Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus
1 Laboratoire Génome, Populations, Interactions, Adaptation, UMR
5000, Equipe Adaptation Ecophysiologique et Ontogenèse,
Université Montpellier II, Place E. Bataillon, CP 092, 34095
Montpellier Cédex 05, France
2 Groupe Biogenèse des Peptides Isomères, UMR CNRS Physiologie
et Physiopathologie, Université Paris VI, CC 256, 7 Quai Saint-Bernard,
75252 Paris Cédex 05, France
* Author for correspondence (e-mail: pierrot{at}univ-montp2.fr)
Accepted 19 December 2002
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Summary |
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Key words: Crustacea, crayfish, Astacus leptodactylus, eyestalk, crustacean hyperglycemic hormone, osmoregulation, neuropeptide, CHH, isoform, D-Phe3 CHH
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Introduction |
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Studies on crayfish osmoregulation have demonstrated that these crustaceans
hyperosmoregulate in FW, and thus maintain a high hemolymph osmolality and ion
content, through three main physiological mechanisms: (1) low permeability of
the chitinoproteic cuticle to prevent water invasion and ion loss; (2) active
uptake of ions (essentially Na+ and Cl-) by specialized
cells, or ionocytes, located in the epithelia of the branchial chambers and
(3) production of hypotonic urine through the excretory antennal glands
(reviewed by Potts and Parry,
1964; Mantel and Farmer,
1983
; Péqueux,
1995
; Wheatly and Gannon,
1995
).
Since the early work of Scudamore
(1947), numerous experiments
have established the existence of neuroendocrine control of hydromineral
metabolism in decapod crustaceans, mainly in marine species. The presence of
active factors has been suggested in neuroendocrine centers located in the
eyestalks, the pericardial organs, the cerebroid ganglia, the thoracic
ganglionic mass and the ventral nervous system (reviewed by Kamemoto,
1976
,
1991
;
Kleinholz, 1976
;
Mantel, 1985
;
Muramoto, 1988
;
Morris, 2001
). Generally,
eyestalk ablation or ligation performed on crustaceans acclimated to dilute
media results in a decrease in hemolymph osmolality, ion content and ionic
(Na+ and Cl-) influx and an increase in water content
(reviewed by Kamemoto, 1976
;
Charmantier et al., 1984
;
Mantel, 1985
;
Muramoto, 1988
). Implantation
of eyestalk tissue or injection of eyestalk extracts restores or enhances
ionic and osmotic regulation in eyestalkless crustaceans
(Kamemoto, 1976
;
Charmantier et al., 1984
;
Mantel, 1985
;
Charmantier-Daures et al.,
1988
; Freire and McNamara,
1992
). These results have suggested the involvement of an eyestalk
neuroendocrine factor(s) in the control of osmoregulatory processes.
In decapod crustaceans, each eyestalk hosts, within the medulla terminalis,
a group of neurosecretory cells, called the X-organ, which synthesize
neurohormones that are subsequently stored in and released from a neurohemal
organ, the sinus gland (SG). Studies on the neuroendocrine control of
osmoregulation point to the involvement of a factor(s) from the SG. For
instance, injection of total extracts of SG into destalked juvenile lobsters
increases the hemolymph osmolality in dilute media in a dose- and
time-dependent manner (Charmantier-Daures
et al., 1988). Studies on the hyperhypo-regulating crab
Pachygrapsus marmoratus have demonstrated that SG extracts perfused
through isolated posterior gills stimulate ionic regulation mechanisms
(Pierrot et al., 1994
;
Eckhardt et al., 1995
).
Several 8-9.5 kDa neuropeptides, forming the so-called crustacean
hyperglycemic hormone family, have been isolated from the X-organSG
complex: the molt inhibiting hormone (MIH) involved in molting, the
vitellogenesis inhibiting hormone (VIH) involved in reproduction, the
mandibular organ inhibiting hormone (MOIH) involved in reproduction and
development, and the crustacean hyperglycemic hormone (CHH) involved in the
regulation of hemolymph glucose level (reviewed by
Keller, 1992;
Van Herp, 1998
). CHH has been
extensively studied and has been purified from the SG of numerous species
(reviewed by Soyez, 1997
;
Lacombe et al., 1999
). This
neuropeptide appears to be an important multifunctional hormone: primarily
involved in carbohydrate metabolism, it also controls other physiological
activities including secretion of digestive enzymes
(Keller and Sedlmeier, 1988
)
and lipid metabolism (Santos et al.,
1997
). In addition, CHH can exhibit MIH, VIH and/or MOIH
activities (reviewed by Van Herp,
1998
). Interestingly, Charmantier-Daures et al.
(1994
) have demonstrated that
one of the CHH isoforms from the SG of Homarus americanus can restore
the osmoregulatory capacity in eyestalkless adult lobsters acclimated to low
salinities. A recent study has shown that CHH purified from the SG of the crab
P. marmoratus increases the Na+ influx and the
transepithelial potential difference in perfused posterior gills from crabs
acclimated to diluted seawater
(Spanings-Pierrot et al.,
2000
). CHH thus seems to be involved in the control of
osmoregulation in marine crustaceans.
However, very little information is available on the neuroendocrine factors involved in osmoregulation in FW crustaceans. The main objective of the present study was to examine the potential involvement of CHH isoforms in the neuroendocrine mediation of osmoregulation in a FW species, the crayfish Astacus leptodactylus. First, the effect of eyestalk ablation on the hemolymph osmolality and ion content was reported, suggesting a neuroendocrine control of osmoregulation. Then, CHH was purified, isolated and characterized by high-performance liquid chromatographic (HPLC) fractionation of SG extracts together with immunochemical tests and bioassays of hemolymph glucose concentration. Finally, the effect of the neuropeptide on osmotic regulation was determined following injection of different CHH isoforms into eyestalkless crayfish.
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Materials and methods |
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Isolation and characterization of the crustacean hyperglycemic
hormone
Sinus gland extraction and RP-HPLC
Sinus glands (SG) from freshly excised eyestalks were isolated with a
minimum of surrounding tissue and were ground in the incubation medium
(ice-cold 10% acetic acid) with a Potter (glassglass) microhomogenizer.
The homogenates were incubated in a water bath at 80°C for 5 min and were
then pooled and stored at -20°C.
A pool of frozen SG homogenate was centrifuged at 12 000 g for 20 min. The supernatant was again centrifuged for 30 min in a centrifugal evaporator. The pellet was reextracted twice with 200 µl of 10% acetic acid and then centrifuged at 12 000 g for 20 min. The pooled supernatants were injected onto a reverse-phase HPLC (RP-HPLC) column (250 mm length x 4.6 mm i.d.) filled with Nucleosil C-18 (5 µm particle size) and eluted using a gradient of solvent B [0.1% trifluoroacetic acid (TFA) in 100% acetonitrile] in solvent A (0.1% TFA in water) at a flow rate of 0.75 ml min-1. UV absorbance was monitored at 220 nm with an LDC-Milton Roy Spectromonitor 3000 spectrophotometer. Fractions were collected every 30 s or 60 s.
For hyperglycemia and osmoregulation bioassays, pools of 50 SG equivalents (SGequiv) were extracted and subjected to HPLC. Fractions with a retention time of 45-48 min (see Fig. 4A) were pooled in three different zones (Z1, Z2 and Z3), lyophilized and stored at -20°C before use.
|
Identification of the hyperglycemic hormone: localization of CHH by
ELISA of RP-HPLC fractions
Direct enzyme-linked immunosorbent assay (ELISA) tests with three different
antibodies were performed on RP-HPLC fractions. 5 µl of each fraction was
deposited into the wells of three microtitration plates and dried under vacuum
before addition of 100 µl of sodium carbonate buffer (0.1 mol
l-1, pH 9.6) to each well. The plates were incubated at 37°C
for 2 h then at 4°C for 12 h and washed three times with 150 µl of
phosphate-buffered saline (PBS)tweenazide (0.2 mol
l-1 PBS, pH 7.2, containing 0.1% Tween 20 and 0.02% Na azide); 100
µl of rabbit specific antisera, diluted 1:500 in
PBStweenazide, were then added to each well. The antisera used
in this study were anti-Astacus CHH antiserum and two hapten-specific
antisera discriminating between the amino-terminal of CHH stereoisomers
(anti-octapeptide antisera). These antisera were raised against two synthetic
octapeptides with a sequence identical to the amino-terminal part of the
isoforms of the lobster Homarus americanus CHH:
pGlu-Val-Phe-Asp-Gln-Ala-Cys-Lys for anti-L antiserum and
pGlu-Val-D-Phe-Asp-Gln-Ala-Cys-Lys for anti-D antiserum. The production and
characterization of the two antisera have been described by Soyez et al.
(1998,
2000
). The plates were
incubated at 37°C for 1.5 h and were then washed three times with 150
µl of PBStweenazide. Incubation with 100 µl of a goat
anti-rabbit antiserum conjugated with alkaline phosphatase and diluted 1:50 in
PBStweenazide was performed at 37°C for 1.5 h. The plates
were washed three times with PBStween. A volume of 100 µl of
substrate (two tablets of p-nitrophenyl phosphate dissolved in 30 ml
of 0.1 mol l-1 carbonate buffer, pH 9.6) was added to each well to
visualize the alkaline phosphatase activity. The absorbance was determined at
405 nm using a Titertek Multiskan Plus reader.
Injection of purified fractions
Preparation of samples. Lyophilized HPLC fractions (Z1, Z2 and Z3)
were resuspended in a Van Harreveld
(1936) saline solution (205.33
mmol l-1 NaCl; 5.36 mmol l-1 KCl; 2.46 mmol
l-1 MgCl2.6H2O; 15.3 mmol l-1
CaCl2.2H2O; 5 mmol l-1 maleic acid; 5 mmol
l-1 Tris; pH 7.4) adjusted to the mean hemolymph osmotic pressure
of the injected animals (approximately 375 mosmol kg-1). Aliquots
of 10 SGequiv in 50 µl saline solution were prepared. Control animals were
injected with 50 µl of Van Harreveld saline. Before injection, samples were
vortexed, sonicated for 5 min and then briefly microcentrifuged.
Injection of samples. Injections were performed into the blood sinus at the base of one cheliped using a heat-sharpened glass micropipette (Drummond microcaps) connected via a polyethylene tubing to a 50 µl calibrated Hamilton syringe.
Measured parameters
The crayfish abdomen was dried with absorbent paper. Hemolymph was sampled
from the abdominal blood sinus using a hypodermic needle mounted on a syringe
and was then deposited on Parafilm.
Osmolality
The osmolality of 50 µl hemolymph samples was measured on a Roebling
micro-osmometer.
Ionic concentration
Hemolymph Na+ was titrated with an Eppendorf flame photometer
following appropriate dilution (3 µl of hemolymph in 2 ml deionised water).
Hemolymph Cl- titration was measured with an amperometric
Aminco-Cotlove chloridimeter (10 µl hemolymph sample diluted in 0.5 ml
deionised water and 3 ml aceticnitric reagent).
Hemolymph glucose quantification
Each 50 µl hemolymph sample was mixed with 50 µl of 0.66 mol
l-1 perchloric acid (PCA), which precipitates proteins, then
vortexed and centrifuged at 13 000 g for 20 min. Hemolymph
glucose concentration was quantified using the glucose oxidase method
(Peridochrom Glucose/GODPAP; Fisher-Osi Biolabo, Fismes, France). A
standard curve was established by twofold serial dilutions ranging from 0 mg
ml-1 to 2 mg ml-1 glucose solution in 0.66 mol
l-1 PCA. Into wells of a microtitration plate, 20 µl of 0.66 mol
l-1 PCA (blank), 20 µl of each standard dilution and 20 µl of
hemolymph samples were deposited in duplicate before the addition of 200 µl
of glucose oxidase peroxidase 4-amino-phenazone (GODPAP) reagent. The
plate was incubated at 37°C for 1 h. The absorbance of each well was
measured at 490 nm by an Elx800 Bio-Tek Instruments reader.
Mass
Animals were blotted with absorbent paper to remove peripheric water and
branchial chambers water and were then weighed on an electronic balance
(±0.1 g).
Statistical analysis
Analysis of variance (ANOVA), Fisher's multiple-range least significant
difference (LSD) post hoc test and Student's t-test were
used for multiple and pairwise statistical comparisons of mean values,
respectively, after appropriate checks of normal distribution and equality of
variance (Scherrer, 1984).
Indicated values represent means ± S.E.M.
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Results |
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Hemolymph osmolality
No significant variation in osmolality was observed among control crayfish
during the 43 days of the experiment (Fig.
1). During the first 21 days following the operation, no
significant difference in osmolality was noted in eyestalk-ablated crayfish.
However, a significant decrease of 52±4 mosmol kg-1
(P<0.001, N=20) in hemolymph osmolality occurred in
eyestalkless crayfish after 28 days (Fig.
1), i.e. several days after the molt of all eyestalkless crayfish.
The osmolality remained significantly lower until the end of the experiment at
day 43, even though a higher osmolality was then noticed compared with that on
days 28 and 35.
|
Statistical comparison of the two groups of crayfish at the same molting stage C was conducted. At the beginning of the experiment, before any molt had occurred (t=0 days and t=14 days), no significant difference was observed between control and eyestalkless crayfish. On the contrary, after all the crayfish had molted, a significant difference (P<0.001) was observed between intermolt controls in stage C at t=43 days and eyestalkless crayfish in stage C at t=35 days (Fig. 1).
Mass
Fourteen days after the beginning of the experiment, the increase in mass
was 2.5±0.5% in controls, which was significantly different from
5.3±1.6% in eyestalkless animals
(Fig. 2). In the latter group,
a sharp increase of 48.9±2.7% was observed 43 days after eyestalk
ablation. This increase was significantly higher (P<0.001) than
the increase in controls (13.4±1.2%) after the molt
(Fig. 2).
|
Na+ and Cl- concentrations
Hemolymph Na+ (Fig.
3A) and Cl- (Fig.
3B) concentrations were not significantly different between
control and eyestalkablated crayfish 14 days after the beginning of the
experiment. After 43 days, hemolymph Na+ concentration was
significantly lower in eyestalkless animals compared with controls
(P<0.005), while no difference was observed in hemolymph
Cl- concentration.
|
Identification and characterization of CHH
RP-HPLC fractionation of 50 SGequiv of A. leptodactylus resulted
in several peaks on the chromatogram (Fig.
4A). A major absorbance zone with two high peaks was observed
between 45 min and 48 min. The first peak was called P1, and the second peak
comprised two peaks, P2 and P3, the latter corresponding to a small peak on
the descending edge of the higher P2 peak (see
Fig. 4A); P2 is approximately
two-thirds and P3 approximately one-third of the total surface of the second
high peak. Three minor peaks with lower absorbance were eluted earlier in the
chromatogram (retention times, 20 min, 29 min and 37 min), and a fourth peak
was observed at a retention time of 53 min. This analysis was repeated several
times with similar results.
RP-HPLC fraction samples were subjected to three different direct ELISA tests in order to localize CHH and related peptides (Fig. 4B-D). Maximal immunoreactivity with the anti-Astacus CHH antiserum coincided with the major absorbance double peak of the chromatogram (retention times 45-48 min; Fig. 4B). The earlier minor immunoreactive peaks are generally considered to be oxidized or degraded forms of CHH. The anti-octapeptide antisera revealed that the most immunoreactive fractions had retention times of 46 min and 48 min for the anti-D antiserum (Fig. 4C) and 45 min and 47 min for the anti-L antiserum (Fig. 4D).
Based on the HPLC fractionation and the results of the three direct ELISA tests, fractions between 45 min and 48 min retention times were pooled in three zones in order to study the effects of CHH isoforms on specific osmoregulatory parameters: Z1 (fractions 45 and 46), Z2 (fraction 47) and Z3 (fraction 48).
Effects of the injection of RP-HPLC fractions on glucose
concentration and osmoregulation
This experiment was conducted on 30 eyestalkless crayfish in early stage C,
28 days after eyestalk ablation. Three groups of eight animals were injected
with aliquots of 10 SGequiv of zones Z1-Z3. The controls (N=6) were
injected with Van Harreveld saline. As loss of material or degradation could
occur during the different steps of sample preparation (extraction, HPLC,
etc.), hyperglycemic activity was measured after injection of RP-HPLC
fractions to assess the presence of bioactive material in the
preparations.
Hemolymph glucose level
The hemolymph glucose level was markedly elevated (P<0.001) 3 h
after the crayfish received an injection of each of zones Z1, Z2 or Z3
(Fig. 5). At 8 h, the effect,
still significant, declined, the increase in glycemia remaining higher in Z3
(P<0.01) compared with the other two zones (P<0.05).
At 24 h, only Z3 maintained a very low, but significant, hyperglycemic
response. Statistical comparisons indicated a significant difference in
hyperglycemia between zones Z2 and Z3, with a higher effect in Z3, 3 h
(P<0.01) and 8 h (P<0.05) after injection.
|
Osmolality
Hemolymph osmolality in eyestalkless crayfish was 370±2 mosmol
kg-1 immediately before injection (t=0). Eight hours after
injection, the osmolality was not significantly different among all injected
groups. A significant increase in hemolymph osmolality (P<0.05)
was observed 24 h after injection of Z3. Injection of Z1 and Z2 did not evoke
a significant increase in hemolymph osmolality
(Fig. 6).
|
Na+ and Cl- concentrations
Hemolymph ionic concentrations in eyestalkless crayfish were 168±1
mequiv Na+l-1 and 184±2 mequiv
Cl-l-1 immediately before injection (t=0).
These ionic concentrations were not significantly different 8 h after
injection. After 24 h, hemolymph Na+ concentration significantly
increased in animals injected with the three different zones
(Fig. 7A). The effect resulting
from the injection of Z3 was significantly higher compared with injection of
Z1 (P<0.01) and Z2 (P<0.05). No significant increase
was noted in hemolymph Cl- concentration after the injections
(Fig. 7B).
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Discussion |
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These results are in agreement with eyestalk-ablation experiments performed
on another Astacidae, Homarus americanus, which also show an effect
only after molting (Charmantier et al.,
1984; Charmantier-Daures et
al., 1994
). Moreover, in vivo experiments conducted on
different species of shrimps
(Nagabhushanam and Jyoti,
1977
; McNamara et al.,
1990
), crayfishes (Kamemoto et
al., 1966
; Kamemoto and Ono,
1969
; Kamemoto and Tullis,
1972
) and crabs (Kamemoto et
al., 1966
; Kamemoto and Ono,
1969
; Kato and Kamemoto,
1969
; Kamemoto and Tullis,
1972
; Heit and Fingerman,
1975
; Davis and Hagadorn,
1982
) have shown that eyestalk ligation or ablation increased
water influx and decreased hemolymph osmolality and/or ions (Na+,
Cl-) concentration in animals acclimated to low salinities. In
eyestalkless crustaceans, the decrease in osmolality and hemolymph ion
concentrations could be due to: (1) a higher influx of water resulting from an
alteration in the permeability of the teguments, (2) a greater loss of ions by
an increase of permeability and/or a modification of the active ion uptake
mechanisms and/or an increase in urine production, (3) a modification of the
drinking rate and/or (4) a modification of the function of aquaporins (if they
exist in crustaceans) caused by the absence of an eyestalk factor. In our
study, the apparent increase in volume and the cuticle softness of the
eyestalk-ablated crayfish following the molt suggested that the increase in
mass is due to an important influx of water. In decapod crustaceans, eyestalk
ablation is known to affect water metabolism, which leads to an increase in
water content after a molt (Muramoto,
1988
). The effects of eyestalk ablation are generally compensated
for by the injection of eyestalk or SG extracts (reviewed by Kamemoto,
1976
,
1991
;
Mantel and Farmer, 1983
;
Mantel, 1985
;
Muramoto, 1988
). The results
of these previous studies suggested the presence of a neuroendocrine factor(s)
in the eyestalk that can influence osmoregulatory processes.
In A. leptodactylus, our results suggest that osmoregulatory
mechanisms might be permanently activated to maintain a high hemolymph
osmolality during the intermolt stage. At ecdysis, to compensate for water
influx and/or loss of ions, factors are probably released from the eyestalks
to stimulate osmotic processes. In this species, a significant increase in the
nucleus volume of the X-organ neuroendocrine cells was measured in postmolt
stage A, suggesting an intensive synthesis of mRNA immediately after ecdysis
(Kallen, 1985). Moreover,
Chung et al. (1999
) have
observed that a hormonal factor regulating water and ion movements is
precisely time-released during exuviation in a crab species. Furthermore, the
activities of both Na+/K+-ATPase and V-ATPase double in
early postmolt in the crayfish Cherax destructor
(Zare and Greenaway, 1998
). It
has been demonstrated that the activity of these transporters is stimulated by
eyestalk or SG extracts (Eckhardt et al.,
1995
; Onken et al.,
2000
). These observations corroborate our hypothesis that the
operated A. leptodactylus devoid of eyestalk neuroendocrine factor(s)
might not be able to prevent the drop of osmolality following molting.
Involvement of CHH in the control of osmoregulation
Identification and characterization of CHH
SG peptides of A. leptodactylus have been separated by RP-HPLC.
Based on the ELISA performed with an anti-Astacus CHH antiserum and
on the bioassay for hyperglycemic activity, two main UV-absorbing peaks
corresponding to the CHH of A. leptodactylus were eluted at retention
times ranging from 45 min to 48 min. Using anti-octapeptide antisera,
different molecular forms of CHH have been identified, including stereoisomers
L-CHH and D-Phe3 CHH (P2 and P3, respectively). P1 may correspond
to another minor CHH form that differs in amino acid residue sequence, as was
demonstrated in the lobster H. americanus
(Tensen et al., 1989;
Soyez et al., 1990
). CHH
polymorphism resulting from isomerization of one amino acid residue in
position 3 of the amino-terminal fragment from the L- to the D-configuration
has been reported in other crayfish species, such as Orconectes
limosus (Soyez et al.,
1998
), Procambarus bouvieri
(Aguilar et al., 1995
) and
Procambarus clarkii (Yasuda et
al., 1994
), and in H. americanus
(Soyez et al., 1994
). A
D-amino acid in the amino-terminal part of a peptide may increase its
resistance to proteolytic enzymes (Kreil,
1997
). Injection of D-Phe3 CHH into A.
leptodactylus (this study) and O. limosus
(Keller et al., 1999
) thus
significantly extends the hyperglycemic response compared with the injection
of L-CHH. Furthermore, changes in the secondary structure of the peptide may
modify the affinity with its receptor, which might account for the difference
observed in the biological activity following injection of the different
isoforms: the D-Phe3 CHH isoform is particularly effective on blood
glucose regulation and osmoregulatory parameters in A. leptodactylus
(this study), as well as in O. limosus, where the D-Phe3
CHH has a hyperglycemic activity ten times higher than the L-CHH isoform 1 h
after injection (Keller et al.,
1999
).
CHH and ionic movements
In the present study, hemolymph osmolality and Na+ concentration
were significantly higher in A. leptodactylus injected with the
HPLC-purified D-Phe3 isoform of CHH (Z3) compared with control
animals after 24 h. The two other CHH forms (Z1 and Z2) did not induce a
significant effect on osmolality but increased the hemolymph Na+
concentration. However, these increases are lower than the one observed with
Z3. These results indicate that the eyestalk hyperglycemic hormone stimulates
osmoregulatory parameters in an FW crustacean.
Our observations are in agreement with a few results reporting effects of
CHH-like peptides on osmoregulation in marine species. Charmantier-Daures et
al. (1994) have demonstrated
that one CHH isoform stimulated hyperosmoregulation in H. americanus
acclimated to low salinities. Chiral analysis performed on HPLC fractions of
H. americanus SGs has identified this peptide as a D-Phe3
CHH isoform (Soyez et al.,
1994
). Chung et al.
(1999
) have also observed a
direct implication of a gut CHH-like peptide in osmoregulating mechanisms of
the crab Carcinus maenas. In addition, Spanings-Pierrot et al.
(2000
) have demonstrated that
purified CHH from the SG of the crab P. marmoratus increases
Na+ influx in perfused posterior gills. Interestingly, another
neurohormone involved in osmoregulatory processes, the ion transport peptide
(ITP), has been isolated from the corpora cardiaca of different orthopteran
insects (Audsley et al.,
1992a
,b
;
Macins et al., 1999
); showing
high structural homology with the hormones of the CHH family
(Soyez, 1997
), ITP, which has
thus been integrated into the CHH family as the first non-crustacean member,
stimulates ileal reabsorption of Na+, Cl-, K+
and fluid (reviewed by Phillips et al.,
1998
).
In our study, injection of Z3 containing 10 SGequiv induced a significant
increase in hemolymph osmolality and Na+ concentration. However,
the corresponding amount of CHH is unknown, as part of the bioactive material
can be lost or degraded during purification, lyophilization and preparative
steps of injected samples
(Spanings-Pierrot et al.,
2000). As discussed by Lin et al.
(1998
), data from the
literature show that, although 0.1-2 endocrine organ equivalents are enough to
induce a significant hyperglycemic response in several species of crustaceans,
most of the in vivo or in vitro experiments reporting a
positive effect of endocrine organ extracts on osmoregulation have been
conducted with 2-10 organ equivalents
(Kamemoto and Ono, 1969
;
Heit and Fingerman, 1975
;
Kamemoto and Oyama, 1985
;
Charmantier-Daures et al.,
1988
; Kamemoto,
1991
; Pierrot et al.,
1994
; Spanings-Pierrot et al.,
2000
).
In A. leptodactylus, our results indicate that fraction Z3
increased both hemolymph osmolality and Na+ concentration, while Z1
and Z2 increased only hemolymph Na+ concentration, with a lower
response than Z3. This implies more than a single target system and a complex
message/receptor for the different isoforms. The activation of
D-Phe3 CHH receptors, which may be more frequent in osmoregulating
organs such as branchial chambers, antennal glands and gut, could modify the
hydromineral balance. Furthermore, as we know that a 3:1 mixture of
L-CHH:D-CHH isoforms is produced by the crayfish X-organSG complex
(Soyez et al., 1998; this
study), we can also postulate that the affinity with the receptors located on
osmoregulating target organs is higher for the D-Phe3 CHH than for
the L-CHH. The mode of action of CHH on osmoregulation could also be indirect.
As suggested by Spanings-Pierrot et al.
(2000
), CHH might increase the
availability of metabolizable energy to the ion-exchange pumps through
increased glycogenolysis. As a matter of fact, osmoregulation is an important
energy-demanding process, especially in FW crustaceans constantly submitted to
osmotic water influx and diffusive ion loss.
Other storage organs for CHH or other (neuro)endocrine factors located
outside the eyestalk might also be involved in osmoregulation in A.
leptodactylus. Several authors have reported the effects of thoracic
ganglion or pericardial organ extracts and catecholamines on water and ion
movements (Kamemoto and Oyama,
1985; Sommer and Mantel,
1988
; Kamemoto,
1991
; Mo et al.,
1998
). As CHH-related molecules have been recently described in
these organs (Dircksen and Heyn,
1998
; Chang et al.,
1999
; Dircksen et al.,
2001
; H. Dircksen and D. Soyez, unpublished data), or very
transiently in other endocrine cells
(Webster et al., 2000
), these
CHH-like peptides could also contribute to the control of hydromineral
regulation. Interestingly, in the crab C. maenas, the pericardial
organ CHH-like peptide appears to regulate neither hemolymph glucose nor
ecdysteroid synthesis (Dircksen et al.,
2001
) but seems to be involved in another physiological regulation
such as osmoregulation (Townsend et al.,
2001
). Moreover, during exuviation of this species, an important
surge of a CHH-like peptide, synthesized in specialized gut endocrine cells,
regulates water and ion uptake but induces only a moderate increase of the
blood glucose level (Chung et al.,
1999
).
The present study shows for the first time in an FW crustacean the effects
of CHH isoforms on osmoregulatory parameters, particularly hemolymph
osmolality and Na+ concentration. Nevertheless, future studies
appear necessary to identify the mode of action of CHH and the cellular
mechanisms in target organs involved in the control of salt and water
movements in FW crayfish. However, a differential effect has been demonstrated
between the different CHH isoforms, with a higher activity of the modified
enantiomer (D-Phe3 CHH). This suggests that the post-translational
isomerization of one amino acid leads to a higher functional diversity of
molecules, generating peptides with different specialized functions from one
gene (Soyez et al., 2000).
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