Pharmacological control of ciliary activity in the young sea urchin larva: chemical studies on the role of cyclic nucleotides

Distinct peaks in cAMP and cGMP content during early development, partly opposite to each other, may be correlated with the two main phases of gastrulation and ciliary activity. Monoamines increases cAMP formation. A transient or extended decrease follows, presumably reflecting some feedback mechanism. Muscarinic agents and Ca2+ interfere. The developmental variation in cyclic nucleotides may reflect a temporal shift in the role of various signal substances as well as feedback regulation related to Ca2+ influx. The opposite changes in cAMP and cGMP during early gastrulation may reflect a mutual dependency of the two nucleotide cyclases related to changes in Ca2+ influx.     

Pharmacological control of ciliary activity in the young sea urchin larva. Studies on the role of Ca2+ and cyclic nucleotides

The ciliary stimulation by monoamines is enhanced by adenylcyclase activators and a phosphodiesterase inhibitor indicating that cAMP is a mediator, a conclusion supported by the effects of db-cAMP. The role of cGMP is also examined. When stimulation exceeds certain levels, it is overpowered by some inhibitory feedback mechanism. The effects of altered Ca2+ concentrations, Ca2+ antagonists and a Ca2+ ionophore suggest that Ca2+ is involved in ciliary excitation as well as in the inhibitory mechanism. These suggestions are examined by experiments on the influence of altered Ca2+ concentrations and of the phosphodiesterase inhibitor on the response to various agents.     

Pharmacological control of muscular activity in the sea urchin larva. I. Effects of nicotinic and muscarinic agents

1. The muscular system of the sea urchin pluteus is stimulated by nicotinic agents. Excessive stimulation is followed by paralysis of the most powerful muscular strands. The effects are counteracted by antinicotinic agents.2. Muscarinic agents keep the activity low, calm down initially unquiet larvae, and counteract the nicotinic effects.3. The effects are quickly elicited and in general are also quickly reversible in sea water, which indicates that most of the receptors involved occur at the larval surface.4. It is suggested that the nicotinic and muscarinic signals control certain ionic fluxes in opposite directions, and that the stimulatory effect on the most powerful muscular strands is amplified by a monoaminergic system.     

Pharmacological control of ciliary activity in the young sea urchin larva. Effects of monoaminergic agents

Ciliary activity is affected by serotonin, dopamine and various alpha- and beta-adrenergic agonists reacting with specific receptors. The effects of various monoamine precursors applied at hatching or later on suggest that the initiation of ciliary activity and its control during early gastrulation reflects a temporary formation of monoamines. Application of monoamines brings about stimulation throughout gastrulation with some exceptions which may reflect side effects or some mechanism of feedback inhibition. Strong stimulation is often followed by conspicuous, transient decrease in ciliary activity around the mid-gastrula stage presumably reflecting feedback inhibition and compensatory mechanisms alleviating this.     

Pharmacological control of muscular activity in the sea urchin larva. II. Role of calcium in nicotinic stimulation and paralysis,and the modulatory role of muscarinic agents

1. Ca²⁺-antagonists counteract the muscular activity of the sea urchin pluteus. Agents that block rapid Na⁺-channels have no effect.2. High muscular activity is induced by increasing the sea water concentration of Ca²⁺ or K⁺ and by a Ca²⁺-ionophore. The stimulatory effects tend to decline.3. Muscarinic agents counteract the effects of Ca²⁺ and K⁺.4. Variation in the concentration of Ca²⁺ or K⁺ has profound effects on the response to nicotinic agents.5. It is suggested that Ca²⁺ plays the role as a charge-carrier and in the release of monoamines from an inner source, and that an excessive Ca²⁺-influx induces an outflux of K⁺ leading to hyperpolarization and abolition of the impulse activity.     

Pharmacological control of muscular activity in the sea urchin larva--IV. Effects of monoamines and adenosine.

1. The muscular activity of the sea urchin pluteus is affected by catecholamines. alpha-Agonists in high concentrations bring about a strong, temporary, stimulation. 2. The stimulation by beta-agonists tends to be masked by different mechanisms. 3. Serotonin brings about a strong stimulation of long duration, even at 10(-7) M. It reactivates larvae where the activity has declined after exposure to catecholamines. A dopamine-effect at 10(-5)-5 x 10(-6) M is similar to that of serotonin. The effect of adenosine is similar to that of dc-c-AMP. 4. The action of the alpha- and beta-agonists and adenosine appears to involve an increased respectively excessive Ca2(+)-influx, directly or indirectly mediated by c-AMP. 5. It is suggested that a strong Ca2(+)-influx induces an outflux of K+ leading to hyperpolarization. Serotonin may decrease the K(+)-outflux.     

Developmental changes in chromatin proteins of the sea urchin from blastula to mature larva.

Chromatin proteins of the sea urchin Lytechinus pictus from blastula to fully grown feeding larva were analyzed by polyacrylamide-gel electrophoresis. Two subspecies of histone protein changed during development. Histone f1g increased progressively relative to f1m and within a few days after the larvae began to feed constituted >99% of the f1 fraction. Two slower-moving subfractions of histone f2b disappeared in the same interval, being replaced by a single fastermoving species. The electrophoretic profile of nonhistone protein changed quantitatively and qualitatively with the largest change, a relative decrease of high molecular weight bands and a new pattern of middle molecular weight bands, also occurring several days after feeding began. The profiles remained virtually the same during subsequent development though the cell number increased at least tenfold. The onset of feeding thus seems to be correlated with major changes in chromatin proteins.     

Ars insulator protects transgenes from long-term silencing in sea urchin larva

Reporter genes have been used as a powerful tool to analyze cis-regulatory elements responsible for temporal and spatial expression in the early development of sea urchin. However, here we show that the transgenes introduced into the sea urchin embryos undergo suppression in larval stage. The transgene silencing could be one of major obstacle in the analysis of regulatory regions in the late stages of development. We previously demonstrated that a DNA fragment (ArsI) located in the upstream region of sea urchin (Hemicentrotus pulcherrimus) arylsulfatase gene has the property of an insulator. We show that tandem ArsI prevents silencing of a transgene in sea urchin larvae when the ArsI is fused to the 5′ end of the reporter gene. Furthermore, we demonstrate that DNA of the reporter gene introduced into the sea urchin eggs is methylated during development and that the ArsI protects the transgene from the DNA methylation.     

Morphology of the organic matrix of the spicule of the sea urchin larva

The morphology of the organic matrix of the skeletal spicule of the sea urchin pluteus larva has been analyzed by light and electron microscopy. Purified isolated spicules can be demineralized, and they reveal lamellae of an irregular fibrillar nature with overall outlines similar to the shape of the intact spicule. Sections through the spicule show the fibrous lamella is probably composed of interconnected concentric sleeves.     

Role of cyclic nucleotides in modulating ovarian hCG action.

The role of cyclic nucleotides in mediating hormonally responsive adenylate cyclase and cAMP-dependent protein kinase was examined in vivo and in vitro when pseudopregnant rats were injected with hCG. Intracellular ovarian levels of cAMP increased, as expected, but no change in cGMP concentrations was observed. However, both cGMP and cAMP activated ovarian CDPK holoenzyme in vitro but cGMP had a lower affinity. The subunits of hCG were without effect. Even though cGMP and cAMP dissociate partially purified ovarian CDPK holoenzyme in vitro, the receptor sites of the regulatory subunit of CDPK would appear to be relatively specific for cAMP. Moreover, cGMP probably does not mediate hCG action in vivo.     

Translational control genes in the sea urchin genome

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.     

Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm.

Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

Effects of egg factors on cyclic nucleotide metabolism in sea urchin sperm.

Cyclic AMP in Strongylocentrotus purpuratus sperm was elevated approximately 2-fold by theophylline or 1-methyl-3-isobutylxanthine. Factors released from sea urchin eggs (FRE) elevated sperm cyclic AMP by about 7-fold within 1 min, and the combination of FRE with theophylline increased sperm cyclic AMP up to 100-fold within 1 min. Cyclic GMP in sea urchin sperm was slightly elevated by theophylline, but was lowered by FRE. Cyclic GMP in sperm treated with FRE plus theophylline was not higher than in sperm treated with theophylline alone. The ability of FRE-containing sea water to increase sperm cyclic AMP in the presence of theophylline was altered only slightly if at all by boiling, but it was decreased by about 50% by dialysis and destroyed by ashing. Filtration of FRE on Sephadex G-50 columns yielded two peaks of cyclic AMP-elevating activity. One peak (peak I) was eluted at the column void volume, and the other (peak II) was retained by the column. The cyclic GMP-lowering activity was located in fractions approximately corresponding to peak I of cyclic AMP-elevating activity. Dialysis of FRE-containing sea water before its application to the G-50 column virtually eliminated peak II of the cyclic AMP-elevating activity. When the cyclic AMP-elevating activity in peak I was filtered on Bio Gel A-5m columns, it also migrated at or near the column void volume. Fractions corresponding to peak I contained material that inhibited both guanylate and adenylate cyclase activities in broken cell preparations of sperm and guanylate cyclase from rat lung. The inhibitory material was stable to boiling, non-dialyzable, and destroyed by ashing. Under a variety of conditions, FRE-containing sea water or cyclic AMP-elevating peaks I or II did not stimulate sperm adenylate cyclase activity in broken cell preparations.