Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): a light and electron microscopic study.

Two peptides, Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe-NH2 (SGYSVLYFamide), recently isolated from the sea cucumber Holothuria glaberrima [Díaz-Miranda et al. (1992) Biol. Bull. 182:241-247] represent the first neuropeptides isolated from holothurians. Using an antibody against GFSKLYFa, we describe here the localization and distribution pattern of GFSKLYFa-like immunoreactivity in H. glaberrima, where immunoreactive fibers form a prominent and extensive peptidergic nervous system component. Neuron-like cells and nerve fibers expressing GFSKLYFa-like immunoreactivity are found in the ectoneural and hyponeural divisions of the radial nerve cords as well as in the digestive, haemal, respiratory, and reproductive systems; in the tentacles; and in tube feet. Neuroendocrine-like cells are found in the mucosal layer of the intestine. Ultrastructure immunocytochemical analysis revealed that, in nerve cells and fibers in the serosal layer of the intestine, the immunoreactivity is concentrated in vesicles. The immunoreactive nerve fibers are found mainly within a dense nerve plexus overlying and in close contact with smooth muscle cells of the intestine. The exclusive expression of GFSKLYFa-like immunoreactivity in neuronal or neuroendocrine tissue together with the close apposition of some fibers to muscle cells suggests that GFSKLYFa acts as a neuromuscular transmitter or neuromodulator in H. glaberrima. The wide occurrence of GFSKLYFa-like immunoreactivity throughout the nervous system of the sea cucumber suggests that GFSKLYFa plays an important role in the control of multiple action systems, including digestion, respiration, circulation, reproduction, and locomotion.

Regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima.

Among higher metazoans, echinoderms exhibit the most impressive capacity for regeneration. Holothurians, or sea cucumbers, respond to adverse stimuli by autotomizing and ejecting their visceral organs, which are then regenerated. Neuronal fibers and cell bodies are present within the viscera, but previous regeneration studies have not accounted for the nervous component. We used light microscopic immunocytochemistry and ultrastructural studies to describe the regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima. This study provides evidence that the enteric nervous system of this echinoderm regenerates after evisceration and that in 3-5 weeks the regenerated system is virtually identical to that of noneviscerated animals. The regeneration of the enteric nervous system occurs parallel to the regeneration of other organ components. Nerve fibers and cells are observed within the mesenterial thickenings that give rise to the new intestine and within the internal connective tissue prior to lumen formation. We also used bromodeoxyuridine incorporation to show that proliferation of the neuronal population occurs in the regenerating intestine. The regeneration of the nervous system commands high interest because members of the closely related phylum Chordata either lack or have a very limited capacity to regenerate their nervous system. Thus, holothurians provide a model system to study enteric nervous system regeneration in deuterostomes.

Visceral regeneration in holothurians.

Holothurians, or sea cucumbers, exhibit two processes that have intrigued biologists for decades: autotomy and regeneration. Autotomy includes the loss of body parts by evisceration or fission, and regeneration is the extraordinary process by which the lost organs are replaced. In this article, we review the literature on evisceration, transection, and visceral regeneration in holothurians and compare these processes in different orders and lower taxa. Focusing mainly on the digestive tube, we analyze regeneration from a cellular perspective, considering especially the origin, migration, and proliferation of the cellular components of the regenerated organ. The data highlight the most interesting aspects of holothurian regeneration and indicate those critical problems requiring new information and new approaches.

Depolarization effects on the peptidergic phenotypes of chick sympathetic and adrenal cells.

Depolarizing stimuli are among the factors known to influence the phenotypic plasticity of nerve cells. In order to determine the prevalence of the depolarization effects in terms of cell and neuropeptide phenotypes, we have analyzed the effect of potassium (K+)-induced depolarization on the avian sympathoadrenal system. The expression of three peptidergic phenotypes, somatostatin (SS), neuropeptide Y (NPY) and enkephalin (Enk) by two cell types, adrenal and sympathetic, was studied under different depolarizing regimens. Cells from the sympathetic paravertebral ganglion and adrenal gland of 10-11-day chick embryos were cultured and the peptide levels were measured by radioimmunoassays. Chronic depolarization causes differential effects on the peptidergic phenotypes increasing NPY and Enk but decreasing SS in both adrenal and sympathetic cultures. However, shorter exposures to depolarizing stimuli revealed diverse effects on NPY and Enk phenotypes and even between adrenal and sympathetic cells. Moreover, the maintenance of the effects after removal of the depolarizing stimuli showed additional differences among the phenotypes. Our results are not compatible with a previously established hypothesis stating that depolarization increases the synthesis of whichever neurotransmitters a neuron is already producing. They provide evidence indicating that the depolarization effect is much more complex than originally thought, and serve to initiate an in depth probe into the effect of depolarization of cellular plasticity.

Descending inhibition of slow spinal reflex in an in vitro preparation of the newborn rat and its possible involvement in pain control.

An isolated brainstem-spinal cord preparation of the newborn rat is described in which brainstem stimulation inhibits a spinal reflex of slow time course possibly associated with nociception. The inhibition-producing area is localized in the rostral medulla and caudal pons. Perfusion of the preparation with phentolamine or strychnine antagonizes the inhibition, suggesting the involvement of alpha-adrenergic and glycine-mediated inhibitory mechanisms. Results are discussed in relation to descending inhibition of nociception.

In vivo and in vitro expression of vasoactive intestinal polypeptide-like immunoreactivity by neural crest derivatives.

Qualitative and quantitative in vivo studies were performed on the development of the neuropeptide vasoactive intestinal polypeptide (VIP) in the peripheral nervous system of quail embryos. VIP-like immunoreactivity (VIPLI) was found by radioimmunoassay (RIA) from the sixth day of embryonic life onward in the sympathetic chain, the esophagus and duodenum, and from day 15 of incubation onward in the adrenal glands and the nodose ganglia. By using immunocytochemistry, we identified cells expressing VIPLI in sensory spinal ganglia of 13- to 15-day-old embryos. In neural crest cultures, cells expressing the VIP phenotype differentiated constantly under various culture conditions, in contrast to other phenotypes which had specific medium requirements, i.e. adrenergic cells or substance P-containing neurons.

Modulation of neuropeptide expression in avian embryonic sympathetic cultures.

Two distinct neuropeptide-related phenotypes are found in avian paravertebral sympathetic ganglia, corresponding to somatostatin- (SS) and vasoactive intestinal polypeptide- (VIP) expressing cells. We have detected the same cell phenotypes in cultures of embryonic quail sympathetic ganglia and have used this system to study the modulation of their expression by the environment. The cell phenotypes were identified using immunocytochemistry and induced catecholamine fluorescence and quantitative data were obtained by radioimmunoassay. Dissociation of the ganglia caused a profound increase in the expression of VIP but had no effect on SS levels. Addition of corticosterone (10(-6) M) increased the expression of SS without modifying VIP levels. In contrast, depolarization of the cells induced changes in levels of both neuropeptides. The modulation of VIP correlates with the modulation of cholinergic properties. The regulation of neuropeptide expression in the avian system shows both similarities and differences to what has been found in the mammalian system.

Differential regulation of NPY mRNA expression in embryonic sympathetic and chromaffin cultures by NGF.

RT-PCR analysis of NPY mRNA expression in chick embryonic sympathoadrenal cells in culture showed that NGF increases sympathetic but not adrenal NPY mRNA content. These results show that the previously reported differential effect at the protein level can also be detected at the mRNA level, suggesting a pre-translational point of regulation. The differential NGF effect in such closely related phenotypes is particularly relevant to studies of plasticity and differentiation.

Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors.

Most neurons and endocrine cells are known to co-express a 'classical neurotransmitter' with one or more neuropeptides. Although their expression has been shown to be modulated by differentiation factors, it is not known if particular combinations of neurotransmitter/neuropeptide(s) are co-regulated. We have analyzed the effect of nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-beta 1) on the modulation of neuroactive substances co-expressed by avian chromaffin cells. The content of the neuropeptides neuropeptide Y (NPY), enkephalin (ENK) and somatostatin (SS) was measured by radioimmunoanalysis, and the content of the catecholamines norepinephrine (NE) and epinephrine (E) by high pressure liquid chromatography-electrochemistry (HPLC-EC). In addition, the morphological differentiation of chromaffin cells in response to the growth factors was assessed. All of the studied factors had distinct effects on the chromaffin content of neuropeptides and catecholamines. Our results show that the modulation of CAs and neuropeptides, and among the neuropeptides themselves is completely dissociated. Moreover, the cellular responses to the different growth factors show that neurochemical properties are modulated independently of morphological ones.

Growth factors effects on the expression of morphological and biochemical properties of avian embryonic sympathetic cells. Emphasis on NGF.

Growth factors are known to be important agents in the differentiation and modulation of neuronal phenotypes. We have analyzed the effect of several growth factors on the modulation of morphological and biochemical properties of avian embryonic sympathetic neurons. The growth factors studied include: nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-beta1). Morphological properties were analyzed by immunocytochemistry to neurofilament proteins and visualization of fibers after glyoxylic acid-induced fluorescence. Biochemical modulation was determined by radioimmunoanalysis for the peptides enkephalin (ENK), somatostatin (SS) and neuropeptide Y (NPY) and by HPLC-electrochemistry quantification of catecholamines. Similar to previous results using chromaffin cell cultures [R. Ramírez-Ordóñez, J.E. García-Arrarás, Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors, Dev. Brain Res., 87 (1995) 160-171], we found a dissociation in the modulation of biochemical and morphological properties, however, the effect of specific factors differed between the chromaffin and sympathetic cultures. We have focused on NGF to analyze its effect on the sympathetic peptide phenotypes and its lack of an effect on the chromaffin cell peptide phenotypes. The results presented here, establish interesting differences between chromaffin cells and sympathetic neurons that are of importance to studies of cell lineage and differentiation.

Enteric nerve fibers of holothurians are recognized by an antibody to acetylated alpha-tubulin.

The distribution of immunoreactivity to 6-11B-1, a monoclonal antibody that labels acetylated alpha-tubulin, was studied in the radial nerve and intestinal system of holothurians. As shown previously for other species, this antibody recognizes cilia and nerve fibers in Holothuria glaberrima and Holothuria mexicana. Thus, anti-acetylated alpha-tubulin can be used as a marker for nerve fibers in the enteric nervous system.

E-type prostaglandins depolarize primary afferent neurons of the neonatal rat.

The mechanism of action of prostaglandins (PGs) to sensitize sensory terminals to noxious stimuli was studied in the isolated spinal cord-tail preparation of the newborn rat. Application of a small amount of capsaicin to the tail induced a nociceptive reflex that was recorded extracellularly from the lumbar ventral root. Pretreatment of the tail with PGE1 or E2 (0.8-4 microM) markedly potentiated the capsaicin-induced nociceptive reflex. In the isolated spinal cord preparation of the newborn rat, application of PGE1 or E2 (10 nM-1 microM) induced a depolarization of the dorsal root. Based on these results we propose a hypothesis that PGs regulate the resting potential of the peripheral terminals of nociceptive primary afferent fibers and that the depolarization is associated with lowering of threshold for various noxious stimuli.

Electroimmunoblotting of neuropeptide Y: application to the rat vas deferens.

In this study we have optimized the electroimmunoblotting conditions for neuropeptide Y (NPY). NPY standards and samples extracted from the rat vas deferens were separated on urea-sodium dodecyl sulphate gels. Densitometric scanning of the Coomassie Blue-stained gels allow a semi-quantitative analysis of NPY in the range of approximately 10(-11) to 10(-8) mol of NPY. Electroimmunoblotting of NPY was also shown to be best achieved overnight at 4 degrees C and with NC membranes of 0.22 micron. Under these conditions NPY extracted from the vas deferens has been efficiently electroimmunoblotted. Higher molecular weight NPY-reactive peptides were also detected that may be related to proteolytic processing of the NPY precursor.

FMRFamide-like immunoreactivity in cells and fibers of the holothurian nervous system.

Immunoreactivity to FMRFamide (FMRFa) was detected in several components of the nervous system of the sea cucumber, Holothuria glaberrima. Neurons and fibers expressing immunoreactivity to this peptide were found in the radial nerves, and in nerve plexuses of the esophagus, and large and small intestine. The neurons in the enteric nervous system were located in the serosal layer and immunoreactive fibers appeared to innervate the longitudinal muscle. Co-existence of immunoreactivities to FMRFa and cholecystokinin was detected in most of the enteric fibers. Therefore, in the holothurians, neurons expressing FMRFa and FMRFa/cholecystokinin might be involved in the physiology of the digestive tract.

Identification of Hox Gene Sequences in the Sea Cucumber Holothuria glaberrima Selenka (Holothuroidea: Echinodermata).

The Echinodermata is a unique animal group forming an early branch in the deuterostomes phylogenetic tree. In echinoids and asteroids a single Hox cluster with nine cognates of the vertebrate Hox paralogous groups has been reported, but no data are available from other echinoderm classes. We report here nine Hox-type sequences from the sea cucumber Holothuria glaberrima, a member of the class Holothuroidea. Partial homeodomain sequences were amplified by polymerase chain reaction from genomic DNA and from a regenerating gastrointestinal tract complementary DNA library. Sequence analyses suggest that the holothuroid cluster has at least three genes of the anterior, one of the medial, and five of the posterior groups. This is the first evidence of five posterior sequences in echinoderms.

Pharmacological action of the heptapeptide GFSKLYFamide in the muscle of the sea cucumber Holothuria glaberrima (Echinodermata).

The holothurian neuropeptide GFSKLYFamide (Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2), GFSKLYFa, was characterized recently and shown to be present in nerve fibers that apparently innervate various muscle systems. We have studied the potential neurotransmitter role of this peptide by assaying its effects on the contractility of visceral and somatic muscles. GFSKLYFa in nanomolar concentrations induces a relaxation of the muscle tension in the intestine. A similar effect is observed on the longitudinal muscle bands of the body wall of the sea cucumber. The relaxing action of GFSKLYFa is dose dependent suggesting that its action is mediated by receptors present in the muscle cells. In addition, GFSKLYFa induces the relaxation of the acetylcholine contracted intestine. Our investigation provides additional evidence indicating that GFSKLYFa might be a neurotransmitter acting at the neuromuscular junctions of the sea cucumber Holothuria glaberrima.

Gut regeneration in holothurians: a snapshot of recent developments.

Visceral regeneration in sea cucumbers has been studied since early last century; however, it is only within the last 15 years that real progress has been made in understanding the cellular and molecular events involved. In the present review, we bring together these recent studies, providing readers with basic information on the anatomy and histology of the normal gut and detailing the changes in tissue organization and gene expression that occur during the regenerative process. We discuss the nature and possible sources of cells involved in the formation of the intestinal regenerate as well as the role of cell death and proliferation in this process. In addition, we compare gut formation during regeneration and during embryogenesis. Finally, we describe the molecular studies that have helped advance regenerative studies in holothurians and integrate the gene expression information with data on cellular events. Studies on visceral regeneration in these echinoderms provide a unique view that complements regeneration studies in other animal phyla, which are mainly focused on whole-animal regeneration or appendage regeneration.