for on-line monitoring of cytosolic iron under normal and abnormal conditions of cell iron supply and for assessing the dynamics of intracellular iron in living cells.

Transport of iron and other transition metals into cells as revealed by a fluorescent probe.

Transport of nontransferrin-bound iron into cells is thought to be mediated by a facilitated mechanism involving either the trivalent form Fe(III) or the divalent form Fe(II) following reduction of Fe(III) at the cell surface. We have made use of the probe calcein, whose fluorescence is rapidly and stoichiometrically quenched by divalent metals such as Fe(II), Cu(II), Co(II), and Ni(II) and is minimally affected by variations in ionic strength, Ca(II) and Mg(II). Addition of Fe(II) salts to calcein-loaded human erythroleukemia K-562 cells elicited a slow quenching response that was markedly accelerated by the ionophore A-23187 and was reversed by membrane-permeant but not by impermeant chelators. These observations were confirmed by fluorescence imaging of cells. Other divalent metals such as Co(II), Ni(II), and Mn(II) permeated into cells at roughly similar rates, and their uptake, like that of Fe(II), was blocked by trifluoperazine, bepridil, and impermeant sulfhydryl-reactive organomercurials, indicating the operation of a common transport mechanism. This method could provide a versatile tool for studying the transport of iron and other transition metals into cells.

Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells.

The epithelial cell line HT-29, which constitutively expresses the cystic fibrosis transmembrane conductance regulator (CFTR), was induced to become drug resistant by cultivation in the presence of colchicine. The gradual acquisition of drug resistance was associated with a corresponding increase in the expression of the multidrug resistance P-glycoprotein (P-gp) and a marked (> 80%) decrease in the constitutive levels of CFTR protein, as determined by immunoblotting. The reduction in CFTR content occurred at the onset of acquisition of drug resistance when P-gp expression was still relatively low. Reversal of drug resistance by removal of colchicine from the culture medium led to a 70% decrease in P-gp levels and a concomitant 40% increase in CFTR. The levels of other membrane proteins such as Na(+)-K(+)-ATPase and alkaline phosphatase remained relatively constant (< 26% variation). We propose that a selective downregulation of CFTR is elicited by acquisition of the multidrug resistance (MDR) phenotype and that induction of P-gp expression leads to a reversible repression of CFTR biosynthesis. These findings provide an experimental foundation for the complementary patterns of expression of the CFTR and MDR1 genes observed in vivo.

Protein kinase C mediates down-regulation of cystic fibrosis transmembrane conductance regulator levels in epithelial cells.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells is known to be down-regulated by the action of phorbol myristate acetate (PMA). We show here that in addition to suppressing the rate of transcription of the CFTR gene, PMA treatment stimulates degradation of the CFTR protein. HT-29 colon epithelial cells and the CFTR-transfected pancreatic cells PLJ-4.7 lost 55-80% of their CFTR protein after 3-6 h of treatment with 100 nM PMA, as analyzed by quantitative Western blotting. In contrast to PMA, actinomycin D and cycloheximide reduced the CFTR protein content by 19 and 9% in HT-29 cells and by 22 and 40% in PLJ-4.7 cells, respectively, while inhibiting total cellular RNA and protein synthesis by over 80%. The PMA-induced loss of CFTR was partially reversed by the protein kinase C inhibitor GF109203X. The PMA-induced degradation of CFTR may represent a regulatory pathway for terminating CFTR-mediated chloride and mucin secretion.

Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl- channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 mumol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells.

Pyridoxal phosphate is a potent probe for exploring the "sidedness" of proteins in the membrane of the intact red blood cell. It reacts with amino groups with a high degree of specificity, forming a Schiff's base that can be fixed as an irreversible bond upon reduction with NaBH4; its binding site can be identified by use of [3-H]pyridoxal phosphate or NaB3-H4; it can be used as a surface probe under conditions of minimal penetration, or it can be used as a probe for proteins on the inside of the membrane under conditions of substantial uptake. Pyridoxal phosphate uptake involves a rapid and a slow component. The former represents the binding to the outer surface of the membrane; it is not substantially affected by pH and temperature, but is reduced considerably by pretreatment of cells by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid, a specific inhibitor of anion transport. The slow component represents penetration into the cell; it is blocked by high pH, low temperature, or pretreatment with the disulfonic stilbene. Pyridoxal phosphate itself is also an effective and specific inhibitor of the permeation of other anions. Under conditions of minimal uptake, the only labeled proteins are three glycoproteins and a protein of apparent molecular weight 95,000. Under conditions of substantial uptake into the cell, the other major protein bands seen by staining on acrylamide gels after electrophoresis are labeled. It is concluded that virtually all of the major membrane proteins interact with pyridoxal phosphate from one side of the membrane or the other. The differences in labeling under conditions of minimal or maximal uptake can, therefore, be attributed to the sidedness in the distribution of the membrane proteins rather than to differences in their reactivity.