The Na⁺,K⁺-ATPase plays a key role in animal cells; however, many insects are refractory to the Na⁺,K⁺-ATPase inhibitor ouabain. In Drosophila Malpighian (renal) tubules ouabain (even at 10^-3 M) is almost without effect (Dow et al. 1994; Linton & O’Donnell, 1999), although tubules contain Na⁺,K⁺-ATPases and the Drosophila Na⁺,K⁺-ATPase is ouabain sensitive in vitro (Lebovitz et al. 1989). We proposed that Malpighian tubules contained a ouabain transport system that protected the Na⁺,K⁺-ATPase. In vertebrates organic anion transporting polypeptides (OATPs) have broad substrate specificities and can, therefore, transport and eliminate a wide variety of structurally unrelated compounds, including cardiac glycosides.

We adopted a functional genomic approach and screened the Drosophila genome in silico, identifying eight genes similar to vertebrate OATP members. These were designated OATPs 26F, 30B, 33Ea, 33Eb, 58Da, 58Db, 58Dc and 74D (named according to their chromosomal location). RT-PCR and in situ hybridisation analysis reveal that six of these (OATPs 30B, 33Ea, 33Eb, 58Db, 58Dc and 74D) are expressed in Drosophila Malpighian tubules.

Fluid secretion assays (Dow et al. 1994) reveal that alternative transporter substrates, such as taurocholate (6 X 10^-4 M), sulphobromophthalein (BSP) (2 X 10^-6 M), prostaglandin E2 (PGE2) (1 X 10^-4 M) and phenol red (6 X 10^-5 M) (but not cAMP (1 X 10^-7 M) or cCMP (4 X 10^-5 M)), unmask the inhibitory action of 10^-3 M ouabain on secretion.

In support of our model, radiolabelled ouabain transport assays reveal Malpighian tubules actively transport ouabain. These transport assays allow ‘transport ratios’ to be calculated (transport ratio = c.p.m. in 10 nl secreted drop/c.p.m. in 10 nl bathing medium) with transport ratios >1 indicative of active transport of ouabain. The data from these experiments show that ouabain is actively transported (transport ratios of 3-4). Consistent with the fluid secretion data, this active transport is abolished (transport ratios Æ<<1) by the addition of the alternative transporter substrates taurocholate, BSP, PGE2 and phenol red (but not cyclic nucleotides).

Heterologous expression of the putative Drosophila oatps in NIH3T3 cells show that cells transfected with OATPs 30B, 33Eb and 58Db transport significantly more (P < 0.05, Student’s unpaired t test) [³H]ouabain than control cells. However, cells transfected with OATPs 33Ea, 58Dc and 74D show no significant difference, compared to control cells, in terms of [³H]ouabain transport. This suggests that some members of the Drosophila OATP family may be important in the excretion of ouabain through the Malpighian tubule.

These data show that the Na⁺,K⁺-ATPase may be as significant in insect tissues as in vertebrates, but is protected by a potent transport system that excretes a range of xenobiotics.

The support of the BBSRC and Syngenta is gratefully acknowledged.