Histidine 186 of the nicotinic acetylcholine receptor alpha subunit requires the presence of the 192-193 disulfide bridge to interact with alpha-bungarotoxin

We have previously shown that two histidine residues of the nicotinic acetylcholine receptor are relevant for alpha-bungarotoxin binding. This paper studies: (1) the interaction between alpha-bungarotoxin and the peptide alpha173-202--synthesized according to the sequence of the Torpedo californica receptor alpha subunit--and between the toxin and the same peptide containing His186 modified with ethoxyformic anhydride or substituted by Ala; (2) the influence of the presence of Cys192-Cys193 disulfide bridge on such interactions. Solid-phase and in-solution competition assays were performed: ethoxyformylation of His186 or its substitution by Ala led to a significant drop in the toxin binding capacity only for peptides containing the bridge. Circular dichroism and fourth derivate spectra of all peptides were also analyzed. Results strongly indicate the involvement of His186 in the toxin binding to those peptides with the bridge--also present in the native receptor molecules--but not to their reduced forms; on the other hand, they give further support to the already established premise that, though the bridge does not participate directly in receptor-toxin binding, its presence is relevant to define the appropriate conformation of the interaction area.     

Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl-myo-inositol 3,4,5-triphosphate in oligodendrocyte cell signaling

There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine, and a [(14)C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P(3)] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P(2)]. When PtdIns(3,4,5)P(3) was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P(3) levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P(3) and is most likely involved in receptor clustering and capping.     

Matrix metalloproteinase-12 inhibitors: synthesis,structure-activity relationships and intestinal absorption of novel sugar-based biphenylsulfonamide carboxylates

MMP-12 is a validated target in pulmonary and cardiovascular diseases. The principal obstacles to clinical development of MMP-12 inhibitors are an inadequate selectivity for the target enzyme and a poor water solubility, with consequent poor oral bioavailability. We recently reported a new class of sugar-based arylsulfonamide carboxylates with a nanomolar activity for MMP-12, a good selectivity and an improved water solubility. In this study, we designed and synthesized new derivatives to characterize the structure-activity relationship (SAR) within this class of glycoconjugate inhibitors. All the new derivatives were tested on human recombinant MMP-12 and MMP-9 in order to evaluate their affinity and the selectivity for the target enzyme. Among them, the four most promising compounds were selected to assess their intestinal permeability using an ex vivo everted gut sac model. Given the high polarity and structural similarity to glucose, compound 3 was demonstrated to cross the intestinal membrane by using the facilitative GLUT2 transport.     

Properties of microtubule sliding disintegration in isolated tetrahymena cilia

Properties of the sliding disintegration response of demembranated tetrahymena cilia have been studied by measuring the spectrophotomeric response or turbidity of cilia suspensions at a wavelength of 350 nm relative to changes in the dynein substrate (MgATP(2-)) concentration. The maximum decrease in turbidity occurs in 20 muM ATP, and 90 percent of the decrease occurs in approximately 5.9 s. At lower ATP concentrations (1-20 muM), both the velocity and magnitude of the turbidity decreases are proportional to ATP concentration. The velocity data for 20 muM ATP permit construction of a reaction velocity curve suggesting that changes in turbidity are directly proportional to the extent and velocity of disintegration. At ATP concentrations more than 20 muM (50muM to 5mM), both velocity and magnitude of the turbidimetric response are reduced by approximately 50 percent. This apparent inhibition results in a biphasic response curve that may be related to activation of residual shear resistance or regulatory components at the higher ATP concentrations. The inhibitory effects of elevated ATP can be eliminated by mild trypsin proteolysis, whereupon the reaction goes to completion at any ATP concentration. The turbidimetric responses of the axoneme-substrate suspensions are consistent with the extent and type of axoneme disintegration revealed by electron microscope examination of the various suspensions, suggesting that the turbidimetric assay may prove to be a reliable means for assessing the state of axoneme integrity.     

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism

The two CHCl₃ activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and β-naphthoflavone). The present results provide direct evidence that CHCl₃ concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl₃ concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10–15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl₃ only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl₃ oxidative bioactivation do not participate in CHCl₃ reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl₃ activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 305–312, 1997.     

Differential regulation of sphingomyelin synthesis and catabolism in oligodendrocytes and neurons

Neurons (both primary cultures of 3-day rat hippocampal neurons and embryonic chick neurons) rapidly converted exogenous NBD-sphingomyelin (SM) to NBD-Cer but only slowly converted NBD-Cer to NBD-SM. This was confirmed by demonstrating low in vitro sphingomyelin synthase (SMS) and high sphingomyelinase (SMase) activity in neurons. Similar results were observed in a human neuroblastoma cell line (LA-N-5). In contrast, primary cultures of 3-day-old rat oligodendrocytes only slowly converted NBD-SM to NBD-Cer but rapidly converted NBD-Cer to NBD-SM. This difference was confirmed by high in vitro SMS and low SMase activity in neonatal rat oligodendrocytes. Similar results were observed in a human oligodendroglioma cell line. Mass-Spectrometric analyses confirmed that neurons had a low SM/Cer ratio of (1.5 : 1) whereas oligodendroglia had a high SM/Cer ratio (9 : 1). Differences were also confirmed by [³H]palmitate-labeling of ceramide, which was higher in neurons compared with oligodendrocytes. Stable transfection of human oligodendroglioma cells with neutral SMase, which enhanced the conversion of NBD-SM to NBD-Cer and increased cell death, whereas transfection with SMS1 or SMS2 enhanced conversion of NBD-Cer to NBD-SM and was somewhat protective against cell death. Thus, SMS rather than SMases may be more important for sphingolipid homeostasis in oligodendrocytes, whereas the reverse may be true for neurons.     

Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides

The organophosphorothioate (OPT) pesticide malathion (MAL) in mammals is readily hydrolyzed by mammalian carboxylesterases (CE). The reaction competes with the CYP-catalyzed formation of malaoxon (MOX), the toxic metabolite. Alterations or individual variations in CE activity may result in increased MOX formation, enhancing MAL toxicity. We have characterized the human hepatic CE activity in a panel of 18 human liver microsomes as well as the inhibitory effect of IsoMAL, a major impurity of MAL commercial formulations, parathion (PAR), chlorpyrifos (CPF), and chlorpyrifos-oxon (CPFO). CE activity showed a low level of variation among individuals (4-fold). The reaction consists of two different phases, differing in their affinity for mal (k(m1)=0.25-0.69 microm; K(m2)=10.3-26.8 microM). The relatively low K(m1) values confirmed that human CE efficiently detoxify MAL. IsoMAL was shown to be a potent noncompetitive inhibitor of MAL detoxification (K(i)=0.6 microM), with a higher inhibitory potency than CPF and PAR (K(i)=7.5 microM and 50 microM, respectively). These two latter compounds very likely act as mixed inhibitors. CPFO showed the highest inhibitory potency toward CE-mediated detoxification, being characterized by a K(i)=22 nM. The present results provide useful information for a better understanding of possible interactions between different OPTs and for assessing the potential cumulative risk for exposure to OPT mixtures.     

Multiple activation of chloroform in kidney microsomes from male and female DBA/2J mice

Microsomes from the renal cortex of DBA/2J mice can metabolize chloroform through oxidative and reductive pathways, similar to hepatic microsomes. The oxidative or reductive nature of CHCl₃ activation is strictly dependent on the oxygenation of the incubation mixture, as indicated by the formation of qualitatively different adducts to phos-pholipids (PLs). The protein and lipid binding levels measured in kidney microsomes from control females differed significantly from the binding levels observed with kidney microsomes from male and testosterone-treated female DBA/2J mice in aerobic conditions only. Therefore, the sex-dependent CHCl₃-induced acute nephrotoxicity seems related only with the oxidative CHCl₃activation. The levels of adducts to PL polar heads and to protein showed a strict correlation with each other. Therefore, the assay of adducts to PL polar heads may be used as a substitute for the assay of adducts to protein. This might be especially convenient when studying the effects of both phosgene and the trichloromethyl radicals.