Neuroendocrine Light Green Cells Research Articles

Comparison of cytotoxic effects of chemotherapeutics against human leukemia and granulosa cells

The formation of neurites in isolated neurones of the snail Lymnaea stagnalis in primary culture was studied. The insulin-related neuropeptide (MIP: Molluscan insulin-related peptide) produced by the neuroendocrine light green cells (LGCs) of Lymnaea stimulated neurite formation, both in isolated unidentified central neurons and in the LGCs. The effect of MIP was dose dependent. It was significant from the second day of culture and amounted up to an 8-fold increase in neurite outgrowth after 3 days. The results add a functional aspect to the evolutionary relationship of MIP with mammalian insulin and insulin-related peptides and suggest that the LGCs, which stimulate growth, are also involved in development of the nervous system.

Biosynthesis and axonal transport of multiple molluscan insulin-related peptides by the neuroendocrine light green cells of Lymnaea stagnalis

The neuroendocrine light green cells (LGCs) control body growth and metabolism of the freshwater snail, Lymnaea stagnalis. These cells, located in the cerebral ganglia, use the peripheries of the median lip nerves (MLNs) as the neurohemal area. The LGCs express four members of a gene family encoding the precursors of distinct though related molluscan insulin-related peptides (MIPs). The molecular characterization and biosynthesis of MIPs have been investigated. MLN extracts were first size fractionated using gel permeation chromatography, and a major peak that coeluted with bovine insulin was further resolved using reverse phase high performance liquid chromatography. Amino acid composition analysis revealed that the four MIPs were present, as encoded by the MIP genes. Biosynthesis and axonal transport of the MIPs were studied in vitro with LGC systems in a series of pulse-chase incubations in combination with gel permeation chromatography analysis. During the pulse period of 20 min MIP precursors were formed in the LGC cell bodies. During the subsequent chase period these precursors were converted to MIPs and C peptides, which were transported to the LGC axon terminals in the MLNs for storage and release. The MIP precursors seem to be processed similarly to the insulin precursor in pancreatic islets.

Differential expression of four genes encoding molluscan insulin-related peptides in the central nervous system of the pond snail Lymnaea stagnalis

In the pond snail Lymnaea stagnalis, the growth regulating system consists of (1) about 200 neuroendocrine light green cells, located in four clusters in the cerebral ganglia, and (2) the paired canopy cells, located in the lateral lobes. These cells express genes encoding the molluscan insulin-related peptides (MIPs). Six MIP genes have previously been identified. Four of these (I, II, III and V) are expressed in the light green cells and the canopy cells. The MIP-VI gene is a pseudogene. In the present in situ hybridization study, using oligonucleotide probes specific to the transcripts of MIP-I, -II, -III, -IV and -V, no signal was obtained with the MIP-IV probe, indicating that gene IV is also a pseudogene. With the other four probes, two types of light green cells were distinguished. Type-A cells express all four MIP genes, whereas type-B cells do not (or only faintly) express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Type-A cells are mainly located in the periphery of the clusters, whereas type-B cells are present in the center. The canopy cell resembles type-A light green cells. The expression levels of the MIP-II and MIP-V genes are low in the canopy cell. The expression pattern of the MIP genes correlates with the staining pattern of the anti-MIP-C antibody, which has been raised to a synthetic C-fragment shared by MIP-I, -II and -V. Type-A cells stain more intensely with the antibody than type-B cells.

Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis.

The growth-controlling neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express a family of genes encoding structurally related, yet distinct, molluscan insulin-related peptides (MIPs). In the present study one of these peptides, MIP II, has been isolated and structurally identified. MIP II is a heterodimer of A and B chains connected by disulfide bonds. Both chains are N-terminally blocked with pyroglutamate. After cleaving of the A and B chains and deblocking with pyroglutamate amino-peptidase their sequences have been determined as: A chain: pQRTTNLVCECCFNYCTPDVVRKYCY and B chain: pQSSCSLSSRPHPRGICGSNLAGFRAFICSNQNSPS. In comparison with the MIP II sequence based on complementary DNA studies, it is clear that the two C-terminal amino acid residues of the B chain are posttranslationally removed. In addition, the glutamic acid residue in A chain was recovered in very low yields during Edman degradation, suggesting that the residue may be posttranslationally modified.

Neurohormonal control of growth and carbohydrate metabolism by the light green cells in Lymnaea stagnalis

The neuroendocrine light green cells (LGCs), 4 clusters of together ∼150 giant neurons in the cerebral ganglia of the freshwater gastropod, Lymnaea stagnalis, have been suggested to be involved in the control of growth. The present study examines in greater detail this role and possible actions on energy metabolism. Growth indices (total body and organ wet and dry weights, as well as protein and DNA contents of the organs) and metabolic indices (tissue lipid, polysaccharide and glucose levels) were compared in LGC extirpated/reimplanted with control groups. LGC extirpation in rapidly growing juvenile snails immediately arrested growth, which was restored by reimplantation of cerebral ganglia with LGCs but not by cerebral ganglia without LGCs, indicating their neuroendocrine control of growth. The LGCs stimulate a pattern of organ growth, which is in proportion to the growth of the whole body except for the shell, which shows a disproportionally faster growth due to calcium deposition over the whole surface. The data on protein and DNA in the organs strongly suggest that the LGCs induce growth by stimulating cell multiplication. The LGCs maintain low tissue glycogen reserves and hemolymph concentrations of both glycogen and glucose. The secretions of these cells stimulate the uptake of glucose by the growing tissues with no apparent effects on lipid metabolism.

Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis

Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis

Characterization of a cDNA clone encoding molluscan insulin-related peptide V of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide V (MIP V) was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnae stagnalis, using a heterologous screening with a previously identified MIP II cDNA. The MIP V cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chai, however, in this case connected by two distinct C peptides, Cα and Cβ, instead of one single C peptide. This phenomenon, which is shared by the MIP II precursor, represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIPs V, I and II, differ remarkably in primary structure; in contrast, the Cα peptide domains are almost identical. MIP V has only limited sequence similarity with insulins and related peptides. Both MIP V and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I, II and V genes are expressed in a single type of neuron: the growth controlling neuroendocrine light green cells of the Lymnaea CNS.

Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C α peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNlVCECCMKPCTLSELRQYCP B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C α peptide has also been isolated and its sequence was determined as NAETDLDDPLRN1KLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.

The Light Green Cells of Lymnaea: a neuroendocrine model system for stimulus-induced expression of multiple peptide genes in a single cell type.

We review recent experiments showing that the cerebral neuroendocrine Light Green Cells (LGCs) of the freshwater snail, Lymnaea stagnalis, express a family of distinct though related molluscan insulin-related peptide (MIP) genes. The LGCs are involved in the regulation of a wide range of interrelated life processes associated with growth, (energy) metabolism and reproduction. We consider the mechanism of generation of diversity among MIPs, and present evidence that conditions with distinct effects on growth, metabolism and reproduction also can induce distinct patterns of expression of the MIP and schistosomin genes. The stimulus-dependent expression of multiple neuropeptide genes enormously increases the adaptive potential of a peptidergic neuron. We suggest that this contributes significantly to the information-handling capacity of the brain.

Characterization of a cDNA clone encoding molluscan insulin‐related peptide II of Lymnaea stagnalis

A cDNA clone encoding molluscan insulin-related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, C alpha and C beta, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the C alpha peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS.

Molluscan insulin-related neuropeptide promotes neurite outgrowth in dissociated neuronal cell cultures

The formation of neurites in isolated neurones of the snail Lymnaea stagnalis in primary culture was studied. The insulin-related neuropeptide (MIP: Molluscan insulin-related peptide) produced by the neuroendocrine light green cells (LGCs) of Lymnaea stimulated neurite formation, both in isolated unidentified central neurons and in the LGCs. The effect of MIP was dose dependent. It was significant from the second day of culture and amounted up to an 8-fold increase in neurite outgrowth after 3 days. The results add a functional aspect to the evolutionary relationship of MIP with mammalian insulin and insulin-related peptides and suggest that the LGCs, which stimulate growth, are also involved in development of the nervous system.

Ultrastructural evidence for synthesis, storage and release of insulin-related peptides in the central nervous system of Lymnaea stagnalis

The cerebral neuroendocrine Light Green Cells of the pulmonate snail Lymnaea stagnate, which control body growth and associated processes, stain positively with an affinity-purified antiserum raised to a large part of the C-chain of pro-molluscan insulin-related peptides. At the ultrastructural level, the rough endoplasmic reticulum is immunonegative, the Golgi apparatus is slightly positive and secretory granules in the process of budding from the Golgi apparatus are strongly positive. These observations indicate that the Light Green Cells synthesize molluscan insulin-related peptides, which are processed before packing by the Golgi apparatus into secretory granules. The two morphologically distinct secretory granule types, i.e. with pale and dark contents, respectively, are equally immunoreactive with antiserum raised to the C-chain of molluscan insulin-related peptides. Secretory granules within lysosomal structures reveal various degrees of immunoreactivity, indicating their graded breakdown. The Light Green Cells release secretory material by the process of exocytosis into the haemolymph from neurohaemal axon terminals located in the periphery of the median lip nerve. The electron-dense (tannic acid method) released contents are clearly immunopositive. The same holds for secretory granule contents released from Light Green Cells axon profiles in the centre of the lip nerve. Some immunoreactivity is also present in the intercellular space between these axon profiles. It is concluded that molluscan insulin-related peptides may act in two ways, namely (1) as neurohormones via the haemolymph at peripheral targets (2) in a non-synaptic (paracrine) fashion at targets within the central nervous system.

Sensory input to growth stimulating neuroendocrine cells of Lymnaea stagnalis.

Several environmental factors influence the growth of the basommatophoran freshwater snail Lymnaea stagnalis. Growth is hormonally controlled by 4 cerebral clusters of ca 50-75 peptidergic, neuroendocrine Light Green Cells (LGC). The present light, transmission, and scanning electron microscopic study shows that the LGC are synaptically contacted by a tentacle sensory system (TSS). The TSS consists of 2 types of primary sensory neurone, viz. ca 150 S1-cells and ca 50-100 S2-cells. A S1-cell has a non-ciliated dendrite and an axon branch that synaptically contacts the soma of a S2-cell. A S2-cell has a branching, ciliated dendrite. Probably, S1- and S2-cells have different sensory modalities and can integrate sensory information by intersensory interaction. The S2-axons run through the tentacular nerves, the cerebral ganglia, and the intercerebral commissure. In each ganglion S2-axons branch and form synaptic contacts on the axons and somata of the LGC and on glial cells that surround the LGC. In an LGC-cluster, 1-3 LGC-somata are particularly strongly innervated. Probably, the TSS is involved in the environmental control of growth in L. stagnalis.

A hormone dependent calcium-binding protein in the mantle edge of the freshwater snail Lymnaea stagnalis.

By means of gel filtration and ion exchange chromatography on calcium-saturated Chelex-100, a calcium-binding fraction was isolated from the mantle edge of the freshwater snail lymnaea stagnalis. This fraction was not present in other tissues. Treatment with trypsin caused a disappearance of the calcium-binding capacity, proving that the active substance in this fraction is a protein (calcium-binding protein; CaBP). Removal of the growth hormone-producing neuroendocrine light green cells resulted in a strong decrease of the amount of CaBP. It is concluded that L. stagnalis possesses a hormone-dependent CaBP, probably responsible for the maintenance of a high calcium concentration in that part of the mantle that produces the outer crystalline layer of the shell.