A putative 'pre-nervous' endocannabinoid system in early echinoderm development.

Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.

[Preneural transmitters as regulators of embryogenesis. Current state of the problem].

Our knowledge about the preneural neurotransmitter systems and their functions were based on the old pharmacological and biochemical data that have recently been confirmed and substantially supplemented. Specific components of the preneural serotoninergic and endocannabinoid systems were identified in developing echinoderm embryos using immunocytochemistry, Western immunoelectroblotting, and HPLC-mass spectroscopy. These data were corroborated by the results of pharmacological experiments: it was found that some ligands of serotonin receptors, as well as the agonist of cannabinoid receptors anandamide induced the appearance of abnormal embryonic phenotypes, whose expression depended on the ligand-teratogen concentration. Their appearance was prevented, correspondingly, by serotonin and its lipophilic (or hydrophilic) analogs and antagonists of cannabinoid (CB1/CB2)-receptors.

[Fluorescent-labeled lipophilic analogues of serotonin, dopamine, and acetylcholine: synthesis, mass spectrometry, and biological activity]. / Fluorestsentnomechenye lipofil'nye analogi serotonina, dofamina i atsetilkholina: sintez, mass-spektrometriia i biologicheskaia aktivnost'.

4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl derivatives of serotonin, dopamine, choline, and N,N-dimethylaminoethanol, with the fluorescence maximum at 512 nm (lambda(exc) 470 nm), and 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl derivatives of choline and N,N-dimethylaminoethanol, with the fluorescence maximum at 554 nm (lambda(exc) 470 nm), were synthesized. These compounds yield protonated molecular ions of 100% intensity upon mass spectrometry with electrospray ionization at atmospheric pressure. The fragmentation of molecular ions under the conditions of secondary mass spectrometry mainly proceeds through the elimination of hydrogen fluoride from the fluorescent core of the molecules. Experiments on sea urchin Lytechinus variegatus embryos and larvae showed that these compounds easily penetrate into the cells and are accumulated in the cytoplasm. They do not differ in their biological activity from similar derivatives of arachidonic acid described previously and are agonists of serotonin or acetylcholine or antagonists of nicotinic acetylcholine receptors. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Effects of arachidonoyl dopamine, haloperidol, and their mixtures on regeneration of freshwater Hydra attenuata]. / Vliianie arakhidonoildofamina, galoperidola i ikh smesei na regeneratsiiu presnovodnoi gidry Hydra attenuata.

Arachidonoyl dopamine and haloperidol, both separately and in different combinations, inhibit regeneration of the gastral and basal regions of hydra. In addition, both substances induce stable anomalies of morphogenesis in the form of outgrowths and additional tentacles in gastral regenerates. In the presence of both substances at different combinations, anomalies either do not appear altogether, or exist for a short time, thus suggesting the normalization of morphogenesis. Possible mechanisms underlying the effects of these substances are discussed.

Serotonin and serotonin-like substances as regulators of early embryogenesis and morphogenesis.

The problem of pre-nervous neurotransmitter systems arose from studies carried out on different groups of invertebrates and vertebrates in the late 1950s to early 1960s. These investigations were motivated by an hypothesis formulated by K. S. Koshtoyants concerning the similarity between pre-nervous control processes and neuronal functions. Here, we review new data related to the embryogenetic and morphogenetic functions of serotonin (5-HT) and 5-HT-like substances in early embryos of sea urchins, mouse, and other species. Accumulating evidence across animal phyla indicates that 5-HT, together with other classical neurotransmitters, regulates basic developmental processes, including cell proliferation, migration, differentiation, and morphogenesis. Future investigations of cellular and molecular mechanisms underlying phylogenetically old functions of neurotransmitters could provide new insights into the evolutionary emergence of the vertebrate nervous system.

An invertebrate model of the developmental neurotoxicity of insecticides: effects of chlorpyrifos and dieldrin in sea urchin embryos and larvae.

Chlorpyrifos targets mammalian brain development through a combination of effects directed at cholinergic receptors and intracellular signaling cascades that are involved in cell differentiation. We used sea urchin embryos as an invertebrate model system to explore the cellular mechanisms underlying the actions of chlorpyrifos and to delineate the critical period of developmental vulnerability. Sea urchin embryos and larvae were exposed to chlorpyrifos at different stages of development ranging from early cell cleavages through the prism stage. Although early cleavages were unaffected even at high chlorpyrifos concentrations, micromolar concentrations added at the mid-blastula stage evoked a prominent change in cell phenotype and overall larval structure, with appearance of pigmented cells followed by their accumulation in an extralarval cap that was extruded from the animal pole. At higher concentrations (20-40 microM), these abnormal cells constituted over 90% of the total cell number. Studies with cholinergic receptor blocking agents and protein kinase C inhibitors indicated two distinct types of effects, one mediated through stimulation of nicotinic cholinergic receptors and the other targeting intracellular signaling. The effects of chlorpyrifos were not mimicked by chlorpyrifos oxon, the active metabolite that inhibits cholinesterase, nor by nonorganophosphate cholinesterase inhibitors. Dieldrin, an organochlorine that targets GABA(A )receptors, was similarly ineffective. The effects of chlorpyrifos and its underlying cholinergic and signaling-related mechanisms parallel prior findings in mammalian embryonic central nervous system. Invertebrate test systems may thus provide both a screening procedure for potential neuroteratogenesis by organophosphate-related compounds, as well as a system with which to uncover novel mechanisms underlying developmental vulnerability.

[Arachidonoylcholine and N,N-dimethylaminoethyl arachidonate are new cholinergic compounds]. / Arakhidonoilkholin i N,N-dimetilaminoétilarakhidonat--novye kholinérgicheskie veshchestva.

Choline and N,N-dimethylaminoethyl esters of arachidonic and some other fatty acids were synthesized. Experiments on the embryos and larvae of sea urchins, sensitive to cholinergic compounds, showed that arachidonoylcholine exhibited cholinomimetic activity similar to that of nicotine whereas N,N-dimethylaminoethyl arachidonate acted as an acetylcholine antagonist. The corresponding esters of docosahexaenoic acid displayed similar biological properties.

[Cholinergic regulation of the sea urchin embryonic and larval development]. / Kholinergicheskaia reguliatsiia razvitiia u zarodyshei i lichinok morskikh ezhei.

Choline esters of polyenoic fatty acids block cleavage divisions of sea urchins and evoke the formation of one-cell multinuclear embryos. If the fatty acids AA-Ch or DHA-Ch are added at the mid or late blastula stage, many cells are extruded, forming extra-embryonic cell clusters near the animal pole of embryos or larvae. Both effects are prevented by dimethylaminoethyl esters of polyenoic fatty acids (AA-DMAE or DHA-DMAE) or their 5-hydroxytryptamides. Nicotinic acetylcholine receptor antagonists, imechine, d-tubocurarine or QX-222 provide partial protection against AA-Ch or DHA-Ch. The organophosphate pesticide, chlorpyrifos, or a combination of (-)-nicotine + phorbol 12-myristate 13-acetate, also evoke the mass extrusion of transformed embryonic cells at the animal pole of larvae. These effects are similarly antagonized by AA-DMAE, DHA-DMAE, or fatty acids 5-hydroxytryptamides. Taking together, these results suggest that AA-Ch and DHA-Ch act on sea urchin embryos and larvae as agonists of acetylcholine receptors, whereas AA-DMAE and DHA-DMAE act as antagonists. The ability of fatty acids 5-hydroxytryptamides to prevent the effects of AA-Ch or DHA-Ch may be due to restoration of the normal dynamic balance of cholinergic and serotonergic signaling during cleavage divisions and gastrulation.

[Polyenic acid 5-hydroxytryptamides and 3-hydroxytyramides as tools for studying of the pre-nervous biogenic monoamine functions]. / 5-Gidroksitriptamidy i 3-gidroksitiramidy polienovykh zhirnykh kislot v izychenii donervnykh funktsii biogennykh monoaminov.

Three main effects of the amides on embryos of opistobranch molluscs, sea urchins and starfish, were revealed. First, a rather independent and clear protective action of 5-HYDROXYTRYPTAMIDES AND 3-HYDROXYTYRAMIDES against cytostatic antagonists of serotonin and dopamine, resp. Second, prevention of developmental abnormalities induced by protein kinase C activators both by hydroxytryptamides and hydroxytyramides. Third, the cytostatic effect of 3-HYDROXYTRIPTAMIDES eliminated or prevented by 5-HYDROXYTRYPTAMIDES. These effects quantitatively depended on the structure of their fatty acids part. Some functionally active regulatory substances similar to 5-HYDROXYTRYPTAMIDES AND 3-HYDROXYTYRAMIDES may exist in the early embryos.

Cholinoreceptors of early (preneural) sea urchin embryos.

Agonists of nicotinic cholinoreceptors (n-AChR) and 1-acetyl-4-methylpiperazine (100 microM) had no effect on early embryogenesis in sea urchins, while in the presence of phorbol-12-myristate-13-acetate (PMA) and various other protein kinase C activators, these agents induced rapid lysis of oocytes or early embryos, as a result of calcium shock. Many n-AChR ligands which do not penetrate into the cytoplasm (not being antagonists of muscarinic cholinoreceptors) protected against this cytotoxic effect. In the presence of PMA, acetylcholine and carbachol had actions which were much weaker than those of nicotine, while muscarine was completely inactive in these conditions. Thus, the surfaces of sea urchin oocytes and early embryos bear receptor structures, presumably n-AChR, which are functionally linked with second messengers which are endogenous protein kinase C activators.

[The possible functional interaction of serotonin and neuropeptides in embryogenic regulatory processes (experiments on embryos of the mollusk Tritona diomedea)]. / O vozmozhnom funktsional'nom vzaimodeistvii serotonina i neiropeptidov v reguliatornykh protsessakh émbriogeneza (opyty na zarodyshakh molliuska Tritona diomeda).

Ritanserin and inmecarb hydrochloride, antagonists of serotonin, act cytostatically and teratogenically on early embryos of Tritonia diomedea, a nudibranch mollusk. On the basis of a pharmacological analysis and the type of developmental abnormalities observed, this action appears to be due to disturbances in the functional activity of endogenous serotonin and is associated with damage of to the cytoskeleton. The effects of ritanserin and inmecarb are prevented or attenuated by lipophilic serotonin analogs (serotoninamides of polyenoic fatty acids), as well as by polypeptides isolated from neurons Pd5 and Pd6 of the pedal ganglia of the adult Tritonia. In late embryos (stage of veligers), serotonin and to a lesser extent its lipophilic analogs strongly increase embryonic motility. This effect of serotonin is potentiated by some neuropeptides and inhibited by others. These results provide evidence for functional interaction between serotonin and neuropeptides in the control processes of embryogenesis.

Action of serotonin antagonists on cytoplasmic calcium levels in early embryos of sea urchin Lytechinus pictus.

Possible interaction of the serotonergic system with intracellular calcium mechanisms was investigated using techniques of ratio imaging measurement of intracellular Ca2+ and confocal microscopy in cleaving embryos of sea urchin Lytechinus pictus. Some serotonin antagonists specifically increase free intracellular Ca2+ and evoke transient regression of the first cleavage furrow, suggesting possible linkage of serotonergic and calcium mechanisms in the regulation of cellular events during cleavage divisions. These effects were more pronounced in the experiments with hydrophilic 5-HT-antagonists, quarternary ammonium salts that do not penetrate the cell membrane. Thus, it appears that 5-HT-receptors which mediate these effects are localised on the cell membrane, whereas previously studied receptors mediating the cytostatic action of lipophilic 5-HT-antagonists are localised intracellularly.

Changes in the physiological roles of neurotransmitters during individual development.

The classical neurotransmitters (acetylcholine and biogenic monoamines) are multifunctional substances involved in intra- and intercellular signaling at all stages of ontogenesis in multicellular animals. A cyclical scheme is proposed to describe age-related changes in neurotransmitter functions at different stages of development from oocyte maturation to neuron formation. This may reflect not only the temporospatial organization of neurotransmitter processes, but also the origin of the functions of acetylcholine and biogenic monoamines from the protosynapses of the cleaved embryo to neuronal synapses.

[Cholinergic receptors in early (pre-nervous) sea urchin embryos]. / Kholinoretseptory rannikh (donervnykh) zarodyshei morskikh ezhei.

Agonists of nicotinic acetylcholine receptors (nAChR) nicotine and 1-acetyl-4-methylpiperazine do not act on the early sea urchin embryogenesis but evoke calcium shock in both oocytes and early embryos under certain conditions. Many nAChR ligands protect both oocytes and embryos against this shock. There seem to exist putative nAChR on the cell surface of the early sea urchin oocytes and early embryos. Pre-nervous acetylcholine seems to be functionally coupled via these receptors with the second messengers, endogenous activators of the protein kinase C.

Functional coupling of neurotransmitters with second messengers during cleavage divisions: facts and hypotheses.

Current data on the role of classical neurotransmitters as multiple and multifunctional regulators of early embryogenesis are reviewed. It is shown that these developmental regulators are coupled with second messengers. Peculiarities of this prenervous coupling emphasized and are used as the basis for discussing the problem of the evolutionary origin of cell regulatory systems.

Nicotinic antagonists (piperidines and quinuclidines) reduce the susceptibility of early sea urchin embryos to agents evoking calcium shock.

1. Some nicotinic antagonists (piperidine and quinuclidine derivatives and bis-quaternary compounds) protect early embryos of the sea urchin Lytechinus pictus against a calcium shock evoked by ionomycin or a mixture of phorbol myristate acetate and nicotine. 2. Maximal protective potency was found for drugs that did not penetrate the plasma membrane. 3. Early sea urchin embryos have nicotinic acetylcholine receptors (nAChR) or nAChR-like structures localized on the cell surface that, apparently, take part in the control of Ca2+ influx.

[A computer study of the relationship between the value and type of their embryotoxicity and the structure of synthetic analogs of biogenic amines]. / Komp'iuternoe issledovanie sviazi mezhdu velichinoi i vidom émbriotoksichnosti i strukturoi sinteticheskikh analogov biogennykh aminov.

We calculated 219 topological, electronic, and steric indices for each of 59 studied embryotoxic benzylalkylamines and indolamines. Statistically significant equations correlating embryotoxicity and five calculated parameters were obtained using the method of step multiple regression. The prognostic power of the equation was 88-90%. Discriminant functions obtained by step discriminant analysis allowed prediction of the superactivity of benzylalkylamines and indolamines with 83-87% probability.

[Artificially functionalized polyenoic fatty acids--a new lipid bioregulators]. / Iskusstvenno funktsionalizirovannye polienovye zhirnye kisloty-- novye lipidnye bioreguliatory.

Dopamine, histamine, serotonin, and serotonin analogs were acylated with arachidonic and eicosapentaenoic acids, and the reaction products were named as artificially functionalized fatty acids (AFFA). The amides of arachidonic acid with serotonin, dopamine, and histamine were found to inhibit human platelet aggregation induced by ADP, arachidonic acid and adrenaline. Amides of arachidonic and eicosapentaeonic acids with serotonin and dopamine protect sea urchin early embryos against cytotoxic action of serotonin and histamine antagonists. These effects are not connected with the possible hydrolytic cleavage of AFFA to their constituent polyenoic fatty acids and amines. Arachidonic acid dopaminamide was shown to be a substrate of soybean 15-lipoxygenase, whereas the arachidonic acid amides with serotonin and its derivatives were resistant to this enzyme. Moreover, arachidonic acid serotoninamide turned out to be an irreversible lipoxygenase inhibitor. Considerable amount of hydroxyl radicals (fluorescent assay) were found for the first time to accompany lipoxygenase oxidation of linoleic acid; arachidonic acid serotoninamide blocked this process completely. Therefore, it was concluded that AFFA possess specific biological activity and can be considered as a novel group of lipid bioregulators.

[The positive effect of pretreatment with serotonin and gangliosides on the development of early mouse embryos after cryopreservation]. / Polozhitel'noe vliianie predobrabotki serotoninom i gangliozidami na razvitie rannikh zarodyshei myshei posle kriokonservatsii.

We have demonstrated that viability of preimplantation mouse embryos F1 (C57B1/6 x CBA) after cryoconservation at the stage of four blastomeres improves after pretreatment with serotonin (5 HT, 5 microM) or total gangliosides of bovine brain gangliosides (TG, 3 microM) added to the cultivation medium. After thawing, 64% of embryos preincubated with total brain gangliosides and 49% of embryos preincubated with serotonin developed to the stage of blastocyst during the cultivation in vitro; in the control, no more than 25% of embryos reaches this stage, and all these embryos were abnormal. Possible mechanisms of protective action of these compounds is discussed. We conclude that mouse embryos subjected to freezing-thawing procedure can be used to examine the role of serotonin and gangliosides in the regulatory processes of mammalian preimplantation development.