Academic journal article Genetics

Fluoxetine-Resistance Genes in Caenorhabditis Elegans Function in the Intestine and May Act in Drug Transport

Academic journal article Genetics

Fluoxetine-Resistance Genes in Caenorhabditis Elegans Function in the Intestine and May Act in Drug Transport

Article excerpt

ABSTRACT

Fluoxetine (Prozac) is one of the most widely prescribed pharmaceuticals, yet important aspects of its mechanism of action remain unknown. We previously reported that fluoxetine and related antidepressants induce nose muscle contraction of C. elegans. We also reported the identification and initial characterization of mutations in seven C. elegans genes that cause defects in this response (Nrf, nose resistant to fluoxetine). Here we present genetic evidence that the known nrf genes can be divided into two subgroups that confer sensitivity to fluoxetine-induced nose contraction by distinct pathways. Using both tissue-specific promoters and genetic mosaic analysis, we show that a gene from one of these classes, nrf-6, functions in the intestine to confer fluoxetine sensitivity. Finally, we molecularly identify nrf-5, another gene in the same class. The NRF-5 protein is homologous to a family of secreted lipid-binding proteins with broad ligand specificity. NRF-5 is expressed in the intestine and is likely secreted into the pseudocoelomic fluid, where it could function to transport fluoxetine. One model that explains these findings is that NRF-5 binds fluoxetine and influences its presentation or availability to in vivo targets.

UNDERSTANDING the molecular mechanisms of \~J the action of drugs is essential for rational development and delivery of compounds with greater efficacy, greater specificity, and fewer adverse side effects. Genetic screens for mutants with altered responses to drugs constitute an approach to identifying drug mechanisms of action that is particularly promising for identifying novel targets and pathways (SCHAFER 1999). Genetic screens using Caenorhabdiiis elegans have been successful in identifying drug targets. For example, screens for resistance to the antihelminthic drug levamisole led to the identification of levamisole-sensitive acetylcholine receptors (LEWIS et al. 1980; FLEMING et al. 1997) and screens for resistance to another antihelminthic drug, ivermectin, led to the identification of a novel class of ivermectin-sensitive glutamate-gated chloride channels (UENT et al. 2000). In addition to identifying direct drug targets, genetic resistance screens can also identify components of signaling pathways that are affected by the drug of interest. For example, the acetylcholinesterase inhibitor aldicarb causes increased synaptic acetylcholine levels and aldicarb-resistant mutants have identified several novel components of synaptic transmission (NGUYEN et al. 1995; MILLER et al. 1996, 2000) Finally, such screens can identify genes involved in drug metabolism, transport, or localization, which are key factors in determining in vivo drug availability and efficacy.

We isolated mutations affecting seven Nrf genes that cause resistance to nose contraction by the antidepressant fluoxetine (Nrf: nose resistant to/luoxetine). Genetic analysis suggested that three of these genes, nrf-5, nrf-6, and ndg-4, function in a common pathway and that mutations in these genes confer their fluoxetineresistant phenotype by a similar mechanism (CHOY and THOMAS 1999). Here, we describe further genetic and molecular characterization of the Nrf genes. We construct double mutants between two classes of Nrf mutants and demonstrate that these two classes confer fluoxetine resistance by different pathways. We have examined the site of action of nrf-6 and found that it is required in the intestine for sensitivity to fluoxetineinduced nose contraction. We have also cloned nrf-5 and found that it is homologous to a family of mammalian secreted lipid-binding proteins. A nrf-5'·'·gfp fusion is expressed in the intestine, suggesting that nrf-5 is secreted into the pseudocoelomic fluid where it could function in drug transport.

MATERIALS AND METHODS

Genetics and pharmacology: General culturing and maintenance of strains was as described (BRENNER 1974). For nrf (Peg: jfede eggs); nrf (non-Peg) double mutants, the Peg mutation was followed by the Peg phenotype and the non-Peg mutation was balanced in irons with flanking double-mutant chromosomes (CnOY and THOMAS 1999). …

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