Characterization of 14,15-epoxyeicosatrienoyl-sulfonamides as 14,15-epoxyeicosatrienoic acid agonists: use for studies of metabolism and ligand binding

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BK(Ca)) channels to cause hyperpolarization and relaxation. We developed a series of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA) as tools to characterize 14,15-EET metabolism and binding. Agonist activities of these analogs were characterized in precontraced bovine coronary arterial rings. All three analogs induced concentration-dependent relaxation and were equipotent with 14,15-EET. Relaxations to these analogs were inhibited by the BK(Ca) channel blocker iberiotoxin (100 nM), the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoylmethylsulfonamide (10 muM), and abolished by 20 mM extracellular K(+). 14,15-EET-PISA is metabolized to 14,15-dihydroxyeicosatrienoyl-PISA by soluble epoxide hydrolase in bovine coronary arteries and U937 cells but not U937 cell membrane fractions. 14,15-EET-P(125)ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4 degrees C and remained unchanged up to 30 min. The estimated K(d) and B(max) were 148.3 +/- 36.4 nM and 3.3 +/- 0.5 pmol/mg protein, respectively. These data suggest that 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full 14,15-EET agonists. 14,15-EET-P(125)ISA is a new radiolabeled tool to study EET metabolism and binding. Our results also provide preliminary evidence that EETs exert their biological effect through a membrane binding site/receptor.     

Role of prostanoids and 20-HETE in mediating oxygen-induced constriction of skeletal muscle resistance arteries.

This study determined the contribution of cytochrome P450 (CP450) 4A enzyme metabolites of arachidonic acid in mediating the constriction of isolated rat skeletal muscle resistance arteries in response to elevated PO2. Gracilis arteries (GA) were viewed via television microscopy and constrictor responses to elevated PO2 were measured with a video micrometer. Endothelium removal and treatment of GA with 17-octadecynoic acid (17-ODYA; suicide substrate inhibitor of CP450 4A enzymes) impaired oxygen-induced constriction of the vessels; treatment of endothelium-denuded GA with 17-ODYA eliminated responses to elevated PO2. NOS inhibition and inhibition of EET production had no effect on oxygen-induced constriction of the vessels, although cyclooxygenase inhibition with indomethacin impaired GA responses to elevated PO2. Treatment of GA with dibromododecenyl methylsulfimide (DDMS; inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) production) or 6(Z),15(Z)-20-HEDE (antagonist for 20-HETE receptors) mimicked the effects of 17-ODYA on GA responses to elevated PO2. Treatment of vessels with iberiotoxin or glibenclamide reduced the constriction of the vessels in response to elevated PO2 while treatment with both K+ channel blockers eliminated oxygen-induced constriction of the vessels. Following treatment of GA with indomethacin and 20-HETE, the vessels failed to respond to elevated PO2. These results suggest that oxygen-induced constriction of skeletal muscle resistance arteries represents the combined effects of reduced prostanoid release from the vascular endothelium and enhanced 20-HETE production in vascular smooth muscle cells.     

Targeted killing of cultured cells by receptor-dependent photosensitization.

This paper describes a method, designated "receptor-dependent photosensitization," by which the receptor-mediated endocytosis of low density lipoprotein (LDL) can be used to deliver a photosensitizing agent, pyrene, to cultured human and animal cells. The hydrophobic core of LDL is extracted and replaced with pyrene covalently coupled to cholesteryl oleate. This reconstituted LDL enters cells in significant amounts only when the cells express LDL receptors, resulting in the accumulation of pyrene cholesteryl oleate within lysosomes. Subsequent exposure of the cells to ultraviolet light leads to cell death. Cells killed by this technique include normal and simian virus 40-transformed human fibroblasts, human A-431 epidermal carcinoma cells, Chinese hamster ovary cells, and mouse L cells, all of which express LDL receptors. Mutant fibroblasts from a patient with homozygous familial hypercholesterolemia, which lack LDL receptors, do not take up significant amounts of the pyrene-containing LDL and are not killed by subsequent exposure to light. The current experiments establish the feasibility of receptor-dependent photosensitization as an efficient and selective method for killing cultured human and animal cells.     

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.     

Chromosomal aberrations in railroad transit workers: Effect of genetic polymorphisms

Complex chemical mixtures are transported by train from Russia to Finland for further shipment. Here, we studied if exposure to genotoxic components among these substances could affect chromosomal aberrations (CAs) in peripheral lymphocytes of workers handling the tank cars. An initial survey among 48 railroad workers and 39 referents (male smokers and nonsmokers) showed an elevation of CAs. A campaign was started to reduce exposures through preventive measures. Five years later, 51 tank car workers and 40 age‐matched referents (all nonsmoking men) were studied for CAs and genetic polymorphisms of xenobiotic metabolism (EPHX1, GSTM1, GSTP1, GSTT1, NAT1, NAT2), DNA repair (ERCC2, ERCC5, XPA, XPC, XRCC1, XRCC3), and folate metabolism (MTHFR, MTR). No increase in CAs was seen in the exposed group, suggesting that the preventive measures had been successful. However, a positive association existed between exposure duration and CA level among the exposed subjects. The level of chromosome‐type breaks was actually lower in the exposed workers than the referents, particularly among MTHFR wild‐type homozygotes or XRCC3 codon 241 variant allele carriers, suggesting modulation of CA frequency by folate metabolism and DNA repair. An interaction was observed between the occupational exposure and MTHFR, EPHX1, and MTR genotypes in determining CA level. The NAT2, ERCC2 exon 10, and XRCC1 codon 194 polymorphisms also affected CA frequency. Our findings suggest that handling of tank cars containing complex chemical mixtures poses a genotoxic risk, which may be reduced by preventive measures. Several genetic polymorphisms seem to modify the genotoxic effect or baseline CA level. Environ. Mal. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.