Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC(50) at 1 and 2.5microM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC(50)=3.8microM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.     

Plasticity of the ecdysone receptor DNA binding domain

Ecdysteroids coordinate molting and metamorphosis in insects via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (Usp) protein. Here we show how the DNA-recognition alpha-helix and the T box region of the EcR DNA-binding domain (EcRDBD) contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. The data indicate a remarkable mutational tolerance with respect to the DNA-binding function of the EcRDBD. This is particularly manifested in the heterocomplexes formed between the EcRDBD mutants and the wild-type Usp DNA-binding domain (UspDBD). Circular dichroism (CD) spectra and protein unfolding experiments indicate that, in contrast to the UspDBD, the EcRDBD is characterized by a lower alpha-helix content and a lower stability. As such, the EcRDBD appears to be an intrinsically unstructured protein-like molecule with a high degree of intramolecular plasticity. Because recently published crystal structures indicate that the ligand binding domain of the EcR is also characterized by the extreme adaptability, we suggest that plasticity of the EcR domains may be a key factor that allows a single EcR molecule to mediate diverse biological effects.     

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.     

Inhibition of brain glutamate decarboxylase by 2-keto-4-pentenoic acid, a metabolite of allylglycine.

Abstract— 2-Keto-4-pentenoic acid, a potent inhibitor of brain glutamate decarboxylase (Orlowski et al., 1977) was prepared by oxidative deamination of l -allylglycine with snake venom l -amino acid oxidase. In the presence of glutamate the keto acid is a competitive inhibitor of the enzyme with respect to glutamate; its K_i is 2.4 ± 10^?6m . After preincubation of brain glutamate decarboxylase with 2-keto-4-pentenoic acid in the absence of glutamate, a slow and incomplete reactivation is obtained by prolonged dialysis, Sephadex gel-filtration, and dilution, suggesting the formation of a slowly dissociating enzyme-inhibitor complex and partial inactivation of the enzyme. In vivo inhibition of brain glutamate decarboxylase after administration of allylglycine is maximal after 2-8 h with activity returning to normal after 16 h. The inhibition of the enzyme after administration of d -allylglycine was greatest in the cerebellum and the medulla-pons area, the sites of the highest activity of d -amino acid oxidase. These results are interpreted as strongly supporting the postulate that allylglycine-induced inhibition of brain glutamate decarboxylase is due to the in vivo formation of 2-keto-4-pentenoic acid.     

Changing pattern of cyclic AMP-binding proteins during germination of Mucor racemosus sporangiospores.

Interation of cyclic AMP with a profoundly changing pattern of specific binding proteins was shown during aerobic germination of sporangiospores from the fungus Mucor racemosus. 32P-labeled 8-azido-cycli AMP, an analogue of cyclic AMP that forms a covalent linkage with the binding proteins under u.v. light, was used as the ligand. Binding proteins carrying this photoaffinity label were separated by polyacrylamide-gel electrophoresis and identified by radioautography. Equibiltrium dissociation constants (Kd) and binding-response curves in the presence of competing nucleotides were identical for both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP. A quantitative binding assay with both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP over the time course of sporangiospore germination indicated a parallel relationship between cyclic AMP-binding capacity and the intracellular concentrations of cyclic AMP reported in a previous study [Paznokas & Sypherd (1975) J. Bacteriol. 124, 134--139]. Both of these parameters attained transient high values at a time of development when addition of exogenous cyclic AMP prevents hyphal-germ-tube emergence. The measured Kd values did not change during sport germination.     

Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores

Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.     

Effects of phospholipids on binding of calcium to (Ca2(+)-Mg2(+)-ATPase

The (Ca2(+)-Mg2(+)-ATPase purified from skeletal muscle sarcoplasmic reticulum binds two Ca2+ ions per ATPase molecule. On reconstitution into bilayers of dioleoylphosphatidylcholine [C18:1)PC) or dinervonylphosphatidylcholine [C24:1)PC) the stoichiometry of binding remains unchanged, but when the ATPase is reconstituted into bilayers of dimyristoleoylphosphatidylcholine [C14:1)PC) the stoichiometry changes to one Ca2+ ion per ATPase molecule. Nevertheless, the level of phosphorylation is the same for the ATPase reconstituted with (C18:1)PC or (C14:1)PC. The effect of (C14:1)PC on the stoichiometry of Ca2+ binding is prevented by androstenol at a 1:1 molar ratio with the phospholipid.     

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (approximately 3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E2) and estrone (E1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E2 and E1 thus demonstrating the presence of similar oxidative and reductive activities for 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) [0.45 and 0.40 pmol/3 h/microgram DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E2 to E1 (0.08 pmol/3 h/microgram DNA) but exhibited an approx 5-fold higher rate of reduction of E1 to E2 (1.81 pmol/3 h/microgram DNA). Estriol (E3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.     

Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.