The iron chelators desferrioxamine and 1-alkyl-2-methyl-3-hydroxypyrid-4-ones inhibit vascular prostacyclin synthesis in vitro.

The iron chelators desferrioxamine (DFO), 1,2-dimethyl(L1)-, 1-ethyl-2-methyl(L1NEt)- and 1-propyl-2-methyl(L1NPr)-3-hydroxypyrid-4-ones inhibited rat aortic prostacyclin (PGI2) synthesis in vitro (rank order of potency: DFO greater than L1 greater than L1NEt greater than L1NPr) when stimulated with adrenaline, arachidonate and the Ca2+ ionophore A23187. The inhibitory action of the chelators was blocked by Fe3+ and Al3+ and reversed by washing and H2O2, but not by ascorbate. These data suggest that iron chelators inhibit prostanoid synthesis in intact tissue through the removal or binding of Fe3+ linked to cyclo-oxygenase. These iron chelators may be of therapeutic value in the treatment of inflammatory and other diseases via two mechanisms: (1) the inhibition of pro-inflammatory prostanoid synthesis and (2) the inhibition of toxic-free-radical generation by cyclo-oxygenase.     

Ferrous ion autoxidation and its chelation in iron-loaded human liver HepG2 cells.

Ferrous ion (Fe(2+)) is long thought to be the most likely active species, producing oxidants through interaction of Fe(2+) with oxygen (O(2)). Because current iron overload therapy uses only Fe(3+) chelators, such as desferrioxamine (DFO), we have tested a hypothesis that addition of a Fe(2+) chelator, 2,2'-dipyridyl (DP), may be more efficient and effective in preventing iron-induced oxidative damage in human liver HepG2 cells than DFO alone. Using ferrozine as an assay for iron measurement, levels of cellular iron in HepG2 cells treated with iron compounds correlated well with the extent of lipid peroxidation (r = 0.99 after log transformation). DP or DFO alone decreased levels of iron and lipid peroxidation in cells treated with iron. DFO + DP together had the most significant effect in preventing cells from lipid peroxidation but not as effective in decreasing overall iron levels in the cells. Using ESR spin trapping technique, we further tested factors that can affect oxidant-producing activity of Fe(2+) with dissolved O(2) in a cell-free system. Oxidant formation enhanced with increasing Fe(2+) concentrations and reached a maximum at 5 mM of Fe(2+). When the concentration of Fe(2+) was increased to 50 mM, the oxidant-producing activity of Fe(2+) sharply decreased to zero. The initial ratio of Fe(3+):Fe(2+) did not affect the oxidant producing activity of Fe(2+). However, an acidic pH (< 3.5) significantly slowed down the rate of the reaction. Our results suggest that reaction of Fe(2+) with O(2) is an important one for oxidant formation in biological system, and therefore, drugs capable of inhibiting redox activity of Fe(2+) should be considered in combination with a Fe(3+) chelator for iron overload chelation therapy.     

Effect of desferrioxamine, rhodotorulic acid and cholylhydroxamic acid on transferrin and iron exchange with hepatocytes in culture

The mechanism of action of the hydroxamate iron chelators desferrioxamine (DFO), rhodotorulic acid (RHA) and cholylhydroxamic acid (CHA) was studied using rat hepatocytes in culture. Each chelator affected both the uptake and, to a much smaller extent, the release of transferrin-125I-59Fe from the cells. All chelators reduced the 59Fe uptake and incorporation into ferritin in a concentration-dependent manner. Uptake of 59Fe into the membrane (stromal-mitochondrial) fraction was also decreased by DFO and RHA but increased by CHA. Transferrin-125I binding was reduced slightly by DFO and RHA and increased by CHA. All chelators released 59Fe transferrin-125I from hepatocytes prelabelled by incubation with rat transferrin-125I-59Fe and washed before reincubation in the presence of the chelators. DFO decreased membrane 59Fe but had little effect on ferritin-59Fe. RHA decreased 59Fe in both membrane and ferritin fractions. CHA decreased hepatocyte-59Fe but increased 59Fe in the hepatocyte membrane fraction. Higher concentrations of the chelators had little further effect on 59Fe release but promoted transferrin-125I release from hepatocytes. All chelators appeared to act on kinetically important iron pools of limited size and hence are likely to be most effective when given by continuous infusion rather than bolus injection.     

Multidentate pyridinones inhibit the metabolism of nontransferrin-bound iron by hepatocytes and hepatoma cells.

The therapeutic effect of iron (Fe) chelators on the potentially toxic plasma pool of nontransferrin-bound iron (NTBI), often present in Fe overload diseases and in some cancer patients during chemotherapy, is of considerable interest. In the present investigation, several multidentate pyridinones were synthesized and compared with their bidentate analogue, deferiprone (DFP; L1, orally active) and desferrioxamine (DFO; hexadentate; orally inactive) for their effect on the metabolism of NTBI in the rat hepatocyte and a hepatoma cell line (McArdle 7777, Q7). Hepatoma cells took up much less NTBI than the hepatocytes (< 10%). All the chelators inhibited NTBI uptake (80-98%) much more than they increased mobilization of Fe from cells prelabelled with NTBI (5-20%). The hexadentate pyridinone, N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine showed comparable activity to DFO and DFP. There was no apparent correlation between Fe status, Fe uptake and chelator activity in hepatocytes, suggesting that NTBI transport is not regulated by cellular Fe levels. The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as at the cell membrane. In conclusion (a) rat hepatocytes have a much greater capacity to take up NTBI than the rat hepatoma cell line (Q7), (b) all chelators bind NTBI much more effectively during the uptake phase than in the mobilization of Fe which has been stored from NTBI and (c) while DFP is the most active chelator, other multidentate pyridinones have potential in the treatment of Fe overload, particularly at lower, more readily clinically available concentrations, and during cancer chemotherapy, by removing plasma NTBI.     

Antioxidant and free radical scavenging activities of the iron chelators pyoverdin and hydroxypyrid-4-ones in iron-loaded hepatocyte cultures: comparison of their mechanism of protection with that of desferrioxamine.

The protective effect on iron-supplemented hepatocyte cultures of three iron chelators, pyoverdin Pa and hydroxypyrid-4-one derivatives CP20 and CP22, was compared to that of the widely known desferrioxamine B (Desferal:DFO), on the basis of two criteria: (a) their effectiveness in inhibiting free malondialdehyde (MDA) production as an index of iron-induced lipid peroxidation; and (b) their ability to reduce intracellular enzyme leakage. In view of these two markers of iron toxicity, the protective effect of these chelators was classified as follows: DFO > CP20 > or = CP22 > Pa. The mechanism of cellular protection was elucidated by investigating both the iron-chelating activity and the free radical scavenging property of these agents. As concerns the iron chelation, DFO and Pa exerted the same rank order as for cytoprotection (DFO > Pa). The free radical scavenging property toward hydroxyl radical .OH and peroxyl radical ROO. was investigated in a cell-free experimental model. The two siderophores, DFO and Pa, appeared to have a lower antiradical activity toward .OH than hydroxypyrid-4-one CP22. This .OH scavenging activity was classified as follows: CP22 > Pa > DFO. Moreover, the chelators exhibited for the quenching of ROO. the same order of effectiveness as that observed for cellular protection: DFO > CP20 > or = CP22 > Pa. These data indicate that, in addition to the iron-chelating activity which represents the most important property for determining the protection capacity of these iron chelators, their free radical scavenging ability also must be taken into account. This direct demonstration of a strong association between the free radical scavenging activity and the protective effect of iron chelators further increases the prospects for the development and clinical applications of new oral chelating drugs.     

In vitro antimalarial activity of ICL670: a further proof of the correlation between inhibition of β-hematin formation and of peroxidative degradation of hemin

Iron chelators such as deferiprone, deferoxamine (DFO) and ICL670 (deferasirox) have previously been shown to display in vitro and/or in vivo antimalarial activities. To gain further insight in their antimalarial mechanism of action, their activities on inhibition of β-hematin formation and on both peroxidative and glutathione (GSH)-mediated degradation of hemin were investigated. Neither deferiprone nor DFO were able to inhibit β-hematin formation while ICL670 activity nearly matched that of chloroquine (CQ). Peroxidative degradation of hemin was also only strongly inhibited by both CQ and ICL670, the latter being significantly more efficient at pH 5.2. All iron chelators displayed minor, if any, inhibitory activity on GSH-mediated degradation of hemin. Discrepancies in the results obtained for the three iron chelators show that iron chelation is not the main driving force behind interference with heme degradation. Deferiprone, DFO and ICL670 share little structural community but both ICL670 and antimalarial ursolic acid derivatives (previously shown to block β-hematin formation and the peroxidative degradation of hemin) have hydrophobic groups and hydroxyphenyl moieties. These similarities in structures and activities further back up a possible two-step mechanism of action previously proposed for ursolic acid derivatives (Mullié et al., 2010) implying (1) stacking of an hydrophobic structure to hemin and (2) additive protection of hemin ferric iron from H₂O₂ by hydroxyphenyl groups through steric hindrance and/or trapping of oxygen reactive species in the direct neighborhood of ferric iron. These peculiar antimalarial mechanisms of action for ICL670 warrant further investigations and development.     

Characterization of MbrC involved in bacitracin resistance in Streptococcus mutans

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease.     

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.     

RAT BRAIN STEM TRYPTOPHAN HYDROXYLASE: MECHANISM OF ACTIVATION BY CALCIUM

Abstract— The activity of soluble tryptophan hydroxylase from rat brain stem was increased in presence of mm concentrations of calcium. Similarly to that observed by treating the enzyme with sodium dodecyl sulphate or trypsin, this activation resulted mainly from an increased affinity of tryptophan hydroxylase for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). In addition, the optimal pH for the enzymic activity was shifted from 7.6 to 7.9 following activation by calcium, sodium dodecyl sulphate or trypsin.
Under the assay conditions used for measuring tryptophan hydroxylase activity, calcium also stimulated a neutral proteinase. This latter enzyme could be eliminated from the solution of tryptophan hydroxylase by filtration through Sephadex G 200. The resulting partially purified tryptophan hydroxylase could be activated by calcium only when the neutral proteinase was included in the assay mixture. In support of this conclusion, the effect of calcium on tryptophan hydroxylase was very small in the new born rat when the activity of the neutral proteinase was low. In addition, the activating effect of Ca²⁺ could be antagonized not only by a chelating agent like EGTA but also (partially) by specific inhibitors of proteinases such as benzethonium and PMSF.
These results strongly suggest that the activation of tryptophan hydroxylase by calcium is the consequence of a partial proteolysis of the enzyme by the calcium-dependent neutral proteinase. Therefore, the physiological significance of this irreversible effect is doubtful.     

Molecular forms of aconitase and their interconversions.

Aconitase, as isolated from mammalian mitochondria by traditional methods, is virtually inactive and contains an oxidized [3Fe-4S]+ cluster. The activation of the enzyme and attendant conformational change have been studied by monitoring the changes in activity, in tryptophan fluorescence, and in the electron paramagnetic resonance of the cluster on incubation with dithionite, with and without added Fe2+. Restoration of the full activity is achieved with one electron per 3Fe cluster and at least 0.6 g-atoms of Fe2+ per mol. The process involves building up of [4Fe-4S]2+ clusters. Other metal ions do not substitute for Fe2+. Reduction alone, in the absence of added Fe2+, yields up to 70% of the maximum activity, but requires approx. 1.8 electrons of reductant per cluster. The results presented are consistent with the view that activation without added Fe2+ involves the destruction of some of the [3Fe-4S] clusters and the incorporation of the Fe so liberated into other clusters to yield a tetra-nuclear one. In particular, the effect of EDTA and of other iron chelators in inhibiting activation by dithionite alone is in accord with this view, although recent magnetic-circular-dichroism studies do not support this interpretation. The rates of increase in activity and tryptophan fluorescence are the same when Fe2+ is present, but in its absence, activation is very much slower than the increase in fluorescence, suggesting that the protein conformational change triggered by reduction of the Fe-S clusters precedes the insertion of the iron. Consistent with this view is the observation that iron chelators inhibit activation by dithionite, but not the increase in fluorescence and, hence, the conformational change. The results are discussed in light of data in the literature on the forms of the cluster and its possible function in catalysis.     

Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.     

Regulation of tryptophan hydroxylase activity in Xenopus laevis photoreceptor cells by cyclic AMP

The aim of this study was to investigate the role of cyclic AMP in the regulation of tryptophan hydroxylase activity localized in retinal photoreceptor cells of Xenopus laevis, where the enzyme plays a key role in circadian melatonin biosynthesis. In photoreceptor-enriched retinas that lack serotonergic neurons, tryptophan hydroxylase activity is markedly stimulated by treatments that increase intracellular levels of cyclic AMP or activate cyclic AMP-dependent protein kinase, including forskolin, phosphodiesterase inhibitors, and cyclic AMP analogues. In contrast, cyclic AMP has no effect on tryptophan hydroxylase mRNA abundance. Experiments using cycloheximide and actinomycin D demonstrate that cyclic AMP exerts its regulatory effect via posttranslational mechanisms mediated by cyclic AMP-dependent protein kinase. The effect of cyclic AMP is independent of the phase of the photoperiod, suggesting that the nucleotide is not a mediator of the circadian rhythm of tryptophan hydroxylase. Cyclic AMP accumulation is higher in darkness than in light, as is tryptophan hydroxylase activity. Furthermore, the stimulatory effect of forskolin and that of darkness are inhibited by H89, an inhibitor of cyclic AMP-dependent protein kinase. In conclusion, cyclic AMP may mediate the acute effects of light and darkness on tryptophan hydroxylase activity of retinal photoreceptor cells.     

Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe(3+)-chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe(3+) complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe(3+) complexes, and toxicity of the chelators increased with oxygen tension. Fe(3+) complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)(2) did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)(2) in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe(3+) complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.     

Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation

Iron is an important contributor to reoxygenation injury because of its ability to promote hydroxyl radical formation. In previous in vivo studies, we demonstrated that iron chelators that underwent glomerular filtration provided significant protection against postischemic renal injury. An in vitro system was employed to further characterize the protection provided by extracellular iron chelators. Primary cultures of rat proximal tubular epithelial cells were subjected to 60 min hypoxia and 30 min reoxygenation (H/R). During H/R, there was a 67% increase in ferrozine-detectable iron in cell homogenates and increased release of iron into the extracellular space. Cells pretreated with either deferoxamine (DFO) or hydroxyethyl starch-conjugated deferoxamine (HES-DFO), an iron chelator predicted to be confined to the extracellular space, were greatly protected against lethal cell injury. To further localize the site of action of DFO and HES-DFO, tracer quantities of 59Fe were added to DFO or HES-DFO, and their distribution after 2 h was quantitated. Less than 0.1% of DFO entered the cells, whereas essentially none of the HES-DFO was cell-associated. These findings suggest that iron was released during hypoxia/reoxygenation and caused lethal cell injury. Iron chelators confined to the extracellular space provided substantial protection against injury.     

Effect of extracellular hemin on hemoglobin and ferritin content of erythroleukemia cells

Mouse (MEL) and human (K-562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme-linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K-562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe-hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 X 10(-14) g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron-saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K-562 cells were induced by 57Fe-hemin in the presence of 56Fe-transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe-hemin in the presence of 57Fe-transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K-562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra- or intracellular iron sources such as transferrin or ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.     

In Vitro Reactivation of Rat Cortical Tryptophan Hydroxylase Following In Vivo Inactivation by Methylenedioxymethamphetamine

The activity of tryptophan hydroxylase (EC 1.14.16.4) from rat brain was significantly decreased 1 h following a single systemic injection of 3,4-methylenedioxymethamphetamine (MDMA) when assessed ex vivo by radioenzymatic assay or in vivo by the quantitation of 5-hydroxytryptophan accumulation following central L-aromatic amino acid decarboxylase inhibition. Recovery of enzymatic activity in vivo, which occurred within 24 h of low-dose MDMA treatment, appeared not to involve synthesis of new enzyme protein, because the return of enzymatic activity was not prevented by prior cycloheximide. Acutely MDMA-depressed cortical tryptophan hydroxylase activity could be completely restored in vitro by a prolonged (20-24 h) anaerobic incubation in the presence of dithiothreitol and Fe2+ at 25 degrees C; partial reconstitution occurred when 2-mercapto-ethanol was substituted for dithiothreitol. Cortical tryptophan hydroxylase acutely inactivated by methamphetamine or p-chloroamphetamine could be similarly reactivated. MDMA-inactivated cortical tryptophan hydroxylase derived from rats killed later than 3 days after drug treatment could not be significantly reactivated under the conditions described above, indicating the development of irreversible enzymatic damage. Kinetic analysis of enzyme reactivation revealed an approximate doubling of enzyme Vmax with no change in enzyme affinity for either substrate, tryptophan, or pterin cofactor. These studies suggest that MDMA and its congeners inactivate central tryptophan hydroxylase by inducing oxidation of key enzyme sulfhydryl groups. The reactivation capacity of drug-inactivated enzyme at various times after MDMA treatment may provide a means of assessing the development of MDMA-induced neurotoxicity.     

Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX

Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin. Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.