Am J Physiol Regul Integr Comp Physiol 307: R828–R836, 2014.
First published July 9, 2014; doi:10.1152/ajpregu.00561.2013.
Serotonin triggers cAMP and PKA-mediated intracellular calcium waves in
Malpighian tubules of Rhodnius prolixus
Paula Gioino, Brendan G. Murray, and Juan P. Ianowski
Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Submitted 23 December 2013; accepted in final form 7 July 2014
Malpighian tubule; Rhodnius prolixus; intracellular calcium; calcium
waves
RHODNIUS PROLIXUS IS A HEMATOPHAGOUS hemipteran vector for
Chagas’ disease. This insect is capable of ingesting blood
meals that may exceed 10 times its unfed body weight (26).
The large blood meal decreases the ability of the animal to
move, thus, increasing the risk of predation. In addition, the
blood meal causes osmotic stress, as the plasma fraction of the
blood meal is hypoosmotic with respect to the hemolymph (24,
26). Not surprisingly, R. prolixus has evolved a rapid diuretic
process that allows the insect to excrete the excess water and
ions ingested with the blood meal within 2 h (26). The main
excretory organ in insects is the Malpighian (renal) tubule,
which produces urine by active ion transport (33, 56). Fluid
and ion transport across fully stimulated R. prolixus Malpighian tubules may be very rapid, with each cell exchanging
the equivalent of its whole volume every 10 s and all intracellular Na⫹ and Cl⫺ content every 5 s (29).
Malpighian tubule secretion is triggered by at least two
diuretic hormones, serotonin and Rhopr-DH, a corticotropinreleasing factor (CRF)-like peptide (12, 50 –53). Both of these
hormones function through activation of the intracellular secAddress for reprint requests and other correspondence: J. P. Ianowski,
Health Science Bldg., Rm. 2D30.4, Univ. of Saskatchewan, 107 Wiggins
Rd., Saskatoon, Saskatchewan, S7N 5E5, Canada. (e-mail: juan.ianowski
@usask.ca).
R828
ond messenger cAMP (8, 53, 25). Until recently, calcium had
been ruled out as a second messenger in serotonin-stimulated
Malpighian tubules from R. prolixus based on the observation
that incubation with Ca2⫹-free saline containing the calcium
chelator EGTA, and treatment with the Ca2⫹ signaling blockers TMB-8 and CoCl2 do not affect serotonin-stimulated secretion (37, 45). However, a recent publication showed that
high doses of TMB-8 block serotonin-stimulated secretion,
suggesting that intracellular calcium may act as an intracellular
second messenger during the diuretic response in Rhodnius
prolixus (36). Thus, we decided to study the role of Ca2⫹ in
serotonin-stimulated fluid secretion by R. prolixus Malpighian
tubules.
Our results show that serotonin triggers intracellular Ca2⫹
waves that can be blocked with the Ca2⫹ chelator BAPTAAM. The Ca2⫹ waves can be triggered by stimulation with the
membrane-permeant cAMP analog, 8-Br-cAMP. Furthermore,
Ca2⫹ waves were blocked by PKA blocker H-89.
MATERIALS AND METHODS
Animals. Rhodnius prolixus were obtained from a laboratory colony maintained at 25–27°C and ⬃60% relative humidity in the
Department of Physiology, University of Saskatchewan. Experiments
were carried out at room temperature (22–25°C). R. prolixus is a
hemipteran species native to the Americas that has five nymphal
instars and an adult stage. During the nymphal stages, the animal
consumes larger blood meals relative to body size and undergoes the
most pronounced diuretic process. Since most of the research on
diuresis in R. prolixus has been carried out in the nymphal stages, we
used fifth- and third-instar R. prolixus 1–3 wk after molting.
Malpighian tubules were dissected from the animals under control
saline with the aid of a dissecting microscope. Each animal has four
Malpighian tubules that are long, thin, blind-ended tubes ⬃100 ␮m in
diameter and ⬃40 mm in length that empty their content into the
rectum (57). We used the fluid-secreting portion of the tubule, which
comprises the two-thirds (⬃25 mm) most distal to the opening of the
tubule to the rectum. The distal tubule comprises a single epithelial
cell type with uniform secretory properties along its length and
lacking contractile cells (10, 57). The tubules are one cell thick and
are made up of one or a few cells encircling the lumen (57).
Saline solutions. Control saline contained (in mmol/l) 122.6 NaCl,
14.5 KCl, 8.5 MgCl2, 2.0 CaCl2, 10.2 NaHCO3, 4.3 NaH2PO4, 8.6
HEPES, and 20 glucose, pH 7.0. Nominal Ca⫹-free saline contained
(in mmol/l) 122.6 NaCl, 14.5 KCl, 8.5 MgCl2, 10.2 NaHCO3, 4.3
NaH2PO4, 8.6 HEPES, and 20 glucose, at pH 7.0.
Secretion assays. Malpighian tubule fluid secretion rates were
measured using a modified Ramsay assay (42), as described previously (16). Briefly, the distal segment of Malpighian tubules from
fifth-instar insects were isolated in 100-␮l droplets of bathing saline
under paraffin oil. The cut end of the tubule was pulled out of the
droplet of bathing saline and wrapped around a fine steel pin pushed
into the Sylgard base of a Petri dish under paraffin oil. After stimulation with serotonin (5-hydroxytryptamine, 1 ␮mol/l) or 8-Br-cAMP
(8-bromoadenosine 3=,5=-cyclic monophosphate, 1 mmol/l), secreted
0363-6119/14 Copyright © 2014 the American Physiological Society
http://www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
Gioino P, Murray BG, Ianowski JP. Serotonin triggers cAMP and
PKA-mediated intracellular calcium waves in Malpighian tubules of Rhodnius prolixus. Am J Physiol Regul Integr Comp Physiol 307: R828–R836,
2014. First published July 9, 2014; doi:10.1152/ajpregu.00561.2013.—Rhodnius prolixus is a hematophagous insect vector of Chagas disease
capable of ingesting up to 10 times its unfed body weight in blood in
a single meal. The excess water and ions ingested with the meal are
expelled through a rapid postprandial diuresis driven by the Malpighian tubules. Diuresis is triggered by at least two diuretic hormones, a CRF-related peptide and serotonin, which were traditionally
believed to trigger cAMP as an intracellular second messenger.
Recently, calcium has been suggested to act as a second messenger in
serotonin-stimulated Malpighian tubules. Thus, we tested the role of
calcium in serotonin-stimulated Malpighian tubules from R. prolixus.
Our results show that serotonin triggers cAMP-mediated intracellular
Ca2⫹ waves that were blocked by incubation in Ca2⫹-free saline
containing the cell membrane-permeant Ca2⫹ chelator BAPTA-AM,
or the PKA blocker H-89. Treatment with 8-Br-cAMP triggered Ca2⫹
waves that were blocked by H-89 and BAPTA-AM. Analysis of the
secreted fluid in BAPTA-AM-treated tubules showed a 75% reduction
in fluid secretion rate with increased K⫹ concentration, reduced Na⫹
concentration. Taken together, the results indicate that serotonin
triggers cAMP and PKA-mediated Ca2⫹ waves that are required for
maximal ion transport rate.
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
The 340 nm/380 nm ratio was calculated using Metafluor version 7.0
software (Molecular Devices, Burlington, ON, Canada).
The possibility of sequestration of fura-2 within cellular organelles,
such as vesicles containing calcium concretion (27), was addressed.
We used a protocol based on selective permeabilization of the cell
membrane with digitonin. We used 20 ␮mol/l digitonin to permeabilize the cell membrane and allow cytosolic fura-2 to diffuse out of the
cells leaving behind fura-2 AM sequestered in organelles. Subsequent
treatment with Triton X permeabilized the membrane of the organelles and allowed fura-2 to leave the organelles (43, 1, 54, 20; for
review, see Ref. 21). The data show that tubules incubated with
digitonin in Ca2⫹-free saline plus 1 mmol/l EGTA saline reduced
fura-2 AM fluorescence 95%, while only 5% of the fura-2 AM was
sequestered in organelles (Fig. 1).
Fura-2 AM, probenecid, and pluronic F-127 were obtained from
Life Technologies. Stock solutions of fura-2 AM and pluronic F-127
were prepared in anhydrous DMSO. The maximal final concentration
of DMSO was ⬍1% (vol/vol). Previous studies have shown that
Malpighian tubule secretion rate is unaffected by DMSO at concentrations ⬍1% (vol/vol) (17). BAPTA-AM and 8-bromoadenosine3=,5=-cyclic monophosphate sodium salt (8-Br-cAMP) were obtained
from Tocris (Bioscience, Bristol, UK). H-89 (H-89 dihydrochloride
hydrate) was obtained from Sigma (St. Louis, MO).
Statistics. Data sets are presented as means ⫾ SE. The data were
tested for normality using Kolmogorov-Smirnov test, and the variances were tested using F test for unequal variances to ensure that the
data could be analyzed using parametric tests. The data were analyzed
using Student’s t-test or ANOVA, as appropriate. All statistical
analysis were two-tailed, and differences were considered significant
when P ⬍ 0.05. Analysis was performed using GraphPad Prism 5
(GraphPad Software, San Diego, CA).
RESULTS
Serotonin triggers cAMP-mediated intracellular Ca2⫹
waves. Stimulation with serotonin (1 ␮mol/l) triggered intracellular Ca2⫹ waves (Fig. 2A; n ⫽ 28 repetitions, all of which
showed serotonin-triggered intracellular Ca2⫹ waves). The
response time and the intensity of the response varied among
cells in each tubule (Fig. 2B). The Ca2⫹ signal was initiated in
a small number of cells, i.e., pioneer cells (48, 58), and
propagated to neighboring cells both toward the distal and
proximal ends of the tubule, showing asynchrony and heterogeneity (Fig. 2B, see supplemental material for a time-lapse
Fig. 1. Fura-2 is not sequestered in intracellular organelles. Malpighian tubules
were incubated with fura-2 AM (5 ␮mol/l), probenecid (1 mmol/l), and
pluronic F-127 (0.1%) in Ringer solution for 60 min at room temperature.
After incubation, the fura-2 AM was washed out with Ca2⫹-free saline plus 1
mmol/l EGTA saline containing probenecid (1 mmol/l). The concentration of
digitonin was 20 ␮mol/l and that of Triton X was 1% (wt/vol). a.u., arbitrary
units.
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
fluid droplets formed at the cut end of the tubule and were pulled away
from the tubule every 5 or 10 min using a fine glass probe. The
secreted droplets were photographed using a digital camera mounted
in a dissecting microscope (MiniVid, LW Scientific, Lawrenceville,
GA). Droplet radius (r) was measured from the stored images using
ImageJ 1.32 software (National Institutes of Health, Bethesda, MD).
The volume (V) of the secreted droplets was calculated using the
formula for the volume of a sphere V ⫽ 4/3␲r3, and the secretion rate
was calculated by dividing droplet volume by the time over which it
formed.
Measurement of K⫹ and Na⫹ in secreted droplets. Distal tubules
from fifth-instar insects were isolated and set up in a secretion assay,
as described above, and secreted droplets were collected. The Na⫹
and K⫹ concentrations in the secreted droplets were measured using
ion-selective microelectrodes, as previously described (17, 18, 19, 23,
34). Borosilicate capillaries (World Precision Instruments, Sarasota,
FL) were pulled using a vertical micropipette puller (PE-2, Narishige,
Japan). The electrodes were silanized with dichlorodimethylsilane
(Fluka) and baked for 20 min at 250°C.
Na⫹-selective electrodes were based on sodium ionophore I, cocktail A (Sigma, St. Louis, MO), and backfilled with 0.5 mol/l NaCl.
The reference electrode was backfilled with 1 mol/l KCl⫺. Na⫹selective electrodes were calibrated in solutions of (in mmol/) 15
NaCl, 135 KCl, and 150 NaCl.
K⫹-selective electrodes were based on potassium ionophore I,
cocktail B (Sigma, St. Louis, MO), and backfilled with 1 mol/l KCl.
The reference electrode was filled at the tip with 1 mol/l Na⫹ acetate,
and the rest with 1 mol/l KCl⫺. K⫹-selective electrodes were calibrated in solutions of (in mmol/l) 15 KCl, 135 NaCl, and 150 KCl.
Only electrodes that displayed a change of ⱖ50 mV per decade
change in ion concentration of the calibration solution were used.
The electrodes were connected through a high-impedance amplifier
(FD223A, World Precision Instruments, Sarasota, FL) to a PC data
acquisition system (LabChart 7pro V.7.2.1, Powerlab 4/35,
ADInstruments, Sydney, Australia). All measurements were taken
inside a Faraday cage.
Ion concentration in secreted droplets was calculated using the
formula: cd ⫽ cc * 10⌬V/S, where cd is the ion concentration in the
secreted droplet, cc is the ion concentration in one of the calibration
solutions, ⌬V is the difference in voltage measured between the
secreted droplet and the same calibration solution, and S is the slope
of the electrode (i.e., mV change in response to a 10-fold change in
ion concentration in the calibration solutions). Although ion-selective
microelectrodes measure ion activity and not concentration, data can
be expressed in terms of concentrations if it is assumed that the ion
activity coefficient is the same in calibration and experimental solutions (32).
Calcium imaging. Distal Malpighian tubules from third-instar insects were isolated and placed in a custom-built chamber with a glass
bottom. The chamber was treated with poly-L-lysine to facilitate
adhesion of the tubule to the bottom of the chamber (Sigma, St. Louis,
MO). The tubules were incubated at room temperature in the dark for
60 min in control saline containing fura-2 AM (5 ␮mol/l), probenecid
(1 mmol/l), and pluronic F-127 (0.1%) following the protocol provided by the supplier (Life Technologies, Carlsbad, CA). After incubation, the fura-2 AM solution was washed out with control saline
solution containing probenecid (1 mmol/l). Calcium was visualized
with an upright fluorescent microscope (BX61WI, Olympus, Tokyo,
Japan) using a water immersion 20⫻ objective with a field of view of
800 ⫻ 700 ␮m (UMPLFLN 20⫻W, Olympus, Tokyo, Japan). The
light source for excitation was an arc lamp (Prior Lumen 200PRO,
Prior Scientific, Cambridge, UK) filtered with 340 nm or 380 nm
excitation filters (BrightLine, Semrock, Rochester, NY). Fluorescent
light filtered with a 510-nm emission filter (BrightLine, Semrock,
Rochester, NY) was collected every 0.5–1 s with an EMCCD camera
(QImaging Rolera EM-C2, QImaging designs, Surrey, BC, Canada).
R829
R830
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
A
1
2
4
B
1
2
3
4
movie of the effect of serotonin on intracellular Ca2⫹). For our
analysis of intracellular Ca2⫹ signal, we selected the pioneer
cells in each tubule.
Serotonin triggered an initial large Ca2⫹ signal followed by
Ca2⫹ waves that persisted for the duration of the experiment.
We stopped the recordings while the waves were ongoing, the
longest measurement was 30 min. Intracellular Ca2⫹ waves
were terminated by washing out serotonin (Fig. 3A). In addition, both the frequency and amplitude of the serotonin-stimulated Ca2⫹ waves were concentration-dependent (Fig. 3, B
and C).
The effect of serotonin on intracellular Ca2⫹ was completely
blocked by preincubation with Ca2⫹-free saline containing the
cell-permeant Ca2⫹ chelator BAPTA-AM (30 ␮mol/l, Fig. 4, A
and B; n ⫽ 11 repetitions, all of which showed blockage of
serotonin-triggered intracellular Ca2⫹ waves). In contrast, incubation in Ca2⫹-free saline containing EGTA (1 mmol/l), a
Ca2⫹ chelator with lower cell membrane permeability, did not
block the intracellular Ca2⫹ waves triggered by serotonin (Fig.
4, C and D; n ⫽ 6 repetitions, all of which showed serotonintriggered intracellular Ca2⫹ waves). However, the frequency of
the intracellular Ca2⫹ waves triggered by serotonin was higher
in tubules incubated in Ca2⫹-free saline plus EGTA compared
with tubules incubated in control saline (Fig. 5A; P ⫽ 0.0003,
n ⫽ 28 for serotonin and n ⫽ 6 for EGTA, two-way repeatedmeasures ANOVA and Tukey-Kramer multiple-comparison
test). The amplitude was not different (Fig 5B; P ⫽ 0.062, n ⫽
28 for serotonin and n ⫽ 6 for EGTA, two-way repeatedmeasures ANOVA and Tukey-Kramer multiple-comparison
test).
Thus, we decided to test whether incubation with Ca2⫹-free
saline containing EGTA or BAPTA-AM block serotonin-stimulated fluid secretion by Malpighian tubules. Incubation of R.
prolixus tubules in Ca2⫹-free saline containing BAPTA-AM
(300 ␮mol/l) significantly reduced the serotonin-stimulated
secretion rate from 57 ⫾ 3 to 15 ⫾ 3 nl/min in control saline
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 2. Serotonin triggers intracellular Ca2⫹
waves. Tubules were incubated in control saline,
fura-2 AM, and probenecid for 60 min. After
incubation, the fura-2 AM was washed out with
saline containing probenecid (1 mmol/l). A: 340
nm/380 nm ratio when serotonin was added (time
0) and 100 and 200 s after serotonin (1 ␮mol/l)
stimulation. We selected four regions of identical
surface area (137 ␮m2) that did not include the
lumen of the tubule, labeled 1 to 4, to measure the
340 nm/380 nm ratio. Scale bar: 90 ␮m, while
the color key represents the 340/380 ratio. See
Supplemental Movie S1 for a time-lapse movie of
this preparation. B: graphs labeled 1 to 4 in the
panels below represent the ratio over time. The
addition of serotonin is marked with an arrow.
Site number 1 corresponds to a pioneer cell where
calcium waves were originated and propagated to
neighboring cells. The dashed lines indicate the
propagation of individual waves. The horizontal
bar represents 50 s, and the vertical bar represents
0.1 ratio 340/380.
3
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
and Ca2⫹-free saline plus BAPTA-AM, respectively (Fig. 6).
Washing BAPTA-AM out for 60 min restored the response to
serotonin to that of tubules never exposed to BAPTA-AM, 43 ⫾ 6
and 42 ⫾ 4 nl/min, respectively. In contrast, EGTA (1 mmol/l)
had no significant effect on serotonin-stimulated fluid secretion
(Fig. 6A; P ⬍ 0.0001, n ⫽ 18 tubules for each group, two-way
repeated-measures ANOVA and Tukey-Kramer multiple-comparison test). BAPTA-AM also affected the ion composition of
the fluid secreted by the serotonin-stimulated tubule. The Na⫹
concentration of the secreted fluid was reduced from 96 ⫾ 3 to
80 ⫾ 3 mmol/l by treatment with BAPTA-AM (Fig. 6B; P ⫽
0.0008, n ⫽ 52 for each group, two-way repeated-measures
ANOVA and Tukey-Kramer multiple-comparison test). The
K⫹ concentration increased from 89 ⫾ 1.3 to 100 ⫾ 2 after
treatment with BAPTA-AM (Fig. 6C; P ⫽ 0.0006, n ⫽ 52 for
each group, two-way repeated-measures ANOVA and TukeyKramer multiple-comparison test). Treatment with BAPTAAM reduced both Na⫹ and K⫹ flux from ⬃5 to 1 nmol/min.
Taken together, these results suggest that serotonin-stimulated
intracellular Ca2⫹ waves persist in the absence of extracellular
Ca2⫹ and that blocking intracellular Ca2⫹ reduces fluid secretion rate and Na⫹ and K⫹ flux.
The observation that serotonin stimulation of R. prolixus
tubules requires intracellular Ca2⫹ seems to contradict evidence showing that serotonin triggers cAMP and that stimulation with cAMP triggers maximal fluid secretion (25, 41).
Thus, we tested the effect of the membrane-permeant cAMP
analog 8-Br-cAMP on intracellular Ca2⫹. The results show that
treatment with 8-Br-cAMP (1 mmol/l) triggered intracellular
Ca2⫹ waves similar to those triggered by serotonin (Fig. 7A;
n ⫽ 8). Tubules stimulated with 8-Br-cAMP produced fluid at
similar rates to those stimulated with serotonin (Fig. 8A). The
amplitude of the intracellular Ca2⫹ waves was not different
from those stimulated by serotonin (Fig. 7B; P ⫽ 0.16, n ⫽ 13
for serotonin and n ⫽ 8 for 8-Br-cAMP, two-way repeatedmeasures ANOVA and Tukey-Kramer multiple-comparison
test). The frequency of intracellular Ca2⫹ wave stimulated by
8-Br-cAMP was initially similar to that triggered by serotonin.
The serotonin-stimulated Ca2⫹ waves decreased its frequency
over time; in contrast, the 8-Br-cAMP-stimulated frequency
remained constant (Fig. 7C; P ⫽ 0.001, n ⫽ 13 for serotonin
and n ⫽ 8 for 8-Br-cAMP, two-way repeated-measures
ANOVA and Tukey-Kramer multiple-comparison test). 8-BrcAMP-stimulated intracellular Ca2⫹ waves was blocked by
incubation in Ca2⫹-free saline plus BAPTA-AM (Fig. 8A; P ⬍
0.0001, n ⫽ 22 for serotonin, n ⫽ 17 for 8-Br-cAMP, and n ⫽
19 for 8-Br-cAMP plus BAPTA-AM, two-way repeated-measures ANOVA and Tukey-Kramer multiple-comparison test).
The intracellular second messenger pathways initiated by
cAMP frequently recruit PKA. Thus, we decided to test the
effect of H-89, a specific PKA blocker known to be effective in
insect tissue, including Malpighian tubules from R. prolixus
(36) and Aedes aegypti (40), the cryptonephric complex of
Manduca sexta (22), midgut epithelium of A. aegypti (30), and
the blowfly salivary gland (47). Treatment with H-89 (10
␮mol/l) blocked serotonin-stimulated Ca2⫹ waves (Fig. 8B;
n ⫽ 13). Incubation with H-89 for 40 min reduced the fluid
secretion rate from 54 ⫾ 4 to 15 ⫾ 1 nl/min in serotoninstimulated tubules and in tubules after 30-min treatment with
H-89 (Fig. 8A; n ⫽ 19 for H-89 and n ⫽ 22 for serotonin; P ⬍
0.0001, two-way repeated-measures ANOVA and TukeyKramer multiple-comparison test). Finally, H-89 blocked 8-BrcAMP-stimulated intracellular Ca2⫹ waves (Fig. 8C; n ⫽ 8).
Taken together, these results indicate that serotonin stimulation
of R. prolixus Malpighian tubules triggers intracellular Ca2⫹
waves mediated by cAMP and PKA activation. This is the first
evidence of cAMP- and PKA-mediated intracellular Ca2⫹
waves in insect epithelia.
DISCUSSION
Serotonin triggers cAMP- and PKA-mediated intracellular
Ca2⫹ waves. The results indicate that serotonin triggers not
only cAMP (25, 33, 41), but also cAMP and PKA-mediated
intracellular Ca2⫹ waves in R. prolixus Malpighian tubule
cells. The intracellular Ca2⫹ waves initiate in pioneer cells and
propagate to neighboring cells, suggesting heterogeneity in the
Malpighian tubule cells (48, 58). This is the first report of
cAMP and PKA-mediated intracellular Ca2⫹ waves in insect
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 3. Serotonin triggers a concentration-dependent Ca2⫹ response that is
terminated when serotonin is removed. A: effect of serotonin is removed by
washing. The addition of a second dose of serotonin 1 ␮mol/l triggers
intracellular calcium waves. The amplitude (B) and frequency (C) of the
calcium waves are concentration-dependent. The frequency was calculated by
counting the number of waves over the whole experiment and dividing by the
time. The amplitude is the average of the amplitudes of all the waves in the
experiment.
R831
R832
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 4. BAPTA-AM blocks serotonin-stimulated intracellular Ca2⫹ waves. Tubules were incubated in
Ca2⫹-free saline plus BAPTA-AM (30 ␮mol/l),
fura-2 AM, and probenecid for 60 min. A: 340 nm/
380 nm ratio when serotonin (1 ␮mol/l) was added
(time 0) and 100, 200, and 300 s after serotonin
stimulation. B: fura-2 AM ratio over time. C: tubules
incubated in Ca2⫹-free saline plus EGTA (1 mmol/l),
fura 2-AM, and probenecid for 60 min. Serotonin (1
␮mol/l) was added (time 0) and 100 and 200 s after
serotonin stimulation. D: fura-2 AM ratio over time.
The addition of serotonin is marked with an arrow.
The scale bar represents 90 ␮m, while the color scale
bar represents the 340/380 ratio.
epithelia that propagate from cell to cell, possibly through gap
junctions (55). Similar cAMP-mediated intracellular Ca2⫹ release has been described in mammalian cells (14, 15, 46).
Treatment with the membrane-permeant Ca2⫹ chelator
BAPTA-AM inhibited intracellular Ca2⫹ signaling, resulting
in a 75% reduction in the secretion rate. In contrast, incubating
the tubules in Ca2⫹-free bathing solution (Ca2⫹-free saline plus
EGTA) altered the frequency, but did not inhibit, serotoninstimulated intracellular Ca2⫹ waves and did not affect secretion rate. This is consistent with previous reports that serotonin-stimulated tubules secrete at maximal rate in Ca2⫹-free
saline plus EGTA (36, 37). The results suggest that Ca2⫹ from
intracellular stores is necessary for serotonin stimulation of R.
prolixus Malpighian tubules.
Serotonin stimulation of R. prolixus tubules involves cAMP
intracellular second messenger pathways, and treatment with
8-Br-cAMP produces the maximal secretion rate (25, 35). Our
results show that 8-Br-cAMP triggers intracellular Ca2⫹ waves
that can be blocked by treatment with BAPTA-AM. In addition, the effects of serotonin and 8-Br-cAMP are blocked
by treatment with the PKA inhibitor H-89 (36). Taken together,
the results suggest that serotonin increases intracellular cAMP
and activates PKA, which subsequently triggers intracellular
Ca2⫹ waves. Even though there is great diversity in the diuretic
factors and intracellular second messenger pathways involved
in the regulation of Malpighian tubule secretion in different
species, including biogenic amines tyramine, serotonin, and
several families of peptides (for review, see Refs. 9 and 38),
there are other species that require both cAMP and Ca2⫹
signaling for stimulation. For example, the Malpighian tubules
from the dipterans Drosophila melanogaster (31, 33), Culex
salinarius (7), and Aedes aegypti (40, 59) respond to diuretic
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
R833
mammalian 5-HT2 and 5-HT7, which stimulate intracellular
Ca2⫹ and cAMP, respectively, triggering intracellular second
messenger pathways with complex cross-talk between them
(13, 44). However, in R. prolixus Malpighian tubules, there
seems to be a single serotonin receptor that triggers cAMP
since 8-Br-cAMP triggers full fluid secretion and intracellular
Ca2⫹ waves, suggesting that the Ca2⫹ waves are not dependent
on serotonin binding to a 5-HT receptor.
signals by increasing both cyclic nucleotides, which stimulate active cation transport, and Ca2⫹ signaling, which
modulates epithelial Cl⫺ permeability. In the cricket, Acheta
domesticus, intracellular cAMP and Ca2⫹ have opposing
effects, with cAMP stimulating secretion and Ca2⫹ inhibiting secretion (49).
Serotonin has been shown to stimulate both cAMP and Ca2⫹
pathways in the salivary gland cells of the blowfly Calliphora
(3, 13, 44). In these salivary glands, cells express two different
populations of serotonin receptors, with high similarity to
Fig. 6. Chelating intracellular calcium blocks the effect of serotonin. A:
Malpighian tubules bathed in control saline (), Ca2⫹-free saline containing
EGTA (1 mmol/l, Œ), and Ca2⫹-free saline containing BAPTA-AM (300
␮mol/l, Œ) were stimulated at time 0 with serotonin (1 ␮mol/l). Treatment with
BAPTA-AM significantly reduced fluid secretion rate (P ⬍ 0.0001; n ⫽ 18
tubules for control, EGTA, and BAPTA-AM groups, two-way repeatedmeasures ANOVA, Tukey-Kramer multiple-comparison test; data points labeled with different letters differ significantly). B: sodium concentration was
significantly reduced in tubules incubated in Ca2⫹-free saline plus
BAPTA-AM (300 ␮mol/l; P ⫽ 0.0008, n ⫽ 14 for control group and n ⫽ 21
for Ca2⫹-free saline plus BAPTA-AM group, two-way repeated-measures
ANOVA, Tukey-Kramer multiple-comparison test, data points labeled with
different letters differ significantly). C: potassium concentration increased in
tubules incubated in Ca2⫹-free saline plus BAPTA-AM (300 ␮mol/l, P ⫽
0.0006, n ⫽ 52 for each group, two-way repeated-measures ANOVA, TukeyKramer multiple-comparison test; data points labeled with different letters
differ significantly).
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 5. Incubation in Ca2⫹-free saline plus EGTA results in higher Ca2⫹ wave
frequency. We calculated the frequency and amplitude of Ca2⫹ waves at
different times after stimulation with serotonin (time 0). A: tubules incubated
in Ca2⫹-free saline plus EGTA saline displayed an increase in frequency (P ⫽
0.0003; n ⫽ 28 for serotonin and n ⫽ 6 for EGTA, two-way repeated-measures
ANOVA and Tukey-Kramer multiple-comparison test). Data points labeled
with different letters differ significantly. B: however, EGTA had no effect on
amplitude of the Ca2⫹ waves (P ⫽ 0.062; n ⫽ 28 for serotonin and n ⫽ 6 for
EGTA, two-way repeated-measures ANOVA and Tukey-Kramer multiplecomparison test).
R834
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 7. 8-Br-cAMP triggers intracellular Ca2⫹ waves. A: stimulation with
8-Br-cAMP (1 mmol/l) triggers intracellular Ca2⫹ waves. The amplitude and
frequency of Ca2⫹ waves were measured at different times after stimulation
with serotonin or 8-Br-cAMP (time 0). B: amplitude of the Ca2⫹ waves was
similar to that triggered by serotonin (P ⫽ 0.16; n ⫽ 13 for serotonin and n ⫽
8 for 8-Br-cAMP, two-way repeated-measures ANOVA and Tukey-Kramer
multiple-comparison test). C: frequency of Ca2⫹ waves was higher in 8-BrcAMP-stimulated tubules (Fig. 7C; P ⫽ 0.001; n ⫽ 13 for serotonin and n ⫽
8 for 8-Br-cAMP). Two-way repeated-measures ANOVA and Tukey-Kramer
multiple-comparison test were performed. Data points labeled with different
letters differ significantly.
Perspectives and Significance
This is the first report of cAMP- and PKA-mediated intracellular Ca2⫹ waves in insect epithelia. Interestingly, a recent
publication has demonstrated that the Malpighian tubules of R.
prolixus display oscillation in the transepithelial potential
(TEP) when stimulated at submaximal concentrations of serotonin (28). Thus, it is possible that Ca2⫹ may regulate Cl⫺ flux
in R. prolixus, thus affecting TEP and producing oscillations.
Similar fluctuations of the TEP driven by modulations of Cl⫺
permeability by intracellular Ca2⫹ oscillations have been reported in the salivary glands of the blowfly Calliphora (2) and
Malpighian tubules of Aedes aegypti (5, 6, 39). These Ca2⫹
oscillations may play an important role in the cross-talk be-
Fig. 8. cAMP-PKA-mediated intracellular Ca2⫹ waves. A: fluid secretion rate
by tubules stimulated with 8-Br-cAMP (1 mmol/l, Œ; n ⫽ 17) or serotonin (1
␮mol/l, ; n ⫽ 22) showed no significant differences. 8-Br-cAMP-stimulated
tubules incubated with Ca2⫹-free saline plus BAPTA-AM (300 ␮mol/l, Œ; n ⫽
19), and serotonin-stimulated tubules treated with H-89 (10 ␮mol/l, downward
arrow, n ⫽ 19) showed decreased secretion rate compared with serotonin- and
8-Br-cAMP-stimulated tubules but showed no differences when compared
with each other (P ⬍ 0.0001, two-way repeated-measures ANOVA, TukeyKramer multiple-comparison test). Data points labeled with different letters
differ significantly. B: intracellular Ca2⫹ waves in tubules stimulated with
serotonin (1 ␮mol/l) are abolished by PKA blocker H-89 (10 ␮mol/l). C:
8-Br-cAMP-stimulated intracellular Ca2⫹ waves are abolished by PKA
blocker H-89 (10 ␮mol/l).
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
tween the different components of the transport machinery to
maintain intracellular homeostasis (3, 4). Such a role for Ca2⫹
in cross-talk has been proposed in the early distal tubule of the
frog nephron (11).
ACKNOWLEDGMENTS
We thank Drs. Nigel West and Michel Desautels for stimulating discussion
and critique.
GRANTS
This work was supported by a Discovery Grant from the Natural Sciences
and Engineering Research Council of Canada to J. P. Ianowski. P. Gioino was
supported by a fellowship from the College of Graduate Studies and Research
at the University of Saskatchewan.
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: P.G., B.G.M., and J.P.I. performed experiments;
P.G., B.G.M., and J.P.I. analyzed data; P.G. and J.P.I. interpreted results of
experiments; P.G. and J.P.I. prepared figures; P.G. and J.P.I. drafted manuscript; P.G. and J.P.I. approved final version of manuscript; J.P.I. conception
and design of research; J.P.I. edited and revised manuscript.
REFERENCES
1. Allen CU, Herman B, Granger A. Fura-2 measurement of cytosolic free
Ca2⫹ concentration in corpus allatum cells of larval Manduca sexta. J.
Exp. Biol .166: 253–266, 1992.
2. Berridge MJ, Rapp PE. A comparative survey of the function, mechanism and control of cellular oscillators. J Exp Biol 81: 217–279, 1979.
3. Berridge MJ. Electrophysiological evidence for the existence of separate
receptor mechanisms mediating the action of 5-hydroxytryptamine. Mol
Cell Endocrinol 23: 91–104, 1981.
4. Berridge MJ. Smooth muscle cell calcium activation mechanisms. J
Physiol 586: 5047–5061, 2008.
5. Beyenbach KW, Aneshansley DJ, Pannabecker TL, Masia R, Gray D,
Yu M. Oscillations of voltage, and resistance in Malpighian tubules of
Aedes aegypti. J Insect Physiol 46: 321–333, 2000.
6. Beyenbach KW. Regulation of tight junction permeability with switchlike speed. Curr Opin Nephrol Hypertens 12: 543–550, 2003.
7. Clark TM, Hayes TK, Holman GM, Beyenbach KW. The concentration-dependence of CRF-like diuretic peptide: mechanisms of action. J
Exp Biol 201: 1753–1762, 1998.
8. Coast GM, Orchard I, Phillips JE, Schooley DA. Insect diuretic and
antidiuretic hormones. Adv Insect Physiol 29: 279 –409, 2002.
9. Coast GM. Serotonin has kinin-like activity in stimulating secretion by
Malpighian tubules of the house cricket Acheta domesticus. Peptides 32:
500 –508, 2011.
10. Collier K, O’Donnell M. Analysis of epithelial transport by measurement
of K⫹, Cl⫺ and pH gradients in extracellular unstirred layers: ion secretion
and reabsorption by Malpighian tubules of Rhodnius prolixus. J Exp Biol
200: 1627–1638, 1997.
11. Cooper GJ, Fowler M, Hunter M. Membrane cross-talk in the early
distal tubule segment of frog kidney: role of calcium stores and chloride.
Pflügers Archiv 442: 243–247, 2001.
12. Donini A, O’Donnell MJ, Orchard I. Differential actions of diuretic
factors on the Malpighian tubules of Rhodnius prolixus. J Exp Biol 211:
42–48, 2008.
13. Fechner L, Baumann O, Walz B. Activation of the cyclic AMP pathway
promotes serotonin-induced Ca2⫹ oscillations in salivary glands of the
blowfly Calliphora vicina. Cell Calcium 53: 94 –101, 2013.
14. Gomes P, Soares-da-Silva P. Role of cAMP-PKA-PLC signaling cascade
on dopamine-induced PKC-mediated inhibition of renal Na⫹-K⫹-ATPase
activity. Am J Physiol Renal Physiol 282: F1084 –F1096, 2002.
15. Hoque KM, Woodward OM, van Rossum DB, Zachos NC, Chen L,
Leung GP, Guggino WB, Guggino SE, Tse CM. Epac1 mediates protein
kinase A-independent mechanism of forskolin-activated intestinal chloride
secretion. J Exp Biol 135: 43–58, 2010.
16. Ianowski JP, Christensen RJ, O’Donnell MJ. Intracellular ion activities
in Malpighian tubule cells of Rhodnius prolixus: evaluation of Na⫹-K⫹2Cl⫺ cotransport across the basolateral membrane. J Exp Biol 205:
1645–1655, 2002.
17. Ianowski JP, Christensen RJ, O’Donnell MJ. Na⫹ competes with K⫹ in
bumetanide-sensitive transport by Malpighian tubules of Rhodnius prolixus. J Exp Biol 207: 3707–3716, 2004.
18. Ianowski JP, O’Donnell MJ. Electrochemical gradients for Na⫹, K⫹,
Cl⫺ and H⫹ across the apical membrane in Malpighian (renal) tubule cells
of Rhodnius prolixus. J Exp Biol 209: 1964 –1975, 2006.
19. Ianowski JP, O’Donnell MJ. Transepithelial potential in Malpighian
tubules of Rhodnius prolixus: lumen-negative voltages and the triphasic
response to serotonin. J Insect Physiol 47: 411–421, 2001.
20. Kao JPY, Harootunian AT, Tsien RY. Photochemically generated
cytosolic calcium pulses and their detection by Fluo-3. J Biol Chem 264:
8179 –8184, 1989.
21. Kao JPY, Li G, Auston DA. Practical aspects of measuring intracellular
calcium signals with fluorescent indicators. Methods Cell Biol 99: 113–
152, 2010.
22. Liao S, Audsley N, Schooley DA. Antidiuretic effects of a factor in
brain/corpora cardiaca/corpora allata extract on fluid reabsorption across
the cryptonephric complex of Manduca sexta. J Exp Biol 203: 605–615,
2000.
23. Maddrell SH, O’Donnell MJ, Caffrey R. The regulation of haemolymph
potassium activity during initiation and maintenance of diuresis in fed
Rhodnius prolixus. J Exp Biol 177: 273–285, 1993.
24. Maddrell SH, O’Donnell MJ. Insect Malpighian tubules: V-ATPase
action in ion and fluid transport. J Exp Biol 172: 417–429, 1992.
25. Maddrell SH, Pilcher DE, Gardiner BO. Pharmacology of the Malpighian tubules of Rhodnius and Carausius: the structure-activity relationship of tryptamine analogues and the role of cyclic AMP. J Exp Biol. 54:
779 –804, 1971.
26. Maddrell SH. Excretion in the blood-suking bug, Rhodnius prolixus Stål.
III. The control of the release of the diuretic hormone. J Exp Biol 41:
459 –472, 1964.
27. Maddrell SHP, Whittembury G, Mooney RL, Harrison JB, Overton
JA, Rodriguez B. The fate of calcium in the diet of Rhodnius prolixus:
storage in concretion bodies in the Malpighian tubules. J Exp Biol 157:
483–502, 1991.
28. Maddrell SHP. Insect homeostasis: past and future. J Exp Biol 212:
446 –451, 2009.
29. Maddrell SHP. The fastest fluid-secreting cell known: The upper Malpighian tubule of Rhodnius. BioEssays 13: 357–362, 1991.
30. Moffett DF, Jagadeshwaran U, Wang Z, Davis HM, Onken H, Goss
GG. Signaling by intracellular Ca2⫹ and H⫹ in larval mosquito (Aedes
aegypti) midgut epithelium in response to serosal serotonin and lumen pH.
J Insect Physiol 58: 506 –512, 2012.
31. O’Donnell MJ, Dow JA, Huesmann GR, Tublitz NJ, Maddrell SH.
Separate control of anion and cation transport in Malpighian tubules of
Drosophila melanogaster. J Exp Biol 199: 1163–1175, 1996.
32. O’Donnell MJ, Fletcher M, Haley CA. KCl reabsorption by the lower
Malpighian tubule of Rhodnius prolixus: inhibition by Cl⫺ channel blockers and acetazolamide. J Insect Physiol 43: 657–665, 1997.
33. O’Donnell MJ, Ianowski JP, Linton SM, Rheault MR. Inorganic and
organic anion transport by insect renal epithelia. Biochim Biophys Acta
1618: 194 –206, 2003.
34. O’Donnell MJ, Maddrell SH. Fluid reabsorption and ion transport by the
lower Malpighian tubules of adult female Drosophila. J Exp Biol 198:
1647–1653, 1995.
35. O’Donnell MJ, Quinlan MC. Anti-diuresis in the blood-feeding insect
Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the
role of organic acid transport. J Insect Physiol. 44: 561–568, 1998.
36. Paluzzi JP, Yeung C, O’Donnell MJ. Investigations of the signaling
cascade involved in diuretic hormone stimulation of Malpighian tubule
fluid secretion in Rhodnius prolixus. J Insect Physiol 59: 1179 –1185,
2013.
37. Paluzzi JP, Young P, Defferrari MS, Orchard I, Carlini CR,
O’Donnell MJ. Investigation of the potential involvement of eicosanoid
metabolites in anti-diuretic hormone signaling in Rhodnius prolixus.
Peptides 34: 127–134, 2012.
38. Paluzzi JP. Anti-diuretic factors in insects: the role of CAPA peptides.
Gen Comp Endocrinol .176: 300 –308, 2012.
39. Pannabecker TL, Hayes TK, Beyenbach KW. Regulation of epithelial shunt
conductance by the peptide leucokinin. J Membr Biol 132: 63–76, 1993.
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
DISCLOSURES
R835
R836
cAMP-MEDIATED INTRACELLULAR CALCIUM WAVES IN RHODNIUS MALPIGHIAN TUBULES
50. Te Brugge V, Ianowski JP, Orchard I. Biological activity of diuretic
factors on the anterior midgut of the blood-feeding bug, Rhodnius prolixus. Gen Comp Endocrinol 162: 105–112, 2009.
51. Te Brugge V, Paluzzi JP, Schooley DA, Orchard I. Identification of
the elusive peptidergic diuretic hormone in the blood-feeding bug
Rhodnius prolixus: a CRF-related peptide. J Exp Biol 214: 371–381,
2011.
52. Te Brugge VA, Miksys SM, Coast GM, Schooley DA, Orchard I. The
distribution of a CRF-like diuretic peptide in the blood-feeding bug
Rhodnius prolixus. J Exp Biol 202: 2017–2027, 1999.
53. Te Brugge VA, Schooley DA, Orchard I. The biological activity of
diuretic factors in Rhodnius prolixus. Peptides 23: 671–681, 2002.
54. von Stockum S, Basso E, Petronilli V, Sabatelli P, Forte MA, Bernardi
P. Properties of Ca2⫹ transport in mitochondria of Drosophila melanogaster. J Biol Chem 286: 41163–41170, 2011.
55. Weng XH, Piermarini PM, Yamahiro A, Yu MJ, Aneshansley DJ,
Beyenbach KW. Gap junctions in Malpighian tubules of Aedes aegypti. J
Exp Biol 211: 409 –422, 2008.
56. Wieczorek H, Weerth S, Schindlbeck M, Klein U. A vacuolar-type
proton pump in a vesicle fraction enriched with potassium transporting
plasma membranes from tobacco hornworm midgut. J Biol Chem 264:
11143–11148, 1989.
57. Wigglesworth VB, Salpeter MM. Histology of the Malpighian tubules in
Rhodnius prolixus Stål (Hemiptera). J Insect Physiol 8: 299 –307, 1962.
58. Yamamoto-Hino M, Miyawaki A, Segawa A, Adachi E, Yamashina S,
Fujimoto T, Sugiyama T, Furuichi T, Hasegawa M, Mikoshiba K.
Apical vesicles bearing inositol 1,4,5-triphosphate receptors in the Ca2⫹
initiation site of ductal epithelium of submandibular gland. J. Cell Biol.
141: 135–142, 1998.
59. Yu MJ, Beyenbach KW. Leucokinin activates Ca2⫹-dependent signal
pathway in principal cells of Aedes aegypti Malpighian tubules. Am J
Physiol Renal Physiol 283: F499 –F508, 2002.
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00561.2013 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on June 17, 2017
40. Petzel DH, Berg MM, Beyenbach KW. Hormone-controlled cAMPmediated fluid secretion in yellow-fever mosquito. Am J Physiol Regul
Integr Comp Physiol 253: R701–R711, 1987.
41. Quinlan MC, Tublitz NJ, O’Donnell MJ. Anti-diuresis in the bloodfeeding insect Rhodnius prolixus Stål: the peptide CAP2b and cyclic GMP
inhibit Malpighian tubule fluid secretion. J Exp Biol 200: 2363–2367,
1997.
42. Ramsay JA. Active transport of water by the Malpighian tubules of the
stick insect, Dixippus Morosus (Orthoptera, Phasmidae). J. Exp. Biol. 31:
104 –113, 1954.
43. Roe MW, Lemasters JJ, Herman B. Assessment of Fura-2 for measurements of cytosolic free calcium. Cell Calcium 11: 63–73, 1990.
44. Röser C, Jordan N, Balfanz S, Baumann A, Walz B, Baumann O,
Blenau W, Roser C. Molecular and pharmacological characterization of
serotonin 5-HT2␣ and 5-HT7 receptors in the salivary glands of the
blowfly Calliphora vicina. PloS One 7: e49459, 2012.
45. Ruiz-Sanchez E, Orchard I, Lange AB. Effects of the cyclopeptide
mycotoxin destruxin A on the Malpighian tubules of Rhodnius prolixus
(Stål). Toxicon 55: 1162–1170, 2010.
46. Schmidt M, Evellin S, Weernink PA, von Dorp F, Rehmann H,
Lomasney JW, Jakobs KH. A new phospholipase-C-calcium signaling
pathway mediated by cyclic AMP and a Rap GTPase. Nat Cell Biol 3:
1020 –1024, 2001.
47. Schmidt R, Baumann O, Walz B. cAMP potentiates InsP3-induced Ca2⫹
release from the endoplasmic reticulum in blowfly salivary glands. BMC
Physiol 8: 10, 2008.
48. Segawa A, Takemura H, & Yamashina S. Calcium signalling in tissue:
diversity and domain-specific integration of individual cell response in
salivary glands. J Cell Sci 115: 1869 –1876, 2002.
49. Spring JH, Clark TM. Diuretic and antidiuretic factors which act on the
malpighian tubules of the house cricket, Acheta domesticus. Prog Clin
Biol Res 342: 559 –564, 1990.