Pharmacological control of muscular activity in the sea urchin larva. III. Role of cyclic nucleotides.

1. The muscular activity of the sea urchin pluteus is strongly affected by dibutyryl-c-AMP, in a stimulatory or inhibitory manner depending on the concentration, the time of exposure, and the spontaneous level of activity. 2. Dibutyryl-c-GMP, like muscarinic agents and the guanylate-cyclase activators biotin and nitrite, keeps the activity low. 3. It is suggested that the effects of muscarinic agents is mediated by c-GMP, the effects of certain monoamines by c-AMP. 4. The two cyclic nucleotides appear to control the cellular influx of Ca2+ in opposite directions. They therefore interfere with the stimulatory and paralytic effects of nicotinic agents.     

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 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 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.     

Sensitivity of sea urchin early embryos to antagonists of acetylcholine and monoamines

Early embryos of A. lixula are 10–800 times more sensitive to several neuropharmaca than are early embryos of 6 other studied species of sea urchins. Of 53 neuropharmaca studied, 25 were found hyperactive; of 19 other inhibitors of development (mitotic and metabolic poisons) only antimycin A was hyperactive for A. lixula. Both hyperactive neuropharmaca and neuropharmaca with normal activity suppress cleavage divisions and inhibit protein biosynthesis acting as antagonists of intracellular acetylcholine and monoamines. The mechanism responsible for the normal sensitivity and hypersensitivity of early sea urchin embryos is discussed.     

Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with triton X-100

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25°C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 µm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 µm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 µmole P_i/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 µmole P_i/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg⁺⁺, although some motility was also obtained with Mn⁺⁺ and Ca⁺⁺. The coupled ATPase activity had a Michaelis constant (K_m) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "K_m" of 0.2 mM.     

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.     

Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella.

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.     

Effects of mycostatin in sea urchin development

Summary Sea urchin development is inhibited in the presence of mycostatin, a fungicidal antibiotic believed to alter cell membranes. Pretreatment of eggs inhibits fertilization inL. pictus, but not inS. purpuratus. The doses resulting in abnormal development inS. purpuratus increase as the treatment is started later in development. AH tissues are sensitive to mycostatin at high concentrations, but the endodermal and mesenchyme derivatives are most sensitive at lower concentrations. The results suggest the heterogeneity of cell membranes and also indicate that membranes change with time of development.This work was supported by San Fernando Valley State College Foundation Grants No 4.267.01, No 4.268.05.     

AP4A-hydrolysing activity in sea urchin embryos

The enzyme activity hydrolysing diadenosine 5,5'-P1, P4-tetraphosphate (AP4A) was demonstrated in the embryonic extract of sea urchin. The enzyme activity was preferentially inhibited by ZnCl2 and by high concentrations of isobutylmethylxanthine, indicating that two types of the enzyme, (AP4A) hydrolase and non-specific phosphodiesterase, are related to the degradation of (AP4A) in sea urchin embryos. The (AP4A)-hydrolysing activity was not detectable in the unfertilized eggs because of the presence of a high-molecular weight (HMW) and thermolabile inhibitory factor. Though the enzymes were activated immediately after fertilization, no cell cycle-dependent fluctuations in their activities were observed.     

Protein synthetic activity in swimming sea urchin spermatozoa

Summary

Free swimming Paracentrotus lividus spermatozoa show a significant rate of protein synthesis which remains nearly linear over a period of 90 min. This protein synthetic activity is scarcely affected by emetine but completely suppressed by chloramphenicol, suggesting its possible mitochondrial origin.     

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.     

Carbonic anhydrase activity in developing sea urchin embryos

A 13-fold increase in carbonic anhydrase specific activity was found during the first 24 h in developing embryos of the sea urchin, Strongylocentrotus purpuratus. Carbonic anhydrase activity was sensitive to inhibition by 10⁻⁴ M acetazolamide. Roles for carbonic anhydrase activity in intracellular pH regulation and spicule formation are discussed.     

A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.