A P-glycoprotein-like mechanism in the nicotine-resistant insect, Manduca sexta.

The phenomenon of multi-drug resistance in tumor cells is mediated by the overexpression of a membrane protein called P-glycoprotein. Since its initial discovery in tumor cells, P-glycoprotein-like molecules have been found to transport a wide range of substrates in organisms from microorganisms to humans. To test the hypothesis that a P-glycoprotein-like mechanism is operating in insects, three insect species were used: Manduca sexta, Periplaneta americana and Rhodnius prolixus, the nicotine-resistant M. sexta was the model toxin-resistant insect. Immunostaining with a monoclonal antibody, C219, which recognizes a highly conserved epitope in all P-glycoprotein molecules, demonstrated positive immunolabelling of the Malpighian tubules of M. sexta and R. prolixus and at the blood-brain barrier of the central nervous systems of M. sexta and P. americana. To assess the extent of P-glycoprotein involvement at the blood-brain barrier of M. sexta, I took advantage of the three different stages of the insect's life cycle, the actively feeding larval stage and the non-feeding pupal and adult stages. Using an extracellular recording technique to monitor levels of neural activity in isolated abdominal nerve cords, I demonstrated that, in contrast with larval M. sexta, the CNS of both the pupal and adult stages are both highly nicotine-sensitive. Immunostaining for P-glycoprotein in the metamorphosing CNS illustrated that the distribution of P-glycoprotein in the barrier region changes dramatically when the need for a barrier to dietary neurotoxins is lost. M. sexta Malpighian tubules provided an ideal in vitro assay system to directly test our hypothesis that a P-glycoprotein-like molecule is involved in nicotine transport. Using an isolated Malpighian tubule preparation, the tubules were assayed for their ability, first, to concentrate nicotine in the tubule lumen (confirming the presence of an active transporter in the tubules) and, second, to determine if various drugs interfere with nicotine transport. Tubules bathed in 0.5 mM nicotine were found to concentrate nicotine in the secreted fluid at least 9-fold. Nicotine transport was inhibited with verapamil, a known inhibitor of P-glycoprotein. In addition, nicotine transport was competitively inhibited by another alkaloid, atropine, suggesting that the M. sexta pump may be a non-selective alkaloid pump. To further confirm that the insect pump is a P-glycoprotein-like multi-drug pump, the tubules were tested for their ability to transport a known P-glycoprotein substrate, vinblastine. The tubules were not only able to transport radiolabelled vinblastine, but this transport was also inhibited by verapamil. P-glycoprotein is localized to the digestive tracts of mammals and of invertebrates, where it is thought to act as a defective mechanism to protect organisms from dietary toxins. Immunohistochemical staining for P-glycoprotein demonstrated that it is also expressed in the M. sexta gut. In a parallel study, a monoclonal antibody against a P450 from a DDT-resistant housefly (Waters et al., 1989) was used to localize a cytochrome P450 monooxygenase enzyme (P450 or PSMO) to the M. sexta midgut. The co-localization of P450 and P-glycoprotein to the midgut suggests that larval M. sexta use a combination of P450-mediated metabolism coupled with transport to process dietary toxins