Egg-laying Hormone Research Articles

Measuring the peptides in individual organelles with mass spectrometry.

New sampling protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allow the assay of single dense core vesicles. Understanding the packaging of vesicles is important as vesicles are the quanta of information for intercellular communication. Using vesicles from the exocrine atrial gland of Aplysia californica as the model, a wide range of bioactive peptides are detected within each vesicle. Although the expression of the egg-laying hormone gene family of type 1 atrial gland cells has been previously examined, chemical characterization of individual 1-2 microm diameter vesicles demonstrates that products from several genes are colocalized. The mass sensitivity of MALDI MS can be further improved to enable the analysis of even smaller subcellular organelles.

In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis.

The ability to directly sequence peptides from biological cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with postsource decay (PSD) and collision-induced dissociation (CID) fragment ion mass analysis is explored. Three different sample preparation methods are described for sequencing peptides in tissue samples and in single neurons from the invertebrate model Aplysia californica. To characterize peptides from the atrial gland, MALDI-PSD/CID is applied directly to a tissue blot covered with the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The resulting fragment ions combined with database searching confirm the structure of several novel peptides encoded by egg-laying hormone genes. Moreover, MS profiling of a single unidentified neuron detects peptides with molecular weights of myomodulins C and E; this assignment is confirmed using MALDI-PSD with the matrix 2,5-dihydroxybenzoic acid (DHB). DHB does not always provide adequate fragmentation for PSD experiments; therefore, a unique dual-matrix sampling method, employing both DHB and CHCA, is developed to directly sequence a decapeptide from a single cerebral ganglion B cell. Mass accuracy of fragment ions from cellular samples is typical for the instrument employed and is not deleteriously affected by the morphology and complexity of the samples.

Egg-laying hormone peptides in the aplysiidae family.

The neuropeptidergic bag cells of the marine mollusc Aplysia californica are involved in the egg-laying behavior of the animal. These neurosecretory cells synthesize an egg-laying hormone (ELH) precursor protein, yielding multiple bioactive peptides, including ELH, several bag cell peptides (BCP) and acidic peptide (AP). While immunohistochemical studies have involved a number of species, homologous peptides have been biochemically characterized in relatively few Aplysiidae species. In this study, a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and electrospray ionization Fourier transform ion cyclotron resonance MS is used to characterize and compare the ELH peptides from related opisthobranch molluscs including Aplysia vaccaria and Phyllaplysia taylori. The peptide profiles of bag cells from these two Aplysiidae species are similar to that of A. californica bag cells. In an effort to characterize further several of these peptides, peptides from multiple groups of cells of each species were extracted, and microbore liquid chromatography was used to separate and isolate them. Several MS-based sequencing approaches are applied to obtain the primary structures of bag cell peptides and ELH. Our studies reveal that (&agr;)-BCPs are 100 % conserved across all species studied. In addition, the complete sequences of (&egr;)-BCP and ELH of A. vaccaria were determined. They show a high degree of homology to their counterparts in A. californica, with only a few amino acid residue substitutions.

The Roles of Transcription and Translation in Mediating the Effect of Electrical Afterdischarge on Neurohormone Synthesis inAplysiaBag Cell Neurons1

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

The Roles of Transcription and Translation in Mediating the Effect of Electrical Afterdischarge on Neurohormone Synthesis in Aplysia Bag Cell Neurons

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

TheLymnaeaCardioexcitatory Peptide (LyCEP) Receptor: A G-Protein–Coupled Receptor for a Novel Member of the RFamide Neuropeptide Family

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Prolonged hormone secretion from neuroendocrine cells of Aplysia is independent of extracellular calcium.

The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co(2+)-substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.

Pheromones linked to sexual behaviors excite the appetitive phase of feeding behavior of Aplysia fasciata II. Excitation of C-PR, a neuron involved in the generation of appetitive behaviors

Pheromones presumably released by conspecifics amplify both the appetitive and the consummatory components of feeding in Aplysia. These effects can be mimicked by administering homogenate of the large hermaphroditic duct containing atrial gland tissue, as well as peptides from the bag cells. Identified cerebro-pedal regulator (C-PR) neuron is thought to command various behaviors that comprise the appetitive phase of feeding. In a reduced preparation, we investigated the effects on the C-PR of applying these substances to the rhinophores, the sensory organs which detect pheromones. Stimuli that excite feeding in the animal were also found to affect the C-PR. Large hermaphroditic duct homogenate caused a doubling in the firing rate of the C-PR, and amplified the response of the C-PR to other excitatory stimuli, such as touch of food to the rhinophores. Bag cell peptides (α, β and γ bag cell peptide, and egg-laying hormone) caused smaller increases in the firing rate of the C-PR. These data are consistent with the hypothesis that pheromones facilitate appetitive feeding behavior in part via their excitation of C-PR.

Proteolytic processing of the Aplysia egg-laying hormone prohormone.

By using matrix-assisted laser desorption/ionization time-of-flight MS, individual peptidergic neurons from Aplysia are assayed. A semiquantitative method is developed for comparing single-cell profiles by using spectral normalization, and peptides are localized to specific cells by mass spectrometric cell mapping. In addition to all previously identified products of the egg-laying hormone (ELH) gene, other peptides are formed from proteolytic hydrolysis of Leu-Leu residues within ELH and acidic peptide (AP). AP exhibits further processing to yield AP1-20 and AP9-27. These peptides appear to be colocalized in vesicles with ELH, transported to specific neuronal targets, and released in a Ca2+-dependent manner. A differential peptide distribution is observed at a specific target cell, and a low-frequency variation of AP, [Thr21]AP, is detected in a single animal.

Seasonal Fluctuations in the Secretory Response of Neuroendocrine Cells ofAplysia californicato Inhibitors of Protein Kinase A and Protein Kinase C

The neuroendocrine bag cells ofAplysiaprovide an excellent model system for exploring the roles of second-messenger pathways regulating peptide hormone secretion. Both the cAMP and diacylglycerol second-messenger systems and their associated protein kinases (PKA and PKC) are involved in regulating membrane excitability in bag-cell neurons ofAplysia.The purpose of the present set of experiments was to determine if PKA and PKC also play roles in regulating egg laying hormone (ELH) secretion from bag-cell neurons. Abdominal ganglia with attached bag-cell clusters and connective nerves were dissected from reproductively matureAplysia,and ELH secretion in response to electrically stimulated afterdischarges was measured by RIA. ELH secretion from bag cells treated with protein-kinase inhibitors (Rp-cAMPS to inhibit PKA; H-7 to inhibit PKC) was compared to that from untreated controls. Our experiments showed that 100 μM Rp-cAMPS significantly attenuated ELH secretion during the nonbreeding seasons (winter and spring) of 2 consecutive years. This suggested a role for PKA in regulating ELH secretion. However, Rp-cAMPS had no effect on ELH secretion during the breeding seasons (summer and fall) of 2 consecutive years, even when the dose of Rp-cAMPS was increased to 200 μM. These findings indicate that there is a seasonal fluctuation in responsiveness to PKA inhibition. We also investigated if there was a seasonal fluctuation in the ability of the PKC inhibitor H-7 to suppress ELH secretion. During the nonbreeding season, 10–100 μM H-7 significantly inhibited ELH secretion, but during the breeding season, only the highest dose (100 μM) of H-7 inhibited ELH release. These results confirm that PKC plays a role in regulating ELH secretion and indicate that there is a seasonal fluctuation in responsiveness to PKC inhibition. Overall, our findings suggest that both the cAMP and diacylglycerol second-messenger pathways are regulated on a seasonal basis.

Consummatory feeding movements in Aplysia fasciata are facilitated by conspecifics with access to mates, by reproductive tract homogenates and by bag cell peptides

In Aplysia fasciata, pheromones released by conspecifics with access to mates increase the quantity of food eaten. This effect is blocked when the chemosensory rhinophores are ablated, indicating that the rhinophores sense pheromones. The modulation of feeding by pheromones can be monitored by an increase in the amplitude of swallowing movements in the presence of conspecifics with access to mates. Atrial gland homogenates and four bag cell peptides (egg-laying hormone, and α, β, and γ bag cell peptides) amplify the swallow amplitude in a manner similar to that caused by conspecifics with access to mates, suggesting that peptides from the bag cell/atrial gland family that are released from the atrial gland into the surrounding water may be pheromones regulating feeding and reproductive behaviors.

Mass spectrometric survey of interganglionically transported peptides in aplysia

The major ganglionic connectives in Aplysia are assayed to determine putative neuropeptides. Matrix-assisted laser desorption/ionization mass spectrometry allows direct measurement of peptides in a nerve. Many previously characterized peptides are observed, including APGWamide, buccalins, small cardioactive peptides, and egg-laying hormone. Several unreported peptides are detected in specific nerves, suggesting they may have important physiological roles. Furthermore, novel processing products of the L5–67 precursor peptide and the APGWamide/cerebral peptide 1 prohormone are strongly suggested, and their interganglionic transport demonstrated.

Leech egg-laying-like hormone: structure, neuronal distribution and phylogeny

Cells immunoreactive to antisera specifically directed against Lymnaea stagnalis caudo dorsal cells egg-laying hormone (CDCH) or against α- and β-peptides (CDCP), encoded on the egg-laying hormone precursor, were detected in central nervous system (CNS) of the rhynchobdellid leech Theromyzon tessulatum. A co-localization of the CDC-like hormone and CDC-like peptides was found in T. tessulatum as in L. stagnalis CNS. ≈45 immunoreactive cells to the anti-CDCH were detected in leech brain but this number varies according to the stage of the animal life cycle, i.e. it reaches a maximum just before egg-laying while after it decreases to 2–3 cells. CDCH and α-CDCP epitopes recognized by anti-CDCH and anti- α-CDCP were contained in neurosecretory granules. Following an extensive purification, including HPGPC and reverse-phase HPLC, the CDC-like hormone contained in the T. tessulatum CNA was isolated. The sequence (GSGVSNGGTEMIQLSHIRERQRYWAQDNLRRRFLEK-amide) was established by a combination of automated Edman degradation, arginyl-endopeptidase digestion, electrospray mass spectrometry measurement and carboxypeptidase A treatment. The results demonstrate that the peptide recognized by the anti-CDCH in the leech CNS possesses 27.8, 37.2 and 47.2% sequence identity with Aplysia parvula, Lymnaea stagnalis and Aplysia californica ELH, respectively. This molecule was named the leech egg-laying-like hormone (L-ELH). The secondary structure prediction of the L-ELH and all mollusks ELH, revealed the existence of a conserved segment (segment 29–34) in a strong helicoidal bend that might be important for receptor recognition and/or activation. This finding constitutes the first biochemical characterization of an egg-laying hormone in other invertebrates than mollusks.

The Fate of Newly Synthesized Hormone from Neuroendocrine Cells ofAplysia

The neuroendocrine bag cells fromAplysiacontrol reproductive functions through the regulated release of egg-laying hormone (ELH). The purposes of this study were to investigate (1) if synthesis of ELH in bag cells compensates for the release and degradation of the hormone, (2) the ratio of released to stored ELH, (3) the fate of newly synthesized ELH and its relative contribution to the overall pattern of ELH secretion, and (4) the consequences of blocking protein synthesis in bag cells on the pattern of ELH secretion and subsequent reproductive function. Excised bag cells were incubated with [3H]leucine to radiolabel newly synthesized ELH and were then electrically stimulated to trigger secretion of the hormone on 4 successive days, mimicking bag cell activities during the breeding season. Samples of bag cell secretion were collected; total (new and old) and radiolabeled (new) ELH levels were determined. The results showed that ELH synthesis could not keep up with the daily loss of ELH through release and degradation, but the ratio of released to stored ELH remained constant over the 4-day study. In addition, releasable ELH was secreted preferentially within 24 hr after it was synthesized. Blocking protein synthesis resulted in a 50% decrease in total ELH released, suggesting that newly synthesized ELH contributes to half of the total ELH secreted. Furthermore, this lowered level of total ELH, i.e., older ELH alone, was not sufficient to stimulate egg laying inAplysia,indicating that the contribution of newly synthesized ELH was physiologically relevant.

The rhinophores sense pheromones regulating multiple behaviors in Aplysia fasciata

Pheromones released during mating and egg laying in Aplysia facilitate various aspects of behavior. We now show that the chemosensory rhinophores sense these pheromones. Ablating the rhinophores causes a significant decrease in the time spent mating. In addition, the lesion blocks the increases of feeding in response to pheromones released by egg cordons and by mating conspecifics. Respiratory pumping is significantly increased in response to egg cordons, mating conspecifics and egg laying hormone (ELH). The increase in response to egg cordons is blocked by ablating the rhinophores, but not by lesioning the osphradium, a second chemosensory organ.

Identification of a Vesicular Pool of Calcium Channels in the Bag Cell Neurons ofAplysia californica

To study the molecular mechanism of calcium current modulation in the bag cell neurons of Aplysia californica, we have identified calcium channel subtypes expressed in these cells and analyzed their distribution using channel-specific antibodies. Using PCR to amplify reverse-transcribed RNA from bag cell clusters, we identified two classes of calcium channel alpha1 subunit. One, BCCa-I, belongs to the ABE subfamily of calcium channels, whereas the other, BCCa-II, belongs to the SCD subfamily. Antibodies generated against the bag cell calcium channels recognize membrane proteins of approximately 210 and 280 kDa on immunoblots. Both channels are expressed in the bag cell clusters as well as in other parts of the Aplysia nervous system. BCCa-II also localizes to glia and muscle. The subcellular distribution of the two channel types is strikingly different. Antibody staining of bag cell neurons in primary culture shows that BCCa-II is present on the plasma membrane, whereas BCCa-I has a punctate, intracellular distribution consistent with a vesicular localization. The BCCa-I-containing vesicles are found in bag cell neuron somata and growth cones and occasionally in neuritic hotspots. Their distribution is similar but not identical to that of LysoTracker Red, a marker for acidic organelles, but unlike that of dense-core vesicles containing egg-laying hormone. The vesicular channels may represent the protein kinase C-sensitive calcium channels of bag cell neurons that are believed to enhance hormonal release during electrical activity.

Expression and genetic variation of theAplysia egg-laying hormone gene family in the atrial gland

We have screened an Aplysia atrial gland cDNA library using an egg-laying hormone (ELH) precursor probe and have isolated and characterized five different clones, four of which are full-length and approximately 0.8 kb in size. The characterization of these cDNA clones firmly established the genetic variation of the ELH-related precursors expressed in the atrial gland and provided a rational basis for their revised nomenclature proposed herein. The five precursor ELH-related cDNA sequences obtained predicted the following genetically distinct polypeptide precursors designated as: A, [Asp143]A, [Glu94,Gln139]A, [Pro25]B, and [Phe96,Asp107]BT. The [Phe96,Asp107]Br cDNA sequence predicted a truncated form of a B-type precursor. Northern blot analysis of atrial gland RNA identified two transcripts of about equal intensity of 0.9 kb and 1.1 kb. Polymerase chain reaction of genomic DNA, together with DNA sequence analysis, resolved previously reported discrepancies between genomic and cDNA sequences of the ELH-related precursors. Taken together the results obtained identified the expression of five ELH-related precursor genes in the atrial gland of Aplysia from at least two genetic loci per haploid genome.

Gene expression and regulation of Aplysia californica prohormone convertases aPC1B and aPC2.

Peptide diversity can be regulated in many ways. One of the possible mechanisms of such a regulation may lie in the differential processing of a precursor in a tissue-specific manner or in a modulated processing within a single cell. To address these questions the enzymes responsible for the release of active neuropeptides first need to be well characterized. In this study, we have established the cellular distributions of the recently cloned prohormone convertases aPC2 and aPC1B in Aplysia californica and found the distribution of these enzymes to be mainly neuronal and endocrine. We then investigated in two sets of neurons (bag cell neurons and sensory neurons) the possible regulation of these two prohormone convertases by a neurotransmitter known to modulate behavior in Aplysia. In these neurons, we found that only aPC2 is regulated by serotonin possibly via the cyclic AMP cascade. Finally, a study of the biosynthesis of the bag cell egg-laying hormone precursor after treatment with serotonin suggests that such a treatment may increase its processing.

Cell Type-Specific Sorting of Neuropeptides: A Mechanism to Modulate Peptide Composition of Large Dense-Core Vesicles

The CNS of Lymnaea stagnalis contains two populations of egg-laying hormone (ELH)-producing neurons that differ in size and topology. In type I neurons, all peptides located C-terminally from the cleavage site Arg-Ser-Arg-Arg180-183 are sorted into secretory large dense-core vesicles (LDCV), whereas N-terminal-located peptides accumulate in a distinct type of vesicle, the large electrondense granule (LEG). Via immunoelectron microscopy, we now show that the second population of ELH-producing neurons, type II neurons, lack LEG and incorporate all proELH-derived peptides into LDCV. This finding provides the first example of a cell type-specific sorting of neuropeptides into LDCV. Furthermore, we provide evidence that LEG are formed through a differential condensation process in the trans-Golgi network and that these bodies are ultimately degraded. Analysis of the endoprotease composition of the two types of proELH-producing neurons suggests that the formation of LEG, and consequently the retention of N-terminal peptides from the secretory pathway, requires the action of a furin-like protein.

Developmental dissociation of excitability and secretory ability in Aplysia bag cell neurons.

1. Despite the considerable progress made in understanding the role of electrical activity in triggering secretion, the developmental relationships between excitability and secretion are not well understood. The well-characterized bag cell neurons of Aplysia provide an advantageous system in which to investigate developmental interactions of these two key properties of neurons. 2. A prolonged afterdischarge triggers egg laying hormone (ELH) secretion in mature bag cell neurons. To investigate secretion in the developmental framework of excitability, we first examined whether immature neurons, which are incapable of the mature form of excitability (afterdischarge), contain ELH and whether this hormone is packaged in vesicles. We used immunoelectron microscopy to compare vesicular localization of ELH and to compare the size and density of ELH-containing vesicles in neurons from adult and juvenile Aplysia. This comparison revealed that immature neurons contain ELH in vesicles in the size range of secretory vesicles. However, they lack a class of large vesicles (> 250 nm in diameter) that is characteristic of mature neurons. 3. To investigate whether the ELH contained in immature bag cell neurons could be secreted in response to electrical activity, we used the potassium channel blocker tetraethylammonium (TEA) combined with nerve stimulation to depolarize neurons from both juvenile animals (ovotestes do not contain eggs) and from adult Aplysia (ovotestes contain eggs). Using radioimmunoassay, we have found that the duration and amount of ELH secreted from bag cell neurons from juvenile Aplysia in response to TEA does not depend on whether or not the cells can be induced to afterdischarge, and the amount and duration of ELH secreted from bag cell neurons of juvenile Aplysia (whether or not they afterdischarged) differed from those secreted by adult neurons. However, by normalizing for body size, we found that the final estimated hemolymph concentration of ELH would be similar in juvenile and adult animals. 4. We investigated the potential functional significance of secretion of bag cell hormones in juvenile Aplysia by attempting to bypass the bag cell neurons and directly activate downstream elements with extract from adult bag cell neurons (BCE), known to contain ELH and other peptides. We found that juvenile Aplysia exhibit at least one component of egg-laying behavior, cessation of locomotion, in response to BCE during a developmental period (as measured by weight) in which they normally would possess neurons incapable of afterdischarge. Thus developmental regulation of excitability in the bag cell neurons may prevent inappropriate hormone release and subsequent premature expression of reproductive behaviors.