Neuropeptides: mediators of behavior in Aplysia.

The Aplysia neuroendocrine system is a particularly advantageous model for cellular and molecular studies because of the relatively small number and large size of its component neurons. Recombinant DNA techniques have been used to isolate the genes that encode the precursors of peptides expressed in identified neurons of known function. The organization and developmental expression of these genes have been examined in detail. Several of the genes encode precursors of multiple biologically active peptides that are expressed in cells which also contain classical transmitters. These studies, as well as immunohistochemical studies and the use of intracellular recording and voltage clamp techniques are the first steps toward revealing the mechanisms by which neuropeptides govern simple behaviors.

Expression of the L11 neuropeptide gene in the Aplysia central nervous system.

Neuron L11 in the abdominal ganglion of Aplysia californica is thought to be both cholinergic and peptidergic. In previous studies, we isolated a cDNA clone encoding the precursor for an L11 secreted protein(s) by differentially screening an abdominal ganglion cDNA library. We now report the isolation of genomic clones encoding the L11 cDNA sequences. Analysis of these clones reveals that the gene is present in a single copy per haploid genome. RNA blotting and cDNA cloning demonstrate that the L11 gene is expressed not only in the abdominal ganglion but in the head ganglia as well. To define the positions of cells expressing this gene and to follow their processes, we raised antibodies to synthetic peptides defined by the cDNA sequence. Histochemistry revealed about 100 neurons containing immunoreactive material. These cells arborize in the neuropil and are distributed throughout the central nervous system, representing about 0.5% of the Aplysia central neurons. In addition, cells in the abdominal ganglion send processes to the mantle floor at the base of the gill via the genital and branchial nerves. Our data suggest that this network of cells expresses the single L11 peptide gene.

Low molecular weight proteins of Aplysia neurosecretory cells.

The proteins of identified cells from the Aplysia californica central nervous system were labeled with radioactive amino acids and fractionated on SDS acrylamide gels containing 6 M urea. Most of the large cells contain prominent, cell-specific protein products in the molecular weight range between 3 and 30 KD. The molecular weights of the largest specific prevalent protein products are in good agreement with the predicted molecular weights of precursors as determined from an analysis of cDNA clones homologous to mRNA's specifically expressed in several of these neurons. Biologically active peptides have been found in many of these cells. These data, and other indirect evidence suggests that the synthesis of a large amount of a particular protein in this molecular weight range is indicative of the synthesis of a neurosecretory product. We conclude that most, if not all, large neurons in the Aplysia central nervous system are peptidergic.

Treatment of experimental autoimmune myasthenia gravis in rabbits with leupeptin, a protease inhibitor.

We injected 12 New Zealand white rabbits intraperitoneally with 15 mg/kg Leupeptin on alternative days for about 4 months. After 1 week of Leupeptin treatment, they were challenged with purified acetylcholine receptor (AChR) from Torpedo californica in Freund's complete adjuvant. All control animals died within 60 days. Six animals treated with Leupeptin did not develop EAMG in spite of repeated AChR injections. Three animals developed clinical signs of EAMG after 65 days. The clinical course was short in the one that survived and prolonged in the 2 that finally died. All animals (Leupeptin-treated and controls) had circulating anti-AChR antibodies. Among the survivors, titers were slightly lower and EMG repetitive stimulation tests were normal. Leupeptin (0.02-200 mM) did not prevent curaremimetic [3H]toxin binding to AChR in membranes or in solution, nor dissociate AChR-toxin-antibody complexes. Immune response to antigens other than receptor remained intact in Leupeptin-treated animals. Leupeptin was not toxic at the doses given. The mechanism of this protection is not well understood. Leupeptin seems to decelerate the turnover rate of AChR induced by anti-AChR antibodies and/or to decrease the complement-mediated immune attack against the muscle end-plate.

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica.

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.

Neuropeptides in identified Aplysia neurons.

Extensive electrophysiological experiments on Aplysia neurons have resulted in an understanding of simple behaviors in terms of the activities of a single identified neurons. Beginning with the work of Kupfermann & Kandel, neuropeptides in Aplysia have become increasingly implicated as chemical agents that control or affect behavior. Several neuropeptides have been isolated and characterized; recently, the genes that code for several of these neuropeptides have been isolated. Studies of neuropeptide gene expression and the behaviors affected thereby have been bridged in the egg-laying hormone neuroendocrine system. The role of polyproteins in coordinating complex, fixed-action patterns is beginning to emerge. The continued investigation of this neuroendocrine system, and the other cell-specific polyproteins that have been characterized more recently, promises to yield further insights into the roles of neuropeptides in governing behavior.

A cDNA clone encoding neuropeptides isolated from Aplysia neuron L11.

Single nerve cells can use more than one substance as extracellular chemical messengers. Classical transmitters have been shown to coexist in the same neuron and possibly even in the same vesicle as neuroactive peptides. Furthermore, multiple neuroactive peptides, which are thought to be coreleased, are often encoded in the same precursor assuring stoichiometric synthesis. The precise organization of multiple message systems and the physiological significance of the coexistence is poorly understood. The abdominal ganglion of the gastropod mollusc Aplysia contains a number of identified neurons that are cotransmitter candidates. One such cell, L11, is cholinergic and probably also uses biologically active peptides. Differential screening with labeled cDNA was used to isolate cDNA clones expressed specifically in the bag cells and abdominal ganglion neurons L11 or R15. Analysis of an L11-specific clone suggests that it encodes a 14.7-kDa protein that is the precursor for the secreted peptides. The poly(A)+ RNA transcript is approximately equal to 1.2 kilobases and there are 1-3 copies of this gene in the Aplysia haploid genome.

Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state.

Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.

Stimulation of lung lysyl oxidase activity in hamsters with elastase-induced emphysema.

Lysyl oxidase activity was measured in lung extracts of hamsters with elastase-induced emphysema 8 days after administration of the enzyme and again after 2, 3, and 4 wk. Levels of activity rose rapidly to 7 times the base values determined in the lungs of saline-injected control animals. In parallel with the increase in lysyl oxidase activity, the rate of 14C-lysine incorporation into desmosine and isodesmosine was at its maximum 1 wk after elastase administration, reflecting the lysyl-oxidase-mediated cross-link formation, which is the final step in the resynthesis of the pulmonary elastin destroyed by the elastolytic insult.

Proteolytic processing of a peptide precursor in Aplysia neuron R14.

The large neurons of the mollusc Aplysia are useful for studying the biogenesis of neuropeptides in single cells. Neuron R14 in the abdominal ganglion synthesizes large quantities of a 10-kDa neuropeptide precursor. The amino acid sequence of this precursor has been defined by analysis of the nucleotide sequence of a cDNA clone. We labeled proteins in vivo by microinjection of radioactive amino acids into individual R14 neurons. The labeled peptides were fractionated by high performance liquid chromatography and subjected to Edman degradation, thus enabling us to determine post-translational processing sites. Cleavage of the signal sequence was observed and at two internal sites. Cleavage at these internal sites occurs at basic amino acids and results in three products, a 2.9-, a 4.9-, and a 1.4-kDa peptide. These studies of protein processing serve as a basis for further investigations of the biogenesis and physiological activities of the neuropeptides.

Aplysia neurons express a gene encoding multiple FMRFamide neuropeptides.

The neuroactive peptide Phe-Met-Arg-Phe-NH2 (FMRF-amide) has a variety of effects on both mammalian and invertebrate tissues; moreover, FMRFamide-like immunoreactivity is found throughout the animal kingdom. Here we describe the isolation and characterization of a cDNA clone from an Aplysia abdominal ganglion cDNA library that encodes a precursor protein that may give rise to as many as 19 individual FMRFamide peptides. Nearly all of the FMRF sequences are flanked on the amino terminus by Lys-Arg residues and on the carboxy terminus by Gly-Lys residues, suggesting that the single lysine residues function to signal cleavage by processing enzymes. The gene is present in a single copy per haploid genome and gives rise to multiple transcripts, at least some of which appear to arise through alternate RNA splicing. Immunohistochemical analysis suggests that the peptide is present in many neurons throughout the Aplysia nervous system and that these neurons send processes to a variety of different tissues.

A continuous-flow, rapid-mixing, photolabeling technique applied to the acetylcholine receptor.

A continuous-flow technique is described in which a photoaffinity label, membrane rich in acetylcholine receptor, and various effectors are rapidly mixed, passed through a delay tube, through a tube in which they are irradiated, and are collected in a tube containing quencher. Delay times as short as 20 ms between mixing and photolysis are achievable. Because the flow is continuous, milliliter volumes of membrane can be labeled in a single run, which is convenient for the analysis of both the functional effects and sites of photolabeling. Using this technique, we have found that receptor in its transitory, active state, in which the channel is open, is more susceptible to photolabeling by the noncompetitive inhibitor analog [3H] quinacrine azide than is receptor in either its resting or desensitized states, in which the channel is closed. This technique should prove generally useful for the photolabeling of transient conformational states of macromolecules.

Identification of the alpha subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site.

Nicotinic acetylcholine receptors contain a readily reducible disulfide bond at the periphery of the acetylcholine binding site. Following reduction of this disulfide, the binding site is susceptible to affinity labeling by electrophilic reagents with quaternary ammonium moieties. We reduced purified receptor from Torpedo californica electric tissue and affinity alkylated it with 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The label was incorporated solely into the alpha subunit of the receptor. Isolated, labeled alpha subunit was cleaved with CNBr, and the fragments were separated by reverse-phase high-performance liquid chromatography. A uniquely labeled CNBr fragment was isolated, and its partial sequence was determined by automated Edman degradation. This CNBr fragment was cleaved at tryptophan residues, the subfragments were separated, and the labeled subfragments were partially sequenced. From our protein sequence information, we identify the labeled CNBr fragment as residues 179 to 207 of the sequence of alpha predicted from the cDNA sequence (Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982) Nature (Lond.) 299, 793-797). From the cycle of the Edman degradation in which radioactive residues are released, we conclude that Cys 192 and, possibly in addition, Cys 193 are the residues specifically labeled by 4-(N-maleimido)benzyltri[3H]methylammonium iodide. They are, therefore, close to the acetylcholine binding site.

Methods for the detection of a specific Mycobacterium leprae antigen in the urine of leprosy patients.

Two methods for detecting the phenolic glycolipid, PGL-1, a Mycobacterium leprae-specific molecule, in the urine of leprosy patients are described. Both methods rely on the 100-fold preconcentration of the urine, which can be accomplished by a single-step ultrafiltration procedure. The equivalent of approximately 2.5 micrograms of PGL-1/ml was detected in the urine of LL patients with an inhibition ELISA. The second method, a direct dot-blot assay on nitrocellulose paper, was much simpler and more sensitive. As little as 3 ng of antigen was detected by the dot-blot technique. PGL-1 was detected in the urine of LL patients.