Fluctuations of adenylate cyclase activity during anterior regeneration in Owenia fusiformis (Polychaete Annelid)

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1 /. Embryol. exp. Morph. Vol. 48, pp , Printed in Great Britain Company of Biologists Limited 1978 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, Marseille Cedex 2, France.

2 74 J. COULON AND M. MARILLEY ISO 160- = I 100- ^ SO - "5 S. 60e I I I I I st I l I nd I I T rd i r i th -w- i r Horns D;ivs Regeneration time 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-mgso 4, 2 mm-atp, 0-1 mci/ml [ 3 H]ATP (Amersham/Searle, specific activity 500 mci/mm) and 10 mm-naf. The separation

3 Adenylate cyclase activity in Owenia oo Timc of Day (h) M G, i 1 '» 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 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

4 76 J. COULON AND M. MARILLEY Table 1. Activation ofowenia adenylate cyclase Addition to incubation medium (01 mm final concentration) Adenylate cyclase activity (cyclic AMP formed: pmoles/mg protein/15 min) None Epinephrine Noradrenaline (NA) 5-hydroxytryptamine (5-HT) GABA (y-aminobutyric acid) 40-8 ± ± ± ± ±2-83 Each result represents the mean value of at least four experiments; each experiment concerning seven pooled animals. day after amputation) reveals a maximal activity in G 2 and G r early 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 determined 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). 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 DNA synthesis (Marilley & Thouveny, 1975). Furthermore, in various cell cultures a close correlation exists between the intracellular level of cyclic AMP and the degree of proliferation (Hadden, Hadden, Haddox & Goldberg, 1972). The cyclic AMP level rises as the cells

5 Adenylate cyclase activity in Owenia 77 approach confluency and the increase of cyclic AMP 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 AMP 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 x and low during S phase like the cyclic AMP level (Raska, 1973); high in G 2 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 AMP 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 NA 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 AMP formation by means of adenylate cyclase acting on target blastema cells, undergoing the cell cycle. REFERENCES COULON, J. & BESSONE, R. (1978). Radioautographic localization of indolamines and catecholamines neurons in the nervous system of Owenia fusiformis (Polychaete Annelid). Cell and Tissue Research. (Submitted.) COULON, J., MARILLEY, M. & THOUVENY, Y. (1976). Variations journalières du taux des sécrétions nerveuses chez Owenia fusiformis. Persistance d'un rythme journalier dans les tissus en culture. C. r. hebd. Séanc. Acad. Sci., Paris 283, DELAAGE, M. A., BELLON, B. N. & CAILLA, H. L. (1974). Rapid assays for adenylate cyclase and 3',5'cyclic AMP phosphodiesterase activities. Simultaneous measurements of other pathways of ATP catabolism. Analyt. Biochem. 62, HADDEN, J. W., HADDEN, E. M., HADDOX, M. K. & GOLDBERG, N. D. (1972). Guanosine 3':5'-Cyclic monophosphate: a possible intracellular mediator of mitogenic influences in Lymphocytes. Proc. natn. Acad. Sci., U.S.A. 69, EMB 48

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