Abstract5V -Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5V -fluoro-2V -deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5V -fluoro-2V -dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5-and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were f9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 Mmol/L

Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

Na Na؉ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMP i on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMP i , elevation of cGMP i by treatment with dibutyryl-cGMP or 8-bromocGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMPdependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.

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Plasma membrane Na+/H+ exchanger (NHE) isoforms NHE1 and NHE3 exhibit very different sensitivities to amiloride and its 5-amino-substituted analogues, benzoyl guanidinium derivatives (e.g. (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE694)), and cimetidine. To define structural domains that confer differential sensitivity to these antagonists, unique restriction endonuclease sites were engineered into cDNAs for each isoform near the regions that encode the putative membrane-spanning domains. These new sites did not modify their pharmacological properties and allowed several chimeric Na+/H+ exchangers to be constructed by exchanging homologous segments. The modified parental (E1' and E3') and chimeric molecules were stably expressed in exchanger-deficient Chinese hamster ovary AP-1 cells and assayed for their sensitivities to amiloride, ethylisopropylamiloride, HOE694, and cimetidine. Most chimeras showed drug sensitivities corresponding to the dominant parental segment. However, interchanging a 66-amino acid segment containing the putative ninth transmembrane (M9) domain and its adjacent loops caused reciprocal alterations in the sensitivities of E1' and E3' to all antagonists. In addition, substituting the first five putative membrane-spanning domains of E3' with the corresponding region of E1' modestly reduced the transporter's sensitivity to cimetidine but not the other compounds. These data indicate that the protein segment between M8 and M10 may be a major site of interaction with these antagonists, although other regions modestly influence sensitivity to certain drugs.