/. Embryol. exp. Morph. Vol. 48, pp. 73-78, 1978
Printed in Great Britain © Company of Biologists Limited 1978
73
Fluctuations of adenylate cyclase
activity during anterior regeneration in
Owenia fusiformis (Polychaete Annelid)
By JOSIANE COULON 1 AND MONIQUE MARILLEY 1
From the Laboratory of Animal Morphology and Institute of Cytology
SUMMARY
Biochemical assays of adenylate cyclase activity were performed during the early phases of
regeneration in Owenia fusiformis (Polychaete Annelid).
The results indicate the existence of a strong stimulation in an early phase following
trauma. This stimulation is then followed by periodic fluctuations exhibiting a diurnal
rhythm correlated with the cell cycle. Adenylate cyclase activity is also shown to be neurotransmitter-dependent.
In this paper it is proposed that neurotransmitters might participate in the regulation of
cyclic AMP formation, by means of adenylate cyclase acting on target blastema cells, undergoing the cell cycle.
INTRODUCTION
Traumatic regeneration involves cellular reactivation and stimulation of the
proliferation process. Cyclic AMP appears to be an important mediator of
cellular proliferation (Pastan, Johnson & Anderson, 1975), especially in controlling the completion of the cell cycle (Zeilig, Johnson, Sutherland & Friedman, 1976), transition phases being correlated with sharp variations in levels
of cyclic nucleotides (Zeilig & Goldberg, 1977).
The intracellular level of cyclic AMP is controlled in part by the adenylate
cyclase which is a plasma membrane-bound enzyme (Sutherland, Rail &
Menon, 1962), whose activity is regulated by different agents such as hormones
or neurotransmitters (Robison, Butcher & Sutherland, 1967).
In Owenia, the start of DNA synthesis and the following cell cycles were
studied after anterior amputation. The syntheses, occurring in all the blastema
cells on the third day, were found to be well synchronized (Marilley & Thouveny,
1978). Thus, this system appeared to be suitable to investigate the membrane
changes during these early phases of regeneration.
Since no data in this field are available concerning Polychaete Annelid, the
1
Authors' address: Laboratoire de Morphogenese animale et Institut de Cytologie,
Departement de Biologie moleculaire et cellulaire, UER Scientifique de Luminy, 13288
Marseille Cedex 2, France.
74
J. COULON AND M. MARILLEY
ISO —
160-
= 140-
5 1 20 I 100^ SO "5
S. 60e404-
20
I I I I I
0 1 3 6 12
1st
I l I
I
IT
i
r i
72 7578
24 2730
46 5154
4th
2nd
3rd
Regeneration time
-w-
i r
96 99 Horns
D;ivs
Fig. 1. Fluctuations of adenylate cyclase activity during the first, second, third
and fourth days following cephalic amputation. (Mean ± standard error.)
present work reports on the biochemical assays of adenylate cyclase activity
performed in regenerating and non-amputated worms.
MATERIAL AND METHODS
The animals were amputated at the first abdominal segment where the
regeneration is always complete. Since the initial state of the cells may be
different according to the time of amputation, all the animals were operated
at the same time: 12 a.m. (Maiilley & Thouveny, 1978). Short fragments
corresponding to the amputation zone were examined from 1 to 96 h after
operation.
Adenylate cyclase activity was determined according to the procedure of
Delaage, Bellon & Cailla (1974). The assays were performed on total homogenate and the incubation medium usually contained 36 mM Tris-HCl pH 7-5,
0-2 mM papaverine, 27 mg/1. albumin, 72 i.u./l. pyruvate kinase, 4-5 mM phosphoenol pyruvate, 12 mM-MgSO4, 2 mM-ATP, 0-1 mCi/ml [3H]ATP (Amersham/Searle, specific activity 500 mCi/mM) and 10 mM-NaF. The separation
75
Adenylate cyclase activity in Owenia
140 -
oo
80-
5 60 2
40-|
20Timc of Day (h)
-i
6
40
10
12
48
M
14
16
G,
18
54
1 '
»-
20
22
Regeneration time (h)
Fig. 2. Detailed study of the well-synchronized cell cycle (blastema growth stage).
Adenylate cyclase contents detected from the 40th to the 58th h after amputation
on regenerating worms (
) and on the unamputated control animals (
).
(Mean ± standard error.)
of all the cyclic nucleotides was obtained by electrophoresis on cellulose
acetate strips (15 min at 50 V/cm) with a fluorescent buffer. All the nucleotides
were located under u.v. light by fluorescent inhibition and each fraction was
dissolved in Bray's solution for scintillation counting. An aliquot of homogenate was sampled for protein determination. Controls performed on cells
after boiling (3 min) and with an inhibitor of adenylate cyclase (Alloxan),
indicate that there was no remaining adenylate cyclase activity.
RESULTS
Adenylate cyclase activity increases strongly 1 h after amputation and then
varies slightly during the first 12 h (Fig. 1), corresponding to the prereplicative
phase (Marilley & Thouveny, 1978).
Periodic fluctuations of this activity occur during the second, third and fourth
days following trauma, and are seen to follow a diurnal rhythm: maxima
located at 10-12 a.m., minima at 4 p.m. Maximal and minimal values increase
gradually until the fourth day (Fig. 1).
Adenylate cyclase activity assayed on the well-synchronized cell cycle (third
76
J. C O U L O N
AND
M.
Table 1. Activation ofOwenia
Addition to
incubation m e d i u m
( 0 1 m M final concentration)
None
Epinephrine
Noradrenaline (NA)
5-hydroxytryptamine (5-HT)
G A B A (y-aminobutyric acid)
MARILLEY
adenylate
cyclase
Adenylate cyclase activity
(cyclic A M P f o r m e d :
p m o l e s / m g p r o t e i n / 1 5 min)
40-8
69-8
75-6
86-1
86-9
± 2-03
± 1-20
±3-00
±2-46
±2-83
E a c h result represents the m e a n value of at least four experiments;
each experiment concerning seven pooled animals.
day after amputation) reveals a maximal activity in G and G e a r l y S phases,
and this activity attains its lowest level in M and S phases (our results concern
only the beginning and the end of S phase) (Fig. 2).
The values of adenylate cyclase being compared with basal activity deter­
mined on non-amputated control animals, show an important stimulation
during the regenerating processes (Fig. 2).
In presence of various neurotransmitters (Catecholamines, Serotonin and
GABA) the basal activity of adenylate cyclase is significantly stimulated
(Table 1).
2
r
DISCUSSION
These results indicate the existence of a strong stimulation of adenylate
cyclase activity during the early phases of cephalic regeneration. However, this
increase appears to be composed of two different stages: the first one being
non-periodic while the second presents a cyclic character.
The early increase in adenylate cyclase content (1 h after amputation)
supports the hypothesis that this enzyme can be activated by alterations in
membrane structures (Perkins & Moore, 1971): it may therefore imply a
non-specific mechanism. It is to be noted that a high level of adenylate cyclase
activity occurred during the whole prereplicative phase determined by Marilley
& Thouveny (1978).
In contrast, the subsequent periodic fluctuations seem to follow a diurnal
rhythm which is well correlated with the cell cycle rhythm in Owenia, described
elsewhere (Marilley & Thouveny, 1978). In regenerating worms active cell
proliferation precedes blastema differentiation occurring on the fourth day,
which is accompanied by a sharp decrease in D N A synthesis (Marilley &
Thouveny, 1975).
Furthermore, in various cell cultures a close correlation exists between the
intracellular level of cyclic A M P and the degree of proliferation (Hadden,
Hadden, Haddox & Goldberg, 1972). The cyclic A M P level rises as the cells
Adenylate
cyclase activity in Owenia
77
approach confluency and the increase of cyclic A M P appears to be due to
increase in adenylate cyclase activity (Pastan et al. 1975; Mackman, 1971;
Zacchello, Benson, Giannelli & McGuire, 1972). Thus, since the cell confluency in blastema formation may be compared with cell culture (Coulon,
Marilley & Thouveny, 1976) the gradual increase in adenylate cyclase content
until the fourth day after amputation might be correlated with increasing cell
population density.
During the cell cycle, periodic changes in intracellular levels of cyclic A M P
have been demonstrated in mammalian cell lines (Millis, Forrest & Pious, 1972;
Zeilig & Goldberg, 1977). Adenylate cyclase has also been reported to vary
with the cell cycle: high in G and low during S phase like the cyclic A M P
level (Raska, 1973); high in G and low in mitosis (Millis, Forrest & Pious,
1974). Similar observations are obtained in Owenia and extend during the
complete cell cycle. Indeed, radioimmunoassays of cyclic A M P performed in
regenerating animals reveal variations which correspond exactly to changes
observed in adenylate cyclase content (data not shown).
The fluctuations of adenylate cyclase activity observed on unamputated
control animals are significant and they may be interpreted as being correlated
with the cell cycle. Indeed, there exists a small percentage of rapidly renewing
cells (ventral cells of digestive duct, splanchopleural cells, somatopleural cells)
which may account for the oscillations observed.
In our experiments in vitro, adenylate cyclase activity is seen to be neurotransmitter-dependent. Since 5-HT and N A have been shown to be present in
the ventral nervous cord at the regenerating level (Coulon & Bessone, 1978),
it is suggested that the early increase in adenylate cyclase (1 h after amputation)
may be correlated with a sharp release in neurotransmitters consecutive to the
section of the nervous cord. Furthermore, neurotransmitters were found to
vary periodically (Coulon et al. 1976); thus, it is possible that they participate
in the regulation of cyclic A M P formation by means of adenylate cyclase acting
on target blastema cells, undergoing the cell cycle.
x
2
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(Received 23 March 1978, revised 21 June 1978)