Dissimilar associations of two secretory peptides with a neurosecretory granule-enriched fraction from the bag cells

The bag cell neurons of Aplysia californica synthesize and secrete several neuropeptides. To gain more detailed information about their posttranslational routing and transport, we have undertaken isolation of the neurosecretory granules (NSG). Extracts of radiolabeled cells were subjected to discontinuous, isosmotic density-gradient centrifugation. Radiolabeled peptides likely to be contained in NSG were found to relocate from the starting zone and to be associated with particulate structures. Assay of enzyme markers for lysosomes and endoplasmic reticulum disclosed gradient distributions that differed from that shown by the peptides. Hence, it is probable that the position of peak concentrations of particulate peptides represents the location of NSG. Of particular interest is the further observation that the known secretory peptides ELH and AP do not evidence strict covariance across the gradient. This deviation from covariance is consistent with hypotheses that the peptides are in different associations with the NSG cores or that more than one type of neurosecretory granule is produced in the bag cells.     

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.     

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.     

Precursor and product processing in the bag cell neurons of Aplysia californica

Posttranslational processing in the biosynthesis of the egg-laying hormone (ELH) by the bag cell neurons of Aplysia californica was studied. The precursor (pro-ELH) to ELH was found to be resistant to solubilization in denaturant-free media throughout its lifetime. Its principle products show a similar insolubility for 3 h, but two of these, ca. 6,000 daltons, subsequently become readily recoverable in the low-speed supernatant of a homogenate of the cells. The remaining product shows no change in solubility characteristics. From studies employing ultracentrifugation and examination of axoplasmic transport, the solubility shift for the lower molecular weight products is interpreted to represent the liberation of secretory vesicles into the cytoplasm from larger membranous associations. This event is accompanied by, but does appear to be dependent upon, a 15% reduction in the molecular weight of one of the products. These findings are considered in the light of the extensively studied posttranslational processing regimen for the production of insulin in the pancreatic beta cell.     

Anti-immunoglobulin-induced proliferation of B cells : Parallelism in the inhibition by chloroquine, monensin and immunoglobulin

Proliferation of rabbit lymphocytes was induced with goat anti-rabbit immunoglobulin. Chloroquine and monensin, known to inhibit internalization-related events, yielded inhibition of proliferation that paralleled the inhibition by a specific competitive ligand, rabbit immunoglobulin (IgG), whereas inhibition by puromycin did not. Moreover, virtually all of the cells that can be activated in freshly isolated populations adhered to anti-immunoglobulin-coated Petri plates, whereas all of the activatable population was recovered in the non-adherent fraction after a brief incubation of the cells with anti-immunoglobulin to induce internalization of surface membrane immunoglobulin. Using immunofluorescence it was further observed that monensin and Chloroquine inhibit the reappearance of surface immunoglobulins on the cell surface to some extent subsequent to their removal induced by anti-immunoglobulin.     

Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia californica

The biosynthesis and processing of low molecular weight protein (presumed neurosecretory protein) in cells R15, R14 and L11 of Aplysia californica was studied at high resolution by polyacrylamide slab gel electrophoresis in sodium dodecylsulfate. The number of low molecular weight proteins detected in each cell ranges from 3 in R14 and L11 to 5 or 6 in R15. In each of the cells studied, the low molecular weight protein consists of a primary precursor of ca. 12,000 daltons, and its proteolytic processing products. In each cell, the smallest protein, or in the case of R14, one of the two smallest proteins, accumulates to a significant extent, suggesting that it might correspond to a final processed neurohormone. In cell R15, the biosynthesis of the primary precursor and its subsequent processing to smaller peptides is largely unaffected by removal of extracellular calcium, by replacement of calcium with cobalt or by inhibition of spontaneous bursting via stimulation of the brachial nerve.     

Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia californica.

The biosynthesis and processing of low molecular weight protein (presumed neurosecretory protein) in cells R15, R14 and L11 of Aplysia californica was studied at high resolution by polyacrylamide slab gel electrophoresis in sodium dodecylsulfate. The number of low molecular weight proteins detected in each cell ranges from 3 in R14 and L11 to 5 to 6 in R15. In each of the cells studied, the low molecular weight protein consists of a primary precursor of ca. 12,000 daltons, and its proteolytic processing products. In each cell, the smallest protein, or in the case of R14, one of the two smallest proteins, accumulates to a significant extent, suggesting that it might correspond to a final processed neurohormone. In cell R15, the biosynthesis of the primary precursor and its subsequent processing to smaller peptides is largely unaffected by removal of extracellular calcium, by replacement of calcium with cobalt or by inhibition of spontaneous bursting via stimulation of the brachial nerve.     

Conduction velocity and spike duration during afterdischarge in neuroendocrine bag cells of Aplysia

Changes in conduction velocity and spike duration during electrically triggered afterdischarges were determined with extracellular recordings from bag-cell neurites of Aplysia. Spikes with high conduction velocity and short duration occurred at the onset of the afterdischarge during the period of high-frequency firing and regular interspike intervals. Later in the afterdischarge, spike frequency and conduction velocity decreased, while spike duration increased. During the short bursts within the later part of the afterdischarge, conduction velocity was highest for the first spike and decreased for successive spikes in the burst. That conduction velocity and spike frequency were both maximal during the first minute of the afterdischarge and lower during the later periods of the spike train supports the hypothesis that changes in the excitability of the bag-cell neurites occur during this firing pattern. Furthermore, the slower conduction velocity and longer duration of spikes from the bag-cell neurites late in the afterdischarge, and late in the individual bursts within the afterdischarge, suggest the hypothesis of enhanced hormone release per action potential during these periods.     

On the effects of weak bases and monensin on sorting and processing of lysosomal enzymes in human cells

The weak bases chloroquine, primaquine, NH4Cl and the ionophore monensin exert similar but not identical effects on sorting, transport and processing of cathepsin D in several human cell lines (fibroblasts, HepG2 cells, U937, monocytes). The drugs inhibit the segregation of newly synthesized cathepsin D from the secretory route. The kinetics of transport of nonsegregated cathepsin D precursor along the secretory route is retarded resulting in a delayed hypersecretion. Higher concentrations of the drugs can arrest the intracellular transport completely. The extent of inhibition of segregation varies among the different human cell types tested. Thus, in fibroblasts the secretion can be stimulated to exceed 80%, while in U937 cells the secretion cannot be enhanced above 50% although both cell types have the same basal rate of secretion (approximately 10% of the synthesized cathepsin D). We suggest that pH-independent sorting mechanisms contribute to the targeting of cathepsin D in U937 cells. Processing of the cathepsin D remaining in cells is characteristically changed depending on the drug. The proteolytic processing is strongly inhibited by chloroquine and is rather insensitive to monensin. Unlike the other drugs, monensin blocks the formation of complex oligosaccharides in cathepsin D and allows for extensive secretion solely of molecules that are sensitive to endo H.     

Development of embryonic cells containing serotonin, catecholamines, and FMRFamide-related peptides in Aplysia californica

This study demonstrates the presence of a relatively extensive but previously unrecognized nervous system in embryonic stages of the opisthobranch mollusc Aplysia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of catecholamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger, additional FMRFamide-LIR and catecholamine-containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embryonic behaviors, some elements may also guide the development of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontogeny and phylogeny of both behavioral and neuronal function in this important model species.     

The morphology and coupling of Aplysia bag cells within the abdominal ganglion and in cell culture

The bag cells in the abdominal ganglion of Aplysia californica control egg-laying behavior by releasing a polypeptide (ELH) during an afterdischarge of synchronous action potentials. We have used intracellular injection of Lucifer Yellow to study the morphology and interconnections of the bag cells. These neurosecretory cells are typically multipolar and their processes extend in all directions out from the bag cell clusters into the surrounding connective tissue, where they branch in a complex manner. In some of the dye injection experiments, dye transfer from the injected cell to neighboring cells was observed. Freeze fracture of the bag cell clusters and their surrounding connective tissue revealed numerous gap junctions on bag cell processes within the clusters as well as on more distal processes. We have also examined the morphology and coupling between bag cells in primary culture. As in the intact ganglion, bag cells in culture were found to be multipolar. All pairs of bag cells whose somata or processes had formed contacts in culture were electrically coupled. The strongest coupling was observed between pairs of cells whose somata appeared closely apposed. In these cases transfer of Lucifer Yellow between cells could also be observed. It is therefore likely that the synchrony of bag cell action potentials during a bag cell afterdischarge is a result of coupling between individual cells in the bag cell cluster.     

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. Accepted: 14 June 1997     

Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.     

Effects of ammonia,chloroquine, and monensin on the vacuolar apparatus of an absorptive epithelium

Summary The distribution of two major immunoreactive forms of somatostatin, somatostatin-14 and somatostatin-34, within the brain, pancreas and intestine of adult lampreys, Petromyzon marinus, was identified using antisera raised against these peptides. Immunostaining of the brain is similar in juveniles and upstream migrants, and somatostatin-14 is the major somatostatin form demonstrated. A few somatostatin-34-containing cells are localized within the olfactory bulbs, thalamus and hypothalamus, but cells immunoreactive to anti-somatostatin-34 in the hypothalamus and thalamus do not co-localize somatostatin-14. Immunostaining of pinealocytes within the pineal pellucida with anti-somatostatin-14 may infer a novel function for this structure. Somatostatin-14 and somatostatin-34 are co-localized within D-cells of the cranial pancreas and caudal pancreas of juveniles and upstream migrants. Numerous somatostatin-34-immunoreactive cells are distributed within the epithelial mucosa of the anterior intestine but not all of these cells cross-react with anti-somatostatin-14. It appears that somatostatin-34 is the major somatostatin in the pancreo-gastrointestinal system of adult lampreys.     

Endogenous peptides work at multiple sites in the nervous system in the control of gill behaviors in Aplysia

The suprafusion of two endogenous neuropeptides, arginine vasotocin (AVT) and small cardioactive peptide B (SCPB), over the abdominal ganglion of Aplysia californica significantly affects the ability of a central gill motor neuron to elicit a gill withdrawal response. Gill motor neurons L7 or LDG1 were depolarized to produce the same number of action potentials (APs) on each trial. When AVT (10(-6)M) was suprafused, the motor neurons' ability to elicit a gill movement was suppressed; while SCPB (10(-6)M) superfusion facilitated the response. Neither peptide altered the passive membrane properties of the motor neurons nor did they affect the duration of their APs. These results are consistent with the hypothesis that the peptides act via central control neurons which exert both suppressive and facilitatory control over gill reflex behaviors and associated neural activity.     

Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.     

Multiple effects of the phenylhydrazone derivative FCCP on the secretory pathway in rat plasma cells

We studied the sensitivity of the last steps of the secretory process of antibody-producing cells to carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and sodium azide (NaN3), agents which lower the cellular ATP content by inhibiting oxidative phosphorylation and mitochondrial electron transport, respectively. Popliteal lymph node cells or purified plasma cells from rats immunized against horseradish peroxidase were incubated with the drugs. The rate of secretion of anti-HRP antibodies was measured by an enzyme-linked immunoadsorbent assay or after biosynthetic labeling with L-[3H]fucose. FCCP as well as NaN3 were shown to rapidly inhibit (in less than 5 min) the secretion of immunoglobulins (Ig) and to partially block the release of fucosylated Ig. This indicates that the drugs inhibit the transport of Ig from the Golgi apparatus (GA) (where fucose is added to Ig) to the plasma membrane. However, the degree of inhibition reached 40 to 50% with NaN3 and 70 to 80% with FCCP, whereas both drugs similarly depleted ATP stores by 45 to 55%. These results are consistent with multiple effects of FCCP on the secretion pathway of Ig. As a tentative explanation, we suggest that FCCP, because of its protonophore properties, not only reduces cellular ATP levels but may also neutralize the Golgi or post-Golgi acidic compartments recently shown to be involved in the transport of plasma membrane and secretory proteins.     

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[3H]Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM. 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.     

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly,Protophormia terraenovae

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum–hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg⁷]-corazonin and [Arg⁷]-corazonin^3–11) in the PL, 13 peptides (including myosuppressin, [Arg⁷]-corazonin and [Arg⁷]-corazonin^3–11) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 °C) and diapause-averting conditions (LD 18:6 at 25 °C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd–10th amino acids were sequenced as –YRKPPFNGS–, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.