Tetraethylammonium and nicotine transport by the Malpighian tubules of insects

ARTICLE IN PRESS

Journal of Insect Physiology 52 (2006) 487–498 www.elsevier.com/locate/jinsphys

Tetraethylammonium and nicotine transport by the Malpighian tubules of insects M.R. Rheault, J.S. Plaumann, M.J. O’Donnell Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ont., Canada L8S 4K1 Received 17 June 2005; received in revised form 23 January 2006; accepted 24 January 2006

Abstract 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. r 2006 Published by Elsevier Ltd. Keywords: Malpighian tubules; Organic cation transport; p-Glycoprotein; Tetraethylammonium; Nicotine

1. Introduction Renal excretion of organic cations (OCs) was first demonstrated in vertebrate tissues nearly 60 years ago (Sperber, 1947; Rennick et al., 1947). OCs represent a structurally diverse group of primary, secondary, tertiary or quaternary amines with a net positive charge on the amine nitrogen at physiological pH (Rennick, 1981) and may include both endogenous compounds, xenobiotics and xenobiotic metabolites. Endogenous OCs include choline, N-methylnicotinamide (NMN) and catecholamines, while xenobiotics include environmental pollutants, drugs such as tetraethylammonium (TEA), animal toxins and plant alkaloids. Excretion of a wide range of organic compounds Corresponding author. The Whitney Laboratory, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080, USA. Tel.: +1 904 461 4070; fax: +1 904 461 4052. E-mail address: [email protected]fl.edu (M.R. Rheault).

0022-1910/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.jinsphys.2006.01.008

by an OC transport system appears to be a common characteristic of the renal tissues of both invertebrates and vertebrates (Miller and Holliday, 1987; Boom et al., 1992; Hawk and Dantzler, 1984; McKinney et al., 1981; Wright et al., 2004). Pritchard and Miller (1993, 1996) have reviewed the physiological evidence for the ‘‘classical’’ OC pathway. This ‘‘classical’’ OC transport pathway includes three steps: (1) carrier-mediated potential driven uptake of OCs through a single pathway at the basolateral membrane, (2) intracellular sequestration of the cation and (3) luminal exit through OC/proton exchange or by a pglycoprotein. A recent review incorporating new physiological and molecular evidence has indicated that the ‘‘classical’’ model is an oversimplification of the OC transport pathway (Wright and Dantzler, 2004). It is now clear that there are multiple OC transporters with different selectivities and affinities for a diverse array of structurally different OCs at both the basolateral and luminal membranes of vertebrate renal cells.

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The insect Malpighian tubules (MTs) along with the hindgut are considered analogous to the vertebrate renal tubule. Although mechanisms of inorganic ion transport in insect MTs and their regulatory control have been extensively studied and reviewed (Dow and Davies, 2001; O’Donnell and Spring, 2000), there is strikingly little known about the mechanisms underlying the transport of OCs by the MTs of insects. Nijhout (1975) has demonstrated that the tubules of larval Manduca sexta secrete the basic (cationic) dyes methyl green and methylene blue. Insect MTs also actively secrete the plant alkaloid nicotine (Maddrell and Gardiner, 1976) and the archetypical pglycoprotein substrate vinblastine (Gaertner et al., 1998). The possibility that nicotine transport in insect MTs is mediated by a p-glycoprotein-like mechanism is suggested by the observations that verapamil, a known p-glycoprotein inhibitor, blocks the transport of nicotine, while nicotine interferes with the transport of vinblastine by the isolated MTs from larval Manduca (Gaertner et al., 1998). We have previously demonstrated both carrier-mediated basolateral uptake (Rheault et al., 2005) and active transepithelial transport (Rheault and O’Donnell, 2004) of the prototypical type I OC TEA by the isolated MTs of Drosophila melanogaster. A recent study by Bijelic and O’Donnell (2005) has proposed that the transport of TEA across the apical membrane in Drosophila MTs occurs through a TEA/H+ exchange mechanism. Thus, it appears that TEA and nicotine excretion by insect MTs are mediated by two separate transport pathways. Although previous studies have demonstrated transport of TEA or nicotine by the MTs of different insects no study has demonstrated the transport of both of these substrates by the MTs of the same insect. In this study, we have used [14C] radiolabelled tracers to examine transepithelial transport of nicotine and TEA in isolated MTs from nine species of insects from six different orders. We have also measured the influence of physiological age and influence of feeding on TEA and nicotine excretion in Rhodnius tubules because an earlier study showed that transport of the organic anion p-aminohippurate is unaffected by age but is stimulated by consumption of a blood meal (Maddrell and Gardiner, 1975). Our findings indicate that active transport of the type I OC TEA and the type II OC nicotine involves separate pathways and that transport is common to the MTs of most but not all of the species studied. 2. Materials and methods 2.1. Insects MTs of the following insects were examined: D. melanogaster Meigen (Diptera) adult females; Rhodnius prolixus Sta˚l (Hemiptera) fifth instar larvae and adults; Oncopeltus fasciatus Dallas (Hemiptera) adults; Aedes aegypti L. (Diptera) fourth instar larvae and adult females; Acheta domesticus L. (Orthoptera) adult; Locusta migra-

toria L. (Orthoptera) adult males; Tenebrio molitor L. (Coleoptera) adult and larvae; Periplaneta americana L. (Dictyoptera) adults; Trichoplusia ni Hu¨bner (Lepidoptera) fourth instar larvae.

2.2. Insect rearing conditions All insects were maintained at temperatures ranging from 20 to 25 1C, and ambient humidity on a 12:12 h light dark cycle unless otherwise stated. D. melanogaster (Oregon R. strain), R. prolixus, A. aegypti and P. Americana were obtained from laboratory cultures maintained in the Department of Biology, at McMaster University. D. melanogaster were maintained according to procedures described previously by Ashburner (1989). R. prolixus were maintained at 25–28 1C and 60% relative humidity. Nymphs and adults were sorted by age into jars and fed en masse on rabbit blood to synchronize development. Eggs of the mosquito A. aegypti were hatched in plastic containers filled with distilled water (65 mmol l
1 Na+). Larvae were maintained in these containers until they reached the pupal stage (10–12 days) and were fed every second day with a solution of liver powder and yeast made up in distilled water. Pupae were then removed and placed in a dish containing fresh distilled water inside a mesh cage and allowed to emerge as adults. The adults were fed on a 2% w/v sucrose solution from a small dish into which a cotton wick had been placed. P. americana were fed on a diet of dry ground Purina cat chowsTM supplemented weekly with lettuce and apple. Water was provided continuously. O. fasciatus and T. molitor were obtained from Ward’s Natural Science (St. Catharine’s, Ont., Canada). First instar to adult O. fasciatus were sorted by age into jars and were fed a diet of dried milkweed seeds and raw sunflower seeds. Water was supplied continuously from a small dish into which a cotton wick had been placed at the bottom of each jar. T. molitor were maintained in dry bran cultures supplemented with apple slices as a source of moisture. Larvae of similar size were used in all experiments. Adult crickets (A. domesticus) were obtained from a local pet supplier and were fed on a paste composed of oats, sugar, skim milk powder and water. Lettuce and water were provided ad libitum. Adult male locusts (L. migratoria) were obtained from a colony in Dr. Angela Lange’s laboratory at the University of Toronto at Mississauga, Canada, where they were raised under crowded conditions on a 12 h light and 12 h dark regime at 30 1C. Locusts were fed fresh wheat seedlings supplemented with bran. Insects were used for experiments immediately upon their arrival at McMaster University. Fourth instar larval cabbage loopers (T. ni) were obtained from Dr. Cam Donly’s laboratory at the Southern Crop Protection and Food Research Centre at London, Canada where they were raised on an artificial diet using standard procedures (16:8 h light:dark photoperiod at

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27 1C; Guy et al., 1985). Insects were used for experiments immediately upon their arrival at McMaster University. 2.3. Ringer’s solutions The Ringer’s solutions used for dissection and experiments on isolated MTs of each species can be found in Table 1. In the absence of published recipes for T. ni saline, dissection and fluid secretion assays of isolated MTs of this species were done in D. melanogaster saline. 2.4. Dissection and fluid secretion rates Procedures for the preparation of isolated MTs have been described previously for D. melanogaster (Dow et al., 1994), R. prolixus (Maddrell, 1969), A. aegypti (Scott et al., 2004), A. domesticus (Coast, 1988), L. migratoria (Anstee et al., 1979), P. Americana (Kay et al., 1992) and larval T. molitor (Wiehart et al., 2002). Tubules from adult T. molitor were isolated as described by Nicolson and Hanrahan (1986) for Onymacris plana. Procedures for dissection of the MTs from Trichoplusia larva were similar to those used for the Tenebrio larval tubules. Tubules of O. fasciatus were dissected as described by Meredith et al. (1984) and the fluid secreting segment (segment II) was used.

489

Rates of transepithelial fluid secretion were measured in a modified Ramsay (1954) assay. Briefly, isolated tubules from all insects were transferred on fine glass probes from the dissecting saline to 20–100 ml droplets of saline in a Sylgard lined Petri dish filled with paraffin oil. Tubules from Oncopeltus, Rhodnius, Drosophila, Aedes and Acheta are blind ended and therefore the distal end of each tubule was immersed in the bathing droplet. Distal ends were removed from tubules of Locusta, Tenebrio, Periplaneta and Trichoplusia during dissection. For these tubules, the cut distal end was pulled out of the bathing saline and sealed by wrapping it around a fine steel pin under paraffin. The open proximal end of all tubules was pulled out of the bathing droplet and wrapped around a second fine steel pin under paraffin. Secreted fluid droplets formed at the open proximal end of the tubule were collected at 30–60 min intervals with a fine glass probe. Droplet diameters (d) were measured using an ocular micrometer, and droplet volume (nl) was calculated as pd 3 =6. Secretion rate (nl min
1) was calculated by dividing droplet volume by the time (min) over which the droplet formed. Only the fluid secreting segments of the tubules from Oncopeltus (segment II), Rhodnius (upper tubule) and Drosophila (main segment) were used in most experiments. Tubules of Rhodnius and Oncopeltus were stimulated to secrete by adding 10
5 mmol l
1 5-HT or 1 mmol l
1 cAMP to their respective

Table 1 Composition of experimental salines (concentrations in mmol l
1) Species Oncopeltus fasciatus

NaCl 20 KCl 24 2 CaCl2 2 MgCl2 MgSO4 — NaHCO3 — NaH2PO4 2.5 3.5 Na2HPO4 NaOH — Arginine — Leucine — Glycine — Proline 5 Alanine 5 Serine — Histidine — Succinic acid 5.5 Malic acid 5.5 Tri-sodium 11 citrate HEPES — Sucrose 193 Glucose 6.7 Glutamine 2.7 pHa 7.0 a

Rhodnius prolixus (8.6 K+)

Rhodnius prolixus (4.0 K+)

Drosophila Aedes melanogaster aegypti (larva)

Aedes aegypti (adult)

Acheta domesticus

Locusta migratoria

Tenebrio molitor

Periplaneta americana

129 8.6 2 8.5 — 10.2 4.3 — — — — — — — — — — — —

133.6 4.0 2 8.5 — 10.2 4.3 — — — — — — — — — — — —

117.5 20 2 8.5 — 10.2 4.3 — — — — — — — — — — — —

33 3.3 5.5 — 0.7 5.5 — — — 3.7 15.8 — 5.5 — — 9.6 — — —

150 3.4 1.7 0.6 — 1.8 — — — — — — — — — — — — —

100 8.6 2 8.5 — 4 4 — 11 — — — 24 — — — — — —

100 8.6 2 8.5 — 4 4 — 11 — — — — — — — — — —

90 50 2 5 — 6 4 — — — — 10 10 — 10 10 — — —

100 8.6 2 8.5 — 4 4 — 11 — — — — — — — — — —

8.6 — 20 10 7.0

8.6 — 20 10 7.0

8.6 — 20 10 7.0

27.5 — 11 10 7.0

25 — 5 — 7.1

10 — 25 — 7.2

25 — 34 10 7.2

pH adjusted using NaOH and HCl for all solutions.

— — 50 10 7.0

25 — 34 10 7.2

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baths. All other tubules secreted fluid spontaneously when immersed in their respective bathing salines. TEA and nicotine transport were measured by adding [14C] labelled TEA or nicotine (100 mmol l
1) to the saline bathing the isolated tubules. After fluid secretion rates had been measured, droplets of secreted fluid were collected and transferred into 4 ml of aqueous counting scintillant for b counting in a TriCarb 2900TR liquid scintillation counter (Perkin-Elmer, Woodbridge, Ont., Canada) for determination of secreted fluid TEA or nicotine concentration. TEA and nicotine transport rates of individual tubules were calculated as the product of fluid secretion rate (nl min
1) and secreted fluid TEA or nicotine concentration (mmol l
1) and were expressed as pmol min
1 tubule
1. In experiments where it was necessary to compare fluid secretion rates, TEA secretion rates or nicotine secretion rates between species, the measured secretion rates (nl min
1) were divided by the length (mm) of tubule immersed in the bathing saline and then multiplied by 60 min. The resultant secretion rates were expressed as nl h
1 mm
1 of tubule. The product of these ‘‘length standardized’’ secretion rates and secreted fluid TEA or nicotine concentration yielded TEA and nicotine transport rates (pmol h
1 mm
1), which could be compared between species. Pharmacological agents were added to the saline after baseline fluid secretion rates and TEA or nicotine transport rates had been established. A modified Ramsay preparation previously described by Haley and O’Donnell (1997) was used in experiments where the transport of TEA by the whole tubule (WT) and main segment of Rhodnius tubules was studied (see inset Fig. 3). Upper MTs (UMTs) were bathed in a droplet of Rhodnius saline containing 8.6 mmol l
1 K+. Lower MTs (LMTs) were bathed in saline containing 4 mmol l
1 K+ (see Table 1). Rhodnius LMT transport of TEA was assessed using a preparation in which the UMT and LMT were each bathed in separate droplets containing 14C labelled TEA. Secreted fluid droplets were collected from WTs after which the LMT was removed and droplets of secreted fluid were collected from the UMT only. Subsequently, the concentration of TEA in the secreted fluid from WTs and UMTs was determined. TEA transport rate for the LMT was calculated by subtracting the transport rate of the UMT from those of the WT. 2.5. Thin-layer chromatography (TLC) TLC was done to determine whether the [14C] labelled nicotine was metabolized by the MTs of the insects used in our experiments. TLC separations were preformed on 20  20 cm2 chromatographic plates coated with silica gel 60 F254 (EMD Chemicals, Fisher Scientific, Nepean, Ont., Canada). [14C] labelled secreted fluid, bathing saline and nicotine (2 ml) were mixed with 1 ml of 1 mmol l
1 cold nicotine and loaded on the origin of the TLC plates. Chromatograms were developed to a distance of 17.5 cm in a vertical chamber (Kontes, Fisher Scientific, Nepean,

Ont., Canada) using ethylacetate:methanol:triethylamine (80:15:5, v/v) as the mobile phase. Chromatographic plates were exposed to X-ray film for 6 days at
80 1C before being developed. Relative retention factors (Rf) were calculated as Rf ¼

MDsample , MDsolvent

(1)

where Rf is the relative retention factor, MDsample the migration distance of the sample of interest measured in centimeters and MDsolvent the migration distance of the solvent front in centimeters. TEA containing samples were not analysed by TLC because a search of the literature indicates that no pathways for the metabolism of TEA have been reported. Moreover, measurement of secreted fluid TEA concentrations using TEA-selective microelectrodes in a previous study (Rheault and O’Donnell, 2004) yield identical results to those published in this study using [14C] radiolabelled TEA.

2.6. Chemicals [14C] labelled TEA (55.6 mCi mmol
1) and nicotine (55 mCi mmol
1) were obtained from American Radiolabelled Chemicals (St. Louis, MO, USA). All other chemicals were purchased from Sigma-Aldrich (Oakville, Ont., Canada). Chemicals were dissolved in standard bathing saline, ethanol or dimethylsulphoxide (DMSO). The final concentration of DMSO or ethanol in the experimental treatments was p1%. This percentage of each solvent was tested and had no effect on any of parameters measured in this study.

2.7. Data and statistics TEA and nicotine concentrations in secreted fluid (mmol l
1) were calculated from disintegrations per minute per volume of secreted fluid and bathing droplet specific activity. Measurements of fluid secretion rates, secreted fluid TEA or nicotine concentrations, secreted fluid-to-bathing medium (S/B) ratios of TEA or nicotine, and TEA or nicotine transport rates are expressed as mean7SEM for (n) number of tubules. Two-sample F-tests were used to compare the variances of the data for the control and experimental groups. Depending on the outcome of each F-test, differences between experimental and control groups were compared using unpaired t-tests assuming either equal or unequal variances. The responses of the same group of tubules before and after an experimental treatment were compared using a paired t-test. Where appropriate, data were analysed by one-way analysis of variance (ANOVA) followed by a Tukey–Kramer multiple comparisons test. In all cases, differences were considered significant if Po0:05.

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In this study, we measured the ability of the MTs of nine species from six different orders of insects, to transport the OCs TEA and nicotine. Fig. 1 shows the fluid secretion rates (Fig. 1A), secreted fluid TEA concentration (Fig. 1B) and the calculated rates of TEA transport (Fig. 1C), for segment II of the MTs of the Milkweed bug, O. fasciatus. The MTs of Oncopeltus secreted fluid at an initial rate of 5.4470.43 nl min
1 (n ¼ 19). The addition of 0.1 mmol
1 TEA to the bathing medium had no effects on fluid secretion rates, measured after 60 min of exposure (P40:05, paired t-test). Similar results were observed for the effects of 0.1 mmol l
1 nicotine on the tubules of Oncopeltus. TEA reached a maximum concentration of 1.5470.15 mmol l
1 (n ¼ 19) in the secreted fluid of the MTs of Oncopeltus (Fig. 1B), which corresponded to an S/ B ratio of 15.5471.22 (n ¼ 19). Fig. 1C shows that TEA transport rates reached a maximum value of 6.9870.56 pmol min
1 tubule
1 (n ¼ 19). Further experiments of TEA or nicotine transport by the MTs from all other insect species were conducted in an identical manner.

6 Fluid secretion rate (nl min-1)

3. Results

(A)

491

5 4 3 2 1 0 0

25

50

75

100

125

150

[TEA]SF (m mol l-1)

2.0

*

1.5

1.0

0.5

0.0

3.1. Rates of fluid secretion of isolated MTs

(B)

75

100

125

150

75 100 Time (min)

125

150

*

6

4

2

0

The concentration of TEA and nicotine (mmol l ) in the fluid secreted by the MTs of all species is shown in Fig. 2B and Fig. 3B, respectively. In all cases, the concentration of TEA or nicotine in the droplet bathing the tubule was 0.1 mmol l
1. TEA concentrations ranged from a high of 3.0870.52 mmol l
1 (n ¼ 23) in fluid secreted by the MTs of Locusta to 0.00370.001 mmol l
1 (n ¼ 12) in the fluid secreted by the MTs of Rhodnius. Nicotine concentrations ranged from a high of 2.1070.15 mmol l
1 (n ¼ 30) in fluid secreted by the MTs of Locusta to 0.2070.02 mmol l
1 (n ¼ 16) in the fluid secreted by the MTs of Oncopeltus.

50

0

3.2. TEA and nicotine concentrations in secreted fluid
1

25

8

Rate of TEA transport (pmol min-1 tubule-1)

In order to compare rates of fluid secretion between species, data were expressed per unit length (mm) of tubule (Fig. 2A and 3A). Addition of 0.1 mmol l
1 TEA or nicotine to the bathing medium had no effects on the rates of fluid secretion by the MTs in any of the species studied (e.g. Fig. 1A). In this study, the isolated tubules from the blood feeding bug R. prolixus stimulated with 5-HT (10
5 mmol l
1) exhibited the highest rates of fluid secretion (167710 nl h
1 mm
1, n ¼ 11), which was 133 times faster than the rate of fluid secretion observed by the unstimulated tubules from the cockroach, P. americana. Surprisingly, 5-HT stimulated tubules from R. prolixus secreted fluid at a rate only 1.6 times faster than unstimulated tubules from the cabbage looper, T. ni, whose secretion rates are reported here for the first time.

0

(C)

25

50

Fig. 1. Fluid secretion rate (A), secreted fluid concentration of TEA (B) and TEA transport rates (C) of segment II of the isolated tubules from O. fasciatus before and after the addition of 100 mmol l
1 [14C] labelled TEA to the bathing medium. The arrow indicates the time at which TEA was added to the bath. Each point represents mean7SEM for n ¼ 13 tubules. The dotted line in panel B indicates the concentration of TEA in the bathing medium. The asterisk (*) denotes that a new steady state for secreted fluid concentration and rates of TEA transport had been reached 60 min after the addition of TEA to the bathing medium. The symbol (y) indicates the values reported in Fig. 2. Where no error bars are apparent, error bars are smaller than the symbol used.

ARTICLE IN PRESS M.R. Rheault et al. / Journal of Insect Physiology 52 (2006) 487–498

492

200 Fluid secretion rate (nl h-1 mm-1)

30 20 10

(A)

50

0 2.5

Secreted fluid nicotine concentration (m mol l-1)

4 Secreted fluid TEA concentration (m mol l-1)

100

(A)

0

3

2

1

0

(B)

150

2.0

1.5

1.0

0.5

(B)

0.0

Rate of nicotine secretion (pmol h-1 mm-1)

Fluid secretion rate (nl h-1 mm-1)

200 170 140 110 80

70

Rate of TEA secretion (pmol h-1 mm-1)

70 60

T. n

T.

R

(C)

Species

R

(C) Fig. 2. Fluid secretion rates (A), secreted fluid concentration of TEA (B) and rate of TEA transport (C) for isolated MTs exposed to 100 mmol l
1 [14C] labelled TEA in the bathing saline. Each bar represents a mean7SEM value where n ¼ 11–55 tubules from each species.

in

in sta

r) e) e) tor ia us na ter tic ator arva oli rica sta gas rva s a e r l m l o e ig r ( T. th i( an om .f am O s (5 mel ypti . d L. m olito P. A . u g m D lix ae T. ro A. .p

s

tu

ia

c as

r)

0

h

pt ii

gy

ae

A.

in

P. am ni eric a (4 th na in sta r)

) s r r) ter e) ria ae ito cu sta gas rva sti rato larv mol a e l o ( T. th an i( om ig r .f O s (5 mel ypti . d L. m olito A . u g m lix D . ae T. ro A .p

s

tu

ia

c as

10

4t

0

20

i(

10

30

pt ii

20

40

gy

30

50

ae

40

60

A.

50

Species

Fig. 3. Fluid secretion rates (A), secreted fluid concentration of nicotine (B) and rate of nicotine transport (C) for isolated MTs exposed to 100 mmol l
1 [14C] labelled nicotine in the bathing saline. Each bar represents a mean7SEM value where n ¼ 10–48 tubules from each species.

3.3. Transport rates of TEA and nicotine by isolated MTs The transport rates for TEA and nicotine for the MTs of all species are shown in Fig. 2C and Fig. 3C, respectively. TEA and nicotine transport rates were calculated by multiplying the ‘‘length standardized’’ fluid secretion rate of the tubule by the concentration of TEA and nicotine in

the secreted fluid. Our results showed that Trichoplusia MTs had the highest rates of TEA secretion while Aedes MTs had the lowest rate of TEA secretion. Rates of nicotine transport were the highest in the MTs of Rhodnius and the lowest in the MTs of Periplaneta.

ARTICLE IN PRESS M.R. Rheault et al. / Journal of Insect Physiology 52 (2006) 487–498

Using unpaired t-tests we compared the ability of the MTs from each species to transport TEA versus nicotine. The MTs of Oncopeltus, Drosophila, larval Tenebrio and adult Tenebrio exhibited a 6.7-, 1.5-, 9.0- and 4.9-fold higher rate of TEA transport relative to their rate of nicotine transport, respectively. In contrast, the MTs of Rhodnius, larval Aedes and adult Aedes, exhibited a 134.8-, 7.3- and 29.1-fold higher rate of nicotine transport relative to their rate of TEA transport, respectively. The transport rates of TEA versus nicotine by the MTs from Acheta, Locusta, Periplaneta and Trichoplusia were not statistically different. 3.4. Steady-state secreted S/B ratios of TEA and nicotine by isolated insect MTs We have used the secreted fluid TEA and nicotine concentrations reported in Figs. 2B and 3B to determine the steady-state secreted S/B concentration ratios for both TEA and nicotine in all of the insects we studied (Table 2). The S/B ratios for TEA ranged from a low value of 0.0370.01 by the 5-HT stimulated MTs of Rhodnius to a high of 25.0374.53 by the MTs of Locusta. The MTs of Locusta also exhibited the highest S/B ratio for nicotine, which was 21.6471.51. The lowest S/B ratio for nicotine observed was 2.0170.26 from the MTs of Oncopeltus. The question arises of whether our observed S/B ratios represent active transport of TEA and nicotine or passive accumulation in the tubule lumen. Both TEA and nicotine exist almost completely in their positively charged form at physiological pH (Pratt, 1990; Maddrell and Gardiner, 1976). We, therefore, calculated the maximum predicted steady-state S/B ratios for TEA and nicotine passive accumulation at a concentration of 0.1 mmol l
1 in the bath using the least positive available transepithelial potentials (TEPs) reported in the literature for each species

493

MTs (Table 2). The least positive reported TEP was used for each species because this allowed us to calculate the highest potential predicted steady-state S/B ratio for passive accumulation of an OC in the tubule lumen. Active transport of TEA or nicotine was indicated if our measured S/B ratio exceeded the maximum predicted S/B ratio for passive accumulation. The isolated MTs of all species, for which TEP measurements were available, demonstrated active transepithelial transport of nicotine. The MTs with the greatest potential for active transport of nicotine were from Locusta, as indicated by an S/B ratio 30 times higher than the S/B ratio predicted by passive accumulation. The insect MTs with the least potential for active transport of nicotine were from Rhodnius and exhibited an S/B ratio that was only 1.3 times greater than that predicted for passive accumulation of nicotine. Unlike nicotine, not all of the insect MTs studied actively transported TEA. The MTs of R. prolixus and A. aegypti had S/B ratios for TEA which could be accounted for by passive accumulation. In fact, the S/B ratio for isolated tubules of Rhodnius was o1% of that predicted on the basis of passive accumulation, indicating that they had the least potential for transepithelial TEA transport. S/B ratios for TEA in MTs of all other species were higher than those consistent with passive accumulation, indicating active transport of TEA. The MTs from Tenebrio larva exhibited the greatest potential for TEA transport with an S/B ratio that was 41 times higher than the maximum predicted steady-state S/B ratio for passive accumulation. 3.5. TEA transport by the lower tubule of R. prolixus Previously, we have shown that the LMT of D. melanogaster actively transports TEA into the lumen at a rate four-fold higher than the main segment (Rheault and O’Donnell, 2004). Given that our results in Table 2

Table 2 Steady-state secreted fluid-to-bath (S/B) TEA and nicotine ratios Species

Transepithelial potential (mV)

Maximum predicted steadystate TEA or nicotine S/B ratios for passive accumulation

Measured steady-state S/B ratios

TEA R. prolixus D. melanogaster A. aegypti (adult) A. aegypti (larva) A. domesticus L. migratoria T. molitor (larva) T. molitor (adult) O. fasciatus P. americana T. ni


35 +32 +15

3.90 0.30 0.54

+4.3 +8.7 +24

0.85 0.71 0.39

NA NA NA

NA NA NA

0.0370.01 8.0770.43 0.2770.02 0.6170.11 8.7970.94 25.0374.53 15.9772.66 14.1372.51 15.5471.22 11.0071.07 5.1070.33

Reference

Nicotine (12) (25) (19) (11) (55) (16) (12) (13) (19) (29) (20)

4.9270.36 4.5570.25 4.64 70.70 5.8370.78 10.0770.48 21.6471.51 2.5570.21 2.5270.35 2.0170.26 8.9970.55 4.7570.24

(11) (10) (13) (29) (54) (27) (13) (19) (16) (11) (24)

Ramsay (1954) O’Donnell et al. (1998) Beyenbach et al. (2000) Coast and Kay (1994) Fathpour et al. (1983) Wiehart et al. (2002)

Values for measured TEA and nicotine S/B ratios are means7SEM (n); where n ¼ number of tubules. Transepithelial potentials were obtained from the corresponding references. Maximum predicted steady-state TEA or nicotine S/B ratios for passive distribution at electrochemical equilibrium are calculated from the transepithelial potential. NA, not available.

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indicate that the UMT of Rhodnius does not actively transport TEA, it was of interest to determine if the LMT of Rhodnius was a site of active TEA transport. Results of experiments to test this hypothesis are shown in Fig. 4. In

order to assess LMT transport, a modified Ramsay preparation was used (see inset Fig. 4). TEA transport rates of the UMT and LMT were similar (paired t-test, P40:05, n ¼ 9). The TEA concentration in the fluid secreted by the WT was 0.00870.001 mmol l
1, only 8% of the TEA concentration present in the fluid bathing the WT. Thus, it appears that neither the UMT nor LMT of Rhodnius actively transported TEA.

WT

3.6. Effects of physiological age and feeding on TEA and nicotine transport by the MTs of R. prolixus

UMT LMT

0.4

In this study, we measured the effect of feeding and the subsequent moult on both TEA and nicotine transport by the UMTs from fifth instar larval Rhodnius (Fig. 5). Consumption of a blood meal had no significant effect on TEA or nicotine transport by isolated MTs from fifth instar Rhodnius (one-way ANOVA, Po0:05, Tukey–Kramer multiple comparisons test). After feeding we measured TEA and nicotine transport by the MTs as the insects aged and subsequently moulted from fifth instar to adults. Rhodnius moulted over a 3-day period beginning on the 22nd day after the blood meal. Twenty-three days after the blood meal, on day 47 of the experiment, fifth instars, which had visibly begun the moulting process, were collected and their tubules ability to transport TEA and nicotine were assessed. No change in nicotine transport by MTs in animals undergoing moult was observed. In contrast, TEA transport by the MTs of insects undergoing moult increased nearly six-fold. On day 54 of the experiment, 5 days after the last Rhodnius had moulted to an adult, TEA and nicotine transport by isolated adult MTs were measured. TEA and nicotine transport by adults

LMT = WT - UMT

0.3

0.2

0.1

0.0 WT

UMT

LMT

Fig. 4. Rates of TEA transport by the whole tubule (WT, filled bar), upper Malpighian tubule (UMT, open bar) and the lower Malpighian tubule (LMT, hatched bar) of fifth instar R. prolixus. Each bar represents mean7SEM value for n ¼ 9 tubules. The inset shows the corresponding arrangement of tubules and bathing droplets when secreted fluid was collected for analysis from WT versus UMT. The rate of TEA transport by the LMT was calculated as indicated. [14C] labelled TEA (100 mmol l
1) was added to droplets of saline bathing both the UMT and LMT.

Rate of nicotine transport (pmol min-1 tubule-1)

40

8 7

30

6 5

20

4 3

10

2 1

0

Rate of TEA transport (pmol min-1 tubule-1)

Rate of TEA transport (pmol tubule-1 )

0.5

0 0

4

8

12

16

20

24

28

32

36

40

44

48

52

56

Day Fig. 5. Effects of physiological age and feeding on TEA (K) and nicotine (J) transport by the upper Malpighian tubule of fifth instar, and adult R. prolixus. All animals received a blood meal on day 24. The shaded region indicates the period from which the first animal began to moult until the point at which the last animal had finished moulting. All points represent mean7SEM for n ¼ 6–12 tubules.

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was not significantly different from the rates observed in fifth instar larva prior to moult. 3.7. Effects of pharmacological agents on nicotine transport by the isolated MTs of Rhodnius The effects of 1 mmol l
1 NMN, cimetidine and quinidine on the rate of transport of nicotine by the UMT of Rhodnius are shown in Fig. 6. In these experiments, nicotine was present in the bath at a concentration of 0.12 mmol l
1. Addition of 1 mmol l
1 NMN, and cimetidine had no significant effect on the rate of nicotine transport by Rhodnius tubules. In contrast, 1 mmol l
1 quinidine caused a 67% inhibition of nicotine transport. 3.8. Thin-layer chromatography The relative retention factor (Rf) for a [14C] labelled nicotine standard was 0.04370.01 when analysed using TLC (data not shown). Rf values of the secreted fluid from tubules bathed in [14C] labelled nicotine did not differ significantly (unpaired t-test, P40:05) from standards run on the same TLC plate. This indicates that there was no significant metabolism of nicotine present in the secreted fluid in any of the insect MTs tested. 60

495

4. Discussion We used conventional radioisotopic techniques and the Ramsay fluid assay to assess transport of the prototypical type I OC TEA and the representative plant alkaloid nicotine by isolated MTs from nine species of insect. We previously demonstrated active transepithelial transport of TEA by the MTs of D. melanogaster using TEAselective microelectrodes and the Ramsay fluid assay (Rheault and O’Donnell, 2004). A subsequent study has demonstrated a carrier-mediated potential-dependent mechanism for TEA uptake at the basolateral membrane (Rheault et al., 2005). Bijelic and O’Donnell (2005) have proposed that the uphill apical transport step for TEA involves TEA/H+ exchange across the apical membrane. Maddrell and Gardiner (1976) demonstrated that isolated MTs from R. prolixus (Hemiptera), Manduca sexta (Lepidoptera), Pieris brassicae (Lepidoptera), Calliphora erythrocephala (Diptera) and Musca domesticus (Diptera) rapidly remove the plant alkaloid nicotine from the bathing medium. In a subsequent study, Gaertner et al. (1998) proposed that the transport of nicotine and other alkaloids by the MTs of Manduca sexta was mediated by a p-glycoprotein-like mechanism. The data presented in this study provide additional support for the presence of separate active transport mechanisms for the transport of TEA and nicotine in the MTs of insects from a number of different insect orders.

Rate of nicotine transport (pmol tubule-1 min-1)

4.1. Use of radioisotopes for study of OC transport by insect MTs

40

* 20

ne ni ui Q

Ci m

et

id

di

in

e

N M N

Co nt

ro l

0

Fig. 6. Effects of N-methylnicotinamide (NMN), cimetidine and quinidine on the rate of nicotine transport by the upper Malpighian tubules of R. prolixus. Mean values7SEM (n ¼ 7–19 tubules). Asterisk (*) indicates a significant difference from control values (one-way ANOVA, Tukey– Kramer multiple comparisons post hoc test, Po0:05).

The use of radiolabelled TEA for the study of OC transport by isolated vertebrate renal tubules has been well documented (e.g. Hawk and Dantzler, 1984; Boom et al., 1992). In a previous study, we have utilized radiolabelled TEA to describe the basolateral uptake of OCs by Drosophila MTs (Rheault et al., 2005). This is the first time that radiolabelled TEA has been used to evaluate transepithelial transport of OCs by insect MTs. Our previous study utilized TEA-selective microelectrodes for measurement of TEA concentration in fluid secreted by Drosophila MTs (Rheault and O’Donnell, 2004). In that study, tubules bathed in 0.1 mmol l
1 TEA secreted fluid containing TEA at a concentration of 0.8470.09 mmol l
1 and the rate of TEA transport by isolated tubules was 0.4670.07 pmol tubule
1 min
1. Measurements of TEA transport in this study using [14C] labelled TEA at the same concentration in the bath are essentially identical with a measured secreted fluid TEA concentration of 0.8570.05 mmol l
1 and a TEA transport rate of 0.4770.05 pmol tubule
1 min
1, independently validating the accuracy of TEA-selective microelectrodes used in our previous study. In addition, we have confirmed a previous finding that tubules from Rhodnius transport the plant alkaloid nicotine (Maddrell and Gardiner, 1976). In our study, the rate of nicotine transport for the UMT of

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Rhodnius was 30.1571.36 pmol tubule
1 min
1 when the bathing medium contained nicotine at a concentration of 0.1 mmol l
1. This is nearly identical to the 30 pmol tubule
1 min
1 rate of nicotine transport reported 19 years earlier for a separate colony of laboratory maintained Rhodnius (see Fig. 5, Maddrell and Gardiner, 1976). 4.2. Active transport of TEA and nicotine by insect MTs For species for which the TEP across the MT is unknown, the TEP values that would be required for the passive accumulation of both TEA and nicotine at our measured S/B ratios can be calculated using the Nernst equation. For nicotine, TEPs of
18,
57 and
40 mV would be required for Oncopeltus, Periplaneta and Trichoplusia, respectively. For TEA, TEPs of
70,
62 and
42 mV, would be required for Oncopeltus, Periplaneta and Trichoplusia, respectively. It is important to note that positive TEP values for MTs of insects are normally expected. R. prolixus is the only insect to date for which a negative TEP has been reliably recorded (Ianowski and O’Donnell, 2001). Thus, we believe that the S/B ratios for TEA and nicotine transport reported here for Oncopeltus, Periplaneta and Trichoplusia represent active transport and not passive accumulation. In addition to confirming the previous findings that Rhodnius and Drosophila tubules actively transport nicotine and TEA, respectively, we have shown that tubules from eight additional species of insects actively secrete nicotine and TEA from bath (haemolymph) to lumen (Fig. 2B and C, Table 2). Four of the species we studied (P. americana, T. molitor, A. domesticus and L. migratoria) represent three orders of insects (Dictyoptera, Coleoptera and Orthoptera) in which nicotine transport by the MTs has not been previously reported. Six of the species we studied (O. fasciatus, A. domesticus, L. migratoria, T. molitor, P. americana and T. ni) represent five orders of insects (Hemiptera, Orthoptera, Coleoptera, Dictyoplera and Lepidoptera) in which TEA transport by the MTs has not been previously reported. Although previous studies (Maddrell and Gardiner, 1976; Gaertner et al., 1998; Rheault and O’Donnell, 2004) have demonstrated the transport of nicotine or TEA in different insect species this is the first time that the transport of both TEA, a representative type I OC pathway substrate and nicotine, a putative p-glycoprotein substrate, by the MTs has been studied in the same insect. Tubules from different species vary in their rates of active transport of TEA or nicotine. Tubules from Oncopeltus, Drosophila and Tenebrio have higher rates of transport of TEA than nicotine per unit length. Tubules of Rhodnius and Aedes have higher rates of transport of nicotine relative to TEA per unit length. Finally, tubules from Acheta, Locusta, Periplaneta and Trichoplusia all exhibit similar rates of transport of TEA and nicotine per unit length. All TEA transport by the tubules of Rhodnius and Aedes, both blood feeding insects, could be accounted for by passive

accumulation. The absence of active transport of the prototypical type I OC TEA by the MTs of these species is puzzling. One explanation is that potentially toxic levels of type I OCs in the haemolymph may be avoided by excretion across other epithelia, such as the gut. The midgut of Drosophila, for example, transports TEA from haemolymph to lumen (Rheault and O’Donnell, 2004). Alternatively, type I OCs in these species may be metabolized into other compounds, which are either non-toxic or are readily excreted by the tubules or other epithelia. Differences in transport rates of TEA and nicotine by the MTs in some species suggests two possibilities: (1) that there is a single transport pathway for the transport of TEA and nicotine in insect MTs and that the specificity of this pathway for individual substrates varies between species, or (2) that there are separate transport pathways for TEA and nicotine transport in the tubules. There is evidence to support the existence of separate pathways for TEA and nicotine transport in renal epithelia of both vertebrates and insects. In vertebrates, the apically localized OC/H+ antiporter has been shown to mediate TEA efflux out of cells in the apical membranes (Inui et al., 1985). Additionally, functional separation of TEA and nicotine transport in the porcine kidney epithelial cell line LLC-PK1 has been demonstrated (Takami et al., 1998). However, the identity of the mechanism responsible for the transport of the nicotine in this cell line remains unclear. In insect MTs, the functional separation of TEA and nicotine transport is suggested by a number of observations in different insects. Transport of vinblastine, a known pglycoprotein substrate, is inhibited by nicotine, while transports of both nicotine and vinblastine are inhibited by verapamil, a p-glycoprotein inhibitor in the MTs of Manduca sexta (Gaertner et al., 1998). In addition, the MTs of Manduca sexta (Murray et al., 1994) and R. prolixus (Murray, 1996) show positive immunostaining for p-glycoprotein. In our previous study, verapamil had no effect on TEA transport by the main segment of Drosophila MTs (Rheault and O’Donnell, 2004). Bijelic and O’Donnell (2005) subsequently proposed TEA transport in the main segment of isolated Drosophila tubules was mediated at the apical membrane by OC/H+ exchange. Moreover, in our studies of the effects of physiological age on TEA and nicotine transport by Rhodnius tubules TEA transport increased nearly six-fold while no change in nicotine transport was observed during the period that animals were moulting. This selective induction of only one substrate would not be possible if both substrates were utilizing the same transport pathway and therefore implies separate transport pathways for TEA and nicotine. In a previous study, we showed that cimetidine blocks transepithelial TEA transport in the tubules of D. melanogaster (Rheault and O’Donnell, 2004). Additionally, studies in a vertebrate renal cell line have shown that quinidine, but not NMN and cimetidine block the apical transport of nicotine but not TEA and have concluded that this demonstrates distinct transepithelial transport

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pathways for TEA and nicotine (Takami et al., 1998). In this study, we also demonstrated that nicotine transport by Rhodnius tubules was blocked by quinidine but not by the OCs NMN or cimetidine. Thus, pharmacological evidence also indicates that there are two distinct pathways for TEA and nicotine transport in insect MTs. Taken together with previous studies on TEA and nicotine transport by insect MTs our current data provide further support for the existence of separate pathways for TEA and nicotine transport in insect MTs. The results of this study and Maddrell and Gardiner’s (1976) observations raise some intriguing questions. The adaptive value of active nicotine secretion by the tubules is clear for species such as the tobacco hornworm (Manduca sexta) and the cabbage looper (T. ni) which feed on Nicotiana species. But what is the value of active nicotine secretion by tubules of species which do not feed primarily on tobacco plants? Although we have used nicotine as a representative plant alkaloid in our study it should be noted that in addition to nicotine the MTs of Rhodnius and Manduca are capable of transporting other plant alkaloids such as atropine and morphine (Maddrell and Gardiner, 1976). It is generally accepted that plant alkaloids have evolved to protect plants from herbivores, including insects. Alkaloids are widespread among plants of the class Magnoliopsida (dicots) and have been demonstrated to act as feeding deterrents and repellents of insects (Bernays and Chapman, 1977; Adams and Bernays, 1978). In some cases, specialized insect herbivores actually sequester and utilize ingested plant alkaloids for their own defence against predation (Boppre´, 1986; Schneider, 1987; Hartmann and Witte, 1995). A mechanism for the excretion of these potentially toxic metabolites may have evolved in insects which might be exposed to plant alkaloids or have adapted to diets containing high levels of alkaloids. The adaptive value of active alkaloid secretion by the tubules is less clear for insects such as locusts which normally feed on plants that do not produce alkaloids, or for mosquitoes or Rhodnius which feed on blood. Although the grasses on which Locusts feed do not produce their own alkaloids they are often infected with the fungus Claviceps purpurea, which produces the toxic ergotine alkaloids that have been responsible for ergot poisoning in livestock and humans back to antiquity (Bennett and Klich, 2003). Thus, insects such as locusts or Tenebrio which feed primarily on grains may also have evolved mechanism for excretion of alkaloids. This may also explain the need for alkaloid excretion in blood feeding insects. Although they do not feed on plants containing alkaloids they may be exposed to such compounds in the blood of livestock and humans who have been exposed to alkaloids in their diets. Alternatively, digestion of a blood meal may produce potentially toxic OCs or their metabolites which the MTs of Rhodnius and Aedes may excrete. Moreover, it is of interest to note that de novo synthesis of a number of alkaloids has been demonstrated in different

497

insects (Daloze et al., 1995; Renson et al., 1994; Attygalle et al., 1999; Braekman et al., 1999). Taken together with previous studies our findings suggest that secretion of type I OCs such as TEA and alkaloids such as nicotine by MTs is common to insects from widely separated orders. In addition, we have added to the current body of evidence which suggests independent pathways for the transepithelial transport of TEA and nicotine in insect MTs. Increasingly, it appears that the mechanisms for the transport of OCs by insect MTs are characteristically similar to those proposed for vertebrate renal epithelia, thus extending the functional analogy between the MTs of insects and the renal tubules of vertebrates. Acknowledgements This work was supported by National Science and Engineering Research Council Grants of Canada to M.J. O’Donnell. References Adams, C.M., Bernays, E, 1978. The effect of combinations of deterrents on the feeding behaviour of Locusta migratoria. Entomologia Experimentalis et Applicata 23, 101–109. Anstee, J.H., Bell, D.M., Fathpour, H., 1979. Fluid and cation secretion by Malpighina tubules of Locusta. Journal of Insect Physiology 25, 373–380. Ashburner, M., 1989. Drosophila: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Attygalle, A.B., Svatos, A., Veith, M., Farmer, J.J., Meinwald, J., Smedley, S., Gonzales, A., Wisner, T., 1999. Biosynthesis of epilachnene, a macrocyclic defensive alkaloid of the Mexican bean beetle. Tetrahedron 55, 955–966. Bennett, J.W., Klich, M., 2003. Mycotoxins. Clinical Microbiology Reviews 16, 497–516. Bernays, E.A., Chapman, R.F., 1977. Deterrent chemicals as a basis of oligophagy in Locusta migratoria (L.). Ecological Entomology 2, 1–18. Beyenbach, K.W., Aneshansley, D.J., Pannabecker, T.L., Masia, R., Gray, D., Yu, M.J., 2000. Oscillations of voltage and resistance in Malpighian tubules of Aedes aegypti. Journal of Insect Physiology 46, 321–333. Bijelic, G., O’donnell, M.J., 2005. Diuretic factors and second messengers stimulate secretion of the organic cation tea by the Malpighian tubules of Drosophila melanogaster. Journal of Insect Physiology 51, 267–275. Boom, S.P.A., Gribnau, F.W.J., Russel, F.G.M., 1992. Organic cation transport and cationic drug interactions in freshly isolated proximal tubular cells of the rat. Journal of Pharmacology and Experimental Therapeutics 263, 445–450. Boppre´, M., 1986. Insect pharmacophagously utlizing defensive plant chemicals (pyrrolizidine alkaloids). Naturwissenshaften 73, 17–26. Braekman, J.C., Charlier, A., Daloze, D., Heilporn, S., Pasteels, J.M., Plasman, V., Wang, S.F., 1999. New piperidine alkaloids from two ladybird beetles of the genus Calvia (Coccinellidae). European Journal of Organic Chemistry, 1749–1755. Coast, G.M., 1988. Fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus and their response to diuretic hormone. Physiological Entomology 13, 381–391. Coast, G.M., Kay, I., 1994. The effects of Acheta diuretic peptide on isolated Malpighian tubules from the house cricket Acheta domesticus. Journal of Experimental Biology 187, 225–243.

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