23
Biol Res 30: 23-26 (1997)
Arginase activity of Bufo arenarum embryos
is sensitive to external osmotic pressure
PATRICIA M CASTANÉ*, MARIA G ROVEDATTI and ALFREDO SALIBIAN
Programa de Ecofisiología Aplicada, Departamento de Ciências Básicas,
Universidad Nacional de Luján, 6700 - Luján (B), Buenos Aires, Argentina.
We investigated the impact of environmental osmotic stress on the arginase
activity in Bufo arenarum embryos. The activity at the first developmental stages
was not sensitive to extreme variations of osmotic pressure of the environment.
Later, at gill circulation, opercular fold and right operculum stages, the enzyme
activity of embryos developed in concentrated solutions decreased
significantly
with respect to control. At complete operculum stage, the arginase
activity
increased 1.3-2.5 fold in all conditions, and was significantly higher in embryos
grown in distilled water than in control animals.
Key terms: anuran embryos, Bufo arenarum, embryonic arginase, osmotic stress.
Among vertebrates, Amphibia is a transition
class in the colonization of land. At present,
all of them remain dependent on free water
availability to a greater or lesser degree
(Duellman & Trueb, 1986; Shoemaker et al,
1992). The possibility of a relationship
between water availability and the nature
and p a t t e r n of the e x c r e t e d n i t r o g e n
metabolism end products, has stimulated
much work on amphibians, that have to
withstand, at some stage of their life cycle,
changes in the accessibility of water.
Adaptive responses of organisms to
changes in their environment can occur at
several organization levels (Hochachka &
Somero, 1984). At the molecular level,
changes can be considered in the function of
the enzyme systems. Thus, the knowledge of
the ontogenetic changes in the function of
the urea cycle associated with water-land
t r a n s i t i o n a p p e a r s to be p a r t i c u l a r l y
important to the study of the biochemical
adaptation
of a n u r a n s to
habitats
characterized by different water availability.
It is well k n o w n that a r g i n a s e
(L-arginine ureohydrolase; EC 3.5.3.1) is a
prominent enzyme in the liver and kidney of
ureotelic amphibians; we have used it as an
indicator of the urea cycle function. In a
previous paper (Rovedatti et al, 1995), we
studied the evolution of the arginase specific
activity during the embryonic development
of Bufo arenarum in standard conditions,
observing a gradual increase in the enzyme
activity along the developmental stages.
Similar findings have been recently reported
by Callery and Elinson (1996) in embryos of
Eleutherodactylus
coqui, an species that
does not metamorphose.
In this study we have extended our
research
investigating
how
the
environmental osmotic stress affects the
a c t i v i t y of a r g i n a s e . W e c h o s e s o m e
representative stages in order to compare
the impact of incubation in different media
on the enzyme activity. The capacity of
Bufo arenarum
e m b r y o s to d e v e l o p in
distilled water has been shown in our
laboratory (Castané et al, 1987).
Bufo
arenarum
embryos were
o b t a i n e d by f e r t i l i z a t i o n in vitro,
as
described previously (Castané et al, 1987).
Correspondence to: Bioq Patricia M Castané, Programa de Ecofisiología Aplicada, Departamento de Ciências Básicas,
Division Biologia, Universidad Nacional de Luján, Casilla de Correo 2 2 1 , 6700 - Luján (B), Buenos Aires, Argentina.
Phone: (54-323) 2 3 1 7 1 , ext 246. Fax: (54-323) 25795. E-mail: prodea@unlu01 .edu.ar
24
Biol Res 30: 23-26 (1997)
F r o m f e r t i l i z a t i o n on, e m b r y o s w e r e
allowed to develop in glass Petri dishes
containing three incubation media: one
group was developed in 10% Holtfreter's
solution (control), a second group was
placed in distilled water and the third group
was
incubated
in a
concentrated
Holtfreter's solution (115 m O s m ) . The
solutions were replaced daily. Throughout
experiments, dishes were kept in a Ghilon
TC 120 i n c u b a t i o n c h a m b e r with the
temperature set constant at 20 ± 1 °C and
12 h light-dark cycle. The experiments
were carried out irrespective of the season.
D e v e l o p m e n t a l stages were determined
according to the tables of Del Conte and
Sirlin (1951).
Groups
of
150-300
embryos
morphologically normal of each selected
stage were homogenized on ice and the
arginase activity and protein content were
determined following the already described
t e c h n i q u e s ( R o v e d a t t i et al; 1995).
Enzymatic activity was expressed as nmoles
urea/min/mg protein. All measurements
were carried out in duplicate or triplicate.
Chemicals used were of analytical grade.
Results are presented in Table I.
Normality in the distribution of the
values was checked by Kolmogorov-Smirnov
test. Enzyme data were transformed as 1000
x log specific activity. Standard least-squares
linear regression analysis of arginase specific
activity on time (hours post fertilization) was
d o n e . A n a l y s i s of c o v a r i a n c e for the
regression slopes was carried out (Draper &
Smith, 1981). For each stage, comparisons
between the three developmental conditions
were performed using one way analysis of
variance (ANOVA), and when significant
variation was found, pairwise comparisons of
means were carried out using the Scheffé
procedure (Zar, 1996). For all tests, the
significance level was p < 0.05.
Table I shows that arginase specific
a c t i v i t y is n o t s e n s i t i v e to e x t r e m e
v a r i a t i o n s of o s m o t i c p r e s s u r e of the
e n v i r o n m e n t at t h e e a r l y s t a g e s of
development. However, the activity of the
group developed in concentrated solution
decreased significantly with respect to
control in gill circulation, opercular fold
Table I
Arginase specific activity of embryos of Bufo arenarum developed in media of different osmolarhy
(nmol urea • min" • mg protein" ). In parentheses, number of assayed homogenates; means ± SEMs.
1
1
Incubation media
Stage
Control
Distilled
water
(11.5 mOsm)
Concentrated
Holtfreter
(115 mOsm)
ND (28)
NM
NM
Tail bud
1.2 ± 0.2 (28)
1.0 ± 0.1 (3)
1.5 ± 0.1 (5)
Muscular response
2.0 ± 0.4 (10)
1.4 + 0.4 (4)
1.2 ± 0.2 (4)
Gill circulation
3.9 ± 0.6 (6)
1.5 ± 0.2 (3)
2.6 ± 0.3 (5)
Opercular fold
5.7 ± 1.8 (3)
1.8 ± 0.1 (3) a
2.3 ± 0.4 (3)
Right operculum
6.8 ± 0.7 (9)
3.0 ± 0.8 (3) a
4.7 ± 1.2 (3)
Complete operculum
8.5 + 1.5 (12)
7.6 ± 2.3 (2) b
11.9 ± 2.0 (6) a,b
NF to gastrula
NF:non fertilized oocytes; ND: not detectable; NM: not measured.
a: Significant differences with respect to control at p < 0.05.
b: Significant differences with respect to the above stage of the same group.
25
Biol Res 30: 23-26 (1997)
and right operculum stages. The beginning
of this r e d u c t i o n c o r r e s p o n d s to t h e
moment at which the gills are exposed to
the external medium and epithelial ion
exchange processes are established. Thus,
the N a / N H
e x c h a n g e c o u l d be a
m e c h a n i s m c o m p l e m e n t a r y to the urea
cycle for the elimination of ammonia.
T h e i n t e r p r e t a t i o n of the l o w e r
specific activity found in e m b r y o s of
concentrated Holtfreter group is not easy. It
is not known if embryos have the capacity
to overcome external increases in osmotic
pressure by means of urea accumulation in
their body fluids, as it has been shown in
adults of other species (Balinsky, 1981;
Shoemaker
& McClanahan, 1982;
Shoemaker et al, 1992).
W e m a y s u g g e s t that e m b r y o s
developed in concentrated Holtfreter (a
condition simulating reduced water
availability) may be induced to change
their c a t a b o l i s m of n i t r o g e n p r o d u c t s
towards the uric acid synthesis pathway,
favored by an increase in the synthesis of
nucleic acids (Bretos et al, 1967).
It is w o r t h w h i l e m e n t i o n i n g that
Balinsky et al (1976) postulated that the
+
ability of amphibians for colonizing new
habitats characterized by a shortage of
environmental
water
requires
the
d e v e l o p m e n t of a d a p t i v e b i o c h e m i c a l
mechanisms, achieved not only through the
acquisition of new enzymes, but by means of
changes in the activities of pre-existing ones.
Arginase activity increased along the
stages of development in all three groups.
The covariance analysis for the comparison
of the slopes of linear regression did not
show significant differences (Table II).
In addition, the ANOVA comparing
the stages of each group revealed significant
differences between right operculum and
complete operculum stages in the embryos
grown in both concentrated Holtfreter's
solution and distilled water (Table I).
In animals grown in concentrated
solutions to the complete operculum stage,
arginase activity reached a value similar to
that found in control embryos, while the
activity was significantly higher in those
i n c u b a t e d in d i s t i l l e d w a t e r than in
c o n t r o l s . This p h e n o m e n o n suggests a
biochemical
compensation
of the
embryonic arginase activity when the gills
are reabsorbed.
+
4
Table II
Linear regression analysis parameters for arginase activity of Bufo arenarum embryos
developed in media of different osmolarities, and statistics of the covariance
analysis for the comparisons of the slopes.
Incubation media
Parameters
Slope
Intercept
Control
3.90xl0-
Concentrated
Holtfreter
3
3.75xl0"
3
2.71
2.88
4
6.78xl0"
Distilled
water
4.48xl0
2.75
4
Standard error of slope
4.62X10"
Correlation coefficient
0.79
0.81
0.88
R-squared (%)
61.93
65.74
78.4
Standard error of estimation
0.230
0.189
0.178
F (comparison of slopes): 0.10
F table: 2.84
3
4.80xl0"
4
26
Biol Res 30: 23-26 (1997)
ACKNOWLEDGMENTS
Dr A l f r e d o S a l i b i á n b e l o n g s to the
Comisión de Investigaciones
Científicas,
Província de Buenos Aires, Argentina. This
research was supported by grants from
Consejo
Nacional
de
Investigaciones
Científicas
y Técnicas and
Universidad
Nacional de Luján.
T h a n k s are d u e to E n g R o b e r t o
Y o s h i h a r a and J a v i e r Katz for t h e i r
technical assistance. Authors are grateful to
Wiener Lab for providing kits for urea
determination.
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