Characterization of transepithelial transport of salicylate by the Malpighian tubules of Drosophila melanogaster and the effects of changes in fluid secretion rate

Abstract.  A radioisotope tracer technique is used to study mechanisms and regulation of transepithelial transport of the plant allelochemical salicylate by the Malpighian tubules of Drosophila melanogaster . Transepithelial transport of salicylate is nearly abolished in Na⁺-free saline, and inhibited by ouabain, low K⁺ or K⁺-free bathing saline. In addition, the carboxylates probenecid, unlabelled salicylate, fluorescein, and p -aminohippuric acid (PAH) significantly inhibit transepithelial transport of salicylate. The sulphonates taurocholate and phenol red also inhibit transepithelial transport of salicylate, whereas amaranth has no effect. Stimulation of fluid secretion by cAMP, cGMP or leucokinin I increases transepithelial transport of salicylate, particularly when the concentration of salicylate in the bathing saline is high. The correlation between the fluid secretion rate and transepithelial transport of salicylate shows that 64% of the changes in salicylate transport can be explained on the basis of changes in fluid secretion rate. The results show that naturally-occurring plant secondary metabolite salicylate is transported into the lumen of the Mapighian tubules of D. melanogaster by a mechanism similar to that previously described for the prototypical organic anions PAH and fluorescein. In addition, the transepithelial transport of salicylate increases in response to increases in fluid secretion rate.     

Purine transport by Malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (p ^p) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.This work was supported by Grant GM22366 from NIH.     

The effects of altered expression of the Keap1/CncC pathway on the secretion of fluid and salicylate by Malpighian tubules of Drosophila melanogaster

The Keap1‐Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila melanogaster Meigen, targeted expression of Keap1 and CncC (the latter the orthologue of human Nrf2) in the Malpighian (renal) tubules is known to confer resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mm ) causes increases in the fluid secretion rate and salicylate flux across Malpighian tubules. In the present study, salicylate‐selective microelectrodes and Ramsay assays are used to determine the role of Keap1/Nrf2 in regulating these responses. Genetic manipulations designed to increase Nrf2 activity (by knockdown of the repressor Keap1 or overexpression of the Nrf2 coactivator MafS) or to decrease Nrf2 activity (by overexpression of Keap1) are also studied. Although the results of the Keap1 manipulations are inconclusive, there is no increase in the fluid secretion rate or salicylate flux in tubules isolated from flies in which MafS expression is increased.     

Short-term hardening effects on survival of acute and chronic cold exposure by Drosophila melanogaster larvae

We quantified the variation and plasticity in cold tolerance among four larval stages of four laboratory strains of Drosophila melanogaster in response to both acute (<2 h of cold exposure) and chronic (7 h of cold exposure) cold exposure. We observed significant differences in basal cold tolerance between the strains and among larval stages. Early larval instars were generally more tolerant of acute cold exposures than third-instar larvae. However, wandering larvae were more tolerant of chronic cold exposures than the other stages. Early stages also displayed a more pronounced rapid cold-hardening response than the later stages. Heat pre-treatment did not confer a significant increase in cold tolerance to any of the strains at any stage, pointing to different mechanisms being involved in resolving heat- and cold-elicited damage. However, when heat pre-treatment was combined with rapid cold-hardening as sequential pre-treatments, both positive (heat first) and negative (heat second) effects on cold tolerance were observed. We discuss possible mechanisms underlying cold-hardening and the effects of acute and chronic cold exposures.     

Transepithelial transport of salicylate by the Malpighian tubules of insects from different orders

The organic anion salicylate is a plant secondary metabolite that protects plants against phytophagous insects. In this study, a combination of salicylate-selective microelectrodes and a radioisotope tracer technique was used to study the transepithelial transport of salicylate by the Malpighian tubules of 10 species of insects from five orders. Our results show that salicylate is transported into the lumen of the Malpighian tubules in all the species evaluated, except Rhodnius prolixus. The transepithelial transport of salicylate by the Malpighian tubules of Drosophila simulans, Drosophila erecta, Drosophila sechellia, and Acheta domesticus was saturable, Na⁺-dependent and inhibited by α-cyano-4-hydroxycinnamic acid. This transport system resembles that previously found in tubules of Drosophila melanogaster. In contrast, transepithelial transport of salicylate by Malpighian tubules of Tenebrio molitor, Plagiodera versicolora, Aedes aegypti, and Trichoplusia ni was unaffected by Na⁺-free bathing saline. The presence of both salicylate and salicylate metabolites in the secreted fluid samples from the Malpighian tubules of A. domesticus, R. prolixus, T. molitor, and T. ni indicates that insect Malpighian tubules may both transport and metabolize salicylate. The highest capacities to rid the hemolymph of salicylate were found in T. molitor, P. versicolora and Drosphila spp. Our results suggest that transport of salicylate by the Malpighian tubules might contribute to elimination of this organic anion from the hemolymph, particularly in some species that encounter high levels of organic anion in the diet.     

Effects of anions, acetazolamide, thiocyanate and amiloride on fluid secretion by the Malpighian tubules of Locusta migratoria L.

The effect of Cl⁻, HCO₃⁻, Br⁻, acetazolamide, thiocyanate and amiloride on urine formation in Locusta migratoria Malpighian tubules have been determined. The rate of fluid secretion depends markedly on the concentration of Cl⁻ in the bathing solution with concentrations less than 90 mM resulting in reduced fluid secretion. Substitution of Br⁻ for Cl⁻ had no significant effect on the rate of the fluid secretion. Replacement of NaHCO₃ with NaCl in Hepes buffered Ringer solution reduced the rate of urine production by 23%. Fluid secretions were reduced in the presence of 10⁻⁴–10⁻² M acetazolamide, a carbonic anhydrase inhibitor. The combined effect of acetazolamide in the absence of HCO₃⁻ appears to be additive. A 1 mM concentration of thiocyanate, an ionic inhibitor, reduced fluid secretion by 35%. Amiloride interferes with the electrogenic entry of Na⁺ into the cell and a 1 mM solution reduced fluid secretion by 94% with secretion completely inhibited in 80% of the tubules tested.     

Characterization of salicylate uptake across the basolateral membrane of the malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [(14)C]salicylate by the Malpighian tubules was active, saturable and Na(+)-dependent; the maximum uptake rate (J(max)) and the half saturation concentration (K(t)) were 12.6 pmoltubule(-1)min(-1) and 30.7micromoll(-1), respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01-1.0 mmoll(-1)) or cis-inhibited by high concentrations (5 mmoll(-1)) of various alpha-keto acids (glutaric acid, alpha-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [(14)C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, alpha-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [(14)C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, alpha-cyano-4-hydroxycinnamic acid-sensitive, Na(+):salicylate cotransport system.     

Fluid and cation secretion by the Malpighian tubules of Locusta

In vitro preparations of Locusta Malpighian tubules are able to transport K⁺ against its concentration gradient. The ‘urine’ is slightly hyper-osmotic with respect to the bathing solution and the rate of secretion is inversely dependent on the osmotic pressure of the latter. The rate of fluid secretion increases with increasing temperature; being maximal at approx 40°C. The ionic composition of the secreted fluid, as indicated by Na⁺/K⁺ ratios, is altered by the presence of 1 mM ouabain in the bathing solution. Fluid secretion is inhibited by 1 mM ouabain. In addition, oxygen consumption by the Malpighian tubules is inhibited by either the presence of 1 mM ouabain or the absence of K⁺ in the bathing solution. The relationship between respiration, active transport and the Na⁺K⁺-activated ATPase is discussed.     

Physiological and molecular characterization of methotrexate transport by Malpighian tubules of adult Drosophila melanogaster

A radioisotope tracer technique and quantitative PCR were used to study the mechanisms and regulation of transepithelial transport of the type II organic anion methotrexate (MTX) by the Malpighian tubules of Drosophila melanogaster. Transport of MTX was saturable and Na⁺-independent; the kinetic parameters J_max and K_t were 437 fmol min⁻¹ and 23.5 μM, respectively. The transport of MTX was competitively inhibited by phenol red and probenecid; non-competitively inhibited by salicylate, verapamil and MK-571; and uncompetitively inhibited by Texas Red. Dietary exposure to 0.1 mM MTX led to dramatic increases in gene expression for several members of the ABC family of transporters in both the Malpighian tubules and the gut. Our results suggest that multiple transporters are upregulated in response to dietary exposure to MTX. Increased levels of the protein products which may result from expression of these genes may enhance elimination of toxic compounds such as MTX or its metabolites.     

Effects of dietary or injected organic cations on larval Drosophila melanogaster: mortality and elimination of tetraethylammonium from the hemolymph

This study of larval Drosophila melanogaster examined the effects of injecting the prototypical organic cation tetraethylammonium (TEA) into the hemocoel or adding TEA and/or other organic cations to the diet. Mortality, hemolymph TEA levels, and Malpighian tubule TEA secretion rates were measured. The LD50 for dietary TEA was 158.4 mM and mortality increased if competitive inhibitors of organic cation transporters were also included in the diet. Mortality increased from 24% on TEA (100 mM) alone to 83 and 67% when the diet contained both TEA and quinidine (10 mM) or cimetidine (100 mM), respectively. TEA-selective microelectrode measurements indicated that hemolymph TEA concentration was approximately 3% of that in the diet for larvae maintained on TEA-enriched diet for 24 h. Malpighian tubules isolated from larvae exposed to dietary TEA excreted more TEA than did tubules from controls fed a TEA-free diet. However, the rate of decline of hemolymph TEA concentration following ingestion or injection of TEA into the hemocoel was greater than that explicable by rates of active transport by the gut and Malpighian tubules (MTs). We propose that TEA concentrations in the hemolymph are reduced not only by active transport across the MTs and gut, but also by diffusion into the gut. The latter pathway is particularly important when larvae previously maintained upon TEA-enriched diet are transferred to a TEA-free diet. The ingestion of TEA-free food not only clears the gut lumen, but also creates a TEA-free compartment into which TEA may passively diffuse from the hemolymph.     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P-glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.     

Effects of biogenic amines and hormones on butterfly Malpighian tubules: Dopamine stimulates fluid secretion

Isolated Malpighian tubules of Papilio demodocus, the citrus swallowtail butterfly, were stimulated by biogenic amines as well as by cyclic AMP and the naturally occuring diuretic hormone. The greatest secretory response was obtained with 5-hydroxytryptamine, and smaller responses with dopamine and noradrenaline, but none of these amines could induce the maximal secretion rates obtained with cyclic AMP and diuretic hormone. Various other biogenic amines, hormones and pharmacological agents, including adrenaline, had no effect on Papilio tubules. The blocking agents cyproheptadine, phentolamine and propranolol did not alter the tubule response to biogenic amines.     

Effects of the microbial metabolite destruxin A on ion transport by the gut and renal epithelia of Drosophila melanogaster

Destruxins have been implicated in the infection process by entomopathogenic fungi and have been also found to be highly toxic when applied topically or ingested by different insect species. To gain insight into the mechanism of action of this toxin on insect internal organs, we have evaluated the effects of destruxin A on Drosophila melanogaster Malpighian tubules and gut tissues. Destruxin A was toxic when injected into adults; the calculated EC₅₀ was 0.11 mM. Destruxin A significantly inhibited fluid secretion rate by Malpighian tubules as well; the calculated IC₅₀ was 0.25 μM. The Na⁺ concentration in the secreted fluid increased significantly when tubules were exposed to 0.25 μM destruxin A, whereas pH and the concentrations of Ca²⁺ and K⁺ did not change. In gut, there was no effect of destruxin on H⁺ flux, but there was a significant decrease in K⁺ and Ca²⁺ absorption. The concentration of Ca²⁺ and K⁺ in the hemolymph of destruxin A‐injected flies was not significantly different from those of control flies after 3 h. Taken together, these results show that destruxin A produces differential effects on ion transport by renal and gut tissues. © 2012 Wiley Periodicals, Inc.     

Tetraethylammonium and nicotine transport by the Malpighian tubules of insects

We examined transepithelial transport of the prototypical type I organic cation (OC) tetraethylammonium (TEA) and the plant alkaloid nicotine by the isolated Malpighian tubules (MTs) of nine insect species from six orders. Isolated tubules were exposed to radiolabelled forms of either TEA or nicotine in the bathing (basal) fluid. Luminal (apical) secreted fluid was collected and TEA or nicotine concentration was determined. Active net transport of nicotine from bath to lumen was observed by the MTs of all the insects studied. TEA was also transported from bath to lumen in MTs of all species except Rhodnius prolixus and Aedes aegypti. MTs of both of these blood feeders did not show active transport of TEA under normal physiological conditions. Transport of TEA but not nicotine increased during the moult in the MTs of Rhodnius, but the concentrations of TEA in the secreted fluid were still consistent with passive accumulation in response to the lumen-negative transepithelial potential. Nicotine transport by Rhodnius MTs was inhibited by the type II OC quinidine, a known p-glycoprotein inhibitor, but not by the type I OCs N-methylnicotinamide or cimetidine. Taken together, the results suggest that active transport of OCs by the MTs is common among species from different orders and that transepithelial TEA and nicotine transport occur through separate pathways.     

Drosophila melanogaster larvae detect low doses of UVC radiation as manifested by a writhing response

Exposure of insects and higher orders of animals to UVC radiation has been shown to result in toxicity with a delayed expression. We have observed an immediate writhing response in slowly wandering third instar larvae of Drosophila melanogaster exposed to UVC radiation at doses that were not lethal. UVB and UVA radiation had no effect. Mutants for the visual and sensory systems appeared to respond normally, but CO₂ anesthesia resulted in reversible inhibition, ebony and silver mutants, which affect different pathways in catecholamine metabolism, showed an absent to reduced response. Using UVC lasers, we were able to demonstrate a response in different regions of the larval body. Furthermore, a 193 nm laser that penetrates only 2–5 μm was able to induce a response but unable to kill the larvae. These results suggest a photochemical reaction occurs in the cuticle which produces free radicals that stimulate the nerves and muscle which are present immediately below the epidermis. Possible targets for the UVC radiation are catecholic compounds secreted and processed into the cuticle of third instar larvae just prior to pupariation whose primary function is to crosslink the protein and carbohydrate components. © 1996 Wiley-Liss, Inc.     

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca²⁺ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca²⁺ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca²⁺ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca²⁺ regulation. Basolateral Ca²⁺ transport across distal tubules of Drosophila varied throughout the life cycle; Ca²⁺ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca²⁺ uptake and Ca²⁺ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca²⁺ transport is bidirectional. Ca²⁺ uptake across the distal segment of tubules of young adults and Ca²⁺ release across the distal segment of tubules of older adults was also suggestive of reversible Ca²⁺ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca²⁺ sequestration during periods of high dietary calcium intake and passive Ca²⁺ release during periods of calcium deficiency.     

Lifespan extension by dietary restriction is not linked to protection against somatic DNA damage in Drosophila melanogaster

Dietary restriction (DR) has been shown to robustly extend lifespan in multiple species tested so far. The pro-longevity effect of DR is often ascribed to an increase in cellular defense against somatic damage, most notably damage by reactive oxygen species (ROS), considered a major cause of aging. Especially irreversible damage to DNA, the carrier of genetic information, is considered a critical causal factor in aging. Using a recently developed transgenic Drosophila melanogaster model system harboring a lacZ-plasmid construct that can be recovered in E. coli , spontaneous DNA mutation frequency in flies under DR and ad libitum conditions are measured. Three different DR conditions, imposed by manipulating levels of different types of yeast sources, were tested in females and males of two lacZ reporter gene lines. Feeding with the ROS producer paraquat at 1 mM resulted in a rapid accumulation of somatic mutations, indicating that the frequency of mutations at the lacZ locus is a reliable marker for increased oxidative stress. However, none of the DR conditions altered the accumulation of spontaneous mutations with age. These results suggest that the beneficial effects of DR are unlikely to be linked to protection against oxidative somatic DNA damage.     

Fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus, and their response to diuretic hormone

ABSTRACT. A simple modification of the Ramsay method is described for the isolation in vitro of Malpighian tubules from Acheta domesticus. Isolated tubules continue to secrete for many hours in a simple Ringer solution. The rate of secretion is linearly related to the length of tubule within the bathing fluid.
Secretion rates during the immediate post-equilibration period relate to the physiological state of the donor insect. Highest rates are recorded in tubules removed from animals during the scotophase when the crickets are most actively feeding. Starvation reduces the rate of secretion and when previously starved insects are given access to food the rate increases. These results are consistent with variations in the haemolymph titre of DH that are induced by feeding.
Tubule fluid secretion is stimulated by either CC extract (1 gland pair/50 μ1) or dibutyryl cAMP (10^-3M). CC extracts increase the rate of fluid secretion by c. 200 pl/mm/min for up to 6 h after the tubules have been isolated. The response varies little with the age or physiological state of the donor insect. However, tubules from newly moulted insects secrete at very low rates and respond poorly to CC extract, although they are stimulated by cAMP. The significance of these results is discussed with particular reference to the use of Acheta tubules as a bioassay preparation for DH.     

Diuretic factors and second messengers stimulate secretion of the organic cation TEA by the Malpighian tubules of Drosophila melanogaster

This study showed that four factors which stimulate transepithelial fluid secretion and inorganic ion transport across the main segment of the Malpighian tubules of Drosophila melanogaster also stimulate transepithelial secretion of the prototypical organic cation tetraethylammonium (TEA). TEA fluxes across the Malpighian tubules and gut were measured using a TEA-selective self-referencing (TEA-SeR) microelectrode. TEA flux across isolated Malpighian tubules was also measured using a TEA-selective microelectrode positioned in droplets of fluid secreted by tubules set up in a modified Ramsay assay. TEA flux was stimulated by the intracellular second messengers cAMP and cGMP, which increase the lumen-positive transepithelial potential (TEP), and also by tyramine and leucokinin-I (LK-I), which decrease TEP. The largest increase was measured in response to 1 micromol l-1 LK-I which increased transepithelial TEA flux by 72%. TEA flux in the lower tubule was stimulated slightly (13%) by 1 micromol l-1 tyramine but not by any of the other factors. TEA flux across the midgut was unaffected by cAMP, cGMP or tyramine. This is the first study to demonstrate the effects of insect diuretic factors and second messengers on excretion of organic cations.