A dopamine- and octopamine-sensitive adenylate cyclase in the nervous system of Octopus vulgaris.

1. Adenylate cyclase activity was assayed in the optic lobe of Octopus vulgaris. 2. Both octopamine and dopamine stimulate the octopus adenylate cyclase, apparently by competing with the same receptor site. 3. (+/-)-2-Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene-HBr (6,7-ADTN) and a number of phenylethanolamine derivatives stimulate the octopus adenylate cyclase activity. 4. The dopamine D-1 antagonists R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl- 2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SCH-23390) and (+/-)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H- 3-benzazepine-HCl (SKF-83566) are unable to antagonize the effects of dopamine and octopamine, and similarly ineffective is the agonist (+/-)-1-phenyl-2,3,4,5-tetrahydro-1H-3- benzazepine-7,8-diol-HCl (SKF-38393). 5. No detectable binding of labelled SCH-23390 occurs on membrane preparations from octopus optic lobe.

Role of dopamine and indolamine derivatives in the regulation of the sea urchin adenylate cyclase.

The adenylate cyclase of the sea urchin egg is stimulated by dopamine in the presence of GTP. The enzyme activity is strongly enhanced when Gpp (NH)p is substituted for GTP, or after cholera toxin treatment. Gramine, an indolamine derivative, brings about non-competitive inhibition of the dopamine-stimulated adenylate cyclase activity. Pertussis toxin causes an attenuation of the gramine-induced inhibition of adenylate cyclase. These results show that dopamine and indolamine derivatives partecipate in the regulation of the adenylate cyclase activity of the sea urchin egg.

Catecholamine secretion and adenylate cyclase activation in sea urchin eggs.

The role of neurotransmitters in sea-urchin eggs was investigated by studying their effect on adenylate cyclase of the egg membrane. Maximal stimulation of enzyme activity occurs in the presence of dopamine and GTP. 5-hydroxytriptamine, 5-methoxytriptamine and acetylcholine have no effect on activity, despite a decrease in intracellular cAMP level in eggs treated with 5-hydroxytriptamine antagonists as previously reported (Renaud et al., 1983). High-performance liquid chromatography (HPLC) revealed that dopamine is released from the sea-urchin egg into the external medium following fertilization.

Bud induction in decapitated Hydra attenuata by 5-azacytidine: a morphological study.

The effect of 5-azacytidine (5-azaCR) on head regeneration and budding in hydra are reported. Hydra attenuata were exposed to various doses of 5-azaCR for 48 h and then decapitated and cultured. Head regeneration and bud formation were observed for 12 days after decapitation. Untreated control hydra regenerated heads within 7 to 8 days of decapitation with a budding index of 0.2. Buds invariably arose in the normal budding zone (below the gastric region). In the group treated with 0.8 mM-5-azaCR, 9 days after decapitation head regeneration was seen in only 13% of animals, and an average of two buds per hydra were formed, most of which were in the vicinity of the distal end. Induction of budding was also seen in the animals that regenerated heads. In animals exposed to 1 mM-5-azaCR three main types of responses were observed 9 days after decapitation. 44% of the animals regenerated normal heads; about half of them developed at least one bud and these buds originated in the budding zone. 17.5% of the animals developed abnormal, long hypostome-like structures with single or bifurcated tentacles at their tips. There were at least two buds per animal and they were invariably at abnormal sites. 32% of the animals failed to regenerate heads, although they developed two buds. 87% of these buds originated in abnormal sites of the body column and a large number (72%) did not detach even by the 12th day after decapitation. Both 5 and 10 mM of 5-azaCR were toxic to the animals; the survivors formed large globe-shaped heads. Bud induction was seen in 60% and 28% of animals in the 5 and 10 mM groups, respectively. These observations demonstrate that 5-azaCR induces bud formation in hydra at doses that inhibit head regeneration. This bud induction might be due to a specific expression of gene products responsible for bud formation.

dCMP-aminohydrolase activity during early sea urchin development. An example of negative enzyme control during embryogenesis.

In sea urchin, unfertilized eggs have a very high level of dCMP-aminohydrolase (dCMPase) activity, which decreases gradually and at the pluteus stage it is only about a quarter of that found in the unfertilized egg. But in abnormal embryos and in disaggregated cells from embryos, no decrease in the dCMPase activity takes place. To understand the control mechanism involved in this enzyme activity during development, we have analyzed the effect of various drugs which interfere with information transfer, such as actinomycin C, puromycin, 5-azacytidine, 2-thio-uracil and p-fluoro-DL-phenylalanine on dCMPase activity in embryos of Paracentrotus lividus and Sphaerechinus granularis. Among these drugs only actinomycin induces a remarkable increase of the dCMPase activity in embryos with respect to unfertilized eggs. Puromycin has a differential and dose-dependent effect. Other drugs, although they affect normal development and macromolecular synthesis, do not significantly alter the dCMPase activity. On the basis of these results we suggest the presence of a repressor mechanism in the control of dCMP-aminohydrolase level during early embryogenesis of sea urchin.

Studies on differentiation without cleavage in Chaetopterus. Requirement for a definite number of DNA replication cycles shown by aphidicolin pulses.

Aphidicolin, a powerful inhibitor of nuclear DNA replication, has been used to establish the level of polyploidisation required for the realization of the late morphogenetic events (segregation, pseudogastrulation and ciliation) of differentiation without cleavage in Chaetopterus-activated eggs. A parallel has been drawn between cytophotometric estimations of the DNA content and cytoplasmic differentiation in eggs treated with aphidicolin under different experimental conditions: either pulses with aphidicolin were followed by culture in sea water or the eggs were treated with aphidicolin after development had taken place in sea water for various lengths of time. The results suggest that a 'quantal' monasterial cycle might take place 3 h after activation, corresponding presumably to the fourth or fifth replication cycle. Moreover, early DNA replication seems to be more important for morphogenesis than late DNA replication.

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin.

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.

Studies on differentiation without cleavage in Chaetopterus variopedatus: requirements for nucleic acid and protein synthesis.

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.

Species specificity and individual variability of sea urchin sperm H2B histones.

Total histones from the sperms and embryos of the sea urchins Paracentrotus lividus, Arbacia lixula, Psammechinus microtuberculatus and Sphaerechinus granularis hae been fractionated into the component molecules by electrophoretic analyses in SDS, in urea-acetic acid and in Triton-urea-acetic acid. Sperm H2B histones are in all cases different from those of the corresponding embryonic chromatins. Each sea urchin species has distinctive variants of the sperm H2B histones that are fractionated by electrophoresis in SDS acrylamide gel into two to four components forming a new class of lower mobility. This analytical method shows that individuals of the same species have different assortments of the H2B components. Electrophoretic analyses in Triton-urea also show multiple components for H2B but the patterns are similar in the different individuals.