Sequence of small cardioactive peptide A: a second member of a class of neuropeptides in Aplysia

Previous studies have shown that the nervous system and other tissues of molluscs contain a number of peptides that potently excite molluscan hearts. Two such peptides, termed small cardioactive peptides A and B (SCPA and SCPB) are present in large quantities in the nervous system of Aplysia. These peptides are widely distributed within the CNS and peripheral tissues and have been found to be potent modulators of synaptic transmission in Aplysia. SCPB has previously been purified from nervous tissue and sequenced. In this paper, we report the purification of SCPA and propose its sequence. This sequence was confirmed by comparing the chromatographic properties of native SCPA (labelled in organ culture) with a synthetic peptide that has the proposed sequence. A significant proportion of the sequence of the two SCPs is conserved, indicating that they are members of the same peptide class, a finding that is consistent with the recent observation that the two peptide sequences are present in a single precursor.     

ADENYLATE CYCLASE IN HELIX AND APLYSIA GANGLIA: CHARACTERISTICS OF ITS STIMULATION BY A PEPTIDE-CONTAINING NERVOUS SYSTEM EXTRACT

Abstract— A crude particulate fraction, prepared from the central ganglia of Helix or Aplysia , contains levels of adenylate cyclase activity comparable to those in mammalian brain. This activity can be stimulated up to 50-fold by NaF, and 4- to 10-fold by guanyl nucleotides such as GTP and guanylylimidodiphosphate (Gpp(NH)p). A peptide-containing extract from Helix or Aplysia nervous system also stimulates the adenylate cyclase, by 50-400°. In contrast, a number of peptides known to occur in vertebrate and invertebrate nervous system are without effect. The adenylate cylase stimulation by the endogenous molluscan peptide-containing extract may be receptor-mediated, but the effect is not enhanced in the presence of guanyl nucleotides: in this respect it differs from many other hormone-sensitive adenylate cyclases. The endogenous extracts prepared from Helix and Aplysia each stimulate both Helix and Aplysia adenylate cyclases, suggesting that the putative cyclase-linked receptors may be similar in the two species. Furthermore, the active components in the extracts from Helis and Aplysia appear to be similar, since preliminary evidence suggests that they may interact with the same adenylate cyclase-linked receptor in particulate fraction from Helix ganglia.     

The central nervous system of Bulla gouldiana: Peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

A vasotocin-like peptide in Aplysia kurodai ganglia: HPLC and RIA evidence for its identity with Lys-conopressin G.

The presence of a vasopressin (VP)- or vasotocin (VT)-like peptide in the central nervous system of the gastropod mollusc Aplysia has been indicated previously. In the case of Aplysia californica, HPLC and RIA evidence suggested the peptide was VT-like but not identical with the nonmammalian vertebrate peptide [Arg8]VT (AVT). In the present study, anterior ganglia extracts from the related species Aplysia kurodai were analyzed by HPLC followed by RIA. Further analysis of the major AVT-IR peak showed it to be indistinguishable, in three distinct solvent systems, from the sea snail venom peptide Lys-conopressin G, but to be different from the vertebrate peptides [Arg8]VP (AVP), [Lys8]VP (LVP), AVT, oxytocin (OT), mesotocin, isotocin, aspargtocin, glumitocin, and valitocin, from the sea snail venom peptide Arg-conopressin S, and from the peptides [Lys8]VT and [Gln8]OT. In addition, the carboxymethylated (CM) A. kurodai peptide had the same HPLC retention time as CM-Lys-conopressin G. The HPLC/RIA results suggest that (i) based on the properties of the solvent systems used, the A. kurodai peptide has two basic amino acids (like the conopressins but unlike the vertebrate peptides), and (ii) there is a high probability that the A. kurodai peptide is identical with Lys-conopressin G.     

Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons.

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

Peptide B induction of bag-cell activity in Aplysia: localization of sites of action to the cerebral and pleural ganglia

The bag cells of the marine mollusc Aplysia are model neuroendocrine cells involved in the initiation of egg laying and its associated behaviors, but the natural stimulus triggering bag-cell activity is not known. The atrial gland of A. californica, an exocrine organ in the reproductive tract, contains two structurally related peptides (A and B) which can induce an afterdischarge in vitro, and these peptides can be used to probe the central nervous system for sites where extrinsic excitatory input onto the bag-cell system might occur. These sites were identified in a series of lesion and ablation experiments. The entire central nervous system was removed from an animal and placed in a chamber with two compartments which could be independently perfused, allowing peptides (atrial gland extract or purified peptide B) to be selectively applied to specific regions of the nervous system while bag-cell activity was monitored using extracellular suction electrodes. Afterdischarges were consistently and reliably induced when peptides were applied to the cerebral ganglion, the pleural ganglia, the cerebropleural connectives, or the rostral 10-15% of the pleurovisceral connectives, provided that an intact neuronal pathway connected the site of peptide application with the bag cells. In contrast, afterdischarges were never observed when peptides were selectively applied to the buccal or pedal ganglia and only rarely observed when applied to the abdominal ganglion and caudal pleurovisceral connectives. These results support the hypothesis that bag-cell processes and/or neuron(s) presynaptically excitatory to the bag cells are located in the pleural and cerebral ganglia and narrow the region of the central nervous system where the critical initiator element(s) can be identified.     

Profiling 26,000 Aplysia californica neurons by single cell mass spectrometry reveals neuronal populations with distinct neuropeptide profiles

Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain–Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones’ previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry–based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.     

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.     

A novel view on the mechanisms of action of insulin and other insulin superfamily peptides: involvement of adenylyl cyclase signaling system

A new signaling mechanism common to mammalian insulin, insulin-like growth factor I, relaxin and mollusc insulin-like peptide, and involving receptor-tyrosine kinase==>G(i) protein (betagamma)==>phosphatidylinositol-3-kinase==>protein kinase Czeta==>adenylyl cyclase==>protein kinase A was discovered in the muscles and some other tissues of vertebrates and invertebrates. The authors' data were used to reconsider the problem of participation of the adenylyl cyclase-cAMP system in the regulatory effects of insulin superfamily peptides. A hypothesis has been put forward according to which the adenylyl cyclase signaling mechanism producing cAMP has a triple co-ordinating role in the regulatory action of insulin superfamily peptides on the main cell processes, inducing the mitogenic and antiapoptotic effects and inhibitory influence on some metabolic effects of the peptides. It is suggested that cAMP is a key regulator responsible for choosing the transduction pathway by concerted launching of one (proliferative) program and switching off (suppression) of two others, which lead to cell death and to the predomination of anabolic processes in a cell. The original data obtained give grounds to conclude that the adenylyl cyclase signaling system is a mechanism of signal transduction not only of hormones with serpentine receptors, but also of those with receptors of the tyrosine kinase type (insulin superfamily peptides and some growth factors).     

Conservatism of the Insulin Signaling System in Evolution of Invertebrate and Vertebrate Animals

The insulin system including hormone insulin and signaling mechanisms realizing a wide spectrum of its regulatory effect is one of the major systems in the animals and human organism. At present the history of origin of this regulatory system in the course of evolution starts to be formed. There are grounds to believe that it appeared in unicellular eukaryotes, developed in multicellular ones, and achieved significant perfection in higher vertebrates. This paper analyzes the structural-functional organization of insulin-like peptides, their receptors, and the corresponding signaling mechanisms in four types of invertebrates (sponges, nematodes, molluscs, arthropods) in comparison with those in higher vertebrates. There is revealed evolutionary conservatism in the common structural-functional organization of insulin-like peptides of invertebrates and insulin of vertebrate animals; receptors of insulin-like peptides of invertebrates and receptors of insulin and insulin-like growth factor 1 of vertebrates that have tyrosine kinase activity; the insulin-like signaling systems including signaling blocks, similar by their primary structure in invertebrate and vertebrate animals (IRS-proteins, G-proteins, adenylyl cyclase, protein kinases A and C, etc.). The point of view is put forward that the conservatism of the functional blocks of the insulin system does not mean the absence of evolutionary changes of this system as a whole. Examples of such evolutionary changes leading to complication of the insulin system organization at supramolecular and cellular levels and to an increase of efficiency of its functioning are presented.     

Stimulation of an Insulin Receptor Activates and Down-Regulates the Ca2+-Independent Protein Kinase C, Apl II, Through a Wortmannin-Sensitive Signaling Pathway in Aplysia

Abstract: Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca²⁺-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca²⁺-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca²⁺-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC₁₆phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca²⁺-independent protein kinase C isoforms.     

Structure of an anti-cholera toxin antibody Fab in complex with an epitope-derived D-peptide: a case of polyspecific recognition

The structure of a complex of the anti-cholera toxin antibody TE33 Fab (fragment antibody) with the D-peptide vpGsqhyds was solved to 1.78 A resolution. The D-peptide was derived from the linear L-peptide epitope VPGSQHIDS by a stepwise transformation. Despite the very similar amino acid sequence-the only difference is a tyrosine residue in position 7-there are marked differences in the individual positions with respect to their contribution to the peptide overall affinity as ascertained by a complete substitutional analysis. This is reflected by the X-ray structure of the TE33 Fab/D-peptide complex where there is an inverted orientation of the D-peptide as compared with the known structure of a corresponding complex containing the epitope L-peptide, with the side chains establishing different contacts within the binding site of TE33. The D- and L-peptide affinities are comparable and the surface areas buried by complex formation are almost the same. Thus the antibody TE33 provides a typical example for polyspecific binding behavior of IgG family antibodies.     

Structural and functional diversities of the Aplysia Mytilus inhibitory peptide-related peptides

Aplysia Mytilus inhibitory peptide-related peptides (AMRPs) are multiple hexapeptides coded on a single precursor. By comparing the AMRP precursors of two species of Aplysia (Aplysia californica and Aplysia kurodai), we found that there are substantial numbers of species-specific AMRPs. We next compared the function of AMRPs on the anterior aorta between A. kurodai and Aplysia juliana. In A. juliana, AMRPs inhibited the contractile activity of the aorta (EC(50)=10(-9) to 10(-8)M), whereas the peptides had no obvious action in A. kurodai up to 10(-7)M. These results indicate that AMRPs are both structurally and functionally diverse neuropeptides even among closely related species.     

Endogenous neurotrophic factors enhance neurite growth by bag cell neurons of Aplysia

Mechanisms that regulate neurite outgrowth are phylogenetically conserved, including the signaling molecules involved. Here, we describe neurotrophic effects on isolated bag cell neurons (BCNs) of substrate-bound growth factors endogenous to the sea slug Aplysia californica. Sheath cells dissociated from the pleural-visceral connectives of the Aplysia CNS and arterial cells dissociated from the anterior aorta enhance neurite outgrowth when compared to controls, i.e., BCNs grown in defined medium alone. In addition, the substrate remaining after sheath cells or arterial cells are killed significantly enhances growth, relative to all other conditions tested. For instance, primary neurites are more numerous and greater in length for BCNs cultured on substrate produced by arterial cells. These results suggest that sheath and arterial cells produce growth-promoting factors, some of which are found in the substrates produced by these cell types. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we found that Aplysia collagen-like peptides are produced by dissociated arterial cells, and therefore likely contribute to the observed growth effects. Collagen-like peptides and other factors produced by sheath and arterial cells likely influence neurite growth in the Aplysia CNS during development, learning and memory, and regeneration after injury.     

Interganglionic axonal transport of neural peptides within the nervous system of Aplysia.

Neurons of the circumesophageal ganglia of Aplysia synthesize 1--2000 dalton peptides and subject them to axonal transport in large quantities in the pleuro-visceral connective and pedal nerves. Most of the protein transported in the connective nerves accumulates in the abdominal ganglion, although some passes out its peripheral nerves. Autoradiography revealed no evidence for terminations of the transporting axons in possible neurohemal areas of this ganglion. It is suggested that these data reflect the existence of a pathway mediating the "directed delivery" of neural peptides in this nervous system.     

The gamma-carboxy glutamic acid content of human and bovine prothrombin following warfarin treatment.

A form of prothrombin induced by Warfarin therapy, has been isolated which is adsorbed onto insoluble barium salts, but has a reduced biological activity. This protein contains, on average, seven out of a possible ten gamma-carboxy glutamic acid residues. A second form of prothrombin is also described, which is not adsorbed into barium slats, and has less than 1% the activity of the normal protein, contains only four gamma-carboxy glutamic acid residues. The significance of these results is discussed.     

Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.