The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti
1 Department of Biomedical Sciences, VRT 8004, Cornell University, Ithaca,
NY 14853, USA
2 Department of Biochemistry, University of Nevada, Reno, NV 89557,
USA
* Author for correspondence (e-mail: KWB1{at}cornell.edu)
Accepted 19 May 2004
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Summary |
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Key words: antidiuresis, isosmotic fluid secretion, cGMP, inhibition of electroneutral transport, Na/H exchange, Na/K/2Cl cotransport, Tenebrio molitor, antidiuretic factor `a' (Tenmo ADFa), Malpighian tubule, Aedes aegypti
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Introduction |
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The diuresis wanes with time due to the excretion and/or inactivation of
diuretic hormones (Laenen et al.,
2001; Williams et al.,
1983
). The utility of antidiuretic factors terminating the
diuresis has also been proposed. Quinlan et al.
(1997
) reported an increase in
cyclic guanosine monophosphate (cGMP) in Rhodnius Malpighian tubules
as the diuresis decreases, and suggested that this may be important in
bringing the diuresis to a conclusion.
Spring et al. (1988) were
first to demonstrate the existence of an antidiuretic mechanism in Malpighian
tubules of an insect. Subjecting house crickets to dehydration, Spring and
coworkers detected antidiuretic activity in the hemolymph that inhibited fluid
secretion in Malpighian tubules. The antidiuretic agent in the hemolymph of
house crickets has not yet been identified.
The first peptide found to have antidiuretic potency, among other
activities, is Manse CAP2b, the cardioacceleratory peptide of
Manduca sexta (Huesmann et al.,
1995). It is an octapeptide with diverse physiological and
species-specific activities. It increases the heart rate in Manduca
and Drosophila (Huesmann et al.,
1995
), and it has antidiuretic effects in Rhodnius
prolixus (Quinlan et al.,
1997
). It is also a weak antidiuretic in Tenebrio
molitor, with an EC50 of 85 nmol l1. In
contrast, it has diuretic activity in Drosophila Malpighian tubules,
where it elevates nitric oxide (NO) and then cGMP to cause diuresis
(Davies et al., 1995
). cGMP
has also been shown to cause diuresis in Manduca sexta although no
peptide has yet been discovered that elevates cGMP in this species
(Skaer et al., 2002
).
Tenmo ADFa is the antidiuretic factor of the beetle Tenebrio
molitor. The peptide inhibits fluid secretion in Tenebrio
Malpighian tubules with an EC50 of 10 fmol l1,
employing cGMP as second messenger
(Eigenheer et al., 2003;
Wiehart et al., 2002
). FopADF
is the antidiuretic factor of the forest ant Formica polyctena
(Laenen et al., 2001
). It
decreases fluid secretion in Malpighian tubules to values 29% of control by
blocking the entry of K+ from the hemolymph into the cell and by
inhibiting the proton pump at the apical membrane of epithelial cells
(Laenen et al., 2001
). The
primary structure of FopADF has not yet been sequenced.
The present study sought to elucidate the mechanism of action of Tenmo ADFa in Malpighian tubules of Aedes aegypti. We found that Tenmo ADFa significantly inhibits the transepithelial secretion of both NaCl and KCl. At the same time the antidiuretic has no effect on transepithelial voltage and resistance, nor on basolateral membrane voltage and input resistance in principal cells. Tenmo ADFa significantly increases intracellular cGMP concentration, and low concentrations of peritubular cGMP (20 µmol l1) mimic the effects of Tenmo ADFa. Higher cGMP concentrations (500 µmol l1) induce electrophysiological effects but without increasing antidiuretic effects. Thus, Tenmo ADFa and physiological concentrations of cGMP reduce isosmotic fluid secretion by inhibiting electroneutral and non-conductive transport pathways.
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Materials and methods |
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Ringer's solution, Tenmo ADFa, cGMP and Ba2+
Ringer solution contained in mmol l1: 150 NaCl, 3.4 KCl,
1.7 CaCl2, 1.8 NaHCO3, 1.0 MgSO4, 25 Hepes,
and 5 glucose. Tenmo ADFa is a 14-amino-acid peptide that was originally
isolated from head extracts of Tenebrio molitor
(Eigenheer et al., 2002).
Tenmo ADFa with the sequence
Val-Val-Asn-Thr-Pro-Gly-His-Ala-Val-Ser-Tyr-His-Val-Tyr was synthesized by K.
Schegg and the gift of D. Schooley. It is unlikely that Tenmo ADFa, the
antidiuretic peptide of Aecheta, is produced in mosquitoes, although
related peptides are probably produced. So far, Malpighian tubules of insects
have been remarkably responsive to heterologous peptides such as CRF-like
peptides and leucokinin (Audsley et al.,
1995
). The doseresponse of the effects of Tenmo ADFa on
fluid secretion of isolated Malpighian tubules in Tenebrio molitor
puts the EC50 at 1014 mol l1
(Eigenheer et al., 2002
).
Accordingly, we prepared a stock solution of 108 mol
l1 in Ringer solution and kept it frozen in 1 ml portions
until use. In all studies, Tenmo ADFa was used at a peritubular concentration
of 109 mol l1. The effect of other
concentrations of ADFa were not explored since the focus of this study was on
physiological mechanisms of action.
Cyclic GMP was obtained from Sigma-Aldrich (St Louis, MO, USA) and used at
peritubular concentrations between 10 µmol l1 and 1000
µmol l1. The effect of Ba2+ on fluid and
electrolyte secretion was tested at a concentration of 5 mmol
l1 BaCl2, which is a saturating concentration for
electrophysiological effects (Masia et
al., 2000). In these experiments, MgCl2 rather than
MgSO4 was used in the Ringer solution. In addition, the control
Ringer solution was fortified with 15 mmol l1 mannitol for
osmotic balance.
Ramsay fluid secretion assay
One advantage of Malpighian tubules of Aedes aegypti is their
spontaneous secretion of fluid in vitro. The tubules do not require
pre-stimulation with a secretagogue before experiments on the rates and
mechanisms of fluid secretion can commence. Rates of transepithelial fluid
secretion were measured in isolated Malpighian tubules using the method of
Ramsay (1953) as described
previously (Hegarty et al.,
1991
). In brief, the isolated Malpighian tubule was transferred to
40 µl Ringer solution under oil. The open end of the tubule was pulled out
of the Ringer droplet into the oil and draped to a needle post that had been
coated with 0.125 mg ml1 poly-lysine for stickiness. Needle
posts with a length of approximately 2 mm and an outer diameter of 0.2mm were
the broken tips of jeweler's brooches (purchased from local cooperative
jewelers), which were also used to trim the ends of Malpighian tubules. The
brooches are made of hardened steel that resists bending when the tips are
filed to sharpness. Two such sharpened jewelers brooches are routinely used to
fray open the end of Malpighian tubules for in vitro microperfusion
and for fluid secretion assays by the method of Ramsay. The use of blunt
instruments tends to crush the tubule and seal the lumen.
The volume of secreted fluid was calculated by measuring the diameter of
the spherical aqueous droplet emerging from the open end of the tubule. Volume
measurements were made every 5 min for at least 30 min each for the control
and experimental periods. After the initial 30 min control period, the
cumulative secreted volume was collected for analysis by electron probe
(Williams and Beyenbach,
1983). Thereafter, 4 µl of Ringer solution was removed and
replaced with Tenmo ADFa or cGMP to yield the desired concentration. Secreted
volume was then measured again as in the control period and the cumulative
volume was collected for electron probe analysis after at least 30 min.
The method of Ramsay yields measures of net transepithelial volume transport that includes secretory and absorptive fluxes. Accordingly, a decrease in secretory flow rate can reflect the inhibition of a secretory flux and/or the stimulation of an absorptive flux. Since the blind (distal) end of Malpighian tubules initiates the formation of urine by tubular secretion, we interpret changes in secretion rates to reflect primarily changes in secretory transport activity.
Electron probe analysis of secretions
The concentrations of Na+, K+ and
Cl in secreted fluid were measured by electron probe
analysis as previously described (Williams
and Beyenbach, 1983). In brief, picoliter volumes of (1) fluid
secreted by tubules in the Ramsay assay, (2) standard solutions of
Na+, K+ and Cl and (3) the peritubular
Ringer solution were transferred in quintuplets onto the polished surface of a
beryllium block. Unknown, standard and Ringer samples were then dehydrated and
irradiated with a beam current of 50 nA in a JEOL 8900 (Tokyo, Japan) electron
microprobe. The X-ray emissions of the Na+, K+ and
Cl present in the standard solutions were used to construct
standard curves, against which the unknown Na+, K+ and
Cl concentrations in secreted fluid were read. The X-ray
emissions from Ringer solutions served as internal controls. For each
measurement we used the mean of five samples.
In vitro microperfusion of Malpighian tubules
The method used for measuring transepithelial and membrane voltage and
resistance in isolated perfused Malpighian tubules is described in Williams
and Beyenbach (1984) and Yu
and Beyenbach (2001
). Briefly,
a 100 µm segment of Malpighian tubule was trimmed open at both ends and
suspended between two holding pipettes in a Ringer bath. The tubule lumen was
cannulated with a double-barreled perfusion pipette. One barrel of the pipette
was used to perfuse the tubule lumen with Ringer solution and to measure the
transepithelial voltage Vt with respect to ground in the
peritubular Ringer bath. The other barrel was used to inject current
(I=50 nA) into the tubule lumen for the measurement of the
transepithelial resistance (Rt) by cable analysis
(Helman, 1972
;
Yu and Beyenbach, 2001
). The
typical experiment began with collecting control data in the presence of
normal Ringer solution. Thereafter the peritubular Ringer solution was
switched to include Tenmo ADFa or cGMP and experimental data were
collected.
Two-electrode voltage clamp of principal cells
The method of voltage-clamping single principal cells in intact Malpighian
tubules is described in Masia et al.
(2000). An isolated Malpighian
tubule was placed on the bottom of a perfusion bath, and a principal cell was
impaled with two conventional microelectrodes filled with 3 mol
l1 KCl (2030 M
). When the basolateral membrane
voltage (Vbl) measured by both electrodes were within 5 mV
of each other, the experiment was continued. Vbl and the
input resistance (Rpc) of principal cells were measured
under control conditions and then in the presence of peritubular Tenmo ADFa or
cGMP. Rpc was determined in currentvoltage plots
obtained from five voltage clamp steps in increments of 10 mV on the positive
and negative side of the spontaneous Vbl.
Ba2+ is known to block K+ channels in Malpighian
tubules of insects (Leyssens et al.,
1993; Masia et al.,
2000
; Nicolson and Isaacson,
1987
; Weltens et al.,
1992
). We used Ba2+ in the peritubular Ringer bath to
block a major conductive pathway of the basolateral membrane of principal
cells in order to uncover small changes in Vbl and
Rpc that might be induced by Tenmo ADFa and cGMP.
Intracellular cGMP concentration
The effect of Tenmo ADFa on intracellular cGMP concentration was measured
by enzyme immunoassay of cGMP using the cyclic GMP EIA kit from Cayman
Chemicals (Ann Arbor, MI, USA). All five tubules were removed from a single
cold-anesthetized female mosquito and placed in 25 µl of Ringer solution
including 100 µmol l1 zaprinast, a phosphodiesterase
inhibitor. To one set of five tubules 25 µl of Ringer solution was added to
serve as control. To another set of five tubules 25 µl of Ringer and
2x109 mol l1 Tenmo ADFa was added.
The two sets of tubules were incubated at room temperature for 15 min.
Thereafter, 200 µl of ice-cold ethanol was added, and the tubules were
sonicated on ice-water for 15 min. After centrifugation at 16 000
g and 4°C for 10 min, 200 µl of the supernatant was
removed and evaporated to dryness in a vacuum centrifuge. The residue was
subsequently reconstituted in 200 µl of buffer solution (EIA kit) and
duplicate or triplicate 50 µl samples transferred into the wells of the kit
plate. Cyclic GMP, AChE (acetylcholinesterase) tracer and cyclic GMP EIA
antiserum were added. After incubation for 18 h, the plates were developed in
Ellman's reagent for 90 min. The absorbance was read at 412 nm
(Broderick et al., 2003;
Eigenheer et al., 2002
;
Quinlan et al., 1997
).
The cytoplasmic cGMP concentration of Malpighian tubules was measured against cGMP standards ranging from 0.24 to 30.0 pmol l1. A sigmoidal regression (SigmaPlot V 8.0) provided the best fit, against which the absorbance of unknown cGMP concentrations was read.
Since cGMP was extracted from whole Malpighian tubules, the intracellular cGMP concentration was estimated from the measured amount of cGMP extracted from five Malpighian tubules and the cellular volume of the tubules. The latter was estimated by subtracting the volume of the tubule lumen from the total tubule volume of the typical female Malpighian tubule with the following dimensions: length 3.5 mm, o.d. 100 µm, i.d. 10 µm.
Statistical data
Control rates of fluid secretion can vary a great deal in isolated
Malpighian tubules, as can values of transepithelial voltage, resistance and
other electrophysiological variables. In the case of transepithelial fluid
secretion, the difference between low and high rates of secretion is more a
difference of magnitude rather than of kind. For example, male Malpighian
tubules secrete fluid at much lower rates (0.093 nl min1)
than female Malpighian tubules (0.64 nl min1), but the kind
of fluid secreted by both is the same, consisting of NaCl, KCl and water
(Plawner et al., 1991). In
female Malpighian tubules, control rates of fluid secretion can be as low as
0.1 nl min1 and as high as 1.0 nl min1,
but NaCl and KCl are consistently the major osmolytes of secreted fluid. To
deal with highly variable secretion data, we perform paired experiments. Each
tubule is used as its own control. First, the control variable of the tubule
under study is measured, then the experimental agent is introduced, and the
experimental variable is measured. The statistical analysis tests the
significance of the difference between control and experimental values
according to the paired Student's t-test. We prefer this treatment of
variable data over the selection of Malpighian tubules according to certain
physiological criteria.
The measurement of intracellular cGMP concentrations precluded paired measurements. Accordingly, the significant difference of population means was tested.
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Results |
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The addition of Tenmo ADFa (109 mol l1) to the peritubular Ringer solution significantly (P<0.01) decreased the rate of transepithelial fluid secretion from 0.94 nl min1 to 0.44±0.08 nl min1 (Fig. 1A). However, Tenmo ADFa had no effect on the concentrations of Na+, K+, and Cl in secreted fluid. In the presence of 109 mol l1 Tenmo ADFa, the Na+ concentration in secreted fluid was 98.6±16.0 mmol l1, K+ concentration 54.9±8.2 mmol l1, and Cl concentration 144.7±20.2 mmol l1 (Fig. 1B). In the absence of significant effects on the concentrations of secreted ions, the rates of transepithelial ion secretion fell proportionally with the decrease in fluid secretion. The rate of transepithelial Na+ significantly dropped from 81.9 pmol min1 to 46.9±12.2 pmol min1. Transepithelial K+ significantly fell from secretion 52.9 pmol min1 to 23.4±4.6 pmol min1, and transepithelial Cl secretion significantly decreased from 131.3 pmol min1 to 65.6±14.5 pmol min1. In the presence of Tenmo ADFa, the sum of the concentrations of Na+, K+, and Cl in secreted fluid was 298.1±42.0 mmol l1 and not significantly different from control. Significant reduction in the rates of transepithelial ion and water secretion without significant effects on the concentrations of secreted Na+, K+ and Cl (and hence osmotic pressure) reveals Tenmo ADFa as an inhibitor of isosmotic fluid secretion.
Effects of Tenmo ADFa on electrophysiological variables of Malpighian tubules
Under perfusion of the tubule lumen with the same Ringer solution present
in the peritubular bath, the transepithelial voltage Vt
was 32.7±9.4 mV (lumen-positive) in seven Malpighian tubules and
remained near that value at 31.3±9.5 mV in the presence of
109 mol l1 Tenmo ADFa
(Fig. 2A). The transepithelial
resistance Rt was 14.3±1.6 kcm in the same
seven Malpighian tubules under control conditions and did not change
significantly (14.6±1.7 k
cm) in the presence of Tenmo ADFa
(Fig. 2B). Thus, Tenmo ADFa
reduces the rate of isosmotic fluid secretion without affecting
transepithelial electrogenic and conductive transport pathways.
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In studies of principal cells of the tubule by the method of two-electrode
voltage clamp, Tenmo ADFa (109 mol l1) had
no effect on the basolateral membrane voltage Vbl and no
effect on the input resistance of principal cells Rpc
(Fig. 2C,D). The small
depolarization of Vbl from 78.9±3.9 mV
(control) to 77.9±4.7 mV in the presence of Tenmo ADFa was not
significant (Fig. 2C).
Likewise, the small increase in Rpc from 450.2±32.2
k (control) to 470.0±20.2 k
was not significant
(Fig. 2D). Higher Tenmo ADFa
concentrations (108, 107 and
106 mol l1) did not elicit significant
effects on Vbl or Rpc (data not
shown).
Effects of cGMP on transepithelial electrolyte and fluid secretion
Since cGMP is thought to be the second messenger of Tenmo ADFa, the effects
of this nucleotide were of interest. In 24 Malpighian tubules, the addition of
500 µmol l1 cGMP to the peritubular Ringer bath
significantly (P<0.01) reduced the rate of transepithelial fluid
secretion from 0.39±0.03 nl min1 to 0.19±0.02
nl min1 (Fig.
3A). In a subset of 12 Malpighian tubules in which secreted ion
concentrations were measured, the rate of fluid secretion again fell
significantly (P<0.05) from 0.37±0.04 nl
min1 to 0.22±0.03 nl min1. In the
same 12 tubules the Na+ concentration in secreted fluid increased
from 112.6±10.5 mmol l1 to 126.7±12.9 mmol
l1, the K+ concentration decreased from
53.0±8.7 mmol l1 to 41.6±6.0 mmol
l1, and the Cl concentration increased
from 140.3±6.9 mmol l1 to 151.1±17.9 mmol
l1 (Fig. 3B).
Only the decrease in the K+ concentration reached statistical
significance (P<0.05). As expected from the reciprocal change in
Na+ and K+ concentration, the sum of the concentrations
of Na+, K+ and Cl in secreted fluid
did not change significantly: 306.0±13.4 mmol l1
under control conditions and 319.4±32.4 mmol l1 in
the presence of 500 µmol l1 cGMP.
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Like Tenmo ADFa, cGMP significantly inhibited transepithelial ion secretion (Fig. 3C). Transepithelial Na+ secretion significantly decreased from 39.0±3.3 pmol min1 to 26.1±2.6 pmol min1; K+ secretion significantly fell from 21.7±5.0 pmol min1 to 8.9±1.8 pmol min1 and Cl secretion significantly fell from 52.1±6.9 pmol min1 to 30.7±3.3 pmol min1 (Fig. 3C).
Effects of cGMP on electrophysiological variables of Malpighian tubules
To evaluate the effects of cGMP on electrogenic transport mechanisms,
Vt and resistance were measured in isolated perfused
Malpighian tubules. In this series of experiments Vt was
20.4±2.8 mV (lumen-positive) in 20 Malpighian tubules and
Rt was 8.2±1.2 kcm
(Fig. 4A,B). The addition of
500 µmol l1 cGMP to the peritubular bath significantly
(P<0.05) hyperpolarized Vt from 20.4 mV to
41.8±6.1 mV and significantly (P<0.05) reduced
Rt from 8.2 k
cm to 6.2±0.7 k
cm.
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At a concentration of 500 µmol l1, cGMP also had
significant effects on Vbl and Rpc
(Fig. 4C,D). Upon addition of
cGMP to the peritubular bath of the tubule, Vbl
significantly (P<0.01) depolarized from 89.0±1.9 mV
to 54.3±6.6 mV(N=20 principal cells). In parallel,
Rpc significantly (P<0.05) decreased from
406.0±34.7 k to 339.2±24.8 k
(N=21
principal cells). Since Vt hyperpolarized by 21.4 mV and
Vbl depolarized by 34.7 mV in the presence of 500 µmol
l1 cGMP, it follows that the apical membrane also
depolarized by 13.3 mV. Thus, high concentrations of cGMP affected both apical
and basolateral membranes of principal cells.
Dose response curve to cGMP
Similar effects of Tenmo ADFa and cGMP on transepithelial electrolyte and
fluid secretion are consistent with the second messenger role of cGMP (Figs
1,
3). However, Tenmo ADFa did not
affect electrophysiological variables (Fig.
2) whereas 500 µmol l1 cGMP did
(Fig. 4). To investigate the
difference, we consulted a doseresponse curve of the effects of cGMP on
Vbl and Rpc.
As shown in Fig. 5, cGMP had no effect on voltage and resistance at peritubular concentrations of less than 300 µmol l1. At concentrations higher than 300 µmol l1, cGMP significantly depolarized Vbl in parallel with significant reductions in Rpc. Typically, Vbl responded to cGMP with an initial steep depolarization that subsequently recovered to a new steady state value (Fig. 5A, insert). A doseresponse curve drawn to peak voltage depolarizations yielded an estimate of 380 µmol l1 as the half maximum (EC50) cGMP concentration (Fig. 5A). The EC50 was 468 µmol l1 for the doseresponse drawn to new steady state values in the presence of cGMP. The EC50 for the effect on Rpc was 351 µmol l1, similar to the EC50 of peak depolarization (Fig. 5B).
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Measurement of intracellular cGMP concentration
Since cGMP is thought to be the second messenger of Tenmo ADFa
(Eigenheer et al., 2002),
measurements of intracellular cGMP concentrations in the absence and presence
of ADFa were of interest. Female Malpighian tubules of Aedes aegypti
have a cytoplasmic volume of approximately 27.2 nl
(Wu and Beyenbach, 2003
),
enabling us to estimate the intracellular cGMP concentration from the cGMP
content of homogenates of Malpighian tubules. Under control conditions, the
mean intracellular cGMP concentration was 2.90±0.77 µmol
l1 (19 measurements of five Malpighian tubules each). In the
presence of 109 mol l1 Tenmo ADFa, the
intracellular cGMP concentration significantly (P<0.05) increased
to 7.43±1.78 µmol l1 (21 determinations of five
Malpighian tubules each).
The intracellular cGMP concentration of 7.4 µmol l1 measured in the presence of antidiuretic concentrations of Tenmo ADFa (Fig. 1) is much lower than 300 µmol l1, the lowest peritubular cGMP concentration that affects Vbl and Rpc (Fig. 5). Suspecting pharmacological effects of high concentrations of cGMP on tubule electrophysiology, it was of interest to investigate whether a low concentration of cGMP could inhibit fluid secretion without inducing effects on voltage and resistance. A peritubular cGMP concentration of 20 µmol l1 was chosen for these experiments.
Effects of a low peritubular concentration of cGMP
The addition of 20 µmol l1 cGMP to the peritubular
Ringer bath significantly decreased the rate of transepithelial fluid
secretion from 0.23±0.01 nl min1 to 0.12±0.01
nl min1 (N=9). The 48% decrease is similar to 51%
measured in the presence of 500 µmol l1 cGMP
(Fig. 3A) and 53% in the
presence of 109 mol l1 Tenmo ADFa
(Fig. 1A). Thus, a low
peritubular cGMP concentration of only 20 µmol l1,
duplicated the antidiuretic effect of Tenmo ADFa and elicited antidiuretic
effects similar to those of 500 µmol l1 cGMP. Like Tenmo
ADFa, 20 µmol l1 cGMP had no significant effects on
Vt and Rt. The control
Vt was 27.4±5.8 mV in eight Malpighian tubules. The
small increase to 28.1±5.4 mV in the presence of 20 µmol
l1 cGMP was not significant, nor was the small increase in
Rt from 13.9±4.0 kcm (control) to
14.2±4.2 k
cm.
A peritubular cGMP concentration of 10 µmol l1 had no
effect on Vbl or Rpc. In these
experiments, Vbl was 90.4±2.5 mV under
control conditions and 91.8±2.3 mV in the presence of 10 µmol
l1 cGMP (N=9 cells). In the same cells
Rpc was 426.7±35.1 k and 431.1±35.4
k
under control and experimental conditions, respectively.
Ba2+ block of K+ channels does not uncover effects of ADFa and low concentrations of cGMP
Concerned that the high K+ conductance of the
basolateral membrane of principal cells
(Beyenbach and Masia, 2002)
might shunt the voltage effects of Tenmo ADFa and low concentrations of cGMP,
we repeated experiments in the presence of barium, a known blocker of
K+ channels in principal cells of Aedes Malpighian tubules
(Masia et al., 2000
).
Under control conditions, Vbl was
85.1±3.9 mV and Rpc was 476.9±23.2
k in six principal cells. The addition of 5 mmol l1
Ba2+ to the peritubular bath immediately and significantly
(P<0.05) hyperpolarized the membrane voltage from 85.1 mV
to 103.0±5.1 mV, and significantly (P<0.05)
increased the input resistance from 476.9 k
to 740.7±94.4
k
(N=6 principal cells). In the presence of Ba2+,
Tenmo ADFa (109 mol l1) had no effect on
Vbl (103.5±6.1 mV) nor on
Rpc (755.5±84.1 k
, N=6 principal
cells).
Similar observations were made with cGMP at a peritubular concentration of
20 µmol l1. In this series of experiments
Vbl was 80.6±4.8 mV under control
conditions, which again significantly (P<0.05) hyperpolarized to
86.9±5.5 mV in the presence of 5 mmol l1
Ba2+ (N=8 principal cells). The control
Rpc was 452.6±41.9 k, which significantly
(P<0.05) increased to 585.3±55.4 k
in the presence
of Ba2+ in same eight principal cells. In the presence of
Ba2+, the addition of 20 µmol l1 cGMP to the
peritubular bath had no significant effects on Vbl
(86.9±5.7 mV) and Rpc (595.9±81.2
k
). Thus, significant electrophysiological effects of Tenmo ADFa and
physiological concentrations of cGMP were not observed in
Ba2+-treated tubules that should have revealed small changes on
voltage and resistance.
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Discussion |
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Diuretic and antidiuretic factors of Tenebrio
The mealworm Tenebrio molitor has so far yielded two diuretic and
two antidiuretic hormones. The diuretic hormones belong to the family of
sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic
peptides (Furuya et al., 1995,
1998
). The two hormones
increase the production of cAMP, the nucleotide that is widely believed to
mediate diuresis in insects (Beyenbach,
2003
; Cady and Hagedorn,
1999
; Hazelton et al.,
2002
; O'Donnell et al.,
1996
; Xu and Marshall,
2000
). Cyclic AMP increases fluid secretion in Malpighian tubules
of Aedes aegypti by increasing a Na+-conductance
and bumetanide-sensitive transport system with the effect of stimulating
transepithelial NaCl secretion, and not KCl secretion
(Hegarty et al., 1991
;
Sawyer and Beyenbach,
1985
).
The two antidiuretic hormones of Tenebrio molitor, Tenmo ADFa and
ADFb, have 14 and 13 amino acids, respectively (Eigenheer et al.,
2002,
2003
). Although they bear no
structural resemblance to each other, they inhibit fluid secretion in
Tenebrio Malpighian tubules, and both are thought to use cGMP as
second messenger. Tenmo ADFa is about 24 000 times more potent than Tenmo
ADFb. In the present study we used synthetic Tenmo ADFa on Malpighian tubules
of female Aedes aegypti. The inhibition of fluid secretion,
concomitant with the increase in intracellular cGMP concentration, indicates
that the mosquito possesses an ADFa-like receptor-mediated mechanism of
antidiuresis.
Diverse effects of intracellular cGMP
The nucleotide cGMP has species-specific effects. It stimulates fluid
secretion in Malpighian tubules of Drosophila
(Coast et al., 2001),
Locusta (Morgan and Mordue,
1985
), Teleogryllus
(Xu and Marshall, 2000
), and
Manduca (Skaer et al.,
2002
), but it inhibits fluid secretion in Malpighian tubules of
Rhodnius (Quinlan et al.,
1997
), Tenebrio
(Eigenheer et al., 2002
) and
Aedes (Fig. 3). Since
it increases fluid secretion in Drosophila and reduces it in
Malpighian tubules of Aedes, cGMP has opposite effects in species of
the same order, Diptera, but similar effects across orders, namely
Aedes (Diptera) and Rhodnius (Hemiptera).
Next to species differences, cGMP exhibits heterogeneities along the length
of Malpighian tubules. The nucleotide stimulates fluid secretion in the main
segment but not in the distal segment of Malpighian tubules of the black field
cricket Teleogryllus (Xu and
Marshall, 2000). Similar heterogeneities have been reported in
Rhodnius Malpighian tubules
(O'Donnell and Quinlan,
1998
).
In the present study we observe dose-dependent effects of cGMP in Malpighian tubules of Aedes aegypti. The nucleotide inhibited fluid secretion at all concentrations tested. At concentrations less than 200 µmol l1 cGMP had no electrophysiological effects (Fig. 5). In contrast, cGMP concentrations above 300 µmol l1 had significant effects on voltage and resistance. Since high concentrations of cGMP did not inhibit fluid secretion any more than low cGMP concentrations, the electrophysiological effects of high cGMP concentrations are apparently independent of the effects on epithelial transport (Fig. 4). The ionic conductance activated by high concentrations of cGMP is unknown.
Dose-dependent effects, functional heterogeneities along the length of
Malpighian tubules, and opposite effects in tubules of different species,
reflect the confusion that can arise in the search for unifying themes. At the
level of epithelial cells, cGMP, cAMP, calcium, inositol trisphosphate, diacyl
glycerol, ATP and prostaglandins are but some intracellular regulators that
not only influence transport but also interact with each other. Interactive
effects of cGMP and cAMP can be antagonistic, as in Tenebrio
Malpighian tubules, where the stimulatory effects of cAMP can be neutralized
by cGMP (Quinlan et al., 1997;
Wiehart et al., 2002
). Quinlan
and O'Donnell (1998
) suggest
that in Rhodnius Malpighian tubules, cGMP activates the
phosphodiesterase of cAMP, thereby reducing intracellular cAMP concentrations
and hence rates of transepithelial electrolyte and fluid secretion
(Quinlan and O'Donnell,
1998
).
Inhibition of isosmotic fluid secretion
The inhibition of transepithelial fluid secretion by Temno ADFa without
changes in the concentrations of secreted Na+, K+ and
Cl signifies the inhibition of isosmotic fluid secretion.
How isosmotic fluid secretion is reduced at mechanistic and molecular levels
is an intriguing question. Since water follows solute by osmosis, the
reduction in transepithelial fluid secretion must reflect the inhibition of
solute secretion. Indeed, Tenmo ADFa reduces transepithelial secretion rates
of Na+, K+ and Cl
(Fig. 1) without affecting
their concentrations in secreted fluid and without affecting
electrophysiological variables of the tubule and its principal cells. Thus,
Tenmo ADFa inhibits the kinetics of transepithelial transport and not the
transepithelial electrochemical potentials of transported ions. Three
hypothetical mechanisms come to mind.
The first hypothesis proposes metabolic control of transepithelial transport. By reducing the energy supply of active transport systems, the rate of isosmotic fluid secretion is reduced without affecting transepithelial concentration differences of transported ions and without affecting the transepithelial and cell membrane voltages and resistances.
The second hypothesis considers a reduction in transepithelial water permeability. Leaky epithelia characteristically transport solute and water in isosmotic proportions due to high transepithelial permeabilities to both. Accordingly, a decrease in water permeability is expected to decrease transepithelial volume flow without affecting solute concentrations.
The third hypothesis suggests an effect on transepithelial
Cl transport. Of the three ions secreted into the tubule
lumen, the transepithelial secretion of Cl correlates more
strongly with the secretion of water than the secretion of Na+ and
K+ (Beyenbach et al.,
1993). Since Cl is the counterion of
transepithelial Na+ and K+ secretion, inhibition of
Cl transport is expected to reduce Na+ and
K+ transport and hence transepithelial solute and water transport.
However, this mechanism would be expected to significantly increase the
transepithelial resistance, which was not observed in the present study.
The three hypotheses illustrate how little we know about Malpighian
tubules. To our knowledge there are no modern investigations of the energetics
of transepithelial transport in Malpighian tubules that might give further
insights into the metabolic inhibition proposed above. The evidence for the
presence of aquaporin water pathways is just now emerging
(Echevarria et al., 2001;
Dow and Davies, 2003
), but
transepithelial water permeabilities in the presence and absence of diuretic
or antidiuretic agents have not yet been measured in Malpighian tubules. As to
possible effects of ADFa on transepithelial Cl transport,
there is no consensus on the mechanism and the route of transepithelial
Cl transport in Malpighian tubules
(O'Donnell et al., 1998
;
Yu and Beyenbach, 2002
).
Inhibition of electroneutral transport systems
Tenmo ADFa and low, physiological concentrations of cGMP reduced
transepithelial NaCl, KCl and water secretion by approximately the same extent
(50%), without effects on voltage and resistance measured across the
tubule or in principal cells. Thus, Tenmo ADFa and low concentrations of cGMP
inhibit electroneutral transport mechanisms. The large contribution of
electroneutral transport to transepithelial secretion surprised us because
Malpighian tubules of Aedes aegypti usually display parallel effects
on epithelial transport and electrophysiology. Stimulators of secretion,
mosquito natriuretic peptide, cAMP and leucokinin all affect voltage and
resistance (Beyenbach, 2003
;
Pannabecker et al., 1993
;
Petzel et al., 1987
;
Sawyer and Beyenbach, 1985
),
and inhibitors of secretion, Ba2+ and bafilomycin, also affect
voltage and resistance (Beyenbach et al.,
2000
; Masia et al.,
2000
). In contrast, Tenmo ADFa and its second messenger elicit no
electrophysiological effects in spite of the significant reduction in
secretion. Accordingly, effects on transport must not always be mirrored in
the electrophysiology of the tubule and its epithelial cells.
We have recently started to investigate the relative contributions of
electrogenic and electroneutral transport systems to transepithelial secretion
in Aedes Malpighian tubules
(Scott et al., 2004).
Transport mechanisms across the basolateral membrane of principal cells
received our first attention. The basolateral membrane is dominated by a
K+-conductance that accounts for 64% of the total membrane
conductance (Beyenbach and Masia,
2002
). Blocking this conductance with Ba2+ inhibits
transepithelial fluid secretion by 80%. The remaining K+ secretion
(20%) is blocked by bumetanide, the blocker of Na/K/2Cl cotransport in
vertebrate tissues (Scott et al.,
2004
). Bumetanide alone blocks transepithelial K+
secretion by 70%, as much as channel block by Ba2+
(Hegarty et al., 1991
;
Scott et al., 2004
). Since
Ba2+ inhibits transepithelial K+ secretion by 80% and
bumetanide by 70%, it appears that electroconductive and electroneutral
transport mechanisms are functionally coupled, where the block of
K+-channels reduces K+ transport via Na/K/2Cl
transport and vice versa (Fig.
6). In the thick ascending limb of the loop of Henle, this kind of
functional coupling serves to recycle K+ across the apical
membrane. Apical membrane K+-channels return intracellular
K+ to the tubule lumen, thereby ensuring a sufficient K+
supply for Na/K/2Cl cotransport across that membrane
(Vuillemin et al., 1992
),
because glomerular filtration may not always present sufficient quantities of
K+ to the luminal face of the Na/K/2Cl cotransporter.
|
A second electroneutral transport system that might be inhibited by ADFa is
Na/H exchange (Giannakou and Dow,
2001; Petzel,
2000
). In Malpighian tubules of Aedes aegypti, 1 mmol
l1 amiloride inhibits transepithelial fluid secretion by 60%
without significant effects on electroconductive transport pathways
(Hegarty et al., 1992
). The
inhibition of Na/H exchange across the basolateral membrane inhibits not only
transepithelial NaCl secretion but also KCl secretion, suggesting again the
coupling of electroconductive and electroneutral transport.
Since Tenmo ADFa and low concentrations of cGMP inhibit electroneutral
transport in Aedes Malpighian tubules, Na/K/2Cl and Na/H transporters
are possible targets of their antidiuretic action
(Fig. 6). Cyclic GMP is
reported to inhibit Na/K/2Cl transport in rabbit cardiomyocytes
(Clemo and Baumgarten, 1995;
Lew et al., 1997
) and HeLa
cells (Kort and Koch, 1990
).
The nucleotide is known to inhibit Na/H exchange in renal proximal tubules
(Roczniak and Burns, 1996
),
small intestine (Fawcus et al.,
1997
), rat mesangial cells
(Schulte et al., 1999
) and in
epithelial cell lines such as Caco-2 (Gill
et al., 2002
) and human intestinal C2/bbe
(McSwine et al., 1998
).
Accordingly, a fourth hypothesis regarding the mechanism of action of ADF and
cGMP proposes the inhibition of Na/H exchange transport and Na/K/2Cl
cotransport.
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Acknowledgments |
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