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1 Department of Zoology, Washington State University, Pullman, Washington 99164; 2 Bayer Corporation, Raleigh, North Carolina 27606; and 3 Section of Physiology, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The mechanism of
action of synthetic Culex
corticotropin-releasing factor (CRF)-like diuretic peptide (CCRF-DP)
was investigated in isolated, perfused Malpighian tubules of the yellow
fever mosquito, Aedes aegypti. Low
concentrations of CCRF-DP
(10
10 and
109 M) caused depolarizing
oscillations of the lumen-positive transepithelial voltage
(Vt) in
Malpighian tubules, whereas high concentrations (108 and
107 M) first depolarized
and then transiently hyperpolarized
Vt; CCRF-DP
always lowered transepithelial resistance
(Rt),
regardless of voltage depolarization or hyperpolarization. The
short-circuit current
(Isc), an
electrical estimate of active transepithelial transport of Na and K,
remained unchanged at low concentrations of CCRF-DP, but
Isc more than
doubled at high concentrations. These effects of CCRF-DP suggest
dose-dependent sites of action: low concentrations of CCRF-DP affect
the paracellular pathway, and high concentrations affect both
paracellular and transcellular pathways.
Aedes aegypti; transepithelial voltage; transepithelial resistance; short-circuit current; regulation of shunt pathway
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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THERE ARE TWO MAJOR ROUTES of transport across epithelia, a transcellular route and a paracellular route (3, 25). The transcellular route is a pathway for active transport through the cell, where the cell provides the energy for transepithelial transport against chemical and electrical potentials (Fig. 1). The paracellular route is a pathway for passive transport between cells, through tight junctions in the case of vertebrate epithelia and through septate junctions in the case of insect Malpighian tubules (22). Passive transport through the paracellular pathway would undo what active transport through cells has just accomplished were it not for selective permeabilities of the paracellular pathway. In general, the paracellular pathway is largely anion selective when transcellular transport is cation specific, and the paracellular pathway is largely cation selective when transcellular transport is anion specific. In Malpighian tubules of the yellow fever mosquito, Aedes aegypti, the paracellular pathway is Cl selective (9, 17), and the transcellular active transport pathway is both Na specific and K specific (3, 27, 28).
In previous work, we have shown that the mosquito natriuretic peptide
(MNP), isolated from A. aegypti,
stimulates active transport of Na through cells (3, 5, 18, 19). As a
result, rates of transepithelial secretion of NaCl and water increase
(5). In contrast, the octapeptide leucokinin isolated from the
cockroach Leucophaea increases the Cl
permeability of the paracellular pathway (3, 13, 17, 26). Thus two
different peptides, MNP and leucokinin, regulate, respectively, the
transcellular pathway and the paracellular transport pathway in
Malpighian tubules (3). In the present study, we show effects on both
paracellular and transcellular transport pathways by a single peptide.
This peptide is Culex
corticotropin-releasing factor (CRF)-like diuretic peptide (CCRF-DP)
that was isolated from a close relative of A. aegytpi, the salt water mosquito Culex
salinarius (F. L. Clottens, G. M. Holman, A. A. Strey,
N. F. Totty, I. Kay, G. M. Coast, A. I. Mallet, O. Truong, V. Johnson,
J. K. Olson, T. M. Clark, K. W. Beyenbach, and T. K. Hayes,
unpublished observations). This peptide derives its name in part from
its structural similarity to vertebrate CRF. In Malpighian tubules of
the yellow fever mosquito, CCRF-DP affects the paracellular pathway at
concentrations less than
108 M and both paracellular
and transcellular pathways at concentrations higher than
108 M.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Mosquitoes and Malpighian tubules. The mosquito colony was maintained as described by Pannabecker et al. (17). On the day of the experiment, a female mosquito (3-7 days posteclosion) was cold anesthetized and then decapitated. Malpighian tubules were removed from the abdominal cavity under Ringer solution. As in previous studies, tubule segments near the blind end of the tubule were used (3, 5). These segments are known to secrete salt and water (27, 28). Isolated tubule segments were between 0.5 and 1 mm long. The blind (closed) end of the tubule segment was opened with small, sharp dissection needles so that it could be perfused in vitro as described previously (4).
Composition of Ringer solution. Aedes Ringer solution contained the following (in mM): 150 NaCl, 25 HEPES, 3.4 KCl, 7.5 NaOH, 1.8 NaHCO3, 1 MgSO4, 1.7 CaCl2, and 5 glucose. The pH was adjusted to 7.1.
Culex CRF-DP. The peptide was isolated from whole body extracts of wild mosquitoes collected in the marshlands of Anahuac National Wildlife Refuge in Southeast Texas near the Gulf Coast. Over 90% of the captured mosquitoes were identified as the salt water mosquito Culex salinarius (10). The isolation and purification of CCRF-DP was accomplished in the laboratories of Drs. T. K. Hayes and G. M. Holman (Texas A & M University) by tracking the peptide via its ability to increase cAMP concentrations in Malpighian tubules of Manduca sexta (Clottens et al., unpublished observations). The amino acid sequence of Culex CRF-DP was determined to be TKPSLSIVNPLDVLRQRIILEMARRQMRENTRQVERNKAILREI-amide (Clottens et al., unpublished observations). This sequence resembles that of vertebrate CRF. The peptide thus belongs to the growing family of CRF-DPs identified in insects (7, 21).
Synthetic CCRF-DP was prepared in the laboratory of T. K. Hayes and G. M. Holman and shipped to us in dry form in four tubes containing the
following quantities of CCRF-DP: 1, 10, 100, and 1,000 pmol. Each tube
further contained 1,000 µg 
-lactoglobulin to minimize binding of
CCRF-DP to the wall of microcentrifuge tubes. To each tube Ringer
solution (200 µl) was added to produce stock solutions. On the day of
the experiment, 10 µl of stock solution was added to the peritubular
Ringer bath (500 µl) holding the tubule to yield the four desired
test concentrations of CCRF-DP: 1010,
109,
108, and
107 M.
Electrophysiological investigations. Immediately after dissection from the abdominal cavity of the mosquito, the Malpighian tubule was suspended in a Ringer bath between two holding pipettes. The tubule lumen was then cannulated with a hand-forged double-barrel pipette with an outer diameter of ~10 µm (Theta-Borosilicate glass, no. 1402401; Hilgenberg, Malsfeld, Germany). One barrel of this pipette was used to perfuse the tubule lumen with Ringer solution at rates less than 5 nl/min and to measure transepithelial voltage (Vt) with respect to ground in the peritubular Ringer bath (4). The other barrel was used to inject 100 nA into the tubule lumen for measurements of transepithelial resistance (Rt) by cable analysis (11). The peritubular bath (500 µl) was perfused with Ringer solution at a rate of 4 ml/min. In view of high perfusion rates of both tubule lumen and peritubular bath, Vt and Rt were measured in the virtual absence of transepithelial ion gradients, i.e., in symmetrical solutions, which is a requirement when the short-circuit current (Isc) is used as a measure of active transport (see below). Vt was measured continuously and recorded on a Gould brush chart recorder and stored in digital form. Rt was measured periodically when of interest.
Malpighian tubules that under control conditions maintained a stable Vt of at least 20 mV (lumen-positive) for at least 5 min were used for further study. After Vt had stabilized, bath flow was stopped with negligible effects on Vt and Rt. CCRF-DP was then added to the peritubular Ringer. The small quantities of synthetic CCRF-DP available did not allow us to test the effects of peptide in a flowing peritubular bath. After the effects of CCRF-DP on Vt and Rt were recorded and stored for data analysis, bath flow was started again to wash out CCRF-DP, which invariably returned Vt and Rt to values observed under control conditions.
A dose-response curve for CCRF-DP was obtained by testing peptide
concentrations of 1010,
109,
108, and
107 M in nine Malpighian
tubules. Each peptide concentration was tested in each Malpighian
tubule, beginning with the lowest and ending with the highest peptide
concentration. This was possible because the effects of CCRF-DP were
fully reversible upon washout. When the addition of CCRF-DP triggered a
voltage response, its concomitant effect on
Rt was measured
as well.
Equivalent electrical circuit of transepithelial ion transport in Malpighian tubules. Since electrolyte secretion is electrogenic in Malpighian tubules of A. aegypti (3, 5, 28), transepithelial transport of ions can be modeled with an electrical equivalent circuit. Ussing and Windhager (25) first used such an electrical model in frog skin to distinguish between the active transport pathway through cells and the passive transport pathway between cells (Fig. 1). They defined the active transport pathway to consist of 1) the electromotive force of the active transport system, Ecell, and 2) the resistance of the active (transcellular) transport pathway, Rcell. They defined the paracellular pathway to consist of only the shunt resistance, Rshunt, since there are no transepithelial electrochemical potentials (Et) when the same Ringer solution is present on both sides of the epithelium (symmetrical solutions as in the present study).
In Malpighian tubules of A. aegypti, Na and K are secreted into the tubule lumen by active transport through principal cells, and Cl is secreted into the tubule lumen by passive transport through septate junctions between principal cells (Fig. 1). Thus the transcellular pathway is cation specific for Na and K, and the paracellular pathway is anion selective for Cl (3, 5, 17). Since transcellular and paracellular pathways span the epithelium in parallel (Fig. 1), they are electrically coupled such that current through the cell equals that between cells. Accordingly, for each cation secreted by active transport through the cell, an anion is secreted into the lumen through the pathway between cells, preserving electroneutrality in the solutions on both sides of the epithelium. Indeed, rates of transepithelial cation secretion (Na and K) equal rates of transepithelial Cl secretion (3).
An analysis of the circuit in Fig. 1 reveals that under open-circuit conditions, i.e., during perfusion of the tubule lumen, the intraepithelial current, Ie, is
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(1) |
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(2) |
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(3) |
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(4) |
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(5) |
Statistical evaluation of data. Each tubule was used as its own control. Accordingly, the data were analyzed for the differences between paired samples, control versus experimental, in each tubule (paired Student t-test). When of interest, ANOVA followed by Student-Newman-Keuls multiple comparisons test, was used (23).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effects of CCRF-DP on Vt. Malpighian tubules generate appreciable Vt values (~40-60 mV) that are lumen positive when the tubule lumen is perfused in vitro with the same Ringer solution that is present in the peritubular bath (3, 5, 28). These voltages are active transport voltages (8) that largely derive from the secretion of Na and K by active transport mechanisms residing in principal cells of Malpighian tubules (Fig. 1).
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10 to
107 M (Fig.
2). Concentrations higher than
1010 M may appear excessive
for native hormones operating in vivo but not for a peptide that has
been isolated from one species (Culex) and is studied in vitro in
another species (Aedes).
Nevertheless, the lowest concentration of CCRF-DP tested
(1010 M) elicited small,
depolarizing oscillations of
Vt that continued as long as the peptide was present in the peritubular bath (Fig. 2a). In eight other tubules where
the effect of 1010 M
CCRF-DP was tested, one tubule showed no voltage response at all, and
another tubule exhibited a single, small, brief hyperpolarization of
Vt in addition to
the usual oscillating depolarizations (data not shown). Observations
from all nine tubules are expressed as percent control
Vt in Fig.
2b. This grouping of the data tends to
obliterate the voltage oscillations seen in single tubules.
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9 M, the amplitude of
voltage oscillations increased to ~30 mV as shown in the
representative experiment illustrated in Fig.
2c. In three of nine tubules, a brief
hyperpolarization of the
Vt was seen in
addition to the depolarizations (data not shown). When the data from
all nine tubules are summarized, only a single transient depolarization of the Vt is seen
after the addition of CCRF-DP to the peritubular bath (Fig.
2d).
At a CCRF-DP concentration of
108 M, the
Vt immediately
depolarized upon the addition of peptide to the peritubular bath (Fig. 2e). This depolarization is followed
by a repolarization that continued with a hyperpolarization of
Vt that lasted
~1 min (Fig. 2e). This biphasic
response of the
Vt, first a
depolarization and then a hyperpolarization of
Vt, was observed
in all nine tubules (Fig. 2f).
The effect of 107 M CCRF-DP
on Vt was similar
to that seen with 108 M,
except for the significant blunting of the initial depolarization of
the Vt, both in
terms of magnitude and duration (Fig.
2g). On average, the duration of the
initial depolarization significantly decreased from 0.9 ± 0.2 min
in the presence of 108 M
CCRF-DP to 0.4 ± 0.1 min in the presence of
107 M CCRF-DP
(P < 0.05, n = 9 tubules, paired
t-test; Fig. 2,
f and
h). In parallel, the magnitude of
the Vt
depolarization significantly decreased from 24.3 ± 3.6 to 15.9 ± 2.4 mV (P < 0.05, n = 9 tubules; Fig. 2,
f and
h) as CCRF-DP concentration
increased from 108 to
107 M. Reductions in both
magnitude and duration suggest that, with increasing concentration of
CCRF-DP, the hyperpolarization of Vt occurs
earlier, thereby reducing magnitude and duration of the depolarization
(Fig. 2, f and
h). Apparently, voltage
depolarizations and hyperpolarizations stem from independent mechanisms
that are additive.
Effects of CCRF-DP on
Vt,
Rt, and
Isc.
Figures 3 and 4 summarize the effects of CCRF-DP on
Vt and
Rt, along with
the Isc that is
used as a measure of active transport. Voltage and resistance were
measured simultaneously at several critical times during the
experiment: first in the absence of CCRF-DP (control), then in the
presence of peritubular CCRF-DP during phases of voltage
depolarization, repolarization, and/or hyperpolarization, and
finally upon removal of CCRF-DP (washout). Concentrations of
1010 and
109 did not affect
Isc, whereas
concentrations of 108 and
107 M did.
10
M, which caused
Vt to oscillate
in a depolarizing direction (Fig. 2a). The magnitude of these voltage
depolarizations was on average 10.8 ± 3.9 mV reaching statistical
significance (means ± SE, P < 0.05, n = 4 tubules, paired
t-test; Fig. 3).
Vt measured in parallel at these voltage depolarizations also decreased significantly from 6.13 ± 1.33 to 4.89 ± 1.00 k
· cm
(P < 0.05, n = 4 tubules, paired
t-test; Fig. 3). Since the percent
decrease of voltage and resistance was similar (27.8% and 20.1%), it
follows that their ratio
(Vt/Rt),
namely the Isc,
remained unchanged (Fig. 3). When the
Vt repolarized to
control values in the presence of CCRF-DP,
Rt invariably
returned to control values as well (Figs. 2a and 3). Thus the oscillations
observed at a CCRF-DP concentration of
1010 M constitute parallel
transient reductions in
Vt and
Rt.
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9 M
CCRF-DP were essentially the same as those of
1010 M: voltage and
resistance decreased but not
Isc. The
Vt significantly depolarized from 40. 9 ± 3.6 to 23.4 ± 5.1 mV in the presence of CCRF-DP (P < 0.02, n = 6 tubules). In parallel, the
Rt significantly decreased from 6.26 ± 1.05 to 3.06 ± 0.57 k · cm (P < 0.05, n = 6 tubules). Since both
Vt and
Rt drop by a
similar percent change, the
Isc remained
unchanged. Even though the reductions of
Vt and Rt in the
presence of 109 M were
greater than those in the presence of
1010 M, there was no effect
on Isc. Again,
Vt and
Rt oscillated in parallel as in the presence of
1010 M. Voltage and
resistance decreased together and then rose together during,
respectively, voltage depolarization and repolarization.
Figure 4 illustrates the effects of CCRF-DP
at a concentration of 108
M, which, as shown in Fig. 2e, is
known to first depolarize and then hyperpolarize
Vt. The first
effect of CCRF-DP was the significant decrease in
Vt from 35.2 ± 2.8 to 20.9 ± 3.3 mV (P < 0.02). In parallel, resistance significantly dropped from 5.21 ± 1.09 to 3.09 ± 0.57 k · cm
(P < 0.05) without an effect on
Isc
(n = 4 tubules, Fig. 4). These effects
on Vt and
Rt are like those
observed in the presence of low doses of CCRF-DP (Figs. 2 and 3).
However, during the subsequent, significant
(P < 0.02) hyperpolarization of the
Vt to 51.0 ± 5.4 mV (Fig. 4), the
Rt remained
significantly low (P < 0.05) at 2.99 ± 0.72 k · cm. As a result, the
Isc increased significantly from 8.0 ± 1.1 to 20.6 ± 4.3 µA/cm during the
voltage hyperpolarization (Fig. 4). Washout of CCRF-DP restored control values of Vt,
Rt, and
Isc.
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7 M were the same as
those of 108 M, except for
larger effects consistent with the 10-fold increase in CCRF-DP
concentration. The first effects of CCRF-DP were again the significant
reductions in Vt
from 35.8 ± 3.7 to 20.9 ± 4.0 mV
(P < 0.02) and
Rt
(P < 0.05) from 4.24 ± 0.90 to
2.70 ± 0.44 k · cm with no effect on the
Isc
(n = 6 tubules). In the subsequent significant (P < 0.01)
hyperpolarization of
Vt to 60.5 ± 7.1 mV, the Rt
remained low at 2.75 ± 0.59 k · cm, with the
effect of significantly increasing (P < 0.01) the Isc
from 9.99 ± 3.62 to 24.9 ± 4.6 µA/cm. Washout of CCRF-DP
returned Vt,
Rt, and
Isc to control
values (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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More than 30 years ago Windhager and Ussing (25) proposed an electrical model for Na transport across the frog skin that featured an active transport pathway in parallel with and coupled to a passive shunt pathway (Fig. 1). The simple elegance of this model has borne many fruits in our understanding of epithelial transport and its regulation. The model is as applicable to secretory transport across Malpighian tubules as it is to absorptive transport across frog skin.
Because of the requirement for energy, the active transport pathway is modeled to pass through the cell and to consist of the electromotive force and the resistance of the active transport pathway, Ecell and Rcell, respectively, (Fig. 1). In the case of Malpighian tubules, Ecell represents the electromotive force of active transepithelial transport systems for Na and K located in principal cells (3, 5, 16, 28), and Rcell represents the total transcellular resistance and includes the resistances of basolateral and apical cell membranes of principal cells, the cytoplasmic resistance, and the internal resistance of active transport pumps (3, 20). The passive transport pathway, the so-called shunt pathway, is dissipative (Fig. 1). It is located outside principal cells and is represented by the simple ohmic resistor Rshunt (3, 17, 25). Previous studies in Malpighian tubules of A. aegypti have shown that Rshunt is Cl selective and probably also permeable to water (12, 17, 26).
Examination of the model reveals that as Rshunt goes to zero, rates of transepithelial secretion of NaCl and KCl increase (Eq. 1). At the same time, Vt goes to zero (Eq. 2). When Rshunt is zero, Vt is zero, which defines the Isc condition that allows active transport through the cell to proceed at maximum (Eq. 5).
Under control conditions, the average calculated
Isc value
(Eq. 5) for all tubules studied in
the present sets of experiments was 8.6 ± 1.6 µA/cm tubule length
(n = 9 tubules). Application the
Faraday constant yields an average transepithelial transport rate of
~5.3
nmol · min1 · cm
tubule length1. Rates of
transepithelial cation secretion measured by chemical methods are on
average 0.7 nmol · min1 · cm
tubule length1 (27). Thus
the Isc is
between seven- and eightfold greater than rates of transepithelial
transport measured in secreting tubules, which is expected, because the
secreting epithelium does not operate under short-circuit conditions.
It operates under open-circuit conditions, i.e., in the presence of a
paracellular Rshunt that
reduces intraepithelial current (Eq. 1) and hence rates of transepithelial transport with
increasing Rshunt
(Fig. 1). When
Rshunt is very
high as in storage epithelia, intraepithelial current is very low, and
rates of transepithelial transport approach zero in these
high-resistance, high-voltage epithelia
(Eqs.
1-3).
Dose-dependent, biphasic effects of
CCRF-DP. Low concentrations of CCRF-DP
(1010 and
109 M) significantly
decrease Vt
concomitant with a decrease in
Rt (Figs.
2-4). The parallel decrease in
Vt and
Rt is significant
in that it strongly suggests an effect on the paracellular
Rshunt. A
decrease in
Rshunt is
expected to decrease both
Vt
(Eq.
2) and Rt
(Eq.
3), but it should have no effect on
the active transport pathway or
Isc
(Eqs.
4 and 5). This is indeed what is found in the experiment: low concentrations of CCRF-DP cause parallel reductions in Vt and
Rt without an
effect on Isc
(Figs. 2-4). Furthermore, the oscillations of
Vt observed in
the presence of low concentrations of CCRF-DP appear to stem primarily
from oscillations of the
Rshunt, since
voltage depolarizations and repolarizations are, respectively, accompanied (or preceded) by parallel decreases and increases in
Rt with short
circuit remaining constant throughout (Figs. 3 and 4). Accordingly, we
conclude that low concentrations of CCRF-DP affect primarily, if not
alone, the paracellular shunt pathway. This selective effect on the
Rshunt is
reminiscent of the effects of the diuretic leucokinin, which is known
to increase paracellular Cl conductance in
Aedes Malpighian tubules (9, 17).
High concentrations of CCRF-DP
(108 and
107 M) elicit a biphasic
voltage response (Figs. 2 and 4). The first response lowers Vt and
Rt, again without
a change in Isc,
mimicking the effects of low concentrations of CCRF-DP (Figs. 3 and 4).
Thus high concentrations of CCRF-DP initially affect the paracellular
pathway. However, during or shortly after the effect on the
paracellular pathway, the
Vt repolarizes
and then hyperpolarizes while
Rt remains low. As a result, the
Isc significantly
increases, indicating the stimulation of active, transcellular
transport (Fig. 2 and 4). Thus high concentrations of CCRF-DP affect
transcellular transport in addition to paracellular transport. The
temporal sequence, first the effect on paracellular and then on
transcellular transport, suggests the activation of different CCRF-DP
receptors and second messenger systems.
Relation of present work to epithelial transport in general and to Malpighian tubules in particular. In view of the primacy of active transport in transepithelial transport, it is obvious why in the past most experimental attention has focused on the mechanisms of active transport through epithelial cells and its regulation by hormones, peptides, and other extracellular signals. However, in recent years, the paracellular pathway is increasingly drawing attention to its role as regulator of transepithelial transport (1, 14). Our observations of the effects of leucokinin (17) and now CCRF-DP on the resistance of the shunt pathway in Malpighian tubules provide clear evidence for acute, rapid, and short-term regulation of the paracellular pathway by native extracellular peptides (3, 17, 26). Acute, rapid regulation requires ease of reversibility. Indeed, the effects of both leucokinin (17) and CCRF-DP on the shunt pathway are quickly and fully reversible upon washout (Figs. 3 and 4).
In recent years two families of diuretic peptides have emerged that stimulate fluid secretion in Malpighian tubules of insects: the CRF-related peptides and the insect kinins (2, 6, 7, 21). The CRF-related peptides are thought to affect primarily the active transport pathway via cAMP, which stimulates transcellular secretion of Na in part by increasing the Na conductance of the basolateral membrane of principal cells that in turn hyperpolarizes the Vt while lowering Rt (3, 20, 21). In terms of function, MNP, which we have purified from A. aegypti, behaves like a CRF-related peptide, but its CRF-like structure remains to be established (18, 19). Insect kinins, particularly the leucokinins, are thought to affect primarily the paracellular shunt transport pathway via Ca by increasing the Cl conductance of the paracellular septate junctional pathway (3, 15, 26). By affecting different transport pathways and employing different second messenger pathways, CRF-related diuretic peptides and insect kinins, acting together, have the potential of eliciting diuretic effects that are greater than the sum of their separate effects (6). An electrophysiological basis for this synergism can be gleaned from an examination of the epithelial transport model in Fig. 1. When transcellular resistance drops, rates of transepithelial secretion increase (Eq. 1). Likewise, when Rshunt drops, rates of transepithelial secretion increase (Eq. 1). However, Eq. 1 also shows that a drop in both transcellular resistance and Rshunt increases secretion appreciably more than the sum of their single effects. Coast (6) presents evidence for such synergism between CRF-related diuretic peptide and insect kinin in Malpighian tubules of the locust. The present study in Malpighian tubules of A. aegypti suggests that CRF-DP alone is capable of synergistic effects but in a dose-dependent way. At low concentrations, the peptide affects only the paracellular shunt pathway, but at high concentrations, it affects both shunt and transcellular pathways with expected synergistic effects on the rates of transepithelial secretion of electrolytes and water.
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ACKNOWLEDGEMENTS |
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K. W. Beyenbach thanks Sandy Hellman for past lessons in epithelial electrophysiology.
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FOOTNOTES |
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We thank the National Science Foundation for making this work possible via Grants IBN-9220464 and IBN-9604394 awarded to K. W. Beyenbach and by Grant IBN-9419990 awarded to T. K. Hayes.
Address for reprint requests: K. W. Beyenbach, Section of Physiology, VRT 8014, Cornell Univ., Ithaca, NY 14853.
Received 2 October 1997; accepted in final form 29 January 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Hayes, T. K.,
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