Isolation and partial characterization of neuropeptides that mimic prolonged inhibition produced by bag cell neurons in Aplysia.

The bag cell neurons of the marine mollusk Aplysia are part of a neural system that utilizes four neuropeptides as neurotransmitters. The peptides, derived from the egg-laying hormone/bag cell peptide (ELH/BCP) precursor protein, are released during a 20-min burst discharge of the bag cells and produce several types of responses in various abdominal ganglion neurons. In the identified neurons L3 and L6, bag cell activity produces prolonged inhibition that lasts for more than 2 h. One of the bag cell peptides, alpha-BCP, mediates an early component of the inhibition in these neurons. To identify the co-transmitter mediating the prolonged component of inhibition, we purified material from an acid extract of abdominal ganglia using molecular sizing high-pressure liquid chromatography (HPLC) on TSK 250-125 followed by two steps of reverse-phase HPLC on C4 or C18. We isolated three inhibitory factors that mimic the prolonged component of inhibition. Mass spectroscopy and partial amino acid sequence analysis indicate one factor is ELH [2-36], that is, ELH that lacks the first, N-terminal amino acid. This inhibitory activity was similar in potency to that of ELH and is the first to be described for an ELH-related peptide. The two other factors were approximately 3,300 and 4,700 Da and were effective at 10- and 50-fold lower concentration, respectively, than ELH or its fragment. Amino acid composition analysis suggests that they are not derived from the ELH/BCP precursor protein. The 4,700 Da factor is effective at the lowest concentration and produces an effect that lasts as long as 100 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local hormonal modulation of neural activity in Aplysia

The abdominal ganglion of Aplysia provides a convenient experimental system for cellular studies on the roles of peptides as chemical messengers in the nervous system. There are indications that the bag cells, a group of neuroendocrine cells, synthesize and release egg laying hormone (ELH), a peptide with an apparent molecular weight of 6000. Our recent investigations indicate that a burst of impulse activity in the bag cells produces five types of long-lasting responses, some excitatory, others inhibitory, in 26 identified neurons and 2 identified cell clusters located near the bag cells in the abdominal ganglion. The responses have slow, smoothly graded onsets, and many of them result in modulation of neuronal activity for 3 hours or more. Physiological and ultrastructural data support the hypothesis that they are induced by a bag cell hormone (or hormones) that is released into vascular and interstitial spaces of the ganglion to act on the target neurons. Local application of purified ELH to one of the target neurons provides evidence that the bag cell effect is mediated by ELH. Many of the target neurons are known to be parts of neuronal circuits that control specific behavioral and homeostatic processes. Since egg laying is initiated by the bag cell discharge and is associated with a stereotyped behavior pattern lasting several hours, the actions of these peptide-secreting neurons on the central nervous system may serve to regulate certain elements of behavior and homeostasis during egg laying.     

Multiple neuropeptides derived from a common precursor are differentially packaged and transported

The ELH prohormone is proteolytically processed into at least nine peptides which govern egg-laying behavior in Aplysia. Quantitative immunocytochemistry demonstrates that peptides derived from the prohormone are packaged into distinct vesicle classes. Further experiments suggest the segregation occurs via a rapid initial proteolytic cleavage of the prohormone followed by sorting at the trans Golgi. Egg-laying hormone (ELH) immunoreactivity is localized to the cell body and processes, while bag cell peptide (BCP) immunoreactivity is greater in the cell body. Steady state levels of the amino-terminal set of peptides including the BCPs are 3- to 8-fold lower than the carboxy-terminal cleavage products, such as ELH. Thus, intracellular packaging and routing of the peptides cleaved from a single prohormone regulate their localization and levels in these neurons.     

The bag cell egg-laying hormones of Aplysia brasiliana and Aplysia californica are identical

Egg laying in the marine molluscan genus Aplysia is elicited by an egg-laying hormone (ELH) which induces ovulation and acts on central neurons to effect egg-laying behavior. ELH, isolated from the A. californica bag cells, and three ELH-related peptides, isolated from the A. californica atrial gland, have been chemically characterized, yet relatively little is known about homologous peptides in other Aplysia species. In these studies, the primary structure of A. brasiliana ELH was determined. Bag cell clusters were extracted in an acidic solution, and the peptides purified by sequential gel filtration and reversed-phase HPLC; ELH was identified by bioassay. Amino acid compositional and sequence analyses demonstrated that the neurohormone was a 36-residue peptide whose sequence was identical to that of A. californica ELH: NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile- Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-COOH .     

Delta-bag cell peptide from the egg-laying hormone precursor of Aplysia. Processing, primary structure, and biological activity

The bag cells of the marine mollusk Aplysia express a gene encoding a 271-residue egg-laying hormone (ELH) precursor that is processed into at least nine peptide products. Four of the peptides have been identified in bag cell releasates and are known to act as nonsynaptic neurotransmitters in the abdominal ganglion. The isolation, primary structure, and proposed biological activity of a fifth peptide product (delta-bag cell peptide (delta-BCP)) from the ELH precursor are described. delta-BCP was established to be a 39-residue peptide: NH2-Asp-Gln-Asp-Glu-Gly-Asn-Phe-Arg-Arg-Phe-Pro-Thr-Asn-Ala-Val-Ser-Met- Ser-Ala-Asp- Glu-Asn-Ser-Pro-Phe-Asp-Leu-Ser-Asn-Glu-Asp-Gly-Ala-Val-Tyr-Gln-Arg- Asp-Leu-COOH. This sequence corresponds to residues 81-119 of the ELH prohormone and shares sequence identity with atrial gland peptides A and B. Significantly, synthetic delta-BCP stimulated Ca2+ uptake into mitochondria of secretory cells in the albumin gland in vitro, suggesting that the peptide regulates the cellular release of perivitelline fluid by the gland. Similar results were obtained with purified peptide A and a shorter version of delta-BCP (delta-BCP-(14-33)). These results indicate that delta-BCP belongs to a family of structurally related peptides with similar pharmacological activities that center at a conserved region of sequence corresponding to delta-BCP-(14-33).     

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.     

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.     

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

Evidence for the expression of three genes encoding homologous atrial gland peptides that cause egg laying in Aplysia

Three peptide complexes which can induce egg laying in Aplysia were isolated from the atrial gland of the marine mollusc Aplysia californica and chemically characterized. Amino acid sequence analyses established the covalent structures, including disulfide assignments, of all three dimeric complexes. Each complex consisted of an identical 18-residue peptide (A-AP) which was disulfide-bonded to a 36-residue peptide that was homologous to bag cell egg-laying hormone (ELH). The primary structure of A-AP was determined to be: NH2-Asp-Ser-Asp-Val-Ser-Leu-Phe-Asn-Gly-Asp-Leu-Leu-Pro-Asn-Gly-Arg-Cys- Ser-COOH. The primary structure of one of the three ELH-related peptides (A-ELH) was determined to be NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile-Gln-Ala-Arg-Arg-Arg-Cys-Leu-Asp-Ala-Leu-Arg-Gln-Arg-Leu-Leu-Asp- -Leu-COOH. The two other ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH, differed from A-ELH at 1 and 2 residues, respectively. Both [Ala27] A-ELH and [Gln23, Ala27]A-ELH were novel peptide sequences representing products of as yet uncharacterized genes within the ELH family. These structural studies provide the first direct chemical evidence that an 18-residue peptide (A-AP) derived from a polypeptide precursor encoded by the A gene, as predicted from nucleotide sequence analysis, occurs in the atrial gland; the Cys17 residue of A-AP is disulfide-bonded to Cys25 of A-ELH; and A-AP also occurs disulfide-bonded to two additional, previously undescribed ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH.     

Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates

The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1,422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC(50) values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract.     

Effect of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B_26–34), and the 8-amino acid alpha-bag cell peptide (α-BCP_1–8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both α-BCP_1–8 and B_26–34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells in mimicked by α-BCP_1–8. The excitatory effect of the atrial gland extract cannot be duplicated with B_26–34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B_26–34 seems to mimick α-BCP_1–8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R3-R14 of Aplysia

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R₃-R₁₄, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R₃-R₁₄ have been postulated to play a role in cardiovascular control, peptide II was without effect at ≤10⁻⁴ M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.     

Subcellular sites of processing of precursors to neurosecretory peptides in the bag cells of Aplysia: Inferences from the effects of monensin,FCCP, and chloroquine

The effects of the sodium ionophore monensin were examined in the bag cells of Aplysia californica in order to identify the subcellular sites of processing of precursors to their neurosecretory products. Incubation of bag cells in media containing 10 μM monensin led to a marked disruption of the morphology of the Golgi apparatus without affecting that of other organelles. Exposure of bag cells to monensin led to a significant impairment of processing of the largest precursor and of an intermediate protein which gives rise to the immediate precursors to the final secreted products, the egg-laying hormone (ELH) and the acidic peptide (AP). Furthermore, ELH and AP were never produced in the presence of monensin during the time course of these experiments. When axonal transport was allowed to proceed, the contents of bag-cell terminals indicated that the intermediate protein is the first to be packaged in Golgi-derived vesicles, and in monensin-treated cells may be transported without being processed further. In contrast to these results, the protonophore FCCP-impaired precursor and intermediate cleavage equally, indicating that monensin and FCCP have different effects on intracellular transport and precursor processing. These data are interpreted to indicate that the largest ELH-AP precursor is normally processed within the Golgi apparatus, and that the disruption of this organelle induced by monensin produces the impairment seen in its processing. The impairment of cleavage of the intermediate species, and the blockade of production of AP and ELH, are probably the result of monensin-induced impairment of production of proteolytically competent secretory granules by the Golgi apparatus.     

Mapping study of Achatina giant neurons sensitive to molluscan peptides

1. Mapping studies of the Achatina identifiable neuron types sensitive to the following 6 molluscan peptides were examined under current-clamp.2. These were Ser-Mytilus inhibitory peptide (Ser-MIP), catch-relaxing peptide (CARP), oxytocin, small cardioactive peptide_b (SCP_b), α-bag cell peptide (α-BCP) and egg-laying hormone (ELH).3. These peptides at 10⁻³ M (3 × 10⁻⁴ M for ELH), with 0.5% Fast Green, were applied locally to the neuron to be tested by pneumatic pressure ejection (2 kg/cm² and 400 msec in duration).4. Ser-MIP showed the inhibitory (hyperpolarizing) effects on the majority of neuron types tested.5. CARP also produced inhibition of the 3 neuron types out of 16 types tested.6. Oxytocin had an excitatory effect on two neuron types.7. SCP_b showed excitatory effects on 4 neuron types: the membrane conductance of 1 neuron type, d-RPeAN, measured under voltage-clamp was reduced by the peptide.8. α-BCP showed no effect.9. ELH produced slight inhibition of the 2 neuron types.     

The bag cell neurons ofAplysia

Egg laying in Aplysia involves a well-characterized series of behaviors that can last for several hours. The behaviors are controlled by two bilateral clusters of peptidergic neurons in the abdominal ganglion. Following brief stimulation, these neurons, which have been termed the bag cell neurons, undergo a sequence of changes in their excitability lasting many hours. The bag cell neurons have served as a model system for studying the molecular mechanisms involved in the synthesis, processing, and release of neuroactive peptides and in the regulation of prolonged changes in neuronal excitability.     

Neuropeptide processing activity in Aplysia bag cell homogenates

The neurosecretory bag cells of the mollusk, Aplysia, generate a peptide egg-laying hormone (ELH) from a 29,000 Dalton precursor protein by proteolytic cleavage to a 6-9,000 Dalton intermediate, followed by cleavage of the intermediate. We report here the initial characterization of these cleavage activities. Homogenates of bag cells in low ionic strength buffer process endogenous precursor to a peptide which is indistinguishable from ELH in molecular weight and isoelectric point. Non-specific proteolysis in the homogenates is not detectable. The pH optimum for cleavage of the precursor and the intermediate is 5.5-6.5. The cleavage activities exhibit a substantial degree of membrane association, and the inhibitor profile of each is characteristic of a thiol protease without a metal cofactor requirement. Precursor cleavage activity differs from that of the intermediate cleaving activity in inhibitor profile, solubility, and slightly, in pH optimum.     

Atrial gland cells synthesize a family of peptides that can induce egg laying inAplysia

Endogenous peptides induce egg laying in the marine mollusc Aplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying. Atrial glands were incubated in a mixture of 3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE. Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparent Mr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparent Mr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm. Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule. Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pI's in peak D.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complex behavior induced by egg-laying hormone in Aplysia

Previous studies have described a pattern of complex behavior that occurs in the marine mollusc Aplysia during egg laying. Egg laying and the behavior are initiated by a burst of impulse activity in the neuroendocrine bag cells of the abdominal ganglion or by injection of bag cell extract. To more precisely identify the factors responsible for inducing the behavior we injected animals with egg laying hormone (ELH), one of the neuropeptides secreted by the bag cells. We found that ELH causes a behavior pattern similar to what occurs during spontaneous egg laying. This includes a temporal pattern of head movements consisting of waves and undulations, followed near the beginning of egg deposition by a transition to head weaves and tamps and inhibition of locomotion. There was also a small decrease in respiratory pumping. Except for respiratory pumping, a similar pattern occurred in a second group of animals injected with atrial gland homogenate, which is presumed to induce bag cell activity, but not in controls. These results further implicate ELH in regulation of the behavior. We discuss possible sites of action of ELH and the neural mechanisms by which the behavior is controlled.Abbreviations ELH egg laying hormone - ASW artificial sea water     

Functional and morphological evidence for the existence of neurites from abdominal ganglion bag cell neurons in the head-ring ganglia of Aplysia

Summary Three lines of evidence are presented indicating that axons of the Aplysia neuroendocrine bag cells extend into the head-ring ganglia of the CNS. When the abdominal ganglion was bisected longitudinally, separating the two bag cell clusters, an afterdischarge induced in one cluster generated an afterdischarge in the other via activity through the head-ring ganglia to which each half abdominal ganglion was attached by connective nerves. This suggests that some axons of bag cells in each cluster communicate through the head-ring ganglia. Retrograde labelling of bag cells occurred when rhodamine-onjugated latex microspheres were injected into the cerebral or either pleural ganglion, a direct demonstration that bag cell axons extend into these ganglia. Finally, cell LP1 in the left pleural ganglion was inhibited during a bag cell afterdischarge, an action mimicked by application of alpha-bag cell peptide (BCP). Since BCP can act only close to its site of release due to susceptibility to peptidase activity, it is likely that LP1 inhibition is dependent on the local release of BCP from bag cell neurites in the pleural ganglion. These results open new possibilities for how bag cell afterdischarges may be initiated and broaden the distribution of their effects.Abbreviations ASW artificial sea water; -BCP -bag cell peptide - ELH egg-laying-hormone - IR immunorective - PB phosphate buffer - PVC pleurovisceral connective