PubMed Health⌕ Search

Biomedical subjects

K J Angelides

Publications and source records attributed to K J Angelides.

At least 37 records · Page 2Linked to original sources
PubMed1991

Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin.

Tetramethylrhodamine-conjugated omega-conotoxin was used as a fluorescent stain (Jones et al., 1989) to determine the spatial distribution of voltage-gated Ca2+ channels along frog motor nerve terminals. Like native omega-conotoxin, the fluorescent toxin blocked neuromuscular transmission irreversibly. The fluorescent staining was confined to the neuromuscular junction and consisted of a series of narrow bands (in face views) or dots (in side views) approximately 1 micron apart. This characteristic staining pattern was prevented by pretreatment with omega-conotoxin and by prior denervation for 5-7 d. Combined fluorescence and phase-contrast optics indicated that the stain was on the synaptic rather than the nonsynaptic side of the nerve terminal. The bands and dots of stain proved to be in spatial register with the postsynaptic junctional folds, as revealed by combined staining of ACh receptors. It is concluded that the voltage-gated Ca2+ channels on frog motor nerve terminals are concentrated at active zones. The findings are consistent with the suggestion (Heuser et al., 1974; Pumplin et al., 1981) that the large intramembraneous particles seen at freeze-fractured active zones are voltage-gated Ca2+ channels.

Animals↗
PubMed1990

The voltage-dependent sodium channel in mammalian CNS and PNS: antibody characterization and immunocytochemical localization.

Monoclonal and polyclonal antibodies were generated against the voltage-dependent sodium channel purified from rat brain, and were used to characterize and localize sodium channels within mammalian central nervous system (CNS) and peripheral nervous system (PNS). These antibodies immunoblot and immunoprecipitate from labeled membrane proteins a 260-kDa polypeptide, as well as immunoprecipitate sodium channels saturated with [3H]saxitoxin. These monoclonal and polyclonal antibodies do not, however, recognize sodium channels in cardiac or skeletal muscle. Immunocytochemical analyses of cultured CNS and PNS neurons and immuno-ultrastructural localization of sodium channel reactivity within CNS tissue in situ indicate that these probes provide a unique tool for studying the level of expression, organization and turnover of sodium channels within the CNS and PNS.

Action Potentials↗
PubMed1990

Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro.

The effect of phosphorylation by cyclic AMP dependent protein kinase on the assembly of the core-forming 68 KDa neurofilament subunit protein (NF-L) was studied in vitro by fluorescence energy transfer and electron microscopy. Phosphorylation of unassembled NF-L in a low ionic strength buffer by cyclic AMP dependent protein kinase led to the incorporation of 1-2 phosphate groups/mole protein. Assembly of this phosphorylated NF-L was inhibited significantly; compared to non-phosphorylated NF-L, the critical concentration of phosphorylated NF-L was raised by greater than 30-fold. Assembled NF-L filaments could also be phosphorylated by cyclic AMP dependent protein kinase indicating that the sites were accessible. Phosphorylation of NF-L in the filamentous state induced their disassembly. The results suggest that phosphorylation by cyclic AMP dependent protein kinase is a possible means to modulate the assembly state of NF-L.

Animals↗
PubMed1990

Sodium channel expression detected with antibody 7493 in A2B5+ and A2B5- astrocytes from rat optic nerve in vitro.

Astrocytes cultured from neonatal rat optic nerve can be classified into two subtypes, distinguished by their morphology (stellate or fibroblast-like) and their ability to bind monoclonal antibody A2B5. The presence of sodium channels in astrocytes cultured from rat optic nerve was demonstrated by indirect immunofluorescence with polyclonal antibody 7493, which is directed against purified rat brain sodium channel protein. Astrocytes cultured from postnatal day 7 (P7) rat optic nerves exhibited sodium channel immunostaining on both A2B5+ and A2B5- astrocytes up to 6 days in vitro (DIV). Staining was distributed throughout the cytoplasm and cell processes, with areas of greater intensity in the perinuclear region. At 6 DIV, the A2B5-/GFAP+ cells exhibited a loss of sodium channel immunostaining, while the A2B5+/GFAP+ cells continued to display 7493 immunoreactivity. This sodium channel staining pattern persisted for up to 28 DIV (the longest time point examined). Astrocyte cultures derived from PO rat optic nerves exhibited sodium channel immunoreactivity during the first 6 DIV. The P0 astrocyte cultures, in which, the vast majority of cells are A2B5-/GFAP+, displayed a similar staining pattern to those astrocytes with corresponding phenotype derived from P7 optic nerves. P0-derived A2B5- astrocytes showed loss of 7493 immunostaining at 6 DIV, while the rare (less than 1% of cells) A2B5+/GFAP+ cells continued to express sodium channels reactive to 7493. The reduction of sodium channel immunoreactivity in A2B5- but not A2B5+, astrocytes from both P0 and P7 optic nerves after a similar latency (approximately 6 DIV) suggests that the loss of immunostaining may result from the absence of neuronal associations in the culture environment, rather than an intrinsic biologically timed change in astrocytic expression of sodium channels.

Animals↗
PubMed1990

Sodium channels in the cytoplasm of Schwann cells.

Immunoblotting, ultrastructural immunocytochemistry, and tritiated saxitoxin ([3H]STX) binding experiments were used to study sodium channel localization in Schwann cells. Polyclonal antibody 7493, which is directed against purified sodium channels from rat brain, specifically recognizes a 260-kDa protein corresponding to the alpha subunit of the sodium channel in immunoblots of crude glycoproteins from rat sciatic nerve. Electron microscopic localization of sodium channel immunoreactivity within adult rat sciatic nerves reveals heavy staining of the axon membrane at the node of Ranvier, in contrast to the internodal axon membrane, which does not stain. Schwann cells including perinodal processes also exhibit antibody 7493 immunoreactivity, localized within both the cytoplasm and the plasmalemma of the Schwann cell. To examine further the possibility that sodium channels are localized within Schwann cell cytoplasm, [3H]STX binding was studied in cultured rabbit Schwann cells, both intact and after homogenization. Saturable binding of STX was significantly higher in homogenized Schwann cells (410 +/- 37 fmol/mg of protein) than in intact Schwann cells (214 +/- 21 fmol/mg of protein). Moreover, the equilibrium dissociation constant was higher for homogenized preparations (1.77 +/- 0.37 nM) than for intact Schwann cells (1.06 +/- 0.29 nM). These data suggest the presence of an intracellular pool of sodium channels or channel precursors in Schwann cells.

Animals↗
PubMed1990

Distribution and lateral mobility of glycine receptors on cultured spinal cord neurons.

Strychnine is one of the most potent antagonists of glycine-mediated inhibitory conductances in the mammalian spinal cord. In order to examine the distribution of glycine receptors (GlyRs) on neuronal cells, 2 novel fluorescent strychnine derivatives were synthesized and characterized chemically, spectroscopically, and biologically. Both compounds retain their biological activity after derivatization and are potent inhibitors of 3H-strychnine binding to bovine spinal cord membranes and membranes from rat spinal cord cultures. Using these fluorescent strychnine analogs, the cellular distribution and lateral mobility of GlyRs on cultured rat spinal cord neurons were studied by digital fluorescence imaging and photobleach recovery microscopy. On these neurons, even in the absence of observable synaptic contact and early in development GlyRs are predominantly localized to cell bodies with sparse labeling of neuritic processes. Although GlyRs are confined to the neuronal cell body, approximately 50% of the receptors are very mobile, with lateral diffusion coefficients of 1.15 +/- 0.05 x 10(-9) cm2/sec, a value which is characteristic of unrestricted protein lateral diffusion. However, the remaining fraction of these receptors are immobile on the neuronal cell body. More than 70% of the GlyRs distributed on neuronal processes are immobile, while 30% are laterally mobile, with diffusion rates of 5.50 +/- 0.1 x 10(-10) cm2/sec. The results indicate that even early in development GlyRs are expressed and segregated to the cell body, where they are confined within a domain that restricts their redistribution.

Animals↗
PubMed1989

Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve.

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.

Animals↗
PubMed1989

Localization and mobility of omega-conotoxin-sensitive Ca2+ channels in hippocampal CA1 neurons.

Voltage-dependent Ca2+ channels (VDCCs) are modulators of synaptic plasticity, oscillatory behavior, and rhythmic firing in brain regions such as the hippocampus. The distribution and lateral mobility of VDCCs on CA1 hippocampal neurons have been determined with biologically active fluorescent and biotinylated derivatives of the selective probe omega-conotoxin in conjunction with circular dityndallism, digital fluorescence imaging, and photobleach recovery microscopy. On noninnervated cell bodies, VDCCs were found to be organized in multiple clusters, whereas after innervation the VDCCs were concentrated and immobilized at synaptic contact sites. On dendrites, VDCC distribution was punctate and was interrupted by extensive bare regions or abruptly terminated. More than 85% of the dendritic VDCCs were found to be immobile by fluorescence photobleach recovery. Thus, before synaptic contact, specific mechanisms target, segregate, and immobilize VDCCs to neuronal cell bodies and to specialized dendritic sites. Regulation of this distribution may be critical in determining the firing activity and integrative properties of hippocampal CA1 neurons.

Animals↗
PubMed1989

Sodium channels in astrocytes of rat optic nerve in situ: immuno-electron microscopic studies.

Immuno-electron microscopic localization of sodium channels within astrocyte somata and processes of adult rat optic nerve was demonstrated with polyclonal antibody 7493. In immunoblots of crude glycoproteins from adult rat optic nerve, antisera 7493, which is directed against purified rat brain sodium channels, recognizes a 260 kDa protein. Antisera 7493 intensely immunostains axon membrane at nodes of Ranvier. Associated perinodal astrocyte processes are also stained with antisera 7493. In addition, astrocyte cell bodies and major processes exhibit immunoreactivity with antibody 7493. Immunostaining with antisera 7493 is heterogeneously distributed within astrocyte cytoplasm and also appears to be associated with some regions of astrocyte plasmalemma. Glial filaments are not immunostained with 7493 antisera. Astrocyte processes forming the glial limitans and surrounding blood vessels display reduced immunoreactivity to 7493 compared to longitudinally oriented or perinodal astrocyte processes. However, some focal regions of the glial limitans exhibit robust 7493 immunostaining. Oligodendrocytes do not display 7493 antisera immunoreactivity. Optic nerve sections incubated with preimmune sera or with 7493 antisera that had been previously adsorbed with purified sodium channel protein, exhibited no immunoreactivity. These results demonstrate localization of sodium channels within astrocytes in situ of rat optic nerve and extend previous electrophysiological and pharmacological findings of sodium channels in cultured astrocytes. Possible functional roles of sodium channels within astrocytes are discussed.

Animals↗
PubMed1989

Resolution and purification of a neurofilament-specific kinase.

Both in vivo and in vitro, neurofilaments (NFs) are among the most highly phosphorylated proteins known. The majority of the NF phosphorylation sites reside on the carboxyl-terminal tails of the proteins. We have isolated and characterized an effector-independent neurofilament-specific protein kinase from bovine spinal cord that is associated with the NF complex and exhibits a marked substrate specificity for NF-H, the largest subunit of the NF triplet. This kinase activity emerges from a NF-conjugated affinity column coincident with a 67-kDa doublet on NaDodSO4/polyacrylamide gels and has a purity of greater than 90%. The purified enzyme exclusively phosphorylates NF-H tails and is dependent on prior phosphorylation of this molecule. The enzyme is also not autophosphorylated. While the molecular properties and substrate specificities of the NF kinase distinguish it from cAMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin kinase, and casein kinases I and II, it exhibits certain properties similar to, but different from, the growth-associated histone H1 kinase. The molecular properties and specific sequence requirements of the NF kinase suggest that this enzyme could play a pivotal role in the phosphorylation of NFs in normal and pathological states such as Alzheimer disease, where NFs are hyperphosphorylated.

Animals↗
PubMed1989

Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.

We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarin-labeled NF-L as fluorescence donor and fluorescein-labeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCl concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38 micrograms/ml or 0.6 microM. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units.

Animals↗
PubMed1989

Distribution and lateral mobility of GABA/benzodiazepine receptors on nerve cells.

The distribution and mobility of GABA/benzodiazepine receptors has been examined on living nerve cells by fluorescence digital imaging and fluorescence photobleach recovery with 2 novel fluorescent derivatives of the benzodiazepines Ro7-1986 and 1012S. These fluorescent derivatives retain their high affinity for the GABA/benzodiazepine receptor complex with Ks of 79 and 85 nM, respectively. Digital fluorescence imaging of GABA/benzodiazepine receptors on rat spinal cord neurons maintained in culture reveal that, even in the absence of observable synaptic contact, GABA/benzodiazepine receptors are localized on the cell body in clusters of very high density. Receptor density on processes is lower than on cell bodies, although occasional areas of intense fluorescence were observed. Fluorescence photobleach recovery measurements show that 70% of the GABA/benzodiazepine receptors on the cell body and greater than 85% on the processes of rat spinal cord neurons are immobile. In comparison, voltage-gated sodium channels are diffusely distributed and very mobile on the cell body (D = 10(-9) cm2/sec). The results suggest that even in the absence of nerve-nerve cell contact, there are specific mechanisms that segregate GABA/benzodiazepine receptors to the neuronal cell body and control their lateral mobility.

4-Chloro-7-nitrobenzofurazan↗
PubMed1988

Distribution and lateral mobility of voltage-dependent sodium channels in neurons.

Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltage-dependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which can be compared with the more diffuse and uniform labeling in the cell body. Using fluorescence photobleaching recovery (FPR) we measured the lateral mobility of voltage-dependent sodium channels over specific regions of the neuron. Nearly all sodium channels labeled with specific neurotoxins are free to diffuse within the cell body with lateral diffusion coefficients on the order of 10(-9) cm2/s. In contrast, lateral diffusion of sodium channels in the axon hillock is restricted, apparently in two different ways. Not only do sodium channels in these regions diffuse more slowly (10(-10)-10(-11) cm2/s), but also they are prevented from diffusing between axon hillock and cell body. No regionalization or differential mobilities were observed, however, for either tetramethylrhodamine-phosphatidylethanolamine, a probe of lipid diffusion, or FITC-succinyl concanavalin A, a probe for glycoproteins. During the maturation of the neuron, the plasma membrane differentiates and segregates voltage-dependent sodium channels into local compartments and maintains this localization perhaps either by direct cytoskeletal attachments or by a selective barrier to channel diffusion.

Animals↗