Myotropic effect of helicokinins, tachykinin-related peptides and Manduca sexta allatotropin on the gut of Heliothis virescens (Lepidoptera: Noctuidae).

Different insect neuropeptides (helicokinins, tachykinin-related and allatoregulating peptides) were investigated with regard to their myostimulatory effects using whole-gut preparations isolated from fifth instar Heliothis virescens larvae. The experiments demonstrated that representatives of all three peptide families are able to induce and amplify gut contractions in this species in a dose-dependent manner. Structure-activity studies (alanine scan, D-amino acid scan and truncated analogues) with the helicokinin Hez-K1 supported the finding, that the core sequence for biological activity of kinins is the amidated C-terminal pentapeptide (FSPWG-amide). Similar investigations with insect tachykinin isolated from Leucophaea madera (Lem-TRP1) revealed that the minimum sequence evoking a physiological gut response in H. virescens is the amidated hexapeptide (GFLGVR-amide), which represents the conserved amino acid sequence for Leucophaea TRPs in general. The peptide concentration causing a half-maximal gut contraction (EC(50)) for Lem-TRP1 was about 26 nM. Although the potency of Lem-TRP1 was 9-fold lower compared with Hez-KI (EC(50): 3 nM), the maximal tension of the gut obtained with Lem-TRP1 was 1.7-fold higher compared with Hez-KI. The EC(50) of Manduca sexta allatotropin (Mas-AT; 79 nM) was of lowest potency among all three peptides tested. In a pharmacological study, co-incubation experiments with Lem-TRP1, Hez-KI or Mas-AT and compounds interfering with signal transduction pathways were employed to investigate the mode of action of the myotropic effects of these peptides. Cadmium and the protein kinase C (PKC) inhibitor tamoxifen attenuated the contractile effects of all three peptides tested. The data suggest that in the gut muscle of H. virescens the myotropic peptides bind to G-protein-coupled receptors that cause contraction by promoting the entry of extracellular calcium mediated by a PKC involved pathway.

BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity.

L-Glutamate (Glu) activates at least eight different G protein-coupled receptors known as metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular domain in which agonists bind and a transmembrane heptahelix region involved in G protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound, BAY36-7620 [(3aS,6aS)- 6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on], on mGlu receptors (mGlu1-8), transiently expressed in human embryonic kidney 293 cells. BAY36-7620 is a potent (IC(50) = 0.16 microM) and selective antagonist at mGlu1 receptors and inhibits >60% of mGlu1a receptor constitutive activity (IC(50) = 0.38 microM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [(3)H]quisqualate binding from the Glu-binding pocket, further indicating that BAY36-7620 is a noncompetitive mGlu1 antagonist. Studies of chimeric receptors containing regions of mGlu1 and regions of DmGluA, mGlu2, or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4 to 7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity.

Novel pluripotential neural progenitor lines exhibiting rapid controlled differentiation to neurotransmitter receptor-expressing neurons and glia.

The immortalization of progenitor cells from embryonic murine hippocampus using oncogene-carrying retroviral vectors is described. Use of a vector encoding the oncogene v-myc results in lines of nestin-positive progenitor cells. Limited differentiation ensues if the cells are cultured in the presence of dibutyryl cyclic adenosine monophosphate. In contrast, use of a vector in which the extracellular portion of the epidermal growth factor (EGF) receptor is fused to the neu tyrosine kinase generates lines of pluripotential nestin-positive progenitor cells, which differentiate upon withdrawal of EGF into neurons and glia. Differentiated neurons expressing action potentials and neurotransmitter receptors make up a high proportion of the cells. These cell lines are useful tools to investigate the characteristics of differentiating neurons and glia, as well as to screen neuroactive drugs. This work has been reported in preliminary form as an abstract (1996 Society for Neuroscience Abstract, #606.20, p. 1537).

Effects of the selective metabotropic glutamate receptor agonist, L-CCG-I, on acquisition of a Morris task by rats.

L-Glutamate, a major excitatory neurotransmitter in the central nervous system, plays an important role in a variety of neuronal events associated with learning and memory, neuronal plasticity, neurotoxicity, and neurodegeneration. We assessed the effects of L-CCG-I ((2S,3S,4S)-alpha-(carboxycyclopropyl)glycine), a conformationally restricted glutamate analogue, in a standard Morris water escape task with young adult rats. L-CCG-I is considered to be a selective agonist of the metabotropic glutamate receptor. Vehicle, 5, 50, or 500 nmol L-CCG-I was injected intra-cerebroventricularly (i.c.v.) into the right lateral ventricle 30 min before the start of each of five daily acquisition sessions. The data indicate that L-CCG-I had a centrally mediated mode of action; rats treated with 500 nmol L-CCG-I were clearly impaired in acquiring the standard Morris water escape task. The no-effect dose was 5 nmol.

Retention of J1/tenascin and the polysialylated form of the neural cell adhesion molecule (N-CAM) in the adult olfactory bulb.

To gain insight into the cellular and molecular mechanisms underlying neurogenesis in adult mouse olfactory bulb, several adhesion molecules expressed by glial cells and neurons were investigated. In the germinal zone of the olfactory bulb, the subependymal layer of the rostral region of the lateral ventricles, two adhesion molecules are detectable that are characteristic of early morphogenetic events: J1/tenascin and the polysialylated form, the so-called embryonic form, of N-CAM. The polysialylated form of N-CAM is expressed by most cells in the subependymal layer, and by some astrocytes and neurons in the granular layer adjacent to the subependymal layer. This suggests that bipotential precursor cells retain expression of the embryonic form during their migration from the subependymal layer and during the first stages of differentiation into neurons and glia. Expression of the polysialylated form of N-CAM is also retained in monolayer cultures of six-day-old olfactory bulbs, 55 days after seeding in vitro. J1/tenascin was detectable in the subependymal layer using two monoclonal antibodies. The immunostaining pattern was different between the two antibodies and more restricted to the subependymal layer than when staining with polyclonal J1 antibodies was performed, indicating that J1/tenascin exists in distinct isoforms. Finally, our observations suggest that, in the adult olfactory bulb, L1 is not only a neuron-neuron adhesion molecule, but it may also be involved in neuron-glia interactions, since it is found at contact sites between these two cell types. L1, therefore, may be a neuron-glia adhesion molecule in some parts of the CNS, while it is not in others.

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase.

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.

Identification of a cDNA clone specific for the neural cell adhesion molecule AMOG.

A cDNA clone of the neural cell adhesion molecule AMOG was isolated from a lambda gt10 library constructed from 8-day-old mouse brain poly(A) + RNA with a 17mer oligonucleotide probe designed from a nonapeptide sequence obtained from tryptic peptides of AMOG. The cDNA clone expressed as a fusion protein that is recognized by polyclonal AMOG antibodies; conversely, polyclonal antibodies prepared against the fusion protein react with AMOG. The clone contains the full sequence derived from the nonapeptide. Of all tissues tested, only brain expresses detectable levels of AMOG by ELISA and Northern blot analyses, indicating a high correlation in expression at the protein and mRNA levels. Both brain and astrocytes express a 3 kb long mRNA, which appears to be encoded by a single gene.

Detection of the L2/HNK-1 carbohydrate epitope on glycoproteins and acidic glycolipids of the insect Calliphora vicina.

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.

The adhesion molecule on glia (AMOG) incorporated into lipid vesicles binds to subpopulations of neurons.

To investigate the functional role of the novel adhesion molecule on glia (AMOG) in cell surface interactions, immunoaffinity-purified AMOG was incorporated into liposomes and measured for its ability to bind to cells in monolayer cultures. AMOG could be incorporated into liposomes in functionally active form after solubilization from membranes in 1% cholate buffer containing soybean lecithin, elution from the AMOG monoclonal antibody column with 4 M MgCl2, containing 1% octylglucoside, and removal of detergent for liposome incorporation by gel filtration. AMOG-containing liposomes bound to neurons, but not to oligodendrocytes, astrocytes, or fibroblasts in early postnatal cerebellar cultures. AMOG-containing liposomes also bound to the pheochromocytoma cell line PC12, but not to neurons in cultures of spinal cord and dorsal root ganglia after various times in vitro. Fab fragments of monoclonal AMOG antibodies, but not of L3 monoclonal antibodies directed against a carbohydrate structure on AMOG, inhibited binding of liposomes. Liposome binding was not reduced by preincubation of cerebellar cells with antibodies to AMOG, to the neuron adhesion molecule L1, the neural cell adhesion molecule N-CAM, or the L3 carbohydrate structure, nor with 2 monoclonal antibodies reacting with neuronal cell surface glycoproteins related to the L2/HNK-1 family. These results show that AMOG is indeed a ligand in adhesion and binds to particular subpopulations of neurons in L1- and N-CAM-independent mechanisms.

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration.

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.

Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein.

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in binding to some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipids of the PNS.

Modulation of granule cell migration by a glia-derived protein.

Cultured explants from early postnatal mouse cerebellum were used to examine the influence of a 43-kDa glia-derived neurite-promoting factor (GdNPF) on the migration of [3H]thymidine-labeled granule cell neurons. GdNPF, which is a potent serine protease inhibitor, significantly reduced the extent of granule cell migration in a dose-dependent manner. This effect could be neutralized by addition of thrombin, which binds GdNPF. Other protease inhibitors such as aprotinin, hirudin, soybean trypsin inhibitor, leupeptin, 6-aminocaproic acid, and D-Phe-Pro-ArgCH2Cl do not show this inhibitory effect. These results demonstrate that a glia-derived protein can regulate the migration of postmitotic neurons, an important cellular event in the development of the nervous system.