Abstract
Insect osmoregulation is subject to highly sophisticated endocrine control. In Drosophila, both Drosophila kinin and tyramine act on the Malpighian (renal) tubule stellate cell to activate chloride shunt conductance, and so increase the fluid production rate. Drosophila kinin is known to act through intracellular calcium, but the mode of action of tyramine is not known. Here, we used a transgenically encoded GFP::apoaequorin translational fusion, targeted to either principal or stellate cells under GAL4/UAS control, to demonstrate that tyramine indeed acts to raise calcium in stellate, but not principal cells. Furthermore, the EC(50) tyramine concentration for half-maximal activation of the intracellular calcium signal is the same as that calculated from previously published data on tyramine-induced increase in chloride flux. In addition, tyramine signalling to calcium is markedly reduced in mutants of NorpA (a phospholipase C) and itpr, the inositol trisphosphate receptor gene, which we have previously shown to be necessary for Drosophila kinin signalling. Therefore, tyramine and Drosophila kinin signals converge on phospholipase C, and thence on intracellular calcium; and both act to increase chloride shunt conductance by signalling through itpr. To test this model, we co-applied tyramine and Drosophila kinin, and showed that the calcium signals were neither additive nor synergistic. The two signalling pathways thus represent parallel, independent mechanisms for distinct tissues (nervous and epithelial) to control the same aspect of renal function.
Highlights
Insect Malpighian tubules play key roles in ion transport and excretion [1], immune function [2,3] and xenobiotic detoxification [4,5]
Stellate cells are activated by Drosophila kinin, or Drosokinin (NSVVLGKKQRFHSWGamide) [21], a member of a neuropeptide family found in most insects [22,23], which signals through a canonical G-protein coupled receptor (GPCR) to raise intracellular calcium [24], and thence to rapidly increase the chloride shunt conductance, effectively removing the ‘brake’ on active cation pumping, resulting in a rapid collapse of transepithelial potential (TEP) and concomitant increase in fluid secretion [25,26]
We have previously described the use of quantitative reporters based on transgenic aequorin [29], as well as imaging reporters based on pericam [33]; here, we generated flies transgenic for a calcium reporter based on a translational fusion of green fluorescent protein (GFP) and apoaequorin, under control of the UAS control region (‘UASGFP::aeq’) by cloning a synthetic cDNA into the transformation vector pPfUASTg and germ-line transforming Drosophila according to standard protocols
Summary
Insect Malpighian tubules play key roles in ion transport and excretion [1], immune function [2,3] and xenobiotic detoxification [4,5]. Stellate cells are activated by Drosophila kinin, or Drosokinin (NSVVLGKKQRFHSWGamide) [21], a member of a neuropeptide family found in most insects [22,23], which signals through a canonical G-protein coupled receptor (GPCR) to raise intracellular calcium [24], and thence to rapidly increase the chloride shunt conductance, effectively removing the ‘brake’ on active cation pumping, resulting in a rapid collapse of TEP and concomitant increase in fluid secretion [25,26]. As well as demonstrating that tyramine does signal through intracellular calcium in only the stellate cells, we report the use of an improved calcium sensor in tubules that is based on a translational fusion of the two jellyfish photoproteins apoaequorin and green fluorescent protein (GFP), resulting in markedly improved sensitivity [30,31]. For EC50 values, best fit was calculated by least-squares nonlinear fit (GraphPad Prism), and the resulting log(EC50) values compared with a t-test
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