Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1.
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The transport of organic anions in proximal convoluted tubules plays an essential role in the active secretion of a variety of small molecules by the kidney. In addition to other anionic substrates, the human renal organic anion transporter 1 (hOATI) is capable of transporting the nucleotide analogs adefovir and cidofovir. To investigate the involvement of hOATI in the mechanism of nephrotoxicity associated with these two clinically important antiviral agents, Chinese hamster ovary (CHO) cells were stably transfected with hOATI cDNA. The resulting CHOhOAT cells showed probenecid-sensitive and pH-dependent uptake of p-aminohippurate (Km = 15.4 FtM, V,,, ..ax = 20.6 pmol/106 cells min), a prototypical organic anion substrate. In addition, the stably expressed hOATI mediated efficient transport of adefovir (Km, = 23.8 tLM, V, a,, = 46.0 pmol/106 cells min) and cidofovir (K, = 58.0 /iM, Vt,ax = 103 pmol/106 cells * min) such that the levels of intracellular metabolites of both nucleotides were > 1 00-fold higher in CHOh OAT cells than in parental CHO. Consequently, adefovir and cidofovir were approximately 500-fold and 400-fold more cytotoxic, respectively, in CHOh OAT cells compared to CHO. The cytotoxicity of both drugs in CHOh OAT cells was markedly reduced in the presence of hOATI inhibitors. The cyclic prodrug of cidofovir, which exhibits reduced in vivo nephrotoxicity, was a poor substrate for hOATI and showed only marginally increased cytotoxicity in CHOh OAT cells. In conclusion, these studies demonstrate that hOATI plays a critical role in the organ-specific toxicity of adefovir and cidofovir, and indicates that CHOh OAT cells may represent a useful in vitro model to investigate the potential nephrotoxicity of clinically relevant organic anion agents. (+info)
Apical endocytosis of ricin in MDCK cells is regulated by the cyclooxygenase pathway.
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Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A(2) activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells. (+info)
Single-dose pharmacokinetics and safety of the oral antiviral compound adefovir dipivoxil in children infected with human immunodeficiency virus type 1. The Pediatrics AIDS Clinical Trials Group.
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The acyclic phosphonate analog adefovir is a potent inhibitor of retroviruses, including human immunodeficiency virus (HIV) type 1, and, unlike some antiviral nucleosides, does not require the initial phosphorylation step for its activity. Two oral dosages of the adefovir prodrug adefovir dipivoxil were evaluated in a phase I study with children with HIV infection. A total of 14 patients were stratified into age groups ranging from 6 months to 18 years of age. Eight patients received 1.5 mg of adefovir dipivoxil per kg of body weight, and six patients received 3.0 mg of adefovir dipivoxil per kg. Serum samples were obtained at intervals during the 8 h postdosing and were analyzed for adefovir concentrations. Patients were monitored for adverse effects. All samples collected resulted in quantifiable levels of adefovir (lower limit of quantitation, 25 ng/ml) from each patient. The areas under the concentration-versus-time curves (AUCs) were similar (P = 0.85) for the 1.5- and 3.0-mg/kg doses, while the apparent oral clearance (CL/F) was significantly higher (P = 0.05) for the 3-mg/kg dose. Pharmacokinetic parameters differed by patient age. In comparing those children older and younger than the median age of 5.1 years, AUC (P = 0.03), maximum concentration of drug in serum (P = 0.004), and the concentration at 8 h postdosing (P = 0.02) were significantly lower for the younger children. There were no significant differences for apparent volume of distribution and CL/F normalized to body surface area, but there was a suggestive difference in half-life (P = 0.07) among the subjects in the older and younger age groups. No significant adverse events were encountered. These data provide the basis for a multidose phase II study of adefovir dipivoxil in HIV-infected infants and children. (+info)
alpha - and beta -phosphorylated amines and pyrrolidines, a new class of low toxic highly sensitive 31P NMR pH indicators. Modeling of pKa and chemical shift values as a function of substituents.
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Fourteen linear and cyclic alpha- and beta-aminophosphonates in which the P-atom is substituted by alkoxy groups have been synthesized and evaluated as (31)P NMR pH markers in Krebs-Henseleit buffer. pK(a) values varied with substitution in the range 1.3-9.1, giving potentially access to a wide range of pH. Temperature had a weak influence on pH and a dramatic increase in ionic strength slightly modified the pK(a) of the pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH). All compounds displayed a 4-fold better NMR sensitivity than inorganic phosphate or other commonly used phosphonates, as assessed by differences delta(b)-delta(a) between the chemical shifts of the protonated and the unprotonated forms. In isolated perfused rat hearts, a non-toxic concentration window of 1.5-15 mm was determined for three representative compounds. Using empirical linear relationships, the experimental values of pK(a), delta(a), and delta(b) have been correlated with the two-dimensional structure, i.e. the chemical nature of substituents bonded to the secondary amine and P-atom. The data suggest that DEPMPH and its cyclic and linear variants are ideal versatile (31)P NMR probes for the study of tenuous pH changes in biological processes. (+info)
Opposite effects of GABA(A) and GABA(C) receptor antagonists on the b-wave of ERG recorded from the isolated rat retina.
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The largest component in the fully dark-adapted ERG is a corneal-positive response, known as the b-wave, and believed to originate from depolarizing (ON-type) bipolar cells. The two types of GABA receptors, GABA(A) and GABA(C) have been reported to exist on bipolar cells in rat retina. The goal of these experiments was to find whether these GABA receptors participate in the generation of the b-wave of electroretinogram (ERG). ERGs were recorded from the isolated rat retinas. The P(2)(t) component, obtained by subtracting the ERGs measured before the application of 50 micrograms APB from those measured after the application of 50 micrograms APB, was used as an indicator of depolarizing bipolar cell activity. Photovoltages, the fast P(3)(t) component of ERG, were registered between the two microelectrodes across the rod outer segments. Bicuculline and 3-aminopropylphosphonic acid (3-APA) were used as selective antagonists of GABA(A) and GABA(C) receptors, respectively. It was found that the GABA(A) and GABA(C) receptors antagonists have opposite effects on the b-wave: bicuculline increased the b-wave amplitude, while 3-APA reduced the amplitude of the b-wave. Neither bicuculline nor 3-APA affect photoreceptors. (+info)
Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation.
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The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B. (+info)
Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.
(47/1594)
Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H...X. (+info)
Investigations concerning the mode of action of 3,4-dihydroxybutyl-1-phosphonate on Escherichia coli.
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Experiments were performed to evaluate the ability of the enzymes of Escherichia coli involved in glycerol 3-phosphate metabolism to recognize phosphonic acid analogues of the natural substrate. Neither the catabolic membrane-bound glycerol-3-phosphate dehydrogenase nor the acyl coenzyme A: glycerol-3-phosphate acyltransferase can use 3,4-dihydroxybutyl-1-phosphnate or 2,3-dihydroxypropyl-1-phosphonate are inhibitors of the reduction of dihydroxyactone phosphate as substrates. The 4-carbon phosphonic acid analogue does not exhibit inhibitory activity for either of these enzymes. While the 3-carbon phosphonic acid analogue has no inhibitory effect upon the catabolic dehydrogenase, it does appear to have a slight but reproducible inhibitory effect on the acyltransferase. Glycerol 3-phosphate and 3,4-dihydroxybutyl-1-phosphonate by glycerol 3-phosphate:NAD (P) oxidoreductase. rac-2,3-Dihydroxypropyl-1-phosphonate does not appear to be recognized by this enzyme. The apparent K-i for snglycerol 3-phosphate is 19 muM and for D-3,4-dihydroxybutyl-1-phosphonate it is 42 muM. In addition the glycerol 3-phosphate:NAD(P) oxidoreductase catalyzes the reduction of 4-hydroxy-3-oxobutyl-1-phosphonate (apparent K-m of 182 muM), a phosphonic acid analogue of dihydroxyacetone phosphate. 3,4-Dihydroxybutyl-1-phosphonate is both a competitive inhibitor (apparent Ki of 740 muM) and a substrate (apparent K-m of 450 muM) for the CDP-diglyceride: glycerol 3 phosphate phosphatidyltransferase but it has no effect upon CDP-diglyceride:L-serine phosphatidyltransferase. The relationship ofthese in vitro studies to in vivo investigations is discussed. (+info)