Binding of tetanus toxin to somatic neural hybrid cells with varying ganglioside composition

125I-labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB-20, followed by NBr-10A, NG108-C15, and SB21-B1 cells. Specific binding of toxin to NCB-20 and SB21-B1 cells is 7- and 60-fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB-20, NBr-10A, and NG108-C15 clones display a complex ganglioside pattern, including the presence of [N-acetyl-neuraminyl]-galactosyl-N-acetylgalactosaminyl[ N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1a) and two unidentified [14C]galactose-labelled lipid-soluble compounds, while the SB21-B1 is most abundant in [N-acetyl-neuraminyl]-galactosylglucosyl-ceramide (GM3) and N-acetyl-galactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosyl-c eramide (GM2) gangliosides. None of the cells tested contain measurable levels of [14C]galactose-labelled or resorcinol-positive bands of galactosyl-N-acetyl-galactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1b) and [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GT1b) gangliosides. After 2 h at 37 degrees C a near plateau of toxin association with NCB-20 cells is seen. Binding in low-ionic-strength medium is 1.35-fold higher at 37 degrees C than at 4 degrees C, but is reduced by 21 and 51% at 4 degrees C and 37 degrees C, respectively, in physiologic medium. Treatment of NCB-20 cells with neuraminidase causes a partial loss (29%) of toxin-binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.     

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.     

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.     

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies

Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC₅₀) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg⁹-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg⁰-HYP³-Thienyl^5,8-D-Phe⁷]-BK (HYP³-antagonist), [D-Arg⁰-HYP³-Thienyl,^5,8-D-Phe⁷]-BK (Thienyl antagonist) and Des-Arg⁹-Leu⁸-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B₂-type of BK receptors. This was confirmed further by the fact that both the B₂-selective Hyp³- and Thienyl-antagonists inhibited BK-induced PI turnover with K_BS of 369±51 nM and 368±118 nM respectively while the B₁-selective antagonist, Des-Arg⁹-Leu⁸-BK, was inactive at 1 M. [³H]BK receptor binding studies revealed two binding sites, one with high affinity (K_d=0.24±0.06 nM; B_max=1.4±0.4 pmol/g tissue) and the other with low affinity (K_d=18.5±0.95 nM; B_max=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [³H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B₂-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [³H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A₂.     

Periodate-Modified Gangliosides Enhance Surface Binding of Tetanus Toxin to PC 12 Pheochromocytoma Cells

The interaction of 125I-labeled tetanus toxin with PC12 pheochromocytoma cells in monolayer cultures has been examined. Under regular growth conditions, the PC12 cells bind 125I-tetanus toxin to a limited degree compared with dissociated cerebral neuron cultures. After exposure to nerve growth factor for 2 days in low serum-containing media with growth factor supplements, binding of toxin increases over twofold compared with untreated PC12 cells. Binding can also be enhanced (greater than 2.5-fold) after treatment of cells with 2 mM sodium metaperiodate for 20 min. Dissociated cerebral neurons but not fibroblasts in cell culture bind more toxin after periodate treatment. The effect of periodate can be abolished by 5 mM sodium borohydride. A ganglioside isolated from periodate-treated PC12 cells and tentatively identified as GT1b [(N-acetylneuraminyl)galactosyl-N-acetylgalactosaminyl(N- acetylneuraminyl-N-acetylneuraminyl)-galactosyl-glucosylceramide] binds 125I-tetanus toxin on silica gel chromatoplates and on nitrocellulose paper. There are no indications to suggest binding to a polypeptide from treated cells after polyacrylamide gel electrophoresis. Cells artificially supplemented with GT1b and subsequently treated with periodate effectively bind the toxin. The data suggest that modified sialyl groups linked to gangliosides, and not to proteins, are preferential targets for tetanus toxin.     

Dihydrocodeinone-hydrazone,dihydrocodeinone-oxime,naloxone-3-ome-oxime,and clocinnamox fail to irreversibly inhibit opioid kappa receptor binding

Previous work from our lab identified two subtypes of the opioid kappa receptor. Whereas the kappa₁ receptor can be labeled by [³H]U69,593 (5, 7, 8-(–)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl]-phenyl-benzeneacetamide), the kappa₂ receptor can be labeled by [¹²⁵I]IOXY (6-¹²⁵iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5-epoxymorphinan). Other data demonstrate that [¹²⁵I]IOXY, like [³H]bremazocine, labels two populations of kappa2 receptors in guinea pig brain: kappa_2a and kappa_2b binding sites. In the present study, we tested the hypothesis that certain dihydrocodeinone and oxicodone derivatives, which have been shown to irreversibly block low affinity [³H]naloxone binding sites, would also bind irreversibly to opioid kappa receptor subtypes. We also tested the novel irreversible mu receptor antagonist, clocinnamox (14-(p-chlorocinnamoylamino)-7,8-dihydro-N-cyclopropylmethylnormorphinone mesylate). Wash-resistant inhibition (WRI) assays were conducted to detect apparent irreversible inhibition. The proportion of WRI attribuable to inhibition of receptor binding, termed receptor inhibition (RI), was calculated by the equation: RI=WRI (wash-resistant inhibition)-SI (supernatant inhibition or inhibition attributable to residual drug.) Dihydrocodeinone-hydrazone, dihydrocodeinone-oxime and naloxone-3-OMe-oxime failed to produce any wash-resistant inhibition of kappa receptor binding. In contrast, preincubating guinea pig membranes with 1 M clocinnamox produced a substantial degree of wash-resistant inhibition (greater than 90%) at kappa₁ and kappa₂ binding sites. However, as indicated by supernatant inhibition values of 70% to 90%, there was a large amount of residual clocinnamox which remained despite the use of an extensive washing procedure. Thus, it is apparent that clocinnamox has essentially no irreversible effect on kappa binding sites. Moreover, these results clearly demonstrate the requirement to determine supernatant inhibition when testing putative irreversible ligands. The apparent inactivity of dihydrocodeinone-hydrazone, dihydrocodeinone-oxime or naloxone-3-OMe-oxime as irreversible inhibitors of kappa receptors suggests that the low affinity [³H]naloxone binding site eliminated by these agents may not be a kappa binding site.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.     

Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1^-1 [-³²P]ATP whereas incubation with 50 mol·1^-1 [-³²P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1^-1 ATP, and around 40 mol·1^-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-³²P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1^-1). The ADP-dependent appearance of ³²P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of ³²P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1^-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.Abbreviations LHCP ligh-harvesting chlorophyll-a/b-binding protein - S_0.5 concentration giving half-maximal phosphorylation - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

M3 muscarinic receptors on murine HSDM1C1 cells: Further functional,regulatory, and receptor binding studies

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M₃-selective radioligand, [³H]4-DAMP, to cell homogenates was characterized. Carbachol (EC₅₀=9 M), (+)muscarine (EC₅₀=4.5 M) and cis-dioxolane (EC₅=0.72 M) were full agonists which stimulated PI turnover by 13.3±1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM–10 M muscarine during the last 24h of [³H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cisdioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. pertussis toxin (5–2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 M) PI turnover. The half-maximal effect of TPA was produced at 1.8±0.3 nM. [³H]4-DAMP binding to cell homogenates was of high affinity (K_d=0.19±0.04 nM) and moderate capacity (B_max=201±22 fmol/mg protein). The pharmacological specificity (4-DAMP>p-FHHSiD>dicyclomine>pirenzepine>methoctramine>AFDX-116 >gallamine) resembled that for [³H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M₃ receptors on HSDM1C1 cells. These receptors have been shown to be influenced by pertussis toxin, an active phorbol ester and to exhibit desensitization.     

Neuronal acquisition of tetanus toxin binding sites: Relationship with the last mitotic cycle

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Using a combined approach of immunocytochemistry and [³H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 ± 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.     

Tetanus toxin receptor Specific cross-linking of tetanus toxin to a protein of NGF-differentiated PC 12 cells

A subclone of rat pheochromocytoma cells expresses high affinity receptors for tetanus toxin on differentiation with NGF [Walton, K.M., Sandberg, K., Rogers, T.B. and Schnaar, R.L. (1988) J. Biol. Chem. 263, 2055–2063]. In the presence of protein cross-linking agents, [¹²⁵I]tetanus toxin, bound to these cells at 0°C, forms a cross-linked product with apparent molecular weight of 120 kDa. The formation of [¹²⁵I]tetanus toxin conjugate involves the heavy chain of the toxin, is prevented by cold toxin and it is largely reduced by pretreating cells with proteases, The cross-linked product is formed only upon incubation of the toxin with NGF-differentiated cells. These results suggest that a protein with apparent molecular weight of 20 kDa is involved in the neurospecific binding of tetanus toxin.     

Killer toxin secretion through the cell wall of the yeastPichia anomala

A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')₂ antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15 gold particles of 15 nm - IEM immunoelectron microscopy - IFA immunofluorescence assay - MAb monoclonal antibody - PBS phosphate buffered saline - SAM/F(ab)₂ sheep antibodies anti-mouse F(ab)₂ - SBB Sabouraud buffered broth     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Histrionicotoxins: Effects on binding of radioligands for sodium,potassium, and calcium channels in brain membranes

A series of eight histrionicotoxins and two synthetic analogs inhibit binding of [³H]batrachotoxinin B to sites on voltage dependent sodium channels in brain membranes. Perhydrohistrionicotoxin (IC₅₀ 0.33 M) and octahydrohistrionicotoxin (IC₅₀ 1.2 M) are comparable in activities to potent local anesthetics. Histrionicotoxin (IC₅₀ 17 M) and the other histrionicotoxins are much less potent. The histrionicotoxins also inhibit binding of [³H]phencyclidine to putative potassium channels in brain membranes. Histrionicotoxin (IC₅₀ 15 M) and the other histrionicotoxins are much more potent than perhydrohistrionicotoxin (IC₅₀ 200 M), but are at least 200-fold less potent than phencyclidine. The histrionicotoxins enhance binding of [³H]nitrendipine to sites on calcium channels in brain membranes, with the exception of perhydrohistrionicotoxin, which inhibits binding. Structure activity relationships at these channel sites and at the sites for noncompetitive blockers on the nicotinic acetylcholine receptor channel (AChR) complex differ. The histrionicotoxins are more potent at the sites on the AChR complex than at sites on other channels with the exception of perhydrohistrionicotoxin, which has comparable potency at the AChR complex and sodium channels.     

Interaction between new neoglycoproteins and thed-Man/l-fuc receptor of rabbit alveolar macrophages

New types of neoglycoproteins, -caseins coupled with ovalbumin-derived asparagine oligosaccharides (AO), aspartate aminotransferase-phosphopyridoxylated AO complex (AAT-PG), and streptavidin-biotinylated AO complex (SA-BAO), were tested for their inhibitory effect on binding of bovine serum albumin derivatized with thiomannoside, Man-AI-BSA [Lee YC, Stowell CP, Krantz MJ (1976) Biochemistry 15:3956–63] by rabbit alveolar macrophages. The -casein derivatives and the AAT-PG complex increased binding affinity as the number of oligosaccharide chains attached was increased. Their inhibitory potencies were closely related to those of the Man-Al-BSA derivatives [Hoppe CA, Lee YC (1983) J Biol Chem 258:14193–99] on the basis of terminal mannose density. The SA-BAO complex containing three AO chains gave stronger inhibitory potency than the -casein derivative with three AO residues, suggesting that proper orientation of the oligosaccharides on the protein can affect the receptor-ligand interaction.Abbreviations BSA bovine serum albumin - Man₄₃-BSA BSA derivative containing on average 43 residues of Man linked through an amidino-linkage [7] - AO asparagine oligosaccharide (Man₅-GlcNAc₂-Asn) from ovalbumin - AAT aspartate aminotransferase - PG phosphopyridoxylated AO - SA streptavidin - BAO biotinylated AO     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.