A search for an 'ouabain-like' substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids.

The electric organ of Electrophorus electricus contains substances which inhibit (Na+ + K+)-ATPase activity, the specific binding of [3H]ouabain to purified (Na+ + K+)-ATPase and 86Rb+ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous 'ouabain-like' activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.

Biosynthesis and posttranslational processing of the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line. Effect of dexamethasone.

Neurotensin (NT) and Neuromedin N (NN) are two biologically active peptides present in one copy each in the C-terminal region of a 169-residue precursor. Four basic Lys-Arg doublets occur within the precursor and represent putative processing sites. We investigated the effects of dexamethasone on the biosynthesis and the posttranslational processing of the NT/NN precursor in the rat medullary thyroid carcinoma 6-23 cell line (rMTC 6-23). Western blot analysis and RIA coupled to HPLC and arginine-directed tryptic cleavage of precursor forms were performed with antisera specific for precursor sequences adjacent to the four basic doublets. These studies revealed that rMTC 6-23 cells synthesized the NT/NN precursor in response to dexamethasone and had the capability to process this precursor at the three Lys-Arg doublets that flank and separate NT and NN, thus yielding authentic NT, NN, and several larger products. The most N-terminal Lys-Arg doublet was not processed in this system. Dexamethasone increased in a concentration- and time-dependent manner the levels of all the NT/NN precursor-derived products. This increase did not affect the relative proportion of the different products. We also showed by Northern blot analysis that both the 1.1-kilobase and 1.5-kilobase NT/NN precursor messenger RNAs were present in the rMTC 6-23 cell line and that the time course and dose response of dexamethasone-induced messenger RNA synthesis were in good agreement with those observed for dexamethasone-induced increase in processing products. The rMTC 6-23 cell line represents a good model to elucidate the steps involved in the posttranslational processing of the NT/NN precursor.

Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel.

Binding studies indicate that ciguatoxin and brevetoxin allosterically enhance in a very similar way the binding of [3H]batrachotoxinin A 20-alpha-benzoate to the neuronal Na+ channel protein. Moreover ciguatoxin competitively inhibits the binding of [3H]brevetoxin-3 to rat brain membranes. The affinity of ciguatoxin for the Na+ channel is at least 20-50-times higher than that of brevetoxin. These results indicate that ciguatoxin and brevetoxins act at the same binding site on the sodium channel.

Charybdotoxin blocks dendrotoxin-sensitive voltage-activated K+ channels.

Charybdotoxin, a short scorpion venom neurotoxin, which was thought to be specific for the blockade of Ca2+-activated K+ channels also blocks a class of voltage-sensitive K+ channels that are known to be the target of other peptide neurotoxins from snake and bee venoms such as dendrotoxin and MCD peptide. Charybdotoxin also inhibits 125I-dendrotoxin and 125I-MCD peptide binding to their receptors. All these effects are observed with an IC50 of about 30 nM.

Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat--I. Biochemical characterization of maturation products.

Neurotensin and neuromedin N are two biologically active related peptides which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are closest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present studies were aimed at determining the extent to which each of these four dibasic sites is cleaved and at identifying and quantifying the intermediate and mature products to which this cleavage gives rise in extracts from whole rat brain, hippocampus and globus pallidus. This was achieved by means of radioimmunoassays specific for sequences of the neurotensin/neuromedin N precursor that are adjacent to the dibasic processing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all tissue extracts, it was found that the three most C-terminal dibasic processing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neuromedin N-like sequence is processed to a lesser extent. As reported previously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

PC12 cells can be induced to produce, but do not process, the neurotensin/neuromedin N precursor.

Neurotensin and neuromedin N are two biologically active, related peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences, which are thought to represent putative processing sites, occur in the precursor molecule. Studies by others have shown that rat pheochromocytoma PC12 cells produced neurotensin and dramatically increased their neurotensin/neuromedin N precursor mRNA content in response to a combination of nerve growth factor, dexamethasone, forskolin, and Li+. Here, we investigated the effects of this combination of inducers on the posttranslational processing of the neurotensin/neuromedin N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and arginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doublets. Thus, mature neurotensin and neuromedin N represented less than 1% of the total precursor content in PC12 cells. The PC12 cell line may represent an interesting model with which one could transfect the recently cloned prohormone convertases PC1 and PC2, thereby allowing the study of the role of these enzymes in the processing of the neurotensin/neuromedin N precursor.

High affinity receptors for the bee venom MCD peptide. Quantitative autoradiographic localization at different stages of brain development and relationship with MCD neurotoxicity.

High densities of MCD receptors were found in the stratum radiatum of Ammon's horn, the neocortex, the molecular layer of the cerebellum, colliculi and pons. Conversely areas such as the stratum lacunosum moleculare of Ammon's horn contained only low levels of MCD binding sites. The density of MCD receptors is low during the perinatal period and increases rapidly by postnatal day 10 with a decrease of the receptor affinity for MCD. The adult distribution of MCD receptors was reached at postnatal day 30. Increases in density of MCD receptors are discussed in relation with increased neurotoxicity of MCD during brain development. Effects of MCD during the perinatal period are very weak. However, the threshold MCD dose to induce seizures drastically decreased after the first postnatal week. The efficient dose corresponding to adult stage is reached after postnatal day 40.

The brain response to the bee venom peptide MCD. Activation and desensitization of a hippocampal target.

The mast cell-degranulating peptide (MCD) isolated from bee venom has been found previously to have receptor sites in rat brain. Behavioral and electrocorticographic responses following intracerebroventricular injections of various doses of MCD have been analyzed. MCD produced a quasi-permanent hippocampal theta rhythm in the motionless animal alternating with epileptiform spike waves and paroxystic seizures. At a dose of 70 pmol seizures occurred for half of the treated rats. At a dose of 100 pmol generalized paroxystic crises were observed for all the rats. These effects were not antagonized by naloxone, morphine, diazepam and progabide. Rats recovered 24 h after a 100 pmol injection of MCD. A second ipsilateral injection to these rats showed the occurrence of a desensitization phenomenon. Desensitization was not observed when the second injection was contralateral. These physiological responses were studied in relation with a biochemical approach on membrane sites of action of MCD using [125I]MCD and their behavior in the desensitization process. The target of [125I]MCD is the ipsilateral hippocampus. Recovery from MCD effects was not due to MCD degradation. Desensitization was not due to down-regulation of the MCD receptor level.

Subtypes of K+ channels differentiated by the effect of K+ channel openers upon K+ channel blocker-induced seizures.

Intracerebroventricular injection of mast-cell degranulating peptide (MCD), dendrotoxin I (DTXI) and 4-aminopyridine (4-AP), 3 blockers of a subclass of K+ channel, produces seizures and convulsions. Three different K+ channel openers are potent blockers of MCD-induced hyperexicitatory effects when they are administered preventively but they are unable to inhibit the epileptogenic effects induced by DTXI and 4-AP which were thought to block the same K+ channel which is blocked by MCD.

Analogies and differences in the mode of action and properties of binding sites (localization and mutual interactions) of two K+ channel toxins, MCD peptide and dendrotoxin I.

Both the bee venom toxin, mast cell degranulating (MCD) peptide, and the snake toxin, dendrotoxin 1 (DTX1) induce epileptiform activity and paroxystic seizures after intracerebroventricular (i.c.v.) injection to rats. Although many of the properties of the two toxins, which are blockers of the same K+ channel, appear to be very similar, a number of differences have been found. (1) Induced seizures have an hippocampal origin for MCD and two different origins, situated in the cortex and in the limbic system, for DTX1. (2) A first i.c.v. administration of DTXI desensitizes against a second ipsilateral injection of the same peptide as we had previously observed for MCD. However no cross-desensitization was observed between the two different toxins. (3) The number of high affinity (Kd = 41 pM) binding sites for 125I-DTXI in synaptic membranes is about 5 times higher than the number of high affinity (Kd = 158 pM) binding sites for 125I-MCD. (4) Autoradiographic analysis of the distribution of high affinity 125I-DTX1 binding sites has been compared to our previous analysis of high affinity 125I-MCD binding sites. High levels of high affinity binding sites for both toxins seem to be localized in synapse-rich areas. However high affinity binding sites for the two toxins are not always co-localized. Analysis of the mutual interactions between DTXI and MCD binding sites has revealed the presence of classes of low affinity binding sites for MCD. In most areas of the brain, a large proportion of high affinity binding sites for DTXI is allosterically related to low affinity binding for MCD.

Neurotensin and neuromedin N brain levels after fornix transection: evidence for an efficient neurotensin precursor processing in subicular neurons.

High levels of neurotensin/neuromedin N precursor mRNA, but few if any NT-positive perikarya have been detected in the dorsal subiculum of the adult rat or human hippocampus. This apparent discrepancy was tentatively ascribed to a lack of precursor mRNA translation or to a poor precursor posttranslational processing in neurons of the hippocampus. Another hypothesis is that in long neuronal pathways, maturation of neuropeptide precursors and derived peptides occurs during axonal transport to terminals, a process which accounts for the lack of peptide detection in cell bodies. In order to test this hypothesis, we performed surgical transection of the fornix to interrupt axonal transport of putative NT/NN products arising from the dorsal hippocampus and measured NT and NN levels in different brain regions. In the mamillary bodies, the main projection area of the dorsal subiculum, NN and NT levels were highly reduced 4 or 14 days after the septo-hippocampal transection which was correlated with a slight increase in NN and NT levels in the dorsal hippocampus and the retrosplenial cortex of 4 days lesioned animals. An increase in hypothalamic NN levels was also detected 14 days after the lesion. These data suggest that the peptide precursor processing can take place during the axonal transport, as shown here for neurotensin and neuromedin N from subicular neurons to their efferent brain areas such as the mamillary bodies.

Ca2+ channel blockers prevent seizures induced by a class of K+ channel inhibitors.

Intracerebroventricular injection into rats of mast-cell degranulating peptide (MCD), dendrotoxin I (DTXI) and 4-aminopyridine (4-AP), three blockers of a subclass of K+ channels, elicited epileptiform wave bursts and convulsions. Three different types of L-type Ca2+ channel inhibitors (+)PN 200-110, a 1,4-dihydropyridine, (-)D888, a phenylalkylamine, and fluspirilene, a diphenylbutylpiperidine, were potent blockers of the convulsant-induced hyperexcitatory effects when they were administered preventively. D-AP5, a N-methyl-D-aspartate antagonist, was active on the 4-AP-induced seizures but was without effect on the MCD- and dendrotoxin-induced seizures.

Biochemical properties of the brain phencyclidine receptor.

This paper gives a detailed account of techniques which can be used to measure [3H]phencyclidine binding to its receptor. The main properties of the binding component are the following: (i) It is rapidly heat-inactivated at temperatures over 50 degrees C. (ii) It is destroyed by proteases like trypsin, pronase or papain suggesting that it is of a protein nature. The receptor structure is resistant to chymotrypsin. (iii) A good correlation was found between the pharmacological activity of 30 different analogs as measured by the rotarod assay and the affinity of these different molecules for the phencyclidine receptor. (iv) Monovalent and divalent cations antagonize [3H]phencyclidine binding to its receptor. The dissociation constant is 15 mM, the same for Na+, Li+, K+, cholinium or Tris. Na+ (and other monovalent cations) and phencyclidines bind to distinct sites. The saturation of the Na+ site by Na+ modulates the affinity of phencyclidine for its receptor. Divalent cations antagonize [3H]phencyclidine binding in the absence of Na+. This antagonism is of the non-competitive type. (v) [3H]phencyclidine binding is also antagonized by histrionicotoxin and by local anaesthetics.

Purification and pharmacological characterization of peptide toxins from the black mamba (Dendroaspis polylepis) venom.

This paper reports the purification of 28 different peptides from the venom of the snake Dendroaspis polylepis. These peptides represent 99% of the total peptide fraction in the venom. The 14 most cationic peptides form a structurally and functionally homogeneous group of analogs of the most abundant dendrotoxin toxin I (DTXI). They recognize antibodies raised against DTXI as well as brain membrane binding sites corresponding to K+ channels that are sensitive to DTXI and the bee venom peptide MCD. Similarly to DTXI these 14 peptides induce convulsions after intracerebroventricular injections in mice and induce GABA release from synaptosomes. However, members in this iso-DTXI family differ widely in their affinity for the DTXI/MCD receptors and in their contractility promoting action on intestinal smooth muscle. The 14 other less cationic peptides do not interact with the DTXI receptor or with DTXI antibodies and they do not evoke GABA release. Their targets seem to be essentially of a peripheral nature. Half of them contract guinea pig ileum. In this group of toxins there might be new tools to study membrane excitability.

[Identification of 4-O-methyldopamine in rat tissues by reverded-phase liquid chromatography (author's transl)]. / Identification de la 4-O-méthyl dopamine dans les tissus de rat par chromatographie liquide en phase inverse.

4-O-Methyldopamine was identified and assayed in tissues from L-dopa treated rats by reversed-phase high-performance liquid chromatography. The initial steps in the separation of catecholamines were performed by alumine, a weak cation-exchange resin, and thin-layer chromatographic techniques. After L-[3 H] dopa administration, the radiochromatogram was superimposed on the fluorochromatogram obtained with authentic marker 4-O-methyldopamine. This metabolite was detected in kidney but not in brain. The 4-O-methyldopamine:3-O-methyldopamine ratio was 0.032 in kidney. The influence of various treatments on this ratio was investigated. A 160% increase was found after L-dopa administration. This effect was potentiated by nialamide pretreatment (550% increase).

[Fluorimetric assay of 3-o-methyldopamine and 4-0-methyldopamine in rat urine and o-methylation of dopamine]. / Dosage fluorimétrique de la 3-O-méthyldopamine et de la 4-O-méthyldopamine dans l'urine de rat et O-méthylation de la dopamine

A new method for the isolation and determination of 3-O-methyldopamine (3 MD) and 4-O-methyldopamine (4 MD) in the urine of Rat has been described. The administration of L-dopa enabled us to detect 4 MD in the urine with a molar ratio 4 MD/3 MD = 0.07. This ratio decreased with the simultaneous treatment of S-adenosylmethionine (SAM). This result might explain the therapeutic L-dopa+SAM in the treatment of parkinsonism, 4 MD being neurotoxic. The low level of 4 MD compared with the level of isovanillic acid, found by other authors, seems to indicate that the 4-O-methylation pathway takes place via dihydroxyphenylacetic acid.